| Objective: To clarify the key mechanism of ADPpS, P2Y receptor agonists, promotes insulin secretion in a glucose dependent manner, and get the reliable evidence of mediating Kv channel on β cell. To provide theoretical basic for the research and development of new anti-diabetic drugs whose targets are P2Y receptor.Method:(1) Opening the enterocoelia under aseptic condition quickly after killing the rat. Slowly injected the collagenase.β solution from common bile-duct, and digested in a water bath for11minutes under38℃. Using density gradient centrifugation to separate islets. To separate pancreatic islets into β cells with Dispase Ⅱ, islets and cells could be used only7days in vitro experiment.(2) Using AO/PI to dye islets for10minutes, fluorescence microscope was used to observe in490-nm excitation light and excitation spectra for510-nm emission.(3) After one night cultivation, radioimmunoassay was used to detect the effect ADPβS on insulin secretion and cAMP intervened by other drugs.(4) Patch-clamp technique was used to test the effect of ADPβS on voltage-gated potassium channel (Kv)、action potential (AP) and voltage-gated calcium channel on β cell.(5) Confocal laser scanning microscope was used to detect the change of calcium ion concentration altered by ADPβS on P cell.Result:(1) The islet and cell were in good condition.(2) More than95percent of islets were dyed green.(3) ADPβS (0.5μM) promoted insulin secretion in a glucose-dependent manner. The regulatory action was blocked when islets were treated with Suramin (50μM), a P2Y receptor antagonist or SQ22536(10μμM), an adenylyl cyclase (AC) inhibitor. The same as AC agonist Forskolin (10μM), ADPβS increased the concentration of cAMP in cells. The effect was eliminated by Suramin and SQ22536. Epac inhibitor ESI-09(10μM) blocked the effect of ADPpS on insulin secretion, but this was not the case for PKA inhibitor PKI (10nM). TEA (20mM) increased insulin secretion, and the effect of ADPβS was interdicted on the basis of TEA.(4) Data showed that ADPβS significantly inhibited Kv currents compared to control at80mV (ADPβS,113.46±10.81pA/pF, n=6; control,142.29±8.64pA/pF, n=6, p<0.05). The effect of ADPβS was reversed by Suramin (50μM) and SQ22536(10μM), which had no effect on Kv current alone. ESI-09abolished the effect induced by ADPβS, whereas PKI did not. On the basis of TEA, ADPβS could not inhibit Kv currents further. In the detection of action potential duration (APD),0.5μM ADPβS significantly prolonged APD (ADPβS,72.9±6.1ms, n=8; control,49.9±2.0ms, n=8;p<0.01).0.5μM ADPβS had no effect on voltage gated calcium channel, the current density was-5.94±0.20pA/pF for ADPβS (n=7) compared to-5.78±0.19pA/pF for control (n=7) at0mV,p>0.05.(5) The confocal laser experiment showed that on the basis of8.3mM glucose,0.5μM ADPβS significantly enhanced the concentration of calcium ion. Suramin (50μM) and SQ22536(10μM) eliminated the effect of ADPβS. TEA (20mM) obviously increased the density of calcium ion in β cell, and ADPβS could not enhance the density of calcium ion on the basis of TEA.Conclusion:(1) The islet and cell were in good condition.(2) The activity of islets were good (AO/PI dyeing).(3) ADPβS increased insulin secretion in a glucose-dependent manner. And the effect of ADPβS could be eliminated by P2Y receptor inhibitor Suramin、AC inhibitor SQ22536、Epac inhibitor ESI-09. The PKA inhibitor PKI had no effect on ADPβS. ADPβS significantly enhanced the concentration of cAMP in β cell and the effect was abolished by Suramin and SQ22536.(4) ADPβS inhibited Kv channel and the effect was eliminated by Suramin、 SQ22536、ESI-09.(5) ADPβS significantly prolonged APD and had no effect on voltage-gated calcium channel.(6) ADPβS increased the concentration of calcium ion and the function was eliminated by Suramin、SQ22536. In summary, ADPβS potentiated insulin secretion in a glucose-dependent manner, mainly via activating of P2Y receptor to activate AC, increased the concentration of intracellular cAMP. Next to activate the Epac to inhibit the openness of Kv channel, prolonged the APD, increased the time of calcium ion enter the cell, enhanced the concentration of calcium ion to augment insulin secretion. |
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