Gonadotropin-releasing hormone-induced Ca2+ transients in single identified gonadotropes require both intracellular Ca2+ mobilization and Ca2+ influx.

Abstract

We examIned the effects of gonadotropinreleasing hormone (GnRH) (10). We now report the use of these techniques to show that GnRH produces Ca2" transients in identified gonadotropes with a very characteristic and complex time course. The role of these transients in the control of gonadotropin release is discussed. MATERIALS AND METHODSCell Culture. Female 35-day-old Sprague-Dawley rats were decapitated, their pituitaries were removed, and adenohypophyses were dissected from neurohypophyses. Anterior pituitaries were then enzymatically dispersed for 20 min with 0.25% trypsin (bovine, type XII-S, Sigma) in a metabolic shaker at 370C. Cells were then filtered through organza cloth (Tetko, Elmsford, NY), diluted to 50 ml in medium 199 with Hanks' salts and L-glutamine (GIBCO), and centrifuged at 350 x g for 10 min. The supernatant was removed, and cells were resuspended by trituration through a Pasteur pipette in 5 ml ofmedium and then were plated on sterile, poly(L-lysine)-coated (10 ,ug/ml, Sigma) etched-grid coverslips (Bellco). The cell density was z'2.5 x 105 cells per coverslip. The cell yield was z4 x 105 cells per rat. Cells were incubated for 2 days in 60-mm Falcon culture dishes, with medium 199 containing 10% (vol/vol) heat-inactivated horse serum (GIBCO) and 2.5% (vol/vol) fetal bovine serum (GIBCO) and supplemented with penicillin (5 units/ml) and streptomycin (50 pg/ml), in a 5% C02/95% air atmosphere in a water-jacketed incubator at 370C.Identification of Isolated Gonadotropes. Sheep erythrocytes (Colorado Serum, Denver) were washed four times in 0.9o NaCl and conjugated to protein A (0.5 mg/ml, Sigma) with CrCl2 (0.2 mg/ml). After further washing, cells were then mixed with LH antiserum (1:5), guinea pig complement (1:10, GIBCO), and GnRH (100 nM, Peninsula Laboratories, San Carlos, CA).

Estradiol Modulation of GNRH-Stimulated LH Release in Rat Anterior Pituitary Cells: Involvement of Dihydropyridine-Sensitive Calcium Channels

“…Some authors, however, do not agree [ 18]. Recently our group has confirmed the implica tion of PKC in the action of GnRH, but this effect was related to treatment of the cells by E2 [19,20], It has also been demonstrated that binding of GnRH to its receptor results in an increase in intracellular calcium ([Ca2 + ]i), which initiates events such as hormone secretion [21,22], This has been confirmed more recently by several studies using fluorescence analysis on gonadotroph populations [23][24][25] or in single gonadotroph cells [26][27][28][29]. Also, GnRH-induced [Ca2 + ]i increase was greater when cells were treated by E2 and this increase was affected by extra cellular calcium [30], [Ca2 + ]i increase in gonadotroph cells occurs through two major mechanisms: (i) mobilization of Ca2+ from in tracellular bound stores [10,31] and (ii) the influx of cal cium [32] across the plasma membrane via the voltagesensitive calcium channels (VSCC).…”

Anterior pituitary thyrotropes are multifunctional cells

“…] c and hormone release (15,20,22,29). To quantify the responses to the HRHs, we measured the changes of [Ca 2ϩ ] c induced in the different cell types.…”

Dependence of hormone secretion on activation-inactivation kinetics of voltage-sensitive Ca2+ channels in pituitary gonadotrophs.

“…Thus, Ca2+ channels could be activated by protein kinases and inactivated by phosphatases (19,22). GnRH receptors are coupled to the phospholipase C pathway and activation of protein kinase C, which could promote phosphorylation of L-type channels as suggested by the ability of phorbol esters to stimulate LH release and modulate Ca2' entry in gonadotrophs (23,24).…”