Changes In Amylase Secretion Research Articles

The role of calcium-binding protein S100g (CalbindinD-9K) and annexin A10 in acute pancreatitis

BackgroundWe reported that the pancreas of the interferon-regulatory factor (IRF) 2 knock-out (KO) mouse represents an early phase of acute pancreatitis, including defective regulatory exocytosis, intracellular activation of trypsin, and disturbance of autophagy. The significantly upregulated and downregulated genes in the IRF2 KO pancreas have been reported. The catalogue of gene transcripts included two types of calcium-binding proteins (S100 calcium binding protein G [S100g] and Annexin A10 [Anxa10]), which were highly upregulated in the IRF2 KO pancreas. As the intracellular calcium signal plays a pivotal role in regulatory exocytosis and its disturbance is related to pancreatitis, we then evaluated the role of S100g and Anxa10 in acute pancreatitis. MethodWe induced cerulein-pancreatitis in wild-type mice and examined the changes in the expression of these genes by qPCR and immunohistochemistry. We constructed S100g-overexpressing or Anxa10-overexpressing AR42J cells (AR42J-S100g, AR42J-Anxa10). We examined the changes in amylase secretion, intracellular calcium ([Ca2+]i), and cell viability in these cells, when incubated with cholecystokinin (CCK). ResultsThe expression of S100g and Anxa10 was increased in cerulean-induced pancreatitis. The acini were patchily stained for S100g and the cytosol of acini was evenly but weakly stained for Anxa10. Stimulation with 100pM CCK-8, decreased amylase secretion and inhibited the [Ca2+]i increase in AR42J-S100g cells. These effects were weak in AR42J-Anxa10 cells. Cell viability was not changed by incubation with cerulein. ConclusionIn cerulean pancreatitis, the expression of S100g and Anxa10 was induced in the acini. S100g may work as a Ca2+ buffer in acute pancreatitis.

Early to late sparing of radiation damage to the parotid gland by adrenergic and muscarinic receptor agonists.

Damage to salivary glands after radiotherapeutic treatment of head and neck tumours can severely impair the quality of life of the patients. In the current study we have investigated the early-to-late pathogenesis of the parotid gland after radiation. Also the ability to ameliorate the damage using pretreatment with adrenergic or muscarinic receptor agonists is studied. Rats were locally irradiated with or without i.p. pretreatment with phenylephrine (α-adrenoceptor agonist, 5 mg kg−1), isoproterenol (β-adrenoceptor agonist, 5 mg kg−1), pilocarpine (4 mg kg−1), methacholine (3.75 mg kg−1) (muscarinic receptor agonists) or methacholine plus phenylephrine. Parotid salivary flow rate, amylase secretion, the number of cells and gland histology were monitored sequentially up to 240 days postirradiation. The effects were described in 4 distinct phases. The first phase (0–10 days) was characterised by a rapid decline in flow rate without changes in amylase secretion or acinar cell number. The second phase (10–60 days) consists of a decrease in amylase secretion and is paralleled by acinar cell loss. Flow rate, amylase secretion and acinar cell numbers do not change in the third phase (60–120 days). The fourth phase (120–240 days) is determined by a further deterioration of gland function but an increase in acinar cell number, albeit with poor tissue morphology. All drug pretreatments used could reduce radiation effects in phase I and II. The protective effects were lost during phase IV, with the exception of methacholine plus phenylephrine pretreatment. The latter combination of drugs ameliorated radiation-damage throughout the entire follow-up time. The data show that combined pre-irradiation stimulation of muscarinic acetylcholine receptors with methacholine plus α-adrenoceptors with phenylephrine can reduce both early and late damage, possibly involving the PLC/PIP2 second messenger pathways. This opens perspectives for the development of clinical applicable methods for long-term sparing of parotid glands subjected to radiotherapy of head and neck cancer patients. © 2001 Cancer Research Campaignhttp://www.bjcancer.com

Early to late sparing of radiation damage to the parotid gland by adrenergic and muscarinic receptor agonists

