Introduction

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It is generally accepted that dopamine is an important regulator of renal function (Lee, 1993). When administered to humans and other species, dopamine has marked natriuretic and diuretic effects (Jose et al., 1992; Lee, 1993). Although the hemodynamic actions of dopamine (increased renal blood flow and glomerular filtration rate) undoubtedly contribute to these effects on renal function, direct tubular actions of dopamine are also likely to be involved (Jose et al., 1992; Holtback et al., 2000). The proximal tubule and inner medullary collecting duct (IMCD) cells in culture synthesize dopamine (Huo et al., 1991; Jose et al., 1992), and the natriuretic effects of this catecholamine appear to be part of an intrarenal paracrine or autocrine system (Siragy et al., 1989; Jose et al., 1992; Holtback et al., 2000). Dopamine receptors have been identified in various segments of the nephron. Results from pharmacological, ligand binding, and reverse transcriptase-polymerase chain reaction studies have provided evidence for D₁, D₂, D₃, and D₄ receptors associated with various tubule segments (Meister et al., 1991; Takemoto et al., 1991; Gao et al., 1994; O'Connell et al., 1995, 1998; Sun et al., 1998). Functional effects attributed to dopamine include inhibition of Na⁺,K⁺-ATPase in a number of tubule segments (Bertorello and Katz, 1993), inhibition of fluid absorption, Na⁺/H⁺ exchange and phosphate transport in the proximal tubule (Kaneda and Bello-Reuss, 1983; Felder et al., 1990; Baum and Quigley, 1998), and inhibition of vasopressin-stimulated osmotic water permeability (Pf) and Na⁺ transport in the cortical collecting tubule (Muto et al., 1985; Sun and Schafer, 1996). Although most of the proximal tubule effects of dopamine appear to be due to activation of D₁ receptors (Felder et al., 1990; Baum and Quigley, 1998; Holtback et al., 2000), a series of recent studies (Sun and Schafer, 1996; Li and Schafer, 1998; Sun et al., 1998) suggests that dopamine's effects on vasopressin-dependent water permeability and Na⁺ transport in the rat cortical collecting tubule are mediated by a D₄-like receptor. This was based on observations that D₄ receptor mRNA and protein are expressed throughout the collecting duct system (Sun et al., 1998), and agonists and antagonists of D₁, D₂, and D₃ receptors failed to mimic or attenuate the inhibitory effects of dopamine on AVP-dependent water, Na⁺ transport (Sun and Schafer, 1996), and AVP-stimulated cAMP accumulation (Li and Schafer, 1998). However, clozapine, an "atypical" neuroleptic with D₄ antagonist activity (Van Tol et al., 1991), did attenuate the dopamine-mediated effects on vasopressin action (Sun and Schafer, 1996; Li and Schafer, 1998). In the present study, the effects of dopamine were examined on AVP-dependent water permeability and cAMP accumulation in the rat IMCD to determine whether a similar system is operable in this segment of the nephron. Since high concentrations of dopamine can activate ₂-adrenoceptors (Phillips, 1980), which are known to inhibit AVP action in the IMCD (Edwards and Gellai, 1988), the effects of norepinephrine were studied in parallel. Contrary to expectations, the results of the present study suggest that the effects of dopamine on AVP action in the IMCD can be attributable to activation of ₂-adrenoceptors.

	Materials and Methods

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Perfused Tubules. Tubules were perfused as previously described in detail (Edwards and Speilman, 1994). Kidneys were removed from anesthetized (pentobarbital, 50 mg/kg i.p.) male Sprague-Dawley rats (250-300 g, Charles River Laboratories, Inc., Wilmington, MA) that had free access to standard laboratory chow and water. Corticomedullary slices were placed in chilled bath solution (see below) containing 0.1% bovine serum albumin to facilitate dissection. IMCDs were dissected from the lower two-thirds of the inner medulla, transferred to a temperature-controlled chamber, and mounted on micropipettes. Perfused tubule length averaged 786 ± 30 µm (n = 33). Tubules were initially perfused and bathed with a hypertonic solution consisting of 210 mM NaCl, 5 mM KCl, 1.5 mM CaCl₂, 1.2 mM MgSO₄, 2.3 mM Na₂HPO₄, 8 mM glucose, 5 mM alanine, and 10 mM HEPES. The pH and osmolality were adjusted to 7.4 with NaOH and 450 mOsmol/kg H₂O with NaCl, respectively. The temperature of the bath was gradually increased and maintained at 37°C. Prewarmed bath solution was continuously pumped through the chamber at 0.5 ml/min. Thirty minutes after the chamber had reached 37°C, the perfusate was changed to an isotonic solution (300 mOsmol/kg H₂O) that was identical with the bath except that it contained less NaCl (135 mM) and dialyzed [³H]inulin, which served as a volume marker. Timed collections of perfusate were made using a constant volume pipette, and Pf (µm/s) was calculated according to Al-Zahid et al. (1977). Tubules were perfused at rates of 20 to 30 nl/min to prevent osmotic equilibrium between the perfusate and bath and did not differ between control and experimental periods.

