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Vol. 283, Issue 3, 1276-1284, 1997
-O-(3-[35S]thio)triphosphate to NG108-15
Cell Membranes: Characterization of Agonist and Inverse Agonist
Effects1
Department of Chemistry, Loughborough University,
Leicester, LE11 3TU, UK (P.G.S., J.R.T.), and
Department of
Pharmacology, University of Michigan Medical School, Ann Arbor,
Michigan (J.R.T.)
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]GTP
S) 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 GDP
S, and Mg++ ions was essential to observe
agonist-mediated stimulation of [35S]GTPS binding,
although the guanine dinucleotides alone had complex inhibitory and
stimulatory effects on [35S]GTPS binding. The relative
ability of the delta antagonists benzylidenenaltrexone
and naltriben to inhibit DPDPE-stimulated [35S]GTPS
binding suggested the opioid receptor involved was of the
delta-2 subtype. Ligand binding assays demonstrated
biphasic binding of these antagonists to this single receptor
type. [35S]GTPS 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]GTPS binding. Pertussis toxin
pretreatment blocked agonist stimulation of [35S]GTPS
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]GTPS 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]GTPS binding to NG108-15 cell
membranes allows a functional measure of delta opioid
activity that can provide systems of differing relative efficacy.
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