RESUMEN
The instantaneous release of energy in a localized area of a gas results in the formation of a low-density region and a series of shock and expansion waves. If this process occurs near a boundary, the shock reflections can interact with the density inhomogeneity, leading to the baroclinic generation of vorticity and the subsequent organization of the flow into several structures, including a vortex ring. By means of numerical simulations we illustrate the qualitative changes that occur in the pressure wave patterns and vorticity distribution as the distance from the area of energy release to the boundary is varied. Those changes are shown to be related to the combined effect of the shock waves that, respectively, initially move away and towards the center of the low-density region. In particular, we describe how for small enough offset distances the shocks internal to the inhomogeneity can make a substantial contribution to the vorticity field, influencing the circulation and characteristics of the resulting flow structures.
Asunto(s)
Modelos Teóricos , Simulación por Computador , PresiónRESUMEN
Insulin resistance is associated with mitochondrial dysfunction, but the mechanism by which mitochondria inhibit insulin-stimulated glucose uptake into the cytoplasm is unclear. The mitochondrial permeability transition pore (mPTP) is a protein complex that facilitates the exchange of molecules between the mitochondrial matrix and cytoplasm, and opening of the mPTP occurs in response to physiological stressors that are associated with insulin resistance. In this study, we investigated whether mPTP opening provides a link between mitochondrial dysfunction and insulin resistance by inhibiting the mPTP gatekeeper protein cyclophilin D (CypD) in vivo and in vitro. Mice lacking CypD were protected from high fat diet-induced glucose intolerance due to increased glucose uptake in skeletal muscle. The mitochondria in CypD knockout muscle were resistant to diet-induced swelling and had improved calcium retention capacity compared to controls; however, no changes were observed in muscle oxidative damage, insulin signaling, lipotoxic lipid accumulation or mitochondrial bioenergetics. In vitro, we tested 4 models of insulin resistance that are linked to mitochondrial dysfunction in cultured skeletal muscle cells including antimycin A, C2-ceramide, ferutinin, and palmitate. In all models, we observed that pharmacological inhibition of mPTP opening with the CypD inhibitor cyclosporin A was sufficient to prevent insulin resistance at the level of insulin-stimulated GLUT4 translocation to the plasma membrane. The protective effects of mPTP inhibition on insulin sensitivity were associated with improved mitochondrial calcium retention capacity but did not involve changes in insulin signaling both in vitro and in vivo. In sum, these data place the mPTP at a critical intersection between alterations in mitochondrial function and insulin resistance in skeletal muscle.
RESUMEN
In luteinizing granulosa cells, prostaglandin E(2) (PGE(2)) can exert luteotrophic actions, apparently via the cAMP signalling pathway. In addition to stimulating progesterone synthesis, PGE(2) can also stimulate oxidation of the physiological glucocorticoid, cortisol, to its inactive metabolite, cortisone, by the type 1 11beta-hydroxysteroid dehydrogenase (11betaHSD1) enzyme in human granulosa-lutein cells. Having previously shown these human ovarian cells to express functional G-protein coupled, E-series prostaglandin (PTGER)1, PTGER2 and PTGER4 receptors, the aim of this study was to delineate the roles of PTGER1 and PTGER2 receptors in mediating the effects of PGE(2) on steroidogenesis and cortisol metabolism in human granulosa-lutein cells. PGE(2)-stimulated concentration-dependent increases in both progesterone production and cAMP accumulation (by 1.9 +/- 0.1- and 18.7 +/- 6.8-fold respectively at 3000 nM PGE(2)). While a selective PTGER1 antagonist, SC19220, could partially inhibit the steroidogenic response to PGE(2) (by 55.9 +/- 4.1% at 1000 nM PGE(2)), co-treatment with AH6809, a mixed PTGER1/PTGER2 receptor antagonist, completely abolished the stimulation of progesterone synthesis at all tested concentrations of PGE(2) and suppressed the stimulation of cAMP accumulation. Both PGE(2) and butaprost (a preferential PTGER2 receptor agonist) stimulated concentration-dependent increases in cortisol oxidation by 11betaHSD1 (by 42.5 +/- 3.1 and 40.0 +/- 3.0% respectively, at PGE(2) and butaprost concentrations of 1000 nM). Co-treatment with SC19220 enhanced the ability of both PGE(2) and butaprost to stimulate 11betaHSD1 activity (by 30.2 +/- 0.2 and 30.5 +/- 0.6% respectively), whereas co-treatment with AH6809 completely abolished the 11betaHSD1 responses to PGE(2) and butaprost. These findings implicate the PTGER2 receptor-cAMP signalling pathway in the stimulation of progesterone production and 11betaHSD1 activity by PGE(2) in human granulosa-lutein cells.