Inositol trisphosphate independent increase of intracellular free calcium and amylase secretion in pancreatic acini

It is generally believed that the activation of various cell surface receptors results in the phospholipase C-catalyzed production of inositol trisphosphate which, in turn, increases the intracellular concentration of free Ca2+ by stimulating its release from nonmitochondrial sources. We have investigated both the production of inositol trisphosphate and changes in intracellular Ca2+ concentration in rat pancreatic acini in response to caerulein and CCK-JMV-180, two analogs of cholecystokinin. Both of these analogs cause comparable increases in the rate of amylase secretion and in intracellular Ca2+ concentration but their effects on inositol phosphate generation are dramatically different; caerulein stimulates significant production of inositol phosphates within 1 min of its addition, whereas no detectable levels of inositol phosphates were generated within the same time after addition of CCK-JMV-180. These results suggest that the CCK-JMV-180 stimulated release of intracellular Ca2+ is not mediated by inositol trisphosphate but some other as yet unidentified messenger.     

Glucocorticoids increase cholecystokinin receptors and amylase secretion in pancreatic acinar AR42J cells

We recently reported in AR42J pancreatic acinar cells that glucocorticoids increased the synthesis, cell content, and mRNA levels for amylase (Logsdon, C.D., Moessner, A., Williams, J.A., and Goldfine, I.D. (1985) J. Cell Biol. 100, 1200-1208). In addition, in these cells glucocorticoids increased the volume density of secretory granules and rough endoplasmic reticulum. In the present study we investigate the effects of glucocorticoids on the receptor binding and biological effects of cholecystokinin (CCK) on AR42J cells. Treatment with 10 nM dexamethasone for 48 h increased the specific binding of 125I-CCK. This increase in binding was time-dependent, with maximal effects occurring after 48 h, and dose-dependent, with a one-half maximal effect elicited by 1 nM dexamethasone. Other steroid analogs were also effective and their potencies paralleled their relative effectiveness as glucocorticoids. Analyses of competitive binding experiments conducted at 4 degrees C to minimize hormone internalization and degradation revealed the presence of a single class of CCK binding sites with a Kd of approximately 6 nM and indicated that dexamethasone treatment nearly tripled the number of CCK receptors/cell with little change in receptor affinity. Treatment with 10 nM dexamethasone increased both basal amylase secretion and the amylase released in response to CCK stimulation. In addition, dexamethasone increased the sensitivity of the cells to CCK. The glucocorticoid decreased the concentration of CCK required for one half-maximal stimulation of amylase secretion from 35 +/- 6 to 8 +/- 1 pM. These data indicate, therefore, that glucocorticoids induce an increase in the number of CCK receptors in AR42J cells, and this increase leads to enhanced sensitivity to CCK.     

Stimulation of pancreatic amylase release is associated with a parallel sustained increase of cytoplasmic calcium

The kinetics of the changes in the cytoplasmic Ca²⁺ concentration (Ca_i²⁺) and amylase release were measured in fura-2-loaded pancreatic acinar cells and perifused pancreatic acini, respectively. Cholecystokinin octapeptide (CCK-8) and its amphibian analogue caerulein induced similar dose-related increases of Ca_i²⁺ and amylase secretion with threshold concentrations of 2–6·10⁻¹² M, and maximal effects at 2·10⁻¹⁰ M. The action of CCK/caerulein on Ca_i²⁺ was complex and similar to that of carbachol and bombesin with a prompt several-fold increase within seconds followed by a gradual decline over more than 5 min to a new sustained suprabasal level. The kinetics of amylase release in response to CCK and carbachol correlated with the changes in Ca_i²⁺. Additions of the antagonists N²,O²-dibutyrylguanosine 3′:5′-cyclic monophosphate and atropine after 30 min of CCK-8 and carbachol stimulation, respectively, were associated with prompt lowerings of Ca_i²⁺ and inhibitions of amylase secretion. The patterns observed with substance P (SP) and eledoisin were different with high concentrations (10⁻⁸–10⁻⁷ M) giving monophasic increases of Ca_i²⁺ and amylase release. An initial stimulation of cells with a high dose of CCK eliminated the Ca_i²⁺ response to further stimulation with CCK, carbachol, bombesin and SP, whereas cells subjected to initial stimulation with SP responded to subsequent exposure to CCK with prolonged elevation of Ca_i²⁺. The data indicate that stimulation with CCK, carbachol and bombesin may be associated with intracellular mobilization of calcium from more than one pool, and that an increase of Ca_i²⁺ is involved even in threshold stimulation of amylase release.     

Tissue-specific and insulin-dependent expression of a pancreatic amylase gene in transgenic mice.

The regulatory properties of mouse pancreatic amylase genes include exclusive expression in the acinar cells of the pancreas and dependence on insulin and glucocorticoids for maximal expression. We have characterized a murine pancreatic amylase gene, Amy-2.2y, whose promoter sequence is 30% divergent from those of previously sequenced amylase genes. To localize sequences required for tissue-specific and hormone-dependent activation, we established two lines of transgenic mice. The first line contained a single copy of the complete Amy-2.2y gene as well as 9 kilobases of 5'-flanking sequence and 5 kilobases of 3'-flanking sequence. The second line carried a minigene which included 208 base pairs of 5'-flanking sequence and 300 base pairs of 3'-flanking sequence. In both lines the transgene was expressed at high levels exclusively in the pancreas. Both constructs were dependent on insulin and induced by dexamethasone. Thus, the transferred genes contained the sequences required for tissue-specific and hormonally regulated expression.     

