A voltage-sensitive transient potassium current in mouse pancreatic acinar cells

Cellular and luminal pH of isolated ant Malpighian tubules were measured in different bath K⁺ concentrations using double-barrelled pH microelectrodes. The electrochemical gradient for H⁺ across the basolateral and the apical cell membranes was estimated. In control Ringer (51 mmol/l K⁺) cell and luminal pH were alkaline with respect to the basolateral solution: 7.77 and 7.36, respectively, versus 7.25. On lowering basolateral K⁺ concentration to 5 mmol/l or increasing it to 113 mmol/l, luminal pH and to a lesser extent cell pH followed: luminal pH changed to 7.14 and 7.43 and cell pH to 7.69 and 7.82, respectively. In all conditions a cell inward electrochemical gradient for protons across both membranes was observed. Increasing basolateral K⁺ concentration, which was positively correlated with secretion rate, decreased the cell inwardly directed apical proton gradient; moreover, the apical membrane potential difference decreased as well, from –93 mV in 5 mmol/l K⁺ to –65 mV in 113 mmol/l K⁺. Therefore the turnover rate of the electrogenic active proton pump at the apical membrane is facilitated in a high basolateral K⁺ concentration. The calculated electromotive force of this pump is –159 mV. Comparing the proton with the K⁺ electrochemical gradient, taken from another study in the same experimental conditions, we find that the apical proton electrochemical gradient can drive K⁺ extrusion into the lumen for each value of secretion rate.     

Reduced cholecystokinin receptor phosphorylation and restored signalling in protein kinase C down-regulated rat pancreatic acinar cells

 Receptor phosphorylation in response to agonist stimulation is a key regulatory principle in signal transduction. Previous work has suggested the concerted action of protein kinase C (PKC) and a staurosporine-insensitive receptor kinase in homologous phosphorylation of the cholecystokinin (CCK) receptor in freshly isolated rat pancreatic acinar cells [Gates, Ulrich, Miller (1993) Am J Physiol 264:G840–G847]. The present study shows that down-regulation of PKC by prolonged (2 h) treatment with 0.1 μM 12-O-tetradecanoylphorbol-13-acetate (TPA) markedly reduced basal CCK receptor phosphorylation as well as that induced by TPA (0.1 μM) and cholecystokinin-(26-33)-peptide amide (CCK₈, 0.1 μM). The phosphorylation level reached was the same with both stimulants and equalled basal phosphorylation in untreated control cells. The absence of any CCK₈-stimulated phosphorylation reflecting the activity of a putative staurosporine-insensitive receptor kinase raises the intriguing possibility that a basal level of PKC-mediated receptor phosphorylation is required for the action of such a receptor kinase. Immunoblot analysis revealed that the decrease in receptor phosphorylation coincided with a marked reduction of PKC-α and, to a lesser extent, PKC-ɛ. In addition, TPA-induced inhibition of the increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) evoked by the high-affinity CCK receptor agonist JMV-180 was completely reversed. The time-course of recovery closely matched that of the reduction of PKC-α. Finally, digital imaging microscopy of individual PKC down-regulated cells revealed a marked increase in the duration of JMV-180-evoked oscillatory changes in [Ca²⁺]_i. Taken together, the present findings are in agreement with the idea that PKC-α-mediated receptor phosphorylation leads to a shortening of the duration of the [Ca²⁺]_i oscillations and eventually to inhibition of high-affinity Ca²⁺ signalling through the native CCK receptor in pancreatic acinar cells. Received: 15 September 1997 / Accepted: 2 October 1997     

Intracellular pH and calcium signalling in rat pancreatic acinar cells

 Intracellular free Ca²⁺ signals, which occur in many secretory cell types after the binding of some secretagogues to their membrane receptors, are due to Ca²⁺ mobilization from internal stores and Ca²⁺ influx from the extracellular space. There is also growing evidence for a modulatory role of intracellular pH in Ca²⁺ metabolism. In fact it has been proposed that Ca²⁺ stores in pancreatic acinar cells may be loaded by Ca²⁺/H⁺ exchange. The aim of this paper was to establish the effect of intracellular pH on Ca²⁺ signalling in pancreatic acinar cells. Application of the proton carrier nigericin impairs Ca²⁺ mobilization in response to cholecystokinin (CCK-8), and application of membrane-permeant bases or acids inhibits CCK-8-evoked intracellular Ca²⁺ oscillations. Both nigericin and a cell-permeant weak base release Ca²⁺ from internal stores. However, cytosolic acidification by removal of extracellular Na⁺ had no effect on the resting or stimulated cytosolic Ca²⁺ concentration. After depletion of Ca²⁺ stores by a maximal concentration of CCK-8, nigericin and ionomycin released a residual Ca²⁺ pool. Taken together, our results show that in pancreatic acinar cells Ca²⁺ signals require the existence of subcellular gradients of pH and indicate the presence of acidic pools of Ca²⁺. Received: 21 March 1997 / Received after revision: 14 May 1997 / Accepted: 15 May 1997     

