Activation of particulate guanylyl cyclase by endothelins in cultured SV-40 transformed cat iris sphincter smooth muscle cells

We investigated the effects of endothelins (ETs) on cGMP production in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. ET-3 increased cGMP formation in a concentration-dependent manner (EC50 = 98nM), which was 2.5 times higher than that of ET-1. The ET(B)receptor agonists sarafotoxin-S6c and IRL 1620 also increased cGMP production, mimicking the effects of the ETs. The ET(B) receptor antagonist BQ 788, but not the ET(A) receptor antagonist BQ610, dose-dependently blocked ET-3-stimulated cGMP formation (IC50=10nM). The phorbol ester, Phorbol 12, 13-dibutyrate (PDBu), which inhibits particulate guanylyl cyclase in smooth muscle, dose-dependently inhibited ET-3-stimulated cGMP accumulation (IC50=66nM). LY83583 and ODQ, inhibitors of soluble guanylyl cyclases, as well as inhibitors of the nitric oxide cascade and of intracellular Ca2+ elevation had no appreciable effect on ET-3-induced cGMP production. ET-3 markedly inhibited carbachol-induced intracellular Ca2+ mobilization. We conclude that ET-3 increases intracellular cGMP levels in SV-CISM-2 cells through activation of the ET(B) receptor subtype and subsequent stimulation of the membrane-bound guanylyl cyclase. Elevation of cGMP by ET and the subsequent inhibition of muscarinic stimulation of intracellular Ca2+ mobilization by the cyclic nucleotide could serve to modulate the contractile effects of Ca2+-mobilizing agonists in the iris sphincter smooth muscle.     

The alpha2beta1 isoform of guanylyl cyclase mediates plasma membrane localized nitric oxide signalling

Nitric oxide (NO) is a mediator of copious biological processes, in many cases through the production of cGMP from the enzyme nitric oxide-sensitive guanylyl cyclase. Natriuretic peptides also elevate cGMP, often with distinct biological effects, raising the issue of how specificity is achieved. Here we show that a recently described alpha(2)beta(1) isoform of guanylyl cyclase is expressed in a number of epithelia, where it is localized to the apical plasma membrane. We measured the functional properties of the alpha(2)beta(1) isoform by utilizing the NO-dependent activation of the ion channel cystic fibrosis transmembrane conductance regulator (CFTR), which occurs by phosphorylation via the membrane-bound type II isoform of cGMP-dependent protein kinase. We found that cGMP generated by NO activation of the alpha(2)beta(1) isoform of guanylyl cyclase is an exceptionally efficient mediator of nitric oxide action on membrane targets, activating CFTR far more effectively than the cytoplasmically located alpha(1)beta(1) guanylyl cyclase isoform. Targeting the alpha(1)beta(1) isoform of guanylyl cyclase to the membrane also dramatically enhanced the effects of nitric oxide on CFTR within the membrane. This was not due to increased enzymatic activity of guanylyl cyclase in a membrane location, but to production of a localised membrane pool of cGMP by membrane-localized NO-dependent guanylyl cyclase that was resistant to degradation by phosphodiesterases. Selective effects of cGMP produced from this enzyme in response to NO are directed at membrane targets and suggest that drugs selectively activating or inhibiting this alpha(2)beta(1) isoform of guanylyl cyclase may have unique pharmacological properties.     

Muscarinic agents modify kinetics properties of membrane-bound guanylyl cyclase activity

Plasma membranes from bovine tracheal smooth muscle show guanylyl cyclase activity, which can be stimulated by muscarinic agonists such carbamylcholine and oxotremorine and blocked by atropine. This stimulation was observed in the presence of 150 mM NaCl. In the absence of this salt, guanylyl cyclase activity was considerably higher but was not affected by muscarinic agonists. Carbamylcholine decreased the apparent Km but did not change the Vmax of this enzyme. When plasma membrane fractions were extracted with 1% octylglucoside, guanylyl cyclase activity was preserved, however the muscarinic activation was abolished, despite a muscarinic receptor capable of [3H]quinuclidinylbenzilate binding being present in the extract. The detergent extraction changed the affinity of guanylyl cyclase for GTP but the Mn2+ kinetics was unaltered. Based on these findings and on current information in the literature, we propose that another component is required to restore the link between the muscarinic receptor and guanylyl cyclase, however the nature of this component remains to be established.     

