Rat Pancreatic Tissue Research Articles

Effect of somatostatin on the growth of gastrointestinal mucosa and pancreas in rats. Role of endogenous gastrin.

This study was undertaken to determine the influence of somatostatin on the growth of gastric, duodenal, and pancreatic tissue in rats placed on liquid diet. In the first part of the study animals were fed an elemental liquid diet for 10 days and then killed, and the growth of the oxyntic gland area of the stomach, 2 cm segments of duodenum and pancreatic tissue was determined. Feeding an elemental diet caused a decrease in organ weight, nucleic acid contents and serum gastrin level. Subsequent addition of pentagastrin prevented this reduction in organ weight and RNA and DNA contents while somatostatin failed to affect the decrease in growth parameters or serum gastrin level in tests with or without addition of pentagastrin. In a second part of the study, sham operated and antrectomised rats were used. Antrectomy caused a significant decrease in serum gastrin concentration and resulted in a significant reduction in the weight and RNA and DNA contents of the tissue tested. As in liquid diet, subsequent administration of pentagastrin prevented the reduction in the growth parameters both in tests with and without somatostatin. These results suggest that somatostatin inhibits the growth of the gastroduodenal mucosa by a mechanism involving, at least in part, the suppression of gastrin release.

Degranulation effect of ferric nitrilotriacetate (Fe3+-NTA) on the pancreatic islet beta-cells: its acute toxic effect on glucose metabolism.

A single injection of ferric nitrilotriacetate (Fe3+-NTA) caused a transitory increase in plasma immunoreactive insulin (IRI) and plasma immunoreactive glucagon (IRG) in rats. They reached maximum levels at 2 days after injection and returned to the normal range at 10 days. At 2 days after Fe3+-NTA injection, blood glucose level was normal but the glucose tolerance test (GTT) was impaired. There was a further increase in plasma IRI level and IRG level was suppressed after glucose loading. At 10 days after Fe3+-NTA injection, glucose tolerance was normal and IRI also returned to the normal range. No degenerative changes were found on H.E.-stained rat pancreatic tissue sections after Fe3+-NTA injection. Histochemical staining, however, showed a reduction in beta-granules and heavy metals (Timm's granules) from islet cells in the central area of the rat pancreatic islet 1 to 3 days after injection of Fe3+-NTA. The fading remained in some islets even at 10 days after injection, but by then the beta-granule distribution was restored in most islet cells. The results indicate a single Fe3+-NTA injection induced transitory instability of the pancreatic islet beta-cell granules and the glucose intolerance with a hyperresponse of IRI.

Ca 2+ and cyclic nucleotide dependence of amylase release from isolated rat pancreatic acinar cells rendered permeable by intense electric fields

Enzyme digestion of rat pancreatic tissue yielded a preparation of isolated acinar cells, over 90% of which excluded trypan blue. These isolated cells responded to a variety of secretagogues, the responses being sensitive to the removal of extracellular calcium, increasing extracellular magnesium, and by trifluoperazine, an antagonist of Ca-dependent processes. When exposed to intense electric fields, isolated acinar cells became permeable to CaEGTA and MgATP, these markers gaining access to over 60% of the intracellular mileu within minutes. The accessability to these markers seemed independent of the ionised Ca 2+ level. Less than 0.5% of the cellular amylase was released when cells were rendered leaky in a medium containing about 10 −9 M Ca 2+, but typically 4% was released when the Ca 2+ level was subsequently raised to 10 −5M levels, the EC 50 for Ca 2+ being 2 μM. This amount of amylase released was comparable to the amounts secreted from intact cells in response to a variety of agonists. The cytosolic marker lactate dehydrogenase was also released from leaky cells, but the extent was independent of Ca 2+ concentration. No amylase was released at 10 −7M Ca 2+ when permeable cells were exposed to cyclic 3′,5′-AMP or cyclic 3′,5′-GMP. The calcium activation curve for amylase release seemed to be independent of cyclic nucleotides, but was markedly increased in both the extent of release and apparent affinity for Ca 2+ in the presence of the phorbol ester 12-O-tetradecanoyl phorbol 13 acetate. These results suggest that when “functionally normal” isolated acinar cells are rendered permeable, Ca 2+ — but not cyclic nucleotides — acts as a second messenger for amylase secretion, and furthermore that protein kinase C may be involved in the secretory process.

