Interrelation of secretory and trophic responses in the exocrine pancreas

This paper was addressed to know whether early events in mitogenesis (activation of the Na+/H+, activation of ornithine decarboxylase and formation of cyclic AMP) are involved in pancreatic cell proliferation and mediate secretory process. The AR4-2J cell line was used. Analogues of amiloride inhibited cell proliferation but had no effect on amylase release. Activation of ornithine decarboxylase was triggered via a CCK B receptor type not involved in pancreatic secretion. Inhibition of cyclic AMP was not involved in inhibition of cell proliferation caused by somatostatin. Specific effectors might be related either to the secretory or to the trophic pathway. Another possibility is that multiple receptor sub-classes are linked to specific pathways.     

Studies on intracellular transport of secretory proteins in the rat exocrine pancreas

Summary The possible role of microtubules and microfilaments in the secretory process of the rat exocrine pancreas was analysed in vitro using isolated pancreatic lobules. Colchicine and vinblastine as microtubule inhibitors, hexylene glycol as a microtubule stabilizer, and cytochalasin B as a disruptive agent for microfilaments were used in increasing concentrations to test their effects on protein synthesis, intracellular transport, zymogen discharge, and cellular respiration.Colchicine only at 10^–2 M concentrations inhibits protein synthesis, while vinblastine inhibits at 10^–6 and 10^–5 M by 20% and at 10^–4 M by 55%. A similar inhibition is observed with 1.5% concentrations of hexylene glycol while cytochalasine B at 1,5 and 10 g/ml is without effect on protein synthesis. Colchicine and vinblastine have their major effects on intracellular transport both in secretion studies and cell fractionation experiments. Colchicine in concentrations between 10^–3 to 10^–5 M inhibits discharge of newly synthesized proteins by 50%, while vinblastine shows a dose-response relationship of 40% inhibition at 10^–6 M to 90% at 10^–4 M. Discharge of amylase is uniformly reduced by 30% by both colchicine and vinblastine in the whole dose range. The pronounced effect of colchicine and vinblastine is evident in cell fractionation studies: both drugs inhibit the disappearance of protein radioactivity from microsomes and its appearance in zymogen granules; similarly the peak radioactivity in smooth microsomes (Golgi) appears delayed. No differential effect on the secretory process was observed with 1.5% concentrations of hexylene glycol or cytochalasin B at 1.5 and 10 g/ml concentrations. A fines tructural analysis of microtubules and microfilaments in the exocrine pancreatic cell reveals their distribution in all parts of the cytoplasm and in relation to all cell organelles. Both systems (microtubules, microfilaments) seem to be connected, at least in certain areas of the cytoplasm and at the plasma membrane.The reduction of transport efficiency by microtubule inhibitors results in a deposition of secretory material in the cisternal space of the rough endoplasmatic reticulum, which leads to the formation of paracrystals. Colchicine at 10^–3 M concentrations leads to an enlargement of condensing vacuoles in the Golgi complex.A short communication on the same subject was presented at a Symposion on Stimulus-Secretion-Coupling in the Gastro-intestinal Tract, Titisee (May 27–29, 1975).Supported by Deutsche Forschungsgemeinschaft (Ke 113/8).     

Ultrastructural responses of rat exocrine pancreas to cholecystokinin octapeptide and secretin

The study examines the ultrastructural changes in the rat pancreas stimulated in vivo to secrete zymogen and fluid by the hormones cholecystokinin and secretin, administered either separately or in combination. The octapeptide of cholecystokinin (CCK-OP) (2.5 X 10(-7) g/kg) 5 min after injection produced discharge of electron-dense zymogen into the acinar lumen and intercellular canaliculi (ICC), leaving misshapen, collapsed zymogen granule profiles around the lumen. Five minutes after secretin (7.5 clinical units/kg), acinar cells were distended, rough endoplasmic reticulum was dilated, acinar lumina and ICC were expanded and filled by electron-lucent and flocculent contents, and there were "halo" zymogen granules and pale "vacuoles." Electron-lucent zones surrounding acinar and duct cell microvilli indicated transcellular fluid secretion. When secretin was administered with CCK-OP, the picture was a composite between zymogen and fluid secretory patterns. Zymogen granules took up fluid producing a halo appearance, pale vacuoles formed in acinar cells, and acinar lumina and discharging zymogen granules were of intermediate electron density. The results demonstrated that, although fluid is secreted by duct cells in response to secretin, a major site of secretin-stimulated fluid secretion is acinar cells. Fluid is transported across both cell types by transcellular routes, and the acinar cell fluid secretion is integrated with zymogen discharge. CCK-OP produces partial discharge of undiluted zymogen by exocytosis.     

