RESUMEN
1 To investigate anti-inflammatory activity of organic germanium, we measured the effect of germanium-concentrated yeast on arachidonic acid release, prostaglandin E(2) (PGE(2)) production, histamine release, and intracellular H(2)O(2) or hydroperoxide generation in RBL 2H3 cells, and carrageenan-induced paw oedema in rats. 2 Germanium-concentrated yeast dose-dependently inhibited carrageenan-induced paw oedema, suggesting that germanium-concentrated yeast has anti-inflammatory activity in acute inflammation. 3 Germanium-concentrated yeast significantly inhibited melittin-induced arachidonic acid release and PGE(2) production in RBL 2H3 cells. 4 Germanium-concentrated yeast did not affect melittin-induced histamine release and silica-induced intracellular H(2)O(2) or hydroperoxide generation in RBL 2H3 cells. 5 These results suggest that anti-inflammatory activity of germanium-concentrated yeast appears partly to be related to the inhibition of arachidonic acid release and PGE(2) production in RBL 2H3 cells.
Asunto(s)
Antiinflamatorios/farmacología , Ácido Araquidónico/metabolismo , Dinoprostona/metabolismo , Edema/prevención & control , Germanio/farmacología , Compuestos Organometálicos/farmacología , Levaduras/química , Administración Oral , Animales , Carragenina , Línea Celular Tumoral , Dinoprostona/biosíntesis , Relación Dosis-Respuesta a Droga , Edema/inducido químicamente , Edema/patología , Germanio/química , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Meliteno , RatasRESUMEN
1. This study was aimed at evaluating the effect of rutin and harmaline (1-methyl-7-methoxy-3,4-dihydro-beta-carboline) on the development of the surgically induced reflux oesophagitis, on gastric secretion, lipid peroxidation, polymorphonucleocytes (PMNs) accumulation, superoxide and hydroxyl radical production in PMNs, cytokine [interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha)] production in blood and [Ca2+]i mobilization in PMNs. 2. Rutin and harmaline significantly prevented the development of reflux oesophagitis and gastric secretion. Treatments of oesophagitis rats with rutin and harmaline inhibited lipid peroxidation, and myeloperoxidase (MPO) in the oesophagus in comparison with untreated rats. 3. Superoxide anion and hydrogen peroxide production in 1 microm formylmethionylleucylphenylalanine (fMLP)- or 0.1 microg ml-1N-phorbol 12-myristate 13-acetate (PMA)-activated PMNs was inhibited by rutin and harmaline in a dose-dependent fashion. Rutin and harmaline effectively scavenged the hydroxyl radical and hydrogen peroxide. Treatments of oesophagitis rats with rutin and harmaline inhibited IL-1beta production in the oesophagus in comparison with untreated rats, but TNF-alpha production was not affected by rutin and harmaline. The fMLP-induced elevation of [Ca2+]i was inhibited by rutin. 4. The results of this study suggest that rutin and harmaline may have beneficial protective effects against reflux oesophagitis by the inhibition of gastric acid secretion, oxidative stress, inflammatory cytokine production (i.e. IL-1beta), and intracellular calcium mobilization in PMNs in rats.
Asunto(s)
Esofagitis Péptica/prevención & control , Harmalina/uso terapéutico , Rutina/uso terapéutico , Animales , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Inducción Enzimática/efectos de los fármacos , Esofagitis Péptica/metabolismo , Esófago/enzimología , Ácido Gástrico/metabolismo , Harmalina/farmacología , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/fisiología , Masculino , Peroxidasa/biosíntesis , Ratas , Ratas Sprague-Dawley , Rutina/farmacologíaRESUMEN
This study investigated the influence of zinc-oxide eugenol (ZOE) temporary restorations on microleakage in composite restorations. Class V cavities were prepared on the buccal surfaces of 32 freshly extracted, non-carious human premolars. The teeth were randomly divided into four groups of eight teeth. Specimens in Group 1 (control) received no temporary restoration. Group 2 and 3 specimens were covered with IRM (Type III ZOE cement) mixed at powder:liquid (P:L) ratio of 10g:1g and 10g:2g, respectively. Specimens in Group 4 were covered with poly-carboxylate cement (eugenol-free) mixed at a P:L ratio of 2.85g:1g. The temporary restorations were removed mechanically with an ultrasonic scaler after one week storage in distilled water at 37 degrees C. The preparations were washed and restored with Scotchbond Multi-Purpose Plus and Z100 according to manufacturers' instructions. The restorations were finished, thermally stressed for 400 cycles and subjected to dye penetration testing. Results were analyzed using Kruskal-Wallis and Mann-Whitney tests at a significance level of 0.05. At both enamel and dentin margins, the microleakage associated with Group 3 was significantly greater than for Groups 1, 2 and 4. For Groups 1 and 4, leakage at the dentin margins was significantly greater than in enamel margins. For the groups pretreated with IRM, no significant difference in dye penetration scores was observed between enamel and dentin. Pre-treatment with IRM mixed at a P:L ratio of 10g:2g significantly increased microleakage and is not recommended clinically.
