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T Michel

Publications and source records attributed to T Michel.

At least 73 records · Page 4Linked to original sources
PubMed1995

Oligomerization of endothelial nitric oxide synthase. Evidence for a dominant negative effect of truncation mutants.

Nitric oxide produced by the endothelial isoform of nitric oxide synthase (ecNOS) is a key determinant of vascular tone. In contrast to other nitric oxide synthase (NOS) isoforms, which have been characterized as soluble homodimeric enzymes, ecNOS is predominantly membrane-associated, a feature that has hindered direct biochemical analyses of its oligomeric structure. We investigated ecNOS oligomerization using co-immunoprecipitation experiments in transiently transfected COS-7 cells. When COS-7 cells co-transfected with constructs encoding wild-type ecNOS and an epitope-tagged myristoylation-deficient mutant were biosynthetically labeled with [3H]myristate, the antibody to the epitope tag specifically immunoprecipitated 3H-labeled ecNOS, reflecting enzyme oligomerization. In COS-7 cells transfected with cDNAs encoding epitope-tagged truncation mutants and untagged full-length ec-NOS, the wild-type enzyme could be immunoprecipitated by the antibody to the epitope tag. Co-immunoprecipitation of ecNOS with truncation mutants documented that both N- and C-terminal domains are involved in ecNOS oligomerization. When these truncation mutants are co-expressed with wild-type ecNOS, they exert a marked dominant negative effect on enzyme activity. Since NOS oligomerization itself may be subject to dynamic modulation, the regulation of ecNOS assembly may have implications for NO signaling in the vascular wall.

Animals↗
PubMed1995

Nitric oxide-dependent parasympathetic signaling is due to activation of constitutive endothelial (type III) nitric oxide synthase in cardiac myocytes.

Nitric oxide synthase (NOS) isoforms are discovered in an increasing variety of cell types with different roles in signaling. The inducible NOS (i.e. iNOS or NOS II) is expressed in cardiac myocytes in response to specific cytokines. Independent of iNOS induction, however, receptor-dependent signaling is modulated by a constitutive nitric oxide (NO) synthase isoform in these cells (Balligand, J. L., Kelly, R.A., Marsden, P.A., Smith, T. W., and Michel, T. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 347-351). We now show that cardiac myocytes constitutively express the endothelial isoform of NO synthase (ecNOS or NOS III). Transcripts for NOS III were detected by Northern blot in myocyte extracts using as a probe a polymerase chain reaction-generated cDNA amplified with isoform and species-specific primers. In subcellular fractionation experiments, a calcium-sensitive NO synthase activity was present primarily in the particulate fraction, coinciding with the distribution of NOS III analyzed by protein immunoblotting. The localization of NOS III within cardiac myocytes was further demonstrated by immunohistochemistry. The functional role of NOS III was explored by analyzing the effects of NOS inhibitors on single myocyte L-type calcium current and contractility. Inhibition of NOS blocked the attenuation by carbamylcholine of the increases in both parameters induced by beta-adrenergic stimulation. We conclude that NO-dependent parasympathetic signaling is mediated by NOS III in cardiac myocytes.

Amino Acid Oxidoreductases↗
PubMed1995

Agonist-modulated palmitoylation of endothelial nitric oxide synthase.

The nitric oxide synthases (NOS) comprise a family of enzymes which differ in primary structure, biological roles, subcellular distribution, and post-translational modifications. The endothelial nitric oxide synthase (ec-NOS) is unique among the NOS isoforms in being modified by N-terminal myristoylation, which is necessary for its targeting to the endothelial cell membrane. The subcellular localization of the ecNOS, but not enzyme myristoylation, is dynamically regulated by agonists such as bradykinin, which promote ecNOS translocation from membrane to cytosol, as well as enhancing enzyme phosphorylation. Using transiently transfected endothelial cells, we now show that a myristoylation-deficient mutant ecNOS undergoes phosphorylation despite restriction to the cytosol, suggesting that phosphorylation may be a consequence rather than a cause of ecNOS translocation. We therefore explored whether other post-translational modifications might regulate ecNOS targeting and now report that ecNOS is reversibly palmitoylated. Biosynthetic labeling of endothelial cells with [3H]palmitic acid followed by immunoprecipitation of ecNOS revealed that the enzyme is palmitoylated; the label is released by hydroxylamine, consistent with formation of a fatty acyl thioester, and authentic palmitate can be recovered from labeled ecNOS following acid hydrolysis. Importantly, pulse-chase experiments in endothelial cells biosynthetically labeled with [3H]palmitate show that bradykinin treatment promotes ecNOS depalmitoylation. We conclude that ecNOS palmitoylation is dynamically regulated by bradykinin and propose that depalmitoylation of the enzyme may result in its cytosolic translocation and subsequent phosphorylation.

