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.     

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.     

Positive inotropic and lusitropic effects of HNO/NO- in failing hearts: independence from beta-adrenergic signaling

Nitroxyl anion (HNONO(-)), the one-electron reduced form of nitric oxide (NO), induces positive cardiac inotropy and selective venodilation in the normal in vivo circulation. Here we tested whether HNO/NO(-) augments systolic and diastolic function of failing hearts, and whether contrary to NO/nitrates such modulation enhances rather than blunts beta-adrenergic stimulation and is accompanied by increased plasma calcitonin gene-related peptide (CGRP). HNO/NO(-) generated by Angelis' salt (AS) was infused (10 microg/kg per min, i.v.) to conscious dogs with cardiac failure induced by chronic tachycardia pacing. AS nearly doubled contractility, enhanced relaxation, and lowered cardiac preload and afterload (all P < 0.001) without altering plasma cGMP. This contrasted to modest systolic depression induced by an NO donor diethylamine(DEA)NO or nitroglycerin (NTG). Cardiotropic changes from AS were similar in failing hearts as in controls despite depressed beta-adrenergic and calcium signaling in the former. Inotropic effects of AS were additive to dobutamine, whereas DEA/NO blunted beta-stimulation and NTG was neutral. Administration of propranolol to nonfailing hearts fully blocked isoproterenol stimulation but had minimal effect on AS inotropy and enhanced lusitropy. Arterial plasma CGRP rose 3-fold with AS but was unaltered by DEA/NO or NTG, supporting a proposed role of this peptide to HNO/NO(-) cardiotropic action. Thus, HNO/NO(-) has positive inotropic and lusitropic action, which unlike NO/nitrates is independent and additive to beta-adrenergic stimulation and stimulates CGRP release. This suggests potential of HNO/NO(-) donors for the treatment of heart failure.     

Nitric oxide: not just a negative inotrope

Nitric oxide (NO) appears to play a role in modulating cardiac function in both health and disease. Early studies in isolated rodent cardiac myocytes demonstrated a depressant effect of NO supplied by NO donors (exogenous) as well as NO generated within myocytes (endogenous). There is increasing evidence for a functional NO generating system within the human myocardium, which appears upregulated in certain disease states. Induction of the high output nitric oxide synthase isoform (iNOS) has been demonstrated in the failing myocardium, though its functional significance remains unproven. More recently published data have contradicted the notion that NO acts solely as a negative inotrope demonstrating positive inotropy in both isolated rodent and human ventricular myocytes in response to a range of NO donors. Different NO donors have different NO release kinetics and generate a range of NO species (NO., NO+ and NO-) which may interact at a number of subcellular targets. The observed response of any cardiac preparation to an NO donor represents the net effect of activation of different effector targets and may explain the contradictory reported effects of NO. To realise the therapeutic potential of NO will require specific targeting at a subcellular level.     

Compartmentalized phosphodiesterase-2 activity blunts beta-adrenergic cardiac inotropy via an NO/cGMP-dependent pathway

beta-Adrenergic signaling via cAMP generation and PKA activation mediates the positive inotropic effect of catecholamines on heart cells. Given the large diversity of protein kinase A targets within cardiac cells, a precisely regulated and confined activity of such signaling pathway is essential for specificity of response. Phosphodiesterases (PDEs) are the only route for degrading cAMP and are thus poised to regulate intracellular cAMP gradients. Their spatial confinement to discrete compartments and functional coupling to individual receptors provides an efficient way to control local [cAMP]i in a stimulus-specific manner. By performing real-time imaging of cyclic nucleotides in living ventriculocytes we identify a prominent role of PDE2 in selectively shaping the cAMP response to catecholamines via a pathway involving beta3-adrenergic receptors, NO generation and cGMP production. In cardiac myocytes, PDE2, being tightly coupled to the pool of adenylyl cyclases activated by beta-adrenergic receptor stimulation, coordinates cGMP and cAMP signaling in a novel feedback control loop of the beta-adrenergic pathway. In this, activation of beta3-adrenergic receptors counteracts cAMP generation obtained via stimulation of beta1/beta2-adrenoceptors. Our study illustrates the key role of compartmentalized PDE2 in the control of catecholamine-generated cAMP and furthers our understanding of localized cAMP signaling.     

