BACKGROUND: Although hypochlorhydria, hypergastrinemia and antiparietal cell antibody have been well documented in the patients with hyperthyroidism, a cause of hypochlorhydria or hypergastrinemia is unknown at the present time. Therefore, in order to clarify an inhibitory mechansim of gastric acid secretion in the patients with hyperthyroidism, interrelationship among hypochlorhydria, hypergastrinemia and antiparietal cell antibody was investigated in this study. METHODS: The gastric secretory function, fasting and postprandial plasma concentrations of gastrin and titer of antiparietal cell antibody in the plasma were determined in the patients with hyperthyroidism and normal subjects. Immunoblot analysis was performed to identify the gastric membrane protein, a possible gastric antigen to antiparietal cell antibody. Using a immunocytochemical technique with electron microscopy, intracellular structure of the parietal cell reacted with antiparietal cell antibody was observed. RESULTS: The basal and pentagastrin-stimulated maximal acid output were reduced in the patients with hyperthyroidism. The fasting and postprandial plasma concentrations of gastrin were markedly elevated in the patients. The plasma gastrin concentration in the patients with the antiparietal cell antibody was higher than that of the norrnal subjects as well as the patients without the antibody not only in the fasting state but also in the postprandial state. However, the plasma gastrin concentration of the patients without the antiparietal cell antibody was elevated in the fasting state only. There was no difference in the gastrin content of the antral mucosa between the norrnal subjects and the patients. The antiparietal cell antibody was detected in 5 (38.5 %) out of 13 patients by using the indirect immunofluorescence method. Patient IgG dose-dependently inhibited rabbit gastric H (+),K (+)-ATPase activity. Among proteins of the rabbit gastric mucosa membrane, four high molecular weight proteins (91, 140, 170 and 210 K dalton) were reacted to the patient IgG. The patient IgG positive peroxidase-antiperoxidase (PAP) activity was electron microscopically detected on the intracellular cannalicular membrane of the parietal cell CONCLUSION: We conclude that hypochlorhydria and hypergastrinemia in the patients with hyperthyroidism are partially related to the antiparietal cell antibody and that the antigen to the antiparietal cell antibody may be H (+),K (+)-ATPase in the intracellular canalicular membrane of the parietal cell.
Achlorhydria ; Fasting ; Fluorescent Antibody Technique, Indirect ; Gastric Acid* ; Gastric Mucosa ; Gastrins ; Humans ; Hyperthyroidism* ; Immunoglobulin G ; Membrane Proteins ; Membranes ; Microscopy, Electron ; Molecular Weight ; Mucous Membrane ; Plasma ; Rabeprazole

A role of endogenous somatostatin in pancreatic exocrine secretion induced by intrapancreatic cholinergic activation was studied in the isolated rat pancreas perfused with modified Krebs-Henseleit solution. Intrapancreatic neurons were activated by electrical field stimulation (EFS: 15 V, 2 msec and 8 Hz). Pancreatic exocrine secretion, including volume flow and amylase output, and release of somatostatin from the pancreas were respectively determined. Somatostatin cells in the islet were stained with an immunoperoxidase method. EFS significantly increased pancreatic volume flow and amylase output, which were reduced by atropine by 59% and 78%, respectively. Intraarterial infusion of either pertussis toxin or a somatostatin antagonist resulted in a further increase in the EFS-evoked pancreatic secretion. EFS also further elevated exocrine secretion in the pancreas treated with cysteamine, which was completely restored by intraarterial infusion of somatostatin. EFS significantly increased not only the number of immunoreactive somatostatin cells in the islet but also the concentration of immunoreactive somatostatin in portal effluent. It is concluded from the above results that intrapancreatic cholinergic activation elevates pancreatic exocrine secretion as well as release of endogenous somatostatin. Endogenous somatostatin exerts an inhibitory influence on exocrine secretion induced by intrapancreatic cholinergic activation via the islet-acinar portal system in the isolated pancreas of the rat.
Amylases ; Animals ; Atropine ; Cysteamine ; Infusions, Intra-Arterial ; Neurons ; Pancreas ; Pertussis Toxin ; Portal System ; Rats ; Somatostatin* ; Somatostatin-Secreting Cells

