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 nM) but vehicle application had no effect. 4. Intracellular dialysis of antibodies to alpha(o), alpha(i)-1-2, or alpha(i)-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-alpha(o) antibodies significantly attenuated the reduction in Ca2+ currents that was obtained upon application of 10 or 100 nM SRIF. Dialysis of neither anti-alpha(i)-1-2 nor anti-alpha(i)-3 antibodies diminished the effect of SRIF on Ca2+ currents. 6. Intracellular dialysis of antisense oligonucleotides directed against the alpha(o) subunit mRNA (alpha(o) ASm, for alpha(o) common) or against the alpha(i)-3 subunit mRNA (alpha(i)-3 AS) blocked expression of alpha(o) or alpha(i)-3 subunits in the cells, respectively, as assessed by fluorescent staining with anti-alpha(o) or anti-alpha(i)-3 antibodies 48 h after dialysis. 7. Dialysis of alpha(o) ASm, but not alpha(i)-3 AS, significantly diminished the inhibitory effect of SRIF on Ca2+ currents. This effect of alpha(o) 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 alpha(o)-1 (alpha(o)-1 AS) or alpha(o)-2 (alpha(o)-2 AS) were dialysed into somatotrophs and only alpha(o)-2 AS significantly attenuated the inhibition of Ca2+ currents by SRIF. 9. We conclude that the G(o)-2 protein mediates the effect of SRIF on Ca2+ currents in ovine somatotrophs in primary culture.