Introduction

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Luminal NHE accounts for the bulk of active Na⁺ reabsorption in the PT (1). The NHE functions in the reabsorption of HCO₃ and secretion of H⁺ (2). Catecholamines, peptide hormones, and a number of pharmacological agents regulate proximal nephron NHE (3, 4).

Numerous studies indicate that antidiuretic and antinatriuretic effects in response to low frequency renal nerve stimulation are mediated by ₁-AR (see Ref. 5 for a review). The ₁-ARs activated during renal nerve stimulation are postulated to be located postsynaptically (6). Stimulation of PT ₁-ARs increase NHE in addition to water, chloride, and bicarbonate reabsorption (7, 8). The tubular effects on transport occur independently of changes in glomerular filtration rate, renal blood flow, or the intrarenal redistribution of blood flow (9).

Three ₁-AR subtypes, designated _1A-, _1B-, and _1D-AR, have been cloned (10, 11). _1A-ARs have a high affinity for 5-methyurapidil, WB-4101, and (+)-niguldipine and are sensitive to the alkylating agent SZL-49 (12). _1B-ARs have a low affinity for these agents but are highly sensitive to CEC and spiperone (11). In comparison, _1D-ARs are selectively antagonized by BMY 7378 and SK&F 105854 (13). In human kidney, expression of ₁-AR protein is quite low relative to that of ₂-ARs. The localization of ₁-ARs in kidney remains somewhat controversial. Studies using in situ hybridization or message expression often arrive at different distributions compared with pharmacological binding or autoradiography experiments (14-16). In general, the consensus for ₁-AR localization seems to be PT and thick ascending limb/early distal convoluted tubule, where there is direct innervation by renal nerves (5, 17). Recent studies identify _1A- and _1B-AR subtypes in PTs (18, 19), whereas others have identified all three subtypes in PT cells (15). The purpose of this investigation was to identify the subtypes of ₁-ARs present in PT cells that regulate NHE.

Message for all three ₁-AR subtypes was observed through the use of RT-PCR and Northern analysis with RNA obtained from mouse PT cells. A comparison of the nucleotide sequences obtained from the partial clones of the RT-PCR products of mouse PT cells with published rat cDNA sequences indicates >94% identity at the nucleotide level and >98% identity at the amino acid level for the three subtypes of ₁-ARs. Use of antisense ODNs selective for _1A and _1B-AR subtypes inhibit protein expression and ₁-AR agonist-induced increases of pH_i in PT cells by 42% and 49%, respectively. Similar levels of inhibition in agonist-induced pHi were observed with the _1A-AR antagonist WB-4101 and the _1B-AR antagonist spiperone. There was no functional reduction of agonist-induced pH_i in cells treated with antisense ODN or the antagonist BMY 7378 to the _1D-AR subtype. Combined treatment with _1A and _1B antisense ODNs resulted in additive inhibition of ₁-AR-induced increases of intracellular pH_i by 90%. In summary, message for three subtypes of ₁-AR is expressed in PT cells, but only _1A- and _1B-AR subtypes regulate NHE.

	Experimental Procedures

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Preparation of Primary Cell Cultures and Immortalized Proximal Convoluted Tubule Cells

Primary cultures of mouse PT (proximal convoluted and straight tubule) cells were prepared as reported previously (20). An immortalized mouse S1 PT cell line was established as described previously (21). The S1 proximal cells exhibit the phenotype of the proximal convoluted S1 portion of the nephron that includes (i) functional Na/P_i cotransport, (ii) formation of cAMP in response to parathyroid hormone, (iii) alkaline phosphatase activity, and (iv) gluconeogenic activity (21).

Primary cultures of mouse proximal cells and immortalized S1 cells were grown in Dulbecco's modified Eagle's medium/Ham's F-12 medium (Sigma Chemical, St. Louis, MO) supplemented with 5% heat-inactivated fetal calf serum (Sigma) and PSN antibiotic mixture (50 µg of penicillin, 50 µg of streptomycin, 100 µg of neomycin/100 ml of media; GIBCO BRL, Gaithersburg, MD) in a humidified atmosphere of 95% O₂/5% CO₂ at 37°. Cells were placed in serum-free Dulbecco's modified Eagle's medium/Ham's F-12 medium for 16 hr before use.

