Constitutive activity of the δ-opioid receptor expressed in C6 glioma cells: identification of non-peptide δ-inverse agonists

摘要

class="enumerated" style="list-style-type:decimal">G-protein coupled receptors can exhibit constitutive activity resulting in the formation of active ternary complexes in the absence of an agonist. In this study we have investigated constitutive activity in C6 glioma cells expressing either the cloned δ-(OP1) receptor (C6δ), or the cloned μ-(OP3) opioid receptor (C6μ).Constitutive activity was measured in the absence of Na⁺ ions to provide an increased signal. The degree of constitutive activity was defined as the level of [³⁵S]-GTPγS binding that could be inhibited by pre-treatment with pertussis toxin (PTX). In C6δ cells the level of basal [³⁵S]-GTPγS binding was reduced by 51.9±6.1 fmols mg⁻¹ protein, whereas in C6μ and C6 wild-type cells treatment with PTX reduced basal [³⁵S]-GTPγS binding by only 10.0±3.5 and 8.6±3.1 fmols mg⁻¹ protein respectively.The δ-antagonists N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174,864), 7-benzylidenenaltrexone (BNTX) and naltriben (NTB), in addition to clocinnamox (C-CAM), acted as δ-opioid receptor inverse agonists. Naloxone, buprenorphine, and naltrindole were neutral antagonists. Furthermore, naltrindole blocked the reduction in [³⁵S]-GTPγS binding caused by the inverse agonists. The inverse agonists did not inhibit basal [³⁵S]-GTPγS binding in C6μ or C6 wild-type cell membranes.Competition binding assays in C6δ cell membranes revealed a leftward shift in the displacement curve of [³H]-naltrindole by ICI 174,864 and C-CAM in the presence of NaCl and the GTP analogue, GppNHp. There was no change in the displacement curve for BNTX or NTB under these conditions.These data confirm the presence of constitutive activity associated with the δ-opioid receptor and identify three novel, non-peptide, δ-opioid inverse agonists.