Estrogen directly acts on osteoclasts via inhibition of inward rectifier K + channels

Abstract

Using the whole-cell patch-clamp technique in freshly isolated rat osteoclasts we examined the effects of estrogen on ionic channels. The predominant current was an inward rectifier K+ current (IKir). In the absence of non-osteoclastic cells, extracellularly applied 17beta-estradiol (>0.1 microM) inhibited IKir, indicating that estrogen acts directly on osteoclasts. Application of 17beta-estradiol (10 microM) for 10 min reduced IKir at the membrane potential of -120 mV to 70 +/- 15% of control. Removal of 17beta-estradiol partially restored the inhibition. The inhibition of IKir was dependent on concentration and application time. Intracellularly applied 17beta-estradiol had no effect on IKir. 17alpha-estradiol also inhibited the IKir, whereas progesterone and testosterone had no effect. The inhibitory action of 17beta-estradiol was not affected by guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS), adenosine 3',5'-cyclic monophosphothioate Rp diastereomer (Rp-cAMPS), okadaic acid, staurosporine and phorbol ester, and was independent of intracellular Ca2+ concentration ([Ca2+]i). With no influence from soluble factors secreted from non-osteoclastic cells, preincubation of the osteoclasts for more than 60 min with much lower concentrations of 17beta-estradiol (1 and 10 nM) caused a reduction of IKir. In current-clamp configuration, application of 17beta-estradiol (10 microM) depolarized the membrane associated with a decrease in a membrane conductance, indicating that 17beta-estradiol inhibits IKir and depolarizes the membrane of osteoclasts. These results suggest that the 17beta-estradiol-induced inhibition of IKir might be mediated via non-genomic mechanisms. This direct action of 17beta-estradiol on osteoclasts may contribute to the regulation of [Ca2+]i and partially account for the protective effects of estrogen against bone loss.

Genistein, a Soybean Isoflavone, Inhibits Inward Rectifier K+ Channels in Rat Osteoclasts.

“…1B), resulting in depolarization of the membrane. We have recently shown that 17␤-estradiol inhibits I Kir and depolarizes the membrane of rat osteoclasts [21]. Therefore genistein appears to cause membrane depolarization in a manner similar to 17␤-estradiol.…”

“…Hyperpolarizing pulses elicited inward current, whereas depolarizing pulses elicited much smaller outward current, typical for inward rectifier K ϩ channels (I Kir ) [21]. Application of genistein (50 M) to the bath for 5 min reduced the amplitude of inward currents evoked by hyperpolarizing pulses, and the currents were restored by the removal of genistein.…”

“…Approximately 50% of osteoclasts studied (nϭ51) exhibited an outward rectifier Cl Ϫ current at positive membrane potentials. In these cells we made whole-cell recordings in the presence of the Cl Ϫ channel blocker DIDS (0.5 mM) to isolate I Kir [21]. DIDS did not change the magnitude of the inhibition by genistein (data not shown).…”

“…Extracellularly applied PMA (10 nM), an activator of protein kinase C (PKC), also had no effect on the inhibition of I Kir by genistein (data not shown). We did not examine the effect of the PKA activator forskolin on the inhibitory action of genistein, since forskolin itself has a weak inhibitory effect on I Kir [21]. The application of these signal modulators did not significantly change the amplitude of I Kir .…”

“…Indeed, mRNA expressing IRK1/Kir2.1 has been detected in rat osteoclasts [20]. We have reported that at physiological concentrations, estrogen acts directly on isolated rat osteoclasts and reduces the activity of inward rectifier K ϩ channels [21]. Therefore in the present study, we applied the whole-cell patch-clamp technique to freshly isolated rat osteoclasts and examined the effects of isoflavones, including genistein on the inward rectifier K ϩ channels and the possible mechanisms underlying their effects.…”