RESUMEN
KEY POINTS: Wnt ligands belonging to both canonical and non-canonical Wnt pathways regulate membrane potential signifying a very early event in the signal transduction. Wnts activate K+ currents by elevating intracellular Ca2+ and trigger Ca2+ release from intracellular stores. Control of potential by Wnt ligands has significant implications for gene transcription and opens up a novel avenue to interfere with this critical pathway. ABSTRACT: The Wnt signalling network determines gene transcription with free intracellular Ca2+ ( Cai2+ ) and ß-catenin as major intracellular signal transducers. Despite its critical importance during development and disease, many of the basic mechanisms of Wnt signal activation remain unclear. Here we show by single cell recording and simultaneous Cai2+ imaging in mammalian prostate cancer cells that an early step in the signal cascade is direct action on the cell membrane potential. We show that Wnt ligands 5A, 9B and 10B rapidly hyperpolarized the cells by activating K+ current by Ca2+ release from intracellular stores. Medium-throughput multi-well recordings showed responses to Wnts at concentrations of 2 nm. We identify a putative target for early events as a TRPM channel. Wnts thus act as ligands for ion channel activation in mammalian cells and membrane potential is an early indicator of control of transcription.
Asunto(s)
Señalización del Calcio , Potenciales de la Membrana , Vía de Señalización Wnt , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Humanos , Células MCF-7 , Canales de Potasio/metabolismo , Canales Catiónicos TRPM/metabolismo , Proteínas Wnt/metabolismo , Proteínas Wnt/farmacologíaRESUMEN
Human induced pluripotent stem cells can be differentiated into dopaminergic neurons (Dopa.4U). Dopa.4U neurons expressed voltage-gated NaV and KV channels and showed neuron-like spontaneous electrical activity. In automated patch clamp measurements with suspended Dopa.4U neurons, delayed rectifier K+ current (delayed KV) and rapidly inactivating A-type K+ current (fast KV) were identified. Examination of the fast KV current with inhibitors yielded IC50 values of 0.4 mM (4-aminopyridine) and 0.1 mM (tetraethylammonium). In manual patch clamp measurements with adherent Dopa.4U neurons, fast KV current could not be detected, while the delayed KV current showed an IC50 of 2 mM for 4-aminopyridine. The NaV channels in adherent and suspended Dopa.4U neurons showed IC50 values for tetrodotoxin of 27 and 2.9 nM, respectively. GABA-induced currents that could be observed in adherent Dopa.4U neurons could not be detected in suspended cells. Application of current pulses induced action potentials in approx. 70 % of the cells. Our results proved the feasibility of automated electrophysiological characterization of neuronal cells.
Asunto(s)
Neuronas Dopaminérgicas/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Canales de Potasio/fisiología , Diferenciación Celular , Neuronas Dopaminérgicas/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , Canales Iónicos , Técnicas de Placa-ClampRESUMEN
Free intracellular calcium ([Ca2+]i), in addition to being an important second messenger, is a key regulator of many cellular processes including cell membrane potential, proliferation, and apoptosis. In many cases, the mobilization of [Ca2+]i is controlled by intracellular store activation and calcium influx. We have investigated the effect of several ion channel modulators, which have been used to treat a range of human diseases, on [Ca2+]i release, by ratiometric calcium imaging. We show that six such modulators [amiodarone (Ami), dofetilide, furosemide (Fur), minoxidil (Min), loxapine (Lox), and Nicorandil] initiate release of [Ca2+]i in prostate and breast cancer cell lines, PC3 and MCF7, respectively. Whole-cell currents in PC3 cells were inhibited by the compounds tested in patch-clamp experiments in a concentration-dependent manner. In all cases [Ca2+]i was increased by modulator concentrations comparable to those used clinically. The increase in [Ca2+]i in response to Ami, Fur, Lox, and Min was reduced significantly (P < 0.01) when the external calcium was reduced to nM concentration by chelation with EGTA. The data suggest that many ion channel regulators mobilize [Ca2+]i We suggest a mechanism whereby calcium-induced calcium release is implicated; such a mechanism may be important for understanding the action of these compounds.