RNA Isolation, RT-PCR, and Design and Introduction of Antisense ODNs

RNA isolation. Culture dishes (100 mm) of PT cells were washed twice with 5 ml of Ca²⁺/Mg²⁺-free Hanks' balanced salt solution. Cells were solubilized and scraped in the presence of 1 ml of 1 M guanidine isothiocyanate layered onto a 1.5-ml CsCl gradient and overlaid with 0.15 ml of 20% sarkosyl. Gradients were centrifuged for 2 hr at room temperature, and pellets were washed with 70% ethanol and resuspended in 100 µl of sterile water. Quantification of yield was determined by absorbance at 260 and 280 nm.

RT-PCR. One microgram of total RNA from PT cells was reverse-transcribed using MuMLV reverse transcriptase and the reverse primers for each subtype (GeneAMP RNA-PCR Kit; Perkin-Elmer Cetus, Norwalk, CT) for 15 min at 42°. The cDNA was then amplified with Taq polymerase. Mouse heart and kidney was used as positive controls for appropriately sized PCR products. Primer sequences specific for each ₁-AR subtype are presented in Table 1. PCR was performed at 94° for 1 min, annealed at the temperature indicated for each primer set in Table 1 (22) for 1 min, and extended for 1 min at 72° for 35 cycles, with a final extension of 5 min. The products were electrophoresed on a 4% low-melting agarose gel and stained with ethidium bromide. Products were cut from a low-melt agarose gel, the cDNA was eluted, and 30 ng of each product was directly sequenced with 3.2 pmol of the forward or reverse PCR primers using the PRISM DyeDeoxy Sequencing Kit (Applied Biosystems, Foster City, CA) as described by the manufacturer. To control for nucleotide incorporation errors introduced by Taq polymerase, multiple RT-PCR reactions were sequenced. The cDNAs from two to four independent reactions were sequenced in both forward and reverse directions. Comparisons between cDNA products and published ₁-AR subtype sequences were carried out with GCG (Genetics Computer Group, Madison, WI) and GeneWorks (Intelligenetics, Mountain View, CA) software.

View this table: [in this window] [in a new window]   TABLE 1 Primers used for amplification of 1-AR subtype transcripts by RT-PCR The sequences of the primers (listed from 5 to 3) used for RT-PCR analysis are shown. The optimized annealing temperature for the amplification of mouse transcripts by each primer pair is also shown. The primer sequences were determined from rat in a previous report (22).

Design and introduction of antisense ODNs. To design antisense ODNs that were highly specific for each ₁-AR subtype, sequences were chosen to span the third intracellular loop of the ₁-AR DNA sequence. This region was chosen since it is the most divergent among the three ₁-AR subtypes. Antisense and sense ODNs were designed from the cDNA sequence obtained for each ₁-AR subtype in the mouse PT cells used in this study. Antisense and sense 18-mers that were phosphorothioate-substituted in each position were prepared using a low pressure reverse-phase purification system (Molecular Resources, Fort Collins, CO). The sequence for each of the subtypes was as follows: _1A-AR antisense ODN (positions 96-113), 5-CTTATTCTTGGCACTGCT-3; _1A-AR sense ODN, 5-AGCAGTGCCAAGAATAAG-3; _1B-AR antisense ODN (positions 36-53), 5-CTTGGTGGTCCTCTTGGC-3; _1B-AR sense ODN, 5-GCCAAGAGGACCACCAAG-3; _1D-AR antisense ODN (positions 212-229), 5-GCGGGAAAACTTGAGCAG-3; and _1D-AR sense ODN, 5-CTGCTCAAGTTTTCCCGC-3. Antisense and sense ODNs were reconstituted in PBS and stored at 20°. Final concentrations of 5 µM ODNs were introduced into cells grown onto 30-mm dishes or glass coverslips with transient streptolysin-O permeabilization (20 units/ml; GIBCO BRL) for 10 min at 37° as described previously (23). Control cells were permeabilized, but no ODNs were added. Time and concentration dependence were determined with Western blot analysis for the _1B-AR subtype. In preliminary studies, we determined that multiple treatments with antisense ODNs (three antisense ODN treatments over 72 hr) were required for maximal inhibition of protein expression.

