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PubMed1999

Serum amylase isoenzymes in patients undergoing operation for ruptured and non-ruptured abdominal aortic aneurysm.

OBJECTIVE: Previous work has suggested that hyperamylasemia in patients who undergo operation for ruptured abdominal aortic aneurysm (AAA) is associated with poor outcome. The aims of this study were to determine, for the first time, the source of serum amylase in such patients and to examine the prognostic significance of amylase isoenzyme expression. METHODS: This study was designed as a prospective clinical and laboratory study. The study consisted of 40 patients who underwent operation for ruptured AAA and 10 patients who underwent operation for non-ruptured AAA. The main outcome measures were serum total and pancreatic and salivary amylase activities determined with enzymatic colorimetric assay before operation and 6 hours after aortic clamp release. RESULTS: Five of 40 patients (12.5%) with rupture and one of 10 patients (10%) with non-rupture had elevated total amylase levels before operation, and seven of 31 patients (23%) with rupture and five of 10 patients (50%) with non-rupture had elevated total amylase levels after operation. The preoperative salivary amylase (P =.05) and postoperative pancreatic amylase (P <.02) levels were significantly lower in ruptured AAA as compared with non-ruptured AAA. The preoperative salivary amylase level was significantly lower in non-survivors of rupture, such that a level equal to or less than 45 U/L was associated with death in 11 of 13 patients (85%). CONCLUSION: These data do not support previous works that suggest that hyperamylasemia is associated with poor outcome in ruptured AAA. By contrast, a low preoperative salivary amylase level was associated with increased mortality in ruptured AAA and may be a marker of the severity of shock.

Aged↗
PubMed2000

SH3 binding sites of ZG29p mediate an interaction with amylase and are involved in condensation-sorting in the exocrine rat pancreas.

ZG29p, a novel pancreas-specific zymogen granule protein, has been proposed to act as a 'helper protein' in granule formation. To address its function in more detail, we searched for putative binding partners of ZG29p. In zymogen complexes isolated by nondenaturing isoelectric focusing, ZG29p was associated with a protein complex consisting of amylase and cationic trysinogen. Amylase also coeluted with ZG29p after immunoaffinity chromatography using an antibody to recombinant ZG29p. Cross-linking experiments with granule content proteins revealed a direct interaction between recombinant ZG29p and amylase. An interaction was also observed when purified amylase was used, whereas no interaction with recombinant or purified cationic trypsinogen was seen. ZG29p could also be cross-linked to three membrane proteins with molecular masses of 40, 18, and 16 kDa. The binding of ZG29p to amylase and to the membrane proteins was inhibited in the presence of synthetic peptides matching the consensus sequence of proline-rich SH3 binding sites present in ZG29p. The synthetic peptides could be cross-linked to amylase and to three yet unidentified acidic content proteins with molecular masses of about 30 kDa. The peptides also interacted with purified or recombinant amylase, but not with recombinant or purified cationic trypsinogen. In a condensation-sorting assay, the binding (sorting) of zymogen complexes to the granule membrane was reduced in the presence of the peptides. Our results indicate that the interaction of ZG29p with amylase is mediated by SH3 binding domains and that these domains are involved in the sorting of amylase to the granule membrane.

Amino Acid Sequence↗
PubMed1980

Interaction of wheat monomeric and dimeric protein inhibitors with alpha-amylase from yellow mealworm (Tenebrio molitor L. larva).