Asunto(s)
Resinas Compuestas/química , Filtración Dental/clasificación , Restauración Dental Permanente , Restauración Dental Provisional , Dióxido de Silicio , Cemento de Óxido de Zinc-Eugenol/química , Circonio , Grabado Ácido Dental , Diente Premolar , Colorantes , Preparación de la Cavidad Dental/clasificación , Esmalte Dental/ultraestructura , Raspado Dental/instrumentación , Dentina/ultraestructura , Recubrimientos Dentinarios/química , Humanos , Ensayo de Materiales , Metilmetacrilatos/química , Cemento de Policarboxilato/química , Polvos , Cementos de Resina/química , Estadísticas no Paramétricas , Temperatura , Termodinámica , Factores de Tiempo , Terapia por Ultrasonido/instrumentación , Agua/químicaRESUMEN
BACKGROUND: Ethanol is generally believed to inhibit extracellular Ca2+ influx, thereby inhibiting gastric muscle contraction. Recently, we observed that verapamil inhibited only the amplitude of spontaneous phasic contractions, whereas ethanol inhibited both amplitude and frequency. In our objective to investigate the mechanism of ethanol's inhibition of gastric motility, the involvement of various protein kinases in ethanol-inhibited spontaneous phasic contractions of the stomach muscle strips was tested. METHODS: Circular muscle strips (2.0 x 0.2 cm) were prepared from the corpus of cat stomach in order to measure isometric contraction in a chamber filled with Krebs-Ringer solution (pH 7.4, temperature 36 degrees C) bubbled with 5% CO2 in O2. RESULTS: Spontaneous phasic contraction was not affected by various receptor antagonists (I microM atropine, 1 microM hexamethonium, 1 microM phentolamine and 1 microM propranolol) or 1 microM tetrodotoxin. EGTA and verapamil dose-dependently inhibited only the amplitude of spontaneous phasic contractions and not the frequency. Ethanol dose-dependently inhibited both the amplitude and frequency of phasic contractions. The amplitude and frequency of spontaneous phasic contractions were significantly inhibited by protein kinase C and tyrosine kinase inhibitors. However, neither protein kinase C activator nor various phosphatase inhibitors blocked the inhibitory effect of ethanol. CONCLUSIONS: Ethanol appears to inhibit spontaneous phasic contractions by a mechanism other than the inhibition of protein kinase C or tyrosine kinase or the inhibition of extracellular Ca2+ influx.
Asunto(s)
Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Contracción Isométrica/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Estómago/efectos de los fármacos , Animales , Gatos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Motilidad Gastrointestinal/efectos de los fármacos , Motilidad Gastrointestinal/fisiología , Contracción Isométrica/fisiología , Masculino , Músculo Liso/fisiopatología , Proteínas Quinasas/farmacología , Estómago/fisiopatología , Factores de TiempoRESUMEN
This study investigated the influence of eugenol-containing temporary restorations on bond strength of composite to dentin. Thirty-two freshly extracted human molars were embedded and horizontally sectioned at a level 2 mm from the central fossa to obtain a flat dentin surface. The teeth were randomly divided into four groups of eight teeth. Specimens in Group 1 (control) received no pre-treatment with any temporary restorations. Group 2 and 3 specimens were covered with IRM (eugenol-containing) mixed at powder: liquid (P:L) ratio of 10 g: 1 g and 10 g: 2 g, respectively. Specimens in Group 4 were covered with polycarboxylate cement (eugenol-free) mixed at a P:L ratio of 2.85 g: 1 g. The temporary restorations were mechanically removed with an ultrasonic scaler after one-week storage in distilled water at 37 degrees C. The dentin surfaces were cleaned with pumice-water slurry and treated with Scotchbond Multi-Purpose Plus bonding system according to manufacturer's instructions. Composite (Z100) columns (3 mm diameter, 2 mm high) were applied and shear bond testing was carried out after 24 hours storage in distilled water at 37 degrees C using an Instron Universal testing machine with a cross-head speed of 0.5 mm/minute. The mode of failure was examined using a stereomicroscope at X40 magnification. Results were analyzed using one-way ANOVA/Scheffes's post-hoc test at significance level 0.05. Ranking of bond strengths was as follows: Group 1 (22.58 MPa) > Group 2 (21.14 MPa) > Group 4 (15.35 MPa) > Group 3 (13.02 MPa). Group 3 had significantly lower bond strength than Groups 1 and 2. No significant difference in dentin bond strength was observed between the Group 1 (control) and Groups 2 and 4. Although the predominant mode of failure for Groups 1, 2 and 4 was cohesive in dentin, all specimens in Group 3 exhibited adhesive failure. Pre-treatment with polycarboxylate cement or IRM mixed at P:L ratio of 10 g: 1 g did not affect shear bond strength of composite to dentin. IRM mixed at a lower P:L ratio of 10 g: 2 g significantly decreased bond strength.
