Activation of distinct cAMP-dependent and cGMP-dependent pathways by nitric oxide in cardiac myocytes.

Nitric oxide (NO) donors were recently shown to produce biphasic contractile effects in cardiac tissue, with augmentation at low NO levels and depression at high NO levels. We examined the subcellular mechanisms involved in the opposing effects of NO on cardiac contraction and investigated whether NO modulates contraction exclusively via guanylyl cyclase (GC) activation or whether some contribution occurs via cGMP/PKG-independent mechanisms, in indo 1-loaded adult cardiac myocytes. Whereas a high concentration of the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 100 micromol/L) significantly attenuated contraction amplitude by 24.4+/-4.5% (without changing the Ca2+ transient or total cAMP), a low concentration of SNAP (1 micromol/L) significantly increased contraction amplitude (38+/-10%), Ca2+ transient (26+/-10%), and cAMP levels (from 6.2 to 8.5 pmol/mg of protein). The negative contractile response of 100 micromol/L SNAP was completely abolished in the presence of the specific blocker of PKG KT 5823 (1 micromol/L); the positive contractile response of 1 micromol/L SNAP persisted, despite the presence of the selective inhibitor of GC 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 micromol/L) alone, but was completely abolished in the presence of ODQ plus the specific inhibitory cAMP analog Rp-8-CPT-cAMPS (100 micromol/L), as well as by the NO scavenger oxyhemoglobin. Parallel experiments in cell suspensions showed significant increases in adenylyl cyclase (AC) activity at low concentrations (0.1 to 1 micromol/L) of SNAP (AC, 18% to 20% above basal activity). We conclude that NO can regulate both AC and GC in cardiac myocytes. High levels of NO induce large increases in cGMP and a negative inotropic effect mediated by a PKG-dependent reduction in myofilament responsiveness to Ca2+. Low levels of NO increase cAMP, at least in part, by a novel cGMP-independent activation of AC and induce a positive contractile response.     

The nitric oxide donors, SNAP and DEA/NO, exert a negative inotropic effect in rat cardiomyocytes which is independent of cyclic GMP elevation.

The role of guanosine 3',5'-cyclic monophosphate (cGMP) in the regulation of cardiac contractility remains controversial. The present study has examined the effects of high concentrations of the nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine (SNAP) and 1,1-diethyl-2-hydroxy-2-nitroso-hydrazine (DEA/NO), on cGMP levels and isoproterenol-induced increases in contractility in rat cardiomyocytes before and after selective inhibition of soluble guanylyl cyclase with 1 H -[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). In control myocytes, 100 microm SNAP or 100 microm DEA/NO increased cGMP levels by more than 15-fold at 2 and 6 min and produced marked attenuations of isoproterenol-mediated increases in maximal cell shortening over the same time period. The NO donors had no significant effect on basal cell shortening (in the absence of isoproterenol). Pretreatment of myocytes with 25 microm ODQ for 30 min resulted in a complete blockade of the SNAP- or DEA/NO-induced increases in cGMP with no reversal of negative inotropy. ODQ did not affect basal contractility, basal cGMP levels or isoproterenol-induced increases in cell shortening. Furthermore, myocytes exposed to the cGMP analog, 8-bromo-cGMP (100 microm), did not exhibit significant differences in basal contractility or isoproterenol-induced increases in cell shortening. These results suggest that attenuation of cardiac contractility by NO donors in rat cardiomyocytes occurs by a mechanism independent of increases in cGMP levels.     

Single L-type Ca(2+) channel regulation by cGMP-dependent protein kinase type I in adult cardiomyocytes from PKG I transgenic mice

