A model beta cell line, R7T1, was used for our initial studies in which we determined the sensitivities of various ion channels of R7T1 cells to the endocannabinoid 2-AG (2- arachidonylglycerol). Upon completion of these experiments in the past year, we began studies in actual mouse pancreatic beta cells. Using conventional whole cell patch clamp approaches in the R7T1 cells, we examined the effects of 2-AG on delayed rectifier potassium currents. A 2-AG concentration-response curve revealed that the EC-50 for inhibition of the delayed rectifier was 20 micromolar. It was also noted in during these experiments that voltage-dependent sodium currents were also present in these cells. The blockade of sodium currents by 2-AG correlated with the block of the delayed rectifier, suggesting a common cause such as activation of a cannabinoid receptor or nonspecific effects of the hydrophobic 2-AG molecule on the lipid membrane. To test whether the block by 2-AG was mediated by a cannabinoid receptor, the cannabinoid agonist WIN55,212-2 was tested and found to have only small effects that were generally opposite to those of 2-AG. Furthermore, an enantiomer of WIN55,212-2 that is inactive at cannabinoid CB1 and CB1 receptors (WIN55,212-3) had similar effects on R7T1 cell activity. Finally, the cannabinoid CB1 receptor antagonist AM251 was ineffective in blocking the effects of 2-AG. Collectively, these findings suggest that 2-AG acts independently of cannabinoid receptors to alter R7T1 cell ion channel activity. We also examined the effects of 2-AG on high voltage activated calcium currents (HVACCs) in R7T1 cells. A concentration-response curve gave an estimated EC-50 of 13 micromolar. As with the delayed rectifier, the cannabinoid agonist WIN55,212-2 gave only a small (15%) block of these currents that was also insensitive to blockade by AM251, again suggesting that the block by 2-AG was not mediated by the CB1 cannabinoid receptor. The L-channel antagonist Nifedipine (10 uM) was used to address the question of whether the high voltage-activated calcium currents in these cells were L channels. The conclusion, complicated by finding three populations of HVACCs, was that 60-80% of the HVACCs were carried by L-type calcium channels. Omega-conotoxin GVIA, a specific antagonist of N-type calcium channels, had no effect on total calcium current, suggesting that N-type channels are absent in these cells. The ATP sensitive potassium channels, K(ATP), are key initiators of insulin secretion. As plasma glucose levels rise, the intracellular ratio of ATP to ADP also rises in beta cells. This raised ATP:ADP blocks the K(ATP) channels, thereby initiating depolarization of the beta cells, opening L-type calcium channels, and causing the vesicular release of insulin. These channels were studied by single ion channel recording in inside-out patches of R7T1 membranes. These currents were verified as K(ATP) by (1) their amplitude of 4.2 pA (at +60 mV) and (2) their inhibition by 1 millimolar ATP. A concentration-response curve using 2-AG showed that these channels displayed an EC-50 of 1 micromolar. Therefore, K(ATP) channels are over ten-fold more sensitive to 2-AG than both the delayed rectifier and the HVACC currents. Because these are outside-out membrane patches, agents acting on a cannabinoid receptor had to be dissolved in the pipette solution. Thus, to test whether the 2-AG block was mediated by CB1 receptors, both 2- AG and a saturating concentration of 10 micromolar AM-251 were included in the pipette solution. No attenuation of the 2-AG block was detected, suggesting that, like the effects on delayed rectifier and HVACCs, the effects of 2-AG on K(ATP) channels were independent of CB1 receptors. Because the K(ATP) channels were most sensitive to blockade by 2-AG, we will repeat this finding acutely dissociated beta cells. Although this approach is technically demanding, we anticipate that we will be able to compare our data obtained in the R7T1 cells with those of acutely isolated beta islet cells during the upcoming project period.
| Spivak, Charles E; Kim, Wook; Liu, Qing-Rong et al. (2012) Blockade of ?-cell K(ATP) channels by the endocannabinoid, 2-arachidonoylglycerol. Biochem Biophys Res Commun 423:13-8 |
