Do antimuscarinic drugs increase cAMP or cGMP

Do antimuscarinic drugs increase cAMP or cGMP

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Activation of muscarinic receptors M2 and M4 inhibits adenylate cyclase which reduces cAMP levels. It would be expected that antimuscarinics such as ipratropium would increase cAMP levels. However, the pharmacological action of ipratropium is said to be mediated through increased cGMP levels [1]. Why is this the case?


Cyclic dinucleotides bind the C-linker of HCN4 to control channel cAMP responsiveness

cAMP mediates autonomic regulation of heart rate by means of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which underlie the pacemaker current If. cAMP binding to the C-terminal cyclic nucleotide binding domain enhances HCN open probability through a conformational change that reaches the pore via the C-linker. Using structural and functional analysis, we identified a binding pocket in the C-linker of HCN4. Cyclic dinucleotides, an emerging class of second messengers in mammals, bind the C-linker pocket (CLP) and antagonize cAMP regulation of the channel. Accordingly, cyclic dinucleotides prevent cAMP regulation of If in sinoatrial node myocytes, reducing heart rate by 30%. Occupancy of the CLP hence constitutes an efficient mechanism to hinder β-adrenergic stimulation on If. Our results highlight the regulative role of the C-linker and identify a potential drug target in HCN4. Furthermore, these data extend the signaling scope of cyclic dinucleotides in mammals beyond their first reported role in innate immune system.


Nitric oxide (NO) was first described by Stuehr and Marletta (1985) 1 as a product of activated murine macrophages. Also, the substance known as endothelium-derived relaxing factor (EDRF), described by Furchgott and Zawadzki (1980), 2, 3 has been identified as NO.

Soluble guanylate cyclase (sGC), responsible for the enzymatic conversion of guanosine-5-triphosphate (GTP) to cyclic guanosine-3′,5′-monophosphate (cGMP), was first identified as a constituent of mammalian cells almost three decades ago. 4

NO and cGMP together comprise an especially wide-ranging signal transduction system when one considers the many roles of cGMP in physiological regulation, including smooth muscle relaxation, visual transduction, intestinal ion transport, and platelet function. 5

Erectile dysfunction (ED) is defined as the consistent inability to achieve or maintain an erection sufficient for satisfactory sexual performance, and is considered to be a natural process of aging. 6 Studies have shown that ED is caused by inadequate relaxation of the corpus cavernosum with defect in NO production. 7

It is clear that NO is the predominant neurotransmitter responsible for cavernasal smooth muscle relaxation and hence penile erection. Its action is mediated through the generation of the second messenger cGMP. Neurally derived NO has been established as a mediator of smooth muscle relaxation in the penis, and it is thought that constitutive forms of nitric oxide synthase (NOS) work to mediate the erection. 8 Released NO activates sGC, which catalyzes the conversion of GTP to the intracellular second messenger cGMP in smooth muscle cells. An increase in cGMP modulates cellular events, such as relaxation of smooth muscle cells. 9

This review will describe current knowledge of cellular events involved in cavernosal relaxation and the range of putative factors involved in NO-mediated relaxation.


Cyclic GMP (cGMP) made in response to atrial natriuretic peptide (ANP) or nitric oxide (NO) is an important regulator of short-term changes in smooth muscle tone and longer-term responses to chronic drug treatment or proliferative signals. The ability of smooth muscle cells (SMCs) to utilize different combinations of phosphodiesterase (PDE) isozymes allows cGMP to mediate these multiple processes. For example, PDE5 as a major cGMP-hydrolyzing PDE effectively controls the development of smooth muscle relaxation. In order for contraction to occur, PDE5 is activated and cGMP falls. Conversely, blockade of PDE5 activity allows the relaxation cycle to be prolonged and enhanced. A recently shown direct activation of PDE5 by cGMP binding to the GAF A domain suggests that this regulatory site might be a target for new drug development. The calcium surge associated with vasoconstrictor initiated contraction also activates a calcium/calmodulin-dependent PDE (PDE1A). Together, PDE5 and PDE1A lower cGMP sufficiently to allow contraction. Longer term, both PDE5 and PDE1A mRNA are induced by chronic stimulation of guanylyl cyclase. This induction is a major cause of the tolerance that develops to NO-releasing drugs. Finally, high levels of cGMP or cAMP also act as a brake to attenuate the proliferative response of SMCs to many mitogens. After vessel damage, in order for SMC proliferation to occur, the levels of cGMP and cAMP must be decreased. In humans, this decrease is caused in large part by induction of another Ca 2+ /calmodulin-dependent PDE (PDE1C) that allows the brake to be released and proliferation to start.

The cyclic nucleotide second messengers, cAMP and cGMP, have been shown to regulate a wide variety of processes in many different tissues of the body and have been suggested to regulate many more. In fact, they have been proposed to modulate so many different processes that, until recently, it has been difficult to understand how these simple, small, second messenger molecules could provide both the specificity of action and the diversity of function necessary for such regulation. Particularly problematic has been an understanding about how both very rapid and very slow processes can be modulated by the same mechanisms.

A major conceptual advance in our understanding of the mechanisms by which such temporally and spatially disparate processes can be controlled was the realization that many different isozymes for synthesis (cyclases) and degradation (phosphodiesterases, PDEs) of cAMP and cGMP are present in the organism. 1 For example, at least 10 different adenylyl cyclase genes and nearly 20 different PDE genes have been identified in mammalian species. Any particular cell type might express two or three different cyclases and three or four different PDEs. More importantly, the number of possible combinations that can be expressed in any one compartment of the cell is very large. Therefore the differential expression and localization of unique combinations of synthetic and degradative enzymes provide each cell with molecular solutions for these problems.

In arterial smooth muscle, a number of processes are controlled by cAMP and cGMP. They include metabolic and mechanical events that are regulated on a relatively rapid time scale. The contractile tone of the muscle is perhaps the best example of this. 2 Both cAMP and cGMP cause smooth muscle relaxation in large part through their effects to lower intracellular calcium or activate myosin phosphatase. Slower changes that are regulated by cyclic nucleotides include altered proliferation in response to injury or even longer-term desensitization to chronic stimulation by drugs or hormones. As with all regulatory messengers, the amplitude and duration of the cyclic nucleotide signals are governed by their rates of synthesis and rates of degradation. In this article, we review some of the current thoughts about the regulation of smooth muscle function by cyclic nucleotides and especially the roles played by the phosphodiesterases that either hydrolyze or respond to cGMP.

Regulation of Smooth Muscle Function by Cyclic Nucleotide Phosphodiesterases

There are now known to be 11 different PDE gene families expressed in mammalian tissues (Figure 1). Most families contain more than one gene and most genes code for more than one mRNA (by alternative splicing or alternative transcriptional start sites). Depending a bit on the species, the major phosphodiesterases present in arterial smooth muscle are PDEs1A, 1B, and 1C, PDEs3A and 3B, and PDE5. In a few species a substantial amount of PDE4 activity is also expressed in smooth muscle. As PDE4 is specific for cAMP and not regulated by cGMP, we will not discuss its role further in this review. Under basal conditions (ie, low calcium levels), it is thought that the most active cGMP hydrolyzing PDE in smooth muscle is the cGMP-specific, cGMP-binding PDE, PDE5. Under higher calcium conditions (eg, during muscle contraction and possibly in cells being stimulated to divide), one or more of the PDE1 variants can become the predominant PDE. Although PDE3 does not have as great a total catalytic capacity as the other two, it may still play a role in controlling cAMP and perhaps cGMP in specific compartments of the cell. This enzyme is strongly inhibited by cGMP and has been termed the cGMP-inhibited PDE in many previous studies. In all cases, it must be remembered that these various PDEs do not necessarily share the same subcellular localization and therefore often subserve, at least in part, different functional compartments in the cell.

Figure 1. Domain organization of 11 PDE gene families. All PDEs share significant homology in their catalytic domain but differ greatly in their N-terminal parts, which contain different types of regulatory domains. UCR domain is an upstream conserved region found only in PDE4. GAF and PAS domains are derived from the first letters of initial members of their corresponding groups. GAF domains (c G MP-regulated phosphodiesterases, several a denylyl cyclases, and F hlA) were originally defined in PDE2, PDE5, and PDE6 and later shown in PDE10 and PDE11. PAS domain ( P er, A RNT, and S im proteins) was found only in PDE8.

PDE5: cGMP-Specific, cGMP-Binding Phosphodiesterase

PDE5 and Smooth Muscle Relaxation

It is well established that nitric oxide (NO), atrial natriuretic peptide (ANP), and several other endogenous vasodilators regulate smooth muscle tone through activation of guanylyl cyclase, elevation of cGMP, and activation of cGMP-dependent protein kinase (PKG) (Figure 2). NO/cGMP effects on contraction in smooth muscle appear to be mediated specifically by PKG but not cAMP-dependent protein kinase (PKA), because in PKG-I-deficient mice, cGMP-induced relaxation of aortic smooth muscle is completely abolished, whereas cAMP-dependent relaxation is not affected. 2 There are several specific physiological substrates for PKG in smooth muscle including the regulatory myosin-binding subunit of myosin phosphatase, 3 calcium-activated maxi K + (BKCa) channels, 4 and IRAG (IP3 receptor associated cGMP kinase substrate). 5 Phosphorylation of all of these targets contributes to a reduction of intracellular Ca 2+ concentration or reduction in sensitivity to Ca 2+ and thereby decreased smooth muscle tone. 6

Figure 2. cGMP is an important regulator of smooth muscle function. Nitric oxide (NO) and other endogenous vasodilators regulate smooth muscle tone through the cGMP/PKG signaling pathway. I, PDE5 effectively controls the development of smooth muscle relaxation. Blocking PDE5 activity allows smooth muscle to enhance and prolong the relaxation cycle. II, Longer-term responses to chronic nitroglycerin treatment (nitro-tolerance) produce changes in PDE1A and PDE5 and, thus, less responsiveness to cGMP. III, PDE1C is induced only in human smooth muscle cells of the proliferative phenotype and its inhibition results in suppression of SMCs proliferation.

PDE5, as a major cGMP-hydrolyzing PDE expressed in smooth muscle cells, is in a position to effectively control this cGMP/PKG signaling pathway, especially under conditions of low calcium. As discussed later, under conditions of higher calcium, PDE1s likely play an increasingly important role. PDE5 has been found in all types of vascular and visceral (uterus, small intestine) SMCs. The physiological importance of PDE5 in regulation of smooth muscle tone has been most effectively demonstrated by the successful clinical use of its specific inhibitor, sildenafil (Viagra, Pfizer Pharmaceuticals), in the treatment of erectile dysfunction. 7 Relaxation of the smooth muscle of the corpus cavernosum is induced by NO release from endothelial cells and noncholinergic nonadrenergic (NANC) neurons surrounding the arteries and sinusoids in the corpora cavernosa. By inhibiting PDE5 hydrolytic activity sildenafil causes a higher rate of accumulation of cGMP in response to the NO, thus enhancing the erectile response. 8

PDE5 and Pulmonary Vasculature

Recently, the treatment of pulmonary hypertension has emerged as a new potential area for clinical application of PDE5 inhibitors. Pulmonary hypertension is a life-threatening disease characterized by high pulmonary arterial pressure and vascular resistance. 9 In the pulmonary vasculature, NO plays an important role as a vasorelaxant. Currently, inhaled nitric oxide is one of the more effective therapies for treatment of pulmonary hypertension. 10 One of the advantages of exogenously administered NO is that it has little effect on systemic blood pressure. However, the half-life of NO is relatively short, and therefore, effective NO treatment requires its multiple administration. Unfortunately, tachyphylaxis commonly is seen within a few days.

The use of sildenafil for treatment for pulmonary hypertension has shown positive results in humans. A clinical study of patients with severe primary pulmonary hypertension showed a dramatic improvement of pulmonary systolic pressure after treatment with oral sildenafil. 11 In another report sildenafil was found to be a potent pulmonary vasodilator and superior to inhaled NO in decreasing pulmonary artery pressure and reducing pulmonary vascular resistance. 12 The combination of sildenafil and NO treatment produced an even larger, synergistic effect.

PDE5 and the Systemic Vasculature

Originally, sildenafil was tested as an antianginal drug, targeting PDE5 in the systemic vasculature. 13 However, in early clinical trials it soon became evident that sildenafil had only a modest effect on reduction of systemic blood pressure. Nevertheless, this small effect may turn into severe hypotension for patients taking a combination of sildenafil and nitroglycerin or other organic nitrates. This is consistent with the widespread occurrence of PDE5 in all smooth muscle beds. Because sildenafil is able to greatly potentiate the effects of NO generating compounds, use of sildenafil is contraindicated for most patients also using any organic nitrate. 14

Thus, as with all PDE5 inhibitors, sildenafil is most effective when the NO/cGMP signaling pathway is activated, suggesting that the clinical use of these inhibitors might be potentially expanded to other diseases associated with changes in cGMP signaling.

Recently, the development of two other PDE5 specific inhibitors, tadalafil (Cialis, Lilly ICOS LLC) and vardenafil (Levitra, Bayer and GlaxoSmithKline), has been reported. 15,16 Each is able to inhibit PDE5 activity with IC50s in the nmol/L concentration range, and currently, both are being used for treatment of erectile dysfunction. They each have very similar affinities for PDE5 but vary somewhat in their pharmacokinetics and selectivity toward other PDEs. 17 It is not yet clear if these differences will provide any advantages in clinical efficacy or appearance of side effects.

