0022-3565/01/2983-1001–1006$3.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics
JPET 298:1001–1006, 2001
Vol. 298, No. 3
4062/925669
Printed in U.S.A.
Dopamine Inhibits Vasopressin Action in the Rat Inner
Medullary Collecting Duct via ␣2-Adrenoceptors
RICHARD M. EDWARDS and DAVID P. BROOKS
Department of Renal/Urology Biology, GlaxoSmithKline, King of Prussia, Pennsylvania
Received April 20, 2001; accepted May 22, 2001
This paper is available online at http://jpet.aspetjournals.org
and norepinephrine-induced decreases in AVP-stimulated Pf.
Dopamine-induced inhibition of AVP-dependent cAMP levels
was antagonized by the ␣2-adrenoceptor antagonists, rauwolscine, idazoxan, and yohimbine, but not by the dopamine receptor antagonists, spiperone, SCH-23390, or raclopride. Clozapine (1–10 ␮M) inhibited the effects of both dopamine and
norepinephrine on AVP-stimulated cAMP levels. We conclude
that the inhibitory effects of dopamine on AVP-induced Pf and
cAMP accumulation in the rat IMCD are mediated via ␣2adrenoceptors.
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 vaso-
pressin-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 (Ed-
ABBREVIATIONS: IMCD, inner medullary collecting duct; AVP, arginine vasopressin; Pf, osmotic water permeability; CPT-cAMP, 8-p-chlorophenylthio-cAMP.
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ABSTRACT
We compared the effects of dopamine and norepinephrine on
vasopressin (AVP)-stimulated increases in osmotic water permeability (Pf) and cAMP accumulation in the rat inner medullary
collecting duct (IMCD). Both dopamine and norepinephrine
inhibited AVP-induced Pf and cAMP accumulation in a concentration-dependent manner; however, norepinephrine was approximately 100-fold more potent than dopamine. The effects
of dopamine on Pf were antagonized by the selective ␣2-adrenoceptor antagonist, rauwolscine (10 nM–1 ␮M). Clozapine (10
␮M), a dopamine D4 receptor antagonist with significant activity
at adrenergic receptors, partially attenuated both dopamine
1002
Edwards and Brooks
wards 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
Results
Dopamine produced a rapid and reversible decrease in
AVP-induced Pf (Fig. 1). In the presence of 10 pM AVP, Pf
averaged 947 ⫾ 89 ␮m/s. Addition of 10 ␮M dopamine to the
bath decreased Pf to 141 ⫾ 27 ␮m/s (p ⬍ 0.001). Upon
removal of dopamine, Pf increased to 890 ⫾ 99 ␮m/s, a value
not different from the initial AVP period. In contrast to its
effect on AVP-induced Pf, dopamine (10 ␮M) had no effect on
Fig. 1.. Effects of dopamine on AVP (䡺) and 8-p-chlorophenylthio-cAMP
(cAMP; f) induced increases on Pf. Tubules were exposed to AVP (10 pM)
or cAMP (100 ␮M) throughout the experiment. In the second period, the
bath was changed to one containing dopamine (10 ␮M) 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.
ⴱ, value significantly different (p ⬍ 0.05) from both AVP periods.
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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 ␮m (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 (␮m/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.
Most experiments consisted of three collection periods: a control
period, an experimental period, and an additional control period at
the end of the experiment. Approximately 20 min after changing to
an isotonic perfusate, the bath was changed to one containing a
near-maximal concentration of AVP, 10 pM (Nadler et al., 1992), to
which the tubule was exposed for the remainder of the experiment.
Thirty to 40 min following the addition of AVP, three to four collections were made to determine AVP-stimulated Pf (control period).
Test compounds (e.g., dopamine) were then added to the AVP-containing bath, and 15 min later, three to four collections were made
(experimental period). Test compounds were then removed from the
bath, and following a 15-min equilibration period, an additional
three to four collections were made in the presence of AVP alone. An
identical protocol was used when the cAMP analog, 8-p-chlorophenylthio-cAMP (CPT-cAMP), was used in place of AVP. Concentration-response experiments were performed in a similar manner except that each tubule was exposed to sequentially higher
concentrations of the compounds, separated by 15-min equilibration
periods. For each experiment, a Pf value for a given period was
determined by averaging the values obtained from three to four
collections. Results are expressed as Pf in absolute terms or as a
percentage of AVP-stimulated Pf.
