INTRODUCTION

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WE AND OTHERS HAVE SHOWN that dopamine inhibits Na⁺ and water reabsorption in the cortical collecting duct (CCD). In the isolated perfused rabbit CCD, Muto et al. (8) showed that dopamine inhibited the increase in osmotic water permeability (P_f) produced by arginine vasopressin (AVP) and ascribed this effect to a D₂-type dopamine receptor, because it was prevented or reversed by metoclopramide. In the rat CCD, we (17) also found that dopamine inhibited AVP-dependent P_f, but, in addition, it inhibited the lumen-to-bath flux of Na⁺ (J_Na) and transepithelial voltage (V_T); neither of these latter parameters was measured in the experiments of Muto et al. (8). However, we found no effect of dopamine on either J_Na or V_T in the absence of AVP (17). 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 GTP binding protein G_i (17).

In contrast, Satoh et al. (12, 13) have shown that dopamine increases cAMP production in isolated, but nonperfused, rat CCD via a D_1A receptor, which in turn inhibits Na-K-ATPase activity and thus would be expected to inhibit J_Na. In our studies, we found no effect of the D_1A 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 (17). Among several dopamine receptor antagonists tested in our studies, only clozapine, a D₄-specific antagonist, reversed the effects of dopamine on AVP-dependent P_f, J_Na, and V_T. Thus we concluded that the inhibition of AVP-dependent transepithelial Na⁺ and water transport in the rat CCD by dopamine is mediated by a D₄-like receptor (17).

In more recent studies, we have used RT-PCR to show that both the D_1A and D₄ receptors are present in the rat CCD at the mRNA level, and, using immunohistochemistry, we were able to show that the D₄ receptor was localized to the CCD in medullary rays within the cortex and in the medullary collecting duct (unpublished observations and Ref. 18). The presence in the CCD of two receptor isoforms that have opposite effects on cAMP production raises the question of which effect dominates in regulating that production and thus AVP-dependent Na⁺ and water reabsorption. Therefore, we undertook these experiments to measure the effect of dopamine and dopamine receptor agonists and antagonists on cAMP production by the rat CCD in the presence and absence of AVP.

	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 (Teklad 8746; Madison, WI) containing 0.53% NaCl (measured in our laboratory) for 2-4 wk, at which time their weight was 70-170 g. CCD segments were microdissected from the kidneys, using a modified collagenase digestion method, which we have recently described in detail (15). Briefly, after the kidney was removed from a rat, the capsule was stripped away, and a Thomas-Stadie-Riggs tissue slicer (Thomas Scientific, Swedeboro, NJ) was used to cut a coronal section of 0.5-1.0 mm thickness or, in some cases, a slice that was tangential to the cortical surface. The slice was torn in 4-5 small pieces, which were incubated in 2 ml of warm Eagle's minimal essential medium (MEM) containing 0.5 mg/ml of class 2 collagenase, 5 mM glycine, 50 U/ml DNase, and 48 µg/ml of soybean trypsin inhibitor in a 20-ml glass scintillation vial. (All reagents were from Sigma Chemical, St. Louis, MO, with the exception of the collagenase, which was obtained from Worthington Biochemical, Freehold, NJ; catalog no. 4176.) The cortical pieces were briefly and gently agitated by hand and incubated without shaking at 37°C. At 10- to 15-min intervals, the suspension was gently swirled, and the tubule-rich supernatant was poured off the pieces of tissue. The tubule segments sedimented rapidly in ice-cold 5-ml test tubes; the supernatant was removed and replaced with 2 ml of ice-cold, enzyme-free MEM, containing 1% bovine serum albumin (BSA) to adsorb any remaining collagenase. The tubule segments were once again suspended and then transferred into the bathing solution (BS), which contained (in mM) 122 NaCl, 25 NaHCO₃, 5 KCl, 1.5 CaCl₂, 0.5 MgCl₂, 8 D-glucose, 4 L-alanine, 5 sodium acetate, and 6 urea, plus 0.05 g/dl of purified BSA. (This is the same bathing solution as used for isolated CCD perfusion in previous studies.) CCD segments were manually sorted in this bathing solution at 4°C, as described previously (15).