Western Blot Analysis

Control and ODN-treated cells were collected in lysate buffer (50 mM Tris·HCl, pH 8.0, 0.5% deoxycholate, 1% Triton X-100, 0.1% SDS, 1 µg/ml aprotinin, 75 µg/ml phenylmethylsulfonyl fluoride) and centrifuged at 15,000 × g for 10 sec. The supernatant was stored at 80°. Samples containing 100 µg of protein/well were boiled in loading buffer (62.5 mM Tris·HCl, pH 6.8, 2% SDS, 10% glycerol, 5% -mercaptoethanol, bromphenol blue) for 5 min and separated on 7.5% SDS-polyacrylamide gels. The fractionated proteins were transferred to a nitrocellulose membrane. The blots were blocked with 5% Blotto in TBS (137 mM NaCl, 20 mM Tris, pH 7.4) at 4° overnight. After three washes, the blots were incubated with 0.5 µg/ml of goat anti-_1B-AR polyclonal antibody (Santa Cruz Biochemicals, Santa Cruz, CA) in 1% Blotto in TBS for 90 min at room temperature. The blot was washed and incubated with 1:3000 peroxidase-conjugated rabbit lgG fraction to goat lgG (CAPPEL, Durham, NC) in TBS for 60 min. The antibody binding was detected using an enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL) and exposed on Kodak X-OMAT film. Mouse anti--actin monoclonal antibody (Sigma) was used as a control. Western blots were scanned with a laser densitometer, and the density of each band from antisense-treated cells was compared with that of control and sense ODN treated cells. The final values were normalized with the density units obtained with -actin in the corresponding sample.

Northern Blot Analysis

Total RNA (20 µg) or mRNA (1 µg) from the PT cells was electrophoresed on a 1% agarose/formaldehyde gel and electrophoretically transferred to GeneScreen Plus membrane (Dupont-NEN, Wimington, DE). The blots were prehybridized in a solution of 1 M NaCl, 1% SDS, and 10% dextran sulfate for 60 min at 60°. The cDNA products for each of the ₁-AR subtypes were randomly primed with 2 × 10⁶ cpm/ml [³²P]dCTP (ICN Pharmaceuticals, Costa Mesa, CA) and used to probe RNA from mouse PT cells. The blots were washed at high stringency with 50 ml of 2× sodium chloride/sodium citrate containing 0.1% SDS three times at room temperature followed by three washes with 0.1× sodium chloride/sodium citrate containing 0.1% SDS at 60° and exposure to Kodak X-AR film for 24-48 hr at 70° (1× sodium chloride/sodium citrate = 150 mM NaCl, 15 mM sodium citrate).

Fluorescent BODIPY FL Prazosin Binding

To determine relative amounts of ₁-AR subtypes and examine antisense ODN inhibition of receptor protein expression, the fluorescent ligand BODIPY FL prazosin (Molecular Probes, Eugene, OR) was used for labeling ₁-ARs. Cells were grown on eight-well Falcon culture slides (Becton Dickinson Labware, Franklin Lakes NJ); when the cells were treated with ODNs, they were permeabilized on the slide, with streptolysin-O and ODNs introduced as described above. BODIPY FL prazosin (30 nM) was incubated with cells on slides for 4 hr at 4°. For competitive binding studies with pharmacological antagonists, cells were preincubated with antagonists at 4° for 15 min before incubation with antagonists plus BODIPY FL prazosin for 4 hr. After binding of BODIPY FL prazosin, cells were rinsed three times with 4° PBS buffer containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1 mM KH₂PO₄, pH 7.40, and fixed for 15 min at room temperature with 3.7% methanol-free paraformaldehyde in PBS. Slides were rinsed three times with PBS, the chamber wells were removed, and a glass coverslip was affixed with Fluorostab mounting solution (ICN, Costa Mesa, CA). Cells were visualized with a Nikon FXA microscope equipped with a B2A filter cube.

