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PubMed1989

The relationship of intracellular free calcium activity to amylase secretion in substance P- and isoprenaline-stimulated rat parotid acini.

The relationship between intracellular free calcium activity ([Ca2+]i) and stimulated amylase secretion was investigated in rat parotid acini by measuring the effects of substance P methyl ester and isoprenaline on quin2 fluorescence and amylase release. Although both of these drugs evoked concentration-dependent increases in [Ca2+]i and amylase release, the tachykinin had a greater effect on [Ca2+]i while the catecholamine was the more effective secretagogue. Whereas isoprenaline exerted equipotent effects on amylase secretion and [Ca2+]i, the dose-response relationship for stimulation of secretion by substance P was dissociated by three orders of magnitude to the right of that for elevation of [Ca2+]i by this peptide. It is concluded that these data do not support the hypothesis that substance P-stimulated amylase secretion is mediated solely through an increase in [Ca2+]i and that other second-messengers may be involved in mediation of this secretory response.

Aminoquinolines↗
PubMed1990

In vitro action of human and porcine alpha-amylases on cyclomalto-oligosaccharides.

The vitro action of human and porcine pancreatic alpha-amylases on cyclomalto-oligosaccharides (cyclodextrins) was investigated both by a high-performance liquid chromatographic analysis and a quantitative analysis of the reducing power of cyclodextrin hydrolyzates. Cyclomalto-octaose (gamma-cyclodextrin) was hydrolyzed to produce mainly maltose, but cyclomalto-hexaose and -heptaose were little affected both by human and porcine alpha-amylases. Quantitative analysis of reducing power revealed that the ring-opening rate of gamma-cyclodextrin catalyzed by human pancreatic alpha-amylase was 2.8 times slower than that catalyzed by the porcine enzyme. The number of multiple attacks on gamma-cyclodextrin and its inhibitor constants for human pancreatic alpha-amylase and porcine pancreatic alpha-amylase were almost the same.

Animals↗
PubMed1993

Syntheses of modified 2-chloro-4-nitrophenyl beta-maltopentaosides as useful substrates for assay of human alpha amylase.

Twenty-three novel 2-chloro-4-nitrophenyl beta-D-maltopentaosides modified at the 6(5) and/or 4(5) position were synthesized as substrates for human alpha amylase. Two human alpha amylases hydrolyzed 6(5)-deoxy-6(5)-, 6(5)-O-, and 4(5),6(5)-di-O-substituted derivatives at essentially a single D-glucosidic linkage, but 4(5),6(5)-O-bridged and 4(5)-O-substituted derivatives were hydrolyzed at two or more linkages. The amylases displayed smaller Km values for the compounds having hydrophobic modifications. In these derivatives, 2-chloro-4-nitrophenyl O-(6-bromo-6-deoxy-alpha-D-glucopyranosyl)-(1-->4)-tris[O-alpha-D- glucopyranosyl-(1-->4)]-beta-D-glucopyranoside (10), 2-chloro-4-nitrophenyl O-(6-azido-6-deoxy-alpha-D-glucopyranosyl)-(1-->4)- tris[O-alpha-D-glucopyranosyl-(1-->4)]-beta-D-glucopyranoside (19), and 2-chloro-4-nitrophenyl O-[6-O-(N-isopropyl)carbamoyl-alpha-D-glucopyranosyl]-(1-->4)- tris[O-alpha-D-glucopyranosyl-(1-->4)]-beta-D-glucopyranoside (30), which were rapidly hydrolyzed by the two amylases at a limited position at an approximately equal rate, were shown to be very useful blocked-type substrates for assay of human alpha amylase.

Carbohydrate Sequence↗
PubMed1993

Syntheses of subtractively modified 2-chloro-4-nitrophenyl beta-maltopentaosides and their application to the differential assay of human alpha-amylases.

