INTRODUCTION

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RENAL ADRENERGIC NERVES and circulating catecholamines are involved in the regulation of Na⁺ and water excretion in the kidney not only by their effects on hemodynamics but also on epithelial transport processes along the nephron. It has long been recognized that renal denervation and -adrenoceptor (-AR) blockade during dietary Na⁺ restriction or volume depletion produce natriuresis and diuresis in the absence of changes in renal hemodynamics or glomerular filtration (e.g., 1, 18, 36). Conversely, increased efferent renal sympathetic nerve activity (ERSNA) and ₁-AR agonists, at levels that have no effect on renal hemodynamics or glomerular filtration, reversibly decrease Na⁺ excretion and produce antidiuresis (e.g., 18, 29, 35).

The anatomical basis for such effects comes from the work of Barajas and co-workers (see Ref. 2 for additional references), who convincingly demonstrated that, in addition to the afferent and efferent arterioles, virtually all segments of the nephron are innervated by adrenergic nerves. The neuroeffector junctions are generally regarded to involve norepinephrine as the neurotransmitter and ₁-postsynaptic receptors (18), which are also the dominant type in the renal vasculature (39). Binding studies with radiolabeled prazosin in microdissected tubule segments have demonstrated the presence of ₁-ARs along most of the nephron, with the exception of the cortical and outer medullary collecting ducts (7). ₂-ARs are also widely distributed. In the proximal tubule (PT), radioligand binding studies show ₁- and ₂-ARs predominantly on the basolateral membrane (25, 41). ₂-ARs are also found in distal nephron segments (41).

In contrast to ₁-ARs, the ₂-ARs are regarded to be primarily extrajunctional and thus responsive to circulating catecholamines (28, 38). These receptors may be the target of norepinephrine that is released from adrenergic nerve endings and which spills over into the circulation. Increased ERSNA at levels that have no hemodynamic effect produces a rise in renal venous concentrations of both norepinephrine and dopamine sufficient to activate their respective receptors, and the magnitude of this spillover is directly correlated with ERSNA (9, 18).

The consequences of renal venous catecholamine spillover and of ₂-AR activation in the regulation of Na⁺ and water excretion remain unclear, but two types of observations suggest they have an important role. First, renal venous norepinephrine spillover increases in human subjects with hypertension, congestive heart failure, hepatic cirrhosis, and Na⁺ depletion (see Ref. 18 for additional references). Second, renal ₂-AR density is increased in most, if not all, animal models of hypertension (18, 40), and ₂-AR density is increased in normal and Dahl salt-sensitive rats when they are given a high-salt diet or when ₁-ARs are blocked by prazosin (27, 37). Because of the predominant effect of ₂-ARs to inhibit adenylyl cyclase via a G_i protein (18, 38), it is generally believed that ₂-ARs exert their predominant influence by opposing the effects of hormones that act on renal epithelial cells through cAMP.

We have been interested in the effects of ₂-ARs on Na⁺ and water transport in the cortical collecting duct (CCD), where their activation has been shown to inhibit cAMP production in response to arginine vasopressin (AVP) (3, 12, 42). In the isolated perfused CCD and inner medullary collecting duct, ₂-AR agonists have been shown to inhibit the osmotic water permeability (P_f) response to AVP (14, 16, 19, 33). In the rat CCD, where AVP produces an increase in Na⁺ reabsorption that is synergistic with the effect of aldosterone or deoxycorticosterone (DOC) (5, 15), we found that the lumen-to-bath Na⁺ flux (J_Na) was reversibly inhibited by clonidine or epinephrine (14, 16). Epinephrine at 100 nM in the bathing solution inhibited net Na⁺ transport almost completely, and this effect was prevented or reversed by 1 µM yohimbine, an ₂-AR antagonist (14). However, three additional findings suggested that the effects of the adrenoceptors involve mechanisms in addition to inhibition of adenylyl cyclase via G_i coupling. First, epinephrine inhibited not only the AVP-dependent increase in J_Na but also the DOC-dependent J_Na (14). Second, when P_f and J_Na were stimulated by forskolin or 8-Br-cAMP plus IBMX, 100 nM epinephrine still produced a 30-40% inhibition of J_Na and P_f. Third, the effect of epinephrine in the CCD could be partially blocked by the ₁-AR antagonist corynanthine (14), although the specificity of this antagonist is now questionable (22). These observations led us to conclude that, although the primary inhibitory effect of epinephrine on P_f and J_Na was mediated by an ₂-AR acting via a G_i protein, there must be an additional second messenger pathway that was either coupled to the same ₂-AR, a second ₂-AR, or possibly an ₁-AR (14). There are now three known ₂-ARs: _2A, _2B, and _2C (45). All three isoforms have been shown to act via G_i to inhibit cAMP production (10). It has also been shown that _2A- and _2C-AR, when expressed in CHO cells, not only inhibit adenylyl cyclase but also stimulate phosphoinositide hydrolysis (8).

