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/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

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

Phenolic compounds affect intracellular free Ca2+ concentration ([Ca2+]i) signaling. The study examined whether the simple phenolic compound octyl gallate affects ATP-induced Ca2+ signaling in PC12 cells using fura-2-based digital Ca2+ imaging and whole-cell patch clamping. Treatment with ATP (100 micrometer) for 90 s induced increases in [Ca2+]i in PC12 cells. Pretreatment with octyl gallate (100 nM to 20 micrometer) for 10 min inhibited the ATP-induced [Ca2+]i response in a concentration-dependent manner (IC50=2.84 micrometer). Treatment with octyl gallate (3 micrometer) for 10 min significantly inhibited the ATP-induced response following the removal of extracellular Ca2+ with nominally Ca2+-free HEPES HBSS or depletion of intracellular Ca2+ stores with thapsigargin (1 micrometer). Treatment for 10 min with the L-type Ca2+ channel antagonist nimodipine (1 micrometer) significantly inhibited the ATP-induced [Ca2+]i increase, and treatment with octyl gallate further inhibited the ATP-induced response. Treatment with octyl gallate significantly inhibited the [Ca2+]i increase induced by 50 mM KCl. Pretreatment with protein kinase C inhibitors staurosporin (100 nM) and GF109203X (300 nM), or the tyrosine kinase inhibitor genistein (50 micrometer) did not significantly affect the inhibitory effects of octyl gallate on the ATP-induced response. Treatment with octyl gallate markedly inhibited the ATP-induced currents. Therefore, we conclude that octyl gallate inhibits ATP-induced [Ca2+]i increase in PC12 cells by inhibiting both non-selective P2X receptor-mediated influx of Ca2+ from extracellular space and P2Y receptor-induced release of Ca2+ from intracellular stores in protein kinase-independent manner. In addition, octyl gallate inhibits the ATP-induced Ca2+ responses by inhibiting the secondary activation of voltage-gated Ca2+ channels.
Adenosine Triphosphate ; Animals ; Calcium ; Constriction ; Extracellular Space ; Gallic Acid ; Genistein ; HEPES ; Indoles ; Maleimides ; Nimodipine ; PC12 Cells ; Phenol ; Protein Kinase C ; Protein-Tyrosine Kinases ; Thapsigargin

Interneuron diversity is one of the key factors to hinder understanding the mechanism of cortical neural network functions even with their important roles. We characterized inhibitory interneurons in layer II/III of the rat primary visual cortex, using patch-clamp recording and confocal reconstruction, and classified inhibitory interneurons into fast spiking (FS), late spiking (LS), burst spiking (BS), and regular spiking non-pyramidal (RSNP) neurons according to their electrophysiological characteristics. Global parameters to identify inhibitory interneurons were resting membrane potential (> -70 mV) and action potential (AP) width (< 0.9 msec at half amplitude). FS could be differentiated from LS, based on smaller amplitude of the AP (< ~50 mV) and shorter peak-to-trough time (P-T time) of the afterhyperpolarization (< 4 msec). In addition to the shorter AP width, RSNP had the higher input resistance (> 200 M omega) and the shorter P-T time (< 20 msec) than those of regular spiking pyramidal neurons. Confocal reconstruction of recorded cells revealed characteristic morphology of each subtype of inhibitory interneurons. Thus, our results provide at least four subtypes of inhibitory interneurons in layer II/III of the rat primary visual cortex and a classification scheme of inhibitory interneurons.
Action Potentials ; Animals ; Classification* ; Interneurons* ; Membrane Potentials ; Neurons ; Rats* ; Visual Cortex*

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