Asunto(s)
Calcio/metabolismo , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Canales Iónicos/metabolismo , Línea Celular Tumoral , Fenómenos Electrofisiológicos/efectos de los fármacos , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Humanos , CinéticaRESUMEN
A number of potassium channels including members of the KCNQ family and the Ca(2+) activated IK and SK, but not BK, are strongly and reversibly regulated by small changes in cell volume. It has been argued that this general regulation is mediated through sensitivity to changes in membrane stretch. To test this hypothesis we have studied the regulation of KCNQ1 and BK channels after expression in Xenopus oocytes. Results from cell-attached patch clamp studies (approximately 50 microm(2) macropatches) in oocytes expressing BK channels demonstrate that the macroscopic volume-insensitive BK current increases with increasing negative hydrostatic pressure (suction) applied to the pipette. Thus, at a pipette pressure of -5.0 +/- 0.1 mmHg the increase amounted to 381 +/- 146% (mean +/- S.E.M., n = 6, P < 0.025). In contrast, in oocytes expressing the strongly volume-sensitive KCNQ1 channel, the current was not affected by membrane stretch. The results indicate that (1) activation of BK channels by local membrane stretch is not mimicked by membrane stress induced by cell swelling, and (2) activation of KCNQ1 channels by cell volume increase is not mediated by local tension in the cell membrane. We conclude that stretch and volume sensitivity can be considered two independent regulatory mechanisms.
Asunto(s)
Membrana Celular/fisiología , Tamaño de la Célula , Mecanotransducción Celular/fisiología , Canales de Potasio/fisiología , Animales , Línea Celular , Estimulación Eléctrica , Fenómenos Electrofisiológicos/fisiología , Femenino , Humanos , Presión Hidrostática , Soluciones Hipertónicas/farmacología , Soluciones Isotónicas/farmacología , Canales de Potasio KCNQ/fisiología , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/fisiología , Potenciales de la Membrana/fisiología , Oocitos/metabolismo , Transfección , Xenopus laevisRESUMEN
In isolated rat pancreatic alpha-cells, glucose, arginine, and the sulfonylurea tolbutamide stimulated glucagon release. The effect of glucose was abolished by the KATP-channel opener diazoxide as well as by mannoheptulose and azide, inhibitors of glycolysis and mitochondrial metabolism. Glucose inhibited KATP-channel activity by 30% (P<0.05; n=5) and doubled the free cytoplasmic Ca2+ concentration. In cell-attached recordings, azide opened KATP channels. The N-type Ca2+-channel blocker omega-conotoxin and the Na+-channel blocker tetrodotoxin inhibited glucose-induced glucagon release whereas tetraethylammonium, a blocker of delayed rectifying K+ channels, increased secretion. Glucagon release increased monotonically with increasing K+ concentrations. omega-Conotoxin suppressed glucagon release to 15 mM K+, whereas a combination of omega-conotoxin and an L-type Ca2+-channel inhibitor was required to abrogate secretion in 50 mM K+. Recordings of cell capacitance revealed that glucose increased the exocytotic response evoked by membrane depolarization 3-fold. This correlated with a doubling of glucagon secretion by glucose in intact rat islets exposed to diazoxide and high K+. In whole-cell experiments, exocytosis was stimulated by reducing the cytoplasmic ADP concentration, whereas changes of the ATP concentration in the physiological range had little effect. We conclude that glucose stimulates glucagon release from isolated rat alpha-cells by KATP-channel closure and stimulation of Ca2+ influx through N-type Ca2+ channels. Glucose also stimulated exocytosis by an amplifying mechanism, probably involving changes in adenine nucleotides. The stimulatory action of glucose in isolated alpha-cells contrasts with the suppressive effect of the sugar in intact islets and highlights the primary importance of islet paracrine signaling in the regulation of glucagon release.
Asunto(s)
Células Secretoras de Glucagón/efectos de los fármacos , Células Secretoras de Glucagón/metabolismo , Glucagón/metabolismo , Glucosa/farmacología , Nucleótidos de Adenina/farmacología , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Diazóxido/farmacología , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Exocitosis/efectos de los fármacos , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Masculino , Concentración Osmolar , Técnicas de Placa-Clamp , Potasio/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , Canales de Potasio/fisiología , Ratas , Ratas Sprague-Dawley , Azida Sódica/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Tapsigargina/farmacologíaRESUMEN
The release of sPLA(2) from single mouse pancreatic beta-cells was monitored using a fluorescent substrate of the enzyme incorporated in the outer leaflet of the plasma membrane. Stimulation of beta-cells with agents that increased cytosolic free Ca(2+) concentration ([Ca(2+)](i)) induced a rapid release of sPLA(2) to the extracellular medium. Exogenous sPLA(2) strongly stimulated insulin secretion in mouse pancreatic islets at both basal and elevated glucose concentrations. The stimulation of insulin secretion by sPLA(2) was mediated via inhibition of ATP-dependent K(+) channels and an increase in [Ca(2+)](i). Measurements of cell capacitance in single beta-cells revealed that sPLA(2) did not modify depolarisation-induced exocytosis. Our data suggest that a positive feedback regulation of insulin secretion by co-released sPLA(2) is operational in pancreatic beta-cells and point to this enzyme as an autocrine regulator of insulin secretion.