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Dinoprostona/farmacología , Células Lúteas/metabolismo , Progesterona/biosíntesis , Receptores de Prostaglandina E/metabolismo , Alprostadil/análogos & derivados , Alprostadil/farmacología , Células Cultivadas , Cortisona/metabolismo , AMP Cíclico/metabolismo , Ácido Dibenzo(b,f)(1,4)oxazepina-10(11H)-carboxílico, 8-cloro-, 2-acetilhidrazida/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Hidrocortisona/metabolismo , Células Lúteas/efectos de los fármacos , Antagonistas de Prostaglandina/farmacología , Prostaglandinas E Sintéticas/farmacología , Subtipo EP1 de Receptores de Prostaglandina E , Subtipo EP2 de Receptores de Prostaglandina E , Xantonas/farmacologíaRESUMEN
Luteinization of follicular granulosa cells leads to an increase in progesterone secretion that is regulated by luteinizing hormone (LH). LH acts mainly by elevating intracellular cyclic 3',5'-adenosine monophosphate (cAMP) and activating cAMP-dependent protein kinase (PKA). In this study, we have examined the role of PKA in relation to progesterone output by luteinizing human granulosa cells. Human granulosa cells were obtained by percoll gradient centrifugation of follicular aspirates of patients undergoing oocyte retrieval for assisted conception. Cells were cultured in serum-supplemented medium for up to 3 days in the presence and/or absence of human (h)LH and other cAMP-elevating agents. Spent medium was assayed for cAMP and progesterone content by specific RIA. Cell lysates were collected and assessed for PKA regulatory (R)IIalpha/catalytic (C)alpha expression by Western blotting. Although basal progesterone secretion increased progressively throughout culture, cAMP levels remained unchanged. Under basal conditions, PKA RIIalpha/Calpha expression appeared to increase throughout the 3-day culture period. In the presence of hLH and other cAMP-elevating agents, progesterone secretion increased in a dose-dependent manner coincident with an increase in cAMP. However, despite the increase in both progesterone secretion and cAMP accumulation, there was a dose-dependent decrease in both PKA RIIalpha and Calpha expression. Thus, data presented in this study show that increases in progesterone secretion in luteinizing human granulosa cells can be dissociated from increases in PKA expression. This notion implies that progesterone secretion may be regulated by PKA-dependent as well as PKA-independent mechanisms.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células Lúteas/metabolismo , Hormona Luteinizante/farmacología , Progesterona/metabolismo , Análisis de Varianza , Western Blotting/métodos , Células Cultivadas , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico , Proteínas Quinasas Dependientes de AMP Cíclico/análisis , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Isoenzimas/análisis , Progesterona/análisis , Radioinmunoensayo/métodos , Estimulación QuímicaRESUMEN
Prostaglandin E(2) (PGE(2)) exerts mainly luteotrophic effects in the corpus luteum. In other tissues, PGE(2) acts via specific PGE(2) receptor subtypes including EP1, which modulates intracellular calcium ([Ca(2+)](i)) and EP2, which is coupled to cyclic AMP (cAMP) generation. We have therefore investigated the presence of functional EP1 and EP2 receptors using human granulosa-lutein (GL) cells. Reverse-transcription PCR revealed that GL cells expressed mRNA transcripts encoding both EP1 and EP2 receptors. When GL cells were challenged with ligands that can bind to both receptor subtypes (PGE(2) and 16,16 dimethyl PGE(2)) or exclusively to EP2 (butaprost), both cAMP formation and progesterone synthesis were stimulated. Furthermore, the cAMP response to these agonists could be significantly blocked by an EP1/2 antagonist AH6809 but not by an EP1-selective antagonist SC19220. Exposure of GL cells to 16,16-dm PGE(2) transiently raised [Ca(2+)](i) levels, which could be prevented by both AH6809 and SC19220. We therefore conclude that human GL cells express functional EP1 and EP2 receptors.
