Effect Of Dopamine On Insulin Secretion From Rat Pancreatic And Possible Mechanism

Objective:Dopamine(DA)not only play an important role in the central nervous system,after acting on the peripheral also affect the certain function of the body.The main purpose of this experiment is to investigate the effects of dopamine on insulin secretion from rat islets and the possible mechanism.Method:(1)After wistar male rat was sacrificed,the pancreas was inflated by injecting medium containing 1g·L-1 of collagenase P,then digested in a 37℃ water bath and density gradient centrifuged by histopaque-1077.5g·L-1 DispaseⅡ was applied to disperse pancreatic islets into single cells and then incubated at 37℃ in 5% CO2 humidified atmosphere.(2)Islets were pooled and allowed to recover from the isolation procedure overnight,intervene pancreatic with various reagents then insulin concentration was measured by Iodine[125I] Insulin Radioimmunoassay Kit.(3)Employing whole cell patch-clamp technique to records the voltage dependent potassium channels(Kv),voltage dependent calcium channels and action potential(AP)from rat islet beta cells intervened by different reagents.(4)Using calcium imaging technique to detect the change of the concentration of intracellular calcium when perfused islet cells with dopamine and Quinpirole.Result:(1)The islet used collagenase P digestion and 1077 gradient centrifugation was form neat,a round or oval,surface smooth and about 100~300μm in diameter,occasionally,a small amount of islets connected with acini.Dispase II digestion of islet cells were surface smooth,single or multiple cells aggregated distribution.(2)Dopamine inhibited the release of insulin in a concentration-dependent manner and the inhibitory effect was more prominent with the peripheral sugar concentration increases.D1-like agonist SKF38393(10μM)have no effect on insulin secretion but D2-like agonist Quinpirole(10μM)significantly inhibition of insulin secretion(control,150.02±19.49μmol·L-1,n = 6;Quinpirole,91.70±6.66μmol·L-1,n = 6,p < 0.01).D2-like antagonist Eticlopride(10μM)has no effect on insulin secretion(control,136.81±5.60μmol·L-1,n = 6;Eticlopride,123.32 ± 11.59μmol·L-1,n = 6,p > 0.05),whereas can eliminate the effect of dopamine on insulin secretion(10μM)to a great extent(dopamine,47.24±6.35μmol·L-1,n=6;dopamine+Eticlopride,117.16± 7.58μmol·L-1,n = 6,p < 0.001).(3)Examined the Kv channel currents in response to dopamine and D2-like agonist Quinpirole on islet beta cells.The result demonstrated that sustained Kv currents were potently promoted by dopamine(10μM)compared to control at 80 m V(control,69.79±2.96 p A/p F,n=6;DA,109.92±6.68 p A/p F,n=6,p < 0.01).D2-like agonist Quinpirole(10μM)also promoted Kv channel currents compared to control at 80 m V(control,84.45±5.39 p A/p F,n=6;Quinpirole,123.85±8.27 p A/p F,n=6,p < 0.01).D2-like antagonist Eticlopride(10μM)did not cause changes of Kv currents compared to control,but reversed the effect of dopamine on Kv channels(control,84.87±3.50 p A/p F,n=6;Eticlopride,76.24±3.76 p A/p F,n=6;Eticlopride+dopamine,81.58±3.85 p A/p F,n=6).dopamine(10μM)reduces the influx of extracellular calcium ion(control,-3.68±0.35 p A/p F,n=6;dopamine,-2.74±0.48 p A/p F,n = 6,p < 0.05),while D2-like agonist Quinpirole(10μM)has no effect on voltage-dependent calcium channel.dopamine(10μM)significantly shortened action potential duration(control,59.58 ±4.69 ms,n = 6;dopamine,32.97±3.17 ms,n = 6,p < 0.001),Eticlopride(10μM)can eliminate the effect of dopamine on action potential duration.(4)The confocal laser experiment showed that on the basis of 2.8 m M glucose,16.7 m M glucose significantly increased the concentration of intracellular calcium ion,dopamine(10μM)and D2-like agonist Quinpirole(10μM)obviously decreased intracellular calcium ion concentration.Conclusion:(1)Pancreas islet and cells using collagenase P and Dispase II digestion were in excellent condition.(2)Dopamine and D2-like receptor agonist Quinpirole inhibiting release of insulin in rats and the effect of the dopamine can be largely eliminated by D2-like receptor antagonist Eticlopride.(3)Dopamine and Quinpirole significantly promoted the Kv channel,and the effect of dopamine can be eliminated by Eticlopride.(4)Dopamine inhibited the voltage dependent calcium channel and reduced the intracellular calcium influx,but Quinpirole had no effect on the calcium influx.(5)Dopamine shortened the duration of action potential,which could be eliminated by Eticlopride.(6)Both dopamine and Quinpirole reducing the concentration of intracellular calcium.