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Activation of Maxi Cl(-) channels by antiestrogens and phenothiazines in NIH3T3 fibroblasts
Miguel A Valverde1, Simon P Hardy, Mario Díaz
1Unitat de Senyalització Cel-lular, Dept. de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, 08003 Barcelona, Spain. miguel.valverde@cexs.upf.es
Abstract:
The identification of alternative estrogen actions has been accumulating steadily over the past two decades. Typically, these novel actions are not directly related to nuclear transcriptional events but related to the interaction of estrogens with sites present at plasma membrane or cytosolic locations. These alternative effects, widely known as non-genomic effects, range from the modulation of plasma membrane ion channel activity to the regulation of different intracellular signalling cascades. In the present study we have investigated the modulation of a large conductance chloride channel (Maxi Cl(-)) by estrogens, non-steroidal triphenylethylene antiestrogens and phenothiazines in NIH3T3 fibroblasts and the dependence on guanosine triphosphate (GTP) of the Maxi Cl(-) activation. Our data identifies the non-steroidal antiestrogens toremifene and tamoxifen, and the phenothiazines chlorpromazine and triflupromazine as activators of Maxi Cl(-) channels. In contrast, 17 beta-estradiol and cAMP, added prior to the exposure to antiestrogens, prevent channel activation. The pure antiestrogen ICI 182780 did not activate the channel nor prevent its activation by non-steroidal antiestrogens. The activation of Maxi Cl(-) channels by toremifene and tamoxifen required the presence of intracellular nucleotides and was inhibited by the stable analog, GDP beta -S, suggesting the participation of a G-protein in the activation process. Little is known about the physiological relevance of Maxi Cl(-) channels. However, that fact that its regulation by estrogens and antiestrogens is shared by different cell types might imply a common role which needs to be identified.
Insights
Non-steroidal antiestrogens like tamoxifen activate Maxi chloride channels via G-protein signaling. Estrogen and cAMP can block this activation, suggesting complex non-genomic estrogen effects.
Area of Science:
- Molecular Pharmacology
- Cell Biology
- Signal Transduction
Background:
- Estrogen exhibits non-genomic effects beyond nuclear transcriptional regulation.
- These non-genomic actions involve plasma membrane and cytosolic interactions, modulating ion channels and signaling cascades.
- The specific role of large conductance chloride channels (Maxi Cl(-)) in these pathways remains largely uncharacterized.
Purpose of the Study:
- To investigate the modulation of Maxi Cl(-) channels by estrogens and related compounds.
- To explore the dependence of Maxi Cl(-) channel activation on guanosine triphosphate (GTP).
- To elucidate the signaling pathways involved in estrogen and antiestrogen-mediated channel regulation.
Main Methods:
- Utilized NIH3T3 fibroblasts to study Maxi Cl(-) channel activity.
- Tested the effects of 17 beta-estradiol, non-steroidal antiestrogens (toremifene, tamoxifen, ICI 182780), phenothiazines (chlorpromazine, triflupromazine), and cAMP.
- Investigated the requirement for intracellular nucleotides and G-protein involvement using GDP beta-S.
Main Results:
- Non-steroidal antiestrogens (toremifene, tamoxifen) and phenothiazines (chlorpromazine, triflupromazine) activated Maxi Cl(-) channels.
- 17 beta-estradiol and cAMP inhibited antiestrogen-induced channel activation.
- ICI 182780 did not activate or inhibit the channel.
- Activation by toremifene and tamoxifen was nucleotide-dependent and inhibited by GDP beta-S, indicating G-protein involvement.
Conclusions:
- Non-steroidal antiestrogens and certain phenothiazines directly activate Maxi Cl(-) channels.
- G-proteins mediate the activation of Maxi Cl(-) channels by non-steroidal antiestrogens.
- Estrogen and cAMP exert inhibitory effects on this activation pathway, highlighting complex non-genomic signaling.