Rapid action of estrogens on intracellular calcium oscillations in primate luteinizing hormone-releasing hormone-1 neurons

Abstract

Feedback controls of estrogen in LHRH-1 neurons play a pivotal role in reproductive function. However, the mechanism of estrogen action in LHRH-1 neurons is still unclear. In the present study, the effect of estrogens on intracellular calcium ([Ca(2+)](i)) oscillations in primate LHRH-1 neurons was examined. Application of 17beta-estradiol (E(2), 1 nm) for 10 min increased the frequency of [Ca(2+)](i) oscillations within a few minutes. E(2) also increased the frequency of [Ca(2+)](i) synchronization among LHRH-1 neurons. Similar E(2) effects on the frequency of [Ca(2+)](i) oscillations were observed under the presence of tetrodotoxin, indicating that estrogen appears to cause direct action on LHRH-1 neurons. Moreover, application of a nuclear membrane-impermeable estrogen dendrimer conjugate, not control dendrimer, resulted in a robust increase in the frequencies of [Ca(2+)](i) oscillations and synchronizations, indicating that effects estrogens on [Ca(2+)](i) oscillations and their synchronizations do not require their entry into the cell nucleus. Exposure of cells to E(2) in the presence of the estrogen receptor antagonist ICI 182,780 did not change the E(2)-induced increase in the frequency of [Ca(2+)](i) oscillations or the E(2)-induced increase in the synchronization frequency. Collectively, estrogens induce rapid, direct stimulatory actions through receptors located in the cell membrane/cytoplasm of primate LHRH-1 neurons, and this action of estrogens is mediated by an ICI 182,780-insensitive mechanism yet to be identified.

Figure 1

Effects of E₂ (A) or vehicle (B) on [Ca²⁺]_i oscillations and synchronization of primate LHRH-1 neurons. Twelve of 41 LHRH-1 neurons from a culture treated with E₂ and 12 of 50 LHRH-1 neurons from a culture treated with vehicle are shown. After 20 min of control recording, cells were exposed to E₂ (1 nm, stippled bar on the top) or vehicle for 10 min (white bar). Changes in [Ca²⁺]_i levels are shown as ratio values of fura-2 dye (nanomolar). A shaded bar over the traces indicates synchronization of [Ca²⁺]_i peaks among LHRH-1 neurons based on the criteria described in Materials and Methods. Note that E₂ induced an increase in the frequency of [Ca²⁺]_i peaks in LHRH-1 neurons as well as frequent synchronization among LHRH-1 neurons.

Figure 2

Changes in the frequency of [Ca²⁺]_i oscillations (A) and the percentage (B) of stimulated LHRH-1 neurons induced by E₂ or vehicle treatment. For changes in the frequency of [Ca²⁺]_i oscillations, normalized data are shown. Note that E₂ stimulated the frequency of [Ca²⁺]_i oscillations and the percentage of excited cells. The n for E₂ and vehicle are 296 and 275, respectively. *, P < 0.05 vs.−20 to 0; #, P < 0.05; ###, P < 0.001 vs. vehicle at the corresponding time.

Figure 3

A representative example of TTX plus E₂ (A) or TTX (B) treatment on [Ca²⁺]_i oscillations. A, After 20 min of control recording, cells were exposed to E₂ (1 nm, stippled bar on the top) for 10 min. Cells were also exposed to TTX (1 μm) 2 min before E₂ through 2 min after E₂. Shaded bars over the traces indicate synchronization of [Ca²⁺]_i peaks among LHRH-1 neurons. B, Note that the E₂-induced increase in the frequency of [Ca²⁺]_i oscillations and synchronizations was observed in the presence of TTX, whereas TTX alone (white bar on the top) did not induce any significant effects. Nine of 26 LHRH-1 neurons from a culture treated with TTX plus E₂ and nine of 50 LHRH-1 neurons from a culture treated with TTX alone are shown.

Figure 4

Changes in the frequency of [Ca²⁺]_i oscillations (A) and the percentage (B) of stimulated LHRH-1 neurons induced by E₂ in the presence of TTX. For changes in the frequency of [Ca²⁺]_i oscillations, normalized data are shown. Note that TTX did not block the E₂-induced increase in the frequency of [Ca²⁺]_i oscillations or the percentage of excited cells. The n for TTX plus E₂ and TTX are 149 and 194, respectively. *, P < 0.05 vs.−20 to 0; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 vs. TTX alone at the corresponding time.

Figure 5

Effects of EDC (A) or DC (B) on [Ca²⁺]_i oscillations and synchronizations of primate LHRH-1 neurons. After a 20-min control recording, cells were exposed to EDC (1 μm, stippled bar on the top) or DC (white bar) for 10 min. DC did not induce any changes in [Ca²⁺]_i oscillations. Note that EDC induced an increase in the frequency of [Ca²⁺]_i peaks in LHRH-1 neurons as well as frequent synchronization among LHRH-1 neurons (shaded bars over the traces). Thirteen of 27 and 10 of 35 LHRH-1 neurons from cultures treated with EDC and DC, respectively, are shown.

Figure 6

Changes in the frequency of [Ca²⁺]_i oscillations (A) and the percentage (B) of stimulated LHRH-1 neurons induced by EDC (1 nm or 1 μm) or DC (1 μm) treatment. Note that EDC at both doses stimulated the frequency of [Ca²⁺]_i oscillations and the percentage of excited cells. EDC at 1 μm resulted in a more prolonged effect than that of EDC at 1 nm. The n for EDC at 1 nm, EDC at 1 μm, and DC are 195, 149, and 182, respectively. *, P < 0.05; **, P < 0.01 vs.−20 to 0; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 vs. DC at the corresponding time.

Figure 7

Effects of ICI (100 nm) on the E₂-induced changes in [Ca²⁺]_i oscillations. A, Cells were treated with ICI for the entire period of the experiment as indicated by the white bar on the top, and E₂ was applied for 10 min starting 20 min after the initiation of ICI treatment (stippled bar on the top). E₂ induced increases in the frequency of [Ca²⁺]_i oscillations and synchronizations in the presence of ICI. B, ICI alone did not cause any significant changes in [Ca²⁺]_i oscillations and synchronization. Thirteen of 30 and eight of 32 LHRH-1 neurons from cultures treated with ICI plus E₂ and ICI alone, respectively, are shown.

Figure 8

Changes in the frequency of [Ca²⁺]_i oscillations (A) and the percentage (B) of stimulated LHRH-1 neurons induced by E₂ in the presence of ICI. Note that ICI did not block the E₂-induced increase in the frequency of [Ca²⁺]_i oscillations or the percentage of excited cells. The n for ICI plus E₂ and ICI are 180 and 186, respectively. *, P < 0.05 vs.−20 to 0; #, P < 0.05; ###, P < 0.001 vs. ICI alone at the corresponding time.