Effects of nerve stimulation on enzyme secretion from the in vitro rat pancreas and 3H-release after preincubation with catecholamines.

In the presence of the cholinergic antagonist atropine, electrical field stimulation (FS) (5-20 Hz) caused a marked, reversible increase in the amylase output from superfused rat pancreatic segments. Adrenaline and noradrenaline evoked dose-dependent increases in amylase output which were similar to those produced by FS. The FS- and catecholamine-evoked amylase secretions were abolished by the beta-adrenergic antagonist propranolol. The FS-evoked secretion could be abolished by either the removal of external Ca2+ or the application of tetrodotoxin (TTX, 2 X 10(-6) M). FS also resulted in a reversible increase in the fractional efflux of tritium (3H) from rat pancreatic tissues preincubated with either 3H-noradrenaline or 3H-adrenaline. The effects of FS (5-20 Hz) on 3H efflux were abolished by TTX (2 X 10(-6) M). TTX had no effect on the enhancement of 3H efflux caused by elevation of external potassium concentration (high K+, 75 mM). Removal of superfusate Ca2+ completely abolished both the FS- and high K+-induced increases in 3H efflux. These observations suggest that intrinsic nerve stimulation (i.e. FS) results in the Ca2+-dependent release of sympathetic neurotransmitter, noradrenaline, which has a direct secretory action on the rat pancreas. Furthermore, the findings suggest that adrenaline can be taken up by nervous elements. This raises the possibility that uptake and re-release of circulating adrenaline might contribute to the control of rat pancreatic enzyme secretion by the adrenergic nervous system.

Hormonal and cholinergic effects on amylase and lysosomal enzyme activities in pancreatic tissue and ascites of rats with acute experimental pancreatitis

The effects of hormonal or cholinergic stimulation on survival and on activities of lysosomal enzymes and amylase in pancreatic tissue and ascites were studied in rats with induced pancreatitis. Pancreatitis per se caused an increase of the activities of cathepsin D, N-acetyl-β- d-glucosaminidase and amylase, and a decrease of acid phosphatase in pancreatic tissue. Pancreatic protein concentration was not influenced. In pancreatitic rats administration of cerulein or carbachol markedly decreased survival rate. Cerulein increased the activities of cathepsin D and amylase in ascites and cathepsin D and acid phosphatase in pancreatic tissue. Carbachol increased the activities of cathepsin D and amylase in ascites and acid phosphatase in pancreatic tissue. Both cerulein and carbachol decreased the activity of amylase in pancreatic tissue. Administration of secretin or the anticholinergic drug Pro-Banthine did not influence survival rate or the activities of lysosomal enzymes and amylase in ascites. In pancreatic tissue the activity of acid phosphatase was slightly increased by secretin or Pro-Banthine. In conclusion, the results show a nonparallel alteration of lysosomal enzyme activities in pancreatic tissue in rats with pancreatitis. Cerulein and cholinergic stimulation decreased survival rate and brought about a marked increase of cathepsin D activity in ascites and, in the case of cerulein, also in pancreatic tissue. The implication of lysosomes and especially the catheptic proteases in the pathogenesis and outcome of acute pancreatitis deserves further attention.

The presence of a substance in the pancreas that inhibits mitochondrial respiration

Evidence is presented which indicates that there is present in the pancreatic tissue of rats a substance, possibly some type of protein or a related substance, which inhibits mitochondrial respiration. The inhibitory substance may be associated or bound to cell membrane and is released on homogenization of the tissue. This substance may play a significant role in pancreatitis and shock.

Successful long-term cryopreservation of neonatal rat islet tissue

Neonatal rat pancreatic tissue was frozen to −196 °C using Me 2SO as a cryoprotectant and a slow freezing rate to −70 °C followed by immersion in liquid nitrogen. Rapid thawing was used. Viability was demonstrated by successful transplantation to streptozotocin-induced diabetic recipients. Long-term preservation, up to 4 weeks, did not demonstrably affect viability. Cryopreservation techniques may afford a method for providing a diabetic recipient the opportunity to receive a large quantity of pooled islet tissue from well-matched donors.