Amino Acid Oxidoreductases↗
PubMed1995

Expression of constitutive endothelial nitric oxide synthase in human blood platelets.

Nitric oxide (NO) activates the soluble isoform of guanylate cyclase in platelets and inhibits platelet function. Several studies suggest the existence of a pathway for NO synthesis in platelets as a form of feedback inhibition, but the identity of the NO synthase (NOS) isoform present within platelets is unknown. We isolated human platelets, and synthesized cDNA from platelet RNA for analysis by PCR. Primers for human neuronal or inducible NOS failed to yield a PCR signal. However, primers specific for endothelial NOS (ecNOS) amplified a DNA band of the expected size. Analysis of nucleotide sequence revealed that the amplified DNA is ecNOS. NOS enzyme activity was detected in the platelet particulate subcellular fraction, as previously demonstrated for ecNOS in other cells. Thus, ecNOS is present in human platelets, and may play a role in the regulation of platelet function by an endogenous NO pathway.

Base Sequence↗
PubMed1995

Induction of NO synthase in rat cardiac microvascular endothelial cells by IL-1 beta and IFN-gamma.

There are important phenotypic differences between endothelial cells of large vessels and the microvasculature and among microvascular endothelial cells isolated from different tissues and organs. In contrast to most macrovascular endothelial cells, we demonstrate that cultured cardiac microvascular endothelial cells (CMEC) have no detectable constitutive NO synthase (NOS) activity but have a robust increase in NOS activity in response to specific inflammatory cytokines. To determine the identity of the inducible NOS (iNOS) isoform(s) induced by cytokines, we used reverse-transcription polymerase chain reaction techniques to clone and sequence a 217-bp cDNA fragment from CMEC cultures pretreated with interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma) that was identical to the corresponding portion of the murine macrophage iNOS cDNA. By use of this CMEC iNOS cDNA as a probe in Northern analyses, IL-1 beta, but not IFN-gamma, increased iNOS mRNA content in CMEC, although IFN-gamma markedly potentiated iNOS induction in these cells. In IL-1 beta- and IFN-gamma-pretreated CMEC, dexamethasone only minimally suppressed the rise in iNOS mRNA, protein abundance, or maximal iNOS enzyme activity in whole cell lysates but suppressed nitrite production by 60% in intact CMEC. Dual labeling of cytokine-pretreated CMEC in primary culture with an anti-iNOS antiserum and a fluorescein-labeled lectin specific for the microvascular endothelium of rat heart (GS-1) confirmed the presence of iNOS expression in these cells. iNOS was also detected in microvascular endothelium in situ in ventricular muscle from lipopolysaccharide-, but not sham-injected, rat hearts.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1995

Contractile responsiveness of ventricular myocytes to isoproterenol is regulated by induction of nitric oxide synthase activity in cardiac microvascular endothelial cells in heterotypic primary culture.

