INTRODUCTION

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DOPAMINE HAS A MARKED natriuretic and diuretic action in human, rats, and other species (14), and the hormone is clinically useful for treatment of shock and heart failure because of these properties (18, 27). Although the hemodynamic effects of dopamine may be the major determinant of excretory changes, direct actions of exogenous and endogenous dopamine on the nephron are also important (2, 14, 23, 37). Renal dopamine excretion has been shown to increase in proportion to the natriuresis produced by a salt load (18, 27), and both the natriuresis and dopamine excretion are diminished by inhibition of renal dopamine synthesis (14). It is now widely accepted that the source of this dopamine is not the renal nerves but that the dopamine is synthesized primarily by proximal tubules, which possess the necessary metabolic pathways (14), and that this dopamine acts as an autocrine or paracrine regulator of Na⁺ reabsorption (14, 36).

Most studies of the tubular effects of dopamine have centered on the proximal tubule where dopamine inhibits volume reabsorption by inhibiting both the activity of the Na⁺/H⁺ antiporter and the Na-K-ATPase (7, 12, 13, 14). These effects of dopamine in the proximal tubule are mediated by the D_1A dopamine receptor isoform in rats and mice (equivalent to the human D₁ receptor). This receptor is coupled to the G protein, G_s, which stimulates cAMP production and inhibits the Na⁺/H⁺ antiporter (6, 12, 14). The D_1A receptor is also coupled to phospholipase A₂ (PLA₂) and phospholipase C activation, which inhibits Na-K-ATPase activity (14, 30).

Several studies have shown that the normal inhibitory action of dopamine on salt and water reabsorption by the kidney is important in the maintenance of normal blood pressure (14). For example, Albrecht et al. (1) showed that defective coupling of the D_1A receptor to the inhibition of the Na⁺/H⁺ antiporter in renal proximal tubules cosegregated with hypertension in the F2 generation of crosses between spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. The same investigators showed that mice lacking functional D_1A receptors had elevated systolic and diastolic blood pressures that were greater in the mice that were homozygous than in those that were heterozygous for the defective receptor gene (1).

Dopamine also inhibits Na⁺ and water reabsorption in the cortical collecting duct (CCD). In the isolated perfused rabbit CCD, Muto et al. (23) showed that dopamine inhibited the increase in osmotic water permeability (P_f) with arginine vasopressin (AVP) and ascribed this effect to a D₂ dopamine receptor, because it could be prevented or reversed by metoclopramide, an inhibitor of members of the D₂ family of dopamine receptors. (At the time, there was no subclassification of D₂ receptors; so any of them, which includes the D₂, D₃, and D₄ isoforms, could have been inhibited.) Binding studies and RT-PCR have demonstrated both D₂-type and D₁-type receptors (D_1A and D_1B in rat and mouse) in glomeruli and medullary tissue as well as in proximal tubules (10, 14); however, it has not been possible to demonstrate the presence of D₂-type receptors by Northern blot of mRNA from renal cortex (11), possibly because of their low relative abundance.

We have recently examined the effects of dopamine in the isolated perfused rat CCD and found that it inhibited AVP-dependent P_f, as well as the lumen-to-bath flux of Na⁺ (J_Na) and transepithelial voltage (V_T) (37). When P_f and J_Na were stimulated by 8-(4-chlorophenylthio)-cAMP, dopamine had no effect, which we interpreted to indicate that the action was mediated by a D₂-type receptor that was coupled to inhibition of adenylyl cyclase by the G_i-type G-protein.

