Biochem Pharmacol 2002 Jan 15;63(2):283-92

Role of mitochondria in Ca(2+) oscillations and shape of Ca(2+) signals in pancreatic acinar cells.

Camello-Almaraz C, Salido GM, Pariente JA, Camello PJ.

Department of Physiology, Faculty of Veterinary Sciences, University of Extremadura, 10071, Caceres, Spain.

We studied the role of mitochondria in Ca(2+) signals in fura-2 loaded exocrine pancreatic acinar cells. Mitochondrial depolarization in response to carbonylcyanide-p-tryfluoromethoxyphenyl hydrazone or rotenone (assessed by confocal microscopy using rhodamine-123) induced a partial but statistically significant reduction in the decay of Ca(2+) signals under different experimental conditions. Spreading of Ca(2+) waves evoked by the pancreatic secretagogue cholecystokinin cholecystokinin octapeptide was accelerated by mitochondrial inhibitors, whereas the cytosolic Ca(2+) concentration ([Ca(2+)](i)) oscillations in response to physiological levels of this hormone were suppressed by rotenone and carbonylcyanide-p-tryfluoromethoxyphenyl hydrazone. Oligomycin, an inhibitor of mitochondrial ATP synthase, did no affect either propagation of calcium waves nor [Ca(2+)](i) oscillations. Individual mitochondria of rhod-2 loaded acinar cells showed heterogeneous matrix Ca(2+) concentration increases in response to oscillatory and maximal levels of cholecystokinin octapeptide. On the other hand, using Ba(2+) for unequivocal study of capacitative calcium entry we found that mitochondrial inhibitors did not affect this process. Our results show that although the role of mitochondria as a Ca(2+) clearing system in exocrine cells is quantitatively secondary, they play an essential role in the spatial propagation of Ca(2+) waves and in the development of [Ca(2+)](i) oscillations.

Cell Signal 2002 Jun;14(6):547-56

Effects of reactive oxygen species on actin filament polymerisation and amylase secretion in mouse pancreatic acinar cells.

Rosado JA, Gonzalez A, Salido GM, Pariente JA.

Department of Physiology, Faculty of Veterinary Sciences, University of Extremadura, Caceres 10071, Spain.

The present study investigates the effect of reactive oxygen species (ROS) on actin filament reorganisation and its relevance to exocytosis in pancreatic acinar cells. Treatment of pancreatic acini with cholecystokinin (CCK-8) induced spatial and temporal changes in actin filament reorganisation with an initial depolymerisation of the apical actin barrier followed by an increase in the actin filament content in the subapical area leading to amylase release. Hydrogen peroxide (H(2)O(2)) increased actin filament content and potentiated the polymerizing effects of CCK-8 in these cells but abolished the disruption of the apical actin layer and amylase release induced by CCK-8. Similar to CCK-8, ROS generated by the oxidation of hypoxanthine (HX) with xanthine oxidase (XOD) induced an initial decrease in actin filaments located under the apical membrane followed by a smaller increase in the content of actin filaments in the subapical area. XOD-generated ROS are able to increase amylase release in pancreatic acini although combination with CCK-8 leads to abnormal exocytosis. We provide evidence that indicates that CCK-8- and ROS-induced actin reorganisation is entirely dependent on Ca(2+) mobilisation and independent of PKC activation. The regulation of the actin cytoskeleton by ROS might be involved in radical-induced cell injury in pancreatic acinar cells.

XOD-catalyzed ROS generation mobilizes calcium from intracellular stores in mouse pancreatic acinar cells.

Gonzalez A, Schmid A, Salido GM, Camello PJ, Pariente JA.

Department of Physiology, Faculty of Veterinary Sciences, University of Extremadura, P.O. Box 643 10071, Caceres, Spain.

In fura-2 loaded isolated mouse pancreatic acinar cells, xanthine oxidase (XOD)-catalyzed reactive oxygen species (ROS) generation caused an increase in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) by release of Ca(2+) from intracellular stores. The ROS-induced Ca(2+) signals showed large variability in shape and time-course and resembled in part Ca(2+) signals in response to physiological secretagogues. ROS-induced Ca(2+) mobilization started at the luminal cell pole and spread towards the basolateral side in a wave manner. ROS-evoked Ca(2+) responses were not inhibited by the phospholipase C (PLC) inhibitor U73122 (10 microM). Neither 2-aminoethoxy-diphenylborate (2-APB) (70 microM) nor ryanodine (50 microM) suppressed ROS-evoked Ca(2+) release. ROS still released Ca(2+) when the endoplasmic reticulum Ca(2+)-ATPase was blocked with thapsigargin (1 microM), or when rotenone (10 microM) was added to release Ca(2+) from mitochondria. Our results suggest that pancreatic acinar cells ROS do not unspecifically affect Ca(2+) homeostasis. ROS primarily affect Ca(2+) stores located in the luminal cell pole, which is also the trigger zone for agonist-induced Ca(2+) signals. Release of Ca(2+) induces Ca(2+) waves carried by Ca(2+)-induced Ca(2+) release and produces thereby global Ca(2+) signals. Under oxidative stress conditions, the increase in [Ca(2+)](i) could be one mechanism contributing to an overstimulation of the cell which could result in cell dysfunction and cell damage.

