Sub-cellular targeting of constitutive NOS in health and disease

Constitutive nitric oxide synthases (NOSs) are ubiquitous enzymes that play a pivotal role in the regulation of myocardial function in health and disease. The discovery of both a neuronal NOS (nNOS) and an endothelial NOS (eNOS) isoform in the myocardium and the availability of genetically modified mice with selective eNOS or nNOS gene deletion have been of crucial importance for understanding the role of constitutive nitric oxide (NO) production in the myocardium. eNOS and nNOS are homologous in structure and utilize the same co-factors and substrates; however, they differ in their subcellular localization, regulation, and downstream signaling, all of which may account for their distinct effects on excitation-contraction coupling. In particular, eNOS-derived NO has been reported to increase left ventricular (LV) compliance, attenuate beta-adrenergic inotropy and enhance parasympathetic/muscarinic responses, and mediate the negative inotropic response to β3 adrenoreceptor stimulation via cGMP-dependent signaling. Conversely, nNOS-derived NO regulates basal myocardial inotropy and relaxation by inhibiting the sarcolemmal Ca(2+) current (I(Ca)) and promoting protein kinase A-dependent phospholamban (PLN) phosphorylation, independent of cGMP. By inhibiting the activity of myocardial oxidase systems, nNOS regulates the redox state of the myocardium and contributes to maintain eNOS "coupled" activity. After myocardial infarction, up-regulation of myocardial nNOS attenuates adverse remodeling and prevents arrhythmias whereas uncoupled eNOS activity in murine models of left ventricular pressure overload accelerates the progress towards heart failure. Here we review the evidence in support of the idea that NOS subcellular localization, mode of activation, and downstream signaling account for the diverse and highly specialized actions of NO in the heart. This article is part of a Special Issue entitled "Local Signaling in Myocytes".     

Arginase modulates myocardial contractility by a nitric oxide synthase 1-dependent mechanism

Cardiac myocytes contain two constitutive NO synthase (NOS) isoforms with distinct spatial locations, which allows for isoform-specific regulation. One regulatory mechanism for NOS is substrate (l-arginine) bioavailability. We tested the hypothesis that arginase (Arg), which metabolizes l-arginine, constrains NOS activity in the cardiac myocyte in an isoform-specific manner. Arg activity was detected in both rat heart homogenates and isolated myocytes. Although both Arg I and II mRNA and protein were present in whole heart, Arg II alone was found in isolated myocytes. Arg inhibition with S-(2-boronoethyl)-l-cysteine (BEC) augmented Ca(2+)-dependent NOS activity and NO production in myocytes, which did not depend on extracellular l-arginine. Arg II coimmunoprecipited with NOS1 but not NOS3. Isolation of myocyte mitochondrial fractions in combination with immuno-electron microscopy demonstrates that Arg II is confined primarily to the mitochondria. Because NOS1 positively modulates myocardial contractility, we determined whether Arg inhibition would increase basal myocardial contractility. Consistent with our hypothesis, Arg inhibition increased basal contractility in isolated myocytes by a NOS-dependent mechanism. Both the Arg inhibitors N-hydroxy-nor-l-arginine and BEC dose-dependently increased basal contractility in rat myocytes, which was inhibited by both nonspecific and NOS1-specific NOS inhibitors N(G)-nitro-l-arginine methyl ester and S-methyl-l-thiocitrulline, respectively. Also, BEC increased contractility in isolated myocytes from WT and NOS3 but not NOS1 knockout mice. We conclude that mitochondrial Arg II negatively regulates NOS1 activity, most likely by limiting substrate availability in its microdomain. These findings have implications for therapy in pathophysiologic states such as aging and heart failure in which myocardial NO signaling is disrupted.     

Heterogeneous basal expression of nitric oxide synthase and superoxide dismutase isoforms in mammalian heart : implications for mechanisms governing indirect and direct nitric oxide-related effects.

