2,5-Di-(tert-butyl)-1,4-benzohydroquinone rapidly elevates cytosolic Ca2+ concentration by mobilizing the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool.
2,5-Di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), a potent inhibitor of liver microsomal ATP-dependent Ca2+ sequestration (Moore, G. A., McConkey, D. J., Kass, G. E. N., O'Brien, P. J., and Orrenius, S. (1987) FEBS Lett. 224, 331-336), produced a concentration-dependent, rapid increase in cytosolic free Ca2+ concentration ([Ca2+]i) in isolated rat hepatocytes (EC50 = 1-2 microM). The amplitude of the [Ca2+]i increase was essentially identical with that produced by vasopressin, but the tBuBHQ-stimulated [Ca2+]i increase remained sustained for 15-20 min. Vasopressin added 2-3 min after tBuBHQ caused [Ca2+]i to rapidly return to basal levels; however, tBuBHQ added after vasopressin resulted in a Ca2+ transient rather than a sustained [Ca2+]i elevation. Ca2+ influx was not stimulated in tBuBHQ-treated hepatocytes, but was markedly enhanced upon addition of vasopressin. Depletion of the endoplasmic reticular Ca2+ pool by the addition of vasopressin to hepatocytes incubated in low Ca2+ medium virtually abolished the tBuBHQ-mediated [Ca2+]i rise and vice versa. In saponin-permeabilized hepatocytes, tBuBHQ released Ca2+ from the same nonmitochondrial, ATP-dependent Ca2+ pool which was released by inositol 1,4,5-trisphosphate. Furthermore, tBuBHQ-induced Ca2+ release in saponin-permeabilized cells was not inhibited by neomycin, and tBuBHQ did not produce any apparent accumulation of inositol phosphates in intact hepatocytes. The rate of passive efflux of Ca2+ from Ca2+-loaded hepatic microsomes was unaltered by tBuBHQ. Thus, tBuBHQ inhibits ATP-dependent Ca2+ sequestration via a direct effect on the endoplasmic reticulum Ca2+ pump, resulting in net Ca2+ release and elevation of [Ca2+]i. Taken together, our results show that in the absence of hormonal stimuli, excess Ca2+ is only slowly cleared from the hepatocyte cytosol, indicating that the basal rate of Ca2+ removal by the plasma membrane Ca2+ pump and mitochondria is slow. Furthermore, Ca2+-mobilizing hormones appear to stimulate an active process of Ca2+ removal from hepatocyte cytosol which does not depend on re-uptake into the endoplasmic reticulum.
Kass
G E
GE
Department of Toxicology, Karolinska Institutet, Stockholm, Sweden.
Duddy
S K
SK
Moore
G A
GA
Orrenius
S
S
D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology; D001572:Benzofurans; D002118:Calcium / Q000378:metabolism*; D002132:Calcium Radioisotopes; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D005456:Fluorescent Dyes; D016257:Fura-2; D006873:Hydroquinones / Q000494:pharmacology*; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008112:Liver Glycogen / Q000378:metabolism; D008297:Male; D008345:Manganese / Q000494:pharmacology; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013403:Sugar Phosphates / Q000378:metabolism*
D001572:Benzofurans; D002132:Calcium Radioisotopes; D005456:Fluorescent Dyes; D006873:Hydroquinones; D007295:Inositol Phosphates; D008112:Liver Glycogen; D013403:Sugar Phosphates; D001127:Arginine Vasopressin; C019359:2,5-di-tert-butylhydroquinone; D008345:Manganese; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium; D016257:Fura-2