cGMP-stimulated Phosphodiesterase Research Articles

Structure of the Branched-chain Amino Acid and GTP-sensing Global Regulator, CodY, from Bacillus subtilis

CodY is a branched-chain amino acid (BCAA) and GTP sensor and a global regulator of transcription in low G + C Gram-positive bacteria. It controls the expression of over 100 genes and operons, principally by repressing during growth genes whose products are required for adaptations to nutrient limitation. However, the mechanism by which BCAA binding regulates transcriptional changes is not clear. It is known that CodY consists of a GAF (cGMP-stimulated phosphodiesterases, adenylate cyclases, FhlA) domain that binds BCAAs and a winged helix-turn-helix (wHTH) domain that binds to DNA, but the way in which these domains interact and the structural basis of the BCAA dependence of this interaction are unknown. To gain new insights, we determined the crystal structure of unliganded CodY from Bacillus subtilis revealing a 10-turn α-helix linking otherwise discrete GAF and wHTH domains. The structure of CodY in complex with isoleucine revealed a reorganized GAF domain. In both complexes CodY was tetrameric. Size exclusion chromatography with multiangle laser light scattering (SEC-MALLS) experiments showed that CodY is a dimer at concentrations found in bacterial cells. Comparison of structures of dimers of unliganded CodY and CodY-Ile derived from the tetramers showed a splaying of the wHTH domains when Ile was bound; splaying is likely to account for the increased affinity of Ile-bound CodY for DNA. Electrophoretic mobility shift and SEC-MALLS analyses of CodY binding to 19–36-bp operator fragments are consistent with isoleucine-dependent binding of two CodY dimers per duplex. The implications of these observations for effector control of CodY activity are discussed.

Endothelial Actions of ANP Enhance Myocardial Inflammatory Infiltration in the Early Phase After Acute Infarction.

In patients after acute myocardial infarction (AMI), the initial extent of necrosis and inflammation determine clinical outcome. One early event in AMI is the increased cardiac expression of atrial natriuretic peptide (NP) and B-type NP, with their plasma levels correlating with severity of ischemia. It was shown that NPs, via their cGMP-forming guanylyl cyclase-A (GC-A) receptor and cGMP-dependent kinase I (cGKI), strengthen systemic endothelial barrier properties in acute inflammation. We studied whether endothelial actions of local NPs modulate myocardial injury and early inflammation after AMI. Necrosis and inflammation after experimental AMI were compared between control mice and littermates with endothelial-restricted inactivation of GC-A (knockout mice with endothelial GC-A deletion) or cGKI (knockout mice with endothelial cGKI deletion). Unexpectedly, myocardial infarct size and neutrophil infiltration/activity 2 days after AMI were attenuated in knockout mice with endothelial GC-A deletion and unaltered in knockout mice with endothelial cGKI deletion. Molecular studies revealed that hypoxia and tumor necrosis factor-α, conditions accompanying AMI, reduce the endothelial expression of cGKI and enhance cGMP-stimulated phosphodiesterase 2A (PDE2A) levels. Real-time cAMP measurements in endothelial microdomains using a novel fluorescence resonance energy transfer biosensor revealed that PDE2 mediates NP/cGMP-driven decreases of submembrane cAMP levels. Finally, intravital microscopy studies of the mouse cremaster microcirculation showed that tumor necrosis factor-α-induced endothelial NP/GC-A/cGMP/PDE2 signaling impairs endothelial barrier functions. Hypoxia and cytokines, such as tumor necrosis factor-α, modify the endothelial postreceptor signaling pathways of NPs, with downregulation of cGKI, induction of PDE2A, and altered cGMP/cAMP cross talk. Increased expression of PDE2 can mediate hyperpermeability effects of paracrine endothelial NP/GC-A/cGMP signaling and facilitate neutrophil extravasation during the early phase after MI.