Asunto(s)
Insulina/metabolismo , Islotes Pancreáticos/enzimología , Islotes Pancreáticos/metabolismo , Fosfolipasas A/metabolismo , Fosfolipasas A/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Adenosina Trifosfato/metabolismo , Animales , Ácido Araquidónico/farmacología , Calcio/metabolismo , Células Cultivadas , Exocitosis , Femenino , Fosfolipasas A2 Grupo IB , Secreción de Insulina , Islotes Pancreáticos/fisiología , Lisofosfatidilcolinas/farmacología , Ratones , Técnicas de Placa-Clamp , Fosfolipasas A2 , Espectrometría de FluorescenciaRESUMEN
The effect of the novel imidazoline compound 2-[2-(4,5-dihydro-1H-imidazol-2-yl)-1-(5-methyl-2,3-dihydrobenzofuran-7-yl)-ethyl]-pyridine (NNC77-0020) on stimulus-secretion coupling and hormone secretion was investigated in mouse pancreatic islets and isolated alpha- and beta-cells. In the presence of elevated glucose concentrations NNC77-0020 stimulated insulin secretion concentration dependently (EC(50) 64 nM) by 200% without affecting the whole-cell K(+) current or cytoplasmic Ca(2+) levels. Capacitance measurements in single mouse beta-cells showed that intracellular application of NNC77-0020 via the recording pipette enhanced Ca(2+)-dependent exocytosis. This action was dependent on protein kinase C (PKC) and cytoplasmic phospholipase A(2) (cPLA(2)) activity and required functional granular ClC-3 Cl(-) channels. In intact islets NNC77-0020 stimulated glucose-dependent somatostatin secretion, an effect that was also dependent on PKC and cPLA(2) activity. NNC77-0020 also inhibited glucagon secretion. In single mouse alpha-cells this action was not associated with a change in spontaneous electrical activity and resulted from a reduction in the rate of Ca(2+)-dependent exocytosis. Inhibition of exocytosis by NNC77-0020 was pertussis toxin sensitive and mediated by activation of the protein phosphatase calcineurin. In conclusion, our data suggest that the imidazoline compound NNC77-0020 modulates pancreatic hormone secretion in a complex fashion, comprising glucose-dependent stimulation of insulin and somatostatin secretion and inhibition of glucagon release. These mechanisms of action constitute an ideal basis for the development of novel imidazoline-containing anti-diabetic compounds.
Asunto(s)
Glucagón/metabolismo , Glucosa/farmacología , Imidazoles/farmacología , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Piridinas/farmacología , Somatostatina/metabolismo , Animales , Agonistas de los Canales de Cloruro , Canales de Cloruro/metabolismo , Codón , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , Electrofisiología , Exocitosis/efectos de los fármacos , Femenino , Técnicas In Vitro , Islotes Pancreáticos/efectos de los fármacos , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Ratones , Oligonucleótidos Antisentido , Técnicas de Placa-Clamp , Fosfolipasas A/metabolismo , Canales de Potasio , Proteína Quinasa C/metabolismoRESUMEN
Insulin secretion is controlled by the beta cell's metabolic state, and the ability of the secretory granules to undergo exocytosis increases during glucose stimulation in a membrane potential-independent fashion. Here, we demonstrate that exocytosis of insulin-containing secretory granules depends on phosphatidylinositol 4-kinase (PI 4-kinase) activity and that inhibition of this enzyme suppresses glucose-stimulated insulin secretion. Intracellular application of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] stimulated exocytosis by promoting the priming of secretory granules for release and increasing the number of granules residing in a readily releasable pool. Reducing the cytoplasmic ADP concentration in a way mimicking the effects of glucose stimulation activated PI 4-kinase and increased exocytosis whereas changes of the ATP concentration in the physiological range had little effect. The PI(4,5)P(2)-binding protein Ca(2+)-dependent activator protein for secretion (CAPS) is present in beta cells, and neutralization of the protein abolished both Ca(2+)- and PI(4,5)P(2)-induced exocytosis. We conclude that ADP-induced changes in PI 4-kinase activity, via generation of PI(4,5)P(2), represents a metabolic sensor in the beta cell by virtue of its capacity to regulate the release competence of the secretory granules.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/metabolismo , Técnicas Biosensibles , Islotes Pancreáticos/metabolismo , Animales , Exocitosis , Inmunohistoquímica , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/enzimología , RatonesRESUMEN
