Defective D1-like receptor-mediated inhibition of the Cl/HCO3 exchanger in immortalized SHR proximal tubular epithelial cells

Rui Pedrosa,1 Pedro A. Jose,2 and P. Soares-da-Silva1

1Institute of Pharmacology and Therapeutics Faculty of Medicine, 4200 Porto, Portugal; and 2Department of Pediatrics, Georgetown University, 20007 Washington, District of Columbia

Submitted 9 December 2003 ; accepted in final form 9 February 2004


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The sensitivity of the Cl/HCO3 exchanger to dopamine D1- and D2-like receptor stimulation in immortalized renal proximal tubular epithelial cells from the spontaneous hypertensive rat (SHR) and Wistar-Kyoto rat (WKY) was examined. The activity of the Cl/HCO3 exchanger (in pH U/s) in SHR cells (0.00191) was greater than in WKY cells (0.00126). The activity of Cl/HCO3 exchanger was exclusively observed at the apical cell side and probably occurs through the SLC26A6 anion transporter that is expressed in both WKY and SHR cells. Stimulation of D1-like receptors with SKF-38393 markedly attenuated the HCO3-dependent intracellular pH recovery in WKY cells but not in SHR cells. Stimulation of D2-like receptors with quinerolane did not alter Cl/HCO3 exchanger activity in both WKY and SHR cells. The selective D1-like receptor antagonist SKF-83566 prevented the effect of SKF-38393. Both WKY and SHR cells responded to dibutyryl-cAMP (DBcAMP) with inhibition of the Cl/HCO3 exchanger, and downregulation of PKA (overnight exposure to DBcAMP) abolished the inhibitory effect of both DBcAMP and SKF-38393 in WKY cells. Both SHR and WKY cells responded to forskolin with increases in the formation of cAMP. However, only WKY responded to SKF-38393 with increases in the formation of cAMP that was prevented by SKF-83566. It is concluded that WKY cells respond to D1-like dopamine receptor stimulation with inhibition of the apical Cl/HCO3 (SLC26A6) exchanger and SHR cells have a defective D1-like dopamine response.

SLC26A6; hypertension; spontaneous hypertensive rat; Wistar-Kyoto rat


A CONSIDERABLE PART of filtered HCO3 is reabsorbed in the renal proximal tubules by the anion exchangers (AEs) (26, 40). The Na+/HCO3 cotransporter, the Na+-dependent Cl/HCO3 exchanger, and the Na+-independent Cl/HCO3 exchanger have been described in the kidney (3, 16, 34, 39). These AEs facilitate the reversible electroneutral exchange of Cl for HCO3 across the plasma membrane and regulate intracellular pH (pHi), intracellular chloride concentration, bicarbonate metabolism, and cell volume. After an intracellular acid load, the cell responds with stimulation of the Na+/H+ exchanger (NHE), the Na+/HCO3 cotransporter, and the Na+-dependent Cl/HCO3 exchanger to mediate the recovery of pHi (10, 14, 28). In contrast, the Na+-independent Cl/HCO3 exchanger usually mediates the recovery from an intracellular alkalinization (46). In kidney proximal tubules, the main mechanism of chloride reabsorption is via apical Cl/base exchange that works in balance with type 3 NHE (NHE3), this being essential to maintain electrolyte and acid-base homeostasis (2, 5, 6). However, none of the AE family members (AE1, AE2, or AE3) are expressed on the apical membrane of renal tubules. Recently, putative anion transporter-1 (or SLC26A6), a member of a highly conserved family of membrane proteins (SLC26A), was shown to be located in the apical membrane of rat renal proximal tubules (24, 34, 42) and mediated Cl/HCO3 and Cl/OH exchange (21, 24, 34, 42, 44).

