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-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⁺], 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 μM. 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 ± 12%, 148 ± 23%, and 167 ± 39% 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.