Muscarinic receptor regulation of cytoplasmic Ca2+ concentrations in human SK-N-SH neuroblastoma cells: Ca2+ requirements for phospholipase C activation

The relationship between muscarinic receptor-mediated inositol lipid hydrolysis and the generation of Ca2+ signals has been examined in human SK-N-SH neuroblastoma cells. The resting cytoplasmic calcium concentration ( Ca2+]i) as determined by fura-2 fluorescence measurements was 59 +/- 2 nM. Upon the addition of oxotremorine-M, there was a 4-fold increase in Ca2+]i (293 +/- 18 nM), with half-maximal stimulation obtained at an agonist concentration of 8 microM, a value similar to that previously observed for the enhancement of phosphoinositide hydrolysis. Addition of partial muscarinic agonists for phosphoinositide turnover (bethanechol, oxo-2, and arecoline) elicited correspondingly smaller increases in Ca2+]i than did oxotremorine-M. Inclusion of EGTA lowered the basal Ca2+]i within 2 min and markedly reduced (greater than 60%) the magnitude of the agonist-induced rise in Ca2+]i. Addition of muscarinic agonists to SK-N-SH cells that had been prelabeled with 3H]inositol led to the rapid (5-15 sec) release of inositol mono-, bis-, and triphosphates. When assayed under conditions similar to those employed for the fluorescence measurements, EGTA also inhibited both the basal and oxotremorine-M-stimulated release of inositol phosphates by 45-61%. Conversely, ionomycin both elevated Ca2+]i and stimulated the release of inositol phosphates. The addition of Ca2+ (10 nM-2 microM) to digitonin-permeabilized cells directly stimulated the release of labeled inositol mono-, bis-, and trisphosphates by 3-4-fold with a half-maximal effect (EC50) observed at 145 nM free Ca2+ (Ca2+f). A further (6-fold) calcium-dependent increase in inositol phosphate release was obtained by inclusion of either guanosine-5-O-(3-thio)-trisphosphate (GTP gamma S) or oxotremorine-M. In the combined presence of agonist and GTP gamma S, a synergistic release of all three inositol phosphates occurred, with half-maximal stimulation observed at 35-40 nM Ca2+f, a value similar to the Ca2+]i in quiescent cells. These results indicate (i) that the magnitude of the initial rise in Ca2+]i is directly related to the production of phosphoinositide-derived second messenger molecules and (ii) that the phospholipase C-mediated breakdown of inositol lipids in SK-N-SH cells is particularly sensitive to regulation by physiologically relevant Ca2+ concentrations. It is concluded that, in SK-N-SH cells, either an elevation above or reduction below basal Ca2+]i can modulate the extent of hydrolysis of inositol lipids and the subsequent generation of calcium signals.