cAMP Measurements. Dissection of IMCDs and incubation for measurement of cAMP were performed as previously described (Edwards and Gellai, 1988). Briefly, the left kidney was perfused via the abdominal aorta with 10 ml of a Krebs-Ringer-bicarbonate buffer equilibrated with 95% O₂/5% CO₂ (pH 7.4) and consisting of 118 mM NaCl, 25 mM NaHCO₃, 4.8 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 1.5 mM CaCl₂, 10 mM glucose, 5 mM alanine, 0.1% bovine serum albumin, and 2 mg/ml collagenase (Sigma type I). Corticomedullary slices were incubated in the same solution with constant bubbling with 95% O₂/5%CO₂ for 30 to 45 min and then extensively rinsed in chilled dissection/incubation buffer consisting of 137 mM NaCl, 5 mM KCl, 0.34 mM Na₂HPO₄, 0.44 mM KH₂PO₄, 1 mM CaCl₂, 1.2 mM MgSO₄, 10 mM glucose, 5 mM alanine 20 mM HEPES, and 1 mg/ml bovine serum albumin. Single IMCDs were dissected from the lower two-thirds of the inner medulla and transferred in 10 µl of the above solution to siliconized test tubes. The samples were preincubated for 10 min at 37°C, after which an additional 10 µl of buffer containing isobutylmethylxanthine (1 mM final concentration) and various compounds, as described under Results, were added. After 5 min, the incubation was stopped by the addition of 10 µl of cold 0.2 N HCl. The samples were neutralized with NaOH and acetylated, and cAMP was measured by radioimmunoassay (Edwards and Gellai, 1988). For each experiment, three to five replicates were averaged to arrive at a single data point for a given experimental condition. cAMP levels were expressed as femtomoles per millimeter of tubule length or as a percentage of control values. Statistical analysis was performed with Student's t test for paired comparisons or by analysis of variance followed by Tukey's test for multiple comparisons. Statistical analysis and curve fitting were performed using GraphPad Prizm software (GraphPad Software, San Diego, CA).

Reagents. [³H]Inulin and cAMP radioimmunoassay kits were obtained from PerkinElmer Life Science Products (Boston, MA). AVP, dopamine HCl, norepinephrine bitartrate, and clozapine were obtained from Sigma (St. Louis, MO). Rauwolscine, yohimbine, idazoxan, SCH-23390, raclopride, and spiperone were obtained from Sigma/RBI (Natick, MA). Dopamine and norepinephrine stock solutions were made up in 0.1% ascorbic acid and protected from light. Concentrated stock solutions (1 mM) of rauwolscine, yohimbine, idazoxan, SCH-23390, spiperone, and raclopride were made up in water. Clozapine (20 mM) was made up in 0.1 N HCl and diluted with buffer. All control and experimental solutions also contained the appropriate vehicle.

