G(o2) and G(i3) proteins mediate the action of somatostatin on membrane Ca2+ and K+ currents in ovine pituitary somatotrophs

1. Growth hormone (GH) secretion from the anterior pituitary gland is mainly regulated by hypothalamic GH-releasing hormone (GHRH) and somatostatin (SRIF). Somatostatin reduces both spontaneous and GHRH-stimulated GH secretion. 2. Exocytosis of GH is mainly determined by the intracellular free Ca2+ concentration ([Ca2+](i)), which is regulated by the influx of Ca2+ via membrane Ca2+ channels. Somatostatin reduces the influx of Ca2+ through two separate mechanisms, namely a direct action on Ca2+ channels and an indirect action on membrane potentials through the activation of K+ channels. 3. In the present experiments, somatotroph-enriched cells were obtained from the ovine pituitary gland by means of collagenase dissociation and Percoll-gradient centrifugation. Further identification was based on the effect of SRIF (10 nmol/L) on Ca2+ or K+ currents. 4. A significant reduction in Ca2+ currents and an increase in K+ currents was obtained in response to local application of SRIF (10 nmol/L), but vehicle application had no effect. The responses of Ca2+ and K+ currents to SRIF were reversible after removal of SRIF. 5. Dialysis of GTP-gamma-s (200 mu mol/L) abolished the recovery phase of K+ current response to SRIF after its removal, whereas GDP-beta-s (200 mu mol/L) totally blocked the response. Pretreatment of the cells with pertussis toxin (100 nmol/L) overnight abolished the Ca2+ current response to SRIF. 6. Intracellular dialysis of antibodies to alpha 0, alpha i(1-3), alpha i(1-2) and alpha i(3) subunits of the G-proteins into cells via whole-cell patch-clamp pipettes was confirmed by immunofluorescent staining of the antibodies. 7. Dialysis of anti-alpha i(1-3) or anti-alpha i(3) antibodies significantly attenuated the increase in the K+ current in response to 10 nmol/L SRIF, whereas neither anti-alpha 0 nor anti-alpha i(1-2) antibodies diminished the effect of SRIF on the K+ current. 8. Dialysis of anti-alpha 0 antibodies significantly attenuated the reduction, in the Ca2+ current that was obtained upon application of 10 nmol/L SRIF. Neither anti-alpha i(1-2) nor anti-alpha i(3) antibody dialysis diminished the effect of SRIF on the Ca2+ current. 9. Dialysis of the alpha 0 common antisense oligonucleotides (ASm) but not the alpha i(3) AS significantly diminished the inhibitory effect of SRIF on the Ca2+ current. This effect of alpha 0 ASm dialysis occurred at 12 h incubation after dialysis, reaching a maximal level at 48 h and partially recovering at 72 h incubation. Antisense oligonucleotides specific for alpha 0(1) (alpha 0(1) AS) or alpha 0(2) (alpha 0(2) AS) were dialysed into somatotrophs and only alpha 0(2) AS significantly attenuated the inhibition of SRIF on the Ca2+ current. 10. It is concluded that the G(i3) protein mediates the effect of SRIF on the K+ current and that the G(o2) protein mediates the effect of SRIF on the Ca2+ current in primary cultured ovine somatotrophs.