Asunto(s)
Resinas Compuestas , Recubrimiento Dental Adhesivo , Fracaso de la Restauración Dental , Restauración Dental Provisional , Recubrimientos Dentinarios , Eugenol , Metilmetacrilatos , Cemento de Óxido de Zinc-Eugenol , Análisis de Varianza , Dentina , Cementos de Ionómero Vítreo , Humanos , Tercer Molar , Distribución Aleatoria , Cementos de Resina , Dióxido de Silicio , Estadísticas no Paramétricas , CirconioRESUMEN
To understand the underlying mechanism of ethanol in tonic contraction, the effect of ethanol on phospholipase A(2) and phospholipase C activities and the effects of phospholipase inhibitors on ethanol-induced contraction of cat gastric smooth muscle were tested. Circular muscle strips (2.0 x 0.2 cm) obtained from the fundus of cat stomach were used to measure isometric contraction. Ethanol elicited tonic contraction and activated phospholipase A(2) activity in a dose-dependent manner. Phospholipase A(2) inhibitors, manoalide (0.1--10 microM) and oleyloxyethyl phosphorylcholine (1--10 microM), significantly inhibited ethanol-induced contraction. Furthermore, 342 mM ethanol-induced contraction was significantly inhibited by cyclooxygenase inhibitors, ibuprofen (10--100 microM) and indomethacin (10--100 microM), but not by lipoxygenase inhibitors. On the other hand, phospholipase C inhibitors had no effect on ethanol-induced contraction, indicating that phospholipase C is not involved in ethanol-induced contraction. It is suggested from the above results that ethanol-induced contraction in cat gastric smooth muscle is, in part, mediated by phospholipase A(2) and cyclooxygenase pathways.
Asunto(s)
Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Fundus Gástrico/efectos de los fármacos , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Fosfolipasas A/efectos de los fármacos , Fosforilcolina/análogos & derivados , Animales , Gatos , Inhibidores de la Ciclooxigenasa/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Fundus Gástrico/metabolismo , Ibuprofeno/farmacología , Indometacina/farmacología , Fosfatos de Inositol/metabolismo , Masculino , Contracción Muscular/fisiología , Músculo Liso/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Fosfolipasas A/metabolismo , Fosforilcolina/farmacología , Terpenos/farmacologíaRESUMEN
BACKGROUND: Smooth muscle relaxation induced by various agents that increase the cellular levels of cyclic nucleotides (cAMP and cGMP) is accompanied by a decrease in intracellular Ca2+ concentration. However, little is known about the differences between the inhibitory effects of cAMP and cGMP on the contraction of smooth muscle. OBJECTIVE: To compare the effects and underlying mechanisms of cAMP and cGMP on the inhibition of gastric smooth muscle contraction, cyclic nucleotide promoting agents, as well as cell membrane permeable cyclic nucleotides were used. METHODS: Isometric contraction was measured from circular muscle strips prepared from the fundus of cat stomach in a cylinder-shaped chamber filled with Krebs-Ringer solution (pH 7.4, temperature 36 degrees C) bubbled with 5% CO2 in O2. The level of inositol phosphates (IPs) was measured. RESULTS: Forskolin and sodium nitroprusside significantly inhibited acetylcholine (ACh)-induced gastric smooth muscle contraction and increased the cellular levels of cAMP and cGMP, respectively. Direct application of 8-Br-cAMP and 8-Br-cGMP also significantly inhibited ACh-induced contraction. Both verapamil and TMB-8 inhibited ACh-induced contraction. The combined inhibitory effect of verapamil and TMB-8 was significantly greater than the effect of either one, separately. Forskolin or sodium nitroprusside similarly augmented the effect of verapamil. However, the inhibitory effect of TMB-8 was augmented only by 8-Br-cGMP or sodium nitroprusside but not by 8-BrcAMP or forskolin. Forskolin and 8-Br-cAMP significantly inhibited the formation of inositol phosphates stimulated by ACh. CONCLUSIONS: cAMP inhibits the contraction mechanism associated with intracellular Ca2+ mobilization as well as extracellular Ca2+ influx, while cGMP inhibits contraction by inhibiting the mechanism associated with extracellular Ca2+ influx.