Effect of xanthine oxidase-catalyzed reactive oxygen species generation on secretagogue-evoked calcium mobilization in mouse pancreatic acinar cells.

Gonzalez-Mateos A, Camello PJ, Salido GM, Pariente JA.

Department of Physiology, University of Extremadura, Faculty of Veterinary Sciences, P.O. Box 643, 10071, Caceres, Spain.

In the present study we have employed fura-2 loaded isolated mouse pancreatic acinar cells to monitor the effect that xanthine oxidase (XOD)-catalyzed reactive oxygen species generation presents on Ca(2+) mobilization by the secretagogue cholecystokinin octapeptide (CCK-8). Our results show that perfusion of pancreatic acinar cells with CCK-8 at a physiological concentration (20 pM) induced low frequency oscillations in intracellular free calcium concentration ([Ca(2+)](i)) at a rate of 1 per minute; this oscillatory pattern was completely inhibited by the introduction in the perifusion medium of 20 mU/mL XOD to generate reactive oxygen species. In addition, perfusion of pancreatic acinar cells with 20 mU/mL XOD in the absence of extracellular calcium led to a transient increase in [Ca(2+)](i,) that blocked the initiation of the Ca(2+) signals in response to 20 pM CCK-8. Similarly, XOD was also able to block acetylcholine evoked Ca(2+) spikes. However, reactive oxygen species had no effect either on Ca(2+) extrusion or on re-uptake into intracellular stores, but CCK-8-evoked Ca(2+) entry was reduced by XOD. In conclusion, our results show that XOD-evoked reactive oxygen species generation leads to a reduction either of Ca(2+) mobilization, following stimulation of pancreatic acinar cells with the Ca(2+)-mobilizing agonists CCK-8 and acetylcholine, and Ca(2+) influx evoked by CCK-8 depletion of intracellular stores. The possible XOD inhibitory mechanism on Ca(2+) mobilization by agonists is discussed.

Vanadate inhibits the calcium extrusion in rat pancreatic acinar cells

Lajas AI, Sierra V, Camello PJ, Salido GM, Pariente JA.

Department of Physiology. Faculty of Veterinary Science, University of Extremadura, PO Box 643, 10071, Caceres, Spain. alajas@unex.es

Our objective was to evaluate the role of vanadate on calcium extrusion in Fura-2-loaded rat pancreatic acinar cells by digital microscopic fluorimetry and spectrofluorimetry. In the absence of extracellular calcium, perfusion of pancreatic acinar cells with 1 nM CCK-8 and 1 mM vanadate did not significantly affect the typical transient calcium spike induced by CCK-8, but the plateau phase of calcium in response to CCK-8 remained elevated. In addition, vanadate was able to inhibit calcium efflux evoked by CCK-8 when we determined directly calcium transport across plasma membrane using Calcium Green-5N hexapotassium salt (cell impermeant form) in cell populations. The effect of vanadate on calcium extrusion was strongly blocked by the sulfhydryl-reducing agent dithiothreitol (DTT). The present results demonstrate that vanadate is able to irreversibly inhibit the calcium extrusion. This effect of vanadate can be blocked using DTT, indicating that its action is probably mediated by oxidation of sulfhydryl groups of Ca2+-ATPases.

Release of Calcium from Mitochondrial and Nonmitochondrial Intracellular Stores in Mouse Pancreatic Acinar Cells by Hidrogen Peroxide.

J.A. Pariente, C.Camello, P.J.Camello, G.M.Salido

Department of Physiology, Faculty of Veterinary Sc, University of Extremadura, 10071-Caceres, Spain.

In the present study we have studied how [Ca2+]i is influenced by H2O2 in collagenase-dispersed mouse pancreatic acinar cells and the mechanism underlying this effect by using a digital microspectrofluorimetric system. In the presence of normal extracellular calcium concentration, perfusion of pancreatic acinar cells with 1 mM H2O2 caused a slow sustained [Ca2+]i increase, reaching a stable plateau after 10-15 min of perfusion. This increase induced by H2O2 was also observed in a nominally calcium-free medium, reflecting the release of calcium from intracellular store(s). Application of 1 mM H2O2 to acinar cells, in which nonmitochondrial agonist-releasable calcium pools had been previously depleted by a maximal concentration of CCK-8 (1 nM) or thapsigargin (0.5 ?M) was still able to induce calcium release. Similar results were observed when thapsigargin was substituted for the mitochondrial uncoupler FCCP (0.5 ?M). By contrast, simultaneous addition of thapsigargin and FCCP clearly abolished the H2O2-induced calcium increase. Interestingly, co-incubation of intact pancreatic acinar cells with CCK-8 plus thapsigargin and FCCP in the presence of H2O2 did not significantly affect the transient calcium spike induced by the depletion of nonmitochondrial and mitochondrial agonist-releasable calcium pools, but was followed by a sustained increased of [Ca2+]i. . In addition, H2O2 was able to block calcium efflux evoked by CCK and thapsigargin. Finally, the transient increase in [Ca2+]i induced by H2O2 was abolished by a addition of 2 mM dithiothreitol (DTT), a sulfhydryl reducing agent. Our results show that H2O2 releases calcium from CCK-8- and thapsigargin-sensitive intracellular stores and from mitochondria. The action of H2O2 is likely mediated by oxidation of sulfhydryl groups of calcium-ATPases.

