This proposal describes experiments designed to characterize the effects of delta opioids on Ca2+ currents (KCa) and intracellular pH (pHi) in the neuronal cell line NG108-15. In these cells activation of the delta-opioid receptor produces an inhibition of ICa via a pertussis toxin-sensitive pathway and an acceleration of Na+/H+ exchange via a pathway which is not sensitive to pertussis toxin. The overall goal of this proposal is to compare the two arms of this divergent receptor effector system. Initially each pathway will be studied independently to characterize pharmacological, G protein and effector properties. Then the desensitization of the two effector systems will be compared.
Four specific aims are proposed. 1) The pharmacological characteristics of delta-opioid modulation of ICa and Na+/H+ exchange will be compared. 2) The Ca2+ channel subtypes inhibited by delta opioids will be determined. 3) Studies will be performed to determine whether the alkalinization induced by delta opioids is mediated by a G protein. 4) The desensitization of delta-opioid inhibition of ICa and the acceleration of Na+/H+ exchange will be measure simultaneously. To achieve thee aims ICa will be measured with the whole-cell patch-clamp and intracellular ion concentrations will be measured optically in single cells with the ion sensitive dyes indo-1 and carboxy SNARF-1. Additionally combined fluorescence electrophysiological experiments will be performed to simultaneously measure pHi and ICa. These studies are predicted to enhance our understanding of how opioid receptors are coupled to various effectors with particular emphasis on receptor desensitization. Because desensitization may in part underlie the process of tolerance and dependence on opioid drugs, these studies may help determine the molecular substrates of drug abuse and addiction.
| Werth, J L; Zhou, B; Nutter, L M et al. (1994) 2',3'-Dideoxycytidine alters calcium buffering in cultured dorsal root ganglion neurons. Mol Pharmacol 45:1119-24 |
| Werth, J L; Thayer, S A (1994) Mitochondria buffer physiological calcium loads in cultured rat dorsal root ganglion neurons. J Neurosci 14:348-56 |
| Lo, T M; Thayer, S A (1993) Refilling the inositol 1,4,5-trisphosphate-sensitive Ca2+ store in neuroblastoma x glioma hybrid NG108-15 cells. Am J Physiol 264:C641-53 |
| Sipahimalani, A S; Werth, J L; Michelson, R H et al. (1992) Lipophilic amino alcohols with calcium channel blocking activity. Biochem Pharmacol 44:2039-46 |
| Lo, T M; Fallert, C J; Piser, T M et al. (1992) HIV-1 envelope protein evokes intracellular calcium oscillations in rat hippocampal neurons. Brain Res 594:189-96 |
| Werth, J L; Hirning, L D; Thayer, S A (1991) omega-Conotoxin exerts functionally distinct low and high affinity effects in the neuronal cell line NG108-15. Mol Pharmacol 40:742-9 |