In our experiments, we measured the rate of cAMP production in the intact (nonpermeabilized) CCD segments isolated by the above procedure. The dissected segments were collected in a clean area of the dissection dish until the required length had been obtained. For each experimental sample, three segments of 0.7-1.4 mm in length were measured with an ocular micrometer, and the total of the segment lengths ranged from 2.5 to 4.0 mm. Each group of three CCD was then transferred in 15 µl of dissection solution to a 150 × 25-mm petri dish with a 20-mm square grid (Falcon 1013; Becton Dickinson Labware, Lincoln Park, NJ) to form a droplet in the center of a square. The petri dish had been humidified in advance by placing a small piece of dressing sponge saturated with water in the corner, and it was kept on ice until subsequent incubations. Each droplet was checked under a dissecting microscope to be sure all three segments of the CCD sample had been transferred successfully.

After all samples had been placed in the petri dish, 5 µl of 5 mM 3-isobutyl-1-methylxanthine (IBMX) in BS were added to each droplet for a final IBMX concentration of 1.25 mM. The samples were incubated for 10 min at 37°C while being gassed with 4% CO₂ in air. Test reagents were then added to the individual samples, together with additional BS, to bring the final volume of each sample droplet to 40 µl. The samples were incubated for an additional 7 min at 37°C. At the end of this incubation, the dish was put on ice, and 30 µl of ice-cold BS containing 1.25 mM IBMX were added immediately to each droplet to stop further cAMP production.

Subsequent extraction and analysis of cAMP was conducted in accordance with the package instructions accompanying the assay kit. Most of our analyses were conducted using the cAMP Enzyme Immunoassay (EIA) kit from Cayman Chemical (Ann Arbor, MI), but one series of measurements was conducted with the cAMP EIA kit (no. RPN 225) from Amersham Life Science (Little Chalfont, UK). An aliquot of 70 µl of ice-cold 10% trichloroacetic acid (TCA), which was diluted in phosphate buffer (PS) from the kit, was placed near the droplet containing the tubule segments, which were then transferred into the TCA droplet in 5 µl of BS. From the TCA extraction mixture, a 65-µl aliquot containing the tubules was transferred to a 500-µl microcentrifuge tube. Each sample was vortexed and placed on ice for 5 min, after which the cells were homogenized in an immersion sonicator (Branson model 1200; Shelton, CT). The samples were centrifuged at 5,000 rpm for 10 min, and 58 µl of the supernatant was pipetted into a 10 × 75-mm glass test tube. TCA was removed by extraction with water-saturated diethyl ether, according to the kit directions. From each extract, 50 µl was removed to a 1.5-ml test tube, and 450 µl of PS was added.

Acetylation of cAMP in unknown and standard samples, all at a volume of 500 µl, was accomplished by adding 100 µl of 4 M KOH and 25 µl of acetic anhydride, vortexing for 15 s, followed by adding 25 µl of 4 M KOH and vortexing. This was done in quick succession with each sample. The acetylated samples were then analyzed on the 96-well enzyme assay plates, as described in the kit directions. Absorbance was measured at 405 nm and compared with standard values to determine the total cAMP in the sample.

Sources of biochemicals. AVP, epinephrine, yohimbine, IBMX, and dopamine were obtained from Sigma. AVP was added to the bathing solution from a stock solution of 20 µM in water to a final concentration of 200 pM. Dopamine was added to BS at varying concentrations from a stock solution of 2.5 mM in water. Fenoldopam (two lots obtained as generous gifts from Dr. P. Jose, Georgetown University, and Dr. R. Felder, Univ. of Virginia) was added to BS from a stock solution of 1 mM in water to a final concentration 100 nM. Clozapine, pimozide, and spiperone were obtained from Research Biochemicals International (Natick, MA). Clozapine was added from a stock solution of 2.5 mM in DMSO, pimozide was added from a stock solution of 1.0 mM in DMSO, and spiperone was added from a stock solution of 1.0 mM in 95% ethanol. Epinephrine and yohimbine were both added from stock solutions of 1.0 mM in water. Chelerythrine chloride and chalphostin C (Calbiochem, La Jolla, CA) were both prepared at 1.0 mM in DMSO. In each experiment, all the samples contained the same concentration of DMSO or ethanol when these were used as vehicles in any samples.