Determination of pH_i

Cells grown onto 25-mm glass coverslips, were rinsed three times with a assay buffer containing 140 mM Na⁺, 148 mM Cl, 5 mM K⁺, 1 mM Ca²⁺, 1 mM Mg²⁺, 28 mM HEPES, 18 mM Tris, and 10 mM glucose, pH 7.4, and adjusted to 295 mOsmol/kg H₂O). Cells were incubated with the pH-sensitive dye BCECF-AM (10 µM; Molecular Probes, Eugene, OR) for 1 hr at 37°. Cells were rinsed twice with buffer and placed in a temperature-controlled chamber of microincubation system at 37° as described previously (24). A Nikon Photoscan-2 (Natick, MA) was used to measure fluorescence intensity. Each experiment was calculated using equilibration with buffers of varying pH values (6.5-7.6) and treatment with the ionophore valinomycin (10 µM; Calbiochem, San Diego, CA).

Materials, Preparation of Drug Solutions, and Statistical Evaluation of Data

The ₁-AR agonist PHE and the antagonists WB 4101, spiperone, and BMY 7378 were prepared so that the molar concentration indicated in text or figures is the final concentration to which the cells were exposed. Solutions containing drugs were prepared fresh daily. All receptor ligands were purchased from Research Biochemicals (Natick, MA).

All results of Western blot analyses and intracellular pH measurements are presented as mean ± standard error. Comparisons between control and drug-treated groups were examined by post hoc analysis of multiple comparisons with the Bonferroni or Newman-Keuls multiple-comparisons tests using the statistical software Instat for MacIntosh (GraphPAD Software, San Diego CA). Values of p  0.05 were considered significant.

	Results

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Analysis of ₁-AR transcripts in PT cells. RNA isolated from primary cultures of PT cells and immortalized S1 cells was reverse-transcribed, and the resulting cDNA was amplified by PCR in separate experiments using ODN primers specific for the three ₁-AR subtypes (22). RT-PCR for each primer set was performed on RNA samples obtained with three or four independent isolations. Primers for the _1A-AR subtype resulted in a product of 212 bp with RNA from primary cultures and immortalized PT cells (Fig. 1). Primers specific for the _1B-AR subtype produced a band at 300 bp, whereas primers for the _1D-AR subtype resulted in a product of 304 bp. Products of similar size were obtained for each ₁-AR subtype using rat kidney as a positive control (data not shown). For all primer sets and RNAs used, samples analyzed in the absence of RT resulted in no product. These findings suggest that mouse PT cells express transcripts encoding all three ₁-AR subtypes.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Analysis of transcripts for 1-AR subtypes in PT cells. Total RNA obtained from primary cultures (PT) or immortalized (S1) proximal convoluted tubule cells was used to detect message for 1-AR subtypes with RT-PCR. Samples were determined in the presence (+) or absence () of reverse transcriptase. The predicted size for 1A-AR message was 212 bp, and the predicted product was 300 bp for 1B-AR and 304 bp for the 1D-AR.

View larger version (58K): [in this window] [in a new window]   Fig. 2.   Northern blot analysis of PT cell RNA for 1-AR subtypes. Total RNA (20 µg; 1B-AR, 1D-AR) or mRNA (1 µg; 1A AR) obtained from S1 PT cells was probed with randomly primed and 32P-dCTP-labeled 1-AR subtype PCR products. Transcripts were observed of 2.3 kb for the 1A subtype, 2.7 kb for the 1B subtype, and a major band at 2.6 kb and a minor band at 2.3 kb for the 1D AR subtype. These sizes are consistent with transcripts reported in kidney and other tissues.

