G(I2) 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 ot her secretion-stimulating hormones trigger an increase in the cytosoli c Ca2+ concentration by two mechanisms. Ca2+ is released from intracel lular stores in response to inositol 1,4,5 -trisphosphate and can ente r 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 re gulatory 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 s uppression of G-protein synthesis. Antisense oligonucleotides were mic roinjected into GH3 cell nuclei, and 48 h after injection the TRH effe ct was tested. Whereas antisense oligonucleotides hybridizing to the m RNA of G(o) or G(i1) alpha-subunit sequences did not affect stimulatio n by TRH, oligonucleotides suppressing the expression of the G(i2) alp ha subunit abolished this effect, and oligonucleotides directed agains t the mRNA of the G(i3) alpha subunit had less effect. The requirement of a concurrent inositol phospholipid degradation and subsequent prot ein kinase C (PKC) activation for the TRH effect on Ca2+-channel activ ity was demonstrated by inhibitory effects of antisense oligonucleotid es directed against G(q)/G11/G(z) alpha-subunit sequences and treatmen t of GH3 cells with PKC inhibitors, respectively. Our results suggest that TRH elevates the cytosoliC Ca2+ concentration in GH3 cells transi ently via Ca2+ release from internal stores, followed by a phase of su stained Ca2+ influx through voltage-dependent Ca2+ channels stimulated by the concerted action of G(i2) (and G(i3)) plus PKC.