Binding of epidermal growth factor in rat pancreatic acini

Specific, saturable EGF receptors were demonstrated in isolated rat pancreatic acini. Binding of EGF to these receptors was one-half maximal at 20 min and maximal at 120 min. Scatchard analyses revealed a single order of binding sites with a Kd of 4.90 nM. Following binding, EGF was rapidly internalized and converted to two acidic species. EGF did not alter either basal amylase release or the rate of [3H]phenylalanine incorporation into TCA-precipitable protein. The finding of high affinity EGF receptors in pancreatic acinar cells supports the hypothesis that EGF participates in the long-term regulation of pancreatic exocrine function.     

对牛胰多肽抑制胰酶分泌作用的离体研究

为探讨胰多肽抑制胰酶分泌的机制,我们利用大鼠离体胰腺泡制备观察了牛胰多肽(BPP)在细胞受体水平对氨甲酰胆碱等促分泌物作用的影响。实验结果显示,BPP 对氨甲酰胆碱诱导的胰腺泡淀粉酶分泌具有抑制作用,并存在剂量反应关系。BPP0.1μmol/L 和0.2μmol/L,可分别使氨甲酰胆碱诱导淀粉酶分泌的效价降低3倍和10倍;BPP 还可抑制氨甲酰胆碱刺激胰腺泡释放~(45)Ca。以上结果提示,BPP 对胰腺泡的胆碱能 M 受体具有拮抗作用。此外,BPP 对促胰液素及其同类激动剂和氨甲酰胆碱协同作用诱导的胰腺泡淀粉酶分泌具有抑制作用,提示胰多肽在整体对促胰液素诱导的胰酶分泌的抑制,可能是通过拮抗胰腺泡细胞上的 M 受体而抑制了促胰液素和胆碱能刺激协同作用引起的胰酶分泌。     

Effects of cycloheximide and tunicamycin on cell surface expression of pancreatic muscarinic acetylcholine receptors

The importance of glycosylation in cell surface expression of muscarinic receptors in cultured guinea pig pancreatic acini was investigated. Recovery of the muscarinic receptor population after carbachol-induced down regulation was blocked by cycloheximide but not by tunicamycin, although tunicamycin reduced [3H]mannose incorporation into acinar macromolecules by up to 90%. Tunicamycin treatment also failed to alter carbachol stimulation of amylase secretion from cultured acini. These results indicate that glycosylation of the glandular subtype of muscarinic receptor in the pancreatic acinar cell is not necessary for its insertion in the plasma membrane or for its functional activity.     

Protective effect of long term high fiber diet consumption on rat exocrine pancreatic function after chronic ethanol intake

The effects of ethanol administration on exocrine pancreas have been widely studied, but little is known about the effect of dietary fiber in combination with chronic ethanol on exocrine pancreatic function. The aim of this work was to examine the chronic effects of a high fiber diet, ethanol ingestion, and a combination of both on the function of the rat exocrine pancreas. Four groups of rats were fed for six months the following diets: 1.- NW: standard laboratory diet; 2.- FW: high fiber diet (15% cellulose); 3.- NE: standard laboratory diet and 20% ethanol in the drinking water; and 4.- FE: high fiber diet and 20% ethanol. Cholecystokinin (CCK) and acetylcholine (Ach) effects on amylase release and intracellular calcium mobilization in pancreatic acini were studied. In rats fed a 20% ethanol (NE), both the basal amylase release and the basal [Ca(2+)](i) were significantly increased; nonetheless, CCK and Ach-induced amylase release were significantly reduced compared with control rats. Ach- but not CCK-stimulated [Ca(2+)](i) increase in NE rats was significantly decreased compared with NW. In rats fed a combination of ethanol and a high fiber diet (FE) all the parameters under study were not significantly affected compared to control rats (NW). In conclusion, high fiber consumption does not alter the function of the exocrine pancreas. However, it ameliorates the deleterious effect of chronic ethanol consumption on pancreatic amylase secretion and, at least partially, reverses the ethanol-induced alterations on [Ca(2+)](i) in the rat exocrine pancreas.     

