Regulation of cGMP-induced relaxation and cGMP-dependent protein kinase in rat myometrium during pregnancy

Increases inguanosine 3',5'-cyclic monophosphate (cGMP) induced bynitric oxide (NO), nitrovasodilators, and atrial peptides correlatewith relaxation of vascular smooth muscle. Relaxation of myometrialsmooth muscle by increases in cGMP, however, has required unusuallyhigh concentrations of the cyclic nucleotide. We tested the hypothesisthat the sensitivity of myometrium to relaxation by cGMP is increasedduring pregnancy. Aortic smooth muscle was more sensitive to relaxationby cGMP than myometrial tissues, and, contrary to our hypothesis,myometrium from pregnant rats was least sensitive. Although levels ofcGMP were elevated after treatment with the NO donor,S-nitroso-N-acetylpenicillamine, relaxation of myometrial tissues obtained from pregnant rats occurred only at extraordinarily high concentrations. The levels ofcGMP-dependent protein kinase (PKG) were significantly decreased inmyometrium from pregnant rats compared with myometrium from nonpregnantcycling animals or aortic smooth muscle. Administration of estradiol to ovariectomized rats increased myometrial PKG expression, andprogesterone antagonized this response. We conclude that1) myometrial tissues from pregnantrats are not sensitive to relaxation by cGMP and 2) this insensitivity to cGMP isaccompanied by progesterone-mediated decreases in the level of PKGexpression.

     

Role of cGMP in relaxation of vascular and other smooth muscle

The hypothesis that the relaxant action of many drugs on vascular and other smooth muscle is mediated by increases in intracellular cGMP, the "cGMP hypothesis," is gaining wide acceptance. While much information supporting this idea can be found in the literature, there is also a significant amount of information indicating that an elevation in the tissue content of cGMP is by itself insufficient to cause smooth muscle relaxation. The literature is reviewed with reference to the criteria that need to be fulfilled to consider cGMP as the second messenger mediating relaxation of smooth muscle by a drug; i.e., activation of guanylate cyclase, elevation of tissue content of cGMP, potentiation by phosphodiesterase inhibitors, antagonism by inhibitors of cGMP synthesis, and production of relaxation by cGMP analogues. For each criterion, key observations supporting the hypothesis are considered, followed by examples of important observations not consistent with the hypothesis. It is concluded that in some smooth muscles, for example, rat myometrium and vas deferens, cGMP is not a mediator of drug-induced relaxation. In other smooth muscles, including vascular smooth muscle, cGMP appears to play an important role in the relaxation process; but current evidence suggests that other factors are also important and that the cGMP hypothesis may need to be modified.     

Cyclic AMP and cyclic GMP independent stimulation of ventricular calcium current by peroxynitrite donors in guinea pig myocytes

We investigated the potential involvement of peroxynitrite (ONOO(-)) in the modulation of calcium current (I(Ca)) in guinea pig ventricular myocytes with the whole-cell patch clamp technique and with cyclic AMP (cAMP) measurements. Because of the short half-life of ONOO(-) at physiological pH, we induced an increase in its intracellular levels by using donors of the precursors, nitric oxide (NO) and superoxide anion (O(2) (-)). High concentrations of NO donors, SpermineNONOate (sp/NO, 300 microM) or SNAP (300 microM) increased basal I(Ca) (50.3 +/- 4.6%, n = 7 and 46.2 +/- 5.0%, n = 13). The superoxide anion donor Pyrogallol (100 microM) also stimulated basal I(Ca) (44.6 +/- 2.8%, n = 11). At lower concentration sp/NO (10 nM) and Pyrogallol (1 microM), although separately ineffective on I(Ca), enhanced the current if applied together (33.5 +/- 0.7%, n = 7). The simultaneous donor of O(2) (-) and NO, SIN-1 (500 microM), also stimulated basal I(Ca) (22.8 +/- 2.1%, n = 13). In the presence of saturating cyclic GMP (cGMP, 50 microM) in the patch pipette or of extracellular dibutyryl cGMP (dbcGMP, 100 microM), I(Ca) was still increased by SIN-1 (32.0 +/- 6.1%, n = 4 and 30.0 +/- 5.4%, n = 8). Both Manganese(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP, 100 microM) a ONOO(-) scavenger, and superoxide dismutase (SOD) (150 U/ml) reversed the stimulatory effect of SIN-1 on I(Ca) (respectively -0.6 +/- 4.1%, n = 4 and 3.6 +/- 4.3%, n = 4). Intracellular cAMP level was unaltered by SIN-1, while it was enhanced by blocking the NO-cGMP pathway with the NO synthase inhibitor L-NMMA. These results suggest that peroxynitrite donors increase cardiac calcium current without the involvement of cAMP and cGMP.     

