Cyclic AMP, Protein Kinase A, and Phosphodiesterases: Proceedings of an International Workshop

Cyclic nucleotides cAMP and cGMP are part of almost all major cellular signaling pathways. Phosphodiesterases (PDEs) are enzymes that regulate the intracellular levels of cAMP and cGMP. Protein kinase A or cAMP-dependent protein kinase mediates most cAMP effects in the cell. Over the last 25 years, various components of this group of molecules have been involved in human diseases, both genetic and acquired. Lately, the PDEs attract more attention. The pharmacological exploitation of the PDE's ability to regulate cGMP and cAMP, and through them, a variety of signaling pathways, has led to a number of new drugs for diverse applications from the treatment of erectile dysfunction to heart failure, asthma, and chronic obstructive pulmonary disease. We present the abstracts (available online) and selected articles from the proceedings of a meeting that took place at the National Institutes of Health (NIH), Bethesda, MD, June 8-10, 2011.     

Regulation of intracellular cyclic GMP levels in olfactory sensory neurons

Cyclic AMP is the primary second messenger mediating odorant signal transduction in mammals. A number of studies indicate that cyclic GMP is also involved in a variety of other olfactory signal transduction processes, including adaptation, neuronal development, and long-term cellular responses in the setting of odorant stimulation. However, the mechanisms that control the production and degradation of cGMP in olfactory sensory neurons (OSNs) remain unclear. Here, we investigate these mechanisms using primary cultures of OSNs. We demonstrate that odorants increase cGMP levels in intact OSNs in vitro. Different from the rapid and transient cAMP responses to odorants, the cGMP elevation is both delayed and sustained. Inhibition of soluble guanylyl cyclase and heme oxygenase blocks these odorant-induced cGMP increases, whereas inhibition of cGMP PDEs (phosphodiesterases) increases this response. cGMP PDE activity is increased by odorant stimulation, and is sensitive to both ambient calcium and cAMP concentrations. Calcium stimulates cGMP PDE activity, whereas cAMP and protein kinase A appears to inhibit it. These data demonstrate a mechanism by which odorant stimulation may regulate cGMP levels through the modulation of cAMP and calcium level in OSNs. Such interactions between odorants and second messenger systems may be important to the integration of immediate and long-term responses in the setting odorant stimulation.     

Human cyclic nucleotide phosphodiesterases possess a much broader substrate-specificity than previously appreciated

Phosphodiesterases (PDEs) capable of degrading cAMP and cGMP are indispensable for the regulation of cyclic nucleotide-mediated signals. The existence of other cyclic nucleotides such as cCMP and cUMP has been discussed controversially in the literature. Despite publications on PDEs hydrolyzing cCMP or cUMP, the molecular identity of such enzymes remained elusive. Recently, we have provided evidence for a role of cCMP as second messenger in vascular relaxation and inhibition of platelet aggregation. Using an HPLC-MS based assay, here, we show that human PDEs belonging to various families hydrolyze not only cAMP and cGMP but also other cyclic nucleotides.     

Cyclic nucleotide phosphodiesterase (PDE) inhibitors: novel therapeutic agents for progressive renal disease

Cyclic nucleotides are recognized as critical mediators of many renal functions, including solute transport, regulation of vascular tone, proliferation of parenchymal cells, and inflammation. Although most studies have linked elevated cAMP levels to activation of protein kinase A, cAMP can also directly activate cyclic nucleotide gated ion channels and can signal through activation of GTP exchange factors. Cyclic AMP signaling is highly compartmentalized through plasma membrane localization of adenylyl cyclase and expression of scaffolding proteins that anchor protein kinase A to specific intracellular locations. Cyclic nucleotide levels are largely regulated through catabolic processes directed by phosphodiesterases (PDEs). The PDE superfamily is large and complex, with over 60 distinct isoforms that preferentially hydrolyze cAMP, cGMP, or both. PDEs contribute to compartmentalized cyclic nucleotide signaling. The unique cell- and tissue-specific distribution of PDEs has prompted the development of highly specific PDE inhibitors to treat a variety of inflammatory conditions. In experimental systems, PDE inhibitors have been employed to demonstrate functional compartmentalization of cyclic nucleotide signaling in the kidney. For example, mitogenesis in glomerular mesangial cells and normal tubular epithelial cells is negatively regulated by an intracellular pool of cAMP that is metabolized by PDE3, but not by other PDEs. In Madin-Darby canine kidney cells, an in vitro model of polycystic kidney disease, an intracellular pool of cAMP directed by PDE3 stimulates mitogenesis. In mesangial cells, an intracellular pool of cAMP directed by PDE4 inhibits reactive oxygen species and expression of the potent proin-flammatory cytokine monocyte chemoattractant protein 1. An intracellular pool of cGMP directed by PDE5 regulates solute transport. PDE5 inhibitors ameliorate renal injury in a chronic renal disease model. In this overview, we highlight recent studies to define relationships between PDE expression and renal function and to provide evidence that PDE inhibitors may be effective agents in treating chronic renal disease.     

