Affiliation:
1. Department of Physiology, University of Texas Southwestern Medical Center, Dallas TX 75235.
2. Laboratory of Membrane Biology, VA Medical Center and UCLA School of Medicine, Los Angeles, CA 90073
3. Department of Biological Sciences, Discovery Research, Allergan Inc./Herbert Labs, Irvine, CA 92715, U.S.A.
Abstract
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.
Subject
Cell Biology,Molecular Biology,Biochemistry
Cited by
38 articles.
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