G02 AND Gi3 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-λ-s (200 μmol/L) abolished the recovery phase of K⁺ current response to SRIF after its removal, whereas GDP-β-s (200 μmol/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 αo, α_{1_3}, ai_1-2 and ai₃summits of the G-proteins into cells via whole-cell patch-clamp pipettes was confirmed by immunofluorescent staining of the antibodies. 7. Dialysis of anti-ai_1-3 or anti-@aLi₃ antibodies significantly attenuated the increase in the K⁺ current in response to 10 nmol/L SRIF, whereas neither anti-αo nor anti-αi_2 antibodies diminished the effect of SRIF on the K⁺ current. 8. Dialysis of anti-αo antibodies significantly attenuated the reduction in the Ca²⁺ current that was obtained upon application of 10 nmol/L SRIF. Neither anti-αi-2 nor anti-αi3 antibody dialysis diminished the effect of SRIF on the Ca²⁺ current. 9. Dialysis of the ao common antisense oligonucleotides (ASm) but not the αi₃ AS significantly diminished the inhibitory effect of SRIF on the Ca²⁺ current. This effect of ao 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 αo₁ (αo₁ AS) or αo₂(α0₂ AS) were dialysed into somatotrophs and only αo₂ 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 G02 protein mediates the effect of SRIF on the Ca²⁺ current in primary cultured ovine somatotrophs.