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Desensitization of insulin secretory response to imidazolines, tolbutamide, and quinine II. Electrophysiological and fluorimetric studies
No full textProlonged in vitro exposure (18 h) of pancreatic islets to insulin secretagogues that block ATP-dependent K+ channels (K-ATP channels). such as sulfonylureas. imidazolines, and quinine, induced a desensitization of insulin secretion (Rustenbeck et al., pages 1685-1694, this issue). To elucidate the underlying mechanisms. KATP channel activity, plasma membrane potential and the cytosolic Ca2+ concentration ([Ca2+](i)) were measured in mouse single B-cells. In B-cells desensitized by phentolamine or quinine (100 muM each) KATP channel activity was virtually absent and could not be elicited by diazoxide. Desensitization by alinidine (100 muM) induced a marked reduction of KATP channel activity, which could be reversed by diazoxide, whereas exposure to idazoxan (100 muM) or tolbutamide (500 muM) had no lasting effect on K-ATP channel activity. Correspondingly. phentolamine, alinidine, and quinine-desensitized B-cells were markedly depolarized, whereas B-cells that had been exposed to tolbutamide or idazoxan had an unchanged resting membrane potential. The increase in [Ca2+](i) normally elicited by phentolamine and alinidine was suppressed after desensitization by these compounds., whereas the [Ca2+](i) increase by re-exposure to quinine was markedly reduced and that by tolbutamide only minimally affected as compared with control-cultured B-cells. The increase in [Ca2+](i) elicited by a K+ depolarization was diminished in secretagogue-pretreated B-cells. the extent depending on the secretagogue. This effect was closely correlated with the degree of depolarization after pretreatment with the respective secretagogue. In conclusion, the apparently uniform desensitization of secretion by K-ATP channel blockers is due to different effects at two stages located distally in the stimulus-secretion coupling: either at the stage of [Ca2+](i), regulation, where the increase is depressed as a consequence of a persistent depolarization (e.g. in the case of phentolamine or alinidine) and/or at the stage of exocytosis. which responds only weakly to substantial increases in [Ca2+](i) (in the case of tolbutamide). (C) 2001 Elsevier Science Inc, All rights reserved
Desensitization of insulin secretory response to imidazolines, tolbutamide, and quinine I. Secretory and morphological studies
No full textThe desensitization of pancreatic B-cells against stimulation by insulin secretagogues that inhibit ATP-dependent K+ channels (K-ATP channels) was investigated by measuring insulin secretion of perifused pancreatic islets. Additionally, the islet insulin content and the number of secretory granules per B-cell were determined. Prior to the measurement of secretion, islets were cultured for 18 h in the presence or absence of the test agents in a cell-culture medium containing 5 mM glucose. The effects of three imidazolines, phentolamine, alinidine, and idazoxan (100 AM each) were compared with those of the well-characterized sulfonylurea. tolbutamide (500 AM), and those of the ion channel-blocking alkaloid, quinine (100 AM). Insulin secretion was strongly reduced upon re-exposure to phentolamine, alinidine, tolbutamide, and quinine, whereas idazoxan, which stimulated secretion only weakly, had no significant effect. The imidazoline secretagogues phentolamine and alinidine induced a cross-desensitization against the stimulatory effect of tolbutamide and quinine. A long-term depolarization with 40 mM KCl was also able to induce a significant reduction of the secretory response to all of the above secretagogues. The insulin content of cultured islets was moderately, but significantly reduced by alinidine, whereas the reduction by phentolamine, tolbutamide, and quinine was not significant. In contrast to these observations, the ultrastructural examination revealed that tolbutamide-treated B-cells had a high degree of degranulation, whereas the other test agents and 40 mM KCl produced only a partial degranulation, except for phentolamine, which produced no significant degranulation at all. These results suggest that the desensitization of insulin secretion is a common property of all agents that stimulate insulin secretion by depolarisation of the plasma membrane. Depending on the specific secretagogue, additional mechanisms, proximal and distal to Ca2+ influx, appear to contribute to the desensitization (see Rustenbeck et al., pages 1695-1703, this issue). (C) 2001 Elsevier Science Inc. All rights reserved
Antagonism of the imidazoline block of ATP-sensitive K- channels by nucleotides and diazoxide
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beta-cell toxicity of ATP-sensitive K+ channel-blocking insulin secretagogues
No full textA prolonged exposure of isolated pancreatic islets to insulin secretagogues, the imidazolines phentolamine, alinidine and idazoxan (100 muM each), the sulfonylurea tolbutamide (500 muM), or the alkaloid quinine (100 muM) resulted in morphological damage of 4-18% of beta-cells compared to less than 2% in controls. Thus, the question arose whether K-ATP channel-blocking insulin secretagogues are beta-cell toxic as has already been suggested for sulfonylureas. The concentration- and time-dependency of the secretagogue-associated toxicity was documented by viability assays in insulin-secreting HIT T15 cells. Treatment for 24 h with idazoxan reduced MTT conversion by 50% at 100 muM and by 98% at 1000 muM. Phentolamine and quinine reduced viability comparably at 1000 muM, but were less toxic at 100 muM. On the other hand, the imidazoline alinidine and the sulfonylurea tolbutamide were only moderately toxic (less than 40% viability loss at 1000 muM). The imidazoline efaroxan appeared even to be non-toxic. Apoptotic DNA fragmentation and DEVD-caspase activation was observed at 100 muM of idazoxan and phentolamine, whereas at 1000 muM signs of necrosis predominated. Alinidine, tolbutamide and quinine treatment did not increase markers of apoptotic cell death. Blocking Ca2+ influx by D600 did not diminish secretagogue-associated toxicity. Electron microscopy confirmed the validity of these observations for beta-cells in intact mouse islets. In summary, beta-cell toxicity of the tested insulin secretagogues varied widely and did not depend on a prolonged Ca2+ influx via L-type Ca2+ channels. Thus, secretagogue-mediated closure of K-ATP channels is apparently not per se beta-cell toxic. (C) 2004 Elsevier Inc. All rights reserved
Antagonism of the imidazoline block of ATP-sensitive K- channels by nucleotides and diazoxide
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Does a verapamil-sensitive, P-glycoprotein-like transport mechanism participate in mitochondrial rhodamine 123-accumulation?
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