PDE5 Splice Variants and Domain Organization

Three different isoforms of PDE5A have been reported, PDE5A1, PDE5A2, and PDE5A3 (Table). All PDE5 variants differ only at the N-terminal end. The first PDE5 to be purified to homogeneity was the soluble enzyme from bovine lung. 18 Originally called cGMP-binding, cGMP-specific PDE, this isoform is now known as PDE5A1. It appears to be the predominant form expressed in most PDE5 containing tissues. Human PDE5A1 is very similar to bovine PDE5A1, except that it contains an additional 10 amino acid insert at the N-terminal end. 19–21 Another variant, PDE5A2, contains a significantly shorter amino acid N-terminal fragment and also has been shown in several species. 22 PDE5A3 has been reported only in human tissues based on RT-PCR data. 23

Table 1. PDE5 Isoforms Differ in Their N-Terminal Ends

PDE5 is highly specific for cGMP hydrolysis and contains two homologous N-terminal regulatory domains, recently defined as GAF A and GAF B based on their sequence homology with similar regulatory motifs now known to be present in a large group of proteins. 24 The initial members of this group included the c G MP-regulated phosphodiesterases (PDE2, PDE5 and PDE6), several a denylyl cyclases, and a bacterial transcription factor called F hlA. The acronym, GAF, is derived from the first letters of these groups. A recent search of the database shows that nearly a thousand other proteins contain GAF domains. Many, however, are expressed only in prokaryotes.

The GAF A domain of PDE5 is most homologous to the GAF B domain of PDE2 that also binds cGMP with high specificity. Very recently the crystal structure of the GAF A/B domains of PDE2 with cGMP bound has been solved. 25 It is thought that the structure of the PDE5 GAF domains will be very similar.

Mechanisms for Regulation of PDE5 Activity

PKG Induced Phosphorylation of PDE5

Several years ago it was shown by in vitro studies that PDE5 is phosphorylated on its N-terminal part (serine 92) and that binding of cGMP to a noncatalytic GAF domain of PDE5 was necessary for maximal rates of phosphorylation by PKA or PKG. 26,27 This phosphorylation site is conserved in all PDE5 isoforms including bovine, human, canine, mouse, and rat PDE5. Nevertheless, questions remained about whether phosphorylation had any physiological significance in vivo. It was soon shown that in cultured rat smooth muscle cells 32 P could be incorporated into PDE5 after prelabeling with 32 P-ATP and treatment with ANP. 28

More recently, a more quantitative approach using antibodies specific for the phosphorylation site of PDE5 showed a good correlation of activity with increased phosphorylation by PKG but not PKA both in vitro and in intact cells. 29 Addition of 8-Br-cGMP to cultured human smooth muscle cells led to a gradual accumulation of the phosphorylated form of PDE5 (Figure 3A). When phosphorylation of PDE5 was compared with phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a well-characterized substrate for PKA and PKG, it was found that the time-course of PDE5 phosphorylation was similar to that of VASP. It was demonstrated that after PKG activation 25% to 30% of the total PDE5 was phosphorylated, and that immunoprecipitated PDE5 had 2 to 2.5 times higher activity over the nonphosphorylated basal form when assayed at a cGMP substrate concentration of 1 μmol/L.

Figure 3. In intact cells, activation of PKG, but not PKA, leads to PDE5 phosphorylation. A, 1 mmol/L 8-Br-cGMP was added to the cultured human uterine smooth muscle cells, and cells were harvested at different times after addition of 8-Br-cGMP. Phospho-PDE5 accumulation was detected by Western blot analysis using a phospho-specific PDE5 antibody. Phosphorylation of PKG-preferable site on VASP, serine 239, was detected by using monoclonal phospho-serine 239-specific VASP antibody. B, Mouse aortic smooth muscle cells from control mice (+/+) and PKG I-deficient mice (−/−) were incubated with 1 mmol/L 8-Br-cGMP (1), 1 mmol/L 8-Br-cAMP (2), or both (3) for 30 and 60 minutes. Phospho-PDE5 accumulation was detected by Western blot analysis using a phospho-specific PDE5 antibody. Adapted from Rybalkin SD, Rybalkina IG, Feil R, Hofmann F, Beavo JA. Regulation of cGMP-specific phosphodiesterase (PDE5) phosphorylation in smooth muscle cells. J Biol Chem. 2002277:3310–3317.

To answer the question of protein kinase specificity for PDE5 in intact SMCs, aortic SMCs from mice having a disruption in the PKG I gene were used. Incubation of PKG I −/− cells with 8-Br-cGMP did not produce any phosphorylation of PDE5, whereas in PKG I +/+ cells a significant phosphorylation of PDE5 was observed (Figure 3B). Addition of 8-Br-cAMP alone or a combination of both 8-Br-cAMP and 8-Br-cGMP did not stimulate PDE5 phosphorylation in PKG I −/− aortic SMCs. These experiments provide unambiguous evidence that PDE5 phosphorylation in vivo is predominately mediated through the cGMP/PKG I, and not through the cAMP/PKA pathway. 29

In related studies, PDE5 has been shown to be an important regulator of cGMP signaling in platelets. In these cells NO donors like GSNO (S-nitrosoglutathione) produce an extremely rapid increase followed by a rapid decline of intracellular cGMP. A time-dependent change of PDE5 activity and phosphorylation correlated with this rapid decline although no desensitization of guanylyl cyclase was observed. 30

CGMP Binding to the GAF Domain Directly Activates PDE5 Catalytic Activity

Because cGMP binding to PDE2 causes activation of its catalytic activity, 31 the question arose whether or not cGMP binding to the GAF domain of PDE5 causes a similar activation. Although suspected, for many years no evidence had been found to support this idea.

One approach to this problem made use of a fluorescent analog of cGMP (Ant-cGMP-2′-O-anthraniloyl cGMP), which bound poorly to the GAF domain but was a reasonable substrate for the catalytic domain. 32 However, activation of catalytic activity by cGMP binding was not directly demonstrated.

More recently, direct PDE5 activation upon cGMP binding to the regulatory GAF A domain was reported in a study using recombinant PDE5. When PDE5 was preincubated with cGMP, a large (up to 10-fold), time-dependent activation of PDE5 was observed (Figure 4A). 33 PDE5 could also be reactivated after addition of another portion of cGMP to the preincubation mixture. cGMP-induced PDE5 activation was the highest when PDE5 activity was assayed at 0.1 μmol/L cGMP (9 to 11 times). At 1.0 μmol/L cGMP, PDE5 activation was only 2- to 3-fold, and at 10 μmol/L cGMP, no activation was detected (Figure 4B). The observed activation of PDE5 was not a result of PDE5 phosphorylation because mutation of the phosphorylation site (serine 92) to alanine did not change the pattern of PDE5 activation by cGMP.

Figure 4. A, PDE5 is directly activated by cGMP binding to the GAF A domain of PDE5. Mouse recombinant PDE5 expressed in HEK 293 cells was preincubated with 50 μmol/L cGMP on ice (filled triangles), and after appropriate dilutions, PDE5 activity was assayed with 0.1 μmol/L cGMP for 5 minutes at 30°C. An additional portion of 50 μmol/L cGMP was added to the preincubation mixture at 60 minutes after the start of preincubation as indicated by an arrow (open triangles). PDE5 activity was expressed as pmol · min −1 · μg −1 of protein. B, Pretreatment of PDE5 with mAb/P3B2 blocks cGMP-induced PDE5 activation and lowers basal PDE5 activity. PDE5 activity was measured at 0.1 μmol/L, 1.0 μmol/L, or 10 μmol/L cGMP. Samples were analyzed without any treatments (control) or after preincubation with 50 μmol/L cGMP on ice (+cGMP), or after preincubation with mAb/P3B2 for 30 minutes on ice (+mAb/P3B2), or with mAb/P3B2 for 30 minutes and then with 50 μmol/L cGMP (+mAb/P3B2+cGMP). PDE5 activity was expressed as percent of control, and control (nonstimulated) PDE5 activity was defined as 100%. Adapted from Rybalkin SD, Rybalkina IG, Shimizu-Albergine M, Tang X-B, Beavo JA. PDE5 is converted to an activated state upon cGMP binding to the GAF A domain. EMBO J. 200322:469–478.

To verify that the effect of PDE5 activation was due to a direct effect of cGMP occupancy of the cGMP-binding sites, a mouse monoclonal antibody (mAb/P3B2), generated against the GAF domain of PDE5, was found to be able to substantially block cGMP binding to the GAF A domain of PDE5. When this cGMP-blocking monoclonal antibody was applied before the cGMP preincubation, it completely prevented PDE5 activation and greatly reduced the hydrolytic activity of PDE5 (Figure 4B). These data strongly indicate that cGMP bound to its GAF domain exhibits a large stimulatory effect on the PDE5 catalytic domain, and without such an effect PDE5 has only a very low intrinsic hydrolytic activity.

Binding of cGMP appears to be necessary and sufficient to achieve full activation of PDE5, because in vitro phosphorylation of the activated PDE5 did not show any additional activation. It is known that cGMP binding to the GAF domain is necessary for phosphorylation by PKG and that phosphorylation increases the apparent affinity for cGMP binding. 27 Therefore, it appears that what phosphorylation of PDE5 really does in vivo is to stabilize the cGMP-bound, activated state of the enzyme. This probably explains why most studies show 2-fold or less activation when assayed at 1 μmol/L substrate. Moreover, because PDE5 is a selective substrate for PKG, the phosphorylation status of PDE5, which is positively correlated with changes in PKG activity, can be used as an in vivo specific marker for PKG activation in tissues such as smooth muscle, platelets, and cerebellum that contain both PKG and PDE5.

Together, these data suggest that PDE5 can exist in at least two different conformational states in vivo: nonactivated and activated upon cGMP-binding (Figure 5). The nonactivated PDE5 is in a state with low intrinsic catalytic activity that can be converted reversibly to an activated state upon cGMP binding. These reversible conformational states of PDE5 possess different kinetic and inhibitory properties. For example, cGMP-activated PDE5 demonstrated a higher sensitivity toward the PDE5 specific inhibitor sildenafil, compared with nonactivated PDE5. The IC50 for sildenafil inhibition declined from 2.1 to 0.63 nmol/L when PDE5 activity was assayed at a substrate concentration of 0.1 μmol/L cGMP.

Figure 5. PDE5 is directly activated upon cGMP binding to its GAF A domain. Without cGMP bound, PDE5 is in a nonactivated state. PDE5 is converted into an activated state after binding of cGMP to the GAF A domain. Only activated PDE5 is phosphorylated by PKG. Adapted from Rybalkin SD, Rybalkina IG, Shimizu-Albergine M, Tang X-B, Beavo JA. PDE5 is converted to an activated state upon cGMP binding to the GAF A domain. EMBO J. 200322:469–478.

Very recently, it has been shown that cGMP binding to the cGMP-binding sites could increase 3 H-sildenafil binding to the catalytic site. 34 Using a 3 H-sildenafil exchange dissociation method two KD values (12 and 0.83 nmol/L) were found, implying the existence of two “conformers” of the PDE5 catalytic site.

Interestingly, it also has been found that the ability of PDE5 to be directly activated by cGMP was limited to relatively fresh preparations. 33 PDE5 gradually lost its responsiveness to cGMP stimulation after a week of storage on ice. Even though the mechanism of this change has not been determined, this effect likely explains why cGMP-induced PDE5 activation had not been reported previously as long multistep purification protocols have traditionally been used for its isolation. Whether PDE5 activated by storage represents a nonreversible “artificial” or another conformational state remains to be determined.

cGMP-induced, PKG-independent PDE5 activation has also been reported in PKG-depleted platelets. 35 After addition of 500 μmol/L cGMP to these cells PDE5 activity, assayed at 1.0 μmol/L cGMP, was increased about 2.5-fold. A similar level of PDE5 activation (2.9-fold) was observed in control samples, where PDE5 was phosphorylated by PKG. Interestingly, the kinetics of cGMP accumulation in the PKG I deficient platelets treated with 300 μmol/L GSNO was the same as in platelets from wild-type mice, ie, a very quick surge in cGMP by 5 seconds followed by the complete disappearance of cGMP within 15 seconds. Whereas PDE5 in control platelets was substantially phosphorylated, PDE5 in platelets from PKG −/− mice was only slightly phosphorylated, probably by PKA.

Is the GAF A Domain of PDE5 a Likely Target for New Drug Development?

All PDE5 inhibitors described to date have been developed as competitive inhibitors at the active site. The fact that cGMP binding to the GAF domain of PDE5 can cause activation suggests that both antagonists and agonists for PDE5 activity might be developed based on binding to this site. Because all PDEs share a high degree of sequence similarity in their catalytic domain, the selectivity profiles of most inhibitors at least partially overlap. For example, zaprinast a known PDE5 catalytic site antagonist, also is a relatively good inhibitor of PDE1s. The greater the homology between PDEs, the higher the probability is that they will bind the same inhibitors. For this reason, many PDE5 inhibitors also are PDE6 inhibitors. Even sildenafil, which is one of the most specific inhibitors of PDE5, can still inhibit the photoreceptor PDE6 in the nmol/L range of concentration. It is likely that this inhibition may explain the visual side effects some patients report after clinical use of sildenafil. 8 Tadalafil, a newer PDE5 inhibitor, has been reported as much more specific for PDE5 than for PDE6, but it is a relatively good inhibitor of PDE11, with an IC50 of 37 nmol/L. One of the newest PDE5 selective inhibitors, vardenafil, has an IC50 for PDE11 inhibition of 162 nmol/L. Sildenafil inhibits PDE11 with an IC50 of 2730 nmol/L. 17

Therefore, one possible advantage of targeting cGMP binding to the GAF A domain of PDE5 (cGMP antagonists) might be a different profile of selectivity than achieved with the catalytic site antagonists. Conversely, a cGMP binding site agonist might also be possible. Such an agent would be expected to keep intracellular cGMP levels very low. This might be advantageous, for example, in treatment of neuronal excitotoxicity or toxicity associated with ischemia and reperfusion.