0116 : Phosphodiesterases regulate cAMP level in the mitochondria of adult cardiomyocytes

The heart is highly energy-dependent with most of its energy provided by mitochondria. Mitochondria also play a role in many essential cellular processes including metabolite synthesis, redox balance, calcium homeostasis and cell death. Therefore, maintaining a functional population of mitochondria is critical for cardiac function and identification of novel regulatory mechanisms. Recently, a soluble adenylyl cyclase (sAC) has been revealed to serve as a local source of the second messenger cAMP in the mitochondrial matrix in response to bicarbonate and calcium. PDE2A, a cGMP-stimulated cAMP-hydrolyzing phosphodiesterase (PDE), was also shown to be expressed in the mitochondrial matrix of liver and brain. To gain new insights into the control of mitochondrial pool of cAMP, we investigated the role of various isoforms of PDEs in isolated cardiac mitochondria. Western blot analysis of PDE protein expression revealed positive bands for PDE2A, 3A, 4A and 4B subtypes in cardiac mitochondria. Basal cAMP-degrading enzymatic activities were determined by radioenzymatic assay in cardiac mitochondrial lysates with 1μm cAMP as substrate. PDE2 represented the largest mitochondrial cAMP PDE activity (35% of total) and its activity was enhanced ~2-fold by 5μM cGMP and was inhibited by the PDE2 inhibitor, Bay 60-7550 (100nm). PDE3 and PDE4 activities represented, respectively, 30% and 25% of total, and they were inhibited by their respective inhibitors, cilostamide (1μm) and Ro 20- 1724 (10 μM). Accordingly, measurements by ELISA of mitochondrial cAMP levels confirmed that inhibition of PDE2, 3 and 4 families leads to an increase in cAMP level. In conclusion, at least three PDE families and four PDE subtypes are located in the cardiac mitochondrial matrix, participating to a local signaling pathway with sAC to control cAMP level. Our findings unravel a cAMP signaling cascade in cardiac mitochondria which may have implications for the metabolic control of cardiac function.

Involvement of PDE2 in a Localized Regulation of the L-Type Ca2+ Channels by Progesterone

We have demonstrated that a nitric oxide production induced by stimulation of progesterone (P4) receptor suppresses cAMP-stimulated L-type Ca2+ currents (ICa,L) through an increase in cytosolic cGMP, which may be related to the sex difference of QT intervals and risks of arrhythmia. We here seek the underlying mechanisms on the regulation of ICa,L in patch-clamped guinea pig ventricular myocytes. An external application of P4 (100 nM) suppressed ICa,L about 30 % (100 nM P4; 58±4 %, n=13, without P4 (time control); 84±2 %, n=5). In the presence of a specific PDE2 inhibitor, EHNA (30 μM), P4 suppressed ICa,L to 84±5 % (n=7), which is the same extent with the time control. Pharmacological studies revealed that hydrolysis of cAMP by a cGMP-stimulated phosphodiesterase (PDE2) involves in the P4-induced ICa,L suppression. However the underlying mechanisms of the ICa,L suppression remain to be solved. Thus, we employed sucrose density gradient fractionation experiments to examine localization of the molecules in this system, which is dependent of lipid-raft composition. We found that substantial fraction of CaV1.2 and PDE2 clustered in caveolae fractions, but PDE3 clustered in extra-caveolae fractions. Furthermore, a proximity ligation assay (Olink) revealed that CaV1.2 interacted both with PDE2 and PDE3, but there was no interaction between PDE2 and PDE3. These results suggest that PDE2 and the L-type Ca2+ channel co-localizes at the caveolae for the non-genomic regulation of ICa,L by P4.