Dopamine produced by renal proximal tubular cells exerts an autocrine/paracrine action via two classes of dopamine receptors, D1-like (D1 and D5) and D2-like (D2, D3, and D4), which are differentially expressed along the nephron (11, 29). The autocrine/paracrine function of dopamine, manifested by tubular rather than by hemodynamic mechanisms, becomes most evident during extracellular fluid volume expansion (22). This renal autocrine/paracrine function is lost in essential hypertension and in some animal models of genetic hypertension (4, 1820, 23, 47). In the spontaneously hypertensive rat (SHR), despite normal or increased renal production of dopamine and receptor density, there is a defective transduction of the D1 receptor signal in renal proximal tubules. This coupling defect is genetic (precedes the onset of hypertension and cosegregates with the hypertensive phenotype), is receptor specific (not shared by other humoral agents), and is organ and nephron segment selective (occurs in proximal tubules but not in cortical collecting ducts or the brain striatum) (11, 22, 23, 30). A consequence of the defective dopamine receptor/adenylyl cyclase coupling in the SHR is a decreased ability of D1-like receptor agonists to inhibit NHE3 activity (1, 18, 22, 45). The relationship between NHE/dopamine has been clearly implicated in hypertension, but the link is less established for dopamine/AEs. However, it has been reported that dopamine inhibits Na+/HCO3 cotransport in proximal tubules from normotensive Wistar-Kyoto rats (WKY) but not in SHR (27). This defective response, however, was only apparent after stimulation of Na+/HCO3 cotransport with norepinephrine (27).

The present study was aimed at determining whether immortalized renal proximal tubular epithelial cells from WKY and SHR are endowed with Cl/HCO3 exchange activity and evaluating the effect of dopamine D1- and D2-like receptor stimulation on the Cl/HCO3 exchanger.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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Cell culture. Immortalized renal proximal tubular epithelial cells from 4- to 8-wk-old WKY and SHR animals (43) were maintained in a humidified atmosphere of 5% CO2-95% air at 37°C. WKY and SHR cells were grown in Dulbecco's modified Eagle's medium nutrient mixture Ham’s F12 (Sigma, St. Louis, MO) supplemented with 100 U/ml penicillin G, 0.25 µg/ml amphotericin B, 100 µg/ml streptomycin (Sigma), 4 µg/ml dexamethasone (Sigma), 5 µg/ml transferrin (Sigma), 5 µg/ml insulin (Sigma), 5 ng/ml selenium (Sigma), 10 ng/ml epidermal growth factor (Sigma), 5% fetal bovine serum (Sigma), and 25 mM HEPES (Sigma). For subculturing, the cells were dissociated with 0.10% trypsin-EDTA, split 1:4, and subcultured in Costar plates with 21-cm2 growth areas (Costar, Badhoevedorp, The Netherlands). For pHi measurement experiments, cells were grown in 96-well plates (Costar), polycarbonate filter supports (internal diameter 6.5 mm, Transwell, Costar), or glass coverslips. For the measurement of cAMP, the cells were seeded in 24-well plastic culture clusters (Costar). The cell medium was changed every 2 days, and the cells reached confluence after 3–5 days of incubation. For 24 h before each experiment, the cells were maintained in fetal bovine serum-free medium. Experiments were generally performed 1–2 days after cells reached confluence and 4–5 days after the initial seeding; each cm2 contained ~50 µg of cell protein.

pHi measurements. In pHi measurement experiments, WKY and SHR cells were grown in 10-mm-wide collagen-coated glass coverslips or in 96-well plates. pHi was measured as previously described (14). At days 4-5 after seeding, glass coverslips were incubated at 37°C for 30 min with 5 µM of the acetoxymethyl ester of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM). Coverslips were then washed twice with prewarmed dye-free modified Krebs buffer before initiation of the fluorescence recordings. The Krebs medium had the following composition (in mM): 115 NaCl, 25 NaHCO3, 5.4 KCl, 2.8 CaCl2, 1.2 MgSO4, 0.3 NaH2PO4, 0.3 KH2PO4, 10 HEPES, 5 glucose, pH 7.4 (adjusted with Tris base). Cells were mounted diagonally in a 1 x 1-cm acrylic fluorometric cuvette that was inserted in a PerkinElmer cuvette holder (model LS 50) and subsequently placed in the sample compartment of a FluoroMax-2 spectrofluorometer (Jobin Yvon-SPEX, Edison, NJ). The cuvette volume of 3.0 ml was constantly stirred and perfused at 5.0 ml/min with modified Krebs buffer prewarmed to 37°C. Under these conditions, the cuvette medium was replaced within 150 s. After 5 min, fluorescence was measured every 5 s alternating between 440- and 490-nm excitation (1-nm slit size) at 525-nm emission (3-nm slit size).