	Results

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Dopamine produced a rapid and reversible decrease in AVP-induced Pf (Fig. 1). In the presence of 10 pM AVP, Pf averaged 947 ± 89 µm/s. Addition of 10 µM dopamine to the bath decreased Pf to 141 ± 27 µm/s (p < 0.001). Upon removal of dopamine, Pf increased to 890 ± 99 µm/s, a value not different from the initial AVP period. In contrast to its effect on AVP-induced Pf, dopamine (10 µM) had no effect on Pf stimulated by the cAMP analog, CPT-cAMP (Fig. 1). Pf in the presence of 100 µM CPT-cAMP was 690 ± 32 µm/s and did not change when 10 µM dopamine was added to the bath (659 ± 50 µm/s). Figure 2 shows the concentration-dependent effects of dopamine and, for comparison, norepinephrine on AVP-stimulated Pf. Both catecholamines produced a concentration-dependent inhibition of vasopressin-stimulated Pf. Although both compounds inhibited vasopressin action to the same extent, norepinephrine was significantly (p < 0.03) more potent than dopamine. The concentration of norepinephrine needed to inhibit vasopressin-induced Pf by 50% (IC₅₀) was 16.6 ± 4.0 nM compared with 1.9 ± 0.7 µM for dopamine. In contrast to their effects on AVP-stimulated Pf, dopamine and norepinephrine had no effects on basal Pf, which was measured in the absence of AVP. Thus, Pf was 107.9 ± 8.8 and 100.3 ± 11.6 µm/s in the absence and presence of dopamine (10 µM, n = 6) and 112.4 ± 16.3 and 120.6 ± 10.3 µm/s in the absence and presence of norepinephrine (10 µM, n = 5).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   .. Effects of dopamine on AVP () and 8-p-chlorophenylthio-cAMP (cAMP; ) induced increases on Pf. Tubules were exposed to AVP (10 pM) or cAMP (100 µM) throughout the experiment. In the second period, the bath was changed to one containing dopamine (10 µM) and AVP or cAMP. Dopamine was absent during the last period. Results are expressed as means ± S.E.M. of five tubules for both the AVP and cAMP experiments. *, value significantly different (p < 0.05) from both AVP periods.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent inhibition of vasopressin-induced Pf (10 pM) by dopamine () and norepinephrine (). Results are expressed as a percentage of vasopressin-induced Pf, which was 905 ± 93 µm/s for the dopamine series (n = 5) and 836 ± 86 µm/s for the norepinephrine series (n = 4).

Since high concentrations of dopamine can activate ₂-adrenoceptors, which are known to inhibit vasopressin action (Edwards and Gellai, 1988; Chen et al., 1991), the effects of the ₂-adrenoceptor antagonist, rauwolscine, on the inhibitory effect of dopamine was examined. In these experiments, 10 µM dopamine decreased AVP-stimulated Pf from 966 ± 38 to 126 ± 29 µm/s (Fig. 3). In the presence of dopamine, the addition of increasing concentrations of rauwolscine led to a progressive attenuation of the inhibitory effect of dopamine. Pf increased to 242 ± 42, 608 ± 45, and 842 ± 83 µm/s in the presence of 10 nM, 100 nM, and 1 µM rauwolscine, respectively. Pf in the presence of 1 µM rauwolscine was not significantly different from AVP alone. Following removal of rauwolscine, Pf decreased to 234 ± 18 µm/s, a value not different from the initial dopamine period. Clozapine, an atypical neuroleptic with some selectivity for the D₄ receptor (Van Tol et al., 1991), has previously been shown to inhibit the effects of dopamine on AVP-stimulated Pf, Na⁺ transport, and cAMP levels in the rat cortical collecting tubule (Sun and Schafer, 1996; Li and Schafer, 1998). Consistent with that observation, clozapine (10 µM) partially attenuated the inhibitory effect of dopamine (10 µM) on AVP-dependent Pf (Fig. 4), however, clozapine also inhibited the effects of norepinephrine (1 µM) on AVP-dependent Pf to the same degree.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Effect of rauwolscine on dopamine-induced inhibition of AVP-dependent Pf. AVP (10 pM) was present throughout the experiment. Tubules (n = 5) were sequentially exposed to dopamine (10 µM), dopamine plus rauwolscine, followed by a second dopamine exposure at the end of the experiment. DA, dopamine; *, significantly different (p < 0.05) from dopamine periods.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Effect of clozapine on dopamine and norepinephrine-induced inhibition of AVP-dependent Pf. Tubules were exposed to AVP (10 pM), followed by dopamine (10 µM, n = 5) or norepinephrine (1 µM, n = 5) and clozapine (10 µM). , AVP; , agonist; , clozapine; *p < 0.05 compared with dopamine or norepinephrine alone.

