RESUMEN
The accumulation of intracellular ionic zinc and pharmaceutical compounds, like the antibiotic sulfamethoxazole, may contribute to various neuropathologies. Sulfamethoxazole and the drug trimethoprim, are inhibitors of enzymes involved in the synthesis of tetrahydrofolate and also of carbonic anhydrases. The inhibition of the latter enzymes, which are localized both intra- and extracellularly and have a key role in pH regulation, causes alkalinization that is associated with higher spontaneous transmitter release. Intense synaptic stimulation causes the entry of released zinc into postsynaptic neurons, through glutamate receptor channels or voltage dependent calcium channels. The aim of this study was to evaluate the effect of sulfamethoxazole (180 µM) on basal postsynaptic zinc and to compare it with that caused by two depolarizing media, containing high potassium or tetraethylammonium, which may induce long term synaptic plasticity. The studies were performed in brain slices from gestating rats, at the mossy fiber synapses from hippocampal CA3 area, using the zinc indicator Newport Green. In the presence of KCl (20 mM) and sulfamethoxazole (180 µM) the zinc signals were enhanced, unlike in tetraethylammonium (25 mM). After sulfamethoxazole the tetraethylammonium evoked zinc signal had reduced amplitude. Thus, the data suggests that sulfamethoxazole enhances transmitter release affecting synaptic zinc physiology.
Asunto(s)
Antiinfecciosos/toxicidad , Fibras Musgosas del Hipocampo/efectos de los fármacos , Sulfametoxazol/toxicidad , Sinapsis/efectos de los fármacos , Zinc/metabolismo , Animales , Femenino , Fibras Musgosas del Hipocampo/metabolismo , Técnicas de Cultivo de Órganos , Embarazo , Ratas , Ratas WistarRESUMEN
Zinc is a transition metal that is particularly abundant in the mossy fibers of the hippocampal CA3 area. Despite the large number of studies about the zinc role in mossy fibers, the action of zinc in synaptic mechanisms is only partly known. The use of computational models can be a useful tool for this study. In a previous work, a model was developed to evaluate zinc dynamics at the mossy fiber synaptic cleft, following weak stimulation, insufficient to evoke zinc entry into postsynaptic neurons. For intense stimulation, cleft zinc effluxes must be considered. Therefore, the initial model was extended to include postsynaptic zinc effluxes based on the Goldman-Hodgkin-Katz current equation combined with Hodgkin and Huxley conductance changes. These effluxes occur through different postsynaptic escape routes, namely L- and N-types voltage-dependent calcium channels and NMDA receptors. For that purpose, various stimulations were assumed to induce high concentrations of cleft free zinc, named as intense (10 µM), very intense (100 µM) and extreme (500 µM). It was observed that the main postsynaptic escape routes of cleft zinc are the L-type calcium channels, followed by the NMDA receptor channels and by N-type calcium channels. However, their relative contribution for cleft zinc clearance was relatively small and decreased for higher amounts of zinc, most likely due to the blockade action of zinc in postsynaptic receptors and channels. Therefore, it can be concluded that the larger the zinc release, the more predominant the zinc uptake process will be in the cleft zinc clearance.
Asunto(s)
Fibras Musgosas del Hipocampo , Zinc , Zinc/metabolismo , Sinapsis/fisiología , Hipocampo/metabolismo , Receptores de N-Metil-D-Aspartato/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
The glucose sensitivity of bursting electrical activity and pulsatile insulin release from pancreatic islets was determined in absence of functional K(ATP) channels. Membrane potential, [Ca(2+)](i) and 5-HT/insulin release were measured by intracellular recording, fura-2 fluorescence and 5-HT amperometry, respectively. Single mouse islets, bathed in tolbutamide or glibenclamide and high extracellular Ca(2+) (Ca(2+)(o)), displayed bursting activity and concomitant fast [Ca(2+)](i) and 5-HT/insulin oscillations. Sulphonylurea block of K(ATP) channel current was unaffected by raising Ca(2+)(o). Raising glucose or alpha-ketoisocaproic acid (KIC) concentration from 3 to 30 mM increased spiking activity and burst plateau duration. Staurosporine did not impair glucose potentiation of electrical activity, ruling out the involvement of serine/threonine kinases. Glucose enhanced both [Ca(2+)](i) and 5-HT/insulin oscillatory activity, causing a approximately 3-fold increase in overall 5-HT release rate. Cells lacking bursting activity in high Ca(2+)(o) and low glucose (or KIC) developed a pattern of intensified spiking in response to 11 mM glucose. It is concluded that beta-cells exhibit graded oscillatory electrical and secretory responses to glucose in absence of functional K(ATP) channels. This suggests that, under physiological conditions, early glucose sensing may involve other channels besides the K(ATP) channel.
