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. 1. Autoradiographic localization of [3H]spiperone binding to longitudinal sections of rat kidney. Autoradiograms were analyzed by computerized image analysis with pseudocolor coding (the highest density is red). (A) Total binding in the presence of [3H]spiperone (10 nM). Note the high density binding in the inner medulla and the diffuse labeling in the cortex. (B) Nonspecific binding of [3Hlspiperone (10 nM) in the presence of spiperone (100 ,uM). Note the complete absence of labeling in the inner medulla or cortex. (C) [3H]Spiperone (10 nM) binding in the presence of the 5-HT2 antagonist ketanserin (100 AM). Note that ketanserin did not compete for [3H]spiperone binding. (D) Higher magnification of [3H]spiperone binding in the inner medulla. Note the longitudinally oriented tubular-like structures converging toward the tip of the papilla. Downloaded by guest on September 28, 2021 3172 Physiology/Pharmacology: Huo et al. Proc. Natl. Acad Sci. USA 88 (1991)

(Fig. 1A) and was seen in autoradiograms as thin tubular-like T Sp Sc H T Sp Sc H I Sc Sp H elements converging toward the tip of the papilla (Fig. 1D). 200 - - 200 Higher-resolution autoradiography using emulsion-coated

coverslips (11) indicated that this pattern of labeling was due 97- -97 to labeling of the inner medullary collecting ducts. [3H]Spip- 68 - . 68 erone binding in the cortex was distributed diffusely over tubular elements. The renal vasculature, including glomeruli, 43 - ip~t43 was not labeled. [3H]Spiperone has been reported to bind to 5-hydroxytryptamine 2 (5-HT2) receptors in the central ner-

t., ...r ..... vous system (23), but addition of the 5-HT2-selective antag- 29 - , 29 onist ketanserin did not diminish [3H]spiperone binding to Striatu~m Pituitary MeLalla kidney sections (Fig. 1C). Further characterization of the [3H]spiperone binding us- FIG. 2. Autoradiograms of striatum, pituitary, and kidney inner ing crude membranes from the inner medulla indicated that medulla membranes labeled with [I25I]N3-NAPS and separated by SDS/PAGE. Note labeling of a major band of Mr 120,000-130,000 in the binding was saturable, specific, and stereoselective. the absence of competitors (total, T) that could be competed for by Scatchard analysis of the [3H]spiperone binding was best fit 100,M spiperone (Sp) or 100,M haloperidol (H) but not by 100,uM to a single binding site with a Kd of 17.2 ± 1.65 nM and a Bm. Sch 23390 (Sc). Numbers to the left and right refer to the relative of 935 ± 83 fmol per mg of protein (n = 4). Spiperone was migration of proteins of known Mr (X10-3). =300-fold more potent at competing for [3H]spiperone bind- ing than the next most potent drug, haloperidol, and 1000- [1251]N3-NAPS, a photosensitive analogue of spiperone, was fold more potent than (+)-butaclamol and cis(Z)-flupentixol incubated with membranes from the inner medulla, striatum, (Table 1). The active enantiomer (+)-butaclamol was slightly and pituitary. The major incorporation of [125I]N3-NAPS was more potent at competing for [3H]spiperone binding than the into a diffuse band ofapparent Mr 120,000-130,000 in all three inactive enantiomer (-)-butaclamol. There was no discrim- tissues (Fig. 2). Labeling was inhibited by spiperone (a D2 ination between (-)-sulpride, a D2 antagonist, and (+)- receptor antagonist) and haloperidol (a D1/D2 receptor an- sulpride, the inactive enantiomer. The mixed agonist 6,7- tagonist) but not by Sch 23390 (a D1 receptor antagonist). ADTN was -3 times more potent than DA at competing for To test whether the [3H]spiperone binding site in the inner [3H]spiperone binding. LY171555, a D2 agonist in the central medulla was homologous with the striatal D2 receptor, RNA nervous system, was not an effective competitor for [3H]spip- (Northern) hybridization analysis was conducted with oligo- erone binding in the inner medulla. nucleotide probes complementary to the brain D2 receptor The unique pharmacological profile of the [3H]spiperone mRNA. A positive hybridization signal of the appropriate binding in the inner medulla led us to determine whether the size was detected with striatal RNA but was not seen with protein being labeled in the kidney inner medulla was the kidney inner medulla RNA (Fig. 3). same size as the D2 receptor in the striatum and pituitary. DA and the DA2 agonist bromocriptine have been reported to increase urinary excretion of PGE2 (24), the highest levels Table 1. Inhibition of specific [3Hlspiperone binding in rat of which are produced in the kidney inner medulla (25). kidney inner medulla membranes Known stimulators of PGE2 synthesis, such as arachidonic Subtype IC50, ,M Slope factor acid and bradykinin, stimulated PGE2 production from pri- DA antagonist mary cultures of IMCD cells (Fig. 4). Bromocriptine also Spiperone DA2 0.035 ± 0.01 0.56 ± 0.05 increased PGE2 production, but the D2 agonist LY171555 Haloperidol DA1/DA2 12.2 ± 4.7 0.92 ± 0.14 failed to stimulate PGE2 release, consistent with the inability (+)-Butaclamol DA1/DA2 38.3 ± 23.2 1.19 ± 0.20 of LY171555 to compete for [3H]spiperone binding (Table 1). cis(Z)- DA (100 ,M) stimulated PGE2 production from IMCD cells Flupentixol DA1/DA2 43.3 ± 21.0 1.09 ± 0.11 -2-fold. The effect was blocked by domperidone, a periph- Domperidone DA2 118.5 ± 17.5 1.21 ± 0.21 eral DA2 antagonist, but not by other antagonists, including (-)-Butaclamol DA1/DA2 128.2 ± 5.5 1.30 ± 0.20 Sch 23390 (DA1), propranolol (,B-adrenergic), or phentol- SCH23390 DA1 >300 amine (a-adrenergic). The cyclooxygenase inhibitor indo- SKF83566 DA1 >300 methacin completely blocked DA-stimulated PGE2 produc- (-)-Sulpride DA2 >1000 tion. (+)-Sulpride DA2 >1000 DA agonist DISCUSSION 6,7-ADTN DA1/DA2 189.5 ± 34.1 0.54 ± 0.12 DA 533.7 ± 169.7 0.60 ± 0.08 [3H]Spiperone has been used extensively to study D2 recep- Fenoldopam DA1 >300 tors in the central nervous system (26). [3H]Spiperone bind- SKF38393 DA1 >300 ing sites have also been reported in kidney cortex by homog- Apomorphine DA1/DA2 >300 enate binding and have been proposed to represent DA2 Norapomorphine DA1/DA2 >300 receptors (14-16). Autoradiographic localization of [3H]spip- LY171555 DA2 >1000 erone binding indicated that the cortex was diffusely labeled, Other agonist/ whereas the inner medullary collecting ducts were heavily antagonist labeled. [3H]Spiperone binding in the kidney inner medulla Prazosin al >100 Yohimbine a2 >100 1 2 Ketanserin 5-HT2 >100 Isoproterenol 11/132 >300 -28S FIG. 3. Autoradiograms of a Northern blot analysis of 20 Ag of total RNA from Rat inner medulla membranes were incubated with [3H]spiperone kidney inner medulla (lane 1) and striatum (final concentration, S nM) in the presence of unlabeled drug. The (lane 2) hybridized with a brain D2 receptor concentration of unlabeled drug that produced 50% inhibition of -18S synthetic 32P-labeled oligonucleotide probe. specific binding was determined as the IC50. Values are means + Note the absence of a positive signal from SEM of three experiments, with each point determined in triplicate. the inner medulla. Downloaded by guest on September 28, 2021 Physiology/Pharmacology: Huo et al. Proc. Natl. Acad. Sci. USA 88 (1991) 3173

