Abstract
The ability of the delta opioid agonist DPDPE ([d-Pen2,d-Pen4]enkephalin) to stimulate binding of the GTP analog guanosine-5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) to pertussis toxin-sensitive G proteins has been characterized in membranes from NG108–15 mouse neuroblastoma X rat glioma cells. The presence of GDP, or its hydrolysis-resistant analog GDPβS, and Mg++ ions was essential to observe agonist-mediated stimulation of [35S]GTPγS binding, although the guanine dinucleotides alone had complex inhibitory and stimulatory effects on [35S]GTPγS binding. The relative ability of the delta antagonists benzylidenenaltrexone and naltriben to inhibit DPDPE-stimulated [35S]GTPγS binding suggested the opioid receptor involved was of thedelta-2 subtype. Ligand binding assays demonstrated biphasic binding of these antagonists to this single receptor type. [35S]GTPγS binding was also stimulated by [d-Ser2,Leu5,Thr6]enkephalin > deltorphin II = DPDPE = etorphine > levallorphan = diprenorphine = nalorphine = naltrindole. The delta antagonists benzylidenenaltrexone, TIPP (Tyr-Tic-Phe-Phe) and naltriben had no effect, but ICI 174864 (N,N-diallyl-Tyr-Aib-Phe-Leu-OH) acted as an inverse agonist and inhibited [35S]GTPγS binding. Pertussis toxin pretreatment blocked agonist stimulation of [35S]GTPγS binding and also reduced basal binding, thus confirming the presence of constitutively active delta receptors. Replacement of Na+ in the assay buffer with K+ afforded an increased level of basal [35S]GTPγS binding and an apparent increase in both the inverse agonist activity of ICI 174864 and the agonist activity of the partial agonist diprenorphine relative to the full agonist [d-Ser2,Leu5,Thr6]enkephalin. The stimulation of [35S]GTPγS binding to NG108–15 cell membranes allows a functional measure of delta opioid activity that can provide systems of differing relative efficacy.
Footnotes
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Send reprint requests to: Dr. J. R. Traynor, Department of Pharmacology, University of Michigan Medical School, 1301 MSRBIII, Ann Arbor, MI 48109-0632. E-mail:jtraynor{at}umich.edu
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↵1 This work was supported by an EPSRC Research Studentship (P.G.S.) and United States Public Health Service Grant DA-00254–25.
- Abbreviations:
- [d-Ala2
- Glu4]deltorphin II, [Tyr-d-Ala-Phe-Glu-Val-Val-Gly-NH2]
- DPDPE
- [d-Pen2,d-Pen5]enkephalin
- DSLET
- [d-Ser2,Leu5]enkephalin-Thr6
- BNTX
- 7-benzylidenenaltrexone
- NTB
- naltriben
- TIPP
- Tyr-Tic-Phe-Phe (Tic = tetrahydroisoquinoline-3-carboxylic acid)
- GTPγS
- guanosine-5′-O-(3-thio)triphosphate
- DAMGO
- [D-Ala2, MePhe4, Glyol5]enkephalin
- CHO
- Chinese hamster ovary
- DMEM
- Dulbecco’s modified Eagle’s medium
- Received February 21, 1997.
- Accepted August 19, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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