Effect of purinergic agonists and antagonists on insulin secretion from INS-1 cells (insulinoma cell line) and rat pancreatic islets

Abstract

The effects of purinergic agonists on insulin release are controversial in the literature. In our studies (mainly using INS-1 cells, but also using rat pancreatic islets), ATP had a dual effect on insulin release depending on the ATP concentration: increasing insulin release (EC50 approximately/= 0.0032 microM) and inhibiting insulin release (EC50 approximately/= 0.32 microM) at both 5.6 and 8.3 mM glucose. This is compatible with the view that either two different receptors are involved, or the cells desensitize and (or) the effect of an inhibitory degradation product such as adenosine (ectonucleotidase effect) emerges. The same dual effects of ATP on insulin release were obtained using rat pancreatic islets instead of INS-1 cells. ADPbetaS, which is less degradable than ATP and rather specific for P2Y1 receptors, had a dual effect on insulin release at 8.3 mM glucose: stimulatory (EC50 approximately/= 0.02 microM) and inhibitory (EC50 approximately/= 0.32 microM). The effectiveness of this compound indicates the possible involvement of a P2Y1 receptor. 2-Methylthio-ATP exhibited an insulinotropic effect at very high concentrations (EC50 approximately/= 15 microM at 8.3 mM glucose). This indicated that distinct P2X or the P2Y1 receptor may be involved in these insulin-secreting cells. UTP increased insulin release (EC50 approximately/= 2 microM) very weakly, indicating that a P2U receptor (P2X3 or possibly a P2Y2 or P2Y4) are not likely to be involved. Suramin (50 microM) antagonized the insulinotropic effect of ATP (0.01 microM) and UTP (0.32 microM). Since suramin is not selective, the data indicated that various P2X and P2Y receptors may be involved. PPADS (100 microM), a P2X and P2Y1,4,6 receptor antagonist, was ineffective using either low or high concentrations of ATP and ADPbetaS, which combined with the suramin data hints at a P2Y receptor effect of the compounds. Adenosine inhibited insulin release in a concentration-dependent manner. DPCPX (100 microM), an adenosine (A1) receptor antagonist, inhibited the inhibitory effects of both adenosine and of high concentrations of ATP. Adenosine deaminase (1 U/mL) abolished the inhibitory effect of high ATP concentrations, indicating the involvement of the degradation product adenosine. Repetitive addition of ATP did not desensitize the stimulatory effect of ATP. U-73122 (2 microM), a PLC inhibitor, abolished the ATP effect at low concentrations. The data indicate that ATP at low concentrations is effective via P2Y receptors and the PLC-system and not via P2X receptors; it inhibits insulin release at high concentrations by being metabolized to adenosine.

Inhibition of Purinoceptors Amplifies Glucose-Stimulated Insulin Release With Removal of its Pulsatility

“…The role of the purinoceptors in the release of insulin is a matter of controversy, and both stimulatory and inhibitory effects of ATP have been reported (17). It was recently observed in ␤-cells from ob/ob mice that a low concentration of the purinoceptor antagonist 2-deoxy-Nmethyladenosine-3,5-bisphosphate (MRS 2179) removes the [Ca 2ϩ ] i transients supposed to coordinate the secretory activity (2).…”

Purinergic receptors in the endocrine and exocrine pancreas

“…Although the P2Y 1 receptor may be the most important P2Y receptor in regulating insulin secretion, there is also evidence for other P2Y receptors [19][20][21], and also P2X receptors [12,[22][23][24], and indeed the P2X 4 receptor was cloned from rat pancreatic islets [25]. Evidence for P2X-type receptors was provided by intracellular Ca 2+ measurements.…”

“…It is possible that the high-concentration effect was due to transport of adenosine into the cell and subsequent metabolic effects. The inhibitory effect of low-concentration adenosine is exerted via A 1 receptors [7,19,40], which via G i proteins would inhibit adenylate cyclase and thus insulin secretion (Fig. 1).…”

“…That is, the stimulatory effect at low ATP concentrations is exerted via P2Y 1 and other receptors as discussed above. In addition, ATP at high concentrations can have inhibitory effects, because it is metabolised to adenosine, which via A 1 receptors would dominate the insulin response [19].…”

Purinergic signalling in endocrine organs

“…In perfused dog pancreas, the adenosine analogue 5′-N-ethylcarboxamidoadenosine (NECA) inhibited insulin release, the effect being concentration-dependent [16]. A 1 receptors mediating inhibition were pharmacologically identified on β-cells [32,226,572] and in INS-1 cells [543]. A 1 receptor antagonism in rat pancreatic islets potentiates insulin secretion [623].…”