Voltage clamp study of stimulant-evoked currents in mouse pancreatic acinar cells

Isolated segments of mouse pancreas were placed in a perspex bath and superfused with physiological saline solution. Acinar units were voltage-clamped with the help of two intracellular microelectrodes. Voltage homogeneity was in some experiments checked with a third microelectrode inserted into the same unit. The currents associated with hyper- or depolarizing voltage jumps were recorded in the absence or presence of sustained stimulation with acetylcholine (ACh), caerulein or bombesin nonapeptide. ACh (2 x 10(-7)-10(-8) M) evoked a dose-dependent inward current and an increase in the membrane conductance. The steady state ACh-evoked current (control current subtracted from total current in presence of ACh) depended linearly on voltage within the range -100 to +20 mV and its polarity reversed at about -25 mV. The effects of caerulein and bombesin nonapeptide were indistinguishable from those of ACh. Voltage homogeneity in the acinar unit was attained earlier than 1 ms after a hyper- or depolarizing voltage jump. The current transients associated with voltage jumps decayed according to single exponential functions both in the absence and presence of ACh. The time constant of the single exponential current decay after a voltage jump was the same (1-3 ms) in the absence or presence of ACh. The amplitude of the current transient was, however, reduced by ACh. The time constant of the current decay following voltage jumps was independent of the voltage in the range +60 to -60 mV, both in the absence and presence of ACh. The ACh-evoked reduction in the amplitude of the transient current following voltage jumps dependent linearly on the voltage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Redox-sensitive modulation of CD45 expression in pancreatic acinar cells during acute pancreatitis

CD45, a transmembrane protein tyrosine phosphatase required for signal transduction in leukocytes, has recently been found in pancreatic acinar cells. We have investigated the relationship between kinetic expression of CD45 on acinar cells during acute pancreatitis (AP) and the ability of these cells to produce tumour necrosis factor-alpha (TNF-alpha) through mechanisms sensitive to the cellular redox state. Flow cytometric analysis showed a significant decrease in the constitutive expression of CD45 in acinar cells from six hours onwards after inducing AP by bile-pancreatic duct obstruction (BPDO) in parallel with a significant increase in acinar TNF-alpha production. Changes in protein expression on the acinar cell surface preceded CD45 mRNA down-regulation, which was not found until 12 hours after BPDO. N-Acetylcysteine treatment delayed and reduced the down-regulation of CD45 expression induced by AP and prevented acinar cells from producing TNF-alpha. Our results show that CD45 expression is down-regulated in acinar cells during acute pancreatitis by redox-sensitive mechanisms, and they support the notion that CD45 negatively controls the production of cytokines in pancreatic acinar cells.     

The properties of the secretagogue-evoked chloride current in mouse pancreatic acinar cells

In this paper we describe the properties of the secretagogue-evoked chloride current from mouse pancreatic acinar cells. Single cells were patch-clamped in the whole-cell configuration with solutions that excluded cation currents and then stimulated with 1 mM carbachol (CCh). This resulted in a current that rose to a peak and then decayed to a plateau level. The current/voltage relationship of the peak current was linear whereas that of the plateau phase was rectified and showed time and voltage dependence. To determine if the CCh evoked current was strictly Ca2+ dependent, we compared the properties of the plateau current with those of currents evoked by directly raising cytosolic [Ca2+] The properties of the two currents were the same, with both currents showing outward rectification, development at depolarised potentials, similar time constants of activation, permeability sequences and sensitivity to the Cl- channel inhibitor 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulphonic acid (DIDS). We conclude that the agonist-evoked rise in Ca2+ is alone a sufficient signal to activate the CL- current.     

Increased calcium influx in the presence of ethanol in mouse pancreatic acinar cells