OBJECTIVE: Calcium entry via the L-type Ca(2+) channel (LTCC) is crucial for excitation-contraction (EC) coupling and activation of Ca(2+)-dependent signal transduction pathways in cardiac myocytes. Both nitric oxide (NO), signaling via cGMP, and acetylcholine, signaling via the muscarinic receptor, have been identified as negative regulators of beta-adrenoreceptor-stimulated LTCC activity in cardiac myocytes. METHODS: To examine the potential role of cGMP-dependent protein kinase type I (PKG I) in the inhibitory effects of NO/cGMP and the muscarinic receptor on LTCC activity, we generated transgenic (TG) mice overexpressing PKG I selectively in cardiac myocytes under the control of the alpha-myocin heavy chain promoter. Single LTCC-gating properties were assessed in isolated ventricular myocytes from adult wild-type (WT) and PKG I transgenic (TG) mice. RESULTS: Basal LTCC activity (peak average current, mean open probability, mean availability) was significantly decreased by the nitric oxide donor DEA-NO (0.1 micromol/l) and the cGMP-analog 8-Br-cGMP (1 mmol/l) in TG but not in WT cardiac myocytes. Conversely, muscarinic (carbachol, 1 micromol/l) stimulation had no significant effect on basal LTCC activity in either WT or TG cardiac myocytes. beta-Adrenergic stimulation with isoproterenol (1 micromol/l) increases single LTCC activity in WT and TG cardiac myocytes to the same extent. The inhibitory effects of DEA-NO and 8-Br-cGMP on isoproterenol activation of the LTCC current were significantly enhanced in TG as compared to WT cardiac myocytes. By contrast, carbachol inhibition of isoproterenol-stimulated single LTCC activity was not enhanced in TG cardiac myocytes. CONCLUSION: Transgenic overexpression of PKG I augments NO/cGMP inhibition but not muscarinic inhibition of single LTCC activity, indicating that PKG I is a downstream target for NO/cGMP, but not the muscarinic receptor in adult cardiac myocytes.     

Vasorelaxing effect of BAY 41-2272 in rat basilar artery: involvement of cGMP-dependent and independent mechanisms

Decreases in intrinsic NO cause cerebral vasospasms because of the dysregulation of cGMP formation by NO-mediated pathways. Because 5-cyclopropyl-2-{1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl}pyrimidin-4-ylamine (BAY 41-2272) is a potent soluble guanylyl cyclase (sGC) stimulator in an NO-independent manner, this study aimed to investigate the mechanisms underlying the relaxant effects of BAY 41-2272 in the rat basilar artery. BAY 41-2272 (0.0001 to 1 micromol/L) induced relaxations in a concentration-dependent manner, with pEC50 values of 8.13+/-0.03 and 7.63+/-0.05 in intact and denuded rings, respectively. The sGC inhibitor 1H-[1,2,4] oxadiazolo [4,3,-a]quinoxalin-1-one (ODQ) markedly displaced the curve for BAY 41-2272 to the right in intact or denuded rings (&10-fold). The NO synthesis inhibitor NG-nitro-L-arginine methyl ester caused a rightward shift in the curve for BAY 41-2272 (4-fold), whereas the phosphodiesterase type 5 inhibitor sildenafil enhanced BAY 41-2272-induced relaxations (3- to 4-fold). The Na+-K+-ATPase inhibitor ouabain caused 3-fold rightward shifts in the curves for BAY 41-2272. Ca2+-induced contractions in K+ depolarized rings were significantly attenuated by BAY 41-2272 in an ODQ-insensitive manner. The NO donor glyceryl trinitrate and BAY 41-2272 caused rightward shifts in the contractile responses to serotonin. Their coincubation caused a synergistic inhibition of serotonin-induced contractions. BAY 41-2272 and glyceryl trinitrate increased cGMP levels (but not cAMP) by 10-fold and 4-fold above baseline, respectively, in an ODQ-sensitive manner. cGMP levels increased by 50-fold after coincubation. BAY 41-2272 potently relaxes the rat basilar artery in a synergistic fashion with NO. Targeting the sGC with selective activators, such as BAY 41-2272, may represent a new therapy to treat cerebrovascular disease.     

Endothelial NO/cGMP system contributes to natriuretic peptide-mediated coronary and peripheral vasodilation

We tested the hypothesis that the endothelial nitric oxide (NO)-soluble guanylyl cyclase system is involved in atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) mediated regulation of coronary and peripheral vascular resistance. Rat hearts were perfused via the aorta at constant flow and the effect of ANP and CNP on coronary perfusion pressure and release of cGMP was determined in the absence and presence of the nitric oxide synthase inhibitor NG-nitro-L-arginine (L-NNA; 0.2 mmol/L) and the specific inhibitor of soluble guanylyl cyclase ODQ (20 micromol/L), respectively (n = 6). ANP (10-300 nmol/L) reduced perfusion pressure from 133 +/- 2 to 53 +/- 2 mm Hg (-60%; control) in the presence of L-NNA from 132 +/- 1 to 71 +/- 1 mm Hg (-46%) and in the presence of ODQ from 133 +/- 1 to 85 +/- 2 (-36%) (n = 6; P < 0.05). Disruption of the coronary endothelium by perfusion of hearts with collagenase reduced the relaxant effect of ANP to a similar extent as L-NNA. Basal release of cGMP was increased up to sixfold by ANP and this increase was reduced by L-NNA and ODQ (n = 6; P < 0.05). The coronary relaxant effect of CNP (0.1-3 micromol/L) was similarly attenuated by L-NNA and ODQ (n = 6). In conscious mice, a low dose of L-NNA (30 nmol) consistently reduced the blood pressure lowering effect of ANP (30 nmol) by approximately 40% (n = 7), whereas the hypotensive effect of nitroprusside (0.15 micromol) was not affected (n = 5). We conclude that the coronary dilatory and hypotensive action of natriuretic peptides involves the endothelium and is partly mediated by soluble guanylyl cyclase. The data may explain previous observations in humans with congestive heart failure showing impaired vascular ANP responses.     

