In the Kidney Inner Medulla (Prostaglandin E2/Collecting Duct) T
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Proc. Nati. Acad. Sci. USA Vol. 88, pp. 3170-3174, April 1991 Physiology/Pharmacology Characterization of a dopamine receptor (DA2K) in the kidney inner medulla (prostaglandin E2/collecting duct) T. Huo, M. Q. YE, AND D. P. HEALY* Department of Pharmacology, Mount Sinai School of Medicine of the City University of New York, New York, NY 10029 Communicated by Karl H. Beyer, Jr., December 18, 1990 (received for review September 17, 1990) ABSTRACT Dopamine (DA) produces a natriuretic/diuretic branes (14-17), although the precise localization of the bind- response in the kidney by mcnisms that are still not well ing sites remains unclear. understood. There is some ination that DA2 receptors may be Therefore, to gain further insight into the possible role of involved in mating the effects ofDA, but little is known regard- DA2 receptors in the kidney, we sought to localize the DA2 ing the nature of this receptor in the kidney. Autoraigaphic receptor by in vitro autoradiography with [3H]spiperone and localation of [3Hspiperone, a DA2 anta t, i ed that then to characterize the receptor pharmacologically, bio- high-density binding was r ic to inner meduilary ting chemically, and physiologically. ducts (IMCDs). [3H]Spiperoe binding was saturable, high affinity (Kd, 17.2 ± 1.65 aM), and high density (Bmq, 935 ± 83 fnol per MATERIALS AND METHODS mg ofprotein). The photosensitive spiperone anogeNp-azido- m-['25j]iodophenethyl)sIperone labeled similr sized proteins of In Vitro Autoradiography. In vitro autoradiography of M - 120,000 in membranes prepared from the kidney inner [3H]spiperone was conducted by a procedure similar to that medulla, sum, and pituitary. However, the rank-order com- described in ref. 11. Male Sprague-Dawley rats (200-250 g; petition profile for the [3llspiperone binding in the kidney inner Charles River Breeding Laboratories) were anesthetized with medulla differed from the DA2 receptor in striatum and pituitary sodium pentobarbital (50 mg/kg; i.p.) and perfused transcar- and, furthrinore, RNA (Northern) blot analyses of kidney inner dially with 200 ml of 0.1% formalin in phosphate-buffered medullary RNA with brain DA2 receptor oligonuleotide probes saline, and the kidneys were excised. Slide-mounted 15-,um were negative. Functionally, DA stimulated pI E2 pro cryostat sections of kidney were incubated with 10 nM duction by IMCD cells, an effect that could be blocked by the DA2 [3H]spiperone (specific activity 75-85 Ci/mmol; 1 Ci = 37 antagonist domperidone. These results indicate that the kidney GBq; Amersham) in 50 mM Tris buffer (pH 7.4) containing inner medulla expresses a functional DA receptor that may rep- 120 mM NaCl, 5 mM KCI, 2 mM CaC12, 1 mM MgCl2, 100 AM resent a newly identified DA receptor subtype (here d pargyline, and 0.1% ascorbic acid for 60 min at room tem- DAmK). Moreover, these rels suggest that the kidney inner perature, rinsed twice for 30 min each in fresh buffer at 40C, medulla may be a s f t site at which DA, either directly or dried, and exposed to Hyperfilm (Amersham) for 2 weeks. indirectly, influences water and electrolyte excretion. Nonspecific binding was determined in the presence of 100 ,M haloperidol. Exogenous dopamine (DA) produces a natriuretic/diuretic re- Homogenate Binding. Crude membranes from the kidney sponse in a variety of species, including humans (1, 2). The inner medulla were prepared as described for kidney cortex natriuretic effects of DA are generally thought to be due to an (11). Assays were conducted by incubating 50 ,ug of crude increase in renal blood flow and glomerular filtration rate (1) but membrane protein at 250C for 30 min in incubation buffer, as may include direct tubular effects since low doses of DA described above, containing dilute radioligand and drug or increase urine output and urinary sodium excretion without vehicle, followed by vacuum filtration through Whatman altering renal hemodynamics (3-5). Although the mechanism of GF/C filters. For saturation binding experiments, 1-100 nM action ofDA within the kidney is not resolved, the effects ofDA [3H]spiperone was used. Nonspecific binding was defined as on the kidney are blocked by DA receptor antagonists (2, 6, 7). binding in the presence of 100 ,uM haloperidol, with a DA receptors have been classified in the central nervous system total/nonspecific binding ratio of =4:1. For competition as D1 and D2 (8) and in the periphery as DA1 and DA2 (9). A studies, 5 nM [3H]spiperone was used together with increas- great deal of evidence indicates that vascular and tubular DA1 ing concentrations ofunlabeled DA agonists and antagonists. receptors are involved in mediating the actions of DA on the Photoaffinity Labeling. Photoaffinity cross-linking with kidney (6, 7). Further support for a