Inhibition of nitric oxide synthase activity in dispersed gastric muscle cells by protein kinase C.
The present study examined whether NO synthase (NOS) activity in gastric muscle cells was inhibited by protein kinase C (PKC). Vasoactive intestinal peptide (VIP) increased L-[3H]citrulline production (a coproduct and index of NO synthesis) in muscle strips (81.9 +/- 11.6%) and dispersed muscle cells (80.9 +/- 4.6%) of rabbit stomach. Cholecystokinin octapeptide (CCK-8), carbachol, and phorbol 12-myristate 13-acetate (PMA) inhibited VIP-induced L-[3H]citrulline production in muscle cells and muscle strips; the inhibition was reversed by pretreatment with the PKC inhibitor, calphostin C. The Ca(2+)-mobilizing agents, CCK-8, acetylcholine, ionomycin, and KCl, all of which increased PKC activity in dispersed muscle cells, did not increase L-[3H]citrulline production. After treatment of the cells with calphostin C, all four agents stimulated L-[3H]citrulline production, although to a lesser extent than VIP (approximately 50%). VIP-induced relaxation of basal but not carbachol-stimulated tension was accompanied by increase in L-[3H]citrulline production and was inhibited by the NOS inhibitor NG-nitro-L-arginine (L-NNA). Preincubation of carbachol-treated muscle strips with calphostin C restored the ability of VIP to stimulate L-[3H]citrulline production and the ability of L-NNA to inhibit VIP-induced relaxation. We conclude that 1) VIP-stimulated NOS activity is inhibited by agents that increase PKC activity in gastric smooth muscle cells, and 2) agents that increase both cytosolic free Ca2+ concentration and PKC activity stimulate NOS activity only when PKC activity is suppressed.
Murthy
K S
KS
Department of Physiology, Medical College of Virginia, Richmond 23298-0711.
Jin
J G
JG
Makhlouf
G M
GM
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002956:Citrulline / Q000096:biosynthesis; D009126:Muscle Relaxation / Q000187:drug effects; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D019001:Nitric Oxide Synthase; D011493:Protein Kinase C / Q000494:pharmacology*; D011817:Rabbits; D013270:Stomach / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology
D002956:Citrulline; D014660:Vasoactive Intestinal Peptide; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D011493:Protein Kinase C; D002118:Calcium