Trypanosome cyclic nucleotide phosphodiesterase 2B binds cAMP through its GAF-A domain

Trypanosoma brucei, the causative agent of sleeping sickness in humans and livestock, expresses at least three cAMP-specific class I phosphodiesterases (PDEs), all of which are essential for survival of the parasite. These PDEs have either one or two N-terminal GAF domains, which in other proteins function as signaling domains. However, neither the functional roles nor ligands for these domains in trypanosome PDEs are known. The present study shows that TbPDE2B, which contains two tandem GAF domains, binds cAMP with high affinity through its GAF-A domain. A purified recombinant N terminus + GAF-A domain binds cAMP with an affinity (Ki) of approximately 16 nM. It also binds cGMP but with a 15-fold lower affinity of approximately 275 nM. The TbPDE2B holoenzyme has a somewhat lower affinity (approximately 55 nM) for cAMP but a greatly lower affinity (approximately 10 microM) for cGMP. This suggests that both the selectivity and affinity for a ligand can be determined not only by the nature of the binding domain but also by the adjacent domains. Additionally, binding of cAMP to the holoenzyme showed positive cooperativity, with a Hill coefficient value of 1.75. However, binding of cGMP to the holoenzyme did not show any cooperativity, suggesting differences in the conformational changes caused by binding of these two cyclic nucleotides with the protein. Point mutation of a key predicted binding site residue (T317A) resulted in a complete loss of high affinity cAMP binding. This mutation increased the apparent Km of the mutant enzyme for substrate without altering the Vmax. A truncated catalytic domain construct of TbPDE2B also exhibited an increased Km, strongly suggesting that cAMP binding to the GAF-A domain can regulate TbPDE2B by allowing the full activity of the enzyme to be expressed. These properties of the GAF-A domain of TbPDE2B thus suggest that it could be a new target for anti-trypanosomal drugs.     

A glutamine switch mechanism for nucleotide selectivity by phosphodiesterases

Phosphodiesterases (PDEs) comprise a family of enzymes that modulate the immune response, inflammation, and memory, among many other functions. There are three types of PDEs: cAMP-specific, cGMP-specific, and dual-specific. Here we describe the mechanism of nucleotide selectivity on the basis of high-resolution co-crystal structures of the cAMP-specific PDE4B and PDE4D with AMP, the cGMP-specific PDE5A with GMP, and the apo-structure of the dual-specific PDE1B. These structures show that an invariant glutamine functions as the key specificity determinant by a "glutamine switch" mechanism for recognizing the purine moiety in cAMP or cGMP. The surrounding residues anchor the glutamine residue in different orientations for cAMP and for cGMP. The PDE1B structure shows that in dual-specific PDEs a key histidine residue may enable the invariant glutamine to toggle between cAMP and cGMP. The structural understanding of nucleotide binding enables the design of new PDE inhibitors that may treat diseases in which cyclic nucleotides play a critical role.     