In IMCDs stimulated with 1 nM AVP, both dopamine and norepinephrine caused a concentration-dependent inhibition of cAMP accumulation (Fig. 5). The IC₅₀ value for dopamine was 1.3 ± 0.39 µM, whereas norepinephrine was more potent (p < 0.05) with an IC₅₀ value of 9.6 ± 1.4 nM. There was a direct correlation between the decrement in AVP-stimulated cAMP levels and Pf produced by norepinephrine (r = 0.99) and by dopamine (r = 0.97). Furthermore, the difference in potency between norepinephrine and dopamine in producing these effects on cAMP levels (135-fold) and Pf (118-fold) was similar. As was the case for dopamine-induced inhibition of AVP-stimulated Pf (Fig. 4), the ₂-adrenoceptor antagonist, rauwolscine, inhibited dopamine effects on AVP-stimulated cAMP accumulation in a concentration-dependent manner (Fig. 6). In the presence of 1 µM rauwolscine, the inhibitory effect of dopamine was totally abolished. To investigate further the receptor involved in dopamine's action, a number of structurally diverse, selective antagonists of ₂-adrenoceptors (rauwolscine, yohimbine, and idazoxan) and dopamine receptors (SCH-23390, D₁; spiperone, D₂; and raclopride, D₂) were tested for their ability to antagonize dopamine-induced inhibition of AVP-stimulated cAMP accumulation. At a concentration of 1 µM, which is well above the K_i values of the dopamine antagonists for their respective receptors (Gingrich and Caron, 1993), only the ₂-adrenoceptor antagonists, rauwolscine, yohimbine, and idazoxan, attenuated dopamine's action on AVP-induced cAMP accumulation (Fig. 7). Clozapine attenuated the effects of both dopamine and norepinephrine on AVP-stimulated cAMP accumulation consistent with its effects on Pf (Fig. 8).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Concentration-dependent inhibition of AVP-induced cAMP accumulation by dopamine () and norepinephrine () in IMCD. Tubules were incubated with 1 nM AVP and various concentrations of dopamine (n = 6) and norepinephrine (n = 7). Results are expressed as a percentage of cAMP levels in the presence of AVP. Basal and AVP-stimulated cAMP levels were 9.7 ± 2.5 and 403 ± 35 fmol/mm for the dopamine experiments and 7.6 ± 1.4 and 430 ± 67 fmol/mm for the norepinephrine experiments.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Effect of rauwolscine on dopamine-induced inhibition of AVP-stimulated cAMP levels. Tubules were incubated with 1 nM AVP alone or in the presence of dopamine and various concentrations of rauwolscine. *p < 0.05 versus dopamine alone. Results are from tubules dissected from five animals.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Effects of various 2-adrenoceptor antagonists and dopamine receptor antagonists on dopamine-induced inhibition of AVP-dependent cAMP levels. Tubules were incubated with 1 nM AVP or AVP and 10 µM dopamine with and without 1 µM of the indicated antagonists. Results are expressed as a percentage of cAMP levels in the presence of AVP alone, which was 371 fmol/mm, n = 5. *p < 0.01 versus dopamine.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Effect of clozapine on dopamine (10 µM) and norepinephrine (1 µM)-induced inhibition of AVP-stimulated cAMP levels. Results are expressed as a percentage of AVP-stimulated (10 pM) levels, which were 419 ± 36 fmol/mm (n = 5) and 386 ± 43 fmol/mm (n = 6) for the dopamine and norepinephrine experiments, respectively. , agonist; , 1 µM clozapine; , 10 µM clozapine; *p < 0.05 versus dopamine or norepinephrine alone.

	Discussion

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Dopamine has a number of effects on water and electrolyte transport in the kidney (Lee, 1993). With respect to the collecting tubule, previous studies have shown that dopamine inhibits AVP-induced increases in Pf in the rabbit cortical collecting tubule (Muto et al., 1985) and the rat cortical collecting tubule (Sun and Schafer, 1996). In the rabbit cortical collecting tubule, the effects of dopamine were inhibited by the nonselective antagonist, metoclopramide (Muto et al., 1985), whereas the effects of dopamine in the rat cortical collecting tubule were antagonized by clozapine, a relatively selective D₄ receptor antagonist, but not by D₁-, D₂-, or D₃-selective antagonists (Sun and Schafer, 1996; Li and Schafer, 1998). We undertook the present series of experiments to determine whether dopamine inhibits AVP action in the IMCD, the segment of the nephron responsible for the final elaboration of the urine, and a segment of the collecting duct system in which D₄ receptor mRNA has been detected (Sun et al., 1998). Furthermore, since dopamine can activate ₂-adrenoceptors (Phillips, 1980), which have well characterized inhibitory effects on AVP action in the rat collecting tubule (Chen et al., 1991; Edwards and Gellai, 1988), we examined the effects of norepinephrine and dopamine in parallel.