Unlike large-vessel endothelial cells in cell culture, cardiac microvascular endothelial cells (CMEC) isolated from adult rat ventricular muscle exhibit little detectable constitutive nitric oxide (NO) synthase activity after isolation in vitro but respond to specific combinations of inflammatory mediators with an increase in inducible NO synthase (iNOS; type 2 NO synthase) activity. CMEC iNOS is induced by soluble inflammatory mediators in lipopolysaccharide-activated rat alveolar macrophage-conditioned medium at 24 hours, and this induction can be partially prevented by either interleukin-1 (IL-1) receptor antagonist or a polyclonal anti-rat tumor necrosis factor-alpha (TNF-alpha) antiserum. Interferon-gamma (IFN-gamma), which by itself does not induce iNOS in CMEC, potentiates and accelerates iNOS induction by IL-1 beta. Transforming growth factor-beta (TGF-beta) decreases iNOS activity, protein content, and mRNA abundance in IL-1 beta- and IFN-gamma-pretreated CMEC. To determine whether NO released by CMEC would affect myocyte contractile function in vitro, freshly isolated ARVM were allowed to settle onto confluent, serum-starved CMEC that had been pretreated for 24 hours with IL-1 beta, a cytokine that alone does not affect myocyte contractile function in vitro. Baseline contractile amplitude, at 2 Hz and 37 degrees C, of myocytes in heterotypic culture with IL-1 beta-pretreated CMEC was not different from that of myocytes in control, homotypic myocyte cultures. However, cocultured myocytes exhibited decreased contractile responsiveness to 2 nmol/L isoproterenol compared with control cells, and this could be reversed by the addition of 1 mmol/L NG-monomethyl-L-arginine, an inhibitor of NOS.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1995

Induction of nitric oxide synthase activity by cytokines in ventricular myocytes is necessary but not sufficient to decrease contractile responsiveness to beta-adrenergic agonists.

Recent evidence has documented that increased activity of an inducible nitric oxide synthase (iNOS; type 2 NO synthase) in primary isolates of adult rat ventricular myocytes after exposure to soluble mediators in medium conditioned by lipopolysaccharide-activated macrophages is associated with a decrease in their contractile responsiveness to beta-adrenergic agonists. It remained unclear which specific inflammatory cytokines in this medium contribute to the induction of iNOS activity in myocytes and whether induction of iNOS would result in an obligatory decline in contractile function. Interleukin (IL)-1 beta and tumor necrosis factor-alpha (TNF-alpha) were both present in the lipopolysaccharide-activated macrophage-conditioned medium. However, only IL-1 receptor antagonist and not an anti-rat TNF-alpha antiserum diminished the extent of iNOS induction in myocytes exposed to this medium and prevented a decline in contractile responsiveness to isoproterenol. When recombinant cytokines were used, IL-1 beta, TNF-alpha, and IFN-gamma each induced iNOS activity in cardiac myocytes at 24 hours. However, only the combination of IL-1 beta and IFN-gamma reproducibly caused contractile dysfunction in cardiac myocytes. Among the constituents of the defined medium routinely used for maintenance of adult rat ventricular myocytes in primary culture, it was noted that insulin (10(-7) mol/L) was required for NO production, as detected by nitrite release in cytokine-pretreated myocytes, although insulin had no effect on the extent of induction of iNOS mRNA or maximal enzyme activity in myocyte cell lysates.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Agonists↗
PubMed1995

Recombinant endothelial nitric oxide synthase: post-translational modifications in a baculovirus expression system.

Nitric oxide synthesized by the endothelial isoform of nitric oxide synthase (ecNOS) is importantly involved in the homeostatic control of blood pressure and platelet aggregation. The different members of the nitric oxide synthase protein family have several biochemical features in common but serve distinct physiological functions and are the products of distinct genes. The ecNOS is further distinguished by its subcellular distribution in the endothelial cell membrane, and the enzyme undergoes several post-translational modifications, including myristoylation, palmitoylation, and phosphorylation. Overall, however, the ecNOS has remained less well characterized because of the challenges involved in isolating sufficient quantities of this membrane-associated protein from native or cultured endothelial cells. In this report, we describe the purification and characterization of ecNOS expressed in a heterologous system in recombinant baculovirus-infected insect Sf9 cells. Recombinant ecNOS is targeted to the Sf9 cell membrane and comprises approximately 10% of the total cellular protein, allowing purification to homogeneity in a single-step procedure to yield a stable protein that retains the essential features of the native enzyme. Using biosynthetic labeling and immunoprecipitation, we show that recombinant ecNOS is myristoylated, palmitoylated, and phosphorylated when expressed in insect Sf9 cells. The interpretation of structural and enzymological studies of recombinant ecNOS will be facilitated by the apparent fidelity of its biosynthesis and post-translational modification in insect Sf9 cells.

