Effector systems involved in the insulin secretory responses to efaroxan and RX871024 in rat islets of Langerhans (original) (raw)
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British Journal of Pharmacology, 1996
1 Microfluorimetry techniques with fura-2 were used to characterize the effects of efaroxan (200 pM), phentolamine (200-500 gM) and idazoxan (200-500 ,M) on the intracellular free Ca2+ concentration ([Ca2 ]+) in mouse isolated islets of Langerhans. 2 The imidazoline receptor agonists efaroxan and phentolamine consistently elevated cytosolic Ca2+ by mechanisms that were dependent upon Ca2+ influx across the plasma membrane; there was no rise in [Ca2+]1 when Ca2+ was removed from outside of the islets and diazoxide (100-250 gM) attenuated the responses. 3 Modulation of cytosolic [Ca2+]i by efaroxan and phentolamine was augmented by glucose (5-10 mM) which both potentiated the magnitude of the response and reduced the onset time of imidazoline-induced rises in [Ca2+],. 4 Efaroxanand phentolamine-evoked increases in [Ca2+], were unaffected by overnight pretreatment of islets with the imidazolines. Idazoxan failed to increase [Ca2+], under any experimental condition tested. 5 The putative endogenous ligand of imidazoline receptors, agmatine (1 gM-1 mM), blocked KATP channels in isolated patches of f-cell membrane, but effects upon [Ca2+], could not be further investigated since agmatine disrupts fura-2 fluorescence. 6 In conclusion, the present study shows that imidazolines will evoke rises in [Ca2+] in intact islets, and this provides an explanation to account for the previously described effects of imidazolines on KATP channels, the cell membrane potential and insulin secretion in pancreatic f-cells.
Diabetes, 1996
A novel imidazoline compound, RX871024, was used to investigate the mechanisms by which imidazoline derivatives promote insulin secretion in rat pancreatic p-cells and HIT T15 cells. RX871024 stimulated insulin release from rat pancreatic P-cells and HIT T15 cells in a glucose-dependent way. This effect was not related to a 2-adrenergic, 1^, and I 2-imidazoline receptors. RX871024 promoted insulin release by at least two modes of action. One included an increase in cytoplasmic free Ca 2+ concentration ([Ca 2+ ]j), subsequent to blocking of ATP-dependent K + channels, membrane depolarization, and activation of voltage-dependent Ca channels. The other, a more distal effect of imidazoline, affected the exocytotic machinery and was unrelated to changes in membrane potential and [Ca 2+ ]j. The mechanism of RX871024-induced insulin release was dependent on protein kinases A and C. The sensitizing effect of a low dose of RX871024 on glucose-induced insulin secretion suggests that imidazoline compounds of this kind may constitute the basis for development of a new class of oral hypoglycemic agents. Diabetes 45:1610-1618, 1996 G lucose-induced insulin secretion involves metabolism of glucose leading to an increase in the ATP-to-ADP ratio and subsequent closure of ATP-dependent K + channels (K ATP channels), membrane depolarization, opening of voltagedependent Ca 2+ channels, an increase in the cytoplasmic From the Department of Molecular Medicine (S.
British Journal of Pharmacology, 1999
1 When isolated rat islets were cultured for 18 h prior to use, the putative imidazoline binding site ligand, RX871024 caused a dose-dependent increase in insulin secretion at both 6 mM and 20 mM glucose. By contrast, a second ligand, efaroxan, was ineective at 20 mM glucose whereas it did stimulate insulin secretion in response to 6 mM glucose. 2 Exposure of islets to RX871024 (50 mM) for 18 h, resulted in loss of responsiveness to this reagent upon subsequent re-exposure. However, islets that were unresponsive to RX871024 still responded normally to efaroxan. 3 The imidazoline antagonist, KU14R, blocked the insulin secretory response to efaroxan, but failed to prevent the stimulatory response to RX871024. 4 By contrast with its eects in cultured islets, RX871024 inhibited glucose-induced insulin release from freshly isolated islets. Efaroxan did not inhibit insulin secretion under any conditions studied. 5 In freshly isolated islets, the eects of RX871024 on insulin secretion could be converted from inhibitory to stimulatory, by starvation of the animals. 6 Inhibition of insulin secretion by RX871024 in freshly isolated islets was prevented by the cyclooxygenase inhibitors indomethacin or¯urbiprofen. Consistent with this, RX871024 caused a marked increase in islet PGE 2 formation. Efaroxan did not alter islet PGE 2 levels. 7 The results suggest that RX871024 exerts multiple eects in the pancreatic b-cell and that its eects on insulin secretion cannot be ascribed only to interaction with a putative imidazoline binding site.