The basal expression patterns of NO synthase (NOS; endothelial [eNOS], neuronal [nNOS], and cytokine-inducible [iNOS]) and superoxide dismutase (SOD; extracellular membrane bound [ECSOD], MnSOD, and CuZnSOD) isoforms in ferret heart (tissue sections and isolated myocytes) were determined by immunofluorescent localization. We demonstrate the following for the first time in the mammalian heart: (1) heterogeneous expression patterns of the 3 NOS and 3 SOD isoforms among different tissue and myocyte types; (2) colocalization of eNOS and ECSOD at both the tissue and myocyte levels; (3) a significant gradient of eNOS and ECSOD expression across the left ventricular (LV) wall, with both enzymes being highly expressed and colocalized in LV epicardial myocytes but markedly reduced in LV endocardial myocytes; and (4) specific subcellular localization patterns of eNOS and the 3 SOD isoforms. In particular, eNOS and ECSOD are demonstrated (electron and confocal microscopy) to be specifically localized to the sarcolemma of ventricular myocytes. Similar heterogeneous eNOS and ECSOD expression patterns were also obtained in human LV tissue sections, underscoring the general importance of these novel findings. Our data suggest a strong functional correlation between the activities of sarcolemmally localized myocyte eNOS and ECSOD in governing NO*/O(2-) interactions and suggest that NO-related modulatory effects on cardiac myocyte protein and/or ion channel function may be significantly more complex than is presently believed.     

Increased susceptibility to development of triggered activity in myocytes from mice with targeted disruption of endothelial nitric oxide synthase

Nitric oxide generated by cardiac myocytes or delivered by drugs has been shown to regulate cardiac contractile function and has been implicated in suppressing some cardiac arrhythmias, although this remains controversial. We examined the ability of the soluble cardiac glycoside, ouabain, to trigger arrhythmic contractions in ventricular myocytes isolated from mice lacking a functional endothelial nitric oxide synthase gene (eNOS(null)). Arrhythmic activity, defined as aftercontractions, was induced with ouabain (50 micromol/L) and recorded using a video-motion detector in isolated, electrically driven single ventricular myocytes from adult eNOS(null)or from their wild-type (WT) littermates. The rate of ouabain-induced arrhythmic contractions was significantly higher in eNOS(null)myocytes than in WT myocytes. Application of the NO donor S-nitroso-acetylcysteine (SNAC) significantly diminished the frequency of arrhythmic contractions in eNOS(null)myocytes. The antiarrhythmic effect of NO, whether generated by eNOS in WT cells or by SNAC, could be partially reversed by 1H-[1,2,4]oxadiazolo-[4, 3-a]- quinoxalin-1-one (ODQ), a specific soluble guanylyl cyclase inhibitor. Ouabain significantly increased intracellular cGMP in WT but not eNOS(null)hearts, and this cGMP response was blocked by ODQ. Since cardiac glycoside- induced aftercontractions are activated by the transient inward current (I(ti)), the role of NO in ouabain (100 micromol/L)- induced I(ti)was examined using the nystatin-perforated patch-clamp technique. The frequency of ouabain-induced I(ti)was significantly higher in eNOS(null)myocytes than in WT myocytes, and this could be suppressed by SNAC. These data demonstrate that NO derived from myocyte eNOS activation suppresses ouabain-induced arrhythmic contractions by a mechanism that might involve activation of guanylyl cyclase and elevation of cGMP.     

Concerted regulation of skeletal muscle contractility by oxygen tension and endogenous nitric oxide