The nitroxyl anion (HNO) is a potent dilator of rat coronary vasculature

OBJECTIVE: The nitroxyl anion (HNO) is the one-electron reduction product of NO(). This redox variant has been shown to be endogenously produced and to have effects that are pharmacologically distinct from NO(). This study investigates the vasodilator and chronotropic effects of HNO in the rat isolated coronary vasculature. METHODS: Sprague-Dawley rat hearts were retrogradely perfused with Krebs' solution (8 ml/min) using the Langendorff technique. Perfusion pressure was raised using a combination of infusion of phenylephrine and bolus additions of the thromboxane mimetic U46619 to attain a baseline perfusion pressure of 100-120 mm Hg. The vasodilator effects of a nitroxyl anion donor, Angeli's salt, were examined in the absence and presence of HNO and NO* scavengers, K+ channel inhibition, and soluble guanylate cyclase (sGC) inhibition. In addition, the inotropic and chronotropic effects of Angeli's salt were examined in hearts at resting perfusion pressure (50-60 mm Hg) and compared to responses evoked by acetylcholine and isoprenaline. RESULTS: Angeli's salt causes a potent and reproducible vasodilatation in isolated perfused rat hearts. This response is unaffected by the NO* scavenger hydroxocobalamin (0.1 mM) but is significantly inhibited by the HNO scavenger N-acetyl-L-cysteine (4 mM), suggesting that HNO is the mediator of the observed responses. Vasodilatation responses to Angeli's salt were virtually abolished in the presence of the sGC inhibitor ODQ (10 microM). The magnitude of the vasodilatation response to Angeli's salt was significantly reduced in the presence of 30 mM K+, 10 microM glibenclamide and in the presence of the calcitonin gene-related peptide (CGRP) antagonist CGRP((8-37)) (0.1 microM). Angeli's salt had little effect on heart rate or force of contraction, whilst isoprenaline and acetylcholine elicited significant positive and negative cardiotropic effects, respectively. CONCLUSIONS: The HNO donor Angeli's salt elicits a potent and reproducible vasodilatation response. The results suggest that the response is elicited by HNO through sGC-mediated CGRP release and K(ATP) channel activation.     

Effects of endotoxin on human myocardial contractility involvement of nitric oxide and peroxynitrite

OBJECTIVES: This study examined the effects of endotoxin on cardiac contractility in human myocardium. BACKGROUND: In animal myocardium, endotoxin and cytokine treatment led to enhanced inducible nitric oxide synthase (iNOS) expression and contractile dysfunction. Effects in human myocardium are unknown. METHODS: Left ventricular myocardial preparations from failing (n = 18) and nonfailing (n = 5) human hearts were incubated for 6 and 12 h in tyrode solution or in tyrode plus lipopolysaccharides (LPS), with LPS plus N(G)-mono-methyl-L-arginine (L-NMMA), with LPS plus hemoglobin or with LPS plus the superoxide scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron). Force of contraction in response to isoprenaline (0.001 to 3 micromol/liter) was determined in electrically stimulated muscle preparations. The iNOS mRNA expression was examined by in situ hybridization and by polymerase chain reaction. The cyclic guanosine monophosphate (cGMP) levels were determined by radioimmunoassay. RESULTS: Isoprenaline concentration dependently increased force of contraction. Six and 12 hours of LPS treatment of failing myocardium decreased maximum inotropic response to isoprenaline by 54% (p = 0.009) and by 69% (p = 0.0023), respectively. In nonfailing myocardium, 12 h of LPS treatment decreased maximum inotropic effect of isoprenaline by 66% (p < 0.001). The LPS effects were attenuated by L-NMMA, hemoglobin and also Tiron. The iNOS mRNA was expressed in all LPS-treated preparations but also in most control myocardial preparations. In situ hybridization revealed iNOS expression within cardiac myocytes. There was no increase in myocardial cGMP content in response to endotoxin. CONCLUSIONS: Endotoxin exposure of human myocardium leads to a depression of cardiac contractility, which is mediated by enhanced iNOS activity and release of nitric oxide (NO). Consecutive reaction of NO with superoxide and formation of peroxynitrite may contribute to the decrease in force of contraction.     

Reduced positive inotropic effects in diseased human ventricular myocardium

In isolated contracting human ventricular myocardium taken from patients undergoing mitral valve replacement the positive inotropic effects of calcium, isoprenaline, dobutamine, dopamine, histamine, milrinone, isobutylmethylxanthine, and theophylline were determined. Calcium (15 mmol X litre-1) produced an increase in force of contraction similar to that of a maximal effective concentration of ouabain (1 X 10(-7) mol X litre-1); significantly lower maximal effects were measured with all the other positive inotropic compounds tested. The addition of these positive inotropic substances after a stable maximum effect with ouabain (1 X 10(-7) mol X litre-1) had been reached increased the incidence of toxicity without producing any further inotropic effects. These results suggest that inotropic substances acting through cyclic adenosine monophosphate give less than the maximum inotropic response in isolated muscle from diseased human hearts, which is not additive to the maximal ouabain induced inotropy.     

