We have investigated the effect of extracellular proteases on the amiloride-sensitive Na � current (I Na ) in Xenopus oocytes expressing the three subunits �, �, and � of the rat or Xenopus epithelial Na � channel (ENaC). Low concentrations of trypsin (2 �g/ml) induced a large increase of I Na within a few minutes, an effect that was fully prevented by soybean trypsin inhibitor, but not by amiloride. A similar effect was observed with chymotrypsin, but not with kallikrein. The trypsin-induced increase of I Na was observed with Xenopus and rat ENaC, and was very large (�20-fold) with the channel obtained by coexpression of the � subunit of Xenopus ENaC with the � and � subunits of rat ENaC. The effect of trypsin was selective for ENaC, as shown by the absence of effect on the current due to expression of the K � channel ROMK2. The effect of trypsin was not prevented by intracellular injection of EGTA nor by pretreatment with GTP-�S, suggesting that this effect was not mediated by G proteins. Measurement of the channel protein expression at the oocyte surface by antibody binding to a FLAG epitope showed that the effect of trypsin was not accompanied by an increase in the channel protein density, indicating that proteolysis modified the activity of the channel present at the oocyte surface rather than the cell surface expression. At the single channel level, in the cell-attached mode, more active channels were observed in the patch when trypsin was present in the pipette, while no change in channel activity could be detected when trypsin was added to the bath solution around the patch pipette. We conclude that extracellular proteases are able to increase the open probability of the epithelial sodium channel by an effect that does not occur through activation of a G protein-coupled receptor, but rather through proteolysis of a protein that is either a constitutive part of the channel itself or closely associated with it. The passage of sodium through the epithelial Na � (1983) observed that trypsin, at a concentration of 1 channel (ENaC) 1 is the rate limiting step in the sodium mg/ml, induced an irreversible inhibition of the sodium transport and this effect could be prevented by reabsorption by the epithelial cells of the distal nephamiloride. They concluded that a component of the ron and colon and in airways (Garty and Palmer, 1997).Na � channel protein could be cleaved by a protease at It thereby plays a key role in the regulation of the sodium balance, extracellular fluid volume, and blood a site protectable by amiloride bound to its receptor.pressure by the kidney, and in the controlled fluid reLewis and Alles (1986) and Lewis and Clausen (1991) studied the effects of proteases such as kallikrein, absorption in the airways. The activity of ENaC has to which is normally present in mammalian urine, on the be tightly regulated with regard to the whole organism sodium balance, but also with regards to the epithelial Na � channel of the rabbit urinary bladder. To briefly cell transport capacity so that th...

GLUT9 (SLC2A9) is a newly described urate transporter whose function, characteristics, and localization have just started to be elucidated. Some transport properties of human GLUT9 have been studied in the Xenopus laevis oocyte expression system, but the type of transport (uniport, coupled transport system, stoichiometry … .) is still largely unknown. We used the same experimental system to characterize in more detail the transport properties of mouse GLUT9, its sensitivity to several uricosuric drugs, and the specificities of two splice variants, mGLUT9a and mGLUT9b. [14C]urate uptake measurements show that both splice variants are high-capacity urate transporters and have a Km of ∼650 μM. The well-known uricosuric agents benzbromarone (500 μM) and losartan (1 mM) inhibit GLUT9-mediated urate uptake by 90 and 50%, respectively. Surprisingly, phloretin, a glucose-transporter blocker, inhibits [14C]urate uptake by ∼50% at 1 mM. Electrophysiological measurements suggest that urate transport by mouse GLUT9 is electrogenic and voltage dependent, but independent of the Na+ and Cl− transmembrane gradients. Taken together, our results suggest that GLUT9 works as a urate (anion) uniporter. Finally, we show by RT-PCR performed on RNA from mouse kidney microdissected tubules that GLUT9a is expressed at low levels in proximal tubules, while GLUT9b is specifically expressed in distal convoluted and connecting tubules. Expression of mouse GLUT9 in the kidney differs from that of human GLUT9, which could account for species differences in urate handling.

Bone infections are difficult to treat and can lead to severe tissue destruction. Acute bone infections are usually caused by Staphylococcus aureus. Osteoclasts, which belong to the monocyte/macrophage lineage, are the key cells in bone infections. They are not well equipped for killing bacteria and may serve as a reservoir for bacterial pathogens. Silver has been known for centuries for its bactericidal activity. Here, we investigated the bactericidal effects of nano-silver particles in bacteria infected human osteoclasts. We found that nano-silver in per se non-toxic concentration enhanced the bactericidal activity in osteoclasts against intracellular Methicillin-resistant, virulent Staphylococcus aureus. The reduced bacterial survival in nano-silver pretreated cells correlated with increased reactive oxygen responses towards the invading pathogens. Overall, these results indicate that nano-silver compounds should be considered as an effective treatment and prevention option for bacterial bone and orthopedic implant infections.