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PubMed1987

Secretagogue-induced changes in intracellular pH and amylase release in mouse pancreatic acini.

The response of the intracellular pH (pHi) to stimulation of enzyme secretion in pancreatic acini was measured using the fluorescent dye 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Acini suspended in pH 7.40 buffer demonstrated cytoplasmic alkalinization of 0.17, 0.14, and 0.15 pH units 2 min after addition of the secretagogues carbachol (10(-5) M), caerulein (10(-10) M), and bromo-A23187 (10(-6) M). Corresponding net stimulated amylase secretion over 30 min was 9.2, 10.3, and 5.6% of total content, respectively. Pretreatment of acini with atropine blocked the pHi rise induced by carbachol; addition of atropine 2 min after the carbachol did not reverse the alkalinization. Acini suspended in Ca2+ free buffer containing 0.1 or 0.2 mM ethylene glycol tetraacetic acid showed 0.21 and 0.10 pH unit alkalinization in response to caerulein (10(-10) M) and carbachol (10(-5) M) but no change in pHi after addition of bromo-A23187. Amylase release in response to increasing concentrations of caerulein was maximal at 10(-10) M, with decreasing rates of amylase release at higher drug concentrations (10(-8), 10(-7) M). Alkalinization in response to stimulation of secretion was maximal at 10(-8) M caerulein (0.30 pH units at 2 min) but was of lesser magnitude at 10(-7) M. Pancreatic acini demonstrated autoregulation of pHi over a range of external pH from 7.4 to 7.1. Net amylase release over 30 min in response to 10(-5) M carbachol was sustained at normal levels in buffers of pH varying between 7.7 and 6.5. In contrast, cytoplasmic alkalinization in response to carbachol occurred only in buffers with pH values between 7.40 and 7.10. These results indicate that amylase release occurs over a wide range of pHi and is not invariably associated with secretagogue-induced alkalinization.

Amiloride↗
PubMed1988

Visualization of amylase secretion from individual pancreatic acini.

To assess the secretion of individual rat pancreatic acini, we developed a reverse hemolytic plaque assay that allows for a direct visualization of amylase release. This release was detected around secreting cells by the presence of hemolytic plaques that resulted from the complement-mediated lysis of red blood cells bearing amylase-antiamylase complexes bound to protein A. Controls showed that these plaques reflected specifically the active secretion of amylase. Quantitation of hemolytic plaques showed that after a 30-min incubation approximately 50% of the acini secreted under basal conditions. Stimulation of amylase release by increasing concentrations of carbamylcholine resulted in a dose-dependent recruitment of secreting acini as well as in a time-dependent enhancement in the response of individual acini. Under all conditions, the wide distribution of hemolytic plaque sizes indicated large differences in the secretory output of individual acini. Thus, using a new method to directly visualize and quantitate amylase secretion, we have provided evidence for a functional heterogeneity of pancreatic acini.

Amylases↗
PubMed1988

Role of terminal fucose residues in clearance of human glycosylated alpha-amylase.

Human glycosylated alpha-amylase contains a single biantennary N-linked oligosaccharide that terminates with the structure Fuc alpha 1,3(Gal beta 1,4)GlcNAc. To examine the role of terminal fucose in the clearance of alpha-amylase, we used lectin affinity chromatography to isolate an alpha-amylase fraction that contains two terminal fucoses (one in each branch of the oligosaccharide) and a fraction from which both terminal fucoses have been enzymatically removed. In the rat, the rate of clearance of the radioiodinated fraction with terminal fucoses is rapid (t1/2 = 12 min) and is slowed by mannosylated but not galactosylated bovine serum albumin. The rate of clearance of the radioiodinated alpha-amylase fraction with no terminal fucoses is also rapid (t1/2 = 9.5 min), but the clearance is now slowed by galactosylated bovine serum albumin. These findings indicate that the fucosylated and defucosylated alpha-amylase fractions are recognized by different carbohydrate-specific receptors. We conclude therefore that terminal fucose is the recognition marker that effects the physiological clearance of this glycoprotein.

Carbohydrate Conformation↗
PubMed1990

Amylase release from streptolysin O-permeabilized pancreatic acini.

