Cyclic AMP and enzyme secretion by the isolated rabbit pancreas

Summary The isolated rabbit pancreas secreted protein and -amylase at a relatively constant rate during an eight-hour period. Both pancreozymin and acetylcholine caused a massive release of protein and -amylase into pancreatic fluid. Theophylline (10^–2 M) stimulated protein secretion in vitro maximally by 155%, while for -amylase secretion a maximal average stimulation of 80% was observed. Theobromine (10^–2 M) exerted a slight increase on enzyme secretion, equal to that given by 10^–3 M theophylline. Cyclic AMP (10^–3M) also increased enzyme secretion. For protein secretion a maximal average stimulation of 64% was observed, while -amylase secretion was maximally stimulated by 31%. Theophylline (10^–2 M) potentiated the stimulatory effect of pancreozymin (8.5 U/l) on pancreatic enzyme secretion. These results indicate that cyclic AMP is a mediator in enzyme secretion.     

Non-parallel enzyme secretion from rat pancreas in vitro studies.

1. The relative variations of rat pancreatic amylase versus lipase and chymotrypsinogen secretions have been studied in vitro with the help of two different techniques: in situ organ perfusion and incubation of pancreatic lobules. 2. In experiments on the perfused pancreas, with 8 C u.kg-1 hr-1 secretin added to the perfusion fluid, cholecystokinin-pancreozymin (8 ID u.kg-1) or pilocarpine (15 mg kg-1) both resulted in a significant change in the enzyme proportions in the juice. 3. In experiments on pancreatic lobules, the addition to the incubation medium of secretin (10(-7) M), alone or associated with cholecystokinin-pancreozymin (8 x 10(-7) M) or pilocarpine (10(-4) M) did not induce any change in the enzyme proportions in secretion. 4. It was concluded that the non-parallelism between enzyme secretions can occur in the rat upon pancreozyminic or cholinergic stimulation in vitro as well as in vivo (Dagorn, 1978) provided basal protein output is low enough. This is not the case when the tissular integrity of the pancreas is lost, such as in experiments on lobules. 5. This work confirms that pancreatic secretion derives from two intrapancreatic pools of different enzymatic composition, and gives a possible explanation for some discrepancies from the literature on the existence of non-parallel secretion.     

Insulin secretion by isolated perfused rat and mouse pancreas.

A method for isolation and perfusion of a pancreas preparation consisting of pancreas, stomach, proximal duodenum, and spleen is described. Basic characteristics of regulation of insulin secretion from the perfused pancreas isolated from rats, albino mice, obese mice (ob/ob), and black mice were identical. Viability and stability of the pancreas preparation during perfusion were maintained as documented by measurements of oxygen consumption of the pancreas preparation, perfusion pressure, and pH of the perfusion medium. The insulin-secretory capacity of the pancreas of different animal species was compared. Insulin secretion by the perfused rat and obese (ob/ob) mouse pancreas was much more potent than that by the pancreas of lean albino and lean black mice. D-Glucose-induced insulin secretion from the pancreas was decreased after fasting of the animals and was dependent on glucose concentration and presence of calcium in the perfusion medium. D-Glyceraldehyde, tolbutamide, D-mannose, dihydroxyacetone, L-leucine, and L-arginine also induced insulin secretion from the pancreas. D-Fructose, D-galactose, L-glucose, 3-O-methyl-D-glucose, N-acetylglucosamine, D-xylose, D,L-glyceric acid, pyruvate, L-lactate, and theophylline did not provoke insulin secretion.     

Relationships between blood flow and secretion in the isolated perfused canine pancreas

1. Stimulation by a continuous intra-arterial infusion of secretin caused a marked vasodilatation in the pancreato-duodenal preparation or in the isolated pancreas perfused with whole heparinized blood. There was no difference in the vasomotor effects of two unequally purified preparations of secretin. The fact that the vasodilatation was still observed when the duodenum was removed proved that the haemodynamic effect of secretin was initiated in the pancreas itself.2. The secretory response of the pancreas to a given dose of secretin was larger when the blood pressure was kept constant by increasing the blood flow than when the blood pressure was allowed to fall by maintaining the blood flow at a fixed value. It is concluded that the vasodilatation plays a role in the functional regulation of the pancreatic response to secretin.3. The correlation between blood flow and arterial pressure on the one hand, and the secretory response to secretin on the other, was not mediated by the control of respiration since there was no variation in oxygen uptake in response to a variation in the blood flow. This correlation was still observed when the venous blood was discarded and therefore could not be explained by variations in the rate of recirculation of secretin. It was observed too in experiments where the duodenum was removed, the pancreas alone being introduced into the perfusion apparatus, which indicated that this effect was not mediated by a control of the release of secretin by the duodenum.     

Effect of prooxidants on insulin secretion by the isolated rat pancreas

Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 116, No 8, pp. 165–167, August, 1993     

Histamine-evoked amylase secretion is associated with small changes in calcium mobilization in isolated guinea-pig pancreas.

