Irene Schulz

Depletion of intracellular calcium stores activates a calcium conducting nonselective cation current in mouse pancreatic acinar cells.

Receptor-mediated Ca2+ release from inositol (1,4,5)-trisphosphate (IP3)-sensitive Ca2+ stores causes “capacitative calcium entry” in many cell types (Putney, J. W., Jr. (1986) Cell Calcium 7, 1-12; Putney, J. W., Jr. (1990) Cell Calcium 11, 611-624). We used patch-clamp and fluorescence techniques in isolated mouse pancreatic acinar cells to identify ion currents and cytosolic calcium concentrations under conditions in which intracellular Ca2+ stores were emptied. We found that depletion of Ca2+ stores activated a calcium-release-activated nonselective cation current (ICRANC) which did not discriminate between monovalent cations. ICRANC possessed a significant conductance for Ca2+ and Ba2+. It was not inhibited by La3+, Gd3+, Co2+, or Cd2+ but was completely abolished by flufenamic acid or genistein. In whole cell and cell-attached recordings, a 40-45 pS nonselective cation channel was identified which was activated by Ca2+ store depletion. Calcium entry as detected by single cell fluorescence measurements with fluo-3 or fura-2, showed the same pharmacological properties as ICRANC. We conclude that in mouse pancreatic acinar cells 40-45 pS nonselective cation channels serve as a pathway for capacitative Ca2+ entry. This entry pathway differs from the previously described ICRAC (Hoth, M., and Penner, R. (1992) Nature 355, 353-356) in its ion-selectivity, pharmacological profile, and single-channel conductance.

Quantification of intraluminal free [Ca] in the agonistmsensitive internal calcium store using compartmentalized fluorescent indicators: some considerations

Many fluorescent Ca indicators, particularly those loaded as acetoxymethyl (AM)-ester derivatives, are known to become compartmentalized into organelles. This property can be exploited to measure changes in free [Ca] in subcellular compartments, including the inositol (1,4,5)-trisphosphate-sensitive store. However, quantitative measurement of free [Ca] within a particular compartment is complicated by the fact that dye may accumulate in a variety of organelles and, in many cases, by the Mg sensitivity of the indicator. Here the issue of the quantification of free [Ca] within the thapsigargin-sensitive store in BHK-21 fibroblasts using the low affinity Ca indicator, Mag-Fura-2, has been re-examined. At least 88 +/- 1.3% (SEM) of the compartmentalized dye was determined to be confined to the thapsigargin-sensitive store, with the remaining fraction accounted for by other compartments where [Ca] was below the detection limit for the dye (< 5 microM). In situ calibrations with ionophores indicated that the apparent free resting intraluminal [Ca] was 260 +/- 43 microM (SEM). Our analysis shows, however, that dye reporting from regions of low [Ca] contributes disproportionately to the Mag-Fura-2 ratio measured over the whole cell, potentially resulting in large underestimations of intraluminal [Ca] in agonist-sensitive stores. Free [Ca] in the agonist-sensitive store was calculated to be as high as 539 +/- 92 microM, assuming 12% of the Mag-Fura-2 to be in compartments where [Ca] was below 5 microM. In comparison, perturbations arising from the presence of Mg in stores are predicted to be relatively minor.

ATP regulates calcium leak from agonist-sensitive internal calcium stores.

ABSTRACT: Under resting conditions, steady‐state [Ca] in agonist‐sensitive Ca stores reflects a balance between active uptake (usually mediated by a thap‐sigargin‐sensitive Ca‐ATPase of the SERCA family) and passive efflux of Ca. Even though this pump‐leak cycle appears to be a common property of Ca‐storing organelles, little is known about the nature of the leak pathway. Ca homeostasis in thapsigargin‐sensitive internal Ca stores of single permeabilized BHK‐21 fibroblasts was examined using digital image processing of compartmentalized mag‐fura‐2 (a low‐affinity Ca indicator). It is shown here that the leak of Ca from internal stores is regulated specifically by the cytosolic ATP concentration. The rate of leak was 3.6 times slower in 0.375 mM [ATP] than in 4 mM [ATP] (Na or Mg salt). These effects were observed in the presence of 0 Ca/EGTA, thapsigargin, heparin, and ruthenium red, and therefore appear to be independent of the Ca‐ATPase, the InsP3 receptor and the ryanodine receptor. The ATP‐stimulated leak was seen in a variety of cell types, including rat basophilic leukemia cells and mouse pancreatic acinar cells. Other nucleotides (ADP, GTP, CTP, and UTP) and nonhydrolyzable ATP analogs (AMP‐PNP and ATPγS) did not reproduce the action of ATP. Changes in cellular metabolism and ensuing alterations in [ATP] will be expected to influence the filling state of internal Ca stores through effects on the passive leak pathway, potentially leading to modulation of Ca signaling and organellar function.—Hofer, A. M., Curci, S., Machen, T. E., Schulz, I. ATP regulates calcium leak from agonist‐sensitive internal calcium stores. FASEB J. 10, 302‐308 (1996)

Enhanced susceptibility to pancreatitis in severe hypertriglyceridaemic lipoprotein lipase-deficient mice and agonist-like function of pancreatic lipase in pancreatic cells

