The endoplasmic reticulum can act as a functional Ca2+ store in all subcellular regions of the pancreatic acinar cell.

Stimulation of pancreatic acinar cells raises [Ca2+]i via Ca2+ release from inositol-1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores, generally considered to reside within the endoplasmic reticulum (ER). However, with physiological doses of cholinergic agonists, the [Ca2+]i increase is localized to the apical (secretory) pole of the cell, leading to suggestions that zymogen (secretory) granules themselves may constitute an InsP3-sensitive Ca2+ store responsible for localized Ca2+ release. We have therefore re-investigated whether the ER in pancreatic acinar cells is capable of acting as a functional Ca2+ store in all, or only some, cellular regions. In streptolysin O-permeabilized cells, the ER accumulated up to 25 mmol of 45Ca2+ per liter ER volume by an ATP-dependent, thapsigargin-sensitive, process. This tracer Ca2+ uptake was dependent on ambient (loading) [Ca2+], as was the intra-ER free [Ca2+], assessed by imaging the fluorescence of Magfura-2 within the Ca2+ stores. Comparison of free and total intra-ER [Ca2+] indicated that 200-300 Ca2+ ions are bound within the ER lumen for every Ca2+ ion remaining free. Subcellular analysis showed that ER stores in all regions of the permeabilized cell took up Ca2+ at loading [Ca2+] between 60 nM and 1 microM. Thapsigargin released Ca2+ from stores in all cellular regions, as did InsP3. Immunofluorescence with antibodies against sarco(endo)plasmic reticulum-2b type Ca2+,Mg2+-ATPase or calreticulin confirmed that ER Ca2+ stores were present throughout the cytoplasm. In summary, these results clearly show that the endoplasmic reticulum can act as a functional Ca2+ store in all regions of the acinar cell, including the apical pole.

Imaging of intracellular calcium stores in individual permeabilized pancreatic acinar cells. Apparent homogeneous cellular distribution of inositol 1,4,5-trisphosphate-sensitive stores in permeabilized pancreatic acinar cells.

Several lines of evidence suggest that the existence of a heterogeneous population of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-sensitive Ca2+ stores underlies the polarized agonist-induced rise in cytosolic Ca2+ concentration ([Ca2+]i) in pancreatic acinar cells (Kasai, H., Li, Y. X., and Miyashita, Y. (1993) Cell 74, 669-677; Thorn, P., Lawrie, A. M., Smith, P. M., Gallacher, D. V., and Petersen, O. H. (1993) Cell 74, 661-668). To investigate whether the apical pole of acinar cells contains Ca2+ stores which are relatively more sensitive to Ins(1,4,5)P3 than those in basolateral areas, we studied Ca2+ handling by Ca2+ stores in individual streptolysin O (SLO) permeabilized cells using the low affinity Ca2+ indicator Magfura-2 and an in situ imaging technique. The uptake of Ca2+ by intracellular Ca2+ stores was ATP-dependent. A steady-state level was reached within 10 min, and the free Ca2+ concentration inside loaded Ca2+ stores was estimated to be 70 microM. Ins(1,4,5)P3 induced Ca2+ release in a dose-dependent, "quantal" fashion. The kinetics of this release were similar to those reported for suspensions of permeabilized pancreatic acinar cells. Interestingly, the permeabilized acinar cells showed no intercellular variation in Ins(1,4,5)P3 sensitivity. Although SLO treatment is known to result in a considerable loss of cytosolic factors, permeabilization did not result in a redistribution of zymogen granules, as judged by electron microscope analysis. These results suggest that Ins(1,4,5)P3-sensitive Ca2+ stores are unlikely to be redistributed as a result of SLO treatment. The effects of Ins(1,4,5)P3 were therefore subsequently studied at the subcellular level. Detailed analysis demonstrated that no regional differences in Ins(1,4,5)P3 sensitivity exist in this permeabilized cell system. Therefore, we propose that additional cytosolic factors and/or the involvement of ryanodine receptors underlie the polarized pattern of agonist-induced Ca2+ signaling in intact pancreatic acinar cells.

Effect of the aminosteroid, U73122, on Ca2+ uptake and release properties of rat liver microsomes.

