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PubMed1996

Induction of amylase release from rat parotid acinar cells by cooling in vitro.

As amylase exocytosis from parotid acinar cells is an energy-dependent process, it was supposed that it could be terminated by cooling on ice. Unexpectedly, however, the cooling itself markedly induced amylase release from parotid acini. The release finished within 1 min and prolonged incubation on ice did not cause further release. The cold-induced amylase release was observed in the absence of extracellular calcium, and calcium ionophore A23187 neither enhanced nor inhibited it. Treatments with cytochalasin D, phalloidin, or taxol did not disturb the release. Cold treatment did not increase the leakage of lactate dehydrogenase, a cytosolic enzyme, and the acini still maintained normal responsiveness to isoproterenol. Electron-microscopic observation revealed that the plasma membrane and zymogen granules of cold-exposed acini were intact, but many acinar lumina were distended with secretory materials. These results suggest that the cold treatment induces transient amylase release by the fusion of plasma membrane and the zymogen granules that have been closely docked at the luminal membrane.

Amylases↗

PubMed1976

Microheterogeneity in porcine pancreatic amylase preparations due to disulfide-sulfhydryl exchange.

There are two masked SH groups in pancreatic amylase (EC 3.2.1.1) which become reactive after removal of Ca ions, and then only due to fluctuation of the polypeptide chain. Carboxymethylation of the masked SH groups and isolation of the tryptic CM-peptides have shown that the reacting SH groups in active amylase preparations are not identical, depending on the method of preparation. The microheterogeneity is attributed to an SH-SS exchange reaction taking place during preparation. This exchange results in a mixture of two types of amylase molecules containing different SH groups. The tryptic peptides containing the SH groups have been detected from the radioautogram of the peptide may of the [14C] carboxymethylated protein digest. We identified the SH peptides present in native amylase, and those cysteinyl peptides which form an easily reducible disulfide in the vicinity of these thiol groups, and take part in the intramolecular SH-disulfide exchange.

Amylases↗

PubMed1985

Amylase secretion from isolated pure acinar cells.

Isolation of pure acinar cells of the rat pancreas was achieved employing counterflow sedimentation filtration technique (CSFT). The preparation of purified acinar cells contained an occasional red blood cell (RBC, 200:1) with total absence of endocrine and duct cells. A significant stimulation of amylase secretion from isolated pure acinar cells was produced by octapeptide of cholecystokinin (CCK8) and insulin produced potentiation of the effect of CCK8. Synthetic glucagon inhibited basal and CCK8 stimulated amylase secretion. Non-synthetic purified glucagon stimulated amylase secretion and potentiated the effect of CCK8. Vasoactive intestinal polypeptide (VIP) did not stimulate amylase secretion but potentiated the effect of CCK8. No leakage of lactic dehydrogenase (LDH) was detected from the cells in any of the secretion studies. Thus a highly purified preparation of isolated pure acinar cells of rat pancreas could be obtained with excellent morphologic and functional integrity.

Amylases↗

PubMed1989

Eicosanoids evoke the release of amylase and increase cytoplasmic calcium in rat parotid cells.

The effects of various eicosanoids on cytoplasmic calcium and the release of amylase were examined in isolated rat parotid cells. Arachidonate and several of its metabolites increased amylase release and elevated cytoplasmic calcium. Melittin, a stimulator of arachidonate mobilization, and lyso-phosphatidylcholine also released amylase and elevated calcium. These results suggest that the metabolites of arachidonate may have an important role in amylase secretion.

Amylases↗

PubMed1983

Effects of amino acids, glucagon, insulin and acetylcholine on cyclic nucleotide metabolism and amylase secretion in isolated mouse pancreatic fragments.