Mechanism of action of magnesium on acetylcholine-evoked secretory responses in isolated rat pancreas.

This study investigates the effects of magnesium (Mg2+) on acetylcholine (ACh)-evoked secretory responses and calcium (Ca2+) mobilization in the isolated rat pancreas. ACh induced marked dose-dependent increases in total protein output and amylase release from superfused pancreatic segments in zero, normal (1 x 1 mM) and elevated (10 mM) extracellular Mg2+. Elevated Mg2+ attenuated the ACh-evoked secretory responses compared to zero and normal Mg2+. In the absence of extracellular Ca2+, but presence of 1 mM-EGTA (ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid), ACh elicited a small transient release of protein from pancreatic segments compared to a larger and more sustained secretion in the absence of both Ca2+ and Mg2+. Incubation of pancreatic segments with 45Ca2+ resulted in time-dependent uptake with maximum influx of 45Ca2+ occurring after 20 min of incubation period. ACh stimulated markedly the 45Ca2+ uptake compared to control tissues. In elevated extracellular Mg2+ the ACh-induced 45Ca2+ influx was significantly (P less than 0.001) reduced compared to zero and normal Mg2+. ACh also evoked dose-dependent increases in cytosolic free Ca2+ concentrations ([Ca2+]i) in pancreatic acinar cells loaded with the fluorescent dye Fura-2 AM. In elevated Mg2+ the ACh-induced cytosolic [Ca2+]i was significantly (P less than 0.001) reduced compared to zero and normal Mg2+. These results indicate that Mg2+ can influence ACh-evoked secretory responses possibly by controlling both Ca2+ influx and release in pancreatic acinar cells.     

Dose effects of cholecystokinin and acetylcholine on phosphorus compounds and secretory responses in isolated perfused pancreas of rat

Phosphorus nuclear magnetic resonance (31P NMR) spectroscopy was used to study energy supply for protein secretion in the isolated perfused rat pancreas. Stimulation with cholecystokinin (CCK-8) increased fluid secretion and protein output. With 10pM of CCK-8, the tissue contents of ATP, inorganic phosphate (Pi), and creatine phosphate (PCr) remained unchanged. With 100pM of CCK-8, which induced the maximum response in fluid secretion and protein output, Pi increased slightly, ATP and PCr remained almost unchanged. A high dose of CCK-8 (1 nM) suppressed the fluid and protein secretory rates, decreased ATP, and increased Pi significantly, but PCr showed a tendency of increase. Significant changes in ATP and Pi occurred on withdrawal of CCK-8 (1 nM), suggesting activation of ATP hydrolysis for recovery from secretory suppression. During stimulation with 0.1, 0.3, and 3.0 microM of acetylcholine, the similar dose-dependent response was observed in the secretion and the phosphorus metabolism. The present study demonstrated that cytosolic energy metabolism for secretory responses in the pancreas is low and the Lohmann reaction showed less contribution than in the salivary gland. The findings suggest that the energy supply for protein secretion may be cytosolic diffusion of ATP and that the Lohmann reaction system may contribute to synthesis and storing of secretory protein at resting state.     

