Calcium In Pancreatic Acinar Cells Research Articles

Pancreatic Stellate Cells Serve as a Brake Mechanism on Pancreatic Acinar Cell Calcium Signaling with Modulation by Methionine Sulfoxide Reductase Expression

Although methionine sulfoxide reductase (Msr) is known to modulate activity of multiple functional proteins, roles of Msr in pancreatic stellate cell physiology have not been reported. In the present work we investigated expression and function of Msr in freshly isolated and cultured rat pancreatic stellate cells. Msr expression was determined by RT‐PCR, Western blot and immunocytochemistry. Msr over‐expression was achieved by transfection with adenovirus vectors. Pancreatic stellate cells were co‐cultured with pancreatic acinar cells AR4‐2J in monolayer culture. Pancreatic stellate and acinar cell function was monitored by Fura‐2 calcium imaging. Rat pancreatic stellate cells were found to express MsrA, B1, B2, their expressions diminished in culture. Over‐expressions of MsrA, B1, or B2 were found to enhance ATP‐stimulated calcium increase but decreased reactive oxygen species production and lipopolysaccharide‐elicited IL‐1 production. Pancreatic stellate cell‐co‐culture with AR4‐2J blunted cholecystokinin‐ and acetylcholine‐stimulated calcium increases in AR4‐2J, depending on acinar / stellate cell ratio, this inhibition was reversed by MsrA, B1 over‐expression in stellate cells or by Met supplementation in co‐culture medium. Different from the pancreatic acinar cells, although pancreatic stellate cells also express the cholecystokinin 1 receptor, the receptor is not coupled to calcium signaling. In addition, Msr‐overexpression only partially restored cholecystokinin 1 receptor coupling to the calcium signaling pathway in pancreatic stellate cells, whereas addition of free Met in the culture medium had no effect. These data suggest that Msr play important roles in pancreatic stellate cell function and the stellate cells may serve as a brake mechanism on pancreatic acinar cell calcium signaling modulated by stellate cell Msr expression. Pancreatic stellate cell cholecystokinin 1 receptor is de‐coupled from calcium signaling, Msr overexpression only partially restores cholecystokinin‐stimulation of calcium increases at high CCK concentrations in the stellate cells.Support or Funding InformationThis work was supported by grants from NSFC (31670856, 31971170).Figure 1

Pancreatic Stellate Cells Serve as a Brake Mechanism on Pancreatic Acinar Cell Calcium Signaling Modulated by Methionine Sulfoxide Reductase Expression.

Although methionine sulfoxide reductase (Msr) is known to modulate the activity of multiple functional proteins, the roles of Msr in pancreatic stellate cell physiology have not been reported. In the present work we investigated expression and function of Msr in freshly isolated and cultured rat pancreatic stellate cells. Msr expression was determined by RT-PCR, Western blot and immunocytochemistry. Msr over-expression was achieved by transfection with adenovirus vectors. Pancreatic stellate cells were co-cultured with pancreatic acinar cells AR4-2J in monolayer culture. Pancreatic stellate and acinar cell function was monitored by Fura-2 calcium imaging. Rat pancreatic stellate cells were found to express MsrA, B1, B2, their expressions diminished in culture. Over-expressions of MsrA, B1 or B2 were found to enhance ATP-stimulated calcium increase but decreased reactive oxygen species generation and lipopolysaccharide-elicited IL-1 production. Pancreatic stellate cell-co-culture with AR4-2J blunted cholecystokinin- and acetylcholine-stimulated calcium increases in AR4-2J, depending on acinar/stellate cell ratio, this inhibition was reversed by MsrA, B1 over-expression in stellate cells or by Met supplementation in the co-culture medium. These data suggest that Msr play important roles in pancreatic stellate cell function and the stellate cells may serve as a brake mechanism on pancreatic acinar cell calcium signaling modulated by stellate cell Msr expression.

Effect of Chaiqin Chengqi Decoction on cholecystokinin receptor 1-mediated signal transduction of pancreatic acinar cells in acute necrotizing pancreatitis rats.