Intracellular mediators of exocytosis were investigated using isolated mouse pancreatic acini permeabilized with the bacterial toxin streptolysin O (SLO). Permeabilization was demonstrated by fluorescent staining with ethidium bromide and fluorescein diacetate and release of cytoplasmic lactate dehydrogenase. When SLO-permeabilized acini were incubated at 37 degrees C in Ca2(+)-EGTA buffers containing MgATP, amylase secretion was Ca2+ dependent with an EC50 of 0.40 microM Ca2+ and a maximally effective Ca2+ concentration of 1 microM. Maximal amylase secretion was 330% of that in Ca2(+)-free buffer (basal). The nonhydrolyzable GTP analogue guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 30 microM) increased the maximal secretion to 451% of basal in the presence of 1 microM Ca2+ and decreased the EC50 to 0.14 microM Ca2+. Removal of ATP plus addition of antimycin A and 2-deoxy-D-glucose inhibited Ca2(+)-dependent, GTP gamma S-enhanced amylase secretion by 56%. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 1 microM) also enhanced maximal secretion to 450% of basal and decreased the EC50 to 0.18 microM Ca2+. Enhancement of amylase secretion by submaximal concentrations of GTP gamma S or TPA was inhibited by the protein kinase C inhibitor staurosporine. These results suggest that Ca2+ stimulation of amylase secretion is potentiated by activation of protein kinase C. However, the enhancement of secretion by GTP gamma S and TPA was additive at their maximally effective concentrations, suggesting that another G protein(s) maybe involved in the terminal steps of exocytosis.

Alkaloids↗
PubMed1990

Characterization of sustained [Ca2+]i increase in pancreatic acinar cells and its relation to amylase secretion.

The sustained increase in cytosolic free Ca2+ concentration ([Ca2+]i) during maximal stimulation of rat pancreatic acini with carbamylcholine (10(-5) M) was investigated in individual acinar cells by microspectrofluorometric analysis of fura-2. After the large initial [Ca2+]i increase from intracellular stores, [Ca2+]i remained significantly elevated as long as the stimulus was applied. The amplitude of this plateau was dependent on the median Ca2+ concentration ([Ca2+]o) being 45-50 nM above prestimulation in medium with 1 mM [Ca2+]o increasing to 90 nM at 10 mM [Ca2+]o. This Ca2+ plateau was completely blocked by 2.5 mM Ni2+ and 0.25 mM La3+ but was unaffected by elevated K+ or the Ca2+ channel blocker D 600. Mn2+ was able to enter the cytosol after the cell stimulation as indicated by intracellular quenching of fura-2, indicating that acinar cells possess a Mn2(+)-permeable Ca2+ channel. Elimination of [Ca2+]o or addition of Ni2+ and Mn2+ to the medium reduced the level of sustained amylase secretion in a reversible manner under superfusion conditions. Increasing [Ca2+]i above the normal level by increasing [Ca2+]o had no effect on amylase secretion. The process for sustained Ca2+ entry was pH sensitive; decreasing extracellular pH (pHo) to 6.5-6.8 during the cell stimulation resulted in a reduction of the sustained [Ca2+]i plateau level and a decrease in sustained amylase secretion. By contrast, increasing pHo to 8.0 enhanced the level of the sustained [Ca2+]i in a Ni2(+)-sensitive manner but did not increase amylase release. Changes in cytosolic pH had only minimal effects on the sustained [Ca2+]i plateau. The results demonstrate a receptor-mediated Ca2+ entry mechanism, which results in a small increase in [Ca2+]i important in the maintenance of sustained amylase release.

Ammonium Chloride↗
PubMed1992

Amylase release from streptolysin O permeabilized fetal pancreatic acini.

Developmental regulation of Ca(2+)-dependent protein discharge was investigated in fetal and neonatal rat pancreatic acini permeabilized with streptolysin O. When incubated at 37 degrees C in a Ca(2+)-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid/K glutamate buffer, permeabilized day 19 and 20 fetal acini demonstrated Ca(2+)-dependent release of amylase, whereas day 21 (term) fetal acini did not. Ca(2+)-dependent amylase release reappeared in day 1, 2, and 6 neonatal pancreases. ATP depletion completely inhibited Ca(2+)-stimulated amylase release from both day 20 fetal and adult acini. Ca(2+)-dependent amylase discharge from day 20 fetal acini was enhanced by the nonhydrolyzable GTP analogue, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), and by the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA). Ca(2+)-independent GTP gamma S-stimulated amylase release was observed from adult but not from day 20 fetal acini. In contrast to its stimulatory effects in permeabilized adult acini, adenosine 3',5'-cyclic monophosphate (cAMP) alone had little effect on release from permeabilized day 20 fetal acini. Our studies indicate that the fetal pancreas is competent to undergo Ca(2+)-dependent protein secretion but that this secretion is suppressed at birth. Our studies also suggest that the fetal gland is sensitive to modulators of exocytosis active in the adult pancreas, such as GTP gamma S, TPA, and cAMP but responds differently to these agents compared with responses in adult glands.