Analysis of protein expression for ₁-AR subtypes. To assess protein expression of ₁-AR subtypes, two complementary methods were used: (i) Western analysis was performed on membrane preparations from primary cultures and immortalized S1 PT cells, and (ii) competition with pharmacological antagonists and antisense inhibition of protein expression was determined with the fluorescent ₁-AR ligand BODIPY FL prazosin. Expression of the _1B-AR subtype was examined using a polyclonal antibody corresponding to amino acids 500-517 of the _1B-AR and maps to the carboxyl terminus (Santa Cruz). As depicted in Fig. 3, a band of 60 kDa was observed with membrane from control cells (streptolysin-O permeabilized but no ODN treatment). No 60-kDa band was observed when membranes were pretreated with _1B-AR antibody control peptide (Santa Cruz) and confirmed the specificity of this antibody for the _1B-AR (data not shown); similarly, no discernible bands were detected with the rabbit anti-goat secondary antibody alone. Proximal cells treated with 5 µM antisense or sense ODNs to the _1B-AR are also shown. We observed a significant reduction in the intensity of the 60-kDa band with membrane samples obtained from three treatments over 72 hr of antisense- but not sense-treated proximal cells. The level of _1B-AR protein expression in the PT cells treated with sense ODNs was similar to that observed in control cells. Nonspecific bands observed with all three treatment groups did not change relative to the 60-kDa band observed with the _1B-AR antisense ODNs treatment and indicate the specificity of the ODNs. To determine whether the effect was specific for _1B-AR protein expression, the blots were stripped and reprobed with mouse anti--actin monoclonal antibody. Similar intensities of the 45-kDa bands were observed with membranes obtained from control and sense- and antisense-treated cells. Fig. 3 (bottom) demonstrates that cells treated with antisense ODNs to _1A- and _1D-AR subtypes had no effect on expression of _1B-AR. The ratio of _1B-AR protein to -actin was equivalent for all three lanes and supports the specificity of antisense ODN treatment for specific subtypes.

View larger version (64K): [in this window] [in a new window]   Fig. 3.   Analysis of 1B-AR protein expression in PT cells in a Western blot with protein obtained from S1 PT cells. Control cells were permeabilized but received no ODNs, whereas antisense and sense were permeabilized and treated with 5 µM 1B-AR ODNs daily for 3 days (top). A band of 60 kDa is observed in control and sense lanes; the blot was stripped and reprobed for -actin, and a 45-kDa band of similar intensity was observed in each lane. When cells were treated with 1A- and 1D-AR antisense ODNs, there was no change in protein expression of the 60-kDa band of 1B-AR relative to control (bottom).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Antisense ODNs inhibit 1B-AR protein expression in PT cells. Protein expression for 1B-AR and -actin was determined with Western blot analysis for membrane protein obtained from control and sense- and antisense-treated cells. The intensity of each band was determined densitometrically, and the ratio for 1B-AR to -actin was calculated for sense ODN-treated cells at 72 hr and antisense to 1B-AR at 48 or 72 hr. Bars, percent change from the ratio determined for control cells represents mean ± standard error for three separate determinations.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   AR subtypes 1A and 1B but not 1D regulate PHE-induced changes in pHi (A) and represent the majority of 1-AR expressed in PT cells (B). Bars, mean ± standard error for four to seven separate experiments for inhibition of the PHE-induced increase of pHi compared with that observed in control cells (no ODN treatment). Sense and antisense ODN-treated cells were permeabilized and incubated with ODNs three times over 3 days. To assess the relative amount of 1-AR subtypes expressed in PT cells, subtype-selective antisense ODNs were used to inhibit expression of 1-AR subtypes, and the reduction in labeled binding sites was measured with BODIPY FL prazosin. Bars, mean of four to six independent experiments with the fluorescence intensity determined in 10-15 cells on each slide.