The effects of valproate and phenytoin on the cAMP and cGMP levels in nervous tissue

Earlier studies have demonstrated that valproic acid (VPA) and phenytoin (PHT) influence the excitability properties of crayfish axons through different mechanisms. PHT was found to antagonize the electrophysiologic effects of VPA. The purpose of the present study was to determine if the electrophysiologic effects of VPA and PHT are correlated with changes in the cellular levels of either cAMP or cGMP as these substances are known to influence membrane excitability. It was found that PHT (0.1 mM) has no effect on the levels of either cAMP or cGMP within crayfish neural tissue. VPA (4.0 mM) also has no effect on cAMP levels. However, it does significantly reduce the levels of cGMP. Pretreatment of neural tissue with PHT has been shown to eliminate the effects of VPA on membrane excitability. It was found that this pretreatment has no influence on VPA's ability to reduce cGMP levels. The effect of VPA on cGMP levels is observed in the absence of spontaneous activity. Therefore, it is concluded that the observed reduction in cGMP levels does not represent the modulation of cGMP levels that is known to accompany activity. Two experiments demonstrate that the 4-mV depolarization of membranes by VPA can not account for its effect on cGMP levels. In the first, pretreatment with PHT abolished the depolarizing effect on VPA but not its effect on cGMP. In the second, a concentration of ouabain which depolarizes crayfish neural tissue by 8-10 mV without producing spike activity had no effect on either cAMP or cGMP levels. These experiments effectively dissociate the electrophysiologic response to VPA and PHT from changes in cyclic nucleotide levels.     

Modulation of glucocorticoid receptor activity by cyclic nucleotides and its implications on the regulation of human skin fibroblast growth and protein synthesis

The modulation of glucocorticoid receptor activity by cyclic nucleotides was studied in cultured human skin fibroblasts. The receptors appeared to be activated in the presence of dibutyryl-cAMP and inactivated by dibutyryl-cGMP. Significantly, the cGMP content of the fibroblasts increased during cell growth, with a concomitant decrease in the glucocorticoid receptor activity, while when the cells reached early confluency the decrease in cGMP content was accompanied by an increase in cAMP and increased activity of the glucocorticoid receptors. In addition, cortisol induced (2'-5')oligoadenylate synthetase in these cells and raised the cellular (2'-5')oligoadenylate concentrations. This resulted in a decrease in both DNA and protein synthesis activity in the cells, a response which correlated with the (2'-5')oligoadenylate concentration. The combination of cortisol and dibutyryl-cAMP had a synergetic stimulatory effect on the (2'-5')oligoadenylate concentration and a synergetic inhibitory effect on protein synthesis. In conclusion, it is demonstrated here that cyclic nucleotides can modulate glucocorticoid receptor activity in cultured human skin fibroblasts, and thus these compounds may indirectly affect cellular metabolism by regulating the cellular responses to glucocorticoids.     

Local changes in fractional saturation of cGMP- and cAMP-receptors in intestinal microvilli in response to cholera toxin and heat-stable Escherichia coli toxin

Cyclic nucleotide modulation of electrolyte transport across intestinal brushborder membranes is initiated by binding of cGMP and cAMP to high-affinity receptors at the interior of the microvilli. Previously these receptors have been identified by photoaffinity-labelling techniques as regulatory domains of cGMP- and cAMP-dependent protein kinases. In the present study, the receptor concentration in isolated brushborder membrane vesicles and their fractional saturation in absorptive and secretory states of the tissue were estimated. In microvillous membrane vesicles isolated from rat small intestine in the absorptive state, about 10% of the total number of cGMP receptors (25.5 pmol/mg protein) and 40% of all cAMP receptors (28.7 pmol/mg protein) were occupied by endogenous cyclic nucleotides. Luminal exposure of the intestinal segments in vivo to heat-stable Escherichia coli toxin for 3-5 min increased the occupancy of cGMP receptors by about 5-fold without affecting receptor-bound cAMP levels. In contrast, incubation with cholera toxin for 2 h increased the fractional saturation solely of cAMP receptors by 2-fold. Addition of heat-stable E. coli toxin to cholera toxin-pretreated segments, again raising the cGMP levels by 5-fold, did not reduce the amount of receptor-bound cAMP. This finding argues against the concept that increased levels of cAMP during cholera would mimick cGMP effects on ion transport by low-affinity binding to microvillar cGMP receptors. This analysis of local changes in cyclic nucleotide levels at the microvillous level might help to explore the mechanism of action of other secretagogues or antidiarrhoeal agents and to delineate a possible compartmentation of cGMP and cAMP pools within the intestinal mucosa responding differently to external signals.     