Amino Acid Oxidoreductases↗
PubMed1994

Endothelial nitric oxide synthase membrane targeting. Evidence against involvement of a specific myristate receptor.

The endothelial isoform of nitric oxide synthase (ec-NOS) is targeted to the particulate subcellular fraction by means of N-terminal myristoylation. However, the association of ecNOS with the particulate subcellular fraction appears to be dynamically regulated, in that agonist treatment of endothelial cells induces translocation of the enzyme from membrane to cytosol (Michel, T., Li, G., and Busconi, L. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6252-6255). cDNA encoding wild-type and myristoylation-deficient mutant (myr-) ecNOS was transcribed and translated in vitro, and we found that the recombinant wild-type but not the myr- mutant protein undergoes myristoylation and is able to associate with biological membranes prepared from diverse cell sources. Treatment of these cell membranes with heat or with trypsin did not affect their ability subsequently to serve as acceptor membranes for the wild-type recombinant enzyme. The wild-type ecNOS, but not the myr- mutant, is able to form stable associations with phospholipid liposomes. We also explored the possibility that a polybasic domain within the ecNOS protein might serve as a secondary structural determinant for ecNOS membrane association and constructed truncation mutants that flank a polybasic domain present in the ecNOS. These truncation mutants, transcribed and translated in vitro or transfected into COS-7 cells, undergo myristoylation and are able to associate with biological membranes in a fashion indistinguishable from the wild type ecNOS. Taken together, these results indicate that ecNOS binding to biological membranes is dependent upon interactions of the N-terminal myristoyl moiety of ecNOS with lipid components of the membrane, and this association does not require a specific membrane protein functioning as a myristate receptor nor the presence of a polybasic domain within the ecNOS.

Amino Acid Oxidoreductases↗
PubMed1994

Isolation and chromosomal localization of the human endothelial nitric oxide synthase (NOS3) gene.

Nitric oxide (NO) is an important intercellular signaling molecule synthesized in diverse human tissues by proteins encoded by a family of NO synthase (NOS) genes. The similarity of sequence and cofactor binding sites has suggested that the NOS genes may also be related to cytochrome P450 reductase, as well as to plant and bacterial oxidoreductases. Endothelial NOS activity is a major determinant of vascular tone and blood pressure, and in several important (and sometimes hereditary) disease states, such as hypertension, diabetes, and atherosclerosis, the endothelial NO signaling system appears to be abnormal. To explore the relationship of the endothelial NOS gene to other similar genes, and to delineate the genetic factors involved in regulating endothelial NOS activity, we isolated the human gene encoding the endothelial NOS. Genomic clones containing the 5' end of this gene were identified in a human genomic library by applying a polymerase chain reaction (PCR)-based approach. Identification of the human gene for endothelial NOS (NOS3) was confirmed by nucleotide sequence analysis of the first coding exon, which was found to be identical to its cognate cDNA. The NOS3 gene spans at least 20 kb and appears to contain multiple introns. The transcription start site and promoter region of the NOS3 gene were identified by primer extension and ribonuclease protection assays. Sequencing of the putative promoter revealed consensus sequences for the shear stress-response element, as well as cytokine-responsive cis regulatory sequences, both possibly important to the roles played by NOS3 in the normal and the diseased cardiovascular system.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1994

Endothelial nitric oxide synthase in the human placenta: regional distribution and proposed regulatory role at the feto-maternal interface.