Asunto(s)
Glucosa/fisiología , Células Secretoras de Insulina/fisiología , Insulina/metabolismo , Potenciales de la Membrana/fisiología , Serotonina/metabolismo , Adenosina Trifosfato/fisiología , Animales , Calcio/fisiología , Células Cultivadas , Gliburida/farmacología , Secreción de Insulina , Cetoácidos/farmacología , Potenciales de la Membrana/efectos de los fármacos , Ratones , Técnicas de Placa-Clamp , Canales de Potasio/efectos de los fármacos , Tolbutamida/farmacologíaRESUMEN
BACKGROUND: 2-Methylthioadenosine 5'-triphosphate (2-MeSATP), formerly regarded as a specific P2Y (metabotropic) purinergic receptor agonist, stimulates Ca2+ influx and evokes catecholamine release from adrenal chromaffin cells. These cells express P2Y and P2X (ionotropic) purinoceptors, with the latter providing an important Ca2+ influx pathway. Using single cell calcium imaging techniques, we have determined whether 2-MeSATP might be a specific P2X receptor agonist in bovine chromaffin cells and assessed the relative role of P2X and P2Y receptors on catecholamine secretion from these cells. RESULTS: ATP raised the [Ca2+]i in ~50% of the cells. Removing extracellular Ca2+ suppressed the [Ca2+]i-raising ability of 2-MeSATP, observed in ~40% of the ATP-sensitive cells. This indicates that 2-MeSATP behaves as a specific ionotropic purinoceptor agonist in bovine chromaffin cells. The 2-MeSATP-induced [Ca2+]i-rises were suppressed by PPADS. UTP raised the [Ca2+]i in ~40% of the ATP-sensitive cells, indicating that these expressed Ca2+-mobilizing P2Y receptors. UTP-sensitive receptors may not be the only P2Y receptors present, as suggested by the observation that ~20% of the ATP-sensitive pool did not respond to either 2-MeSATP or UTP. The average sizes of the ATP- and 2-MeSATP-evoked [Ca2+]i responses were identical in UTP-insensitive cells. 2-MeSATP stimulated Ca2+ influx and evoked catecholamine release, whereas UTP elicited Ca2+ release from intracellular stores but did not evoke secretion. 2-MeSATP-induced secretion was strongly inhibited by Cd2+ and suppressed by extracellular Ca2+ or Na+ removal. TTX inhibited 2-MeSATP-evoked secretion by ~20%. CONCLUSION: 2-MeSATP is a specific P2X purinoceptor agonist and a potent secretagogue in bovine chromaffin cells. Activation of 2-MeSATP-sensitive receptors stimulates Ca2+ influx mainly via voltage-sensitive Ca2+ channels. For the most part, these are activated by the depolarization brought about by Na+ influx across P2X receptor pores.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Glándulas Suprarrenales/metabolismo , Catecolaminas/metabolismo , Células Cromafines/metabolismo , Agonistas del Receptor Purinérgico P2 , Receptores Purinérgicos P2/fisiología , Tionucleótidos/farmacología , Adenosina Trifosfato/administración & dosificación , Adenosina Trifosfato/farmacología , Glándulas Suprarrenales/citología , Animales , Calcio/metabolismo , Bovinos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Membranas Intracelulares/metabolismo , Concentración Osmolar , Tionucleótidos/administración & dosificación , Uridina Trifosfato/farmacologíaRESUMEN
BACKGROUND: Adrenal chromaffin cells mediate acute responses to stress through the release of epinephrine. Chromaffin cell function is regulated by several receptors, present both in adrenergic (AD) and noradrenergic (NA) cells. Extracellular ATP exerts excitatory and inhibitory actions on chromaffin cells via ionotropic (P2X) and metabotropic (P2Y) receptors. We have taken advantage of the actions of the purinergic agonists ATP and UTP on cytosolic free Ca2+ concentration ([Ca2+]i) to determine whether P2X and P2Y receptors might be asymmetrically distributed among AD and NA chromaffin cells. RESULTS: The [Ca2+]i and the [Na+]i were recorded from immunolabeled bovine chromaffin cells by single-cell fluorescence imaging. Among the ATP-sensitive cells ~40% did not yield [Ca2+]i responses to ATP in the absence of extracellular Ca2+ (Ca2+o), indicating that they expressed P2X receptors and did not express Ca2+- mobilizing P2Y receptors; the remainder expressed Ca2+-mobilizing P2Y receptors. Relative to AD-cells approximately twice as many NA-cells expressed P2X receptors while not expressing Ca2+- mobilizing P2Y receptors, as indicated by the proportion of cells lacking [Ca2+]i responses and exhibiting [Na+]i responses to ATP in the absence and presence of Ca2+o, respectively. The density of P2X receptors in NA-cells appeared to be 30-50% larger, as suggested by comparing the average size of