Determination of ₁-AR subtypes that regulate NHE. As reported previously, ₁-ARs increase NHE in PT cells (29). Proximal cells treated with selective ₁-AR agonists exhibit a rapid increase in intracellular pH relative to the resting intracellular pH. Although pharmacological receptor antagonists provide some estimate of subtype, they are much less selective than the specific and transient knockout of receptor proteins achieved with antisense oligonucleotides. To assess the ₁-AR subtypes that increase NHE in PT cells, we treated cells with antisense specific for _1A-, _1B-, and _1D-AR subtypes. Cells were treated with 5 µM concentrations of antisense ODNs three times over 72 hr because this treatment produced a maximal reduction in _1B-AR protein expression. As shown in Fig. 5, proximal cells exposed to varying concentrations of the ₁-AR agonist PHE produced concentration-dependent increases in pH_i. Basal pH_i of immortalized proximal cells was 7.08 ± 0.01, and on exposure to 1 µM PHE, a maximal increase of 0.35 ± 0.02 pH units was observed (17 independent experiments). The dose-dependent increase in pH_i was similar for cells treated with _1B-AR sense ODNs compared with control cells but was reduced by 46% in cells treated with _1B-AR antisense ODNs.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Dose-response curves for the 1-AR agonist PHE in control (no ODNs) and 1B antisense and sense ODN-treated cells. Cells were grown onto glass coverslips, permeabilized, and treated with ODNs three times over 3 days. Points, pHi measured with BCECF-AM in response to PHE addition (mean ± standard error for three separate sets of experiments). Maximum increases of pHi occurred with 1 µM final concentrations of PHE.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Tracings of pHi responses to PHE in control and antisense ODN-treated PT cells. Control cells were permeabilized but received no ODNs. Proximal S1 cells were treated three times over 72 hr with 1A, 1B, 1D, or 1A plus 1B antisense ODNs at final concentrations of 5 µM. A basal pHi was determined for 2 min before the addition of 1 µM PHE. After exposure to PHE, calibration was performed with 10 µM valinomycin and buffers of pH 6.5-7.6.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Summary of basal and PHE-induced pHi changes in control and antisense (AS) ODN-treated cells (A) and treatment with subtype-selective 1-AR antagonists (B). Cells exposed to antisense ODNs were treated three times over 3 days. Bars, mean ± standard error for six to nine separate experiments for basal pHi and the change of pHi that occurred with addition of 1 µM PHE. B, Cells were treated with the 1A-antagonist WB-4101 (1 µM), the 1B-antagonist spiperone (1 µM), or the 1D-AR antagonist BMY 7378 (1 µM). Bars, mean of four or five independent experiments with each treatment. *, p < 0.01 compared with PHE-induced change in control cells. **, p < 0.05 compared with PHE-induced change in cells treated with 1A or 1B antisense ODNs or antagonists alone.

	Discussion

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Catecholamines bind and activate adrenergic receptors in the kidney, where they mediate effects on tubular transport, metabolism, blood flow, and release of renin (see Ref. 5 for a review). The localization of ₁-AR expression in the kidney has resulted in conflicting reports concerning the distribution of ₁-AR subtypes. Meister et al. (14) report that mRNA for _1A-AR is localized primarily to vessels of the renal parenchyma and _1B-AR mRNA is confined to outer and inner stripe of the medulla in S3 proximal segments and thick ascending limb. Feng et al. (15) report that mRNAs for all three ₁-AR subtypes are expressed in PTs. Gopalakrishnan el al. (18) identify only _1A- and _1B-AR subtypes with radioligand binding in PTs; subsequently, they reported that _1B-ARs increase Na⁺/K⁺-ATPase activity in the PT, whereas _1A-ARs are linked to tubular inositol trisphosphate production and protein kinase C activation (19). Earlier studies that demonstrate ₁-ARs increase NHE in PT cells do not identify the particular receptor subtypes that mediate enhanced exchange activity (29). The purpose of the current study was to determine the ₁-AR subtypes that regulate NHE in PT cells.