Ultrastructure of the secretory compartment of the Rana catesbeiana exocrine pancreas

In the Rana catesbeiana exocrine pancreas 2 types of serozimogenic cells could be observed. They were called light and dark cells. The light cells were observed in a greater number and presented the GER cisterns predominantly located in the basal region of the cell and the vesicular forms of the GER mainly in the supranuclear region. The Golgi complex was well developed and the secretory granules were grouped in the cell apex. The mitochondrion were distributed by the cytoplasm and the nucleous appeared in the basal region of the cell and presented a pale and vesiculous profile that made the nucleolus evident. The dark cells were observed in smaller quantity and in this cell occurred a predominance of GER vesicles. The cytoplasmic matrix presented an electron-dense appearance and a large quantity of free ribosomes. The Golgi complex is poorly developed, the secretory granules and the mitochondria are scarce. We could observe besides the light and dark cells, the centroacinar and the intermediate cells. The centroacinar cells lay in the luminal border of the acinar cells. They presented an electron-lucent cytoplasmic matrix where could be noted a few GER profiles and the secretory granules were absent. The intermediate cells showed, intermingled within the same cell, the zimogenic and the endocrine-like alfa and beta Langerhans granules.     

Effect of age and long-term diet on exocrine pancreas of the rat.

Effects of age and long-term nutritional treatment on pancreas composition and digestive function were determined in rats fed a cereal-type chow after weaning (experiment 1) or diets with 30% casein and 34% butterfat, 54% starch, or 54% sucrose after 9 mo (experiment 2). The rats were adapted for 1-2 wk to a 15% whole-egg protein diet before killing. In experiment 1, pancreas size, nucleic acid, and digestive enzyme content increased significantly with age up to 3 mo. Values for pancreatic weight and DNA were significantly greater in 28-mo-old rats than in 12-mo-old rats. Pancreatic digestive enzyme content was 65-100% lower in rats with and without gross pathologic lesions. In experiment 2, mortality was higher and pathologic changes were more pronounced by 24 mo in rats fed the butterfat or sucrose diet. Usually, pancreatic enzymes were not reduced as much as in experiment 1, although chymotrypsinogen and trypsinogen concentrations were significantly and negatively correlated with the degree of pathologic change. Apparent digestibility of dietary nitrogen and food energy content was not reduced in rats with reduced enzyme reserves. The rate of incorporation in vitro of label into pancreatic protein and RNA did not differ significantly among aged and control rats.     

Effect of short-chain fatty acids on the secretory response of the ovine exocrine pancreas

The secretory response of the exocrine pancreas to short-chain fatty acids has been studied in anesthetized sheep and in isolated lobules. Butyrate, propionate, and acetate stimulated pancreatic juice flow and protein and amylase output in the anesthetized sheep. The secretory response to butyrate was significantly greater than that of propionate or acetate. Rapid intravenous injection of butyrate (625 mumol/kg) caused a 13-fold rise in the juice flow, 26-fold in protein output, and 37-fold in amylase output above the basal levels within 5 min and declined to basal levels over a period of 30 min. Responses to butyrate (625 mumol/kg) were comparable with those obtained with 2 U/kg pancreozymin (Boots). Detectable responses were obtained with 15 mu/kg butyrate, 125 mumol/kg propionate, and 312.5 mumol/kg acetate. The secretory response to butyrate (625 mumol/kg) was not affected by pretreatment with atropine and hexamethonium. In the isolated lobule preparation, amylase release increased in response to butyrate in a concentration-dependent manner, reaching a maximal level at 1 mM and declining at 100 mM. It is concluded that short-chain fatty acids act directly on pancreatic acinar cells to stimulate secretion. The physiological implications of these findings are considered.     

Development of secretory mechanisms in rat pancreas.

Mechanisms and development of secretory function were studied in rat pancreas in vitro. Amylase release from term fetal pancreas was refractory to stimulation by carbamylcholine chloride (carbachol) and cholecystokinin-octapeptide (CCK-OP), but was significantly augmented by calcium ionophore (A23187), DBcAMP, 8-Br-cGMP, and theophylline. The latter agent when combined with either cyclic nucleotide analogue further increased secretory responses. At 1 day and 8 days postnatally, responsiveness to carbachol and CCK-OP had been acquired because amylase secretion stimulated by these agents was brisk and at a level comparable to that found in mature tissue. Increasing extracellular calcium concentrations from 1.23 to 5.28 mM had no effect on basal amylase release in either the fetal or 8-day pancreas. No changes in intracellular cAMP concentrations were found at any age under experimental conditions used. Similarily, in fetal tissue, no changes in cGMP concentrations were found in response to carbachol or A23187. However, at 8 days of age, both agents produced two- to four-fold increases in tissue cGMP levels at 1, 2, and 5 min of incubation. These studies confirm that responsiveness to carbachol and CCK-OP is a maturational process in the pancreas that lags behind the development of intracellular processes involved in stimulus-secretion coupling.     