Statistics. Average parameter values were calculated for duplicate samples in each experimental group. These averages from individual experiments were then used to compute the average parameter values (means ± SE) for all experiments in each protocol. Because each experimental protocol used more than one experimental treatment, effects among experimental periods within the same protocol were compared by ANOVA (SuperANOVA program; Abacus Concepts, Berkeley, CA), with appropriate post hoc tests to determine significance. Differences were considered significant for P < 0.05.

	RESULTS

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As an initial set of experiments to test the cAMP assay method in our intact (nonpermeabilized) rat CCD segments, we examined the effect of epinephrine on AVP-dependent cAMP generation, expecting to see an inhibitory effect, as had been shown previously in permeabilized rat CCD segments (2). As shown in Fig. 1, a dose of 200 pM AVP elevated the rate of cAMP production in the intact (nonpermeabilized) CCD from an average of 10 ± 2 (control) to 153 ± 17 fmol · mm¹ · 7 min¹. (In all experiments, the "background cAMP production," i.e., the measured rate of cAMP production in the absence of tubules in the reaction mixture, averaged 5.4 ± 0.8 fmol · mm¹ · 7 min¹, and this value was subtracted from the experimental values.) The addition of 100 nM epinephrine (Fig. 1) reduced cAMP production to 73 ± 6 fmol · mm¹ · 7 min¹, and this inhibition was reversed to 139 ± 13 fmol · mm¹ · 7 min¹ by the ₂-adrenergic receptor antagonist yohimbine at 1 µM.

View larger version (60K): [in this window] [in a new window]   Fig. 1.   Effect of epinephrine (Epineph.) on arginine vasopressin (AVP)-dependent cAMP production by intact rat cortical collecting duct (CCD). Experimental groups had agents added to bathing solution as follows: control, none; 200 pM AVP; 200 pM AVP + 100 nM epinephrine; and 200 pM AVP + 100 nM epinephrine + 1.0 µM yohimbine (Yohim.). Results are from 6 experiments. * P < 0.01, statistically significant difference compared with control group.  P < 0.01, statistically significant difference compared with preceding and following groups.

In the next set of experiments, shown in Fig. 2, we tested the effect of dopamine on cAMP generation in the presence and absence of AVP. In the presence of 10 µM dopamine, cAMP production was 7.3 ± 1.0 fmol · mm¹ · 7 min¹, which was not significantly different from the control rate of 6.9 ± 1.0 fmol · mm¹ · 7 min¹. AVP at 200 pM elevated production to 88 ± 7 fmol · mm¹ · 7 min¹, which was inhibited to 46 ± 4 fmol · mm¹ · 7 min¹ by 10 µM dopamine, and the latter effect was reversed to 72 ± 7 fmol · mm¹ · 7 min¹ with the further addition of the D₄-specific dopamine receptor antagonist clozapine at 10 µM.

View larger version (52K): [in this window] [in a new window]   Fig. 2.   Effect of dopamine on cAMP production by intact rat CCD. Agents added to bathing solution were as follows: control, none; 10 µM dopamine; 200 pM AVP; 200 pM AVP + 10 µM dopamine; and 200 pM AVP + 10 µM dopamine + 10 µM clozapine (cloz.). Results are from 17 experiments. * P < 0.01, statistically significant difference compared with control group.  P < 0.01, statistically significant difference compared with preceding and following groups.

Figure 3 presents the effect of different dopamine concentrations on AVP-dependent cAMP production. In presence of 200 pM AVP alone, cAMP production was 125 ± 10 fmol · mm¹ · 7 min¹. It was not significantly reduced by 0.1 µM dopamine, but doses of 1, 10, and 100 µM all produced significant inhibition (P < 0.05). Although there was no statistically significant difference among the inhibitions produced by 1, 10, and 100 µM dopamine, the pattern of response suggested that a maximal effect was produced by 10 µM, which was the concentration used in our previous study (17) and in other experiments in the present study.

