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)