The effects of amino acids, exogenous islet hormones and acetylcholine on cyclic nucleotide metabolism and amylase secretion in the isolated mouse pancreas have been investigated. The changes in levels of adenosine 3',5'-cyclic monophosphate (cyclic AMP) and guanosine 3',5'-cyclic monophosphate (cyclic GMP) were measured at different times during exposure of pancreatic fragments to amino acids (L-alanine and L-arginine), islet hormones (insulin and glucagon) or acetylcholine (ACh). L-Alanine (1-20 mM) evoked a transient increase in cyclic AMP concentration accompanied by an initial decrease and subsequent increase in the tissue concentration of cyclic GMP. L-Arginine (1-20 mM) induced a complex triphasic change in cyclic AMP concentrations involving an initial rise and a delayed sustained elevation. The changes in levels of cyclic GMP increased only transiently. The effects of insulin (10(-6) M) and to some extent glucagon (5 X 10(-7) M) resembled those seen with L-arginine. The effects of amino acids and islet hormones were all dose-dependent. ACh (10(-7) M) elicited a marked reduction in cyclic AMP concentration and this was accompanied by a concomitant increase in the level of cyclic GMP. The amino acids and the islet hormones had no significant effect on amylase secretion whereas ACh, of course, evoked a large increase in amylase output. The results with the amino acids and islet hormones reveal a clear dissociation between cyclic nucleotide changes and amylase secretion and further suggest that the marked reciprocal changes in cyclic AMP and cyclic GMP concentrations may constitute an important physiological role for the cyclic nucleotides to regulate amino acid transport in the pancreas.

Acetylcholine↗

PubMed1990

Inhibitory effects of loading with the calcium-chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) on amylase release and cellular ATP level in rat parotid cells.

Rat parotid cells were loaded with the Ca2(+)-chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), by incubation with the acetoxymethyl ester of BAPTA (BAPTA-AM). The BAPTA loading inhibited amylase release induced by beta-adrenergic receptor stimulation without affecting the basal release, and the IC50 value was 25 microM. Incubation of cells with BAPTA-AM also suppressed cellular ATP levels considerably. At 100 microM BAPTA-AM, the ATP level fell to about 50% of the control. A decrease in ATP levels by incubation with oligomycin, a mitochondrial inhibitor, correlated well with the inhibition of amylase release. Although these results do not exclude the possibility that cytosolic free Ca2+ is involved in the regulation of cyclic AMP-mediated amylase release, they suggest that the amylase release inhibition with BAPTA loading may be due, in part, to a decrease in cellular ATP levels. Therefore, the exact mode of the BAPTA action must be interpreted with caution.

Adenosine Triphosphate↗

PubMed1992

Interaction between the calcium and cyclic AMP messenger systems in perifused rat parotid acinar cells. Possible mechanism for potentiation of amylase secretion.

Potentiation of amylase secretion induced by a combination of isoproterenol and carbamylcholine was examined in perifused rat parotid acinar cells. The time course of changes in the augmented amylase secretion induced by isoproterenol plus carbamylcholine was similar to that induced by carbamylcholine alone, but not to that caused by isoproterenol. Concentration-response analysis showed that isoproterenol increased the apparent affinity for carbamylcholine to stimulate amylase secretion with the maximum effect attained by isoproterenol plus carbamylcholine being higher than that attained by isoproterenol or carbamylcholine. 8-Bromo cyclic AMP, forskolin and 3-isobutyl-1-methylxanthine mimicked the effect of isoproterenol. Calcium ionophores (A23187 and ionomycin), but not phorbol 12,13-dibutyrate, mimicked the effect of carbamylcholine. Chelation of intracellular free calcium with 1,2-bis-[2-aminophenoxyl]-ethane-N,N,N',N'-tetraacetic acid, but not that of extracellular calcium with [ethylenebis(oxyethylenenitrile)]-tetraacetic acid (EGTA), abolished the potentiation. Calmodulin antagonists inhibited amylase secretion induced by isoproterenol plus carbamylcholine or carbamylcholine alone, but not that induced by isoproterenol alone. These results suggest that the potentiation is mainly, if not completely, caused by a coordinated interaction between the cyclic AMP system and the Ca2+ system at a step distal to second messenger generation, probably via a cyclic AMP-induced increase in the sensitivity of the Ca2+ response element to calcium.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1976

Antiserum raised against human pancreatic alpha-amylase.

Isozymes of human pancreatic alpha-amylase were used as immunogens to raise antisera in rabbits. These antisera were unable to differentiate between the two isoamylases but produced an almost total inhibition of serum amylase activity when used at a dilution of 1 : 5000. Similar inhibition patterns were produced with the salivary and urinary enzymes as well as with serum from a patient with acute pancreatitis. No cross reactivity of the antisera with hog pancreatic amylase was observed and only at antiserum concentrations of 1 : 5000 and above was ther any inhibition of monkey serum amylase. The degree of inhibition observed varied with the size of the substrate but was not increased by the use of a second antibody.