We have previously demonstrated that the novel imidazoline compound (+)-2-(2-(4,5-dihydro-1H-imidazol-2-yl)-thiopene-2-yl-ethyl)-pyridine (NNC77-0074) increases insulin secretion from pancreatic beta-cells by stimulation of Ca(2+)-dependent exocytosis. Using capacitance measurements, we now show that NNC77-0074 stimulates exocytosis in clonal INS-1E cells. NNC77-0074-stimulated exocytosis was antagonised by the cytoplasmic phospholipase A(2) (cPLA(2)) inhibitors ACA and AACOCF(3) and in cells treated with antisense oligonucleotide against cPLA(2)alpha. NNC77-0074-evoked insulin secretion was likewise inhibited by ACA, AACOCF(3), and cPLA(2)alpha antisense oligonucleotide treatment. In pancreatic islets NNC77-0074 stimulated PLA(2) activity. We propose that cPLA(2)alpha plays an important role in the regulation of NNC77-0074-evoked exocytosis in insulin secreting beta-cells.
Asunto(s)
Señalización del Calcio , Exocitosis , Imidazoles/farmacología , Islotes Pancreáticos/enzimología , Fosfolipasas A/fisiología , Piridinas/farmacología , Animales , Células Cultivadas , Inhibidores Enzimáticos , Fosfolipasas A2 Grupo IV , Imidazoles/antagonistas & inhibidores , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/fisiología , Masculino , Ratones , Oligonucleótidos Antisentido/genética , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A/genética , Fosfolipasas A2 , Piridinas/antagonistas & inhibidores , Células Tumorales CultivadasRESUMEN
We have investigated the effects of the novel imidazoline compound (+)-2-(2-(4,5-dihydro-1H-imidazol-2-yl)-thiopene-2-yl-ethyl)-pyridine (NNC77-0074) on stimulus-secretion coupling in isolated pancreatic alpha- and beta-cells. NNC77-0074 stimulated glucose-dependent insulin secretion in intact mouse pancreatic islets. No effect was observed at =2.5 mM glucose and maximal stimulation occurred at 10-15 mM glucose. NNC77-0074 produced a concentration-dependent stimulation of insulin secretion. Half-maximal (EC(50)) stimulation was observed at 24 microM and at maximally stimulatory concentrations insulin release was doubled. The stimulatory action of NNC77-0074 on insulin secretion was not associated with membrane depolarisation or a change in the activity of ATP-sensitive K(+) channels. Using capacitance measurements, we found that NNC77-0074 stimulated depolarisation-induced exocytosis 2.6-fold without affecting the whole-cell Ca(2+) current when applied via the extracellular medium. The concentration dependence of the stimulatory action was determined by intracellular application of NNC77-0074 through the recording pipette. NNC77-0074 stimulated exocytosis half-maximal at 44 nM and at maximally stimulatory concentrations the rate of exocytosis was increased twofold. NNC77-0074 stimulated depolarised-induced insulin secretion from islets exposed to diazoxide and high external KCl (EC(50)=0.45 microM). The stimulatory action of NNC77-0074 was dependent on protein kinase C activity. NNC77-0074 potently inhibited glucagon secretion from rat islets (EC(50)=11 nM). This was not associated with a change in spontaneous electrical activity and ATP-sensitive K(+) channel activity but resulted from a reduction of the rate of Ca(2+)-dependent exocytosis in single rat alpha-cells (EC(50)=9 nM). Inhibition of exocytosis by NNC77-0074 was pertussis toxin-sensitive and mediated by activation of the protein phosphatase calcineurin. In rat somatotrophs, PC12 cells and mouse cortical neurons NNC77-0074 did not stimulate Ca(2+)-evoked exocytosis, whereas the other imidazoline compounds phentolamine and efaroxan produced 2.5-fold stimulation of exocytosis. Our data suggest that the imidazoline compound NNC77-0074 constitutes a novel class of antidiabetic compounds that stimulates glucose-dependent insulin release while inhibiting glucagon secretion. These actions are exclusively exerted by modulation of exocytosis of the insulin- and glucagon-containing granules.
