BACKGROUND: NO (nitric oxide), derived from L-arginine through the action of nitric oxide synthase (NOS), is a short-lived free radical transmitting cellular signals for vasodilation, neurotransmission, and cytotoxicity. Recently, this molecule has been reported to be involved in the various glandular secretion. Although the relationship between NO and the pancreatic endocrine secretion has been widely investigated, the role of NO on insulin secretion has not been elucidated. Therefore, the present study was designed to reveal the precise action of NO on the secretion and synthesis of insulin following administration of NAME (L-NG -nitroarginine methyl ester) or L-arginine using immunocytochemistry and in situ hybridization techniques. METHODS: NAME or L-arginine was administered into jugular vein of the male Sprague-Dawley rat (180~200 g, b,w.) exhibiting normoglycemia (80~120mg/dL). Blood glucose concentrations were measured at intervals of 30 minutes for 2 hours after drug treatment. The pancreatic tissues were taken out at 30 and 90 minutes following drugs administration for insulin immunocytochemistry and in situ hybridization. RESULTS: Both NAME and L-arginine treatments diminished blood glucose levels. The decrease of blood glucose level was more prominent in NAME-treated rats than that of L-arginine. Insulin immunoreactivity in drugs-treated rat pancreas decreased compared to that in normal control, while the expression of insulin mRNA was significantly increased. CONCLUSION: On the basis of present study, it is concluded that the transient changes of NO con-centration, regardless of increase or decrease, in Langerhans islet might act as a potent stimulant in insulin secretion and its synthesis.
Animals ; Arginine ; Blood Glucose ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Insulin* ; Jugular Veins ; Male ; Nitric Oxide Synthase ; Nitric Oxide* ; Pancreas* ; Rats* ; Rats, Sprague-Dawley ; RNA, Messenger ; Synaptic Transmission ; Vasodilation

BACKGROUND: The incidence of atherosclerosis is well correlated with the progression of type 2 diabetes mellitus. High plasma glucose in uncontrolled diabetic patients evokes many vascular complications such as atherosclerosis. Specifically, high glucose was reported to induce thrombospondin-1 (TSP-1), which activates matrix metalloproteinase-2 (MMP-2) and leads to the invasion of vascular smooth muscle cells (VSMCs) into the intima. Catechins with antioxidant effects are known to inhibit MMP-2 activity. Therefore, this study was aimed at revealing the effect of epicatechin, one of catechins, on high glucose-induced TSP-1 and the invasiveness of VSMCs. METHODS: VSMCs were primarily isolated from Sprague-Dawley rat aorta. The VSMCs were incubated with different doses (30, 100 and 300 micrometer) of epicatechin under high glucose concentration (30 mM). The TSP-1 protein and mRNA expressions were analyzed by performing Western blotting and Northern blot analyses, respectively. RT-PCR was performed to observe the MMP-2 mRNA expression. Gelatin zymography was performed for the measurement of MMP-2 activity. Invasion assays were performed to evaluate the invasiveness of VSMCs. RESULTS: Epicatechin inhibited the high glucose-induced TSP-1 expression and the MMP-2 activity in a dose-dependent manner. Also, epicatechin inhibited the high glucose-induced invasiveness of VSMCs across the matrix barrier in a dose-dependent fashion. CONCLUSION: Collectively, epicatechin may prevent the high glucose-induced proliferation and invasion of VSMCs by inhibiting the TSP-1 expression and the MMP-2 activity. Therefore, epicatechin appears to play a protective role in the development of atherosclerosis.
Animals ; Antioxidants ; Aorta ; Atherosclerosis ; Blood Glucose ; Blotting, Northern ; Blotting, Western ; Catechin* ; Diabetes Mellitus, Type 2 ; Gelatin ; Glucose ; Humans ; Incidence ; Matrix Metalloproteinase 2 ; Muscle, Smooth, Vascular* ; Rats* ; Rats, Sprague-Dawley ; RNA, Messenger ; Thrombospondin 1*