Effect of dephostatin on intracellular free calcium concentration and amylase secretion in isolated rat pancreatic acinar cells.

Lajas AI, Pozo MJ, Camello PJ, Salido GM, Singh J, Pariente JA

Department of Physiology, Faculty of Veterinary Sciences, University of
Extremadura, Caceres, Spain.

This study investigates the effects of dephostatin, a new tyrosine phosphatase inhibitor, on intracellular free calcium concentration ([Ca2+]i) and amylase secretion in collagenase dispersed rat pancreatic acinar cells. Dephostatin evoked a sustained elevation in [Ca2+]i by mobilizing calcium from intracellular calcium stores in either the absence of extracellular calcium or the presence of lanthanium chloride (LaCl3). Pretreatment of acinar cells with dephostatin prevented cholecystokinin-octapeptide (CCK-8)-induced signal of [Ca2+]i and inhibited the oscillatory pattern initiated by aluminium fluoride (AlF4), whereas co-incubation with CCK-8 enhances the plateau phase of calcium response to CCK-8 without modifying the transient calcium spike. The effects of dephostatin on calcium mobilization were reversed by the presence of the sulfhydryl reducing agent, dithiothreitol. Stimulation of acinar cells with thapsigargin in the absence of extracellular Ca2+ resulted in a transient rise in [Ca2+]i. Application of dephostatin in the continuous presence of thapsigargin caused a small but sustained elevation in [Ca2+]i. These results suggest that dephostatin can mobilize Ca2+ from both a thapsigargin-sensitive and thapsigargin-insensitive intracellular stores in pancreatic acinar cells. In addition, dephostatin can stimulate the release of amylase from pancreatic acinar cells and moreover, reduce the secretory response to CCK-8. The results indicate that dephostatin can release calcium from intracellular calcium pools and consequently induces amylase secretion in pancreatic acinar cells. These effects are likely due to the oxidizing effects of this compound.

Effects of antioxidants on calcium signal induced by cholecystokinin in mouse pancreatic acinar cells

C.Camello, P.J.Camello, J.A.Pariente, G.M.Salido

Department of Physiology, Faculty of Veterinary Sc, University of Extremadura, 10071-Caceres, Spain.

Digital imaging fluorescence microscopy was used to study the effect of two antioxidants, N-acetyl-cysteine (NAC) and glutathione, on the cytosolic free calcium concentration ([Ca2+]i) induced by cholecystokinin-octapeptide (CCK-8) of mouse pancreatic acinar cells. When acinar cells were preincubated with either NAC or glutathione, subsequent stimulation with CCK-8 in the presence of each antioxidant had no significant effect on the typical pattern of [Ca2+]i transient evoked by the gastrointestinal hormone. However, application of NAC to acinar cells pretreated during 60 min with the same antioxidant, strongly blocked the oscillatory pattern initiated by CCK-8, inhibiting both amplitude and frequency of calcium oscillations. By the contrast, glutathione was without effect on the oscillatory pattern evoked by CCK-8. The present results allow us to speculate that during [Ca2+]i oscillation there is a production of oxidants that facilitate oscillations by enhancing release of calcium from internal stores.

Biochem Biophys Res Commun 2000 May 10;271(2):311-7

Differential involvement of vacuolar H(+)-ATPase in the refilling of thapsigargin- and agonist-mobilized Ca(2+) stores.

Camello-Almaraz C, Pariente JA, Salido G, Camello PJ

Department of Physiology, University of Extremadura, Caceres, 10071, Spain.

Our objective was to evaluate the role of vacuolar H(+)-ATPase and proton gradients in the refilling of Ca(2+) stores in fura-2-loaded pancreatic acinar cells. Once depleted with a high level of ACh, the Ca(2+) stores were replenished with a Ca(2+)-containing solution. The degree of refilling was estimated with a second release in response to either ACh (ACh-releasable store) or thapsigargin (thapsigargin-releasable store), a specific inhibitor of the endoplasmic reticulum Ca(2+) pumps. Both the protonophore nigericin and folimycin, a specific inhibitor of the vacuolar H(+)-ATPase, reduced reuptake into the ACh-mobilized stores but not into the thapsigargin-releasable pools. These treatments effectively dissipated the subcellular pH gradients (revealed by confocal observation of the distribution of a marker for acidic compartments), and did not impair the [Ca(2+)](i) response to ACh in control cells. Our results indicate that thapsigargin and ACh release heterogeneous Ca(2+) stores which are differently operated by vacuolar proton ATPase.

Curr Biol. 2000 Feb 10;10(3):161-4.

Role of proton gradients and vacuolar H+-ATPases in the refilling of intracellular calcium stores in exocrine cells.

