Endoplasmic Reticulum Of Acinar Cells Research Articles

Drug D, a Diosgenin Derive, Inhibits L-Arginine-Induced Acute Pancreatitis through Meditating GSDMD in the Endoplasmic Reticulum via the TXNIP/HIF-1α Pathway.

Acute pancreatitis (AP) is one of the most common causes of hospitalization for gastrointestinal diseases, with high morbidity and mortality. Endoplasmic reticulum stress (ERS) and Gasdermin D (GSDMD) mediate AP, but little is known about their mutual influence on AP. Diosgenin has excellent anti-inflammatory and antioxidant effects. This study investigated whether Diosgenin derivative D (Drug D) inhibits L-arginine-induced acute pancreatitis through meditating GSDMD in the endoplasmic reticulum (ER). Our studies were conducted in a mouse model of L-arginine-induced AP as well as in an in vitro model on mouse pancreatic acinar cells. The GSDMD accumulation in ER was found in this study, which caused ERS of acinar cells. GSDMD inhibitor Disulfiram (DSF) notably decreased the expression of GSDMD in ER and TXNIP/HIF-1α signaling. The molecular docking study indicated that there was a potential interaction between Drug D and GSDMD. Our results showed that Drug D significantly inhibited necrosis of acinar cells dose-dependently, and we also found that Drug D alleviated pancreatic necrosis and systemic inflammation by inhibiting the GSDMD accumulation in the ER of acinar cells via the TXNIP/HIF-1α pathway. Furthermore, the level of p-IRE1α (a marker of ERS) was also down-regulated by Drug D in a dose-dependent manner in AP. We also found that Drug D alleviated TXNIP up-regulation and oxidative stress in AP. Moreover, our results revealed that GSDMD-/- mitigated AP by inhibiting TXNIP/HIF-1α. Therefore, Drug D, which is extracted from Dioscorea zingiberensis, may inhibit L-arginine-induced AP by meditating GSDMD in the ER by the TXNIP /HIF-1α pathway.

L-ORNITHINE INDUCES ACUTE NECROTIZING PANCREATITIS IN RATS

Introduction: We have recently shown in a pilot study that L-ornithine (L-Orn), a downstream product of L-arginine, causes severe acute pancreatitis in rats. Aim: To determine dose- and time-responses of biochemical and histological parameters of L-Orn-induced pancreatitis. Methods: Male Wistar rats were injected i.p. with 1-6 g/kg L-Orn or 3 g/kg D-ornithine (D-Orn) and sacrificed 0-168 h or 1 month afterwards. Results: 3 g/kg L-Orn significantly increased the serum and ascitic amylase and pancreatic trypsin activities, whereas pancreatic amylase activity was decreased. We observed massive interstitial edema, inflammatory cell infiltration, apoptosis and necrosis of acinar cells. Electron microscopy of pancreatitic tissue revealed large autophagic vacuoles containing zymogen granules, lipid droplets, severe nuclear damage and dilated endoplasmic reticulum in acinar cells. L-Orn induced degradation of IkB proteins (associated with elevated interleukin-1β levels), oxidative stress and heat shock protein 72 expression in pancreas. Administration of 1-2 g/kg L-Orn or 3 g/kg D-Orn did not cause pancreatic damage, whereas 4-6 g/kg L-Orn killed the animals within hours (not due to pancreatitis). Conclusions: We have characterized dose- and time-responses of this new, non-invasive and reproducible model of acute pancreatitis. Supported by OTKA, MTA.

Caffeine-induced calcium release from the endoplasmic reticulum of acinar cells of the submandibular salivary gland

Ryanodine receptors (RyRs) play a key role in the generalization and spreading of calcium waves in excitable cells; however, the question of the existence of functionally active RyRs in nonexcitable cells demonstrating the capacity for exocytosis (e.g., salivary gland acini) remains open. We studied changes in the total amount of calcium stored in the endoplasmic reticulum (ER) of acinar cells of the submandibular salivary gland of rats and changes in the concentration of ionized Ca2+ inside the ER ([Ca2+]ER) using, respectively, a metallochrome dye, arsenazo III, and a low-affinity fluorescent dye, mag-fura 2/AM. In permeabilized cells, caffeine caused dose-dependent decreases in the total amount of calcium and concentration of ionized calcium. The effective concentration of caffeine providing a 50% drop in the [Ca2+]ER (EC50) was, on average, 7.3 ± 1.1 mM. The caffeine-induced drop in the [Ca2+]ER was insensitive to heparin; in addition, it was blocked by high concentrations (100 µM) of ryanodine, potentiated by ryanodine applied in mild concentrations (10 µM), and also demonstrated a bell-shaped dependence on the concentration of cytoplasmic Ca2+. Such peculiarities are typical characteristics of the RyR-mediated reaction. Therefore, functional RyRs whose activation results in a transient release of calcium from the ER are present in acinar cells of the submandibular salivary gland.