Antimuscarinic effects of chloroquine in rat pancreatic acini

Chloroquine inhibited carbachol-induced amylase release in a dose-dependent fashion in rat pancreatic acini; cholecystokinin- and bombesin-induced secretory responses were almost unchanged by the antimalarial drug. The inhibition of carbachol-induced amylase release by chloroquine was competitive in nature with a Ki of 11.7 microM. Chloroquine also inhibited [3H]N-methylscopolamine binding to acinar muscarinic receptors. The IC50 for chloroquine inhibition of [3H]N-methylscopolamine binding was lower than that for carbachol or the other antimalarial drugs, quinine and quinidine. These results demonstrate that chloroquine is a muscarinic receptor antagonist in the exocrine pancreas.     

The actions of tetraethylammonium on dispersed acini from guinea pig pancreas

In dispersed acini from guinea pig pancreas, replacing extracellular sodium by tetraethylammonium (1) abolished carbamylcholine-stimulated amylase secretion but did not alter the increase in amylase secretion caused by the C-terminal octapeptide of cholecystokinin, bombesin, ionophore A23187, vasoactive intestinal peptide or 8-bromoadenosine 3':5' monophosphate, (2) caused a parallel rightward shift in the dose-response curve for carbamylcholine-stimulated amylase secretion and (3) inhibited binding of N-[3H]methyl scopolamine to muscarinic cholinergic receptors. Detectable inhibition of carbamylcholine-stimulated amylase secretion and binding of N-[3H]methyl scopolamine occurred with 300 microM tetraethylammonium, and half-maximal inhibition of these functions occurred with 1-2 mM tetraethylammonium. Replacing extracellular sodium by Tris did not alter the stimulation of enzyme secretion caused by any secretagogue tested. These results indicate that the tetraethylammonium is a muscarinic cholinergic receptor antagonist and that enzyme secretion from pancreatic acini does not depend on extracellular sodium.     

Expression of NK-1 and NK-3 tachykinin receptors in pancreatic acinar cells after acute experimental pancreatitis in rats

Activation of neurokinin (NK)-1 receptors but not of NK-3 stimulates amylase release from isolated pancreatic acini of the rat. Immunofluorescence studies show that NK-1 receptors are more strongly expressed than NK-3 receptors on pancreatic acinar cells under basal conditions. No studies have examined the expression of the two NK receptor populations in pancreatic acini during pancreatitis in rats. We therefore investigated the relationships between expression of these two tachykinin receptors and experimental acute pancreatitis induced by stimulating pancreatic amylase with caerulein (CK) in rats. Hyperstimulation of the pancreas by CK caused an increase in plasma amylase and pancreatic water content and resulted in morphological evidence of cytoplasmic vacuolization. Immunofluorescence analysis revealed a similar percentage of NK-1 receptor antibody immunoreactive acinar cells in rats with pancreatitis and in normal rat tissue but a larger percentage of NK-3 receptor immunoreactive cells in acute pancreatitis than in normal pancreas. Western blot analysis of NK-1 and NK-3 receptor protein levels after CK-induced pancreatitis showed no change in NK-1 receptors but a stronger increase in NK-3 receptor expression in pancreatic acini compared with normal rats thus confirming the immunofluorescence data. These new findings support previous evidence that substance P-mediated functions within the pancreas go beyond sensory signal transduction contributing to neurogenic inflammation, and they suggest that substance P plays a role in regulating pancreatic exocrine secretion via acinar NK-1 receptors. The significant increase in NK-3 receptors during pancreatic stimulation suggests that NK-3 receptors also intervene in the pathogenesis of mild acute pancreatitis in rats.     

