ANTISENSE OLIGODEOXYNUCLEOTIDE TO THE G(I2) 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 li ne, activation of delta-opioid receptors induced an increase in the in tracellular free calcium concentration ([Ca2+](i)) through dihydropyri dine-sensitive calcium channels. This effect was mediated by pertussis toxin-sensitive G proteins. The G protein alpha subunits alpha(12), a lpha(13), alpha(q), and alpha(s) were detected using Western blots, wh ereas alpha(o) and alpha(11) were not found in ND8-47 cell membranes. To identify the specific G protein alpha subunit(s) responsible for th e increase in [Ca2+](i), we treated ND8-47 cells with antisense oligad eoxynucleotides (AS) complementary to the mRNA for each G protein alph a subunit (alpha(12), alpha(13), or alpha(s)), at a concentration of 1 0 mu M, for up to 6 days and examined their effects on opioid-induced increases in [Ca2+](i) and on the levels of G protein a subunits. [Ca2 +](i) was measured in adherent cells using the fluorescent dye fura-2. Treatment of cells with alpha(12)-AS (10 mu M, for 6 days) resulted i n a 73% inhibition of the [D-Ser(2),Leu(5)]-enkephalin-Thr-induced inc rease in [Ca2+](i). In contrast, pretreatment of cells with alpha(13)- AS (10 mu M, for 6 days) or alpha(s)-AS (10 mu M, for 6 days) had no e ffect on the [D-Ser(2),Leu(5)]-enkephalin-Thr-induced responses. Weste rn blots indicated that the levels of alpha(12) were decreased when ce lls were exposed to alpha(12)-AS (10 mu M) for 6 days, whereas the lev els of alpha(13), alpha(s), and alpha(q) were not affected by this tre atment. Treatment of the cells with alpha(13)-AS or alpha(s)-AS for 6 days significantly reduced alpha(13), 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 12).