Amylase In Acinar Cells Research Articles

Using CRISPR/Cas9 to Knock out Amylase in Acinar Cells Decreases Pancreatitis-Induced Autophagy.

Pancreatic cancer is a malignant neoplasm that originates from acinar cells. Acinar cells get reprogrammed to become duct cells, resulting in pancreatic cancer. Pancreatitis is an acinar cell inflammation, leading to “impaired autophagy flux”. Pancreatitis promotes acinar-to-ductal transdifferentiation. Expression of amylase gets eliminated during the progression of pancreatic cancer. Amylase is considered as an acinar cell marker; however, its function in cells is not known. Thus, we investigated whether amylase affects the acinar cell autophagy and whether it plays any role in development of pancreatitis. Here, we knocked out ATG12 in a pancreatic cancer cells and acinar cells using CRISPR/Cas9. Autophagy inhibition led to an increase in the expression of duct cell markers and a simultaneous decrease in that of acinar cell markers. It also caused an increase in cell viability and changes in mitochondrial morphology. Next, we knocked out amylase in acinar cells. Amylase deficiency decreased autophagy induced by pancreatitis. Our results suggest that amylase controls pancreatitis-induced autophagy. We found that eliminating amylase expression contributes to pancreatic cancer etiology by decreasing autophagy. Furthermore, our results indicate that amylase plays a role in selective pancreatitis-induced autophagy of pancreatic enzyme vesicles.

THE HSP70-COCHAPERONE, CYSTEINE STRING PROTEIN, INTERACTS WITH THE SNARE PROTEIN VAMP 8 AND REGULATES ACINAR CELL SECRETION

We recently identified VAMP 8/endobrevin as an important zymogen granule SNARE protein that mediates acinar cell exocytosis. Investigation of potential protein interaction with VAMP 8 by coimmunoprecipitation, GST-pull-down assay and immunofluorescence microscopy revealed that VAMP 8 is present in a complex with a member of the DnaJ family: cysteine string protein (CSP). In contrast, the related pancreatic SNARE isoform VAMP 2 did not interact with CSP. In neurons, CSP acts as a cochaperone for the heat shock proteins HSC70 and HSP70 via interaction through its J-domain, which hydrolyses ATP and thereby activates HSC70 chaperone activity. In acini, CSP was found to interact with HSC70 under normal conditions and with HSP70 following its induction by heat shock. To identify a role for CSP in exocytosis, a CSP or a CSP truncation construct containing the J domain was introduced into perfringolysin O (PFO) permeabilized acinar cells and their effects on Ca2+-stimulated secretion was determined. In contrast to soluble constructs of VAMP 8, which inhibit exocytosis by 50%, CSP augmented acinar cell amylase secretion in a time and concentration-dependent manner, with an 80% maximum enhancement at 200 ug/ml of protein. These data demonstrate a specific role for CSP in mediating acinar cell secretion and moreover, implicate a role for HSC70 in modulating dynamic interactions of SNARE proteins during exocytosis.

Endotoxin can decrease isolated rat parotid acinar cell amylase secretion in a nitric oxide-independent manner

Salivary mucus and amylase have an anti-bacterial nature. Bacterial endotoxin is considered to decrease mucus secreting cell activity by nitric oxide-dependent mechanisms. In this study, the actions of endotoxin on amylase secreting cell activity have been studied. Endotoxin ( Escherichia coli lipopolysaccharide; 3 mg/kg, i.v., 5 h) evoked nitric oxide synthase 2 (NOS2) induction in the rat whole parotid tissue (assessed by Western blot and the citrulline assay) and in rat isolated parotid acinar cells (assessed by Western blot and immunohistochemistry), and reduced basal and acetylcholine-stimulated amylase secretion from these isolated cells. However, N G-nitro- l-arginine methyl ester (0.1 mg/ml, 4 days in drinking water, yielding a dose of 25 mg/kg/day) did not affect amylase release under basal or acetylcholine-stimulated conditions, either in control acinar cells or those from endotoxin challenged rats. Thus, basal, acetylcholine-evoked or endotoxin-decreased cellular amylase secretion from rat isolated parotid acinar cells does not appear to be modulated by endogenous nitric oxide.

