RESUMO
The present study was undertaken to characterize the effect of extracellular Na+ removal on 86Rb outflow from perifused rat pancreatic islets. Complete Na+ omission inhibited 86Rb outflow whether the islets were perifused in the presence or in the absence of extracellular Ca2+. Ouabain (1 mM) did not reduce the inhibitory effect of Na+ deprivation, whilst diphenylhydantoin (72.9 microM) mimicked the Na+-removal-induced fall in 86Rb outflow. Glucose (16.7 mM) lost its capacity to inhibit 86Rb outflow when the perifusate was deprived of extracellular Na+. These results indicate that Na+ omission reproduces the inhibitory effect of glucose on 86Rb outflow. The reduction in 86Rb outflow recorded after Na+ deprivation could be mediated by an intracellular acidification and/or a decrease in the intracellular Na+ activity. It is tempting to speculate that the capacity of glucose to reduce the B-cell Na+ content may participate in the process by which the sugar decreases K+ permeability.
Assuntos
Ilhotas Pancreáticas/metabolismo , Radioisótopos de Rubídio/metabolismo , Sódio/fisiologia , Cloreto de Amônio/farmacologia , Animais , Cálcio/farmacologia , Espaço Extracelular/metabolismo , Glucose/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Cinética , Ouabaína/farmacologia , Fenitoína/farmacologia , Quinina/farmacologia , Ratos , Tetraetilamônio , Compostos de Tetraetilamônio/farmacologiaRESUMO
Na/Ca exchange may play a role in Ca2+ extrusion from the pancreatic B cell. The role played by the exchanger was examined by characterizing the effects of 3'-4'-dichlorobenzamil on ionic fluxes and insulin release in normal rat pancreatic islet cells. 3',4'-Dichlorobenzamil potently inhibited 45Ca uptake mediated by reverse Na/Ca exchange (IC50: 18 microM) in islet cells. The drug failed to decrease intracellular pH but reduced 86Rb outflow from perifused islets. The effects of glucose and 3',4'-dichlorobenzamil on 86Rb outflow were not additive. The drug potently blocked 45Ca uptake through voltage-sensitive Ca2+ channels (IC50: 7.5 microM). In the presence of extracellular Ca2+ and 3',4'-dichlorobenzamil, glucose lost part of its ability to reduce 45Ca outflow. The drug failed to affect the secondary rise in 45Ca outflow induced by the sugar. In the absence of extracellular Ca2+, 3',4'-dichlorobenzamil induced a delayed inhibition of 45Ca outflow, the effect of the sugar and the drug being not additive. This effect of 3',4'-dichlorobenzamil and its ability to impair the inhibitory effect of glucose were reproduced by the removal of extracellular Na+ and disappeared under the latter experimental condition. 3',4'-Dichlorobenzamil did not affect insulin release in the absence of glucose but significantly increased glucose-induced insulin release when used at a high concentration. It is concluded that 3',4'-dichlorobenzamil is a potent inhibitor of the process of Na/Ca exchange in the pancreatic B cell. Unfortunately, the drug is of poor specificity and blocks, in the same range of concentrations, both K+ channels and voltage-sensitive Ca2+ channels. The data also indicate that glucose inhibits 45Ca outflow from pancreatic islets to a great extent (at least 75%) by inhibiting Na/Ca exchange. The type of Na/Ca exchange that is inhibited by glucose, remains to be elucidated.
Assuntos
Amilorida/análogos & derivados , Cálcio/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Sódio/metabolismo , Amilorida/farmacologia , Animais , Células Cultivadas/efeitos dos fármacos , Relação Dose-Resposta a Droga , Glucose/farmacologia , Concentração de Íons de Hidrogênio , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , RatosRESUMO
The present study aimed at comparing the effects of nifedipine, a dihydropyridine "Ca2+ antagonist", and of BAY K 8644, a dihydropyridine "Ca2+ agonist", on the short term (5 min) 45Ca uptake and the cytosolic Ca2+ concentration of rat pancreatic islet cells incubated in the presence of physiological concentrations of glucose. Nanomolar concentrations of nifedipine increased the short term 45Ca uptake while micromolar concentrations decreased it. Cd2+, an inorganic Ca2+ channel blocker, reduced the stimulatory effect of nifedipine. Low concentrations of BAY K 8644 stimulated 45Ca uptake whereas high concentrations decreased it. In contrast, verapamil, a phenylalkylamine type calcium antagonist, only provoked a dose-dependent reduction in 45Ca uptake. Lastly, low concentrations of both nifedipine and BAY K 8644 raised the fluorescence intensity of fura 2 loaded islet cells. These findings indicate that nifedipine and BAY K 8644 may exhibit agonistic and antagonistic actions on B-cell voltage-dependent Ca2+ channels. This dualistic behaviour is markedly concentration-dependent and appears to be inherent to the 1,4-dihydropyridine compounds.