The highly purified alpha-amylase from Tenebrio molitor L. larva (yellow mealworm) reversibly combines with two closely related homogeneous glycoprotein inhibitors, one dimeric (termed 'inhibitor 0.19') and one monomeric (termed 'inhibitor 0.28'), from wheat flour. As established by means of difference spectroscopy and kinetic studies, molar combining ratios for the amylase--inhibitor-0.19 and amylase-inhibitor-0.28 complexes were 1:1 and 1:2 respectively. Two amylase--inhibitor-0.19 complexes with slightly different retention volumes on Bio-Gel P-300 and only one amylase--inhibitor-0.28 complex were observed. Dissociation constants of the amylase--inhibitor-0.19 and amylase--inhibitor-0.28 complexes were 0.85 nM and 0.13 nM respectively. A strong tendency of both complexes to precipitate under an ultracentrifugal field was observed; the minimum molecular weight calculated for the two complexes under such conditions was approx. 95 000. The two complexes showed difference spectra indicating involvement of structurally related or identical tryptophyl side chains in the binding of inhibitors 0.28 and 0.19 to the amylase. A model summarizing the main features of the inhibition of the insect amylase by the two wheat protein inhibitors is proposed.

Animals↗
PubMed1994

Expression of alpha-amylases, carbohydrate metabolism, and autophagy in cultured rice cells is coordinately regulated by sugar nutrient.

A rice suspension cell culture system has been established to study how sugar depletion regulates alpha-amylase expression, carbohydrate metabolism, and other physiological and cellular changes. It is shown here that a group of 44 kDa alpha-amylases are constitutively expressed whether or not the cells are starved of sucrose. However, expression of a new group of alpha-amylases of 46 kDa is dramatically induced when cells are starved of sucrose. Cellular sugar and starch were rapidly consumed and metabolic activity was decreased in the starved cells. Extensive autophagy also occurred in the starved cells, which caused an increase in vacuolar volume and degradation of cytoplasmic constituents including amyloplasts. Immunocytochemical studies revealed that alpha-amylases are localized in starch granules within amyloplasts, in cell walls, and in some of the vacuoles. The presence of putative signal sequences in the N-termini of nine rice alpha-amylases suggests hitherto unidentified pathways for import of alpha-amylases into amyloplasts. The studies show that differential alpha-amylase expression, carbohydrate metabolism, metabolic activity, and vacuolar autophagy are coordinately regulated by the sugar level in the medium. As the starved suspension cells exhibit some sugar-regulated characteristics of alpha-amylase expression in germinating rice embryos as well as physiological changes similar to those in senescing cells, this system represents an ideal tool for studying cellular, biochemical, and molecular biological aspects of alpha-amylase gene regulation, carbohydrate metabolism, senescence, and protein targeting in plants.

Amino Acid Sequence↗
PubMed2003

Relation between domain evolution, specificity, and taxonomy of the alpha-amylase family members containing a C-terminal starch-binding domain.

The alpha-amylase family (glycoside hydrolase family 13; GH 13) contains enzymes with approximately 30 specificities. Six types of enzyme from the family can possess a C-terminal starch-binding domain (SBD): alpha-amylase, maltotetraohydrolase, maltopentaohydrolase, maltogenic alpha-amylase, acarviose transferase, and cyclodextrin glucanotransferase (CGTase). Such enzymes are multidomain proteins and those that contain an SBD consist of four or five domains, the former enzymes being mainly hydrolases and the latter mainly transglycosidases. The individual domains are labelled A [the catalytic (beta/alpha)8-barrel], B, C, D and E (SBD), but D is lacking from the four-domain enzymes. Evolutionary trees were constructed for domains A, B, C and E and compared with the 'complete-sequence tree'. The trees for domains A and B and the complete-sequence tree were very similar and contain two main groups of enzymes, an amylase group and a CGTase group. The tree for domain C changed substantially, the separation between the amylase and CGTase groups being shortened, and a new border line being suggested to include the Klebsiella and Nostoc CGTases (both four-domain proteins) with the four-domain amylases. In the 'SBD tree' the border between hydrolases (mainly alpha-amylases) and transglycosidases (principally CGTases) was not readily defined, because maltogenic alpha-amylase, acarviose transferase, and the archaeal CGTase clustered together at a distance from the main CGTase cluster. Moreover the four-domain CGTases were rooted in the amylase group, reflecting sequence relationships for the SBD. It appears that with respect to the SBD, evolution in GH 13 shows a transition in the segment of the proteins C-terminal to the catalytic (beta/alpha)8-barrel(domain A).

