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.     

Effect of ischemia on soluble and particulate guanylyl cyclase-mediated cGMP synthesis in cardiomyocytes

The effect of simulated ischemia [hypoxia, no glucose, extracellular pH (pH(o)) 6.4] on cGMP synthesis induced by stimulation of soluble (sGC) or particulate guanylyl cyclase (pGC) was investigated in adult rat cardiomyocytes. Intracellular cGMP content was measured after stimulation of sGC by S-nitroso-N-penicillamine (SNAP) or stimulation of pGC by natriuretic peptides [urodilatin (Uro), atrial natriuretic peptide (ANP), or C-type natriuretic peptide (CNP)] for 1 min in the presence of phosphodiesterase inhibitors. After 2 h of simulated ischemia, a decrease of >50% was observed in pGC-dependent cGMP synthesis, but no significant change was observed in sGC-dependent cGMP synthesis. The reduction in cGMP synthesis caused by simulated ischemia was mimicked by extracellular acidosis (pH(o) 6.4), which decreased pGC-mediated cGMP synthesis without altering sGC-mediated cGMP synthesis. An extreme sensitivity of pGC activity to low pH was also observed in membrane cell fractions. Hypoxia without acidosis (pH(o) 7.4) profoundly depressed cellular ATP content but did not change the response to SNAP, Uro, or ANP (selective agonists of pGC type A receptor). Only cGMP synthesis in response to CNP (a selective agonist of pGC type B receptor) was significantly reduced by ATP depletion. These data support the relevance of intracellular pH as a modulator of cGMP and suggest that, in ischemic cardiomyocytes, synthesis of cGMP would be mainly nitric oxide dependent.     

Differential effects of soluble and particulate guanylyl cyclase on Ca(2+) sensitivity in airway smooth muscle.

Maximal relaxation of airway smooth muscle (ASM) in response to atrial natriuretic peptide (ANP), which stimulates particulate guanylyl cyclase (pGC), is less than that produced by nitric oxide (NO) and other compounds that stimulate soluble guanylyl cyclase (sGC). We hypothesized that stimulation of pGC relaxes ASM only by decreasing intracellular Ca(2+) concentration ([Ca(2+)](i)), whereas stimulation of sGC decreases both [Ca(2+)](i) and the force developed for a given [Ca(2+)](i) (i.e., the Ca(2+) sensitivity) during muscarinic stimulation. We measured the relationship between force and [Ca(2+)](i) (using fura 2) under control conditions (using diltiazem to change [Ca(2+)](i)) and during exposure to ANP, diethylamine-NO (DEA-NO), sodium nitroprusside (SNP), and the Sp diastereoisomer of beta-phenyl-1,N(2)-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothionate (Sp-8-Br-PET-cGMPS), a cell-permeant analog of cGMP. Addition of DEA-NO, SNP, or Sp-8-Br-PET-cGMPS decreased both [Ca(2+)](i) and force, causing a significant rightward shift of the force-[Ca(2+)](i) relationship. In contrast, with ANP exposure, the force-[Ca(2+)](i) relationship was identical to control, such that ANP produced relaxation solely by decreasing [Ca(2+)](i). Thus, during muscarinic stimulation, stimulation of pGC relaxes ASM exclusively by decreasing [Ca(2+)](i), whereas stimulation of sGC decreases both [Ca(2+)](i) and Ca(2+) sensitivity.     

Co-operation between particulate and soluble guanylyl cyclase systems in the rat renal glomeruli.

ANP and NO act via different receptors, although inducing the common intracellular messenger - cyclic GMP. However, interaction between both factors remains unclear. Our observations suggested that in the rat kidney glomeruli, activities of the ANP- and NO-dependent guanylyl cyclase systems may be mutually compensated. To check this, we have tested effects of ANP and sodium nitroprusside (SNP) on cGMP synthesis and relaxation of glomeruli contracted with angiotensin II. The glomeruli were isolated from Wistar rats receiving saline (Control), dexamethasone (DEX), deoxycorticosterone (DOCA) or N-c-nitro-L-arginine methyl ester (NAME) for 1 or 2 days. In the DEX glomeruli exposed to 100 microM SNP, rate of cGMP synthesis was significantly higher then in the Control (26.3 vs 16.0 pmol/mg.prot./2 min., P<0.05), while 1 microM ANP was markedly less effective (2.8 vs 16.7 pmol/mg.prot./2 min in Control, P<0.01). On the contrary, in NAME group 1 microM ANP stimulated cGMP synthesis up to 35.6 pmol/mg.prot./2 min whereas efficacy of SNP was slightly suppressed. High correlation coefficient (r = 0.979, p<0.01) indicates interrelationship between NO- and ANP-dependent cGMP synthesis. Ability of the glomeruli to relax in response to ANP or SNP was in accord to their ability to cGMP generation. This was confirmed by high correlation (r = 0.845, p<0.001) between degree of relaxation and rate of cGMP synthesis. Our results support strongly the hypothesis that both, ANP and NO dependent systems co-operate in regulation of the function of kidney glomeruli.     