In contrast, Satoh et al. (30, 31) have shown that dopamine increases cAMP production in isolated but nonperfused rat CCD via a D₁-type receptor, which in turn inhibits Na-K-ATPase activity and might be expected to inhibit J_Na. In our studies, we found no effect of the D₁ agonists fenoldopam or SKF-81297 on P_f or J_Na in the presence or absence of AVP, and the effects of dopamine in the presence of AVP were not reversed by the D_1A antagonist Sch-23390 (37). The D₂ receptor-specific agonist quinpirole also had no effect on the AVP-dependent V_T, and the D₂ and D₃ antagonists domperidone and pimozide did not reverse the dopamine inhibition of V_T. Only clozapine, a D₄-specific antagonist, reversed the effects of dopamine on AVP-dependent P_f, J_Na, and V_T (37). In more recent studies in which cAMP generation was measured in intact rat CCD segments, we found that neither dopamine nor fenoldopam stimulated cAMP production in the absence of AVP. On the other hand, dopamine inhibited AVP-dependent cAMP production by ~50%, and this inhibition was reversed by clozapine but not by other D₂- and D₁-type antagonists (16). Thus we concluded that the inhibition of AVP-dependent transepithelial Na⁺ and water transport in the rat CCD by dopamine is mediated via a D₄ receptor.

The present studies were undertaken to determine, using RT-PCR and immunohistochemistry, whether D_1A and D₄ dopamine receptors are expressed in the rat CCD and thus, ultimately, to identify the alternate signaling pathways that may be involved in the regulation of Na⁺ and water transport in this nephron segment.

	METHODS

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Male Sprague-Dawley rats obtained from Harlan Sprague Dawley (Indianapolis, IN) were maintained on a regular 16% protein rodent diet (diet 8746; Teklad, Madison, WI) containing 0.35% NaCl (measured in our laboratory) for 2-4 wk, at which time they were 5-7 wk of age and weighed 70-170 g. Other rats, used for isolated perfused CCD experiments, were maintained on this diet and also were treated with deoxycorticosterone (DOC) to increase the Na⁺ reabsorptive rate and thus increase the ability to observe change is V_T with luminal dopamine treatment (4). A slow-release (3-wk) pellet containing 2.5 mg of DOC was implanted subcutaneously in the dorsum of these rats. It should be noted that DOC treatment was used only for experiments with isolated perfused renal tubules and not for those involving RT-PCR, immunohistochemistry, or immunoblotting.

In the RT-PCR experiments we initially microdissected CCD segments from slices of kidney that had been digested with collagenase according to standard methods (see Ref. 34) and obtained samples of 2-20 mm total tubule length. Although we have been able to amplify mRNA from 2 mm of CCD for several targets, we could not easily obtain sufficient quantities to detect mRNA for the apparently less abundant dopamine D_1A and D₄ receptors. During this time, we developed a modified collagenase digestion technique for dissecting larger numbers of nephron segments, which has been described in detail (34).

Here, we will describe the method in brief. After a kidney was removed and the capsule was stripped away, a Thomas-Stadie-Riggs tissue slicer (Thomas Scientific, Swedeboro, NJ) was used to cut sections 0.5- to 1.0-mm thick tangential to the cortical surface. The slices were then torn into small chunks. These pieces of tissue were incubated in 2 ml of warm MEM containing 0.5 mg/ml of class 2 collagenase, 5 mM glycine, 50 U/ml DNase, and 48 µg/ml soybean trypsin inhibitor in a 20-ml scintillation vial. [All reagents were from Sigma Chemical (St. Louis, MO), with the exception of the collagenase (catalog no. 4176), which was obtained from Worthington Biochemical (Freehold, NJ)]. The cortical pieces were briefly (<5 s) and very gently agitated by hand and incubated without shaking at 37°C. At ~15-min intervals, the suspension was gently agitated, and the cloudy, tubule-rich supernatant was poured off the sedimented tissue into a 5 ml ice-cold test tube. The tubule segments sedimented rapidly in the test tube, and the supernatant was removed with a Pasteur pipette and replaced with 2 ml of ice-cold, enzyme-free MEM containing 1% BSA. CCD segments were manually sorted at 4°C from the resulting suspension of nephron segments. Using this method, one investigator could sort over 100 mm of CCD segments from a single kidney within 1-2 h.

