Susan L.F. Chan

The α2-adrenoceptor antagonist efaroxan modulates K+ATP channels in insulin-secreting cells

Abstract The actions of efaroxan, a highly selective and potent α 2 -adrenoceptor antagonist, on insulin secretion, cAMP levels, 86 Rb4 + efflux and ATP-regulated potassium (K + ATP ) channels have been studied using isolated pancreatic islets of Langerhans and RINm5F cells. In the absence of an adrenoceptor agonist, efaroxan (1–100 μM) potentiated glucose-induced secretion over the range 4–10 mM glucose, but was without effect upon the maximal rate of secretion induced by 20 mM glucose. Efaroxan did not affect cAMP levels. Suppression of insulin release by the potassium channel opener diazoxide, was partially alleviated by efaroxan and was associated with an inhibition of the diazoxide-induced increase in the rate of 86 Rb + efflux. Using isolated patches of membrane we found efaroxan to be an effective blocker of k + ATP channels, with a K 1 value of 12 μm and a Hill coefficient of approximately 1. These data indicate that efaroxan promotes insulin secretion, in the absence of exogenous agonists, by a mechanism that involves inhibition of ATP-regulated K + channels.

The imidazoline site involved in control of insulin secretion : characteristics that distinguish it from I1- and I2-sites

1 The nature of the binding site mediating the insulin secretagogue activity of certain imidazoline compounds remains unclear and the pharmacology of the I1‐ and I2‐imidazoline sites, described in many tissues, does not correlate with the observed responses to imidazolines in islets. In the present paper, we describe further results which support the concept that the islet imidazoline site may represent a novel subtype of imidazoline receptor. 2 Culture of rat isolated islets in the presence of imidazoline secretagogues (either efaroxan or phentolamine) resulted in loss of responsiveness on subsequent re‐exposure to these agents. However, culture of islets with either idazoxan or UK 14,304 (imidazoline ligands that do not stimulate insulin secretion) did not lead to any loss of response when the islets were subsequently exposed to efaroxan. By contrast, islets cultured with UK 14,304 (a potent α2‐adrenoceptor agonist), displayed loss of sensitivity to noradrenaline, consistent with down‐regulation of α2‐adrenoceptors. 3 In order to characterize the imidazoline site further, radioligand binding studies were performed in membranes from RINm5F insulinoma cells using [3H]‐RX821002, an imidazoline insulin secretagogue that does not interact significantly with imidazoline sites in other tissues. [3H]‐RX821002 labelled α2‐adrenoceptors with high affinity (2.01 ± 0.7 nM) but also labelled a second, non‐adrenoceptor site with much lower affinity. 4 Under conditions of α2‐adrenoceptor blockade (in the presence of adrenaline), efaroxan displaced [3H]‐RX821002 binding to the low affinity site, in a dose‐dependent manner. Competition studies employing additional imidazoline compounds of varying secretagogue activity revealed that the pharmacological profile of the low affinity site correlates well with that observed in secretion experiments. 5 The results obtained from the down‐regulation experiments with isolated islets and from the radioligand binding studies suggest that the low affinity [3H]‐RX821002 binding site may represent the functional receptor responsible for the secretagogue activity of imidazoline compounds in the endocrine pancreas and that it has a pharmacological profile distinct from those of I1‐ and I2‐sites.

Imidazolines and Pancreatic Hormone Secretiona

ABSTRACT: 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 β‐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.

Antagonism of the stimulatory effects of efaroxan and glibenclamide in rat pancreatic islets by the imidazoline, RX801080.

1 The imidazoline α2‐adrenoceptor antagonist, efaroxan, stimulates insulin secretion from rat isolated islets and antagonizes the ability of diazoxide to inhibit glucose‐induced insulin secretion. These effects result from closure of ATP‐sensitive potassium channels although the mechanisms involved have not been elucidated. 2 In the present work, we have examined the effects of a close structural analogue of efaroxan, RX801080, in rat isolated islets of Langerhans. RX801080 was found to be ineffective as a stimulator of insulin secretion and did not prevent the inhibition of insulin secretion mediated by diazoxide. 3 RX801080 acted as an antagonist of the actions of several imidazolines (efaroxan, phentolamine and midaglizole) in rat islets. It dose‐dependently inhibited the ability of efaroxan to antagonize the effects of diazoxide in islets and also completely inhibited the direct stimulation of insulin secretion mediated by efaroxan. 4 RX801080 also antagonized the effects of the non‐imidazoline, ATP‐sensitive potassium channel blocker, glibenclamide, in rat islets. It inhibited both the capacity of glibenclamide to stimulate insulin secretion and the ability of glibenclamide to overcome the inhibitory effects of diazoxide in rat islets. 5 Antagonism of glibenclamide responses by RX801080 was not due to inhibition of binding of the sulphonylurea to its receptor on the pancreatic β‐cell. 6 The results suggest that imidazoline compounds and sulphonylureas interact with distinct binding sites on islet cells, but that these sites can interact functionally to control islet cell ATP‐sensitive potassium channel activity and insulin secretion.

