Possible role of phosphatidylinositol 4,5, bisphosphate in luteinizing hormone releasing hormone-mediated M-current inhibition in bullfrog sympathetic neurons

Luteinizing hormone releasing hormone (LHRH) is a physiological modulator of neuronal excitability in bullfrog sympathetic ganglia (BFSG). Actions of LHRH involve suppression of the noninactivating, voltage-dependent M-type K+ channel conductance (g(M)). We found, using whole-cell recordings from these neurons, that LHRH-induced suppression of g(M) was attenuated by the phospholipase C (PLC) inhibitor U73122 (10 muM) but not by the inactive isomer U73343 (10 muM). Buffering internal Ca2+ to 117 nM with intracellular 20 mM BAPTA + 8 mM Ca2+ or to < 10 nM with intracellular 20 mM BAPTA + 0.4 mM Ca2+ did not attenuate LHRH-induced g(M) suppression. Suppression of g(M) by LHRH was not antagonized by the inositol 1,4,5 trisphosphate (InsP(3)) receptor antagonist heparin (similar to 300 muM). Preventing phosphatidylinositol-4,5-bisphosphate (PIP2) synthesis by blocking phosphatidylinositol-4-kinase with wortmannin (10 muM) or with the nonhydrolysable ATP analogue AMP-PNP (3 mM) prolonged recovery of LHRH-induced g(M) suppression. This effect was not produced by blocking phosphatidyl inositol-3-kinase with LY294002 (10 muM). Rundown of g(M) was attenuated when cells were dialysed with 240 muM di-octanoyl PIP2 or 240 muM di-octanoyl phosphatidylinositol-3,4,5-trisphosphate (PIP3) but not with 240 muM di-octanoyl phosphatidylcholine. LHRH-induced g(M) suppression was competitively antagonized by dialysis with 240 muM di-octanoyl PIP2, but not with di-octanoyl phosphatidylcholine. These results would be expected if LHRH-induced g(M) suppression reflects a PLC-mediated decrease in plasma membrane PIP2 levels.