Gi2 and protein kinase C are required for thyrotropin-releasing hormone-induced stimulation of voltage-dependent Ca2+ channels in rat pituitary GH3 cells.
In rat pituitary GH3 cells, thyrotropin-releasing hormone (TRH) and other secretion-stimulating hormones trigger an increase in the cytosolic Ca2+ concentration by two mechanisms. Ca2+ is released from intracellular stores in response to inositol 1,4,5-trisphosphate and can enter the cell through voltage-dependent L-type Ca2+ channels. Stimulation of these channels is sensitive to pertussis toxin, indicating that a pertussis toxin-sensitive heterotrimeric guanine nucleotide-binding regulatory protein (G protein) is involved in functional coupling of the receptor to the Ca2+ channel. We identified the G protein involved in the stimulatory effect of TRH on the Ca2+ channel by type-selective suppression of G-protein synthesis. Antisense oligonucleotides were microinjected into GH3 cell nuclei, and 48 h after injection the TRH effect was tested. Whereas antisense oligonucleotides hybridizing to the mRNA of G(o) or Gi1 alpha-subunit sequences did not affect stimulation by TRH, oligonucleotides suppressing the expression of the Gi2 alpha subunit abolished this effect, and oligonucleotides directed against the mRNA of the Gi3 alpha subunit had less effect. The requirement of a concurrent inositol phospholipid degradation and subsequent protein kinase C (PKC) activation for the TRH effect on Ca(2+)-channel activity was demonstrated by inhibitory effects of antisense oligonucleotides directed against Gq/G11/Gz alpha-subunit sequences and treatment of GH3 cells with PKC inhibitors, respectively. Our results suggest that TRH elevates the cytosolic Ca2+ concentration in GH3 cells transiently via Ca2+ release from internal stores, followed by a phase of sustained Ca2+ influx through voltage-dependent Ca2+ channels stimulated by the concerted action of Gi2 (and Gi3) plus PKC.
Institute für Pharmakologie, Freie Universität Berlin, Federal Republic of Germany.
M
M
Gollasch
C
C
Kleuss
J
J
Hescheler
B
B
Wittig
G
G
Schultz
D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000187:drug effects* / Q000378:metabolism; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000502:physiology*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010902:Pituitary Gland / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology*
D015220:Calcium Channels; D013973:Thyrotropin-Releasing Hormone; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D002118:Calcium