Opioid-induced increase in [Ca2+]i in ND8-47 neuroblastoma x dorsal root ganglion hybrid cells is mediated through G protein-coupled delta-opioid receptors and desensitized by chronic exposure to opioid.
delta-Receptor agonists induce a concentration-dependent increase in intracellular calcium concentration ([Ca2+]i) in ND8-47 cells by activating dihydropyridine-sensitive Ca2+ channels. The role of G proteins in transducing the opioid effect has been studied. Pretreatment of cells with pertussis toxin (100 ng/ml, 24 h) almost completely blocked [D-Ser2,Leu5]enkephalin-Thr (DSLET)-induced increase in [Ca2+]i. Cholera toxin (10 nM, 24 h) had no effect on DSLET-induced response. Pretreatment of the cells with 1 microM DSLET for 1 h resulted in a 30% inhibition of DSLET-induced increase in [Ca2+]i and a 78% inhibition after exposure for 24 h. After 1 h of exposure to DSLET, there was a decrease in agonist affinity with no significant changes in receptor density. Cells exposed to 1 microM DSLET for 24 h demonstrate a nearly 90% decrease in [3H]diprenorphine binding, with a decrease in affinity for agonist at the remaining binding sites. G protein subunits alpha i2, alpha i3, alpha s, and alpha q were detected in ND8-47 cell membranes by western blot; alpha o and alpha i1 were not present. Chronic DSLET treatment had no significant effect on the quantity of each of the alpha-subunits. These results suggest that the DSLET-induced increase in [Ca2+]i mediated through pertussis toxin-sensitive G proteins (probably Gi2 or Gi3) and the attenuation of this response in chronically treated cells is associated with a relatively rapid reduction in receptor affinity to DSLET and a slow reduction in receptor density.
Tang
T
T
Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 21814-4799, USA.
Kiang
J G
JG
Cote
T
T
Cox
B M
BM
D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism; D002772:Cholera Toxin / Q000494:pharmacology; D004095:Dihydropyridines / Q000494:pharmacology; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005727:Ganglia, Spinal / Q000166:cytology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006822:Hybrid Cells / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism*; D009294:Narcotics / Q000494:pharmacology*; D009447:Neuroblastoma / Q000473:pathology; D009994:Osmolar Concentration; D037342:Pertussis Toxin; D011957:Receptors, Opioid / Q000378:metabolism; D017465:Receptors, Opioid, delta / Q000502:physiology*; D013997:Time Factors; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D015220:Calcium Channels; D004095:Dihydropyridines; D009294:Narcotics; D011957:Receptors, Opioid; D017465:Receptors, Opioid, delta; D010566:Virulence Factors, Bordetella; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004743:Enkephalin, Leucine; C034318:enkephalin, Ser(2), Leu(5), Thr(6)-; D002772:Cholera Toxin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D002118:Calcium