The Effects And Mechanism Of Cyclic AMP On Insulin Secretion Of Rat Islets

Objective: We discuss that adenylate cyclase(AC)/cyclic adenosine monophosphate(c AMP)signaling pathway plays the impotant role of insulin secretion in rats. The goal of this study was to examine the effects of c AMP on insulin secretion and underlying mechanisms by using AC agonist Forskolin and inhibitor SQ22536.Method:(1)We opened rats abdomen after decapitation.The cold Collagenase P was injected into the pancreas, then we quickly cut the pancreas and put it into 38℃ water bath for 11 min. We separated islets by density gradient centrifugation using histopaque. Single islet cells were digested by Dispase II. We used beta cells for 7 days at most in order to make the results much more reliable.(2) Rat islets were dyed by DTZ for 10 minutes. We detected the activity of islets by AO/PI.(3) Islets were incubated in 37 ℃.Radioactive immunity analysis method was used to inspect insulin secretion in rats and c AMP content under the stimulus of the AC agonist Forskolin and inhibitors SQ22536.(4) We detected KV、APD on islet beta cells under the stimulus of Forskolin and SQ22536 with patch clamp.(5) The Ca2+fluorescence imaging were used to detect islet beta intracellular Ca2+concentration in the influence of Forskolin and SQ22536.Result:(1) Pancreatic islets were isolated from male SD rats by collagenase p and separated by density gradient centrifugation using histopaque as described previously.The isletswere round and smooth. We isolated islet beta cells by Dispase Ⅱ.The cells were smooth,some cells organized into committees.(2) Islets were dyed scarlet by DTZ, showing that the quality of islets was qualified. We stained islets green fluorescence by AO/PI, showing that the sactivity of islets was good.(3) The islets had glucose-stimulate insulin secretion in insulin concentration measurement with 2.8 m M, 8.3 m M and 16.7 m M glucose, insulin secretion normalized value were(1.00 ± 0.38)u IU/ml、(2.33 ± 0.52)u IU/ml、(5.55 ±0.12)u IU/ml.(4) In insulin concentration measurement, islets were incubated for 30 min in KRB solution containing 8.3 m M glucose in the presence or absence of AC agonist Forskolin and AC inhibitor SQ22536, Forskolin increased insulin secretion(8.3G,n=6vs8.3G+ Forskolin, 10 μM, n=6, P<0.05); SQ 22536 did not exhibit significant effect on insulin secretion; SQ22536 can block the action of Forskolin’s increasing the insulin secretion(8.3G+Forskolin vs 8.3G+ SQ22536+Forskolin,10μM,n=6,P<0.05).(5) In c AMP measurements, the results of c AMP level were 2.8G(999.68±95.82fmol/10 islets/hr,8.3G(1021.77 ± 73.73 fmol/10 islets/hr), 8.3G+SQ22536(1187.23 ± 133.13 fmol/10islets/hr), 8.3G+Forskolin(3682.14 ± 395.38fmol/10islets/hr), 8.3G+SQ22536+ Forskolin(2947.21±157.63fmol/10 islets /hr). Forskolin has increased islet beta cells biosynthesis of c AMP level(8.3 G, n=6 vs 8.3G+ Forskolin,10μM, n=6, P<0.01); SQ 22536 did not change c AMP level as well under these conditions(8.3 G,n=6vs8.3G+ SQ22536, 10μM,n=6, P>0.05); But SQ22536 could partially block the function of Forskolin increasing c AMP level(8.3G+Forskolin, 10μMn=6vs8.3G+SQ22536+Forskolin, 10μM, n=6, P<0.05).(6)In the patch clamp, KV channels could be potently inhibited by Forskolin, SQ22536 had no effect on KV channels. It was found that SQ22536 can partially block Forskolin inhibiting KV current. In the stimulation of 80 mv, we recorded the current density as follows: Forskolin(n=6,76.67±2.78 p A/p F), SQ22536+Forskolin(n=6,109.89± 4.58 p A/p F,control(n=6, 139.62±4.54 p A/p F), SQ22536+Forskolin could significantly inhibite the KV current; Forskolin(n=6, 76.67 ±2.78 p A/p F)was statistically significant compared to SQ22536+Forskolin(n=6,109.89±4.58 p A/p F);but SQ22536(n=6,132.43±2.78 p A/p F)had no statistical difference on KV current. It had not been found that DMSO have an effect on KV channels compared to control.(7) Patch clamp was used to detect action potential(APD).Forskolin(70.28±5.18 ms,10μM,n=8)compared to control(40.61±3.77 ms, n=8,p<0.01) was significantly prolonged APD; Forskolin+SQ22536(52.73±2.19, 10μM, n=8,p<0.05)could reduce APD compared to Forskolin.(8)In Ca2+fluorescence imaging, it had not been found Forskolin(10 μM,n=8)make a change of [Ca2+]i concentration with 2.8 m M glucose.When giving Forskolin(10 μM, n=8)on the basis of the 8.3 m M glucose, Forskolin could change [Ca2+]i concentration. In the same conditions, firstly, it was no obvious change by giving SQ22536(SQ, 10μM, n= 8). For a while, [Ca2+]i concentration had a small change by giving Forskolin(FSK, 10 μM, n = 8).Conclusion:(1) We isolated islets from male SD rats by collagenase p and separated by density gradient centrifugation using histopaque. Single islet cells were digested from rat islets by Dispase II.(2)It showed high purity by DTZ. It showed good activity by AO/PI in the experiments.(3)Rat islets had glucose-stimulate insulin secretion meat that islets function were good. Forskolin had effect on promoting insulin secretion, which can be weakened by the SQ22536. SQ22536 has no effect on insulin secretion.(4) Forskolin could obviously increase the c AMP level, and SQ 22536 could eliminate the function of Forskolin.(5)Forskolin significantly inhibited KV channels, but this effect could be elimination by SQ22536.(6) Forskolin significantly prolonged APD.(7) Forskolin significantly increased the concentration of[Ca2+]i level. Above all, Forskolin increased c AMP level on beta cells,greatly inhibited the KV channels、prolonged APD、increasd[Ca2+]i level then increased the secretion of insulin in rats; SQ22536 had no effect on insulin secretion and KV current, but weakened the function of Forskolin on insulin secretion、c AMP level 、KV current、ADP、[Ca2+]i level. Our findings clearly demonstrate that c AMP could prolong APD following KV inhibition to an increased insulin secretion as a result of increase in[Ca2+]i level.