PI3K Is Involved In P2Y Receptor-Regulated CaMP/Epac/Kv Channel Signaling Pathway In Pancreatic β Cells

Objective:Previously, we reported that P2YR-modulated insulin secretion is mediated by a c AMP/Epac/Kv channel pathway. However, the interaction between Epac and the Kv channel in P2YR-modulated insulin secretion remains unclear. In this study, we used patch-clamp technique and insulin secretion assay to investigate the potential molecules that may link Epac to Kv channel inhibition induced by P2 YR activation.We identified that phosphatidylinositide 3-kinase, which mediates P2YR-regulated insulin secretion, is a critical mediator between Epac and the Kv channel.Methods:(1)Pancreases were separated from male SD rats weighing 180-250 g by injection of collagenase P at 1 mg/ml into the common bile duct, and incubated at 37℃ for 11 min. After the density gradient centrifugation with histopaque-1077, islets of Langerhans were isolated and hand collected under a stereoscopic microscope. To obtain dispersed islet cells, pancreatic islets were dispersed into single cells by DispaseⅡ digestion. Intact islets or dispersed islet cells were incubated in Hyclone RPMI 1640 medium, at 37℃ in an atmosphere of humidified air(95%) and CO2(5%). islets and cells could be used only a week in vitro experiment.(2)We intervene pancreatic β-cells with different drugs and test the impact of each drug on the voltage dependent potassium channel(Kv) in pancreatic β-cells.(3)After 24 hours incubation, radioimmunoassay was used to test the effect of different drugs on insulin secretion.Results:(1)The islets were textured and light transmittance was small. The pancreaticβ-cells were roud and smooth. Most of the islets and β-cells were in good conditions.(2)We monitored KV currents by using whole-cell voltage-clamp technique respectively with Ro 31-8220(PKC inhibitor) 、 FTS(RAS inhibitor) and PD98059(MEK inhibitor) and we found all of them did not influence the effect of ADPbS on Kv currents(Ro31-8220+ADP b S,117.04±4.61 p A/p F, n=7 vs ADP b S,111.35±2.39 p A/p F, n=7, P<0.05; FTS+ADPbS, 117.50±2.53 p A/p F, n=7 vs ADPbS, 111.35±2.39 p A/p F, n=7,P<0.05; PD98059+ADPbS,112.58±3.36 p A/p F, n=7 vs ADPbS,111.35±2.39 p A/p F, n=7, P<0.05).We observed that PI3 K inhibitor, wortmannin,abolished the effect of ADPbS on Kv currents(wortmannin+ADPbS,143.51±3.91 p A/p F, n=7 vs control, 145.24±2.51 p A/p F, n=7, P>0.05). 8-p CPT-AM(Epac specific activator)blocked Kv currents(8-p CPT-AM,111.36±4.96 p A/p F, n=7 vs control, 145.31±3.21 p A/p F, n=7,P<0.01)and this effect could be erased by wortmannin(CPT+Wortmannin,132.19±5.30 p A/p F, n=7 vs CPT,111.36±4.96 p A/p F, n=7,P<0.01).Another PI3 K inhibitor, BKM 120, which also inhibited the effect of 8-p CPT-AM on Kv currents.(3) Our data of insulin secretion showed that ADPbS potentiated insulin secretion at 8.3m M glucose compared with control, but notably, the potentiated insulin secretion by ADPbS was blocked by wortmannin. In addition, another PI3 K inhibitor,BKM120, also significantly blocked the effect of ADPbS on insulin secretion.Conclusions:(1)Ro31-8220(PKC inhibitor)、FTS(RAS inhibitor) and PD98059(MEK inhibitor)did not interdict the effect of ADPbS on Kv currents.(2)PI3K inhibitor, wortmannin, abolished the effect of ADPbS on Kv currents.8-p CPT-AM(Epac specific activator) blocked Kv currents and this effect could be erased by PI3 K inhibitor(wortmannin or BKM120).(3)Both wortmannin and BKM120 blocked the potentiated insulin secretion by ADPbS.(4)PI3K is a critical molecular link between Epac and the Kv channel.(5) P2YR-regulated Kv channel inhibition is dependent on the PI3 K pathway.In summary, PI3 K is involved in P2 Y receptor-regulated c AMP /Epac/Kv channel signaling pathway in pancreatic b cells.