cGMP Accumulation In Response Research Articles

Changes in nerve- and endothelium-mediated contractile tone of the corpus cavernosum in a mouse model of pre-mature ageing.

Erectile dysfunction (ED) is very prevalent in the older population, although the ageing-related mechanisms involved in the development of ED are poorly understood. We propose that age-induced differences in nerve- and endothelium-mediated smooth muscle contractility in the corpus cavernosum (CC) could be found between a senescent-accelerated mouse prone (SAMP8) and senescent-accelerated mouse resistant (SAMR1) strains. We analysed the changes in muscle tension induced by electrical field stimulation (EFS) or agonist addition 'in vitro', assessing nerve density (adrenergic, cholinergic and nitrergic), the expression of endothelial nitric oxide synthase (eNOS), cGMP accumulation and the distribution of interstitial cells (ICs) by immunofluorescence. We observed no change in both the nerve-dependent adrenergic excitatory contractility at physiological levels of stimulation and in the nitrergic inhibitory response in SAMP8 animals. Unlike cholinergic innervation, the density of adrenergic and nitrergic nerves increased in SAMP8 mice. In contrast, smooth muscle sensitivity to exogenous noradrenaline (NA) was slightly reduced, whereas cGMP accumulation in response to EFS and DEA/NO, and relaxations to DEA/NO and sildenafil, were not modified. No changes in the expression of eNOS and in the distribution of vimentin-positive ICs were detected in the aged animals. The ACh induced atropine-sensitive biphasic endothelium-dependent responses involved relaxation at low concentrations that turned into contractions at the highest doses. CC relaxation was mainly because of the production of NO together with some relaxant prostanoid, which did not change in SAMP8 animals. In contrast, the contractile component was considerably higher in the aged animals and it was completely inhibited by indomethacin. In conclusion, a clear imbalance towards enhanced production of contractile prostanoids from the endothelium may contribute to ED in the elderly. On the basis of these data, we propose the senescence-accelerated mouse model as a reliable tool to analyse the basic ageing mechanisms of the CC.

Melatonin inhibits nitric oxide signaling by increasing PDE5 phosphorylation in coronary arteries

Melatonin inhibits nitric oxide (NO)-induced relaxation of coronary arteries. We tested the hypothesis that melatonin increases the phosphorylation of phosphodiesterase 5 (PDE5), which increases the activity of the enzyme and thereby decreases intracellular cGMP accumulation in response to NO and inhibits NO-induced relaxation. Sodium nitroprusside (SNP) and 8-Br-cGMP caused concentration-dependent relaxation of isolated coronary arteries suspended in organ chambers for isometric tension recording. In the presence of melatonin, the concentration-response curve to SNP, but not 8-Br-cGMP, was shifted to the right. The effect of melatonin on SNP-induced relaxation was abolished in the presence of the PDE5 inhibitors zaprinast and sildenafil. Melatonin markedly inhibited the SNP-induced increase in intracellular cGMP in coronary arteries, an effect that was also abolished by zaprinast. Treatment of coronary arteries with melatonin caused a nearly fourfold increase in the phosphorylation of PDE5, which increased the catalytic activity of the enzyme and thereby increased the degradation of cGMP to inactive 5'-GMP. Melatonin-induced PDE5 phosphorylation was markedly attenuated in the presence of the PKG1 inhibitors DT-2 or Rp-8-Br-PET-cGMPS and in those arteries in which PKG1 expression was first downregulated by 24-h incubation with SNP before exposure to melatonin. The selective MT(2) receptor antagonist 4-phenyl-2-propionamidotetralin completely blocked the stimulatory effect of melatonin on PDE5 phosphorylation as well as the inhibitory effect of melatonin on SNP-induced relaxation and intracellular cGMP. Thus, in coronary arteries, melatonin acts via MT(2) receptors and PKG1 to increase PDE5 phosphorylation, resulting in decreased cGMP accumulation in response to NO and impaired NO-induced vasorelaxation.

