Chloride channel activity is essential for osteoclast function. Consequently, inhibition of the osteoclastic chloride channel should prevent bone resorption. Accordingly, we tested a chloride channel inhibitor on bone turnover and found that it inhibits bone resorption without affecting bone formation. This study indicates that chloride channel inhibitors are highly promising for treatment of osteoporosis. Introduction:The chloride channel inhibitor, NS3736, blocked osteoclastic acidification and resorption in vitro with an IC 50 value of 30 M. When tested in the rat ovariectomy model for osteoporosis, daily treatment with 30 mg/kg orally protected bone strength and BMD by ϳ50% 6 weeks after surgery. Most interestingly, bone formation assessed by osteocalcin, mineral apposition rate, and mineralized surface index was not inhibited. Materials and Methods: Analysis of chloride channels in human osteoclasts revealed that ClC-7 and CLIC1 were highly expressed. Furthermore, by electrophysiology, we detected a volume-activated anion channel on human osteoclasts. Screening 50 different human tissues showed a broad expression for CLIC1 and a restricted immunoreactivity for ClC-7, appearing mainly in osteoclasts, ovaries, appendix, and Purkinje cells. This highly selective distribution predicts that inhibition of ClC-7 should specifically target osteoclasts in vivo. We suggest that NS3736 is inhibiting ClC-7, leading to a bone-specific effect in vivo. Results and Conclusion:In conclusion, we show for the first time that chloride channel inhibitors can be used for prevention of ovariectomy-induced bone loss without impeding bone formation. We speculate that the coupling of bone resorption to bone formation is linked to the acidification of the resorption lacunae, thereby enabling compounds that directly interfere with this process to be able to positive uncouple this process resulting in a net bone gain.

Planar silicon chips with 1-2-microm etched holes (average resistance: 2.04 +/- 0.02 MOmega in physiological buffer, n = 274) have been developed for patch-clamp recordings of whole-cell currents from cells in suspension. An automated 16-channel parallel screening system, QPatch 16, has been developed using this technology. A single-channel prototype of the QPatch system was used for validation of the patch-clamp chip technology. We present here data on the quality of patch-clamp recordings and from actual drug screening studies of human potassium channels expressed in cultured cell lines. Using Chinese hamster ovary (CHO) and human embryonic kidney cells (HEK), gigaseals of 4.1 +/- 0.4 GOmega (n = 146) and high-quality whole-cell current recordings were obtained from hERG and KCNQ4 potassium channels. Success rates for gigaseal recordings varied from 40 to 95%, and 67% of the whole-cell configurations lasted for >20 min. Cells were maintained in suspension up to 4 h in a cell storage facility that is integrated in the QPatch 16. No decline in patchability was observed during this time course. A series of screens was conducted with known inhibitors of the hERG and KCNQ4 potassium channels. Dose-response relationship characterizations of verapamil and rBeKm-1 blockage of hERG currents provided IC(50) values similar to values reported in the literature.

The endogenous volume-regulated anion channel (VRAC) from HEK293 cells was pharmacologically characterized using the whole-cell patch-clamp technique. Under isotonic conditions a small (1.3 nS), Ca(2+)-independent Cl conductance was measured. However, swelling at 75% tonicity activated a VRAC identified as an outward-rectifying anion current ( P(l) > P(Cl) > P(gluconate)), which was ATP-dependent and showed inactivation at positive potentials. Activation of this current followed a sigmoid time course, reaching a plateau conductance of 42.6 nS after 12-15 min ( t(1/2) = 7 min). The pharmacology of this VRAC was investigated using standard Cl(-)-channel blockers (NPPB, DIDS, and tamoxifen) as well as a new group (acidic di-aryl ureas) of Cl(-)-channel blockers (NS1652, NS3623, NS3749, and NS3728). The acidic di-aryl ureas were originally synthezised for inhibition of the human erythrocyte Cl(-) conductance in vivo. NS3728 was the most potent VRAC blocker in this series ( IC(50) = 0.40 micro M) and even more potent than tamoxifen (2.2 micro M). NS3728 accelerated channel inactivation at positive potentials. These results show that acidic di-aryl ureas constitute a promising starting point for the synthesis of potent inhibitors of VRAC.

The effect of external ATP on intracellular pH (pHi) was investigated using a pH imaging system in a human bronchial epithelial cell line (16HBE14o‐) loaded with BCECF‐AM. The steady‐state pHi of 16HBE14o‐ epithelial monolayers was 7.137 ± 0.027 (n= 46). Apical addition of ATP (10−4m) to epithelial monolayers induced a rapid and sustained pHi decrease of 0.164 ± 0.024 pH units (n= 17; P < 0.001). The intracellular acidification was rapidly reversed upon removal of external ATP. In contrast, the non‐hydrolysable ATP analogue AMP‐PNP did not produce any significant change in pHi. Inhibition of purinoreceptors by suramin did not affect the acidification induced by apical ATP. Inhibition of Na+‐H+ exchange by apical Na+ removal or addition of amiloride (0.5 mm) reduced the apical ATP‐induced pHi decrease, suggesting the involvement of a Na+‐H+ exchanger or surface pH effects on the ATP‐induced pHi response. Inhibitors of proton channels such as ZnCl2 (10−4m) also partially inhibited the ATP response. The pHi response to ATP was dependent on the external pH (pHo), with increasing acidification produced at lower pHo values. Neither the basal pHi nor the ATP‐induced intracellular acidification was affected by thapsigargin (a Ca2+‐ATPase inhibitor), chelerythrine chloride (a protein kinase C (PKC) inhibitor), RpcAMP (a protein kinase A (PKA) inhibitor) or PMA (a PKC activator). Therefore, the intracellular acidification of human bronchial epithelial cells induced by apical ATP does not involve signalling via Ca2+, PKC or PKA nor binding to a purinoreceptor. We interpret the effect of ATP to produce an intracellular acidification as a three step process: activation of H+ channels, inhibition of Na+‐H+ exchange and influx of protonated ATP.