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Oocyte NH4+ transport activity was measured from the rate of acidification after NH4Cl application. The rate of pHi change (i.e. dpHi/dt) was calculated by linear regression from the initial pHi value over the first 2 min after NH4Cl exposure. All experiments were performed at room temperature. Data are reported as means �� SEM. The level of significance was assessed using Student's unpaired, two-tailed?t?test for comparison of dpHi/dt,?Vm and?I?between control oocytes and NBCn1-expressing oocytes. One-way ANOVA with Bonferroni?post hoc?test was used to analyse daily?I?values for 5 days. A?P?value of less than 0.05 was considered significant. To assess the effect of NBCn1 cotransport activity on NH4+ transport, we expressed the rat renal splice variant [http://www.selleckchem.com/products/DAPT-GSI-IX.html www.selleckchem.com/products/DAPT-GSI-IX.html] NBCn1-E (hereafter, NBCn1) in?Xenopus?oocytes and simultaneously measured pHi and?Vm during NH4Cl application in the presence of 25 mm HCO3?, 5% CO2 using the proton-selective pH microelectrode and the voltage electrode. Figure 1A shows representative pHi and?Vm traces in a water-injected control oocyte. The oocyte was bathed initially in the Hepes-buffered (HCO3?/CO2-free) solution to maintain stable pHi. [http://www.selleckchem.com/products/Adriamycin.html Doxorubicin solubility dmso] Applying HCO3?/CO2-buffered medium caused pHi to decrease as CO2 entered the oocyte, hydrated and produced H+. The pHi then reached steady state as intracellular CO2 rose to equal extracellular CO2. A slight increase in pHi was often observed (a?in the figure), possibly due to endogenous Na+�CH+ exchange (Busch, 1997). In these conditions, subsequent exposure to 20 mm NH4Cl induced intracellular acidification (b?in the figure). Ammonium chloride also caused the oocyte membrane to be depolarized. These responses to NH4Cl (i.e. intracellular acidification [https://en.wikipedia.org/wiki/Sulfatase Sulfatase] and membrane depolarization) are comparable to the previous observations by others (Cougnon?et al.?1996; Burckhardt & Burckhardt, 1997; Nakhoul?et al.?2010). In an NBCn1-expressing oocyte (Fig. 1B), the application of HCO3?/CO2 also caused pHi to decrease. However, the pHi recovered from an initial CO2-induced acidification (a') as HCO3? transported via NBCn1 associated with intracellular H+. Applying NH4Cl in the continued presence of HCO3?/CO2 induced a rapid acidification (b'). NH4Cl-dependent intracellular acidification was evaluated by subtracting dpHi/dt?values before NH4Cl application from values after NH4Cl application (i.e.?b�Ca?or?b'�Ca'). The acidification rate was ?10.33 �� 0.47 �� 10?4s?1 (n?= 6) for NBCn1-expressing oocytes, which was 2.8-fold higher than ?3.73 �� 0.49 �� 10?4s?1 (n?= 7) for control oocytes (P?