Asunto(s)
AMP Cíclico/fisiología , GMP Cíclico/fisiología , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Óxido Nítrico/fisiología , Estómago/efectos de los fármacos , Acetilcolina/farmacología , Animales , Gatos , Colforsina/farmacología , AMP Cíclico/farmacología , GMP Cíclico/farmacología , Femenino , Fosfatos de Inositol/biosíntesis , Contracción Isométrica/efectos de los fármacos , Contracción Isométrica/fisiología , Masculino , Contracción Muscular/fisiología , Músculo Liso/fisiología , Nitroprusiato/farmacología , Estómago/fisiologíaRESUMEN
Histamine and arachidonic acid (AA) release was measured using the P2-purinoceptor antagonists, phospholipase A2 (PLA2) and cyclooxygenase (COX)/lipoxygenase (LOX) inhibitors to determine whether or not ATP-induced histamine release is associated with arachidonic acid (AA) release in rat peritoneal mast cells. ATP increased histamine release in a dose dependent manner, whereas adenosine did not. PPADS (a selective P2X-purinoceptor antagonist) and suramin (a nonselective P2X,2Y-purinoceptor antagonist) inhibited ATP-induced histamine release in a dose dependent manner. However, RB-2 (a P2Y-purinoceptor antagonist) did not block ATP-induced histamine release. Manoalide and oleyloxyethyl phosphorylcholine (OPC), secretory PLA2 inhibitors, also inhibited ATP-induced histamine release dose-dependently. Both COX inhibitors (ibuprofen and indomethacin) and LOX inhibitors (baicalein and caffeic acid) inhibited ATP-induced histamine in a dose dependent manner. ATP significantly increased [3H]AA release by 54%. PPADS and suramin significantly inhibited ATP-induced [3H]AA release by 81% and 39%, respectively. ATP-induced histamine release was significantly inhibited by a variety of protein kinase inhibitors, such as bisindolmaleimide, genistein, methyl 2,5-dihydroxycinnamate, W-7 and trifluoperazine. Overall, the results suggest that ATP-induced histamine release is in part related to the PLA2-mediated AA metabolism and P2X-purinoceptors.
Asunto(s)
Adenosina Trifosfato/farmacología , Ácido Araquidónico/metabolismo , Liberación de Histamina/efectos de los fármacos , Mastocitos/metabolismo , Fosfolipasas A/fisiología , Fosfato de Piridoxal/análogos & derivados , Animales , Lipooxigenasa/fisiología , Masculino , Cavidad Peritoneal/citología , Fosfolipasas A2 , Proteína Quinasa C/fisiología , Fosfato de Piridoxal/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P2/fisiologíaRESUMEN
It has been proposed that neurotransmitters and neuromodulators may function as neurotrophic factors during the development of the nervous system. Somatostatin (SS) was known to increase neurite outgrowth in PC12 cells, rat pheochromocytoma cell line, and cerebellar granule cells as well as Helisoma neuron. To further investigate a neurotrophic role of SS, voltage-dependent K+ and Ca2+ channel expression was studied using whole-cell patch-clamp in PC12 cells and the effect of SS was compared to that of nerve growth factor (NGF). Cyclic AMP (cAMP) level and mitogen-activated protein (MAP) kinase phosphorylation were also studied following the treatment with SS and/or NGF. Whereas NGF (50 ng/ml) increased continually the current density of the voltage-dependent K+ channel throughout 8 days treatment, SS (1 microM) increased the K+ current density on day 2 to the peak. K+ current density was decreased thereafter and was not different on day 6 from that of undifferentiated cells. Although SS did not increase voltage-dependent Ca2+ current density, it potentiated NGF-induced increase of voltage-dependent Ca2+ channel current density as well as the K+ current density. cAMP level was decreased by NGF and/or SS treatment. An increased phosphorylation of MAP kinase induced by NGF was not changed by SS treatment. These results support functionally that SS may function as a neurotrophic factor in developing nervous system.
