Search

We have previously demonstrated that altered exocrine pancreatic stimulus-secretion coupling is associated with ovariectomy and chronic estradiol administration. To elucidate possible mechanisms underlying those effects we examined the ability of chronic administration of different doses of estradiol to regulate the CCK signal transduction pathway in isolated rat pancreatic acini. Doses of estradiol ranging from 0.5 to 119 μg/day were administered to ovariectomized rats for 18 days. Ovariectomy was associated with enhanced CCK-stimulated pancreatic amylase release, whereas estradiol dose dependently decreased the magnitude of CCK-stimulated amylase release. Ovariectomy was also associated with enhanced CCK receptor numbers on acinar cell membranes. Estradiol administration was associated with dose-dependent decreases in CCK receptor numbers. Neither ovariectomy nor estradiol administration affected CCK receptor affinity. Moreover, estradiol administration was associated with increased expression of the α-subunit of the heterotrimeric G protein Gq/11(Gαq/11). Recent findings (H. Ohnishi, S. A. Ernst, D. I. Yule, C. W. Baker, and J. A. Williams. J. Biol. Chem. 272: 16056–16061, 1997) demonstrate that Gαq/11may exert a tonic inhibitory effect on pancreatic enzyme release. In view of these findings, the increased expression of Gαq/11 induced by estradiol likely contributes to the inhibition of pancreatic enzyme release. We conclude that the effect of estradiol to decrease pancreatic secretion is mediated through regulation of CCK receptor density and Gαq/11 expression.

Cholecystokinin (CCK) receptors on rat pancreatic acinar cells display two binding affinity states in the presence of adeninine and guanine triphosphates with the effect of ATP mediated by the enzyme nucleoside diphosphate kinase. To determine whether this behavior was intrinsic to a single receptor protein we studied the binding affinity of CHO cells stably transfected with a cloned rat CCKA receptor. 125I-CCK binding to intact cells at 37 degrees C revealed two affinity states for CCK of Kd values 20 pM and 2.4 nM. Membranes prepared from these cells displayed a single affinity state for CCK but two affinity states could be restored in the presence of GTP[gamma S], ATP and ATP[gamma S] but not AMP-PCP. ATP and ATP[gamma S] but not AMP-PCP were substrates for nucleoside diphosphate kinase present in CHO cell membranes and transferred their terminal phosphate to GDP. These findings indicate that the interconvertible affinity states of the CCK receptor are inherent in a single receptor protein and that nucleoside diphosphate kinase mediates the effect of ATP to regulate these two affinity states.

The protein components of highly purified secretory granule membranes and the granule contents from rat exocrine pancreas were characterized by two-dimensional polyacrylamide gel electrophoresis, protein staining, lectin absorption, and Western blotting with anti-secretory protein antibodies. NH2-terminal amino acid sequence was obtained for a approximately 53-kDa glycoprotein denoted GP-3, present only in granule membrane preparations where it was resistant to washing with Na2CO3 and KBr. The sequence of this protein showed homology to pancreatic lipase but was distinct from the NH2-terminal sequence of a 50-kDa content protein presumed to be secretory lipase. Polymerase chain reaction amplification with degenerate oligonucleotide primers to GP-3 and secretory lipase gave partial length subclones that were used to isolate clones from a rat pancreas cDNA library. Dideoxy sequencing of full-length subclones of GP-3 revealed the predicted amino acid sequence for a mature protein of 452 amino acids with a potential N-linked glycosylation site and a deglycosylated molecular weight of 50,860. The GP-3 sequence possesses the serine esterase consensus sequence G-X-S-X-G centered around Ser154 and the catalytic state triad Asp178-His265-Ser154 characteristic of pancreatic lipases. Northern blot analysis of various rat tissues showed GP-3 expression solely in pancreas. Comparison of GP-3 nucleotide and amino acid sequence, along with pancreatic lipases of various species including rat, shows extensive homologies to both proteins and reveals an underlying diversity in the pancreatic lipase family. Close homology is observed between GP-3 and a lipase molecule previously isolated from mouse cytotoxic T cells.