In the experiments performed in SHR and WKY cells cultured in 96-well plates or in polycarbonate filter supports (Transwell), pHi measurements were performed after the cells were loaded with 5 µM BCECF-AM at 37°C for 30 min. The test compounds were added to the extracellular fluid 25 min before the start of the pHi recovery period after the alkaline load (CO2/HCO3 removal). Cells were placed in the sample compartment of a dual-scanning microplate spectrofluorometer (Spectramax Gemini XS, Molecular Devices, Sunnyvale, CA), and fluorescence was measured every 19 or 8 s in plastic clusters or polycarbonate filters, respectively, alternating between 440- and 490-nm excitation at 535-nm emission with a cutoff filter of 530 nm. In both types of experiments, the ratio of intracellular BCECF fluorescence at 490 and 440 nm was converted to pHi values by comparison with values from an intracellular calibration curve using the nigericin (10 µM) and high-K+ method (41).

Cl/HCO3 exchanger activity. The Na+-independent HCO3 transport system activity was assayed as the initial rate of pHi recovery after an alkaline load (CO2/HCO3 removal), in the presence of Na+, as previously described (31). In the Krebs HCO3-free medium used, NaHCO3 was replaced by an equimolar concentration of sodium gluconate. To define the initial rate of pHi recovery dependence for Na+ or Cl, the apical side of the monolayers was bathed with a modified Krebs-Hensleit solution containing choline or gluconate, respectively, without affecting the concentrations of other ions. To distinguish the HCO3 exchangers located in the apical and basal cell sides, the cells were cultured in permeable polycarbonate filters. The activity of the apical HCO3 transport system was assayed as the initial rate of pHi recovery after an alkaline load in the apical side (CO2/HCO3 removal), in the absence of HCO3 in the basal cell side. The activity of the basolateral HCO3 transport system was assayed as the initial rate of pHi recovery after an alkaline load in the basal cell side (CO2/HCO3 removal), in the absence of HCO3 in the apical side.

Downregulation studies. PKA downregulation was performed by overnight exposure to cAMP (100 µM), as previously described (13, 36).

Detection of SLC26A6 transcript in WKY and SHR cells. Cells were homogenized (Diax, Heidolph) in TRIzol Reagent (75 mg/ml; Invitrogen) and total RNA was extracted according to the manufacturer's instructions. The RNA obtained was dissolved in diethylpyrocarbonate-treated water and quantified by spectrophotometry at 260 nm. One microgram total RNA was reverse-transcribed to cDNA with SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) according to the manufacturer's instructions. The reverse transcription was performed at 50°C and using 5 µg/µl random hexamers.

Rat expressed sequence tag (EST) database was blast-searched against the human SLC26A6 sequence (GenBank accession no. AF279265). An EST (GenBank accession no. XM217275), which is highly similar to human SLC26A6, was identified. On the basis of the SLC26A6 cDNA sequence, the following oligonucleotide primers (forward: 5'-CGG GAG GCA ACA CGC AGA T-3', and reverse: 5'-GGT GGC TGA GGA ACG GAA GAC-3') corresponding to nucleotides 1403 and 1795 of the rat cDNA (GenBank accession no. XM217275) were designed and used for RT-PCR on RNA isolated from immortalized renal proximal tubular epithelial cells and kidney cortex from WKY and SHR rats. PCR was performed with Platinum TaqPCRx DNA Polymerase (Invitrogen). Amplification conditions were as follows: hot start of 3 min at 95°C; 30 cycles of denaturing (95°C for 30 s), annealing (60°C for 1 min), and extension (72°C for 45 s); and a final extension of 7 min at 72°C.