An investigation was made of the effect of histamine on amylase secretion and calcium mobilization in isolated guinea-pig pancreatic segments. The effect of acetylcholine (ACh) was also examined for comparison. Histamine evoked a dose-dependent increase in amylase output from superfused segments. It had small transient effects on both 45Ca influx and efflux, and elevated cytosolic free Ca2+ concentration in acini. The equivalent dose of ACh evoked a greater amylase output and greater changes in Ca2+ mobilization. The results suggest that histamine may have a physiological role in exocrine secretion of the guinea-pig pancreas but is less potent than ACh.     

The relationship between free cytosolic calcium and amylase release in rat pancreatic acini

We have studied the effects of various pancreatic secretagogues on free cytosolic calcium ([Ca2+]i) and amylase release in dispersed rat pancreatic acini, to determine the role of [Ca2+]i in stimulated enzyme secretion from the exocrine pancreas. Dispersed rat pancreatic acini were loaded with the new Ca2+-sensitive fluorescent indicator, fura-2. Resting [Ca2+]i was 110 +/- 2 nM (a mean +/- S.E.). Carbachol, caerulein, bombesin, and neuromedin B and C each caused a rapid increase in [Ca2+]i; maximal increases of 100 to 400-500 nM were reached within 20s following the secretagogue addition, and this was followed by a return to a lower sustained level within 2 min. When enzyme secretion from the acini was monitored as a function of time using a perifusion system, secretagogue-induced amylase release took a biphasic pattern consisting of an initial burst phase for a several minutes and a second sustained phase during stimulation. Although sustained amylase secretion occurred at near resting [Ca2+]i, the peak [Ca2+]i correlated with the amount of stimulated amylase release as well as with the initial release, during submaximal and maximal stimulation by these agents. At supramaximal concentrations of carbachol and caerulein, amylase release, but not the increase in [Ca2+]i, was attenuated. On the other hand, in response to supramaximal concentrations of bombesin, and neuromedin B and C, both the amount of amylase released and the peak [Ca2+]i were similar to those obtained in response to maximal concentrations. From a standpoint of time course analysis of enzyme secretion, both the first burst phase and the second sustained phase were inhibited during stimulation by 10(-3) M carbachol, compared with 10(-5) M carbachol, while supramaximal stimulation by neuromedin C caused a pattern of amylase release similar to that produced by maximal stimulation. These data suggest that in pancreatic acinar cells an increase in [Ca2+]i plays an important role in stimulus-secretion coupling; however, other factors may be indispensable in regulating enzyme secretion. Furthermore, it is suggested that there is a difference in the intracellular messenger system between carbachol and caerulein, and neurotransmitters belonging to the bombesin family, especially during supramaximal stimulations.     

Effect of acetylcholine stimulation on calcium and sodium uptake by the isolated rat pancreas.

1. The role of Ca2+ and Na+ ions in 'stimulus-secretion' coupling in the isolated uncinate pancreas of 4-week-old rats has been examined using radioisotope tracer techniques. The amount of 45Ca2+ and 22Na+ (mumole/g wet wt.) taken up by unstimulated glands was compared to that of glands in which amylase release was stimulated by ACh(10(-5) M) at various incubation times. 2. The amount of 45Ca taken up by the glands within 60 min of incubation was not found to be increased by the presence of ACh(10(-5) M). In fact, during short incubation periods the 45Ca uptake was significantly less in the stimulated glands than the unstimulated glands. 3. Presence of ACh(10(-5) M) did cause a significant initial increase in 22Na uptake lasting up to 20 min from onset of stimulation. 4. These results indicate that the rise in intracellular Ca2+ concentration which is suggested by an increase in 45Ca efflux during the action of pancreatic secretagogues, is not a consequence of increased 45Ca uptake by the pancreas; but they indicate that an initial action of ACh could be to elicit an increase in Na+ influx and that Na+ ions are likely to be involved in the action of ACh on pancreatic acinar cells.     

Phosphatidylinositol turnover and calcium movement in the rat pancreas

Carbamylcholine, bombesin, pancreozymin, and pentagastrin elicited a similar increase in amylase secretion and phosphatidylinositol turnover in rat pancreatic fragments. The concentration of each secretagogue that provoked half-maximal stimulation of amylase secretion was three to six times lower than that which induced half-maximal stimulation of phosphatidylinositol turnover. The increased turnover of phosphatidylinositols due to carbamylcholine or pancreozymin, but not the secretory response, persisted in a calcium-free medium or in 90% heavy water. The replacement of the media Na+ with Li+ increased an atropine-resistant turnover of phosphatidylinositols, but did not stimulate secretion. The ionophore A-23187 (in a medium containing 2.5 mM Ca2+) and 10 mM NaF induced a high secretory response, but exerted no effect on phosphatidylinositol turnover. K+ at a 70 mM concentration provoked a phosphatidylinositol effect and hypersecretion. Secretin, vasoactive intestinal peptide, dibutyryl cAMP, dibutyryl cGMP, 8-bromo cGMP, and N2-monobutyryl cGMP stimulated amylase secretion without an increased turnover of phosphatidylinositols. It is concluded that, in the rat pancreas, the increased turnover of phosphatidylinositols was directly associated with secretagogues inducing calcium movements.     

