Expression of α-transducin in Chinese hamster ovary cells stably transfected with the human δ-opioid receptor attenuates chronic opioid agonist-induced adenylyl cyclase superactivation

Marc Rubenzik, Eva Varga, Dagmar Stropova, William R. Roeske, Henry I. Yamamura

Research output: Contribution to journalArticle

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Abstract

To investigate the role of G-protein βγ subunits in δ-opioid signal transduction, we have transfected Chinese hamster ovary (CHO) cells stably expressing the human δ-opioid receptor (hDOR/CHO cells) with the Gα-subunit of transducin-1 (hDOR/T1/CHO). Inhibition of forskolin-stimulated adenylyl cyclase and phospholipase Cβ (PLCβ) activation was measured in each of these cell lines. Because PLCβ3 activation in CHO cells has been shown to be mediated by free Gβγ subunits derived from Gαi/o, the action of transducin was confirmed by measuring a significant attenuation of (+)-4-[(αR)-α-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3- methoxybenzyl]-N-N-diethylbenzamide (SNC80)-mediated maximal inositol-1,4,5-trisphosphate formation in transducin-expressing cells of 59 ± 12% compared with control cells. The acute inhibition of cAMP formation was unchanged between control and transducin- expressing cells. We show that cells stably expressing the human δ-opioid receptor exhibited a pertussis toxin-sensitive cAMP overshoot in response to chronic application of SNC80. After 4 h of pretreatment and washout with 100 nM SNC80, maximal forskolin-stimulated cAMP formation in hDOR/CHO cells increased by 229 ± 37% compared with buffer-treated cells. Expression of transducin in hDOR/CHO cells diminished this response: hDOR/T1/CHO cells showed no significant change in maximal forskolin-stimulated cAMP formation after pretreatment and washout. These data indicate that the expression of α-transducin scavenges free Gβγ subunits and, furthermore, that free Gβγ subunits play a role in opioid-mediated PLCβ activation and adenylyl cyclase superactivation, but not acute inhibition of forskolin-stimulated cAMP formation in hDOR/CHO cells.

Original languageEnglish (US)
Pages (from-to)1076-1082
Number of pages7
JournalMolecular Pharmacology
Volume60
Issue number5
StatePublished - 2001
Externally publishedYes

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Transducin
Opioid Receptors
Cricetulus
Adenylyl Cyclases
Opioid Analgesics
Ovary
Colforsin
Type C Phospholipases
Gastrin-Secreting Cells
Inositol 1,4,5-Trisphosphate
Pertussis Toxin
Protein Subunits
GTP-Binding Proteins
Signal Transduction
Buffers

ASJC Scopus subject areas

  • Pharmacology

Cite this

Expression of α-transducin in Chinese hamster ovary cells stably transfected with the human δ-opioid receptor attenuates chronic opioid agonist-induced adenylyl cyclase superactivation. / Rubenzik, Marc; Varga, Eva; Stropova, Dagmar; Roeske, William R.; Yamamura, Henry I.

In: Molecular Pharmacology, Vol. 60, No. 5, 2001, p. 1076-1082.

Research output: Contribution to journalArticle

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abstract = "To investigate the role of G-protein βγ subunits in δ-opioid signal transduction, we have transfected Chinese hamster ovary (CHO) cells stably expressing the human δ-opioid receptor (hDOR/CHO cells) with the Gα-subunit of transducin-1 (hDOR/T1/CHO). Inhibition of forskolin-stimulated adenylyl cyclase and phospholipase Cβ (PLCβ) activation was measured in each of these cell lines. Because PLCβ3 activation in CHO cells has been shown to be mediated by free Gβγ subunits derived from Gαi/o, the action of transducin was confirmed by measuring a significant attenuation of (+)-4-[(αR)-α-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3- methoxybenzyl]-N-N-diethylbenzamide (SNC80)-mediated maximal inositol-1,4,5-trisphosphate formation in transducin-expressing cells of 59 ± 12{\%} compared with control cells. The acute inhibition of cAMP formation was unchanged between control and transducin- expressing cells. We show that cells stably expressing the human δ-opioid receptor exhibited a pertussis toxin-sensitive cAMP overshoot in response to chronic application of SNC80. After 4 h of pretreatment and washout with 100 nM SNC80, maximal forskolin-stimulated cAMP formation in hDOR/CHO cells increased by 229 ± 37{\%} compared with buffer-treated cells. Expression of transducin in hDOR/CHO cells diminished this response: hDOR/T1/CHO cells showed no significant change in maximal forskolin-stimulated cAMP formation after pretreatment and washout. These data indicate that the expression of α-transducin scavenges free Gβγ subunits and, furthermore, that free Gβγ subunits play a role in opioid-mediated PLCβ activation and adenylyl cyclase superactivation, but not acute inhibition of forskolin-stimulated cAMP formation in hDOR/CHO cells.",
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AU - Roeske, William R.

AU - Yamamura, Henry I.

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