Rapid nitric oxide-induced desensitization of the cGMP response is caused by increased activity of phosphodiesterase type 5 paralleled by phosphorylation of the enzyme

Most of the effects of the signaling molecule nitric oxide (NO) are mediated by cGMP, which is synthesized by soluble guanylyl cyclase and degraded by phosphodiesterases. Here we show that in platelets and aortic tissue, NO led to a biphasic response characterized by a tremendous increase in cGMP (up to 100-fold) in less than 30 s and a rapid decline, reflecting the tightly controlled balance of guanylyl cyclase and phosphodiesterase activities. Inverse to the reported increase in sensitivity caused by NO shortage, concentrating NO attenuated the cGMP response in a concentration-dependent manner. We found that guanylyl cyclase remained fully activated during the entire course of the cGMP response; thus, desensitization was not due to a switched off guanylyl cyclase. However, when intact platelets were incubated with NO and then lysed, enhanced activity of phosphodiesterase type 5 was detected in the cytosol. Furthermore, this increase in cGMP degradation is paralleled by the phosphorylation of phosphodiesterase type 5 at Ser-92. Thus, our data suggest that NO-induced desensitization of the cGMP response is caused by the phosphorylation and subsequent activity increase of phosphodiesterase type 5.     

Calcium-independent and cAMP-dependent modulation of soluble guanylyl cyclase activity by G protein-coupled receptors in pituitary cells

It is well established that G protein-coupled receptors stimulate nitric oxide-sensitive soluble guanylyl cyclase by increasing intracellular Ca(2+) and activating Ca(2+)-dependent nitric-oxide synthases. In pituitary cells receptors that stimulated adenylyl cyclase, growth hormone-releasing hormone, corticotropin-releasing factor, and thyrotropin-releasing hormone also stimulated calcium signaling and increased cGMP levels, whereas receptors that inhibited adenylyl cyclase, endothelin-A, and dopamine-2 also inhibited spontaneous calcium transients and decreased cGMP levels. However, receptor-controlled up- and down-regulation of cyclic nucleotide accumulation was not blocked by abolition of Ca(2+) signaling, suggesting that cAMP production affects cGMP accumulation. Agonist-induced cGMP accumulation was observed in cells incubated in the presence of various phosphodiesterase and soluble guanylyl cyclase inhibitors, confirming that G(s)-coupled receptors stimulated de novo cGMP production. Furthermore, cholera toxin (an activator of G(s)), forskolin (an activator of adenylyl cyclase), and 8-Br-cAMP (a permeable cAMP analog) mimicked the stimulatory action of G(s)-coupled receptors on cGMP production. Basal, agonist-, cholera toxin-, and forskolin-stimulated cGMP production, but not cAMP production, was significantly reduced in cells treated with H89, a protein kinase A inhibitor. These results indicate that coupling seven plasma membrane-domain receptors to an adenylyl cyclase signaling pathway provides an additional calcium-independent and cAMP-dependent mechanism for modulating soluble guanylyl cyclase activity in pituitary cells.     

Natriuretic peptides and nitric oxide stimulate cGMP synthesis in different cellular compartments

