Release of nonmitochondrial sequestered Ca2+ from permeabilized muscle cells in culture

SK Ambler and P Taylor

Department of Biology, University of California, San Diego, La Jolla 92093.

Activation of alpha 1-adrenergic receptors in BC3H-1 muscle cells results in the rapid elevation of intracellular Ca2+, accompanied by an unusually slow and small increase in inositol 1,4,5-trisphosphate (IP3) formation [J. Biol. Chem. 263: 1952-1959 (1988); Mol. Pharmacol. 32: 376-383 (1987)]. To further assess the role of IP3 in receptor- stimulated Ca2+ mobilization, we have examined Ca2+ disposition in saponin-permeabilized BC3H-1 cells. Permeabilized cells loaded with tracer 45Ca2+ in a buffer containing 100 nM free Ca2+ accumulated greater than 75% of their Ca2+ into an ATP-sensitive compartment and were insensitive to inhibitors of mitochondrial Ca2+ uptake. Application of IP3 resulted in a rapid increase in 45Ca2+ efflux. Under isotopic equilibrium, approximately 90% of the total membrane-enclosed 45Ca2+ was released by 10 microM IP3 within 30 sec. Maximally and half- maximally effective concentrations of IP3 were 22 microM and 0.9 microM, respectively. Application of 10 microM GTP, but not guanine triphosphate-gamma-sulfate, resulted in a slight increase in 45Ca2+ efflux, which reflected a loss in total cellular Ca2+. The GTP-mediated response was slower and of far smaller magnitude than that mediated by IP3. A Ca2+-triggered Ca2+ release mechanism appears not to amplify the receptor response in BC3H-1 cells, inasmuch as 45Ca2+ efflux was not appreciably increased by elevated concentrations of free Ca2+. Furthermore, caffeine and ryanodine had no effect on basal, IP3- mediated, or alpha 1-adrenergic-stimulated Ca2+ release from intact or permeabilized cells. In conclusion, BC3H-1 cells, although showing small and slow increases in IP3 formation upon agonist stimulation, exhibit normal sensitivity to IP3-elicited release of Ca2+ and low sensitivity to other candidate Ca2+-mobilizing agents. The IP3- sensitive Ca2+ stores may be localized within specialized compartments and may play a greater role in the maintenance of elevated cytosolic Ca2+ than in the initial response to receptor activation.

Volume 35, Issue 3, pp. 369-374, 03/01/1989
Copyright © 1989 by American Society for Pharmacology and Experimental Therapeutics