Pendrin protein abundance in the kidney is regulated by nitric oxide and cAMP

Pendrin is a Cl(-)/HCO(3)(-) exchanger, expressed in the apical regions of some intercalated cell subtypes, and is critical in the pressor response to angiotensin II. Since angiotensin type 1 receptor inhibitors reduce renal pendrin protein abundance in mice in vivo through a mechanism that is dependent on nitric oxide (NO), we asked if NO modulates renal pendrin expression in vitro and explored the mechanism by which it occurs. Thus we quantified pendrin protein abundance by confocal fluorescent microscopy in cultured mouse cortical collecting ducts (CCDs) and connecting tubules (CNTs). After overnight culture, CCDs maintain their tubular structure and maintain a solute gradient when perfused in vitro. Pendrin protein abundance increased 67% in CNT and 53% in CCD when NO synthase was inhibited (N(G)-nitro-L-arginine methyl ester, 100 μM), while NO donor (DETA NONOate, 200 μM) application reduced pendrin protein by ∼33% in the CCD and CNT. When CNTs were cultured in the presence of the guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (10 μM), NO donors did not alter pendrin abundance. Conversely, pendrin protein abundance rose when cAMP content was increased by the application of an adenylyl cyclase agonist (forskolin, 10 μM), a cAMP analog (8-bromo-cAMP, 1 mM), or a phosphodiesterase inhibitor (BAY60-7550, 50 μM). Since NO reduces cellular cAMP in the CNT, we asked if NO reduces pendrin abundance by reducing cAMP. With blockade of cGMP-stimulated phosphodiesterase II, NO did not alter pendrin protein abundance. We conclude that NO acts through cAMP to reduce pendrin total protein abundance by enhancing cAMP degradation.

Mechanisms of Carbon Monoxide Attenuation of Tubuloglomerular Feedback

Carbon monoxide (CO) is a physiological messenger with diverse functions in the kidney, including controlling afferent arteriole tone both directly and via tubuloglomerular feedback (TGF). We have reported that CO attenuates TGF, but the mechanisms underlying this effect remain unknown. We hypothesized that CO, acting via cGMP, cGMP-dependent protein kinase, and cGMP-stimulated phosphodiesterase 2, reduces cAMP in the macula densa, leading to TGF attenuation. In vitro, microdissected rabbit afferent arterioles and their attached macula densa were simultaneously perfused. TGF was measured as the decrease in afferent arteriole diameter elicited by switching macula densa NaCl from 10 to 80 mmol/L. Adding a CO-releasing molecule (CORM-3, 5 × 10(-5) mol/L) to the macula densa blunted TGF from 3.3 ± 0.3 to 2.0 ± 0.3 μm (P<0.001). The guanylate cyclase inhibitor LY-83583 (10(-6) mol/L) enhanced TGF (5.8 ± 0.6 μm; P<0.001 versus control) and prevented the effect of CORM-3 on TGF (LY-83583+CORM-3, 5.5 ± 0.3 μm). Similarly, the cGMP-dependent protein kinase inhibitor KT-5823 (2 × 10(-6) mol/L) enhanced TGF and prevented the effect of CORM-3 on TGF (KT-5823, 6.0 ± 0.7 μm; KT-5823+CORM-3, 5.9 ± 0.8 μm). However, the phosphodiesterase 2 inhibitor BAY-60-7550 (10(-6) mol/L) did not prevent the effect of CORM-3 on TGF (BAY-60-7550, 4.07 ± 0.31 μm; BAY-60-7550+CORM-3, 1.84 ± 0.31 μm; P<0.001). Finally, the degradation-resistant cAMP analog dibutyryl-cAMP (10(-3) mol/L) prevented the attenuation of TGF by CORM-3 (dibutyryl-cAMP, 4.6 ± 0.5 μm; dibutyryl-cAMP+CORM-3, 5.0 ± 0.6 μm). We conclude that CO attenuates TGF by reducing cAMP via a cGMP-dependent pathway mediated by cGMP-dependent protein kinase rather than phosphodiesterase 2. Our results will lead to a better understanding of the mechanisms that control the renal microcirculation.

The roles of cyclic nucleotide phosphodiesterases (PDEs) in steroidogenesis

The second messenger, cAMP, is one of the most important regulatory signals for control of steroidogenesis. This review focuses on current knowledge about regulation of cyclic nucleotides by phosphodiesterases (PDEs) in steroidogenic tissues. The first PDE known to directly regulate steroidogenesis was PDE2, the cGMP-stimulated PDE. PDE2 mediates ANP/cGMP-induced decreases in aldosterone production. Recently, the PDE8 family has been shown to control steroidogenesis in two tissues. Specifically, PDE8A regulates testosterone production by itself and in concert with additional IBMX-sensitive PDEs. PDE8B modulates basal corticosterone synthesis via acute and chronic mechanisms. In addition to cAMP-dependent pathways, cGMP signaling also can promote steroidogenesis, and PDE5 modulates this process. Finally, PDE mutations may lead to several human diseases characterized by abnormal steroid levels.