The PCR products were separated by electrophoresis in a 2% agarose gel and visualized under UV light in the presence of ethidium bromide.

cAMP measurement. cAMP was determined with an enzyme immunoassay kit (Amersham Pharmacia Biotech, Little Chalfont, UK), as previously described (8). Cells were preincubated for 15 min at 37°C in Hanks' medium (in mM: 137 NaCl, 5 KCl, 0.8 MgSO4, 0.33 Na2HPO4, 0.44 KH2PO4, 0.25 CaCl2, 1.0 MgCl2, 0.15 Tris·HCl, and 1.0 sodium butyrate, pH 7.4), containing 100 µM 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor. Cells were then incubated for 25 min with forskolin, SKF-38393 (D1-like agonist) or SKF-38393 (D1-like antagonist), and SKF-83566. At the end of the experiment, cells were lysed by the addition of 200-µl lysis reagent. Aliquots were taken for the measurement of total cAMP content.

Drugs. DIDS, dibutyryl-cAMP (DBcAMP), forskolin, (±)-SKF-83566 hydrochloride, (±)-SKF-38393 hydrochloride, and quinerolane hydrochloride were purchased from Sigma. BCECF-AM and nigericin were obtained from Molecular Probes (Eugene, OR).


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cl/HCO3 exchanger activity in WKY and SHR immortalized cells. The activity of the HCO3 transport system was assayed as the initial rate of pHi recovery after an alkaline load (CO2/HCO3 removal). In an HCO3-containing medium, removal of CO2/HCO3 caused an initial cell alkalinization, as a result of CO2 loss from the cell with subsequent return of pHi toward basal values (Fig. 1). The Na+-independent Cl/HCO3 exchanger (1 external Cl exchanged for 1 internal HCO3) mediated the pHi recovery process after the alkalinization. As shown in Figs. 1 and 2A, the HCO3-dependent recovery of pHi in SHR cells was steeper than that observed in WKY cells. The HCO3-dependent pHi recovery rates (in pH U/s) during the linear phase of pHi recovery after intracellular alkalinization in SHR cells (0.00191 ± 0.00013) were greater than those in WKY cells (0.00126 ± 0.00006; Fig. 2B).



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Fig. 1. Assessment of intracellular pH (pHi) before and after an alkaline load (CO2/HCO3 removal) in perfused spontaneous hypertensive rat (SHR) and Wistar-Kyoto rat (WKY) cells. Traces represent means of 3 experiments per group.

 


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Fig. 2. Assessment of pHi recovery (A) and Cl/HCO3 exchanger activity (B) under Vmax conditions as the initial rate of pHi recovery after an alkaline load (CO2/HCO3 removal). Traces represent means of 8 experiments per group. Columns represent mean of 22 or 23 independent determinations; vertical lines show SE. *Significantly different from WKY cells (P < 0.05).

 
If pHi recovery from intracellular alkalinization involves the Na+-independent Cl/HCO3 exchange, then removal of Cl, but not Na+, should inhibit pHi recovery. To test this hypothesis, Cl and Na+ in the perfusion medium were replaced by gluconate and choline, respectively. Figure 3A shows that in the absence of extracellular Cl, the HCO3-dependent pHi recovery rate was markedly attenuated in both WKY and SHR cells. In contrast, removal of Na+ from the extracellular medium failed to inhibit the pHi recovery after CO2/HCO3 removal. DIDS inhibited the Na+/HCO3 cotransporter, the Na+-dependent Cl/HCO3, and the Cl/HCO3 exchanger in a variety of cell types, including renal cells (3, 5, 7, 34, 38). As shown in Fig. 3B, DIDS markedly reduced the pHi recovery rate in a concentration-dependent manner, in both WKY and SHR cells.



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Fig. 3. A: effect of sodium or chloride removal on Cl/HCO3 exchanger activity in WKY and SHR cells. B: effect of DIDS (0.1–1 mM) on the Cl/HCO3 exchanger activity in WKY and SHR cells. Traces represent means of 7–16 experiments per group. Columns represent mean of 8–23 independent determinations; vertical lines show SE. *Significantly different from corresponding control values (P < 0.05). Ct, control.