Regulation of cyclic nucleotide phosphodiesterase activity in myometrium from pregnant and spayed rhesus monkeys.

The activities of myometrial cyclic nucleotide phosphodiesterases (PDEs) and the sensitivity of these enzymes to the effector molecules, cGMP and cAMP, were determined in the 100,000 g supernatant of homogenates from pregnant and spayed rhesus monkeys. The specific activities (per mg nitrogen) of the myometrial cyclic nucleotide PDEs in the supernatant from spayed monkeys were higher than those from pregnant monkeys at all substrate levels studied. However, when calculated on the basis of the DNA content of the myometrium, which was 8 times higher in the spayed than in the pregnant animals, the specific activities were lower in the tissue from spayed animals. At substrate levels of 2 . 5 micron-cAMP, low levels of cGMP (0 . 1-1 . 0 micron) caused the same percentage increase in cGMP-PDE activity in both tissues. At high substrate levels of 100 micron-cAMP, 1 micron-cGMP inhibited only the cAMP-PDE from spayed monkeys, and the enzyme from spayed monkeys was more effectively inhibited by 10 and 40 micron-cGMP than was the enzyme from pregnant animals. The cGMP-PDE activity was inhibited by cAMP (1 . 0-50 . 0 micron), and the percentage inhibition with increasing levels of cAMP appeared to be similar in the two series. The levels of cGMP and cAMP that modify the rate of hydrolysis of the other nucleotide in rhesus myometrium seem to be within the physiological range for these compounds in situ. It therefore appears possible that cAMP and cGMP are each involved in regulating the degradation of the other nucleotide in rhesus myometrium.     

Cyclic nucleotide phosphodiesterase 3 signaling complexes

The superfamily of cyclic nucleotide phosphodiesterases is comprised of 11 gene families. By hydrolyzing cAMP and cGMP, PDEs are major determinants in the regulation of intracellular concentrations of cyclic nucleotides and cyclic nucleotide-dependent signaling pathways. Two PDE3 subfamilies, PDE3A and PDE3B, have been described. PDE3A and PDE3B hydrolyze cAMP and cGMP with high affinity in a mutually competitive manner and are regulators of a number of important cAMP- and cGMP-mediated processes. PDE3B is relatively more highly expressed in cells of importance for the regulation of energy homeostasis, including adipocytes, hepatocytes, and pancreatic β-cells, whereas PDE3A is more highly expressed in heart, platelets, vascular smooth muscle cells, and oocytes. Major advances have been made in understanding the different physiological impacts and biochemical basis for recruitment and subcellular localizations of different PDEs and PDE-containing macromolecular signaling complexes or signalosomes. In these discrete compartments, PDEs control cyclic nucleotide levels and regulate specific physiological processes as components of individual signalosomes which are tethered at specific locations and which contain PDEs together with cyclic nucleotide-dependent protein kinases (PKA and PKG), adenylyl cyclases, Epacs (guanine nucleotide exchange proteins activated by cAMP), phosphoprotein phosphatases, A-Kinase anchoring proteins (AKAPs), and pathway-specific regulators and effectors. This article highlights the identification of different PDE3A- and PDE3B-containing signalosomes in specialized subcellular compartments, which can increase the specificity and efficiency of intracellular signaling and be involved in the regulation of different cAMP-mediated metabolic processes.     