Feto-placental vessels lack innervation, hence control of this circulation is dependent on locally produced and circulating vasoactive factors. Functional studies have presented evidence that nitric oxide, a potent vasodilator and platelet anti-aggregating agent, may be generated into the feto-placental circulation, contributing to control of vascular tone. In view of the absence of nerves supplying the placenta the source of NO is likely to be endothelial. We have therefore investigated the localization of endothelial constitutive nitric oxide synthase (ecNOS) in human normal full-term placentae, using immunocytochemistry, with rabbit antiserum to a synthetic peptide, corresponding to amino acid residues 1172-1186 of human and bovine ecNOS. On Western blots of partially purified NO synthase extracted from placenta, the peptide antiserum reacted exclusively with a single protein band of approximately 135kDA. Immunoreactivity in tissue sections was localized to endothelium of umbilical artery and vein, and appeared uniform in sections at different levels along the cord. Staining in chorionic vessels was much more variable; it was present mainly in the larger vessels close to the cord where it had a patchy distribution. Staining was not seen in the endothelium of small feto-placental vessels. Strong immunoreactivity was evident in the syncytiotrophoblast of the placenta, although the intensity of staining was variable, being weaker along stem villi and strongest along terminal villi. The differential distribution and intensity of nitric oxide synthase immunoreactivity in the human placenta might indicate that locally produced, and in particular trophoblast-derived nitric oxide may play a pivotal role both in control of feto-placental vascular tone and as a platelet anti-aggregating agent in the utero-placental circulation.

Amino Acid Oxidoreductases↗
PubMed1994

Endothelial nitric oxide synthase is expressed in cultured human bronchiolar epithelium.

Nitric oxide (NO) is an important mediator of physiologic and inflammatory processes in the lung. To better understand the role of NO in the airway, we examined constitutive NO synthase (NOS) gene expression and function in NCI-H441 human bronchiolar epithelial cells, which are believed to be of Clara cell lineage. NOS activity was detected by [3H]arginine to [3H]citrulline conversion (1,070 +/- 260 fmol/mg protein per minute); enzyme activity was inhibited 91% by EGTA, consistent with the expression of a calcium-dependent NOS isoform. Immunoblot analyses with antisera directed against neuronal, inducible, or endothelial NOS revealed expression solely of endothelial NOS protein. Immunocytochemistry for endothelial NOS revealed staining predominantly in the cell periphery, consistent with the association of this isoform with the cellular membrane. To definitively identify the NOS isoform expressed in H441 cells, NOS cDNA was obtained by degenerate PCR. Sequencing of the H441 NOS cDNA revealed 100% identity with human endothelial NOS at the amino acid level. Furthermore, the H441 NOS cDNA hybridized to a single 4.7-kb mRNA species in poly(A)+ RNA isolated from H441 cells, from rat, sheep, and pig lung, and from ovine endothelial cells, coinciding with the predicted size of 4.7 kb for endothelial NOS mRNA. Guanylyl cyclase activity in H441 cells, assessed by measuring cGMP accumulation, rose 6.6- and 5.4-fold with calcium-mediated activation of NOS by thapsigargin and A23187, respectively. These findings indicate that endothelial NOS is expressed in select bronchiolar epithelial cells, where it may have autocrine effects through activation of guanylyl cyclase. Based on these observations and the previous identification of endothelial NOS in a kidney epithelial cell line, it is postulated that endothelial NOS may be expressed in unique subsets of epithelial cells in a variety of organs, serving to modulate ion flux and/or secretory function.

Amino Acid Oxidoreductases↗
PubMed1993

Nitric oxide synthases and cardiovascular signaling.