Groups of sorted CCD segments were transferred in 5-10 µl of the enzyme-free MEM into 1.0 ml guanidinium/acid phenol solution for RNA extraction according to the TRIzol (Life Technologies, Gaithersburg, MD) protocol. RNA was also extracted from rat brain and atria to serve as positive controls, because it is known that both D_1A and D₄ isoforms are expressed in these tissues (22, 25, 44). The total RNA was treated with DNase I to remove any contaminating DNA. One aliquot of the RNA was reverse transcribed with SuperScript II (Life Technologies) using random hexamer primers. Another aliquot of the RNA was treated in the same way but in the absence of the reverse transcriptase. This sample served as a control for genomic contamination in subsequent PCR reactions.

Samples of the cDNA were initially amplified by PCR with primers to glyceraldehyde-3-phosphate dehydrogenase to verify the integrity and relative quantity of cDNA. Each 50-µl PCR reaction used 5% of the cDNA obtained from reverse transcription reactions, representing total mRNA from 5-10 mm (equivalent to ~4,000 to 8,000 cells) of the microdissected tubules. Specific primers were designed for the rat dopamine D_1A and D₄ receptor isoforms based on their published sequences (25, 44). As shown in Fig. 1, we used three primer pairs to amplify D₄ regions covering most of the coding sequence: Transmembrane 2 (tm2) to tm3, the second cytoplasmic loop (c2) to tm5, and c3 to tm7, respectively. Each primer set spanned an intron to ensure genomic DNA was not amplified. (Note that extracted RNA samples were also treated with DNase I.) Two primer pairs were designed for the D_1A dopamine receptor (Fig. 1, bottom): 1) tm7 into the 3'-untranslated region and 2) the third cytoplasmic loop (tm5 to tm6). The D_1A coding region has a single short intron in the 5'-untranslated region (not shown in Fig. 1), which was not reported in the original sequence but which was identified subsequently (22, 44, 45). The PCR products were initially verified by restriction enzyme mapping (Table 1) and subsequently by sequencing.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Location of PCR primer pairs for D4 and D1A dopaminergic receptor isoform cDNA. Diagrams show the organization of the corresponding receptor mRNA. The seven transmembrane coding regions are shown as solid black, the 3'-untranslated region is shaded in gray, and the location of genomic introns is shown by vertical lines. The coding region of the D1A gene has a single intron in the 5'-untranslated region (45), which is not shown. Forward primers are shown by "" with a number indicating the 5' end of the amplified region. Reverse primers are shown by "" accompanied by a number indicating the 3' end of the amplified region. The total lengths of the products predicted from the published mRNA sequences are indicated by the numbers in parentheses.

PCR reaction mixtures contained 2.5 mM MgCl₂, 50 mM KCl, 20 mM Tris · HCl (pH 8.4), 1.7% BSA, 0.05 mM of each deoxynucleoside 5'-triphosphate, 25 pmol of each oligonucleotide primer, and 1 U of Taq DNA polymerase (Promega, Madison, WI; and Fisher Scientific, Pittsburgh, PA) per 50 µl of total reaction volume. To amplify dopamine receptor cDNA, we used 48 cycles (94°C for 1 min, 60°C for 1 min, and 72°C for 1 min) followed by a final 7-min extension at 72°C. When PCR products were to be cloned into the pCR 2.1 plasmid (see below), extension at 72°C was increased to 60 min to increase cloning efficiency (17). The PCR products were resolved on a 2% agarose gel. For restriction analysis, PCR products were ethanol precipitated, then resuspended in sterile water. Purified products were digested with the restriction endonucleases (Table 1) according to the manufacturer's instructions.

When the presence of an isoform was indicated by restriction enzyme analysis, the PCR product was cloned into the plasmid vector pCR 2.1 by TA cloning (Invitrogen, San Diego, CA) according to manufacturer's instructions. Bidirectional sequence analysis was performed using the T7 and M13 universal primers and dye-termination reactions at the University of Alabama DNA Sequencing Core Facility (Dr. S. Hollingshead, Director). The sequences were aligned with the appropriate GenBank sequences using the GAP program of the Wisconsin Sequence Analysis Package (version 8; Genetics Computer Group, Madison, WI).

