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 α_o, α_i-1-2, or α_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-α_o antibodies significantly attenuated the reduction in Ca²⁺ currents that was obtained upon application of 10 or 100 nM SRIF. Dialysis of neither anti-α_i-1-2 nor anti-α_i-3 antibodies diminished the effect of SRIF on Ca²⁺ currents. 6. Intracellular dialysis of antisense oligonucleotides directed against the α_o subunit mRNA (α_o ASm, for α_o common) or against the α_i-3 subunit mRNA (α_i-3 AS) blocked expression of α_o or α_i-3 subunits in the cells, respectively, as assessed by fluorescent staining with anti-α_o or anti-α_i-3 antibodies 48 h after dialysis. 7. Dialysis of α_o ASm, but not α_i-3 AS, significantly diminished the inhibitory effect of SRIF on Ca²⁺ currents. This effect of α_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 α_o-1 (α_o-1 AS) or α_o-2 (α_o-2 AS) were dialysed into somatotrophs and only α_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.