Effects of the phospholipase-C inhibitor, U73122, on signaling and secretion in pituitary gonadotrophs.
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.
Zheng
L
L
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Paik
W Y
WY
Cesnjaj
M
M
Balla
T
T
Tomić
M
M
Catt
K J
KJ
Stojilkovic
S S
SS
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D004075:Diglycerides / Q000378:metabolism; D004963:Estrenes / Q000494:pharmacology*; D005260:Female; D006062:Gonadotropins / Q000378:metabolism*; D007295:Inositol Phosphates / Q000378:metabolism; D007986:Luteinizing Hormone / Q000378:metabolism; D010903:Pituitary Gland, Anterior / Q000187:drug effects* / Q000378:metabolism* / Q000502:physiology; D011760:Pyrrolidinones / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors*
D004075:Diglycerides; D004963:Estrenes; D006062:Gonadotropins; D007295:Inositol Phosphates; D011760:Pyrrolidinones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D007986:Luteinizing Hormone; D010738:Type C Phospholipases; D002118:Calcium