C‐type natriuretic peptide regulation of guanosine‐3′,5′‐cyclic monophosphate production in human endothelial cells

In vascular smooth muscle cells, relaxant actions of guanosine--3',5'-cyclic monophosphate (cGMP) are well recognized, but there is increasing evidence that cGMP also plays regulatory roles in vascular endothelium. However, the autacoid and endocrine mechanisms controlling cGMP production in endothelium are not well understood. The objective of these studies was to examine the mechanisms of cGMP accumulation in human umbilical vein endothelial cells (HUVEC) in response to natriuretic peptides. Expression in HUVEC of natriuretic peptide receptors, particulate guanylyl cyclases (GC)-A and GC-B, was confirmed by RT-PCR and Western blot analysis. In the presence of the phosphodiesterase inhibitor IBMX 500 microM, 3 h incubation of HUVEC with B-type natriuretic peptide (BNP) (preferential GC-A agonist) or C-type natriuretic peptide (CNP) (preferential GC-B agonist) stimulated concentration-dependent increases in cGMP production. At 10 and 100 nM, we observed two to three-fold greater potency of CNP compared to BNP. In the absence of IBMX, CNP-stimulated cGMP accumulation was significantly less than cGMP accumulation in response to sodium nitroprusside 1 mM. This greater sensitivity of GC-B-derived cGMP to phosphodiesterases suggests compartmentalization of two pools of cGMP from particulate and soluble guanylyl cyclases. Although CNP 100 nM and 1 microM was observed to increase nitrite + nitrate (stable metabolites of NO) production in HUVEC two-fold above basal level, the soluble guanylyl cyclase inhibitor ODQ 10 microM did not significantly modify CNP-stimulated cGMP accumulation suggesting that endothelial actions of CNP may be NO-independent. In conclusion, these studies indicate functional signaling by natriuretic peptides in endothelial cells, supporting possible roles of these mediators in regulating endothelial cell function.

Melatonin inhibits NO‐induced cGMP accumulation by increasing PDE5 phosphorylation in coronary arteries

Melatonin impairs NO‐induced relaxation by inhibiting the NO‐induced increase in cGMP in coronary arteries. We tested the hypothesis that the inhibitory effect of melatonin on NO‐signaling is due to increased phosphodiesterase type‐5 (PDE5) activation. Melatonin (10−7M) inhibited sodium nitroprusside (SNP) induced relaxation of isolated coronary arteries but had no effect on relaxation induced by 8‐Br‐cGMP. The inhibitory effect of melatonin on SNP‐induced relaxation was abolished in the presence of either the PDE inhibitor, zaprinast (10−5M) or the MT2‐receptor antagonist, 4P‐PDOT (10−7 M). Melatonin (10−7M) markedly reduced the SNP‐induced increase in cGMP levels in coronary arteries, and this effect of melatonin was significantly attenuated in the presence of either zaprinast (10−5M) or 4P‐PDOT (10−7 M). Immunohistochemical analysis confirmed localization of MT2‐receptors in the vascular smooth muscle cell layer. Immunoblot analysis demonstrated PDE5 expression in coronary arteries. Incubation of the tissues with melatonin (10−7M) caused a significant increase in PDE5 phosphorylation, which was abolished in the presence of 4P‐PDOT (10−7 M). The data indicate that melatonin increases the catalytic activity of PDE5 by activating MT2‐receptors in coronary arteries, resulting in decreased cGMP accumulation in response to NO and impaired NO‐induced vasorelaxation. (Supported by NIH HL77204)

Melatonin inhibits NO‐induced cGMP accumulation in porcine coronary arteries

Recent studies in our laboratory indicate that melatonin modulates vascular tone by interfering with nitric oxide (NO) signaling in coronary arteries. Here we tested the hypothesis that melatonin impairs NO‐induced vasorelaxation by inhibiting the NO‐induced increase in cGMP levels in coronary arteries. In organ bath studies, sodium nitroprusside (SNP) (10−9‐10−5 M) caused relaxation of isolated porcine coronary artery rings. Incubation of the tissues with melatonin (10−7 M) caused a rightward shift in the SNP concentration‐response curve. In the presence of the phosphodiesterase inhibitor, zaprinast (10−5 M), the inhibitory effect of melatonin on SNP‐induced vasorelaxation was abolished. Melatonin (10−7 M) had no effect on basal cGMP levels but markedly inhibited the SNP‐induced increase in cGMP. The effect of melatonin on cGMP accumulation was significantly diminished by incubation of the tissues with either zaprinast (10−5 M) or the MT2‐receptor antagonist, 4P‐PDOT (10−7 M). The expression of MT2‐receptors in coronary arteries was confirmed by immunoblot analysis. Taken together, these results suggest that the inhibitory effect of melatonin on NO signaling in coronary arteries is most likely due to impaired cGMP accumulation in response to NO. The inhibitory effect of melatonin is mediated by MT2‐receptors, perhaps via a mechanism that is coupled to increased phosphodiesterase activity.

