HeLa cells were transiently transfected with rat NK 1 subunit cDNA and co transfected with a total of 2 mg cDNA of rat NK?1 NK 1 to over express rat Na,K ATPase, or with a total of 2 mg cDNA of HK?2 HK 2 to over express rat ngH,K ATPase, using PolyFect reagent following the protocol described by the vendor. 86Rb uptake measurements were performed to ensure that we achieved high levels of functional expression of both Bufo ngH,K and Na,K ATPase in Xenopus oocytes and rat ngH,K and Na,K ATPase in HeLa cells. Xenopus oocytes expressing Bufo bladder H,K ATPase, Na,KATPAse or 2 subunit alone were loaded with Na by 2 h incubation in a K free Ca2 free solution containing : 90 NaCl and 0.5 EGTA. Na loaded oocytes were transferred to a solution containing : 5 KCl, 90 NaCl, 1 CaCl2, 10 HEPES, pH 7.4, 0.2 M ouabain , and 10 M bumetanide . Oocytes were incubated 12 minutes with 86Rb at room temperature, and washed with a solution containing : 90 NaCl, 1 CaCl2, 1 MgCl2, and 10 HEPES, pH 7.4. Individual oocytes were then dissolved in 0.
5 % SDS and 86Rb uptake was determined by scintillation counting. HeLa cells grown in 24 well cluster dishes at 60 80% confluency were transiently transfected as described above. Three days later, SB 203580 86Rb uptake measurements were performed using a wash tray according to Sangan, et al After drilling a hole in each cover well , we inserted a plastic test tube and then glued it into position. This allowed us to fill each tube with wash solutions and invert the whole assembly on the 24 well culture dish containing transfected HeLa cells. The solution obtained in each tube of the wash tray was transferred into individual test wells. Steady state Voltage Clamp measurements Xenopus oocytes were microinjected with Bufo NK?1 NK 2, HK?2 NK 2, and NK 2 cRNAs encoding for Bufo Na,K ATPase, bladder ngH,K ATPase, and Na,K ATPase 2 subunit respectively. Three or four days later, the steady state current activated by 10 mM extracellular K was measured at holding potential of 50mV using the two electrode voltage clamp technique.
The experimental solution contained : 100 Na gluconate, 0.82 MgCl2, 0.41 CaCl2, 10 NMDG HEPES, 5 BaCl2, 10 TEA Cl2, and 0.2 M ouabain . Ba2 and TEA were present to block passive K channels peptide synthesis so that the current produced by ouabain resistant Bufo Na,K pumps could be measured upon addition of extracellular K . An aliquot of 100 M PTX was thawed just prior to each experiment and diluted to a final concentration of 5 nM in the external K free solution containing 0.002% BSA to minimize PTX binding to non glass surfaces. All solutions used in two microelectrode voltage clamp experiments had a pH of 7.4 0.05 and osmolality of approximately 200 mOsm kg.