Adenovirus-mediated gene transfer of RasN17 inhibits specific CCK actions on pancreatic acinar cells.

CCK stimulates pleiotrophic responses in pancreatic acinar cells; however, the intracellular signaling pathways involved are not well understood. To evaluate the role of the ras gene product in CCK actions, a strategy involving in vitro adenoviral-mediated gene delivery of a dominant-negative mutant Ras (RasN17) was utilized. Isolated acini were infected with various titers of either a control adenovirus or an adenoviral construct expressing RasN17 for 24 h before being treated with CCK. Titer-dependent expression of RasN17 in the acini was confirmed by Western blotting. Infection with control adenovirus [10(6)-10(9) plaque-forming units/mg acinar protein (multiplicity of infection of approximately 1-1,000)] had no effect on CCK stimulation of acinar cell amylase release, extracellular-regulated kinase (ERK) or c-Jun kinase (JNK) kinases, or DNA synthesis. In contrast, infection with adenovirus bearing rasN17 increased basal amylase release, inhibited CCK-mediated JNK activation, had no effect on CCK activation of ERK, and inhibited DNA synthesis. These data demonstrate important roles for Ras in specific actions of CCK on pancreatic acinar function.

Bile-pancreatic juice replacement not cholinergic-and cholecystokinin-receptor blockade reverses acinar cell hyperstimulation after bile-pancreatic duct ligation

Acinar cell inhibitors (eg, atropine) fail to ameliorate clinical and experimental acute pancreatitis. We hypothesized that amelioration of pancreatic acinar cell hyperstimulation after bile and pancreatic duct ligation is better with gut replacement of bile and pancreatic juice than with cholinergic- and cholecystokinin (CCK)-receptor blockade. Using acinar cell amylase activity as an index of hyperstimulation, we studied 63 rats in two sets of experiments. Bile-pancreatic juice exclusion from gut--without (set one) and with (set two) bile and pancreatic duct obstruction--was treated with atropine and CCK-receptor antagonist L-364,718, or with enteral replacement of bile-pancreatic juice. In the set one experiment, acinar cell hyperstimulation after bile-pancreatic juice exclusion was reversed by combined L-364,718 and atropine pretreatment. In set two, acinar cell hyperstimulation after bile and pancreatic duct ligation was reversed by enteral bile and pancreatic juice replacement, but not by combined L-364,718 and atropine pretreatment. According to this experimental corollary of early gallstone impaction, prevention of acinar cell hyperstimulation after duct occlusion should be aimed at the source of the response to bile-pancreatic juice exclusion, namely, the gut, rather than at the target of the response, the pancreatic acinar cell.

Estrogens influence cholecystokinin stimulated pancreatic amylase release and acinar cell membrane cholecystokinin receptors in rat

This study examines the influence of ovariectomy and administration of a pharmacologic dose of estradiol on amylase release from isolated-dispersed rat pancreatic acini and cholecystokinin receptors on rat acinar cell membranes. Rats were sham ovariectomized (intact) or ovariectomized (Ovx) and 21 day timed release pellets containing either estradiol (2.5 mg) or vehicle, were implanted subcutaneously. Eighteen days later, pancreatic acini were isolated from rats by collagenase digestion and differential centrifugation. Total cellular amylase, basal and cholecystokinin octapeptide (CCK8) stimulated amylase release and CCK membrane receptors were measured. Acini isolated from estradiol treated Ovx rats had significantly greater total cellular amylase, compared to acini isolated from either intact or Ovx rats. The amplitude of both total stimulated amylase release and percent total stimulated amylase release were significantly greater for acini isolated from vehicle treated Ovx rats, than acini isolated from either intact or estradiol treated Ovx rats. The magnitude of percent total amylase release of acini isolated from estradiol treated Ovx rats was significantly lower than that of acini isolated from intact rats. Cholecystokinin receptor concentration was significantly greater on membranes prepared from vehicle treated Ovx rats, compared to membranes prepared from either intact or estradiol treated Ovx rats. These data indicate that ovariectomy is associated with increased responsiveness of pancreatic acini to CCK stimulation, while chronic estradiol treatment of ovariectomized rats is associated with increased total cellular amylase and decreased acinar cell responsiveness to CCK8. Estrogen mediated alterations in acinar cell amylase content and amylase release may play a role in estrogen related pancreatitis.