Interactions between cell surface protein disulphide isomerase and S-nitrosoglutathione during nitric oxide delivery

In this study, we investigated the role of protein disulphide isomerase (PDI) in rapid metabolism of S-nitrosoglutathione (GSNO) and S-nitrosoalbumin (albSNO) and in NO delivery from these compounds into cells. Incubation of GSNO or albSNO (1 μM) with the megakaryocyte cell line MEG-01 resulted in a cell-mediated removal of each compound which was inhibited by blocking cell surface thiols with 5,5′-dithiobis 2-nitrobenzoic acid (DTNB) (100 μM) or inhibiting PDI with bacitracin (5 mM). GSNO, but not albSNO, rapidly inhibited platelet aggregation and stimulated cyclic GMP (cGMP) accumulation (used as a measure of intracellular NO entry). cGMP accumulation in response to GSNO (1 μM) was inhibited by MEG-01 treatment with bacitracin or DTNB, suggesting a role for PDI and surface thiols in NO delivery. PDI activity was present in MEG-01 conditioned medium, and was inhibited by high concentrations of GSNO (500 μM). A number of cell surface thiol-containing proteins were labelled using the impermeable thiol specific probe 3-( N-maleimido-propionyl) biocytin (MPB). Pretreatment of cells with GSNO resulted in a loss of thiol reactivity on some but not all proteins, suggesting selective cell surface thiol modification. Immunoprecipitation experiments showed that GSNO caused a concentration-dependent loss of thiol reactivity of PDI. Our data indicate that PDI is involved in both rapid metabolism of GSNO and intracellular NO delivery and that during this process PDI is itself altered by thiol modification. In contrast, the relevance of PDI-mediated albSNO metabolism to NO signalling is uncertain.

Hydrogen peroxide restrains endothelium-derived nitric oxide bioactivity—Role for iron-dependent oxidative stress

Vascular diseases are characterized by impairment of endothelial-derived nitric oxide (NO) bioactivity and increased vascular levels of hydrogen peroxide (H 2O 2). Here we examined the implications of H 2O 2 for agonist-stimulated endothelial NO bioactivity in rabbit aortic rings and cultured porcine aortic endothelial cells (PAEC). Vessels pre-treated with H 2O 2 exhibited impaired endothelial-dependent relaxation induced by acetylcholine or calcium ionophore. In contrast, H 2O 2 had no effect on endothelium-independent relaxation induced by a NO donor, indicating a defect in endothelium-derived NO. This defect was not related to eNOS catalytic activity; treatment of PAEC with H 2O 2 enhanced agonist-stimulated eNOS activity indicated by increased eNOS phosphorylation at Ser-1177 and de-phosphorylation at Thr-495 and enhanced conversion of [ 3H]-L-arginine to [ 3H]-L-citrulline that was prevented by inhibitors of Src and phosphatidylinositol-3 kinases. Despite activating eNOS, H 2O 2 impaired endothelial NO bioactivity indicated by attenuation of the increase in intracellular cGMP in PAEC stimulated with calcium ionophore or NO. The decrease in cGMP was not due to impaired guanylyl cyclase as H 2O 2 treatment increased cGMP accumulation in response to BAY 41-2272, a NO-independent activator of soluble guanylyl cyclase. At concentrations that impaired endothelial NO bioactivity H 2O 2 increased intracellular oxidative stress and size of the labile iron pool in PAEC. The increase in oxidative stress was prevented by the free radical scavenger's tempol or tiron and the iron chelator desferrioxamine and these antioxidants reversed the H 2O 2-induced impairment of NO bioactivity in PAEC. This study shows that despite promoting eNOS activity, H 2O 2 impairs endothelial NO bioactivity by promoting oxidative inactivation of synthesized NO. The study highlights another way in which oxidative stress may impair NO bioactivity during vascular disease.