To investigate the effect of Chaiqin Chengqi Decoction (,CQCQD) on cholecystokinin receptor 1 (CCKR1)-mediated signal transduction of pancreatic acinar cell in rats with acute necrotic pancreatitis (ANP). Twenty-seven Sprague-Dawley rats were randomized into three groups: the control group, the ANP group, and the CQCQD group (9 in each group). ANP rats were induced by two intraperitoneal injections of 8% L-arginine (pH=7.0, 4.4 g/kg) over a 2-h period. Rats were treated with 1.5 mL/100 g body weight of CQCQD (CQCQD group) or physiological saline (control and ANP groups) at 2 h interval. And 6 h after induction, pancreatic tissues were collected for histopathological examination. Pancreatic acinar cells were isolated for determination of CCKR1 mRNA and protein expression, phospholipase C (PLC) and inositol-1,4,5-triphosphate (IP3), and determination of fluorescence intensity (FI) as a measure of intracellular calcium ion concentration [Ca(2+)]i. The pancreatic histopathological score (6.2 ± 1.1) and the levels of PLC (1,187.2 ± 228.2 μg/mL) and IP3 (872.2 ± 88.4 μg/mL) of acinar cells in the ANP group were higher than those in the control (2.8 ± 0.4, 682.5 ± 121.8 μg/mL, 518.4 ± 115.8 μg/mL) and the CQCQD (3.8 ± 0.8, 905.3 ± 78.5 μg/mL, 611.0 ± 42.5 μg/mL) groups (P<0.05). [Ca(2+)]i FI for the ANP group (34.8±27.0) was higher than that in the control (5.1 ± 2.2) and CQCQD (12.6 ± 2.5) groups (P<0.05). The expression of pancreatic acinar cell CCKR1 mRNA in the ANP group was up-regulated (expression ratio=1.761; P=0.024) compared with the control group. The expression of pancreatic acinar cell CCKR1 mRNA in the CQCQD group was down-regulated (expression ratio=0.311; P=0.035) compared with the ANP group. The ratio of gray values of the CCKR1 and β-actin in the ANP group (1.43 ± 0.17) was higher than those in the control (0.70 ± 0.15) and CQCQD (0.79 ± 0.11) groups (P<0.05). Pancreatic acinar cell calcium overload of ANP induced by L-arginine was related to the up-regulated expressions of pancreatic acinar cell CCKR1 mRNA and protein. CQCQD can down-regulate expressions of pancreatic acinar cell CCKR1 mRNA and protein to reduce the PLC and IP3 of pancreatic acinar cells, relieving the calcium overload and reducing the pathological changes in rats with ANP.

Chaiqin Chengqi Decoction decreases pancreatic acinar cell calcium overload in rats with acute pancreatitis

To investigate the mechanism of Chaiqin Chengqi Decoction (CQCQD), a compound of traditional Chinese herbal medicine, acting on the pancreatic acinar cell calcium overload in rats with acute pancreatitis (AP). A total of 30 SD rats were randomly divided into normal control group, untreated group and CQCQD group (n=10, respectively). AP was induced in rats by caerulein (5x50 mug/kg) intraperitoneal injection within 4 h. The pancreatic tissue SERCA1 and SERCA2 mRNA expressions were detected by fluorescent quantization polymerase chain reaction method; intracellular calcium fluorescence intensity (FI) of pancreatic acinar cells and the pancreatic pathological score were measured by laser scanning confocal microscopy and light microscopy respectively. There were no SERCA1 mRNA expressions in pancreatic acinar cells of rats in the normal control group and the untreated group. The expression of pancreatic SERCA2 mRNA in the untreated group was down-regulated compared with that in the normal control group (expression ratio=0.536; P=0.001); the expression of pancreatic SERCA2 mRNA in the CQCQD group was up-regulated compared with that in the untreated group (expression ratio=2.00; P=0.012). The pancreatic pathological score in the CQCQD group was lower than that in the untreated group and the FI of Ca(2+) was also lower. CQCQD can up-regulate the expression of pancreatic SERCA2 mRNA, release the calcium overload, and hence reduce the pathological changes in pancreatic tissue.

Role of calcium in substance P‐induced chemokine synthesis in mouse pancreatic acinar cells

Substance P (SP) and chemokines play critical roles in acute pancreatitis. SP elevates cytosolic calcium in pancreatic acinar cells and elevated cytosolic calcium is thought to be an early event in the pathogenesis of acute pancreatitis. SP induces production of chemokines MCP-1, MIP-1alpha and MIP-2 in pancreatic acinar cells, however the exact mechanism by which SP stimulates the production of these pro-inflammatory mediators remain undetermined. The aim of the present study is to investigate the role of calcium in SP-induced chemokine production in pancreatic acinar cells and to establish the signal transduction mechanisms involved. An in vitro model of isolated mouse pancreatic acinar cells was used. Western blotting analysis, ELISA and calcium measurement were performed. SP increased chemokine secretion through the activation of PKCalpha/betaII, MAPKinases (ERK and JNK), NFkappaB and AP-1 in pancreatic acinar cells. These effects were blocked by pretreatment of the cells with the specific calcium chelator BAPTA-AM. Moreover, SP-induced activation of PKCalpha/betaII, ERK, JNK, NF-kappaB, AP-1 and chemokine production was inhibited by the specific phospholipase C inhibitor U73122. SP-induced chemokine production in pancreatic acinar cells resulted from PLC-induced elevated intracellular calcium and PKCalpha/betaII activation, subsequently leading to the activation of MAPKinases (ERK and JNK) and transcription factors NF-kappaB and AP-1. The present study demonstrates the critical role of calcium in SP-induced chemokine production in pancreatic acinar cells. Drugs targeting the SP-calcium mediated signaling pathways could prove beneficial in improving the treatment of acute pancreatitis.