View larger version (66K): [in this window] [in a new window]   Fig. 3.   Effect of dopamine dose on AVP-dependent cAMP production by intact rat CCD. AVP was present at 200 pM in all groups, and dopamine was added at the indicated concentrations. Results from 5 experiments are presented as a percent of the rate of cAMP production in the presence of 200 pM AVP alone (125 ± 10 fmol min1 · 7 min1). * P < 0.05, statistically significant difference compared with AVP alone.

We also tested the effects of the D_1A-specific agonist fenoldopam in a third series of experiments shown in Fig. 4. There was no significant difference between cAMP production in the absence (control) or presence of 0.1 µM fenoldopam (7 ± 2 and 12 ± 2 fmol · mm¹ · 7 min¹, respectively). (We also performed two additional experiments with 1 µM fenoldopam, both of which showed AVP production rates of 8 fmol · mm¹ · 7 min¹.) We then examined the effect of fenoldopam on AVP-dependent cAMP production, as shown in Fig. 4. In these experiments, cAMP production was 141 ± 16 fmol · mm¹ · 7 min¹ in the presence of AVP and was unaltered by the presence of either 0.1 or 1.0 µM fenoldopam. Thus fenoldopam had no significant effect on cAMP production, either in the presence or absence of AVP stimulation.

View larger version (56K): [in this window] [in a new window]   Fig. 4.   Lack of effect of the D1 agonist fenoldopam on cAMP production by intact rat CCD. Results are of 6 experiments with the following additions to bathing solution: control, none; 0.1 µM fenoldopam (Fen.); 200 pM AVP; 200 pM AVP + 0.1 µM fenoldopam; and 200 pM AVP + 1.0 µM fenoldopam. * P < 0.05, statistically significant difference compared with control group and group with 0.1 µM fenoldopam only but not among each other.

In the experiments shown in Fig. 5, we also examined the ability of other dopamine receptor antagonists to inhibit AVP-stimulated cAMP production, including pimozide, which inhibits D₂ and D₃ receptor isoforms to about the same extent, and spiperone, which inhibits D₃ receptors to a greater extent than D₂ receptors (for affinities, see Ref. 4); however, neither significantly reduced the inhibition of cAMP production that was produced by dopamine. In the group treated with AVP alone, cAMP production was 118 ± 17 fmol · mm¹ · 7 min¹, and it was reduced to 53 ± 8 fmol · mm¹ · 7 min¹ by 10 µM dopamine. This inhibition was not reversed by either 25 nM or 10 µM pimozide [cAMP production was 53.8 ± 8.9 (n = 6) and 55.8 ± 8.5 (n = 7) fmol · mm¹ · 7 min¹, respectively] or by 0.1 or 10 µM spiperone [cAMP production was 74.4 ± 16.0 (n = 6) and 59.4 ± 10.4 (n = 7) fmol · mm¹ · 7 min¹, respectively]. Figure 5 presents the cAMP production data for the higher antagonist concentrations.

View larger version (57K): [in this window] [in a new window]   Fig. 5.   Lack of effect of dopamine D2 and D3 receptor antagonists on dopamine inhibition of AVP-dependent cAMP production in intact rat CCD. Agents added to bathing solution were 200 pM AVP, 200 pM AVP + 10 µM dopamine (DA), 200 pM AVP + 10 µM dopamine + 10 µM pimozide, and 200 pM AVP + 10 µM dopamine + 10 µM spiperone. Results are from 7 experiments. * P < 0.001, statistically significant difference compared with group treated with AVP alone.

Finally, in the experiments shown in Fig. 6, we tested whether activation of protein kinase C (PKC) might be involved in the inhibition of cAMP production, as has been demonstrated in other systems. We used two specific PKC inhibitors, chelerythrine and calphostin C, at, respectively, 100 and 150 nM. These concentrations had been shown to reverse PKC-dependent inhibition of AVP effects in the rat medullary thick ascending limb and inner medullary collecting duct (see DISCUSSION and Refs. 5 and 7). In these experiments, 200 pM AVP plus 10 µM dopamine was present in all samples; thus, if PKC activation were involved in the inhibition of AVP-dependent cAMP production, the PKC inhibitors should have increased cAMP production. Neither PKC inhibitor did, but addition of the D₄ receptor antagonist clozapine did significantly increase cAMP production in the presence of either chelerythine or calphostin C. 