Real-time monitoring of the PDE2 activity of live cells: hormone-stimulated cAMP hydrolysis is faster than hormone-stimulated cAMP synthesis

Cyclic nucleotide phosphodiesterases (PDEs) are the enzymes that catalyze the hydrolysis of cAMP and cGMP, thereby restricting the activity of these second messengers in cells. A unique ability to shape gradients of cyclic nucleotides and compartmentalize their signaling implies a high potency and a rapid action of PDEs. However, it has not been demonstrated how fast PDEs can hydrolyze cAMP in a living system. Here we perform a real-time monitoring of PDE2 activity in aldosterone-producing adrenal cells using a recently developed genetically encoded, fluorescent cAMP sensor, which reveals enormously rapid kinetics of cAMP degradation. Activation of PDE2 results in a rapid decrease of intracellular cAMP from high micromolar to the sub-micromolar range within a few seconds. Moreover, the kinetics of atrial natriuretic peptide-stimulated PDE2 activity (measured as decline of cAMP) are much faster than the speed of ACTH and isoprenaline-induced cAMP-synthesis (measured as cAMP accumulation) in the cells, revealing high catalytic activity and fast action of PDEs in regulating cAMP signaling in a physiological system.     

Real-time monitoring of phosphodiesterase inhibition in intact cells

Phosphodiesterases (PDEs) are hydrolytic enzymes, which convert cyclic AMP (cAMP) and cyclic GMP (cGMP) into their corresponding monophosphates. PDE-dependent hydrolysis shape gradients of these second messengers in cells, which may form the basis of their compartmentation and play a key role in a vast number of physiological and pathological processes. Here, we present a novel approach for real-time monitoring of local cAMP and cGMP levels associated with particular PDEs. We used HEK 293 cells expressing genetic constructs encoding a PDE of interest (PDE3A, PDE4A1 or PDE5A) fused to cAMP and cGMP sensors, which allow to directly visualize changes in cyclic nucleotide concentrations in the vicinity of PDE molecules by fluorescence resonance energy transfer (FRET). FRET was detected by imaging of single cells on 96-well plates and demonstrated specific effects of PDE inhibitors on local cyclic nucleotide levels. In addition, this approach reported physiological regulation of PDE3A activity, its activation by PKA-dependent phosphorylation and inhibition by cGMP. In conclusion, our assay provides a unique and highly sensitive method to analyze PDE activity in living cells. It allows to sense cAMP gradients around particular PDE molecules and to study the pharmacological effects of selective inhibitors on localized cAMP signalling.     

Specific localized expression of cGMP PDEs in Purkinje neurons and macrophages

As cGMP hydrolyzing cyclic nucleotide phosphodiesterases (PDEs) have diverse regulatory and catalytic properties, the specific cGMP PDEs a cell expresses will determine the duration and intensity of a cGMP signal. This, in turn, results in different cellular responses between cell types and tissues. Therefore, identifying which cGMP PDEs are expressed in different tissues and cell types could increase our understanding of physiological and pathological processes. The brain is one area where large numbers of diverse cGMP PDEs are expressed in specific regions and cell types. A case in point is differential expression of cGMP PDEs in neuronal cells. For example, we have recently found that PDE5 is expressed in all Purkinje neurons while PDE1B is expressed in only a subset of these neurons. The expression of PDE2 has also been found to be selective for discrete populations of neurons. Another example of selective cGMP PDE expression is seen with cytokine-induced differentiation of monocytes to macrophages. We have recently discovered that monocyte differentiation with the cytokine macrophage colony-stimulating factor (M-CSF) causes an upregulation of PDE2 and a small increase in PDE1B while granulocyte-macrophage colony-stimulating factor (GM-CSF) causes a large increase in PDE1B but a decrease in PDE2. These same cytokines can influence the phenotype of microglial cells and are likely to affect their expression of cGMP PDEs. In this report, we present recent results from our laboratory and review earlier findings illustrating the concept of highly specific expression of cGMP PDEs and discuss how this may be important for understanding brain function and dysfunction.     

Involvement of cGMP in cellular melatonin responses.

Melatonin can enhance and suppress constitutive protein secretion from murine melanoma M2R cells in vitro in a cholera-toxin (CTX) sensitive process. In a number of tissues melatonin has been shown to modulate cGMP levels. The involvement of cGMP in melatonin responses in the melanoma cells was investigated. The effects of melatonin on melanoma cells cGMP and cGMP-phosphodiesterase activity and the effects of cGMP analogs on the melatonin-mediated modulation of protein secretion were studied. Melatonin reduced cGMP levels in the melanoma cells. CTX treatment had a similar and non-additive effect. The effects of melatonin on protein secretion were abrogated by activation of cGMP-dependent protein kinases. In addition, melatonin inhibited cGMP phosphodiesterase activity in these cells. The data presented indicate that inhibition of cGMP via a CTX sensitive G protein may be a major signal transduction pathway used by melatonin in melanoma cells.     

