Kinetics of a cellular nitric oxide/cGMP/phosphodiesterase-5 pathway

Rat platelets served as a model to evaluate quantitatively how guanylate cyclase (GC)-coupled nitric oxide (NO) receptors and phosphodiesterases (here phosphodiesterase-5) interact to transduce NO signals in cells. The platelets expressed mRNA only for the alpha(1) and beta(1) GC-coupled receptor subunits. In intact platelets, the potency of NO for elevating cGMP (EC(50) = 10 nm) was lower than in lysed platelets (EC(50) = 1.7 nm). The limiting activities of GC and phosphodiesterase in intact platelets were both very high, being equivalent to about 100 microm/s. With low phosphodiesterase activity (imposed by 100 microm sildenafil), the cGMP response over time was hyperbolic in shape for a range of NO concentrations or GC activities due to GC desensitization. Without a phosphodiesterase inhibitor, NO generated only brief cGMP transients, peaking after 2-5 s but amounting maximally to about 150 microm cGMP. The transients were caused partly by GC desensitization, which varied in rate (half-time up to 3 s) and extent (up to 80%) depending on the NO concentration, and partly by an enhancement of the phosphodiesterase catalytic activity with time, which was deduced to be up to 30-fold and to occur with a half-time of up to 5 s. The results were simulated by a quantitative model, which also explains the varied shapes of cGMP responses to NO found in other cells. Downstream phosphorylation in platelets was detectable within 2 s, and, with continuous exposure (1 min), this pathway could be engaged by subnanomolar NO concentrations (EC(50) = 0.5 nm).     

In vivo reconstitution of the negative feedback in nitric oxide/cGMP signaling: role of phosphodiesterase type 5 phosphorylation

Most effects of the messenger molecule nitric oxide (NO) are mediated by cGMP, which is formed by NO-sensitive guanylyl cyclase (GC) and degraded by phosphodiesterases (PDEs). In platelets, NO elicits a spike-like cGMP response and causes a sustained desensitization. Both characteristics have been attributed to PDE5 activation caused by cGMP binding to its regulatory GAF domain. Activation is paralleled by phosphorylation whose precise function remains unknown. Here, we report reconstitution of all features of the NO-induced cGMP response in human embryonic kidney cells by coexpressing NO-sensitive GC and PDE5. The spike-like cGMP response was blunted when PDE5 phosphorylation was enhanced by additional overexpression of cGMP-dependent protein kinase. Analysis of PDE5 activation in vitro revealed a discrepancy between the cGMP concentrations required for activation (micromolar) and reversal of activation (nanomolar), indicating the conversion of a low-affinity state to a high-affinity state upon binding of cGMP. Phosphorylation even increased the high apparent affinity enabling PDE5 activation to persist at extremely low cGMP concentrations. Our data suggest that the spike-like shape and the desensitization of the cGMP response are potentially inherent to every GC- and PDE5-expressing cell. Phosphorylation of PDE5 seems to act as memory switch for activation leading to long-term desensitization of the signaling pathway.     

Direct activation of PDE5 by cGMP: long-term effects within NO/cGMP signaling

In platelets, the nitric oxide (NO)-induced cGMP response is indicative of a highly regulated interplay of cGMP formation and cGMP degradation. Recently, we showed that within the NO-induced cGMP response in human platelets, activation and phosphorylation of phosphodiesterase type 5 (PDE5) occurred. Here, we identify cyclic GMP-dependent protein kinase I as the kinase responsible for the NO-induced PDE5 phosphorylation. However, we demonstrate that cGMP can directly activate PDE5 without phosphorylation in platelet cytosol, most likely via binding to the regulatory GAF domains. The reversal of activation was slow, and was not completed after 60 min. Phosphorylation enhanced the cGMP-induced activation, allowing it to occur at lower cGMP concentrations. Also, in intact platelets, a sustained NO-induced activation of PDE5 for as long as 60 min was detected. Finally, the long-term desensitization of the cGMP response induced by a low NO concentration reveals the physiological relevance of the PDE5 activation within NO/cGMP signaling. In sum, we suggest NO-induced activation and phosphorylation of PDE5 as the mechanism for a long-lasting negative feedback loop shaping the cGMP response in human platelets in order to adapt to the amount of NO available.     