Asunto(s)
Canales de Calcio/metabolismo , Factores de Crecimiento Nervioso/farmacología , Células PC12/efectos de los fármacos , Células PC12/metabolismo , Canales de Potasio/metabolismo , Somatostatina/farmacología , Animales , Canales de Calcio/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , AMP Cíclico/metabolismo , Sinergismo Farmacológico , Conductividad Eléctrica , Electrofisiología , Activación Enzimática/fisiología , Concentración Osmolar , Técnicas de Placa-Clamp , Canales de Potasio/fisiología , RatasRESUMEN
Gastric smooth muscle of cats was used to investigate the involvement of protein kinase in vanadate-induced contraction. Vanadate caused a contraction of cat gastric smooth muscle in a dose-dependent manner. Vanadate-induced contraction was totally inhibited by 2 mM EGTA and 1.5 mM LaCl3 and significantly inhibited by 10 microM verapamil and 1 microM nifedipine, suggesting that vanadate-induced contraction is dependent on the extracellular Ca2+ concentration. and the influx of extracellular Ca2+ was mediated through voltage-dependent Ca2+ channel. Both protein kinase C inhibitor and tyrosine kinase inhibitor significantly inhibited the vanadate-induced contraction and the combined inhibitory effect of two protein kinase inhibitors was greater than that of each one. But calmodulin antagonists did not have any influence on the vanadate-induced contraction. On the other hand, both forskolin (1 microM) and sodium nitroprusside (1 microM) significantly inhibited vanadate-induced contraction. Therefore, these results suggest that both protein kinase C and tyrosine kinase are involved in the vanadate-induced contraction which required the influx of extracellular Ca2+ in cat gastric smooth muscle, and that the contractile mechanism of vanadate may be different from that of agonist binding to its specific receptor.
Asunto(s)
Músculo Liso/efectos de los fármacos , Proteína Quinasa C/fisiología , Proteínas Tirosina Quinasas/fisiología , Vanadatos/farmacología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Gatos , Colforsina/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Técnicas In Vitro , Masculino , Contracción Muscular/efectos de los fármacos , Neurotransmisores/antagonistas & inhibidores , Neurotransmisores/farmacología , Nitroprusiato/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Estómago/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
Cat gastric smooth muscle strips were used to investigate the involvement of protein kinases in the steady-state contraction induced by 1 microM acetylcholine or 20 mM KCI. The steady-state contraction induced by acetylcholine or KCl was inhibited by EGTA dose dependently. Voltage-dependent Ca2+ channel antagonists dose dependently inhibited the contractions induced by KCI as well as by acetylcholine. Inhibitory effects of voltage-dependent Ca2+ channel antagonists were significantly more prominent on KCI-induced contractions than on acetylcholine-induced contractions. The acetylcholine-induced contraction was dose dependently inhibited by 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8, a blocker of intracellular Ca2+ release), but the KCl-induced contraction was not inhibited at all. Therefore both intracellular Ca2+ release and extracellular Ca2+ influx seem to be necessary for the acetylcholine-induced contraction, but intracellular Ca2+ release is not necessary for the KCl-induced contraction. Protein kinase C inhibitors, 10 microM 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine 2HCl (H-7) and 1 microM staurosporine, significantly inhibited the contraction induced by acetylcholine or KCl. Calmodulin antagonists, 30 microM trifluoperazine and 50 microM N-(6-aminohexyl)-5-chloro-2-naphthalenesulfonamide HCI (W-7), however, significantly inhibited the contraction induced by acetylcholine but not by KCl. A tyrosine kinase inhibitor, 50 microM genistein, did not affect the acetylcholine-induced contraction but significantly inhibited the KCl-induced contraction. These results strongly suggest that the involvement of protein kinases in regulation of the steady-state contraction may be agonist-dependent.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Calcio/metabolismo , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Acetilcolina/antagonistas & inhibidores , Animales , Calmodulina/efectos de los fármacos , Gatos , Femenino , Masculino , Músculo Liso/metabolismo , Cloruro de Potasio/antagonistas & inhibidores , EstómagoRESUMEN
