G Protein Modulation of ω‐Conotoxin Binding Sites in Neuroblastoma X Glioma NG 108‐15 Hybrid Cells

Abstract

Electrophysiological evidence shows that voltage-dependent calcium channel (VDCC) activity can be regulated by a large number of neurotransmitters. In particular, guanine nucleotide binding regulatory protein (G protein)-mediated inhibitory modulation of the channel activity has been deduced from evidence that GTP analogues and purified G proteins are able to mimic this effect. The G proteins involved are pertussis toxin (PTx) sensitive. The purpose of the present study was to investigate, using biochemical techniques, whether G protein activation modulates the recognition site for omega-conotoxin GVIA (CgTx), a peptide neurotoxin that selectively labels a population of high-threshold VDCC. Undifferentiated and differentiated (1 mM dibutyryl cyclic AMP, 4 days) NG 108-15 cells were used. In both crude cellular extracts specific binding of 125I-CgTx was characterized. Differentiation induced a sixfold increase in the number of binding sites and doubled the KD value. The in vitro addition of guanylylimidodiphosphate (GMP-PNP; a nonhydrolyzable analogue of GTP) to extracts prepared from differentiated cells reduced the 125I-CgTx binding by 48%. This effect, observed in undifferentiated cells as well, was also caused by other triphosphate guanine nucleotides, such as GTP, but not by guanosine 5'-O-(2-thiodiphosphate) or adenine nucleotides. Treatment of the cells with PTx prevented the GMP-PNP effect. Moreover, the results obtained after preincubation with specific antisera raised against the alpha subunits of Gi1-2 and Go suggest that Go is the G protein responsible for the observed effect.

Characterization of Acetylcholinesterase Secretion in Neuronal Cultures and Regulation by High K+ and Soluble Factors from Target Cells

“…The difference between the levels of AChE release was significant by Student's t analysis (" .p < 0 .01) . toxin, a ligand for a specific neuronal Ca" channel, which is developmentally regulated in neuroblastoma cells (Bergamaschi et al ., 1992) . Although in differentiated cells a concentration-dependent, saturable binding was observed, treatment of Nl8TG2 cells with 0 .2 nmol/L w-conotoxin did not abolish high K -'-induced ACNE secretion (data not shown) .…”

Characterization of a rat clonal [beta]-cell line, RINm5F, in a passage- and morphology-dependent manner

“…Dibutyryl cAMP (BtjCAMP), an analogue of cAMP, induces elongation of CHO (Chinese hamster ovary) cells (Hsie and Puck, 1971), Swiss 3T3 cells (Johnson and Pastan, 1972), cultured iris epithelial cells (Ortiz et al, 1973), cultured vascular smooth muscle cells (Nabika et al, 1988), primary cultured astrocytes (Baorto et al, 1992), NG 108-15 cells (Bergamaschi et al, 1992), NCB-20 cells (Mienville, 1992) and BHK21 cells (Edwards et al, 1993). N®-substituted derivatives of adenosine can produce the processes without affecting cAMP levels.…”