BACKGROUND: The incidence of atherosclerosis is well correlated with the progression of type 2 diabetes mellitus. High plasma glucose in uncontrolled diabetic patients evokes many vascular complications such as atherosclerosis. Specifically, high glucose was reported to induce thrombospondin-1 (TSP-1), which activates matrix metalloproteinase-2 (MMP-2) and leads to the invasion of vascular smooth muscle cells (VSMCs) into the intima. Catechins with antioxidant effects are known to inhibit MMP-2 activity. Therefore, this study was aimed at revealing the effect of epicatechin, one of catechins, on high glucose-induced TSP-1 and the invasiveness of VSMCs. METHODS: VSMCs were primarily isolated from Sprague-Dawley rat aorta. The VSMCs were incubated with different doses (30, 100 and 300 micrometer) of epicatechin under high glucose concentration (30 mM). The TSP-1 protein and mRNA expressions were analyzed by performing Western blotting and Northern blot analyses, respectively. RT-PCR was performed to observe the MMP-2 mRNA expression. Gelatin zymography was performed for the measurement of MMP-2 activity. Invasion assays were performed to evaluate the invasiveness of VSMCs. RESULTS: Epicatechin inhibited the high glucose-induced TSP-1 expression and the MMP-2 activity in a dose-dependent manner. Also, epicatechin inhibited the high glucose-induced invasiveness of VSMCs across the matrix barrier in a dose-dependent fashion. CONCLUSION: Collectively, epicatechin may prevent the high glucose-induced proliferation and invasion of VSMCs by inhibiting the TSP-1 expression and the MMP-2 activity. Therefore, epicatechin appears to play a protective role in the development of atherosclerosis.
Animals ; Antioxidants ; Aorta ; Atherosclerosis ; Blood Glucose ; Blotting, Northern ; Blotting, Western ; Catechin* ; Diabetes Mellitus, Type 2 ; Gelatin ; Glucose ; Humans ; Incidence ; Matrix Metalloproteinase 2 ; Muscle, Smooth, Vascular* ; Rats* ; Rats, Sprague-Dawley ; RNA, Messenger ; Thrombospondin 1*

BACKGROUND: Activation of G-protein coupled-somatostatin receptors induces the release of calcium from inositol 1, 4, 5-trisphosphate-sensitive intracelluar stores. G-protein-coupled receptor signaling decreases with prolonged exposure to an agonist. SEBJECTS and METHODS: Fura-2-based digital Ca2+ imaging was used to study the effects of prolonged exposure to an agonist on the somatostatin-induced intracellular Ca2+ concentration([Ca2+]i) increases in NG108-15 cells, which were differentiated with CO2-independent medium and 10micrometer forskolin. RESULTS: Exposure to somatostatin(1micrometer) for 30 min completely desensitized the NG108-15 cells to a second somatostatin-induced response. The cells recovered gradually over 20 min following washout of the somatostatin. The desensitization was not due to depletion of the intracellular Ca2+ stores, and pretreatment for 30 min with bradykinin(100nM), which activates phospholipase C, or DADLE(D-Ala2-D-Leu5 enkephalin, 1microM), which activates phospholipase C, failed to cross-desensitize the somatostatin-evoked [Ca2+]i increases. Treatment with 8-cpt-cAMP(0.1mM) for 30min did not influence the somatostatin-induced[Ca2+]i increases. Phorbol 12, 13-dibutyrate(PdBu, 1microM) blocked the response completely. Down-regulation of PKC due to 24 h exposure of PdBu (1microM) inhibited the somatostatin-induced desensitization. CONCLUSION: Prolonged exposure of somatostatin to NG108-15 cells desensitized the somatostatin-induced release of Ca2+ from the intracelluar store, with protein kinase C also involved in the desensitization.
Calcium* ; Colforsin ; Down-Regulation ; Enkephalins ; GTP-Binding Proteins ; Inositol ; Protein Kinase C* ; Protein Kinases* ; Somatostatin ; Type C Phospholipases