the [Na+]i and [Ca2+]i responses to ATP. Conversely, approximately twice as many AD-cells expressed Ca2+-mobilizing P2Y receptors, and they appeared to exhibit a higher (~20%) receptor density. UTP raised the [Ca2+]i in a fraction of the cells and did not raise the [Na+]i in any of the cells tested, confirming its specificity as a P2Y agonist. The cell density of UTP-sensitive P2Y receptors did not appear to vary among AD- and NA-cells. CONCLUSION: Although neither of the major purinoceptor types can be ascribed to a particular cell phenotype, P2X and Ca2+-mobilizing P2Y receptors are preferentially located to noradrenergic and adrenergic chromaffin cells, respectively. ATP might, in addition to an UTP-sensitive P2Y receptor, activate an UTP-insensitive P2Y receptor subtype. A model for a short-loop feedback interaction is presented whereby locally released ATP acts upon P2Y receptors in adrenergic cells, inhibiting Ca2+ influx and contributing to terminate evoked epinephrine secretion.
Asunto(s)
Glándulas Suprarrenales/metabolismo , Calcio/metabolismo , Células Cromafines/metabolismo , Epinefrina/metabolismo , Norepinefrina/metabolismo , Receptores Purinérgicos P2/metabolismo , Adenosina Trifosfato/farmacología , Glándulas Suprarrenales/citología , Animales , Bovinos , Células Cultivadas , Células Cromafines/clasificación , Células Cromafines/efectos de los fármacos , Células Cromafines/fisiología , Membranas Intracelulares/metabolismo , Concentración Osmolar , Fenotipo , Agonistas Purinérgicos , Sodio/metabolismo , Distribución Tisular , Uridina Trifosfato/farmacologíaRESUMEN
OBJECTIVES: Glucose-induced insulin secretion from pancreatic beta cells is modulated by several hormones and transmitters, namely adenosine triphosphate (ATP) via purinergic receptors. Although P2Y receptors are well documented in beta cells, the presence of P2X receptors remains elusive. We present the first electrophysiological evidence for the presence of P2X receptors in single beta cells of different species. METHODS: Ionic currents were recorded from voltage-clamped beta cells near their resting potential using the perforated (nystatin) whole-cell patch-clamp configuration. Receptors were detected by immunocytochemistry. RESULTS: When bathed in substimulatory (2 mM) glucose, mouse beta cells, isolated from islets displaying immunochemical colocalization of P2X1 or P2X3 receptors and insulin, developed large (approximately 250 pA/pF), rapidly activating, and then biexponentially decaying (tau1, approximately 20 milliseconds/tau2, approximately 1 second) inward currents on exposure to micromolar concentrations of ATP and alpha,beta-methylene ATP. The ATP also evoked inward currents (100-300 pA/pF) from porcine and human beta cells, albeit with a slower and more complex inactivation pattern. CONCLUSIONS: The ATP-gated ion channels are present in pancreatic beta cells from different species. Specifically, mouse beta cells express rapidly desensitizing P2X1 and P2X3 receptors. Paracrine or neural activation of these receptors may contribute to the initial outburst of glucose- or acetylcholine-evoked insulin release, thus enhancing the islet secretory response.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Receptores Purinérgicos P2/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Electrofisiología , Femenino , Glucosa/farmacología , Humanos , Inmunohistoquímica , Técnicas In Vitro , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Activación del Canal Iónico/efectos de los fármacos , Ratones , Técnicas de Placa-Clamp , Receptores Purinérgicos P2X , Receptores Purinérgicos P2X3 , PorcinosRESUMEN
Thymeleatoxin (TMX), an activator of Ca2+-sensitive protein kinase C (cPKC) isoforms, was used to assess the PKC isoform specificity of cholinergic potentiation of glucose (11 mM)-induced pulsatile 5-HT/insulin release (PIR) from single mouse pancreatic islets. TMX (100 nM) and carbachol (Cch, 50 microM) enhanced PIR approximately 3-fold while reducing the underlying [Ca2+]i oscillations (duration and amplitude) by approximately 40-50%. Both effects were ablated by the specific PKC inhibitor bisindolylmaleimide and chronic TMX pretreatment. Cch also evoked an initial transient [Ca2+]i rise and surge of 5-HT release, which remained unaffected by chronic TMX pretreatment. It is concluded that the immediate cholinergic responses are insensitive to cPKC. In contrast, specific activation of a cPKC isoform mediates sustained cholinergic potentiation of glucose-induced insulin secretion.