PDE1: Calmodulin-Stimulated Cyclic Nucleotide Phosphodiesterases

PDE1 Splice Variants and Domain Organization

Ca 2+ /calmodulin-stimulated PDEs (CaM-PDEs) constitute a large family of enzymes, encoded by three genes, PDE1A, PDE1B, and PDE1C. 36 Multiple amino-terminal or carboxy-terminal splice variants have been identified within each gene. CaM-PDEs contain two Ca 2+ /calmodulin binding domains and binding of both Ca 2+ and calmodulin is required for full activation of these PDEs. The degree of activation by Ca 2+ /CaM varies from 3- to 10-fold or even higher depending on the source, tissue, and purity of the enzyme preparation. In vitro, a basal nonstimulated state of PDE1s is obtained by removing Ca 2+ with a Ca 2+ chelator (eg, EGTA).

CaM-PDEs are able to hydrolyze both cGMP and cAMP, but substrate specificity differs among the different genes. PDE1A and PDE1B share the same high affinity for cGMP, but have different and lower affinities for cAMP. The affinity of PDE1B for cAMP is higher than that of PDE1A. PDE1C differs from both PDE1A and PDE1B by its ability to hydrolyze cGMP and cAMP equally well. Splice variants from the same gene family retain similar substrate characteristics. However, variations at the amino-terminal end have been found to have profound effects on the ability of calmodulin to induce PDE1 activation. For example, bovine PDE1A1 and PDE1A2 share identical protein sequence except for the very N-terminal 18 amino acids, but half maximal activation of PDE1A1 by calmodulin is 0.1 nmol/L, whereas for PDE1A2 it is 10 times higher. 37

PDE1A and Nitrate Tolerance

Ca 2+ /CaM-stimulated PDE enzyme activity has been shown previously to be important for the regulation of vascular cGMP levels and reactivity (Figure 2). 38 Most vasoconstrictors, such as norepinephrine (NE), angiotensin II (Ang II), and endothelin-1 (ET-1) increase intracellular Ca 2+ , which is thought to be the major mechanism of vasoconstrictor-mediated smooth muscle contraction. Accordingly, a Ca 2+ /CaM-stimulated PDE was found to be the major enzyme responsible for hydrolysis of cGMP in rabbit aorta stimulated with vasoconstrictors such as NE. 39 Activation of PDE1A1 by increases in Ca 2+ concentration has been demonstrated in cultured rat aortic SMCs. For example, it has been found that Ang II stimulates PDE1A1 activity in rat aortic SMCs, probably via an Ang II-mediated increase in Ca 2+ concentration. 40 Inhibition of PDE1A1 blocked the Ang II-mediated attenuation of ANP-evoked cGMP accumulation, suggesting that PDE1A1 mediates the inhibitory effect of Ang II on cGMP accumulation.

In addition to the rapid allosteric regulation of PDE1A1 and PDE5A1 by Ca 2+ and cGMP, respectively, the longer-term expression levels of PDE1A1 and 5A1 are regulated by various pharmacological reagents or pathophysiological settings. For example, PDE1A1 enzyme activity, protein level, and mRNA expression are selectively upregulated in the nitrate tolerant rat model induced by chronic nitroglycerin (NTG) treatment. 40 NTG remains one of the foremost drugs in the treatment of stable and unstable angina pectoris. 41 When given acutely, NTG has potent vasodilator capacities on arteries, veins, and coronary collateral vessels. Chronic administration of NTG, however, is limited due to the rapid development of nitrate tolerance. 42,43 Several mechanisms have been proposed to account for this phenomenon such as neurohormonal counter regulation (so-called pseudotolerance), 44 or mechanisms intrinsic to the vascular tissue itself such as intracellular SH-group depletion, desensitization of the soluble guanylyl cyclase (sGC), 45 increases in vascular production of reactive oxygen species, 46 or increases in PDE activity (so-called true vascular tolerance). 47 Chronic NTG treatment also has been shown to be associated with an increase in sensitivity to vasoconstrictors such as catecholamines, Ang II, KCl, and serotonin 48 all of which may compromise the vasodilator capacity of NTG, thereby contributing to tolerance.

NTG induces vasorelaxation by releasing NO. NO can activate sGC and increase tissue levels of cGMP. 49 As described earlier, cGMP in turn activates PKG, which has been shown to mediate vasorelaxation via phosphorylation of proteins that regulate contractility. Regardless of the mechanisms of tolerance, it appears to be associated not only with diminished cGMP elevation in response to subsequent nitrate exposure but also in response to vasoconstrictors such as NE. 45 Interestingly, the functional consequences of decreased intracellular cGMP levels would nicely explain both phenomena observed in the setting of tolerance, ie, decreased sensitivity to NTG as well as increased sensitivity to vasoconstrictors. Thus, as mentioned earlier, the activity and expression of Ca 2+ /CaM-stimulated PDE1A1 but not PDE5A1 was selectively induced in rat aortas treated for 3 days with a clinically relevant dose (10 μg · kg −1 · min −1 ) of NTG. 40 The PDE inhibitor vinpocetine partially restored the sensitivity of the tolerant vasculature to subsequent NTG exposure. In the vasculature, PDE1A1 is primarily present in SMCs. 50 Therefore, changes in PDE1A1 expression in intact aortas after NTG treatment most likely occurs in the SMCs. In nitrate tolerant vessels, an increase in sensitivity to NE has been found to be due to a greater cGMP lowing effect of NE in nitrate tolerant vessels. 45 These observations together strongly support the idea that induction of PDE1A1 in nitrate tolerant vessels may be one of the mechanisms by which NO/cGMP-mediated vasodilation is desensitized and Ca 2+ -mediated vasoconstriction is sensitized. 40 Selective inhibition of PDE1A1 expression and/or activity therefore could be a novel therapeutic approach to limit nitrate tolerance.

Finally, in cultured rat SMCs, Ang II treatment rapidly and transiently upregulates both PDE1A1 and PDE5A1 mRNA expression, which is followed by increased protein levels and enzyme activities. 51 The molecular mechanism for Ang II regulation of PDE1A1 and PDE5A1 expression appears very similar to that of the immediate early gene, c-fos. These observations suggest that alteration of PDE1A1 and PDE5A1 mRNA expression is an important mechanism for regulation of the cGMP-hydrolyzing activity in vasculature SMCs under physiological and pathological conditions. However, as discussed in the next section, human and nonhuman (eg, rat or monkey) SMCs have different PDE expression profiles. Thus, because nitrate tolerant models are mostly based on experiments with nonhuman tissues, studies in human vessels need to be conducted.

PDE1C and Human Arterial Smooth Muscle Cell Proliferation
PDE1C Is Induced in Proliferating Human SMCs

As mentioned earlier, all of the PDE1 isoforms, PDE1A, PDE1B, and PDE1C, can be expressed in arterial SMCs under different conditions and in different species. Expression of PDE1A and PDE1B, however, does not appear to be regulated by the proliferative state of the SMCs. 52 This section will focus on PDE1C, an isoform that is activated by Ca 2+ /calmodulin like all other PDE1s but, unlike PDE1A and PDE1B, has the ability to hydrolyze both cGMP and cAMP with equal efficiency. 53

Smooth muscle of intact human thoracic aorta from adult or newborn donors does not express PDE1C. 52 In contrast, PDE1C is highly expressed in smooth muscle of intact human fetal aorta, which contains abundant proliferating cells as measured by expression of proliferating cell nuclear antigen (Figure 2). 54 Furthermore, there is a marked induction of PDE1C expression and activity in human adult and newborn aortic SMCs that have been stimulated to proliferate in culture. 52 Thus, in striking contrast to the intact adult or newborn human aorta, when SMCs were cultured and analyzed by HPLC, high activity of PDE1C was observed (Figure 6A). As expected, PDE1C hydrolyzes both cGMP and cAMP in the presence of Ca 2+ /calmodulin (denoted by solid symbols in Figure 6A), and the PDE1C protein is recognized by antibodies generated against the C-terminus of recombinant PDE1C (Figure 6A), but not by antibodies against PDE1A or PDE1B. The PDE1C induced in proliferative human SMCs in culture constitutes as much as 85% of the total cGMP-hydrolyzing activity and 80% of the total cAMP-hydrolyzing activity (at low substrate concentrations) in the presence of Ca 2+ /calmodulin. The presence of high PDE1C activity in cultured human aortic SMCs has recently been confirmed by Palmer and Maurice. 55 Abundant PDE1C activity also has been found in cultured human SMCs isolated from the carotid artery, and a number of human proliferating SMCs from different smooth muscle containing organs, showing that PDE1C induction is not restricted to cultured aortic SMCs. 52

Figure 6. Human and monkey SMCs have different PDE expression patterns: PDE1C is induced in human SMCs in primary culture but not in monkey SMCs. High-performance liquid chromatography (HPLC)-elution profile of PDE activity in the absence or presence of Ca 2+ /calmodulin from human (A) and monkey (B) aortic SMCs in primary culture. PDE activity in each fraction was assayed with either 1 μmol/L cAMP (triangles) or 1 μmol/L cGMP (squares) without Ca 2+ (in the presence of 1 mmol/L EDTA open symbols) or with 1 mmol/L CaCl2 and 4 μg/mL calmodulin (filled symbols). Immunoblots with detectable bands of PDE1A, PDE1B, PDE1C, and PDE5 are shown above their respective chromatographic fractions. Adapted from Rybalkin SD, Bornfeldt KE, Sonnenburg WK, Rybalkina IG, Kwak KS, Hanson K, Krebs EG, Beavo JA. Calmodulin-stimulated cyclic nucleotide phosphodiesterase (PDE1C) is induced in human arterial smooth muscle cells of the synthetic, proliferative phenotype. J Clin Invest. 1997100:2611–2621.

The above studies showed a clear correlation between induction of PDE1C expression and activity, on the one hand, and the proliferative capacity of human SMCs in fetal aorta and cultured human SMCs. To further verify that PDE1C expression is regulated by the proliferative state of the cell, human SMCs were plated on fibrillar collagen type I. This type of collagen results in complete growth arrest of SMCs 56 and a phenotype similar to SMCs found in the intact aorta. 57 Consistent with the hypothesis that PDE1C expression is regulated by the proliferative state, cultured human SMCs plated onto fibrillar collagen type I have a markedly reduced PDE1C expression. 54 On the other hand, cultured human SMCs exposed to low serum conditions do not downregulate PDE1C expression to a significant extent. Interestingly, the quiescence induced by fibrillar collagen type I is more pronounced than that induced by serum withdrawal. Thus, cells that have been plated onto fibrillar collagen require more time to enter S phase after release from the fibrillar collagen compared with cells that have not been previously exposed to fibrillar collagen. It is likely that the fibrillar structure of the collagen results in exit of the cells from the cell cycle (most cells would be in G0), whereas serum withdrawal results in G1 arrest. Thus, PDE1C expression is downregulated first when the SMC exits the cell cycle to G0.

Finally, there is a clear correlation between time required for induction of DNA synthesis and induction of PDE1C expression in SMCs that have been released from fibrillar collagen type I and replated onto tissue culture plastic in the presence of serum (Figure 7A). Together, these experiments show that PDE1C expression not only is upregulated when SMCs enter the cell cycle, but also is downregulated when cellular quiescence is induced, and suggest that either complete quiescence or exit of SMCs from the cell cycle into G0 is required to reduce PDE1C expression.

Figure 7. A, PDE1C induction correlates with cell cycle progression. Immortalized human aortic SMCs (FLTR cells) were plated onto fibrillar collagen for 2 days. Cells were then replated in uncoated 24-well trays in the presence of 10% FBS. DNA synthesis was measured as [ 3 H]thymidine incorporation and expressed as mean±SD of triplicate samples. PDE1C expression was measured in duplicate samples by Western blot analysis. B, PDE1C antisense oligonucleotides inhibit SMC proliferation. FLTR cells plated onto fibrillar collagen for 2 days to reduce basal PDE1C expression were treated with PDE1C antisense or control (reversed) oligonucleotides. PDE1C expression was measured by Western blot analysis in cells before replating (control) or cells replated for 3 days on uncoated plates. Cells treated with PDE1C antisense oligonucleotides or control oligonucleotides (reversed) were also counted. Control (cells on fibrillar collagen) was set to 100%. Adapted from Rybalkin SD, Rybalkina I, Beavo JA, Bornfeldt KE. Cyclic nucleotide phosphodiesterase 1C promotes human arterial smooth muscle cell proliferation. Circ Res. 200290:151–157.