Nitric Oxide-Dependent Reduction in Soluble Guanylate Cyclase Functionality Accounts for Early Lipopolysaccharide-Induced Changes in Vascular Reactivity

We investigated the role of soluble guanylate cyclase in lipopolysaccharide-induced hyporesponsiveness to phenylephrine. The effects of phenylephrine on the blood pressure of female Wistar rats were evaluated at 2, 8, and 24 h after lipopolysaccharide injection (12.5 mg/kg i.p.). Vasoconstrictive responses to phenylephrine were reduced 40 to 50% in all time periods. Methylene blue, a soluble guanylate cyclase inhibitor (15 micromol/kg i.v.) restored the reactivity to phenylephrine in animals injected with lipopolysaccharide 2 and 24 h earlier. However, it failed to do so in animals injected with lipopolysaccharide 8 h earlier. Incubation with sodium nitroprusside (SNP) increased lung and aorta cGMP levels in control animals and in tissues of rats treated with lipopolysaccharide 24 h earlier. However, SNP failed to increase tissue cGMP in rats injected 8 h earlier. Lipopolysaccharide reduced the vasodilatory response to NO donors 8 h after injection. This effect and the decreased lung cGMP accumulation in response to SNP were reversed by an NO synthase blocker. Guanylate cyclase protein levels were lower than controls in lungs harvested from rats injected 8 h earlier and were back to normal values in lungs of rats injected 24 h earlier with lipopolysaccharide. Thus, data indicate that there is a temporal window of 8 h after lipopolysaccharide injection in which soluble guanylate cyclase is not functional and that this loss of function is NO-dependent. Thus, the putative use of soluble guanylate cyclase inhibitors in the treatment of endotoxemia may be beneficial mainly at early stages of this condition.

Evidence for a role for the Dictyostelium Rap1 in cell viability and the response to osmotic stress.

The Dictyostelium genome contains a single rapA gene, which encodes a Rap1 monomeric G protein. As attempts at generating rapA-null Dictyostelium cells had been unsuccessful, expression of antisense RNA from the rapA gene under control of the folate repressible discoidin promoter was used to reduce cellular levels of the Rap1 protein. As Rap1 levels gradually decreased following antisense rapA RNA induction, growth rate and cell viability also decreased, a result consistent with the idea that rapA is an essential gene. The Rap1-depleted cells exhibited reduced viability in response to osmotic shock. The accumulation of cGMP in response to 0.4 M sorbitol was reduced after rapA antisense RNA induction and was enhanced in cells expressing the constitutively activated Rap1(G12V) protein, suggesting a role for Rap1 in the generation of cGMP. Dictyostelium Rap1 formed a complex with the Ras-binding domain of RalGDS only when it was in a GTP-bound state. This assay was used to demonstrate that activation of Rap1 in response to 0.4 M sorbitol occurred with initial kinetics similar to those observed for the accumulation of cGMP. Furthermore, the addition of 2 mM EDTA to osmotically shocked cells, a treatment that enhances cGMP accumulation, also enhanced Rap1 activation. These results suggest a direct role for Rap1 in the activation of guanylyl cyclase during the response to hyperosmotic conditions. Rap1 was also activated in response to low temperature but not in response to low osmolarity or high temperature.

The shaping of nitric oxide signals by a cellular sink.

1. The functioning of nitric oxide (NO) as a biological messenger necessitates that there be an inactivation mechanism. Cell suspensions from a rat brain region rich in the NO signalling pathway (cerebellum) were used to investigate the existence of such a mechanism and to determine its properties. 2. The cells consumed NO in a manner that could not be explained by reaction with O(2), superoxide ions or contaminating red blood cells. Functionally, the mechanism was able to convert constant rates of NO formation into low steady-state NO concentrations. For example, with NO produced at 90 nM min(-1), the cells (20 x 10(6) ml(-1)) held NO at 20 nM. Various other cell types behaved similarly. 3. The influence of NO inactivation on the ability of NO to access its receptor, soluble guanylyl cyclase, was explored by measuring cGMP accumulation in response to the clamped NO concentrations. The extrapolated steady-state EC(50) for NO was 2 nM, a concentration readily achieved by low NO release rates, despite inactivation. 4. When confronted by higher NO release rates for several minutes, the clamping mechanism failed, resulting in a progressive rise in NO concentration. While the clamp was maintained, cellular respiration was unaffected but, as it failed, respiration became inhibited by NO. The IC(50) was measured to be 120 nM (at 100-140 microM O(2)). 5. It is concluded that cerebellar (and other) cells possess a powerful NO inactivation mechanism that, extrapolated to the whole tissue, would impose on NO a half-life of around 100 ms. This and other properties of the device appear ideal for shaping low-level NO signals for activating its receptor, soluble guanylyl cyclase, whilst avoiding adverse effects on mitochondrial function. The exhaustibility of the mechanism provides a scenario for NO to become toxic.