Expression and activity of cGMP-dependent phosphodiesterases is up-regulated by lipopolysaccharide (LPS) in rat peritoneal macrophages

It has been shown that cyclic GMP (cGMP) modulates the inflammatory responses of macrophages, but the underlying molecular mechanisms are still poorly understood. Looking for proteins potentially regulated by cGMP in rat peritoneal macrophages (PMs), in this study we analyzed expression and activity of cGMP-hydrolyzing and cGMP-regulated phosphodiesterases (PDEs). It was found that freshly isolated peritoneal exudate macrophages (PEMs) express enzymes belonging to families PDE1-3, PDE5, PDE10, and PDE11. Analysis of substrate specificity, sensitivity to inhibitors, and subcellular localization showed that PDE2 and PDE3 are the main cGMP-regulated PDE isoforms in PEMs. The profile of PDE expression was altered by maintaining PEMs in culture and treatment with bacterial endotoxin (LPS). After 24 h culture, PDE5 was not present and the levels of PDE2, PDE3, and PDE11 were markedly decreased. However, their expression and activity was recovered after treatment of cultured cells with LPS. A similar pattern of changes was observed for the expression of TNFalpha, but not for guanylyl cyclase A (GC-A). LPS up-regulated PDE expression also in resident peritoneal macrophages (RPMs), although not all PDEs present in PEMs were detected in RPMs. Taken together, our results show that in rat PMs expression of cGMP-dependent PDEs positively correlates with the activation state of cells. Moreover, the fact that most of these PDEs hydrolyze also cAMP indicates that cGMP can play a role of potent regulator of cAMP signaling in macrophages.     

Seven Dictyostelium discoideum phosphodiesterases degrade three pools of cAMP and cGMP

The Dictyostelium discoideum genome uncovers seven cyclic nucleotide PDEs (phosphodiesterases), of which six have been characterized previously and the seventh is characterized in the present paper. Three enzymes belong to the ubiquitous class I PDEs, common in all eukaryotes, whereas four enzymes belong to the rare class II PDEs that are present in bacteria and lower eukaryotes. Since all D. discoideum PDEs are now characterized we have calculated the contribution of each enzyme in the degradation of the three important pools of cyclic nucleotides: (i) extracellular cAMP that induces chemotaxis during aggregation and differentiation in slugs; (ii) intracellular cAMP that mediates development; and (iii) intracellular cGMP that mediates chemotaxis. It appears that each cyclic nucleotide pool is degraded by a combination of enzymes that have different affinities, allowing a broad range of substrate concentrations to be degraded with first-order kinetics. Extracellular cAMP is degraded predominantly by the class II high-affinity enzyme DdPDE1 and its close homologue DdPDE7, and in the multicellular stage also by the low-affinity transmembrane class I enzyme DdPDE4. Intracellular cAMP is degraded by the DdPDE2, a class I enzyme regulated by histidine kinase/phospho-relay, and by the cAMP-/cGMP-stimulated class II DdPDE6. Finally, basal intracellular cGMP is degraded predominantly by the high-affinity class I DdPDE3, while the elevated cGMP levels that arise after receptor stimulation are degraded predominantly by a cGMP-stimulated cGMP-specific class II DdPDE5. The analysis shows that the combination of enzymes is tuned to keep the concentration and lifetime of the substrate within a functional range.     