An improved method of isolation of rat pancreatic prokallikrein: Characterization of the zymogen and of the kallikrein produced by trypsin activation

A prokallikrein was purified 1600-fold from rat pancreatic tissue in an overall yield of 40% by a simple four-stage procedure. The final and crucial step was immunoaffinity chromotography utilizing antibody raised to a very small amount of prokallikrein. Both the pure zymogen and the active kallikrein generated from it by trypsin activation are single chain species with M r values of 38 400±300 and 35 500±400, respectively. Valine is the N-terminal amino acid residue of prokallikrein. The zymogen Was comparatively stable both to autoactivation and denaturation with respect to temperature and pH. The kallikrein produced by trypsin activation of the zymogen was similar in some of its catalytic properties to the kallikrein purified from autolyzed rat pancreas but the two species differed in their susceptibility to substrate activation.

Function of pancreatic endocrine cells in experimental acute pancreatitis.

AbstractThis experimental study was carried out to clarify changes in pancreatic endocrine function in acute pancreatitis. Male Donryu rats were divided into 2 groups, consisting of a group with acute pancreatitis induced by Block's method, and another group with simple laparotomy as control. Histological examination in the former group revealed an interstitial edematous pancreatitis, but there was no marked alteration of endocrine cells in spite of inflammatory changes in the exocrine region. Insulin and glucagon levels in the pancreatic tissue of rats with acute pancreatitis were significantly lower than those of control rats. Autoradiographic studies using3H‐leucine showed a significant decrease of grain counts for endocrine granules in rats with acute pancreatitis. The release of insulin from isolated islets of Langerhans was not reduced until 72 hours after the development of acute pancreatitis, while the release of glucagon from isolated islets was reduced 6 hours after the development of acute pancreatitis. These findings indicate that there is a reduction of synthesis and secretion function in endocrine cells of the pancreas in acute pancreatitis, and suggest that the damage to A cells occurs more rapidly than damage to B cells.

Kallikrein in rat pancreatic tissue after beta cell destruction or acinar cell atrophy.

The purpose of this study was to determine whether glandular kallikrein in rat pancreas is located in the beta cells of the endocrine pancreas or in the acinar cells of the exocrine pancreas. Kallikrein was measured by radial immunodiffusion and a direct radioimmunoassay in homogenates of pancreas obtained from 1) control rats, 2) rats with pancreatic beta cells selectively destroyed by streptozotocin, and 3) rats with acinar cell atrophy induced by pancreatic duct occlusion. Beta cell destruction was confirmed by the presence of hyperglycemia and by an almost total depletion of insulin-producing cells as demonstrated immunohistochemically. Acinar cell atrophy was confirmed histologically and by an almost total depletion of trypsin-like enzymes in pancreatic homogenates. The concentration of kallikrein in pancreatic homogenates was unchanged after beta cell destruction, whereas it was greatly decreased following acinar cell atrophy. Kallikrein was, by immunohistochemistry, demonstrated in the acinar cell only. The immunohistochemical localization of kallikrein agrees with the above results. These studies strongly indicate that kallikrein is predominantly located in the acinar cells of the exocrine pancreas.

Distribution of amylase and chymotrypsin in four regions of the rat pancreas.

Recent observations have confirmed the existence of more than one pool of secretory proteins in the rat pancreatic tissue. To determine if these different pools could be located in the different regions of the rat pancreas, the amylase and chymotrypsin contents have been measured in the biliary, duodenal, gastric, and splenic regions. On a tissue DNA content, protein content, or a fresh weight basis, the proportions of these two enzymes are comparable in the four regions. It is therefore postulated that heterogeneity of enzyme composition exists either within these regions or within the acinar cell itself.

Sizing of protein A-colloidal gold probes for immunoelectron microscopy.