Nitric oxide regulates the levels of cGMP accumulation in the cricket brain

Cricket brains were incubated in a saline containing nitric oxide (NO)-donor and phosphodiesterase inhibitor IBMX, which could activate soluble guanylate cyclase (sGC) to increase cGMP levels in the targets of NO. The increase of cGMP was detected by immunohistochemistry and enzyme linked immunosorbent assay. NO-induced cGMP immunohistochemistry revealed that many cell bodies of cricket brain showed cGMP immunoreactivity when preparations were treated with a saline containing 10 mM NO-donor SNP and phosphodiesterase inhibitor IBMX, but only a few cell bodies showed immunoreactivity when preparations were incubated without NO-donor. The concentration of cGMP in cricket brains were then measured by using cGMP-specific enzyme linked immunosorbent assay. Cricket brains were treated with a saline containing 1 microM of NO-donor NOR3 and 1 mM IBMX. The cGMP levels in the brain were increased about 75% compared to control preparations that was treated with a cricket saline containing IBMX. The level of cGMP decreased about 40% when preparations were incubated NOR3 saline containing sGC inhibitor ODQ. These results indicate that NO activates sGC and increases the levels of cGMP in particular neurons of the cricket brain and that the level of cGMP would be kept a particular level, which might regulate synaptic efficacy in the neurotransmission.     

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.     

Stimulation of cyclic GMP synthesis in human cultured glomerular cells by atrial natriuretic peptide

Recently a stimulatory effect of atrial natriuretic peptide (ANP) on the particulate guanylate cyclase system has been reported in the glomeruli from different species. Using cultures of homogeneous human glomerular cell lines, we found that rat and human ANP stimulated markedly cGMP formation in epithelial cells with a threshold dose of 1 nM. A 20-fold increase was obtained at 5 microM. Stimulation was also present but less substantial (2-fold at 5 microM) in mesangial cells. cGMP was formed rapidly and released in the medium. ANP and sodium nitroprusside, an activator of soluble guanylate cyclase, had additive effects on cGMP formation. ANP did not inhibit cAMP formation in both cell lines. These results demonstrate that, at least in the human species, epithelial cells represent the main target of ANP in the glomerulus. Synthesis of cGMP in the glomerular epithelial cells in response to ANP also suggests that the excess of urinary cGMP produced by the kidney which is observed after ANP administration is of glomerular rather than of tubular origin.     

Guanylyl cyclase mediates ANP-induced vasoconstriction of murine splenic vessels

We have previously shown that ANP causes differential constriction of the splenic vasculature of the rat (veins greater than arteries), which may be inhibited by blocking the production of cGMP with A7195. In this paper, we report experiments done on vessels derived from guanylyl cyclase (GC)-A knockout mice. Small splenic arteries ( approximately 150-microm diameter) and veins ( approximately 250-microm diameter) were dissected from male GC-A-deficient 129sv mice or age-matched wild-type controls and mounted in a wire myograph. In the wild-type mice, ANP exhibited higher potency in the veins than in the arteries (EC(50) values wild-type mice: artery, 8 +/- 3 x 10(-9) M, n = 5 vs. vein, 6 +/- 4 x 10(-10) M, n = 5; P < 0.05). The concentration-response curve for ANP-induced vasoconstriction was also shifted leftward in denuded compared with intact arteries (EC(50) values: denuded artery: 5 +/- 3 x 10(-10) M, n = 5 vs. intact artery, 8 +/- 3 x 10(-9) M, n = 5; P < 0.05), i.e., the denuded vessels were more reactive. By contrast, ANP caused no significant change in tension from baseline in intact splenic arteries, intact splenic veins, or denuded splenic arteries derived from the GC-A-deficient mice, although these vessels did show normal concentration-dependent increases in tension to phenylephrine. We conclude that ANP causes vasoconstriction in the splenic vasculature by an endothelium-independent mechanism, mediated via guanylyl cyclase.     

