Volume 53, Issue 5, Pages 1269-1277 (May 1998) Polarized distribution of Na+/H+ exchanger isoforms in rabbit collecting duct cells  Lorenzo Guerra, Francesca Di Sole, Giovanna Valenti, Pierre M. Ronco, Elda Perlino, Valeria Casavola, Stephan J. Reshkin  Kidney International  Volume 53, Issue 5, Pages 1269-1277 (May 1998) DOI: 10.1046/j.1523-1755.1998.00897.x Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 1 Electron micrograph of a RC.SV3 monolayer grown on permeable filter (×1500). The cells appear to be polarized as illustrated by apical microvilli and lateral digitation. Kidney International 1998 53, 1269-1277DOI: (10.1046/j.1523-1755.1998.00897.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 2 Representative traces depicting the Na+-dependence and polarity of intracellular pH (pHi) recovery after an acid load and the effect of DMA in RC.SV3 monolayers in (A) basolateral and (B) apical membranes. The acid load was imposed by exposing the cells for five minutes to 20mm NH4Cl in Na+-medium followed by Na+-free tetramethylammonium (TMA) medium (Methods). Each trace begins at the start of perfusion of the monolayer with TMA-Cl solution. There was no recovery of pHi under this condition. When the monolayer was perfused with either basolateral (first trace) or apical (third trace) 135mm Na+ nominally bicarbonate-free HEPES solution, a rapid recovery of pHi commensed. The monolayer was then acidified again and after a two minute preincubation with 2 μm DMA in TMA-medium, the monolayer was perfused with 135mm Na+ medium plus 2 μm DMA. The ordinate is the pHi corresponding to the nigericin calibration performed as described in the Methods section. Kidney International 1998 53, 1269-1277DOI: (10.1046/j.1523-1755.1998.00897.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 3 External Na+dependence of intracellular pH (pHi) recovery. To examine the effect of a series of different Na+ concentrations within a monolayer, cells were repeatedly acidified to the same pHi level and the initial recovery rate measured in response to 6.7, 27, 34, 67.5, 100 and 135mm external sodium added to either the (A) basolateral or (B) apical perfusion solution. For each experiment a least-squares fit was performed on the initial rate of pHi recovery (15 data points for each recovery), and the slopes normalized to the pHi recovery rate observed in the presence of 135mm Na+. Na+ activation of basolateral Na+/H+ exchange followed simple Michaelis-Menten kinetics. The apparent Km values were 24.5 ± 5.8mm for the basolateral and 47.6 ± 9.8mm Na+ for the apical exchangers, respectively. The curved line is a non-linear least-squares fit of the Michaelis-Menten equation. Kidney International 1998 53, 1269-1277DOI: (10.1046/j.1523-1755.1998.00897.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 4 Dixon plot analysis of DMA inhibition of (A) basolateral or (B) apical Na+/H+exchange activities. In experiments conducted as in Figure 2, the initial recovery after an acid load was measured in monolayers perfused after with either 34mm (▪) or 135mm (•) Na+ medium in the presence of 0, 0.2, 2 or 10 μm DMA. The half-inhibition constant (Ki) was calculated from the intersection point of the two lines. Kidney International 1998 53, 1269-1277DOI: (10.1046/j.1523-1755.1998.00897.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 5 Dixon plot analysis of HOE-694 inhibition of (A) basolateral and (B) apical Na+/H+exchange activities. In experiments conducted as in Figure 4, initial recovery after an acid load was measured in monolayers perfused after with either 34mm (▪) or 135mm (•) Na+ medium in the presence of 0, 0.01, 0.1 or 0.2 μm HOE-694. The half-inhibition constant (Ki) was calculated from the intersection point of the two lines. Kidney International 1998 53, 1269-1277DOI: (10.1046/j.1523-1755.1998.00897.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 6 Influence of pharmacological activation of protein kinases on the rate of Na+/H+exchange activity. Either the basolateral or apical Na-dependent intracellular pH (pHi) recovery was measured in response to 34mm Na+ medium both in the absence and in the presence of either 10-7m TPA or 10-4m 8-bromo-cAMP. Both the agents were added 10minutes before analyzing the pHi recoveries. Values are the mean percent change in pHi recovery rate of the treated cells with respect to untreated cells ± se. N = 5, **P < 0.02, ***P < 0.001. Kidney International 1998 53, 1269-1277DOI: (10.1046/j.1523-1755.1998.00897.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 7 Western blot analysis and immunofluorescence localization of NHE-1 in RC.SV3 monolayers grown on filters. (A) Confluent monolayers were grown in DMEM plus 10% serum and total membrane protein was isolated, separated, blotted and Western blot performed as described in the Methods section. (B) Immunofluoresence localization of NHE-1 in RC.SV3 cells grown on coverslips (×650). The protein for NHE-1 is expressed in the basolateral membrane of the cells (arrows). Kidney International 1998 53, 1269-1277DOI: (10.1046/j.1523-1755.1998.00897.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 8 Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of NHE-1, NHE-2 and NHE-3 RNA expression. In a representative experiment, ethidium bromide stained RT-PCR amplified products of RC.SV3 total cDNA. The molecular size of amplified bands are indicated on the left and are assigned on the basis of the co-migration of a 1kb DNA ladder (Gibco BRL). Lane 1, RT-PCR amplification product with NHE-1 primers (250bp); lane 2, RT-PCR amplification product with NHE-2 primers (550bp); lane 3, RT-PCR amplification product with NHE-3 primers; and lane 4, RT-PCR amplification product with primers for aquaporin (AQP4) and plasmid borne AQP4 cDNA (800bp). Kidney International 1998 53, 1269-1277DOI: (10.1046/j.1523-1755.1998.00897.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Kidney International 1998 53, 1269-1277DOI: (10. 1046/j. 1523-1755 Kidney International 1998 53, 1269-1277DOI: (10.1046/j.1523-1755.1998.00897.x) Copyright © 1998 International Society of Nephrology Terms and Conditions