Abstract
Abstract: :
Purpose:To determine whether inhibitory amino acid evoked currents (GABA and Gly) of wide field amacrine cells (WFACs) are modulated by intracellular Ca++ release by caffeine. Methods: Isolated WFACs from white bass retina were recorded from using standard whole-cell voltage clamp techniques. Results: Intracellular Ca++ release by 10 mM caffeine reversibly blocked approximately 80% of both GABA and Gly (200 µM) evoked currents ([Ca++]i: 1 mM, [EGTA]i: 0.2 mM). Elevation of [EGTA]i, or replacement with 10 mM BAPTA had no effect on the caffeine inhibition of the Gly current, while it substantially reduced the caffeine inhibition of the GABA current. Application of the endoplasmatic reticulum Ca-ATPase blocker thapsigargin and Ca++-store depletor ryanodine inhibited the GABA current, and completely eliminated the caffeine effect upon it, but the Gly current remained unaffected, as well as the action of caffeine on it. High [BAPTA]i eliminated the inhibitory effect of ryanodine on the GABA current. Including the Ca++/Calmodulin dependent protein phosphatase inhibitor calmidazolium in the pipette also reduced the caffeine effect, but only on the GABA evoked current, while Ca++-dependent protein kinase inhibitor staurosporin (20nM) was ineffective. Exogenous 8-Br-cAMP and 8-Br-cGMP (100µM) had no effect on either current. Dose-response curves for Gly obtained in the presence of caffeine revealed caffeine was acting as a competitive antagonist. They also showed that Gly had about x100’s greater affinity for the receptor than did caffeine. Conclusion: Our results indicate, that both GABA and Gly evoked currents of the WFAC can be inhibited reversibly by caffeine, however, the mechanisms are completely different. Caffeine inhibits the Gly current by directly binding to the Gly receptor, thus competing with Gly for binding sites. The GABA current is inhibited by caffeine through release of Ca++ from intracellular stores, which in turn activaties protein phosphatases. Dephosphorylation of the GABAA receptor complex or a related regulatory protein resulted in substantial decrease of the GABA evoked current.
Keywords: 312 amacrine cells • 490 neurotransmitters/neurotransmitter systems • 334 calcium