Antisense oligodeoxynucleotide to the Gi2 protein alpha subunit sequence inhibits an opioid-induced increase in the intracellular free calcium concentration in ND8-47 neuroblastoma x dorsal root ganglion hybrid cells.
In ND8-47 cells, a neuroblastoma x dorsal root ganglion hybrid cell line, activation of delta-opioid receptors induced an increase in the intracellular free calcium concentration ([Ca2+]i) through dihydropyridine-sensitive calcium channels. This effect was mediated by pertussis toxin-sensitive G proteins. The G protein alpha subunits alpha i2, alpha i3, alpha q, and alpha s were detected using Western blots, whereas alpha o and alpha i1 were not found in ND8-47 cell membranes. To identify the specific G protein alpha subunit(s) responsible for the increase in [Ca2+]i, we treated ND8-47 cells with antisense oligodeoxynucleotides (AS) complementary to the mRNA for each G protein alpha subunit (alpha i2, alpha i3, or alpha s), at a concentration of 10 microM, for up to 6 days and examined their effects on opioid-induced increases in [Ca2+]i and on the levels of G protein alpha subunits. [Ca2+]i was measured in adherent cells using the fluorescent dye fura-2. Treatment of cells with alpha i2-AS (10 microM, for 6 days) resulted in a 73% inhibition of the [D-Ser2,Leu5]-enkephalin-Thr-induced increase in [Ca2+]i. In contrast, pretreatment of cells with alpha i3-AS (10 microM, for 6 days) or alpha s-AS (10 microM, for 6 days) had no effect on the [D-Ser2,Leu5]-enkephalin-Thr-induced responses. Western blots indicated that the levels of alpha i2 were decreased when cells were exposed to alpha i2-AS (10 microM) for 6 days, whereas the levels of alpha i3, alpha s, and alpha q were not affected by this treatment. Treatment of the cells with alpha i3-AS or alpha s-AS for 6 days significantly reduced alpha i3 or alpha s levels, respectively. These results indicate that the opioid-induced increase in [Ca2+]i in ND8-47 cells is mediated by G alpha i2.
Tang
T
T
Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799, USA.
Kiang
J G
JG
Côté
T E
TE
Cox
B M
BM
D000700:Analgesics / Q000037:antagonists & inhibitors; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects; D002478:Cells, Cultured; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors; D019204:GTP-Binding Proteins / Q000235:genetics*; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects*; D006822:Hybrid Cells; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000473:pathology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D017465:Receptors, Opioid, delta / Q000187:drug effects
D000700:Analgesics; D015220:Calcium Channels; D016376:Oligonucleotides, Antisense; D017465:Receptors, Opioid, delta; D004743:Enkephalin, Leucine; C034318:enkephalin, Ser(2), Leu(5), Thr(6)-; D019204:GTP-Binding Proteins; D002118:Calcium