Cyclic nucleotide-gated (CNG) channels are a family of ion channels activated by the binding of cyclic nucleotides. Endogenous channels have been used to measure cyclic nucleotide signals in photoreceptor outer segments and olfactory cilia for decades. Here we have investigated the subcellular localization of cGMP signals by monitoring CNG channel activity in response to agonists that activate either particulate or soluble guanylyl cyclase. CNG channels were heterologously expressed in either human embryonic kidney (HEK)-293 cells that stably overexpress a particulate guanylyl cyclase (HEK-NPRA cells), or cultured vascular smooth muscle cells (VSMCs). Atrial natriuretic peptide (ANP) was used to activate the particulate guanylyl cyclase and the nitric oxide donor S-nitroso-n-acetylpenicillamine (SNAP) was used to activate the soluble guanylyl cyclase. CNG channel activity was monitored by measuring Ca2+ or Mn2+ influx through the channels using the fluorescent dye, fura-2. We found that in HEK-NPRA cells, ANP-induced increases in cGMP levels activated CNG channels in a dose-dependent manner (0.05-10 nM), whereas SNAP (0.01-100 microM) induced increases in cGMP levels triggered little or no activation of CNG channels (P < 0.01). After pretreatment with 100 microM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor, ANP-induced Mn2+ influx through CNG channels was significantly enhanced, while SNAP-induced Mn2+ influx remained small. In contrast, we found that in the presence of IBMX, both 1 nM ANP and 100 microM SNAP triggered similar increases in total cGMP levels. We next sought to determine if cGMP signals are compartmentalized in VSMCs, which endogenously express particulate and soluble guanylyl cyclase. We found that 10 nM ANP induced activation of CNG channels more readily than 100 muM SNAP; whereas 100 microM SNAP triggered higher levels of total cellular cGMP accumulation. These results suggest that cGMP signals are spatially segregated within cells, and that the functional compartmentalization of cGMP signals may underlie the unique actions of ANP and nitric oxide.     

Nitric oxide-evoked glutamate release and cGMP production in cerebellar slices: control by presynaptic 5-HT1D receptors

We previously reported that pre- and postsynaptic 5-hydroxytryptamine (5-HT) receptors effectively control glutamatergic transmission in adult rat cerebellum. To investigate where 5-HT acts in the glutamate ionotropic receptors/nitric oxide/guanosine 3',5'-cyclic monophosphate (cGMP) pathway, in the present study 5-HT modulation of the cGMP response to the nitric oxide donor S-nitroso-penicillamine (SNAP) was studied in adult rat cerebellar slices. While cGMP elevation produced by high-micromolar SNAP was insensitive to 5-HT, 1 microM SNAP, expected to release nitric oxide in the low-nanomolar concentration range, elicited cGMP production and endogenous glutamate release both of which could be prevented by activating presynaptic 5-HT1D receptors. Released nitric oxide appeared responsible for cGMP production and glutamate release evoked by 1 microM SNAP, as both the effects were mimicked by the structurally unrelated nitric oxide donor 2-(N,N-diethylamino)-diazenolate-2-oxide (0.1 microM). Dependency of the 1 microM SNAP-evoked release of glutamate on external Ca2+, sensitivity to presynaptic release-regulating receptors and dependency on ionotropic glutamate receptor functioning, suggest that nitric oxide stimulates exocytotic-like, activity-dependent glutamate release. Activation of ionotropic glutamate receptors/nitric oxide synthase/guanylyl cyclase pathway by endogenously released glutamate was involved in the cGMP response to 1 microM SNAP, as blockade of NMDA/non-NMDA receptors, nitric oxide synthase or guanylyl cyclase, abolished the cGMP response. To conclude, in adult rat cerebellar slices low-nanomolar exogenous nitric oxide could facilitate glutamate exocytotic-like release possibly from parallel fibers that subsequently activated the glutamate ionotropic receptors/nitric oxide/cGMP pathway. Presynaptic 5-HT1D receptors could regulate the nitric oxide-evoked release of glutamate and subsequent cGMP production.     

Control of Trypanosoma cruzi Epimastigote Motility through the Nitric Oxide Pathway

ABSTRACT. Trypanosoma cruzi epimastigote motility can be enhanced by addition of L-arginine, to the culture. This effect is blocked by N-methyl-L-arginine, a competitive inhibitor of the nitric oxide synthase. N-methyl-D-aspartate and L-glutamate, two agonists of the NMDALglutamate receptor, also enhanced motility. This stimulation is blocked by MK-801 a noncompetitive antagonist of the NMDA receptor. In addition, sodium nitroprusside, a guanylyl cyclase stimulator and 8-Br-cyclic GMP, an analog of cyclic GMP, also stimulated epimastigote motility. It is suggested that an increase of intracellular cyclic GMP levels mediated by nitric oxide may be responsible for the increase in epimastigote motility.     

