Expression of purinergic P2Y receptor subtypes by INS-1 insulinoma beta-cells: a molecular and binding characterization.

Purinergic P2Y-receptor agonists amplify glucose-induced insulin secretion from pancreatic beta-cells, thus offering new opportunities for the treatment of type 2 diabetes. However, little is known about which subtypes of purinergic P2Y receptors are expressed in these cells. The INS-1 beta-cell line is used as a model of pancreatic beta-cells, expressing most of their properties. Therefore, we investigated the expression of different molecular subtypes in this cell line by means of real time Polymerase Chain Reaction and Western blot. We also performed a characterization of the binding of a prototypic purinergic P2Y agonist, Adenosine-5'-O-(1-[(35)S]thiotriphosphate) (ATP-alpha-[(35)S]), to cell membrane homogenates. The molecular analysis evidenced the presence of five different purinergic P2Y receptor subtypes (P2Y(1), P2Y(2), P2Y(4), P2Y(6) and P2Y(12)), which were expressed at similar levels. The Western blot analysis allowed detecting corresponding proteins. The binding assay demonstrated a specific ATP-alpha-[(35)S] interaction on high (40%) and low (60%) affinity components. The analysis of ATP-alpha-[(35)S] pharmacological profile on both sites permitted to classify the high affinity binding site as representative of the purinergic P2Y(1) receptor subtype and the low affinity binding site of the P2Y(4) and/or P2Y(6) receptor subtypes. ATP-alpha-S and Adenosine-5'-O-(2-thiodiphosphate) (ADP-beta-S) exhibited opposite selectivity on high and low affinity binding sites. Although purinergic P2Y(1) receptor, or a P2Y(1)-like subtype, has been generally considered as that implicated in the modulation of glucose-induced insulin release, the present data show that the beta-cell expresses a complex profile of purinergic P2Y receptor subtypes, the functional implication of which remains to be fully elucidated.

P2Y receptor mediated modulation of insulin release by a novel generation of 2-substituted-5'-O-(1-boranotriphosphate)-adenosine analogues.

PURPOSE: A series of C2-substituted ATP analogues was previously shown to have potent insulin-secreting properties, yet with poor tissue-selectivity for the pancreatic beta-cell. The present study was designed to evaluate the binding profile on beta-cell membranes and the effects on insulin release and pancreatic vascular resistance of a second generation of P2Y(1) receptor agonists, based on C2-substitution of the adenosine 5'-O-(1-boranotriphosphate) scaffold. MATERIALS AND METHODS: Functional experiments were performed in the rat isolated pancreas model; binding studies with ATP-alpha-[(35)S] were performed in membrane homogenates from the rat insulinoma INS-1 cell line. The diastereoisomers of the compounds are designated by A and B. RESULTS: Under 8.3 mmol l(-1) glucose, 2-methylthio-ATP-alpha-B, A isomer, induced a biphasic and concentration dependent insulin response; its maximal efficacy reaches ninefold the baseline secretion and its EC(50) is 28.1 nmol l(-1). No significant effect of this isomer was observed on vascular resistance, whereas the B isomer, which was a less potent insulin secretagogue, consistently induced a transient vasoconstriction. Interestingly, the insulin response induced by 2-methylthio-ATP-alpha-B, A isomer, was clearly glucose-dependent. This drug competes with ATP-alpha-[(35)S] binding in a complex two sites interaction model, with a K(0.5) value of 17.7 nmol l(-1). 2-Chloro-ATP-alpha-B had a similar insulin-secreting profile as 2-methylthio-ATP-alpha-B, with a lower tissue-selectivity. The non-substituted ATP-alpha-B analog, A isomer, was less potent than the C2-substituted derivatives (A isomers) and had a vasorelaxant effect. CONCLUSIONS: We conclude that 2-methylthio-ATP-alpha-B, A isomer, is a potent and tissue-selective P2Y receptor agonist with high efficacy. Its insulin-releasing action is glucose-dependent, which gives interest to this compound as a drug candidate for treating type 2 diabetes.