The present studies were undertaken to determine which -ARs are expressed in the rat CCD and thus ultimately to identify alternate signaling pathways that may be involved in the regulation of Na⁺ and water transport in this nephron segment. We also performed experiments with isolated perfused CCD to obtain evidence of a functional ₁-adrenoceptor effect.

	METHODS

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Male Sprague-Dawley rats were obtained from Harlan Sprague-Dawley, (Indianapolis, IN). The rats were maintained on a regular 16% protein rodent diet (diet 8746; Teklad, Madison, WI) containing 0.53% NaCl (measured in our laboratory) for 2-4 wk at which time their weight was from 70 to 170 g.

RT-PCR experiments were performed with total RNA from microdissected CCD and PT fragments (~20 mm total length per RT reaction) using a guanidinium/acid phenol extraction procedure (TRIzol; Life Technologies, Gaithersburg, MD). RNA was also extracted from rat brain to serve as positive controls. The RNA samples were treated with DNase I and then reverse transcribed with SuperScript II using oligo(dT)_12-18 primers (Life Technologies) to form single-strand cDNA. The cDNA was initially amplified in PCR reactions with primers to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), to verify the integrity and relative quantity of cDNA. Aliquots of RNA that were not reverse transcribed were included in PCR reactions to verify the absence of genomic DNA. Each 50-µl PCR reaction used 5% of the cDNA obtained from RT reactions, representing total mRNA from 1 mm (~800 cells) of the microdissected tubules.

Specific primers were designed for each of the three rat ₂-AR isoforms, (Table 1). These primers amplify from the junction of the third cytoplasmic loop and the sixth transmembrane (TM) region (sense) to the 3'-untranslated regions (UTR) (antisense). A second pair of primers for _2B-AR () span the third cytoplasmic loop (sense) to TM-7 (antisense). Degenerate ₁-AR primers were designed to amplify from the conserved region of TM-1 (sense) to TM-5 (antisense) of _1A-, _1B-, and _1D-AR. The predicted products of the degenerate primer PCR reactions could be distinguished by unique restriction sites (Table 1).

View this table: [in this window] [in a new window]   Table 1.   PCR amplification and restriction enzyme analysis of rat -adrenergic isoforms

Unique primers were subsequently designed to amplify the ₁-AR isoforms for cloning purposes. The _1A primers amplify a region within the carboxy terminus, whereas the _1B primers were designed from the same region as the degenerate ₁ primers described above (Table 1).¹ Two sets of primers were used to detect _1D; the first set was designed to amplify from the carboxy terminus (sense) to the 3'-UTR (antisense), whereas the second set (), designed by Feng et al. (11), spanned the third intracellular loop.

PCR reactions contained 2.5 mM MgCl₂, 50 mM KCl, 20 mM Tris · HCl (pH 8.3), 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) per 50 µl of total reaction volume. Forty-eight cycles of PCR amplification followed by a final 7-min extension at 72°C were performed in a Perkin-Elmer DNA Thermal Cycler with the following protocol: 94°C for 1 min, 60°C (56°C for degenerate ₁-AR primers, 58°C for _1A-, _1B-, and -AR primers) for 1 min, and 72°C for 1 min. The PCR reaction products were resolved on a 2% agarose gel.