Asunto(s)
Alprostadil/análogos & derivados , Células de la Granulosa/metabolismo , Receptores de Prostaglandina E/biosíntesis , Xantonas , 16,16-Dimetilprostaglandina E2/farmacología , Alprostadil/farmacología , Calcio/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Ácido Dibenzo(b,f)(1,4)oxazepina-10(11H)-carboxílico, 8-cloro-, 2-acetilhidrazida/farmacología , Dinoprostona/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Células de la Granulosa/efectos de los fármacos , Humanos , Líquido Intracelular/metabolismo , Luteína/metabolismo , Progesterona/biosíntesis , Antagonistas de Prostaglandina/farmacología , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Receptores de Prostaglandina E/antagonistas & inhibidores , Receptores de Prostaglandina E/genética , Subtipo EP1 de Receptores de Prostaglandina E , Subtipo EP2 de Receptores de Prostaglandina E , Xantenos/farmacologíaRESUMEN
CCAAT/enhancer-binding protein-alpha (C/EBP alpha) is a basic leucine zipper protein that controls transcription of genes important for liver function, white adipose tissue development, and granulocyte differentiation. In addition to its function in controlling gene expression in differentiated tissues, C/EBP alpha is also associated with an antimitotic activity. We have previously demonstrated that C/EBP alpha interacts with p21, a cyclin-dependent kinase (CDK) inhibitor, and that C/EBP alpha inhibits proliferation when expressed in several different cell types (Timchenko, N. A., Harris, T. E., Wilde, M., Bilyeu, T. A., Burgess-Beusse, B. L., Finegold, M. J., and Darlington, G. J. (1997) Mol. Cell. Biol. 17, 7353--7361). Here we define the regions of C/EBP alpha required for interaction with p21 and demonstrate that CDK2 also interacts with C/EBP alpha. We show that C/EBP alpha can cooperate with p21 to inhibit CDK2 activity in vitro. The effect of C/EBP alpha on CDK2 activity requires the p21 and CDK2 interaction sites within C/EBP alpha. C/EBP alpha mutants incapable of inhibiting CDK2 activity in vitro do not inhibit proliferation in cultured cells. However, C/EBP alpha mutants defective in DNA binding inhibit proliferation as effectively as the wild-type protein. These findings show that C/EBP alpha-mediated growth arrest occurs through protein interactions and is independent of its transcriptional activity.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Quinasas CDC2-CDC28 , Ciclo Celular/fisiología , Ciclina E/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Ciclinas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Sustitución de Aminoácidos , Animales , Proteínas Potenciadoras de Unión a CCAAT/aislamiento & purificación , División Celular , Línea Celular , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/aislamiento & purificación , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/metabolismo , Fibroblastos/citología , Fibroblastos/fisiología , Humanos , Cinética , Ratones , Mutagénesis Sitio-Dirigida , Fosforilación , Mutación Puntual , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Spodoptera , TransfecciónRESUMEN
Twenty pairs of cadaveric humeri were used to compare the rotational stability of proximal humeral prostheses fixed by proximal cementation with the stability offered by press fit or full cementation. For each proximally cemented specimen, only the upper portion of the prosthesis was coated with cement. For the fully cemented specimens, a cement restrictor was used just distal to the prosthesis, and a finger-packing technique was used to fill the proximal humeral medullary canal. Torque was applied to the Morse taper of the prostheses, and rotational micromotion was measured at the level of the osteotomy. In each of 11 pairs of cadaveric humeri, one side was press fit and the contralateral side was proximally cemented; in each of 9 pairs, proximal cementation was compared with full cementation. Proximally cemented prostheses' micromotion was significantly less than that of press-fit prostheses (P = .0016). There was no difference in micromotion between proximal cementation and full cementation (P = .82). Proximal cementation increased initial fixation over press fit. Full cementation did not increase rotational stability.
