Agonist-sensitive and -insensitive intracellular Ca2+ pools. Separate Ca(2+)-releasing mechanisms revealed by manoalide and benzohydroquinone.
The mechanism of action of a novel compound, 2,5-di-(t-butyl)-1,4-benzohydroquinone (BHQ), used to modulate cell free cytosolic Ca2+ concentration ([Ca2+]i) was studied in AR42J cells and pancreatic acini by using single-cell fluorescence techniques applied to Fura-2-loaded cells. In the presence of extracellular Ca2+ (Ca(2+)out), BHQ induced a biphasic [Ca2+]i increase, an initial and rapid transient followed by a sustained increase. The initial increase was due to Ca2+ release from intracellular stores, being independent of Ca(2+)out. The sustained response was due to Ca2+ entry, being dependent on Ca(2+)out, blocked by La3+ and correlated with an increased rate of Mn2+ entry, all indicative of increased plasma-membrane permeability to Ca2+. Treatment of AR42J cells with BHQ for about 5 min reversibly blocked agonist-dependent Ca2+ release and oscillations, whereas agonist pretreatment decreased, but did not prevent, the effects of BHQ on [Ca2+]i. Accordingly, depletion of the Ins(1,4,5)P3-mobilizable pool in permeabilized AR42J cells by BHQ required 5 min of incubation, although inhibition of the internal Ca2+ pump by BHQ was rapid. These observations suggest that BHQ mobilized an additional intracellular Ca2+ pool that did not respond to changes in Ins(1,4,5)P3. Manoalide, an inhibitor of Ca2+ channels, inhibited agonist-evoked [Ca2+]i oscillation and [Ca2+]i increase in a dose- and time-dependent manner without significant effect on internal Ca2+ pumps and Ca2+ content of the internal stores. Manoalide also inhibited the BHQ-evoked [Ca2+]i increase in the absence and presence of Ca(2+)out. Neither BHQ nor manoalide affected Ins(1,4,5)P3 levels in resting or stimulated cells. Therefore, the effect of BHQ appears to involve unmasking of passive Ca(2+)-permeation pathways in the plasma and intracellular membranes that do not respond to cholecystokinin octapeptide, following its described inhibition of the internal-store Ca2+ pumps responsible for accumulating Ca2+ in these pools.
Muallem
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Department of Physiology, University of Texas Southwestern Medical Center, Dallas 75235.
Loessberg
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Sachs
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Wheeler
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LA
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000187:drug effects / Q000502:physiology; D002463:Cell Membrane Permeability / Q000187:drug effects; D006873:Hydroquinones / Q000494:pharmacology*; D007811:Lanthanum / Q000494:pharmacology; D008345:Manganese / Q000378:metabolism; D010179:Pancreas / Q000187:drug effects / Q000378:metabolism*; D010190:Pancreatic Neoplasms / Q000378:metabolism*; D051381:Rats; D013729:Terpenes / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D002121:Calcium Channel Blockers; D006873:Hydroquinones; D013729:Terpenes; C019359:2,5-di-tert-butylhydroquinone; D008345:Manganese; D007811:Lanthanum; C045673:manoalide; D000252:Calcium-Transporting ATPases; D002118:Calcium