title: Depletion of intracellular Ca2+ stores activates nitric-oxide synthase to generate cGMP and regulate Ca2+ influx.
abstract: The mechanism of activation of the agonist-stimulated Ca2+ entry pathway in the plasma membrane is not known. To determine the role of nitric-oxide synthase (NOS) and cGMP in the regulation of this pathway, we used intact and streptolysin O (SLO)-permeable pancreatic acini and measured the relationship between Ca2+ release from internal stores, the NO metabolic pathway, generation of cGMP, and activation of Ca2+ entry. We found that agonist- or thapsigargin (Tg)-activated Ca2+ entry is inhibited by L-NA, a specific inhibitor of NOS, and by LY83583, an inhibitor of guanylyl cyclase. Inhibition of Ca2+ entry by inhibition of NOS was reversed by the NO releasing molecules NO2- and sodium nitroprusside (SNP) and by Bt2cGMP. Inhibition of Ca2+ entry by inhibition of guanylyl cyclase was reversed by Bt2cGMP, but not by the NO releasing agents. The use of L-NA-treated cells and different concentrations of SNP revealed that cGMP has a dual effect on Ca2+ entry. Increasing cGMP up to 10-fold above control activated Ca2+ entry. Further increase in cGMP up to 80-fold above control inhibited Ca2+ entry in a concentration-dependent manner. Measurement of cellular cGMP in intact cells showed that carbachol, Tg, and NO2- increased cGMP to similar levels. The effects of carbachol and Tg were inhibited by L-NA and LY83586, whereas the effect of NO2- was inhibited only by LY83583. SLO-permeabilized cells were shown to be agonist-competent in that the agonist induced Ca2+ release from the inositol 1,4,5-trisphosphate (IP3) pool and activated a NO-dependent generation of cGMP. These cells were used to study the regulation of NOS by Ca2+ and by Ca2+ content of the internal stores. When internal stores were maintained loaded with Ca2+, increasing medium [Ca2+] up to 2.5 microM only modestly increased NOS activity. In contrast, the depletion of Ca2+ from internal stores markedly increased NOS activity independent of medium [Ca2+]. Thus, NOS senses both cytosolic [Ca2+]i and internal store Ca2+ load. We propose that activation of Ca2+ entry involves an agonist-mediated Ca2+ release from internal stores which activates a cellular pool of NOS to generate cGMP, which then modulates Ca2+ entry pathway in the plasma membrane. This mechanism can explain the capacitative nature of Ca2+ entry. The biphasic effect of cGMP provides the cells with a negative feedback mechanism which inhibits Ca2+ entry during periods of high cell [Ca2+]i. This could allow oscillatory behavior of Ca2+ entry.
authors.affiliation: Department of Physiology, University of Texas Southwestern Medical Center, Dallas 75235-9040.
authors.forename: X
authors.initials: X
authors.lastname: Xu
authors.forename: R A
authors.initials: RA
authors.lastname: Star
authors.forename: G
authors.initials: G
authors.lastname: Tortorici
authors.forename: S
authors.initials: S
authors.lastname: Muallem
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000187:drug effects / Q000378:metabolism*; D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D006152:Cyclic GMP / Q000096:biosynthesis*; D004789:Enzyme Activation; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase; D019335:Nitroarginine; D010179:Pancreas / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction
chemical_list: D000634:Aminoquinolines; D019335:Nitroarginine; D009569:Nitric Oxide; C041715:6-anilino-5,8-quinolinedione; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D006152:Cyclic GMP; D002118:Calcium