Previously, we have reported that p-chlorophenylalanine (PCPA), a serotonin depletor, profoundly increased pancreatic fluid and bicarbonate secretion but remarkably inhibited pancreatic amylase secretion in anesthetized rats. The present study was performed to verify the detailed effects of PCPA on pancreatic amylase synthesis that is directly related to amylase exocrine secretion. PCPA significantly decreased pancreatic RNA and protein contents as well as the amylase activity. However, pancreatic DNA content, trypsin and chymotrypsin activities were not influenced by the treatment of PCPA. The rate of pancreatic amylase synthesis, which was assessed by the amount of incorporated (35S)-methionine into amylase for 1 h, was also significantly decreased by 44% in PCPA-treated rats. In order to determine whether the PCPA-induced decrease of amylase synthesis resulted from change in the level of amylase mRNA, Northern blot analysis was performed. The mRNA expression level of amylase was also decreased by 48% in the PCPA-treated rats, indicating that the inhibitory effect of PCPA on the synthesis of pancreatic amylase was mainly regulated at a step prior to translation. It was also revealed in SDS-polyacrylamide gel electrophoresis that the qualitative change of amylase was induced by PCPA. The 54 KDa amylase band seems to be degraded into small molecular weight protein bands in PCPA-treated rats, suggesting that the PCPA-induced decrease of amylase may be partly attributed to the degradation of synthesized amylase.
Amylases* ; Animals ; Blotting, Northern ; Chymotrypsin ; DNA ; Electrophoresis ; Fenclonine* ; Molecular Weight ; Pancreas ; Rats* ; RNA ; RNA, Messenger ; Serotonin ; Trypsin

Previously, we have isolated authentic bombesin and another bombesin like peptide named bombesin like immunoreactivity (BLI)-K2 from the skin of Korean fire-bellied toad, Bombina orientalis. In the present study, we have newly purified three heterogeneous forms of BLI named BLI-K3, BLI-K4, and BLI-K5 from side fractions obtained in previous isolation of bombesin like peptide. The BLIs were separated into five peaks on a column of C18 preparative HPLC. Among them, three minor peaks containing BLI-K3, K4, and K5 were purified by means of sequential chromatography on the columns of SP cation exchange HPLC and C18 reverse phase HPLC. The purified BLI-K3 and K4 showed high binding affinity to an anti-bombesin serum (LBE 2G-2) with binding potency of 72 and 95%, respectively, relative to that of bombesin. However, they did not possess any distinctive biological activity of bombesin like peptide. On the contrary, the biological activity of BLI-K5 was similar to that of bombesin but its binding affinity to an anti-bombesin serum was low. The results indicate that three heterogeneous forms of BLI were coexpressed with bombesin and BLI-K2 in the skin of B. orientalis. All forms of the purified BLI in the present study were immunologically active but only BLI-K5 possessed the distinctive biological activity of bombesin like peptide.
Anura* ; Bombesin* ; Chromatography ; Chromatography, High Pressure Liquid ; Population Characteristics ; Skin*

Aim of this study was to investigate if pancreatic polypeptide (PP) reduced the insulin action via the intra-pancreatic cholinergic nerves in the isolated rat pancreas. The pancreas was isolated from rats and perfused with intra-arterial infusion of modified Krebs-Henseleit solution containing 2.5 mM glucose at a flow rate of 1.2 ml/min. Simultaneous intra-arterial infusion of insulin (100 nM) resulted in potentiation of the pancreatic flow rate and amylase output which were stimulated by cholecystokinin (CCK, 14 pM). These potentiating actions of insulin on the CCK-stimulated pancreatic exocrine secretion were completely abolished by administration of rat PP. Vesamicol, a potent inhibitor of vesicular acetylcholine storage, and tetrodotoxin (TTX) also significantly reduced the combined actions of insulin and CCK. Administration of carbamylcholine, an acetylcholine agonist, completely restored the vesamicol-or TTX-induced inhibition of the potentiation between insulin and CCK. Also rat PP failed to attenuate the restoring effect of carbamylcholine. Electrical field stimulation (15-30 V, 2 msec and 8 Hz) resulted in a significant increase in the pancreatic flow rate and amylase output in voltage-dependent manner. Effects of electrical field stimulation were augmented by endogenous insulin. Rat PP also suppressed the pancreatic exocrine secretion stimulated by electrical field stimulation. These observations strongly suggest that PP inhibits the potentiating actions of insulin on CCK-stimulated pancreatic exocrine secretion by suppression of the intra-pancreatic cholinergic activity in the isolated rat pancreas.
Acetylcholine ; Amylases ; Animals ; Carbachol ; Cholecystokinin ; Cholinergic Agonists ; Glucose ; Infusions, Intra-Arterial ; Insulin ; Pancreas* ; Pancreatic Polypeptide* ; Rats* ; Tetrodotoxin

