Pfltigers Arch (1994) 427:325-331 E6i:i i n Journal of Physiology 9 Springer-Verlag 1994 The thapsigargin-evoked increase in [Ca2+]i involves an InsP3-dependent Ca 2+ release process in pancreatic acinar cells Emil C. Toescu, Ole H. Petersen The Physiological Laboratory, Crown Street, Liverpool University, P.O. Box 147, Liverpool L69 3BX, Merseyside, UK Received October 26, t993/Received after revision and accepted December 15, 1993 Abstract. In pancreatic acinar cells, as in many other cell types, the tumour promoter thapsigargin (TG) evokes a significant increase of intracellular free Ca 2+ ([Ca2+]3. The increases of [Ca2+]~ evoked by TG was associated with significant changes of plasma membrane Ca z+ permeability, with [Ca2+]i values following changes in extracellular [Ca2+]. Plasma membrane Ca> extrusion is activated rapidly as a consequence of the rise in [Ca2+]i evoked by TG and the rate of extrusion is linearly dependent on [Ca2+]~ up to 1 pM Ca 2+. In con- trast, the activation of the Ca 2+ entry pathway is delayed and the apparent rate of Ca2+ entry is independent of [Ca2+]i. In the presence of 20 mM caffeine, which re- duces the resting levels of inositol trisphosphate (InsP3), the increase of [Ca2+]~ evoked by TG was significantly reduced. The reduction was manifest both as a decrease of the amplitude of the [Ca>It peak (30% reduction) and, more importantly, as a reduction of the apparent maximal rate of [Ca2+]~ increase (from 12.3 + 1.0 to 6.1 _+ 0.6 nM Ca>/s). The inhibition evoked by caffeine was reversible and the removal of caffeine in the con- tinuous presence of TG evoked a further increase of [Ca2+]i. The amplitude of the [Ca2+]~ increase upon caf- feine removal was reduced as a function of the time of TG exposure. Addition of TG in the presence of 1 mM La3+, which is known to inhibit the plasma membrane CaZ+-activated adenosine triphosphatase, induced a much higher peak of [Ca2+]i. This increase was associ- ated with an augmentation of the apparent rate of [Ca>]~ increase (from 12.3-+ 1.2 to 16.1_ 1.9 nM Ca2§ The inhibitory effect of caffeine, as well as the increase in [Ca:+]~ observed on caffeine removal was not affected by the presence of I mM La 3+. These data indicate that an important component of the TG-evoked [Ca2+]~ in- crease is due to InsP3-sensitive Ca 2+ release which is probably mediated by the resting levels of InsP3. Correspondence to: E. C. Toescu Key words: InsP3 - Ca 2+ entry - Ca 2+ extrusion - Thapsigargin - Exocrine cells Introduction Many agonists which evoke an increase of intracellular Ca 2+ ([Ca2+]i) by stimulating inositol triphosphate (InsP3) production induce, at submaximal concen- trations, periodic intracellular Ca 2+ spikes (Ca2+ oscil- lations [1]). A major component of the Ca> increase during the rising phase of each Ca2+ transient is repre- sented by the Ca 2+ released from the InsP3- and/or Ca2+- sensitive Ca 2+ stores. Following each peak, at least part of the cytosolic Ca 2+ is returned into the intracellular pools by an ATP-dependent Ca2+ pump, which belongs to the sarco(endo)plasmic reticulum Ca2+-activated ad- enosine triphosphatase (SERCA) family of proteins [13]. Important information regarding the nature of the Ca 2+ pools involved during the agonist-evoked Ca > os- cillations has been obtained in the last few years through the use of specific inhibitors of endoplasmic reticulum (ER) Ca>ATPase such as thapsigargin (TG [27]). As a consequence of inhibition of the ER Ca>ATPase, TG induces an elevation of [Ca2+]~ in a variety of cell types [5-8, 10-12, 19, 22, 24-25]. What renders TG a very useful pharmacological tool is the fact that the rise in [Ca2+]i, which sometimes mimics the agonist-evoked response [5], is not associated with measurable changes of intracellular levels of InsP3 [5, 7, 10, 11, 24]. In most cells, TG appears to mobilize Ca 2+ from a pool similar to the one accessed by the agonists which generate InsP3 [19, 24]. This feature of TG action has been used extensively to investigate the relationship be- tween intracellular Ca 2+ stores and plasma membrane Ca ~+ entry. According to the "capacitative Ca> entry" model [24], depletion of intracellular Ca ~+ stores acti- vates, through a mechanism still unclear, the entry of Ca2+ from the extracellular medium. This component of