The effects of alcohol on Ca²⁺ signalling remains poorly understood. Here we have investigated the effects of acute ethanol exposure on Ca²⁺ influx in mouse pancreatic acinar cells. Cells were loaded with fura‐2 and the changes in fluorescence were monitored by spectrofluorimetry and imaging analysis. Stimulation of cells with 20 pM cholecystokinin evoked an oscillatory pattern in [Ca²⁺]_c, both in the presence and in the absence of extracellular Ca²⁺. Stimulation of cells with cholecystokinin in the presence of 50 mM ethanol led to a transformation of physiological oscillations into a single transient increase in [Ca²⁺]_c. This effect was observed when Ca²⁺ was present in the extracellular medium, and did not appear in its absence. Addition of 1 mM CaCl₂ to the extracellular medium, following release of Ca²⁺ from intracellular stores by stimulation of cells with 1 nM cholecystokinin or 1 μM thapsigargin in the absence of extracellular Ca²⁺, was followed by an increase in [Ca²⁺]_c. Ca²⁺ influx was increased in the presence of 50 mM ethanol. The anti‐oxidant cinnamtannin B‐1 (10 μM) or inhibition of alcohol dehydrogenase by 4‐MP (1 mM), significantly reduced Ca²⁺ influx evoked by cholecystokinin in the presence of ethanol. In summary, intoxicating concentrations of ethanol may lead to over stimulation of pancreatic acinar cells by cholecystokinin. This might be partially explained by the generation of reactive oxygen species and an increased Ca²⁺ entry in the presence of ethanol. Potentially ethanol might lead to Ca²⁺ overload, which is a common pathological precursor that is implicated in pancreatitis.     

Bile acids induce calcium signals in mouse pancreatic acinar cells: implications for bile-induced pancreatic pathology

The effect of the natural bile acid, taurolithocholic acid 3-sulfate (TLC-S), on calcium signalling in pancreatic acinar cells has been investigated. TLC-S induced global calcium oscillations and extended calcium transients as well as calcium signals localised to the secretory granule (apical) region of acinar cells. These calcium signals could still be triggered by TLC-S in a calcium-free external solution. TLC-S-induced calcium signals were not inhibited by atropine, but were abolished by caffeine or by depletion of calcium stores, due to prolonged application of ACh. Global calcium signals, produced by TLC-S application, displayed vectorial apical-to-basal polarity. The signals originated in the apical part and were then propagated to the basal region. Other natural bile acids, taurocholate (TC) and taurodeoxycholate (TDC), were also able to produce local and global calcium oscillations (but at higher concentrations than TLC-S). Bile, which can enter pancreas by reflux, has been implicated in the pathology of acute pancreatitis. The calcium releasing properties of bile acids suggest that calcium toxicity could be an important contributing factor in the bile acid-induced cellular damage.     

Regulation of early response genes in pancreatic acinar cells: external calcium and nuclear calcium signalling aspects

Nuclear calcium signalling has been an important topic of investigation for many years and some aspects have been the subject of debate. Our data from isolated nuclei suggest that the nuclear pore complexes (NPCs) are open even after depletion of the Ca(2+) store in the nuclear envelope (NE). The NE contains ryanodine receptors (RyRs) and Ins(1,4,5)P(3) receptors [Ins(1,4,5)P(3)Rs], most likely on both sides of the NE and these can be activated separately and independently: the RyRs by either NAADP or cADPR, and the Ins(1,4,5)P(3)Rs by Ins(1,4,5)P(3). We have also investigated the possible consequences of nuclear calcium signals: the role of Ca(2+) in the regulation of immediate early genes (IEG): c-fos, c-myc and c-jun in pancreatic acinar cells. Stimulation with Ca(2+)-mobilizing agonists induced significant increases in levels of expression. Cholecystokinin (CCK) (10 nm) evoked a substantial rise in the expression levels, highly dependent on external Ca(2+): the IEG expression level was lowest in Ca(2+)-free solution, increased at the physiological level of 1 mm [Ca(2+)](o) and was maximal at 10 mm [Ca(2+)](o), i.e.: 102 +/- 22% and 163 +/- 15% for c-fos; c-myc -73 +/- 13% and 106 +/- 24%; c-jun -49 +/- 8% and 59 +/- 9% at 1 and 10 mm of extracellular Ca(2+) respectively. A low CCK concentration (10 pm) induced a small increase in expression. We conclude that extracellular Ca(2+) together with nuclear Ca(2+) signals induced by CCK play important roles in the induction of IEG expression.     

Cell type-specific dependence of muscarinic signalling in mouse hippocampal stratum oriens interneurones

Cholinergic signalling is critically involved in learning and memory processes in the hippocampus, but the postsynaptic impact of cholinergic modulation on morphologically defined subtypes of hippocampal interneurones remains unclear. We investigated the influence of muscarinic receptor (mAChR) activation on stratum oriens interneurones using whole-cell patch clamp recordings from hippocampal slices in vitro . Upon somatic depolarization, mAChR activation consistently enhanced firing frequency and produced large, sustained afterdepolarizations (ADPs) of stratum oriens–lacunosum moleculare (O-LM) interneurones. In contrast, stratum oriens cell types with axon arborization patterns different from O-LM cells not only lacked large muscarinic ADPs but also appeared to exhibit distinct responses to mAChR activation. The ADP in O-LM cells, mediated by M₁/M₃ receptors, was associated with inhibition of an M current, inhibition of a slow calcium-activated potassium current, and activation of a calcium-dependent non-selective cationic current ( I _CAT). An examination of ionic conductances generated by firing revealed that calcium entry through I _CAT controls the emergence of the mAChR-mediated ADP. Our results indicate that cholinergic specializations are present within anatomically distinct subpopulations of hippocampal interneurones, suggesting that there may be organizing principles to cholinergic control of GABA release in the hippocampus.     