Asunto(s)
Húmero/cirugía , Implantación de Prótesis , Articulación del Hombro/patología , Artroplastia/métodos , Cadáver , Cementación , Humanos , Húmero/patología , Inestabilidad de la Articulación , Rango del Movimiento Articular , Articulación del Hombro/cirugíaRESUMEN
The presence and functional significance of the extracellular calcium-sensing receptor (CaR) on human pancreatic beta-cells were investigated. Reverse transcriptase-polymerase chain reaction with primers for the extracellular domain of the CaR expressed in human parathyroid-secreting cells identified a product of the expected size in human pancreatic mRNA. Immunocytochemistry using an antibody against the extracellular region of CaR showed extensive immunoreactivity in insulin- and glucagon-containing cells but not in somatostatin-containing cells. In perifusion experiments, elevations in extracellular Ca2+ produced initial transient increases in insulin secretion, followed by a concentration-dependent and prolonged, but reversible, inhibition of secretion. Microfluorometric measurements of intracellular Ca2+ ([Ca2+]i) in isolated human beta-cells demonstrated that elevations in extracellular Ca2+ (0.5-10 mmol/l) caused rapid elevations in [Ca2+]i. Increases in extracellular Ca2+ caused small increases in the cyclic AMP content of whole human islets. These studies demonstrated that human beta-cells express an extracellular CaR and that activation of the receptor inhibits basal and nutrient-stimulated insulin secretion. The transduction mechanism that mediates this inhibitory effect is unknown, but our results suggest that it is unlikely to be through the adenylate cyclase-cyclic AMP pathway or through the phospholipase C-IP3 pathway. This CaR-mediated inhibitory mechanism may be an important autoregulatory mechanism in the control of insulin secretion.
Asunto(s)
Espacio Extracelular/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/fisiología , Receptores de Superficie Celular/fisiología , Calcio/fisiología , AMP Cíclico/metabolismo , Humanos , Técnicas In Vitro , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Páncreas/metabolismo , ARN Mensajero/metabolismo , Receptores Sensibles al Calcio , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismoRESUMEN
Elevations in intracellular Ca(2+) ([Ca(2+)](i)) initiate insulin secretion from pancreatic beta-cells, but the secretory responses become rapidly desensitised to maintained elevations in [Ca(2+)](i). We have investigated the mechanisms underlying the Ca(2+) desensitization of insulin secretion using electrically permeabilized rat islets of Langerhans. Measurements of Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) enzyme activity and immunoreactivity in permeabilized islets demonstrated Ca(2+)-induced reductions in enzyme activity which could not be attributed to reductions in CaMK II immunoreactive protein. Measurements in intact islets demonstrated that the Ca(2+)-induced reduction of CaMK II activity was also operative in intact cells, suggesting that this mechanism may have pathophysiological implications for beta-cell function.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Calcio/fisiología , Insulina/metabolismo , Islotes Pancreáticos/fisiología , Animales , Calcio/farmacología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Permeabilidad de la Membrana Celular , Electrofisiología , Glucosa/farmacología , Técnicas In Vitro , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/enzimología , Potenciales de la Membrana , Cloruro de Potasio/farmacología , Ratas , Tolbutamida/farmacologíaRESUMEN
Inhibitors of protein kinases are widely used to study stimulus-response pathways in pancreatic beta-cells. Synthetic peptides modelled on the pseudosubstrate sites of protein kinases, or of their endogenous inhibitor proteins, offer potentially specific inhibitors of individual protein kinases or kinase isoforms. However, the use of these inhibitors in studies of beta-cell physiology has been limited, since such peptide sequences are usually poorly membrane permeant. Myristoylation of these peptides enhances their ability to cross intact plasma membranes and thus inhibit intracellular protein kinases, and this approach is becoming increasingly common in identifying the cellular role(s) of particular protein kinases. In this study, using insulin-secreting beta-cells, we demonstrate that myristoylation alters the specificity of pseudosubstrate peptides such that all myristoylated peptides tested, even those lacking pseudosubstrate domains, acted as protein kinase C (PKC) inhibitors. This effect of myristoylation was limited to the inhibition of PKC, since the specificity of peptide inhibitors towards beta-cell protein kinase A activity was not affected by myristoylation. These results demonstrate that myristoylated pseudosubstrate peptides have value as protein kinase inhibitors in intact beta-cells, but emphasise that studies using them to ascribe role(s) for protein kinases in beta-cells must be interpreted with caution.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Islotes Pancreáticos/enzimología , Islotes Pancreáticos/metabolismo , Fragmentos de Péptidos/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Bucladesina/farmacología , Línea Celular , Células Cultivadas , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Cinética , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The role(s) played by protein tyrosine kinases (PTKs) in the regulation of insulin secretion from pancreatic beta cells is not clear. We have examined the effects of glucose, the major physiological insulin secretagogue, on the tyrosine phosphorylation state of islet proteins, and assessed beta cell insulin secretory responses in the presence of PTK inhibitors. Under basal conditions islets contained many proteins phosphorylated on tyrosine residues, and glucose (20 mM; 5-15 min) was without demonstrable effect on the pattern of tyrosine phosphorylation, in either the absence or presence of the protein tyrosine phosphatase (PTP) inhibitor, sodium pervanadate (PV). PV alone (100 microM) increased tyrosine phosphorylation of several islet proteins. The PTK inhibitors genistein (GS) and tyrphostin A47 (TA47) inhibited islet tyrosine kinase activities and glucose-, 4alpha ketoisocaproic acid (KIC)- and sulphonylurea-stimulated insulin release, without affecting glucose metabolism. GS and TA47 also inhibited protein serine/threonine kinase activities to a limited extent, but had no effect on Ca2+, cyclic AMP- or phorbol myristate acetate (PMA)-induced insulin secretion from electrically permeabilised islets. These results suggest that PTK inhibitors exert their inhibitory effects on insulin secretion proximal to Ca2+ entry and it is proposed that they act at the site of the voltage-dependent Ca2+ channel which regulates Ca2+ influx into beta cells following nutrient- and sulphonylurea-induced depolarisation.