PDE1C Regulates Proliferation of Human SMCs

The studies described above show that PDE1C expression is markedly regulated by the cell cycle in human SMCs. Does this mean that induction of PDE1C is required for cell cycle progression and cell proliferation? Currently, there are two means available to address this question: inhibition of PDE1C activity using pharmacological inhibitors and reduction of PDE1C expression by PDE1C antisense or RNA interference techniques. Specific PDE isoforms show different responsiveness to pharmacological inhibitors. 53 However, because specific PDE1C inhibitors were not available, 8-methoxymethyl 3-isobutyl-1-methylxanthine (8MM-IBMX) at concentrations of 10 to 30 μmol/L was used to inhibit PDE1C activity. Treatment of cultured human SMCs with 8MM-IBMX resulted in a significant reduction of DNA synthesis. 54 These effects of 8MM-IBMX are likely to be due to PDE1C inhibition, because the same concentrations do not significantly inhibit the other cAMP PDEs (PDE3 and PDE4). Furthermore, the effects of 8MM-IBMX were not mimicked by PDE5 inhibitors. To further verify a regulatory role for PDE1C in human SMC proliferation, PDE1C antisense studies were performed. Treatment with PDE1C antisense oligonucleotides resulted in decreased expression of PDE1C without affecting expression of PDE5, and also significantly inhibited SMC proliferation (Figure 7B). Together, these results strongly suggest that induction of PDE1C promotes proliferation of human arterial SMCs and may be required for it to occur.

Is the effect of PDE1C on SMC proliferation mediated by hydrolysis of cAMP or cGMP, or both? In vivo, SMCs are believed to be exposed to both cAMP- and cGMP-elevating agents released by the endothelium. Prostacyclin (PGI2) is an example of a cAMP-inducing agent released from the endothelium, whereas NO acts, at least in part, by elevating cGMP levels in target cells (Figure 2). Both cGMP and cAMP are known to inhibit SMC proliferation in vitro and in vivo. 58 Accordingly, human SMC proliferation can be inhibited by pharmacological inhibitors selective for cAMP-hydrolyzing PDEs or for cGMP-hydrolyzing PDEs. 54 The effect of cGMP on SMC proliferation appears to be more complex than that of cAMP. 59,60 In contrast to the complete suppression of cell cycle traverse induced by 8-Br-cAMP (a cAMP analog partially resistant to PDE-mediated hydrolysis), 8-Br-cGMP appears to delay, but not block, the G1-to-S transition in human neonatal umbilical artery SMCs. 60 These cells express both PKA and PKG, effectors of cAMP and cGMP signaling. Thus, the molecular pathways regulated by cAMP and cGMP may be distinct. This concept was further supported by the findings that both 8-Br-cAMP and 8-Br-cGMP completely suppressed platelet-derived growth factor (PDGF)-stimulated cdk4 activity, but that only 8-Br-cAMP resulted in induction of the cdk inhibitor p27 Kip1 and a sustained suppression of cdk2 activation. 60 Alternatively, 8-Br-cAMP and 8-Br-cGMP may be hydrolyzed at different rates by PDEs expressed in the human umbilical artery SMCs, resulting in a more transient effect of 8-Br-cGMP as compared with 8-Br-cAMP. In this context, it may also be relevant that cAMP analogs and subsequent activation of PKA can exert a negative feedback on PDE1C activity. 61

In human SMCs, PDE1C inhibition results in increased levels of both cAMP and cGMP, 54 as would be expected based on the enzymatic characteristics of PDE1C. In fact, the actions of cAMP and cGMP are not always unrelated. For example, a recent study shows that PDE5 inhibitors reduce proliferation of bovine coronary artery SMCs via cGMP elevation and subsequent inhibition of PDE3, a cGMP-inhibitable cAMP-hydrolyzing PDE. 62 Through this mechanism, increased intracellular levels of cGMP can result in elevation of cAMP levels (see section on PDE3). The effects of PDE1C on SMC proliferation may therefore be due to hydrolysis of both cGMP and cAMP.

Why Is PDE1C Induced in Proliferating Human SMCs but not in SMCs From Other Species?

To date, detectable PDE1C activity has only been observed in proliferating human SMCs and, curiously, not in proliferating aortic SMCs from nonhuman primates (pigtail monkey, Macaca nemestrina, and baboon), bovine SMCs, porcine SMCs, rat SMCs, or ovine SMCs. 52,63 On the other hand, all of these species express PDE1A and/or PDE1B. As shown by Figure 6B, the PDE activity profile in proliferating aortic SMCs isolated from pigtail monkey is strikingly different from that of SMCs isolated from human aorta (Figure 6A). Both human and monkey SMCs express PDE5 and PDE3/4, but whereas human SMCs express PDE1C, monkey SMCs express PDE1B (Figure 6). These findings show that there are marked differences in expression and activities of PDE1C in SMCs from different species. We can only speculate on the reason for induction of PDE1C in proliferating human SMC, as opposed to SMCs derived from other species. It is possible that human SMCs have the need for a more extensive and controlled cAMP and cGMP hydrolysis during cell cycle progression than SMC from many other species. Intracellular calcium levels are tightly regulated during cell cycle progression, 64 and induction of PDE1C may serve as a means to coordinate mitogenic calcium signaling with a concomitant decrease in levels of the growth-inhibiting actions of both cGMP and cAMP. Regardless of the reason for the induction of PDE1C in proliferating human SMCs, special care must be taken when extrapolating results obtained with selective PDE inhibitors in animal studies to clinical trials in humans.

Given the key role apparently played by PDE1C in the regulation of human smooth muscle proliferation, it would not be surprising if therapeutic agents can be developed that target PDE1C activity. Such agents would be expected to minimize the excess smooth muscle proliferative response that occurs in response to injury and inflammation that is caused by balloon angioplasty or stenting and perhaps even hypertension. They likely also would be less toxic than many currently used agents.

PDE3: cGMP-Inhibited PDE

Both PDE3A and 3B are expressed in most vascular smooth muscle beds. Although they may not be the predominant contributors to hydrolysis of cGMP in these vascular beds, or for that matter in the cardiocyte, the PDE3s probably are regulated by cGMP in vivo. PDE3s do not have separate allosteric cGMP-binding domains, and are not regulated by cGMP in the same way as the GAF domain containing PDEs (PDE2 and PDE5). However, the PDE3 catalytic sites have similar high affinity for cAMP and cGMP, but the Vmax for cAMP is much higher (4 to 10 times) than for cGMP. Therefore, the mechanism by which cGMP inhibits PDE3 catalytic activity is through competition with cAMP at the catalytic site. Thus, cGMP acts as a transient switch causing inhibition of cAMP hydrolytic activity by PDE3 until it is itself hydrolyzed.

PDE3 Domain Organization and Function

The PDE3 family contains two genes, PDE3A and PDE3B. The domain organization of both is quite similar and includes a conserved catalytic domain, a divergent N-terminal region with its membrane association domain and a C-terminal hydrophilic end (Figure 1). PDE3A and PDE3B share most homology in the catalytic domain, including a unique 44 amino acid insertion, not found in the catalytic domains of PDEs from any other family. However, these isoforms differ greatly at both their N-terminal and C-terminal ends. 65 Studies of different truncated PDE3 forms revealed that the N-terminal end was not required for maintaining full catalytic activity and sensitivity to PDE3 specific inhibitors, although it might be important for localization. 65

The two PDE3 isoforms are differentially expressed. PDE3A is expressed in vascular smooth muscle, platelets, cardiocytes, and oocytes. In rat and human vascular smooth muscle, both PDE3A and PDE3B are expressed, but they have distinct subcellular localizations. 55,66 PDE3A was found mostly in the soluble fraction, whereas PDE3B was associated with the particulate fractions. PDE3B also is highly expressed in adipose cells, hepatocytes, and spermatocytes. 67

PDE3 is usually thought to mediate mostly cAMP regulated processes such as cardiac contractility, platelet aggregation, smooth muscle relaxation, and hormonal regulation. 67 Much less is known about PDE3 involvement in regulation of cGMP signaling. Some studies have shown that cGMP elevating agents can produce an increase in cAMP levels by inhibiting PDE3 activity. For example, NO-induced inhibition of rabbit platelet aggregation was caused in part by cAMP accumulation as a result of PDE3 inhibition. 68 It also has been suggested that in human atrial myocytes, the stimulatory effects of NO-donors on cardiac calcium current were due to cGMP inhibition of PDE3 activity. 69 PDE3 has also been suggested as an important determinant of NO effects on renal vasculature. 70

However, recent studies of PKG I-deficient mice showed that high concentrations of NO donors were able to produce enough cGMP to get direct PKA activation, whereas low concentrations of cGMP induced smooth muscle relaxation exclusively through the cGMP signaling pathway. 71 Therefore, further studies are needed to determine under what conditions cGMP enhances PKA activity by direct activation as opposed to indirectly through PDE3 inhibition.

PDE3 and Cardiovascular Drug Development

The first generation of PDE3 inhibitors (milrinone, vesnarinone, enoximone) were found to have significant vasodilatory and inotropic effects in vitro and in animal studies. 53 In initial clinical trials these inhibitors were believed to have a positive effect in the treatment of chronic congestive heart failure. 72 However, long-term effects of oral administration of milrinone revealed an increase the morbidity and mortality of patients with severe chronic heart failure. 73 Although it is not known if the correct doses were used or even if the cardiotoxic effect was due only to PDE3 inhibition, this unsuccessful clinical trial presented an additional challenge for the development of PDE3 inhibitors. Despite these results, short-term clinical use of milrinone has been approved for the treatment of patients with acute decompensated heart failure. 74 Intravenous injection of milrinone lactate (Primacor, Sanofi-Synthelabo Inc) can provide a significant, but strongly time-limited clinical effect (no longer than 48 hours) and requires close observation of the electrocardiographic parameters of these patients.

Another PDE3 inhibitor, cilostazol (Pletal, Otsuka America Pharmaceutical, Inc/Pharmacia Corporation), has been approved for the treatment of intermittent claudication, a vascular disease characterized by pain in the legs. 75 Again, this drug is contraindicated for patients with congestive heart failure. Newer generations of PDE3 specific inhibitors probably will have to demonstrate tissue or isoform specificity that should minimize effects on cardiac tissue in order to meet approval.

Conclusions and Perspective

It is expected that in the next few years we will learn much more about what specific roles individual PDE isozymes play in smooth muscle function. We also should be able to identify how the transcription of these PDEs is regulated, what role(s) may be played by some of the newly discovered PDEs (eg, PDE9), and particularly how different species utilize different combinations of PDEs to regulate their cyclic nucleotide dependent processes. It seems quite possible that new uses for the “traditional” smooth muscle PDE inhibitors like Viagra will be identified and perhaps new drugs will be developed that act on other sites of PDE5 or on the active sites of other PDEs expressed in vascular smooth muscle.

Original received May 6, 2003 revision received July 7, 2003 accepted July 8, 2003.

This work was supported in part by a Grant-in-Aid from the American Heart Association Washington Affiliate and NIH Grant HL62887 to Karin E. Bornfeldt, NIH grants DK21723 and HL44948 to Joseph A. Beavo, and American Heart Association Grant 0030302T to Chen Yan.

Drug now in clinical trials for Parkinson's strengthens heart contractions in animals

A drug currently in clinical trials for treating symptoms of Parkinson's disease may someday have value for treating heart failure, according to results of early animal studies by Johns Hopkins Medicine researchers.

The drug, a member of a class of compounds known as phosphodiesterase (PDE) type I inhibitors, shows promising effects on dog and rabbit hearts, as well as on isolated rabbit heart cells, most notably an increase in the strength of the heart muscle's contractions, the researchers say.

Human heart failure is a chronic condition often marked by weakening of the heart muscle and its subsequent failure to pump enough blood. Currently, dozens of drugs are available to treat or manage heart failure symptoms, but drugs that improve the strength of the heart muscle's contractions, such as dobutamine, carry the risk of dangerous complications such as developing an irregular heartbeat.

However, in their study, described in a report published in the journal Circulation on July 20, the Johns Hopkins researchers demonstrate that the new compound works differently than current drugs, suggesting its use may be a safer way to increase heart contraction strength.

Heart failure affects about 5.7 million U.S. adults, according to the Centers for Disease Control and Prevention, and contributes to an estimated one in nine deaths. Standard treatment includes diuretics that increase urine production to keep the heart from becoming enlarged angiotensin-converting enzyme (ACE) inhibitors that lower blood pressure and reduce the workload on the heart and beta blockers that protect against heart damage from high levels of the stress hormone adrenaline that are common with heart failure, and that help reduce the heart's workload. There is no cure.

"Our results are intriguing because so far it's been largely uncharted territory to come up with a way of increasing contractility that doesn't ultimately hurt patients," says David Kass, M.D., the Abraham and Virginia Weiss Professor of Cardiology at the Johns Hopkins University School of Medicine and principal investigator of the study.

The drug explored in the new study, ITI-214, inhibits the enzyme PDE1, which is part of the larger phosphodiesterase (PDE) family of over 100 such proteins. All PDEs work by breaking down one or both of two molecules: cAMP and cGMP, each of which serve as molecular messengers inside cells. Each PDE has very specific features, including the type of cell they exist in and their location inside that cell, allowing them to adjust cAMP and/or cGMP very precisely.

