Functional expression of NOS 1 in vascular smooth muscle

CM Brophy, L Knoepp, J Xin… - American Journal of …, 2000 - journals.physiology.org
CM Brophy, L Knoepp, J Xin, JS Pollock
American Journal of Physiology-Heart and Circulatory Physiology, 2000journals.physiology.org
Substances that increase intracellular calcium concentration ([Ca2+] i), such as serotonin,
are known to induce vascular smooth muscle (VSM) contraction. However, increases in
[Ca2+] i also activate Ca2+/calmodulin-dependent nitric oxide synthases (NOS), which leads
to increases in cGMP and activation of cGMP-dependent protein kinase (PKG). One recently
identified substrate protein of PKG is the small heat shock protein, HSP20. The purpose of
this study was to determine if serotonin activates a Ca2+-dependent NOS in VSM. Strips of …
Substances that increase intracellular calcium concentration ([Ca2+]i), such as serotonin, are known to induce vascular smooth muscle (VSM) contraction. However, increases in [Ca2+]i also activate Ca2+/calmodulin-dependent nitric oxide synthases (NOS), which leads to increases in cGMP and activation of cGMP-dependent protein kinase (PKG). One recently identified substrate protein of PKG is the small heat shock protein, HSP20. The purpose of this study was to determine if serotonin activates a Ca2+-dependent NOS in VSM. Strips of bovine carotid arterial smooth muscle denuded of endothelium were stimulated with serotonin in the presence and absence of the nonspecific NOS inhibitorN-monomethyl-l-arginine (l-NMMA). Activation of NOS was determined by increases in cGMP and in the phosphorylation of HSP20. Immunohistochemical and Western blotting techniques were performed to identify specific NOS isoforms in bovine carotid arterial smooth muscle preparations. Serotonin stimulation led to significant increases in cGMP and in the phosphorylation of HSP20, which were inhibited by pretreatment with l-NMMA. Antibodies against NOS 1 stained the media of bovine carotid and human renal arteries, whereas antibodies against NOS 3 stained only the endothelium. Additionally, the conversion of radiolabeledl-arginine to l-citrulline NOS activity demonstrated a consistent amount of activity present in the endothelium-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 demonstrates that NOS 1 is present in VSM and may effect physiological contractile responses.
American Physiological Society