The products of PCR amplification were ethanol precipitated, then resuspended in sterile water. Aliquots of the purified products were treated with the restriction endonucleases, as described in Table 1. Digestion products were analyzed by electrophoresis on 2% agarose gels. Bands were visualized with ultraviolet transillumination, and the gel images were digitized and stored on computer disk using a Foto/Analyst Archiver (Fotodyne, Hartland, WI).

Digitized agarose gel images were examined using Collage Image Analysis Software (Fotodyne). Each agarose gel was run with sample lanes bracketed by 154 to 2,176 bp molecular weight standard ladders (MW Weight Marker VI; Boehringer-Mannheim, Indianapolis, IN). These ladders were used to calibrate a standard curve from which the size of PCR products and restriction digests were determined using the Collage software.

When the presence of an isoform was indicated by restriction enzyme analysis, one PCR product of each isoform was subcloned into the plasmid vector pCR 2.1 using TA cloning (Invitrogen, San Diego, CA) according to manufacturer's instructions. Sequence analysis was performed using the T7 and/or M13 universal primers and dye-termination reactions at the Univ. of Alabama at Birmingham, 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).

Western blot analysis with an _2B-specific antibody was performed on protein extracted from manually sorted CCDs and PTs, as well as from whole kidney cortex. CCD and PT segments were collected to total ~100-200 mm. They were subsequently washed twice with PBS, pelleted, resuspended in 100 µl extraction buffer (20 mM Tris · HCl, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 0.5% Triton X-100, and 25 µg/ml each of aprotinin and leupeptin), and incubated on ice for 20 min. The supernatant was retained after centrifugation at 12,000 rpm for 5 min, and the protein concentration was determined by Micro BCA protein assay (Pierce Chemical, Rockford, IL). Whole kidney cortex was prepared as described by Huang et al. (17).

Samples, 10-50 µg, of protein from CCD, PT, and whole kidney cortex were resolved by electrophoresis on an 8% SDS-polyacrylamide gel (16), transferred to a nitrocellulose membrane, and stained with Ponceau S for molecular weight determination. The membranes were incubated in a blocking solution of 5% nonfat powdered milk in T-TBS (Tris-buffered saline, pH 7.4, containing 0.1% Tween 20) 25°C for 1 h. This was followed by a incubation with the primary antibody diluted 1:250 in blocking solution for 1 h at 25°C. After six 5-min washes with T-TBS, the membrane was incubated for 1 h at 25°C with donkey anti-rabbit IgG linked to horseradish peroxidase at a 1:3,000 dilution in blocking solution. Following six 5-min washes in T-TBS, the membrane was incubated with enhanced chemiluminescence substrate (ECL, Amersham) and exposed to autoradiography on X-ray film for 3 min.

For in vitro perfusion experiments, approximately one-half of the rats were implanted with a subcutaneous pellet containing either 2.5 mg DOC or 1 mg d-aldosterone (Innovative Research of America, Toledo, OH) 48-72 h after receipt. This pharmacological dose of DOC, in the presence of AVP, leads to maximum stimulation of Na⁺ absorption by the CCD. The rats were anesthetized by brief exposure to CO₂, and the pellets were implanted subcutaneously in the interscapular region with a 15-gauge trocar designed for this purpose. The rats were then used for experiments 3-8 days after pellet implantation. The remaining rats, which were left untreated, were used at the same time after receipt. All rats weighed 80-110 g at the time of tubule dissection. Segments of CCD were dissected from fresh kidneys and then were placed in an isotonic artificial bathing solution similar to rat serum and perfused with a hypotonic solution resembling early distal tubular fluid (5). Perfusion at 10-20 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. At least 5 min of equilibration was allowed between sequential additions of adrenergic agonists or antagonists to the bathing solution.