PDE inhibitors work by stopping the breakdown of cAMP and cGMP, causing these molecules to build up so they can influence proteins to alter the cell. In heart disease, PDE activity can limit the beneficial effects of cAMP or cGMP, so inhibitors have the potential to act as a therapy.

In mice, Kass notes, PDE1 inhibitors had been reported to shrink abnormally thick heart muscle caused by high blood pressure and dilate blood vessels. However, in mice the heart mostly has a different form of the PDE1 enzyme than found in humans, so PDE1 inhibitors likely affect mice differently than humans.

Dogs and rabbits, which this research focused on, have a PDE1 composition more similar to humans, Kass says.

For their experiments, the researchers used six dogs surgically outfitted with sensors and heart pacemakers, and tested ITI-214's effects on them before and after inducing heart failure by running the pacemaker rapidly for approximately three weeks. The drug was tested at different doses, both orally and intravenously. The dogs were given at least a day between tests.

When given at an oral dose of 10 milligrams for every kilograms via a peanut butter-covered pill, ITI-214 increased the amount of blood pumped out by the heart each minute by 50 percent in the healthy hearts and by 32 percent in the failing hearts. It did this, Kass says, by increasing the strength of the heart's contractions by almost 30 percent and by dilating the blood vessels. Intravenous administration of the drug resulted in similar, but more rapid, effects.

"We were pretty agnostic about what we would find and didn't necessarily expect anything that novel," says Kass. "To my knowledge, no study had reported increased heart contraction strength from a PDE1 inhibition before. But then, all of the prior studies where this might have been tested had used mice, and we knew that a different PDE1 form was found in larger mammals and humans. So, we just had to try it, and the results were very interesting."

In healthy dogs, Kass cautions, the drug also raised their heart rate by approximately 40 beats per minute on average, which can be dangerous for heart failure patients. However, the dogs with failing hearts had no significant difference in heart rate before and after the drug was given.

Even with these promising results, there was a major concern. Other heart failure drugs designed to strengthen heart contractions have potentially fatal complications, such as developing wildly irregular heartbeats. Inhibitors of a different PDE, PDE3, including amrinone and milrinone, are especially infamous for this.

"This was the boogeyman in the room," says Kass. "The new drug produced many of the same heart and artery changes that PDE3 inhibitors do, so we naturally worried whether it worked in a similar way and might also have complications. So we tested them side by side."

When they compared the effects of ITI-214 to a PDE3 inhibitor in isolated muscle cells from 13 rabbit hearts, the way the two drugs acted looked different.

One of the major ways that PDE3 inhibitors are thought to work is by increasing the amount of calcium inside the muscle cell, which triggers key proteins to exert more force on the cell, and causes the cell to contract more strongly.

As expected, when the researchers applied a PDE3 inhibitor to the heart cells, calcium levels rose and the cells contracted more strongly than without the inhibitor.

By itself, inhibiting PDE1 had no effect on the muscle cells, but the researchers thought this might be because PDE1 activity is too low in a resting cell. So they used a drug to first slightly increase cAMP levels, and this increased PDE1 activity enough for them to observe ITI-214's effects.

With the added drug, ITI-214 caused the cell to contract more strongly. However, the cell's calcium levels didn't rise, strongly indicating that ITI-214 increases muscle contractions through a different mechanism than the PDE3 inhibitors.

"Our results show that inhibiting PDE1 produces different changes than blocking PDE3, and so we hope that we can bypass the calcium-mediated and potentially deadly arrhythmias that have plagued PDE3 inhibitors," says Grace Kim, a lead co-author and a postdoctoral fellow in Kass' lab. "We are anticipating similar positive benefits on heart function but with much less toxicity."

Kass says ITI-214 also appears to function differently than dobutamine, which strengthens heart contractions in people with heart failure but also can cause fatal irregular heart rhythms. Dobutamine works by stimulating the beta adrenergic system, the same system that is activated by adrenaline. Dobutamine acts on the same pool of messenger molecules that increase the cAMP that PDE3 degrades, so its heart effects are similar to those of a PDE3 inhibitor.

When the researchers blocked the beta adrenergic receptors in 11 healthy, anesthetized rabbits and then applied ITI-214, all of the effects -- except for its impact on heart rate -- remained. If ITI-214 were acting through the beta adrenergic system, blocking the receptors should have blocked its actions.

Instead, it appears the drug might be working on cAMP generated by a different signaling system in the heart that uses adenosine. When the researchers used a drug to block receptors in the adenosine system in a separate set of seven anesthetized rabbits, all of the effects of the drug, including increased heart rate, were eliminated.

Other studies have demonstrated that the adenosine pathway can have protective effects on the heart, Kass says. In the same issue of Circulation, other investigators at the University of Rochester also found that PDE1 controls the adenosine pathway, and that inhibiting PDE1 could protect the heart from toxicity of some cancer drugs.

ITI-214 is now in early clinical trials and is being tested in heart failure patients at Johns Hopkins Medicine and Duke University. It has already passed phase 1 safety trials in healthy individuals.

Other researchers involved in the study include Toru Hashimoto, Richard Tunin, Tolulope Adesiyun, Steven Hsu, Ryo Nakagawa, Guangshuo Zhu and Dong Lee of Johns Hopkins, and Jennifer O'Brien, Joseph Hendrick, Robert Davis, Wei Yao, David Beard, Helen Hoxie and Lawrence Wennogle of Intra-Cellular Therapies.

This work was supported by the National Heart, Lung and Blood Institute (HL135827-01, P01HL107153, HL119012), the Japanese Circulation Society Overseas Research Fellowship and Uehara Memorial Foundation Research Fellowship, the T32 Training Program, an American Heart Association fellowship grant and Intra-Cellular Therapies, which provided the drug as well as funding for the research.

Funding and drugs for the study were provided by Intra-Cellular Therapies. Kass is a paid consultant to Intra-Cellular Therapies. This arrangement has been reviewed and approved by The Johns Hopkins University in accordance with its conflict of interest policies.

The roles of cAMP–PKA–CREB signaling pathway in tumors

cAMP–PKA–CREB signaling has paradoxical effects in tumors, acts as a tumor-suppressor or tumor-promoter in different tumor types (Table 1). The role of cAMP–PKA–CREB signaling pathway in liver cancer and other tumors is described below.

Liver cancer

Whereas some studies suggest that increasing cAMP levels may inhibit HCC cells growth [51,52,53], it has been reported that PKA promotes HCC invasion and metastasis by phosphorylating multiple substrates such as CIP4 [29]. Fibrolamellar hepatocellular carcinoma (FL-HCC) is a primary liver cancer that occurs mainly in children and young adults. 80%-100% FL-HCC patients have DNAJB1–PRKACA gene fusion, resulting in the deletion of a 400 kb gene fragment on chromosome 19 and the production of a chimeric protein that retains PKA kinase activity [54]. DNAJB1–PRKACA knock-in mice can develop tumors characteristics of FL-HCC [55]. In addition, PRKACA is overexpressed in about 80% of BAP1 gene (encoding BRCA1-associated protein 1)-mutated HCC, which exhibit similar clinical manifestations and histological characteristics to DNAJB1–PRKACA fusion-related FL-HCC [56]. PKA pathway dysfunction plays an important role in the development and progression of these two kinds of HCC. Hepatitis B virus (HBV) infection is a major risk factor for the development of HCC. HBV X protein plays an important role in HBV-related HCC. Mechanistically, HBVx can promote liver carcinogenesis through CREB-miR-3188 and ZHX2-Notch signaling pathway [57], promote HCC cell growth by activating CREB-YAP axis [58], and promote the invasion and metastasis of HBV-related HCC by up-regulating FOXM1 expression through Erk-CREB pathway [59]. Collectively, these studies indicate that the PKA-CREB pathway may promote HCC progression. Indeed, analysis of rat HCC tumors and paired normal liver samples showed significant increase in CREB and CREB phosphorylation levels in HCC [60].

However, the roles of cAMP in HCC appear to be somewhat paradoxical. As we described above, increasing cAMP levels by PDE inhibitors may arrest HCC cells growth. Treatment of HepG2 cells with cAMP analogues significantly reduce the transcription and protein levels of cyclin A and induce cell cycle arrest [61]. In addition, PDE4 inhibitor rolipram and DC-TA-46 can up-regulate the expression of p21, p27 and p53 and down-regulate cyclin A expression, thereby inhibiting the proliferation and promoting apoptosis of HepG2 cells [52]. In contrast, vasoactive intestinal peptide could reduce cAMP concentration, CREB expression and Ser 133 phosphorylation, and inhibit Bcl-xL expression, leading to Huh7 cell apoptosis [62]. The versatile roles of cAMP in HCC may be due to the multiplicity of its targets with diverse functions. Hence, whether cAMP promotes or inhibits HCC may be context-dependent. The homeostasis in cAMP levels may be critical for HCC progression.

Brain cancer

The roles of the CREB-activating kinase PKA in brain tumors are also paradoxical. Several studies have shown that PKA plays a tumor-suppressive role in glioblastoma cell line A-172. Activation of PKA by increasing cAMP levels or supplying cAMP analogues (dcAMP and 8-Br-cAMP) can reduce the proliferation rate of A-172 cells, promote differentiation, and induce apoptosis [63]. Increasing the intracellular cAMP levels by rolipram, a PDE inhibitor, can up-regulate the expression of p21 and p27, and activate PKA and Epac1-Rap1 signaling, leading to A-172 cell growth arrest and apoptosis [64, 65]. cAMP pathway also plays anticancer role in medulloblastoma. Pituitary adenylyl cyclase inhibits the proliferation of medulloblastoma cells through PKA-Gli1 pathway [66], and neurociliary proteins inhibit the growth of medulloblastoma through PDE4D-PKA-Hedgehog pathway [67]. ARHGAP36 protein, a member of RhoGAP family, can bind to PKA catalytic subunit and inhibit its activity, thereby activating Hedgehog pathway and promoting the growth of medulloblastoma [68]. The tumor-suppressive effects of cAMP and PKA may be mediated by Epac1, Rap1 and Gli1, rather than CREB.

Phosphorylation of CREB can be directly inhibited by PTEN, an anticancer protein phosphatase that is frequently mutated or inactivated in many cancers, including the most aggressive types of brain cancer, glioblastoma multiforme and astrocytoma [69, 70]. PTEN deficiency can enhance CREB activity and induce the expression of PAX7, thereby promoting the conversion of human neural stem cells to glioblastoma stem-like cells [71]. In addition, CREB can reciprocally down-regulate PTEN. Tan et al. reported that CREB was highly expressed in clinical samples and cell lines of glioma, and CREB could inhibit PTEN expression through miR-23, thus promoting the development of glioma [72]. Moreover, EGFR can activate CREB through MAPK-RSK2 pathway, and then promote glioma cells growth and invasion [73].

Epidermal growth factor receptor (EGFR), EGFRvIII mutant and platelet derived growth factor receptor (PDGFR) can promote the development and invasion of glioblastoma through the PKA-Dock180 signaling pathway [74, 75]. Also, miR-33a enhances cAMP–PKA signaling by inhibiting PDE8A expression, thus promoting the growth and self-renewal of initial glioma stem cells [76]. In human glioblastoma cell line MGR3, activation of PKA leads to a significant increase in GSTP1 phosphorylation and activity, which may lead to drug resistance and treatment failure [77].

Lung cancer

In non-small cell lung cancer (NSCLC), there are significant up-regulation of CREB expression and phosphorylation in tumor tissues compared with adjacent normal tissues. Increased CREB expression is correlated with short survival period of patients [78]. cAMP could down-regulate SIRT6 expression and thus reduce the apoptosis of NSCLC cells induced by radiotherapy [79]. In the highly malignancy small cell lung cancer (SCLC), increased activity of CREB helps maintain its neuroendocrine characteristics and proliferation [80]. RGS17 is increased in 80% of lung cancer tissues compared with matched normal lung tissue and promotes cell proliferation through the cAMP–PKA–CREB pathway [81]. In addition, PKA-Smurf1-PIPKIγ signaling transduction promotes the progression of lung cancer in vivo [82]. cAMP–PKA–CREB pathway could regulate the hypoxia response in lung cancer cells [83]. PKA inhibitors H-89 and PKACA knockdown antagonize hypoxia-induced epithelial-mesenchymal transformation, cell migration, and invasion of lung cancer cells [84]. Moreover, oxidative stress plays an important role in the pathogenesis of lung diseases, including pulmonary fibrosis and lung cancer. Interactions among PKA, Erk1/2 and CREB mediate cell survival in oxidative stress [85].

Contradictory to the inhibition of radiotherapy-induced NSCLC cell apoptosis by cAMP-Sirt6 pathway [79], cAMP–PKA–CREB pathway seems to play an anticancer role in radiotherapy. BALB/c mice pretreatment with forskolin could inhibit ATM and NF-κB by PKA-induced PP2A phosphorylation, resulting in an increase in radiotherapy-induced apoptosis [86]. In lung cancer cell line H1299, Gs protein promotes Bak expression through PKA-CREB-AP1 pathway and increases apoptosis induced by radiotherapy [87]. These studies suggest that the cAMP–PKA–CREB pathway may also exert opposite effects under different circumstances of the same type of tumor.