Relative significance of the nitric oxide (NO)/cGMP pathway and K+ channel activation in endothelium-dependent vasodilation in the femoral artery of developing piglets.

Mechanisms mediating endothelium-dependent vasodilation were investigated in femoral artery rings from <2-day-old (newborn) and 2-week-old piglets. Based on previous results we hypothesized an age difference in the relative contribution of nitric oxide(NO)-cyclic 3',5'-guanosine monophosphate (cGMP) and K+ channel-activation to acetylcholine (ACh)-induced vasodilation. Changes in vascular tone were studied in organ baths in the absence or presence of NO synthase(NOS) inhibition or K+ channel blockade and the intra-arterial accumulation of cGMP in response to ACh was measured with radioimmunoassay (RIA). In control experiments, relaxant responses to ACh were equal in the two age groups. In the presence of the NOS-inhibitors N G-monomethyl-L-arginine acetate (L-NMMA; 100 microM) or NG-nitro-L-arginine (L-NOARG; 1-100 microM), however, relaxation was significantly more reduced in femoral artery rings from 2-week-old than from newborn, with lower pD2 values in the older age group. Inhibition of large (BKCa) conductance calcium-sensitive K+ channels with tetraethylammonium chloride (TEA; 1 mM), gave a significant rightward shift in the concentration-response curves to ACh which was of the same magnitude in both age groups. The ACh-induced vasodilation was abolished in both age groups by high K+ (20 mM) in combination with L-NOARG (100 microM). The relative increase in cGMP levels after addition of ACh (10 nM) was significantly larger in rings from newborn compared with 2-week-old piglets (12- vs. four-fold). In summary, sensitivity to NOS inhibition increased with age while the effect of K+ channel blockade with TEA was the same in femoral artery rings from newborn to 2-week-old piglets. Lower sensitivity to NOS inhibition and a larger increase in cGMP in response to ACh could indicate a higher efficacy of the NO/cGMP pathway in this vessel in the newborn piglet.

Desensitization of soluble guanylate cyclase in renal cortex during endotoxemia in mice.

Acute endotoxemic renal failure involves renal vasoconstriction, which presumably occurs despite increased nitric oxide (NO) generation by inducible NO synthase in the kidney. The present study examined the hypothesis that the renal vasoconstriction during endotoxemia occurs in part because of desensitization of soluble guanylate cyclase (sGC). Endotoxic shock was induced in male B6/129F2/J mice by an intraperitoneal injection of Escherichia coli lipopolysaccharide. The endotoxemia resulted in shock and renal failure as evidenced by a decrease in mean arterial pressure and an increase in serum creatinine and urea nitrogen. Serum NO increased in a time-dependent manner, reaching the highest levels at 24 h, in parallel with induction of inducible NO synthase protein in the renal cortex. In renal cortical slices obtained from endotoxemic mice, cyclic guanosine monophosphate (cGMP) increased significantly at 6 h and 15 h as compared with control but normalized at 24 h after injection of lipopolysaccharide. Incubation of renal cortical slices in the presence of a phosphodiesterase inhibitor isobutylmethylxantine did not alter the pattern of changes in cGMP. Incubation of renal cortical slices with 2 mM sodium nitroprusside resulted in a similar accumulation of cGMP in slices taken from control and endotoxemic mice at 6 h and 15 h. However, in slices from 24-h endotoxemic mice, accumulation of cGMP in response to sodium nitroprusside was significantly lower. This lower stimulability of sGC was not paralleled by a decrease in its abundance in renal cortex on immunoblot. Taken together, these results demonstrate a desensitization of sGC in renal cortex during endotoxemia, which may contribute to the associated renal vasoconstriction.