Asunto(s)
Glucosa/farmacología , Insulina/metabolismo , Islotes Pancreáticos/enzimología , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Quinasas/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Animales , Calcio/fisiología , Canales de Calcio/fisiología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Línea Celular , AMP Cíclico/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Inhibidores Enzimáticos/farmacología , Genisteína/farmacología , Glucosa/metabolismo , Secreción de Insulina , Transporte Iónico , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Isoflavonas/farmacología , Leupeptinas/farmacología , Fluoruro de Fenilmetilsulfonilo/farmacología , Fosforilación/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/fisiología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/fisiología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Sistemas de Mensajero Secundario/fisiología , Acetato de Tetradecanoilforbol/farmacología , Tirfostinos/farmacología , Vanadatos/farmacologíaRESUMEN
CCAAT/enhancer binding protein alpha (C/EBP alpha) is expressed at high levels in quiescent hepatocytes and in differentiated adipocytes. In cultured cells, C/EBP alpha inhibits cell proliferation in part via stabilization of the p21 protein. The role of C/EBP alpha in regulating hepatocyte proliferation in vivo is presented herein. In C/EBP alpha knockout newborn mice, p21 protein levels are reduced in the liver, and the fraction of hepatocytes synthesizing DNA is increased. Greater than 30% of the hepatocytes in C/EBP alpha knockout animals continue to proliferate at day 17 of postnatal life when cell division in wild-type littermates is low (3%). p21 protein levels are relatively high in wild-type neonates but undetectable in C/EBP alpha knockout mice. The reduction of p21 protein in the highly proliferating livers that lack C/EBP alpha suggests that p21 is responsible for C/EBP alpha-mediated control of liver proliferation in newborn mice. During rat liver regeneration, the amounts of both C/EBP alpha and p21 proteins are decreased before DNA synthesis (6 to 12 h) and then return to presurgery levels at 48 h. Although C/EBP alpha controls p21 protein levels, p21 mRNA is not influenced by C/EBP alpha in liver. Using coimmunoprecipitation and a mammalian two-hybrid assay system, we have shown the interaction of C/EBP alpha and p21 proteins. Study of p21 stability in liver nuclear extracts showed that C/EBP alpha blocks proteolytic degradation of p21. Our data demonstrate that C/EBP alpha regulates hepatocyte proliferation in newborn mice and that in liver, the level of p21 protein is under posttranscriptional control, consistent with the hypothesis that protein-protein interaction with C/EBP alpha determines p21 levels.
