CYTOKINE-INDUCIBLE NITRIC-OXIDE SYNTHASE (INOS) EXPRESSION IN CARDIACMYOCYTES - CHARACTERIZATION AND REGULATION OF INOS EXPRESSION AND DETECTION OF INOS ACTIVITY IN SINGLE CARDIAC MYOCYTES IN-VITRO

Cellular constituents of heart muscle contain both constitutive and in ducible nitric oxide (NO) signaling pathways that modulate the contrac tile properties of cardiac myocytes. The identities of the inducible N O synthase (iNOS) isoform(s) expressed in cardiac muscle, and of the s pecific cell types expressing iNOS activity, remain poorly characteriz ed. We amplified a 217-base pair cDNA by reverse transcriptase-polymer ase chain reaction from primary cultures of inflammatory cytokine pret reated adult rat ventricular myocytes (ARVM) that was nearly identical to other NOS cDNA sequences. Using this 217-base pair cDNA as a probe in Northern blots, we found no evidence of iNOS mRNA in control myocy tes, but both interleukin-1 beta and interferon-gamma individually inc reased MOS mRNA abundance in primary cultures of ARVM, with maximal ex pression 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 b y IL-1 beta and INF gamma. Pretreatment with dexamethasone also abolis hed the induction of iNOS mRNA, but not the increase in GTP cyclohydro lase mRNA in purified cardiac myocytes from lipopolysaccharide-injecte d rats, In order to further characterize the specific cell type produc ing NO, we used a NO-specific porphyrinic/Nafion-coated microsensor to record NO release from a single, isolated ARVM pretreated with IL-1 b eta and IFN gamma in L-arginine-depleted medium. NO release could be d etected following microinjection of L-arginine in the vicinity of the cell juxtaposed to the NO microsensor, but not following microinjectio n of D-arginine, and not from ARVM pretreated with L-N-monomethylargin ine. Cytokine-pretreated ARVM that had been maintained in L-arginine-d epleted medium also exhibited a depressed contractile response to isop roterenol after addition of L-arginine, but not D-arginine. These resu lts indicate that altered contractile function of cardiac myocytes fol lowing exposure to specific inflammatory cytokines is due to induction of myocyte iNOS.