Three novel maltopentaosides, 2-chloro-4-nitrophenyl O-(6-deoxy-alpha-D-xylo-hex-5-enopyranosyl)-(1-->4)-tris[O-alpha-D - glucopyranosyl-(1-->4)]-beta-D-glucopyranoside (3), 2-chloro-4-nitrophenyl O-(6-deoxy-alpha-D-glucopyranosyl)-(1-->4)-tris[O- alpha-D-glucopyranosyl-(1-->4)]-beta-D-glucopyranoside (10), and 2-chloro-4-nitrophenyl O-(3,6-anhydro-alpha-D-glucopyranosyl)-(1-->4)-tris[O-alpha-D-glucopyran osyl- (1-->4)]-beta-D-glucopyranoside (26) were synthesized by chemical and enzymatic reactions. Two human alpha-amylases, salivary alpha-amylase (HSA) and pancreatic alpha-amylase (HPA), hydrolyzed 3 and 10 with the same specificity, almost entirely at a single D-glucosidic linkage, but had no hydrolytic activity for 26. Compound 3 was hydrolyzed by each of these amylases at an approximately equal rate, while 10 was hydrolyzed by HSA 4-fold faster than by HPA. Taking advantage of the difference in the hydrolytic rate of 10, we developed a new method for the differential assay of these two human alpha-amylases.

Carbohydrate Sequence↗
PubMed1975

Separation of human alpha-amylase isozymes by electro-focusing and their immunological properties.

Human alpha-amylase (alpha-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) was separated by electrofocusing. The amylases in serum and urine were seperated into two major isozymes with isoelectric points of pH 6.4-6.5 and pH 6.9-7.0, respectively, and one minor isozyme with an isoelectric point of pH 5.9. The amylases in saliva were separated into one major isozyme with an isoelectric point of pH 6.5 and two minor isozymes with isoelectric points of pH 6.0 and 6.9. The amylases in pancreatic juice gave one major peak at pH 7.0 and two minor peaks at pH 6.0 and pH 6.5. Repeated electrofocusing of each isozyme have only a single peak with a constant isoelectric point. The antigenicites of these three isozymes were also investigated using antisera to rat pancreatic and parotid amylases.

Amylases↗
PubMed1977

Ectopic production of a salivary type amylase by adenocarcinoma cells: demonstration by a culture technique.

Characterization of an elevated amylase activity in ascitic fluid obtained from a patient with carcinomatous peritonitis is described; ninety-one percent of the increased amylase activity in the fluid was of salivary type and the remainder of pancreatic type, when studied by ion-exchange chromatography. Culture of ascitic cells successfully demonstrated morphologically characterized tumor cells surviving for at least 31 days. During that period, significant amylase activities were detected in the culture media, and the isozyme pattern was a single band whose electrophoretic mobility corresponded to salivary amylase. The data obtained indicate that the ascites amylase of salivary type was produced ectopically by the tumor cells.

Adenocarcinoma↗
PubMed1977

Effects of albumin and other proteins during assay of amylase activity.

Addition of albumin, gamma-globulin, alpha-casein or submaxillary mucin to the assay system for chromogenic measurement of human or rat amylase with blue starch increased the amylase activity, albumin having the most effect. These proteins seemed to increase the activity by protecting amylase from inactivation. Amylase activity was higher in urine samples showing proteinuria than in urine samples without detectable protein. It is concluded that amylase assay is more reliable when a final concentration of albumin of 1 mg per ml is added at the dilution step and at the incubation step.

Albumins↗
PubMed1996

Production and certification of an enzyme reference material for pancreatic alpha-amylase (CRM 476).

We describe the preparation of a lyophilized material containing purified human pancreatic alpha-amylase and the certification of its catalytic concentration. The enzyme was purified from human pancreas by ammonium sulphate precipitation and chromatography successively on DEAE-Sephacel, CM-Sepharose and Sephadex G-75. The purified enzyme had a specific activity of 52.9 kU/g protein and was > 99% pure on polyacrylamide gel electrophoresis. Only trace amounts of lipase and lactate dehydrogenase were detected in the purified fraction. The purified pancreatic alpha-amylase had a molar mass of 57,500 g/mol and an isoelectric point at 7.1. The material was prepared by diluting the purified alpha-amylase in a matrix containing PIPES buffer 25 mmol/l, pH 7.0, sodium chloride 50 mmol/l, calcium chloride 1.5 mmol/l, EDTA 0.5 mmol/l and human serum albumin 30 g/l, dispensing in ampoules and freeze-drying. The ampoules were homogeneous and the yearly loss of activity on the basis of accelerated degradation studies was less than 0.01% at -20 degrees C. The certified value for alpha-amylase catalytic concentration in the reconstituted reference material is 555 U/l +/- 11 U/l when measured by the specified method at 37 degrees C. The material can be used to verify the comparability of results from laboratories, for intra-laboratory quality control or for calibration of alpha-amylase catalytic concentration measurements.

