Abstract
Thyrotropin-releasing hormone (TRH), like numerous other Ca2+-mobilizing agonists, has been found to stimulate polyphosphoinositide hydrolysis in responsive cells. The present studies further clarify the mechanism of action of this peptide hormone by demonstrating direct in vitro effects of TRH on polyphosphoinositide hydrolysis in GH3 pituitary cell membranes. Membranes from [3H]myoinositol-labeled cells were found to generate inositol bis- and tris- but not monophosphate upon incubation. Inositol polyphosphate generation was stimulated 2-3-fold by nanomolar concentrations of TRH in a reaction which was potentiated by micromolar concentrations of GTP; hormone-stimulated hydrolysis observed in the absence of GTP was fully antagonized by guanosine 5'-O-(2-thiodiphosphate). Guanosine 5'-O-(3-thiotriphosphate), Ca2+, and sodium fluoride also activated phosphoinositide hydrolysis in vitro. Stimulated inositol polyphosphate generation was accompanied by stimulated 1,2-diacylglycerol formation. Evidence that both phosphatidylinositol 4,5-bisphosphate as well as phosphatidylinositol 4-phosphate served as substrates for the activated phosphoinositide phosphodiesterase is presented. Pretreatment of GH3 cells with cholera or pertussis toxin did not influence stimulated hydrolysis in membranes. It is concluded that the TRH receptor directly regulates polyphosphoinositide hydrolysis in GH3 cell plasma membranes by a GTP-dependent process. The GTP dependence does not appear to be mediated through a cholera or pertussis toxin substrate and may involve a novel GTP-binding protein (NP).
Highlights
Thyrotropin-releasing hormone (TRH), like numer- hormone secretion from normal pituitary cells or from GH, ous other Ca2+-mobilizingagonists, has been found to rat pituitary tumor cells [1].Specific high-affinity receptors stimulate polyphosphoinositide hydrolysis in respon- for TRH have been identified in ligand-binding studies [3], sive cells
We show that TRH-stimulated polyphosphoinositide turnover occurs in purified GH3 cell plasma membranes by a receptor-associated pathway which is dependent upon guanine nucleotides
Previous studies from several laboratories (5-8, 10, 12, 1521,54) have suggested second messenger roles forthe metabolites derived from PtdIns-4,5-Pz catabolism (InsP, and 1,2sn-diacylglycerol) in mediating the prolactin-releasing actions of TRH in GHc3ells
Summary
Membranes were prepared from [3H]inositol-labeledGH3cells and incubated at 37 “C at 50 nM free Ca2+.Incubations containing no further additions generated InsP2 and InsP3(Fig. 1).Addition of TRH stimulated InsP2 and InsP3generation whereas addition of GTP had little influence. TRH stimulation of InsP2 InsP3 generation was halfmaximal at -30 nM TRH in the absence [22] or presence of GTP (Fig. 2). W - Methylhistidine TRH promoted phosphoinositide hydrolysis at 10-foldlower concentrations than TRHin accord with the reported [3] greaterpotencyof this analog (Fig.). GTP alone stimulated InsPz InsP3 generation only marginally in the experiment of Fig. l. Partial stimulation by GDP may result from in uitro conversion to GTP since GDP-p-S, an analog reported to notundergo phosphorylation [35],was less stimulatory (Fig. 3).
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