The influence of methylene blue on the spontaneous contractity of the non-pregnant human myometrium and on the myometrial response to DEA/NO

Nitric oxide (NO) is a potent inhibitor of spontaneous contractions of the human non-pregnant myometrium; however, the precise mechanism by which NO causes the myometrial smooth muscles to relax remains unclear. The aim of this study was to determine the influence of methylene blue (MB) on myometrial contractions and the response of the myometrium to DEA/NO in vitro. Concentration-response curves to DEA/NO were constructed in the absence and presence of MB (5x10(-6), 10(-4) and 10(-2) mol/l) and 5x10(-3) mol/l cystamine. Cystamine did not counteract the DEA/NO-induced relaxation of the myometrial strips. MB itself, excluding the lowest concentration, caused noticeable changes in spontaneous activity. The changes involved a concentration-dependent increase in the frequency of contractions, and a decrease in their amplitude. In conclusion, our results confirm that NO relaxes the human myometrium via a cGMP-independent mechanism. The results obtained in the presence of MB may be misleading because of its complex influence on myometrial contractile activity.     

Effect of NO donors on protein phosphorylation in intact vascular and nonvascular smooth muscles

It is generally well accepted that nitrovasodilator-induced relaxation of vascular smooth muscle involves elevation of cGMP and activation of a specific cGMP-dependent protein kinase [protein kinase G (PKG)]. However, the protein targets of PKG and the underlying mechanisms by which this kinase leads to a relaxant response have not been elucidated. Several types of smooth muscle, including rat myometrium and vas deferens, are not relaxed by sodium nitroprusside, even at concentrations that produce marked elevation of cGMP and activation of PKG. The main objective of our studies was to compare PKG-mediated protein phosphorylation in intact rat aorta, rat myometrium, and rat vas deferens using two-dimensional gel electrophoresis. In intact rat aorta, seven PKG substrates were detected during relaxation of the tissue. None of the PKG substrates identified in the rat aorta appeared to be phosphorylated in the myometrium or vas deferens after administration of various cGMP-elevating agents. Thus the failure of the rat myometrium and rat vas deferens to relax in the face of cGMP elevation and PKG activation may be due to a lack of PKG substrate phosphorylation.     

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.     

8Br-cGMP mediates relaxation of tracheal smooth muscle through PKA

In this study, guinea pig tracheal smooth muscle pre-contracted with histamine was relaxed by the addition of 100microM 8Br-cGMP, a non-hydrolyzable and cell-permeable analog for cGMP. This effect was not sensitive to cGMP-dependent protein kinase (PKG) inhibitors, whereas it was partially blocked by cAMP-dependent protein kinase (PKA) inhibitors. The relaxation observed was also reverted up to 50+/-8.5% by iberiotoxin, a selective inhibitor of large conductance, calcium-activated potassium channels (BK(Ca)). Our results indicate that there exists a crosstalk mechanism between cAMP and cGMP signaling pathways which lead to relaxation of guinea pig tracheal smooth muscle and also that BK(Ca) channels are involved to a certain extent in this phenomenon.     