Aging↗
PubMed1993

Effects of valinomycin on osmotic lysis of zymogen granules and amylase exocytosis from parotid acini.

The role of osmotic swelling of the secretory granules in adenosine 3',5'-cyclic monophosphate (cAMP)-mediated amylase exocytosis was evaluated by use of isolated zymogen granules and saponin-permeabilized acini of the rat parotid gland. The osmotic lysis of the isolated granules was markedly enhanced by the addition of valinomycin (> 10(-9) M) in the presence of isosmotic KSCN or KI medium. However, valinomycin (up to 10(-5) M) did not increase the granule lysis in KCl medium, although the granules were slightly less stable in KCl medium than in K2SO4 or potassium gluconate medium. Guanosine 5'-O-(3-thiotriphosphate) did not affect the granule lysis. Valinomycin alone had no effect on amylase release from saponin-permeabilized parotid acini incubated in KCl medium, but completely abolished cAMP-mediated amylase release in all K+ media used. The inhibition was clearly detected at 0.1 microM valinomycin in KCl medium, not blocked by the addition of 1 mM MgATP to the medium, and was greatly reduced in NaCl medium. cAMP-evoked amylase release was completely inhibited by SCN- and I- (permeant anions), the mean inhibitory dosages of which were approximately 25 and 50 mM, respectively. These results suggest that 1) the membrane of parotid zymogen granules has no detectable Cl- channels responsible for osmotic swelling of the granules, and 2) increase in K+ or anion conductance of the granule does not enhance but inhibits cAMP-mediated amylase exocytosis from parotid acini.

Amylases↗
PubMed1998

Crucial role of milk-borne insulin in the development of pancreatic amylase at the onset of weaning in rats.

The development of pancreatic amylase activity was examined in rats fed in regular cages or in special cages, designed so the pups could not reach solid food to prevent weaning. In both groups, the amylase activity in zymogen granules increased in rat pups aged 14 days, peaked at 18 days, and thereafter remained at a 1.6-fold higher level than at 14 days of age. An increase in the plasma concentration of immunoreactive insulin preceded the increase of amylase activity, whereas the plasma concentration of C-peptide, indicating the secretion rate of endogenous insulin, remained unchanged. The administration of insulin at 20 ng/ml (the physiological concentration) in the milk formula caused an increase in the plasma insulin concentration of 17-day-old pups. In addition, increased pancreatic amylase activity was observed in 17-day-old rats raised on milk formula to which insulin was added. We propose that the increase of amylase activity at the beginning of weaning is dependent on the milk-borne insulin and not on the dietary change in rats.

Amylases↗
PubMed2002

The ascites to serum amylase ratio identifies two distinct populations in acute pancreatitis with ascites.

BACKGROUND/AIM: While the characteristics of ascites in the setting of chronic pancreatitis are well established, little has been written about the characteristics of spontaneous, clinically apparent ascites in the setting of acute pancreatitis. Our aim was to define the characteristics of clinically apparent ascites complicating acute pancreatitis, particularly with regard to outcomes. METHODS: We performed a search of our hospital's discharge records for ICD codes 577.0 (acute pancreatitis) and 789.5 (ascites). Clinical and laboratory variables in survivors and nonsurvivors were compared using a Mann-Whitney U test. RESULTS: We identified 59 records of which 25 cases had ascites fluid analysis. Only the ascites amylase (p = 0.033) and the ascites to serum amylase ratio (p = 0.002) correlated with mortality. Setting a cutoff of 1, the ascites to serum amylase ratio achieved a sensitivity of 83% and a specificity of 92% as a predictor of mortality. CONCLUSIONS: The ascites to serum amylase ratio identifies 2 sets of patients with ascites complicating acute pancreatitis. In patients with a high ratio, ascites may result from a localized duct disruption. In patients with a low ratio ascites may be secondary to comorbid conditions or a capillary leak. In acute pancreatitis with clinically apparent ascites, the ascites to serum amylase ratio may be a predictor of mortality.

Acute Disease↗
PubMed1987

Serum and peritoneal fluid amylase levels in CAPD. Normal values and clinical usefulness.

The mean serum amylase of 42 asymptomatic CAPD patients was elevated but was not significantly different from that of a group of chronic hemodialysis patients. Serum amylase levels in CAPD patients with peritonitis were not elevated with respect to asymptomatic patients. Amylase activity was not detectable in the peritoneal fluid of 38/42 asymptomatic patients and 6/13 peritonitis patients and was present at low levels in the other 11 patients. Patients with other abdominal conditions (pancreatitis, cholecystitis and small bowel perforation) had very marked elevations of serum and/or peritoneal fluid amylase which differentiated them from the asymptomatic and peritonitis patients. Although hyperamylasemia is common in asymptomatic CAPD patients and in those with peritonitis, measurement of serum and peritoneal fluid amylase levels is useful in the evaluation of CAPD patients presenting with abdominal symptoms.