 
To evaluate whether the Cl/HCO3 exchanger activity is located in the apical or basal cell side, it was decided to measure the Cl/HCO3 exchanger activity in cells cultured in polycarbonate filters with exclusive access to the apical or basal cell side of the monolayer. As shown in Fig. 4, A and C, the Na+-independent Cl/HCO3 exchanger activity mediated the pHi recovery process after the alkalinization from the apical cell side only, in both WKY and SHR cells. Furthermore, the activity of the Cl/HCO3 exchanger and the inhibition mediated by DIDS (0.5 mM) in cells cultured in polycarbonate filters were similar to that obtained in cells cultured in plastic clusters (Fig. 4, B and D).



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Fig. 4. Assessment of Cl/HCO3 exchanger activity at the apical or basolateral HCO3 transport system as the initial rate of pHi recovery after an alkaline load from the apical or basal cell side (CO2/HCO3 removal) in WKY (A) and SHR (C) cells cultured in polycarbonate filters. B and D: effect of DIDS (0.5 mM) added from the apical cell side on the activity of the apical Cl/HCO3 exchanger (CO2/HCO3 removal from the apical cell side) in WKY (B) and SHR (D) cells cultured in plastic clusters or polycarbonate filters. Symbols or columns represent means of 3 to 8 experiments per group; vertical lines show SE. *Significantly different from control values (P < 0.05).

 
The addition of the D1-like dopamine agonist SKF-38393 (1 µM) before and during the intracellular alkalinization markedly attenuated (from 0.00126 ± 0.00006 to 0.00085 ± 0.00005 pH U/s) the HCO3-dependent recovery of pHi in WKY cells but not in SHR cells (Fig. 5A). By contrast, the D2-like dopamine agonist quinerolane (1 µM) did not affect the HCO3-dependent pHi recovery in both SHR and WKY cells. The effect of SKF-38393 (1 µM) on Na+-independent Cl/HCO3 exchanger activity in WKY cells was abolished by the D1-like-selective receptor antagonist SKF-83566 (1 µM; Fig. 5B).



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Fig. 5. A: effect of SKF-38393 (1 µM) and quinerolane (1 µM) on Cl/HCO3 exchanger activity in WKY and SHR cells. B: effect of SKF-38393 (1 µM) on Cl/HCO3 exchanger activity in WKY cells in the absence and presence of SKF-83566 (1 µM). Each column represents the mean of 5–23 experiments per group; vertical lines indicate SE. Significantly different from values for vehicle (*P < 0.05) and values for SKF-38393 (#P < 0.05).

 
Previous studies demonstrated that second messenger pathways involved in inhibition of Cl/HCO3 exchanger activity include stimulation of protein kinase A (PKA) (15). To evaluate whether this was the case in WKY and SHR cells, we examined the effect of DBcAMP, a direct activator of PKA. Treatment of WKY and SHR cells with DBcAMP (500 µM; Fig. 6) significantly reduced Na+-independent Cl/HCO3 exchanger activity in both WKY and SHR cells. To confirm the involvement of PKA in D1-like dopamine-induced inhibition of Cl/HCO3 exchanger activity, the effects of D1-like dopamine receptor stimulation studies were tested in cells in which PKA was downregulated. Downregulation of PKA was performed by incubating overnight WKY and SHR cells in the presence of DBcAMP (100 µM). Under this experimental condition, the effect of both DBcAMP and SKF-38393 was abolished (Fig. 6A) in WKY cells. Similarly, the DBcAMP-induced inhibition of Cl/HCO3 exchanger activity in SHR cells was also prevented or markedly attenuated by downregulation of PKA (Fig. 6B).