cGMP-mediated negative-feedback regulation of endothelial nitric oxide synthase expression by nitric oxide

Earlier studies have demonstrated that nitric oxide (NO) exerts a fast-acting inhibitory influence on endothelial NO synthase (eNOS) enzymatic activity in isolated vascular tissue preparations. The present study was designed to examine the possible effect of NO on eNOS protein expression in cultured endothelial cells and intact animals. Human coronary endothelial cells were incubated with S-nitroso-N-acetyl-penicillamine (SNAP, an NO donor), oxyhemoglobin (HGB, an NO trapping agent), SNAP plus HGB, or inactive vehicle (control). In other experiments, cells were treated with 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor), 1H-[1,2, 4]oxadiazolo-[4,3-2]quinoxalin-1-one (ODQ, a guanylate cyclase inhibitor), SNAP plus ODQ, 8-bromo-cGMP (8-Br-cGMP, a cell-permeable cGMP compound), 8-Br-cGMP plus HGB, or inactive vehicle in order to discern the effect of cGMP. The incubations were conducted for 24 hours, and total nitrate plus nitrite production and eNOS protein abundance (Western analysis) were measured. To determine the effect of NO on eNOS expression in vivo, rats were treated with either the NO donor isosorbide dinitrate or placebo by gastric gavage for 48 hours, and aortic eNOS protein expression was examined. The NO donor SNAP markedly depressed, whereas the NO scavenger HGB significantly raised, eNOS protein expression. The downregulatory action of SNAP was completely abrogated by HGB. Phosphodiesterase inhibitor and 8-Br-cGMP downregulated, whereas the guanylate cyclase inhibitor ODQ upregulated eNOS protein expression. The downregulatory action of SNAP was completely overcome by the guanylate cyclase inhibitor ODQ, and the upregulatory action of the NO scavenger HGB was abrogated by 8-Br-cGMP. Administration of NO donor resulted in a marked downregulation of aortic eNOS protein expression in intact animals, thus confirming the in vitro findings. NO serves as a negative-feedback regulator of eNOS expression via a cGMP-mediated process.     

Nicorandil inhibits oxidative stress-induced apoptosis in cardiac myocytes through activation of mitochondrial ATP-sensitive potassium channels and a nitrate-like effect

The anti-anginal drug nicorandil has been shown to inhibit apoptosis by activating mitochondrial ATP-sensitive potassium (K(ATP)) channels. The possible contribution of the nitrate moiety of this drug to its anti-apoptotic effect has now been investigated in neonatal rat ventricular myocytes subjected to oxidative stress. Exposure of cultured myocytes to 100 micromol/l hydrogen peroxide (H(2)O(2)) increased the number of nuclei stained by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique as well as induced internucleosomal DNA fragmentation, loss of mitochondrial membrane potential, cytochrome c release into the cytosol, and activation of caspases-3 and -9, all of which are characteristics of apoptosis. Pretreatment of cells with nicorandil (100 micromol/l) inhibited these effects of H(2)O(2). Both the mitochondrial K(ATP) channel antagonist 5-hydroxydecanoate (5-HD) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, attenuated the anti-apoptotic effect of nicorandil in concentration-dependent manners. Coapplication of ODQ (10 micromol/l) and 5-HD (500 micromol/l) completely abolished nicorandil-induced cytoprotection. The effect of nicorandil was also reduced by an inhibitor of cGMP-dependent protein kinase (KT5823, 1 micromol/l). The nitric oxide donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP, 50 micromol/l) mimicked the protective effect of nicorandil in a manner sensitive to ODQ but not to 5-HD. A cell-permeable cGMP analog, 8-bromo-cGMP, also reduced H(2)O(2)-induced apoptosis. The inhibition of the H(2)O(2)-induced activation of caspase-3, but not that of caspase-9, by nicorandil in the presence of 5-HD or by SNAP was reversed by the addition of dithiothreitol to the enzyme assay. Nicorandil inhibits oxidative stress-induced apoptosis in cardiac myocytes through a nitric oxide/cGMP-dependent mechanism as well as by activating mitochondrial K(ATP) channels.     