The interaction between thymine DNA glycosylase and nuclear receptor coactivator 3 is required for the transcriptional activation of nuclear hormone receptors

The cellular counterpart of the “soluble” guanylyl cyclase found in tissue homogenates over 30 years ago is now recognized as the physiological receptor for nitric oxide (NO). The ligand-binding site is a prosthetic haem group that, when occupied by NO, induces a conformational change in the protein that propagates to the catalytic site, triggering conversion of GTP into cGMP. This review focuses on recent research that takes this basic information forward to the beginnings of a quantitative depiction of NO signal transduction, analogous to that achieved for other major transmitters. At its foundation is an explicit enzyme-linked receptor mechanism for NO-activated guanylyl cyclase that replicates all its main properties. In cells, NO signal transduction is subject to additional, activity-dependent modifications, notably through receptor desensitization and changes in the activity of cGMP-hydrolyzing phosphodiesterases. The measurement of these parameters under varying conditions in rat platelets has made it possible to formulate a cellular model of NO-cGMP signaling. The model helps explain cellular responses to NO and their modification by therapeutic agents acting on the guanylyl cyclase or phosphodiesterase limbs of the pathway.     

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.     

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.     

Nitric Oxide Regulates Cyclic GMP-Dependent Protein Kinase Phosphorylation in Rat Brain

Abstract: Nitric oxide (NO) acts via soluble guanylyl cyclase to increase cyclic GMP (cGMP), which can regulate various targets including protein kinases. Western blotting showed that type II cGMP-dependent protein kinase (cGK II) is widely expressed in various brain regions, especially in the thalamus. In thalamic extracts, the phosphorylation of several proteins, including cGK II, was increased by exogenous NO or cGMP. In vivo pretreatment with a NO synthase inhibitor reduced the phosphorylation of cGK II, and this could be reversed by exogenous NO or cGMP. Conversely, brainstem electrical stimulation, which enhances thalamic NO release, caused a NO synthase-dependent increase in the phosphorylation of thalamic cGK II. These results indicate that endogenous NO regulates cGMP-dependent protein phosphorylation in the thalamus. The activation of cGKII by NO may play a role in thalamic mechanisms underlying arousal.     

Expression and regulation of the cGMP-binding, cGMP-specific phosphodiesterase (PDE5) in human colonic epithelial cells: role in the induction of cellular refractoriness to the heat-stable enterotoxin peptide

Stable toxin (ST) peptides are the causative agents for a severe form of watery diarrhea. These peptides bind to a membrane-associated form of guanylyl cyclase, guanylyl cyclase C. The result is an accumulation of cyclic guanosine monophosphate (cGMP) in the intestinal cell, regulating protein kinase activity and the phosphorylation of a number of proteins involved in ion transport across the intestine. Using the human T84 colonic cell line as a model system, we show that cGMP accumulation in these cells after ST application is regulated by the activity of the cGMP-binding, cGMP-specific phosphodiesterase (PDE5). The presence of human PDE5 in this cell line was confirmed by Western blot analysis, using an antibody raised to the bovine enzyme, and by the observation that cGMP hydrolytic activity detected in T84 cell lysates was almost completely inhibited by low concentrations of zaprinast, a specific inhibitor of PDE5. An increase in activity of PDE5 was observed in T84 cell lysates on exposure to the ST peptide and prolonged exposure of T84 cells to the ST peptide led to the induction of cellular refractoriness in these cells, which was largely contributed in terms of an increased rate of degradation of cGMP in desensitized cells as a result of PDE5 activation. This activation was correlated with an increase in the affinity of the enzyme for the substrate cGMP, as well as an increased affinity for zaprinast. We provide evidence for the first time that cGMP levels in the human colonocyte are regulated by the cGMP-hydrolytic activity of PDE5 and suggest that the expression and regulation of PDE5 in the intestine could therefore be important in controlling cGMP-mediated signaling in this tissue.     

Localization of cGMP immunoreactivity and of soluble guanylyl cyclase in antennal sensilla of the hawkmoth Manduca sexta