The putative phospholipase C inhibitor, U73122, transiently increases the cytosolic free Ca2+ concentration in rabbit pancreatic acinar cells by stimulating the release of Ca2+ from intracellular stores [Willems, Van de Put, Engbersen, Bosch, Van Hoof & De Pont (1994) Pflügers Arch. 427, 233-243]. In order to elucidate the exact mechanism of action of U73122 we studied its effects on both Ca(2+)-stimulated Mg(2+)-dependent ATPase activity and Ca(2+)-stimulated ATP-dependent Ca2+ uptake in rat liver microsomes. In addition, we studied its effects on Ca2+ release from steady-state loaded microsomes. The effects of U73122 were compared with those of thimerosal, described in the literature as inhibiting Ca(2+)-ATPases and sensitizing inositol 1,4,5-trisphosphate-operated Ca2+ release channels, and thapsigargin, a specific inhibitor of sarcoplasmic and endoplasmic reticulum Ca(2+)-ATPases. Both U73122 (IC50 = 9 microM) and thimerosal (IC50 = 11 microM) dose-dependently inhibited Ca(2+)-stimulated Mg(2+)-dependent ATPase activity, without significantly affecting Mg(2+)-stimulated ATPase activity. Similarly, both U73122 (IC50 = 9 microM) and thimerosal (IC50 = 14 microM) dose-dependently inhibited ATP-dependent Ca2+ uptake. At concentrations beyond 20 microM, U73122 stimulated Ca2+ release from steady-state loaded microsomes at a rate considerably higher than obtained with a maximally inhibitory concentration of thapsigargin (1 microM). This observation, which was not reached with equally inhibitory concentrations of thimerosal, demonstrates that higher U73122 concentrations cause an additional increase of passive Ca2+ leak. The data presented demonstrate that U73122 stimulates the release of actively stored Ca2+ primarily through inhibition of the internal Ca2+ pump.

Differences in uptake, storage and release properties between inositol trisphosphate-sensitive and -insensitive Ca2+ stores in permeabilized pancreatic acinar cells.

Rabbit pancreatic acinar cells, permeabilized by saponin treatment, were used to study the kinetics of ATP-dependent Ca2+ uptake and release in inositol 1,4,5-trisphosphate (Ins-1,4,5-P3)-sensitive and -insensitive stores. Permeabilized acinar cells rapidly accumulated Ca2+ to steady-state. At steady state, approximately 60% of actively stored Ca2+ resided in the Ins-1,4,5-P3-sensitive store. Kinetic analysis of the Ca2+ uptake process revealed that the initial Ca2+ uptake rate was 1.7 times higher in the Ins-1,4,5-P3-insensitive store as compared to the Ins-1,4,5-P3-sensitive store. On the other hand, the Ca2+ uptake capacity was 1.6 times higher in the Ins-1,4,5-P3-sensitive store as compared to the Ins-1,4,5-P3-insensitive store. The Ca2+ uptake rate in the Ins-1,4,5-P3-sensitive store remained virtually constant for at least 4 min, whereas in the Ins-1,4,5-P3-insensitive Ca2+ store this rate progressively declined with time. These observations are compatible with: (i) an Ins-1,4,5-P3-sensitive store containing relatively few Ca2+ pumps but possessing a relatively high Ca2+ uptake capacity, which may reflect the presence of a substantial amount of Ca2+ binding protein; and (ii) an Ins-1,4,5-P3-insensitive Ca2+ store containing relatively many Ca2+ pumps but possessing a relatively low Ca2+ uptake capacity, which may reflect the presence of little if any Ca2+ binding protein. The data presented are consistent with the idea of a heterogeneous distribution of Ca2+ pumps, Ca2+ binding proteins and Ca2+ release channels between intracellular Ca2+ storage organelles.

Induction of Ca2+ oscillations by selective, U73122-mediated, depletion of inositol-trisphosphate-sensitive Ca2+ stores in rabbit pancreatic acinar cells.

The effect of the putative inhibitor of phospholipase C activity, U73122, on the Ca2+ sequestering and releasing properties of internal Ca2+ stores was studied in both permeabilized and intact rabbit pancreatic acinar cells. U73122 dose dependently inhibited ATP-dependent Ca2+ uptake in the inositol (1,4,5)-trisphosphate-[Ins(1,4,5)P3]-sensitive, but not the Ins(1,4,5)P3-insensitive, Ca2+ store in acinar cells permeabilized by saponin treatment. In a suspension of intact acinar cells, loaded with the fluorescent Ca2+ indicator, Fura-2, U73122 alone evoked a transient increase in average free cytosolic Ca2+ concentration ([Ca2+]i,av), which was largely independent of external Ca2+. Addition of U73122 to cell suspensions prestimulated with either cholecystokinin octapeptide or JMV-180 revealed an inverse relationship in size between the U73122- and the agonist-evoked [Ca2+]i,av transient. Moreover, thapsigargin-induced inhibition of intracellular Ca(2+)-ATPase activity resulted in a [Ca2+]i,av transient, the size of which was not different following maximal prestimulation with either U73122 or agonist. These observations suggest that U73122 selectively affects the Ins(1,4,5)P3- casu quo agonist-sensitive internal Ca2+ store, whereas thapsigargin affects both the Ins(1,4,5)P3-sensitive and -insensitive Ca2+ store. Digital-imaging microscopy of Fura-2-loaded acinar cells demonstrated that U73122, in contrast to thapsigargin, evoked sustained oscillatory changes in [Ca2+]i. The U73122-evoked oscillations were abolished in the absence of external Ca2+. The ability of U73122 to generate external Ca(2+)-dependent Ca2+ oscillations suggests that depletion of the agonist-sensitive store leads to an increase in Ca2+ permeability of the plasma membrane and that the Ins(1,4,5)P3-insensitive Ca2+ pool is necessary for the Ca2+ oscillations.