Specific PKG inhibitors: do they really exist?

Background Cellular cGMP effects can be mediated by a number of effectors including cGMP-dependent protein kinases (PKGs), cGMP-stimulated phosphodiesterase (PDE2), cGMP-inhibited phosphodiesterase (PDE3), and cGMPgated channels. Pharmacological inhibitors of PKG are often used to discriminate between these diverse cGMP effects. Currently used PKG inhibitors can be divided into three classes: cyclic nucleotide binding site inhibitors like Rp-phosphorothioate analogs, ATP binding site inhibitors like KT5823, and substrate binding site inhibitors like the recently described DT-oligopeptides. However, several studies have observed no PKG inhibition by KT5823 in intact cells [1] or by Rp-cGMPS analogs in smooth muscle cells [2], as well as unspecific (PKG-independent) effects of Rp-cGMPS analogs in platelets [3]. Results We tested the inhibitory effects of (D)-DT2 and DT3 on PKG and its effects on intact cells. Our data show that (D)-DT2 selectively inhibited 2 nM purified PKG Ia and Ib with an IC50 of 8n M, and that up to 1µ M (D)-DT2 did not inhibit PKG II or PKA. In broken platelet cell experiments, PKG activity was inhibited by (D)-DT2 starting at 5 µM, with complete inhibition at 20 µM, but we also observed inhibition of PKA activity at these concentrations. However, concentrations of up to 200 µM of compounds failed to inhibit PKG activity (assessed by phosphorylation of the established PKG substrates VASP, PDE5 and GRP2) in intact human platelets, rat mesangial cells and neonatal mouse cardiac myocytes. It should be noted that the measured PKG concentration is about 7 µM in platelets and 0.1 – 0.5 µM in all other tested cells. (D)-DT2 effects on platelet function did not correlate with PKG activity. Preincubation of platelets with 10 nM (D)-DT2 strongly inhibited thrombin-induced platelet aggregation and calcium mobilization, whereas it potentiated these effects in collagen-stimulated platelets. Conclusion Interpretations of results based on PKG inhibitors require caution. None of the commercially available PKG inhibitors should be used without control experiments in intact cells since they may have unpredictable functional effects not mediated by PKG activity.

Nitric oxide signaling via cGMP‐stimulated phosphodiesterase in striatal neurons

Nitric oxide (NO) acts in the nervous system to activate guanylyl cyclase and increase cGMP. One target for cGMP appears to be the cGMP-stimulated phosphodiesterase (PDE2A), which is widely expressed in the brain and provides a molecular mechanism for NO to regulate cAMP levels. We have found that PDE2A is highly expressed in the medium spiny neurons of the striatum, which project to the pallidum and substantia nigra. These cells express dopamine-stimulated adenylyl cyclase, and we have found that increases in cAMP in these neurons, produced by activation of the D1-type dopamine receptor, are dramatically enhanced by the general phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and the PDE2A-selective inhibitor erythro-p-(2-hydroxyl-3-nonyl)adenine (EHNA). These results indicate that PDE2A plays a major role in regulating dopamine-stimulated cAMP production in striatal neurons. EHNA also enhances NO-induced increases in striatal cGMP. In addition, dopamine appears to act via another receptor, activated by the agonist SKF83959, to increase striatal cGMP in a NO-dependent manner. Together, these observations indicate that striatal NO producing interneurons can act via the PDE2A in the medium spiny neurons to regulate the cAMP response to dopamine stimulation.