Hypoxia enhances a cGMP-independent nitric oxide relaxing mechanism in pulmonary arteries

Nitric oxide (NO) donors generally relax vascular preparations through cGMP-mediated mechanisms. Relaxation of endothelium-denuded bovine pulmonary arteries (BPA) and coronary arteries to the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) is almost eliminated by inhibition of soluble guanylate cyclase activation with 10 microM 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), whereas only a modest inhibition of relaxation is observed under hypoxia (PO2 = 8-10 Torr). This effect of hypoxia is independent of the contractile agent used and is also observed with NO gas. ODQ eliminated SNAP-induced increases in cGMP under hypoxia in BPA. cGMP-independent relaxation of BPA to SNAP was not attenuated by inhibition of K+ channels (10 mM tetraethylammonium), myosin light chain phosphatase (0.5 microM microcystin-LR), or adenylate cyclase (4 microM 2',5'-dideoxyadenosine). SNAP relaxed BPA contracted with serotonin under Ca2+-free conditions in the presence of hypoxia and ODQ, and contraction to Ca2+ readdition was also attenuated. The sarcoplasmic reticulum Ca2+-reuptake inhibitor cyclopiazonic acid (0.2 mM) attenuated SNAP-mediated relaxation of BPA in the presence of ODQ. Thus hypoxic conditions appear to promote a cGMP-independent relaxation of BPA to NO by enhancing sarcoplasmic reticulum Ca2+ reuptake.     

Nitric oxide induces dose-dependent CA(2+) transients and causes temporal morphological hyperpolarization in human neutrophils

We exposed adherent neutrophils to the nitric oxide (NO)-radical donors S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO), and sodium nitroprusside (SNP) to study the role of NO in morphology and Ca(2+) signaling. Parallel to video imaging of cell morphology and migration in neutrophils, changes in intracellular free Ca(2+) ([Ca(2+)](i)) were assessed by ratio imaging of Fura-2. NO induced a rapid and persistent morphological hyperpolarization followed by migrational arrest that usually lasted throughout the 10-min experiments. Addition of 0.5-800 microM SNAP caused concentration-dependent elevation of [Ca(2+)](i) with an optimal effect at 50 microM. This was probably induced by NO itself, because no change in [Ca(2+)](i) was observed after treatment with NO donor byproducts, i.e. D-penicillamine, glutathione, or potassium cyanide. Increasing doses of SNAP (>/=200 microM) attenuated the Ca(2+) response to the soluble chemotactic stimulus formyl-methionyl-leucyl-phenylalanine (fMLP), and both NO- and fMLP-induced Ca(2+) transients were abolished at 800 microM SNAP or more. In kinetic studies of fluorescently labeled actin cytoskeleton, NO markedly reduced the F-actin content and profoundly increased cell area. Immunoblotting to investigate the formation of nitrotyrosine residues in cells exposed to NO donors did not imply nitrosylation, nor could we mimic the effects of NO with the cell permeant form of cGMP, i.e., 8-Br-cGMP. Hence these processes were probably not the principal NO targets. In summary, NO donors initially increased neutrophil morphological alterations, presumably due to an increase in [Ca(2+)](i), and thereafter inhibited such shape changes. Our observations demonstrate that the effects of NO donors are important for regulation of cellular signaling, i.e., Ca(2+) homeostasis, and also affect cell migration, e.g., through effects on F-actin turnover. Our results are discussed in relation to the complex mechanisms that govern basic cell shape changes, required for migration.     

Effects of endothelin-1 and nitric oxide on proliferation of cultured guinea pig bronchial smooth muscle cells