Sources of biochemicals. Amplification grade DNase I, and SuperScript II reverse transcriptase were from Life Technologies (Gaithersburg, MD). Oligonucleotide primers were from Integrated DNA Technologies (Coralville, IA) and Oligos Etc. (Guilford, CT). Taq DNA polymerase (catalog no. M1861) was from Promega (Madison, WI). Restriction endonucleases BamH I, Ava II, and Kpn I were from Stratagene (La Jolla, CA); Pst I, Sac II, Ban II, and Ban I were from Promega; and Aat II and Xmn I were from New England Biolabs (Beverly, MA). Nitrocellulose membranes were from MSI (Westborough, MA), and the ECL system was from Amersham (Arlington Heights, IL). AVP was from Sigma (St. Louis, MO). Rauwolscine and phenylephrine were from Research Biochemicals International (Natick, MA), and propranolol was from Solo Pak Laboratories (Elk Grove Village, IL).

	RESULTS

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RT-PCR analyses were conducted for -AR isoforms in the CCD, and, for comparison, in PT segments. Based on previous functional results in isolated, perfused CCD, the presence of one or more ₂-ARs was expected. PCR amplification with specific ₂-AR primers followed by restriction enzyme analysis indicated the presence of _2A and _2B in both CCDs and PTs. Figure 1A is representative of the findings summarized in Table 2.

View larger version (58K): [in this window] [in a new window]   Fig. 1.   A: restriction enzyme analysis of PCR products, from a pair of specific primers for 2A-AR and two pairs of specific primers for 2B-AR, reveal the presence of both isoforms in cortical collecting duct (CCD). B: brain tissue was used to demonstrate the ability of the 2C-AR primers to detect this isoform. Sequence analysis of the 2A- and 2B-AR PCR products indicated nucleotide identity with the published sequences. Sizes are listed in base pairs.

View this table: [in this window] [in a new window]   Table 2.   Summary of 2-AR isoforms detected in rat tubules by RT-PCR

The 264-bp _2A-AR product restricted as anticipated from the published sequence with Ban I and was detected in CCD in 7 of 9 experiments, whereas _2B was detected with both primer pairs in 10 of 10 total experiments (Table 2). The 395-bp _2B-AR product restricted with BamH I, whereas the 381-bp product of the primers restricted with Xmn I. Restriction analysis of PCR products from PTs indicated the presence of the same isoforms in this region of the nephron (Table 2). _2C-AR was never observed in CCD or PTs, although it was readily detectable in brain (Fig. 1B). Sequence determination of both the _2A and _2B-AR PCR products indicated nucleotide identity with published sequences from rat liver (20) and kidney (48), respectively.

We extended our examination of adrenoceptors to include ₁-AR isoforms, initially using degenerate primers. Unexpectedly, _1A and _1B-AR were readily visualized using our standard quantity of cDNA obtained from ~1 mm of microdissected rat CCD (Fig. 2) and PTs (Table 3). Using techniques newly designed in our laboratory to microdissect larger quantities of tubules (34), we were able to detect _1D-AR using cDNA derived from ~5 mm of CCD in the RT-PCR reaction (Fig. 2). The results of these experiments are summarized in Table 3.

View larger version (100K): [in this window] [in a new window]   Fig. 2.   Restriction enzyme analysis of the PCR products of degenerate 1-AR primers reveals the presence of 1D-, 1B-, and 1A-AR (Table 1). Sizes are listed in base pairs.

View this table: [in this window] [in a new window]   Table 3.   Summary of 1-AR isoforms detected in rat tubules by RT-PCR

Specific primers were designed to amplify the ₁-AR isoforms (Table 1) for cloning purposes. The _1A- and _1B-AR PCR products were identical with the sequences isolated from rat brain (21, 43). The first set of specific _1D primers detected this isoform in rat brain (Fig. 3); however, we obtained no evidence of this isoform in CCD or in PTs. With the primers used by Feng et al. (11) for the detection of _1D-AR () in proximal convoluted tubules (PCT) and medullary thick ascending limbs of the loop of Henle, we were able to demonstrate the presence of this isoform in brain as well as in CCD (Fig. 3), but not in PTs (data not shown). The PCR product with the primers was identical with the sequence originally obtained in rat brain (23).