Crystal Structure of SpoVT, the Final Modulator of Gene Expression during Spore Development in Bacillus subtilis

Endospore formation in Bacillus subtilis is orchestrated by five developmental sigma factors and further modulated by several auxiliary transcription factors. One of these, SpoVT, regulates forespore-specific σ G-dependent genes and plays a key role in the final stages of spore formation. We have determined the crystal structure of the isolated C-terminal domain of SpoVT at 1.5 Å by experimental phasing techniques and used this model to solve the structure of the full-length SpoVT at 2.6 Å by molecular replacement. SpoVT is a tetramer that shows an overall significant distortion mediated by electrostatic interactions. Two monomers dimerize via the highly charged N-terminal domains to form swapped-hairpin β-barrels. These asymmetric dimers further tetramerize through the formation of mixed helix bundles between their C-terminal domains, which themselves fold as GAF (c GMP-specific and cGMP-stimulated phosphodiesterases, Anabaena adenylate cyclases, and Escherichia coli FhlA) domains. The combination of a swapped-hairpin β-barrel with a GAF domain represents a novel domain architecture in transcription factors. The occurrence of SpoVT homologs throughout Bacilli and Clostridia demonstrates the ancestral origin of this factor in sporulation.

CGMP decreases surface NKCC2 levels in the thick ascending limb: role of phosphodiesterase 2 (PDE2)

NaCl absorption in the medullary thick ascending limb of the loop of Henle (THAL) is mediated by the apical Na/K/2Cl cotransporter (NKCC2). Hormones that increase cGMP, such as nitric oxide (NO) and natriuretic peptides, decrease NaCl absorption by the THAL. However, the mechanism by which cGMP decreases NaCl absorption in THALs is not known. We hypothesized that cGMP decreases surface NKCC2 levels in the THAL. We used surface biotinylation to measure surface NKCC2 levels in rat THAL suspensions. We tested the effect of the membrane-permeant cGMP analog dibutyryl-cGMP (db-cGMP) on surface NKCC2 levels. Incubating THALs with db-cGMP for 20 min decreased surface NKCC2 levels in a concentration-dependent manner (basal=100%; db-cGMP 100 microM=77+/-7%; 500 microM=54+/-10% and 1,000 microM=61+/-8%). A different cGMP analog 8-bromo-cGMP (8-Br-cGMP) also decreased surface NKCC2 levels by 25%, (basal=100%; 8-Br-cGMP=75+/-5%). Incubation of isolated, perfused THALs with db-cGMP decreased apical surface NKCC2 labeling levels as measured by immunofluorescence and confocal microscopy. cGMP-stimulated phosphodiesterase 2 (PDE2) mediates the inhibitory effect of NO on NaCl absorption by THALs. Thus we examined the role of PDE2 and found that PDE2 inhibitors blocked the effect of db-cGMP on surface NKCC2. Also, a nonstimulatory concentration of db-cAMP blocked the cGMP-induced decrease in surface NKCC2. Finally, db-cGMP inhibited THAL net Cl absorption by 48+/-4%, and this effect was completely blocked by PDE2 inhibition. We conclude that cGMP decreases NKCC2 levels in the apical membrane of THALs and that this effect is mediated by PDE2. This is an important mechanism by which cGMP inhibits NaCl absorption by the THAL.

Neuronal nitric oxide synthase gene transfer decreases [Ca 2+] i in cardiac sympathetic neurons

Gene transfer of neuronal nitric oxide synthase (nNOS) can decrease cardiac sympathetic outflow and facilitate parasympathetic neurotransmission. The precise pathway responsible for nitric oxide (NO) mediated inhibition of sympathetic neurotransmission is not known, but may be related to NO–cGMP activation of cGMP-stimulated phosphodiesterase (PDE2) that enhances the breakdown of cAMP to deactivate protein kinase A (PKA), resulting in a decrease in Ca 2+ influx mediated exocytosis of the neurotransmitter. We investigated depolarization evoked Ca 2+ influx in nNOS gene transduced sympathetic neurons from stellate ganglia with a noradrenergic cell specific vector (Ad.PRS-nNOS or empty vector), and examined how nNOS gene transfer affected cAMP and cGMP levels in these neurons. We found that targeting nNOS into these sympathetic neurons reduced amplitudes of voltage activated Ca 2+ transients by 44%. nNOS specific inhibition by N-[(4 S)-4-Amino-5-[(2-aminoetyl](amino] pentyl]- N′-nitroguanidine (AAAN) reversed this response. nNOS gene transfer also increased intracellular cGMP (47%) and decreased cAMP (29%). A PDE2 specific inhibitor Bay60-7557 reversed the reduction in cAMP caused by Ad.PRS-nNOS. These results suggest that neuronal NO modulates cGMP and PDE2 to regulate voltage gated intracellular Ca 2+ transients in sympathetic neurons. Therefore, we propose this as a possible key step involved in NO decreasing cardiac sympathetic neurotransmission.