This study was undertaken to investigate the effect of stimulation of the dorsal motor nucleus of the vagus (DMV) on myoelectric activity and motility of the gastric antrum and duodenum in normal and in vagotomized cats. 37 cats were starved for 24 h and then anesthetized with alpha-chloralose (70-80 mg/kg, iv). Electrical stimulation (0.1 mA, 0.2 ms, 50 Hz) of the left DMV was performed through a stereotaxically inserted electrode in 19 of the cats. The remaining 18 cats were injected in the left DMV with a glutamate solution (1 M, 200 nl) through an inserted 3-barreled micropipette. The myoelectric activity (slow wave) and the motility of the gastric antrum (2 cm proximal to the pylorus) and duodenum (3 cm distal to the pylorus) were measured using serosal bipolar electrodes and intraluminal balloons. Both the electrical and the glutamate stimulations of the DMV markedly increased the occurrence of spike potentials on the antral and duodenal myoelectric activity; however, the stimulations significantly decreased the frequency of the antral slow wave. The stimulations also produced increases in the motility of the antrum and duodenum which corresponded to the changes in the myoelectric activity. All the changes in the myoelectric activity and the motility were not observed after the ipsilateral vagotomy. Thus, these results strongly suggest that the dorsal motor nucleus of the vagus has a stimulatory influence on antral and duodenal motility through myoelectromechanical coordination via the vagus nerve in cats.
Asunto(s)
Duodeno/fisiología , Motilidad Gastrointestinal/fisiología , Nervio Vago/fisiología , Animales , Gatos , Cricetinae , Estimulación Eléctrica , Femenino , Masculino , VagotomíaRESUMEN
The effects of electrical stimulation of the central (CE) and basolateral (BL) amygdaloid nuclei on pancreatic exocrine secretion were tested in urethane-anesthetized rats. Electrical stimulation (0.1 mA, 1 ms, 40 Hz) of the CE significantly increased basal and hormone (secretin+cholecystokinin-8)-infused pancreatic exocrine secretion. These increases in pancreatic secretion were abolished by bilateral vagotomy. Electrical stimulation of the BL had no significant effect on basal and hormone-infused secretion. We conclude that the CE plays a stimulatory role in pancreatic exocrine secretion via the vagus nerve but the BL does not affect pancreatic secretion.
Asunto(s)
Amígdala del Cerebelo/fisiología , Páncreas/metabolismo , Animales , Estimulación Eléctrica , Electrodos , Masculino , Proteínas/metabolismo , Ratas , VagotomíaRESUMEN
Effects of medial amygdaloid stimulation on pancreatic exocrine secretion were investigated in anesthetized rats with pyloric ligation. During intraduodenal 0.01 N HCl perfusion, the electrical stimulation of the medial amygdaloid nucleus augmented significantly the HCl-induced pancreatic secretion of water and bicarbonate output. The duodenal acidification significantly increased secretin concentration in plasma. The effects of medial amygdaloid stimulation were also tested in the rats that received a small dose of porcine secretin. Although the amount of secretin infused was not enough to increase the pancreatic exocrine secretion, the stimulation of the medial amygdaloid nucleus significantly increased flow and bicarbonate output. The augmented pancreatic secretory responses to electrical stimulation of the medial amygdaloid nucleus were abolished by bilateral truncal vagotomy. However, the medial amygdaloid stimulation was not effective in stimulating pancreatic secretion in the rats perfused with isotonic saline. From these results, it is suggested that the medial amygdaloid nucleus plays a stimulatory role in pancreatic exocrine secretion via vagus nerves and that a certain level of plasma secretin is needed for the medial amygdaloid stimulation to be effective.