In agreement with the studies cited above, we found that dopamine caused a concentration-dependent inhibition of AVP-induced Pf and cAMP accumulation in the rat IMCD. Also consistent with the rat cortical collecting tubule study (Sun and Schafer, 1996) was our observation that dopamine had no effect on the increase in Pf caused by CPT-cAMP. These data, coupled with the inhibition of AVP-stimulated cAMP accumulation, suggest that dopamine inhibits AVP action at the level of adenylate cyclase. However, unlike the conclusions derived from the rat cortical collecting tubule study (Sun and Schafer, 1996), our results are more consistent with dopamine acting via ₂-adrenoceptors to inhibit AVP action in the IMCD. This conclusion is based primarily on the following observations. First, norepinephrine produced identical effects to that of dopamine but was more than 100-fold more potent at inhibiting both AVP-induced increases in Pf and cAMP levels. This is the opposite of what one would expect if the D₄ receptor was involved, since norepinephrine is greater than 100-fold less potent than dopamine in activating this receptor (Van Tol, 1998). Second, a number of selective ₂-adrenoceptor antagonists, including rauwolscine, yohimbine, and idazoxan, antagonized the effects of dopamine on AVP-induced increases in Pf and/or cAMP levels in a concentration-dependent manner. These compounds have been shown to have affinities for ₂-adrenoceptors in the low-nanomolar range (Bylund et al., 1998), although they are approximately 1000-fold less potent at antagonizing various dopamine receptor subtypes (Scatton et al., 1980; Walter et al., 1984; Dearry et al., 1990). Finally, the relatively selective antagonists of the D₁ (SCH-23390) and D₂ receptors (spiperone, raclopride) had no effect on dopamine-induced inhibition of AVP-stimulated cAMP levels, thus, ruling out a role for these dopamine receptor subtypes. This latter observation is consistent with the findings of Sun and Schafer (1996) and Li and Schafer (1998) in the rat cortical collecting tubule in which D₁, D₂, or D₃ antagonists had no effect on dopamine-induced inhibition of Pf, sodium transport, or cAMP levels stimulated by AVP.

Of the various dopamine receptor antagonists tested, only clozapine inhibited dopamine's effect on AVP action in the IMCD. This is also consistent with previous observations in the rat cortical collecting tubule (Sun and Schafer, 1996; Li and Schafer, 1998). However, we also found that clozapine attenuated norepinephrine-induced inhibition of AVP-dependent Pf and cAMP levels. Clozapine is a so-called atypical neuroleptic, which has activity at a number of different receptors, including various subtypes of the dopamine, serotonin, and adrenergic families (Millan et al., 1998). Although clozapine does show some selectivity for the D₄ receptor over other dopamine receptor subtypes (Millan et al., 1998), of pertinence to the present study are the findings that the K_i for clozapine at the rat cerebral cortex ₂-adrenoceptor (~67 nM) (Millan et al., 1998) is similar to that found at the rat cloned D₄ receptor (~90 nM) (Gazi et al., 2000). Therefore, the partial attenuation of both dopamine and norepinephrine effects on AVP-induced Pf and cAMP accumulation in the IMCD by clozapine is probably due to antagonism of ₂-adrenoceptors. Furthermore, spiperone, which had no effect on dopamine-induced inhibition of AVP-stimulated cAMP levels in this study or in the rat cortical collecting tubule (Li and Schafer, 1998), has a higher affinity for the rat cloned D₄ receptor (~4 nM) (Gazi et al., 2000) than does clozapine (~90 nM). A greater affinity for spiperone (0.07 nM) than for clozapine (22 nM) has also been observed with the human cloned D₄ receptor (Asghari et al., 1994). Thus, if dopamine was acting via the D₄ receptor, spiperone should have demonstrated some antagonist activity. Our data therefore indicate that dopamine inhibits AVP action in the rat IMCD by activating ₂-adrenoceptors. Whether or not a similar situation occurs in the rat cortical collecting tubule is not known, since the effects of ₂-adrenoceptor antagonists on dopamine-induced inhibition of AVP action have not been studied (Sun and Schafer, 1996; Li and Schafer, 1998).

Although our results indicate that dopamine acts through ₂-adrenoceptors to inhibit AVP action in the IMCD, we do not rule out a possible role for this catecholamine in the regulation of salt and water transport in this nephron segment. Should dopamine concentrations reach high enough levels in the inner medulla from local synthesis (Huo et al., 1991) or from the proximal tubule via the postglomerular circulation, dopamine could modulate AVP action by activating ₂-adrenoceptors in the IMCD or alter electrolyte transport by action at D₄ receptors or other dopamine receptor subtypes yet to be localized to this nephron segment.