This study was conducted to investigate the role of humoral factors in pancreatic alterations induced by obstructive jaundice (OJ) in rats. OJ in male Sprague-Dawley rats induced significant increases in pancreatic weight, DNA content, and RNA content of acinar cells. These changes were accompanied by enlargement of eosinophilic granules and compressed nuclei. Protein, amylase, and trypsinogen contents of pancreas were also increased in OJ rats. In addition, plasma levels of bilirubin, cholecystokinin (CCK), and estradiol increased in OJ rats and were correlated positively with each other and with pancreatic weights. Administration of a specific CCK receptor, L-364,718, to OJ rats partly attenuated the changes of the pancreas, indicating that CCK is involved in these changes. These findings suggest that estradiol may be involved in regulating the pancreatic changes induced by OJ in rats.

This study was conducted to investigate pancreatic exocrine function and pancreatic growth in rats with obstructive jaundice (OJ). OJ was produced in adult male Sprague-Dawley rats by bile duct ligation; control rats underwent laparotomy only. Induction of OJ was associated with significant hyperplasia and hypertrophy of the pancreas in rats as shown by increased DNA and RNA contents of pancreatic tissue. Factors associated with pancreatic growth in OJ rats were further examined in isolated dispersed pancreatic acini from OJ rats and the data were compared with those for control rats. Studies with isolated dispersed acini from OJ rats showed that pancreatic growth was accompanied by significant increases in total cellular amylase content; however, amylase release (percentage of initial) in response to cholecystokinin octapeptide was significantly decreased in OJ rats compared to control rats. Total amylase output in response to 100 pM cholecystokinin (CCK) was higher in the OJ group when compared to the control group (8.6 U/mg protein versus 6.4 U/mg protein), as calculated from the total amylase content and percentage of amylase released. Receptor binding data showed that the capacity of CCK receptors in OJ rats was significantly lower when it was compared with control. In addition, plasma levels of CCK were significantly elevated in OJ rats when compared to controls. These results suggest that obstructive jaundice induces pancreatic growth that is associated with alteration of exocrine pancreatic function. Abnormally high levels of stored amylase in pancreatic acini may be implicated in the development of pancreatitis as often seen in obstructive jaundice patients.

In individual fura-2 loaded cells of rat pancreatic acini endothelin-1 (ET-1) (10-50 nM) induced sustained oscillations in [Ca2+]i. At higher concentrations a larger, but transient increase in [Ca2+]i was observed, which was largely unaffected by removal of extracellular Ca2+. ET-1 induced the release of Ca2+i from the same store as cholecystokinin (CCK), but with less potency. At concentrations of endothelin which transiently increased Ca2+, ET-1 increased the accumulation of inositol phosphates. Specific binding sites for 125I-endothelin were demonstrated on rat pancreatic acini. A single class of binding sites was identified with an apparent Kd 108 +/- 12 pM and Bmax of 171 +/- 17 fmol/mg for ET-1. The relative potency order for displacing [125I]ET was ET-1 greater than ET-2 greater than ET-3. In contrast to CCK and the non-phorbol ester tumour promoter Thapsigargin (TG) which induce both transient and sustained components of [Ca2+]i elevation, ET-1 failed to increase amylase release over the range 100 pM-1 microM.

To investigate the mechanisms by which dietary compositions regulate the exocrine pancreas, we examined the effects of no-fat diet (NFD) and high-fat diet (HFD) on cholecystokinin (CCK)-stimulated amylase secretion from rat pancreatic acini. Rats were maintained for 4 weeks on NFD or HFD, which contained 0 or 45% fat and 58 or 29% carbohydrates, respectively. Pancreatic acini were isolated and stimulated by graded doses of CCK for 30 min. Maximal CCK-stimulated amylase secretion by pancreatic acini from rats on NFD (23.1 +/- 4.3 U/mg protein at 10(-10) M) was significantly higher than that of HFD (5.5 +/- 1.6 U/mg protein at 10(-10) M). In contrast, expressed as a percentage of the initial content, maximal CCK-stimulated amylase secretion by pancreatic acini from rats on NFD (15.0 +/- 0.8%) tended to be lower than that from rats on HFD (28.1 +/- 3.5%). To study further the effects of the diets on amylase mRNA levels, another group of rats was maintained on the respective diets for 4 weeks, sacrificed, and total pancreatic RNA isolated. Amylase mRNA levels in rats on NFD were 2.5 times higher than in rats on HFD. The results suggest that alterations in CCK-stimulated amylase secretion by pancreatic acini, as well as modifications in pancreatic amylase expression, may be involved in the mechanisms by which the exocrine pancreas adapts to diet.