Asunto(s)
Amígdala del Cerebelo/fisiología , Páncreas/metabolismo , Anestesia , Animales , Bicarbonatos/metabolismo , Estimulación Eléctrica , Masculino , Páncreas/inervación , Ratas , Ratas Sprague-Dawley , Secretina/sangre , Nervio Vago/fisiologíaRESUMEN
NIH 3T3 fibroblasts were stably transfected with rat brain inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) 3-kinase to explore the relationship between increased production of Ins(1,3,4,5)P4 and the formation of InsP5 and InsP6. Mass measurements of InsP5 and InsP6 revealed no significant difference between kinase- and vector-transfected fibroblasts. However, such 3-kinase-transfected cells, when labeled with [3H]inositol for 48-72 h, showed lower levels of [3H]InsP5 and [3H]InsP6, as well as [3H]Ins(1,3,4,6)P4 and D/L[3H]Ins(1,4,5,6)P4, than their vector-transfected counterparts. Because Ins(1,4,5)P3 3-kinase-transfected cells grew less rapidly than vector-transfected controls, we determined whether the synthesis of InsP5 and InsP6 was related to a specific phase of the cell cycle. When NIH 3T3 cells prelabeled with [3H]inositol were synchronized by serum deprivation followed by stimulation with platelet-derived growth factor (PDGF), the amounts of labeled InsP5 and InsP6 began to increase only after 12 h of stimulation, when cells entered the S-phase as indicated by increased [3H]thymidine incorporation. The enhanced synthesis of these inositol polyphosphates was preceded by an early increase in Ins(1,4,5)P3 and its metabolites that was no longer evident by the fifth hour of PDGF action. There was also a prominent and biphasic increase in the level of D/L-Ins(1,4,5,6)P4 with an early peak at approximately 3 h and a second rise that paralleled the increases in InsP5 and InsP6. These results indicate that the formation of highly phosphorylated inositols is not tightly coupled to the receptor-mediated formation of Ins(1,4,5)P3 and its metabolites but is mainly determined by other factors that operate at specific points of the cell cycle.
Asunto(s)
Células 3T3/metabolismo , Ciclo Celular , Fosfatos de Inositol/biosíntesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/biosíntesis , Factor de Crecimiento Derivado de Plaquetas/farmacología , Células 3T3/efectos de los fármacos , Animales , Encéfalo/enzimología , División Celular , Inducción Enzimática , Ratones , Fosforilación , Ratas , Proteínas Recombinantes/farmacología , TransfecciónRESUMEN
The effect of histamine on [3H]-inositol phosphates (IPs) formation was investigated with [3H]-inositol-labeled gastric smooth-muscle cells in guinea pigs. Histamine (10(-5) M) increased the formation of [3H]-IPs in the muscle cells. The increase was significantly inhibited by pyrilamine (10(-5) M) but not by cimetidine (10(-5) M). The contractile response to histamine was also completely inhibited by pyrilamine but not by cimetidine. Phorbol ester 12-myristate 13-acetate (10 microM) significantly inhibited histamine-stimulated [3H]-IPs formation by 56%, whereas forskolin (10 microM) decreased it by 18%. This study demonstrates that the response of [3H]-IPs formation and contraction to histamine is mediated through H1 receptor, and the formation of [3H]-IPs is negatively regulated by protein kinase C in gastric smooth muscle of guinea pigs.
Asunto(s)
Fosfatos de Inositol/biosíntesis , Contracción Muscular/fisiología , Músculo Liso/fisiología , Receptores Histamínicos H1/fisiología , Estómago/fisiología , Animales , Células Cultivadas , Cobayas , Histamina/farmacología , Técnicas In Vitro , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Proteínas Quinasas/fisiologíaRESUMEN
The proposed Ca(2+)-signaling actions of inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), formed by phosphorylation of the primary Ca(2+)-mobilizing messenger, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), were analyzed in NIH 3T3 and CCL39 fibroblasts transfected with rat brain Ins(1,4,5)P3 3-kinase. In such kinase-transfected cells, the conversion of Ins(1,4,5)P3 to Ins(1,3,4,5)P4 during agonist stimulation was greatly increased, with a concomitant reduction in Ins(1,4,5)P3 levels and attenuation of both the cytoplasmic Ca2+ increase and the Ca2+ influx response. This reduction in Ca2+ signaling was observed during activation of receptors coupled to guanine nucleotide-binding proteins (thrombin and bradykinin), as well as with those possessing tyrosine kinase activity. Single-cell Ca2+ measurements in CCL39 cells revealed that the smaller averaged Ca2+ response of enzyme-transfected cells was due to a marked increase in the number of cells expressing small and slow Ca2+ increases, in contrast to the predominantly large and rapid Ca2+ responses of vector-transfected controls. There was no evidence that high Ins(1,3,4,5)P4 levels promote Ca2+ mobilization, Ca2+ entry, or Ca2+ sequestration. These data indicate that Ins(1,4,5)P3 is the major determinant of the agonist-induced Ca2+ signal in fibroblasts and that Ins(1,3,4,5)P4 does not appear to contribute significantly to this process. Instead, Ins(1,4,5)P3 3-kinase may serve as a negative regulator of the Ca(2+)-phosphoinositide signal transduction mechanism.