Damage to salivary glands after radiotherapeutic treatment of head and neck tumours can severely impair the quality of life of the patients. In the current study we have investigated the early-to-late pathogenesis of the parotid gland after radiation. Also the ability to ameliorate the damage using pretreatment with adrenergic or muscarinic receptor agonists is studied. Rats were locally irradiated with or without i.p. pretreatment with phenylephrine (α-adrenoceptor agonist, 5 mg kg–1), isoproterenol (β-adrenoceptor agonist, 5 mg kg–1), pilocarpine (4 mg kg–1), methacholine (3.75 mg kg–1) (muscarinic receptor agonists) or methacholine plus phenylephrine. Parotid salivary flow rate, amylase secretion, the number of cells and gland histology were monitored sequentially up to 240 days postirradiation. The effects were described in 4 distinct phases. The first phase (0–10 days) was characterised by a rapid decline in flow rate without changes in amylase secretion or acinar cell number. The second phase (10–60 days) consists of a decrease in amylase secretion and is paralleled by acinar cell loss. Flow rate, amylase secretion and acinar cell numbers do not change in the third phase (60–120 days). The fourth phase (120–240 days) is determined by a further deterioration of gland function but an increase in acinar cell number, albeit with poor tissue morphology. All drug pretreatments used could reduce radiation effects in phase I and II. The protective effects were lost during phase IV, with the exception of methacholine plus phenylephrine pretreatment. The latter combination of drugs ameliorated radiation-damage throughout the entire follow-up time. The data show that combined pre-irradiation stimulation of muscarinic acetylcholine receptors with methacholine plus α-adrenoceptors with phenylephrine can reduce both early and late damage, possibly involving the PLC/PIP2 second messenger pathways. This opens perspectives for the development of clinical applicable methods for long-term sparing of parotid glands subjected to radiotherapy of head and neck cancer patients. © 2001 Cancer Research Campaign http://www.bjcancer.com

Cyclic AMP potentiates substance P-induced amylase secretion by augmenting the effect of calcium in the rat parotid acinar cells

Potentiation of amylase secretion by the combination of isoproterenol and substance P was examined in perfused rat parotid acinar cells. Combined additions of substance P and isoproterenol evoked biphasic changes in amylase secretion, an initial large peak and the following sustained plateau: the magnitudes of the both responses were higher than the sum of the responses induced by each agonist alone. Isoproterenol also increased the maximum response and the apparent affinity (EC 50) for substance P to evoke the initial peak response; the EC 50 values were about 20 and 0.8 nM, respectively, in the absence and the presence of isoproterenol. On the other hand, 1 nM substance P was sufficient for evoking the maximum potentiation of the sustained plateau response. Substance P did not change the EC 50 for isoproterenol. The effect of isoproterenol was mimicked with dibutyryl cyclic AMP and agonists that increase parotid cyclic AMP. Omission of Ca 2+ or addition of 5 mM nickel chloride almost completely abolished the potentiation of the sustained plateau, but little decreased that of the initial peak. Depletion of Ca 2+ in InsP 3-sensitive intracellular stores with thapsigargin, on the other hand, decreased the initial peak response, but not the sustained plateau, to substance P. The potentiation was also observed between isoproterenol and Ca 2+ ionophores. Switching to the solutions containing higher concentrations of Ca 2+ during the continuous stimulation with isoproterenol or IBMX evoked a large, but transient, response of amylase secretion. Time course of changes in amylase secretion induced by isoproterenol and substance P in combination was very similar to that of substance P, but not of isoproterenol. Isoproterenol did not enhance the effect of substance P on [Ca 2+] i. These results show that the potentiation is mainly, if not totally, caused by cyclic AMP-induced enhancement of the potency and the efficacy in the pathway regulated by Ca 2+.

