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

Abstract

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 × 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 δ-opioid receptor-Ca²⁺ channel coupling was attained by 1 μM DADLE. The EC₅₀ value for desensitization was estimated to be 78 nM.

4. RNA blot hybridization analysis and immunoblot analysis revealed the expression of β-adrenoceptor kinase-1 (βARK1) in NG108-15 cells.

5. Heparin, an inhibitor of βARK, 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 βARK, but neither PKA nor PKC, is involved in desensitization, while serine/threonine phosphatases mediate resensitization.

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