Modulation by estrogens and xenoestrogens of recombinant human neuronal nicotinic receptors

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Abstract

The effects of estrogens and xenoestrogens on human neuronal nicotinic acetylcholine receptor/channels were examined by expressing recombinant channels in Xenopus oocytes. When functional channels were expressed with α3 and β4 subunits, estrogens (17β-estradiol, 17α-estradiol, 17α-ethynylestradiol and diethylstilbestrol) and xenoestrogens (bisphenol A, p-nonylphenol and p-octylphenol) inhibited an ionic current activated by acetylcholine at concentrations up to 100 μM. When the subunit combination was changed to α4β2, diethystilbestrol and the xenoestrogens inhibited the acetylcholine-activated current, but 17β-estradiol or 17α-estradiol did not. For 17α-ethynylestradiol, the current through the α4β2 receptor/channel was inhibited at 1 μM, but it was markedly enhanced at 10 and 100 μM. Tamoxifen (10 μM), an antiestrogen, itself inhibited the acetylcholine-activated current but did not antagonize the current modulations induced by the estrogens and the xenoestrogens. These and additional results suggest that human neuronal nicotinic acetylcholine receptors are the targets of non-genomic actions of estrogens and xenoestrogens.

Introduction

The hormone 17β-estradiol regulates gene transcription through intracellular estrogen receptors. A number of environmental agents are known or suspected to act on estrogen receptors. These compounds (except for those structurally related to 17β-estradiol) are called xenoestrogens, and scientists have warned of their influence on humans and animals Corbone et al., 1993, Korach, 1993, Stone, 1994. In addition to a genomic action through intracellular estrogen receptors, 17β-estradiol has been shown to exhibit relatively rapid non-genomic actions which may be mediated through receptors or other functional proteins in the cell membrane (see reviews, Moss et al., 1997, Kelly and Lagrange, 1998, Kelly and Wagner, 1999). It has scarcely been clarified whether or not xenoestrogens produce similar non-genomic actions or not, though Nadal et al. (2000) recently suggested that catecholamine receptors may serve as non-genomic receptors for both estrogens and xenoestrogens.

Nicotinic acetylcholine receptors are channel-forming receptors which promote excitatory cellular functions in postsynaptic cells, including spike generation in neurons, the contraction of skeletal muscle and catecholamine secretion from the adrenal medulla, by allowing extracellular cations to enter cells. It has been reported that nicotinic acetylcholine receptors are the targets of the non-genomic actions of 17β-estradiol. Ke and Lukas (1996) showed that 17β-estradiol as well as other steroids such as progesterone inhibited 86Rb+ efflux induced by carbamylcholine in both human neuronal and muscle cell lines expressing nicotinic acetylcholine receptors. Uki et al. (1999) found that steroids including 17β-estradiol and 17α-estradiol suppressed an ionic current activated by acetylcholine in rat superior cervical ganglionic neurons.

We previously showed that Xenopus oocytes are suitable for pharmacological studies of cloned human neuronal nicotinic acetylcholine receptors (Nakazawa and Ohno, 1999). In the present study, we used this Xenopus oocyte expression system to examine the effects of estrogens and xenoestrogens on the cloned human neuronal nicotinic acetylcholine receptor.

Section snippets

Methods

Cloned human neuronal nicotinic receptors Kuryatov et al., 1997, Olale et al., 1997 were expressed in Xenopus oocytes as described previously (Nakazawa and Ohno, 1999). Defolliculated Xenopus oocytes were injected with in vitro transcribed cRNAs encoding α and β subunits, and incubated for 2 to 6 days at 18 °C. Membrane current was measured by the conventional two-microelectrode voltage-clamp technique under the conditions described before (Nakazawa et al., 1994a). Oocytes were bathed in an

Modulation by estrogens and xenoestrogens of acetylcholine-activated current through α3β4 and α4β2 receptor/channels

Fig. 2 illustrates examples of recordings of acetylcholine-activated currents in the absence and the presence of estrogens. Among the estrogens tested, diethylstilbestrol was the most potent in inhibiting the acetylcholine-activated current through both α3β4 and α4β2 receptor/channels Fig. 2, Fig. 3. The current was almost abolished at 100 μM, and IC50 values were about 100 nM for α3β4 receptor/channels and about 300 nM for α4β2 receptor/channels. 17β-Estradiol and 17α-estradiol partially

Discussion

We have demonstrated that estrogens and xenoestrogens affect human recombinant neuronal nicotinic acetylcholine receptor/channels. Most of the modulation by these compounds was inhibitory, and diethylstilbestrol exhibited the highest potency in inhibiting both α3β4 and α4β2 receptor/channels. The modulation by estrogens or related compounds of nicotinic acetylcholine receptors has scarcely been reported, except for that elicited by 17β-estradiol. Ke and Lukas (1996) reported that 17β-estradiol

Acknowledgements

This work was supported by a Health Science Research Grant for Research on Environmental Health from the Ministry of Health, Labour and Welfare, Japan. We thank H. Ojima for helping with some of the current recordings.

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