Asunto(s)
Calcio/metabolismo , Inhibidores Enzimáticos/farmacología , Glucagón/metabolismo , Imidazoles/farmacología , Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Piridinas/farmacología , Animales , Canales de Calcio/fisiología , Células Cultivadas , AMP Cíclico/metabolismo , Exocitosis , Femenino , Glucagón/antagonistas & inhibidores , Glucosa/metabolismo , Técnicas In Vitro , Secreción de Insulina , Islotes Pancreáticos/enzimología , Islotes Pancreáticos/fisiología , Ratones , Ratones Endogámicos , Técnicas de Placa-Clamp , Canales de Potasio/fisiología , Proteína Quinasa C/fisiologíaRESUMEN
Using capacitance measurements, we investigated the effects of intracellularly applied recombinant human cytosolic phospholipase A2 (cPLA2alpha) and its lipolytic products arachidonic acid and lysophosphatidylcholine on Ca2+-dependent exocytosis in single mouse pancreatic beta-cells. cPLA2alpha dose dependently (EC50 = 86 nM) stimulated depolarization-evoked exocytosis by 450% without affecting the whole cell Ca2+ current or cytoplasmic Ca2+ levels. The stimulatory effect involved priming of secretory granules as reflected by an increase in the size of the readily releasable pool of granules from 70-80 to 280-300. cPLA2alpha-stimulated exocytosis was antagonized by the specific cPLA2 inhibitor AACOCF3. Ca2+-evoked exocytosis was reduced by 40% in cells treated with AACOCF3 or an antisense oligonucleotide against cPLA2alpha. The action of cPLA2alpha was mimicked by a combination of arachidonic acid and lysophosphatidylcholine (470% stimulation) in which each compound alone doubled the exocytotic response. Priming of insulin-containing secretory granules has been reported to involve Cl- uptake through ClC-3 Cl- channels. Accordingly, the stimulatory action of cPLA2alpha was inhibited by the Cl- channel inhibitor DIDS and in cells pretreated with ClC-3 Cl- channel antisense oligonucleotides. We propose that cPLA2alpha has an important role in controlling the rate of exocytosis in beta-cells. This effect of cPLA2alpha reflects an enhanced transgranular Cl- flux, leading to an increase in the number of granules available for release, and requires the combined actions of arachidonic acid and lysophosphatidylcholine.
Asunto(s)
Ácido Araquidónico/biosíntesis , Citosol/enzimología , Exocitosis/fisiología , Islotes Pancreáticos/metabolismo , Lisofosfolípidos/biosíntesis , Fosfolipasas A/metabolismo , Animales , Calcio/metabolismo , Calcio/fisiología , Canales de Calcio/metabolismo , Canales de Cloruro/metabolismo , Gránulos Citoplasmáticos/metabolismo , Citosol/efectos de los fármacos , Exocitosis/efectos de los fármacos , Femenino , Fosfolipasas A2 Grupo IV , Técnicas In Vitro , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/enzimología , Inhibidores de la Lipooxigenasa/farmacología , Lisofosfatidilcolinas/farmacología , Potenciales de la Membrana/fisiología , Ratones , Oligonucleótidos Antisentido , Técnicas de Placa-Clamp , Fosfolipasas A2 , Estimulación QuímicaRESUMEN
The glycosphingolipid sulfatide is present in secretory granules and at the surface of pancreatic beta-cells, and antisulfatide antibodies (ASA; IgG1) are found in serum from the majority of patients with newly diagnosed type 1 diabetes. Here we demonstrate that sulfatide produced a glucose- and concentration-dependent inhibition of insulin release from isolated rat pancreatic islets. This inhibition of insulin secretion was due to activation of ATP-sensitive K(+)-(K(ATP)) channels in single rat beta-cells. No effect of sulfatide was observed on whole-cell Ca(2+)-channel activity or glucose-induced elevation of cytoplasmic Ca(2+) concentration. It is interesting that sulfatide stimulated Ca(2+)-dependent exocytosis determined by capacitance measurements and depolarized-induced insulin secretion from islets exposed to diazoxide and high external KCl. The monoclonal sulfatide antibody Sulph I as well as ASA-positive serum reduced glucose-induced insulin secretion by inhibition of Ca(2+)-dependent exocytosis. Our data suggest that sulfatide is important for the control of glucose-induced insulin secretion and that both an increase and a decrease in the sulfatide content have an impact on the secretory capacity of the individual beta-cells.