Asunto(s)
Glucosa/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Ésteres del Forbol/farmacología , Proteína Quinasa C/efectos de los fármacos , Serotonina/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Carbacol/farmacología , Agonistas Colinérgicos/farmacología , Electroquímica , Fluorometría , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Ratones , Proteína Quinasa C/metabolismo , Flujo Pulsátil/efectos de los fármacosRESUMEN
The development of type 2 diabetes is associated with the impairment of insulin secretion. To evaluate the evolution of the secretory response, a chronological study comparing normal Wistar (W) vs Goto-Kakizaki (GK) rats, an animal model of non obese type 2 diabetes, was done. Glucose and arginine were tested in collagenase isolated islets of Langerhans with perfusion and ELISA immunoassay techniques. Fasting glycaemia and insulinemia and glucose tolerance were also evaluated. We have seen, in W rats, a mild glucose intolerance in the first two weeks of age. GK rats were always glucose intolerant with hyperglycaemia and hyperinsulinemia at fasten after one month old. Wistar islets had a characteristic biphasic response to glucose after the first two weeks of age. GK islets were always glucose irresponsive. Arginine induced an increase in insulin secretion in both animal models, independent of age, although GK rats had always a smaller response when compared to W rats. We concluded that 1) in W rats, a biphasic insulin secretion in response to glucose is observed after the first two weeks of age, simultaneously with glycaemia stabilization 2) in GK rats, both first and second phases of glucose-induced insulin release are significantly reduced and a smaller reduction in response to arginine is observed. This beta-cell disfunction is a primary event in this model of type 2 diabetes, preceding fasting hyperglycaemia and hyperinsulinemia.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Modelos Animales de Enfermedad , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/fisiopatología , Animales , Secreción de Insulina , Ratas , Ratas WistarRESUMEN
The impairment of insulin secretion, a major feature of type 2 diabetes, is caused by beta-cell mass reduction and functional failure. Pancreatic beta-cell mass reduction is variable in humans, not exceeding 50%, and has been associated with amyloid deposits. In the present study, we have chronologically compared the endocrine pancreas morphology of Wistar control rats (W) and Goto-Kakizaki (GK) rats, an animal model of non obese type 2 diabetes. We have also characterised and compared their body weight, glycaemia (fasting and after oral glucose load) as well as other biochemical parameters. GK rats were always glucose intolerant and fasting hyperglycaemia arised at four week of age. Wistar rats had mild glucose intolerance in their first two weeks of life. GK rats had a total beta-cell mass always decreased when compared to controls, but above 40%. In adult GK rats (12 weeks old) alterations in the architecture of a sub-population of islets occurred which displayed signs of prominent fibrosis, with cluster of beta-cells widely separated by strands of connective tissue and deposits of PAS positive material. Our findings demonstrate that, using GK rats from the Coimbra colony, beta-cell mass reduction is one of the primary features in the pathological sequence leading to diabetes. Structural lesions of the islets, that will further increase beta-cell mass reduction and compromise beta-cell function, will appear latter mainly due to hyperglycaemia.
Asunto(s)
Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 2/patología , Modelos Animales de Enfermedad , Islotes Pancreáticos/patología , Animales , Femenino , Masculino , Ratas , Ratas WistarRESUMEN
Thymeleatoxin (TMX), an activator of Ca2+-sensitive protein kinase C (cPKC) isoforms, was used to assess the PKC isoform specificity of cholinergic potentiation of glucose (11 mM)-induced pulsatile 5-HT/insulin release (PIR) from single mouse pancreatic islets. TMX (100 nM) and carbachol (Cch, 50 mM) enhanced PIR ~ 3-fold while reducing the underlying [Ca2+]i oscillations (duration and amplitude) by ~ 40-50 percent. Both effects were ablated by the specific PKC inhibitor bisindolylmaleimide and chronic TMX pretreatment. Cch also evoked an initial transient [Ca2+]i rise and surge of 5-HT release, which remained unaffected by chronic TMX pretreatment. It is concluded that the immediate cholinergic responses are insensitive to cPKC. In contrast, specific activation of a cPKC isoform mediates sustained cholinergic potentiation of glucose-induced insulin secretion.