Related Gα subunits play opposing roles during Dictyostelium development

Of the several known Dictyostelium G protein subunits, the Gα4 and Gα5 subunits are the most closely related pair based on phylogenetic analysis and expression patterns, but these subunits perform different roles during development. To investigate potential relationships between these subunits with respect to cell differentiation, chimeric organisms composed of strains lacking or overexpressing either subunit were created and examined for developmental morphogenesis and spore production. Chimeras of gα4 null and gα5 null strains or Gα4 and Gα5 overexpression strains displayed compensatory morphogenesis, implying that the subunits promote complementary developmental processes. However, chimeras composed of gα4 null and Gα5 overexpression strains or gα5 null and Gα4 overexpression strains displayed distorted tip morphogenesis, suggesting the strains of these chimeras share common developmental deficiencies. Cells lacking the Gα5 subunit localized to the prespore region of chimeras similar to the pattern observed for cells overexpressing the Gα4 subunit, and cells overexpressing the Gα5 subunit displayed localization patterns similar to gα4 null mutants. A strain overexpressing both subunits displayed a partial suppression of morphology, gene expression, and cell localization phenotypes associated with the overexpression of the individual Gα subunit genes, suggesting that each Gα subunits can inhibit signaling mediated by the other subunit. Overexpression of the Gα5 subunit inhibited chemotaxis and cGMP accumulation in response to folic acid, indicating that the Gα5 subunit can inhibit early steps in the Gα4-mediated signal transduction pathway. The contrasting phenotypes of the Gα mutants suggest the Gα4 and Gα5 subunits provide opposing functions in cell differentiation, localization, and chemotactic responses to folic acid.

Nitric Oxide Activation of p38 Mitogen-activated Protein Kinase in 293T Fibroblasts Requires cGMP-dependent Protein Kinase

An increase in cellular levels of cyclic nucleotides activates serine/threonine-dependent kinases that lead to diverse physiological effects. Recently we reported the activation of the p38 mitogen-activated protein kinase (MAPK) pathway in neutrophils by a cGMP-dependent mechanism. In this study we demonstrated that exogenously supplied nitric oxide leads to activation of p38 MAPK in 293T fibroblasts. Phosphorylation of p38 corresponded with an increase in ATF-2-dependent gene expression. The effect of nitric oxide was mimicked by addition of 8-bromo-cGMP, indicating that activation of soluble guanylyl cyclase was involved. The importance of cGMP-dependent protein kinase in the activation of p38 MAPK by nitric oxide in 293T cells was assessed in a transfection based assay. Overexpression of cGMP-dependent protein kinase-1alpha caused phosphorylation of p38 in these cells and potentiated the effectiveness of cGMP. Overexpression of a catalytically inactive mutant form of this enzyme (T516A) blocked the ability of both nitric oxide and 8-bromo-cGMP to activate p38 as measured by both p38 phosphorylation and ATF-2 driven gene expression. Together, these data demonstrate that nitric oxide stimulates a novel pathway leading to activation of p38 MAPK that requires activation of cGMP-dependent protein kinase.

Effects of L-arginine on cyclosporin-induced alterations of vascular NO/cGMP generation.

Cyclosporin (CsA)-induced vascular dysfunction has been attributed to a diminished role of the nitric oxide (NO)/cGMP-mediated vasodilator mechanism. The present study was aimed at investigating whether L-arginine, the substrate of NO synthesis, ameliorates CsA-induced vascular dysfunction. Male Sprague-Dawley rats were used throughout the study. The thoracic aorta was isolated from normal rats and acutely treated with CsA (10(-4) mol/l, 60 min) in vitro, or the aorta was taken from rats treated with CsA (25 mg/kg/day, i.m., 1 week). The vascular relaxation response to acetylcholine, and tissue levels of NO metabolites and cGMP were determined. The vascular expression of NO synthase (NOS) isoforms was also determined by western blot analysis. Acute treatment with CsA in vitro markedly attenuated the vasorelaxation response to acetylcholine, which was completely restored by L-arginine. The vascular accumulation of NO metabolites in response to acetylcholine was decreased significantly by CsA, which was prevented by cotreatment with L-arginine. CsA decreased the cGMP accumulation in response to both acetylcholine and sodium nitroprusside. L-Arginine restored, although not completely, acetylcholine-stimulated cGMP generation, whereas it did not affect sodium nitroprusside-stimulated cGMP generation. Following chronic CsA treatment in the whole animal, the vasorelaxation response to acetylcholine was decreased significantly along with tissue levels of NO metabolites; this was preserved by L-arginine-supplementation. Vascular expression of iNOS protein was decreased by CsA treatment along with decreased tissue accumulation of NO metabolites. L-Arginine supplementation did not modify the altered expression of NOS proteins. These results suggest that CsA causes an L-arginine-sensitive vascular dysfunction which is associated with impaired generation of NO and cGMP.

