1. Acid regulation of NaDC-1 requires a functional endothelin B receptor
Liping Liu, Miriam Zacchia, Xuefei Tian, Laxiang Wan, Aiji Sakamoto, Masashi Yanagisawa, Robert J. Alpern, Patricia A. Preisig 
Kidney International 
Volume 78, Issue 9, Pages (November 2010)
DOI: /ki
Copyright © 2010 International Society of Nephrology Terms and Conditions

2. Figure 1
Opossum kidney cell line (OKP) cells transfected with OKP Na-dependent dicarboxylate cotransporter-1 (oNaDC-1) show acid-regulated Na-dependent citrate uptake. OKP cells were exposed to control (pH 7.4) or acid (pH 6.8) media × 6 h. oNaDC-1 activity was assayed using [14C]-citrate uptake and expressed as pmol of citrate/mg protein/5 min. *P<0.05 versus minus Na+; #P<0.05 versus plus Na+ control. n=3 for green fluorescent protein (GFP) only; n=12 for GFP-oNaDC-1 Con (control) and acid groups.
Kidney International  , DOI: ( /ki )
Copyright © 2010 International Society of Nephrology Terms and Conditions

3. Figure 2
Opossum kidney cell line (OKP) cells transfected with OKP Na-dependent dicarboxylate cotransporter-1 (oNaDC-1) show pH- and time-dependent increases in citrate uptake after media acidification. (a) pH is noted on the x-axis; y-axis: citrate uptake expressed as pmol/mg protein/5 min. *P<0.05 versus pH 7.4. n=9 for each pH. (b) x-axis: length of exposure to acidic media; y-axis: acid:control ratio at each time point. *P<0.05 versus no percentage change in the ratio (100). n=9 for all time points except 12 h, where n=8. oNaDC-1 activity was assayed using [14C]-citrate uptake.
Kidney International  , DOI: ( /ki )
Copyright © 2010 International Society of Nephrology Terms and Conditions

4. Figure 3
Opossum kidney cell line (OKP) cells transfected with OKP Na-dependent dicarboxylate cotransporter-1 (oNaDC-1) and endothelin B (ET)B show both dose- and time-dependent ET-1 stimulation of oNaDC-1 activity. (a) Dose-dependent study. oNaDC-1 activity was assayed as [14C]-citrate uptake after 35 min exposure to the indicated ET-1 concentrations. *P<0.05 versus vehicle. n=9 for all ET-1 concentrations. (b) Time-dependent study after exposure to 10-8 mol/l ET-1. *P<0.05 versus vehicle; #P=0.09 versus vehicle. n=12 for studies at 5, 10, 15, and 25 min; n=9 for studies at 35 min and 1 h; n=6 for studies at 4 and 8 h.
Kidney International  , DOI: ( /ki )
Copyright © 2010 International Society of Nephrology Terms and Conditions

5. Figure 4
Endothelin-1 (ET-1) stimulation of opossum kidney cell line Na-dependent dicarboxylate cotransporter-1 (oNaDC)-1 activity is dependent on the ETB receptor. Cells were co-transfected with oNaDC-1 alone (left set of bars) or oNaDC-1+ETB (right set of bars). oNaDC-1 activity was assayed as [14C]-citrate uptake and reported as pmol/mg protein/5 min. (a) Cells were exposed to vehicle or 10-8 mol/m ET-1 × 35 min. n=8 for vehicle and n=9 for BQ788 studies. *P<0.01 versus vehicle. (b) Cells were exposed to media of pH 7.4 (con) or 6.8 (acid) × 6 h. n=8 for all studies. *P<0.05 versus pH 7.4; #P=0.06 versus pH 7.4.
Kidney International  , DOI: ( /ki )
Copyright © 2010 International Society of Nephrology Terms and Conditions

6. Figure 5
Endothelin-1 (ET-1) does not stimulate opossum kidney cell line Na-dependent dicarboxylate cotransporter-1 (oNaDC-1) activity in opossum kidney cells transfected with ETA. Cells were cotransfected with oNaDC-1 or oNaDC-1+ETA. oNaDC-1 activity was assayed as [14C]-citrate uptake. *P<0.001 versus vehicle; **P<0.01 versus vehicle n=9 for all studies.
Kidney International  , DOI: ( /ki )
Copyright © 2010 International Society of Nephrology Terms and Conditions