Mechanisms Underlying Leakage of Calcium from the Endoplasmic Reticulum of Acinar Cells of the Submandibular Salivary Gland

In the resting state, the Ca2+ concentration in agonist-sensitive intracellular stores reflects the balance between active uptake of Ca2+, which is mediated by Ca2+-ATPase (SERCA), and passive leakage of Ca2+. The mechanisms underlying such a leakage in cells of the submaxillary salivary gland were not studied. In our experiments, we examined possible pathways of passive leakage of Ca2+ from the endoplasmic reticulum (ER) of acinar cells obtained from the rat submaxillary salivary gland; direct measurements of the concentration of Ca2+ in the ER ([Ca2+]ER) using a low-affinity calcium-sensitive dye, mag-fura 2/AM, were performed. The cellular membrane was permeabilized with the help of β-escin (40 μg/ml); the Ca2+ concentration in the cytoplasm ([Ca2+] i ) was clamped at its level typical of the resting state (∼100 nM) using an EGTA/Ca2+ buffer. Incubation of permeabilized acinar cells in a calcium-free intracellular milieu, as well as application of thapsigargin, resulted in complete inhibition of the uptake of Ca2+ with the involvement of SERCA. This effect was observed 1 min after the beginning of superfusion of the cells with the corresponding solutions and was accompanied by the leakage of Ca2+ from the ER; this is confirmed by a gradual drop in the [Ca2+]ER. Such a leakage of Ca2+ remained unchanged in the presence of thapsigargin, heparin, and ruthenium red; therefore, it is not mediated by SERCA, inositol 1,4,5-trisphosphate-sensitive receptors (InsP3R), or ryanodine receptors (RyRs). At the same time, an antibiotic, puromycin (0.1 to 1.0 mM), which disconnects polypeptides from the ER-ribosome translocon complex, caused intensification of passive leakage of Ca2+ from the ER. This effect did not depend on the functioning of SERCA, InsP3R, or RyR. Therefore, passive leakage of Ca2+ from the ER in acinar cells of the submaxillary salivary gland is realized through pores of the translocon complex of the ER membrane.

Ultrastructural Study of the Effects of Stress on the Pancreas in Rats

We studied morphologic changes in a rat model of acute hemorrhagic pancreatitis in order to investigate the mechanism by which water immersion stress injures the pancreas. Acute hemorrhagic pancreatitis was induced by two intraperitoneal injections of 40 micrograms/kg body weight of caerulein at 1-h intervals under water immersion stress for 5 h at 23 degrees C. Light microscopy showed interstitial edema with inflammatory cell infiltration, degeneration and necrosis of acinar cells, and bleeding. Electron microscopy showed large autophagic vacuoles, decreased zymogen granules, and dilated rough endoplasmic reticulum in acinar cells. Basolateral exocytosis of large vacuoles and phagocytosis of the degenerated acinar cells were observed. In addition, microvascular damage, including the destruction of the capillary endothelial cells, capillary thrombosis, and the extravasation of blood cells, was seen. In contrast, in a pancreatitis model induced by caerulein injection alone, there was no bleeding, no remarkable vascular change, and no thrombosis. Degeneration and necrosis of acinar cells were less severe. In the pancreas under stress alone, microvascular damage and degeneration of acinar cells were observed. These findings demonstrate that stress injures the pancreas and worsens the pancreatitis by causing microcirculatory disturbances, such as vascular damage, thrombosis, increased vascular permeability, and bleeding. These results suggest that chemical mediators, such as free radicals and platelet-activating factor (PAF), which are produced by vascular damage and thrombosis, may accelerate the activation of zymogen proteases in acinar cells in caerulein-induced pancreatitis, leading to hemorrhagic pancreatitis.

Alcohol and the integrity of the pancreas.

Alcohol may affect the integrity of the pancreatic parenchyma, as seen in alcoholic pancreatitis, some cases of chronic alcoholism without clinical pancreatitis, and experimental studies. The composition of pancreatic juice may reflect some of these changes. One type of parenchymal alteration is the loss of differentiative features of acinar cells, so that they take on the characteristics of ductular cells. Concomitant fibrosis completes the formation of the tubular complexes found in association with alcoholic chronic pancreatitis. Sustained alcohol intake may produce the accumulation of lipid droplets in parenchymal cells, some of which may be shown to be within the rough endoplasmic reticulum of acinar cells. Epithelial cells may undergo mucous metaplasia. The epithelial-basal lamina barrier frequently is breached in the area of intraluminal aggregates, with or without obvious inflammation in the immediate area. Loss of barrier function may lead to interaction among components of the external compartment (lumen) and the internal compartment (stroma). Increased levels of blood proteins and glycosaminoglycans in the juice, enzyme activation, fibrin formation, and complement activation are potential consequences of barrier loss. Increased lactoferrin levels could result in part from the activity and degranulation of polymorphonuclear leukocytes.