Binding Sites↗
PubMed1996

Character of a wheat amylase inhibitor preparation and effects on fasting human pancreaticobiliary secretions and hormones.

BACKGROUND & AIMS: Amylase inhibition induces carbohydrate tolerance, satiety, and weight loss and prolongs gastric emptying, effects that may be useful in the treatment of obesity and non-insulin-dependent diabetes mellitus. The aim of this study was to determine (1) purity of a wheat amylase inhibitor preparation, (2) intraduodenal concentration of the wheat amylase inhibitor preparation that inhibits > 90% amylase activity (which causes carbohydrate malabsorption), and (3) if the inhibitor alters pancreaticobiliary secretions or intraluminal pH. METHODS: High-performance liquid chromatography followed by electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used for characterization. Groups of 3 subjects received intraduodenal infusions of 3.0, 4.5, or 6.0 mg/mL of the inhibitor for 90 minutes during the middle of a 270-minute essential amino acid solution infusion (which stimulates 50% maximal pancreatic enzyme secretion). Pancreatic enzyme and bile acid delivery to the duodenum were measured for a 270-minute period. RESULTS: The inhibitor is 96% protein, 59% containing 0.19, 0.28, 0.38, and 0.53 inhibitors. The 0.38 inhibitor has the most antipancreatic alpha-amylase activity. The inhibitor reduced amylase activity in the duodenum dose dependently (r = 0.7; P = 0.04); > 4 mg/mL inhibited > 90% amylase activity but did not affect delivery of other enzymes or bile acids to the duodenum or gastric or duodenal pH. CONCLUSIONS: The preparation has a high protein purity and a high specific activity against alpha-amylase activity and effectively inhibits human pancreatic amylase activity secreted into the duodenum.

Adult↗
PubMed2001

Pain at 24 hours associated with amylase levels greater than 5 times the upper normal limit as the most reliable indicator of post-ERCP pancreatitis.

BACKGROUND: The frequency of post-ERCP/sphincterotomy pancreatitis is between 1.3% and 7.6% in prospective studies. This range likely reflects differences in definitions of pancreatitis and methods of data collection. METHODS: To identify clinical findings and enzymatic values consistent for clinical pancreatitis at 24 hours, the post-ERCP/sphincterotomy course of 1185 procedures was prospectively recorded. Patients were evaluated for pancreatic-type pain, white blood cell count, and serum amylase before and 24 hours after the procedure; pain and amylase levels were also recorded 6 to 8 hours after the procedure. CT was performed in all patients with pain associated with amylase levels greater than 3 times normal. All patients were evaluated clinically at 48 hours. RESULTS: Pancreatic-type pain never occurred in cases with amylase levels lower than 3 times normal; it was significantly (p < 0.001) associated with amylase levels greater than 5 times normal, either 6 to 8 hours or 24 hours after the procedure. Leukocytosis and CT findings consistent with pancreatitis were observed only in patients (41.7% and 29.5%, respectively) with 24-hour amylase levels greater than 5 times normal. None of the 18 patients with pain at 24 hours and serum amylase lower than 5 times normal had symptoms that persisted at 48 hours. Twenty-five (41.7%) of the 60 patients with pain at 24 hours and amylase higher than 5 times normal had 48-hour pain at 48 hours and hyperamylasemia. CONCLUSIONS: Features consistent with clinical pancreatitis were present only among patients with pancreatic-type pain at 24 hours and amylase levels higher than 5 times normal. Additional follow-up is required for these patients.

Adolescent↗
PubMed1985

A 78-kilobase region of mouse chromosome 3 contains salivary and pancreatic amylase genes and a pseudogene.

