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Published on: January 27, 2014
Desensitization and resensitization of delta-opioid receptor-mediated Ca2+ channel inhibition in NG108-15 cells
1Department of Anesthesia, Kyoto University Hospital, Japan.
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 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.
Insights
Opioid receptor desensitization involves beta-adrenergic receptor kinase (betaARK) in NG108-15 cells. Serine/threonine phosphatases mediate the recovery of opioid receptor function after desensitization.
Area of Science:
- Neuroscience
- Pharmacology
- Cell Signaling
Background:
- Opioid receptors mediate crucial physiological functions, but their signaling can desensitize with prolonged exposure.
- Understanding the molecular mechanisms of opioid receptor desensitization is vital for developing effective pain management strategies.
- NG108-15 neuroblastoma x glioma hybrid cells provide a model system to study G protein-coupled receptor signaling.
Purpose of the Study:
- To elucidate the mechanisms underlying the desensitization of opioid receptor-mediated calcium channel inhibition.
- To investigate the role of specific kinases and phosphatases in the desensitization and resensitization processes.
- To characterize the time course and concentration dependence of opioid-induced calcium channel current inhibition.
Main Methods:
- Analysis of barium currents (I(Ba)) through calcium channels in NG108-15 cells using prolonged application of [D-Ala2, D-Leu5]enkephalin (DADLE).
- Assessment of desensitization kinetics and concentration-dependence (EC50).
- RNA blot and immunoblot analyses to detect beta-adrenoceptor kinase-1 (betaARK1) expression.
- Pharmacological inhibition of betaARK (heparin), protein kinase A (PKA; Rp-cyclic AMPS, PKI), protein kinase C (PKC; phorbol ester), and serine/threonine phosphatases (okadaic acid).
Main Results:
- DADLE-induced inhibition of I(Ba) desensitized by 57% with a time constant of 4.4 min, with maximal desensitization at 1 microM DADLE and an EC50 of 78 nM.
- betaARK1 was expressed in NG108-15 cells.
- Heparin significantly reduced desensitization magnitude and rate, while PKA and PKC inhibitors had no significant effect.
- Recovery from desensitization (resensitization) occurred with a time constant of 6.7 min and was attenuated by okadaic acid.
Conclusions:
- Opioid receptor desensitization in NG108-15 cells is a reversible process.
- Beta-adrenergic receptor kinase (betaARK) plays a significant role in the desensitization of opioid receptor-mediated calcium channel inhibition.
- Serine/threonine phosphatases are implicated in the resensitization process, while PKA and PKC are not directly involved.