We have previously demonstrated in permeabilized rat pancreatic acini that the existence of two affinity states of the pancreatic cholecystokinin (CCK) receptor seen in intact cells depends on the presence of ATP. In the present study, we demonstrate that this effect of ATP is mediated by the enzyme nucleoside diphosphate kinase (NDPK). Northern blot hybridization analysis demonstrated NDPK mRNA in pancreas. Furthermore, pancreatic membranes possessed NDPK activity, which transferred high-energy phosphate groups to [8-3H]GDP. This enzyme also utilized UTP and ITP as a source of gamma-phosphate for GTP formation while guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was formed in the presence of adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S). However, adenylyl (beta, gamma-methylene)-diphosphate (AMP-PCP) did not serve as a substrate for NDPK. Analysis of 125I-Bolton-Hunter-labeled CCK octapeptide ([125I]BH-CCK-8) binding data in the absence of nucleotides was consistent with a single affinity state with dissociation constant (Kd) equal to 80 pM and maximal binding equal to 50.8 fmol/mg. ATP, UTP, ITP, ATP gamma S, and GTP gamma S all induced two CCK binding affinity states, which in the presence of 1 mM ATP were Kd = 74 pM for high-affinity sites and Kd = 4.3 nM for low-affinity sites: AMP-PCP did not induce two affinity states. GDP at 10 microM had no effect on CCK binding but potentiated the effect of ATP. GTP gamma S, in addition to inducing high- and low-affinity states, also elicited a significant concentration-dependent reduction in the total number of measurable CCK receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

We previously demonstrated that the existence of two affinity states of the pancreatic cholecystokinin (CCK) receptor depends on the presence of ATP.' Subsequently, we established that the effect of ATP to induce two CCK binding affinity states was biochemically mediated by the enzyme nucleoside diphosphate kinase (NDPK) (manuscript in preparation). Nucleoside diphosphate kinase catalyzes the transfer of high energy gamma-phosphate groups from nucleoside triphosphates to nucleoside diphosphates. In that study the ability of an assortment of nucleoside triphosphates to serve as substrate for NDPK was compared with their ability to induce two binding affinity states of the CCK receptor on rat pancreatic membranes for which previous studies have observed only a single binding affinity state. Nucleoside triphosphates capable of serving as a substrate for NDPK could also induce two CCK binding affinity states on pancreatic membranes, whereas those that could not serve as substrate for NDPK could not induce two CCK binding affinity states. Furthermore, GDP potentiated the effect of ATP on binding (manuscript in preparation). To conclusively identify NDPK we found it necessary to clone and functionally express this enzyme. We report here that the characteristics of the cloned and expressed enzyme are similar to those of pancreatic membrane NDPK and consistent with the ability of this enzyme to induce two CCK binding affinity states.

This study was conducted in rats to investigate the influence of exogenously administered estradiol (ESD) and/or cholecystokinin (CCK) on components and secretions of the pancreatic acini. Intact male rats were treated for 14 d with exogenous administration of ESD, CCK, or ESD+CCK. After 14 d CCK treatment induced significant increases in DNA and RNA contents, and DNA/RNA ratio in the pancreas, indicating hyperplasia and hypertrophy of the pancreas, however, ESD treatment did not have these effects. Both ESD treatment and CCK treatment induced significant increases in amylase and trypsinogen contents in pancreatic acini and each decreased secretion from acini in response to CCK. Combined treatment with ESD plus CCK augmented these effects on enzyme contents and secretion. The results indicate that exogenous administration of CCK has trophic effects on the exocrine pancreas, increasing effects on enzyme contents and inhibitory effects on amylase secretion. In contrast, exogenous administration of ESD had no trophic effects on pancreas, but had increasing effects on enzyme contents and inhibitory effects on amylase secretion. The results suggest that the effects of exogenous ESD and CCK on pancreas are not similar to each other, but both ESD and CCK may be involved in regulating pancreatic exocrine functions.