It is generally accepted that inhibition of nitric oxide synthase (NOS) facilitates, and thus nitric oxide (NO) inhibits, contractility of skeletal muscle. However, standard assessments of contractility are carried out at a nonphysiological oxygen tension [partial pressure of oxygen (pO2)] that can interfere with NO signaling (95% O2). We therefore examined, in normal and neuronal NOS (nNOS)-deficient mice, the influence of pO2 on whole-muscle contractility and on myocyte calcium flux and sarcomere shortening. Here, we demonstrate a significant enhancement of these measures of muscle performance at low physiological pO2 and an inhibitory influence at higher physiological pO2, which depend on endogenous nNOS. At 95% O2 (which produces oxidative stress; muscle core pO2 approximately 400 mmHg), force production is enhanced but control of contractility by NO/nitrosylation is greatly attenuated. In addition, responsivity to pO2 is altered significantly in nNOS mutant muscle. These results reveal a fundamental role for the concerted action of NO and O2 in physiological regulation of skeletal muscle contractility, and suggest novel molecular aspects of myopathic disease. They suggest further that the role of NO in some cellular systems may require reexamination.     

A defect of neuronal nitric oxide synthase increases xanthine oxidase-derived superoxide anion and attenuates the control of myocardial oxygen consumption by nitric oxide derived from endothelial nitric oxide synthase

Endothelial nitric oxide synthase (eNOS) plays an important role in the control of myocardial oxygen consumption (MVO2) by nitric oxide (NO). A NOS isoform is present in cardiac mitochondria and it is derived from neuronal NOS (nNOS). However, the role of nNOS in the control of MVO2 remains unknown. MVO2 in left ventricular tissues from nNOS-/- mice was measured in vitro. Stimulation of NO production by bradykinin or carbachol induced a significant reduction in MVO2 in wild-type (WT) mice. In contrast to WT, bradykinin- or carbachol-induced reduction in MVO2 was attenuated in nNOS-/-. S-methyl-L-thiocitrulline, a potent isoform selective inhibitor of nNOS, had no effect on bradykinin-induced reduction in MVO2 in WT. Bradykinin-induced reduction in MVO2 in eNOS-/- mice, in which nNOS still exists, was also attenuated. The attenuated bradykinin-induced reduction in MVO2 in nNOS-/- was restored by preincubation with Tiron, ascorbic acid, Tempol, oxypurinol, or SB203850, an inhibitor of p38 kinase, but not apocynin. There was an increase in lucigenin-detectable superoxide anion (O2-) in cardiac tissues from nNOS-/- compared with WT. Tempol, oxypurinol, or SB203850 decreased O2- in all groups to levels that were not different from each other. There was an increase in phosphorylated p38 kinase normalized by total p38 kinase protein level in nNOS-/- compared with WT mice. These results indicate that a defect of nNOS increases O2- through the activation of xanthine oxidase, which is mediated by the activation of p38 kinase, and attenuates the control of MVO2 by NO derived from eNOS.     

Conditional neuronal nitric oxide synthase overexpression impairs myocardial contractility

The role of the neuronal NO synthase (nNOS or NOS1) enzyme in the control of cardiac function still remains unclear. Results from nNOS(-/-) mice or from pharmacological inhibition of nNOS are contradictory and do not pay tribute to the fact that probably spatial confinement of the nNOS enzyme is of major importance. We hypothesize that the close proximity of nNOS and certain effector molecules like L-type Ca(2+)-channels has an impact on myocardial contractility. To test this, we generated a new transgenic mouse model allowing conditional, myocardial specific nNOS overexpression. Western blot analysis of transgenic nNOS overexpression showed a 6-fold increase in nNOS protein expression compared with noninduced littermates (n=12; P<0.01). Measuring of total NOS activity by conversion of [(3)H]-l-arginine to [(3)H]-l-citrulline showed a 30% increase in nNOS overexpressing mice (n=18; P<0.05). After a 2 week induction, nNOS overexpression mice showed reduced myocardial contractility. In vivo examinations of the nNOS overexpressing mice revealed a 17+/-3% decrease of +dp/dt(max) compared with noninduced mice (P<0.05). Likewise, ejection fraction was reduced significantly (42% versus 65%; n=15; P<0.05). Interestingly, coimmunoprecipitation experiments indicated interaction of nNOS with SR Ca(2+)ATPase and additionally with L-type Ca(2+)- channels in nNOS overexpressing animals. Accordingly, in adult isolated cardiac myocytes, I(Ca,L) density was significantly decreased in the nNOS overexpressing cells. Intracellular Ca(2+)-transients and fractional shortening in cardiomyocytes were also clearly impaired in nNOS overexpressing mice versus noninduced littermates. In conclusion, conditional myocardial specific overexpression of nNOS in a transgenic animal model reduced myocardial contractility. We suggest that nNOS might suppress the function of L-type Ca(2+)-channels and in turn reduces Ca(2+)-transients which accounts for the negative inotropic effect.     