The effect of the putative endogenous imidazoline receptor ligand, clonidine-displacing substance, on insulin secretion from rat and human islets of Langerhans

The effects of a rat brain extract containing clonidine‐displacing substance (CDS), a putative endogenous imidazoline receptor ligand, on insulin release from rat and human isolated islets of Langerhans were investigated. CDS was able to potentiate the insulin secretory response of rat islets incubated at 6 mm glucose, in a dose‐dependent manner. The magnitude of this effect was similar to that in response to the well‐characterized imidazoline secretagogue, efaroxan. CDS, like other imidazoline secretagogues, was also able to reverse the inhibitory action of diazoxide on glucose‐induced insulin release, in both rat and human islets. These effects of CDS on secretion were reversed by the imidazoline secretagogue antagonists, RX801080 and the newly defined KU14R, providing the first evidence that imidazoline‐mediated actions of CDS can be blocked by specific imidazoline antagonists. The effects of CDS on insulin secretion were unaffected when the method of preparation involved centri‐filtration through a 3,000 Da cut‐off membrane or when the extract was treated with protease. These results confirm that the active principle is of low molecular weight and is not a peptide. Overall, the data suggest that CDS behaves as a potent endogenous insulin secretagogue acting at the islet imidazoline receptor.

Affinity isolation of imidazoline binding proteins from rat brain using 5-amino-efaroxan as a ligand

We have employed an amino derivative of the imidazoline ligand, efaroxan, to isolate imidazoline binding proteins from solubilised extracts of rat brain, by affinity chromatography. A number of proteins were specifically retained on the affinity column and one of these was immunoreactive with an antiserum raised against the ion conducting pore component of the ATP‐sensitive potassium channel. Patch clamp experiments confirmed that, like its parent compound, amino‐efaroxan blocks ATP‐sensitive potassium channels in human pancreatic β‐cells and can stimulate the insulin secretion from these cells. The results reveal that a member of the ion conducting pore component family is strongly associated with imidazoline binding proteins in brain and in the endocrine pancreas.

Characterisation of new efaroxan derivatives for use in purification of imidazoline-binding sites

The insulin secretagogue activity of certain imidazoline compounds is mediated by a binding site associated with ATP-sensitive K+ (K(ATP)) channels in the pancreatic beta-cell. We describe the effects of a series of structural modifications to efaroxan on its activity at this site. Substitution of amino-, nitro- or azide- groups onto the 5-position of the benzene ring of efaroxan did not significantly affect the functional interaction of the ligand with the islet imidazoline binding site. Modification of the imidazoline ring to an imidazole to generate 2-(2-ethyl-2,3-dihydrobenzo[b]furan-2-yl)-1H-imidazole (KU14R) resulted in loss of secretagogue activity. Indeed, this reagent appeared to act as an imidazoline antagonist since it blocked the secretory responses to imidazoline compounds and also inhibited the blockade of beta-cell K(ATP) channels by efaroxan in patch clamp experiments. Application of KU14R alone resulted in a modest reduction in K(ATP) channel opening, suggesting that it may display weak partial agonism, at least in patch-clamp experiments.

Evidence that the ability of imidazoline compounds to stimulate insulin secretion is not due to interaction with σ receptors

Recent studies have suggested that a variety of ion channels possess a binding site for ligands such as phencyclidine (PCP), dizocilpine and certain sigma 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 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(+)-ATP channel in pancreatic B-cells and show that their effects are not reproduced by PCP or sigma receptor ligands.

Mechanisms involved in stimulation of insulin secretion by the hypoglycaemic alpha-adrenergic antagonist, DG-5128

The selective alpha 2-antagonist DG-5128 provoked a dose-dependent stimulation of insulin release from isolated rat islets. DG-5128 was only weakly effective as an antagonist of noradrenaline-induced inhibition of insulin secretion but, surprisingly, was able to reverse the suppression of secretion and increase in 86Rb efflux from preloaded islets, mediated by diazoxide. These effects were not reproduced with more effective alpha-antagonists, suggesting that stimulation of insulin secretion by DG-5128 is independent of alpha-receptor blockade.

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

Imidazoline α2‐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 α2‐adrenoceptor antagonism is involved. 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. 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 α2‐selective‐(+)‐enantiomer; the imidazoline receptor‐selective‐(−)‐enantiomer was ineffective. 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. Incubation of rat islets under conditions designed to minimize the extent of α2‐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. 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.