Asunto(s)
Ciclinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Hígado/citología , Hígado/metabolismo , Proteínas Nucleares/metabolismo , Animales , Animales Recién Nacidos , Proteínas Potenciadoras de Unión a CCAAT , División Celular/genética , División Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , ADN/biosíntesis , Proteínas de Unión al ADN/genética , Técnicas In Vitro , Regeneración Hepática/genética , Regeneración Hepática/fisiología , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , RatasRESUMEN
Synthetic peptides derived from the endogenous protein kinase A inhibitor (PKI) offer a specific means of inhibiting cyclic AMP-dependent protein kinase A (PKA), but their use in whole cells is restricted by the plasma membrane. We have now modified PKI sequences by N-terminal myristoylation to enhance their membrane permeability, and have used the myristoylated (myr) peptides to investigate the role of PKA activation in glucose-induced insulin secretion from intact pancreatic beta-cells. The myristoylated PKI peptides, myr PKI14-22 and myrPKI6-22, were effective inhibitors in vitro of PKA activity extracted from rat islets of Langerhans. In experiments using intact islets, myr PKI14-22 caused a concentration-dependent inhibition of insulin secretion in response to the PKA activators dibutyryl cyclic AMP and forskolin, suggesting that it gained access to the cytosolic compartment of intact beta-cells and inhibited PKA in situ. However, these concentrations of myr PKI14-22 did not inhibit insulin secretion in response to glucose suggesting that the activation of PKA is not required for the initiation of glucose-induced insulin secretion.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular , Islotes Pancreáticos/enzimología , Animales , Proteínas Portadoras/farmacología , AMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Glucosa/metabolismo , Técnicas In Vitro , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Masculino , Fragmentos de Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Especificidad por SustratoAsunto(s)
Calcio/farmacología , Carbazoles/farmacología , Diglicéridos/farmacología , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Insulina/metabolismo , Islotes Pancreáticos/fisiología , Isoenzimas/antagonistas & inhibidores , Proteína Quinasa C/antagonistas & inhibidores , Animales , Células Cultivadas , Cricetinae , Glucosa/farmacología , Secreción de Insulina , Insulinoma , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/enzimología , Cinética , Neoplasias Pancreáticas , Ratas , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
The involvement of protein kinase C (PKC) isoforms in glucose-induced insulin secretion was investigated by comparing the effects of the PKC inhibitors Gö 6976, which is PKC specific and selective for the Ca(2+)-dependent isoforms, and Ro 31-8220, a specific PKC inhibitor which does not discriminate between isoforms. Gö 6976 inhibited the Ca(2+)- and diacylglycerol (DAG)-dependent PKC activities in beta-cell extracts in vitro and fully inhibited insulin secretory responses of rat islets of Langerhans to the PKC activator 4 beta phorbol myristate acetate (PMA), suggesting that it was an effective inhibitor of the DAG-dependent isoforms of PKC in situ. However, glucose-induced insulin secretion from rat islets was not inhibited by Gö 6976, whereas secretory responses to glucose were partially inhibited by the non-isoform selective PKC inhibitor, Ro 31-8220. The simplest explanation of these results is that glucose-induced insulin secretion is dependent, at least in part, upon the activation of an atypical isoform(s) of PKC within the beta-cell.
Asunto(s)
Carbazoles/farmacología , Indoles/farmacología , Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Animales , Inhibidores Enzimáticos/farmacología , Glucosa/farmacología , Secreción de Insulina , Islotes Pancreáticos/enzimología , Islotes Pancreáticos/metabolismo , Masculino , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Ratas Sprague-DawleyRESUMEN
We have used synthetic pseudosubstrate peptide inhibitors of protein kinase C (PKC) to re-examine the role of conventional isoforms of PKC in the insulin secretory response of intact rat islets of Langerhans to glucose and to the cholinergic agonist carbachol (CCh). One peptide was modified by N-terminal myristoylation (PKC-myr20-28) to allow its use in intact beta-cells. Maximal inhibition of PKC activity in vitro required 10-fold less of this peptide (PKC-myr20-28) than of its non-myristoylated analogue. The maximum inhibitory concentration of PKC-myr20-28 had little effect on islet protein kinase A or Ca2+/calmodulin kinase activities. PKC-myr20-28 (25-100 microM) caused a dose-dependent inhibition of phorbol myristate acetate (PMA)-induced insulin secretion from intact rat islets but non-myristoylated peptides had little effect on the secretory response to PMA. A concentration of PKC-myr20-28 (100 microM) which maximally inhibited PMA-induced insulin secretion, also inhibited the secretory response to CCh, but did not affect glucose-stimulated insulin secretion from intact islets. These results indicate that myristoylation of pseudosubstrate peptides increases their potency as inhibitors and that PKC-myr20-28 is a selective and cell-permeant inhibitor of PMA-sensitive isoforms of PKC. They also suggest that the activation of PMA-sensitive PKC isoforms mediates the stimulatory effects of CCh, but is not obligatory for glucose-induced insulin secretion from pancreatic beta-cells.