Study or the effects of estrogen on voltage-dependent ionic channels in osteoclast cells

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Basic experiments : Osteoclasts were obtained from the leg long bones of neonatal Wistar rats (1-2 day old). The tibiae and femora were removed and then placed in culture medium (minimum essential medium alpha medium ; Gibco BRL), and finely chopped. The suspension was gently agitated with a wide bore pipette and osteoclasts were dissociated from the bone surface. This suspension was seeded on glass coverslips. A cell with three or more nuclei was used for the whole-cell recordings. These cells were determined to be osteoclasts because such cells stained positive for tartrate-resistant acid phosphatase (TRAP) and formed resorption pits when the cells were cultured on dentine slices for 24 hr. In the condition using modified Tyrode solution as an intracellular solution, and high-potassium solution as a pipette solution, inwardly rectifer type potassium current could be recorded in osteoclasts by whole-cell voltage clamp. l7 β-Estradiol at the concentration above 1μM inhibited this inwardly rectifer type potassium current in dose-dependent manner. This result suggests that estradiol inhibits bone absorption by osteoclasts by the inhibition of inwardly reclifer type potassium current in osteoclast cells.Clinical research : In oophorectomized women, hormone replacement therapy using conjugated equine estrogen (CEE ; 0.625mg/day) was done, and the bone mineral density was calculated by means of dual energy X-ray absorptiometry in these women. We found that in the initial 2-year period after oophorectomy, 0.625 mg/day of CEE alone could not prevent acute bone loss suggesting that additional therapy for the prevention of osteoporosis may be needed. Furthermore we found that concomitant use of ipriflavone with CEE from an early stage after oophorectomy inhibited bone loss and was considered to be effective in maintaining bone mass after oophorectomy.