Three ₁-AR subtypes have been cloned (see Ref. 31 for a review); all three cloned subtypes bind prazosin (11). Drugs with selectivity for _1A-AR over _1B-AR include 5-methyl-urapidil, (+)-niguldipine, SZL-49, and WB 4101. There is some indication that BMY 7378 may exhibit selectivity for _1D- over _1A- and _1B-AR, whereas only CEC seems to exhibit selectivity for _1B relative to _1A and _1D, with the profile of alkylation and inactivation: _1B > _1D > _1A (11). The pharmacological agents currently available do not sufficiently discriminate ₁-AR subtypes. Two agents that bind with selectivity to _1A-AR (i.e., niguldipine and 5-methyl-urapadil) also bind L-type Ca²⁺ channels and 5-HT_1A receptors, respectively (31). Decreases in urine flow rate and Na⁺ excretion induced by PHE in Sprague-Dawley rats are abolished by pretreatment with CEC but not SZL-49 and suggest that these effects are mediated by _1B-AR (32). In comparison, PHE-induced reductions in urine volume and absolute and fractional sodium excretion in Wistar and stroke-prone spontaneously hypertensive rats are blocked by 5-methyl-urapadil (33). These findings suggest that _1A-AR mediate the increase in Na⁺ and water absorption. Other subtype-selective effects of ₁-AR in kidney have also been reported (34).

In human kidney, detection of mRNAs for ₁-AR subtypes is somewhat controversial. Some studies discern _1A-AR message by RNase protection assays but not RT-PCR (35, 36). It is estimated that the _1A-AR subtype may constitute up to 45% of all ₁-AR mRNA in the kidney (see Ref. 37 for a review). In rats, message for _1B-AR is detected in outer and inner stripes and PT (14, 15). In comparison, several binding studies detect _1A- and _1B-AR protein in kidney, with a predominate localization on the PT (15, 18). The _1D-AR subtype is the least abundant form in human kidney (37). In rats, message expression of the _1D-AR subtype is detected only in intrarenal blood vessels (14).

As demonstrated in Figs. 1 and 2, we detected transcripts for all three ₁-AR subtypes in primary cultures of PT cells and in the immortalized proximal S1 cell line. These findings are consistent with those reported by Feng et al. (15). Through the use of RT-PCR and CEC-sensitive and -insensitive binding, they concluded all three ₁-AR subtypes are expressed in rat PTs. One must note that the findings of Feng et al. (15) do not demonstrate protein for the _1D-AR subtype in PT cells, so it is difficult to determine whether protein for this subtype is expressed in PT cells. In comparison, Gopalakrishnan et al. (18) provide binding data that only _1A- and _1B-ARs are present in rat renal PTs. Based on [³H]prazosin binding and competition studies with selective antagonists, they report equal distributions of _1A- and _1B-ARs. Although transcripts for all three ₁-AR subtypes are observed, the results of the functional studies support the presence of only _1A- and _1B-ARs.

To identify the ₁-AR subtypes that regulate NHE in PT cells, two strategies were used and involved (i) antisense ODNs designed to regions of poor conservation among the ₁-AR subtypes to inhibit expression of selected receptor subtypes and (ii) subtype-selective ₁-AR antagonists. Antisense ODNs were used to inhibit gene expression of specific ₁-AR subtypes and circumvent the relative specificity of pharmacological antagonists. This problem is noted as being particularly significant for _1A- and _1B-ARs because few ligands exhibit sufficient selectivity for these receptor subtypes to permit unambiguous detection with radioligand binding techniques (10). In the current study, phosphorothioate ODNs were used because the phosphorothioate linkage affords resistance to intracellular nuclease degradation (38). Antisense and sense ODNs of 18 nucleotides were chosen because reductions in length result in both decreased activity and affinity (38). Oligonucleotides were introduced into PT cells using a transient permeabilization with streptolysin-O as reported previously (23). In addition, the use of cell-permeabilization reagents may help to release ODNs from endosomal vesicles and enhance entry to the nucleus (38). Based on preliminary dose and time course studies, we determined that a final concentration of 5 µM for ODNs and multiple treatments was necessary to achieve a maximal inhibition of protein expression (Figs. 3 and 4) with a minimal loss of cell number or viability. In general, minimal toxicity is associated with phosphorothioate-substituted ODNs and only at concentrations well above those needed to produce specific effects (38). Based on the unavailability of antibodies selective for _1A- and _1D-ARs and the degree of inhibition observed on _1B-AR protein expression, equivalent ODN treatments were performed for _1A- and _1D-AR subtypes. Preliminary studies for each subtype indicated that additional treatments or increased concentrations did not enhance the degree of functional inhibition observed for each subtype.