Role of cGMP-inhibited phosphodiesterase and sarcoplasmic calcium in mediating the increase in basal heart rate with nitric oxide donors

Nitric oxide (NO) donors increase heart rate (HR) through a guanylyl cyclase-dependent stimulation of the pacemaker current I(f), without affecting basal I(Ca-L). The activity of I(f)is known to be enhanced by cyclic nucleotides and by an increase in cytosolic Ca(2+). We examined the role of cGMP-dependent signaling pathways and intracellular Ca(2+)stores in mediating the positive chronotropic effect of NO donors. In isolated guinea pig atria, the increase in HR in response to 1-100 micromol/l 3-morpholino-sydnonimine (SIN-1; with superoxide dismutase, n=6) or diethylamine-NO (DEA-NO, n=8) was significantly attenuated by blockers of the cGMP-inhibited phosphodiesterase (PDE3; trequinsin, milrinone or Ro-13-6438, n=22). In addition, the rate response to DEA-NO or sodium nitroprusside (SNP) was significantly reduced following inhibition of PKA (KT5720 or H-89, n=15) but not PKG (KT5728 or Rp-8-pCPT-cGMPs, n=16). Suppression of sarcoplasmic (SR) Ca(2+)release by pretreatment of isolated atria with ryanodine or cyclopiazonic acid (2 micromol/l and 60 micromol/l, n=16) significantly reduced the chronotropic response to 1-100 micromol/l SIN-1 or DEA-NO. Moreover, in isolated guinea pig sinoatrial node cells 5 micromol/l SNP significantly increased diastolic and peak Ca(2+)fluorescence (+13+/-1% and +28+/-1%, n=6, P<0.05). Our findings are consistent with a functionally significant role of cAMP/PKA signaling (via cGMP inhibition of PDE3) and SR Ca(2+)in mediating the positive chronotropic effect of NO donors.     

Concentration-response curves of positive inotropic agents before and after ouabain pretreatment

Current therapy for congestive heart failure (diuretics, digitalis, vasodilators) may be insufficient. Addition of a second positive inotropic substance to digitalised patients has been previously shown to increase cardiac index and decrease vascular resistance. To test the hypothesis that the positive inotropy of ouabain can be increased by other inotropic agents, the following studies were performed. Firstly, concentration-response curves of positive inotropic agents (ouabain, dobutamine, dopamine, orciprenaline, phenylephrine, theophylline, amrinone, sulmazole and histamine) were measured in contracting left atria and papillary muscles from cat and guinea pig hearts. The maximal increase in force of contraction was similar for all compounds except histamine and phenylephrine which gave decreased effects in guinea pig heart muscle. Secondly, these positive inotropic agents were added to the contracting heart muscles after maximal inotropy without toxicity of a ouabain concentration which gave more than 90% of the maximal increase in force of contraction. In guinea pig left atria, dobutamine was the only compound to give a significant, although transient, increase in force of contraction above the maximal ouabain response. Theophylline (2 X 10(-4) mol X litre-1, EC25) produced significant decreases in force of contraction. In papillary muscles, low concentrations of all positive inotropic compounds, except amrinone, significantly increased force of contraction after a submaximal ouabain concentration. However, the maximal increase in force of contraction after combined addition of ouabain and a second inotropic agent was not different from the maximal increase with ouabain, dobutamine or dopamine alone. Addition of higher concentrations of the second inotropic agents after ouabain pretreatment led to a markedly increased incidence of toxicity with only transient positive inotropic effects. These results indicate that any haemodynamic improvement observed in adequately digitalised patients after combined positive inotropic therapy is unlikely to result from directly additive inotropic effects but is probably a result of other cardiovascular effects such as vasodilatation.     

Reversal of angiotensin II effect on the cyclic adenosine 3',5' monophosphate response to isoprenaline in cardiac hypertrophy.