Assuntos
Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Cálcio/metabolismo , Ilhotas Pancreáticas/metabolismo , Nifedipino/farmacologia , Animais , Cálcio/farmacocinética , Radioisótopos de Cálcio , Células Cultivadas , Meios de Cultura , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Fluorescência , Fura-2 , Glucose/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ratos , Verapamil/farmacologiaRESUMO
Na+/Ca2+ exchange in the B cell was recently characterized by measuring intracellular-Na(+)-dependent 45Ca2+ uptake in isolated rat pancreatic islet cells. The aim of the present study was to investigate the regulation of this process. Extracellular pH (pHo) and intracellular pH (pHi) markedly affected Na+/Ca2+ exchange. A fall of 0.04 unit in pHi decreased the exchange by 45%, whereas a rise of 0.13 unit increased the uptake by 70%. Mitochondrial poisons (oligomycin, antimycin A and 2,4-dinitrophenol) inhibited reverse Na+/Ca2+ exchange by about 25-50%. The exchanger displayed a low Q10 (temperature coefficient), indicating that it is only indirectly dependent on metabolic energy. The phorbol ester phorbol 12-myristate 13-acetate did not affect Na+/Ca2+ exchange. Likewise, lowering the extracellular K+ concentration did not inhibit 45Ca2+ uptake. In conclusion, the pHi and the metabolic state of the cell may represent important modulatory signals by which insulin secretagogues such as glucose could regulate reverse Na+/Ca2+ exchange in the B cell. The process does not appear to co-transport K+ nor to be influenced by protein kinase C.
Assuntos
Cálcio/metabolismo , Ilhotas Pancreáticas/metabolismo , Sódio/metabolismo , 2,4-Dinitrofenol , Animais , Antimicina A/farmacologia , Transporte Biológico/efeitos dos fármacos , Cátions Bivalentes , Dinitrofenóis/farmacologia , Concentração de Íons de Hidrogênio , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Monensin/farmacologia , Oligomicinas/farmacologia , Potássio/farmacologia , Ratos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
S 9795 (1-methyl-3-isobutyl-8-[2-ethyl 1-(4-diphenylmethylpiperazinyl)]- 3,7-dihydro(1H)purine-2,6-dione) is a new xanthine derivative displaying antiasthmatic properties in animals. The drug might exert its pharmacological actions either by inhibiting phosphodiesterases, or by inhibiting cellular Ca2+ movements or antagonizing purinoreceptors. Since the process of glucose-induced insulin release is markedly influenced by xanthine derivatives, the effect of S 9795 was compared to that of two other xanthine derivatives (theophylline and 3-isobutyl-1-methylxanthine (IBMX] on glucose-induced insulin release and ionic fluxes in rat pancreatic islets. Theophylline and IBMX potentiated glucose-induced insulin release, as expected, while S 9795 inhibited the insulinotropic effect of glucose. The effect of S 9795 was observed at a concentration of 10(-5) mol/l, lower concentrations (10(-6) to 10(-9) mol/l) failing to affect glucose-induced insulin release. At 10(-5) mol/l, the drug also inhibited the secondary rise in 45Ca efflux evoked by glucose from preloaded islets and the uptake of 45Ca by incubated islets stimulated either by glucose or potassium. The drug failed to alter 86Rb fluxes in stimulated and unstimulated islets labelled with the radioisotope. These data show that S 9795 inhibits glucose-induced insulin release, possibly by blocking glucose-stimulated Ca2+ inflow into the B-cell.
Assuntos
Insulina/metabolismo , Pâncreas/efeitos dos fármacos , Xantinas/farmacologia , Animais , Cálcio/metabolismo , Células Cultivadas , Glucose/farmacologia , Pâncreas/metabolismo , Potássio/farmacologia , Ratos , Teofilina/farmacologiaRESUMO
The presence of different types [long lasting (L) and transient (T)] of active voltage-operated Ca2+ channels in islet cells was investigated by comparing the effects of Cd2+, Ni2+, and 1,4-dihydropyridines on 45Ca uptake, 45Ca efflux, and insulin release in intact rat pancreatic islets. In several other excitable cells the L-channel has been shown to be modulated by 1,4-dihydropyridines and Cd2+, whereas the T-channel was reported to be sensitive to Ni2+. Nifedipine and Cd2+ inhibited whereas BAY K 8644 enhanced the glucose (11.1, 22.2 mM)-stimulated short-term 45Ca uptake, 45Ca efflux, and insulin release. In contrast, the stimulatory effects of glucose (11.1, 22.2 mM) on 45Ca uptake, 45Ca efflux, and insulin release were unaffected by Ni2+. These findings confirm that glucose provokes Ca2+ entry mainly by activating voltage-sensitive Ca2+ channels of the L-type and suggest that the B-cell plasma membrane is not equipped with active T-type Ca2+ channels.