The proliferative effects of endothelin-1 (ET-1), both alone and in combination with epidermal growth factor (EGF), and the effect of nitric oxide (NO) on the cell proliferation were investigated in cultured guinea pig bronchial smooth muscle cells. ET-1 (10-100 nM) alone augmented cell proliferation, and was additive to the effect of EGF (0.48 nM) in a concentration-dependent manner. An ET(A) antagonist, BQ-123 (10 microM), reduced the cell-proliferative effect of ET-1, whereas an ET(B) antagonist, BQ-788 (10 microM), did not influence the effect. A NO donor, SIN-1 (10 nM-1 microM), reduced the cell-proliferative effect of ET-1 in a concentration-dependent manner. The effect of SIN-1 (1 microM) was partly, but significantly, reversed by a soluble guanylyl cyclase inhibitor, ODQ (1 microM). These results suggest that ET-1 acts not only as a co-mitogen with EGF but also as a mitogen alone, and that its action is mediated through activation of ET(A) receptors. Therefore, ET-1 may contribute to airway remodeling, a pathophysiological hallmark of asthma. In addition, NO, which is produced mainly in the airway epithelium and is partly mediated through cGMP-dependent pathway, may reduce the phenomenon.     

Specificity of antioxidant enzyme inhibition in skeletal muscle to reactive nitrogen species donors

Nitric oxide (*NO) and its by-products modulate many physiological functions of skeletal muscle including blood flow, metabolism, glucose uptake, and contractile function. However, growing evidence suggests that an overproduction of nitric oxide contributes to muscle wasting in a number of pathologies including chronic heart failure, sepsis, COPD, muscular dystrophy, and extreme disuse. Limited data point to the potential of inhibition various enzymes by reactive nitrogen species (RNS), including (.)NO and its downstream products such as peroxynitrite, primarily in purified systems. We hypothesized that exposure of skeletal muscle to RNS donors would reduce or downregulate activities of the crucial antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX). Diaphragm muscle fiber bundles were extracted from 4-month-old Fischer-344 rats and, in a series of experiments, exposed to either (a) 0 (control), 1, or 5 mM diethylamine NONOate (DEANO: *NO donor); (b) 0, 100, 500 microM, or 1 mM sodium nitroprusside (SNP: *NO donor); (c) 0 or 2 mM S-nitroso-acetylpenicillamine (SNAP: *NO donor); or (d) 0 or 500 microM SIN-1 (peroxynitrite donor) for 60 min. DEANO resulted in a 50% reduction in CAT, GPX, and a dose-dependent inhibition of Cu, Zn-SOD. SNP resulted in significantly lower activities for total SOD, Mn-SOD isoform, Cu, Zn-SOD isoform, CAT, and GPX in a dose-dependent fashion. Two millimolar SNAP and 500 microM SIN-1 also resulted in a large and significant inhibition of total SOD and CAT. These data indicate that reactive nitrogen species impair antioxidant enzyme function in an RNS donor-specific and dose-dependent manner and are consistent with the hypothesis that excess RNS production contributes to skeletal muscle oxidative stress and muscle dysfunction.     

NO(+) but not NO radical relaxes airway smooth muscle via cGMP-independent release of internal Ca(2+)

We compared the effects of two redox forms of nitric oxide, NO(+) [liberated by S-nitroso-N-acetyl-penicillamine (SNAP)] and NO. [liberated by 3-morpholinosydnonimine (SIN-1) in the presence of superoxide dismutase], on cytosolic concentration of Ca(2+) ([Ca(2+)](i); single cells) and tone (intact strips) obtained from human main stem bronchi and canine trachealis. SNAP evoked a rise in [Ca(2+)](i) that was unaffected by removing external Ca(2+) but was markedly reduced by depleting the internal Ca(2+) pool using cyclopiazonic acid (10(-5) M). Dithiothreitol (1 mM) also antagonized the Ca(2+) transient as well as the accompanying relaxation. SNAP attenuated responses to 15 and 30 mM KCl but not those to 60 mM KCl, suggesting the involvement of an electromechanical coupling mechanism rather than a direct effect on the contractile apparatus or on Ca(2+) channels. SNAP relaxations were sensitive to charybdotoxin (10(-7) M) or tetraethylammonium (30 mM) but not to 4-aminopyridine (1 mM). Neither SIN-1 nor 8-bromoguanosine 3',5'-cyclic monophosphate had any significant effect on resting [Ca(2+)](i), although both of these agents were able to completely reverse tone evoked by carbachol (10(-7) M). We conclude that NO(+) causes release of internal Ca(2+) in a cGMP-independent fashion, leading to activation of Ca(2+)-dependent K(+) channels and relaxation, whereas NO. relaxes the airways through a cGMP-dependent, Ca(2+)-independent pathway.     