Camello C, Pariente JA, Salido GM ,Camello PJ

University of Extremadura, Dept Physiology, Fac. Veterinary Science,10071, Caceres, Spain

Numerous hormones and neurotransmitters activate cells by increasing cytosolic calcium concentration ([Ca2+]i), a key regulatory factor for many cellular processes. A pivotal feature of these Ca2+ signals is the release of Ca2+ from intracellular stores, which is followed by activation of extracellular calcium influx, allowing refilling of the stores by SERCA pumps associated to the endoplasmic reticulum. While the mechanisms of calcium release and calcium influx have been extensively studied, the biology of Ca2+ stores is poorly understood. The presence of heterogeneous calcium pools has been previously reported (1-3). Although recent technical improvements have confirmed this heterogeneity (see 4), knowledge about transport of Ca2+ in the stores and its underlying mechanisms is very scarce and rather speculative. A recent report (5) has described in polarized exocrine cells the existence of Ca2+ tunneling from basolateral stores to luminal pools, where Ca2+ is initially released upon cell activation. We present here evidence that, during stimulation, Ca2+ transported into basolateral stores by SERCA pumps is conveyed toward the luminal pools with the cooperation of proton gradients driven by vacuolar H-ATPases. This finding unveils a new aspect of the machinery of Ca2+ stores.

Cell Signal 1999 Oct;11(10):727-34

Phenylarsine oxide evokes intracellular calcium increases and amylase secretion in isolated rat pancreatic acinar cells.

Lajas AI, Pozo MJ, Camello PJ, Salido GM, Pariente JA

Department of Physiology, Faculty of Veterinary Science, University of Extremadura, Caceres, Spain.

The effects of the thiol reagent, phenylarsine oxide (PAO, 10(-5)-10(-3) M ), a membrane-permeable trivalent arsenical compound that specifically complexes vicinal sulfhydryl groups of proteins to form stable ring structures, were studied by monitoring intracellular free calcium concentration ([Ca2+]i) and amylase secretion in collagenase dispersed rat pancreatic acinar cells. PAO increased [Ca2+]i by mobilizing calcium from intracellular stores, since this increase was observed in the absence of extracellular calcium. PAO also prevented the CCK-8-induced signal of [Ca2+]i and inhibited the oscillatory pattern initiated by aluminium fluoride (AlF-4). In addition to the effects of PAO on calcium mobilization, it caused a significant increase in amylase secretion and reduced the secretory response to either CCK-8 or AlF-4. The effects of PAO on both [Ca2+]i and amylase release were reversed by the sulfhydryl reducing agent, dithiothreitol (2 mM). Pretreatment of acinar cells with high concentration of ryanodine (50 microM) reduced the PAO-evoked calcium release. However, PAO was still able to release a small fraction of Ca2+ from acinar cells in which agonist-releasable Ca2+ pools had been previously depleted by thapsigargin (0.5 microM) and ryanodine receptors were blocked by 50 microM ryanodine. We conclude that, in pancreatic acinar cells, PAO mainly releases Ca2+ from the ryanodine-sensitive calcium pool and consequently induces amylase secretion. These effects are likely to be due to the oxidizing effects of this compound.

Biochem Pharmacol 1999 Jul 1;58(1):77-84

Oxidizing effects of vanadate on calcium mobilization and amylase release in rat pancreatic acinar cells.

Pariente JA, Lajas AI, Pozo MJ, Camello PJ, Salido GM

Department of Physiology, Faculty of Veterinary Sciences, University of Extremadura, Caceres, Spain.

The effects of vanadate were examined by monitoring intracellular free calcium concentration ([Ca2+]i) and amylase secretion in collagenase-dispersed rat pancreatic acinar cells. Vanadate increased [Ca2+]i by mobilizing calcium from agonist-releasable intracellular calcium stores, since this increase was observed in the absence of extracellular calcium and vanadate failed to increase [Ca2+]i after treatment with thapsigargin in calcium-free medium. Moreover, pretreatment of acinar cells with vanadate prevented the cholecystokinin octapeptide (CCK-8)-induced signal of [Ca2+]i, whereas co-incubation with CCK-8 potentiated the plateau phase of calcium response to CCK-8 without modifying the transient calcium spike. The effects of vanadate on calcium mobilization were reversed by the presence of the sulfhydryl reducing agent dithiothreitol. Vanadate also activated the calcium influx, since an additional enhancement of calcium influx induced by thapsigargin-evoked intracellular store depletion was observed and vanadate reversed the inhibitory effect of lanthanum (an inhibitor of calcium entry) into acinar cells. In addition, vanadate evoked a concentration-dependent release of amylase from pancreatic acinar cells and moreover, reduced the secretory response to CCK-8. We conclude that, in pancreatic acinar cells, vanadate releases calcium from the agonist-releasable intracellular calcium pool and consequently induces amylase secretion. These effects are likely due to the oxidizing effects of this compound.

Exp Physiol 1999 Mar;84(2):299-318

Interaction of islet hormones with cholecystokinin octapeptide-evoked secretory responses in the isolated pancreas of normal and diabetic rats.