Background and aims: Recurrent pancreatitis is a common complication of severe hypertriglyceridaemia in patients with various gene mutations in lipoprotein lipase (LPL) or apolipoprotein CII. However, the exact pathogenetic mechanism has not yet been defined. Methods: Susceptibility to pancreatitis in LPL-deficient mice was compared with that of wild-type mice after intraperitoneal injections of caerulein by determination of amylase release and pancreatic pathological scores. The effect of chylomicrons and fatty acids on enzyme release, Ca2+ signalling and cell injury in isolated pancreatic acinar cells from wild-type and LPL-deficient mice was investigated. Results: Caerulein induced higher levels of serum amylase and more severe inflammation in the pancreas of LPL-deficient mice than in wild-type mice. Addition of free fatty acids or chylomicrons to isolated pancreatic acinar cells led to the release of amylase and caused cell injury at higher concentrations. The effect of chylomicrons was partially blocked by orlistat, an inhibitor of pancreatic lipase. These results suggest that increased concentrations of free fatty acids from chylomicron hydrolysis by pancreatic lipase can induce acinar cell injury. Surprisingly, pancreatic lipase, whether in its active or inactive state could act like an agonist by inducing amylase secretion without cell injury. It caused an increase in cGMP levels and conversion of cell-damaging sustained elevations of [Ca2+] to normal Ca2+ oscillations. Conclusions: LPL-deficient mice with severe hypertriglyceridaemia display enhanced susceptibility to acute pancreatitis. High levels of chylomicrons and free fatty acids result in pancreatic cell injury. Pancreatic lipase has a dual effect: generating free fatty acids from chylomicrons and preventing Ca2+ overload in pancreatic acinar cells.

Low Cytoplasmic [Ca2+] Activates ICRACIndependently of Global Ca2+ Store Depletion in RBL-1 Cells

Release of Ca2+ from inositol (1,4,5)-trisphosphate-sensitive Ca2+ stores causes “capacitative calcium entry,” which is mediated by the so-called “Ca2+ release-activated Ca2+ current” (ICRAC) in RBL-1 cells. Refilling of the Ca2+stores or high cytoplasmic [Ca2+] ([Ca2+]cyt) inactivate ICRAC. Here we address the question if also [Ca2+]cyt lower than the resting [Ca2+]cyt influences store-operated channels. We therefore combined patch clamp and mag fura-2 fluorescence methods to determine simultaneously both ICRAC and [Ca2+] within Ca2+ stores of RBL-1 cells ([Ca2+]store). We found that low [Ca2+]cyt in the range of 30–50 nm activates ICRAC and Ca2+ influx spontaneously and independently of global Ca2+ store depletion, while elevation of [Ca2+]cyt to the resting [Ca2+]cyt (100 nm) resulted in store dependence of ICRAC activation. We conclude that spontaneous activation of ICRAC by low [Ca2+]cyt could serve as a feedback mechanism keeping the resting [Ca2+]cyt constant.

Cell Side-specific Sensitivities of Intracellular Ca2+ Stores for Inositol 1,4,5-Trisphosphate, Cyclic ADP-ribose, and Nicotinic Acid Adenine Dinucleotide Phosphate in Permeabilized Pancreatic Acinar Cells from Mouse

In pancreatic acinar cells hormonal stimulation leads to a cytosolic Ca2+ wave that starts in the apical cell pole and subsequently propagates toward the basal cell side. We used permeabilized pancreatic acinar cells from mouse and the mag-fura-2 technique, which allows direct monitoring of changes in [Ca2+] of intracellular stores. We show here that Ca2+ can be released from stores in all cellular regions by inositol 1,4,5-trisphosphate. Stores at the apical cell pole showed a higher affinity to inositol 1,4,5-trisphosphate (EC50 = 89 nm) than those at the basolateral side (EC50 = 256 nm). In contrast, cADP-ribose, a modifier of Ca2+-induced Ca2+ release, and nicotinic acid adenine dinucleotide phosphate (NAADP) were able to release Ca2+ exclusively from intracellular stores located at the basolateral cell side. Our data agree with observations that upon stimulation Ca2+ is released initially at the apical cell side and that this is caused by high affinity inositol 1,4,5-trisphosphate receptors. Moreover, our findings allow the conclusion that in Ca2+ wave propagation from the apical to the basolateral cell side observed in pancreatic acinar cells Ca2+-induced Ca2+ release, modulated by cADP-ribose and/or NAADP, might be involved.

Characterization of single potassium channels in mouse pancreatic acinar cells.

