Ca+2 dependence of gonadotropin-releasing hormone-stimulated luteinizing hormone secretion: in vitro studies using continuously perifused dispersed rat anterior pituitary cells.

A continuously perifused dispersed rat anterior pituitary cell system was used to determine the importance of calcium (Ca+2) on the release of LH by GnRH. In response to continuous exposure to 10 nM GnRH, LH was released in a biphasic fashion; arbitrarily, phase I was defined as being the LH released during the initial 40 min and phase II as the subsequent release. Withdrawal of Ca+2 from the perifusion medium during phases I or II of LH release led to a rapid inhibition of the LH secretion. Cells were exposed to GnRH for 2.5 min, washed with medium for 30 min, and then reexposed to GnRH for 30 min. This sequence was repeated 1 h later under identical conditions in the presence of a Ca+2 blocking agent; D600 (20 or 100 microM). D600 inhibited both the 2.5- and the 30-min GnRH-stimulated LH release. The results were expressed as the ratio obtained by dividing the total LH released during the second GnRH exposure (either 2.5 or 30 min) by the total LH released during the respective initial GnRH exposure of same duration. For the cells perifused with 20 microM D600 the ratios +/- SE (D600 vs. control) were 0.48 +/- 0.06 vs. 1.28 +/- 0.13 (P = 0.0001) and 0.29 +/- 0.05 vs. 1.01 +/- 0.08 (P less than or equal to 0.0001) for the 2.5- and 30-min exposures, respectively. For the cells perifused with 100 microM D600 the ratios +/- SE (D600 vs. control) were 0.18 +/- 0.05 vs. 1.28 +/- 0.13 (P less than or equal to 0.00001) and 0.12 +/- 0.03 vs. 1.01 +/- 0.08 (P = 0.002) for the 2.5- and 30-min exposures, respectively, revealing an even more profound inhibitory effect of D600 on GnRH stimulated LH secretion. Our data both confirm previous reports that Ca+2 is involved in LH release and demonstrate that Ca+2 is an essential requirement during both phases of GnRH-stimulated LH release in perifused dispersed rat anterior pituitary cells.