Genetic studies have demonstrated that salivary and pancreatic amylase genes are closely linked in human and mouse. To analyze the arrangement of genes within the amylase cluster, a library of YBR mouse genomic DNA was cloned in the cosmid vector pJB8. Clones containing amylase genes were identified by hybridization with amylase cDNA probes. Salivary and pancreatic amylase genes were isolated on separate cosmid clones, but no overlapping clones were evident from the initial screening. A strategy for the rapid isolation of terminal noncoding fragments from the cosmid clones was developed. By using these terminal fragments for chromosome "walking," a map of 78 kilobases of the amylase gene region was constructed. The salivary and pancreatic amylase genes are present within this region in the same 5'-to-3' orientation, separated by 22 kilobases of genomic DNA. A truncated amylase pseudogene is located 10 kilobases downstream from the pancreatic amylase gene.

Amylases↗
PubMed1966

Distribution of newly synthesized amylase in microsomal subfractions of guinea pigs pancreas.

Amylase distribution was studied in guinea pig pancreas microsomes fractionated by centrifuging, for 2 hr at 57,000 g in a linear 10 to 30% sucrose gradient, a resuspended high speed pellet obtained after treating microsomes with 0.04% deoxycholate (DOC).(1) Amylase appeared in the following positions in the gradient: (a) a light region which contained approximately 35% of total enzymic activity and which coincided with a monomeric ribosome peak; (b) a heavy region which contained approximately 10% of enzymic activity in a sharp peak but which had very little accompanying OD(260) absorption; (c) a pellet at the bottom of the centrifuge tube which contained approximately 20% of the enzymic activity. After 5 to 20 min' in vivo labeling with leucine-1-C(14), radioactive amylase was solubilized from these three fractions by a combined DOC-spermine treatment and purified by precipitation with glycogen, according to Loyter and Schramm. In all cases, the amylase found in the pellet had five to ten times the specific activity (CPM/enzymic activity) of the amylase found in the light or heavy regions of the gradient. The specific radioactivity (CPM/mg protein) of the proteins or peptides not extracted by DOC-spermine was similar for all three fractions. Hypotonic treatment of the fractions solubilized approximately 80% of the total amylase in the fraction from the heavy region of the gradient, but only approximately 20% of the amylase in the monomer or pellet fraction. Electron microscope observation indicates that the monomer region of the gradient contained only ribosomes, that the heavy region of the gradient contained small vesicles with relatively few attached ribosomes, and that the pellet was composed mostly of intact or ruptured microsomes with ribosomes still attached to their membranes. It is concluded from the above, and from other evidence, that most of the amylase activity in the monomer region is due to old, adsorbed enzyme; in the heavy region mostly to enzyme already inside microsomal vesicles; and in the pellet to a mixture of newly synthesized and old amylase still attached to ribosomes. Furthermore, the ribosomes with nascent, finished protein still bound to them are more firmly attached to the membranes than are ribosomes devoid of nascent protein.

Amylases↗
PubMed1986

Interstrain variation in amylase gene copy number and mRNA abundance in three mouse tissues.

Amylase expression in strain YBR differs in several respects from the standard mouse phenotype. The synthesis of salivary amylase is elevated twofold in YBR mice and the synthesis of pancreatic amylase is reduced to one-half the normal rate. We have compared the concentrations of amylase mRNA in the parotid, liver and pancreas of YBR mice with those in strains A/J and C3H. We observed differences in amylase mRNA abundance which can account for the levels of amylase protein synthesis in the parotid and pancreas of these strains. Unexpectedly, the concentration of amylase mRNA in the liver of YBR mice was also higher than in the other strains. Since liver amylase is transcribed from the same gene as parotid amylase, duplication of the Amy-1 locus could account for the elevated mRNA concentration in both tissues. Quantitative analysis of genomic DNA by Southern blotting provided direct evidence for duplication of Amy-1 in strain YBR.

Animals↗
PubMed2002

Dialysis with icodextrin interferes with measurement of serum alpha-amylase activity.