Assuntos
Cádmio/farmacologia , Canais de Cálcio/fisiologia , Cálcio/metabolismo , Ilhotas Pancreáticas/fisiologia , Níquel/farmacologia , Animais , Canais de Cálcio/efeitos dos fármacos , Glucose/farmacologia , Técnicas In Vitro , Cinética , RatosRESUMO
Na(+)-Ca2+ exchange may play a role in Ca2+ extrusion from the pancreatic B-cell. The characteristics of the process working in its reverse mode were examined in normal rat pancreatic islet cells. Isosmotical replacement of extracellular Na+ by sucrose induced a concentration-dependent increase in 45Ca uptake, displaying a pharmacological sensitivity compatible with an uptake mediated by Na(+)-Ca2+ exchange. Glucose, up to 2.8 mM, stimulated reverse Na(+)-Ca2+ exchange. Likewise, membrane depolarization activated the process but only under raised intracellular Na+ activity. In conclusion, the B-cell Na(+)-Ca2+ exchange displays properties similar to those observed in other cells: reversibility and sensitivity to membrane potential. When working in its reverse mode the exchanger displays a quite large capacity. The role played by the exchanger in the process of insulin release warrants further investigation.
Assuntos
Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Ilhotas Pancreáticas/metabolismo , Sódio/metabolismo , Amilorida/farmacologia , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Radioisótopos de Cálcio , Gliclazida/farmacologia , Glucose/farmacologia , Técnicas In Vitro , Ilhotas Pancreáticas/efeitos dos fármacos , Cetoácidos/farmacologia , Cinética , Lantânio/farmacologia , Nifedipino/farmacologia , Ouabaína/farmacologia , Potássio/farmacologia , Ratos , Trocador de Sódio e Cálcio , Tetrodotoxina/farmacologiaRESUMO
In cultured neonatal islet cells, glucose (16.7 mM) and K+ (50 mM) increased cytosolic free Ca2+ ([Ca2+]i). The increments in [Ca2+]i induced by either glucose or K+ were similar to those obtained in cultured adult islet cells but only half of that recorded in freshly isolated adult islet cells. These data indicate that, in neonatal islet cells, the reduced insulin release in response to glucose is associated with a diminished increase in [Ca2+]i. This reduced insulin response may not solely be due to an impaired regulation of the ATP-sensitive K+ channels as previously suggested. It may also result from some alteration in the process of Ca2+ inflow through voltage-sensitive Ca2+ channels.
Assuntos
Cálcio/farmacocinética , Citosol/metabolismo , Glucose/farmacologia , Ilhotas Pancreáticas/citologia , Potássio/farmacologia , Trifosfato de Adenosina/farmacologia , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos/metabolismo , Animais Recém-Nascidos/fisiologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Cálcio/análise , Cálcio/metabolismo , Células Cultivadas , Citosol/química , Relação Dose-Resposta a Droga , Galopamil/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/química , Ilhotas Pancreáticas/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/fisiologia , RatosRESUMO
The possible relevance of changes in extracellular and/or intracellular pH to the insulinotropic action of L-arginine and L-homoarginine was investigated in rat pancreatic islets. A rise in extracellular pH from 7.0 to 7.4 and 7.8 augmented the secretory response to these cationic amino acids whilst failing to affect the uptake of L-arginine by islet cells and whilst decreasing the release of insulin evoked by D-glucose. Under these conditions, a qualified dissociation was also observed between secretory data and 45Ca net uptake. Moreover, at high extracellular pH, the homoarginine-induced increase in 86Rb outflow from prelabelled islets rapidly faded out, despite sustained stimulation of insulin release. The cationic amino acids failed to affect the intracellular pH of islet cells, whether in the absence or presence of D-glucose and whether at normal or abnormal extracellular pH. These findings argue against the view that the secretory response to L-arginine would be related to either a change in cytosolic pH or the accumulation of this positively charged amino acid in the beta-cell. Nevertheless, they suggest that the yet unidentified target for L-arginine and its non-metabolized analogue in islet cells displays pH-dependency with optimal responsiveness at alkaline pH.
Assuntos
Arginina/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Animais , Arginina/farmacologia , Cálcio/metabolismo , Glucose/metabolismo , Homoarginina/metabolismo , Concentração de Íons de Hidrogênio , Secreção de Insulina , Ratos , Rubídio/metabolismoRESUMO
L-arginine, L-ornithine, L-lysine and L-histidine (each 10 mM) stimulated amylase release from rat parotid cells. The secretory response to the cationic amino acids was suppressed in the absence of extracellular Ca2+ and, at physiological Ca2+ concentration, coincided with stimulation of 45Ca net uptake by the parotid cells. All cationic amino acids also accumulated inside the parotid cells. Nevertheless, the concept that the stimulation of amylase release is merely attributable to depolarization of the plasma membrane, secondary to the accumulation of these positively charged amino acids in the parotid cells, is questioned in view of both the inverse correlation found between their secretory effects and degree of ionization and the knowledge that parotid cells are electrically inexcitable.