Pancreatic enzyme secretion in the conscious rat

1. A method for collecting duodenal juice and gastric content separately, in conscious rats, is described. Metal cannulas were implanted into the stomach fundus. For the main experiment a double lumen tube was inserted through the cannula via the pylorus into the duodenum. 2. The following secretion patterns were observed: a) In the resting state there was a constant flow rate of duodenal volume, bicarbonate, trypsin and amylase. b) Cholinergic stimuli were capable of increasing enzyme secretion as much as fourfold for a period of 30 to 40 min when administered as a single subcutaneous injection. This effect was annulled by atropine. c) Secretin and cholecystokinin-pancreozymin given together in a single injection s.c. or i.v., elicited a similarly strong response. d) Identical ranges of the secretion maxima were found with a tendency to decrease after the first hour, when the hormones were infused either s.c. or i.v. e) Doses from 0.5 to 25 U/100 g b.w. /hr showed identical responses. Doses below 0.2 U/100 g/hr were without effect. 3. Narcosis (pentobarbital) inhibited markedly the resting and stimulated enzyme secretion. 4. The method is suitable for examination of physiological and pharmacological effects on resting and stimulated enzyme secretion of the rat pancreas.     

The distribution and exchange of brain calcium in vivo in the rat

Summary Calcium exhibits a heterogenous distribution among the various brain areas studied. The pituitary gland contains 25.97 meq./kg of wet tissue, followed by the pons containing 9.91 meq. and the hypothalamus 5.35 meq. The calcium content in the other areas is as follows: 4.23 meq. in medulla, 4.0 meq. in cerebral white matter, 3.60 meq. in cerebellum and about 3.0 meq./kg of wet tissue in the cerebral cortex. No difference in calcium concentration was found between the right and left corresponding brain areas.The distribution of brain calcium between the extracellular and cellular compartments was derived, and indicates that the concentration of cellular calcium is several-fold that in the extracellular space.Three hours after intraperitoneal injection of ⁴⁷Ca Cl₂, the maximum uptake of ⁴⁷Ca was reached in all tissues except the pituitary gland where maximum uptake occurred 1 h after injection. The radioactive calcium in the brain was resolved into two components: A fast component having a short half time ranging between 0.5 and 4.25 h and a relatively slow component having a half time of 4.5–18 h.In the pituitary gland, pons, hypothalamus and medulla the calcium exchanges partially with serum calcium whereas, in cerebellum, cerebral cortex and white matter of cerebrum, tissue calcium is completely exchangeable.     

Food, duodenal extracts, and enzyme secretion by the pancreas

Previous studies have reported that injection of duodenal extracts from rats fed different meals into the celiac artery of recipient rats elicited the secretion of related pancreatic enzymes. We have been unable to reproduce the enzyme-specific increases in the average output of particular enzymes that were observed but did find changes similar in direction, although not magnitude, to those reported previously. The outputs of amylase and trypsinogen were compared by plotting individual data points and performing a regression analysis on them. The injection of duodenal extracts from lactalbumin hydrolysate-fed rats led to trypsinogen secretion being favored over that of amylase and vice versa for extracts from rats fed a glucose meal. In addition, it was found that cholecystokinin-pancreozymin produced a dramatic nonparallel transport of these two enzymes with amylase secretion being augmented to a greater degree than trypsinogen secretion. The relation between their outputs was curvilinear, i.e., the amylase dominance of secretion became more pronounced as overall enzyme output (not dose of hormone) increased. Thus, this nonparallel secretion does not seem to be the results of a discontinuous switch in the character of enzyme secretion produced by the hormone but a graded effect reflecting the magnitude of the response.     

Inhibitory influence of amino acids on secretagogues induced exocrine secretion in isolated perfused rat pancreas

Influence of amino acids upon pancreatic exocrine secretion has been investigated in the isolated perfused pancreas of rats. Arg produced significant and dose-related inhibition of pancreatic juice flow, protein output and amylase output evoked by CCK-PZ (1.25 pM). The secretory response evoked by CCK-PZ was inhibited by other amino acids (Ala, Asp, Asn, Gly, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Val, in each 20 mM). A similar inhibitory pattern was observed using 10 mixed amino acids of 2 mM each (Pro, Phe, Thr, Met, Lys, Asp, Leu, Trp, Val, Gly). Gly at a concentration of 20 mM produced significant inhibition of exocrine secretion evoked by ACh (50 nM) or GRP (36 pM). The inhibitory response induced by amino acids could not be repeated by using exogenous insulin (1 microM) and glucagon (280 nM). The inhibitory response was also not changed by increased extracellular Ca (5 or 10 mM). However, Gly (20 mM) produced inhibition of exocrine secretion evoked by Ca reintroduction into a pancreas which was pretreated with A 23187. It was suggested that the inhibitory effects of some amino acids on exocrine secretion are mainly caused by suppression of Ca influx in a stimulus-secretion coupling process.