Complex oscillations and waves of calcium in pancreatic acinar cells

We perform a bifurcation analysis of a model of Ca 2+ wave propagation in the basal region of pancreatic acinar cells. The model we consider was first presented in Sneyd et al. [J. Sneyd, K. Tsaneva-Atanasova, J.I.E. Bruce, S.V. Straub, D.R. Giovannucci, D.I. Yule, A model of calcium waves in pancreatic and parotid acinar cells, Biophys. J. 85 (2003) 1392–1405], where a partial bifurcation analysis was given of the model in the absence of diffusion. We obtain more complete information about bifurcations of the diffusionless model via numerical studies, then analyse the spatially extended model by numerical investigation of the travelling wave equations and direct numerical solution of the model equations. We find solitary waves in the model equations arising from homoclinic bifurcations in the travelling wave equations. The solitary waves exist and appear to be stable for a significant interval of the primary bifurcation parameter (i.e., the concentration of inositol trisphosphate) but are eventually replaced by irregular spatio-temporal behaviour. The homoclinic bifurcations are related to a number of complicated mathematical structures in the travelling wave equations, including an anomalous homoclinic-Hopf bifurcation, heteroclinic bifurcations between an equilibrium and a periodic orbit, and homoclinic bifurcations of periodic orbits.

Do cholecystokinin antagonists increase cytosolic calcium in pancreatic acinar cells and thereby promote pancreatitis?

In a number of studies using animal models, and in a human study, cholecystokinin (CCK) antagonists ameliorated pancreatitis. In a recent report of a study in a rat model of obstructive acute pancreatitis, however, it was suggested that a potent CCK1 antagonist, L364,718, had proved harmful. This effect was attributed to an increase in free cytosolic calcium levels in pancreatic acinar cells. Our understanding of obstructive pancreatitis now rests on feedback mechanisms that control CCK levels and are disrupted when obstruction is present. CCK antagonism might interrupt the process of pancreatitis by reducing the increase in CCK levels that promotes enzyme release. This article reviews the findings obtained with CCK antagonists in several experimental models of pancreatitis and assesses the recent findings with L364,718 in that light.

Early Changes in Pancreatic Acinar Cell Calcium Signaling after Pancreatic Duct Obstruction

Intracellular Ca(2+)-changes not only participate in important signaling pathways but have also been implicated in a number of disease states including acute pancreatitis. To investigate the underlying mechanisms in an experimental model mimicking human gallstone-induced pancreatitis, we ligated the pancreatic duct of Sprague-Dawley rats and NMRI mice for up to 6 h and studied intrapancreatic changes including the dynamics of [Ca(2+)](i) in isolated acini. In contrast to bile duct ligation, pancreatic duct obstruction induced intra-pancreatic trypsinogen activation, leukocytosis, hyperamylasemia, and pancreatic edema and increased lung myeloperoxidase activity. Although resting [Ca(2+)](i) in isolated acini rose by 45% to 205 +/- 7 nmol, the acetylcholine- and cholecystokinin (CCK)-stimulated calcium peaks as well as the amylase secretion declined, but neither the [Ca(2+)](i)-signaling pattern nor the amylase output in response to the Ca(2+)-ATPase inhibitor thapsigargin nor the secretin-stimulated amylase release were impaired by pancreatic duct ligation. On the single cell level pancreatic duct ligation reduced the percentage of cells in which submaximal secretagogue stimulation was followed by a physiological response (i.e. Ca(2+) oscillations) and increased the percentage of cells with a pathological response (i.e. peak plateau or absent Ca(2+) signal). Moreover, it reduced the frequency and amplitude of Ca(2+) oscillation as well as the capacitative Ca(2+) influx in response to secretagogue stimulation. Serum pancreatic enzyme elevation as well as trypsinogen activation was significantly reduced by pretreatment of animals with the calcium chelator BAPTA-AM. These experiments suggest that pancreatic duct obstruction rapidly changes the physiological response of the exocrine pancreas to a Ca(2+)-signaling pattern that has been associated with premature digestive enzyme activation and the onset of pancreatitis, both of which can be prevented by administration of an intracellular calcium chelator.