Prostate cancer

PKA subunit can be a biomarker to predict the response of prostate cancer to radiotherapy and chemotherapy. Analysis of 456 clinical prostate cancer specimens found that PKA RIα is highly expressed in 80 cases (17.5%), and PKA RIα overexpression is associated with poor efficacy of radiotherapy and short-term or long-term androgen deprivation therapy, distant metastasis and abnormal biochemical index [88]. In prostate cancer cell line PC3M cells, the overexpression of wild type PKA RIIβ or mutant type PKA RIα-P (functionally similar with PKA RIIβ) leads to growth inhibition and apoptosis in vitro, and inhibits tumor growth in vivo [89]. Androgen receptor (AR) signaling is critical for prostate carcinogenesis. Testosterone directly stimulates GPR56 and then activates the cAMP/PKA pathway, which promotes AR signaling [90]. PKA also phosphorylates the Thr 89 residue in HSP90, leading to the release of AR from HSP90 and the binding of AR to HSP27, which transfers AR into the nucleus to transactivate its targets [91]. In addition, PKA signaling pathway is involved in the neuroendocrine differentiation of prostate cancer, an early marker for the development of androgen independence [92, 93]. Moreover, high expressions of osteocalcin and ostesialin in androgen independent prostate cancer cell line C4-2B are dependent on the cAMP–PKA signaling pathway [94]. PAK4 can be activated by cAMP–PKA to enhance CREB transcription activity independent of phosphorylation at Ser 133 residue, and PAK4 knockdown in PC-3 and DU145 cells inhibit tumor formation in nude mice [95]. Moreover, RGS17 is overexpressed in prostate cancer samples and promotes cell proliferation through the cAMP–PKA–CREB pathway [81]. In addition, it has been reported that depression and behavioral stress can accelerate the progression of prostate cancer through PKA kinase [30, 96].

Ovarian cancer

About 54% of epithelial-derived human ovarian tumors in tissue microarray have moderate or high levels of CREB expression, while no expression was observed in normal ovarian samples. Knockdown of CREB expression significantly reduces proliferation of ovarian cancer cells, but had no effect on apoptosis [97]. Epithelial ovarian cancer is one of the most deadly gynecologic malignant tumors. PKA RIα is highly expressed in epithelial ovarian cancer [98]. During metastatic spread of epithelial ovarian cancer cell line SKOV3, extracellular matrix invasion needs PKA activity and AKAPs anchor, and inhibition of PKA activity or PKA RI and RII anchor can block matrix invasion [99]. In addition, PKA reduces the intensity of tight junction in epithelial ovarian cancer cell line OVCA433 by phosphorylating claudin-3 [100]. Also, gonadotropin promotes the metastasis of epithelial ovarian cancer cells through PKA and PI3K pathways [101]. Whereas the above-mentioned studies suggest that PKA-CREB plays a tumor-promoting role in epithelial ovarian cancer, one study suggests that PKA can phosphorylate EZH2 at Thr 372 residue, leading to mitochondrial dysfunction, the binding of EZH2 to STAT3 and then inhibiting STAT3 phosphorylation and epithelial ovarian cancer cell growth [102].

Breast cancer

Overexpression of R subunit especially RI of PKA is associated with cell proliferation in normal breast, malignant transformation of breast epithelial cells, poor prognosis of breast cancer, and tolerance to anti-estrogen therapy. Recent study demonstrates that nuclear localization of activated PKA is correlated with breast cancer metastasis [103]. Integrin α9 maintains the stability of β-catenin through ILK/PKA/GSK3 signaling and, thereby promotes the growth and metastasis of triple negative breast cancer cells [104]. Moreover, successful anti-estrogen therapy is associated with reduced RI mRNA expression, and RI antisense oligonucleotides can reduce the growth rate of breast cancer cells [105]. In estrogen receptor positive breast cancer, PKA-induced ERα Ser 305 phosphorylation and PAK1 are associated with tamoxifen resistance and breast cancer progression [106, 107]. In Her-2 positive breast cancer cells, PKA activation is associated with trastuzumab resistance [108]. In addition, cAMP–PKA–CREB pathway also plays an important role in the metabolic regulation of breast cancer. Serotonin promotes mitochondrial biosynthesis through the AC-PKA pathway in breast cancer cells [109]. Cytoplasmic G-protein coupled estrogen receptor promotes aerobic glycolysis through cAMP–PKA–CREB pathway [110]. Contrary to above studies, it is reported that IL-24 induces breast cancer cells apoptosis by activating TP53 and endoplasmic reticulum stress through PKA [111]. Hence, PKA may also have paradoxical roles in breast cancer depending on the context.


The role of cAMP is quite different in diverse types of lymphoma. PDE4 inhibitors block intracellular TLR signaling and promote apoptosis of chronic lymphocytic leukemia (CLL) cells through increasing cAMP concentration [112, 113]. Interestingly, PDE4 inhibitors induce apoptosis in B cell CLL but not in T cell CLL or normal circulating hematopoietic cells, probably due to that PDE4 inhibitors only augment glucocorticoid receptor and cAMP levels in B cell CLL [114, 115]. cAMP–PKA could promote apoptosis through mitochondria dependent pathways, reducing expression of anti-apoptotic proteins Bcl-2 and survivin, and increasing expression of pro-apoptotic protein Bax in lymphoma cells [116,117,118]. The chemokines CXCR4 and CXCL12 released from the microenvironment can bind to Gαi-conjugated GPCRs on CLL cells, reducing cAMP synthesis and increasing survival rate of CLL cells [119, 120]. PDE7B is overexpressed in CLL, and inhibitors of PDE7 (BRL-50481 and IR-202) and a dual PDE4/PDE7 inhibitor IR-284 increase apoptosis of CLL cells, which is attenuated by PKA inhibition [121].

CREB is overexpressed in the bone marrow of most leukemia cell lines and patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) [122]. Previous studies also show that CREB is highly expressed in the majority of myeloid leukemia cells in AML patients and associated with poor prognosis [123]. The jmjd3/UTX inhibitor GSKJ4 can promote CREB degradation and inhibit AML cell growth [123]. Overexpression of CREB can promote AML cells proliferation by up-regulating cyclin A1 expression. In addition, the CREB transgenic mice shows myeloproliferative diseases but not leukemia, suggesting that CREB is involved in the leukemia phenotype during the leukemia germination, but is not sufficient to completely transform into leukemia [124]. In contrast, cAMP can protect acute promyelocytic leukemia cells from apoptosis induced by arsenic trioxide and anthracyclines [124]. In AML cell line IPC-81, cAMP can induce apoptosis through up-regulation of Bim by CREB and CDK [125].

Other tumors

The mRNA and protein levels of PKA RIα and AKAP10 are significantly increased in colorectal cancer tissues, correlating with invasion depth, differentiation degree and short survival [126]. Type-I insulin-like growth factor receptor (IGF-IR) is tightly involved in tumorigenesis and drug resistance [127]. IGF-IR signaling induces ezrin phosphorylation at Thr 567 residue and thereby promotes cAMP-dependent PKA activation and colorectal cancer cell survival [128]. Overexpression of PKA RIα and AKAP10 in several colorectal cancer cell lines is directly correlated to metastasis. In addition, the resistance of colorectal cancer cells to a selective MEK1/2 inhibitor selumetinib is induced by PKA activation [129], and the resistance of colorectal cancer cells to methotrexate can be induced by cAMP signaling [130]. PKA antagonists could inhibit the nuclear translocation of β-catenin and expression of c-myc and COX2 in APC mutant colorectal cancer, thereby inhibiting tumor development [131].

Immunohistochemical experiments found that normal melanocytes did not express PKA RIα proteins, but it is highly expressed in human melanoma samples and some melanoma cell lines. RII activation or RIα silence can inhibit proliferation and increase caspase 3-promoted apoptosis [132]. PKA can promote the migration of melanoma cells. Studies have found that hypoxia can induce the expression of scaffold protein AKAP12 in melanoma, and PKA-regulated phosphorylation during hypoxia is dependent on the presence of AKAP12. Inactivation of AKAP12 leading to the reduction of tumor growth, migration, and invasion in melanoma mouse models [133]. PKA pathway also plays an important role in the synthesis of melanin. Diethylstilbestrol can promote melanin production through cAMP–PKA mediated up-regulation of tyrosinase and MITF in mouse melanoma cell line B16 [134], and gingerol inhibits melanin production by down-regulating MAPK and PKA [135].



Drosophila melanogaster Meigen were reared on standard medium at 25°C, with a 12 h:12 h ligh:dark photoperiod and 55% relative humidity. Lines used in this study were: wild-type, Canton S white mutants: w 1118 (partial deletion, loss of function: w H (hypomorphic allele)(Zachar and Bingham, 1982)tubule principal cell-specific GAL4 driver, c42(Broderick et al., 2004)transgenic w::eYFP lines D4, E5 and H8 (generated for this study).

Mutant w Drosophila (w 1118 and w H ) were `cantonized' by crossing white-eyed flies with isogenized Canton S wild-type flies. The offspring were collected and interbred to produce recessively white-eyed offspring. This process was repeated five times, thus removing 97% of any compensatory mutations that might have accumulated since the mutant stocks were first isolated.

For dissection, flies were anaesthetized by chilling on ice, and decapitated, before removing the tubules in Schneider's medium (Invitrogen Ltd, Paisley, Scotland). All chemicals and drugs were obtained from Sigma(Sigma-Aldrich, Gillingham, Dorset, UK), unless otherwise stated.

Generation of transgenic Drosophila

Over-expression lines containing White tagged with enhanced yellow fluorescent protein (eYFP) at the C terminus were generated as follows:

The coding sequence for eYFP (Clontech UK Ltd, Basingstoke, Hampshire, UK)was amplified using primers that incorporated NotI and KpnI sites at the 5′ and 3′ ends, respectively(GCGGCCGCCATGGTGAGCAAGGGCGAGG/GGTACCCTACTTGTACAGCTCGTCCATGC). The resulting fragment was cloned into the NotI and KpnI sites of pP using standard methods to form pP. The white open reading frame, excluding the stop codon, was PCR amplified from tubule cDNA template using primers:AGATCTATGGGCCAAGAGGATCAGGAG/GCGGCCGCCTCCTTGCGTCGGGCCCGAAG, that incorporated EcoRI and NotI sites at the 5′ and 3′ ends,respectively. This fragment was cloned into pP using the EcoRI and NotI restriction sites to form plasmid pP<w – eYFP-UAST>. The insert was sequenced to check for PCR errors, and the plasmid injected into wDrosophila embryos(w 1118 ) by standard techniques (Vanedis Drosophila injections service, Transformants were selected and maintained using standard Drosophilagenetic techniques.

Fluid transport assays

Fluid transport assays were performed as previously described(Dow et al., 1994b) on intact tubules from Canton S, w 1118 and cantonised w 1118 7-day-old adult flies. Basal rates of fluid transport were established for 30 min, after which 100 μmol l –1 cGMP was added to tubules and fluid transport rates measured for a further 60 min. Data are shown as mean fluid transport rates± s.e.m., N=7.

Cyclic nucleotide transport assays

Transport assays for cGMP and cAMP were based on a modified fluid transport assay transport rates ratios were calculated as previously described(Day et al., 2006). The transport rate provides a linear measure of basal to apical unidirectional flux, whereas a secreted:bathing ratio of >1 indicates that the transport substrate is being concentrated by the tubules(Maddrell et al., 1974). Maximal rates of cGMP and cAMP transport occurred at 100 μmol l –1 (Evans,2007) thus, all transport assays were conducted with a final concentration of 100 μmol l –1 of cyclic nucleotide. The maximal rates of transport of cAMP are significantly higher than that of cGMP:a transport ratio of ∼5 at 100 μmol l –1 cAMP, vs ∼3 for cGMP at 100 μmol l –1 cGMP.

Tubules were dissected into saline (Dow et al., 1994a) and allowed to recover for 30 min prior to addition of cyclic nucleotides: `cold' cGMP or cAMP at 100 μmol l –1 , and tritiated cGMP or cAMP added as tracer (Amersham Pharmacia, Biotech UK Ltd, Amersham, Bucks, UK). Where competitors or drugs were included, these were added 30 min before the radiolabelled substrate. Where the removal of amino acids and citrate was investigated, a minimal Drosophila saline was used(Linton and O'Donnell, 1999)to which the missing ingredients of Drosophila saline(Dow et al., 1994a) were reintroduced at the concentrations normally used.

In all the transport assays, the ratio and rate of transport was measured 1 h after the radiolabelled cyclic nucleotide was added(Evans, 2007). The tubules were allowed to secrete for 1 h before the secreted droplet was measured and removed to Eppendorf tubes containing scintillation fluid (Fisher Scientific,Loughborough, UK). A 1 μl sample of each reservoir droplet was also removed and radioactivity measured in the scintillation counter (Beckman, High Wycombe, UK).

CGMP-dependent kinase bioassay for secreted cGMP

In order to determine if unaltered cGMP is transported through the tubule from the bathing droplet into the lumen, secreted fluid was tested for its ability to stimulate cGMP-dependent protein kinase (cGK) activity in vitro. A secretion assay was carried out with 80 tubules in the standard bathing droplet of Drosophila saline/Schneiders' medium (control) or saline/Schneiders' medium with 100 μmol l –1 cGMP. After allowing the tubules to secrete for 1 h, secreted droplets were pooled (∼2 ml in total), removed from the secretion assay dish and placed into an Eppendorf tube. To remove any residual mineral oil derived from the secretion assay, samples were centrifuged and the oil (top layer) was discarded. A cGK assay was then carried out using the Drosophila cGK, DG2(MacPherson et al., 2004),which had been expressed in S2 cells as a source of DG2 and therefore, cGK activity (MacPherson et al.,2004). Standard kinase reactions were set up in a total volume of 44 μl with 5 μl of DG2 protein sample, 39 μl of kinase assay buffer(MacPherson et al., 2004) and either 1 μl of secreted fluid from control samples or 1 μl of secreted fluid from tubules incubated in 100 μmol l –1 cGMP. Positive controls were set up by adding 1 μl of 100 μmol l –1 cGMP (final concentration 2.2 mmol l –1 )to the assay mix as described above. Three separate experiments were performed for each condition and the results expressed in pmol ATP min –1 mg –1 protein (mean ±s.e.m.).