7. Figure 6
Apical membrane opossum kidney cell line Na-dependent dicarboxylate cotransporter-1 expression increases after either media acidification or exposure to 10-8 mol/l endothelin-1 (ET-1). Cells were prepared for confocal microscopy as described in the Methods section. (a) Green fluorescent protein (GFP) expression comparing exposure to control (pH 7.4) and acid (pH 6.8) media × 6 h. (b) GFP expression comparing exposure to vehicle and 10-8 mol/l ET-1 × 35 min. Typical pictures for both panels.
Kidney International  , DOI: ( /ki )
Copyright © 2010 International Society of Nephrology Terms and Conditions

8. Figure 7
Stimulation of opossum kidney cell line Na-dependent dicarboxylate cotransporter-1 activity by acid feeding is dependent on the (endothelin B) ETB receptor. ETB+/+ and ETB-/- mice were provided with plain drinking water or drinking water containing 0.3 mol/l NH4Cl ad lib × 7 days. Kidney cortex was harvested, brush border membrane vesicles prepared, and Na-dependent [14C]-citrate uptake measured as described in the Methods section. All assays were carried out in triplicate and meaned to obtain a transport rate for this study. *P<0.05 versus mice drinking plain water. n=6 for all groups.
Kidney International  , DOI: ( /ki )
Copyright © 2010 International Society of Nephrology Terms and Conditions

9. Figure 8
Endothelin B (ETB) C-terminal tail required for ET-1 stimulation of opossum kidney cell line Na-dependent dicarboxylate cotransporter-1 (oNaDC-1) activity. Cells were transfected with oNaDC-1 and the wild-type or chimeric receptor as indicated on the x-axis and [14C]-citrate uptake was measured. ETA: wild-type ETA receptor; ETB: wild-type ETB receptor; ETB no tail: ETB receptor without a C-terminal tail; ETB TM/ETA tail: chimeric receptor including the ETB transmembrane (TM) domains and ETA C-terminal tail; ETA TM/ETB tail: chimeric receptor including the ETA transmembrane domains and ETB C-terminal tail. Statistical results from the one-way analysis of variance are included in the figure. *P<0.05 for the ET-1:vehicle ratio being statistically greater than 100. n=9 for ETA and ETB no tail studies; n=18 for ETA TM/ETB tail and ETB TM/ETA tail studies; n=23 for wild-type ETB studies. Abbreviation: NS, not significant.
Kidney International  , DOI: ( /ki )
Copyright © 2010 International Society of Nephrology Terms and Conditions

10. Figure 9
Endothelin B (ETB) C-terminal truncations alter ET-1 regulation of opossum kidney cell line Na-dependent dicarboxylate cotransporter-1 activity. (a) The wild-type (WT) human ETB (hETB) C-terminal sequence and five truncations are shown. The underlined and italized residues (YLV) represent the end of the last transmembrane domain. The Δ43 truncation is the construct used for the studies in Figure 8 labeled ETB no tail. (b) Cells were transfected with oNaDC-1 and the WT or truncated receptor as indicated on the x-axis and [14C]-citrate uptake was measured. *P<0.05 versus vehicle; #P=0.051 versus vehicle. n=27 for ETB; n=9 for Δ11, n=18 for Δ23, n=21 for Δ37 and Δ43, and n=23 for Δ47.
Kidney International  , DOI: ( /ki )
Copyright © 2010 International Society of Nephrology Terms and Conditions

11. Figure 10
Endothelin B (ETB) C-terminal Tyr430 is not involved in ET-1 stimulation of opossum kidney cell line Na-dependent dicarboxylate cotransporter-1 (oNaDC-1) activity. Cells were cotransfected with oNaDC-1 and either the wild-type ETB receptor or ETBY430F mutant receptor, and [14C]-citrate uptake was measured. *P<0.01 versus vehicle. n=8 for both groups.
Kidney International  , DOI: ( /ki )
Copyright © 2010 International Society of Nephrology Terms and Conditions