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. 1999 Oct;128(3):556-62.
doi: 10.1038/sj.bjp.0702816.

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

C L Neilan  1 , H Akil, J H Woods, J R Traynor
Affiliations

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

C L Neilan et al. Br J Pharmacol. 1999 Oct.

Abstract

1. 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 delta-(OP1) receptor (C6delta), or the cloned mu-(OP3) opioid receptor (C6mu). 2. 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]-GTPgammaS binding that could be inhibited by pre-treatment with pertussis toxin (PTX). In C6delta cells the level of basal [35S]-GTPgammaS binding was reduced by 51.9+/-6.1 fmols mg-1 protein, whereas in C6mu; and C6 wild-type cells treatment with PTX reduced basal [35S]-GTPgammaS binding by only 10.0+/-3.5 and 8.6+/-3.1 fmols mg-1 protein respectively. 3. The delta-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 delta-opioid receptor inverse agonists. Naloxone, buprenorphine, and naltrindole were neutral antagonists. Furthermore, naltrindole blocked the reduction in [35S]-GTPgammaS binding caused by the inverse agonists. The inverse agonists did not inhibit basal [35S]-GTPgammaS binding in C6mu; or C6 wild-type cell membranes. 4. Competition binding assays in C6delta 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. 5. These data confirm the presence of constitutive activity associated with the delta-opioid receptor and identify three novel, non-peptide, delta-opioid inverse agonists.

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Figures

Figure 1
Figure 1
Agonist-independent [35S]-GTPγS binding in C6δ (739 fmols receptor mg−1 protein) and C6μ (1703 fmols receptor mg−1 protein) cell membranes in the presence of either 100 mM NaCl or 100 mM KCl. Values represent mean±s.e.mean for three experiments performed in triplicate. *P<0.05, **P<0.01, significantly different from basal binding in the presence of NaCl, Student's unpaired t-test.
Figure 2
Figure 2
Agonist-independent [35S]-GTPγS binding in membranes prepared from control and pertussis toxin pre-treated (100 ng ml−1, 24 h) C6 glioma cells expressing the δ-opioid receptor at 739 fmols mg−1 protein, untransfected (W-T) C6 cells, or C6 cells expressing the μ-opioid receptor at 427 and 1703 fmols mg−1 protein. Values represent the mean±s.e.mean of at least three experiments performed in duplicate. **P<0.01, *P<0.05, compared to non-PTX treated cells, Student's unpaired t-test.
Figure 3
Figure 3
Inhibition of basal [35S]-GTPγS binding to membranes of C6δ cells by (a) ICI 174,864, (b) C-CAM, (c) BNTX and (d) NTB. Values represent mean±s.e.mean for at least three experiments performed in duplicate.
Figure 4
Figure 4
Reversal of inverse agonist activity of 1 μM of BNTX, CCAM or ICI 174864 by the neutral δ-antagonist naltrindole (10 μM) in C6δ cell membranes. Values represent mean±s.e.mean for three experiments performed in duplicate. *P<0.05, Student's unpaired t-test.
Figure 5
Figure 5
The displacement of [3H]-naltrindole (0.2 nM) binding to membranes of C6δ cells by (a) ICI 174,864, (b) C-CAM, (c) BNTX and (d) NTB in the absence or presence of NaCl (100 mM) and the GTP analogue, GppNHp (50 μM). Values represent mean±s.e.mean for three experiments performed in duplicate.
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