Chronic hypoxia inhibits postnatal maturation of porcine intrapulmonary artery relaxation.

Neonatal pulmonary hypertension is associated with increased pulmonary vascular reactivity. We studied the responses of isolated porcine intrapulmonary arteries after exposure of piglets to chronic hypobaric hypoxia (CHH) from 0 to 2.5, 3 to 6, or 14 to 17 days of age. CHH inhibited the postnatal development of endothelium-dependent vasorelaxation to acetylcholine (ACh) and the calcium ionophore A-23187. Basal accumulation of guanosine 3', 5'-cyclic monophosphate (cGMP) was unaffected, but cGMP response to ACh was inhibited. Endothelium-independent relaxation to nitric oxide (NO) and zaprinast (a phosphodiesterase inhibitor) was also inhibited, but cGMP accumulation in response to these agonists was normal. The ability of sodium nitroprusside (SNP) to cause vasorelaxation and increase cGMP accumulation was unaffected. Contractile responses to potassium chloride and prostaglandin F2 alpha (PGF2 alpha) were similar to normal after exposure from birth and 3 days and were decreased in the older group, but the ability of NG-monomethyl-L-arginine acetate to increase PGF2 alpha-induced contractions decreased. Thus exposure of newborn piglets to CHH causes 1) no increase in contractile responses and 2) impairment of endothelium-dependent and -independent relaxation by impairing signal transduction mechanisms involved in the release of NO and the effectiveness of cGMP.

CGMP accumulation and gene expression of soluble guanylate cyclase in human vascular tissue.

Gene expression of soluble guanylate cyclase (sGC) and cGMP accumulation in response to sodium nitroprusside (SNP) were studied in cultured human vascular cells and freshly harvested vascular tissue. As revealed by reverse transcriptase-polymerase chain reaction, cultured smooth muscle and endothelial cells, as well as freshly isolated human vascular tissue, express mRNA for the alpha 3 and beta 3 subunits but not for the alpha 2 and beta 3 subunits is evident even in the absence of increased cGMP accumulation in response to SNP. cGMP accumulation in human cells cultured from different vascular beds typically increased two- to five-fold (maximum of 11.4-fold) over baseline following stimulation with 100 microM SNP. Bovine, murine, canine, and avian vascular smooth muscle cells accumulated similar or lower amounts of cGMP than human cells, whereas porcine, rat, and rabbit smooth muscle cells accumulated greater amounts of cGMP. In freshly harvested human vessels, cGMP accumulation in response to SNP was found to increase fifteen-fold over baseline. In contrast to the SNP-induced cGMP accumulation, cGMP levels in response to particulate guanylate cyclase activator atriopeptin II were equal to or greater in cultured human cells than in fresh human vascular tissue. We conclude that human vascular cells (fresh and cultured) express the mRNA for both a large (alpha 3) and a small (beta 3) sGC subunit and that fresh human cells are more sensitive to SNP stimulation.

Effects of C‐Type Natriuretic Peptide on Rat Astrocytes: Regional Differences and Characterization of Receptors

We have compared the effects of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) on the accumulation of cyclic GMP (cGMP) in secondary cultures of rat astrocytes. The order of potency of these peptides was CNP > ANP > BNP, which would be compatible with a predominance of guanylate cyclase B (GC-B)- versus guanylate cyclase A (GC-A)-type receptors in these cells. Accordingly, we found by northern blot analysis that the mRNA transcripts of GC-B were much more abundant in astrocytes than the transcripts of GC-A. In addition, astrocytes from diencephalon accumulated two times more cGMP in response to CNP than astrocytes from cortex. Binding experiments with 125I-labeled ANP or [Tyro]-CNP established that these ligands recognized only clearance-type receptors on astrocytes. However, the number of binding sites was approximately 100 times higher in astrocytes from cortex than in astrocytes from diencephalon and thus was inversely correlated to the amplitude of the cGMP response in the same cells. We found no further evidence for differences in the levels of GC-B receptors in astrocytes from the two regions because (a) the abundance of GC-B mRNA was similar and (b) there was no difference in particulate guanylate cyclase activity in astrocytes from each region. In addition, occupancy of clearance receptors with C-ANP4-23 did not affect the accumulation of cGMP in response to CNP; this makes it unlikely that the differences in cGMP responsiveness can be accounted for by binding and sequestration of CNP to the clearance receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Tolerance to Sodium Nitroprusside