STUDY OBJECTIVE--The aim was to examine the effect of angiotensin II on the response of hypertrophic cardiac myocytes to the beta adrenergic receptor agonist isoprenaline. DESIGN--Viable ventricular myocytes were isolated from hypertrophic hearts of Dahl S rats and hearts of Dahl R rats. Myocytes were stimulated with isoprenaline. cAMP content was measured by competitive binding radioimmunoassay. MEASUREMENTS AND MAIN RESULTS--Hypertrophic Dahl S myocytes had smaller cAMP response to isoprenaline than Dahl R myocytes (p less than 0.05). Angiotensin II inhibited cAMP stimulation in Dahl R myocytes. In contrast, angiotensin II increased cAMP accumulation in hypertrophic Dahl S myocytes compared to the effect of isoprenaline alone (p less than 0.05). Enhancement of isoprenaline stimulation of cAMP accumulation by angiotensin II was positively related to the degree of cardiac hypertrophy. Angiotensin II treatment in the absence of isoprenaline had no effect on cAMP levels. CONCLUSION--The reversal of the normal inhibitory action of angiotensin II is evidence of a unique alteration in the signal transduction of beta receptor stimulation and is of potential importance in defining the role of angiotensin II in cardiac hypertrophy.     

Effect of positive inotropic agents on myosin isozyme population and mechanical activity of cultured rat heart myocytes.

To examine whether catecholamines have a direct effect on myosin heavy chain expression of heart myocytes or whether they act via an altered work load, myocytes from neonatal rat hearts were cultured in thyroid hormone-free media containing various positive inotropic and chronotropic agents. The velocity and frequency of contraction of the myocytes were monitored using an optoelectronic system. After 3-5 days of culture, myosin isozyme populations, cellular cAMP content, and 2-deoxy-D-glucose uptake of the myocytes were determined. Compared with myocytes cultured in the absence of inotropic agents (32.6 +/- 3.5% V1), the proportion of myosin V1 was significantly (p less than 0.05) increased in the case of 1 microM isoproterenol (48.2 +/- 5.9% V1), 1 microM forskolin (57.1 +/- 11.7% V1), and 1 mM dibutyryl cAMP (79.1 +/- 2.0% V1). Dibutyryl cAMP increased V1 to a similar level as 30 nM triiodothyronine did (70.2 +/- 13.0% V1). Only a small increase was observed in myocytes cultured in the presence of 10 microM phenylephrine (40.4 +/- 8.4% V1), 10 microM ouabain (40.6 +/- 11.9% V1), or 10 microM Bay K 8644 (40.7 +/- 11.7% V1). The agents with a marked effect on myosin heavy chain expression resulted in a higher cAMP content; isoproterenol and forskolin also stimulated 2-deoxy-D-glucose uptake. All agents resulted in a higher velocity of contraction; with the exception of ouabain, frequency of contraction was also increased. A change in Ca2+ concentration in the medium from 1.3 to 2.4 mM resulted in a small increase in V1 (40.7 +/- 5.2% V1) but had the same effect on contraction velocity as dibutyryl cAMP did. Furthermore, 10 nM isoproterenol also increased V1 in myocytes that were arrested with 10 microM verapamil. The increase in V1 in the case of dibutyryl cAMP, isoproterenol, and forskolin is thus most probably not a correlate of the increased mechanical activity but of the high cellular cAMP content.     

Nitric oxide prevents oxidised LDL-induced p53 accumulation, cytochrome c translocation, and apoptosis in macrophages via guanylate cyclase stimulation

BACKGROUND: Oxidatively modified low density lipoprotein (OxLDL) induces apoptosis in vascular cells including macrophages, while NO exerts antiapoptotic effects. Here we studied the impact of nitric oxide (NO) on OxLDL-induced cytochrome c release, apoptosis, and expression of the proapoptotic p53 in macrophages. METHODS: Human LDL was oxidised by Cu(2+), and monocytes were prepared from human buffy coats. Differentiation to macrophages was achieved by culturing cells in the presence of human serum and was followed by detecting monocyte chemoattractant protein 1 (MCP-1) expression (RT-PCR). Cytochrome c release and p53 expression of macrophages were detected by immunoblotting, and apoptosis by visualisation of nuclear condensation. RESULTS: OxLDL dose-dependently (50-200 microg/ml) induced cytochrome c release that was prevented by preincubation with the NO-donor S-nitrosoglutathione (GSNO) (100 microM) or with the cGMP analogue 8-br-cGMP (100 microM) for 15 h. In cells co-treated with GSNO and the soluble guanylate cyclase (sGC) inhibitor oxadialoquinoxalione (ODQ, 10 microM, 15 h), OxLDL-evoked cytochrome c release remained effective, indicating that NO acted via sGC-dependent cGMP formation. Parallel incubation of macrophages with 8-br-cGMP (100 microM) and ODQ (10 microM) for 15 h left the protective effect of 8-br-cGMP unaltered. Short pre-incubation (30 min) with GSNO or 8-br-cGMP was ineffective in preventing OxLDL-elicited cytochrome c release. Initiation of cytochrome c release in macrophages was paralleled by a dose-dependent accumulation of the proapoptotic factor p53, and by enhanced rate of nuclear condensation. Stabilisation of p53 was prevented by preincubation with the NO-donor GSNO or 8-br-cGMP, thus implying a downmodulatory effect of cGMP on pathways that upregulate the tumor suppressor p53. CONCLUSIONS: OxLDL induces cytochrome c release and apoptosis in human macrophages in close association with p53 accumulation. NO attenuates OxLDL-induced cytochrome c release and p53 accumulation via activation of sGC and cGMP formation. These effects may be of particular importance in arterial tissue with reduced NO activity.     