BACKGROUND: The glucose polymer icodextrin has gained a widespread use in peritoneal dialysis especially in patients with low ultrafiltration and high peritoneal transport properties. In patients using a once-daily exchange with icodextrin, a decreased serum amylase activity has been reported. We explored the potential underlying mechanisms of this effect. METHODS: Using standard chromolytic methods, serum amylase activity was measured in blood samples from 11 patients on icodextrin treatment and from 11 patients on conventional glucose treatment. Samples were additionally supplemented with alpha-amylase and unused icodextrin dialysis fluid. Potential complex formation between icodextrin and alpha-amylase was studied by SDS-gel electrophoresis with protein silver staining and fluorophore-assisted carbohydrate staining with the AMAC (FACE) method, which provides oligosaccharide labelling. Lipase activity was measured in parallel in all samples by two standard methods. RESULTS: Amylase activity was reduced by 90% in serum from patients using icodextrin for the long dwell (15.9+/-10.9 U/l) vs patients using standard glucose (157.1+/-23.7 U/l; P<0.001). Addition of icodextrin to serum samples from patients using conventional glucose solutions induced a dose-dependent decrease in amylase activity. The assay results indicated a substrate competition between ET7-G7PNP and icodextrin. AMAC fluorophore staining of icodextrin and subsequent gel electrophoresis failed to demonstrate complex formation between icodextrin and alpha-amylase. Unlike the amylase findings, icodextrin did not affect lipase activity. CONCLUSIONS: The present findings indicate that icodextrin competitively interacts as a substrate in the amylase assay. In support of this, fluorophore-assisted oligosaccharide electrophoresis on SDS gel failed to reveal the formation of an 'amylase/icodextrin complex'. Lipase measurement should provide an alternative and unconfounded method for diagnosing pancreatitis in icodextrin patients.

Adult↗
PubMed1996

Amylase gene structures in primates: retroposon insertions and promoter evolution.

Amylase transcription in the human salivary gland results from the evolutionary juxtaposition of two inserted elements, a gamma-actin pseudogene and an endogenous retrovirus, to create an unusual salivary-specific promoter. We utilized these structures as molecular tags to characterize the amylase genes in extant primates by polymerase chain reaction amplification of promoter fragments from genomic DNA. Six distinct amylase promoter structures were identified, which allowed us to infer the structures of common ancestors and trace the evolution of the modern human amylase promoters. Our data show that integration of the pseudogene and retrovirus were evolutionarily recent events. The gamma-actin pseudogene integrated after the divergence of the New World monkeys from the primate ancestral tree, and the retrovirus integrated later, after the divergence of the Old World monkeys. The New World monkey amylase promoter represents the mammalian amylase precursor structure before integration of the two retroposons. Two distinct amylase genes were identified in the Old World monkeys, one with a complete gamma-actin pseudogene insert and another novel structure with a truncation of the gamma-actin sequences. We demonstrated abundant amylase expression in the saliva of an Old World monkey, indicating that the endogenous retrovirus is not required for amylase transcription in the primate salivary gland.

Actins↗
PubMed1998

Effect of chronic amylase inhibition on pancreatic growth and acinar cell secretory function in rats.

Wheat amylase inhibitor (WAI) was given to growing rats to determine whether chronic inhibition of intraluminal amylase activity alters pancreatic growth, pancreatic enzyme composition, and secretory responsiveness to cholecystokinin octapeptide (CCK-OP) and carbachol. For 21 days 13 rats were fed amylase inhibitor (AI) as 2.72% of the weight of their food; 13 were pair-fed controls (PFC), and 12 were controls with free access to food (FAC). Amylase and lipase secretion was measured from isolated pancreatic acini in response to CCK-OP (10(-12)-10(-8) M) and carbachol (10(-8)-10(-3) M). AI and PFC rats had similar food intakes and weight gains, pancreatic weights, and contents of enzymes (amylase, lipase, trypsin, chymotrypsin), protein, and RNA, but these measurements were significantly reduced compared to those of FAC rats. DNA contents per milligram of pancreas and per gram of body weight and amylase/DNA and trypsin/DNA were similar among all groups. Lipase/DNA and chymotrypsin/DNA in AI rats were the same as in PFC rats but significantly lower than in FAC rats. In response to CCK-OP, amylase secretion was similar in all three groups, but in response to carbachol amylase secretion was significantly less in AI compared to PFC and FAC rats. Lipase secretion increased in response to CCK-OP in AI compared to PFC and FAC rats but was similar in all three groups in response to carbachol. Long-term inhibition of intraluminal amylase activity suppresses pancreatic growth and content of enzymes and RNA by reducing food intake and weight gain and also decreases acinar cell secretion of amylase in response to carbachol and increases lipase secretion in response to CCK-OP.