Disruption of inhibitory G-proteins mediates a reduction in atrial beta-adrenergic signaling by enhancing eNOS expression

OBJECTIVE: Cardiac parasympathetic nerve activity is reduced in most cardiovascular disease states, and this may contribute to enhanced cardiac sympathetic responsiveness. Disruption of inhibitory G-proteins (Gi) ablates the cholinergic pathway and increases cardiac endothelial nitric oxide (NO) synthase (eNOS) expression, suggesting that NO may offset the impaired attenuation of beta-adrenergic regulation of supraventricular excitability. To test this, we investigated the role of endogenous NO production on beta-adrenergic regulation of rate (HR), contraction (CR) and calcium (Ca2+) handling in atria following blockade of Gi-coupled muscarinic receptors. METHODS: Mice were administered pertussis toxin (PTx, n=105) or saline (C, n=100) intraperitoneally. After 3 days, we measured CR, HR, and NOS protein levels in isolated atria. Intracellular calcium (Ca2+) transients and Ca2+ current density (I(Ca)) were also measured in atrial myocytes. RESULTS: PTx treatment increased atrial myocyte eNOS protein levels compared to C (P<0.05). This did not affect basal atrial function but was associated with a significant reduction in the CR and HR response to isoprenaline (ISO) compared with C. NOS inhibition normalized responses in PTx atria with respect to responses in C atria (P<0.05), which were unaffected. Furthermore, PTx did not affect ISO-stimulated HR and CR in eNOS gene knockout mice (n=40). In agreement with these findings, the ISO-mediated increase in Ca2+ transient was suppressed in PTx-treated myocytes (P<0.05), whereas I(Ca) did not differ between groups. CONCLUSION: eNOS-derived NO inhibits beta-adrenergic responses following disruption of Gi signaling. This suggests that increased eNOS expression may be a compensatory mechanism which reduces beta-adrenergic regulation of heart rate when cardiac parasympathetic control is impaired.     

Differential Central NOS‐NO Signaling Underlies Clonidine Exacerbation of Ethanol‐Evoked Behavioral Impairment

Background: The molecular mechanisms that underlie clonidine exacerbation of behavioral impairment caused by ethanol are not fully known. We tested the hypothesis that nitric oxide synthase (NOS)‐derived nitric oxide (NO) signaling in the locus coeruleus (LC) is implicated in this phenomenon. Methods: Male Sprague–Dawley rats with intracisternal (i.c.) and jugular vein cannulae implanted 6 days earlier were tested for drug‐induced behavioral impairment. The latter was assessed as the duration of loss of righting reflex (LORR) and rotorod performance every 15 minutes until the rat recovered to the baseline walk criterion (180 seconds). In a separate cohort, we measured p‐neuronal NOS (nNOS), p‐endothelial NOS (eNOS), and p‐ERK1/2 in the LC following drug treatment, vehicle, or NOS inhibitor. Results: Rats that received clonidine [60 Ig/kg, i.v. (intravenous)] followed by ethanol (1 or 1.5 g/kg, i.v.) exhibited synergistic impairment of rotorod performance. Intracisternal pretreatment with nonselective NOS inhibitor N^ω‐nitro‐l ‐arginine methyl ester (l ‐NAME, 0.5 mg) or selective nNOS inhibitor N‐propyl‐l ‐arginine (1 μg) exacerbated the impairment of rotorod performance caused by clonidine–ethanol combination. Exacerbation of behavioral impairment was caused by l ‐NAME enhancement of the effect of ethanol, not clonidine. l ‐NAME did not influence blood ethanol levels; thus, the interaction was pharmacodynamic. LORR caused by clonidine (60 μg/kg, i.v.)–ethanol (1 g/kg, i.v.) combination was abolished by selective inhibition of central eNOS (l ‐NIO, 10 μg i.c.) but not by nNOS inhibition under the same conditions. Western blot analyses complemented the pharmacological evidence by demonstrating that clonidine–ethanol combination inhibits phosphorylation (activation) of nNOS (p‐nNOS) and increases the level of phosphorylated eNOS (p‐eNOS) in the LC; the change in p‐nNOS was paralleled by similar change in LC p‐ERK1/2. NOS inhibitors alone did not affect the level of nitrate/nitrite, p‐nNOS, p‐eNOS, or p‐ERK1/2 in the LC. Conclusions: Alterations in NOS‐derived NO in the LC underlie clonidine–ethanol induced behavioral impairment. A decrease in nNOS activity, due at least partly to a reduction in nNOS phosphorylation, mediates rotorod impairment, while enhanced eNOS activity contributes to LORR, elicited by clonidine–ethanol combination.     