Comparative relaxant effects of YC-1 and DEA/NO on the sheep sphincter of Oddi.

BACKGROUND/AIMS: Nitric oxide (NO) is a major inhibitor in various parts of the gastrointestinal tract. This study was designed to compare the effects of YC-1, NO-independent soluble guanylate cyclase (sGC) activator, and DEA/NO, NO-nucleophile adduct, on sheep sphincters of Oddi (SO). METHODS: SO rings were mounted in a tissue bath and tested for changes in isometric tension in response to 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1, 10(-10)-10(-5)M), diethylamine/NO complex (DEA/NO, 10(-8)-10(-4)M). We also evaluated the effect of YC-1 (10(-6) and 10(-5)M) and DEA/NO (10(-5) and 10(-4)M) on the levels cyclic GMP (cGMP) in isolated SO. RESULTS: YC-1 (10(-10)-10(-5) M) and DEA/NO (10(-8)-10(-4)M) induced concentration-dependent relaxation of isolated SO rings precontracted with carbachol (10(-6)M). The pEC(50) value of DEA/NO was significantly lower than those for YC-1 (p < 0.05), with no change of E(max) values. YC-1 increased cGMP levels more than control, carbachol and DEA/NO groups (p < 0.05). CONCLUSION: These results show that YC-1 is a more potent relaxant than DEA/NO and causes more elevation of cGMP levels in isolated SO rings.     

Nitric oxide effects depend on different mechanisms in different regions of the rat heart

The important role of nitric oxide (NO) in regulating cardiac functions has been investigated in prior research. However, NO-induced signaling mechanisms in the different regions of the heart have not been explored until now. In this study, the mechanism of NO effects on the spontaneously beating right atrium and left papillary muscle isolated from the rat heart was examined. The NO donor diethylamine NONOate (DEA/NO) (0.1–100 μM) depressed the resting and developed tensions, as well as the sinus rate, of the right atrium. The effect of DEA/NO on contractions of the right atrium was blocked by the soluble guanylate cyclase (sGC) inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one) (10 μM). The ATP-sensitive potassium channel (K_ATP) blocker glyburide (3 μM) reversed DEA/NO-induced decreases in the resting tension. The suppressor effect of DEA/NO on the sinus rate was inhibited only by the superoxide radical scavenger superoxide dismutase (25 U/ml). Neither the cGMP-dependent protein kinase (PKG) inhibitor KT5823 (0.1 μM) nor the cAMP-dependent protein kinase (PKA) inhibitor KT5720 (1 μM) changed DEA/NO responses in the right atrium. While the resting tension of the right atrium was decreased by the NO precursor l-arginine (1–100 μM), it was increased by the nitric oxide synthase inhibitor l-NMMA (0.1–100 μM). The sinus rate was not affected by l-arginine or l-NMMA. The left papillary muscle contraction was not influenced by any of these NO-related agents. These results show that high concentration NO-induced depression of the contraction of the right atrium is due to sGC and K_ATP channel activation, but suppression of the sinus rate depends on redox regulation. Our results may have important implications for the region-dependent functional disability of cardiac myocytes, as well as the regulation of heart performance in high NO-induced pathological conditions.     

