In order to study the dependence of GnRH-stimulated LH release on inositol phosphate (IP) turnover, this study used an inhibitor of phospholipase C activity, 1-[6-[[17 beta-3- methoxyestra-1,3,5(10)-triene-17-yl]amino]hexyl]-1H-pyrrole-dione (U-73122) and an inactive analog 1-[6[[17 beta-3-methoxyestra-1,3,5(10)- triene-17-yl]amino]hexyl]2,5-pyrrolidine-dione (U-73343). U-73122 (10 microM) decreased GnRH-provoked (1 microM, 45 min) IP accumulation from 873 +/- 61 dpm to 365 +/- 50 dpm (basal accumulation also was decreased from 420 +/- 18 dpm to 207 +/- 16 dpm) while LH release was not inhibited (30.2 +/- 1.4% of cellular LH in control compared to 30.3 +/- 1.1% in U-73122 pretreated cells). GnRH provoked increased IP3 accumulation (123% of basal) after 15 sec of stimulation, IP2 accumulation (131% of basal) after 30 sec, and IP1 (121% of basal) after 1 min. Pretreatment with U-73122 blocked accumulation of IPs at these early timepoints. Sodium fluoride (NaF)-stimulated IP accumulation was also inhibited by U-73122 (from 1539 +/- 132 dpm to 414 +/- 21 dpm) while LH release increased from 22.9 +/- 1.4% total cellular LH to 28.0 +/- 2.2%. In contrast, GnRH- and NaF-stimulated IP accumulation were not significantly decreased in U-73343 pretreated cells (GnRH: 817 +/- 43 dpm compared to 873 +/- 61 dpm in control; NaF: 1133 +/- 74 dpm compared to 1539 +/- 132 dpm in control cells). Results of a perifusion study showed that U-73122 did not block the initial phase of GnRH-stimulated LH release or interfere with the development of desensitization to the releasing hormone. In addition, GnRH-stimulated intracellular Ca2+ fluctuations were similar in magnitude and duration in U-73122-pretreated compared to U-73343-pretreated cells. These results demonstrate that GnRH- as well as NaF-stimulated LH release can be uncoupled from IP production calling to question the role of IP3 as a second messenger for GnRH-stimulated LH release.