Acute and chronic effects of opioids on delta and mu receptor activation of G proteins in NG108-15 and SK-N-SH cell membranes.
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.
Breivogel
C S
CS
Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27157, USA.
Selley
D E
DE
Childers
S R
SR
D000700:Analgesics / Q000494:pharmacology; D000818:Animals; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D004745:Enkephalins / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism*; D005910:Glioma; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D006822:Hybrid Cells / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D009270:Naloxone / Q000494:pharmacology; D009271:Naltrexone / Q000031:analogs & derivatives / Q000494:pharmacology; D009292:Narcotic Antagonists / Q000494:pharmacology; D009294:Narcotics / Q000494:pharmacology*; D009447:Neuroblastoma; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D017465:Receptors, Opioid, delta / Q000819:agonists / Q000187:drug effects / Q000378:metabolism*; D017450:Receptors, Opioid, mu / Q000819:agonists / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013462:Sulfur Radioisotopes; D013997:Time Factors
D000700:Analgesics; D004745:Enkephalins; D008565:Membrane Proteins; D009292:Narcotic Antagonists; D009294:Narcotics; D017465:Receptors, Opioid, delta; D017450:Receptors, Opioid, mu; D013462:Sulfur Radioisotopes; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D009270:Naloxone; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004743:Enkephalin, Leucine; D009271:Naltrexone; C034318:enkephalin, Ser(2), Leu(5), Thr(6)-; D019204:GTP-Binding Proteins; C055382:naltrindole