The intracellular messenger cGMP (cyclic guanosine monophosphate) has been suggested to play a role in olfactory transduction in both invertebrates and vertebrates, but its cellular location within the olfactory system has remained elusive. We used cGMP immunocytochemistry to determine which antennal cells of the hawkmoth Manduca sexta are cGMP immunoreactive in the absence of pheromone. We then tested which antennal cells increase cGMP levels in response to nitric oxide (NO) and to long pheromonal stimuli, which the male encounters close to a calling female moth. In addition, we used in situ hybridization to determine which antennal cells express NO-sensitive soluble guanylyl cyclase. In response to long pheromonal stimuli with NO donors present, cGMP concentrations change in at least a subpopulation of pheromone-sensitive olfactory receptor neurons. These changes in cGMP concentrations in pheromone-dependent olfactory receptor neurons cannot be mimicked by the addition of NO donors in the absence of pheromone. NO stimulates sensilla chaetica type I and II, but not pheromone-sensitive trichoid sensilla, to high levels of cGMP accumulation as detected by immunocytochemistry. In situ hybridizations show that sensilla chaetica, but not sensilla trichodea, express detectable levels of mRNA coding for soluble guanylyl cyclase. These results suggest that intracellular rises in cGMP concentrations play a role in information processing in a subpopulation of pheromone-sensitive sensilla in Manduca sexta antennae, mediated by an NO-sensitive mechanism, but not an NO-dependent soluble guanylyl cyclase.     

HIV-1 coat protein gp120 decreases NO-dependent cyclic GMP accumulation in rat brain astroglia by increasing cyclic GMP phosphodiesterase activity

The human immunodeficiency virus type-1 (HIV-1) coat glycoprotein gp120 has been proposed as a likely etiologic agent of HIV-associated dementia (HAD). The pathogenic mechanisms underlying HAD have not yet been fully elucidated, but different evidences indicate that glial cells play an essential role in the development and amplification of the disease. The NO/cyclic GMP (cGMP) system is a widespread signal transduction pathway in the CNS involved in numerous physiological and pathological functions. Increased expression of NO synthase has been reported in the brain of AIDS patients and in cultured rodent glial cells exposed to gp120. The aim of this study was to investigate if gp120 could cause alterations in the metabolism of the NO physiological messenger cGMP that could contribute to the pathogenesis of HAD. Here, we show that long-term treatment (more than 24 h) of rat cerebellar astrocyte-enriched cultures with gp120 (10 nM) induces changes in the cultured cells--astrocyte stellation and proliferation of ameboid microglia--compatible with the acquisition of a reactive phenotype and reduces the capacity of the astrocytes to accumulate cGMP in response to NO in a time-dependent manner (maximal after 72 h). Measurements in cell extracts show that gp120 enhances Ca2+-independent cGMP phosphodiesterase activity by 80-100% without significantly affecting soluble guanylyl cyclase (sGC). Experiments in whole cells using specific phosphodiesterase inhibitors indicate that the viral protein increases the activity of cGMP specific phosphodiesterase 5.     

Modulation of soluble guanylate cyclase activity by phosphorylation

The levels of the cGMP in smooth muscle of the gut reflect continued synthesis by soluble guanylate cyclase (GC) and breakdown by phosphodiesterase 5 (PDE5). Soluble GC is a haem-containing, heterodimeric protein consisting alpha- and beta-subunits: each subunit has N-terminal regulatory domain and a C-terminal catalytic domain. The haem moiety acts as an intracellular receptor for nitric oxide (NO) and determines the ability of NO to activate the enzyme and generate cGMP. In the present study the mechanism by which protein kinases regulate soluble GC in gastric smooth muscle was examined. Sodium nitroprusside (SNP) acting as a NO donor stimulated soluble GC activity and increased cGMP levels. SNP induced soluble GC phosphorylation in a concentration-dependent fashion. SNP-induced soluble GC phosphorylation was abolished by the selective cGMP-dependent protein kinase (PKG) inhibitors, Rp-cGMPS and KT-5823. In contrast, SNP-stimulated soluble GC activity and cGMP levels were significantly enhanced by Rp-cGMPS and KT-5823. Phosphorylation and inhibition of soluble GC were PKG specific, as selective activator of cAMP-dependent protein kinase, Sp-5, 6-DCl-cBiMPS had no effect on SNP-induced soluble GC phosphorylation and activity. The ability of PKG to stimulate soluble GC phosphorylation was demonstrated in vitro by back phosphorylation technique. Addition of purified phosphatase 1 inhibited soluble GC phosphorylation in vitro, and inhibition was reversed by a high concentration (10 microM) of okadaic acid. In gastric smooth muscle cells, inhibition of phosphatase activity by okadaic acid increased soluble GC phosphorylation in a concentration-dependent fashion. The increase in soluble GC phosphorylation inhibited SNP-stimulated soluble GC activity and cGMP formation. The results implied the feedback inhibition of soluble GC activity by PKG-dependent phosphorylation impeded further formation of cGMP.     