The influence of adenine and guanine nucleotides on cholecystokinin (CCK) receptor binding was examined in streptolysin O-permeabilized rat pancreatic acini. Specific binding of tracer to intact acini was 12.1 +/- 0.4% per milligram protein, while permeabilized acini bound 34.6 +/- 2.9% (n = 7). The increase in binding was also seen when normalized to DNA. Binding to permeabilized acini was reduced by the presence of 1 mM ATP to 23.0 +/- 1.3%. Analysis of competitive inhibition of tracer binding by unlabeled CCK-8 was consistent with binding to two affinity states on intact acini, with the equilibrium dissociation constants for the high (KdH)- and low (KdL)-affinity states equal to 41 +/- 5 pM and 5.2 +/- 0.4 nM, respectively; permeabilized acini displayed a single binding site with Kd = 598 +/- 40 pM. In the presence of 1 mM ATP, two states were seen on permeabilized acini with KdH = 85 +/- 11 pM and KdL = 2.7 +/- 0.6 nM. ATP, ATP gamma S, GTP, and GTP gamma S all inhibited binding, with half-maximal inhibition occurring at greater than 1 mM, 21 microM, 5 microM, and 0.4 microM, respectively. GTP gamma S (1 microM) also induced two affinity states with KdH = 112 +/- 7 pM and KdL = 1.5 +/- 0.2 nM (n = 3). Binding of CCK to pancreatic membranes was also decreased by ATP, and a similar regeneration of two binding affinity states was observed. ATP also decreased binding of [125I-Tyr4]bombesin to permeabilized acini, but in contrast did not generate two measurable binding affinity states.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Adaptation of exocrine pancreas by dietary fats and its resulting effects on morphology and function of the exocrine pancreas remain controversial. In this study we have examined the influence of dietary fats on pancreatic pathophysiology by examining the basal and stimulated pancreatic secretion of enzymes in response to high fat (HFD) and no fat (NFD) diets. Methods: Male Sprague Dawley rats (N=10) were fed ad libitum with high fat diets containing 45% fat (vegetable oil) and 29% carbohydrates. An identical group was fed with diets containing 58% carbohydrates but no fat. The animals were maintained in screen bottomed cages at 22°C on a 12 hr light-dark cycle with free access to water. After four weeks, the animals were fasted for 24 hrs, anesthetized, bile pancreatic duct was cannulated to collect pancreatic juice. Bile pancreatic juice was collected for 30 min before and after infusion of CCK for 30 min via the jugular vein. The animals were maintained for additional 24 hrs with recirculation of pancreatic juice to the duodenum and then sacrificed to isolate pancreatic acini by collagenase digestion. Amylase content and release in response to CCK-8 by isolated acini were measured. Histopathologic studies of pancreas, fixed in 10% buffered formalin, were evaluated by light microscopy. Results: Outputs of enzymes (amylase, lipase and trypsinogen) in bile pancreatic juice of rats on high fat diets under basal and stimulated conditions were significantly higher than that of rats on no fat diet. Amylase release measured in isolated acini of rats on high fat diet in response to CCK-8 was significantly lower than that of rats on no-fat diet. Histopathology revealed significant difference in cellular changes (cytoplasmic vacuolation; swelling and pyknosis) in rats on high fat diet. Conclusions: Dietary fats, especially high fat, alters pancreatic morphology and function. The results indicate that high fat diets enhanced the outputs of enzymes in bile pancreatic juice, down regulated CCK stimulated amylase response in isolated pancreatic acini and induced cellular injury.