Release of nonmitochondrial sequestered Ca2+ from permeabilized muscle cells in culture.
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.
Ambler
S K
SK
Department of Biology, University of California, San Diego, La Jolla 92093.
Taylor
P
P
D000255:Adenosine Triphosphate / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000494:pharmacology; D009132:Muscles / Q000378:metabolism*; D012433:Ryanodine / Q000494:pharmacology
D007295:Inositol Phosphates; D012433:Ryanodine; D015544:Inositol 1,4,5-Trisphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D002118:Calcium