Antibodies for immunoblotting and immunohistochemistry of D₄ and D_1A dopamine receptors. We examined the distribution of the D₄ dopamine receptor using an anti-D₄ polyclonal antibody (catalog no. AS-3545G, lot 7805; R & D Antibodies, Berkeley, CA), which had been raised in rabbit against a peptide corresponding to the second extracellular loop (amino acids 176-185) of the human receptor. [This corresponds to amino acids 169-178 of the published rat D₄ sequence (accession no. 231977), also in the second extracellular loop.] The D₄ antibody was supplied and used as the IgG serum fraction. According to the manufacturer, it was not possible to affinity purify this antibody, because removal from the affinity column results in denaturation. Specificity of antibodies was verified by the supplier (R & D Antibodies) using immunoblotting and immunohistochemical localization to rat brain areas known to express the D₄ receptor. We have further confirmed the specificity of this D₄ receptor antibody by immunoblotting (see below).

Measurement of V_T in isolated perfused CCD. For these experiments, CCD segments were dissected from kidneys without collagenase treatment and were perfused in vitro using an isotonic artificial bathing solution similar to rat serum and a hypotonic perfusate resembling early distal tubular fluid. [Methoxy-³H]inulin was added to the perfusate as the volume marker. Perfusion at 10 to 20 nl/min was carried out at 38°C. V_T was measured between Ag/AgCl electrodes in the perfusate and bathing solution and was recorded continuously on a strip chart recorder.

	RESULTS

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RT-PCR analyses were conducted for dopamine D_1A and D₄ isoforms in the rat CCD with, respectively, seven and eight separate groups of CCD from which RNA was extracted. Based on previous results in isolated, perfused CCD in our laboratory (37) and in nonperfused CCD segments in the laboratory of Katz and colleagues (30, 31, 32), mRNA for both D_1A and D₄ dopamine receptors was expected to be present. In our early experiments, in which RNA was extracted from <20 mm of CCD, we detected either isoform only infrequently. In subsequent studies, we extracted RNA from 100-200 mm of CCD, and thus RNA from ~4,000 to 8,000 cells was amplified. As summarized in Table 2, single PCR products of the expected size were readily observed for both isoforms, with a single exception in the case of each.

The first set of D₄ primers (161-bp product, Table 1) restricted as anticipated with Acc I and Ban II and was detected in CCDs in seven of eight RNA extractions. The PCR product was cloned into the pCR 2.1 plasmid vector, and bidirectional sequence analysis indicated 100% nucleotide identity with the published sequence from rat atria (25); however, there was an additional 6-bp (GTCCAG) insert within our PCR product precisely at the site of the first intron. As shown in Fig. 2, our PCR sequence is identical to the mouse and human D₄ receptor sequences in this region, and our 6-bp sequence matches the beginning of exon 2 in those species (9, 40). Our sequence was also confirmed by the fact that the extra six bases introduce an Ava II restriction site in the PCR product, and we obtained products of the size predicted by the human and mouse sequences (9, 40). We used the same primer pair to amplify cDNA from rat atria and brain and confirmed the presence of the same 6-bp insert by Ava II restriction and sequence analysis (data not shown). In the rat sequence published by O'Malley et al. (25), the same nucleotide sequence was included as the ending of the first intron, but subsequent sequencing of the rat D₄ cDNA by Dr. O'Malley's laboratory revealed the presence of the same additional 6 bases (personal communication) in the exon (see DISCUSSION).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Comparison of a region of the rat D4 receptor obtained from our sequenced PCR product with the rat D4 receptor sequence published by O'Malley et al. (25) and with the published mouse and human sequences (9, 40). Regions of difference among the sequences are given in the 2 boxes. The amino acid sequence is given in the last line. In all 3 of the previously published genomic sequences, there is an intron near the end of the second transmembrane region (underlined nucleotides) as indicated by the arrow. The sequence of the PCR product obtained in our experiments is identical in the region shown to the mouse and human sequences (with the exception of the C-for-T substitution in the human sequence that results in a conservative alanine-for-valine amino acid substitution).