Alterations in ventricular myocyte contraction caused by C-type natriuretic peptide and nitric oxide in eNOS-/- mice

Lack of endothelial nitric oxide synthase (eNOS) may affect the sensitivity of cyclic GMP signaling through soluble guanylyl cyclase (sGC). We hypothesized that in eNOS knockout (eNOS-/-) mice, stimulation of guanylyl cyclase would have enhanced effects inhibiting cardiac contraction. We measured cell shortening and calcium transients in isolated ventricular myocytes from adult eNOS-/- and wild-type (WT) mice after stimulating particulate guanylyl cyclase (pGC) with C-type natriuretic peptide (CNP, 10(-8) and 10(-7) M) or sGC with S-nitroso-N-acetyl-penicillamine (SNAP, NO donor, 10(-6) and 10(-5) M). Although sGC activity was increased by +71% in eNOS-/-, SNAP had similar effects in the two groups (%shortening -39% control vs. -37% eNOS-/-), suggesting that the cyclic GMP pathway was desensitized in eNOS-/- myocytes. CNP had significantly smaller effects on cell contraction (%shortening -34% control vs. -14% eNOS-/-) and pGC activity was not changed in eNOS-/- myocytes. Similar effects were also produced by guanylin and carbon monoxide, stimulators of pGC and sGC. CNP's effects on Ca(2+) transients were also attenuated in eNOS-/- myocytes. SNAP did not alter Ca(2+) transients in eNOS-/- or control cells. In the eNOS-/- mice, cyclic GMP-dependent protein kinase and cyclic AMP phosphodiesterase activity were reduced. This study demonstrated that the downstream cyclic GMP pathway was attenuated in eNOS-/- mice and this was partially compensated for by increased sGC, but not pGC activity in ventricular myocytes.     

cGMP mediates the vascular and platelet actions of nitric oxide: confirmation using an inhibitor of the soluble guanylyl cyclase.

The L-arginine:nitric oxide (NO) pathway is believed to exert many of its physiological effects via stimulation of the soluble guanylyl cyclase (SGC); however, the lack of a selective inhibitor of this enzyme has prevented conclusive demonstration of this mechanism of action. We have found that the compound 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) inhibits the elevation of cGMP induced by the NO donor S-nitroso-DL-penicillamine in human platelets and rat vascular smooth muscle (IC50 = 10-60 nM and <10 nM, respectively) and that this is accompanied by prevention of the platelet inhibitory and vasodilator actions of NO donors. ODQ also inhibited the antiaggregatory action of NO generated by the platelets but did not affect the action of prostacyclin or that of a cGMP mimetic. In addition, ODQ inhibited the vasodilator actions of endogenously released NO and of NO generated after induction of NO synthase in vascular preparations. It did not, however, affect the increase in vascular smooth muscle cGMP or the dilatation induced by atrial natriuretic factor. ODQ had no effect on NO synthase activity, nor did it react with NO. It did, however, potently (IC50 approximately 10 nM) inhibit the activity of the SGC in cytosol obtained from crude extract of rat aortic smooth muscle. Thus ODQ prevents the actions of NO on platelets and vascular smooth muscle through its potent inhibitory effect on the SGC.     

Lysozyme binding to endocardial endothelium mediates myocardial depression by the nitric oxide guanosine 3',5' monophosphate pathway in sepsis

Inflammatory mediators have been implicated as a cause of reversible myocardial depression in septic shock. We previously reported that the release of lysozyme-c (Lmz-S) from leukocytes from the spleen or other organs contributes to myocardial dysfunction in Escherichia coli septic shock in dogs by binding to a cardiac membrane glycoprotein. However, the mechanism by which Lzm-S causes this depression has not been elucidated. In the present study, we tested the hypothesis that the binding of Lzm-S to a membrane glycoprotein causes myocardial depression by the formation of nitric oxide (NO). NO generation then activates soluble guanylyl cyclase and increases cyclic guanosine monophosphate (cGMP), which in turn triggers contractile impairment via activation of cGMP-dependent protein kinase (PKG). We examined these possibilities in a right ventricular trabecular preparation in which isometric contraction was used to measure cardiac contractility. We found that Lzm-S's depressant effect could be prevented by the non-specific NO synthase (NOS) inhibitor N(G)-monomethyl-l-arginine (l-NMMA). A guanylyl cyclase inhibitor (ODQ) and a PKG inhibitor (Rp-8-Br-cGMP) also attenuated Lzm-S's depressant effect as did chemical denudation of the endocardial endothelium (EE) with Triton X-100 (0.5%). In EE tissue, we further showed that Lzm-S caused NO release with use of 4,5 diaminofluorescein, a fluorescent dye that binds to NO. The present study shows that the binding of Lzm-S to EE generates NO, and that NO then activates the myocardial guanosine 3',5' monophosphate pathway leading to cardiac depression in sepsis.     