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Fig. 6. Effect SKF-38393 (1 µM) and dibutyryl DBcAMP (500 µM) on Cl/HCO3 exchanger activity after overnight treatment with vehicle [active protein kinase A (PKA)] or DBcAMP (100 µM) followed in WKY (A) and SHR (B) cells. Each column represents the mean of 6–15 experiments per group; vertical lines indicate SE. Significantly different from corresponding control values (*P < 0.05) and values for SKF-38393 or DBcAMP (#P < 0.05).

 
Detection of SLC26A6 transcripts in WKY and SHR cells. The presence of SLC26A6 transcripts in WKY and SHR cells and kidney cortex of 12-wk-old WKY and SHR animals was examined by RT-PCR with specific primers for SLC26A6 rat cDNA sequences. The kidney cortex from 12-wk-old WKY and SHR animals was used as positive control. The expected 413-bp fragment corresponding to SLC26A6 was identified in total RNA from the kidneys and immortalized cells of both SHR and WKY rats (Fig. 7).



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Fig. 7. Detection of SLC26A6 in total RNA extracted from kidney cortexes of WKY and SHR or WKY and SHR immortalized cells with SLC26A6 rat-specific primers. MW, molecular weight.

 
cAMP measurements. Activation of the D1-like dopamine receptor is frequently accompanied by increases in cAMP levels (1, 8, 11, 12, 14, 18, 19, 22, 23, 30). Thus it was decided to measure the accumulation of cAMP in response to the SKF-38393 (1 µM). Because a defective transduction pathway from the D1 dopamine receptor to adenylyl cyclase has been clearly demonstrated in renal proximal tubules from the SHR (12, 22, 23, 30), the effect of forskolin, an adenylyl cyclase, activity was also evaluated. Both WKY and SHR cells responded to forskolin with increases in the formation of cAMP (Fig. 8A). However, only WKY responded to SKF-38393 with increases in the formation of cAMP (Fig. 8A), this increase being completely prevented by pretreatment with the D1-like receptor antagonist SKF-83566 (Fig. 8B).



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Fig. 8. A: effect of forskolin (30 µM) and SKF-38393 (1 µM) on cAMP accumulation in WKY and SHR cells. B: effect of SKF-38393 (1 µM) on cAMP accumulation in WKY cells in the absence and presence of SKF-83566 (1 µM). Each column is the mean of 4 or 5 separate experiments; vertical lines indicate SE. Significantly different from corresponding control values (*P < 0.05) and values for SKF-38393 (#P < 0.05).

 

    DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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The present study was designed to evaluate the activity of the Cl/HCO3 exchanger in immortalized renal proximal tubular epithelial cells from the SHR and their normotensive controls (WKY) and its sensitivity to inhibition by dopamine D1- and D2-like receptor stimulation. The results presented here demonstrate that in SHR cells, the activity of the Cl/HCO3 exchanger was greater than that observed in WKY cells. It is also shown that SHR cells, but not WKY cells, have a defective signaling from the D1-like dopamine receptor downstream to the Cl/HCO3 exchanger.

In the absence of extracellular Cl, but not that of Na+, the HCO3-dependent pHi recovery rate was almost abolished in both WKY and SHR cells. The Na+ independence of this exchanger and the sensitivity by DIDS indicate that this transporter is distinct from the Na+-dependent Cl/HCO3 exchange and Na+/HCO3 cotransporter and most likely corresponds to the Na+-independent Cl/HCO3 exchanger. This type of response is consistent with the Na+-independent Cl/HCO3 exchanger previously characterized in rat and rabbit proximal tubules (25, 34, 37). In fact, it is quite likely that the Cl/HCO3 exchange response is provided from apical membrane, because in these experimental conditions there is exclusive access to the apical side. Therefore, the Cl/HCO3 exchange activity observed in the present study might be consistent with the SLC26A family of AEs, many of which have been shown to transport anions. Recently, SLC26A6, a member of the SLC26A proteins, was shown to be located in the apical membrane of rat kidney proximal tubules (24, 34, 42) and mediated Cl/HCO3 and Cl/OH exchange (21, 34, 42, 44). By contrast, this view excludes the contribution of AEs (AE1, AE2, or AE3) that are not expressed in the apical membrane. Taken together, these studies and the data presented here suggest that Cl/HCO3 exchange might occur at the apical membrane of proximal tubular epithelial cells through the SLC26A6 protein that is expressed in both SHR and WKY immortalized renal proximal tubular epithelial cells.