Amylase expression in human parotid neoplasms: evidence by in situ hybridization for lack of transcription of the amylase gene.

Salivary alpha-amylase (EC 3.2.1.1) is the major protein component of human parotid gland secretion. We studied amylase gene structure and expression in tissue from a series of normal and neoplastic parotid glands by Southern blot analysis, in situ hybridization, and immunohistochemistry. Thirty-two tumors were examined. Southern blot analysis of DNA extracted from a Warthin tumor, an adenoid cystic carcinoma, and a mucoepidermoid carcinoma showed no evidence of structural rearrangement of amylase genes. Eleven parotid Warthin tumors were negative for amylase protein and mRNA by immunocytochemistry and in situ hybridization. One pleomorphic adenoma in the group of 10 examined showed focal staining for amylase protein, although amylase mRNA could not be demonstrated in the same population of cells by in situ hybridization in serial tissue sections. Five mucoepidermoid carcinomas and three acinar cell carcinomas were devoid of amylase protein and mRNA. Normal parotid tissue obtained from all patients studied revealed abundant acinar cell amylase mRNA and protein. In situ hybridization, in conjunction with immunocytochemistry, allows precise cellular localization of mRNA and protein, thereby establishing the site of production of specific transcripts. We conclude that the interruption in amylase gene expression in parotid gland neoplasms occurs at the transcriptional level.

Pancreatic Acinar Cell Amylase Gene Expression: Selective Effects of Adrenalectomy and Corticosterone Replacement*

To examine the role of glucocorticoids in the regulation of the acinar pancreas, adult male rats were adrenalectomized (Adx) and replaced with no corticosterone (B), normal B, or high B. Plasma B concentration, body weight gain, and thymus weight were used as independent measures of treatment efficacy. Compared to controls, Adx animals had a 75 +/- 0.5% (n = 30) reduction in pancreatic amylase content; a 50% decrease occurred within 1 day and the maximal 75% decrease was observed after 5 days. In Adx animals, amylase content was normalized by normal B replacement and was increased to 235 +/- 39% (n = 30) of control by high B replacement. Furthermore, in all Adx rats, pancreatic content of amylase and plasma B concentration was significantly correlated (r = 0.81, n = 30). The effect of adrenalectomy was selective for amylase; contents of ribonuclease, chymotrypsin, and elastase were not altered. However, the effects of high B replacement were not selective, and increased the content of all digestive enzymes. To determine whether the changes in enzyme content were associated with changes in messenger RNA (mRNA), pancreatic RNA was probed with 32P-labeled complementary DNAs for amylase, ribonuclease, and chymotrypsin. After adrenalectomy and B replacement there was a significant correlation only between amylase mRNA (r = 0.87, n = 13) and plasma B concentration. These data indicate that physiological levels of B have a selective effect on pancreatic amylase gene expression. In contrast, high levels of B have the separate, nonselective effect of increasing the content of all digestive enzymes without increasing corresponding mRNA levels.

