Characterization of a KATP channel-independent pathway involved in potentiation of insulin secretion by efaroxan.

Efaroxan, like several other imidazoline reagents, elicits a glucose-dependent increase in insulin secretion from pancreatic beta-cells. This response has been attributed to efaroxan-mediated blockade of KATP channels, with the subsequent gating of voltage-sensitive calcium channels. However, increasing evidence suggests that, at best, this mechanism can account for only part of the secretory response to the imidazoline. In support of this, we now show that efaroxan can induce functional changes in the secretory pathway of pancreatic beta-cells that are independent of KATP channel blockade. In particular, efaroxan was found to promote a sustained sensitization of glucose-induced insulin release that persisted after removal of the drug and to potentiate Ca2+-induced insulin secretion from electropermeabilized islets. To investigate the mechanisms involved, we studied the effects of the efaroxan antagonist KU14R. This agent is known to selectively inhibit insulin secretion induced by efaroxan, without altering the secretory response to glucose or KCl. Surprisingly, however, KU14R markedly impaired the potentiation of insulin secretion mediated by agents that raise cAMP, including the adenylate cyclase activator, forskolin, and the phosphodiesterase inhibitor isobutylmethyl xanthine (IBMX). These effects were not accompanied by any reduction in cAMP levels, suggesting an antagonistic action of KU14R at a more distal point in the pathway of potentiation. In accord with our previous work, islets that were exposed to efaroxan for 24 h became selectively desensitized to this agent, but they still responded normally to glucose. Unexpectedly, however, the ability of either forskolin or IBMX to potentiate glucose-induced insulin secretion was severely impaired in these islets. By contrast, the elevation of cAMP was unaffected by culture of islets with efaroxan. Taken together, the data suggest that, in addition to effects on the KATP channel, imidazolines also interact with a more distal component that is crucial to the potentiation of insulin secretion. This component is not required for Ca2+-dependent secretion per se but is essential to the mechanism by which cAMP potentiates insulin release. Overall, the results indicate that the actions of efaroxan at this distal site may be more important for control of insulin secretion than its effects on the KATP channel.

Effects of imidazoline binding site ligands on the growth and viability of clonal pancreatic beta-cells.

Pancreatic beta-cells express imidazoline binding sites which play a role in the regulation of insulin secretion, but it is not known whether ligands for these sites also affect other aspects of beta-cell physiology. In the present study, we have investigated the effects of a range of imidazoline reagents on the growth and viability of clonal pancreatic beta-cells (RINm5F and HIT-T15). Three imidazoline compounds (idazoxan, phentolamine and antazoline) were found to cause marked inhibition of beta-cell growth in a time and concentration dependent manner. Idazoxan was the most potent of these with as little as 0.5 microM causing a significant decrease in beta-cell viability (EC50 approximately 10 microM). All three imidazolines also decreased the viability of clonal beta-cells in parallel with their inhibitory effects on cell growth. These effects were not reproduced by any of a wide-range of other imidazoline compounds, including effective insulin secretagogues such as efaroxan and RX821002. The effects of the three ligands did not correlate with their relative potencies for binding to any of the well-characterised imidazoline binding sites nor to alpha2-adrenoceptors. In addition, the inhibitory responses were not antagonised by other imidazoline binding site ligands. The inhibitory effects of idazoxan on the growth of RINm5F and HIT-T15 beta-cells required as little as 3-h exposure to the imidazoline and were not readily reversible when the reagent was removed. Reductions in growth rate were accompanied by marked alterations in the morphology of the cells, which could be detected before loss of viability. Cells exposed to phentolamine showed the characteristic features of apoptosis in that the nuclei were condensed (as judged by acridine orange staining) and electrophoresis of DNA revealed the presence of oligonucleosomal fragmentation. These changes could not be detected in cells exposed to idazoxan despite the more profound reduction in viability induced by this agent. We conclude that a sub-group of imidazoline compounds can exert profoundly detrimental effects on the growth and viability of clonal beta-cells but that these effects do not correlate with their binding affinity at imidazoline binding sites or alpha2-adrenoceptors.