Pseudomonas aeruginosa exotoxin Y is a promiscuous cyclase that increases endothelial tau phosphorylation and permeability

Exotoxin Y (ExoY) is a type III secretion system effector found in ~ 90% of the Pseudomonas aeruginosa isolates. Although it is known that ExoY causes inter-endothelial gaps and vascular leak, the mechanisms by which this occurs are poorly understood. Using both a bacteria-delivered and a codon-optimized conditionally expressed ExoY, we report that this toxin is a dual soluble adenylyl and guanylyl cyclase that results in intracellular cAMP and cGMP accumulation. The enzymatic activity of ExoY caused phosphorylation of endothelial Tau serine 214, accumulation of insoluble Tau, inter-endothelial cell gap formation, and increased macromolecular permeability. To discern whether the cAMP or cGMP signal was responsible for Tau phosphorylation and barrier disruption, pulmonary microvascular endothelial cells were engineered for the conditional expression of either wild-type guanylyl cyclase, which synthesizes cGMP, or a mutated guanylyl cyclase, which synthesizes cAMP. Sodium nitroprusside stimulation of the cGMP-generating cyclase resulted in transient Tau serine 214 phosphorylation and gap formation, whereas stimulation of the cAMP-generating cyclase induced a robust increase in Tau serine 214 phosphorylation, gap formation, and macromolecular permeability. These results indicate that the cAMP signal is the dominant stimulus for Tau phosphorylation. Hence, ExoY is a promiscuous cyclase and edema factor that uses cAMP and, to some extent, cGMP to induce the hyperphosphorylation and insolubility of endothelial Tau. Because hyperphosphorylated and insoluble Tau are hallmarks in neurodegenerative tauopathies such as Alzheimer disease, acute Pseudomonas infections cause a pathophysiological sequela in endothelium previously recognized only in chronic neurodegenerative diseases.     

Nitric oxide-evoked transient kinetics of cyclic GMP in vascular smooth muscle cells

Cyclic-3',5'-guanosine monophosphate (cGMP) mediates the intracellular signaling cascade responsible for the nitric oxide (NO) initiated relaxation of vascular smooth muscle (VSM). However, the temporal dynamics, including the regulation of cGMP turnover, are largely unknown. Here we report new mechanistic insights into the kinetics of cGMP synthesis and hydrolysis in primary VSM cells by utilizing FRET-based cGMP-indicators [A. Honda, S.R. Adams, C.L. Sawyer, V. Lev-Ram, R.Y. Tsien, W.R. Dostmann, Proc. Natl. Acad. Sci. U S A 98 (5) (2001) 2437.]. First, 2-(N,N-Diethylamino)-diazenolate 2-oxide (DEA/NO) and 2,2'-(Hydroxynitrosohydrazono)-bis-ethanimine (DETA/NO) induced NO-concentration dependent, transient cGMP responses ("peaks") irrespective of their rates of NO release. The kinetic characteristics of these cGMP peaks were governed by the concerted action of the NO-sensitive guanylyl cyclase (GC) and phosphodiesterase type V (PDE5) as shown by their respective inhibition using 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and Sildenafil. These responses occurred in the presence of moderately elevated cGMP (5-15% FRET ratio), and thus activated PKG and phosphorylated PDE5, suggesting a prominent role for GC in the maintenance and termination of cGMP peaks. Furthermore, cGMP transients could be elicited repeatedly without apparent desensitization of GC or by suppression of cGMP via long-term PDE5 activity. These results demonstrate a continuous sensitivity of the NO/cGMP signaling system, inherent to the phasic nature of smooth muscle physiology.     

The atrial natriuretic peptide receptor (NPR-A/GC-A) is dephosphorylated by distinct microcystin-sensitive and magnesium-dependent protein phosphatases