Amylases↗
PubMed1998

Identification and analysis of a gene (abpA) encoding a major amylase-binding protein in Streptococcus gordonii.

Oral streptococci such as Streptococcus gordonii bind the abundant salivary enzyme alpha-amylase. This interaction may be important in dental plaque formation and metabolism, thus contributing to the initiation and progression of dental caries and periodontal disease, the two most common plaque-mediated diseases. The conjugative transposon Tn916 was used to insertionally inactivate gene(s) essential to the expression of amylase-binding components of S. gordonii Challis, and a mutant deficient in amylase-binding (Challis Tn1) was identified. While wild-type strains of S. gordonii released both 20 kDa and 82 kDa amylase-binding proteins into culture supernatants, Challis Tn1 expressed the 82 kDa but not the 20 kDa protein. The 20 kDa amylase-binding protein was isolated from culture supernatants of S. gordonii Challis by hydroxyapatite chromatography. A partially purified, functionally active 20 kDa protein was sequenced from blots, and the N-terminal sequence obtained was found to be DEP(A)TDAAT(R)NND. A novel strategy, based on the single-specific-primer polymerase chain reaction technique, enabled the gene inactivated by Tn916 to be cloned. Analysis of the resultant nucleotide sequence revealed an open reading frame of 585 bp, designated amylase-binding protein A (abpA), encoding a protein of 20 kDa (AbpA), immediately downstream from the insertion site of Tn916. This protein possessed a potential signal peptide followed by a region having identity with the N-terminal sequence of the 20 kDa amylase-binding protein. These results demonstrate the role of the 20 kDa protein in the binding of amylase to S. gordonii. Knowledge of the nature of amylase-binding proteins may provide a better understanding of the role of these proteins in the colonization of S. gordonii in the oral cavity.

Amino Acid Sequence↗
PubMed1994

Sequence similarities and evolutionary relationships of microbial, plant and animal alpha-amylases.

Amino acid sequence comparison of 37 alpha-amylases from microbial, plant and animal sources was performed to identify their mutual sequence similarities in addition to the five already described conserved regions. These sequence regions were examined from structure/function and evolutionary perspectives. An unrooted evolutionary tree of alpha-amylases was constructed on a subset of 55 residues from the alignment of sequence similarities along with conserved regions. The most important new information extracted from the tree was as follows: (a) the close evolutionary relationship of Alteromonas haloplanctis alpha-amylase (thermolabile enzyme from an antarctic psychrotroph) with the already known group of homologous alpha-amylases from streptomycetes, Thermomonospora curvata, insects and mammals, and (b) the remarkable 40.1% identity between starch-saccharifying Bacillus subtilis alpha-amylase and the enzyme from the ruminal bacterium Butyrivibrio fibrisolvens, an alpha-amylase with an unusually large polypeptide chain (943 residues in the mature enzyme). Due to a very high degree of similarity, the whole amino acid sequences of three groups of alpha-amylases, namely (a) fungi and yeasts, (b) plants, and (c) A. haloplanctis, streptomycetes, T. curvata, insects and mammals, were aligned independently and their unrooted distance trees were calculated using these alignments. Possible rooting of the trees was also discussed. Based on the knowledge of the location of the five disulfide bonds in the structure of pig pancreatic alpha-amylase, the possible disulfide bridges were established for each of these groups of homologous alpha-amylases.

Amino Acid Sequence↗
PubMed1980

Effects of carbachol on isoprenaline evoked amylase release from the rabbit parotid gland in vitro.

