Abstract
To compare activation of G proteins by opioid receptors, opioid agonist-stimulated guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTP gamma S) binding in the presence of excess GDP was assayed in membranes from NG108-15 (delta) and SK-N-SH (primarily mu) cells. Basal [35S]GTP gamma S binding consisted of a single class of low-affinity sites (KD 400-500 nM). Addition of agonists produced a high-affinity site 100-300-fold higher in affinity than the basal site. The receptor/transducer amplification factor (ratio of activated G protein Bmax to opioid receptor Bmax) was 10-fold higher for SK-N-SH mu receptors than for NG108-15 delta receptors. Chronic delta agonist ([D-Ser2]-Leu-enkephalin-Thr; DSLET) treatment of NG108-15 cells resulted in an 80% loss of DSLET-stimulated [35S]-GTP gamma S binding within 1 h. Morphine treatment of SK-N-SH cells decreased mu agonist ([D-Ala2, N-Me-Phe4,Gly5-ol]-enkephalin; DAMGO)-stimulated [35S]GTP gamma S binding by 45% after 16 h, with no effect after 1 h. Loss of agonist response was due to a decrease in the Bmax of activated G proteins with no change in the KD. These results provide a quantitative description of G protein activation occurring on acute and chronic exposure to opioid agonists.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Analgesics / pharmacology
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Animals
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Enkephalin, Ala(2)-MePhe(4)-Gly(5)-
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Enkephalin, Leucine / analogs & derivatives
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Enkephalin, Leucine / pharmacology
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Enkephalins / pharmacology
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GTP-Binding Proteins / drug effects
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GTP-Binding Proteins / metabolism*
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Glioma
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Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
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Humans
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Hybrid Cells / chemistry
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Hybrid Cells / drug effects
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Hybrid Cells / metabolism
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Membrane Proteins / drug effects
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Membrane Proteins / metabolism
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Mice
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Naloxone / pharmacology
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Naltrexone / analogs & derivatives
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Naltrexone / pharmacology
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Narcotic Antagonists / pharmacology
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Narcotics / pharmacology*
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Neuroblastoma
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Protein Binding / drug effects
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Rats
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Receptors, Opioid, delta / agonists
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Receptors, Opioid, delta / drug effects
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Receptors, Opioid, delta / metabolism*
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Receptors, Opioid, mu / agonists
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Receptors, Opioid, mu / drug effects
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Receptors, Opioid, mu / metabolism*
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Signal Transduction / drug effects
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Sulfur Radioisotopes
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Time Factors
Substances
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Analgesics
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Enkephalins
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Membrane Proteins
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Narcotic Antagonists
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Narcotics
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Receptors, Opioid, delta
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Receptors, Opioid, mu
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Sulfur Radioisotopes
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Enkephalin, Ala(2)-MePhe(4)-Gly(5)-
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Naloxone
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Guanosine 5'-O-(3-Thiotriphosphate)
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Enkephalin, Leucine
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Naltrexone
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enkephalin, Ser(2), Leu(5), Thr(6)-
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GTP-Binding Proteins
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naltrindole