Production and role of extracellular guanosine cyclic 3', 5' monophosphate in sodium uptake in human proximal tubule cells

The present study was designed to determine the capability of human renal proximal tubule (RPT) to generate and export guanosine cyclic 3', 5' monophosphate (cGMP) in response to direct stimulation of soluble guanylyl cyclase by nitric oxide (NO) donors. In addition, we investigated whether cGMP extrusion from human RPT cells is required for inhibition of cellular sodium uptake. RPT cells were cultured from fresh human kidneys (normotensive subjects, n=4, mean age 65+/-4.7 years, 3 men, 1 woman; hypertensive patients, n=6, mean age 64+/-6.1 years, 4 men, 2 women) after unilateral nephrectomy. The fluorescence dye Sodium Green was employed to determine cytoplasmic Na+ concentration. In the presence of the Na+/K+ ATPase inhibitor ouabain, fluorescence was monitored at the appropriate wavelength (excitation 485 nm, emission 535 nm). Nitric oxide donor, S-nitroso-N-acetylpenicillamine (SNAP, 10(-4) M), increased both intracellular and extracellular cGMP (from 1.26+/-0.21 to 88.7+/-12.6 pmol/mg protein and from 0.58+/-0.10 to 9.24+/-1.9 pmol/mL, respectively, P<0.01) and decreased cellular Na+ uptake by 37.4+/-6.8% (P<0.05) compared with control. The effects of SNAP on cGMP production were similar in normotensive and hypertensive subjects. The increases in intracellular and extracellular cGMP concentration because of SNAP were blocked completely by soluble guanylyl cyclase inhibitor ODQ (1-H-[1,2,4] oxadiazolo [4,2-alpha] quinoxalin-1-one). Probenecid, an organic anion transport inhibitor, augmented the SNAP (10(-6) M)-induced increase in intracellular cGMP accumulation (from 4.9+/-0.9 to 9.8+/-1.5 pmol/mg protein, P<0.05), abrogated the SNAP-induced increase in extracellular cGMP extrusion (from 1.07+/-0.4 to 0.37+/-0.1 pmol/L, P<0.05) and blocked the SNAP-induced reduction in cellular Na+ uptake. Neither intracellular nor extracellular cGMP were influenced by l-arginine, the metabolic precursor of NO, or N(G)-nitro-L-arginine methyl ester, an inhibitor of NO synthase. After exogenous administration of cGMP (10(-5) M) or its membrane-permeable analogue 8-Br-cGMP (10(-5) M), only 8-Br-cGMP crossed the cell membrane to increase intracellular cGMP (from 1.36+/-0.19 to 289.7+/-29.4 pmol/mg protein, P<0.01). However, both cGMP and 8-Br-cGMP were effective in decreasing cellular Na+ uptake. In conclusion, human RPT cells contain soluble guanylyl cyclase and are able to generate and export cGMP in response to NO. Because human RPT cells do not themselves contain constitutive NO synthase, the NO-generating cGMP must be derived from sources outside the human RPT. The cGMP cellular export system is critical in the regulation of RPT cellular Na+ absorption in humans.     

An endothelium-derived hyperpolarizing factor distinct from NO and prostacyclin is a major endothelium-dependent vasodilator in resistance vessels of wild-type and endothelial NO synthase knockout mice