Amylase secretion from dispersed lobules of the parotid gland of the rabbit was studied in response to isoprenaline (10(-8)-10(-5) M) and to carbachol (10(-8)-10(-5) M). The effects of each agent were investigated along and in combination at certain concentrations. Isoprenaline produced a dose-related amylase secretion with an average maximum of 688 units/100 mg, at 10(-5) M. The amylase secretion produced by submaximal concentrations of isoprenaline could be further increased by carbachol, already in low concentrations (10(-8)-10(-7) M), that were subthreshold for amylase secretion. This potentiating effect was seen not only as a larger secretion but also as a greater depletion of amylase from the tissue. In contrast to what is known from experiments in vivo, carbachol in a high concentration (10(-5) M), by activating muscarinic receptors only, released amylase to the same extent as that released by isoprenaline. This concentration of carbachol regularly decreased the amylase secretion evoked by isoprenaline, and may be regarded as unphysiologically high. The increase in isoprenaline evoked amylase secretion, brought about by carbachol in lower concentrations, may be due to an improved transport of amylase, because of secretion of fluid, but could also be caused by augmentation of the beta-adrenoceptor mediated effects.

Amylases↗
PubMed1982

The effects of manganese, cobalt and calcium on amylase secretion and calcium homeostasis in rat pancreas.

1. Mn(2+) evoked an atropine-resistant secretion of amylase from the isolated pancreas of the young rat. The lowest effective concentration of Mn(2+) was 10(-3)m. The response to 10(-2)m-Mn(2+) was biphasic, an initial peak being followed by a slow sustained rise in amylase output. The maximal effect of 10(-2)m-Mn(2+) was to double the basal rate of amylase secretion after 70 min incubation.2. Co(2+) (10(-2)m) also stimulated amylase secretion. The maximal rate, about three times the basal value, was attained after 20 min incubation. Atropine partially inhibited this effect.3. Ca(2+) (10(-2)m) evoked an atropine-resistant amylase secretion similar in both magnitude and time course to the sustained phase observed with 10(-2)m-Mn(2+).4. Mn(2+) (10(-4)-10(-2)m) also increased the rate of (45)Ca efflux from the gland. Maximal efflux rates were attained after 30 min incubation and thereafter declined to basal values. A small increase was also observed with 10(-2)m-Co(2+), but not with 10(-2)m-Ca(2+). The effect of Co(2+) was almost completely abolished by atropine.5. Reducing the extracellular Ca(2+) concentration from 2.5 x 10(-3) to 10(-5)m did not reduce amylase secretion in response to 10(-2)m-Mn(2+), but secretion was abolished in a Ca(2+)-free medium containing EGTA. The increase in (45)Ca efflux rate evoked by Mn(2+) was inversely related to the extracellular Ca(2+) concentration.6. Mn(2+) (10(-2)m) increased the concentration of cyclic 3',5'-guanosine monophosphate (cyclic GMP) within the pancreas. Also, Mn(2+) accumulated within the cellular pool of the gland. The time course of both these effects was similar to the time course of (45)Ca efflux.7. Mn(2+) displaced Ca(2+) bound to isolated pancreatic microsomal membranes. The cation-binding sites on these membranes probably have a higher affinity for Mn(2+) than Ca(2+).8. We conclude that Mn(2+) stimulates enzyme secretion by displacing membrane-bound Ca(2+), the resulting increase in cytosolic Ca(2+) concentration activating the secretory mechanism.9. Mn(2+) partially inhibited amylase secretion stimulated by optimal doses of either acetylcholine (ACh) or caerulein. Maximal inhibition (about 60%) occurred with 10(-3)m-Mn(2+) (i.e. the lowest concentration required to stimulate secretion in the absence of secretagogues). Decreasing the extracellular Ca(2+) concentration reduced the inhibitory effect of Mn(2+).10. When glands were exposed to ACh and Mn(2+) simultaneously, the time required for inhibitory effects to develop was inversely related to the dose of ACh and the concentration of Mn(2+).11. Mn(2+) did not alter the acceleration of (45)Ca efflux evoked by ACh or by caerulein in a medium containing 2.5 x 10(-3)m-Ca(2+). However, under conditions of Ca(2+) deprivation ACh-stimulated (45)Ca efflux was greatly enhanced.12. Mn(2+) reduced the total amount of Ca(2+) accumulated into the cellular pool of the pancreas after 60 min incubation, but had no effect on the initial, rapid phase of Ca(2+) uptake.13. The effects of Mn(2+) on the relationship between ACh dose, amylase release and the extracellular Ca(2+) concentration suggest that the inhibitory actions of Mn(2+) cannot be explained by a simple, competitive interaction with the stimulant or with extracellular Ca(2+). However, the time course of inhibition is consistent with a requirement for Mn(2+) to accumulate within the acinar cells.14. Mn(2+) partially inhibited amylase secretion stimulated by hyperosmolarity and also increased the (45)Ca efflux rate under these conditions.15. Our results are not consistent with Mn(2+) exerting its inhibitory effect on secretagogue-stimulated enzyme secretion solely by blocking Ca(2+) influx from the extracellular space. We conclude that inhibition probably depends on the ability of Mn(2+) to displace Ca(2+) from binding sites involved in secretion, presumably coupled with a reduced ability of Mn(2+) to replace Ca(2+) in the secretory process.