Inhibition of agonist-stimulated aldosterone production from adrenal zona glomerulosa cells by ANP is mediated by GC-A

Atrial natriuretic peptide (ANP) is a potent inhibitor of agonist-induced aldosterone synthesis in zona glomerulosa (ZG) cells of the adrenal gland. Cellular ANP effects are mediated by two receptors: GC-A, a membrane guanylyl cyclase, and GC-C that lacks guanylyl cyclase activity. To identify which one of these two receptors is involved in inhibitory pathway of aldosterone production we developed a new model of isolation and primary culture of mouse ZG cells. Aldosterone concentration in the blood was not different in wild type and GC-A knockout mice. ANP (10 nM) significantly inhibited basal, forskolin, and Angiotensin II-stimulated aldosterone production from wild type mice, while in GC-A knockout mice ANP had no inhibitory effect on aldosterone production. Inhibition of forskolin or ACTH stimulated aldosterone production by ANP was connected with increased concentration of cGMP, stimulation of cGMP-stimulated phosphodiesterase (PDE2), and decrease of intracellular cAMP content. IBMX and the PDE2-selective inhibitor MEP-1 completely prevented ANP inhibitory effect on cAMP-stimulated aldosterone production. In contrast, ANP-dependent inhibition of Ang II-stimulated aldosterone production was not connected with PDE2 or PKG activity indicating that other cGMP-dependent mediator(s)/mechanism(s) are involved in regulation of Ang II-stimulated aldosterone production from ZG cells by cGMP.

Phosphodiesterases 1 and 2 regulate cellular cGMP level in rabbit submandibular gland cells

In rabbit salivary glands, stimulation of muscarinic cholinergic receptors causes production of cGMP through intracellular Ca 2+ and nitric oxide. In this study, we investigated a role of cyclic nucleotide phosphodiesterase (PDE) in regulating the cellular cGMP level by using cells dispersed from the submandibular gland. Methacholine, a cholinergic agonist, rapidly elevated the cGMP level. The elevation was greatly enhanced by IBMX, a non-specific inhibitor for most isoforms of the 11 PDEs. The cGMP level was also elevated by MM-IBMX and EHNA, which inhibit the activities of PDE1 and PDE2, respectively. The elevation by the simultaneous application of the two drugs corresponded to 90% of that by IBMX. Therefore, PDE1 and PDE2 are the main PDEs that act to degrade cGMP in methacholine-stimulated cells. The presence of the two PDEs was confirmed by assaying their activities of the cell lysate. In unstimulated cells, the cGMP level was elevated by MM-IBMX and little elevated by EHNA. While the PDE2 activity was thus low, it was estimated that methacholine increases its activity approximately 50-fold. The strong activation can be explained by the elevation of the cGMP level because PDE2 is a cGMP-stimulated PDE. SNAP, a nitric oxide donor, causes production of cGMP without a receptor-operated increase in intracellular Ca 2+ concentration. In SNAP-stimulated cells, MM-IBMX elevated the cGMP level higher than in methacholine-stimulated cells although the PDE1 activity is dependent on Ca 2+/calmodulin. Besides Ca 2+, other factors may regulate the PDE1 activity in living cells.