Gold particles in colloidal solutions often vary considerably in size. The finest sols (diameter less than 15 nm), especially, are very heterogeneous, as is indicated by coefficients of variance (CV) of 25-35%. We have complexed staphylococcal protein A with gold particles (PA/Au) and then fractionated the preparations by glycerol or sucrose gradient centrifugation into very homogeneous subfractions. In this way, PA/Au probes of almost any size between 4.5 and 15 nm could be prepared. The variation of the gold particles in these fractions resulted in CV's between 9 and 16%. The reactivity of the PA/Au complex was not affected by the gradient procedure, as was shown by single- and double-labeling immunocytochemistry of ultrathin cryosections of rat pancreatic tissue.

Digestive end products mobilize secretory proteins from subcellular stores in the pancreas.

Two digestive end products, D-glucose and L-lysine, produced substantial concentration-dependent release of amylase and trypsinogen, respectively, from subcellular storage pools into a postmicrosomal supernatant fraction of rat pancreatic tissue homogenate. This process was selective in that D-glucose did not lead to trypsinogen release, while L-lysine did not effect amylase. An analogue of D-glucose, 2-deoxy-D-glucose, was much less potent than D-glucose on an equimolar basis. Half-maximal release for both end-product enzyme pairs occurred at concentrations within the range of normal plasma values for these end products in the rat. Although amylase release reached an apparent plateau when the concentration of glucose was increased beyond the maximally effective level, lysine concentrations higher than that maximally effective resulted in a fall in trypsinogen release that ultimately returned (at 3.0 mM L-lysine) to the level seen in its absence. When isolated zymogen granules were exposed to the same concentrations of D-glucose or L-lysine, a similar pattern of release was seen, indicating that the zymogen granules are a source of the enzymes released from the particulate phase of the homogenates. These findings can be explained most simply by the selective movement of digestive enzymes across zymogen granule membranes in response to the presence of appropriate end products. They are also consistent with the concept that digestive end products can act rapidly and directly on the pancreatic acinar cell to regulate the mixture of enzymes secreted in response to the specific hydrolytic needs of a meal.

Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue.

The recently developed low temperature embedding procedure with the resin Lowicryl K4M (Carlemalm E, Garavito M, Villiger W: Proc 7th Eur Cong Electron Microsc, 1980, p 656; Garavito M, Carlemalm E, Villiger W: Proc 7th Eur Cong Electron Microsc, 1980, p 658) was tested for its suitability for embedding of glutaraldehyde-fixed rat pancreatic tissue and for postembedding staining of thin sections with the protein A-gold (pAg) technique (Roth J, Bendayan M, Orci L: J Histochem Cytochem 26:1074, 1978) for amylase. Compared to conventional Epon embedding of glutaraldehyde fixed tissue, the low temperature embedding method with Lowicryl K4M resulted in a superior preservation of the general cellular fine structure, particularly in the Golgi apparatus. For low temperature embedded tissue, the quantitative evaluation of the immunocytochemical labeling for amylase showed a more specific staining of the rough endoplasmic reticulum, the Golgi apparatus, and the zymogen granules. This was due to a significant lowering of the background staining over all cellular organelles. The use of Lowicryl K4M at low temperature, due to the superior preservation, yields improved resolution and specificity in immunocytochemical postembedding staining.

Tween lipase 証明法の再吟味

Histochemical method of Tween lipase was studied on pancreatic tissues of male rats by means of reaction medium containing various amounts of glycerin, dimethylsulfoxide (DMSO)or triton X-100, at pH 6-8, for 3-40 hours. The medium (5% Tween 80; 1.0 ml,0.2M Tris HC1 buffer; 2.5m1,10% calcium chloride; 1. Oml, ad Aq. dist.; 25. Oml) containing 5%DMSO and 0.25% sodium taurocholate, final pH 6.2, was followed by the best demonstrati on of lipase activity. Holczinger's copper-rubeanic acid technique was also available for the visualizationSmall blocks of rat pancreatic tissues were fixed in buffered 2.5% glutaraldehyde for 3 hours, washed in cacodylate buffer overnight, cut 40μ by Vibratom. These tissue slices were incubated in above-mentioned medium at 37°C for 5 hours, rinsed in cacodylate buffer, immersed in 0.1% lead nitrate for 15 minutes, washed thoroughly in cacodylate buffer, post-fixed in osmium buffer, dehydrated in cold ethanols, and embedded in epon. The reaction products for lipase activity were observed in the cisternae of ER and Golgi apparatus, around the zymogen granules and lysosomes, and within the secretory canaliculi of pancreatic acini and the pancreatic ductules.