Tolerance to nitrovasodilators, including sodium nitroprusside (SNP), is a clinical problem. SNP causes vasodilatation by releasing nitric oxide, which stimulates intracellular guanosine 3':5'-cyclic monophosphate (cGMP) accumulation in smooth muscle cells. This study examined tolerance to SNP in coronary smooth muscle cells by measuring intracellular cGMP formation. Smooth muscle cells were isolated from pig coronary vessels. Intracellular cGMP formation in response to SNP, nitroglycerine, and atrial natriuretic peptide (ANP) was quantitated by radioimmunoassay before and after chronic pretreatment. The detection of tolerance of cells rechallenged with SNP required exposure to a minimum concentration of 1 microM for SNP for at least 60 min. In untreated cells, acutely applied 100 microM SNP increased the cGMP concentration to 300 +/- 8.6 pmol/mg protein (mean +/- SEM). The cGMP concentration in cells pretreated with 1 microM SNP for 60 min increased to 213 +/- 11 pmol/mg in response to 100 microM SNP (P < 0.01, compared to untreated cells). The pretreatment of cells for 60 min with increasing concentrations of SNP caused a sequential reduction in the acute concentration-response curves of cGMP to SNP. After a 6 h washout period, cGMP accumulation in response to a rechallenge with SNP returned to control levels. The acute concentration-response curve of cGMP to SNP in cells pretreated with 100 microM nitroglycerine for 60 min was reduced significantly. Pretreatment with 100 microM SNP for 60 min did not change the response of cAMP accumulation to isoproterenol or forskolin. However, the response of cGMP formation to ANP was potentiated after pretreatment. In addition, elevation of basal cAMP or cGMP concentrations, induced with isoproterenol or ANP, respectively, did not alter the response of cGMP formation to SNP. The study yielded the following findings: (1) reversible tolerance to SNP was induced in cultured vascular smooth muscle cells; (2) chronic exposure to nitroglycerine induced cross-tolerance to SNP; (3) cells that were tolerant to SNP showed a potentiated response to ANP.

Electrical field stimulation causes endothelium-dependent and nitric oxide-mediated relaxation of pulmonary artery.

The objective of this study was to ascertain the mechanism by which electrical field stimulation (EFS) of bovine pulmonary arterial rings causes endothelium-dependent smooth muscle relaxation. Like acetylcholine-elicited relaxation, EFS-elicited relaxation was endothelium-dependent and accompanied by accumulation of guanosine 3',5'-cyclic monophosphate (cGMP) in the vascular smooth muscle. Relaxation in response to EFS was unaltered by tetrodotoxin, guanethidine, atropine, propranolol, chlorpheniramine, cimetidine, indomethacin, aminophylline, alpha, beta-methylene ATP, nifedipine, capsaicin, and certain antioxidants and free radical scavengers. Thus the relaxation was not neurogenically mediated and was not attributed to free radical formation during EFS. Like nitric oxide-elicited relaxation, EFS-elicited relaxation was antagonized by oxyhemoglobin and methylene blue. Relaxation was also antagonized by the three NG-substituted L-arginine analogues: NG-methyl-L-arginine, NG-nitro-L-arginine, and NG-amino-L-arginine. NG-amino-L-arginine also inhibited the tissue cGMP accumulation in response to EFS. The inhibitory effect of the NG-substituted L-arginine analogues was reversed by addition of excess L-arginine but not D-arginine. Relaxation in response to EFS was dependent on the presence of extracellular calcium and intracellular calmodulin, as removal of extracellular calcium or addition of trifluoperazine nearly abolished relaxation. EFS-elicited relaxation was inhibited also by tetraethylammonium chloride and elevated extracellular potassium concentration. These observations indicate that EFS-elicited relaxation of bovine pulmonary artery is mediated by neuronally independent, but endothelium- and calcium-dependent, stimulation of nitric oxide and cGMP formation.