Palmitic acid (PAM), one of the most common saturated fatty acid (SFA) in animals and plants, has been shown to induce apoptosis in exocrine pancreatic AR42J cells. In this study, we investigated cellular mechanisms underlying protective effects of oleic acid (OLA) against the lipotoxic actions of PAM in AR42J cells. Exposure of cells to long-chain SFA induced apoptotic cell death determined by MTT cell viability assay and Hoechst staining. Co-treatment of OLA with PAM markedly protected cells against PAM-induced apoptosis. OLA significantly attenuated the PAM-induced increase in the levels of pro-apoptotic Bak protein, cleaved forms of apoptotic proteins (caspase-3, PARP). On the contrary, OLA restored the decreased levels of anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-xL, and Mcl-1) in PAM-treated cells. OLA also induced up-regulation of the mRNA expression of Dgat2 and Cpt1 genes which are involved in triacylglycerol (TAG) synthesis and mitochondrial beta-oxidation, respectively. Intracellular TAG accumulation was increased by OLA supplementation in accordance with enhanced expression of Dgat2 gene. These results indicate that restoration of anti-apoptotic/pro-apoptotic protein balance from apoptosis toward cell survival is involved in the cytoprotective effects of OLA against PAM-induced apoptosis in pancreatic AR42J cells. In addition, OLA-induced increase in TAG accumulation and up-regulation of Dgat2 and Cpt1 gene expressions may be possibly associated in part with the ability of OLA to protect cells from deleterious actions of PAM.
Animals ; Apoptosis ; bcl-2 Homologous Antagonist-Killer Protein ; Cell Death ; Cell Survival ; Gene Expression ; Humans ; Oleic Acid ; Palmitic Acid ; Proteins ; RNA, Messenger ; Triglycerides ; Up-Regulation

Dexamethasone converts pluripotent pancreatic AR42J cells into exocrine cells expressing digestive enzymes. In order to address molecular mechanism of this differentiation, we have investigated the role of mitogen-activated protein (MAP) kinase pathway and gene expressions of p21(waf1/cip1)and nuclear oncogenes (c-fos and c-myc) during AR42J cell differentiation. Dexamethasone markedly increased the intracellular and secreted amylase contents as well as its mRNA level. However, cell growth and DNA content were significantly decreased. With these phenotypic changes, AR42J cells induced transient mRNA expression of p21(waf1/cip1)gene, which reached maximal level by 6 h and then declined gradually toward basal state. In contrast to p21(waf1/cip1), c-fos gene expression was transiently inhibited by 6 h and then recovered to basal level by 24 h. Increased c-myc expression detected after 3 h, peaked by 12 h, and remained elevated during the rest of observation. Dexamethasone inhibited epidermal growth factor-induced phosphorylation of extracellular signal regulated kinase. Inhibition of MAP kinase pathway by PD98059 resulted in further elevation of the dexamethasone-induced amylase mRNA and p21(waf1/cip1)gene expression. These results suggest that p21(waf1/cip1)and nuclear oncogenes are involved in dexamethasone-induced differentiation and inhibition of MAP kinase pathway accelerates the conversion of undifferentiated AR42J cells into amylase-secreting exocrine cells.
Amylases/genetics ; Animals ; Cell Differentiation/*drug effects ; Cell Division/drug effects ; Cell Line, Tumor ; Cyclins/genetics/*metabolism ; Dexamethasone/*pharmacology ; Gene Expression Regulation/drug effects ; Genes, fos/genetics ; Genes, myc/genetics ; MAP Kinase Signaling System/*drug effects ; Mitogen-Activated Protein Kinases/*metabolism ; Pancreas/cytology/*drug effects/enzymology/metabolism ; RNA, Messenger/genetics/metabolism ; Rats ; Support, Non-U.S. Gov't

