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Abstract
Cellular constituents of heart muscle contain both constitutive and inducible nitric oxide (NO) signaling pathways that modulate the contractile properties of cardiac myocytes. The identities of the inducible NO synthase (iNOS) isoform(s) expressed in cardiac muscle, and of the specific cell types expressing iNOS activity, remain poorly characterized. We amplified a 217-base pair cDNA by reverse transcriptase-polymerase chain reaction from primary cultures of inflammatory cytokine-pretreated adult rat ventricular myocytes (ARVM) that was nearly identical to other iNOS cDNA sequences. Using this 217-base pair cDNA as a probe in Northern blots, we found no evidence of iNOS mRNA in control myocytes, but both interleukin-1 beta and interferon-gamma individually increased iNOS mRNA abundance in primary cultures of ARVM, with maximal expression at 12 h. The half-life of iNOS mRNA in actinomycin C1-treated cells was 4 h. Both dexamethasone and transforming growth factor-beta attenuated the induction of iNOS mRNA abundance and enzyme activity by IL-1 beta and INF gamma. Pretreatment with dexamethasone also abolished the induction of iNOS mRNA, but not the increase in GTP cyclohydrolase mRNA in purified cardiac myocytes from lipopolysaccharide-injected rats. In order to further characterize the specific cell type producing NO, we used a NO-specific porphyrinic/Nafion-coated microsensor to record NO release from a single, isolated ARVM pretreated with IL-1 beta and IFN gamma in L-arginine-depleted medium. NO release could be detected following microinjection of L-arginine in the vicinity of the cell juxtaposed to the NO microsensor, but not following microinjection of D-arginine, and not from ARVM pretreated with L-N-monomethylarginine. Cytokine-pretreated ARVM that had been maintained in L-arginine-depleted medium also exhibited a depressed contractile response to isoproterenol after addition of L-arginine, but not D-arginine. These results indicate that altered contractile function of cardiac myocytes following exposure to specific inflammatory cytokines is due to induction of myocyte iNOS.
Highlights
From the Cardiovascular Division, Brigham and Women’sHospital and Harvard Medical School, Boston, Massachusetts 02115, the lllepartment of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, the Wepartmentof Chemistry and Instituteof Biotechnology, Oakland Uniuersity, Rochester, Michigan 48309, and the **Wayne State University School of Medicine, Detroit, Michigan 48201
Using this 217-base pair cDNA as a probe in Northern established that both neonatal and adult carmdiyaoccytes conblots, we found no evidence ofiNOSmRNA in control tain a constitutive nitric oxide (NO) synthase activity that mediates myocytes, but both interleukin-lp and interferon-yindi- the responsivenessof these two myocyte phenotypes to muscavidually increased induction of myosynthase (iNOS) mRNA abundance in primary rinic andP-adrenergic agonists, respectively [1].Han et al [2]
In this report,we describe the isolation and sequencing of a partial iNOS cDNA from cytokine-treated primary ARVM isolates that isidentical to the other iNOS isoforms cloned from other cell types, as well as theregulation of iNOS mRNA and protein content and enzyme activity by specific cytokines and glucocorticoids.To determine whether cardiac myocytes werea source ofNO in these primary cultures, we employed an NOselectivemicrosensor
Summary
From the Cardiovascular Division, Brigham and Women’sHospital and Harvard Medical School, Boston, Massachusetts 02115, the lllepartment of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, the Wepartmentof Chemistry and Instituteof Biotechnology, Oakland Uniuersity, Rochester, Michigan 48309, and the **Wayne State University School of Medicine, Detroit, Michigan 48201. Cellular constituents of heart muscle contain both proterenol after addition of L-arginine, but not ~ - a r g i - constitutive and inducible nitric oxide (NO) signaling nine.These results indicate that altered contractile pathways that modulate the contractile properties of function of cardiac myocytes followingexposure to specardiac myocytes. The identities of the inducible NO cific inflammatory cytokineis due to induction of myosynthase (iNOS) isoform(s)expressed in cardiac muscle, cyte iNOS. Of the specific cell types expressing iNOS activity, remain poorly characterized. Pair cDNAby reverse transcriptase-polymerasechain reaction from primary cultures of inflammatory cytokine-pretreated adult rat ventricular myocytes ( A R V M ) that was nearly identical to other iNOS cDNAsequences. Recent evidence indicates that nitric oxide (N0)l signaling pathways play a direct role in regulating the contractileproperties of cardiac muscle in vitro and in vivo.
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