Effect of dopamine on amylase secretion from guinea pig pancreatic acinar cells in vitro

Dopamine has been shown to effect pancreatic flow, protein output and amylase secretion in a variety of species. However, there is conflicting evidence regarding the role of dopamine on amylase release in vitro. Specific studies were conducted to evaluate the effect of dopamine and to compare its effects with other substances on basal- and secretagogue-stimulated amylase secretion in a guinea pig dispersed pancreatic acinar cells preparation. Dopamine (10(-6) M) induced a small, but significant (P less than 0.05) increase of amylase secretion. Established secretagogues (10(-6) M) including bombesin, cholecystokinin-octapeptide (CCK-8) and carbachol as anticipated induced significantly larger responses. Other substances tested (10(-6) M) including thyrotropin-releasing hormone (TRH) and muscimol were without effect. Complete dose-response studies (10(-11)-10(-3) M) in the presence of bombesin, CCK-8 and carbachol revealed that dopamine does not affect amylase release in response to these secretagogues. These findings suggest that dopamine is a weak stimulant of amylase secretion in vitro, and that it may therefore play a minor role in regulation of pancreatic enzyme secretion. Several factors including vascular, hormonal and neural have been implicated in regulation of pancreatic exocrine secretion. In particular, autonomic nervous system activity, notably cholinergic, has been shown to affect the secretory status of the pancreatic acinar cell. In addition, several biologically active peptides including bombesin, cholecystokinin (CCK), secretin, vasoactive intestinal peptide (VIP), substance P, gastrin and stimulation of cholinergic (muscarinic) receptors with carbachol have been shown to stimulate pancreatic enzyme secretion both in vivo and in vitro. Certain controversy regarding the role of the sympathetic nervous system in regulation of pancreatic exocrine secretion does exist. For example, several studies with agonists and antagonists of noradrenergic and dopaminergic receptor subtypes suggest a stimulatory effect on pancreatic fluid, electrolyte and enzyme secretion. However, these responses are species-specific and variations inherent to the model have been described. Dopamine administration has been shown to stimulate pancreatic bicarbonate and enzyme secretion in a variety of species including mice, dogs, and man. Radioligand binding studies with 3H-dopamine have revealed the presence of high- and low-affinity dopamine binding sites in dog pancreatic acinar cells. Stimulation of these receptors has been correlated with dose-dependent increases in intracellular cAMP levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

电离辐射后大鼠离体胰腺腺泡淀粉酶释放及M受体数量变化

我们曾观察到大鼠经γ-射线照射后胰淀粉酶活性降低和分泌减少[1],为进一步探讨照射后胰酶分泌减少的机制,本研究制备出分散的大鼠胰腺腺泡悬液并以不同浓度的~3H-二苯羟乙酸-3-喹咛环酯(~3Hquinuclidinyll benzilatc,简称~3H-QNB)进行M受体结合测定,同时观察胆碱能介质氨甲酰胆碱刺激腺泡所引起的淀粉酶释放反应。结果表明,γ-射线10Gy照射后3天,大鼠分散的胰腺腺泡在氨甲酰胆碱刺激时淀粉酶释放量减少到对照的50％,腺泡M受体与~3H-QNB最大结合量(Bmax)减少到对照的38％,伋M受体与~3H-QNB结合的解离常数(K_D)无改变,说明胰腺腺泡细胞M受体数量的减少可能是照射后胰腺腺泡分泌淀粉酶减少的原因之一。     

PACAP-38, a novel peptide from ovine hypothalamus, is a potent modulator of amylase release from dispersed acini from rat pancreas.

Despite studies indicating the presence of specific pancreatic acinar receptors for PACAP-38, a peptide that was recently isolated from ovine hypothalamus, the actions of the new peptide on pancreatic enzyme secretion have not been examined. The present study demonstrates that in terms of cAMP production and amylase release from dispersed acini from rat pancreatic acini, PACAP-38 and an N-terminal fragment, PACAP-27, have the same potency and efficacy as vasoactive intestinal peptide (VIP). As with VIP, these actions are potentiated by adding an inhibitor of cyclic nucleotide phosphodiesterase, and combination of PACAP-38 with bombesin, CCK-8, carbachol or the calcium ionophore A23187 results in 2-fold augmentation of the secretory actions of these agents. Inhibition of PACAP-38-induced cAMP production and amylase release by two VIP-receptor antagonists indicates that the secretory effects of PACAP-38 are mediated by interaction with VIP receptors. PACAP-38, a new brain-gut peptide, may be a physiological modulator of pancreatic enzyme secretion.     