Effects of atrial natriuretic factor, nitroprusside and acetylcholine on cGMP immunostaining in the isolated perfused rat kidney.

In the present study the localization of the cGMP production in response to the vasodilators acetylcholine (ACh) and sodium nitroprusside (SNP) and to atrial natriuretic factor (ANF) was studied in the isolated perfused rat kidney using cGMP immunocytochemistry. After ACh (0.3 microM) infusion increased cGMP immunoreactivity was found in kidney interlobar and segmental arteries and in glomeruli. SNP (1 microM) and ANF (0.01 microM) elevated cGMP staining in the same elements of the kidney as ACh. In the glomeruli ACh and SNP stimulated cGMP production in mesangial cells whereas ANF stimulated cGMP production in mesangial cells whereas ANF stimulated cGMP production in epithelial cells (podocytes). However, SNP at higher doses (10 microM) stimulated cGMP production not only in glomeruli, but also in interstitial cells throughout the cortex. In addition SNP and ANF increased cGMP production in the medulla.     

Resveratrol inhibits MAPK activity and nuclear translocation in coronary artery smooth muscle: reversal of endothelin-1 stimulatory effects.

In porcine coronary arteries, short-term treatment with resveratrol (RSVL) substantially inhibited MAPK activity (IC50 = 37 microM); and immunoblot analyses revealed consistent reduction in the phosphorylation of ERK-1/-2, JNK-1 and p38, at active sites. Endothelin-1 (ET-1), a primary antecedent in coronary heart diseases, enhanced MAPK activity, phosphorylation and nuclear translocation in a concentration-responsive but RSVL-sensitive manner. RSVL had no effect on basal or forskolin-stimulated cAMP levels, a known downregulator of the MAPK cascade. Likewise, inhibition of MAPK by RSVL was not reversed by the estrogen receptor blockers tamoxifen and ICI-182,780. Conversely, RSVL remarkably attenuated basal and ET-1-evoked protein tyrosine phosphorylation. Because MAPKs promote smooth muscle proliferation and contraction, their current inhibition may contribute to the putative protection by RSVL against coronary heart diseases. These effects apparently do not involve interaction with estrogen receptors.     

Metabolism of cyclic GMP in peritoneal macrophages of rat and guinea pig

The aim of our studies was to establish which enzymes constitute the "cGMP pathway" in rat and guinea pig peritoneal macrophages (PM). We found that in guinea pig PM synthesis of the nucleotide was significantly enhanced in response to activators of soluble guanylyl cyclase (sGC) and it was only slightly stimulated by specific activators of particulate guanylyl cyclases (pGC). In contrast, rat PM responded strongly to atrial natriuretic peptide (ANP), the activator of pGC type A. The rat cells synthesized about three-fold more cGMP than an equal number of the guinea pig cells. The activity of phosphodiesterases (PDE) hydrolyzing cGMP was apparently regulated by cGMP itself in PM of both species and again it was higher in the rat cells than in those isolated from guinea pig. However, guinea pig PM revealed an activity of Ca(2+)/calmodulin-dependent PDE1, which was absent in the rat cells. Using Western blotting analysis we were unable to detect the presence of cGMP-dependent protein kinase 1 (PKG1) in PM isolated from either species. In summary, our findings indicate that particulate GC-A is the main active form of GC in the rat PM, while in guinea pig macrophages the sGC activity dominates. Since the profiles of the PDE activities in rat and guinea pig PM are also different, we conclude that the mechanisms regulating cGMP metabolism in PM are species-specific. Moreover, our results suggest that targets for cGMP other than PKG1 should be present in PM of both species.     

Reciprocal regulation of cGMP-mediated vasorelaxation by soluble and particulate guanylate cyclases

Nitric oxide (NO) and atrial natriuretic peptides (ANP) activate soluble (sGC) and particulate guanylate cyclase (pGC), respectively, and play important roles in the maintenance of cardiovascular homeostasis. However, little is known about potential interactions between these two cGMP-generating pathways. Here we demonstrate that sGC and pGC cooperatively regulate cGMP-mediated relaxation in human and murine vascular tissue. In human vessels, the potency of spermine-NONOate (SPER-NO) and ANP was increased after inhibition of endogenous NO synthesis and decreased by prior exposure to glyceryl trinitrate (GTN). Aortas from endothelial NO synthase (eNOS) knockout (KO) mice were more sensitive to ANP than tissues from wild-type (WT) animals. However, in aortas from WT mice, the potency of ANP was increased after pretreatment with NOS or sGC inhibitor. Vessels from eNOS KO animals were less sensitive to ANP after GTN pretreatment, an effect that was reversed in the presence of an sGC inhibitor. cGMP production in response to SPER-NO and ANP was significantly greater in vessels from eNOS KO animals compared with WT animals. This cooperative interaction between NO and ANP may have important implications for human pathophysiologies involving deficiency in either mediator and the clinical use of nitrovasodilators.     