Multiple effector pathways regulate the insulin secretory response to the imidazoline RX871024 in isolated rat pancreatic islets.

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 ineffective at 20 mM glucose whereas it did stimulate insulin secretion in response to 6 mM glucose. Exposure of islets to RX871024 (50 microM) 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. The imidazoline antagonist, KU14R, blocked the insulin secretory response to efaroxan, but failed to prevent the stimulatory response to RX871024. By contrast with its effects in cultured islets, RX871024 inhibited glucose-induced insulin release from freshly isolated islets. Efaroxan did not inhibit insulin secretion under any conditions studied. In freshly isolated islets, the effects of RX871024 on insulin secretion could be converted from inhibitory to stimulatory, by starvation of the animals. Inhibition of insulin secretion by RX871024 in freshly isolated islets was prevented by the cyclo-oxygenase inhibitors indomethacin or flurbiprofen. Consistent with this, RX871024 caused a marked increase in islet PGE2 formation. Efaroxan did not alter islet PGE2 levels. The results suggest that RX871024 exerts multiple effects in the pancreatic beta-cell and that its effects on insulin secretion cannot be ascribed only to interaction with a putative imidazoline binding site.

Imidazolines and pancreatic hormone secretion.

A range of imidazoline derivatives are known to be effective stimulators of insulin secretion, and this response correlates with closure of ATP-sensitive potassium channels in the pancreatic beta-cell. However, mounting evidence indicates that potassium channel blockade may form only part of the mechanism by which imidazolines exert their effects on insulin secretion. Additionally, it remains unclear whether members of this class of drugs can bind directly to potassium channel components and whether occupation of a single binding site accounts for their functional activity. This review considers recent developments in the field and highlights evidence that does not fit readily with the concept that a single mechanism of action is sufficient to mediate the effects of imidazolines on pancreatic hormone secretion.

Effector systems involved in the insulin secretory responses to efaroxan and RX871024 in rat islets of Langerhans.

One component of the mechanism by which imidazoline compounds promote insulin secretion involves closure of ATP-sensitive K+ channels in the beta-cell plasma membrane. Recently, however, it has also been proposed that these compounds may exert important effects on more distal effector systems. In the present work, we have investigated the contribution played by protein kinases A and C to the insulin secretory responses of isolated rat islets of Langerhans treated with efaroxan and RX871024 (1-phenyl-2-(imidazolin-2-yl) benzimidazole). Removal of extracellular Ca2+ or blockade of voltage-sensitive Ca2+ channels prevented stimulation of insulin secretion by efaroxan, confirming a critical role for increased Ca2+ influx in the secretory response. By contrast, inhibition of protein kinases A or C failed to alter efaroxan-induced insulin secretion. RX871024 dose-dependently increased insulin secretion from cultured islets incubated with 20 mM glucose. This effect was unaffected by modulation of protein kinase C, but was significantly attenuated by a selective inhibitor of protein kinase A (Rp-cAMPs). Measurements of cAMP revealed that RX871024 increased the islet cAMP content by more than 3-fold; reaching values similar in magnitude to those elicited by 50 microM 3-isobutyl-1-methyl xanthine. The results reveal that neither protein kinase A nor protein kinase C is obligatory for stimulation of insulin secretion by imidazolines. However, they suggest that a rise in cAMP may contribute to the amplified secretory response observed when cultured islets are incubated with RX871024 in the presence of a stimulatory glucose concentration.

Insulin secretagogues with an imidazoline structure inhibit arginine-induced secretion from isolated glucagon secretion from isolated rat islets of Langerhans.