Amylases↗

PubMed1977

A new serum alpha-amylase assay of high sensitivity.

The use of chemically modified starch substrates for measurement of serum alpha-amylase activity is described. Action of alpha-amylase on such substrates, in the presence of excess fungal glucoamylase, results in the production of glucose in direct proportion to the amount of alpha-amylase present. The glucose produced is measured by a specific enzymic assay. Results obtained by using this new assay correlate well with activities determined by a conventional saccharogenic assay. The new method is of much higher sensitivity, and is less susceptible to interference, than are most other alpha-amylase assay methods.

Amylases↗

PubMed1980

Preparation and application of Procion Yellow starch for amylase assay.

A new starch derivative for the determination of amylase activity has been synthesized by coupling Procion Yellow dye with starch. The product of this reaction is intensely yellow and is easily suspended in water, in neutral buffer solution, and also in acidic solution. Amylase from pancreatin, saliva, urine and serum readily hydrolyzes this chromogenic substrate. A method for determining amylase activity based on the use of Procion Yellow Starch substrate is described. The procedure requires 0.1 ml of sample and an incubation time of 30 min. The soluble chromogen, which is liberated by enzymatic hydrolysis, is measured at 420 nm and the amylase activity-absorbance relationship at this wavelength is linear. The normal range for this method is 50--150 Somogyi units/dl.

Amylases↗

PubMed1982

Serum and urinary amylase isoenzymes in carcinoma of the prostate.

Serum and urinary amylase isoenzyme activities were evaluated in 70 patients with carcinoma of the prostate. The results were compared with amylase isoenzyme activities fo 20 healthy man and 30 patients with benign prostatic hypertrophy. Increased serum S-type amylase activity was found in 13 patients (18%) with carcinoma of the prostate, whereas no changes of this isoenzyme was found in benign prostatic hypertrophy. No significant changes of serum P-type amylase activities were observed in our patients.

Aged↗

PubMed1985

Determination of alpha-amylase in biological fluids using a new substrate (beta-2-chloro-4-nitrophenyl-maltopentaoside).

A novel substrate, beta-2-chloro-4-nitrophenylmaltopentaoside (beta CNPG5), was used for the enzyme-coupled determination of alpha-amylase in biological fluids. It was hydrolyzed specifically by alpha-amylase to about 90% producing beta-2-chloro-4-nitrophenylmaltoside (beta CNPG2) and maltotriose. Under the assay conditions, the absorption of 2-chloro-4-nitrophenol (CNP) generated by the secondary reaction of alpha-glucosidase and beta-glucosidase as auxiliary enzymes is about twice the absorption of 4-nitrophenol (PNP), which is the end product currently measured in some alpha-amylase assay methods. The sensitivity of the assay using beta CNPG5 was thus much higher than that using 4-nitrophenyl-maltopentaoside (PNPG5) as substrate. The absorption of CNP did not fluctuate with temperature or with pH between 6.8 and 7.2, which are the conditions normally used for determination of amylase activity in biological fluids.

Body Fluids↗

PubMed1986

Impaired amylase activities caused by binding of abnormal immunoglobulin A in patients with macroamylasemia.

Eight of thirty analyzed samples of immunoglobulin A-linked macroamylases showed significantly higher enzyme activity with the low molecular (short-chain amylose) substrate than with the macromolecular (blue starch polymer) substrate. Immunoglobulin A, which had been separated from one macroamylase, was found to bind to normal amylases from different organs resulting in decreased affinity for the macromolecular substrate. These findings suggest that abnormal immunoglobulin A is stoichiometrically associated with a specific site adjacent to the active site of the amylase molecule. In addition, alpha-amylase inhibitor failed to inhibit the enzyme activity of some macroamylases with macromolecular substrate, suggesting that immunoglobulin A blocked the binding site for the inhibitor. We conclude that immunoglobulin A-linked macroamylases have heterogeneous binding sites for immunoglobulin A, resulting in functional abnormality of amylase activity.