View larger version (57K): [in this window] [in a new window]   Fig. 3.   Specific primers are used to verify the presence of the 1-AR isoforms and for sequence analysis. Brain tissue was used to demonstrate the ability of the 1D-AR primers to detect this isoform. A second pair of 1D-AR primers, , detected this isoform in CCD. Sequence analysis of the 1A-, 1B-, and -AR PCR products indicated nucleotide identity with the published sequences. Sizes are listed in base pairs.

The presence of _2B-AR at the protein level was assessed by Western blot analysis. As illustrated in Fig. 4, a labeled protein with a mobility of 62 kDa was expressed in CCD. A protein of the same mobility was also dominant in the PT, although minor bands of 67 and 48 kDa were also variably detected. Whole kidney cortex displayed a dominant doublet of 62 and 65 kDa and an additional band at 48 kDa.

View larger version (51K): [in this window] [in a new window]   Fig. 4.   Western blot analysis of rat tissue for 2B-AR. Twenty micrograms of total protein extracted from rat kidney cortex, CCD, and proximal tubule were resolved by 8% SDS-PAGE, transferred to nitrocellulose membranes, and probed with 2B-AR-specific antibody. Migration of labeled proteins (kDa) are indicated.

We studied the effects of an ₁-agonist, phenylephrine, on transepithelial voltage (V_T) after treatment with AVP in isolated perfused CCD segments from both DOC-treated and untreated rats. Rauwolscine and propranolol were used to block, respectively, ₂- and -adrenergic effects. Phenylephrine was subsequently added as an ₁-AR agonist, followed by the antagonist phentolamine. Examination of the data by ANOVA indicated that the only significant ₁-AR inhibitory effect on V_T (P < 0.002) was in the non-DOC-treated rats, and phentolamine tended to reverse this effect (P = 0.056) (Fig. 5). Although the means reflected the same trend in the CCD from DOC-treated rats, there were no statistically significant differences.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   In vitro perfusion experiments are used to detect 1-AR effects on transepithelial voltage (VT) in CCD from untreated and deoxycorticosterone (DOC)-treated rats. The treatments were: 1) arginine vasopressin (AVP, 22 pM), 2) addition of rauwolscine (Rauw, 300 nM) and propranolol (Propr, 1 µM), 3) addition of phenylephrine (10 µM), 4) addition of phentolamine (20 µM), and 5) return to AVP alone.

	DISCUSSION

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The results of our RT-PCR studies of ₂-receptor isoforms summarized in Table 2 revealed the presence of mRNA for both _2A- and _2B-isoforms in CCD and PT, but neither segment expressed _2C. The PCR products were verified both by restriction and sequence analysis. Based on our previous functional studies (14, 16), we expected to find one or more ₂-AR in the CCD. We did not, however, anticipate finding the three ₁-AR mRNAs. We initially used a degenerate primer pair (Table 1) to amplify the ₁-isoforms and, by restriction enzyme analysis of the product, obtained evidence for _1A- and _1B-AR in the CCD and PT (Table 3). The presence of _1D was also detected in two of nine CCD mRNA extractions but not in any PT samples. Subsequently, we designed ₁-AR isoform-specific primer pairs and detected the presence of _1A, _1B, and _1D in all of the CCD samples. The ₁ PCR products from the CCD were verified by restriction and sequence analysis.

