U-73122, AN AMINOSTEROID PHOSPHOLIPASE-C ANTAGONIST, NONCOMPETITIVELY INHIBITS THYROTROPIN-RELEASING-HORMONE EFFECTS IN GH3 RAT PITUITARY-CELLS

TRH increases cytosolic-free calcium ([Ca2+]i) by activating phospholipase C(PL-C), which induces phosphoinositol hydrolysis, leading to Ca2+ mobilization from inositol trisphosphate (IP3) sensitive stores, and by increasing Ca2+ influx. Increases in [Ca2+]i stimulate PRL secretion. We investigated the effects of U-73122, an aminosteroid inhibitor of PL-C dependent processes, on TRH-stimutated second messenger pathways and on PRL secretion in GH3 rat pituitary cells. [Ca2+]i was monitored by Indo-1 fluorescence, and IP3 and metabolites separated on ion exchange columns. In Ca2+-free buffer, [Ca2+]i was 96 +/- 6 nm and increased to 323 +/- 23 nm (P < 0.001) after TRH (100 nm). U-73122 dose dependently inhibited the TRH effect (IC50 = 967 nm; complete inhibition at 3-5 mum). Subsequent addition of monensin (100 mum) increased [Ca2+]i from 107 +/- 4 to 142 +/- 4 nm (P < 0.001), confirming our previous findings of a non-TRH regulated Ca2+ pool in GH3 cells. Pretreatment (15 sec) with U-73122 partly inhibited the TRH effect on [Ca2+]i; Complete suppression occurred with 70 sec of pretreatment. An inactive analog (U-73343) had no inhibitory effect at 5 mum. U-73122 acted noncompetitively, as the mean maximum velocity (expressed as percent increase in [Ca2+]i after TRH) was reduced from 225 to 91 while the Michaelis-Menten constant for TRH was unchanged (15.4 vs. 13.8 nm, n = 3). Of note, U-73122, at 3-5 muM, increased basal [Ca2+]i from 109 +/- 5 to 120 +/- 5 nm (P < 0.001). In 1.3 mm Ca2+ buffer containing nifedipine (1 mum) and verapamil (50 mum), similar effects of U-73122 (5 mum) were observed on basal and TRH-stimulated [Ca2+]i. IP3, IP2, and IP1 increased to 241 +/- 12%, 148 +/- 23%, and 167 +/- 39% of control, 30 sec after TRH (100 nm); these responses were prevented by 1 mum U-73122. At 5 mum, U-73122 also significantly increased IP3 levels. TRH (100 nM) increased 4-h PRL secretion from 16.3 +/- 1.4 to 27.6 +/- 3.2 ng/well (P < 0.05). U-73122 (5 mum) increased basal PRL secretion to 35.9 +/- 3.2 ng/well (P < 0.05), but abolished the TRH effect. In contrast, U-73343 (with Ca2+ channel blockers) did not inhibit the TRH effect on PRL (control: 24.3 +/- 2.1; TRH: 51.0 +/- 6.3 ng/well). Conclusion: U-73122 is a potent inhibitor of TRH stimulated IP3 production, intracellular Ca2+ mobilization, and PRL secretion, and is a useful tool for studying PL-C mediated processes in GH3 cells.