View larger version (56K): [in this window] [in a new window]   Fig. 6.   Lack of effect of protein kinase C (PKC) inhibitors on dopamine inhibition of AVP-dependent cAMP production in intact rat CCD. AVP was present at 200 pM and dopamine at 10 µM in all groups together with 1) nothing (control), 2) 150 nM calphostin C (Calph.), 3) 150 nM calphostin C + 10 µM clozapine (Cloz.), 4) 100 nM chelerythrine (Chel.), and 5) 100 nM chelerythrine + 10 µM clozapine. Results are from 4 experiments. * P < 0.05, statistically significant difference compared with preceding group.

	DISCUSSION

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The results presented above show that the basal rate of cAMP production by intact rat CCD segments is low, on the order of 5-10 fmol · mm¹ · 7 min¹, and is increased to the range of 85 to 160 fmol · mm¹ · 7 min¹ by 200 pM AVP. Because we used intact, nonpermeabilized CCD segments, we performed an initial set of experiments to confirm that we could reproduce measurements in permeabilized rat CCD segments. As observed previously by Charbardès et al. (2) using the latter preparation, epinephrine inhibited cAMP production by ~50%, and the effect was completely reversed by the ₂-adrenergic receptor antagonist yohimbine (Fig. 1). Both the magnitude of the effect of AVP on cAMP production and of the epinephrine inhibition confirmed our technique for measuring cAMP production in intact CCD segments in comparison with that of Charbardès et al. (2) and others (e.g., Refs. 10 and 13) in permeabilized segments.

In further experiments, we found no effect of 10 µM dopamine on cAMP production when it was added in the absence of AVP (Fig. 2). However, dopamine inhibited AVP-dependent cAMP production in a dose-dependent manner with a maximal inhibition of 50% occurring at 10 µM (Figs. 2 and 3). The inhibition by 10 µM dopamine was reversed by 10 µM clozapine (Fig. 2), but the D₂ and D₃ dopaminergic receptor antagonists pimozide and spiperone were ineffective (Fig. 5). In our previous study (17), we also found that only clozapine, among a variety of dopamine receptor antagonists (including Sch-23390, specific to D₁ receptors; domperidone, specific to D₂ receptors; and pimozide, specific to D₂ and D₃ receptors), reversed the inhibitory effect of dopamine on Na⁺ transport in isolated perfused CCD (for affinities, see Ref. 4). Clozapine is unique among the dopamine receptor antagonists in having a 10- to 30-fold higher affinity for the D₄ receptor than for any other dopamine receptor isoform (4). Clozapine is also known to bind to serotonin (5-HT) receptors of the 5-HT2 isoform family, but these receptors inhibit phosphoinositide turnover rather than cAMP generation (1). Furthermore, dopamine should not be an agonist for serotonin receptors, and thus clozapine would not be expected to reverse the effects of dopamine by any antagonist effect on 5-HT receptors.

We also tested the effect of fenoldopam, which is an agonist specific to D_1A and D_1B receptors, and has previously been shown to exert a half-maximal effect at 1.5 to 30 nM (4, 10). Fenoldopam at 0.1 or 1.0 µM had no significant effect on cAMP production in the absence or presence of AVP (Fig. 4).

These and our previous results (17) would suggest that dopamine is acting via a receptor coupled to the GTP binding protein G_i that inhibits adenylyl cyclase; however, PKC is an alternative intermediate of adenylyl cyclase inhibition. AVP-dependent cAMP generation is inhibited by prostaglandin E₂ in the rat medullary thick ascending limb and by purinergic receptor agonists in the inner medullary collecting duct, but this inhibition is reversed by chalphostin C or chelerythrine, indicating that PKC is the intracellular mediator (5, 7). To test this possibility in the rat CCD, we conducted the set of experiments shown in Fig. 6, in which 10 µM dopamine was used to inhibit AVP-dependent cAMP generation. Neither calphostin C nor chelerythrine had any significant effect on cAMP production in the presence of dopamine, but the further addition of 10 µM clozapine increased cAMP production significantly. Therefore, we conclude that dopamine inhibits AVP-dependent cAMP generation via a D₄ receptor coupled to G_i.