Phosphorylation of phospholamban correlates with relaxation of coronary artery induced by nitric oxide,adenosine, and prostacyclin in the pig

The intracellular mechanisms underlying the action of the endogenous vasodilators such as NO/EDRF, adenosine, and prostacyclin acting through cGMP and cAMP, respectively, are not well understood. One important action of cyclic nucleotides in smooth muscle relaxation is to lower the cytosolic Ca²⁺ concentration by enhanced sequestration into the sarcoplasmic reticulum. The present study was undertaken to elucidate the potential role of phosphorylation of phospholamban, the regulator of sarcoplasmic reticulum Ca²⁺ pump, for the control of coronary vascular tone by NO/EDRF, adenosine, and prostacyclin. Phospholamban was identified in pig coronary artery preparations by immunofluorescence microscopy, Western blotting and in vitro phosphorylation. Segments of pig coronary artery, with either intact or denuded endothelium, were precontracted with prostaglandin F_2α (PGF_2α). In endothelium-denuded preparations 3-morpholinosydnonimine (SIN-1), 5′-N-ethylcarboxiamidoadenosine (NECA), and iloprost (ILO) caused both relaxation and phospholamban phosphorylation with the potency: SIN-1 > NECA > ILO. The regulatory myosin light chain was significantly dephosphorylated only by SIN-1. In endothelium-intact pig coronary artery, L-NAME caused additional vasoconstriction and a decrease in phospholamban phosphorylation, while phosphorylation of myosin light chain remained unchanged. An inverse relationship between phospholamban phosphorylation and vessel tone was obtained. Our findings demonstrate significant phospholamban phosphorylation during coronary artery relaxation evoked by NO, prostacyclin, and adenosine receptor activation. Because of the close correlation between phosphorylation of phospholamban and vessel relaxation, we propose that phospholamban phosphorylation is an important mechanism by which endogenous vasodilators, especially endothelial NO/EDRF, control coronary vascular smooth muscle tone. J. Cell. Biochem. 70:49–59, 1998. © 1998 Wiley-Liss, Inc.     

YC-1 enhances the responsiveness of tolerant vascular smooth muscle to glyceryl trinitrate

A major limitation of the use of organic nitrates in cardiovascular medicine is the development of tolerance, which has been attributed, in part, to a decrease in their metabolic activation in the vascular smooth muscle cell. Recently, 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) was shown to potentiate vascular smooth muscle responsiveness to glyceryl trinitrate (GTN), sodium nitroprusside, and the nitric oxide donor NOC 18, in organic nitrate-naive vascular smooth muscle. We used GTN-tolerant rabbit aortic rings (RARs) to test the hypothesis that a non-vasorelaxant concentration of YC-1 enhances the ability of the prototypical organic nitrate GTN to relax vascular smooth muscle and elevate intravascular cGMP under conditions of GTN tolerance. Treatment with YC-1 (3 microM) produced a left shift of the GTN concentration-response curve and decreased the EC50 value for GTN-induced relaxation in both GTN-tolerant and non-tolerant RARs (P < 0.05). Intravascular cGMP elevation induced by GTN was enhanced in the presence of YC-1 in GTN-tolerant and non-tolerant RARs (P < 0.05). These observations indicate that YC-1, or similarly acting drugs, may be useful in overcoming the tolerance that develops during sustained GTN therapy, and that its mechanism may involve enhanced cGMP formation.     

