Carbachol-induced accumulation of inositol-1-phosphate in neurohybridoma NCB-20 cells: effects of lithium and phorbol esters

Abstract

Clonal neurohybridoma NCB-20 cells expressed muscarinic cholinergic receptors coupled to phospholipase C. Addition of carbachol in the presence of Li+ to cells prelabeled with 3H-inositol increased 3H-inositol-l-phosphate (3H-IP1) accumulation by more than 4-fold with an EC50 of about 50 microM. This carbachol-induced response was blocked by atropine and pirenzepine with a Ki of 0.5 and 25 nM, respectively. The EC50 of Li+ for the carbachol-induced phosphoinositide turnover was 17 +/- 1.2 mM compared with a value of 1.8 +/- 0.2 mM in brain slices, suggesting the presence of an unusual type of inositol-l-phosphatase in NCB-20 cells. Carbachol-induced IP1 accumulation in these cells was potently and noncompetitively inhibited by the biologically active phorbol esters, phorbol dibutyrate (PDB) and phorbol myristate diacetate (PMA), while the biologically inactive phorbol, 4 beta-phorbol, failed to affect this phosphoinositide breakdown. The basal IP1 accumulation was also significantly attenuated by PDB and PMA but not by 4 beta-phorbol.