BACKGROUND/AIMS: Pancreatic acini of streptozotocin (STZ)-induced diabetic rats release amylase less than normal acini on cholecystokinin (CCK) stimulation. Pancreatic enzyme secretion has been closely related to the intracellular calcium concentration ([Ca2+]i) of the acinar cell. In the present study, sequential changes of the intracellular calcium signal which probably underlie the altered enzyme secretion in response to CCK-8 were investigated using pancreatic acini from diabetic rats. METHODS: Diabetic rats were prepared by single intravenous injection of STZ (70 mg/kg). Stimulating experiments with CCK-8 were performed 7 days later. Pancreatic acini were isolated by collagenase digestion. Amylase release and [Ca2+]i were measured by colorimethod and calcium imaging, respectively. The geometry of intracellular calcium signal was analyzed. RESULTS: Normal acini exhibited concentration-dependent [Ca2+]i increase and regular oscillatory calcium signal on CCK-8 stimulation. Amylase release was also concentration-dependent. However, diabetic acini showed significantly less [Ca2+]i increase, prolonged time to peak [Ca2+]i, decreased calcium spikes number, and decreased amylase release compared with normal acini. The decreased [Ca2+]i in diabetic acini was restored significantly by insulin treatment. CONCLUSIONS: Relatively decreased amylase release in diabetic pancreatic acini in response to CCK, appears to be associated with altered calcium signal due to insulin deficiency.
Amylases/*secretion ; Animals ; Calcium Signaling/*drug effects ; Diabetes Mellitus, Experimental/*physiopathology ; Pancreas/cytology/metabolism/*secretion ; Rats ; Rats, Sprague-Dawley ; Sincalide/*pharmacology

To investigate the mechanism of smooth muscle contraction induced by emptying of intracellular Ca2+ stores, we measured isometric contraction and 45Ca2+ influx. CaCl2 increased Ca2+ store emptying- induced contraction in dose-dependent manner, but phospholipase C activity was not affected by the Ca2+ store emptying-induced contraction. The contraction was inhibited by voltage-dependent Ca2+ channel antagonists dose dependently, but not by TMB-8 (intracellular Ca2+ release blocker). Both PKC inhibitors (H-7 and staurosporine) and tyrosine kinase inhibitors (genistein and methyl 2,5-dihydroxycinnamic acid) significantly inhibited the contraction, but calmodulin antagonists (W-7 and trifluoperazine) had no inhibitory effect on the contraction. The combined inhibitory effects of protein kinase inhibitors, H-7 and genistein, together with verapamil were greater than that of each one alone. In Ca2+ store-emptied condition, 45Ca2+ influx was significantly inhibited by verapamil, H-7 or genistein but not by trifluoperazine. However combined inhibitory effects of protein kinase inhibitors, H-7 and genistein, together with verapamil were not observed. Therefore, this kinase pathway may modulate the sensitivity of contractile protein. These results suggest that contraction induced by emptying of intracellular Ca2+ stores was mediated by influx of extracellular Ca2+ through voltage-dependent Ca2+ channel, also protein kinase C and/or tyrosine kinase pathway modulates the Ca2+ sensitivity of contractile protein.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ; Animals ; Calmodulin ; Cats* ; Genistein ; Isometric Contraction ; Muscle, Smooth* ; Phosphotransferases ; Protein Kinase C ; Protein Kinase Inhibitors ; Protein-Tyrosine Kinases ; Trifluoperazine ; Type C Phospholipases ; Verapamil

Serotonin (5-hydroxytroptamine, 5-HT) has been shown to affect the induction of long-term potentiation (LTP) in the cortex such as the hippocampus, the visual cortex and the prefrontal cortex. Fluoxetine, as a selective serotonin reuptake inhibitor, is used in the management of a wide variety of psychological diseases. To study the effect of fluoxetine on the induction of LTP, we recorded the field potential in layer II/III of the frontal cortex from 3-wk-old. LTP was induced in horizontal input by theta burst stimulation (TBS). TBS with two-folds intensity of the test stimulation induced LTP, which was blocked by application of D-AP5 (50microM), an NMDA receptor antagonist. Whereas bath application of 5-HT (10microM) inhibited the induction of LTP, treatment with the 5-HT depleting agent, para-chloroamphetamine (PCA, 10microM), for 2hr did not affect the induction of LTP. Bath application of fluoxetine (1, 3, and 10microM) suppressed the induction of LTP in concentration-dependent manner, however, fluoxetine did not inhibit the induction of LTP in 5-HT-depleted slices. These results indicate that fluoxetine may inhibit the induction of LTP by modulating serotonergic mechanism in the rat frontal cortex.
Animals ; Baths ; Fluoxetine* ; Hippocampus ; Long-Term Potentiation* ; N-Methylaspartate ; p-Chloroamphetamine ; Prefrontal Cortex ; Rats* ; Serotonin ; Visual Cortex