Effect of leukocytic endogenous mediators on endocrine pancreas secretory responses.

Crude mediators from stimulated rabbit peritoneal leukocytes (LEM) engender numerous physiologic alterations in rats, which are similar to those observed during infection. One hour after the intraperitoneal injection of crude LEM, plasma insulin and glucagon concentrations are elevated; at 2 h the hormonal alterations are manifested by a 30% increase in hepatic cyclic adenosine 3',5'-monophosphate (cAMP), glycogen depression, and uptake of 14C-labeled nonmetabolizable amino acid analogues (AA). Plasma hormone concentrations reach maximum levels by 5 h and decline by 24 h. The hepatic concentrations of AA parallel the insulin and glucagon responses and correlate with the inverse of insulin/glucagon molar ratio. In spite of mobilization of hepatic glycogen evident at 5 h, plasma glucose concentrations were transiently depressed. Plasma insulin, glucagon, and hepatic AA concentrations were dose dependent. Plasma insulin and glucagon responses to crude LEM may explain increases in hepatic cAMP, uptake of AA, and glycogenolysis as well as hypoglycemia. These data partially characterize the role of crude LEM, provide an explanation for the stimuli-inducing hyperglucagonemia and hyperinsulinemia during infection. They implicate the endocrine pancreas as a factor regulating the host's metabolic response to infection.     

Differentiation between the calcium-dependent effects of cholecystokinin-pancreaozymin and the bicarbonate-dependent effects of secretin in exocrine secretion of the rat pancreas.

1. Differentiation between the secretory effects of pure natural cholecystokinin-pancreozymin (CCK-PZ) and those of synthetic secretin was studied in the isolated pancreas of the rat perfused with a standard solution containing both Ca2+ and HCO2-, a Ca2+ deficient solution, or a HCO3-deficient solution. 2. Secretin induced a dose-dependent increase in the flow of pancreatic juice and a slight but definite increase in amylase output which was also dependent upon the dose of secretin. 3. The increase in the flow of pancreatic juice, induced by continuous stimulation with secretin (5 Ivy dog m-u./ml.), was completely abolished during perfusion with a CHO3- deficient solution, but was only slightly suppressed during perfusion with a Ca2+-deficient solution. 4. The increase in flow, induced by continuous stimulation with CCK-PZ (5 Ivy dog m-u./ml.), was partly affected by the deprivation of HCO3-, but was strongly inhibited by the deprivation of Ca2+. The CCK-PZ-induced amylase output was not affected by the deprivation of HCO3-, but was significantly inhibited by the deprivation of Ca2+. 5. The present results favour the view that CCK-PZ acts on the acinar cells to increase both amylase output and juice flow, whereas secretin acts on centro-acinar and terminal duct cells to increase mainly juice flow.     

Calcium and secretagogues-induced conductances in rat exocrine pancreas

The electrical properties of single acinar cells isolated from rat pancreas were studied with the whole-cell tight-seal recording method. Under resting conditions, the relative permeabilities of Cl and K wereP _cl/P _K3. At 1 M internal calcium, a Ca and voltage-dependent Cl conductance was activated. At 10 M internal calcium, the major conductance was selective for cations. It was not voltage-dependent. Acetylcholine and cholecystokinin induced an increase of internal Ca which in turn activated either only a Cl conductance or both Cl and cationic conductances. The secretagogue-induced conductance was increased to a variable extent by depolarisation. The absence of K channels activated by internal calcium indicates that, in pancreatic acinar cells, the mechanism of fluid secretion differs from that observed in other exocrine glands.     