Singh J, Adeghate E, Salido GM, Pariente JA, Yago MD, Juma LO

Department of Applied Biology, University of Central Lancashire, Preston, Lancashire PR1 2HE, UK

This study investigates the effects of the islet hormones, insulin (Ins), glucagon (Glu) and somatostatin (Som) with cholecystokinin octapeptide (CCK-8) on amylase secretion and intracellular free calcium concentration [Ca2+]i and their pattern of distribution in the isolated pancreas of normal and diabetic rats. Ins and Glu evoked small increases in amylase output from pancreatic segments compared with a much enhanced effect of CCK-8. In contrast, Som induced a biphasic response comprising an initial decrease followed by a secondary increase and this biphasic response may be dependent upon the concentration. Combining the islet hormones with CCK-8 resulted in marked potentiation in amylase output compared with either CCK-8 alone or the individual hormone. Genistein and tyrphostin A25, the tyrosine kinase inhibitors, evoked a small decrease in amylase output from pancreatic segments. They had no effect on the CCK-8-evoked secretory response but markedly inhibited the potentiation of the islet hormones with CCK-8. In pancreatic acini and acinar cells Ins, Glu and Som individually evoked small increases in amylase output compared with a much larger response with CCK-8. When the islet hormones were combined with CCK-8 there was no potentiation of amylase output. Similarly, when rats were rendered diabetic by prior treatment with streptozotocin Ins, Glu and Som failed to potentiate the secretory response of CCK-8. In fura-2-loaded pancreatic acinar cells Ins or Glu evoked small increases in [Ca2+]i compared with a much larger elevation with CCK-8. Ins, Glu and Som each enhanced the CCK-8-evoked [Ca2+]i. Genistein elicited a decrease in [Ca2+]i both in the absence and presence of the islet hormones. It also decreased the elevation in [Ca2+]i resulting from the combined presence of CCK-8 with either Ins or Glu but it had no effect on CCK-8 in combination with Som. In pancreatic acinar cells from diabetic rat Ins, Glu and Som had no detectable effect on CCK-8-evoked elevation in [Ca2+]i compared with the response obtained with CCK-8 alone. CCK-8-immunopositive cells were distributed around the walls of blood vessels, numerous Ins-positive cells in the central and peripheral parts of the islets of Langerhans, Glu-immunoreactive cells in the periphery of islets and Som-positive cells in the outer part of the islets. During diabetes, the number of CCK-immunopositive cells remained unchanged whereas the number of Ins-positive cells decreased coupled with an increase in the number of Glu-positive cells. The results indicate that both tyrosine kinase and cellular Ca2+ seem to be the intracellular mediators involved with the enhanced secretory responses obtained with a combination of the islet hormones with CCK-8. Moreover, the presence of viable pancreatic islets of Langerhans seems to be associated with the potentiation of the islet hormones with CCK-8.

J Physiol (Lond) 1999 Apr 15;516 ( Pt 2):399-408

Sequential activation of different Ca2+ entry pathways upon cholinergic stimulation in mouse pancreatic acinar cells.

Camello C, Pariente JA, Salido GM, Camello PJ

University of Extremadura, Department of Physiology, Faculty of Veterinary Science, 10071 Caceres, Spain.

1. We have studied capacitative calcium entry (CCE) under different experimental conditions in fura-2-loaded mouse pancreatic acinar cells by digital microscopic fluorimetry. CCE was investigated during [Ca2+]i decay after cell stimulation with a supramaximal concentration of ACh (10 microM) or during Ca2+ readmission in Ca2+-depleted cells (pretreated with thapsigargin or ACh). 2. La3+ and Zn2+ (100 microM) inhibited CCE during Ca2+ readmission but had negligible effects during ACh decay. In contrast flufenamic acid (100 microM), an inhibitor of non-selective cation channels, genistein (10 microM), a broad-range tyrosine kinase inhibitor, and piceatannol (10 microM), an inhibitor specific for non-receptor Syk tyrosine kinase, inhibited CCE during ACh decay but not during Ca2+ reintroduction. 3. Simultaneous detection of Mn2+ entry and [Ca2+]i measurement showed that, in the presence of extracellular calcium, application of 100 microM Mn2+ during ACh decay resulted in manganese influx without alteration of calcium influx, whilst when applied during Ca2+ readmission, Mn2+ entry was significantly smaller and induced a clear inhibition of CCE. 4. Application of the specific protein kinase C inhibitor GF109293X (3 microM) reduced CCE in Ca2+-depleted cells, whereas the activator phorbol 12-myristate, 13-acetate (3 microM) increased Ca2+ entry. 5. Based on these results we propose that cholinergic stimulation of mouse pancreatic acinar cells induces Ca2+ influx with an initial phase operated by a non-specific cation channel, sensitive to flufenamic acid and tyrosine kinase inhibitors but insensitive to lanthanum and divalent cations, followed by a moderately Ca2+-selective conductance inhibited by lanthanum and divalent cations.

Biochem Pharmacol 1998 Mar 15;55(6):903-908

Effect of basic fibroblast growth factor on cholecystokinin-induced amylase release and intracellular calcium increase in male rat pancreatic acinar cells.

Lajas AI, Pozo MJ, Salido GM, Pariente JA

Department of Physiology, Faculty of Veterinary Science, University of Extremadura, Caceres, Spain.