In addition to nitric oxide (NO) and prostacyclin (PGI(2)), the endothelium generates the endothelium-derived hyperpolarizing factor (EDHF). We set out to determine whether an EDHF-like response can be detected in wild-type (WT) and endothelial NO synthase knockout mice (eNOS -/-) mice. Vasodilator responses to endothelium-dependent agonists were determined in vivo and in vitro. In vivo, bradykinin induced a pronounced, dose-dependent decrease in mean arterial pressure (MAP) which did not differ between WT and eNOS -/- mice and was unaffected by treatment with N(omega)-nitro-l-arginine methyl ester and diclofenac. In the saline-perfused hindlimb of WT and eNOS -/- mice, marked N(omega)-nitro-l-arginine (l-NA, 300 micromol/liter)- and diclofenac-insensitive vasodilations in response to both bradykinin and acetylcholine (ACh) were observed, which were more pronounced than the agonist-induced vasodilation in the hindlimb of WT in the absence of l-NA. This endothelium-dependent, NO/PGI(2)-independent vasodilatation was sensitive to KCl (40 mM) and to the combination of apamin and charybdotoxin. Gap junction inhibitors (18alpha-glycyrrhetinic acid, octanol, heptanol) and CB-1 cannabinoid-receptor agonists (Delta(9)-tetrahydrocannabinol, HU210) impaired EDHF-mediated vasodilation, whereas inhibition of cytochrome P450 enzymes, soluble guanylyl cyclase, or adenosine receptors had no effect on EDHF-mediated responses. These results demonstrate that in murine resistance vessels the predominant agonist-induced endothelium-dependent vasodilation in vivo and in vitro is not mediated by NO, PGI(2), or a cytochrome P450 metabolite, but by an EDHF-like principle that requires functional gap junctions.     

Positive inotropic effects of NO donors in isolated guinea-pig and human cardiomyocytes independent of NO species and cyclic nucleotides

OBJECTIVE: To characterise the inotropic response of isolated myocytes to a range of structurally unrelated NO donors and to assess the role of NO release kinetics, NO species and cyclic nucleotides in mediating the observed changes. METHOD: Guinea-pig (GP) and human myocytes were prepared by enzymatic digestion. Paced contractile amplitude was recorded at 37 degrees C. NO release was measured by reduction of oxyhaemoglobin and using an NO electrode. Cyclic nucleotides were measured using a tritium labelled competitive binding assay. RESULTS: The NO donors S-nitrosoglutathione (GSNO) and diethylamine/NO (DEA/NO) produced positive inotropic effects in GP myocytes at (10(-5) M) (25 and 111% increases of contraction amplitude).The response to GSNO was significantly enhanced in the presence of a low concentration of isoprenaline (3x10(-10) M). Positive inotropy was observed with a range of both thiol and non-thiol donors, amongst which a fast rate of NO release was associated with positive inotropy. The response to GSNO was abolished by the free NO scavenger oxyhaemoglobin, but not by ODQ (soluble guanylyl cyclase [sGC] inhibitor), Rp-cAMPS (protein kinase A inhibitor) or thapsigargin (sarcoplasmic reticulum Ca(2+) uptake blocker). Direct measurement of cyclic nucleotides showed a rise in cGMP but not cAMP. Human ventricular myocytes showed a significant increase of contraction with GSNO (48+/-15.8%, n=7, P<0. 05) in the presence of isoprenaline and a marked response to DEA/NO alone. CONCLUSIONS: Isolated GP and human myocytes show a positive inotropic effect with certain NO donors. This is independent of sGC and cAMP. The rate of NO release from donors appears important in mediating the effect.     

Preeclampsia is associated with impaired regulation of the placental nitric oxide-cyclic guanosine monophosphate pathway by corticotropin-releasing hormone (CRH) and CRH-related peptides

During pregnancy, CRH and CRH-related peptides appear to regulate the fetoplacental circulation via activation of the nitric oxide (NO)/cGMP pathway. Pregnancies with abnormal placental function such as preeclampsia (PE) are characterized by increased maternal plasma CRH concentrations and reduced placental CRH-receptor 1alpha (CRH-R1alpha) expression. In this study, we investigated the actions of CRH/CRH-related peptides on the NO/cGMP system in normal and PE placentas (n = 8 for each group). Fluorescent in situ hybridization, RT-PCR, and immunofluorescence experiments in human term placenta detected mRNAs expression for both R1 and R2 types of CRH-R, as well as urocortin (UCN) II and UCN III and showed CRH-R protein expression mainly in syncytiotrophoblast, whereas the endothelial NO synthase (eNOS) expression was confined within the cytoplasm of the chorionic villi. In placental explants, CRH and UCN induced mRNA and protein expression of eNOS, but not inducible NOS, and also caused an acute increase in cGMP levels (maximal stimulation, 80-90% above basal; P < 0.05). UCN II also induced a modest induction of cGMP (42% above basal; P < 0.05). These responses were attenuated by the NOS and soluble guanylyl cyclase inhibitors, l-N(G)-nitro-l-arginine methyl ester and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. In PE placental explants there was a significant reduction in CRH/CRH-related peptide-induced cGMP response; however, changes in the mRNA content of eNOS, inducible NOS, and soluble guanylyl cyclase (assessed by quantitative RT-PCR) between normal and PE placentas were not altered. In conclusion, we demonstrated that CRH and CRH-related peptides can positively regulate the placental NO/cGMP system. This pathway appears to be impaired in PE and may contribute toward dysregulation of the balance controlling vascular resistance.     