The nonselective cation channel in the basolateral membrane of rat exocrine pancreas

Nonselective Ca²⁺-sensitive cation channels in the basolateral membrane of isolated cells of the rat exocrine pancreas were investigated with the patch clamp technique. With 1.3 mmol/l Ca²⁺ on the cytosolic side, the mean openstate probabilityP _o of one channel was about 0.5. In insideout oriented cell-excised membrane patches the substances diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 3,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) were applied to the cytosolic side. These compounds inhibited the nonselective cation channels by increasing the mean channel closed time (slow block). 100 mol/l of NPPB or DPC decreasedP _o from 0.5 (control conditions) to 0.2 and 0.04, respectively, whereas 100 mol/l of DCDPC blocked the channel completely. All effects were reversible. 1 mmol/l quinine also reducedP _o, but in contrast to the abov mentioned substances, it induced fast flickering. Ba²⁺ (70 mmol/l) and tetraethylammonium (TEA⁺; 20 mmol/l) had no effects. We investigated also the stilbene disulfonates 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS), 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) and 4,4-dinitro-2,2-stilbenedisulfonate (DNDS). 10 mol/l SITS applied to the cytosolic side increasedP _o from 0.5 to 0.7 and with 100 mol/l SITS the channels remained nearly permanently in its open state (P _o1). A similar activation of the channels was also observed with DIDS and DNDS. These effects were poorly reversible. The stilbene disulfonates acted by increasing the channel mean open time. When the channel was inactivated by decreasing bath Ca²⁺ concentration to 0.1 mol/l, addition of 100 mol/l of SITS had no effect. Similarly, reducing bath Ca²⁺ concentration from 1.3 mmol/l in presence of 100 mol/l SITS (channels are maximally activated) to 0.1 mol/l, inactivated the channels completely. These results demonstrate, that SITS can only activate the channels in the presence of Ca²⁺. SITS had no effects, when applied to the extracellular side in outside out patches. In summary, the substances DPC, NPPB and DCDPC inhibit nonselective cation channels, where DCDPC has the most potent and NPPB the smallest effect; whereas SITS, DIDS and DNDS activate the channel when applied from the cytosolic side in the presence of Ca²⁺ ions.     

Influence of pancreatic duct ligation on endocrine and exocrine rat pancreas

A parallel investigation into endocrine and exocrine pancreatic function, after duct-ligation in the rat, was performed to study the effect of reduced intestinal trypsin levels on insulin secretion and glucose tolerance. Animals with only a slight exocrine insufficiency displayed a normal insulin secretion and a normal glucose tolerance 4 weeks after operation. At 4-5 month s these animals showed a slight increase in glucose-induced insulin release when compared with control rats. However, animals operated on with a more complete ligation of the pancreatic ducts, who showed a marked exocrine insufficiency accompanied by decreased levels of intestinal trypsin, displayed a markedly increased insulin secretory response to intravenous glucose and an increased glucose tolerance. The results lend further support to our previous suggestion that, in the rat, the levels of intestinal trypsin may influence insulin secretory processes via complex feed-back mechanisms which may involve cholecystokinin and/or other intestinal hormones.     

Differences in stimulatory effects between rat pancreatic secretory trypsin inhibitor-61 and -56 on rat pancreas

Two types of pancreatic secretory trypsin inhibitors (PSTIs) were recently purified from rat pancreatic juice. One consisted of 61 (PSTI-61) and the other of 56 (PSTI-56) amino acid residues. PSTI-61 has been reported to elicit cholecystokinin (CCK) release when injected into the duodenum. Since no information has been available about the action of PSTI-56 on CCK release, the two PSTIs were compared for their stimulatory effect on CCK release and pancreatic exocrine secretions in conscious rats after intraduodenal administration. Rats were prepared with bile and pancreatic fistulae and with two duodenal cannulae. Pancreatic juice was excluded from the duodenum for 48 h prior to the experiment because rat PSTIs were trypsin sensitive. PSTI-61 significantly stimulated pancreatic secretions and increased plasma CCK concentrations from 3.6 to 6.5 pM, whereas PSTI-56 had no effect on either CCK release or pancreatic secretions. It is suggested that the action as a regulator for CCK release and pancreatic secretions is possessed only by PSTI-61, but not by PSTI-56.