Antisense ODNs designed to a specific sequence of the _1B-AR selectively inhibit the expression of this receptor subtype protein by 90% (Figs. 3 and 4). The fact that antisense but not sense ODNs inhibit protein expression and functional changes of pH_i is consistent with the selectivity of the antisense ODN approach. The observation that a virtually complete inhibition of the _1B-AR subtype reduces NHE by only 50% suggests more than one ₁-AR subtype mediates stimulation of NHE. Nonselective antagonists, such as prazosin, that completely inhibit ₁-AR agonist-induced increases of NHE presumably do so through actions on more than one ₁-AR subtype (29). The finding that _1A- and _1B-ARs each regulate 50% of ₁-AR stimulated NHE (Fig. 7) is consistent with stimulation of Na⁺/K⁺-ATPase by these subtypes in the PT (19). The finding of _1D-AR message expression in proximal by Feng et al. (15) agrees with our observations that _1D-AR transcripts are present in this segment. The data presented in Fig. 7 indicate that antisense ODNs and pharmacological antagonists for the _1D-AR subtype have minimal effects on ₁-AR-stimulated changes in NHE. To estimate the relative expression of ₁-AR subtypes, we treated cells with antisense ODN for each receptor subtype and measured binding of the fluorescent ligand BODIPY FL prazosin. Changes in fluorescence intensity were estimated with image analysis of 10-15 separate cells on four independent slides. Background fluorescence was determined with 100-fold competition with unlabeled prazosin. As depicted in Fig. 8, antisense ODNs reduced BODIPY FL prazosin fluorescence by 41%, 34%, and 18% of _1A-, _1B-, and _1D-AR subtypes, respectively. When cells were treated with combined _1A/_1B AR antisense ODNs, approximately two thirds of labeled sites were reduced. Although these studies do not demonstrate conclusively the presence or absence of _1D-AR protein expression, we provide compelling evidence (Figs. 7 and 8) with antisense ODN inhibition of expression and pharmacological antagonists that this subtype does not seem to have a significant effect on NHE. Whether protein for this subtype is expressed in the PT remains to be determined. Studies in human kidney indicate this is the least abundant subtype and is detected only in intrarenal blood vessels of rat kidney with in situ hybridization (14). Hence, the apparent lack of effect of _1D-ARs in regulation of NHE may relate to the very low or lack of protein expression for this subtype in the PT.

The mechanism through which ₁-ARs activate NHE in PT cells is likely to be increases in intracellular Ca²⁺ and inositol trisphosphate formation that lead to activation of protein kinase C (39). Several studies show that NHE in PT cells is regulated by protein kinase C (40). The increase of ₁-AR agonist-induced intracellular second messengers is abolished with the ₁-AR antagonist prazosin or the phospholipase C inhibitor U-73122 but not pertussis toxin (39).

In summary, several studies demonstrate that ₁-ARs increase NHE in PT cells (29). The particular ₁-AR subtypes that regulate NHE have not been identified. We provide pharmacological and molecular classification of ₁-AR subtypes present in mouse PT cells. Although we identified transcripts for all three subtypes in PT cells, the use of antisense ODNs to inhibit protein expression and subtype-selective pharmacological antagonists indicate only _1A- and _1B-ARs regulate NHE. We conclude that message and protein for _1A- and _1B-ARs are expressed in PT cells and activation of these receptors lead to increased NHE. Furthermore, the two subtypes seem to contribute equally to regulation of NHE. Finally, the observation that _1D -ARs do not regulate NHE in these cells probably relates to the absence of protein expression for this subtype in PT cells.