Our group and others showed that the NHE3 activity and expression in renal proximal tubules from the SHR (immortalized and freshly isolated) were greater than in WKY cells (17, 32, 48). A sustained activation of the NHE is only possible in the presence of a parallel increase of an acidifying pathway, such as the AE, because sustained NHE activity will alkalinize the cell, which inactivates NHE through a cytosolic modifier site (9). This view is in line with the findings that the Na+-independent Cl/HCO3 exchanger activity in SHR cells was greater than in WKY cells. This finding is also in agreement with previous reports showing that in the myocardium of SHR the AE activity increases in parallel with the increases of NHE1 activity (33). In proximal tubular cells, the parallel increase in NHE3 and Cl/HCO3 activity justifies the role of the Cl/HCO3 exchanger in apical membrane and does not conflict with the role of the renal proximal tubules in HCO3 absorption. In fact, apical Cl/HCO3 exchange results in the secretion of HCO3 and absorption of Cl that is crucial to Na+ absorption through NHE3 activity. Furthermore, NHE3 activity is the major mechanism for Na+ and HCO3 reabsorption in the proximal tubule (35), although the stoichiometry of NHE3 and SLC26A6 may not be one to one.

The results presented here show that the D1-like dopamine agonist SKF-38393 significantly reduced the activity of Cl/HCO3 exchanger in WKY but not in SHR cells. In WKY cells, the D1-like dopamine agonist-induced inhibition of Cl/HCO3 exchange was completely prevented by the selective D1-like dopamine receptor antagonist SKF-83566. By contrast, the D2-like dopamine agonist quinerolane did not affect the HCO3-dependent pHi recovery in both SHR and WKY cells. These findings strongly suggest that dopamine-induced inhibition of the Na+-independent Cl/HCO3 exchanger is mediated through the activation of D1-like dopamine receptors. The effect mediated by the D1-like dopamine receptor is in agreement with a previous study (27) that showed inhibition of HCO3 transport system (Na+/HCO3 cotransporter) only in WKY rats. The defective dopamine receptor/adenylyl cyclase coupling in the SHR fits well with the view that both WKY and SHR cells respond to DBcAMP with inhibition of Cl/HCO3 exchange and forskolin was equipotent in increasing the formation of cAMP in WKY and SHR cells. However, only WKY cells responded to SKF-38393 with increases in the formation of cAMP. Altogether, these findings suggest that D1-like dopamine receptor-mediated inhibition of Cl/HCO3 exchange is associated with activation of PKA. This suggestion is in line with the finding that downregulation of PKA completely abolished the inhibitory effects of SKF-38393 and DBcAMP on Cl/HCO3 exchange. In this respect, it is interesting to underline that forskolin and 8-bromo-cAMP were found to reduce Cl/HCO3 exchange in osteoblasts (15). This would agree with the view that inhibition of Cl/HCO3 exchanger activity probably occurs as a result of phosphorylation by PKA.

In conclusion, the results presented here, to our knowledge, are the first to demonstrate that D1-like dopamine receptor stimulation inhibits the activity of the Na+-independent Cl/HCO3 exchanger in WKY cells but not in SHR cells. Most likely SHR cells have a defective transduction of the D1 receptor signal downstream to the Cl/HCO3 exchanger, as shown for the NHE3 and Na+-K+-ATPase.


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 ABSTRACT
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This work was supported by Grant POCTI/35474/FCB/2000 from Fundação para a Ciência e a Tecnologia (Portugal).


    FOOTNOTES
 

Address for reprint requests and other correspondence: P. Soares-da-Silva, Institute of Pharmacology and Therapeutics, Faculty of Medicine, 4200-319 Porto, Portugal (E-mail: psoaresdasilva{at}netcabo.pt).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 REFERENCES
 

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