Because of the ability to obtain larger quantities of protein from PT, in comparison with CCD, radioligand binding studies have successfully demonstrated ₂-AR as well as ₁-AR in that nephron segment (40, 41). Neither in situ hybridization studies (26) nor binding studies (7, 11, 13) have provided evidence for the presence of ₁-AR subtype in the CCD. The CCD presents considerable technical difficulties for such studies. Because of the short length of the CCD and the fact that it derives from multiple proximal nephrons, it comprises only ~1% of the mass of the renal cortex. Although Cohen et al. (7) demonstrated specific binding of the ₁ ligand [¹²⁵I]iodoprazosin in the rat PT, thick ascending limb of the loop of Henle, and the distal convoluted tubule, they did not find significant binding in the CCD or outer medullary collecting duct. Clarke and Garg (6) reported specific [³H]prazosin binding in the inner medullary collecting duct of the rabbit but at a rather low activity of 30 fmol/mg of protein. In comparison, the ₁-receptor density in the PCT is 140 amol/mm of tubule length or ~500 fmol/mg. It appears that the limit of detection with [¹²⁵I]iodoprazosin of high specific activity is ~30 amol/mm in samples of 2 mm tubule length (7). Using CCD obtained from rat kidney by our collagenase digestion method (34), we also conducted such binding studies with [¹²⁵I]iodoclonidine and [³H]prazosin but were unable to detect specific binding even with membranes isolated from samples of 100-200 mm of CCD (data not shown). However, because even this large sample of CCD provides only 10-20 µg of protein, we would have been unable to detect specific binding at low receptor density. Immunohistochemical analysis of the individual ₁-ARs is further limited by the lack of subtype-specific antibodies.

Binding studies indicating the presence of ₂-receptors in individual rat nephron segments are also limited. Compounding this lack of information is the marginal subtype selectivity of the ₂ ligands, as noted by MacDonald et al. (24). However, several antibodies for the ₂-AR subtypes have been recently developed, and they appear to be highly selective when examined in COS cells transfected with the ₂-ARs (17, 30, 31). An antibody developed against the third cytoplasmic loop of the _2A-AR allowed immunohistochemical localization of this subtype in brain stem neurons in the rat (31); however, specific immunoreactivity was not detected in kidney sections (M. D. Okusa, personal communication). The lack of detection, in view of functional evidence of this subtype in rat kidney, likely reflects a low density of receptors in this tissue, in comparison with the brain, which displays high levels of expression in discrete regions. In addition, the antibody did not appear to recognize the denatured receptor and was thus unsuitable for Western blot analysis (D. L. Rosin, personal communication).

We have, however, been able to obtain evidence for the presence of the _2B-receptor protein in the CCD using an antibody developed by Okusa and his collaborators (17). This antibody, targeted to the third cytoplasmic loop of _2B-AR, was found, on immunohistochemical analysis, to label the basolateral membrane of the PT but not other nephron segments. However, it was suggested that the sensitivity of the antibody may not be sufficient to detect the receptor if it is present at a lower density in the CCD (M. D. Okusa, personal communication). For that reason, we tried Western blot analysis of proteins extracted from microdissected CCD and PT and from whole kidney cortex. We found that the _2B antibody consistently labeled a band of the expected size (48 kDa) only in the whole cortex extract and, occasionally, very faintly in the PT but not the CCD. The antibody, however, invariably labeled a protein migrating with a mobility of 62 kDa in all three tissues. Additional bands migrating at 65 and 67 kDa were seen in the cortex and PT samples, respectively. A primary band of ~60 kDa would be expected for the _2A and _2C isoforms because, although all three receptors contain a similar number of amino acids, only the _2A and _2C isoforms are glycosylated (47). Immunohistochemical and Western blot studies in COS cells transfected with _2A and _2C confirmed there is no cross-reactivity of the _2B antibody with the other isoforms (17). On Western blot analysis, a minor band migrating at ~66 kDa was also seen by Huang et al. (see figures 3 and 5 of Ref. 17), and preabsorption with the glutathione S-transferase (GST)/_2B fusion protein against which the antibody was developed, prevented labeling of both the 66- and 45-kDa bands. We have no explanation for the larger protein except for the possibility of alternate processing of _2B.