Exogenous secretory proteins do not inhibit carbamylcholine-stimulated release of pulse-labelled secretory proteins from rat pancreatic tissue slices

Experiments were conducted to test the concept that the results of secretion studies employing pancreatic tissue slices are significantly biased by the distribution of exocrine secretory proteins between the tissue and the incubation medium. The findings demonstrate (1) that the kinetics of release of pulse-labelled secretory proteins from cholinergically-stimulated tissue slices are independent of the concentration of exogenous exocrine secretory proteins and (2) that if there are bidirectional fluxes of secretory proteins across the cell membrane of pancreatic exocrine cells, these fluxes do not account for the fractional release of pulse-labelled secretory proteins.

Ultrastructural studies on the pancreatic polypeptide cell of the rat with special reference to pancreatic regional differences and changes induced by alloxan diabetes.

Rat pancreatic tissues were fixed with our modification of Dalton's fixative and the islets from various pancreatic regions were examined by electron microscopy. In addition, the effect of the alloxan-diabetic state on the pancreatic polypeptide cell was examined at the ultrastructural level. The results are as follows. (1) The islets of the rat contain four types of glandular cells: A, B, D and PP cells. (2) The PP cell is characterized by approximately 170-millimicron-sized secretory granules with a rather ill-defined core. The ultrastructure of this type cell is described in some detail. (3) In the pancreatic portion which hooks around posterior to the superior mesenteric vessels, PP cells are abundant and some of them are markedly degranulated. Their cell organelles, such as the nucleolus, granular endoplasmic reticulum and Golgi apparatus are well developed. (4) In alloxan-diabetic rats, B and PP cells show disappearance and numerical increase, respectively. In the PP cells, hypertrophy of the Golgi apparatus and appearance of extremely electron-translucent secretory granules are noted.

DL-ethionine treatment of adult pancreatic donors. Amelioration of diabetes in multiple recipients withe tissue from a single donor.

Transplantation of adult rat pancreatic islet tissue as a free graft requires the separation of islet from exocrine tissue to avoid host injury or graft destruction by digestive enzymes. The poor yield from islet isolation techniques currently necessitates the use of multiple donors to ameliorate diabetes in a single recipient. DL-ethionine (DLE) is an agent selectively toxic to the exocrine pancreas. We examined the effect of DLE administration on pancreatic digestive enzyme content and islet mass in adult Lewis rats and the ability of such pancreatic tissue dispersed by collagenase digestion without specific islet isolation to ameliorate diabetes when transplanted to the portal vein of syngeneic rats with streptozotocin induced diabetes. Rats fed normal chow supplemented with 0.5% DLE for 14-20 days showed a logarithmic loss of pancreatic mass. Total pancreatic amylase content declined to 0.3 + 0.1 mg, less than 3% of control values (14.3 +/- 1.0 mg). Total insulin content in DLE treated rats was 87 +/- 8 microg, not significantly different from control rats (101 +/- 7 microg). Histological examination confirmed the selective atrophy of exocrine tissue in DLE treated rats. Fresh pancreatic tissue prepared from a single DLE treated donor ameliorated diabetes 75% of the time when transplanted to one or two recipients and 65% of the time when divided between three of four recipients. Tissue prepared from a single DLE treated donor and stored for 24-48 hours ameliorated diabetes 91% of the time when divided between one or two recipients. Only four of 31 diabetic rats transplanted with fresh pancreatic tissue from untreated adult donors became normoglycemic. Pretreatment of adult rats with DLE induces selective exocrine atrophy, permits dispersed pancreatic tissue from a single donor to ameliorate experimental diabetes in up to four recipients, and allows tissue to be preserved by culture for up to 48 hours without specific islet isolation.