Does nitric oxide modulate cardiac ryanodine receptor function? Implications for excitation-contraction coupling

Nitric oxide (NO) is a highly reactive, free radical signalling molecule that is constitutively released in cardiomyocytes by both the endothelial and neuronal isoforms of nitric oxide synthase (eNOS and nNOS, respectively). There are increasing data indicating that NO modulates various proteins involved in excitation-contraction coupling (ECC), and here we discuss the evidence that NO may modulate the function of the ryanodine receptor Ca(2+) release channel (RyR2) on the cardiac sarcoplasmic reticulum (SR). Both constitutive isoforms of NOS have been shown to co-immunoprecipitate with RyR2, suggesting that the channel may be a target protein for NO. eNOS gene deletion has been shown to abolish the increase in spontaneous Ca(2+) spark frequency in cardiomyocytes exposed to sustained stretch, whereas the effect of nNOS-derived NO on RyR2 function remains to be investigated. Single channel studies have been performed with RyR2 reconstituted in planar lipid bilayers and exposed to various NO donors and, under these conditions, NO appears to have a dose-dependent, stimulatory effect on channel open probability (P(open)). We discuss whether NO has a direct effect on RyR2 via covalent S-nitrosylation of reactive thiol residues within the protein, or whether there are downstream effects via cyclic nucleotides, phosphodiesterases, and protein kinases. Finally, we consider whether the proposed migration of nNOS from the SR to the sarcolemma in the failing heart may have consequences for the nitrosative vs. oxidative balance at the level of the RyR2, and whether this may contribute to an increased diastolic Ca(2+) leak, depleted SR Ca(2+) store, and reduced contractility in heart failure.     

Beta 3-adrenoreceptor regulation of nitric oxide in the cardiovascular system

The presence of a third β-adrenergic receptor (β3-AR) in the cardiovascular system has challenged the classical paradigm of sympathetic regulation by β1- and β2-adrenergic receptors. While β3-AR's role in the cardiovascular system remains controversial, increasing evidence suggests that it serves as a “brake” in sympathetic overstimulation — it is activated at high catecholamine concentrations, producing a negative inotropic effect that antagonizes β1- and β2-AR activity. The anti-adrenergic effects induced by β3-AR were initially linked to nitric oxide (NO) release via endothelial NO synthase (eNOS), although more recently it has been shown under some conditions to increase NO production in the cardiovascular system via the other two NOS isoforms, namely inducible NOS (iNOS) and neuronal NOS (nNOS). We summarize recent findings regarding β3-AR effects on the cardiovascular system and explore its prospective as a therapeutic target, particularly focusing on its emerging role as an important mediator of NO signaling in the pathogenesis of cardiovascular disorders.     

