Two important mediators of endothelium-dependent regulation of vascular smooth muscle tone and proliferation are nitric oxide (NO) and endothelin (ET-1). An imbalance between NO and ET-1 may contribute to the alterations in vascular tone characteristic of cardiovascular disease. The objective of this study was to determine whether NO regulates ET receptors in cultured rat superior mesenteric artery vascular smooth muscle cells (RVSMC). Chronic treatment of quiescent RVSMC with any one of three chemically dissimilar NO-generating drugs, S-nitroso-N-acetyl penicillamine (SNAP), sodium nitroprusside (SNP), and isosorbide dinitrate (ISDN) produced a significant dose-and time-dependent increase in the number of ET-A receptors, while concomitantly increasing the affinity of ET-1 for this receptor. This effect was mimicked by both 8-bromo-cGMP and 8-bromo-CAMP. The requirement of both protein and RNA synthesis and activation of a CAMP-dependent protein kinase (A-kinase) was demonstrated following inhibition of this regulation by cycloheximide, actinomycin D and KT5720 (a specific A-kinase inhibitor), respectively. In addition, the cytokine interleukin 1 p (IL-1 p) which induced NOS activity with subsequent NO synthesis in vascular smooth muscle, also caused a similar upregulation of ET receptors. This effect was attenuated in the presence of the specific NOS inhibitor, L-NAME. To assess the possible functional consequences of this NO-mediated upregulation, the effect of SNAP pretreatment on isolated vessel reactivity was determined. In both superior mesenteric artery and thoracic aorta rings, SNAP pretreatment caused a significant increase in the maximal force of contraction to ET-1. Collectively, these data suggest that NO regulates ET-A receptors in vitro through a cGMPdependent mechanism via activation of the CAMP-dependent protein kinase. We conclude that a similar interaction between NO and ET-1 may be operational in Vi VO. 0 1996 Wiley-Liss, Inc Endothelin (ET) is a 21-amino acid peptide with potent vasoconstrictive and mitogenic properties (Rubanyi and Polokoff, 1994 for review). ET is produced by several cell types including vascular endothelial cells, epithelial cells, macrophages, and fibroblasts (Rubanyi and Polokoff, 1994). The cellular action of ET on vascular tissue is initiated by peptide binding to specific cell surface receptors (Rubanyi and Polokoff, 1994). Two different receptors for ET have been identified, cloned and characterized (Arai et al., 1990;Sakurai et al., 1990). The ET-A receptor on vascular tissue which preferentially binds ET-1, is linked to phospholipase C enzyme activity, subsequent phosphoinositide metabolism and the mobilization of intracellular calcium via a Gq protein (Reynolds et al., 1989;Simonson and Dunn, 1990). More recent studies have suggested that ET-A receptors on vascular tissue are also coupled to adenylyl cyclase activity via a Gs protein (Eguchi et al., 1992). The ET-B receptor, which equally binds ET-1 and ET-3, respectively, is functionally coupled to nitric 0