Nitric oxide decreases a calcium-activated potassium current via activation of phosphodiesterase 2 in Helix U-cells

In the present study, we investigated the underlaying mechanism of nitric oxide (NO) and cGMP on the decline of a Ca 2+-activated potassium (K Ca) current in U-cells of the right parietal ganglion of the pulmonate snail, Helix pomatia. Using a two-electrode voltage-clamp technique, we activated a K Ca-current either by opening of endogenous voltage-gated Ca 2+-channels during depolarizing voltage steps or by ionophoretic injection of Ca 2+ via a third electrode containing 100 mM Ca 2+. K Ca-current amplitude in U-cells was sensitive to Ba 2+, TEA, iberiotoxin, kaliotoxin and charybdotoxin (ChTX), but not to 4-aminopyridine (4-AP) (up to 30 mM) and apamin (up to 300 nM). Thus, the biophysical and pharmacological profile of the K Ca-current in U-cells shares similarities with the large-conductance K Ca channel (BK(Ca)). The NO-donor sodium nitroprusside (SNP) or S-nitro- N-acetylpenicillamine (SNAP) as well as NO-gas decreased the K Ca-current amplitude and decreased the rate of K Ca-current activation elicited by Ca 2+-injection. Decline of the current amplitude and decrease of activation of K Ca-current were qualitatively mimicked by the membrane-permeable cGMP analogue dibutyryl-cGMP (db-cGMP). NO-induced decrease of K Ca-current was blocked by methylene blue (50 μM), an inhibitor of the guanylyl-cyclase, and by erytho-9-(2-hydroxyl-3-nonyl) adenine (EHNA) (100 μM), an inhibitor of the cGMP-stimulated phosphodiesterase 2 (PDE2). These experiments suggest that the NO-mediated decrease of K Ca-current in U-cells results from synthesis of cGMP by activation of a guanylyl-cyclase and subsequent activation of PDE2.

CGMP-phosphodiesterase activity is up-regulated in response to pressure overload of rat ventricles.

Although expression of natriuretic peptides in cardiac tissues is up-regulated in response to pressure overload, no significant change in cGMP level in hypertrophied ventricles was observed. Activities of two cyclic nucleotide phosphodiesterase (PDE) isoforms, Ca2+/calmodulin-stimulated PDE (PDE1) and cGMP-stimulated PDE (PDE2), were significantly higher in rat left ventricles 14 days after aortic banding. The absence of significant changes in PDE1A and PDE2A mRNA levels indicated that the two PDE activities were post-transcriptionally up-regulated. These results suggested that the increased cGMP-PDE activity in response to pressure overload plays an important role in neutralizing cGMP action in cardiac tissue.

Olfactory Signal Transduction in the Mouse Septal Organ

The septal organ, a distinct chemosensory organ observed in the mammalian nose, is essentially a small island of olfactory neuroepithelium located bilaterally at the ventral base of the nasal septum. Virtually nothing is known about its physiological properties and function. To understand the nature of the sensory neurons in this area, we studied the mechanisms underlying olfactory signal transduction in these neurons. The majority of the sensory neurons in the septal organ express olfactory-specific G-protein and adenylyl cyclase type III, suggesting that the cAMP signaling pathway plays a critical role in the septal organ as in the main olfactory epithelium (MOE). This is further supported by patch-clamp recordings from individual dendritic knobs of the sensory neurons in the septal organ. Odorant responses can be mimicked by an adenylyl cyclase activator and a phosphodiesterase inhibitor, and these responses can be blocked by an adenylyl cyclase inhibitor. There is a small subset of cells in the septal organ expressing a cGMP-stimulated phosphodiesterase (phosphodiesterase 2), a marker for the guanylyl cyclase-D subtype sensory neurons identified in the MOE. The results indicate that the septal organ resembles the MOE in major olfactory signal transduction pathways, odorant response properties, and projection to the main olfactory bulb. Molecular and functional analysis of the septal organ, which constitutes approximately 1% of the olfactory epithelium, will provide new insights into the organization of the mammalian olfactory system and the unique function this enigmatic organ may serve.

Kinetics of nitric oxide-cyclic GMP signalling in CNS cells and its possible regulation by cyclic GMP.

