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G02 AND G i3 PROTEINS MEDIATE THE ACTION OF SOMATOSTATIN ON MEMBRANE Ca 2+ AND K + CURRENTS IN OVINE PITUITARY SOMATOTROPHS
Author(s) -
Chen Chen
Publication year - 1997
Publication title -
clinical and experimental pharmacology and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.752
H-Index - 103
eISSN - 1440-1681
pISSN - 0305-1870
DOI - 10.1111/j.1440-1681.1997.tb02105.x
Subject(s) - endocrinology , medicine , somatostatin , somatotropic cell , chemistry , anterior pituitary , percoll , pituitary gland , pertussis toxin , g protein , biology , hormone , receptor , centrifugation , biochemistry
SUMMARY 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 Ca 2+ concentration ([Ca 2+ ] i ), which is regulated by the influx of Ca 2+ via membrane Ca 2+ channels. Somatostatin reduces the influx of Ca 2+ through two separate mechanisms, namely a direct action on Ca 2+ 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 Ca 2+ or K + currents. 4. A significant reduction in Ca 2+ 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 Ca 2+ 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 Ca 2+ current response to SRIF. 6. Intracellular dialysis of antibodies to αo, α 1_3 , ai 1‐2 and ai 3 summits 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‐@aLi 3 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 Ca 2+ 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 Ca 2+ current. 9. Dialysis of the ao common antisense oligonucleotides (ASm) but not the αi 3 AS significantly diminished the inhibitory effect of SRIF on the Ca 2+ 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 αo 1 (αo 1 AS) or αo 2 (α0 2 AS) were dialysed into somatotrophs and only αo 2 AS significantly attenuated the inhibition of SRIF on the Ca 2+ 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 Ca 2+ current in primary cultured ovine somatotrophs.