Cardiomyocytes as effectors of nitric oxide signalling

Nitric oxide (NO) generated constitutively within the heart has long been known to influence myocardial function; however, the precise nature of these effects has been controversial--at least in part--because of the experimental use of non-isoform-selective inhibitors of NO synthases (NOS) and unwarranted extrapolation from results obtained with NO donors. Recent studies using NOS-selective inhibitors and genetically modified models are beginning to redress the balance. It is well established that agonist-stimulated release of NO from eNOS in the coronary endothelium exerts paracrine effects on cardiomyocytes, predominantly affecting the timing of relaxation as well as myocardial oxygen consumption. A significant recent advance has been the finding that both eNOS and nNOS are constitutively expressed in distinct subcellular locations within cardiomyocytes. The relative autocrine role of these isoforms in the cardiomyocyte remains to be fully clarified but evidence suggests that the autocrine effects of nNOS may include the modulation of basal inotropy and relaxation, beta-adrenergic responsiveness, and the force-frequency relationship. Myocardial eNOS, on the other hand, may be involved in mediating the inotropic response to sustained stretch. These effects may change significantly in the diseased heart where the expression, activity and/or coupling of NOS isoforms to downstream effectors may be altered. In this article, we review the current understanding of this important but complex field, focussing particularly on contractile function and on recent advances in knowledge regarding the autocrine functions of nNOS-derived NO.     

自发性高血压大鼠心脏血管紧张素Ⅱ受体与β肾上腺素受体的交互作用

目的　研究 14周龄自发性高血压大鼠 (SHR)与 14周龄SD大鼠心脏血管紧张素Ⅱ (AngⅡ )受体与β肾上腺素受体 (β AR)之间的交互作用。 方法　采用放射免疫法测定异丙基肾上腺素 (Iso)及AngⅡ诱导的环磷酸腺苷(cAMP)、环磷酸鸟苷 (cGMP)蓄积水平 ;采用体外左心房收缩功能实验观察AngⅡ对Iso激动 β AR介导正性变力效应的影响。结果　 14周龄SHR ,基础cAMP水平明显降低 ,Iso刺激后两组大鼠心脏cGMP水平明显下降 ,cAMP水平明显升高 ,AngⅡ加Iso联合刺激后cAMP水平更明显升高 ;AngⅡ使SD大鼠心脏 (n =6 )最大收缩效应增强。结论　AngⅡ对Iso诱导的心脏cAMP蓄积有明显的正协同作用 ;AngⅡ可增强SD大鼠心脏Iso激动 β AR介导的正性变力效应 ,而在SHR这种增强作用消失。     

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.     

An inhibitor of nitric oxide synthase does not increase contraction or beta-adrenoceptor sensitivity of ventricular myocytes from failing human heart

OBJECTIVE: Nitric oxide (NO) has been implicated in the depression of cardiac function in human heart failure. Some reports have identified iNOS (inducible nitric oxide synthase) within the myocyte component of the failing human heart, and NO is known to decrease the contraction amplitude of isolated ventricular myocytes. We have treated myocytes from failing human ventricle with a NOS inhibitor, NG-monomethyl-L-arginine (L-NMMA), in an attempt to restore contractile function. METHODS AND RESULTS: Myocytes were isolated from failing and non-failing human ventricles and their contraction amplitude was measured during superfusion (32 degrees C, 1-2 mmol/l Ca2+) and electrical stimulation (0.1-2 Hz). The contraction amplitude of myocytes from failing hearts was depressed in a frequency-dependent manner. At 1 Hz, the contraction amplitude of cells from non-failing heart was 4.70 +/- 0.53% cell shortening (mean +/- SEM, n = 13 subjects), compared with 2.18 +/- 0.27% (P < 0.01, 11 patients) from patients with ischaemic heart disease (IHD) or 2.56 +/- 0.74% (P < 0.02, six patients) with dilated cardiomyopathy (DCM). Superfusion with 0.1 mmol/l L-NMMA did not increase contraction amplitude in myocytes from failing heart at either 0.2 Hz (n = 11) or 1 Hz (n = 7). Responses to beta-adrenoceptor stimulation were reduced in myocytes from failing human heart, with contraction amplitude in maximum isoprenaline 0.47 +/- 0.11 of that in high Ca2+ in the same cell (n = 6), compared to 0.99 +/- 0.07 in non-failing heart (n = 14, P < 0.01). The presence of 0.1 mmol/l L-NMMA did not increase the isoprenaline/Ca2+ ratio in myocytes from failing heart (0.40 +/- 0.09, P = NS). CONCLUSION: These results do not suggest a functional role for tonic NO production in the frequency-dependent depression of contraction or beta-adrenoceptor desensitisation in myocytes from failing human ventricle.