Role of intracellular Ca2+ mobilization in muscarinic and histamine receptor-mediated activation of guanylate cyclase in N1E-115 neuroblastoma cells: assessment of the arachidonic acid release hypothesis.

This study evaluates the role of intracellular levels of Ca2+ [Ca2+]i in cyclic GMP formation mediated by muscarinic and histamine receptors in the mouse neuroblastoma clone N1E-115. Muscarinic agonists activated the turnover of phosphoinositides with a relative maximal response similar to that observed previously for cyclic GMP formation. Carbamylcholine induced a transient increase in inositol trisphosphate with a time course similar to that of cyclic GMP formation. In cells loaded with the fluorescent Ca2+ probe fura-2/acetoxymethyl ester, carbamylcholine as well as histamine induced a rapid and transient rise in [Ca2+]i. The time course of the changes in [Ca2+]i induced by agonists as well as by ionomycin closely paralleled that of cyclic GMP formation. Chelation of [Ca2+]i by loading of N1E-115 cells with quin 2/acetoxymethyl ester inhibited cyclic GMP formation induced by agonists in a dose-dependent manner. When cyclic GMP formation induced by agonists was assayed after the cells were exposed to 3 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) for 2 min, the formation of cyclic GMP was not inhibited significantly; however, it was completely abolished after 30-min exposure to EGTA. Treatment of cells with phospholipase A2 had no effect on resting [Ca2+]i and only slightly increased cyclic GMP formation, in spite of the induction of a marked release of [3H]arachidonate. Moreover, the formation of cyclic GMP induced by ionomycin was inhibited by the addition of phospholipase A2. Melittin contaminated with phospholipase A2 activity induced a rapid and sustained increase in cyclic GMP formation, as well as unesterified [3H]arachidonate release. However, after inactivation of the phospholipase A2 activity of melittin, its ability to stimulate cyclic GMP formation was enhanced. Our data indicate that receptor agonists stimulate cyclic GMP formation in N1E-115 cells by activating the formation of inositol trisphosphate, which is followed by the release of Ca2+ from intracellular stores. The evidence obtained does not support a major role for arachidonate release in receptor-mediated activation of guanylate cyclase. Conversely, it is consistent with an inhibitory role for arachidonic acid or its metabolites in this process.