Isolated rat pancreatic acinar cells were used to investigate the effect of basic fibroblast growth factor (bFGF) on both amylase secretion and intracellular free calcium concentration ([Ca2+]i) in response to the calcium-mobilizing secretagogue cholecystokinin-octapeptide (CCK-8). Our data show that bFGF inhibited CCK-8-induced amylase release in a concentration-dependent manner and decreased the CCK-8-induced rise in [Ca2+]i. This inhibitory effect of bFGF on both amylase secretion and [Ca2+]i increase in response to CCK-8 was reverted when acinar cells were pretreated with 100 microM tyrphostin A25, a tyrosine kinase inhibitor. Tyrphostin A25 also inhibited Ca2+ influx induced by CCK-8. These results show that bFGF inhibits CCK-8-induced pancreatic response by a tyrosine kinase-dependent mechanism. A role for tyrosine phosphorylation in capacitative Ca2+ entry is suggested.

Pflugers Arch 1997 Sep;434(5):609-614

Intracellular pH and calcium signalling in rat pancreatic acinar cells.

Gonzalez A, Pariente JA, Salido GM, Camello PJ

Departamento de Fisiologia, Facultad de Veterinaria, Universidad de Extremadura, Caceres, Spain.

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 ofmembrane-permeant bases or acids inhibits CCK-8-evoked intracellular Ca2+ oscillatio ns. 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+.

Gen Pharmacol 1997 Aug;29(2):211-215

Histamine does not potentiate cyclic AMP-mediated amylase secretion in the guinea-pig pancreatic acinar cells.

Camello C, Lajas AI, Gonzalez A, Camello PJ, Pariente JA

Department of Physiology, Faculty of Veterinary Science, University of Extremadura, Caceres, Spain.

1. This study investigates the interaction between histamine and the adenylate cyclase systems involved in the secretion of amylase in isolated guinea-pig pancreatic acinar cells. 2. Histamine caused does-related enhancement of amylase release. Similarly, incubation of acini with increasing concentrations of vasoactive intestinal peptide (VIP) resulted in a typical dose-dependent increase in amylase output. 3. When pancreatic acinar cells were incubated with histamine in combination with VIP, amylase secretion did not differ statistically from secretion induced by histamine or VIP alone and was significantly lower than theoretical additivity. Additionally, amylase secretion in the presence of histamine plus forskolin was significantly less than additive. The action of histamine was equally effective as VIP in causing cyclic adenosine monophosphate (cAMP) increase. 4. These results indicate that histamine may exert its secretory effects via the cyclic AMP pathway in the exocrine guinea-pig pancreas.

Inflamm Res 1997 May;46(5):159-165

The physiological role of histamine in the exocrine pancreas.

Singh J, Pariente JA, Salido GM

Department of Applied Biology, University of Central Lancashire, Preston, UK.

In addition to the autonomic nervous system and gut hormones, the mast cell mediator histamine has also been associated with exocrine pancreatic secretion. This review is concerned with the distribution and the physiological role of histamine in the control of pancreatic juice secretion. Histamine is distributed widely around blood vessels and acinar tissues in the pancreas and it is released in pancreatic juice during secretagogue stimulation. Histamine has a marked secretagogue effect in the exocrine pancreas of several animal species but in many cases the secretory effect is gender-related. The paracrine hormone exerts its secretory response via activation of H1 and H2 receptors onpancreatic acinar cells to mobilize potassium ions (K+) and cellular calcium (Ca2+) and through  elevation of endogenous adenosine 3',5' cyclic monophosphate (cyclic AMP) levels, respectively. A physiological role for H3 receptors has also been associated with exocrine pancreatic secretion. H3 receptors are located presynaptically on parasympathetic nerve terminals to control the release of acetylcholine via restriction of Ca2+ access into nerve terminal through the N-type Ca2+ channel. Taken together, the results presented in this review strongly support histamine as a potential modulator of exocrine pancreatic function.

Biochem Biophys Res Commun 1997 Jan 23;230(3):652-656

Free cytosolic calcium levels modify intracellular pH in rat pancreatic acini.

Gonzalez A, Camello PJ, Pariente JA, Salido GM

Department of Physiology, University of Extremadura, Caceres, Spain.

We have used BCECF- or Fura-2-loaded rat pancreatic acinar cells to investigate the relationship between Ca2+ mobilization and intracellular pH (pHi). Ca2+-mobilizing agonists CCK-8 and ACh induced a transient acidification totally dependent on release of Ca2+ from internal stores. Employment of different physiological tools including ionomycin and thapsigargin to increase the cytosolic Ca2+ concentration and capacitative calcium influx also induced cellular acidification. Application of 1mM LaCl3 reduced the CCK-8-evoked acidification. These data indicate that the mobilization of intracellular Ca2+ stores by CCK-8 decreases cellular pH by Ca2+/H+ exchanger.

Can J Physiol Pharmacol 1996 Dec;74(12):1342-1350

Secretin potentiates guinea pig pancreatic response to cholecystokinin by a cholinergic mechanism.

Alcon S, Rosado JA, Garcia LJ, Pariente JA, Salido GM, Pozo MJ

Department of Nursing, University of Extremadura, Caceres, Spain.

