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.
Chen
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Prince Henry's Institute of Medical Research, Clayton, Victoria, Australia.
Clarke
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D000818:Animals; D001483:Base Sequence; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D019085:Fluorescent Antibody Technique, Direct; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000502:physiology*; D013006:Growth Hormone / Q000502:physiology*; D007150:Immunohistochemistry; D017551:Microdialysis; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense; D018408:Patch-Clamp Techniques; D010902:Pituitary Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D012756:Sheep; D013004:Somatostatin / Q000494:pharmacology*
D015220:Calcium Channels; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D013004:Somatostatin; D013006:Growth Hormone; D019204:GTP-Binding Proteins