tubular site ofaction for DA N-(p-azido-m-[251I]iodophenethyl)spiperone ([125I]N3-NAPS) comes from autoradiographic localization of DA1 receptors in was conducted similar to the procedure ofAmlaiky and Caron rat kidney, which indicates that the highest levels are found in (18) and Senogles et al. (19) with only minor modifications. In the proximal tubules (10, 11). particular, membranes from the striatum, pituitary, and kid- Much less is known regarding the role of DA2 receptors in ney inner medulla were prepared in 25 mM Tris-HCl, pH 7.2 mediating the renal responses to DA. Spiperone, a central (250C)/250 mM sucrose containing the following protease nervous system D2 antagonist, blocks the DA-stimulated inhibitors: 10 mM EDTA, 10 mM EGTA, benzamidine (15 increase in renal blood flow and sodium excretion in isolated Ag/ml), soybean trypsin inhibitor (5 jg/ml), leupeptin (5 perfused rat kidney (4). Bromocriptine, a DA2 agonist, has Ag/ml), 1 mM phenylmethylsulfonyl fluoride, pepstatin (100 been reported to increase renal blood flow and single- nephron glomerular filtration rate in the rat (12, 13). [3H]Spip- Abbreviations: DA, dopamine; [1251]N3-NAPS, N-(p-azido-m- erone binding has been reported in kidney cortical mem- [125I]iodophenethyl)spiperone; IMCD, inner medullary collecting duct; PGE2, prostaglandin E2; 6,7-ADTN, 6,7-dihydroxy-1,2,3,4- tetrahydronaphthalene; 5-HT2, 5-hydroxytryptamine 2. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" Pharmacology, Box 1215, Mount Sinai School of Medicine, One in accordance with 18 U.S.C. §1734 solely to indicate this fact. Gustave L. Levy Place, New York, NY 10029. 3170 Downloaded by guest on September 28, 2021 Physiology/Pharmacology: Huo et al. Proc. Natl. Acad. Sci. USA 88 (1991) 3171 ng/ml), aprotinin (5 ,ug/ml), 1 mM phenanthroline. Mem- were synthesized and labeled to high specific activity with branes (100-300 jig of protein for all three tissues) were [y-32P]ATP and T4 polynucleotide kinase. incubated in the dark with [1251]N3-NAPS (0.1-0.25 nM for Inner Medullary Collecting Duct (IMCD) Cell Isolation and striatum and pituitary and 0.25-0.5 nM for kidney inner Culture. IMCD cells were isolated from renal inner medulla medulla) for 60 min at room temperature. The membranes ofSprague-Dawley rats (200-250 g) according to the methods were then washed as described above and resuspended in of Sato and Dunn (22) and are described in detail elsewhere buffer. (T.H. and D.P.H., unpublished data). Cells were maintained incubation Photolysis was performed by exposing the 1 day in Dulbecco's modified Eagle's medium/F-12 medium incubation mixtures for 90 s at 12 cm from a high-pressure supplemented with 10% fetal calf serum and then changed to mercury lamp. The membranes were pelleted and resus- a fully defined K-1 medium. pended in SDS/PAGE sample buffer [50 mM Tris HCl, pH Prostaglandin E2 (PGE2) Production. IMCD cells were 6.8/10% SDS/10% (vol/vol) glycerol/5% 2-mercaptoethanol/ maintained in 24-well culture dishes and incubated in tripli- 0.003% bromophenol blue] containing protease inhibitors and cate for 60 min with medium containing 100 ,AM pargyline, were electrophoresed on a discontinuous SDS/10%o polyacryl- 0.01% ascorbic acid, and the appropriate drugs, and the amide gel. An equivalent amount of protein was loaded onto medium was assayed for PGE2 content by radioimmunoas- each lane. After electrophoresis, the gels were dried and say. Values were expressed as means ± SEM of triplicate exposed to autoradiographic film. culture wells based on experiments repeated 3-12 times. RNA (Northern) Blot Analysis. Total cellular-RNA was iso- Pharmacological Agents. The following drugs were used: lated from fresh rat tissues according to the method of Chom- LY171555 (Lilly Research Laboratories); ketanserin (Janssen czynski and Sacchi (20). Twenty micrograms oftotal RNA from Pharmaceutica); SKF 83566 and SKF 38393 (Smith Kline & each tissue was fractionated by electrophoresis on a 1% agarose French); cis(Z)-flupentixol (H. Lundbech A/S, Copenhagen); formaldehyde gel, transferred to nitrocellulose membranes, I-sulpride and d-sulpride (Ravizza, Milan); Sch 23390 (Scher- hybridized overnight with the 32P-labeled oligonucleotide probe ring); spiperone, 6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene at 590C, and washed the following day three times for 30 min (6,7-ADTN), (+)- and (-)-butaclamol, Sch 23388, and pargyline each in 3 x NET (lx NET = 150 mM NaCI/1 mM EDTA/15 (Research Biochemicals, Natick, MA). mM Tris HCI, pH 8) at 55°C. The nitrocellulose paper was then dried and exposed 2 days at -80°C with intensifying screens. RESULTS Two synthetic oligonucleotide probes complementary to the rat Autoradiographic localization of [3H]spiperone binding sites striatal D2 receptor mRNA at bases 478-522 and 1213-1254 (21) indicated that binding was concentrated in the inner medulla I .M' FIG.