Atypical soluble guanylyl cyclases in Drosophila can function as molecular oxygen sensors

Conventional soluble guanylyl cyclases are heterodimeric enzymes that synthesize cGMP and are activated by nitric oxide. Recently, a separate class of soluble guanylyl cyclases has been identified that are only slightly activated by or are insensitive to nitric oxide. These atypical guanylyl cyclases include the vertebrate beta2 subunit and examples from the invertebrates Manduca sexta, Caenorhabditis elegans, and Drosophila melanogaster. A member of this family, GCY-35 in C. elegans, was recently shown to be required for a behavioral response to low oxygen levels and may be directly regulated by oxygen (Gray, J. M., Karow, D. S., Lu, H., Chang, A. J., Chang, J. S., Ellis, R. E., Marletta, M. A., and Bargmann, C. I. (2004) Nature 430, 317-322). Drosophila contains three genes that code for atypical soluble guanylyl cyclases: Gyc-88E, Gyc-89Da, and Gyc-89Db. COS-7 cells co-transfected with Gyc-88E and Gyc-89Da or Gyc-89Db accumulate low levels of cGMP under normal atmospheric oxygen concentrations and are potently activated under anoxic conditions. The increase in activity is graded over oxygen concentrations of 0-21%, can be detected within 1 min of exposure to anoxic conditions and is blocked by the soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ). Gyc-88E and Gyc-89Db are co-expressed in a subset of sensory neurons where they would be ideally situated to act as oxygen sensors. This is the first demonstration of a soluble guanylyl cyclase that is activated in response to changing oxygen concentrations.     

Spontaneous and receptor-controlled soluble guanylyl cyclase activity in anterior pituitary cells

Nitric oxide (NO)-dependent soluble guanylyl cyclase (sGC) is operative in mammalian cells, but its presence and the role in cGMP production in pituitary cells have been incompletely characterized. Here we show that sGC is expressed in pituitary tissue and dispersed cells, enriched lactotrophs and somatotrophs, and GH(3) immortalized cells, and that this enzyme is exclusively responsible for cGMP production in unstimulated cells. Basal sGC activity was partially dependent on voltage-gated calcium influx, and both calcium-sensitive NO synthases (NOS), neuronal and endothelial, were expressed in pituitary tissue and mixed cells, enriched lactotrophs and somatotrophs, and GH(3) cells. Calcium-independent inducible NOS was transiently expressed in cultured lactotrophs and somatotrophs after the dispersion of cells, but not in GH(3) cells and pituitary tissue. This enzyme participated in the control of basal sGC activity in cultured pituitary cells. The overexpression of inducible NOS by lipopolysaccharide + interferon-gamma further increased NO and cGMP levels, and the majority of de novo produced cGMP was rapidly released. Addition of an NO donor to perifused pituitary cells also led to a rapid cGMP release. Calcium-mobilizing agonists TRH and GnRH slightly increased basal cGMP production, but only when added in high concentrations. In contrast, adenylyl cyclase agonists GHRH and CRF induced a robust increase in cGMP production, with EC(50)s in the physiological concentration range. As in cells overexpressing inducible NOS, the stimulatory action of GHRH and CRF was preserved in cells bathed in calcium-deficient medium, but was not associated with a measurable increase in NO production. These results indicate that sGC is present in secretory anterior pituitary cells and is regulated in an NO-dependent manner through constitutively expressed neuronal and endothelial NOS and transiently expressed inducible NOS, as well as independently of NO by adenylyl cyclase coupled-receptors.     

Stimulatory roles of nitric-oxide synthase 3 and guanylyl cyclase in platelet activation

Nitric oxide (NO) stimulates soluble guanylyl cyclase and, thus, enhances cyclic guanosine monophosphate (cGMP) levels. It is a currently prevailing concept that NO inhibits platelet activation. This concept, however, does not fully explain why platelet agonists stimulate NO production. Here we show that a major platelet NO synthase (NOS) isoform, NOS3, plays a stimulatory role in platelet secretion and aggregation induced by low doses of platelet agonists. Furthermore, we show that NOS3 promotes thrombosis in vivo. The stimulatory role of NOS is mediated by soluble guanylyl cyclase and results from a cGMP-dependent stimulation of platelet granule secretion. These findings delineate a novel signaling pathway in which agonists sequentially activate NOS3, elevate cGMP, and induce platelet secretion and aggregation. Our data also suggest that NO plays a biphasic role in platelet activation, a stimulatory role at low NO concentrations and an inhibitory role at high NO concentrations.     