The second set of D₄ receptor primers produced the expected 346-bp product, corresponding to the region from c3 to tm7, which restricted with Ava I, Ban I and Pst I. Identity of the PCR product was verified by bidirectional sequencing. The third set of primers produced the expected 213-bp product, from the c2 to the tm5 region, which restricted as predicted with Acc I, Nru I, and Pvu II (Fig. 3), and was verified by bidirectional sequencing.

View larger version (77K): [in this window] [in a new window]   Fig. 3.   Restriction analysis of the PCR product obtained using primers for the 3rd transmembrane region and the 3rd cytoplasmic loop of the D4 receptor isoform. Product was of the correct size and restriction pattern predicted. Values on left are band sizes in bp.

In experiments with the first set of D_1A receptor primers, the 527-bp product, corresponding to nucleotides from tm7 to the 3'-untranslated region, restricted with Ava I and Ban II (Fig. 4) as anticipated from the published sequence, and was detected in four of four experiments. The second primer set, corresponding to the third cytoplasmic loop (tm5-tm6) of the D_1A receptor isoform, was 258 bp and restricted as expected with Ban I and Bcl I. It was detected in three of four experiments. Both PCR products were verified by bidirectional sequencing.

View larger version (91K): [in this window] [in a new window]   Fig. 4.   Restriction analysis of the PCR product obtained using primers for the 7th transmembrane region and the 3'-untranslated region of the D1A receptor isoform. Product was of the correct size and was cut into the predicted fragments by the three restriction enzymes used. Values on left are band sizes in bp.

We initially attempted to confirm the presence of both receptors at the protein level by immunoblotting of protein extracted from microdissected CCD segments. Extraction of 100 mm of CCD yields slightly more than 10 µg of protein, and we have been successful in using such samples to demonstrate the presence of specific protein kinase C isoform proteins in the rat and rabbit CCD (41). However, despite repeated attempts using a variety of antibody dilutions, and blocking and development procedures, we were unable to obtain acceptable immunoblots of protein extracted from 100-140 mm of CCD segments using the D₄ antibody. (We did not examine the D_1A antibody from Dr. Carey, because its specificity had already been confirmed.) However, using protein extracted from rat heart, which has a 20-fold higher expression of the D₄ receptor mRNA than other tissues (24), and a greater amount of total protein extracted from whole rat kidney cortex than could be obtained from the microdissected CCD segments, the D₄ antibody reacted strongly with a band of the expected 41-42 kDa size of the nonglycosylated protein as shown in Fig. 5. There was also a heavy band of 58-59 kDa and a very faint band of ~48 kDa in the heart protein, but only the heavier band was present in the kidney cortex. Labeling of all three bands was blocked by preadsorption of the antibody to its antigenic peptide. Because the D₄ receptor protein has a glycosylation site, it is likely that the heavier band represents the glycosylated form of the protein and that this is the primary form found in the kidney cortex.

View larger version (68K): [in this window] [in a new window]   Fig. 5.   Immunoblot of D4 dopamine receptor probed with D4-specific serum IgG fraction (D4 Ab) from R & D Antibodies. A: three specific bands of 42, 49, and 59 kDa were observed for rat heart protein (lanes 1 and 2, loaded with 30 µg and 50 µg of protein, respectively) using 1:2,000 dilution of D4 Ab and 1:6,000 of the secondary antibody. For lanes 3 and 4, the D4 antibody was preadsorbed with a 5-fold molar excess of the peptide against which the antibody had been raised. Lane 5 shows labeling of 50 µg of protein extract with a 1:6,000 dilution of the secondary antibody (2°; donkey anti-rabbit IgG linked to horseradish peroxidase). B: immunoblot of protein extracted from rat heart (19 µg total protein loaded) and rat kidney cortex (45 µg). D4 Ab was used at 1:400. Lane 3 was exposed to the secondary antibody only. (The two dots in the middle of both images are reference marks for the 45- and 66-kDa molecular mass standard proteins.) Molecular weights of the bands were determined from a calibration curve plotted from the 45-, 66-, and 97-kDa bands of the Bio-Rad high-range molecular mass standards.