cAMP modulates cGMP-mediated cerebral arteriolar relaxation in vivo

No studies have specifically addressed whether cAMP can influence nitric oxide (NO)/cGMP-induced cerebral vasodilation. In this study, we examined whether cAMP can enhance or reduce NO-induced cerebral vasodilation in vivo via interfering with cGMP efflux or through potentiating phosphodiesterase 5 (PDE5)-mediated cGMP breakdown, respectively, in cerebral vascular smooth muscle cells (CVSMCs). To that end, we evaluated, in male rats, the effects of knockdown [via antisense oligodeoxynucleotide (ODN) applications] of the cGMP efflux protein multidrug resistance protein 5 (MRP5) and PDE5 inhibition on pial arteriolar NO donor [S-nitroso-N-acetyl penicillamine (SNAP)]-induced dilations in the absence and presence of cAMP elevations via forskolin. Pial arteriolar diameter changes were measured using well-established protocols in anesthetized rats. In control (missense ODN treated) rats, forskolin elicited a leftward shift in the SNAP dose-response curves (approximately 50% reduction in SNAP EC50). However, in MRP5 knockdown rats, cAMP increases were associated with a substantial reduction in SNAP-induced vasodilations (reflected as a significant 35-50% lower maximal response). In the presence of the PDE5 inhibitor MY-5445, the repression of the NO donor response accompanying forskolin was prevented. These findings suggest that cAMP has opposing effects on NO-stimulated cGMP increases. On the one hand, cAMP limits CVSMC cGMP loss by restricting cGMP efflux. On the other, cAMP appears to enhance PDE5-mediated cGMP breakdown. However, because increased endogenous cAMP seems to potentiate NO/cGMP-induced arteriolar relaxation when MRP5 expression is normal, the effect of cAMP to reduce cGMP efflux appears to predominate over cAMP stimulation of cGMP hydrolysis.     

Cyclic nucleotide levels during carbachol-induced smooth muscle contractions.

Cyclic nucleotide levels and tension were measured at various times during carbachol-induced smooth muscle contractions. Cyclic GMP levels were markedly increased during contractions of rat vas deferens, guinea pig myometrium and guinea pig taenia coli, but were unchanged during contractions of rat uterus or guinea pig ileum. No significant changes in cyclic GMP levels could be detected in estrogen-primed rat uteri at any of the times or drug concentrations studied. Even in tissues in which large increases in cyclic GMP levels could be detected during carbachol-induced contractions (i.e. guinea pig myometrium and taenia coli) the contractions appeared to precede the cyclic GMP increases by several seconds. No significant changes in cyclic AMP levels were observed during carbachol-induced contractions in any of the smooth muscles studied. Thus, changes in tissue levels of the cyclic nucleotides do not appear to be responsible for the initiation of carbachol-induced smooth muscle contractions.     

Complex effects of imatinib on spontaneous and oxytocin-induced contractions in human non-pregnant myometrium

Human myometrium includes two important cell populations involved in its contractility: smooth muscle fibers and interstitial cells. The pacemaking mechanism is not yet identified, but it is possible that myometrial smooth muscle cells contract in response to a signal generated by c-kit positive interstitial cells. The aim of this study was to investigate the effects of imatinib as a c-kit receptor antagonist on the spontaneous or oxytocin (OT) induced contractions of human non-pregnant myometrium in vitro. Myometrial strips were obtained from non-pregnant women (reproductive age) undergoing hysterectomy for benign indications. The strips were suspended in organ baths for recording of isometric tension. Imatinib effects were assessed on spontaneous contraction and after preexposure to OT.Direct exposure of myometrial strips to imatinib inhibits both amplitude and frequency of contractions (80-320 μM) in a dose dependent manner. Amplitude reverted back to 90% of the baseline amplitude by consequent addition of imatinib (until 480 μM). Total inhibition of myometrial contraction was obtained after addition of OT 60 nM. If myometrium was pre-exposed to OT (320 nM), imatinib 80-160 μm increased amplitude, while decreasing frequency. These data provide evidence that telocytes may be involved as modulators of the spontaneous contractions of the non-pregnant human uterus, via a tyrosine-kinase independent signaling pathway.     

Selected contribution: NO released to flow reduces myogenic tone of skeletal muscle arterioles by decreasing smooth muscle Ca(2+) sensitivity.