Asunto(s)
Canales de Calcio/fisiología , Calcio/fisiología , Exocitosis/fisiología , Glucolípidos/farmacología , Insulina/metabolismo , Islotes Pancreáticos/fisiología , Sulfoglicoesfingolípidos/farmacología , Transportadoras de Casetes de Unión a ATP , Animales , Canales de Calcio/efectos de los fármacos , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Canales KATP , Cinética , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Canales de Potasio/efectos de los fármacos , Canales de Potasio/fisiología , Canales de Potasio de Rectificación Interna , Ratas , Ratas Endogámicas LewRESUMEN
OBJECTIVE: GH causes insulin resistance, impairs glycemic control and increases the risk of vascular diabetic complications. Sulphonylureas stimulate GH secretion and this study was undertaken to investigate the possible stimulatory effect of repaglinide and nateglinide, two novel oral glucose regulators, on critical steps of the stimulus-secretion coupling in single rat somatotrophs. METHODS: Patch-clamp techniques were used to record whole-cell ATP-sensitive K(+) (K(ATP)) and delayed outward K(+) currents, membrane potential and Ca(2+)-dependent exocytosis. GH release was measured from perifused rat somatotrophs. RESULTS: Both nateglinide and repaglinide dose-dependently suppressed K(ATP) channel activity with half-maximal inhibition being observed at 413 nM and 13 nM respectively. Both compounds induced action potential firing in the somatotrophs irrespective of whether GH-releasing hormone was present or not. The stimulation of electrical activity by nateglinide, but not repaglinide, was associated with an increased mean duration of the action potentials. The latter effect correlated with a reduction of the delayed outward K(+) current, which accounts for action potential repolarization. The latter effect had a K(d) of 19 microM but was limited to 38% inhibition. When applied at concentrations similar to those required to block K(ATP) channels, nateglinide in addition potentiated Ca(2+)-evoked exocytosis 3.3-fold (K(d)=3 microM) and stimulated GH release 4.5-fold. The latter effect was not shared by repaglinide. The stimulation of exocytosis by nateglinide was mimicked by cAMP and antagonized by the protein kinase A inhibitor Rp-cAMPS. CONCLUSION: Nateglinide stimulates GH release by inhibition of plasma membrane K(+) channels, elevation of cytoplasmic cAMP levels and stimulation of Ca(2+)-dependent exocytosis. By contrast, the effect of repaglinide was confined to inhibition of the K(ATP) channels.
Asunto(s)
Ciclohexanos/farmacología , Hormona del Crecimiento/metabolismo , Hipoglucemiantes/farmacología , Fenilalanina/análogos & derivados , Fenilalanina/farmacología , Hipófisis/efectos de los fármacos , Bloqueadores de los Canales de Potasio , Canales de Potasio con Entrada de Voltaje , Canales de Potasio , Animales , Calcio/farmacología , Carbamatos/farmacología , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Canales de Potasio de Tipo Rectificador Tardío , Exocitosis/efectos de los fármacos , Hormona Liberadora de Hormona del Crecimiento/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Nateglinida , Técnicas de Placa-Clamp , Piperidinas/farmacología , Hipófisis/citología , Hipófisis/metabolismo , Potasio/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Membrane homeostasis is maintained by exocytosis and endocytosis. The molecular mechanisms regulating the interplay between these two processes are not clear. Inositol hexakisphosphate (InsP(6)) is under metabolic control and serves as a signal in the pancreatic beta cell stimulus-secretion coupling by increasing Ca(2+)-channel activity and insulin exocytosis. We now show that InsP(6) also promotes dynamin I-mediated endocytosis in the pancreatic beta cell. This effect of InsP(6) depends on calcineurin-induced dephosphorylation and is accounted for by both activation of protein kinase C and inhibition of the phosphoinositide phosphatase synaptojanin and thereby formation of phosphatidylinositol 4,5-bisphosphate. In regulating both exocytosis and endocytosis, InsP(6) thus may have an essential integral role in membrane trafficking.