We then used this D₄ antibody and the D_1A antibody from Dr. Carey for immunohistochemistry on coronal slices of rat kidney. We found that the latter antibody labeled the tissue as described by O'Connell et al. (24). Labeling was observed primarily in the cortex and not the medulla. The cortical labeling was diffuse and light throughout proximal tubules, distal tubules, and CCD (data not shown). In contrast, the D₄ antibody labeled exclusively in the medulla and medullary rays in the cortex, where it is was specific to CCD segments and to the whole length of the medullary collecting ducts (Fig. 6, A and B). No staining was seen in any other segments. At high magnification (Fig. 6C), it appeared that the immunoreactivity was greater on the luminal surface than on the basolateral membrane.

View larger version (207K): [in this window] [in a new window]   Fig. 6.   Immunohistochemistry to localize binding of antibody reactive with the D4 receptor isoform in the rat kidney. A: low-power (×2) magnification of a section from near the cortical surface (top) to the inner medulla (bottom). Note staining of medullary collecting ducts and medullary rays extending into the cortex. B: localization of labeling to cortical collecting duct (CCD) in medullary rays (×12). Region shown is just above the corticomedullary junction. C: high-power (×60) magnification of staining in a CCD within a medullary ray. Section has been rotated 90° from the orientation in A and B.

In our previous studies (37), we had examined the functional effects of dopamine only when added to the bathing solution of isolated perfused rat CCD and not the lumen. Because of the apparent localization of immunoreactive D₄ protein on the luminal membrane, we conducted a series of experiments in which we examined the effect of luminal dopamine on V_T. In the five experiments shown in Fig. 7, AVP produced a significant hyperpolarization of V_T in the CCD from DOC-treated rats; however, the further addition of 100 µM dopamine to the luminal perfusate had no significant effect. Addition of 10 µM dopamine to the bathing solution significantly depolarized V_T, and the effect was reversed by the further addition of 10 µM clozapine to the bathing solution as reported previously (37). Finally, the removal of dopamine and clozapine from the bathing solution, thus returning to the conditions of the second period, had no effect on V_T. In summary, the only effect of dopamine on V_T was observed when it was added to the bathing solution, and that effect was reversed by clozapine.

View larger version (35K): [in this window] [in a new window]   Fig. 7.   Effect of dopamine added to the luminal versus bathing solutions on transepithelial voltage (VT). Voltages are presented as VT; i.e., all are lumen negative, although they are presented as positive values. Arginine vasopressin (AVP) was added (+) to the bathing solution in the 2nd period through the last period at 22 pM. Luminal dopamine (DA) was present at 100 µM from the third to the last period. Dopamine was added to the bathing solution at 10 µM in the 4th and 5th period, with 10 µM clozapine (cloz.) added in the 5th period. Average values for 5 experiments are presented. * Significantly different from preceding period mean at P < 0.01, determined by ANOVA with Scheffé posthoc test for significance.

