Functional expression of NOS 1 in vascular smooth muscle

Substances that increase intracellular calcium concentration ([Ca2+](i)), s uch as serotonin, are known to induce vascular smooth muscle (VSM) contract ion. However, increases in [Ca2+](i) also activate Ca2+/calmodulin-dependen t nitric oxide synthases (NOS), which leads to increases in cGMP and activa tion of cGMP-dependent protein kinase (PKG). One recently identified substr ate protein of PKG is the small heat shock protein, HSP20. The purpose of t his study was to determine if serotonin activates a Ca2+ dependent NOS in V SM. Strips of bovine carotid arterial smooth muscle denuded of endothelium were stimulated with serotonin in the presence and absence of the nonspecif ic NOS inhibitor N-monomethyl-L-arginine (L-NMMA). Activation of NOS was de termined by increases in cGMP and in the phosphorylation of HSP20. Immunohi stochemical and West ern blotting techniques were performed to identify spe cific NOS isoforms in bovine carotid arterial smooth muscle preparations. S erotonin stimulation led to significant increases in cGMP and in the phosph orylation of HSP20, which were inhibited by pretreatment with L-NMMA. Antib odies against NOS 1 stained the media of bovine carotid and human renal art eries, whereas antibodies against NOS 3 stained only the endothelium. Addit ionally, the conversion of radiolabeled L-arginine to L-citrulline NOS acti vity demonstrated a consistent amount of activity present in the endotheliu m-denuded smooth muscle preparations that was reduced by 99% with an NOS 1 specific inhibitor. Finally, an NOS 1 specific inhibitor, 7-nitroindazole, augmented contractions induced by high extracellular KCl. This study demons trates that NOS 1 is present in VSM and may effect physiological contractil e responses.