Heterogeneity between intracellular Ca2+ stores as the underlying principle of quantal Ca2+ release by inositol 1,4,5-trisphosphate in permeabilized pancreatic acinar cells.

Permeabilized rabbit pancreatic acinar cells were used to study the effects of Ca2+ pump inhibition and Ca2+ store depletion on the sensitivity of internal Ca2+ stores to emptying by inositol 1,4,5-trisphosphate (Ins-1,4,5-P3). Complete inhibition of pump activity by thapsigargin resulted in the monoexponential loss of 92% of the actively stored Ca2+ with a half-time of 6.2 min. Under these conditions, Ca2+ release evoked by a submaximal concentration of Ins-1,4,5-P3 did not cease after 1.5 min, as was observed in the absence of thapsigargin, but continued for at least 5 min. This observation suggests that under normal conditions of Ca2+ pumping, a substantial part of the internal Ca2+ stores is not depleted by the action of Ins-1,4,5-P3 due to compensatory Ca2+ uptake. Evidence in support of the idea of compensatory Ca2+ pumping was obtained in exchange experiments performed in the absence of thapsigargin. The slow kinetics of sustained Ca2+ release in the absence of Ca2+ pump activity suggests that Ca2+ is released from stores containing either relatively few or less sensitive Ins-1,4,5-P3-operated Ca2+ release channels. Gradual emptying of the internal Ca2+ stores by thapsigargin did not affect the potency with which Ins-1,4,5-P3 released Ca2+, indicating that the intravesicular Ca2+ content does not control the sensitivity of the Ins-1,4,5-P3-operated Ca2+ channel to activation by Ins-1,4,5-P3. This was confirmed using ruthenium red, which preferentially depleted the Ins-1,4,5-P3-releasable store without affecting the EC50 for Ins-1,4,5-P3-stimulated Ca2+ release. The data presented indicate that the quantal type of Ca2+ release observed with Ins-1,4,5-P3 requires compensatory Ca2+ pumping. Moreover, they support the idea that internal Ca2+ stores display differential sensitivities toward Ins-1,4,5-P3 rather than responding uniformly to this internal Ca(2+)-mobilizing messenger.

Basal Mg(2+)-dependent ATPase activity of rat liver microsomes is not influenced by ambient free Ca2+.

The potent inhibitor of Ca2+, Mg(2+)-ATPase activity, thapsigargin, has been used to investigate the effect of ambient free Ca2+ on basal Mg(2+)-dependent ATPase activity in rat liver microsomes. Thapsigargin non-competitively inhibited both Ca(2+)-stimulated ATP-dependent Ca2+ uptake and Ca(2+)-stimulated Mg(2+)-dependent ATPase activity. At a concentration of 1 microM, thapsigargin completely inhibited the Ca(2+)-pump activity, without affecting Mg(2+)-dependent ATPase activity measured in the absence of Ca2+. Increasing the ambient free Ca2+ concentration did not alter the basal Mg(2+)-dependent ATPase activity. The data presented indicate that ATPase activity measured in the absence of Ca2+ is a reliable measure for the basal Mg(2+)-dependent ATPase activity and that, consequently, the Ca(2+)-stimulated Mg(2+)-dependent ATPase activity can indeed be defined as the difference between the ATPase activity measured in the presence and the absence of Ca2+.

Ruthenium red selectively depletes inositol 1,4,5-trisphosphate-sensitive calcium stores in permeabilized rabbit pancreatic acinar cells.