Exocrine Effects of the CCK Antagonist L-364,718 in Canine Pancreatic Autografts

This study evaluated the effect of the cholecystokinin antagonist L364,718 on exocrine secretion in canine pancreatic autografts with pancreaticocystostomies. Urinary (autograft) amylase (U/min) and bicarbonate (mmole/min) secretion, over a 6 hr interval, were determined in the basal state (Group A), after a bolus injection of 20 nmoles/kg of L-364,718 (Group B), during a continuous cholecystokinin octapeptide (OP-CCK) infusion at 125 ng/kg/hr either alone (Group C), with a bolus injection of 20 nmoles/kg (Group D), or 30 nmoles/kg (Group E), of L-364,718 1 hr before initiating OP-CCK, or 20 nmoles/kg of L-364,718 1 hr after initiating OP-CCK (Group F). L-364,718 had no effect on basal or OP-CCK-stimulated secretion of bicarbonate. Basal amylase secretion was decreased 1 hr after L-364,718 and remained significantly lower than controls throughout the study interval. When compared to Group C (280.3 ± 48.6), OP-CCK-stimulated amylase secretion was significantly lower for the first hour after L-364,718 in both Group D (157 ± 46.7) and Group E (31.9 ± 11.6). In Group E, 2, 3, and 4 hr post-L-364,718 amylase releases were 60.2 ± 19.7, 77.7 ± 25.1, and 87.2 ± 28.3 compared to 335.5 ± 85.9, 291.0 ± 21.8, and 289.9 ± 45.7 in Group C indicating a sustained significant inhibition of stimulated autograft amylase secretion with the higher L-364,718 dosage. In Group F, no significant change in amylase secretion was demonstrated, indicating that L-364,718 must be administered prior to CCK stimulation to be effective. These studies demonstrate that L-364,718 has a dose dependent, inhibitory effect on basal, and OP-CCK-stimulated amylase secretion in a denervated autograft model. The therapeutic potential of L-364,718 and other CCK receptor antagonists in pancreatic transplantation warrants further study.

Influence of thyroid status on Ca 2+ mobilization and amylase secretion in rat pancreatic acini

Thyroid hormones influence Ca 2+ homeostasis in both skeletal and cardiac muscle. Since secretory cells, like muscle cells, store and use Ca 2+ in stimulus-response coupling, we have studied the effects of thyroid status on Ca 2+ mobilization and secretion in a model secretory tissue, the pancreatic acinar cell. Hyperthyroidism was induced in rats by daily, subcutaneous injections of triiodothyronine for 8 days and hypothyroidism by adding 6- n-propyl-2-thiouracil to the drinking water for 14 days. Pancreatic acini were prepared by collagenase digestion of pancreatic tissue from hyper- and hypo-thyroid animals and from euthyroid controls. Ca 2+-mobilization was assessed using Quin-2 fluorescence and secretion by assaying amylase release. The data indicate that the amount of Ca 2+ mobilized by the muscarinic agonist carbachol or by cholecystokinin octapeptide increases with increasing thyroid hormone concentrations. Only in hypothyroidism was this change in Ca 2+ homeostasis reflected by a parallel change in amylase secretion. This implies the existence of some compensatory mechanism which stabilizes secretory rate in the face of stimulus-evoked increases in intracellular Ca 2+ concentration.

Irreversible inhibition by acetaldehyde of cholecystokinin-induced amylase secretion from isolated rat pancreatic acini

Acetaldehyde inhibited both amylase secretion induced by maximal concentrations (300 pM) of cholecystokinin octapeptide and the binding of radioiodinated cholecystokinin to receptors on isolated rat pancreatic acini. This inhibition was concentration dependent (10 mM to 1M for amylase secretion and 100 mM to 1M for binding). However, a correlation between the two inhibitory effects could not be obtained. Furthermore, the inhibitory effects were not reversible. Acetaldehyde did not alter the basal amylase secretion between 6 and 45 mM concentrations. However, 60, 100 and 300 mM acetaldehyde significantly decreased basal amylase secretion; no significant change in amylase secretion was observed at 600 mM and 1M. Higher concentrations of acetaldehyde produced a 2- to 10-fold increase in basal amylase secretion. 51Cr release from prelabeled acini revealed no significant cell membrane damage between 10 and 600 mM acetaldehyde. These data suggest that acetaldehyde inhibition of cholecystokinin-induced amylase secretion is intracellularly mediated.

Beta-Adrenoceptor alterations coupled with secretory response in rat parotid tissue.

Simultaneous studies on the secretory response of amylase and the neurotransmitter receptors of rat parotid gland, after brief treatment with agonists, showed selective alteration in beta-adrenoceptors with specific change in amylase secretion, suggesting a regulatory role of the receptors in the secretory response. The beta-adrenergic agonist (+/-)-isoprenaline (IPR) stimulated amylase secretion from rat parotid tissues much more than did the same concentration of an alpha-adrenergic or cholinergic agonist. The stimulatory effects of IPR were studied by pre-treating rat parotid tissues with IPR for 10 min and then incubating the tissue in fresh medium for 10 min. Pre-treatment with 10 microM-IPR for 10 min resulted in increased amylase secretion during further incubation with IPR and also in a lower EC50 value of amylase secretion for IPR. This treatment also resulted in selective changes in the number and affinity of beta-adrenoceptors, assessed by measuring binding of [3H]dihydroalprenolol (DHA): the maximal binding sites increased from 286/357 f-mole to mg protein and the IC50 value (the concentration for 50% inhibition of specific [3H]DHA binding) of beta-agonists, not antagonists, decreased significantly. An increase in the period of pre-treatment with IPR to 30 min resulted in a decrease in the maximal binding sites of beta-adrenoceptors and a decrease in amylase secretion during further incubation with IPR. Experiments with other agonists showed that supersensitivity of the secretory response was induced specifically by beta-agonists. Binding studies with [3H]WB-4101 and [3H]quinuclidinyl benzilate showed that alpha-adrenoceptors and muscarinic ACh receptors in rat parotid did not change under the conditions tested. The alteration in beta-adrenoceptors was parallel with a change in amylase secretion after IPR pre-treatment, but not with a change in cyclic AMP content.