Although there has been little direct information from binding studies for the presence of ₂-AR in the CCD, there is abundant functional evidence of their presence. Several laboratories have reported that epinephrine and clonidine inhibit AVP-dependent cAMP production in the isolated rat and rabbit CCD and that this inhibition is reversed by phentolamine or yohimbine but not by prazosin (3, 20, 28). We have confirmed this effect of epinephrine on cAMP production in our laboratory (21a). In the isolated perfused CCD, we have also shown that clonidine and epinephrine inhibit the AVP-dependent increase in P_f and J_Na (4, 14, 16) and that the effect of 100 nM epinephrine could be completely reversed by 1 µM yohimbine (14).

Despite the consistency of these findings with an ₂-AR mechanism mediated through G_i-dependent adenylyl cyclase activation, other findings suggested there was an additional inhibitory mechanism. We found that 1 µM corynanthine partially reversed the inhibition of P_f and J_Na produced by 100 nM epinephrine (14). Although corynanthine is generally considered an ₁-AR antagonist, it can also block ₂-AR at this concentration (48). More importantly, when P_f and J_Na were stimulated by 50 µM forskolin or a cAMP analog in the presence of IBMX, 100 nM epinephrine still inhibited transport, although it was only 30-40% of the inhibition produced in the presence of AVP (14). The smaller inhibitory effect in the presence of abundant cAMP could be mediated by an ₁-AR, which may explain the partial reversal of inhibition by corynanthine, or the effect could be mediated by an alternate second messenger pathways coupled to an ₂-AR. For example, _2A and _2C-ARs have also been shown to couple to phospholipase C (PLC) to extents which vary with the isoform (8). Although previous experiments in this laboratory have not detected an involvement of the PLC pathway in regulating the -AR effect in rat CCD (as discussed below), we do not rule out this possibility.

To determine whether there was a functional effect of the ₁-ARs, we performed experiments to measure V_T in isolated perfused rat CCD segments. In these experiments rauwolscine and propranolol were present to block ₂- and -adrenergic receptors, respectively. Phenylephrine was used as a general ₁-agonist, followed by phentolamine as an antagonist (Fig. 5). We observed a small depolarization of V_T with phenylephrine, but the effect was statistically significant only in the case of CCD from rats that had not been treated with DOC. Although phentolamine tended to reverse the effect, the reversal did not achieve significance. The effect of phenylephrine was markedly less than the yohimbine-reversible effects produced by epinephrine or clonidine. Thus the functional effect of the ₁ receptors on Na⁺ transport is small in comparison with ₂-AR-mediated effects.

₁-ARs are primarily coupled to PLC (8, 44) and to a lesser extent to PLA₂ (23) and PLD (43). Findings from our laboratory have failed to demonstrate an inhibitory effect of PGE₂ on transport in the rat CCD (4), indicating that at least this product of PLA₂ activation is not involved. We have also previously shown that activation of protein kinase C (PKC) by phorbol myristate acetate or oleoylacetylglycerol, or elevation in intracellular Ca²⁺, appear to have no effect on AVP-dependent P_f or J_Na in the CCD of DOC-treated rats (32). Although these results suggested that PLC is not involved in the inhibition of salt and water transport in this segment, the small effect that could be attributed to ₁-AR activity may not have been detected, particularly in view of DOC pretreatment. It is also possible that the correct PKC isoform was not activated in our previous studies. Using RT-PCR and Western blot analysis, we have detected the presence of five PKC isoforms in the rat CCD (46), most of which are insensitive to phorbol esters and/or Ca²⁺.

We conclude that the primary adrenoceptor functionally involved in regulating CCD Na⁺ and water transport is an ₂-AR, either _2A or _2B, which decreases AVP-dependent cAMP generation by coupling to a G_i protein that inhibits adenylyl cyclase. A small inhibitory effect is also mediated by an ₁-AR activation. The multiple roles of the -adrenoceptors in the CCD as well as the variety of intracellular coupling mechanisms with which they are associated remain an important objective for future studies, especially with regard to the possible effects of diet and salt balance on the relative expression of these receptor isoforms and the consequences of those receptor changes.