Abstract
Opioid receptors often couple to multiple effectors within the same cell. To examine potential mechanisms that contribute to the specificity by which δ-receptors couple to distinct intracellular effectors, we stably transfected rat pituitary GH3 cells with cDNAs encoding for δ-opioid receptors. In cells transfected with a relatively low δ-receptor density of 0.55 pmol/mg of protein (GH3DOR), activation of δ-receptors produced inhibition of adenylyl cyclase activity but was unable to alter L-type Ca2+ current. In contrast, activation of δ-receptors in a clone that contained a higher density of δ-receptors (2.45 pmol/mg of protein) and was also coexpressed with μ-opioid receptors (GH3MORDOR), resulted in not only the expected inhibition of adenylyl cyclase activity but also produced inhibition of L-type Ca2+ current. The purpose of the present study was to determine whether these observations resulted from differences in δ-opioid receptor density between clones or interaction between δ- and μ-opioid receptors to allow the activation of different G proteins and signaling to Ca2+ channels. Using the δ-opioid receptor alkylating agent SUPERFIT, reduction of available δ-opioid receptors in GH3MORDOR cells to a density similar to that of δ-opioid receptors in the GH3DOR clone resulted in abolishment of coupling to Ca2+ channels, but not to adenylyl cyclase. Furthermore, although significantly greater amounts of all G proteins were activated by δ-opioid receptors in GH3MORDOR cells, δ-opioid receptor activation in GH3DOR cells resulted in coupling to the identical pattern of G proteins seen in GH3MORDOR cells. These findings suggest that different threshold densities of δ-opioid receptors are required to activate critical amounts of G proteins needed to produce coupling to specific effectors and that δ-opioid receptors couple more efficiently to adenylyl cyclase than to L-type Ca2+channels.
Footnotes
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Send reprint requests to: Paul L. Prather, Ph.D., Department of Pharmacology and Toxicology, Mail Slot 611, University of Arkansas for Medical Sciences, 4301 W. Markham St., Little Rock, AR 72205. E-mail: pratherpaull{at}exchange.uams.edu
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↵1 This work was supported in part by National Institute on Drug Abuse Grants DA10936 (to P.L.P.), DA07234-07 (to P.L.P), DA05627-01 (to E.T.P.), DA05010 (to T.G.H.), DA07339 (to P.Y.L.), DA05695 (to P.Y.L.), and an intramural pilot study grant from the University of Arkansas for Medical Sciences (to P.L.P.).
- Abbreviations:
- CHO
- Chinese hamster ovary
- GABA
- γ-aminobutyric acid
- SUPERFIT
- cis-(+)-3-methylfentanyl isothiocyanate
- ECL
- enhanced chemiluminescence
- DPDPE
- [d-Pen2,5]-enkephalin
- CTOP
- d-Pen-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH2
- DMEM
- Dulbecco's modified Eagle's medium
- β-FNA
- β-funaltrexamine
- DAMGO
- [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin
- [α-32P]AA-GTP
- [α-32P]azidoanilido-GTP
- PAGE
- polyacrylamide gel electrophoresis
- TBS
- Tris-buffered saline
- Received April 3, 2000.
- Accepted August 3, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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