Traveling waves of calcium in pancreatic acinar cells: model construction and bifurcation analysis

We construct and study a model for intracellular calcium wave propagation, with particular attention to pancreatic acinar cells. The model is based on a model of the inositol trisphosphate (IP 3) receptor, which assumes that calcium modulates the binding affinity of IP 3 to the receptor. Two versions of the model, one simpler than the other, are studied numerically. In both versions, solitary waves in the excitable regime arise via homoclinic bifurcations in the traveling wave equations. As the background concentration of IP 3 is increased, the wave speed increases, and for some values of the IP 3 concentration, the initial pulse gives rise to secondary pulses that travel in both directions. This can give rise to irregular spatio-temporal behavior, or to trains of pulses. In the simpler model, these secondary waves are related to the presence of a T-point, a heteroclinic cycle, and an associated spiral of homoclinic orbits, which terminate the branch of homoclinic orbits.

Progressive disruption of acinar cell calcium signaling is an early feature of cerulein-induced pancreatitis in mice

BACKGROUND & AIMS: Disruption of pancreatic exocrine secretion is an important feature of acute pancreatitis. Because cytosolic calcium is a key intracellular messenger controlling pancreatic secretion, this study examined patterns of calcium signaling during the early stages of cerulein-induced pancreatitis. METHODS: Mice were administered hourly intraperitoneal injections of cerulein (50 micrograms/kg), and paired controls were administered saline. Acini were isolated by collagenase from pancreatic tissue harvested after injections 1, 3, 5, and 7 and were loaded with Fura-2. Individual cellular calcium responses to acetylcholine and cholecystokinin were studied using digital imaging. RESULTS: The proportion of cells maintaining a normal oscillatory calcium response to physiological secretagogue stimulation diminished progressively after increasing cerulein injections. Also, the normal polarized spatial pattern of calcium Increase within individual acinar cells was progressively lost. A sustained response to high-dose stimulation was maintained but with diminishing amplitude. The characteristic calcium response to the Ca(2+)-adenosine triphosphatase inhibitor thapsigargin was maintained, implying that calcium reuptake and extrusion were not impaired. CONCLUSIONS: Progressive disruption of physiological patterns of pancreatic acinar cell calcium signaling, notably in the secretory pole of the cell, is an early feature of pancreatitis induced by cerulein hyperstimulation. These changes may be important in contributing to the disruption of exocrine secretion in acute pancreatitis. (Gastroenterology 1996 Aug;111(2):481-91)

Dual regulation of cGMP formation by calcium in pancreatic acinar cells.

Regulation of guanosine 3',5'-cyclic monophosphate (cGMP) formation by calcium and calcium-binding proteins was studied at the levels of nitric oxide synthase (NOS) and guanylyl cyclase (GC) in dispersed pancreatic acini isolated from guinea pig. In intact cells, in the cytosol, and on diethylaminoethyl fractions from cytosolic proteins, GC activity was negatively regulated by Ca2+. An increase in Ca2+ concentration ([Ca2+]) from 25 to 950 nM suppressed cGMP formation by 85%. On the other hand, NOS was stimulated by agents increasing cytosolic [Ca2+] and inhibited by intracellular Ca2+ chelators. Thus Ca2+ regulates cGMP production in opposite directions by activating NOS and inhibiting GC. Calmodulin antagonists W-7, trifluoperazine, and R-24571 inhibited NOS, suggesting that the enzyme is regulated by calmodulin as in other cell types. Calmodulin antagonists appeared to inhibit GC. In particular, 200 microM W-7 completely abolished the cGMP rise evoked by the nitric oxide donor, nitroprusside. The effect was not reversed by addition of excess calmodulin. The findings suggest that the negative regulation of GC by Ca2+ is due to factors other than calmodulin but affected by calmodulin antagonists.

Fluorescent probe detects redistribution of cell calcium during stimulus–secretion coupling

STIMULUS-secretion coupling in pancreatic acinar cells is believed to be mediated by a rise in intracellular Ca2+ activity1–5. Based on the observation that physiological secretagogues trigger a rapid increase of Ca2+ efflux3,4 and stimulate enzyme release even in the absence of extracellular calcium2,4,5, it has been proposed that stimulus–secretion coupling involves a redistribution of intracellular calcium3,5,6. Since most calcium in pancreatic acinar cells is bound to membranes or is sequestered in mitochondria7, we have attempted to monitor these calcium stores during secretion by using the fluorescent probe chlorotetracycline (CTC). This probe forms highly fluorescent complexes with Ca2+ and Mg2+ in the presence of biological membranes or detergent miceles8,9 and has been successfully used in isolated mitochondria10,11 and sarcoplasmic reticulum vesicles12 to monitor calcium movements induced by substrates and inhibitors. In this report, we show that fluorescence of CTC associated with pancreatic acinar cell membranes decreases rapidly on stimulation of enzyme secretion and that this intensity change is preferential for CTC complexed to calcium.