Effects of the phospholipase-C inhibitor, U73122, on signaling and secretion in pituitary gonadotrophs.

Abstract

The effects of inhibition of phosphoinositide hydrolysis by U73122 [1-(6-[17 beta-3-methoxyestra-1,3,5- (10) triene-17-yl] amino/hexyl) 1H-pyrroledione] and neomycin on agonist-stimulated intracellular signaling and secretory responses were analyzed in cultured pituitary cells and alpha T3-1 gonadotrophs. GnRH (100 nM)- and endothelin-1 (ET-1; 100 nM)-induced inositol (1,4,5)-trisphosphate and diacylglycerol formation in normal cells and immortalized gonadotrophs were reduced by U73122 in a concentration-dependent manner, with IC50 values of about 2 microM and complete inhibition at 10 microM U73122. Neomycin also reduced GnRH- and ET-induced inositol phosphate production in both cell types. Agonist-induced intracellular Ca2+ responses were also inhibited in both cell types by U73122 and neomycin at the same concentrations that inhibited their inositol phosphate responses. In cultured pituitary cells, agonist-induced LH release was inhibited by U73122 and neomycin in a dose-dependent manner. In perifused pituitary cells, U73122 completely inhibited GnRH- and ET-1-induced LH release, but after 10 min caused a progressive and substantial increase in basal LH release. In static cultures, U73122 inhibited agonist-induced LH response at low concentrations (up to 3 microM), but stimulated LH release at higher concentrations due to direct activation of exocytosis by the compound. When added alone, U73122 caused a concentration-dependent increase in LH release with an EC50 of about 7 microM and a maximum response similar that that elicited by GnRH. The stimulatory action of U73122 on LH release was not reduced in the absence of extracellular Ca2+. In contrast to cultured pituitary cells, alpha T3-1 gonadotrophs showed only constitutive exocytosis that was not affected by either neomycin or U73122. These results demonstrate that GnRH and ET(A) receptors are coupled to the phosphoinositide/Ca2+ transduction system in pituitary gonadotrophs, and provide evidence for the dependence of agonist-regulated exocytosis on this signaling pathway. The ability of U73122 to stimulate LH release could reflect an additional action of the compound at late steps in the exocytic pathway.

Regulation of Blastocoele Formation by Intracellular Calcium Release is Mediated through a Phospholipase C-Dependent Pathway in Mice1

“…Although we did not directly measure the intracellular level of PLC or IP 3 after ethanol exposure in this study and cannot be certain that their intracellular levels are increased, the presumed inhibition of PLC by U73122 resulted in both the delay of cavitation and the prevention of intracellular Ca 2 + release after ethanol or LPA exposure. In support of the inhibitory effect of U73122 on PLC activity, Zheng et al [42] demonstrated in agonist-stimulated oT3-1 pituitary gonadotroph cells that diacylglycerol and IP 3 formation, as well as intracellular Ca 2 + release, was significantly reduced with 2 M U73122 and completely inhibited at 10 .M U73122. Lysophosphatidic acid and ethanol both up-regulate phosphoinositide-specific PLC and increase intracellular levels of P 3 [9,43].…”

Pharmacology of Phosphoinositides, Regulators of Multiple Cellular Functions

“…An additional and important effect of U-73122 on secretion has been reported in the concentration range (5-10 µM) that is usually required to fully inhibit PLC activation. In this concentration range, the compound was found to stimulate the secretion of LH in pituitary gonadotrophs [128], and the secretion of prolactin in GH3 cells [129]. This stimulatory effect of U-73122 on secretion did not require the presence of external Ca 2+ and could indicate an additional target, such as a small GTP-binding protein.…”

Exogenous sphingosine 1-phosphate and sphingosine kinase activated by endothelin-1 induced myometrial contraction through differential mechanisms

“…Calcium influx is regulated by membrane hyperpolarization resulting from K ϩ conductance inhibition. A recent study indicated that intracellular S1P suppress K ϩ conductance and then increases calcium influx (60,61). Alternatively, the contribution of SphK activation is not the result of consecutive S1P production but rather because of S1P metabolites obtained via S1P lyase activity as reported for the regulation of mitogenesis in mouse F9 embryonic carcinoma cell lines (18).…”