To clarify the contribution of intracellular Ca(2+) concentration ([Ca(2+)](i))-dependent and -independent signaling mechanisms in arteriolar smooth muscle (aSM) to modulation of arteriolar myogenic tone by nitric oxide (NO), released in response to increases in intraluminal flow from the endothelium, changes in aSM [Ca(2+)](i) and diameter of isolated rat gracilis muscle arterioles (pretreated with indomethacin) were studied by fluorescent videomicroscopy. At an intraluminal pressure of 80 mmHg, [Ca(2+)](i) significantly increased and myogenic tone developed in response to elevations of extracellular Ca(2+) concentration. The Ca(2+) channel inhibitor nimodipine substantially decreased [Ca(2+)](i) and completely inhibited myogenic tone. Dilations to intraluminal flow (that were inhibited by N(omega)-nitro-L-arginine methyl ester) or dilations to the NO donor S-nitroso-N-acetyl-DL-penicillamine (that were inhibited by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) were not accompanied by substantial decreases in aSM [Ca(2+)](i). 8-Bromoguanosine cGMP and the cGMP-specific phosphodiesterase inhibitor zaprinast significantly dilated arterioles yet elicited only minimal decreases in [Ca(2+)](i). Thus flow-induced endothelial release of NO elicits relaxation of arteriolar smooth muscle by a cGMP-dependent decrease of the Ca(2+) sensitivity of the contractile apparatus without substantial changes in the pressure-induced level of [Ca(2+)](i).     

Inhibitory effect of nitric oxide on voltage-dependent calcium currents in rat dorsal root ganglion cells

The effect of nitric oxide (NO) on calcium current (I(Ca)) and intracellular calcium concentration ([Ca(2+)](i)) in primarily cultured dorsal root ganglion (DRG) neurons was investigated from neonatal rats. I(Ca) and [Ca(2+)](i) were simultaneously recorded using perforated-patch technique in combination with fluorescence measurement from single DRG neurons. NO donors, sodium nitroprusside (SNP) and S-nitro-N-acetylpenicillamine (SNAP), inhibited I(Ca) in small-diameter neurons without significant change in voltage-dependence of activation and activation time constants. SNP and SNAP also reduced the transient [Ca(2+)](i) peak accompanied by I(Ca). Inhibition by NO was reproducible, but gradually desensitized. In some DRG neurons, SNP and SNAP increased basal [Ca(2+)](i) in concentration of 10 microM with little effect on NO-induced inhibition of I(Ca). 8-Br-cGMP, a permeable cGMP analog, mimicked the effects of SNP and SNAP. These results suggest that, in DRG neurons, NO has inhibitory effect on I(Ca), which is independent of NO-induced increase of basal [Ca(2+)](i), through cGMP-dependent pathway.     

Nitric-oxide-dependent activation of pig oocytes: the role of the cGMP-signalling pathway

Pig oocytes matured in vitro were parthenogenetically activated (78%) after treatment with 2 mM nitric oxide-donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP) for 24 h. Inhibition of soluble guanylyl cyclase with the specific inhibitors 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or 6-anilino-5,8-quinolinequinone (LY83583) suppressed the SNAP-induced activation in a dose-dependent manner (23% of activated oocytes after treatment with 400 microM ODQ; 12% of activated oocytes after treatment with 40 microM LY83583). 8-Bromo-cyclic guanosine monophosphate (8-Br-cGMP), a phosphodiesterase-resistant analogue of cGMP, enhances the effect of suboptimal doses (0.1 or 0.5 mM) of the NO donor SNAP. DT3, a specific inhibitor of cGMP-dependent protein kinase (PKG, PKG), is also able to inhibit the activation of pig oocytes after NO donor treatment. Involvement of the cGMP-dependent signalling pathway is specific for NO-induced oocyte activation, because both the guanylyl cyclase inhibitor ODQ and the PKG inhibitor DT3 are unable to inhibit activation in oocytes treated with the calcium ionophore A23187. These data indicate that the activation of pig oocytes with an NO donor is cGMP-dependent and that PKG plays an important role in this mode of oocyte activation.