Primary structure of two distinct rat pancreatic preproelastases determined by sequence analysis of the complete cloned messenger ribonucleic acid sequences.

The mRNA sequences for two rat pancreatic elastolytic enzymes have been cloned by recombinant DNA technology and their nucleotide sequences determined. Rat elastase I mRNA is 1113 nucleotides in length, plus a poly(A) tail, and encodes a preproelastase of 266 amino acids. The amino acid sequence of the predicted active form of rat elastase I is 84% homologous to porcine elastase 1. Key amino acid residues involved in determining substrate specificity of porcine elastase 1 are retained in the rat enzyme. The activation peptide of the zymogen does not appear related to that of other mammalian pancreatic serine proteases. The mRNA for elastase I is localized in the rough endoplasmic reticulum of acinar cells, as expected for the site of synthesis of an exocrine secretory enzyme. Rat elastase II mRNA is 910 nucleotides in length, plus a poly(A) tail, and encodes a preproenzyme of 271 amino acids. The amino acid sequence is more closely related to porcine elastase 1 (58% sequence identity) than to the other pancreatic serine proteases (33-39% sequence identity). Predictions of substrate preference based upon key amino acid residues that define the substrate binding cleft are consistent with the broad specificity observed for mammalian pancreatic elastase 2. The activation peptide is similar to that of the chymotrypsinogens and retains an N-terminal cysteine available to form a disulfide link to an internal conserved cysteine residue.

Pathologic Findings In the Pancreas of Mice Infected With Coxsackievirus B4

Pathologic changes in the exocrine and endocrine portions of the pancreas were observed in suckling and young adult mice infected with coxsackievirus B4. Thirty-two of 56 infected mice studied histologically showed significant cellular alterations. Generally, the changes ranged from focal and mild degeneration to widespread necrosis and inflammation. Electron micrographs of other infected mice showed derangement of the endoplasmic reticulum in acinar cells and various degrees of degranulation and cellular disruption in the islets. It seems likely that viruses may produce similar damage to the pancreas in man, and it is postulated that viral pancreatitis may cause subclinical diabetes as well as overt diabetes mellitus of varying degrees. For some time we have been interested in the pathologic changes in a variety of organs of animals infected with the coxsackievirus B4. Our attention has been primarily directed toward the myocardium and associated structures, ie, endocardium and valves, 1-6 and to

Cytotoxic effects of puromycin on the Golgi apparatus of pancreatic acinar cells, hepatocytes and ameloblasts.

A single injection of puromycin was given to rats in a dose sufficient to produce almost total inhibition of protein synthesis in pancreas and liver. By 10 min after injection, Golgi saccules in pancreatic acinar cells, hepatocytes and ameloblasts appeared grossly and irregularly distended, and almost devoid of content. In pancreas the condensing vacuoles near the inner face of the Golgi were often altered, and those normally present in ameloblasts were lacking. Between 2 and 3 hr after injection, protein synthesis had started anew. At this time, granules without limiting membranes appeared within cisternae of the rough endoplasmic reticulum in acinar cells and ameloblasts. These intracisternal granules are believed to consist of newly synthesized secretory protein which could not be transported through the disrupted Golgi apparatus to be packaged into secretory granules. Indeed, by 3 hr postinjection the secretory granules which normally abound in the apical processes of ameloblasts were sparce or absent. Thus, while biochemical evidence indicates that puromycin blocks protein synthesis on the ribosomes, the use of this antibiotic in vivo results in alterations in the Golgi apparatus and interruption of the packaging of protein into secretory granules.

Acinar-islet cells in the exocri pancreas of the adult cat.

Observations on a human pancreas in a case of the Zollinger-Ellison syndrome led us to postulate that secretin is capable of inducing nesidioblastosis (neoformation of islets). In order to test this hypothesis, a group of adult cats was given Pansecrin (Squibb secretin); a control group received normal saline. Acinar-islet cells containing in some cases both zymogen and islet-type secretory granules as well as the endoplasmic reticulum of acinar cells were seen in pancreases of secretin-treated cats. The islet-type secretory granule seen in acinar-islet cells resembled those of alpha-type islet cells.