MULTIPLE MECHANISMS OF ARACHIDONIC-ACID RELEASE IN CHINESE-HAMSTER OVARY CELLS TRANSFECTED WITH CDNA OF SUBSTANCE-P RECEPTOR

We investigated the release of [H-3]arachidonic acid ([H-3]AA) and its relationship to the formation of [H-3]inositol trisphosphate ([H-3]IP 3) elicited by substance P (SP) in prelabeled Chinese hamster ovary ce lls stably expressing the SP receptor. Activation of the SP receptor r esulted in a concentration- and time-dependent stimulation of [H-3]AA release. Half-maximal release was obtained at 10(-9)M, comparable to t hat for [H-3]IP, formation reported previously, and the maximal releas e effected by 0.1 mu M SP was 8 to 10-fold above the basal value. Both the [H-3]AA release and the [H-3]-IP3 accumulation stimulated in the cells by 0.1 mu M SP were concentration-dependently blocked with the s pecific SP receptor antagonist CP-96,345, with IC50 values of 2.5 and 0.4 mu M, respectively. The time course of [H-3]AA release showed a bi phasic pattern: an initial rapid release essentially independent of Ca 2+, followed by a sustained release markedly suppressed by removal of extracellular Ca2+ or chelation of intracellular Ca2+ with ,2-bis(2-am inopbenoxyethane)-N,N,N',N'-tetraacetic acid (BAPTA). While pretreatme nt with pertussis toxin (200 ng/mL, 6 hr) did not block [3H]IP3 format ion, it did reduce [H-3]AA release by 50% at 1 and 10 min after SP sti mulation. Treatment of the cells with a phorbol ester, a protein kinas e C activator, augmented the SP-stimulated [H-3]AA release, and sphing osine, a protein kinase C inhibitor, reversed the phorbol ester-potent iated [H-3]AA release, but not the release stimulated by SP alone, sug gesting a synergistic effect of protein kinase C on SP-stimulated AA r elease. These results demonstrate that SP, acting at the SP receptor, stimulates [H-3]AA release via mechanisms that are (I)mediated by a pe rtussis toxin-sensitive G-protein, (2) dependent on extracellular Ca2, and (3) enhanced by activation of protein kinase C.