Effect of β-estradiol on voltage-gated Ca2+ channels in rat hippocampal neurons: a comparison with dehydroepiandrosterone
Introduction
Estrogen is one of the sex steroids that are mainly produced in the ovaries and is well known to have several important physiological functions in the central nervous system. It has been observed that estrogen increases the dendric spine density in hippocampal neurons Gould et al., 1990, Murphy and Segal, 1996 and these neurotrophic effects are blocked by tamoxifen, an estrogen receptor antagonist, suggesting that estrogen exerts effects by mediating classical genomic pathways (Murphy and Segal, 1996). On the other hand, a number of studies suggest that estrogen also changes the electrical activity within milliseconds to minutes in neurons by mediating non-transcriptional mechanisms (Moss et al., 1997). During neuronal development, estrogen modulates neuronal and sexual differentiation by influencing the organization of neural circuits and neuroendocrine systems Arnold and Gorski, 1984, Arnold and Breedlove, 1985. The estrogen-synthesizing enzyme, aromatase, is expressed in many regions of the brain during neuronal development, including the hippocampus, hypothalamus and cortex. Aromatase activity is significantly greater during development than at any subsequent age Harada and Yamada, 1992, Lephart et al., 1992, MacLusky et al., 1994, suggesting that estrogen plays an important role, especially in the developing brain.
The major precursors of estrogen, dehydroepiandrosterone and its sulfate ester, dehydroepiandrosterone sulfate, are members of neurosteroid families that are expressed in the central nervous system. Levels of dehydroepiandrosterone and dehydroepiandrosterone sulfate decline with age, and the enzymes for their synthesis are more abundant in the developing brain than in adult brain (Compagnone et al., 1995). Recent studies have shown that many neurosteroids modulate the functions of γ-aminobutyric acid type A (GABAA) receptors allosterically Majewska et al., 1986, Puia et al., 1990, Schmid et al., 1998. Several neurosteroids, such as progesterone, pregnenolone and its metabolizes, play an important role in promoting cell growth (Schumacher et al., 1996). In addition, dehydroepiandrosterone and dehydroepiandrosterone sulfate have been reported to provide neuroprotective effects in hippocampal neurons against excitatory amino acid-induced neurotoxicity as well as estrogen Singer et al., 1996, Kimonides et al., 1998, Mao and Barger, 1998. Neurosteroids, including dehydroepiandrosterone and dehydroepiandrosterone sulfate, may be involved in neuronal plasticity through the modulating functions of GABAA receptors in the developing brain.
It is well known that γ-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the adult mammalian central nervous system. In contrast, GABA is reported to act as an excitatory neurotransmitter in the developing brain, including embryonic and postnatal stages Cherubini et al., 1991, Ben-Ari et al., 1994. The excitatory effect of GABA in rat neurons results from its depolarization of the neuron through GABAA receptors Segal, 1993, Ben-Ari et al., 1994, Takebayashi et al., 1996. GABA has not only an inhibitory effect on neuronal transmission, but also neurotrophic effects on developing neurons, such as promoting neurite outgrowth, differentiation and synaptogenesis Spoerri, 1988, Barbin et al., 1993, Ben-Ari et al., 1994. These neurotrophic effects are thought to be mediated by GABAA receptors, because similar effects are obtained using GABAA receptor agonists and are blocked by GABAA receptor antagonists (Barbin et al., 1993). The mechanisms of these neurotrophic effects were due to GABA-induced [Ca2+]i increases induced by GABA, because they are diminished by reducing [Ca2+]i increases with either L-type Ca2+ channel blockers or Ca2+-chelating Berninger et al., 1995, Behar et al., 1996. Furthermore, we reported that GABA depolarizes cultured neurons through GABAA receptors and induce Ca2+ influx through voltage-gated calcium channels (VGCCs), resulting in an increase in [Ca2+]i (Takebayashi et al., 1996).
In this context, we examined whether an acute treatment with estrogen would affect [Ca2+]i induced by GABA, high K+ and N-methyl-d-aspartate acid (NMDA). We also compared effects of estrogen with dehydroepiandrosterone and dehydroepiandrosterone sulfate in order to elucidate the regulatory effect of these steroids on intracellular Ca2+ signaling systems and to examine the possibility of neurotrophic effects by these steroids.
Section snippets
Cell culture
Primary cultures were prepared according to a method we have previously described (Takebayashi et al., 1996). Wister rats were removed from the mother at embryonic day 19 under ether anesthesia. Hippocampal tissue was dissected out and incubated in 0.25% trypsin and 0.02% deoxyribonuclease I for 25 min at 37°C on a shaker, followed by inactivation of the enzymes with fetal bovine serum. The hippocampal cells were mechanically dispersed by pipetting, and rinsed twice with culture medium,
The effect of β-estradiol on the GABA-induced increases in [Ca2+]i in primary cultured rat hippocampal neurons
The application of GABA for 30 s induced a concentration-dependent increase in [Ca2+]i in primary cultures of rat hippocampal neurons. The mean increase in [Ca2+]i induced by 50 μM GABA was 70.6±5.5 nM, as shown in Fig. 1A. In all of our current experiments, we routinely used a response to 50 μM GABA. More than 80% of the monitored cells were responsive. The response was fully recovered in 15–20 min after the previous application with GABA. Therefore, 25 min after measuring the first response
Discussion
We have demonstrated that β-estradiol as well as its precursors, dehydroepiandrosterone and dehydroepiandrosterone sulfate, inhibited the GABA-induced [Ca2+]i increases in primary hippocampal cultures. While β-estradiol inhibited the high K+-induced [Ca2+]i increases, both dehydroepiandrosterone and dehydroepiandrosterone sulfate inhibited to a lesser extent. β-Estradiol also inhibited the NMDA-induced [Ca2+]i increases.
Acute treatment with β-estradiol inhibited the GABA-induced [Ca2+]i
Acknowledgements
The authors are grateful to Dr. M. Inagaki for excellent technical advice on this work. This work was supported by a Grant-in-Aid for Nervous and Mental Disorders from the Ministry of Health and Welfare of Japan and Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Corporation (JST).
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