Generation and characterization of a stable soluble guanylate cyclase-overexpressing CHO cell line.

A stably transfected soluble guanylate cyclase (sGC, alpha1 and beta1 subunits of the rat lung enzyme)-overexpressing CHO cell line was generated for the characterization of different types of activators of the soluble guanylate cyclase. Polyclonal antibodies directed against both subunits of the rat enzyme were used to detect both subunits in the cytosol of the transfected CHO cells. We studied the effects of different nitric oxide (NO) donors like SNP and DEA/NO and, in particular, the direct, NO-independent stimulator of the soluble guanylate cyclase 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1), on intracellular guanosine 3',5'-cyclic monophosphate (cGMP) production. DEA/NO (0.01-3 microM), SNP (1-10 microM), and YC-1 (1-10 microM) induced a concentration-dependent intracellular cGMP increase with maximal effects of 16-fold (3 microM DEA/NO), 8-fold (10 microM SNP), and 6-fold (10 microM YC-1) stimulation compared to controls, respectively. In addition, a synergistic effect of the combination of the NO donor and YC-1 could be observed with a maximal stimulation of 64-fold by SNP (10 microM) and YC-1 (10 microM). 1H-(1,2,4)-Oxadiazolo-(4,3-a)-6-bromo-quinoxazin-1-one (ODQ, 10 microM), a potent and selective inhibitor of sGC, inhibited both the single effects of NO donors [DEA/NO (3 microM), 77%; SNP (3 microM), 83%] and YC-1 [YC-1 (3 microM), 82%], but moreover the synergistic effects between NO donors and YC-1 [DEA/NO (3 microM) + YC-1 (3 microM), 81%; SNP (3 microM) + YC-1 (3 microM),89%] on intracellular cGMP production. In summary,we have generated a simple, sensitive, and useful bioassay method to characterize all types of sGC activators on the cellular level without the need of primary cell culture, several transfections, or purifying enzyme from biological materials.     

Effects of manipulating the nitric oxide/cyclic GMP pathway on bovine oocyte meiotic resumption in vitro

The objective of this study was to examine the effects of manipulating the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway on bovine oocyte nuclear maturation in vitro. Cumulus-enclosed oocytes (CEO) were recovered from abattoir-derived ovaries and cultured in M199+FCS for 7 or 21h in the presence of various molecules affecting the NO/cGMP pathway, and then fixed and stained for evaluation of the stage of nuclear maturation. Cyclic GMP levels were also measured in cumulus-oocyte complexes after 3 and 6 h of culture. The iNOS inhibitor, aminoguanidine (AG, 10 and 50 mM) and the NO donor sodium nitroprusside (SNP, 100 and 500 microM) significantly inhibited GVBD after 7h of culture. However, a lower concentration of SNP (0.01 microM) stimulated GVBD. The inhibitory effects of AG and SNP were reversible, indicating that they were not toxic effects. Although SNP (500 microM) increased cGMP levels in cumulus-oocyte complexes after 3 h of culture, the inhibitor of soluble guanylate cyclase ODQ and the protein kinase G (PKG) inhibitor KT5823 did not reverse the inhibitory effect of SNP on meiosis, suggesting that SNP does not inhibit meiosis through the cGMP/PKG pathway. Similarly, an analogue of cGMP (8-Bromo-cGMP 0.5, 1, 3, and 6 mM), as well as activation of guanylate cyclase with Protoporphyrin IX or atrial natriuretic peptide, or inhibition of the enzyme with ODQ, did not have any significant effect on GVBD after 7 h of culture, supporting the idea that the effects of AG and SNP were not due to altered cGMP levels. Atrial natriuretic peptide, Protoporphyrin IX and SNP 500 microM increased cGMP levels after 3 h but not 6 h of culture. In conclusion, soluble and particulate guanylate cyclases could be activated in bovine cumulus-oocyte complexes, but accumulation of cGMP was probably not responsible for the effects of NO on meiosis.     