Estrogens influence cholecystokinin stimulated pancreatic amylase release and acinar cell membrane cholecystokinin receptors in rat.

This study examines the influence of ovariectomy and administration of a pharmacologic dose of estradiol on amylase release from isolated-dispersed rat pancreatic acini and cholecystokinin receptors on rat acinar cell membranes. Rats were sham ovariectomized (intact) or ovariectomized (Ovx) and 21 day timed release pellets containing either estradiol (2.5 mg) or vehicle, were implanted subcutaneously. Eighteen days later, pancreatic acini were isolated from rats by collagenase digestion and differential centrifugation. Total cellular amylase, basal and cholecystokinin octapeptide (CCK8) stimulated amylase release and CCK membrane receptors were measured. Acini isolated from estradiol treated Ovx rats had significantly greater total cellular amylase, compared to acini isolated from either intact or Ovx rats. The amplitude of both total stimulated amylase release and percent total stimulated amylase release were significantly greater for acini isolated from vehicle treated Ovx rats, than acini isolated from either intact or estradiol treated Ovx rats. The magnitude of percent total amylase release of acini isolated from estradiol treated Ovx rats was significantly lower than that of acini isolated from intact rats. Cholecystokinin receptor concentration was significantly greater on membranes prepared from vehicle treated Ovx rats, compared to membranes prepared from either intact or estradiol treated Ovx rats. These data indicate that ovariectomy is associated with increased responsiveness of pancreatic acini to CCK stimulation, while chronic estradiol treatment of ovariectomized rats is associated with increased total cellular amylase and decreased acinar cell responsiveness to CCK8. Estrogen mediated alterations in acinar cell amylase content and amylase release may play a role in estrogen related pancreatitis.     

Activation of m3 muscarinic receptors induces rapid tyrosine phosphorylation of p125(FAK), p130(cas), and paxillin in rat pancreatic acini

Tyrosine phosphorylation plays a key role in transmembrane and cytoplasmic signal transduction mechanisms stimulated by oncogenes, integrins, growth factors, neuropeptides, and bioactive lipids. Moreover, recent studies show that stimulation of odd-numbered muscarinic receptors increases the tyrosine phosphorylation of several proteins in different cellular types. The present study was aimed at examining whether activation of m3 muscarinic receptors in rat pancreatic acini evokes tyrosine phosphorylation of p125(FAK), and its substrates, p130(cas) and paxillin. Results show that stimulation of pancreatic acini with carbachol resulted in a rapid and transient increase in tyrosine phosphorylation of p125(FAK), p130(cas), and paxillin. Tyrosine phosphorylation of these proteins occurred in a time- and concentration-dependent manner. Simultaneous blockage of both PKC activation and increases in [Ca(2+)](i) partially decreased p125(FAK), p130(cas), and paxillin tyrosine phosphorylation stimulated by carbachol. Pretreatment of pancreatic acini with Clostridium botulinum C3 transferase, which specifically inactivates p21(rho), partially inhibited carbachol-induced p125(FAK), p130(cas), and paxillin tyrosine phosphorylation. In contrast, this treatment had no effect on amylase release stimulated by carbachol. Cytochalasin D, which disrupts actin microfilaments network, completely inhibited carbachol stimulated tyrosine phosphorylation of these proteins without having significant effects in carbachol-stimulated amylase secretion. These results dissociate tyrosine phosphorylation of p125(FAK), p130(cas), and paxillin from amylase secretion after m3 muscarinic receptors occupation in rat pancreatic acini. Taken together, these data suggest that (a) activation of m3 muscarinic receptors in rat pancreatic acini increases tyrosine phosphorylation of p125(FAK) and its substrates, p130(cas) and paxillin by diacylglycerol-activated PKC- and calcium- dependent, and independent pathways, (b) these responses require activation of p21(rho) and an intact actin cytoskeleton, and (c) p125(FAK), p130(cas), and paxillin are unlikely related to secretion in rat pancreatic acinar cells.     

