[Testosterone could induce a rapid rise in intracellular free Ca2+ concentration through binding to the membrane surface of bone marrow-derived macrophages].
OBJECTIVE
To investigate the ways testosterone influences the murine bone marrow-derived macrophages (BMMs) and how testosterone affects the function of BMMs after bound to their membrane surface.


METHODS
BMMs were cultured in vitro, their total RNA and proteins isolated, and the expression of intracellular androgen receptor (AR) detected through RT-PCR and Western blotting. The binding site of testosterone (T) to the membrane surface of BMMs was observed by confocal laser scanning microscopy after T-BSA-FITC incubation. Moreover, the intracellular Ca2+ was tested by Fura-2 method, and the influence of ionic currents on BMMs plasma membrane induced by testosterone was examined by the whole cell patch-clamp.


RESULTS
RT-PCR and Western blotting failed to detect intracellular ARs in BMMs, but confocal laser scanning microscopy showed testosterone to be bound to the membrane surface of BMMs by impermeable T-BSA-FITC, inducing a rapid rise in the intracellular free Ca2+ concentration ([Ca2+]i) of Fura-2 loaded BMMs, predominantly due to the influx of extracellular Ca2+ through Ni2+ -blockable Ca2+ channels in the plasma membrane. Similarly, the patch-clamp technique revealed T-induced calcium influx in BMMs.


CONCLUSIONS
It is reasonable to assume that the testosterone receptor exists on the plasma membranes, and testosterone act through unconventional plasma membrane receptors, induce Ca2+ influx and a rapid rise in the intracellular Ca2+ concentration, and influence the function of BMMs.
Chen
Xin-lai
XL
Laboratory of Biochemistry and Molecular Biology, School of Medicine, Shanghai Jiaotong University, Shanghai 200240, China.
Liu
Li-min
LM
Wang
Ya-jing
YJ
Zhang
Zhi-ming
ZM
Wang
Zhao-xia
ZX
Xu
Wen-xie
WX
Qiao
Zhong-dong
ZD
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D005260:Female; D008264:Macrophages / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D008564:Membrane Potentials; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D011485:Protein Binding; D011944:Receptors, Androgen / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013739:Testosterone / Q000378:metabolism*
D015220:Calcium Channels; D011944:Receptors, Androgen; D013739:Testosterone; D002118:Calcium