Direct evidence for cross-activation of cGMP-dependent protein kinase by cAMP in pig coronary arteries.

Elevation of either cAMP or cGMP causes smooth muscle relaxation. Whether these effects are mediated through cAMP-dependent protein kinase (cAK), cGMP-dependent protein kinase (cGK), or both is unknown. Pig coronary arteries were treated with sodium nitroprusside (SNP) or atrial natriuretic factor (ANF), relaxants which elevate cGMP, and with isoproterenol or forskolin, relaxants which elevate cAMP. Incubation of the arteries with 10 microM SNP produced a 3.3-fold increase in cGMP without altering cAMP; the cGK activity ratio (-cGMP/+cGMP) in these extracts was increased by 2.6-fold as determined by a newly developed assay, while the cAK activity ratio (-cAMP/+cAMP) was unchanged. The increase in cGK activity ratio by SNP was concentration-dependent and was nearly maximal at 30 s. Treatment of the tissue with 10 nM ANF also increased the cGK activity ratio (2.3-fold), but not that of cAK. 100 microM isoproterenol caused a 2.9-fold elevation of cAMP with no change in cGMP, but both cAK and cGK activity ratios were increased (2.3- and 1.6-fold, respectively). The increase in the cGK activity ratio could be mimicked by cAMP addition to control tissue extracts at the concentration measured in extracts of the isoproterenol-treated tissue. Forskolin (1 and 10 microM) also increased the cGK activity ratio (1.9- and 4.9-fold). The increases in cGK activity observed in extracts suggest that moderate elevation of either cGMP or cAMP causes intracellular cGK activation, thus producing relaxation of vascular smooth muscle.     

Chronic hypoxic decreases in soluble guanylate cyclase protein and enzyme activity are age dependent in fetal and adult ovine carotid arteries.

The present study tests the hypothesis that chronic hypoxia enhances reactivity to nitric oxide (NO) through age-dependent increases in soluble guanylate cyclase (sGC) and protein kinase G (PKG) activity. In term fetal and adult ovine carotids, chronic hypoxia had no significant effect on mRNA levels for the beta1-subunit of sGC, but depressed sGC abundance by 16% in fetal and 50% in adult arteries, through possible depression of rates of mRNA translation (15% in fetal and 50% in adult) and/or increased protein turnover. Chronic hypoxia also depressed the catalytic activity of sGC, but only in fetal arteries (63%). Total sGC activity was reduced by chronic hypoxia in both fetal (69%) and adult (37%) carotid homogenates, but this effect was not observed in intact arteries when sGC activity was measured by timed accumulation of cGMP. In intact arteries treated with 300 microM 3-isobutyl-1-methylxanthine (IBMX), chronic hypoxia dramatically enhanced sGC activity in fetal (186%) but not adult (89%) arteries. This latter observation suggests that homogenization either removed an sGC activator, released an sGC inhibitor, or altered the phosphorylation state of the enzyme, resulting in reduced activity. In the absence of IBMX, chronic hypoxia had no significant effect on rates of cGMP accumulation. Chronic hypoxia also depressed the ability of the cGMP analog, 8-(p-chlorophenylthio)-cGMP, to promote vasorelaxation in both fetal (8%) and adult (12%) arteries. Together, these results emphasize the fact that intact and homogenized artery studies of sGC activity do not always yield equivalent results. The results further suggest that enhancement of reactivity to NO by chronic hypoxia must occur upstream of PKG and can only be possible if changes in cGMP occurred in functional compartments that afforded either temporal or chemical protection to the actions of phosphodiesterase. The range and age dependence of hypoxic effects observed also suggest that some responses to hypoxia must be compensatory and homeostatic, with reactivity to NO as the primary regulated variable.     

Differential effects of cGMP produced by soluble and particulate guanylyl cyclase on mouse ventricular myocytes