Natriuretic peptide receptor (NPR)-A is the primary signaling receptor for atrial natriuretic peptide and brain natriuretic peptide. Ligand binding to NPR-A rapidly activates its guanylyl cyclase domain, but its rate of cGMP synthesis declines with time. This waning of activity is called homologous desensitization and is mediated in part by receptor dephosphorylation. Here, we characterize two distinct NPR-A phosphatase activities. The serine/threonine protein phosphatase inhibitor, microcystin, inhibited the desensitization of NPR-A in membrane guanylyl cyclase assays in the absence of magnesium. EDTA also inhibited the desensitization, whereas MgCl(2) stimulated the desensitization. Because the effects of microcystin and EDTA were additive, and microcystin did not block the magnesium-dependent desensitization, the targets for these agents appear to be distinct. Incubation of membranes at 37 degrees C stimulated the dephosphorylation of NPR-A, and microcystin blocked the temperature-dependent dephosphorylation. The addition of MgCl(2) or MnCl(2), but not CaCl(2), further stimulated the dephosphorylation of NPR-A, and microcystin failed to inhibit this process. The desensitization required changes in the phosphorylation state of NPR-A because the guanylyl cyclase activity of a receptor variant containing glutamate substitutions at all six phosphorylation sites was unaffected by MgCl(2), EDTA, or microcystin. Together, these data indicate that NPR-A is regulated by two distinct phosphatases, possibly including a member of the protein phosphatase 2C family. Finally, we observed that the desensitization of NPR-A in membranes from mouse kidneys and NIH3T3 cells was increased by prior exposure to atrial natriuretic peptide, suggesting that hormone binding enhances receptor dephosphorylation.     

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.     

Functional effects of C-type natriuretic peptide and nitric oxide are attenuated in hypertrophic myocytes from pressure-overloaded mouse hearts

Increases in the myocardial level of cGMP usually exert negative inotropic effects in the mammalian hearts. We tested the hypothesis that the negative functional effects caused by nitric oxide (NO) or C-type natriuretic peptide (CNP) through cGMP would be blunted in hypertrophied cardiac myocytes. Contractile function, guanylyl cyclase activity, cGMP-dependent protein phosphorylation, and calcium transients were assessed in ventricular myocytes from aortic stenosis-induced hypertrophic and age-matched control mice. Basal percentage shortening was similar in control and hypertrophic myocytes. S-nitroso-N-acetyl-penicillamine (SNAP, an NO donor, 10(-6) and 10(-5) M) or CNP (10(-8) and 10(-7) M) reduced percentage shortening in both groups, but their effects were blunted in hypertrophic myocytes. Maximal rates of shortening and relaxation were depressed at the basal level, and both reagents had attenuated effects in hypertrophy. Similar results were also found after treatment with guanylin and carbon monoxide, other stimulators of particulate, and soluble guanylyl cyclase, respectively. Guanylyl cyclase activity was not significantly changed in hypertrophy. Addition of Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine (an inhibitor of cGMP-dependent protein kinase, 5 x 10(-6) M) blocked SNAP or the effect of CNP in control mice but not in hypertrophy, indicating the cGMP-dependent kinase (PKG) may not mediate the actions of cGMP induced by NO or CNP in the hypertrophic state. Calcium transients after SNAP or CNP were not significantly changed in hypertrophy. These results suggest that in hypertrophied mice, diminished effects of NO or CNP on ventricular myocyte contraction are not due to changes in guanylyl cyclase activity. The data also indicated that PKG-mediated pathways were diminished in hypertrophied myocardium, contributing to blunted effects.     

Chronic hypoxia attenuates cGMP-dependent pulmonary vasodilation

Chronic hypoxia (CH) augments endothelium-derived nitric oxide (NO)-dependent pulmonary vasodilation; however, responses to exogenous NO are reduced following CH in female rats. We hypothesized that CH-induced attenuation of NO-dependent pulmonary vasodilation is mediated by downregulation of vascular smooth muscle (VSM) soluble guanylyl cyclase (sGC) expression and/or activity, increased cGMP degradation by phosphodiesterase type 5 (PDE5), or decreased VSM sensitivity to cGMP. Experiments demonstrated attenuated vasodilatory responsiveness to the NO donors S-nitroso-N-acetylpenicillamine and spermine NONOate and to arterial boluses of dissolved NO solutions in isolated, saline-perfused lungs from CH vs. normoxic female rats. In additional experiments, the sGC inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, blocked vasodilation to NO donors in lungs from each group. However, CH was not associated with decreased pulmonary sGC expression or activity as assessed by Western blotting and cGMP radioimmunoassay, respectively. Consistent with our hypothesis, the selective PDE5 inhibitors dipyridamole and T-1032 augmented NO-dependent reactivity in lungs from CH rats, while having little effect in lungs from normoxic rats. However, the attenuated vasodilatory response to NO in CH lungs persisted after PDE5 inhibition. Furthermore, CH similarly inhibited vasodilatory responses to 8-bromoguanosine 3'5'-cyclic monophosphate. We conclude that attenuated NO-dependent pulmonary vasodilation after CH is not likely mediated by decreased sGC expression, but rather by increased cGMP degradation by PDE5 and decreased pulmonary VSM reactivity to cGMP.     