	DISCUSSION

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The data presented above demonstrate the presence of mRNA for both the D_1A and D₄ dopamine receptor isoforms in the rat CCD (Table 2) and of protein immunoreactive with a D₄ antibody in the CCD and medullary collecting duct (Figs. 5 and 6). We also observed labeling of all cortical nephron segments with the D_1A antibody as described by O'Connell et al. (24). Other investigators have demonstrated the presence of D₁-type and specific D_1A and D₂ receptors by binding studies and RT-PCR of digests of rat renal cortex and medulla (10, 21, 25), as well as in microdissected and sieved rat proximal tubules and juxtaglomerular cells (8, 42, 43). A D₁-type receptor has also been found by binding studies in the rat CCD (39). RT-PCR has also revealed D₃ and D₄ receptor isoform mRNA in rat juxtaglomerular cells (29) and the D₄ isoform in human kidney mRNA (20). The fact that we had to use a relatively large sample of CCD segments to observe expression of either the D_1A or D₄ isoform by RT-PCR suggests that the mRNA is present in low copy number. This would also explain why the D₄ isoform was not observed by Northern blot analysis of RNA from whole kidney, especially considering that the isoform is confined to the CCD and medullary collecting duct (see Fig. 6).

We expected that the age of the rats used in these studies (5-7 wk) was appropriate for detecting at least the D_1A receptor. Kansra et al. (15) reported that there is a ~50% decrease in renal D_1A receptor density in old (23-24 mo) rats compared with adult rats (6 mo), and that there was also decreased expression of receptor-linked G protein in the older rats. Felder et al. (8) reported that there was no age-related difference in the maximum D₁ receptor density in proximal convoluted tubules from SHR and WKY rats examined at 3, 8, and 20 wk of age; however, there was an increase in dopamine-stimulated adenylyl cyclase activity and in the natriuretic effect of dopamine with age in the WKY rats. If the D₄ receptor density also declines with age, then this may explain why our functional studies with CCD from young rats have indicated the presence of this receptor (37), whereas there is no functional evidence for its presence in other studies in which older animals were used (e.g., Refs. 30, 38).

Sequencing with the first set of D₄ primers showed an extra 6 bp in comparison with the published rat D₄ sequence (25), corresponding to the beginning of the second exon in the mouse and human sequences (Fig. 2). Sequencing of the RT-PCR products from rat atria and brain RNA also showed the same 6-bp insert. We have since learned from Dr. K. O'Malley (personal communication) that her group has examined rat D₄ cDNA, and they also observed this 6-bp insert, which was originally overlooked, probably as the consequence of a mis-assigned splice acceptor site in the genomic sequence.

Immunohistochemistry supported the presence of the D₄ receptor isoform in the CCD and also in the medullary collecting duct (Fig. 6). We verified the specificity of the D₄ antibody by immunoblotting (Fig. 5), and we also supplied a sample of the same lot of the antibody to Dr. K. O'Malley (Washington Univ., St. Louis, MO) for additional testing. Dr. O'Malley's laboratory found (personal communication) that the antibody stained structures in the retina, primarily in the inner nuclear layer, in a pattern which was similar to that which they observed previously for the D₄ receptor mRNA by in situ hybridization (5). (The D₄ receptor in the retina has been demonstrated to be coupled to the modulation of the dark level of light-sensitive cAMP production in photoreceptors; Ref. 5). O'Malley's group (personal communication) also found that our D₄ antibody labeled a ~41-kDa protein on immunoblot analysis of proteins extracted from a cell line that had been stably transfected with the D₄ receptor message, but did not react with proteins from nontransfected cells.

Cortical labeling was seen only in the medullary rays and was associated exclusively with the CCD. Labeling of the medullary collecting duct was, if anything, more dense than in the CCD, and the entire length of the collecting duct showed the presence of the receptor. Maeda et al. (19) have examined the effect of dopamine of AVP-dependent cAMP production in a limited number (n = 3) of inner medullary collecting duct (IMCD) segments. Although they found no effect of dopamine at 0.01 or 1 µM, 100 µM produced a significant inhibition of cAMP production in the presence of 100 pM AVP. Maeda et al. (19) suggested that the effect of such a high dose might have been mediated by the ₂-adrenergic receptors that they observed in the IMCD in the same study; however, they did not test the effect of concentrations in the range of 1-10 µM that we have found to be the most effective (16, 37).