Hydrogen peroxide differentially modulates cardiac myocyte nitric oxide synthesis

Nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) are synthesized within cardiac myocytes and play key roles in modulating cardiovascular signaling. Cardiac myocytes contain both the endothelial (eNOS) and neuronal (nNOS) NO synthases, but the differential roles of these NOS isoforms and the interplay of reactive oxygen species and reactive nitrogen species in cardiac signaling pathways are poorly understood. Using a recently developed NO chemical sensor [Cu(2)(FL2E)] to study adult cardiac myocytes from wild-type, eNOS(null), and nNOS(null) mice, we discovered that physiological concentrations of H(2)O(2) activate eNOS but not nNOS. H(2)O(2)-stimulated eNOS activation depends on phosphorylation of both the AMP-activated protein kinase and kinase Akt, and leads to the robust phosphorylation of eNOS. Cardiac myocytes isolated from mice infected with lentivirus expressing the recently developed H(2)O(2) biosensor HyPer2 show marked H(2)O(2) synthesis when stimulated by angiotensin II, but not following β-adrenergic receptor activation. We discovered that the angiotensin-II-promoted increase in cardiac myocyte contractility is dependent on H(2)O(2), whereas β-adrenergic contractile responses occur independently of H(2)O(2) signaling. These studies establish differential roles for H(2)O(2) in control of cardiac contractility and receptor-dependent NOS activation in the heart, and they identify new points for modulation of NO signaling responses by oxidant stress.     

Nitric oxide causes a cGMP-independent intracellular calcium rise in porcine endothelial cells-a paradox?

This study was undertaken to investigate the influence of exogenous NO on intracellular calcium levels of porcine aortic endothelial cell culture monolayers. Spontaneous NO liberating substances with different half-life periods (NOC-9 [10 micromol/L] approximately 1 min, SNAP [10 micromol/L] approximately 4 h), and an aqueous NO gas solution [130 nmol/L] were added onto the monolayers. All three solutions induced a rapid and similar calcium rise in the endothelial cells. NOC-9 as a rapidly NO releasing compound was selected to be investigated more thoroughly. The NOC-9 calcium rise is not dependent on the activation of the guanylate cyclase since preincubation with a specific guanylate cyclase inhibitor [ODQ, 10 micromol/L] did not alter the effect and a cGMP analogue [8-bromo-cGMP 10 micromol/L] did not significantly elevate calcium levels. The NOC-9 induced calcium rise could be completely blocked by removal of extracellular calcium and partly blocked by SKF 96365 [10 micromol/L], an unspecific inhibitor of the receptor operated calcium channels. Incubation with N-nitroarginine [100 micromol/L] slightly but significantly reduced basal calcium levels in the cell cultures. Therefore, we conclude that exogenous NO elevates [Ca(2+)](i) in cultured porcine aortic endothelial cells. This effect is not dependent on cGMP, and a calcium influx is involved. Moreover, constitutively formed endogenous NO seems to be necessary to maintain basal calcium levels.     

Heme oxygenase-1 is a cGMP-inducible endothelial protein and mediates the cytoprotective action of nitric oxide

Inducible heme oxygenase (HO-1) has recently been recognized as an antioxidant and cytoprotective gene. By use of Western blotting, cell viability analysis, and antisense technique, the present study investigates the involvement of HO-1 in endothelial protection induced by the clinically used nitric oxide (NO) donor molsidomine (specifically, its active metabolite 3-morpholinosydnonimine [SIN-1]) and the second messenger cGMP. In bovine pulmonary artery endothelial cells, SIN-1 and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) at 1 to 100 micromol/L induced the synthesis of HO-1 protein in a concentration-dependent fashion up to 3-fold over basal levels. HO-1 induction by SIN-1 was inhibited in the presence of the NO scavenger phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide and the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazole[4, 3-a]quinoxalin-1-one. 8-Bromo-cGMP (1 to 100 micromol/L) and dibutyryl cGMP (1 to 100 micromol/L) as well as the activator of particulate guanylyl cyclase atrial natriuretic peptide (1 to 100 nmol/L) produced increases in HO-1 protein similar to those produced by SIN-1. SIN-1 and 8-bromo-cGMP increased heme oxygenase activity (bilirubin formation). Cytoprotection by NO donors was abrogated in the presence of the heme oxygenase inhibitor tin protoporphyrin IX. Pretreatment of cells with a phosphorothioate-linked HO-1 antisense oligonucleotide prevented protection by SIN-1 or 8-bromo-cGMP against tumor necrosis factor-alpha cytotoxicity, whereas sense and scrambled HO-1 were without effect under these conditions. Our results show for the first time that HO-1 is a cGMP-sensitive endothelial gene and establish conclusively a causal relationship between HO-1 induction and endothelial protection by the NO/cGMP system. By targeting cytoprotective HO-1, NO donors may therefore be expected to induce antioxidant, antiatherogenic, and anti-inflammatory effects.     