Binding of cholecystokinin to high affinity receptors on isolated rat pancreatic acini

The binding of cholecystokinin (CCK) to its receptors on isolated rat pancreatic acini was investigated employing high specific activity, radioiodinated CCK (125I-BH-CCK), prepared by the conjugation of 125I-Bolton-Hunter reagent (125I-BH) to CCK. Binding was specific, time-dependent, reversible, and linearly related to the acinar protein concentration. After incubation for 30 min at 37 degrees C, the 125I-BH-CCK both in the incubation medium and bound to acini remained intact, as judged by gel filtration and trichloroacetic acid precipitation studies. Scatchard analysis was compatible with two classes of binding sites on acini: a very high affinity site (Kd, 64 pM) and a lower affinity site (Kd, 21 nM). 125I-BH-CCK binding to acini was competitively inhibited by CCK and four of its analogues in proportion to their biological potencies but not by unrelated hormones. Stimulation of amylase secretion by CCK and inhibition of 125I-BH-CCK binding by the same analogues carried out under identical conditions revealed a correlation (r = 0.99) between binding potency and amylase secretion. Stimulation of amylase secretion by CCK closely paralleled the occupancy of the high affinity CCK binding sites. It is concluded that the high affinity CCK binding sites most likely are the receptors mediating the stimulation of amylase secretion by CCK.     

Mechanism of Ca2+ wave propagation in pancreatic acinar cells.

An increase in cytosolic Ca2+ often begins as a Ca2+ wave, and this wave is thought to result from sequential activation of Ca(2+)-sensitive Ca2+ stores across the cell. We tested that hypothesis in pancreatic acinar cells, and since Ca2+ waves may regulate acinar Cl- secretion, we examined whether such waves also are important for amylase secretion. Ca2+ wave speed and direction was determined in individual cells within rat pancreatic acini using confocal line scanning microscopy. Both acetylcholine (ACh) and cholecystokinin-8 induced rapid Ca2+ waves which usually travelled in an apical-to-basal direction. Both caffeine and ryanodine, at concentrations that inhibit Ca(2+)-induced Ca2+ release (CICR), markedly slowed the speed of these waves. Amylase secretion was increased over 3-fold in response to ACh stimulation, and this increase was preserved in the presence of ryanodine. These results indicate that 1) stimulation of either muscarinic or cholecystokinin-8 receptors induces apical-to-basal Ca2+ waves in pancreatic acinar cells, 2) the speed of such waves is dependent upon mobilization of caffeine- and ryanodine-sensitive Ca2+ stores, and 3) ACh-induced amylase secretion is not inhibited by ryanodine. These observations provide direct evidence that Ca(2+)-induced Ca2+ release is important for propagation of cytosolic Ca2+ waves in pancreatic acinar cells.     

Assessing the Secretory Capacity of Pancreatic Acinar Cells

Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work demonstrates how the secretory capacity of these cells can be assessed by culture of isolated acini. The setup is advantageous since isolated acini, which retain many characteristics of the intact exocrine pancreas can be manipulated and monitored more readily than in the whole animal. Proper isolation of pancreatic acini is a key requirement so that the ex vivo culture will represent the in vivo nature of the acini. The protocol demonstrates how to isolate intact acini from the mouse pancreas. Subsequently, two complementary methods for evaluating pancreatic secretion are presented. The amylase secretion assay serves as a global measure, while direct imaging of pancreatic secretion allows the characterization of secretion at a sub-cellular resolution. Collectively, the techniques presented here enable a broad spectrum of experiments to study exocrine secretion.     

Effects of synthetic human pancreastatin on pancreatic secretion and blood flow in rats and dogs.