Amylases↗

PubMed1988

Alpha-amylase assay with use of a benzyl derivative of p-nitrophenyl alpha-maltopentaoside, BG5P.

p-Nitrophenyl O-(6-O-benzyl)-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl- (1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1-- --4)-alpha-D-glucopyranoside (BG5P) is hydrolyzed by both human salivary alpha-amylase (HSA) and human pancreatic alpha-amylase (HPA) to O-(6-O-benzyl)-alpha-D-glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl- (1----4)-alpha-D-glucopyranose (BG3) and p-nitrophenyl alpha-maltoside (G2P). Glucoamylase and alpha-glucosidase cannot hydrolyze BG5P because of the modification of the OH group of the 6-position of the non-reducing-end glucose residue with the benzyl group. Taking advantage of these characteristics of the substrate, BG5P, we developed a method to assay the total alpha-amylase activity in human fluids using glucoamylase and alpha-glucosidase as the coupled enzymes. This method is simple and can be used as the standard method for routine clinical assays of alpha-amylase activity.

Chromatography, High Pressure Liquid↗

PubMed1991

Determination of alpha-amylase using a new blocked substrate (3-ketobutylidene beta-2-chloro-4-nitrophenyl-maltopentaoside).

A new substrate, 3-ketobutylidene beta-2-chloro-4-nitrophenylmaltopentaoside (3KB-CNPG5), was used for the determination of alpha-amylase (EC 3.2.1.1) in serum and urine. Under this alpha-amylase assay condition, 3KB-CNPG5 is resistant to glucoamylase and alpha-glucosidase, which are auxiliary enzymes, because the 4- and 6-positions of the non-reducing-end glucose residue are modified by the 3-ketobutylidene group. The assay using 3KB-CNPG5 for alpha-amylase activity is a highly sensitive method that uses 2-chloro-4-nitrophenol (CNP) as an aglycone, and is a stable method for determination of alpha-amylase activity in biological fluids.

Chromatography, High Pressure Liquid↗

PubMed1993

Total pancreatic and salivary serum iso-amylase activities in alcohol misusers in relapse and remission and in alcoholic liver disease.

By means of immunoinhibition by specific salivary monoclonal antibodies in combination with a chromogenic substrate, assays of serum amylase were performed in control subjects, in chronic alcohol misusers in relapse or remission and in patients with alcoholic liver disease (ALD). There was a selective increase in the salivary isoenzyme in the ALD group. There were no significant changes in either of the alcohol misusing groups, compared with control subjects. It is suggested that the increase in salivary iso-amylase observed in patients with ALD is related to the previously reported functional and histological abnormalities in the parotid glands of this group of patients. It is also suggested that assay of pancreatic iso-amylase may be more discriminatory than total amylase levels in detecting pancreatic disease in patients with alcoholic cirrhosis.

Adult↗

PubMed1982

Effects of neonatal sympathetic denervation on amylase secretion in the adult rat parotid gland: difference in beta 1- and beta 2-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 denervated glands exhibited enhanced maximal enzyme discharge in response to beta-adrenoceptor agonists. However, the selective beta 1-agonist, prenalterol was not effective in this respect whereas an enhanced maximal secretory response to the beta 2-selective agonist, terbutaline, was particularly prominent. DBcAMP was also more efficient in inducing amylase release from the denervated gland. The result of the present study demonstrate that the usual dominance of the beta 1-adrenoceptor subtype in eliciting amylase release is lost, implying that the differentiation of the beta-adrenoceptor into its subtypes is altered by neonatal sympathetic denervation.

Amylases↗

PubMed1986

Phorbol ester stimulates amylase secretion from rat parotid cells.

Phorbol myristate acetate (PMA), a potent activator of Ca2+- and phospholipid-dependent protein kinase (protein kinase C), evoked amylase release from rat parotid cells. In dose-response studies, PMA stimulated amylase release independently of db-cAMP, but potentiated the effect of carbachol. PMA and A23187, a Ca2+ ionophore, synergistically increased amylase release. The maximum effect of carbachol was further enhanced by PMA but not by A23187, suggesting that protein kinase C is not fully activated by the muscarinic-cholinergic agonist under the condition where calcium is fully utilized for amylase secretion.

Amylases↗