G(o)-2 protein mediates the reduction in Ca2+ currents by somatostatin in cultured ovine somatotrophs

1. Somatotroph-enriched cells (up to 85%) were obtained from ovine pituitary glands by means of collagenase dissociation and Percoll-gradient centrifugation. Further identification was based on the reduction in Ca2+ currents by 10 nM somatostatin (SRIF). 2. The whole-cell configuration of the patch-clamp technique was employed to study the membrane Ca2+ currents with K+ ions replaced by Cs+ and the addition of K+ and Na+ channel blockers in bath and pipette solutions. 3. A significant reduction in Ca2+ currents was obtained in response to local application of SRIF (10 mM) but vehicle application had no effect. 4. Intracellular dialysis of antibodies to αo, αi-1-2, or αi-3 subunits of G proteins into the cells via patch-clamp pipettes was confirmed by immunofluorescent staining of the antibodies. Antibody dialysis did not modify resting voltage-gated Ca2+ currents across the cell membrane. 5. Dialysis of anti-αo antibodies significantly attenuated the reduction in Ca2+ currents that was obtained upon application of 10 or 100 nM SRIF. Dialysis of neither anti-αi-1-2 nor anti-αi-3 antibodies diminished the effect of SRIF on Ca2+ currents. 6. Intracellular dialysis of antisense oligonucleotides directed against the αo subunit mRNA (αo ASm, for αo common) or against the αi-3 subunit mRNA (αi-3 AS) blocked expression of αo or αi-3 subunits in the cells, respectively, as assessed by fluorescent staining with anti-αo or anti-αi-3 antibodies 48 h after dialysis. 7. Dialysis of αo ASm, but not αi-3 AS, significantly diminished the inhibitory effect of SRIF on Ca2+ currents. This effect of αo ASm dialysis occurred within 12 h after dialysis and reached a maximum at 48 h; partial recovery was seen at 72 h. 8. Antisense oligonucleotides specific for αo-1 (αo-1 AS) or αo-2 (αo-2 AS) were dialysed into somatotrophs and only αo-2 AS significantly attenuated the inhibition of Ca2+ currents by SRIF. 9. We conclude that the Go-2 protein mediates the effect of SRIF on Ca2+ currents in ovine somatotrophs in primary culture.