Real-time quantitative PCR (Q-PCR)

Q-PCR was performed as described previously(McGettigan et al., 2005),using mRNA prepared from tubules from 7-day-old adult Drosophila. Where the effect of cGMP on gene expression was being investigated, tubules were incubated with or without 100 μmol l –1 cGMP in Schneider's medium for 3 h before the mRNA was extracted. Reverse transcription was carried out using Superscript II (Invitrogen) using oligo(dT) primers. For each sample, 500 ng of cDNA was added to 25 μl of SYBR Green reaction mix (Finnzyme, Oy Espoo, Finland) with an appropriate concentration of the primers – WhiteF: GCCACCAAAAATCTGGAGAAGC/WhiteR:CACCCACTTGCGTGAGTTGTTG. Reactions were carried out in an Opticon 2 thermocycler (MJ Research Inc., Waltham, MA, USA). The ribosomal rp49(rpl32) gene (primers rp49F: TGACCATCCGCCCAGCATAC/rp49R:TTCTTGGAGGAGGACGCCGTG) was used as a reference standard in all experiments(McGettigan et al., 2005).


Immunocytochemistry on intact Malpighian tubules was carried out as previously described (MacPherson et al.,2001). A mouse monoclonal primary anti-GFP antibody recognising GFP variants (Zymed, Invitrogen Ltd) diluted 1:1000 in PAT [0.05% (v/v) Triton X-100 and 0.5% (w/v) BSA in PBS with14 mmol l –1 NaCl, 0.2 mmol l –1 KCl, 1 mmol l –1 Na2HPO4 and 0.2 mmol l –1 KH2PO4, pH 7.4], was used followed by addition of secondary antibody, Alexa Fluor® 568-labelled anti-mouse IgG (Molecular Probes, Invitrogen Ltd), diluted 1:500 in PAT. The nuclear stain 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) was applied to the tubules for 1 min at 500 ng ml –1 in PBS. Samples were viewed using a Zeiss 510 Meta confocal system and images processed using LMS image software. All images were taken at the same gain and exposure.

What is cAMP Boost?

Nutraville describes cAMP Boost as a safe and powerful fat-burning formula that uses the strongest PDE-4 inhibitors available. These inhibitors remove the enzymes blocking fat loss while boosting the fat-burning messages sent from cAMP molecules. Through these mechanisms, cAMP Boost can purportedly tell your cells &ldquoto burn fat hour by hour, nonstop.&rdquo

cAMP Boost is priced at $67 per bottle and backed by a 365-day moneyback guarantee. Each bottle should last for one month. However, the company recommends doubling the dose to maximize the fat-burning effectiveness of the formula.

How Does cAMP Boost Work?

cAMP Boost uses six active ingredients to help you lose weight, including grapefruit extract, guarana seed extract, grains of paradise extract, and black pepper extract.

The manufacturer recommends taking one capsule twice daily (morning and mid-day) to help you lose a significant amount of weight. The manufacturer also recommends doubling the dose to maximize the fat-burning effects of the formula.

Here&rsquos how Nutraville, the maker of cAMP Boost, describes the effects of cAMP Boost:

&ldquocAMP Boost is a powerful fat loss formula that uses ingredients shown in studies to increase cyclic amp signals to fat cells. Those cyclic amp alerts cells to release and burn fat so that you can effortlessly lose fat hour by hour.&rdquo

Every one of us has an enzyme in our bodies called PDE-4. This enzyme blocks your body from sending fat-burning signals to your cyclic adenosine monophosphate (cAMP). cAMP is the most important messenger for sending signals to your cells to burn fat. When PDE-4 is present in your body, it prevents you from maximizing fat burning.

Here&rsquos how the c-AMP Boost website explains the effects of cAMP and PDE-4 on your weight loss:

&ldquoThere&rsquos a certain &ldquosignaling molecule&rdquo in the body called cAMP… And this signaling molecule sends messages to your cells to burn fat. However… there&rsquos also a &ldquosignal-muting enzyme&rdquo in the body called PDE-4… that targets and blocks the fat-burning signals sent from cAMP molecules.&rdquo

When PDE-4 blocks cAMP signals, it makes it hard to burn fat – even if you&rsquore eating right and exercising.

Unfortunately, as we get older, the body creates more PDE-4, which is one cause of unwanted weight gain with age. Nutraville claims cAMP Boost stops PDE-4, inhibiting the enzyme and unlocking effective weight loss.

How Does cAMP Boost Stop PDE-4?

cAMP Boost works as a PDE-4 inhibitor, helping to unlock your fat-burning capabilities.

To block PDE-4, cAMP Boost combines Sinetrol with natural ingredients. The Sinetrol in cAMP Boost deactivates PDE-4 enzymes and boosts fat-burning cAMP signals to your fat cells.

cAMP Boost also contains Bioperine, a black pepper extract shown to enhance the absorption of ingredients.

  • Lose weight safely and rapidly
  • Flatten your belly
  • Burn calories
  • Turn unsightly white body fat into fat-burning brown fat
  • Help your cells burn fat hour by hour, non-stop

Nutraville claims the ingredients in their formula act as &ldquothe strongest PDE-4 inhibitors available,&rdquo removing the enzymes responsible for blocking fat loss. These ingredients also boost the fat-burning messages sent from cAMP molecules, telling your cells to burn fat hour by hour, non-stop.

The Importance of Brown Fat

cAMP Boost also targets brown fat within your body, turning your white body fat into fat-burning brown fat. Yes, there&rsquos a difference between types of fat within your body. Some types of fat are better than others.

White fat, for example, contains only one or two mitochondria. Brown fat, meanwhile, contains a dozen mitochondria, making it a fat-burning powerhouse.

By increasing the amount of brown fat activity within your body, you can make it easier to lose weight. The more brown fat you have, the more calories your body burns.

cAMP Boost contains an African spice that purportedly &ldquotoasts&rdquo white fat tissue within your body. Here&rsquos how Nutraville explains the effects of that ingredient:

&ldquoI like to think of it like roasting a marshmallow over a crackling fire because there&rsquos a certain hot African spice that &ldquotoasts&rdquo white fat tissue… And we&rsquove added this to our cAMP Boost formula to help literally transform stubborn body fat into this body-slimming fat that reshapes your figure and gives you tons of energy.&rdquo

This ingredient and other ingredients within cAMP Boost can purportedly reactivate this fat-burning brown fat, making it easier to lose weight.

How Much Weight Can You Lose With cAMP Boost?

According to Nutraville, you can wake up looking leaner, younger, and sexier in the mirror each morning. Instead of starting each day frustrated by your weight loss progress, you can wake up feeling motivated.

Here&rsquos how much weight you can lose, according to the website:

&ldquoYou&rsquoll suddenly lose 10, 20, even 30 pounds or more of heavy, ugly body fat and amaze your family with a mind-blowing transformation that actually came pretty darn easy…Pull your favorite pair of jeans up past your thighs again… and look bootylicious (in a sexy way).&rdquo

Other weight-loss claims made on the cAMP Boost sales page include:

In one study on an ingredient within cAMP Boost, participants lost up to 677% more body fat than the placebo group – just by taking a PDE-4 inhibitor supplement

In another study, participants lost fat 6x faster than the placebo group while losing 5.53% of their body fat after just 4 weeks, dropping a total of 15.6% of their body fat after 12 weeks researchers in this group took a sour fruit compound that works as a PDE-4 inhibitor

In another study, researchers found that participants were burning an extra 180 calories &ldquowith literally no effort&rdquo after taking one of the ingredients in cAMP Boost participants in that study lost 63% of their fat mass over a 4 month period

One woman introduced on the website claims she lost 22lbs of body fat after 8 weeks while taking cAMP Boost, or around 2.5 to 3.5lbs per week

Some diet pills make unrealistic weight-loss claims, suggesting you can lose 50lbs in a month without dieting or exercising. The cAMP Boost website is more realistic, citing scientific studies, peer-reviewed research, and real customer experiences. Like other diet pills, cAMP Boost should complement healthy diet and exercise habits – not replace them.

CAMP Boost Ingredients

cAMP Boost uses Sinetrol and other ingredients to help you lose weight. Some of these ingredients inhibit PDE-4. Other ingredients increase cAMP signaling. Together, these effects can help you burn more fat 24 hours a day.

Here are the ingredients in cAMP Boost and how they work, according to Nutraville:


Sinetrol is a proprietary formula that targets the root cause of stubborn belly fat: the enzyme called PDE-4. This enzyme blocks the fat-burning activity of cAMP, making it difficult to lose weight. Sinetrol has been studied for its weight loss benefits, and multiple studies have shown Sinetrol can have powerful effects on weight loss.

Grains of Paradise

Grains of paradise is an herb native to southern Ethiopia. This is the &lsquohot African spice&rsquo mentioned in the brown fat section above. According to Nutraville, grains of paradise can activate brown fat within your body. Brown fat burns more energy than white fat. Turning your white fat into brown fat, grains of paradise can purportedly have powerful effects on weight loss. Participants taking grains of paradise burned 100 calories per day more than a placebo group in one study.

Guarana Seed

cAMP Boost also contains guarana seed, a natural extract linked to weight loss in multiple studies. In one study, participants taking guarana seed lost 16.3lbs and 5.1% of their body fat over an 8 week period. Guarana appears to work by helping you burn more calories, leading to &ldquosafe and rapid fat loss,&rdquo according to Nutraville.

Grapefruit Extract

cAMP Boost contains grapefruit extract, a sour superfruit popular for weight loss. Dr. Oz recently praised grapefruit&rsquos weight loss benefits, claiming &ldquothis ingredient is back for weight loss…and better than ever.&rdquo According to Nutraville, grapefruit extract helps control body weight by increasing cAMP signals sent out to cells, creating a safe and rapid fat loss. In one study, participants taking grapefruit extract lost 333% more weight than the placebo group.


The final ingredient in cAMP Boost is Bioperine, a special type of black pepper extract. Nutraville added this ingredient to enhance the absorption and bioavailability of other ingredients in cAMP Boost. According to Nutraville, Bioperine &ldquoworks like a shield around each ingredient,&rdquo helping each ingredient deliver its maximum benefits to your body.

The Story Behind cAMP Boost

cAMP Boost was created by Carly Johnson, a wife, and mother who was frustrated with her weight gain. Carly&rsquos husband left her for a skinnier woman. That woman later made fun of Carly&rsquos weight, teaching her son to call Carly fat.

Humiliated and annoyed, Carly decided to take action. She started researching natural cures for weight loss. She felt like she did everything right, including dieting and exercising, yet she was not losing weight.

Eventually, Carly stumbled upon PDE-4 inhibitors and the ingredients above. She teamed up with her friend, John, to create the ingredients in supplement form. After taking the formula, Carly lost 22lbs in 8 weeks.

Motivated by her weight loss success, Carly decided to sell the supplement online in the form of cAMP Boost.

Carly is careful to explain she&rsquos not a doctor or a nutritionist: she&rsquos just an ordinary woman who decided to create a weight loss supplement. Today, anyone can buy cAMP Boost online through the official website, potentially enjoying similar weight loss results to Carly.

CAMP Boost Ingredients Label

Nutraville publishes its full list of ingredients and dosages upfront, making it easy to compare the supplement to scientific studies and competing for weight loss aids.

  • 450mg of grapefruit extract, guarana seed extract, citrus Sinensis extract, and blood orange concentrate (in the form of Sinetrol Xpur)
  • 40mg of grains of paradise extract (in the form of CaloriBurn)
  • 5mg of black pepper extract (in the form of Bioperine)
  • Other ingredients, including vegetable cellulose, titanium dioxide, rice powder, magnesium stearate, and silicon dioxide

Scientific Evidence for cAMP Boost

Nutraville claims every ingredient within cAMP Boost &ldquohas been proven in studies to be safe and effective.&rdquo The company cites dozens of studies on supporting its advertised benefits.

In this 2006 study, for example, researchers discussed the effects of phosphodiesterase (PDE) inhibitors. Phosphodiesterases are a diverse family of enzymes that play a key role in regulating intracellular levels of the second messengers cAMP and cGMP, making PDEs crucial for cell function. There are three main types of PDE inhibitors, including PDE-3 inhibitors (for congestive heart failure), PDE-4 inhibitors (for inflammatory airway disease), and PDE-5 inhibitors (for erectile dysfunction).

It&rsquos also true that PDE-4 activity drops with age. In this 2016 study, researchers observed age-related differences in PDE activity, leading to noticeable changes in cellular signaling. Researchers were analyzing the cardiovascular health effects of this PDE-4 activity – not the weight loss effects. However, it&rsquos true that PDE-4 activity changes with age.