The guanylyl cyclase receptor family

Cyclic GMP (cGMP) signals through protein kinases, ion channels, and possibly other effector systems as a second messenger. Its synthesis is regulated by guanylyl cyclase, whose activity is found in various cellular compartments including the plasma membrane and cytosol. A soluble form of guanylyl cyclase, which occurs as a heterodimer, appears to serve as a receptor for nitric oxide or nitrosothiols, or both. Recent research suggests the presence of multiple subtypes of the soluble form of guanylyl cyclase and tissue-specific expression of the different forms. At least two different forms of the plasma membrane guanylyl cyclase are known to occur in various mammalian tissues. One form, GC-A, is a receptor for atrial natriuretic peptide, and the binding of ligand causes marked increases in cGMP production. The other form, GC-B, is stimulated more effectively by a brain natriuretic peptide than by atrial natriuretic peptide, but its natural ligand remains in question. Both plasma membrane forms of the enzyme contain a single, putative transmembrane domain. The intracellular region of both forms contains a protein kinase-like domain just within the transmembrane domain. The protein kinase-like domain is followed by a cyclase catalytic region near the carboxyl terminus that is homologous to two internally homologous domains found in a bovine brain adenylyl cyclase. The possibility that other guanylyl cyclase receptor subtypes exist is now being explored. If they do, we may subsequently find that a diversity of specific ligands signals through cGMP.     

cGMP-mediated facilitation in nerve terminals by enhancement of the spike afterhyperpolarization

cGMP has long been suspected to play a role in synaptic plasticity, but the inaccessibility of nerve terminals to electrical recording has impeded tests of this hypothesis. In posterior pituitary nerve terminals, nitric oxide enhanced Ca(2+)-activated K+ channel activity by activating guanylate cyclase and PKG. This enhancement occurred only at depolarized potentials, so the spike threshold remained unaltered but the afterhyperpolarization became larger. During spike trains, the enhanced afterhyperpolarization promoted Na+ channel recovery from inactivation, thus reducing action potential failures and allowing more Ca(2+) to enter. Activating guanylate cyclase, either with applied nitric oxide, or with physiological stimulation to activate nitric oxide synthase, increased action potential firing. Thus, the cGMP/nitric oxide cascade generates a short-term, use-dependent enhancement of release.     

eNOS, nNOS, cGMP and protein kinase G mediate the inhibitory effect of pancreastatin, a chromogranin A-derived peptide, on growth and proliferation of hepatoma cells

Pancreastatin (PST), a chromogranin A-derived peptide, has an anti-insulin metabolic effect and inhibits growth and proliferation by producing nitric oxide (NO) in HTC rat hepatoma cells. When NO production is blocked, a proliferative effect prevails due to the activation a Galphaq/11-phospholipase C-beta (PLC-beta) pathway, which leads to an increase in [Ca2+]i, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation. The aim of the present study was to investigate the NO synthase (NOS) isoform that mediates these effects of PST on HTC hepatoma cells and the possible roles of cyclic GMP (cGMP) and cGMP-dependent protein kinase. DNA and protein synthesis in response to PST were measured as [3H]-thymidine and [3H]-leucine incorporation in the presence of various pharmacological inhibitors: N-monomethyl-L-arginine (NMLA, nonspecific NOS inhibitor), L-NIO (endothelial nitric oxide synthase (eNOS) inhibitor), espermidine (neuronal nitric oxide synthase (nNOS) inhibitor), LY83583 (guanylyl cyclase inhibitor), and KT5823 (protein kinase G inhibitor, (PKG)). L-NIO, similarly to NMLA, reverted the inhibitory effect of PST on hepatoma cell into a stimulatory effect on growth and proliferation. Nevertheless, espermidine also prevented the inhibitory effect of PST, but there was no stimulation of growth and proliferation. When guanylyl cyclase activity was blocked, there was again a reversion of the inhibitory effect into a stimulatory action, suggesting that the effect of NO was mediated by the production of cGMP. PKG inhibition prevented the inhibitory effect of PST, but there was no stimulatory effect. Therefore, the inhibitory effect of PST on growth and proliferation of hepatoma cells may be mainly mediated by eNOS activation. In turn, the effect of NO may be mediated by cGMP, whereas other pathways in addition to PKG activation seem to mediate the inhibition of DNA and protein synthesis by PST in HTC hepatoma cells.     