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 ␮l of the above solution to siliconized test tubes. The
samples were preincubated for 10 min at 37°C, after which an
additional 10 ␮l 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 ␮l 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.
Dopamine and Vasopressin Action
Fig. 2. Concentration-dependent inhibition of vasopressin-induced Pf (10
pM) by dopamine (E) and norepinephrine (F). Results are expressed as a
percentage of vasopressin-induced Pf, which was 905 ⫾ 93 ␮m/s for the
dopamine series (n ⫽ 5) and 836 ⫾ 86 ␮m/s for the norepinephrine series
(n ⫽ 4).
Fig. 3. Effect of rauwolscine on dopamine-induced inhibition of AVPdependent Pf. AVP (10 pM) was present throughout the experiment.
Tubules (n ⫽ 5) were sequentially exposed to dopamine (10 ␮M), dopamine plus rauwolscine, followed by a second dopamine exposure at the
end of the experiment. DA, dopamine; ⴱ, significantly different (p ⬍ 0.05)
from dopamine periods.
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 ␮M, n ⫽ 5) or norepinephrine (1 ␮M, n ⫽ 5) and
clozapine (10 ␮M). 䡺, AVP; f, agonist; o, clozapine; *p ⬍ 0.05 compared
with dopamine or norepinephrine alone.
norepinephrine (1 ␮M) on AVP-dependent Pf to the same
degree.
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 ␮M, 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 ␮M 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
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Pf stimulated by the cAMP analog, CPT-cAMP (Fig. 1). Pf in
the presence of 100 ␮M CPT-cAMP was 690 ⫾ 32 ␮m/s and
did not change when 10 ␮M dopamine was added to the bath
(659 ⫾ 50 ␮m/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 ␮M 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 ␮m/s in the absence and
presence of dopamine (10 ␮M, n ⫽ 6) and 112.4 ⫾ 16.3 and
120.6 ⫾ 10.3 ␮m/s in the absence and presence of norepinephrine (10 ␮M, n ⫽ 5).
Since high concentrations of dopamine can activate ␣2adrenoceptors, 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 ␮M dopamine decreased AVP-stimulated Pf from 966 ⫾ 38
to 126 ⫾ 29 ␮m/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 ␮m/s in the
presence of 10 nM, 100 nM, and 1 ␮M rauwolscine, respectively. Pf in the presence of 1 ␮M rauwolscine was not significantly different from AVP alone. Following removal of
rauwolscine, Pf decreased to 234 ⫾ 18 ␮m/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 ␮M) partially attenuated
the inhibitory effect of dopamine (10 ␮M) on AVP-dependent
Pf (Fig. 4), however, clozapine also inhibited the effects of
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Edwards and Brooks
Fig. 6. Effect of rauwolscine on dopamine-induced inhibition of AVPstimulated 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.
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 ␮M, which is well above the Ki 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).
Discussion
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
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 ␮M
dopamine with and without 1 ␮M 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.
Fig. 8. Effect of clozapine on dopamine (10 ␮M) and norepinephrine (1
␮M)-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. 䡺, agonist; p, 1 ␮M clozapine; f, 10 ␮M clozapine; *p ⬍ 0.05 versus dopamine or norepinephrine
alone.
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 D3selective 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 ␣2adrenoceptors (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
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Fig. 5. Concentration-dependent inhibition of AVP-induced cAMP accumulation by dopamine (F) and norepinephrine (E) 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.
Dopamine and Vasopressin Action
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 dopamineinduced 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.
Acknowledgments
We thank Maria C. McDevitt for assistance in preparing the
manuscript.
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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 dopamineinduced 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 Ki
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 ␣2adrenoceptors. 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
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Address correspondence to: Dr. Richard M. Edwards, GlaxoSmithKline, Department of Renal/Urology Biology, UW2521, 709 Swedeland Road, P.O. Box 1539, King
of Prussia, PA 19406-0939. E-mail: [email protected]
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