One of the biggest ingredients within cAMP Boost is Sinetrol. Sinetrol is a proprietary blend of citrus extracts backed by multiple clinical trials. As explained on, the compound has been proven to enhance body composition and fat loss in three clinical trials. In one study involving 77 overweight and obese subjects, subjects dropped an average of 63% of their excess fat body mass and burned an extra 180 calories per day after taking Sinetrol. Subjects also significantly gained lean mass, leading to healthier body composition.

It&rsquos also true that brown fat is more metabolically active than white fat, possibly helping you burn more calories at rest. As explained in this 2009 study published in Diabetes, metabolically active brown adipose tissue (brown fat) contributes to energy expenditure. In fact, brown fat is a survival mechanism. It dissipates energy as heat and impacts daily energy expenditure, making it value for weight loss.

cAMP Boost contains a patented form of grains of paradise extract called CaloriBurn. As you may guess from the name, CaloriBurn is designed specifically to enhance the fat-burning effects of grains of paradise. In this 2014 study, researchers gave grains of paradise to participants for a 4 week period. Although researchers did not observe significant weight loss compared to a placebo, researchers did find that grains of paradise increased whole-body energy expenditure. Based on these results, researchers concluded that grains of paradise extract &ldquomay be an effective and safe tool for reducing body fat.&rdquo Participants took 30mg of grains of paradise extract per day, which is slightly less than the 40mg of grains of paradise extract in each capsule of cAMP Boost.

Overall, cAMP Boost contains ingredients and dosages proven to help with weight loss. Although Nutraville has not completed clinical trials specifically on cAMP Boost, the company has linked to many studies proving the ingredients in cAMP Boost can help you lose weight.

CAMP Boost Pricing

cAMP Boost is priced at $67 per bottle, although the price drops as low as $41 per bottle when ordering multiple units.

  • 1 Bottle: $67 + $9.95 Shipping
  • 3 Bottles: $171 + Free Shipping
  • 6 Bottles: $246 + Free Shipping

Each bottle contains a 30 day supply (60 capsules). You take one capsule twice per day to inhibit PDE-4, activate cAMP, and lose weight.

CAMP Boost Refund Policy

You can request a complete refund on your purchase (minus original shipping costs) within 365 days of your original purchase date.

If you did not lose a significant amount of weight while taking cAMP Boost, or if you&rsquore unhappy with the results of the supplement for any reason, then you can request a full refund with no questions asked and no hassle.

About Nutraville

cAMP Boost is made by a nutritional supplement company named Nutraville. Nutraville is based in Valencia, California. The company makes three supplements, including Amyl Guard, Gluta Raise, and cAMP Boost.

Materials and Methods

Protein Expression and Purification

A DNA sequence encoding Human PKG Iβ (92�) was cloned into pQTEV [33]. The protein was produced in BL21 (DE3) E. coli which were grown at 37ଌ until OD600 of 0.6 then induced with 0.4 mM IPTG. The cultures were grown for an additional 18 hours at 18ଌ. Cells were suspended in 50 mM Tris, 150 mM NaCl, 1 mM DTT (pH 7.9) and lysed using a cell disruptor (Constant Systems). His-tagged PKG Iβ (92�) was purified with a BioRad IMAC resin on a Bio-Rad Profinia™ purification system. The protein was eluted with cell suspension buffer containing 250 mM imidazole. The sample was incubated with 1.0 mg/ml TEV protease at 4ଌ for 48 hours to remove the His-tag. The protein was purified further with a Q sepharose HP followed by gel filtration on a Hi-load 16/60 Superdex-75 column (GE Healthcare) in 25 mM Tris-HCl, pH 8.0, NaCl 150 mM and 1 mM TCEP-HCl.


For the crystallization of the partial apo crystals, the protein sample was concentrated to 20 mg/ml using a 10 kDa cutoff Amicon Ultra (Millipore). The partial apo crystals were obtained using the vapor diffusion method in 1.4 M sodium/potassium phosphate (pH 5.6) at 22ଌ. Crystal optimization was done using an Orxy6™ robot (Douglas Instruments LTD). The bipyramidal crystals appeared in 1.4 M sodium/potassium phosphate (pH 8.1) at 22ଌ in 2 days. Co-crystallization with cGMP was accomplished by adding cGMP (Aral Biosynthetics) to a final concentration of 5 mM to the purified protein sample, which was then concentrated to 33 mg/ml using a 10 kDa cutoff Amicon Ultra (Millipore). The crystals of the PKG Iβ:cGMP complex were obtained using the vapor diffusion method in 0.1 M sodium malonate (pH 5.0), 12% PEG 3350 at 4ଌ. Similarly, co-crystallization with cAMP was accomplished by adding cAMP to a final concentration of 5 mM to the protein sample, which was concentrated with a 10 kDa cutoff Amicon Ultra (Millipore) to 17 mg/ml. The PKG Iβ:cAMP complex crystals were obtained using the vapor diffusion method in 1.4 M sodium/potassium phosphate (pH 5.6) at 4ଌ.

All crystals were transferred to a cryoprotectant solution (25% glycerol) and flash cooled in liquid nitrogen. X-ray diffraction data were collected at beamline 8.2.1 (Advanced Light Source, Berkeley, CA, USA). Diffraction data were processed and scaled using HKL2000, resulting in acceptable data set with satisfactory summary statistics ( Table 1 ).

The crystal structure of PKG Iβ (92�):cAMP was determined by molecular replacement using a truncated model of PKA RIα (91�) (PDB: 1RGS) as a molecular replacement probe [8]. Subsequent phasing, density modification and model building were carried out with phenix.autosol [34]. The resulting model was manually completed in Coot [35] and restrained-structure-refinement implementing TLS refinement [36] resulted in cAMP model with Rwork and Rfree of 20.6% and 23.0% respectively. Refinement of the 2.9 Å PKG Iβ(92�):cGMP complex was carried out in PHENIX (dev-403) [10] using reference dihedral restraints derived from the higher resolution cAMP complex, as described in the following section. Use of the higher resolution reference model in refinement improved the R and R-free values, as well as MolProbity validation criteria, resulting a final model with Rwork and Rfree of 20.4% and 26.0%, respectively [37]. For all of the Fo-Fc omit maps shown in the figures, we generated simulated annealing omit maps, omitting a region with a border of 2 Å around each ligand as described in Terwilliger et al.[38].

Reference model refinement in phenix.refine

To improve refinement stability and associated model quality in low resolution refinement, the cGMP and partial apo structures were refined with phenix.refine using dihedral restraints obtained from the higher resolution PKG Iβ:cAMP structure. Dihedral restraints obtained from the reference model were imposed on the working model if the absolute angular deviation fell within a user-defined threshold. For this refinement, a threshold value of 15° was used. These restraints served to direct the overall topology of the model while avoiding unjustified bias to the high-resolution model. The refinement scheme is similar in concept to non-crystallographic symmetry restraints adopted in SHELXL and the deformable elastic network approach introduced in the following reference [39].

Isothermal Titration calorimetry

To remove residual cAMP, all samples were denatured by incubating in 6 M guanidine HCl for 24 h at 4ଌ, then renatured by step-wise dialysis against first 2 M and then 0.5 M guanidine HCl over 48 h. The samples were then purified in 10 mM Tris (pH 8.0) and 150 mM NaCl on a Hi-load 16/60 Superdex 75 column (GE Healthcare). The calorimetric measurements for cAMP and of cGMP binding to PKG Iβ (92�) were carried out using a VP-ITC calorimeter (MicroCal LLC, Northampton, MA). The protein was placed in the sample cell at a concentration of 15 µM in the column buffer. Cyclic nucleotides were placed in the injection syringe at a concentration of 250 µM. The injection volume was 5 μl. The data was processed using the Origin software with a manufacturer-supplied custom-addon ITC sub-routine. The reported results were repeated in at least duplicate.

Protein data bank accession codes

The coordinates for the structures described herein have been deposited in the Protein Data Bank under the accession codes 3OD0, 3OCP and 3OGJ for PKG Iβ:cGMP, PKG Iβ:cAMP and the partial apo structures, respectively.

Role and Functions of Second Messengers | Pharmacodynamics

After reading this article you will learn about the role and functions of second messengers.

Many hormones, neurotransmitters, autacoids and drugs act on specific membrane receptors, the immediate consequence of which is activation of a cytoplasmic component of the receptor, which may be an enzyme such as adenylate cyclase, guanylate cyclase or activation of a transport systems or opening of an ion-channel.

These cytoplasmic components which carry forward the stimulus from the receptors are known as second messengers the first messenger being the receptor itself. Examples of second messengers are-cAMP, cGMP, ca 2+ , G-proteins, IP3, DAG, etc.

The role of cAMP as a second messenger was first revealed by the work of Sutherland in late 1950’s. This discovery demolished the barriers that existed between biochemistry and pharmacology. cAMP is a nucleotide synthesised within the cell from ATP by the action of adenylate cyclase in response to activation of many receptors. It is inactivated by hydrolysis to 5′-AMP, by the action of enzyme phosphodiesterase.

cAMP has varied regulatory effects on cellular functions, for example, energy metabolism, cell division and cell differentiation, ion-transport, ion-channel function, smooth muscle contractility etc. These varied effects are brought about by a common mechanism, namely the activation of various protein kinases by cAMP.

Many different drugs, hormones of neurotransmitters produce their effects by increasing or decreasing the catalytic activity of adenylate cyclase and thus lowering or raising the concentration of cAMP within the cell. The cAMP levels in the cell can also be raised by inhibiting the metabolizing enzyme phosphodiesterase.

Cyclic guanosine monophosphate is another intercellular messenger synthesised by the enzyme guanylate cyclase from GTP. It has been identified in cardiac cells, bronchial smooth muscle cells, and other tissues. For most of the effects produced, cAMP seems to be stimulatory while cGMP seems to be inhibitory in nature.

When the cAMP and cGMP systems are both present in a single cell or tissue, they are linked to receptors through which drugs produce opposite effects. For examples in cardiac tissue cells, β-adrenoceptors increase the frequency and force of contraction by increasing cAMP levels, whereas cholinergic receptors have opposite effect by increasing cGMP levels.

The IP3 and DAG system is another important intracellular second messenger system, and was identified first by Michell in 1975. Both are degradation products of membrane phospholipids by an enzyme phospholipase C. IP3 acts very effectively to release calcium from intracellular stores. This Ca 2+ is known to regulate the function of various enzymes, contractile proteins and ion- channels.

DAG directly activates protein kinase C and controls phosphorylation of ammo acids of a variety of intracellular proteins. This causes release of hormones from endocrine glands or modulates neuro­transmitter release or modulates smooth muscle contractibility or inflammatory responses or ion-transport or tumour promotion etc. There exist at least six different types of PKC distributed unequally in different cells.

Activation of another enzymes phospholipase A2 leads to production of arachidonic acid from the membrane phospholipids, which are further broken down to prostaglandins, leukotrienes, thromboxanes etc.

They are well known for their role as local hormones, but it is of interest that arachidonic acid itself and its metabolites have recently been shown to function as intracellular, messengers, controlling potassium channel function in certain neurons.

Calcium ions are of great importance amongst many other intracellular second messengers. Many regulatory actions are mediated by Ca 2+ bound to its intracellular regulatory protein, calmodulin. Ca 2+ ions are also involved in release of arachidonic acid from membrane phospholipids by activated phospholipases and so initiate the synthesis of prostaglandins and leukotrienes. Ca 2+ in synergism with PKC have been shown to activate cellular function like hepatocyte glycogenolysis, insulin release from pancreas. Ca 2+ also plays an important role in contraction and relaxation of skeletal and smooth muscles of body.

G-proteins represent the level of middle management in the cellular organisation and are able to communicate between the receptors and the effector enzymes or ion-channels. They were called G-proteins because of their interaction with the guanine nucleotides, GTP and GDP.

The G proteins are bound to the cytoplasmic surface of the plasma membrane. They are heterotrimeric molecules consisting of 3 subunits α, β and γ (fig 3.10). Their classification as stimulatory or inhibitory is based on the identity of their distinct α subunit.

The β and γ subunits remain associated as β γ complex with the cytoplasmic surface of the membrane when the system is inactive or in resting state, GDP is bound to the α subunit.

Whenever an agonist interacts with the receptor, this facilitates GTP binding to α subunit and promotes dissociation of GDP from its place. Binding of GTP activates the α subunit and α-GTP is then thought to dissociate from β and interact with a membrane bound effector.

The process is terminated when the hydrolysis of GTP to GDP occurs through the GTpase activity of the α-subunit. The resulting α-GDP then dissociates from the effector, and reunites with β γ completing the response cycle. Attachment of the subunit to an effector molecule actually increases its GTpase activity, the magnitude of this increase varies for different types of effector.

Mechanisms of this type in general result in amplification because a single agonist receptor complex can activate several G-protein molecules in turn, and each of these can remain associated with the effector enzyme for long enough to produce many molecules of product.

The product is often a second messenger, and further amplification occurs before the final cellular response is produced. It is the biological adaptation of an organism for judicious use of its transmitter substances.

G-proteins are not all identical, the α-subunit in particular shows variability. It is believed that there are three main varieties of G-protein viz. Gs, Gi and Gq. Gs and Gi produce respectively stimulation and inhibition of the effector system (fig. 3.11). It is not unusual for several receptors in an individual cell to activate a single G protein and a single receptor regulating more than one G-proteins.


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