It is well documented that imidazoline compounds such as efaroxan and phentolamine act as potent insulin secretagogues both in vivo and in vitro, an effect which is mediated principally by blockade of ATP-sensitive potassium channels in the pancreatic B-cell. However, little is known about the effects of these drugs on the secretion of other pancreatic hormones and, in the present work, we have investigated the effects of selective imidazoline compounds on glucagon release from isolated rat islets of Langerhans. None of several imidazoline compounds tested (efaroxan, phentolamine, idazoxan, antazoline) affected glucagon secretion from islets incubated with 4 mM glucose. However, when the rate of glucagon release was stimulated by L-arginine (20 mM) efaroxan caused a rapid, sustained and dose-dependent inhibition of the secretory response (EC50 approximately 30 microM). This effect was seen under both static incubation and islet perifusion conditions. Antazoline and phentolamine also inhibited arginine-induced glucagon secretion, whereas idazoxan (an imidazoline which does not affect insulin secretion) failed to alter glucagon release. The inhibitory effects of imidazolines on glucagon release were not secondary to changes in insulin secretion. Taken together, the results indicate that pancreatic A-cells express functional imidazoline receptors which can regulate the secretory activity of the cells.

Interactions between imidazoline compounds and sulphonylureas in the regulation of insulin secretion.

1. Imidazoline alpha 2-antagonist drugs such as efaroxan have been shown to increase the insulin secretory response to sulphonylureas from rat pancreatic B-cells. We have investigated whether this reflects binding to an islet imidazoline receptor or whether alpha 2-adrenoceptor antagonism is involved. 2. Administration of (+/-)-efaroxan or glibenclamide to Wistar rats was associated with a transient increase in plasma insulin. When both drugs were administered together, the resultant increase in insulin levels was much greater than that obtained with either drug alone. 3. Use of the resolved enantiomers of efaroxan revealed that the ability of the compound to enhance the insulin secretory response to glibenclamide resided only in the alpha 2-selective-(+)-enantiomer; the imidazoline receptor-selective-(-)-enantiomer was ineffective. 4. In vitro, (+)-efaroxan increased the insulin secretory response to glibenclamide in rat freshly isolated and cultured islets of Langerhans, whereas (-)-efaroxan was inactive. By contrast, (+)-efaroxan did not potentiate glucose-induced insulin secretion but (-)-efaroxan induced a marked increase in insulin secretion from islets incubated in the presence of 6 mM glucose. 5. Incubation of rat islets under conditions designed to minimize the extent of alpha 2-adrenoceptor signalling (by receptor blockade with phenoxybenzamine; receptor down-regulation or treatment with pertussis toxin) abolished the capacity of (+)- and (+/-)-efaroxan to enhance the insulin secretory response to glibenclamide. However, these manoeuvres did not alter the ability of (+/-)-efaroxan to potentiate glucose-induced insulin secretion. 6. The results indicate that the enantiomers of efaroxan exert differential effects on insulin secretion which may result from binding to effector sites having opposite stereoselectivity. Binding of (-)-efaroxan (presumably to imidazoline receptors) results in potentiation of glucose-induced insulin secretion, whereas interaction of (+)-efaroxan with a second site leads to selective enhancement of sulphonylurea-induced insulin release.

[Curing trial of complicated oncologic pain by D-phenylalanine (author's transl)]. / Les douleurs oncologiques complexes. Essais de traitement par la D-phénylalanine.

UNLABELLED: Aim of investigations: Very often, chronic pain treatments used for the management of terminal ill cancer patients do not prevent acute or incident pain from coming up. For twenty months D-phenylalanine (DPA), an enkephalinase inhibitor, has been investigated in order to forestall this pain. METHODS: Nine caucasian patients, three males and six females, between forty-nine and seventy-eight, were selected for this trial after informed consent. They were all undergoing severe incident pains related to complications (scabies, osteoporosis, painful cough or colic, Charley-Horse, RX-necrosis of skin or mucous membranes, etc) in spite of having their chronic pain component cured: phanol-rhizotomy: two cases, neuro-adrenolysis by alcohol: four cases, Brampton mixture: three cases. They were administered DPA, 250 mg three times a day for fifteen days, followed by a ten days pause, resumption and so on. RESULTS: Seven patients out of nine were alleviated and they never claimed for more or other analgesics until they died. Four of them got very good ataraxia during the same time (survival mean x = 99,33 days). No side effect was reported, even in patients taking Brampton mixture. CONCLUSIONS: DPA seems a useful drug to prevent acute or incident pain in malignant diseases. Our data point out the consequences the enkephalinases inhibitors will take up for the cure of intractable cancer pain.