Rabbit pancreatic acinar cells, permeabilized by saponin treatment, rapidly accumulated 3.5 nmol of Ca2+/mg protein in an energy-dependent pool when incubated at an ambient free Ca2+ concentration of 100 nM. Maximal loading of the internal stores was reached at 10 min and remained unchanged thereafter. Complete inhibition of the Ca2+ pump with thapsigargin revealed that this plateau was the result of a steady-state between slow Ca2+ efflux and ATP-driven Ca2+ uptake. Sixty percent of the pool could be released by Ins(1,4,5)P3, whereas GTP released another twenty percent. The striking finding of this study is that the energy-dependent store could also be released by ruthenium red. Uptake experiments in the presence of ruthenium red revealed that the dye, at concentrations below 100 microM, selectively reduced the size of the Ins(1,4,5)P3-releasable pool. Ruthenium red had no effect on the half-maximal stimulatory concentration of Ins(1,4,5)P3. At concentrations beyond 100 microM, the dye also affected the GTP-releasable pool. Comparison with thapsigargin revealed that ruthenium red released Ca2+ from stores loaded to steady-state at a rate markedly faster than can be explained by inhibition of the ATPase alone. From the data presented, we concluded that ruthenium red selectively releases Ca2+ from the Ins(1,4,5)P3-sensitive store by activating a Ca2+ release channel, whereas Ca2+ release from the GTP-sensitive store is predominantly caused by inhibition of the Ca2+ pump. The postulated ruthenium red-sensitive Ca2+ release channel might be similar to the ryanodine-receptor in muscle.

GTP-sensitivity of the energy-dependent Ca2+ storage pool in permeabilized pancreatic acinar cells.

Isolated rabbit pancreatic acinar cells, permeabilized by saponin treatment and incubated in the presence of 0.1 microM free Ca2+, accumulated 3.3 nmol of Ca2+/mg of acinar protein in an energy-dependent pool. Part of this energy-dependent pool could be released by GTP in a polyethylene glycol-dependent manner. The kinetics of GTP-induced release of Ca2+ showed a biphasic pattern with an initial rapid phase followed by a sustained slower phase. In contrast, IP3-induced release of Ca2+ was completed within 30 s following addition of IP3. No reuptake of Ca2+ was observed following GTP- or IP3-induced release of Ca2+. The GTP effect was independent of IP3 and not inhibited by Ca2+, indicating that the IP3-operated Ca2+ channel is not involved in GTP-induced release of Ca2+. The size of the IP3-releasable pool was not affected by GTP, indicating that GTP, when added to permeabilized acinar cells, does not promote the coupling between IP3-insensitive and IP3-sensitive Ca2+ accumulating organelles. Thus, in permeabilized acinar cells, GTP and IP3 act on different Ca2+ sequestering pools. Interestingly, however, comparison of the size of the GTP-releasable pool with that of the IP3-releasable pool for the cell preparations used in the present study, revealed an inversed relationship, indicating that at the time of permeabilization the GTP-releasable pool can be coupled to a greater or lesser extent to the IP3-releasable pool. This suggests that, in the intact cell, a GTP-dependent mechanism may exist that controls the size of the IP3-releasable pool by coupling IP3-insensitive to IP3-sensitive organelles. Moreover, this suggests that the extent of coupling is preserved during permeabilization.

Electrophysiological evidence for the existence of GABAA receptors in cultured frog melanotrophs.

The neurotransmitter GABA exerts a biphasic effect on alpha-melanocyte-stimulating hormone (alpha-MSH) secretion from pars intermedia cells: GABA induces a rapid and transient stimulation followed by a sustained inhibition of alpha-MSH release. In the present study, we have investigated the effect of GABA on the electrophysiological properties of frog melanotrophs in primary culture using the patch-clamp technique in the whole cell configuration. In all cells tested, GABA stimulated an inward current and induced depolarization. A transient period of intense firing was consistently observed at the onset of GABA administration. During the depolarization phase, the membrane potential reached a plateau corresponding to the Cl- equilibrium potential. When repeated hyperpolarizing pulses were applied, an increase of membrane conductance was observed throughout the response evoked by GABA. The effect of GABA was abolished by the chloride channel blocker picrotoxin, and by antagonists of GABAA receptors (bicuculline and SR 95531). The depolarizing action of GABA was mimicked by muscimol, an agonist of GABAA receptors. Taken together, our results indicate that the rapid and transient stimulation of alpha-MSH release induced by GABA can be accounted for by activation of a chloride conductance which causes membrane depolarization. These data support the notion that the transient stimulation of alpha-MSH secretion induced by GABA can be accounted for by membrane depolarization which provokes activation of voltage-operated calcium channels. Since no evidence was found for GABA-induced hyperpolarization, the intracellular mechanisms leading to the strong inhibitory effect of GABA on alpha-MSH secretion remain to be elucidated.