Action of cholera toxin on dispersed acini from rat pancreas: Post-receptor modulation involving cyclic AMP and calcium

In dispersed acini from rat pancreas, cholera toxin caused a significant increase in cellular cyclic AMP but little or no change in amylase secretion. The presence of a secretagogue that causes mobilization of cellular calcium (e.g., cholecystokinin, carbamylcholine, bombesin or ionophore A23187) caused a substantial increase in the effect of cholera toxin on enzyme secretion. Cholera toxin did not alter calcium transport or the changes in calcium transport caused by other secretagogues, and secretagogues that mobilize cellular calcium did not alter cellular cyclic AMP or the increase in cyclic AMP caused by cholera toxin. These results indicate that in dispersed acini from rat pancreas there is post-receptor modulation of the action of cholera toxin by secretagogues that mobilize cellular calcium and that this modulation is a major determinant of the effect of the toxin on enzyme secretion.

Effects of neonatal sympathetic denervation on amylase secretion in the adult rat parotid gland: Difference in β1- and β2-adrenoceptor response

Changes in amylase secretion and cyclic AMP accumulation in response to various secretagogues were studied in parotid glands of adult rats subjected to neonatal sympathetic denervation by unilateral excision of the superior cervical ganglion. Denervation decreased the gland content of amylase and both basal and the stimulated levels of cyclic AMP were elevated. The secretory cells of neonatally devervated glands exhibited enhanced maximal enzyme discharge in response to β-adrenoceptor agonists. However, the selective β 1-agonist, prenalterol, was not effective in this respect whereas an enhanced maximal secretory response to the β 2-selective agonist, terbutaline, was particularly prominent. DBcAMP was also more efficient in inducing amylase release from the denervated gland. The results of the present study demonstrate that the usual dominance of the β 1-adrenoceptor subtype in eliciting amylase 3elease is lost, implying that the differentiation of the β-adrenoceptor into its subtypes is altered by neonatal sympathetic denervation.

Secretory capacity of isolated pancreatic acinar cells.

Summary: In order to characterize the secretory capacity of isolated acinar cells, the effects of various secretagogues on amylase release were studied in guinea pigs. The effect of carbachol on amylase secretion reached a maximal level after a 30-min incubation and was dose dependent. At 10 −4 M, carbachol induced a 33% increase in amylase secretion over control values (P < 0.001); at 10−6 M, there was an 8% increase over control (P < 0.05); and, at 10 −8 M, there was no change in amylase secretion. Muscarinic blockade with equimolar concentrations of atropine completely blocked the expected carbachol-induced increase in amylase secretion after both 30− and 60-min incubations. The response to secretin was neither significantly different from that to carbachol nor dose dependent over the concentration range tested. After a 30-min incubation, secretin induced significant increases in amylase secretion of 23%, 19%, and 23% at concentrations of 10 −5 M, 10−7 M, and 10−9 M, respectively. As with carbachol, the response to secretin was blocked by atropine. Secretion of amylase in response to cholecystokinin-pancreozymin octapeptide (CCK-PZ) was maximal after a 30-min incubation and was dose dependent. CCK-PZ, at concentrations of 10−7 M and 10−9 M, induced significant increases in amylase secretion of 18% (P < 0.001) and 17% (P < 0.001), respectively. At 1011 M, CCK-PZ did not evoke a significant increase in amylase secretion. The CCK-PZ-induced increase in amylase secretion was significantly less than that observed with carbachol (P < 0.05), was not significantly different from that observed with secretin, and was not potentiated by the simultaneous exposure of the acinar cells to carbachol.