Differential effects of luteinizing hormone on intracellular free Ca2+ in small and large bovine luteal cells

The effect of LH on the intracellular free Ca2+ concentration ([Ca2+]i) was investigated in highly purified small and large bovine luteal cell populations. Luteal cells were obtained from midcycle corpora lutea dispersed with collagenase and separated by flow cytometry into large and small cells. Resting levels of Ca2+ were higher (P less than 0.05) in the large than small cells [314 +/- 25 nM (n = 5) vs. 186 +/- 13 nM (mean +/- SE; n = 13) for large and small cells, respectively]. LH rapidly increased [Ca2+]i in both small and large cells loaded with the fluorescent Ca2+ probe fura-2. In the small cells, [Ca2+]i was immediately increased 2- to 6-fold (from 176 +/- 8 to 468 +/- 8 nM; n = 5) after adding LH. The LH induced [Ca2+]i rise occurred in two phases: an initial peak due to intracellular Ca2+ mobilization and a secondary rise due to Ca2+ influx from extracellular sources. Preincubation of the small cells with EGTA reduced the initial phase and abolished the secondary rise in [Ca2+]. Both forskolin and 8-bromo-cAMP increased [Ca2+]i in the small cells. In contrast, only a single phase of [Ca2+]i rise was observed in LH-treated large cells, and the response was 1.5- to 2-fold greater than the resting Ca2+ levels [314 +/- 25 vs. 435 +/- 60 nM (n = 4), for resting vs. LH-treated values, respectively]. The addition of both LH and prostaglandin F2 alpha (PGF2 alpha) to the large cells resulted in increases in [Ca2+]i that were greater than those induced by each hormone separately (2.0-fold for LH and 2.7-fold for PGF2 alpha vs. 7- to 9-fold in the presence of both hormones). These findings demonstrate that LH induces rapid increases in intracellular [Ca2+]i that differ in magnitude and profile between the small and large bovine luteal cells. Furthermore, LH and PGF2 alpha interacted to promote increases in [Ca2+]i in the large cells, that were higher than the sum of [Ca2+]i induced by each hormone separately.