Calcium/calmodulin-dependent nitric oxide synthase activity in the CNS of Aplysia californica: biochemical characterization and link to cGMP pathways

We characterized enzymatic activity of nitric oxide synthase (NOS) in the central nervous system of Aplysia californica, a popular experimental model in cellular and system neuroscience, and provided biochemical evidence for NO-cGMP signaling in molluscs. Aplysia NOS (ApNOS) activity, determined as citrulline formation, revealed its calcium-/calmodulin-(Ca/CaM) and NADPH dependence and it was inhibited by 50% with 5mM of W7 hydrochloride (a potent Ca/CaM-dependent phosphodiesterase inhibitor). A representative set of inhibitors for mammalian NOS isoforms also suppressed NOS activity in Aplysia. Specifically, the ApNOS was inhibited by 65-92% with 500 microM of L-NAME (a competitive NOS inhibitor) whereas d-NAME at the same concentration had no effect. S-Ethylisothiourea hydrobromide (5mM), a selective inhibitor of all NOS isoforms, suppressed ApNOS by 85%, l-N6-(1-iminoethyl)lysine dihydrochloride (L-NIL, 5mM), an iNOS inhibitor, by 78% and L-thiocitrulline (5mM) (an inhibitor of nNOS and iNOS) by greater than 95%. Polyclonal antibodies raised against rat nNOS hybridized with a putative purified ApNOS (160 kDa protein) from partially purified central nervous system homogenates in Western blot studies. Consistent with other studies, the activity of soluble guanylyl cyclase was stimulated as a result of NO interaction with its heme prosthetic group. The basal levels of cGMP were estimated by radioimmunoassay to be 44.47 fmol/microg of protein. Incubation of Aplysia CNS with the NO donors DEA/NONOate (diethylammonium (Z)-1-(N,N-diethylamino) diazen-1-ium-1,2-diolate - 1mM) or S-nitroso-N-acetylpenicillamine (1mM) and simultaneous phosphodiesterase inhibition with 3-isobutyl-1-methylxanthine (1mM) prior to the assay showed a 26-80 fold increase in basal cGMP levels. Addition of ODQ (1H-[1,2,4]oxadiazolo[4,3-a] quinoxaline-1-one - 1mM), a selective inhibitor of soluble guanylyl cyclase, completely abolished this effect. This confirms that NO may indeed function as a messenger in the molluscan CNS, and that cGMP acts as one of its effectors.     

Altered regulation of renal nitric oxide and atrial natriuretic peptide systems in angiotensin II-induced hypertension

The present study was aimed to determine whether there is an altered role of local nitric oxide (NO) and atrial natriuretic peptide (ANP) systems in the kidney in association with the angiotensin (Ang) II-induced hypertension. Male Sprague-Dawley rats were used. Ang II (100 ng·min?1·kg?1) was infused through entire time course. Thirteenth day after beginning the regimen, kidneys were taken. The protein expression of NO synthase (NOS) and nitrotyrosine was determined by semiquantitative immunoblotting. The mRNA expression of components of ANP system was determined by real-time polymerase chain reaction. The activities of soluble and particulate guanylyl cyclases were determined by the amount of cGMP generated in responses to sodium nitroprusside and ANP, respectively. There developed hypertension and decreased creatinine clearance in the experimental group. The protein expression of eNOS, nNOS and nitrotyrosine was increased in the cortex, while that of iNOS remained unaltered. The urinary excretion of NO increased in Ang II-induced hypertensive rats. The catalytic activity of soluble guanylyl cyclase was blunted in the glomerulus in Ang II-induced hypertensive rats. The mRNA expression of ANP was increased in Ang II-induced hypertensive rats. Neither the expression of NPR-A nor that of NPR-C was changed. The protein expression of neutral endopeptidase was decreased and the activity of particulate guanylyl cyclase was blunted in the glomerulus and papilla in Ang II-induced hypertensive rats. In conclusion, the synthesis of NO and ANP was increased in the kidney of Ang II-induced hypertension, while stimulated cGMP response was blunted. These results suggest desensitization of guanylyl cyclase in the kidney of Ang II-induced hypertensive rats, which may contribute to the associated renal vasoconstriction and hypertension.