Catalysis↗
PubMed1996

Thermostability of purified human pancreatic alpha-amylase is increased by the combination of Ca2+ and human serum albumin.

Pancreatic fluid from a patient with a post operative pancreatic fistula was used to isolate human alpha-amylase by means of acarbose affinity chromatography. Amylase thermostability was measured in 4 solutions: (1) EDTA-dialyzed; (2) dialyzed solution plus 0.15 mmol/l (1.0 g/dl) human serum albumin; (3) dialyzed solution plus 0.25 mmol/l (1.0 mg/dl) calcium ions; and (4) dialyzed solution with both human serum albumin and calcium ions. Amylase activity was measured at predetermined times in samples heated to 60 degrees C. Thermostability was characterized by t1/2, the time to 50% initial amylase enzyme activity. In the dialyzed solution t1/2 was 0.75 +/- 0.19 min. This rose to 1.62 +/- 0.34 min with added human serum albumin, and to 8.24 +/- 0.13 min with added calcium ions. The combination of human serum albumin and calcium ions resulted in a synergistic increase of t1/2 to 180 +/- 26 min. These findings support our contention that human serum albumin, calcium ions and possibly other body fluid constituents must be considered in any utility involving amylase thermostability as a clinically relevant diagnostic marker.

Calcium↗
PubMed1983

Diphenylhydantoin: effects on amylase release, cyclic nucleotides and ion fluxes in mouse parotid acini.

The effects of diphenylhydantoin were investigated on isolated mouse parotid acini. Diphenylhydantoin inhibited carbachol-stimulated amylase release at concentrations of 5 X 10(-4) M and greater; isoproterenol-stimulated amylase release was not affected. The inhibition of carbachol-stimulated amylase release was attributable to inhibition of 45Ca2+ uptake. The ability of diphenylhydantoin to inhibit carbachol-stimulated cyclic GMP (c-GMP) accumulation was also related to inhibition of 45Ca2+ uptake. Diphenylhydantoin (5 X 10(-4)M) alone, was found to stimulate c-GMP accumulation, an effect related to enhanced 45Ca2+ efflux. Diphenylhydantoin alone, however, failed to enhance amylase release. These findings suggest that the effects of diphenylhydantoin on carbachol-stimulated amylase release and on c-GMP accumulation are related to its effects on transmembrane calcium movements.

Amylases↗
PubMed1983

Modulation of rat pancreatic amylase secretion and muscarinic receptor populations by chronic bethanechol treatment.

F8P6effect of a chronic bethanechol treatment (12 mg X kg-1 X day-1 i.p., for 14 days) was investigated on pancreatic amylase secretion and muscarinic cholinergic receptors in the rat. Dispersed pancreatic acini were used to evaluate enzyme secretion and binding of [3H]N-methylscopolamine, [( 3H]NMS). The bethanechol treatment caused a 4 fold decrease in sensitivity of the pancreas for amylase release in the presence of carbamylcholine, the EC50 being shifted from 0.69 microM to 2.9 microM. Receptor concentration was reduced by 42%, from 3360 to 1930 fmol/mg DNA. The equilibrium dissociation constant (KD) of the receptors for the ligand remained unchanged at 0.17 nM. Binding analysis of carbachol on the muscarinic receptors in terms of two classes of binding sites indicated that the shift in the dose-response curve of amylase secretion was accompanied by modifications to the high and low affinity forms. The maximal number of high affinity sites remained the same while their affinity was greatly decreased from 0.24 to 6.1 microM. The low affinity form showed a moderate decrease in affinity from 34 to 150 microM but a large drop in their numbers from 2620 to 890 fmol/mg DNA. These results suggest that the shift in the amylase dose-response curve to carbamylcholine, noted following bethanechol treatment in vivo, could be coupled with the observed change in affinity of the two agonist forms of muscarinic receptor in the pancreas. However, gradual occupancy or formation of the high affinity form of muscarinic receptors by cholinergic agonist corresponds well with the gradual stimulation of amylase release.

Amylases↗
PubMed1993

Galanin: structure-dependent effect on pancreatic amylase secretion and jejunal strip contraction.