Immunocytochemical studies of pancreatic acinar cells in normal and streptozotocin-induced diabetic rats

Amylase and chymotrypsinogen in pancreatic tissue from normal and diabetic rats were revealed by immunocytochemistry and analyzed biochemically. In acinar cells of control animals, both enzymes were localized with high resolution in the rough endoplasmic reticulum, Golgi apparatus, immature and mature secretory granules. Quantitative evaluations of the intensities of labelings have demonstrated, for both enzymes, the presence of an increasing gradient which followed precisely their secretory pathway. This gradient reflects the normal processing of both proteins through secretion. In streptozotocin-induced diabetic animals, labeling for amylase in acinar cells was markedly reduced (remaining about 11% of the normal values). The gradient along the secretory pathway was abolished, indicating an alteration in the processing and secretion of amylase. On the other hand, labeling for chymotrypsinogen was significantly increased (to 170% p less than 0.0005), and its processing remained normal. In insulin-treated diabetic animals, immunolabeling for amylase was restored and the gradient re-established, indicating a normalization of the secretion. Labeling for chymotrypsinogen was reduced towards normal values. These results were found to be in agreement with those obtained by biochemical approaches and demonstrate that, in the diabetic condition, secretion of amylase is selectively impaired.

Pancreatic polypeptide inhibits amylase release by rat pancreatic acini

Pancreatic polypeptide (PP), a mammalian islet cell peptide, is released after a meal. Exogenously administered PP in physiologic doses inhibits cholecystokinin and secretin-stimulated pancreatic secretion in vivo. Whether PP acts directly on exocrine pancreatic cells is unknown. We studied the effects of PP on dispersed acini prepared from rat pancreas. Amylase release was determined after 30-min incubations of pancreatic acini with octapeptide of cholecystokinin (OP-CCK), secretin, and PP. The response to OP-CCK was augmented by secretin. PP decreased optimal amylase release evoked by OP-CCK and OP-CCK plus secretin. These observations demonstrate that PP acts directly on pancreatic cells to reduce acinar cell amylase release evoked by potent secretagogues.

The relation between amylase secreted in vitro by rat parotid tissue and the amylase concentration in the glands

The assumption that the amount of amylase secreted by parotid glands is dependent on the glandular content is often made but seldom substantiated. This association was tested in vitro in parotid tissue. Fasting, feeding and feeding after pre-ganglionic cervical sympathectomy provided contrasting levels of glandular amylase. The subsequent release into fortified Krebs' medium was measured. Secretion was induced by 4 × 10 −7 M carbachol or isoprenaline. Only when the combined results from fasted and fed animals or fed sympathectomized and fed control rats were analysed were the total amounts of amylase released into the medium positively related to the initial amounts in the glands. This applied to spontaneously secreting, carbachol- and isoprenaline-stimulated tissue. When the results from each group were considered separately, this relationship was absent. When the secretion of amylase was expressed as a percentage of the total amylase in the gland, less was released from repleted glands, particularly in those stimulated by isoprenaline. Pre-treatment of tissues in vitro for 4 h with 1.25 × 10 −4 M colchicine impaired amylase release in response to isoprenaline only. Possible explanations for these findings relate to the different distribution and rate of synthesis of amylase in acinar cells of fasted and fed rats.

AMYLASE AND ELECTROLYTE CHANGES AFTER POSTGANGLIONIC PARASYMPATHECTOMY OF PAROTID GLAND.

Severance of the postganglionic parasympathetic innervation to the parotid gland of the rat resulted, at 4 weeks, in appreciable reduction in the size of the whole gland and of individual acinar cells, without evidence of an increase in the total number of these cells. Some increase in connective tissue was also observed in the denervated glands. Concentration of sodium in the denervated gland was increased, probably reflecting the increase in connective tissue. Sodium and potassium concentrations of the secretion and rate of secretory flow were essentially unchanged. Amylase was appreciably reduced in total amount and in concentration in the unstimulated parotid gland after denervation. Calculation of the concentration of amylase in acinar cells of the denervated gland showed that there was marked reduction in the concentration of this enzyme intracellularly. It was concluded that the parasympathetic innervation to the parotid gland is involved in maintenance of normal concentrations of amylase in the acinar cells but the mechanism of this regulation is not yet established.