It was puzzling that in both the cortex and the medulla the labeling with the D₄ antibody appeared to be heavier on the luminal surface than on the basolateral surface. For that reason, we undertook experiments with isolated perfused CCD to see whether we could obtain an effect of luminal dopamine. As shown in Fig. 7, we were unable to detect any inhibition of AVP-dependent V_T in these experiments, so the function of the luminal receptors remains unclear.

The effects of dopamine on the distal regions of the nephron have been a matter of controversy. Muto et al. (23) showed that dopamine inhibited AVP-stimulated osmotic water permeability in the isolated perfused rabbit CCD presumably by a D₂-type receptor. On the other hand, Satoh et al. (30, 31, 32) have shown that dopamine decreases Na-K-ATPase activity in nonperfused rat CCD segments via a D₁-type receptor, which increases the production of cAMP and activates protein kinase A. Despite the fact that dopamine appears to decrease Na-K-ATPase activity in the basolateral membrane, it seems improbable to us that dopamine could inhibit transepithelial Na⁺ transport by such a mechanism, because AVP is known to stimulate Na⁺ reabsorption in the rat CCD by increasing intracellular cAMP, which thereby increases the Na⁺ conductance of the luminal membrane (4, 28, 33, 35). Thus it would seem that any effect of dopamine to decrease Na-K-ATPase activity through a D_1A receptor is superseded by the effect of cAMP on the luminal membrane Na⁺ channels.

As discussed in the introduction, we have demonstrated that dopamine inhibited the Na⁺ and water transport in isolated perfused rat CCD and the inhibition was mediated by a D₄-like receptor (37). In more recent experiments (16), we also examined the effect of dopamine and fenoldopam on cAMP generation in microdissected, intact CCD segments. We found that neither fenoldopam nor dopamine had any significant effect on cAMP generation in the absence of AVP. However, when cAMP generation was increased by AVP, dopamine but not fenoldopam inhibited this increase, and this effect was reversed by 10 µM clozapine (16). Clozapine also has limited affinity for serotonin (5-HT) receptors; however, the 5-HT₁ subgroup of these receptors, the subgroup that inhibits cAMP production, is not affected by dopamine.

There are two caveats to our interpretation of these results. First, our studies were conducted in young (5- to 7-wk-old) rats. Although Felder et al. (8) observed no difference in D_1A receptor density in proximal tubules from the WKY rat, they did observe diminished cAMP production in response to dopamine in the younger rats, which might explain our inability to demonstrate an increase in cAMP generation with fenoldopam or dopamine. The second caveat is that the CCD used in our previous studies with isolated perfused segments came primarily from DOC-treated rats (37). One set of experiments in that study did examine CCD from rats that were untreated with DOC and showed the same pattern of response to dopamine and clozapine, with no effect of fenoldopam. Our studies on cAMP production were also conducted on CCD from rats receiving no DOC treatment (16), as were all of the experiments in the present study, with the exception of those reported in Fig. 7.

It is also possible that the D₄ receptors could also be associated with alternative intracellular coupling mechanisms. Although the D₄ receptor has been linked to inhibition of adenylyl cyclase (5) as have the other D₂-type receptors, when the D₂ receptor is expressed in CHO cells, dopamine and the D₂ receptor agonist quinpirole stimulate PLA₂ (26). It seems unlikely, however, that this is the mechanism of dopamine action in the rat CCD. Quinpirole has no effect on transepithelial transport process stimulated by AVP (37), and we have found that prostaglandin E₂ is not inhibitory to either Na⁺ transport or water permeability in the rat CCD (3).

Based on the present data as well as our past studies, (16, 37), we conclude that both the D_1A and D₄ dopamine receptors are expressed in the rat CCD. The effects mediated by the D_1A receptor have been observed only in nonperfused preparations in which transepithelial transport was not measured, and any effects of this receptor do not override the effect of dopamine on transepithelial transport in the presence of AVP. Thus we conclude from the current data and previous functional studies (16, 37) that the diuretic and natriuretic effects of dopamine in the CCD are primarily mediated by the D₄ receptor.