Acute Disease↗
PubMed1975

Amylase, isoamylase and macroamylase.

Hyperamylasaemia has long been regarded as pathognomonic of acute pancreatitis. However, recent work has revealed a number of conditions where a gross elevation may be an incidental finding, notably diabetic ketoacidosis. The recent discovery of 'macroamylase', a high molecular weight amylase-protein complex capable of producing hyperamylasaemia with low urine amylase, has further complicated diagnosis and has led to the introduction of the ratio of amylase clearance to creatinine clearance as a diagnostic aid. Serum amylase may be resolved, by most electrophoretic media, into bands which correspond to those obtained when pancreatic homogenates or saliva are electrophoresed. The initial promise of this technique has not been realised at the routine diagnostic level. Duodenal juice amylase has been the classical enzyme used in assessing exocrine pancreatic function and although it is still of value it is being amplified by other enzyme tests.

Acute Disease↗
PubMed1985

Effect of antigastrin (SC-15396) on amylase release and 2-deoxyglucose uptake in mouse pancreatic acini.

The effects of antigastrin (SC-15396) on amylase release and 2-deoxyglucose uptake were studied in dispersed acini from mouse pancreas. Antigastrin at concentrations between 0.5 and 2 mM inhibited cholecystokinin (CCK)-stimulated amylase release and 2-deoxyglucose uptake in a dose-dependent fashion. Antigastrin at concentrations between 0.5 and 2 mM inhibited carbachol-stimulated amylase release and 2-deoxyglucose uptake as well. In addition, the drug (2 mM) inhibited bombesin-stimulated amylase release and 2-deoxyglucose uptake. In contrast, the stimulation of amylase release by Ca2+ ionophore A23187 was not inhibited by antigastrin. The results, therefore, suggest that antigastrin nonselectively inhibits the actions of these secretagogues probably at their receptor sites.

Amylases↗
PubMed1985

Measurement of pancreatic and salivary alpha-amylase in serum: comparison of methods with five different substrates.

Pancreatic and salivary alpha-amylase in human serum have been determined with the inhibitor method with 5 different substrates. An end concentration of 60 inhibitor units per liter reagent is sufficient for inhibition of the salivary alpha-amylase. The recovery of known amounts of pancreatic and salivary alpha-amylase was excellent. A mean +/- SD of 53.1 +/- 3.8% of pancreatic alpha-amylase as percentage of total alpha-amylase in serum has been observed for the 5 different substrates used. Moreover, it seems that standards obtained from crude saliva or duodenal fluid can be used as well as pure enzymes obtained from a commercial supplier.

Glucosides↗
PubMed1974

A method for quantitating amylase and its use in the investigation of various body fluids.

A plate-diffusion technique for the quantitative assay of amylase in body fluids is described which is dependent on the incorporation of a soluble amylopectin-dye complex into agar. The technique correlates well with other established amylase assays, and has a coefficient of variation of 1.45 for amylase levels within the normal range for serum. Normal levels of amylase in serum and urine are reported which agree with those recorded by previous workers, and in addition values of amylase in semen, saliva, and tears are quoted.

Amylases↗
PubMed1985

Contribution of extracellular and intracellular calcium to the enhanced effect of an alpha-adrenergic agonist on amylase release from dispersed rat parotid cells.

The role of calcium in the potentiation of amylase release by the alpha-adrenergic agonist methoxamine (MTX) was examined using dispersed rat parotid acinar cells. The stimulatory effect of MTX (10 microM) on the beta-adrenergic agonist isoproterenol (ISP, 1 microM)-induced amylase release was blocked by the alpha-adrenergic antagonist phentolamine (10 microM). In Ca-free (1 mM EGTA) medium, the increment of amylase release by MTX in normal medium was decreased by about 70%, but MTX still potentiated ISP-induced amylase release. MTX did not affect the cyclic AMP accumulation activated by ISP in either normal or Ca-free medium. MTX enhanced the ISP-stimulated uptake and efflux of 45Ca2+. These results suggest that both extracellular Ca2+ and intracellular stored Ca2+ may play an important role in the potentiation of amylase release from rat parotid acinar cells.

