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-stimulated 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 µM). Subsequent addition of monensin (100 µM) 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 pM. 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 µM, increased basal [Ca2+]i from 109 ± 5 to 120 ± 5 nM (P < 0.001). In 1.3 mM Ca2+ buffer containing nifedipine (1 µM) and verapamil (50 µM), similar effects of U-73122 (5 µM) were observed on basal and TRH-stimulated [Ca2+]i. IP3, IP2, and IP1 increased to 241 ± 127, 148 ± 237, and 167 ± 397 of control, 30 sec after TRH (100 nM); these responses were prevented by 1 µM U-73122. At 5 µM, 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 µM) 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.
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