Desensitization and resensitization of δ-opioid receptor-mediated Ca2+ channel inhibition in NG108-15 cells

1 To approach the mechanisms underlying desensitization of the opioid receptor-mediated Ca2+ channel inhibition, the effects of prolonged application of [D-Ala(2), D-Leu(5)]enkephalin (DADLE) on Ba2+ currents (I-Ba) through Ca2+ channels were analysed in NG108-15 neuroblastoma x glioma hybrid cells. 2 Inhibition of I-Ba by 100 nM DADLE desensitized by 57% with st time constant of 4.4 min. 3 Maximal desensitization of the delta-opioid receptor-Ca2+ channel coupling was attained by 1 mu M DADLE. The EC50 value for desensitization was estimated to be 78 nM. 4 RNA blot hybridization analysis and immunoblot analysis revealed the expression of beta-adrenoceptor kinase-1 (beta ARK1) in NG108-15 cells. 5 Heparin, an inhibitor of beta ARK, significantly reduced the magnitude and rate of desensitization, whereas Rp-cyclic AMPS and PKI (14-24)amide, inhibitors of cyclic AMP-dependent protein kinase (PKA), or long-term treatment with phorbol 12-myristate 13-acetate to induce down-regulation of protein kinase C (PKC) had no significant effect. 6 Recovery from desensitization (resensitization) proceeded with a time constant of 6.7 min. Okadaic acid, an inhibitor of serine/threonine phosphatases 1 and 2A, significantly attenuated the degree of resensitization. 7 In summary, we have characterized the time course and concentration-dependence of the desensitization of DADLE-induced I-Ba inhibition in NG108-15 cells. This desensitization was reversible after removal of DADLE. It is suggested that beta ARK, but neither PKA nor PKC, is involved in desensitization, while serine/threonine phosphatases mediate resensitization.