Reduced phospholamban phosphorylation is associated with impaired relaxation in left ventricular myocytes from neuronal NO synthase-deficient mice

Stimulation of nitric oxide (NO) release from the coronary endothelium facilitates myocardial relaxation via a cGMP-dependent reduction in myofilament Ca2+ sensitivity. Recent evidence suggests that NO released by a neuronal NO synthase (nNOS) in the myocardium can also hasten left ventricular relaxation; however, the mechanism underlying these findings is uncertain. Here we show that both relaxation (TR50) and the rate of [Ca2+]i transient decay (tau) are significantly prolonged in field-stimulated or voltage-clamped left ventricular myocytes from nNOS-/- mice and in wild-type myocytes (nNOS+/+) after acute nNOS inhibition. Disabling the sarcoplasmic reticulum abolished the differences in TR50 and tau, suggesting that impaired sarcoplasmic reticulum Ca2+ reuptake may account for the slower relaxation in nNOS-/- mice. In line with these findings, disruption of nNOS (but not of endothelial NOS) decreased phospholamban phosphorylation (P-Ser16 PLN), whereas nNOS inhibition had no effect on TR50 or tau in PLN-/- myocytes. Inhibition of cGMP signaling had no effect on relaxation in either group whereas protein kinase A inhibition abolished the difference in relaxation and PLN phosphorylation by decreasing P-Ser16 PLN and prolonging TR50 in nNOS+/+ myocytes. Conversely, inhibition of type 1 or 2A protein phosphatases shortened TR50 and increased P-Ser16 PLN in nNOS-/- but not in nNOS+/+ myocytes, in agreement with data showing increased protein phosphatase activity in nNOS-/- hearts. Taken together, our findings identify a novel mechanism by which myocardial nNOS promotes left ventricular relaxation by regulating the protein kinase A-mediated phosphorylation of PLN and the rate of sarcoplasmic reticulum Ca2+ reuptake via a cGMP-independent effect on protein phosphatase activity.     

Interactions between NO and O2 in the microcirculation: a mathematical analysis

Biotransport of nitric oxide (NO) and of oxygen (O(2)) in the microcirculation are inherently interdependent, since all nitric oxide synthase (NOS) isoforms (eNOS, nNOS, and iNOS) require O(2) to produce NO. Furthermore, tissue O(2) consumption is reversibly inhibited by NO. To investigate these complex interactions, a mathematical model was developed for coupled mass transport of NO and O(2) around a cylindrical arteriole using finite element computational methods. Steady-state radial NO and O(2) gradients in the bloodstream, plasma layer, endothelium, vascular wall, and surrounding tissue were simulated for different conditions. Special cases of the model were solved, including O(2)-dependent NO production from eNOS alone, and with additional NO production from either nNOS or iNOS at specified locations. The model predicts that (a) concentration changes in one species can have significant effects on transport of the other species with the degree of interaction dependent on spatial gradients; (b) eNOS NO production rates required to maintain the concentration of NO in the vascular wall are more dependent on NO scavenging in blood than in tissue; (c) relatively low rates of NO production in tissue from either nNOS or iNOS can elevate vascular wall NO, compensating for possible reductions in NO production from eNOS; (d) depending on their physical location, nNOS and iNOS can be very sensitive to O(2); and (e) increased tissue NO can increase O(2) delivery to more distal regions by inhibiting O(2) consumption in other regions.     

Expression, localization, and regulation of NOS in human mast cell lines: effects on leukotriene production