Our results would appear to directly contradict previous reports that both dopamine and fenoldopam increased cAMP production in the absence of AVP and that this stimulatory effect was reversed by the D_1A-specific antagonist Sch-23390 (10, 12, 13). Katz and co-workers (13, 19) subsequently showed that dopamine acted via the D_1A receptor to inhibit Na-K-ATPase activity in permeabilized rat CCD segments, which they proposed would be expected to inhibit transepithelial Na⁺ reabsorption in the intact CCD. Satoh et al. (12, 13) have also shown that the D_1A receptor is linked to inhibition of Na-K-ATPase by activation of phospholipase A₂ and the production of eicosinoids. However, our data are not necessarily in disagreement with those of Satoh et al. (12, 13) and Ohbu and Felder (10) or with their conclusions, for the following reasons.

Using permeabilized nephron segments, these groups showed that the basal rate of cAMP production was in the range of 2 to 3 fmol · mm¹ · 7 min¹ (10, 13), only slightly less than observed in our own studies. Ohbu and Felder (10) found that 0.1 µM fenoldopam increased cAMP production to 6 fmol · mm¹ · 7 min¹. In the experiments of Satoh et al. (13), addition of 10 µM dopamine or 0.1 µM fenoldopam increased cAMP production (in the absence of AVP) significantly to, respectively, 5.5 ± 0.4 and 5.6 ± 0.6 fmol · mm¹ · 7 min¹. We could have easily failed to detect such a small effect of dopamine or fenoldopam on cAMP production in our experiments, given the fact that the means ± SE of our control cAMP measurements were of almost the same magnitude as the increase in cAMP production observed by either group (10, 13). Nevertheless, the important point is that, in contrast to the small inhibition observed by others in the absence of AVP (10, 13), in our experiments dopamine produced a very dramatic stimulation of AVP-dependent cAMP production. Satoh et al. (13) and Ohbu et al. (10) also found, as we did, that AVP increased cAMP production by 20- to 30-fold, but they did not test the effect of dopamine on cAMP production in the presence of AVP.

In other studies, we have shown that the D₄ dopamine receptor isoform is expressed in the rat CCD at both the mRNA and protein levels (unpublished observations and Ref. 18). O'Connell et al. (9) have clearly shown that the D_1A receptor protein is expressed in the rat CCD, and our own RT-PCR experiments confirm the presence of D_1A at the mRNA level (18). Thus an effect of dopamine through the D_1A receptor might be expected, although, in the absence of AVP, we observed no significant effect of dopamine or fenoldopam on cAMP production or Na⁺ transport in the isolated perfused rat CCD (17). It seems to us relevant to ask whether the D_1A receptor effect on cAMP production is an important component of dopamine action in the CCD. As is the case for all of the dopamine D₁-type receptors, the D_1A isoform is linked via G_s to stimulate cAMP production, but this increase in intracellular cAMP should increase transepithelial Na⁺ transport in the rat CCD, due to the increase in luminal membrane Na⁺ conductance it produces (3, 14, 16). Thus functionally increased cAMP production in the rat CCD, as produced by AVP, would be expected to increase Na⁺ reabsorption (3, 6, 11).

Based on the effect of dopamine to significantly reduce AVP-dependent cAMP production in the rat CCD, we conclude that this is the primary mechanism by which it inhibits transepithelial Na⁺ and water reabsorption in the CCD. Dopamine may also act through the D_1A receptor by coupling through phospholipase A₂ to inhibit Na-K-ATPase as proposed by Satoh et al. (12, 13). However, it does not appear that this effect is of significance in inhibiting transepithelial transport of Na⁺ or water. Thus the role of the D_1A receptor isoform in the rat CCD remains to be determined.