Circulatory turnover of pancreatic amylase.

Radiolabeled amylase and unlabeled pancreatic amylase were infused into pigs in order to determine the plasma half-life of the enzyme. Regardless of the parameter measured (radioactivity, enzyme activity or concentration), the plasma removal curves could be resolved into three components when subjected to tracer analysis. The plasma half-life was estimated to be approximately 3 hr. Through the use of a recently developed radioimmunoassay specific for porcine pancreatic amylase, the plasma concentration of amylase was calculated at 2.4 mug/ml. Knowing the plasma concentration and half-life of amylase we determined the circulatory turnover of the enzyme. Over a 2.4-hr period, 9.6 mug of pancreatic amylase/ml of plasma must re-enter the circulation to maintain the enzyme at constant levels.     

Cyclooxygenase-2 transcription is stimulated and amylase secretion is inhibited in pancreatic acinar cells after induction of acute pancreatitis

Cyclooxygenases as the key enzymes of prostaglandin synthesis have an important role in regulation of inflammation. We describe that Cox-1 and Cox-2 are synthesized in rat pancreatic acinar cells. Upon induction of pancreatitis, Cox-2 mRNA increases while Cox-1 expression remains constant. However, the cyclooxygenase inhibitor indomethacin has no influence by a feed-back mechanism on the expression of the two isoforms. We have previously shown that prostaglandins of the E-type inhibit cholecytoskinin-stimulated amylase secretion. Consistent with this observation, we find here that pancreatitis inhibits CCK-stimulated amylase secretion from isolated acini. In agreement with this result, the effect is neutralized by indomethacin inhibition of prostaglandin synthesis. In summary, we have found that both cyclooxygenases are synthesized in pancreatic acinar cells and that their expression is differentially regulated which in turn influences amylase secretion.     

Hibernation reduces pancreatic amylase levels in ground squirrels

Pancreatic enzyme levels in mammals are influenced by food intake and dietary composition. In this study, we examined the activity and expression of pancreatic amylase in a hibernating mammal, a natural model for long-term fasting. Pancreatic tissues were obtained from summer-active 13-lined ground squirrels and hibernating squirrels that had not eaten for at least 6 weeks. Amylase specific activity was reduced by approximately 50% in the torpid hibernators compared with summer squirrels, and immunoblot analysis revealed that amylase protein expression was reduced by approximately 40% in the hibernators. Similar reductions in amylase specific activity were observed in interbout euthermic hibernators. These results support a strong influence of food intake on pancreatic enzyme expression in hibernating mammals. The maintenance of basal levels of this key digestive enzyme at approximately 50% of summer values despite the extended winter fast likely facilitates the rapid resumption of digestive function after terminal arousal in the spring.     

Expression of amylase and other pancreatic genes in Xenopus

To better understand the relationship between the endocrine and exocrine cell types in the Xenopus pancreas, we have cloned the Xenopus amylase cDNA and compared its expression profile with that of four other pancreatic markers: insulin, glucagon, elastase and trypsinogen. Our results demonstrate that the first pancreatic marker to be expressed is insulin, exclusively in the dorsal pancreas. These insulin-expressing cells form small groups which resemble islets, but no insulin is detected in the ventral pancreas until stage 47. In contrast, the exocrine markers, amylase, elastase and trypsinogen are first expressed only in the ventral pancreas beginning at stage 41; by stage 45 their expression extends into the dorsal pancreas. Glucagon, on the other hand, is not expressed in the pancreas until stage 45. In the endocrine cell clusters we do not find glucagon-expressing cells surrounding insulin-expressing cells, either in the tadpole or in the mature frog pancreas.     

Immunogold determination of amylase concentrations in pancreatic subcellular compartments

We used the immunogold method on ultrathin cryosections to measure intracellular amylase (Am) concentrations in subcellular compartments of rat exocrine pancreatic cells. Previously, the quantitation procedure was characterized in a model system consisting of Am dispersed at known concentration in a matrix of gelatin. Variations in labeling efficiency, due to differences in matrix density, were equalized by embedding in 30% polyacrylamide (PAA). Here we applied these model conditions to rat pancreas and established intracellular Am concentrations [Am]. Specimen blocks were composed of tissue and a reference layer of gelatin mixed with a known Am concentration ([Am]r), both fixed in glutaraldehyde. Cryosections of the PAA embedded blocks were immunogold labeled for Am. The labeling density was measured in the reference layer (LDr) and in structures in exocrine cells that were involved in Am synthesis and transport (LDs). In each of these structures the Am concentration ([Am]s) was calculated from: [Am]s = [Am]r. LDs/LDr In this way we measured average concentrations ranging from 63 mg/ml in rough endoplasmic reticulum to 261 mg/ml in secretory granules. Concentration of Am appeared to occur mainly in the most cis- and the most trans-Golgi cisternae. To check whether sterical hindrance was an inherent bias to the [Am] measurements in compartments that contained high concentrations of the enzyme, the labeling efficiency for Am in intact isolated secretory granules in gelatin and embedded in PAA, was compared with the efficiency when the granules were lysed and approximately 50 times diluted in gelatin before PAA embedment. It appeared that Am was detected with similar efficiency under both conditions. This demonstrated that sterical hindrance did not cause errors in the measurements of cellular Am concentrations.