Muscarinic cholinergic regulation of cardiac myocyte ICa-L is absent in mice with targeted disruption of endothelial nitric oxide synthase

Cardiac myocytes have been shown to express constitutively endothelial nitric oxide synthase (eNOS) (nitric oxide synthase 3), the activation of which has been implicated in the regulation of myocyte L-type voltage-sensitive calcium channel current (I_Ca-L) and myocyte contractile responsiveness to parasympathetic nervous system signaling, although this implication remains controversial. Therefore, we examined the effect of the muscarinic cholinergic agonist carbachol (CCh) on I_Ca-L and contractile amplitude in isoproterenol (ISO)-prestimulated ventricular myocytes isolated from adult mice, designated eNOS^null mice, with targeted disruption of the eNOS gene. Although both eNOS^null and wild-type (WT) ventricular myocytes exhibited similar increases in I_Ca-L in response to ISO, there was no measurable suppression of I_Ca-L by CCh in cells from eNOS^null mice, in contrast to cells from WT mice. These results were reflected in the absence of an effect of CCh on the positive inotropic effect of ISO in eNOS^null myocytes. Also, unlike myocytes from WT animals, eNOS^null myocytes failed to exhibit an increase in cGMP content in response to CCh. Nevertheless, the pharmacologic nitric oxide donors 3-morpholino-sydnonimine and S-nitroso-acetyl-cystein increased cGMP generation and suppressed ISO-augmented I_Ca-L in eNOS^null cells, suggesting that the signal transduction pathway(s) downstream of eNOS remained intact. Of importance, activation of the acetylcholine-activated K⁺ channel by CCh was unaffected in atrial and ventricular eNOS^null myocytes. These results confirm the obligatory role of eNOS in coupling muscarinic receptor activation to cGMP-dependent control of I_Ca-L in cardiac myocytes.     

Thrombospondin-1, endothelium and systemic vascular tone

Evaluation of: Bauer EM, Qin Y, Miller TW et al.: Thrombospondin-1 supports blood pressure by limiting eNOS activation and endothelial dependent vasorelaxation. Cardiovasc. Res. 88, 471-481 (2010). Several lines of evidence, both in vivo and ex vivo, suggest that thrombospondin-1 (TSP-1) is important in maintaining systemic vascular tone. Recently published papers demonstrate that TSP-1 can inhibit vascular smooth muscle relaxation by interfering with the interaction between nitric oxide (NO) and soluble guanylyl cyclase, providing a possible mechanism of action to explain this observation. While these in vitro experiments in vascular smooth muscle cells were provocative, it is not clear how such a large protein circulating in the plasma could cross the intact endothelial basal membrane and regulate NO/cGMP signaling in smooth muscle in vivo. This raised the question of whether TSP-1 could modulate NO/cGMP signaling through another mechanism. Herein, we evaluate a recently published paper by Bauer and colleagues that examined whether TSP-1 could exert vasoactive effects without directly accessing the vascular smooth muscle. In their studies they found that TSP-1 could inhibit the NO/cGMP signaling pathway through an alternate mechanism: inhibiting the activation of endothelial NO synthase (eNOS), and therefore NO production in endothelial cells. These findings, combined with previous results from these investigators, suggest that TSP-1 can blunt NO/cGMP signaling through two different mechanisms: inhibiting NO production in endothelial cells by preventing the agonist-induced influx of Ca(2+) required to activate endothelial NO synthase and blunting the ability of endothelial-derived NO to activate soluble guanylyl cyclase in vascular smooth muscle cells. The importance of these two pathways in supporting systemic and pulmonary vascular tone in health and disease is unclear.