Acetylcholine↗
PubMed1985

Mechanism of action of extracellular calcium on isoprenaline-evoked amylase secretion from isolated rat parotid glands.

The effects of extracellular Ca2+ deprivation on amylase secretion, 45Ca efflux and cyclic adenosine 3',5'-monophosphate (cyclic AMP) metabolism were investigated in incubated parotid glands of young rats. Reducing the extracellular Ca2+ concentration from 2.5 X 10(-3) M to 10(-6) M had no effect on amylase secretion but did increase the rate of 45Ca efflux from unstimulated glands. Isoprenaline (10(-5) M) increased amylase secretion and the rate of 45Ca efflux in media containing 2.5 X 10(-3) M-Ca2+, and also increased the parotid cyclic AMP content. When glands were pre-incubated for either 10, 20, or 30 min in media containing either 10(-3) or 10(-6) M-Ca2+ there was no effect on the peak rate of amylase secretion stimulated by isoprenaline but the response took longer to develop. The effects of the same experimental manoeuvre on the net change in 45Ca efflux stimulated by isoprenaline were consistent with the idea that the beta-adrenergic agonist mobilizes Ca2+ from a number of intracellular pools. When glands were pre-incubated for 100 min in Ca2+-depleted buffers and then exposed to isoprenaline a triphasic effect on amylase secretion was observed. The secretory response was depressed when the extracellular Ca2+ concentration was lowered from 2.5 X 10(-3) M to 10(-3) M; remained unaffected when the extracellular Ca2+ was further reduced to 10(-5) M; and was virtually abolished when the extracellular Ca2+ concentration was decreased to 10(-6) M. When glands were pre-incubated for 100 min in media containing Ca2+ at concentrations above 10(-5) M and then stimulated with isoprenaline parallel changes in cyclic AMP content and amylase secretion were observed. At lower Ca2+ concentrations there was no further reduction in cyclic AMP content. The lipophilic cyclic AMP analogue N6,O2-dibutyryl adenosine 3',5'-monophosphate (dibutyryl cyclic AMP) mimicked the effects of isoprenaline on amylase secretion and 45Ca efflux. However, the amylase secretory response took longer to develop and the 45Ca efflux response was of shorter duration. These responses were reduced after pre-incubation of glands for 100 min in a medium containing 10(-6) M-Ca2+. Ca2+ at concentrations up to 10(-7) M enhanced parotid adenylate cyclase activity but higher concentrations were inhibitory. The same pattern of Ca2+ sensitivity was seen for basal and also isoprenaline and F--stimulated activities. The results show that isoprenaline can mobilize Ca2+ from a number of intracellular pools which may exchange at different rates with the extracellular medium. This Ca2+ may be required at a minimum of two sites in the amylase secretory pathway.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylyl Cyclases↗