Asunto(s)
Calcio/metabolismo , Fosfatos de Inositol/biosíntesis , Fosfotransferasas (Aceptor de Grupo Alcohol) , Transducción de Señal , Células 3T3 , Animales , Western Blotting , Bombesina/farmacología , Encéfalo/enzimología , Encéfalo/metabolismo , Cromatografía Líquida de Alta Presión , ADN/genética , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Pulmón/metabolismo , Ratones , Fosfatidilinositol Diacilglicerol-Liasa , Hidrolasas Diéster Fosfóricas/metabolismo , Fosforilación , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/farmacología , Ratas , Transducción de Señal/efectos de los fármacos , Trombina/farmacología , TransfecciónRESUMEN
The Ca2(+)-mobilizing second messenger D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) is converted to the putative messenger D-myo-inositol 1,3,4,5-tetrakisphosphate by Ins(1,4,5)P3 3-kinase. We found that cAMP-dependent protein kinase and protein kinase C phosphorylate, and thereby modulate, the activity of Ins(1,4,5)P3 3-kinase. cAMP-dependent kinase introduced a stoichiometric amount of phosphate at serine 109 of the 53-kDa polypeptide and caused a 1.8-fold increase in Vmax, whereas the protein kinase C-dependent phosphorylation reduced the Vmax to one-fourth of that of the unphosphorylated enzyme. Upon prolonged incubation, protein kinase C introduced phosphate at multiple sites in Ins(1,4,5)P3 3-kinase, and the resulting inactivation of the enzyme appeared to be well-correlated with the simultaneous phosphorylation of two major sites, serine 109 and serine 175. The Km for Ins(1,4,5)P3 was not affected significantly after phosphorylation by either protein kinase. We propose, therefore, that the phosphorylation of Ins(1,4,5)P3 3-kinase by cAMP-dependent kinase and protein kinase C constitutes mechanisms of cross-talk between cellular signaling pathways that use various second messengers such as inositol phosphates, diacylglycerol, Ca2+, and cAMP.
Asunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol) , Fosfotransferasas/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Quinasas/metabolismo , Sistemas de Mensajero Secundario , Secuencia de Aminoácidos , Animales , Encéfalo/enzimología , Cinética , Datos de Secuencia Molecular , Mapeo Peptídico , Fosfopéptidos/aislamiento & purificación , Fosforilación , Fosfotransferasas/aislamiento & purificación , Ratas , TripsinaRESUMEN
A new, rapid method for purification of inositol(1,4,5)P3 3-kinase in high yield from rat brain is described. Purified enzyme exhibited a polypeptide of Mr = 53,000 on sodium dodecyl sulfate-polyacrylamide gel and a specific activity of 29 mumol/min/mg at 37 degrees C in the absence of calmodulin. Inclusion of calpain inhibitors was critical for obtaining the 53-kDa protein as the major product and 0.1% of the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylamino]-2-propanesulfonate, was necessary to stabilize enzyme activity. In the absence of calpain inhibitors, the 53-kDa protein degraded progressively during purification and yielded a mixture containing polypeptides of various sizes. Relative intensity of these degradation products on sodium dodecyl sulfate-polyacrylamide gel varied from one preparation to another. However, broad band(s) at the 42-45 kDa region and a band at 35 kDa were always weak, while bands of 53, 51, 40 (sometimes doublets), 33, and 32 KDa were usually strong. The fact that all of these polypeptides including the weak bands of 42-45 and 35 kDa were derived from the 53 kDa form was confirmed by their immunocross-reactivity with monoclonal antibodies to the 53 kDa form. When the 51, 40, and a mixture of the 33 and 32 kDa forms were obtained separately and nearly free from other forms, each of them exhibited catalytic activity. Nevertheless, calmodulin binds to polypeptides larger than 35,000 but not to the 33 and 32 kDa forms. Incubation of the purified 53 kDa form with calpain generated a fragmentation pattern nearly identical to that generated during purification in the absence of calpain inhibitors. Incubation with five other endoproteases produced proteolytic fragments slightly different from those by calpain. However, the general fragmentation patterns generated by the proteases were similar, suggesting that inositol(1,4,5)P3 3-kinase contains several motifs susceptible to a variety of proteases.