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 o f large vessels and the microvasculature and among microvascular endot helial cells isolated from different tissues and organs. In contrast t o most macrovascular endothelial cells, we demonstrate that cultured c ardiac microvascular endothelial cells (CMEC) have no detectable const itutive NO synthase (NOS) activity but have a robust increase in NOS a ctivity in response to specific inflammatory cytokines. To determine t he identity of the inducible NOS (iNOS) isoform(s) induced by cytokine s, we used reverse-transcription polymerase chain reaction techniques to clone and sequence a 217-bp cDNA fragment from CMEC cultures pretre ated with interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gam ma) that was identical to the corresponding portion of the murine macr ophage 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 i n CMEC, although IFN-gamma markedly potentiated iNOS induction in thes e cells. In IL-1 beta- and IFN-gamma-pretreated CMEC, dexamethasone on ly minimally suppressed the rise in NOS mRNA, protein abundance, or ma ximal iNOS enzyme activity in whole cell lysates but suppressed nitrit e production by 60% in intact CMEC. Dual labeling of cytokine-pretreat ed CMEC in primary culture with an anti-iNOS antiserum and a fluoresce in-labeled lectin specific for the microvascular endothelium of rat he art (GS-1) confirmed the presence of iNOS expression in these cells. i NOS was also detected in microvascular endothelium in situ in ventricu lar muscle from lipopolysaccharide-, but not sham-injected, rat hearts . The induction of iNOS in the endothelium of the cardiac microvascula ture may have important implications for understanding the pathophysio logy of some forms of inflammatory cardiomyopathies.