Amylases↗
PubMed1995

Effects of dopamine on adenylyl cyclase activity and amylase secretion in rat parotid tissue.

Several previous studies have shown that dopamine causes amylase secretion from rat parotid tissue. However, the mechanism of this dopamine action is still unclear. The present study was designed to characterize dopamine action in rat parotid gland tissue by examining the effects of dopamine on cyclic AMP accumulation, adenylyl cyclase activity, and amylase release. Dopamine significantly enhanced accumulation of cyclic AMP in parotid slices and stimulated adenylyl cyclase activity in parotid membrane preparations. It also significantly stimulated amylase release from parotid slices. The stimulatory effects of dopamine on cyclic AMP accumulation, adenylyl cyclase activity, and amylase release were effectively blocked with propranolol, a beta-adrenergic antagonist, but not by either SCH 23390, a preferential D1 antagonist, or butaclamol, a preferential D2 antagonist. No substantial specific binding sites for D1 receptors were detectable by [3H]SCH 23390 binding in parotid membranes. These results suggest that the stimulatory effect of dopamine on amylase secretion in rat parotid tissue is not mediated through specific D1 dopamine receptors but rather through beta-adrenergic receptors.

Adenylyl Cyclases↗
PubMed2006

Effect of insulin on acetylcholine-evoked amylase release and calcium mobilization in streptozotocin-induced diabetic rat pancreatic acinar cells.

This article investigated the effect of acetylcholine (ACh) on amylase secretion and cellular calcium homeostasis [Ca2+]i in streptozotocin (STZ; 60 mg kg(-1), intraperitoneally)-induced diabetic rats compared to age-matched controls in an attempt to understand the cellular mechanism of exocrine pancreatic insufficiency. ACh-evoked marked dose-dependent increases in amylase release from isolated pancreatic acini and acinar cells in healthy control rats. In diabetic acini and acinar cells, the ACh-evoked amylase release was significantly (P < 0.05) reduced compared to healthy acini and acinar cells. Insulin (10(-6)M) stimulated amylase release in both control and diabetic acini and acinar cells but with a much reduced effect in diabetic tissues. Combining insulin with ACh had no significant effect on amylase release compared to the effect of ACh alone. In fura-2 loaded pancreatic acinar cells of normal rats, ACh (10(-5)M) evoked a large initial rise (peak) in [Ca2+]i followed by a decline into a plateau phase. This effect of ACh was significantly (P < 0.05) reduced in fura-2 loaded diabetic acinar cells. In control cells, insulin had no significant effect on either basal or ACh evoked [Ca2+]i compared to the effect of ACh alone. In contrast, in diabetic acinar cells, insulin significantly (P < 0.05) attenuated the effect of ACh. In a normally free extracellular Ca2+ medium [Ca2+]o containing 1 mM EGTA, the ACh-evoked [Ca2+]i in normal healthy fura-2 loaded acini was similar to the response obtained with ACh in fura-2 loaded diabetic acini. Together, the results indicated that exocrine pancreatic insufficiency is associated with decreased [Ca2+]i due to less Ca2+ released from internal stores and less Ca2+ entering the cell from the extracellular medium.

Acetylcholine↗
PubMed1983

Mammary amylase: a possible alternate pathway of carbohydrate digestion in infancy.

Mammary amylase is a possible alternate pathway of digestion of glucose polymers and starches, that is most important in early infancy when pancreatic amylase is low or absent in duodenal fluid and responds poorly to stimuli. Human breast milk contains 1000-5000 units of amylase/liter. In order to evaluate the likelihood that a significant proportion of mammary amylase activity would withstand passage through the stomach, purified and unpurified mammary amylase were exposed to acid and pepsin in vitro to simulate the gastric environment found in young infants. Both purified and unpurified enzymes were stable at pH 7.5 with little or no activity lost after 4 h, and approximately 80% retained at 6 h. When incubated at pH 3.5, one-third of unpurified enzyme activity was retained for 6 h; in contrast, the purified enzyme was acid labile losing 80% by 2 h. Addition of bovine serum albumin or breast milk proteins to purified enzyme protected the activity. When unpurified enzyme was exposed to a stepwise decline in pH from 6.5 to 3.5 over 4 h, 50% of the original activity was retained. Unless the concentration was greater than or equal to 3750 units/ml, the addition of varying concentrations of pepsin to defatted breast milk incubated at pH 3.5 did not affect any greater decay of enzyme activity despite evidence of peptic digestion of proteins in the reaction mixture. This study supports the possibility that ingested mammary amylase could retain a significant proportion of its original activity after exposure to acid and pepsin in the stomach of young infants.

Amylases↗