Nitric oxide (NO) synthesized from L-arginine is a ubiquitous intercellular chemical messenger involved in signal transduction in diverse mammalian cells, including vascular endothelium and neuronal tissues. The recent isolation of molecular clones for NO synthases has permitted the characterization of several distinct enzyme isoforms and has allowed us to identify a family of related genes. NO synthesized in vascular endothelial cells appears to play an important role in the control of vascular tone and platelet aggregation, apparently through the activation of guanylate cyclase activity in target tissues mediated by NO. The role of the NO signaling pathway in the direct modulation of cardiac function is less well characterized. We have found that inhibitors of NO synthase can modulate the response of neonatal or adult rat ventricular myocytes exposed to muscarinic or adrenergic agonists. The effects of carbachol on the inhibition of the spontaneous beating rate of cultured neonatal rat cardiac myocytes are blocked by L-N-monomethylarginine, an L-arginine analog that inhibits NO synthase, and by methylene blue, an inhibitor of NO; these agents have no effect on the basal beating rate of these cells. The negative chronotropic effect of carbachol is also mimicked by analogs of cyclic guanosine monophosphate (cGMP), a second messenger implicated in mediating the action of NO in other cell types. Production of NO can be detected directly in carbachol-stimulated neonatal myocytes using a reporter cell bioassay.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1993

Phosphorylation and subcellular translocation of endothelial nitric oxide synthase.

In the vascular endothelium, diverse cell surface receptors are coupled to the Ca2+/calmodulin-dependent activation of nitric oxide (NO) synthase. We now report that, in intact cultured endothelial cells, several drugs and agonists are associated with increased serine phosphorylation of the endothelial NO synthase. We biosynthetically labeled bovine aortic endothelial cells with [32P]orthophosphoric acid, exposed the cells to various drugs and hormones, and then immunoprecipitated the enzyme from cell extracts using a highly specific anti-peptide antibody. The marked endothelial NO synthase phosphorylation induced by bradykinin is maximal only after 5 min of agonist exposure and is stable for at least 20 min. Basal and agonist-induced phosphorylation of the NO synthase in endothelial cells is completely inhibited by the calmodulin antagonist compound W-7. We prepared subcellular fractions of endothelial cells that had been biosynthetically labeled with [35S]methionine or [32P]orthophosphoric acid and immunoprecipitated the endothelial NO synthase from untreated (basal) and bradykinin-treated cells. In the basal state, [35S]methionine-labeled endothelial NO synthase is associated primarily with the particulate cellular fraction, but the phosphorylated enzyme is primarily cytosolic. Following exposure to bradykinin, a substantial fraction of the [35S]methionine-labeled NO synthase is now found in the cytosolic fraction, associated with a marked increase in the level of cytosolic enzyme phosphorylation. We propose that agonist-induced phosphorylation of NO synthase is associated with translocation of the enzyme from membrane to cytosol and may thereby regulate the biological effects of endothelial NO synthesis in situ.

Amino Acid Oxidoreductases↗
PubMed1993

Endothelial nitric oxide synthase. N-terminal myristoylation determines subcellular localization.

Nitric oxide synthases in diverse mammalian tissues catalyze the oxidation of L-arginine to L-citrulline plus nitric oxide (NO). In the vascular endothelium, synthesis of NO yields a labile intercellular messenger molecule with potent biological activities, including vascular smooth muscle relaxation. We have recently documented that the endothelial cell NO synthase (EC-NOS) constitutes a genetically distinct tissue-specific enzyme isoform. In further contrast to the soluble NO synthases found in neural tissues and in macrophages, the endothelial enzyme is associated primarily with the particulate fraction. Analysis of molecular clones for the endothelial NO synthase reveals no obvious transmembrane-spanning region, but a consensus motif for N-terminal myristoylation was identified; such a consensus sequence is not evident in the primary sequence of the soluble macrophage and neural NO synthases. We performed oligonucleotide-directed mutagenesis of the myristoylation consensus sequence in the endothelial NO synthase cDNA, and studied the pattern of expression of the wild-type and mutant EC-NOS cDNAs in transient transfection experiments in COS-7 cells. The subcellular localization of heterologous endothelial NO synthase was determined using analyses of enzyme activity as well as immunoprecipitation of biosynthetically labeled NO synthase with a highly specific antipeptide antibody. Expression of the wild-type endothelial NO synthase cDNA in COS-7 cells results in targeting of both enzyme activity and NO synthase immunoreactivity primarily to the particular fraction. By contrast, transient expression of the myristoylation- mutant cDNA in COS-7 cells yields NO synthase enzyme activity and immunoreactivity associated exclusively with the cytosol fraction. Following biosynthetic labeling with [3H]myristate, the NO synthase can be specifically immunoprecipitated from the particulate fraction in endothelial and in COS-7 cells transfected with the wild-type cDNA, but not in cells transfected with the myristoylation- mutant EC-NOS cDNA. N-terminal myristoylation of the endothelial NO synthase may provide a potential point of regulation of the biological functions of endothelium-derived NO in situ.