The effects of secretin and cholecystokinin on exocrine pancreas secretion in the guinea pig were investigated. The putative potentiating effect of these two hormones was studied in various settings to elucidate the effect of cholinergic stimuli in such interaction. In anesthetized guinea pig, intravenous infusion of cholecystokinin (0.75 pmol.kg-1.min-1) or secretin (0.5 pmol.kg-1.min-1) resulted in a marked and rapid increase of pancreatic juice flow and protein output. When cholecystokinin was combined with secretin, there was a significant increase in pancreatic, compared with cholecystokinin alone. This increase in pancreatic juice secretion and protein output was significantly suppressed by the prior administration of 100 micrograms/kg atropine. Similar results were obtained when trypsinogen release from pancreatic segments was measured in response to cholecystokinin (32 nM-32 pM) and (or) secretin (1 microM-32 nM). When we assayed the hormonal interaction on amylase release from dispersed pancreatic acini, we found that secretin (32 nM) failed to influence the secretory response to cholecystokinin (1 pM-10 nM). Thus we conclude that a combination of cholecystokinin and secretin resulted in a marked potentiation of the secretory responses in the exocrine guinea pig pancreas by a mechanism that involves cholinergic interactions present at the tissue level but not at the dispersed secretory cell level.

Can J Physiol Pharmacol 1996 Jun;74(6):744-752

Control of exocrine secretion in the guinea-pig pancreas by histamine H3 receptors.

Jennings LJ, Salido GM, Pariente JA, Davison JS, Singh J, Sharkey KA

Department of Applied Biology, University of Central Lancashire, Preston, England, UK.

The aim of this study was to investigate the possible involvement of the histamine H3 receptor in control of exocrine pancreatic secretion from the guinea-pig. In in vitro experiments, the H3 receptor agonist (R)-alpha-methylhistamine (0.01-10 microM) elicited a concentration-dependent decrease in the release of alpha-amylase. (R)-alpha-Methylhistamine concentrations above 10 microM evoked aconcentration-dependent increase in alpha-amylase secretion. Application of mepyramine (1 mi croM) partially blocked this increase. The H3 receptor antagonist thioperanide (1 microM) blocked the effects of (R)-alpha-methylhistamine below 10 microM. Histamine and (R)-alpha-methylhistamine attenuated both protein release elicited during electrical-field stimulation and the release of tritiated choline, and these effects were reversed by thioperamide. In an in vivo study, (R)-alpha-methylhistamine increased juice secretion and total protein content of the juice by 40%. Histamine H1 and H2 receptor antagonists blocked this increase and uncovered an attenuation of the secretory parameters (juice flow 28%, total protein content 44%). This attenuation was blocked by thioperamide. These observations suggest that stimulation of the histamine H3 receptor in the pancreas results in a decreased fluid and enzyme release by inhibition of acetylcholine release from intrinsic pancreatic nerves.

Arch Physiol Biochem 1996;104(3):293-299

Secretory activity and trophic effects of epidermal growth factor in the rat pancreas.

Lajas AI, Pozo MJ, Salido GM, Singh J, Pariente JA

Department of Physiology, Faculty of Veterinary Sciences, University of Extremadura, Caceres, Spain.

This study was to investigate whether epidermal growth factor (EGF) may induce any long-term effect on pancreatic exocrine function in vivo as well to evaluate the chronic effects of EGF on pancreatic growth in rats. Rats were treated with EGF (10 micrograms/kg) for 5 or 7 days. EGF infused intravenously (2 micrograms/kg/h) in anaesthetized and pretreated rats for 5 or 7 days with EGF caused a slight decline flow rate after 1 h of EGF infusion compared to control values. In contrast, EGF evoked a increase in amylase secretion. This stimulatory effect was much larger in EGF-pretreated rats for 7 days, whereas the total protein output was unchanged. The trophic parameters which include pancreatic weight, total protein and total contents of DNA and RNA relative to body weight were not significantly different in any treated group. Only the pancreatic amylase content was increased significantly after 7 days of treatment with EGF. The present study fails to observe a stimulatory role of EGF on pancreatic growth in rats, but may participate in the regulation of pancreatic exocrine function in vivo.

Magnes Res 1995 Sep;8(3):215-222

Reciprocal changes in intracellular and extracellular magnesium in rat pancreatic acinar cells in response to different secretagogues.

Gonzalez A, Pariente JA, Salido GM, Wisdom D, Singh J

Department of Physiology, University of Extremadura, Caceres, Spain.