Potassium Channels, Imidazolines, and Insulin-Secreting Cells
Annals of the New York Academy of Sciences, 1995
Leicester LE2 7LX. UK It has been recognized for many years that certain imidazoline-derived a-adrenergic receptor antagonists will promote insulin secretion from the p-cells of the pancreatic islets of Langerhans. These effects occur both in vivo and in vitro and are mediated through interaction with the P-cell membrane at a site dissimilar to the a-adrenoceptor. Through the combined approaches of radiolabeled tracer studies and electrophysiology, there is good evidence that many of these compounds will inhibit potassium channels. Most electrophysiological studies have focused on a particular type of K' channel, the adenosine-triphosphate (ATP)-regulated K+ (K+ATp) channel. These channels are inhibited by imidazolines, and because they also play a major role in governing changes in P-cell membrane potential, channel block accounts for the fact that such compounds will depolarize the membrane, promote the generation of Ca2+ action potentials, elevate intracellular Ca", and elicit insulin secretion. In this short review we provide some background on the effects of imidazolines on insulinsecreting cells, and we present recent data from our own laboratory which show that imidazolines block other types of K ' channels in the p-cell (including human tissue), that they elevate intracellular Ca2+, and finally that the site of interaction with the K+ 'Human pancreatic P-cell work in our laboratories is funded by the British Diabetic Association (M.D., R.F.L.J., N.J.M.L.) and the Wellcome Trust (M.D.).
Biochemical and Biophysical Research Communications, 2001
Effects of the imidazoline compound RX871024 on cytosolic free Ca(2+) concentration ([Ca(2+)]i) and insulin secretion in pancreatic beta-cells from SUR1 deficient mice have been studied. In beta-cells from wild-type mice RX871024 increased [Ca(2+)]i by blocking ATP-dependent K(+)-current (K(ATP)) and inducing membrane depolarization. In beta-cells lacking a component of the K(ATP)-channel, SUR1 subunit, RX871024 failed to increase [Ca(2+)]i. However, insulin secretion in these cells was strongly stimulated by the imidazoline. Thus, a major component of the insulinotropic activity of RX871024 is stimulation of insulin exocytosis independently from changes in K(ATP)-current and [Ca(2+)]i. This means that effects of RX871024 on insulin exocytosis are partly mediated by interaction with proteins distinct from those composing the K(ATP)-channel.
British Journal of Pharmacology, 1992
Islets from normal mice were used to study the mechanisms by which imidazoline antagonists of a2-adrenoceptors increase insulin release in vitro. 2 Alinidine, antazoline, phentolamine and tolazoline inhibited 86Rb efflux from islets perifused with a medium containing 3 mM glucose, i.e. under conditions where many adenosine 5'-triphosphate (ATP)sensitive K+ channels are open in the a-cell membrane. They also reduced the acceleration of 86Rb efflux caused by diazoxide, an opener of ATP-sensitive K+ channels. 3 ATP-sensitive and voltage-sensitive K+ currents were measured in single P-cells by the whole-cell mode of the patch-clamp technique. Antazoline more markedly inhibited the ATP-sensitive than the voltage-sensitive current, an effect previously observed with phentolamine. Alinidine and tolazoline partially decreased the ATP-sensitive K+ current. 4 The four imidazolines reversed the inhibition of insulin release caused by diazoxide (through opening of ATP-sensitive K+ channels) or by clonidine (through activation of M2-adrenoceptors) in a concentration-dependent manner. Only the former effect correlated with the ability of each drug to increase control insulin release stimulated by 15 mm glucose alone. 5 It is concluded that the ability of imidazoline antagonists of a2-adrenoceptors to increase insulin release in vitro can be ascribed to their blockade of ATP-sensitive K+ channels in P-cells rather than to their interaction with the adrenoceptor.
Naunyn-Schmiedeberg's Archives of Pharmacology, 2003
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 αand β-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 β-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 Clchannels. 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 α-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.