To examine the localization pattern of phospholipase D2 (PLD2) in the pancreatic islet (the islet of Langerhans) depending on species, we conducted a morphological experiment in the rat and guinea pig. Since individual islets display a typical topography with a central core of B cell mass and a peripheral boundary of A, D, and PP cells, double immunofluorescent staining with a panel of antibodies was performed to identify PLD2-immunoreactive cells in the islets PLD2 immunoreactivity was mainly present in A and PP cells of the rat pancreatic islets. And yet, in the guinea pig, PLD2 immunoreactivity was exclusively localized in A cells, and not in PP cells. These findings suggest a possibility that PLD2 is mainly located in A cells of rodent pancreatic islets, and that the existence of PLD2 in PP cells is not universal in all species. Based on these results, it is suggested that PLD2 may play a significant role in the function of A and/or PP cells via a PLD-mediated signaling pathway.
Animals ; Antibodies ; Guinea Pigs* ; Guinea* ; Islets of Langerhans* ; Phospholipases* ; Rats* ; Rodentia

Muscle strips and muscle cells from cat stomach were used to investigate whether spontaneously formed cyclic nucleotides were involved in the inhibition of gastric smooth muscle contraction. A phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), increased the levels of both cyclic GMP (cGMP) and cyclic AMP (cAMP) in resting state cells, while decreasing acetylcholine-induced muscle contraction. Under the influence of IBMX, SQ22536, an adenylyl cyclase inhibitor and methylene blue, a guanylyl cyclase inhibitor completely blocked increases in cAMP and cGMP respectively, without any effect on contraction. However, the combination of SQ22536 and methylene blue completely blocked increases in both cAMP and cGMP levels and stimulated contractions markedly even in the presence of IBMX. Muscle contraction inhibitors such as isoprenaline, vasoactive intestinal polypeptide and sodium nitroprusside also appeared to increase cyclic nucleotide levels which decreased contraction. Which nucleotide increased the most was dependent on the agonist used. Therefore, irrespective of the cyclic nucleotide class, the spontaneous formation of cyclic nucleotides should be considered in evaluating the mechanism of gastric smooth muscle relaxation.
1-Methyl-3-isobutylxanthine ; Adenylyl Cyclases ; Animals ; Cats* ; Cyclic AMP ; Cyclic GMP ; Guanylate Cyclase ; Isoproterenol ; Methylene Blue ; Muscle Cells ; Muscle Contraction ; Muscle Relaxation* ; Muscle, Smooth ; Nitroprusside ; Nucleotides, Cyclic* ; Relaxation ; Stomach ; Vasoactive Intestinal Peptide

This study was performed to investigate the pancreatic exocrine dysfunction in streptozotocin- induced diabetic rats. Changes in pancreatic enzymes secretion and in pancreatic enzymes content were observed. The output and the tissue content of amylase were significantly reduced in diabetic rats, while the output and the content of lipase were increased. Plasma secretin and cholecystokinin (CCK) concentrations of diabetic rats were significantly increased compared to those of normal rats. The altered pancreatic exocrine function was abolished by the exogenous insulin administration. The exogenous insulin also restored the increased plasma secretin and CCK concentrations. From the above results, it is suggested that, in streptozotocin-induced diabetic rats, anticoordinated changes in pancreatic enzymes secretion as well as pancreatic enzymes content are attributable to insulin deficiency and that the insulin deficiency is responsible for the increased plasma concentrations of both secretin and CCK. However, it is not clear whether the elevated plasma secretin and CCK concentrations played a direct role in changes of pancreatic exocrine function.
Amylases ; Animals ; Cholecystokinin ; Gastrointestinal Hormones* ; Insulin ; Lipase ; Plasma ; Rats* ; Secretin ; Streptozocin