Amino Acid Oxidoreductases↗
PubMed1993

Control of cardiac muscle cell function by an endogenous nitric oxide signaling system.

Nitric oxide (NO) synthesized from L-arginine is a ubiquitous intracellular chemical messenger and is involved in signal transduction in diverse mammalian cells, including vascular endothelium and neuronal tissues. The role of the NO-signaling pathway in the direct modulation of cardiac function is less well characterized. In this report, the effects of inhibitors of NO synthase (NOS) were examined in isolated neonatal and adult rat ventricular myocytes exposed to either muscarinic or adrenergic agonists. Carbachol (10 microM) caused a 91% inhibition of the spontaneous beating rate of cultured neonatal rat cardiac myocytes. N omega-monomethyl-L-arginine, an L-arginine analog that inhibits NOS, and methylene blue, an inhibitor of NO, blocked the negative chronotropic effect of carbachol but had no effect on the basal beating rate of these cells. The inhibition by N omega-monomethyl-L-arginine of the negative chronotropic effect of carbachol was reversed by adding excess L-arginine. The negative chronotropic effect of carbachol was also mimicked by analogs of cGMP, a second messenger implicated in mediating the action of NO in other cell types. Production of NO could be detected directly in carbachol-stimulated neonatal myocytes by using a reporter cell bioassay. The regulation of adrenergic responsiveness by the NO signaling system was also documented in studies of adult cardiac myocyte contractility. The NOS inhibitor N omega-nitro-L-arginine significantly increased the inotropic effect of the beta-adrenergic agonist isoproterenol on electrically stimulated adult rat ventricular myocytes, whereas this inhibitor had no effect on basal contractility. Inhibition of NO production by N omega-monomethyl-L-arginine in these cells, as measured by reporter cell bioassay, was also reversible with excess L-arginine. Thus, the physiologic response of isolated neonatal and adult ventricular myocytes to both muscarinic cholinergic and beta-adrenergic stimulation is mediated, at least in part, by products of an endogenous NOS.

Aging↗
PubMed1993

Abnormal contractile function due to induction of nitric oxide synthesis in rat cardiac myocytes follows exposure to activated macrophage-conditioned medium.

The mechanism by which soluble mediators of immune cell origin depress myocardial contractility, either globally as in systemic sepsis, or regionally in areas of inflammatory myocardial infiltrates, remains unclear. When freshly isolated ventricular myocytes from adult rat hearts were preincubated for at least 24 h in medium conditioned by endotoxin (LPS)-activated rat alveolar macrophages, their subsequent inotropic response to the beta-adrenergic agonist isoproterenol was reduced from 225 +/- 19% to 155 +/- 10% of the baseline amplitude of shortening (mean +/- SEM, P < 0.05). Neither baseline contractile function nor the contractile response to high extracellular calcium were affected. To determine whether an endogenous nitric-oxide (NO)-signaling pathway within ventricular myocytes was responsible for their decreased responsiveness to isoproterenol, the L-arginine analogue L-NMMA was added to the preincubation medium. While L-NMMA did not affect baseline contractile function or the response of control myocytes to isoproterenol, it completely restored the positive inotropic response to isoproterenol in myocytes preincubated in LPS-activated macrophage medium. Release of NO by ventricular myocytes following exposure to activated macrophage medium was detected as an increase in cGMP content in a reporter-cell (RFL-6) bioassay and also as increased nitrite content in myocyte-conditioned medium. Thus, the depressed contractile response of adult rat ventricular myocytes to beta-adrenergic agonists by a 24-h exposure to soluble inflammatory mediators is mediated at least in party by induction of an autocrine NO signaling pathway.

Animals↗