Receptor-controlled phosphorylation of alpha 1 soluble guanylyl cyclase enhances nitric oxide-dependent cyclic guanosine 5'-monophosphate production in pituitary cells

It is generally accepted that G protein-coupled receptors stimulate soluble guanylyl cyclase (sGC)-mediated cGMP production indirectly, by increasing nitric oxide (NO) synthase activity in a calcium- and kinase-dependent manner. Here we show that normal and GH(3) immortalized pituitary cells expressed alpha(1)beta(1)-sGC heterodimer. Activation of adenylyl cyclase by GHRH, pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal peptide, and forskolin increased NO and cGMP levels, and basal and stimulated cGMP production was abolished by inhibition of NO synthase activity. However, activators of adenylyl cyclase were found to enhance this NO-dependent cGMP production even when NO was held constant at basal levels. Receptor-activated cGMP production was mimicked by expression of a constitutive active protein kinase A and was accompanied with phosphorylation of native and recombinant alpha(1)-sGC subunit. Addition of a protein kinase A inhibitor, overexpression of a dominant negative mutant of regulatory protein kinase A subunit, and substitution of Ser(107)-Ser(108) N-terminal residues of alpha(1)-subunit with alanine abolished adenylyl cyclase-dependent cGMP production without affecting basal and NO donor-stimulated cGMP production. These results indicate that phosphorylation of alpha(1)-subunit by protein kinase A enlarges the NO-dependent sGC activity, most likely by stabilizing the NO/alpha(1)beta(1) complex. This is the major pathway by which adenylyl cyclase-coupled receptors stimulate cGMP production.     

Redistribution of cyclic GMP in response to sodium butyrate in colon cells

The effect of butyrate on the response to guanylin and Escherichia coli heat-stable enterotoxin, STa, was assessed in T84 cells and Caco-2 cells, cultured colon cell lines possessing the guanylyl cyclase C which is the receptor for these peptides. Butyrate treatment of these cells resulted in an apparent increase in cyclic GMP (cGMP) accumulation when the cGMP content of cells and the supernatant medium was measured. Butyrate treatment did not change the guanylyl cyclase activity or (125)I-STa binding parameters in T84 cells, but the butyrate effect was completely blocked by cycloheximide. Butyrate did not have any effect on STa-stimulated cGMP accumulation in COS cells transfected with the human or porcine GC-C. Further experiments showed that butyrate treatment caused a large increase in the cGMP released into the culture medium, and in cells grown in polarized fashion in Transwell inserts, cGMP efflux was predominantly from the basolateral surface of the cell; intracellular cGMP was actually lowered by butyrate treatment. Exposure of T84 cells to butyrate had no effect on the disposition of cyclic AMP generated in response to forskolin. The effects of butyrate on cGMP were reversible within 24 h of butyrate withdrawal. In colon cells, butyrate treatment induced a previously undescribed, cGMP-specific efflux mechanism which lowered intracellular cGMP and elevated extracellular cGMP in response to peptide agonists such as guanylin and STa.     