Nitric oxide (NO) is a potent radical produced by nitric oxide synthase (NOS) and has pleiotrophic activities in health and disease. As mast cells (MCs) play a central role in both homeostasis and pathology, we investigated NOS expression and NO production in human MC populations. Endothelial NOS (eNOS) was ubiquitously expressed in both human MC lines and skin-derived MCs, while neuronal NOS (nNOS) was variably expressed in the MC populations studied. The inducible (iNOS) isoform was not detected in human MCs. Both growth factor-independent (HMC-1) and -dependent (LAD 2) MC lines showed predominant nuclear eNOS protein localization, with weaker cytoplasmic expression. nNOS showed exclusive cytoplasmic localization in HMC-1. Activation with Ca(2+) ionophore (A23187) or IgE-anti-IgE induced eNOS phosphorylation and translocation to the nucleus and nuclear and cytoplasmic NO formation. eNOS colocalizes with the leukotriene (LT)-initiating enzyme 5-lipoxygenase (5-LO) in the MC nucleus. The NO donor, S-nitrosoglutathione (SNOG), inhibited, whereas the NOS inhibitor, N(G)-nitro-l-arginine methyl ester (L-NAME), potentiated LT release in a dose-dependent manner. Thus, human MC lines produce NO in both cytoplasmic and nuclear compartments, and endogenously produced NO can regulate LT production by MCs.     

A connecting hinge represses the activity of endothelial nitric oxide synthase

In mammals, endothelial nitric oxide synthase (eNOS) has the weakest activity, being one-tenth and one-sixth as active as the inducible NOS (iNOS) and the neuronal NOS (nNOS), respectively. The basis for this weak activity is unclear. We hypothesized that a hinge element that connects the FMN module in the reductase domain but is shorter and of unique composition in eNOS may be involved. To test this hypothesis, we generated an eNOS chimera that contained the nNOS hinge and two mutants that either eliminated (P728IeNOS) or incorporated (I958PnNOS) a proline residue unique to the eNOS hinge. Incorporating the nNOS hinge into eNOS increased NO synthesis activity 4-fold, to an activity two-thirds that of nNOS. It also decreased uncoupled NADPH oxidation, increased the apparent K(m)O(2) for NO synthesis, and caused a faster heme reduction. Eliminating the hinge proline had similar, but lesser, effects. Our findings reveal that the hinge is an important regulator and show that differences in its composition restrict the activity of eNOS relative to other NOS enzymes.     

Effects of the β3-Adrenergic Agonist BRL 37344 on Endothelial Nitric Oxide Synthase Phosphorylation and Force of Contraction in Human Failing Myocardium

BackgroundIn nonfailing myocardium, β₃-adrenergic signaling causes a decrease in contractility via endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) release. This study investigates the hypothesis that β₃-adrenergic signaling undergoes alterations in failing myocardium.MethodsWe compared eNOS- and β₃-adrenoceptor expression using Western blot analysis in human nonfailing myocardium versus failing myocardium. With the use of immunohistochemistry, we investigated the distribution of the β₃-adrenoceptor protein and eNOS translocation and phosphorylation under basal conditions. β₃-adrenergic, eNOS activation, and inotropy were measured in failing myocardium using BRL37344 (BRL, a β₃-adrenoceptor agonist).Resultsβ₃-adrenoceptor expression was increased in failing myocardium. Under basal conditions, Akt- and eNOS^Ser1177 phosphorylation were reduced in failing myocardium. During stimulation with BRL in failing myocardium, a further dephosphorylation of eNOS^Ser1177 and Akt was observed, whereas eNOS^Ser114 phosphorylation was increased. These results suggest a deactivation of eNOS via β₃-adrenergic stimulation. Nevertheless, BRL decreased contractility in failing myocardium, but this effect was not observed in the presence of the NO blocker L-NMA. In failing myocardium, the β₃-adrenoceptor was predominantly expressed in endothelial cells. In the cardiomyocytes, the β₃-adrenoceptor was mainly located at the intercalated disks.ConclusionIn failing cardiomyocytes, β₃-adrenergic stimulation seems to deactivate rather than activate eNOS. At the same time, β₃-adrenergic stimulation induced a NO-dependent negative inotropic effect. Because β₃-adrenoceptors are expressed mainly in the endothelium in failing myocardium, our observations suggest a paracrine-negative inotropic effect via NO liberation from the cardiac endothelial cells.     