Nitric oxide‐induced signalling in rat lacrimal acinar cells

The aim of the present study was to investigate the physiological role of nitric oxide (NO) in mediating secretory processes in rat lacrimal acinar cells. In addition, we wanted to determine whether the acinar cells possess endogenous nitric oxide synthase (NOS) activity by measuring NO production using the fluorescent NO indicator 4,5‐diaminofluorescein (DAF‐2). We initiated investigations by adding NO from an external source by means of the NO‐donor, S‐nitroso‐N‐acetyl‐penicillamine (SNAP). Cellular concentrations of cyclic guanosine 5′‐phosphate (cGMP) ([cGMP]) were measured by radioimmunoassay (RIA), and we found that SNAP induced a fast increase in the [cGMP], amounting to 350% of the [cGMP] in resting cells. Moreover, addition of SNAP and elevating [cGMP] in fura‐2 loaded lacrimal acinar cells, resulted in a cGMP‐dependent protein kinase‐mediated release of Ca²⁺ from intracellular stores, leading to a rise in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i). The Mn²⁺ quenching studies revealed that the Ca²⁺ release was not accompanied by Ca²⁺ influx. Finally, we demonstrate that lacrimal acinar cells possess endogenous NOS activity, which is activated by β‐adrenergic stimulation and not by a rise in [Ca²⁺]_i alone. We show that in rat lacrimal acinar cells, NO and cGMP induce Ca²⁺ release from intracellular stores via G kinase activation. However, the changes in [Ca²⁺]_i are relatively small, suggesting that this pathway plays a modulatory role in Ca²⁺ signalling, thus not by itself causing fast transient increases in [Ca²⁺]_i. In addition, we suggest that endogenously produced NO activated by β‐adrenergic receptor stimulation, plays an important role in signalling to the surrounding tissue.     

Inhibitors of Bcl-2 protein family deplete ER Ca2+ stores in pancreatic acinar cells

Physiological stimulation of pancreatic acinar cells by cholecystokinin and acetylcholine activate a spatial-temporal pattern of cytosolic [Ca⁺²] changes that are regulated by a coordinated response of inositol 1,4,5-trisphosphate receptors (IP₃Rs), ryanodine receptors (RyRs) and calcium-induced calcium release (CICR). For the present study, we designed experiments to determine the potential role of Bcl-2 proteins in these patterns of cytosolic [Ca⁺²] responses. We used small molecule inhibitors that disrupt the interactions between prosurvival Bcl-2 proteins (i.e. Bcl-2 and Bcl-xl) and proapoptotic Bcl-2 proteins (i.e. Bax) and fluorescence microfluorimetry techniques to measure both cytosolic [Ca⁺²] and endoplasmic reticulum [Ca⁺²]. We found that the inhibitors of Bcl-2 protein interactions caused a slow and complete release of intracellular agonist-sensitive stores of calcium. The release was attenuated by inhibitors of IP₃Rs and RyRs and substantially reduced by strong [Ca²⁺] buffering. Inhibition of IP₃Rs and RyRs also dramatically reduced activation of apoptosis by BH3I-2′. CICR induced by different doses of BH3I-2′ in Bcl-2 overexpressing cells was markedly decreased compared with control. The results suggest that Bcl-2 proteins regulate calcium release from the intracellular stores and suggest that the spatial-temporal patterns of agonist-stimulated cytosolic [Ca⁺²] changes are regulated by differential cellular distribution of interacting pairs of prosurvival and proapoptotic Bcl-2 proteins.     

Downstream from calcium signalling: mitochondria, vacuoles and pancreatic acinar cell damage

Ca(2+) is one of the most ancient and ubiquitous second messengers. Highly polarized pancreatic acinar cells serve as an important cellular model for studies of Ca(2+) signalling and homeostasis. Downstream effects of Ca(2+) signalling have been and continue to be an important research avenue. The primary functions regulated by Ca(2+) in pancreatic acinar cells--exocytotic secretion and fluid secretion--have been defined and extensively characterized in the second part of the last century. The role of cytosolic Ca(2+) in cellular pathology and the related question of the interplay between Ca(2+) signalling and bioenergetics are important current research lines in our and other laboratories. Recent findings in these interwoven research areas are discussed in the current review.