Soluble guanylyl cyclase contributes to ventilator-induced lung injury in mice

High tidal volume (HV(T)) ventilation causes pulmonary endothelial barrier dysfunction. HV(T) ventilation also increases lung nitric oxide (NO) and cGMP. NO contributes to HV(T) lung injury, but the role of cGMP is unknown. In the current study, ventilation of isolated C57BL/6 mouse lungs increased perfusate cGMP as a function of V(T). Ventilation with 20 ml/kg V(T) for 80 min increased the filtration coefficient (K(f)), an index of vascular permeability. The increased cGMP and K(f) caused by HV(T) were attenuated by nitric oxide synthase (NOS) inhibition and in lungs from endothelial NOS knockout mice. Inhibition of soluble guanylyl cyclase (sGC) in wild-type lungs (10 muM ODQ) also blocked cGMP generation and inhibited the increase in K(f), suggesting an injurious role for sGC-derived cGMP. sGC inhibition also attenuated lung Evans blue dye albumin extravasation and wet-to-dry weight ratio in an anesthetized mouse model of HV(T) injury. Additional activation of sGC (1.5 muM BAY 41-2272) in isolated lungs at 40 min increased cGMP production and K(f) in lungs ventilated with 15 ml/kg V(T). HV(T) endothelial barrier dysfunction was attenuated with a nonspecific phosphodiesterase (PDE) inhibitor (100 muM IBMX) as well as an inhibitor (10 muM BAY 60-7550) specific for the cGMP-stimulated PDE2A. Concordantly, we found a V(T)-dependent increase in lung cAMP hydrolytic activity and PDE2A protein expression with a decrease in lung cAMP concentration that was blocked by BAY 60-7550. We conclude that HV(T)-induced endothelial barrier dysfunction resulted from a simultaneous increase in NO/sGC-derived cGMP and PDE2A expression causing decreased cAMP.     

Guanylyl cyclase structure, function and regulation

Nitric oxide, bicarbonate, natriuretic peptides (ANP, BNP and CNP), guanylins, uroguanylins and guanylyl cyclase activating proteins (GCAPs) activate a family of enzymes variously called guanyl, guanylyl or guanylate cyclases that catalyze the conversion of guanosine triphosphate to cyclic guanosine monophosphate (cGMP) and pyrophosphate. Intracellular cyclic GMP is a second messenger that modulates: platelet aggregation, neurotransmission, sexual arousal, gut peristalsis, blood pressure, long bone growth, intestinal fluid secretion, lipolysis, phototransduction, cardiac hypertrophy and oocyte maturation. This review briefly discusses the discovery of cGMP and guanylyl cyclases, then nitric oxide, nitric oxide synthase and soluble guanylyl cyclase are described in slightly greater detail. Finally, the structure, function, and regulation of the individual mammalian single membrane-spanning guanylyl cyclases GC-A, GC-B, GC-C, GC-D, GC-E, GC-F and GC-G are described in greatest detail as determined by biochemical, cell biological and gene-deletion studies.     

Nitric oxide regulation of cGMP production in osteoclasts

Bone resorption by osteoclasts is modified by agents that affect cyclic guanosine monophosphate (cGMP), but their relative physiological roles, and what components of the process are present in osteoclasts or require accessory cells such as osteoblasts, are unclear. We studied cGMP regulation in avian osteoclasts, and in particular the roles of nitric oxide and natriuretic peptides, to clarify the mechanisms involved. C-type natriuretic peptide drives a membrane guanylate cyclase, and increased cGMP production in mixed bone cells. However, C-type natriuretic peptide did not increase cGMP in purified osteoclasts. By contrast, osteoclasts did produce cGMP in response to nitric oxide (NO) generators, sodium nitroprusside or 1-hydroxy-2-oxo-3,3-bis(3-aminoethyl)-1-triazene. These findings indicate that C-type natriuretic peptide and NO modulate cGMP in different types of bone cells. The activity of the osteoclast centers on HCI secretion that dissolves bone mineral, and both NO generators and hydrolysis-resistant cGMP analogues reduced bone degradation, while cGMP antagonists increased activity. NO synthase agonists did not affect activity, arguing against autocrine NO production. Osteoclasts express NO-activated guanylate cyclase and cGMP-dependent protein kinase (G-kinase). G-kinase reduced membrane HCI transport activity in a concentration-dependent manner, and phosphorylated a 60-kD osteoclast membrane protein, which immunoprecipitation showed is not an H+-ATPase subunit. We conclude that cGMP is a negative regulator of osteoclast activity. cGMP is produced in response to NO made by other cells, but not in response to C-type natriuretic peptide. G-kinase modulates osteoclast membrane HCI transport via intermediate protein(s) and may mediate cGMP effects in osteoclasts.