Modulation of NMDA receptor function by cyclic AMP in cerebellar neurones in culture

The signal transduction pathways involved in NMDA receptor modulation by other receptors remain unclear. cAMP could be involved in this modulation. The aim of this work was to analyse the contribution of cAMP to NMDA receptor modulation in cerebellar neurones in culture. Forskolin increases cAMP and results in increased intracellular calcium and cGMP that are prevented by blocking NMDA receptors. Similar effects were induced by two cAMP analogues, indicating that cAMP leads to NMDA receptor activation. It has been reported that phosphorylation of Ser897 of the NR1 subunit of NMDA receptors by cAMP-dependent protein kinase (PKA) activates the receptors. Forskolin increases Ser897 phosphorylation. Neither Ser897 phosphorylation nor cGMP increase induced by forskolin are prevented by four inhibitors of PKA, suggesting that NMDA receptor activation is dependent on cAMP but not on PKA. Inhibition of Akt prevents forskolin-induced phosphorylation of Ser897, suggesting a role for Akt in the mediation of the modulation of NMDA receptors by cAMP. Pituitary adenylate cyclase-activating polypeptide (PACAP) activates its receptors, increasing cAMP and also leading to phosphorylation of Ser897 of NR1 and activation of NMDA receptors. These results indicate that cAMP modulates NMDA receptor in cerebellar neurones and may play a role in NMDA receptor modulation by other receptors.     

Prostaglandins and cyclic nucleotides in the urinary bladder of a rabbit model of partial bladder outlet obstruction

Bladder outlet obstruction (BOO) is a common disorder that is associated with altered bladder structure and function. For example, it is well established that BOO results in hypertrophy and hyperplasia of the bladder smooth muscle as well as detrusor instability. Since prostaglandins (PGs) and cyclic nucleotides (cyclic AMP [cAMP] and cyclic GMP [cGMP]) mediate both smooth muscle tone and proliferation, it is reasonable to suggest that changes in their levels may be involved in the pathophysiology of BOO-associated bladder disorders. Hence, the objective of this study was to investigate cyclic AMP, cyclic GMP and prostaglandins in the bladder of a rabbit model of BOO. BOO was induced in adult male New Zealand White rabbits. After 3 weeks, urinary bladders were excised, weighed and cut into segments. They were then incubated with stimulators of PGs, cAMP and cGMP and the formation of PGs, cAMP and cGMP were measured using radioimmunoassays. There was a significant increase in the obstructed bladder weights (P=0.002). The formation of PGE2, PGI2, cAMP and cGMP was significantly diminished in the detrusor (P<0.05) and bladder neck (P<0.05) in the BOO bladders compared to age-matched controls. Since PGE2, PGI2, cAMP and cGMP are known to inhibit the proliferation of smooth muscle cells (SMCs), the decreased synthesis of these factors, in BOO, may play a role in bladder SMC hypertrophy/hyperplasia. Our study points to the possible use of drugs that modulate the NO-cGMP and/or PG-cAMP axes in BOO-associated bladder pathology.     

Intracellular pH on Protein Kinase C and Ionomycin Potentiation of Isoproterenol-Stimulated Cyclic AMP and Cyclic GMP Production in Rat Pinealocytes

In rat pinealocytes, alpha 1-adrenergic activation, which leads to cytoplasmic alkalinization, also potentiates the beta-adrenergic stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) responses. Both elevation of intracellular calcium ([Ca2+]i) and activation of protein kinase C are involved in the potentiation mechanism. Recently, intracellular pH has also been found to modulate the adrenergic-stimulated cyclic nucleotide responses, suggesting intracellular pH may also affect the potentiation mechanism. This possibility was examined in the present study. Cytoplasmic alkalinization by ammonium chloride had an enhancing effect on the isoproterenol and ionomycin-stimulated cAMP and cGMP accumulation. In comparison, cytoplasmic acidification by sodium propionate reduced the isoproterenol and ionomycin-stimulated cAMP and cGMP responses. Direct measurement of [Ca2+]i indicated that neither ammonium chloride nor sodium propionate had an effect on the ionomycin-stimulated elevation of [Ca2+]i, suggesting their effects on cyclic nucleotide responses may be independent of [Ca2+]i. In cells stimulated by isoproterenol and an activator of protein kinase C, ammonium chloride had an enhancing effect on both cAMP and cGMP responses, whereas sodium propionate had no effect. Taken together, these results suggest that a site distal to elevation of [Ca2+]i and activation of protein kinase C, of importance to the potentiation mechanism, is modulated by intracellular pH.