Co-expression and modulation of neuronal and endothelial nitric oxide synthase in human endothelial cells

Despite originally identified in neurones, the neuronal type of nitric oxide synthase (nNOS) is present also in cardiac and skeletal myocytes. Whether nNOS is functionally expressed in human endothelial cells--as the endothelial enzyme (eNOS)--is unknown. Human umbilical vein endothelial cells (HUVEC) were studied under control culture conditions and after 48 h treatment with cytomix (human tumour necrosis factor-alpha, interferon-gamma and E. coli endotoxin). We tested: (i) localisation and expression of nNOS and eNOS proteins by immunostaining and immunoblotting; (ii) activity of nNOS and eNOS by measuring L-arginine to L-citrulline conversion with 1-(2-trifluoromethylphenyl)imidazole (TRIM), a specific nNOS antagonist, in sub-cellular fractions; (iii) intracellular cGMP levels, as a marker for nitric oxide production, after TRIM pre-treatment, by radioimmunoassay. nNOS protein was expressed in the cytosolic fraction and immunolocalised in cultured HUVEC, and co-localised with the eNOS protein in frozen sections of the human umbilical cord. nNOS protein contributed to total L-citrulline production as TRIM selectively and dose-dependently reduced L-citrulline synthesis in the cytosolic but not particulate fraction of HUVEC. Similarly, TRIM reduced intracellular cGMP content both at baseline and after stimulation with a calcium ionophore. Cytomix down-regulated the expression and function of both nNOS and eNOS while no inducible NOS (iNOS) was detected. In conclusion, a functional neuronal type of NOS is co-expressed with the endothelial NOS type in HUVEC, suggesting a possible role for nNOS in regulation of blood flow.     

Dynamic denitrosylation via S-nitrosoglutathione reductase regulates cardiovascular function

Although protein S-nitrosylation is increasingly recognized as mediating nitric oxide (NO) signaling, roles for protein denitrosylation in physiology remain unknown. Here, we show that S-nitrosoglutathione reductase (GSNOR), an enzyme that governs levels of S-nitrosylation by promoting protein denitrosylation, regulates both peripheral vascular tone and β-adrenergic agonist-stimulated cardiac contractility, previously ascribed exclusively to NO/cGMP. GSNOR-deficient mice exhibited reduced peripheral vascular tone and depressed β-adrenergic inotropic responses that were associated with impaired β-agonist-induced denitrosylation of cardiac ryanodine receptor 2 (RyR2), resulting in calcium leak. These results indicate that systemic hemodynamic responses (vascular tone and cardiac contractility), both under basal conditions and after adrenergic activation, are regulated through concerted actions of NO synthase/GSNOR and that aberrant denitrosylation impairs cardiovascular function. Our findings support the notion that dynamic S-nitrosylation/denitrosylation reactions are essential in cardiovascular regulation.     

Caveolins and the regulation of endothelial nitric oxide synthase in the heart

Virtually all cell types within the myocardium express caveolae, where cell-specific isoforms of caveolin both maintain the structural organisation of these cholesterol-rich of the plasmalemma and serve as scaffolds for the dynamic constitution of "signalosomes", or hubs concentrating numerous transmembrane signaling proteins and their effectors. Analysis of the phenotype of mice with genetic deletion or overexpression of specific caveolin isoforms has provided key evidence for the importance of caveolins and caveolae in several aspects of the cardiovascular biology, including vascular contractility, lipid metabolism, angiogenesis, or the control of cardiac hypertrophy. Among specific protein-protein interactions involving caveolins in cardiac tissue, these genetic models unequivocally confirmed the functional importance of the dynamic association of the endothelial isoform of nitric oxide synthase (eNOS) for its post-translational regulation in endothelial cells and cardiac myocytes, which bears on the enzyme's capacity to modulate nitric oxide (NO)-dependent endothelial function, angiogenesis, and excitation-contraction coupling. We will review the current understanding of this regulation of eNOS (and potentially other NOS isoforms) through protein-protein interactions involving several G-protein-coupled receptors and other allosteric modulators in the context of emerging paradigms on the regulation of cardiac function by NO.