Phagocytosis of serum- and IgG-opsonized zymosan (SOZ and IOZ, respectively) particles into J774A.1 macrophages induced apoptosis of the cells, accompanied by the expression of p21(WAF1), one of cyclin-dependent protein kinase (CDK) inhibitors. Furthermore, phagocytosis of SOZ and IOZ particles into macophages induced superoxide formation. Tat-superoxide dismutase (SOD), which is readily transduced into the cells using Tat-domain, protected the cells from the apoptosis induced by phagocytosis of SOZ and IOZ particles. lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma) also caused the apoptosis of the cells. However, Tat-SOD could not protect the cells from LPS/IFN-gamma induced apoptosis, suggesting that apoptosis mechanisms involved are different from each other. In the present study, we determined the amounts of nitric oxide (NO) produced by SOZ, IOZ, and LPS/IFN-gamma, and found that SOZ and IOZ did not induce the generation of NO in macrophages, whereas LPS/ IFN-gamma did. The apoptosis due to phagocytosis was accompanied with the release of cytochrome c from mitochondrial membrane to cytosolic fraction. Furthermore, SOZ and IOZ induced the cleavage of procasapase-3 (35 kDa) to give rise to an active caspase-3 (20 kDa), which was blocked by Tat- SOD but not by 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), a scavenger of NO. On the other hand, LPS/IFN-gamma caused the activation of procaspase-3, which was blocked by PTIO but not by Tat-SOD. Taken together, phagocytosis of SOZ and IOZ particles induced apoptosis through superoxide but not NO in macrophages, accompanied with the release of cytochrome c and the activation of caspase-3.
Apoptosis/*immunology ; Caspases/metabolism ; Cell Line ; Cyclins/biosynthesis ; Cytochromes c/metabolism ; Immunoglobulin G/*immunology ; Interferon Type II/pharmacology ; Lipopolysaccharides/pharmacology ; Macrophages/*immunology/metabolism ; Nitric Oxide/*metabolism ; Opsonins/immunology ; Phagocytosis/*physiology ; Superoxide Dismutase/metabolism ; Superoxides/*metabolism ; Zymosan

Essential fatty acid (EFA) is known to be required for the body to function normally and healthily. However, the effect of EFA on glucose uptake in skeletal muscle has not yet been fully investigated. In this study, we examined the effect of two EFAs, linoleic acid (LA) and alpha-linolenic acid (ALA), on glucose uptake of C2C12 skeletal muscle cells and investigated the mechanism underlying the stimulatory effect of polyunsaturated EFAs in comparison with monounsaturated oleic acid (OA). In palmitic acid (PA)-induced insulin resistant cells, the co-treatment of EFAs and OA with PA almost restored the PA-induced decrease in the basal and insulin-stimulated 2-NBDG (fluorescent D-glucose analogue) uptake, respectively. Two EFAs and OA significantly protected PA-induced suppression of insulin signaling, respectively, which was confirmed by the increased levels of Akt phosphorylation and serine/threonine kinases (PKCtheta and JNK) dephosphorylation in the western blot analysis. In PA-untreated, control cells, the treatment of 500 microM EFA significantly stimulated 2-NBDG uptake, whereas OA did not. Phosphorylation of AMP-activated protein kinase (AMPK) and one of its downstream molecules, acetyl-CoA carboxylase (ACC) was markedly induced by EFA, but not OA. In addition, EFA-stimulated 2-NBDG uptake was significantly inhibited by the pre-treatment of a specific AMPK inhibitor, adenine 9-beta-D-arabinofuranoside (araA). These data suggest that the restoration of suppressed insulin signaling at PA-induced insulin resistant condition and AMPK activation are involved at least in the stimulatory effect of EFA on glucose uptake in C2C12 skeletal muscle cells.
Acetyl-CoA Carboxylase ; Adenine ; alpha-Linolenic Acid ; AMP-Activated Protein Kinases* ; Blotting, Western ; Fatty Acids, Essential* ; Glucose* ; Insulin ; Linoleic Acid ; Muscle, Skeletal* ; Oleic Acid ; Palmitic Acid ; Phosphorylation ; Phosphotransferases

The reactive oxygen species (ROS) are considered to be an important mediator in pancreatic beta cell destruction, thereby triggering the development of insulin-dependent diabetes mellitus. In the present study, HIV-1 Tat-mediated transduction of Cu, Zn-superoxide dismutase (SOD) was investigated to evaluate its protective potential against streptozotocin (STZ) -induced cytotoxicity in insulin-producing MIN6N cells. Tat-SOD fusion protein was successfully delivered into MIN6N cells in a dose-dependent manner and the transduced fusion protein was enzymatically active for 48 h. The STZ induced-cell destruction, superoxide anion radical production, and DNA fragmentation of MIN6N cells were significantly decreased in the cells pretreated with Tat-SOD for 1 h. Furthermore, the transduction of Tat-SOD increased Bcl-2 and heat shock protein 70 (hsp70) expressions in cells exposed to STZ, which might be partly responsible for the effect of Tat-SOD. These results suggest that an increased of free radical scavenging activity by transduction of Tat-SOD enhanced the tolerance of the cell against oxidative stress in STZ-treated MIN6N cells. Therefore, this Tat-SOD transduction technique may provide a new strategy to protect the pancreatic beta cell destruction in ROS-mediated diabetes.
Diabetes Mellitus, Type 1 ; DNA Fragmentation ; HIV-1 ; HSP70 Heat-Shock Proteins ; Insulin-Secreting Cells ; Oxidative Stress ; Reactive Oxygen Species ; Streptozocin ; Superoxide Dismutase ; Superoxides