Effects of synthetic human pancreastatin-52 and human pancreastatin-29 on pancreatic secretion and blood flow were examined in rats and dogs. Synthetic human pancreastatin-52 and human pancreastatin-29 were equally potent in suppressing the release of amylase stimulated by cholecystokinin in rats in vivo. However, neither human pancreastatin-52 nor human pancreastatin-29 altered basal and cholecystokinin-stimulated amylase release from isolated dispersed rat pancreatic acini. In studies in dogs, human pancreastatin-29 suppressed releases of amylase and protein stimulated by cholecystokinin, but did not alter pancreatic blood flow. These results suggest that the inhibitory effects of pancreastatin on pancreatic secretion do not involve a direct action on pancreatic acinar cells nor alteration of pancreatic blood flow. Pancreastatin probably is important in regulating exocrine pancreatic secretions as well as endocrine pancreatic secretions.     

Stimulation of rat pancreatic exocrine secretion by urocortin and corticotropin releasing factor

Neural and hormonal mechanisms control pancreatic secretion. The effects of the corticotropin releasing factor (CRF) related neuropeptide urocortin (UCN) on pancreatic exocrine secretion were examined. In anesthetized male rats, pancreatic secretion volume and total protein were assayed. UCN increased pancreatic secretory volume and protein secretion and potentiated cholecytokinin-stimulated protein secretion. Astressin, a non-specific CRF receptor antagonist, inhibited UCN-stimulated protein output while CRF(2) receptor antagonist, antisauvagine-30, was without effect. Atropine, but not subdiaphragmatic vagotomy, inhibited UCN-mediated secretion. In acinar cells, UCN did not stimulate release of amylase nor intracellular cAMP. UCN is a pancreatic exocrine secreatagogue with effects mediated through cholinergic intrapancreatic neurons.     

Insulin receptors in isolated mouse pancreatic acini.

Specific insulin receptors were measured in isolated mouse pancreatic acini. Scatchard analyses revealed a high affinity binding site with a K_d of 1.67 nM and a lower affinity site with a K_d of 83 nM. Binding of insulin to these receptors was rapid, one-half maximal binding occurring at 2 min and maximal binding at 30 min. Insulin stimulated the uptake of the glucose analogue 2-deoxy-D-glucose; maximum effects were detected at 1.67 μM. Insulin, in contrast, had no direct effects on alpha-aminoisobutyric acid uptake. The finding of high affinity insulin receptors in pancreatic acinar cells supports the hypothesis that insulin may directly regulate specific functions in the exocrine pancreas.     

Down-regulation and recycling of high affinity cholecystokinin receptors on pancreatic acinar cells

First incubating dispersed acini from rat pancreas with monensin, a cation ionophore that can inhibit recycling of receptors, inhibited binding of 125I-cholecystokinin 8 (125I-CCK-8) measured during a second incubation by as much as 50%. A maximal effect of monensin required 90 min of first incubation. Detectable inhibition of binding of 125I-CCK-8 occurred with 300 nM monensin, and inhibition increased progressively with concentrations of monensin up to 25 microM. Pancreatic acini possess two classes of receptors that bind 125I-CCK-8. One class has a high affinity (Kd = 461 pM) and a low capacity for CCK (512 fmol/mg DNA); the other class has a low affinity (Kd = 47 nM) and a high capacity for CCK (18 pmol/mg DNA). First incubating acini with monensin caused an 84% decrease in the number of high affinity CCK receptors with no change in the number of low affinity CCK receptors or the values of Kd for either class of receptors indicating that there is recycling of high affinity CCK receptors but not low affinity CCK receptors. First incubating acini with monensin did not alter CCK-stimulated amylase secretion indicating that in contrast to previous conclusions, occupation of low affinity CCK receptors mediates CCK-stimulated enzyme secretion. Moreover, the biphasic dose-response curve for CCK-stimulated enzyme secretion from monensin-treated acini suggests that pancreatic acini also possess a third, previously unrecognized class of very low affinity CCK receptors.