1. Single K(+)‐selective channels with a conductance of about 48 pS (pipette, 145 mM KCl; bath, 140 mM NaCl + 4.7 mM KCl) were recorded in the patch‐clamp whole‐cell configuration in isolated mouse pancreatic acinar cells. 2. Neither application of the secretagogues acetylcholine (second messenger, inositol 1,4,5‐trisphosphate) or secretin (second messenger, cAMP), nor addition of the catalytic subunit of protein kinase A to the pipette solution changed the activity of the 48 pS K+ channel. 3. Intracellular acidification with sodium propionate (20 mM) diminished activity of the 48 pS channel, whereas channel open probability was increased by cytosolic alkalization with 20 mM NH4Cl. 4. BaCl2 (5 mM), TEA (10 mM) or apamin (1 microM) added to the bath solution had no obvious effect on the kinetics of the 48 pS channel. Similarly, glibenclamide and diazoxide failed to influence the channel activity. 5. When extracellular NaCl was replaced by KCl, whole‐cell recordings revealed an inwardly rectifying K+ current carried by a 17 pS K+ channel. 6. The inwardly rectifying K+ current was not pH dependent and could largely be blocked by Ba2+ but not by TEA. 7. Since the 48 pS K+ channel is neither Ca2+ nor cAMP regulated, we suggest that this channel could play a role in the maintenance of the negative cell resting potential.

Inwardly rectifying, volt age‐dependent and resting potassium currents in rat pancreatic acinar cells in primary culture

1 In exocrine pancreatic acinar cells in primary culture an inwardly rectifying, a voltage‐dependent and a permanent resting K+ current were characterized. 2 Inwardly rectifying K+ currents could be elicited by elevation of the extracellular K+ concentration. The K+ inward currents were almost completely blocked by 5 mm Ba2+, whereas 10 mm TEA+ had only a partial effect. 3 Depolarizing voltage steps from negative clamp potentials evoked transient activation of a voltage‐dependent K+ current. This voltage‐dependent current could be blocked by 10 mm TEA+ and 1 mm 4‐aminopyridine, but not by 5 mm Ba2+. 4 Neither the K+ inward rectifier nor the voltage‐dependent K+ conductance produced a significant negative cell potential. Stable membrane potentials (–38.7 ± 2.3mV, n= 38) could only be recorded on cell clusters (≥ 5 cells). 5 Cell clusters, in contrast to single cells, had a permanent resting K+ conductance in addition to the inward rectifier and the voltage‐dependent current. This resting K+ conductance was not blocked by TEA+, Ba2+, 4‐aminopyridine or by the chromanol 293B. 6 Cytosolic alkalization by addition of NH4Cl to the bath solution decreased the resting K+ current. In parallel, electrical uncoupling of the cells and breakdown of the resting potential could be observed. The same effects could be produced when the cells were uncoupled by 0.2–1.0 mm n‐octanol. It can be concluded that cell coupling is essential for maintenance of stable resting membrane potentials in pancreatic acinar cells.

Capacitative Ca2+ influx and a Ca2u+-dependent nonselective cation pathway are discriminated by genistein in mouse pancreatic acinar cells

We have investigated the effect of genistein on the hormone-stimulated Ca2+ influx and on a 28 pS nonselective cation channel in mouse pancreatic acinar cells using the Ca2+ indicator fluo-3 and the patch-clamp technique. The identity of the Ca2u+ influx pathway has not been established in this cell type so far. Therefore we have investigated the Ca2+-dependent nonselective cation channel as a potential pathway for Ca2+ influx. “Capacitative” Ca2+ entry was induced by depletion of intracellular Ca2+ stores with 500 nM acetylcholine or with the Ca2+ ATPase inhibitor 2,5-di-tert- butylhydroquinone. In the presence of 100 μM genistein, Ca2+ release was unimpaired, whereas Ca2+ influx was reversibly suppressed. Patch-clamp experiments demonstrated that genistein had no effect on Ca2+-activated nonselective cation channels, the activity of which was measured in excised membrane patches (inside/out) or in the whole-cell configuration. Therefore we conclude that this 28 pS nonselective cation channel does not contribute to Ca2+ influx into mouse exocrine pancreatic cells. With the exception of genistein and tyrphostin 25, other tyrosine kinase inhibitors such as methyl-2,5-dihydroxycinnamate, lavendustin A, herbimycin A, and tyrphostin B56 were without effect on Ca2+ signalling. Thus, the involvement of tyrosine phosphorylation in the activation of the Ca2+ entry mechanism in mouse pancreatic acinar cells is unclear.

Inhibition of amylase secretion from differentiated AR4‐2J pancreatic acinar cells by an actin cytoskeleton controlled protein tyrosine phosphatase activity

Disruption of the actin cytoskeleton in AR4‐2J pancreatic acinar cells led to an increase in cytosolic protein tyrosine phosphatase activity, abolished bombesin‐induced tyrosine phosphorylation and reduced bombesin‐induced amylase secretion by about 45%. Furthermore, both tyrosine phosphorylation and amylase secretion induced by phorbol ester‐induced activation of protein kinase C were abolished. An increase in the cytosolic free Ca2+ concentration by the Ca2+ ionophore A23187 had no effect on tyrosine phosphorylation but induced amylase release. Only when added together with phorbol ester, the same level of amylase secretion as with bombesin was reached. This amylase secretion was inhibited by about 40% by actin cytoskeleton disruption similar to that induced by bombesin. We conclude that actin cytoskeleton‐controlled protein tyrosine phosphatase activity downstream of protein kinase C activity regulates tyrosine phosphorylation which in part is involved in bombesin‐stimulated amylase secretion.