Rat and porcine galanin and their fragments inhibited cholecystokinin-8 (CCK-8)-stimulated amylase secretion with the following activities: rat galanin-(1-29) = porcine galanin-(1-29) = galanin-(1-15) = rat galanin-(3-29) > rat galanin-(2-29) = porcine galanin-(2-29) > galanin-(1-10). Fragments of rat galanin-(9-29) and N alpha-acetyl-galanin-(9-29) were able to inhibit CCK-8-stimulated pancreatic amylase secretion but only at higher dose levels. Porcine galanin-(15-29) and rat galanin-(21-29) were unable to produce significant inhibition. Rat and porcine galanin-(1-29), galanin-(1-15) and rat N alpha-acetyl-galanin-(9-29) also inhibited basal pancreatic amylase secretion. In the rat jejunal strip contraction model, rat galanin-(1-29) and porcine galanin-(1-29) have similar potencies. Galanin-(1-15) and galanin-(1-10) stimulate rat jejunal strip contraction with decreasing potencies. Elimination of Gly1 from the N-terminus of both rat and porcine galanin had no significant effect either on pancreatic amylase secretion or on jejunal strip contraction. The rat galanin-(3-29) and (9-29) are not active in the stimulation of rat jejunal strip contraction. Acetylation of porcine galanin-(9-29) created a peptide that was a powerful stimulator of rat jejunal strip contraction. The present data indicate that N-terminal rat galanin amino acid residues are crucial for rat jejunal strip contraction but are not required for inhibition of pancreatic amylase. These results suggest that the galanin amino acid sequence contains several specific domains, which can be recognized by specific galanin receptor subsets.

Amino Acid Sequence↗
PubMed1985

Phorbol ester (TPA) potentiates noradrenaline and acetylcholine-evoked amylase secretion in the rat pancreas.

The effect of the phorbol ester 12-O-tetradeconyl-phorbol-13-acetate (TPA) on noradrenaline (NA) and acetylcholine (ACh)-evoked amylase secretion in isolated segments of rat pancreas was investigated. TPA alone evoked a relatively small increase in amylase output. However, when combined with either noradrenaline or ACh, the phorbol ester markedly enhanced the secretagogue-induced amylase secretion. These effects were dose related. TPA also enhanced the amylase secretion evoked by either the Ca2+ ionophore A23187 or dibutyryl cyclic AMP. This potentiation by TPA of noradrenaline, ACh, Ca2+ ionophore A23187 and dibutyryl cyclic AMP-evoked amylase output may suggest the existence of a third pathway controlling enzyme secretion in the pancreas.

Acetylcholine↗
PubMed1991

Okadaic acid inhibits amylase exocytosis from parotid acini stimulated by cyclic AMP.

To evaluate the role of protein phosphorylation in amylase exocytosis, we studied the effects of okadaic acid, a potent inhibitor of protein phosphatase types 1 and 2A, on amylase release and protein phosphorylation in rat parotid acini. Although okadaic acid by itself weakly stimulated amylase release, it did not potentiate amylase release stimulated by half-maximum doses of isoproterenol or cAMP, and markedly inhibited their maximum effects. Okadaic acid dose-dependently increased cAMP-independent phosphorylation of some proteins and enhanced cAMP-dependent phosphorylation of 21- and 26-kDa proteins. These results indicate that increase in protein phosphorylation does not necessarily enhance the exocytosis of amylase from parotid acini.

Amylases↗
PubMed1985

Expression of mouse Amy-2a alpha-amylase genes is regulated by strong pancreas-specific promoters.

Three types of Amy-2-related DNA sequences, Amy-2a I, Amy-2a II and Amy-X, exist in the genome of mice of the inbred strain A/J. Amy-2a I and Amy-X are single copy sequences. Amy-2a II occurs as three copies per haploid genome. DNA sequence analysis reveals that both classes of Amy-2a genes specify the same unique pancreatic alpha-amylase mRNA species, since they share common exon sequences. Four independently cloned Amy-2a II isolates were found to be identical in all regions sequenced. This suggests that most, if not all, chromosomal Amy-2a II copies are identical. Amy-X is presumably a pseudogene, since its exon sequences, which are distinct from those of Amy-2a, are not detected in pancreatic alpha-amylase mRNA. We have determined the transcriptional activities of the Amy-2a genes by mapping in vitro elongated nascent transcripts to Amy-2a restriction fragments. Transcription initiation occurs at or close to the cap site. The expression of Amy-2a in vivo is under control of strong promoters, which are active exclusively in the pancreas. The accumulation of alpha-amylase mRNA in cells of the exocrine pancreas is regulated mainly at the transcriptional level. We have searched for pancreatic transcripts of Amy-1a, which specifies both parotid gland and liver-type alpha-amylase mRNAs. Surprisingly, the weak Amy-1a promoter, which directs the synthesis of the mRNA containing the liver-type leader sequence, also is active in the pancreas and, hence, in all alpha-amylase-producing tissues.

