Desensitization and resensitization of delta-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-Ala2, D-Leu5]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 a time constant of 4.4 min. 3. Maximal desensitization of the delta-opioid receptor-Ca2+ channel coupling was attained by 1 microM 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 (betaARK1) in NG108-15 cells. 5. Heparin, an inhibitor of betaARK, 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 betaARK, but neither PKA nor PKC, is involved in desensitization, while serine/threonine phosphatases mediate resensitization.
Morikawa
H
H
Department of Anesthesia, Kyoto University Hospital, Japan.
Fukuda
K
K
Mima
H
H
Shoda
T
T
Kato
S
S
Mori
K
K
D000818:Animals; D019556:COS Cells; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D016308:Enkephalin, Leucine-2-Alanine / Q000494:pharmacology*; D006822:Hybrid Cells; D011493:Protein Kinase C / Q000378:metabolism; D017465:Receptors, Opioid, delta / Q000187:drug effects* / Q000502:physiology; D014407:Tumor Cells, Cultured; D051552:beta-Adrenergic Receptor Kinases
D002121:Calcium Channel Blockers; D017465:Receptors, Opioid, delta; D016308:Enkephalin, Leucine-2-Alanine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D051552:beta-Adrenergic Receptor Kinases