British Journal of Pharmacology, 1997
1 The MIN6 cell line derived from in vivo immortalized insulin-secreting pancreatic b cells was used to study the insulin-releasing capacity and the cellular mode of action of S-21663, a newly synthesized imadizoline compound known for its antidiabetic eect in vivo and its ability to release insulin from perfused pancreas. 2 S-21663, at concentrations ranging from 10 75 M to 10 73 M was able to release insulin from MIN6 cells; its activity peaked at 10 74 M, a drop in the stimulant factor being noted between 10 74 and 10 73 M. Its ecacy, which did not dier whatever the glucose concentration (stimulant or not), was higher than that of the other secretagogues tested, glucose, sulphonylureas or the peptide tGLP-1.
European Journal of Pharmacology, 1997
. Recent studies have suggested that a variety of ion channels possess a binding site for ligands such as phencyclidine PCP , dizocilpine and certain s ligands and that some imidazoline compounds can also bind to this site. We have investigated whether interaction with this binding site could account for the ability of imidazolines to stimulate insulin secretion from rat islets. Neither PCP nor dizocilpine shared the insulin secretory activity of the imidazoline efaroxan in rat islets suggesting that they do not have similar actions in the pancreatic Ž Ž . . B-cell. Further, we were able to define a new antagonist, KU14R 2 2-ethyl 2,3-dihydro-2-benzofuranyl -2-imidazole , which selectively blocks the insulin secretory response to imidazolines. The results suggest that imidazolines do not stimulate insulin secretion by causing physical blockade of the K q -ATP channel in pancreatic B-cells and show that their effects are not reproduced by PCP or s receptor ligands.
American Journal of Physiology-endocrinology and Metabolism, 2010
Potential insulin secretagogue properties of an acetoxymethyl ester of a cAMP analog (8-pCPT-2=-O-Me-cAMP-AM) that activates the guanine nucleotide exchange factors Epac1 and Epac2 were assessed using isolated human islets of Langerhans. RT-QPCR demonstrated that the predominant variant of Epac expressed in human islets was Epac2, although Epac1 was detectable. Under conditions of islet perifusion, 8-pCPT-2=-O-Me-cAMP-AM (10 M) potentiated firstand second-phase 10 mM glucose-stimulated insulin secretion (GSIS) while failing to influence insulin secretion measured in the presence of 3 mM glucose. The insulin secretagogue action of 8-pCPT-2=-O-Me-cAMP-AM was associated with depolarization and an increase of [Ca 2ϩ ]i that reflected both Ca 2ϩ influx and intracellular Ca 2ϩ mobilization in islet -cells. As expected for an Epac-selective cAMP analog, 8-pCPT-2=-O-Me-cAMP-AM (10 M) failed to stimulate phosphorylation of PKA substrates CREB and Kemptide in human islets. Furthermore, 8-pCPT-2=-O-Me-cAMP-AM (10 M) had no significant ability to activate AKAR3, a PKA-regulated biosensor expressed in human islet cells by viral transduction. Unexpectedly, treatment of human islets with an inhibitor of PKA activity (H-89) or treatment with a cAMP antagonist that blocks PKA activation (Rp-8-CPT-cAMPS) nearly abolished the action of 8-pCPT-2=-O-Me-cAMP-AM to potentiate GSIS. It is concluded that there exists a permissive role for PKA activity in support of human islet insulin secretion that is both glucose dependent and Epac regulated. This permissive action of PKA may be operative at the insulin secretory granule recruitment, priming, and/or postpriming steps of Ca 2ϩdependent exocytosis. protein kinase A; exocytosis; adenosine-3=,5=-cyclic monophosphate; exchange protein directly activated by adenosine-3=,5=-cyclic monophosphate; calcium ADENOSINE-3=,5=-CYCLIC MONOPHOSPHATE (cAMP) is a cytosolic second messenger that potentiates the glucose metabolismdependent secretion of insulin from pancreatic -cells located within the islets of Langerhans (58). Although it is generally accepted that the insulin secretagogue action of cAMP results from its ability to activate protein kinase A (PKA), studies of rodent islets and insulin-secreting cell lines provide evidence for the existence of an alternative mechanism of cAMP signal transduction, one that is mediated by cAMP-regulated guanine nucleotide exchange factors known as the exchange proteins