Animals↗
PubMed1995

Effect of adenosine and adenosine agonists on amylase release from rat pancreatic lobules.

The effects of adenosine on the amylase secretion from rat pancreatic lobules have been studied. Adenosine induces a dose-dependent stimulation on amylase release, which is maximal at a concentration of 10(-4) M. It has been observed a clear inhibition of this secretory action when atropine was added whereas no amylase release was seen in isolated acini after adenosine. The effect of adenosine is completely blocked by the adenosine receptors antagonist theophylline (10(-4) M), but not by dipyridamole (10(-3) M), a drug that inhibits the transport of adenosine into the cell. The increase of amylase secretion induced by adenosine is inhibited by either the relatively selective A1 receptor antagonist PD116,948 (10(-6) M) and by the A2 receptor antagonist PD115,199 (10(-6) M). Significant increases of amylase release are observed after the relatively selective A1 receptor agonist R-PIA (10(-5) M) and after the relatively selective A2 receptor agonist NECA (10(-4) M). Finally, the effect of R-PIA is not modified by coincubation with PD115,199 and the effect of NECA is not affected by coincubation with PD116,948. These results suggest that the action of adenosine is mediated through the release of acetylcholine and probably by the simultaneous occupation of both A1 and A2 adenosine receptors, whereas the intracellular action of adenosine could be discarded.

Adenosine↗
PubMed1996

Effect of morphine on secretion of amylase from isolated parotid acini.

The effect of morphine on amylase secretion was studied in isolated rat parotid acinar cells. It was found that aluminum fluoride (AlCl3/NaF) and dibutyryl-cyclic AMP but not by cyclic AMP, enhanced amylase secretion. Cyclic AMP was effective in enhancing secretion following permeabilization of cells with alpha-toxin. Following treatment of cells with alpha-toxin, both GTP and GTP-gamma-S also enhanced secretion. Morphine reduced AlCl3/NaF- or GTP-induced secretion of amylase, but was without effect on GTP-gamma-S-induced secretion. Photoaffinity labeling by the use of [32P] 4-azidoanilido GTP revealed its incorporation into 43 kDa and 31 kDa proteins. Incorporation was further enhanced with AlCl3/NaF. Morphine reduced labeling of the 43 kDa protein. Immunoblot analysis identified the 43 kDa GTP binding protein as Gs. When [gamma 32P] GTP was preloaded into permeabilized acinar cells and its hydrolysis measured, morphine stimulated and AlCl3/NaF inhibited GTPase activity. These results suggested the involvement of Gs in secretion of amylase. Furthermore, morphine reduced secretion of amylase by stimulating GTPase activity and by reducing the incorporation of GTP into Gs.

Affinity Labels↗
PubMed1984

Decreased pancreatic CCK receptor binding and CCK-stimulated amylase release in Zucker obese rats.

In Zucker obese rats the response to the effects of CCK on food intake and pancreatic exocrine function are decreased. However, it is unknown whether the decreased responsiveness is due to decreased receptor number and/or sensitivity or abnormal circulating concentrations of CCK. In these experiments percent total binding of 125I-CCK-33 to pancreatic acini from obese rats was one-half that in lean rats when data was expressed on a per microgram DNA basis (19.6 +/- 5.1 vs. 47.4 +/- 11.4, p less than 0.01). In a second experiment while the maximally effective dose of CCK for stimulating amylase secretion from dispersed pancreatic acini was similar in obese and lean rats (10(-10) M), less amylase was secreted in obese rats across the dose range tested (p less than 0.001). In contrast, carbachol had similar potency and efficacy in stimulating amylase release from obese and lean pancreatic acini. The increase of pancreas size by use of a trypsin inhibitor was greater in lean than obese rats (p less than 0.03). In addition, stimulation of amylase release by CCK from obese trypsin inhibitor-treated compared with control obese rats was greater than that from lean trypsin inhibitor-treated compared with control lean rats (p less than 0.002). However, overall, stimulation of amylase secretion by CCK was only 36% of control (p less than 0.001) and by carbachol was only 20% of control (p less than 0.001). Thus, increased size by increased cell number was associated with decreased response per cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Amylases↗