Immediate and Time-Dependent Effects of Glucose on Insulin Release from Rat Pancreatic Tissue

Glucose-induced insulin secretion is enhanced by a preceeding glucose stimulus. The characteristics of this action of glucose were investigated in perfused pancreas and collagenase-isolated islets of Langerhans. A 20- to 30-min pulse of 27.7 mM glucose enhanced both the first and second phase of insulin release in response to a second glucose stimulus by 76-201%. This enhancement was apparent as an augmented maximal insulin release response to glucose. The effect of priming with glucose was seen irrespective of whether the pancreatic tissue was obtained from fed or fasted rats. Separating the two pulses of hexose by a 60-min time interval of exposure to 3.3 mM glucose did not abolish the potentiation of the second pulse. Omission of Ca(++) as well as the inclusion of somatostatin or mannoheptulose during the first pulse abolished insulin secretion during this time period; however, only the inclusion of mannoheptulose deleted the potentiation of the second pulse. d-Glyceraldehyde, but not pyruvate, d-galactose, or 3-isobutyl-1-methylxanthine, could substitute for glucose in inducing potentiation. In islets labeled with [2-(3)H]adenine, the [(3)H]cyclic AMP response to glucose was increased by 35% when measured after 1 min, but was increased only marginally after 2-10 min of stimulation with a second pulse of glucose. The production of (3)H(2)O from glucose was not affected by glucose priming. It is concluded that (a) the induction of the glucose-induced, time-dependent potentiation described here is dependent on glucose metabolism but not on stimulation of cyclic AMP, calcium fluxes, or insulin release per se; (b) the mechanisms that mediate the pancreatic "memory" for glucose are unknown but do not seem to involve to a major extent an increased activity of the adenylate cyclase-cyclic AMP system of the beta-cell; (c) the evidence presented supports the hypothesis of a dual role of glucose for insulin release.

The cytochemical localization of adenylate cyclase: fact or artifact?

In a study of the location of adenylate cyclase activity in rat pancreas with the method of Reik et al. (Science 168:382, 1970), as modified by Howell and Whitfield (J Histochem Cytochem 20:873, 1972) it was found that (a) unspecific staining occurs in rat pancreatic tissue fragments incubated in the Reik-Howell medium in the absence of substrate; (b) addition of adenylyl-imidodiphosphate (AMP-PNP) as substrate, either alone or together with stimulants of rat pancreas adenylate cyclase (secretin. NaF), does not result in increased precipitation; (c) cytochemical incubation of isolated rat pancreatic acinar cells and of rat liver and kidney fragments does not lead to substrate-specific precipitation. In subsequent chemical studies we have found that cyclic adenosine monophosphate (AMP) formation from [alpha32P]AMP-PNP in the presence of rat pancreatic particulate matter is very low in the Reik-Howell medium without lead ions, but is stimulated by addition of lead nitrate (4 mM). Whereas heat-treatment of the particulate matter abolishes all cyclic AMP formation in the absence of lead ions, it actually increases cyclic AMP production in the presence of 4 mM lead nitrate. This indicates that the cyclic AMP formation in the complet Reik-Howell medium occurs by a nonenzymatic mechanism. In addition, this medium shows a tendency to become turbid, particularly when calcium ions are added to the medium, suggesting a possible explanation for the apparently specific cytochemical detection observed by other authors. A revised cytochemical medium, with barium replacing lead and with a pH of 8.9 (optimal for adenylate cyclase with AMP-PNP substrate), leaves rat pancreatic adenylate cyclase activity intact and hormone sensitive, while it is still able to precipitate imidodiphosphate. However, cytochemical incubation of isolated rat pancreatic acinar cells in this revised medium in the presence of AMP-PNP and secretin does not yield an electron-dense precipitate, showing that the enzyme activity is to low to produce sufficient imidodiphosphate. These findings throw further doubt on the validity of the cytochemical detection of adenylate cyclase, reported by other investigators, notwithstanding the alleged positive results.