Particulate guanylyl cyclase (pGC) and soluble guanylyl cyclase (sGC) are cGMP-generation systems distributed in different intracellular locations. Our aim was to test the hypothesis that the functional effects of cGMP produced by pGC and sGC on contraction and Ca2+ transients would differ in ventricular myocytes. We measured myocyte shortening from adult mice using a video edge-detector and investigated the functional changes after stimulating pGC with C-type natriuretic peptide (CNP; 10(-8) M and 10(-7) M) or sGC with S-nitroso-N-acetyl-penicillamine (SNAP; nitric oxide donor; 10(-6) M and 10(-5) M). Significant concentration-dependent decreases in percentage shortening (PCS), maximal rate of shortening (RSmax), and relaxation (RRmax) were produced by CNP. To a similar degree, SNAP concentration-dependently reduced PCS, RSmax, and RRmax. The addition of Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine (cGMP-dependent protein kinase inhibitor; 5 x 10(-6) M) or erythro-9-(2-hydroxy-3-nonyl) adenine (cGMP-stimulated cAMP phosphodiesterase inhibitor; 10(-5) M) reduced the responses induced by CNP or SNAP, suggesting that their actions were through cGMP-mediated pathways. While SNAP significantly increased intracellular cGMP concentration by 57%, CNP had little effect on cGMP production. We also found that CNP markedly decreased the amplitude of Ca2+ transients while SNAP had little effect, suggesting the cGMP generated by sGC may decrease myofilament Ca2+ sensitivity. The small amount of cGMP generated by pGC had a major effect in reducing Ca2+ level. This study suggested the existence of compartmentalization for cGMP in ventricular myocytes.     

High-fat diet-induced obesity leads to increased NO sensitivity of rat coronary arterioles: role of soluble guanylate cyclase activation

The impact of obesity on nitric oxide (NO)-mediated coronary microvascular responses is poorly understood. Thus NO-mediated vasomotor responses were investigated in pressurized coronary arterioles ( approximately 100 microm) isolated from lean (on normal diet) and obese (fed with 60% of saturated fat) rats. We found that dilations to acetylcholine (ACh) were not significantly different in obese and lean rats (lean, 83 +/- 4%; and obese, 85 +/- 3% at 1 microM), yet the inhibition of NO synthesis with N(omega)-nitro-l-arginine methyl ester reduced ACh-induced dilations only in vessels of lean controls. The presence of the soluble guanylate cyclase (sGC) inhibitor oxadiazolo-quinoxaline (ODQ) elicited a similar reduction in ACh-induced dilations in the two groups of vessels (lean, 60 +/- 11%; and obese, 57 +/- 3%). Dilations to NO donors, sodium nitroprusside (SNP), and diethylenetriamine (DETA)-NONOate were enhanced in coronary arterioles of obese compared with lean control rats (lean, 63 +/- 6% and 51 +/- 5%; and obese, 78 +/- 5% and 70 +/- 5%, respectively, at 1 microM), whereas dilations to 8-bromo-cGMP were not different in the two groups. In the presence of ODQ, both SNP and DETA-NONOate-induced dilations were reduced to a similar level in lean and obese rats. Moreover, SNP-stimulated cGMP immunoreactivity in coronary arterioles and also cGMP levels in carotid arteries were enhanced in obese rats, whereas the protein expression of endothelial NOS and the sGC beta1-subunit were not different in the two groups. Collectively, these findings suggest that in coronary arterioles of obese rats, the increased activity of sGC leads to an enhanced sensitivity to NO, which may contribute to the maintenance of NO-mediated dilations and coronary perfusion in obesity.     

NO donor sodium nitroprusside inhibits excitation-contraction coupling in guinea pig taenia coli

In guinea pig taenia coli, the nitric oxide (NO) donor sodium nitroprusside (SNP, 1 microM) reduced the carbachol-stimulated increases in muscle force in parallel with a decrease in intracellular Ca(2+) concentration ([Ca(2+)](i)). A decrease in the myosin light chain phosphorylation was also observed that was closely correlated with the decrease in [Ca(2+)](i). With the patch-clamp technique, 10 microM SNP decreased the peak Ba(2+) current, and this effect was blocked by an inhibitor of soluble guanylate cyclase. Carbachol (10 microM) induced an inward current, and this effect was markedly inhibited by SNP. SNP markedly increased the depolarization-activated outward K(+) currents, and this current was completely blocked by 0.3 micorM iberiotoxin. SNP (1 microM) significantly increased cGMP content without changing cAMP content. Decreased Ca(2+) sensitivity by SNP of contractile elements was not prominent in the permeabilized taenia, which was consistent with the [Ca(2+)](i)-force relationship in the intact tissue. These results suggest that SNP inhibits myosin light chain phosphorylation and smooth muscle contraction stimulated by carbachol, mainly by decreasing [Ca(2+)](i), which resulted from the combination of the inhibition of voltage-dependent Ca(2+) channels, the inhibition of nonselective cation currents, and the activation of Ca(2+)-activated K(+) currents.