This study employs the fluorescent bioprobe Mag-fura-2 to measure the mobilisation of magnesium (Mg2+) in rat pancreatic acinar cells at rest and during stimulation with either cholecystokinin-octapeptide (CCK-8; 10(-8) M), acetylcholine (ACh; 10(-5) M) or histamine (10(-4) M). In Mag-fura-2 loaded acini suspended in normal Krebs-Ringer-Hepes (KRH) solution containing (mMol/litre) NaCL, 130; KCI, 5; HEPES, 20; KH2PO4, 1.2; CaCl2, 1.0; MgSO4, 1.0; glucose, 10; minimum essential medium, 1.0 ml/Litre and pH 7.4, both CCK-8 and ACh evoked a transient rise in intracellular free Mg2+ concentration ([Mg2+]i) followed by a sustained and prolonged decrease. Addition of CCK-8 to acinar cell suspension previously stimulated with ACh resulted in little or no change in [Mg2+]i indicating that the two secretagogues are mobilising magnesium from the same intracellular pool. Histamine had no significant effect on [Mg2+]i or on the CCK-8 evoked response. When acini were challenged in the presence of the cell-impermeant form of Mag-fura-2 (the tetrapotassium salt) in a Ca(2+)-free KRH solution containing 1-mM EGTA, CCK-8 and ACh, stimulation resulted in a gradual release of magnesium reaching a maximum after 300-350 s following application of the secretagogues (magnesium efflux). Again histamine had no effect on magnesium release. Incubation of acinar cells in a nominally magnesium-free KRH solution had no effect on basal [Mg2+]o. When challenged with CCK-8, [Mg2+]i rose transiently, followed by a sustained decrease. In contrast, ACh stimulation resulted in a triphasic response comprising an initial rise followed by a decrease, superceded by a small rise in [Mg2+]i. Again histamine had no effect on [Mg2+]i in nominally magnesium-free solution. Addition of either ACh or CCK-8 to cells placed in KRH solution with no added Mg2+ caused a small efflux of magnesium, as measured in the presence of the tetrapotassium salt of Mag-fura-2 (cell-impermeant form). These results, employing two different forms of a fluorescent bioprobe for the measurement of magnesium, indicate that unlike histamine,CCK-8 and ACh can evoke marked changes in intracellular free magnesium concentration dur ing the stimulus-secretion coupling process.

Gen Pharmacol 1995 Jul;26(4):843-850

Cholinergic dependence of pancreatic response to cholecystokinin in rats and guinea pigs.

Pozo MJ, Estevez MJ, Alcon S, Camello PJ, Pariente JA, Salido GM

Department of Nursing, Faculty of Veterinary Sciences, University Extremadura, Caceres, Spain.

1. Lorglumide and atropine were used to examine the role of cholinergic mechanisms in the pancreatic secretory response to cholecystokinin in two animal species. 2. Anaesthetized rats and guinea pigs with jugular vein and pancreatic cannulae were used and the bile juice was recirculated. In the rat, the treatment with lorglumide (3 mumol/kg) as well as atropine (100 micrograms/kg) did not have effects on basal interdigestive secretion, whereas in guinea pigs only atropine decreased the protein output (41%) and the juice flow (47%) of the basal pancreatic secretion. 3. Infusion of cholecystokinin (150 pmol/kg/hr in the rat and 50 pmol/kg/hr in the guinea pig) induced a marked increase in pancreatic juice flow and protein output compared to saline controls. Pretreatment of both rat and guinea pig with lorglumide resulted in a marked attenuation of the cholecystokinin-evoked secretory response. 4. In the rats, atropine decreased the response to infusion of cholecystokinin octapeptide while this antimuscarinic agent had no effect in the response to cholecystokinin in the guinea pigs. 5. This study supports the concept that the influence of cholinergic system in pancreatic response to cholecystokinin shows interspecific differences.

Inflamm Res 1995 May;44(5):207-211

Histaminergic modulation of hormonal control in the exocrine guinea-pig pancreas.

Alcon S, Pozo MJ, Salido GM, Pariente JA

Department of Physiology, Faculty of Veterinary Science, University of Extremadura, Caceres, Spain.

The effects of histamine upon secretin- or cholecystokinin (CCK)-evoked exocrine pancreatic secretion were investigated in the anaesthetised guinea pig. Histamine (0.1 mumol/kg/min) induced a slight increase in pancreatic juice flow and total protein release compared to saline controls. Secretin (0.5 pmol/kg/min) and CCK-8 (0.75 pmol/kg/min) evoked marked time course increases in both the rate of pancreatic juice flow and total protein output in the anaesthetised guinea pig. Administration of either secretin or CCK-8 simultaneously with histamine elevated the exocrine pancreatic secretion compared to the smaller response obtained when administered separately. These results indicate that histamine may play an important physiological role in modulating the hormonal control of exocrine guinea pig pancreas.

Cell Signal 1995 Jan;7(1):57-60

Histamine and the cAMP pathway in the guinea-pig pancreas.

Lajas AI, Pariente JA, Salido GM

Department of Physiology, Faculty of Veterinary Science, University of Extremadura, Caceres, Spain.

This study investigates the interaction between histamine and the adenylate cyclase systems involved in the secretion of amylase from the guinea-pig pancreatic lobules. Histamine increased amylase release, reaching a maximum response at 10(-5) M. Similarly, vasoactive intestinal peptide (VIP) evoked significant increase in amylase release, though not in a dose-dependent fashion. When the pancreatic lobules were incubated with histamine in combination with VIP, forskolin or 3-isobutyl-1-methylxanthine (IBMX), amylase secretion was increased as compared to histamine alone. The stimulatory effect of VIP was also increased by the presence of forskolin or IBMX. These findings suggest that in guinea-pig pancreatic lobules, VIP, forskolin and IBMX, agents involved in the cyclic adenosine monophosphate (cAMP) pathway, potentiate histamine stimulated amylase release.