Physiologically, nitric oxide (NO) signal transduction occurs through soluble guanylyl cyclase (sGC), which catalyses cyclic GMP (cGMP) formation. Knowledge of the kinetics of NO-evoked cGMP signals is therefore critical for understanding how NO signals are decoded. Studies on cerebellar astrocytes showed that sGC undergoes a desensitizing profile of activity, which, in league with phosphodiesterases (PDEs), was hypothesized to diversify cGMP responses in different cells. The hypothesis was tested by examining the kinetics of cGMP in rat striatal cells, in which cGMP accumulated in neurones in response to NO. Based on the effects of selective PDE inhibitors, cGMP hydrolysis following exposure to NO was attributed to a cGMP-stimulated PDE (PDE 2). Analysis of NO-induced cGMP accumulation in the presence of a PDE inhibitor indicated that sGC underwent marked desensitization. However, the desensitization kinetics determined under these conditions described poorly the cGMP profile observed in the absence of the PDE inhibitor. An explanation shown plausible theoretically was that cGMP determines the level of sGC desensitization. In support, tests in cerebellar astrocytes indicated an inverse relationship between cGMP level and recovery of sGC from its desensitized state. We suggest that the degree of sGC desensitization is related to the cGMP concentration and that this effect is not mediated by (de)phosphorylation.

Attenuation of cAMP accumulation in adult rat cardiac fibroblasts by IL-1beta and NO: role of cGMP-stimulated PDE2.

Treatment of cultured adult rat cardiac fibroblasts with interleukin-1beta (IL-1beta) induces the inducible nitric oxide synthase (iNOS) expression, increases nitric oxide (NO) and cGMP production, and attenuates cAMP accumulation in response to isoproterenol by ~50%. Reduced cAMP accumulation is due to NO production: the effect is mimicked by NO donors and prevented by N(G)-monomethyl-L-arginine, an NOS inhibitor. Effects of NO are not restricted to the beta-adrenergic response; the response to forskolin is similarly diminished. NO donors only slightly (12%) decrease forskolin-stimulated adenylyl cyclase (AC) activity in cardiac fibroblast plasma membranes, suggesting that the main effect of NO is not a direct one on AC. An inhibitor of soluble guanylyl cyclase inhibits the effects of IL-1beta and NO donors; inhibition of cGMP-dependent protein kinase is without effect. 3-Isobutyl-1-methylxanthine, a nonspecific phosphodiesterase (PDE) inhibitor, and erythro-9-(2-hydroxy-3-nonyl)adenine, a specific inhibitor of the cGMP-stimulated PDE (PDE2), completely restore cAMP accumulation in sodium nitroprusside-treated fibroblasts and largely reverse the attenuated response in IL-1beta-treated fibroblasts. Although NO reportedly acts by reducing AC activity in some cells, in cardiac fibroblasts NO production decreases cAMP accumulation largely by the cGMP-mediated activation of PDE2.

Changes in phosphodiesterase activity in the developing rat submandibular gland

Developmental changes (from 2 to 26 weeks) in phosphodiesterase (PDE) activity in the rat submandibular gland were investigated. Major activities for both cAMP- and cGMP-PDE were present in the 100,000× g supernatant fractions (70–90% of total activities), but not in the pellet fractions, during development. The effects of stimulators (Ca 2+/calmodulin and cGMP) and inhibitors (cGMP, cilostamide, rolipram and zaprinast) were investigated in the supernatant fractions. During development, PDE4 (cAMP-specific PDE) was a major PDE, indicating that the majority of cAMP is hydrolysed by PDE4. In the young rat, PDE1 hydrolysed cGMP three-fold more than the control, and PDE2 (cGMP-stimulated PDE) was present, indicating that the concentration of intracellular cGMP may be enhanced, and cGMP may function in the growth pathway in the submandibular gland. Chromatograms eluted on a Mono Q HR5/5 ion-exchange column supported the results of the inhibition studies: PDE1, PDE2, PDE3, PDE4 and PDE5 were present in the young submandibular gland, and PDE1, PDE3, PDE4 and PDE5 in the adult gland. Expression of PDE5 was detected by inhibition studies, reverse transcriptase-polymerase chain reaction and Western blotting in the submandibular gland.