400

co m FIG. 4. Effects of DA agonists on PGE2 300 production by IMCD cells. IMCD cells in culture were incubated for 30 min with or without antagonists and then incubated with -I.0 T Aagonists d alone or in the presence ofantagonists 0 200 ± So for 60 min. Results shown are means SE of triplicate culture wells (each experiment was 0._ repeated 3-12 times). Values are expressed as 0~ percentage basal (basal = 29.3 + 3.1 pg per ug 100 of protein). Statistical analysis of 100 juM DA we alone vs. 100 ,uM DA in the presence of 0w antagonists was performed by analysis of vari- 0XX** ance and Scheffe's F test; *, P < 0.05 vs. control; **, P < 0.05 vs. 100 AM DA. AA, ° arachidonic acid; BK, bradykinin; BM, bro- 6S 0 0 ° 0 0O0 0 0 0 0 mocriptine; LY, LY171555; DP, domperi- ggg done; SC, Sch 23390; PR, propranolol; PT, X.$ t <, phentolamine; IM, indomethacin. was moderately high affinity (Kd, 17 nM), similar to values hybridization results: (i) The level of the D2 receptor mRNA reported for the kidney cortex, with a maximum binding in the inner medulla was simply below the level of detection. density that was 3-6 times higher in the inner medulla than This would appear unlikely since the density of the [3H]spip- in the renal cortex (14-17). [3H]Spiperone binding in the inner erone binding site in the kidney inner medulla was compa- medulla was similar to the maximum binding densities re- rable to the density of the D2 receptor in the striatum. (ii) The ported for rat striatum (17, 26), although the binding affinity kidney DA receptor is an alternatively spliced variant of the was 15-500 times lower in the inner medulla than in rat brain D2 receptor and the probes used here were not com- striatum (17, 26). The specificity ofthe [3H]spiperone binding plementary. Although the D2 receptor gene has an intron/ in the inner medulla also differed from the pharmacological exon structure (28) and two alternatively spliced subtypes of profile of the brain D2 receptor. In particular, dopaminergic the D2 receptor in brain and pituitary have been reported (21, drugs that compete for [3H]spiperone binding at nanomolar 28-30), several observations make this possibility unlikely. concentrations in the brain were competitive at micromolar First, Monsma et al. (21) and Giros et al. (29), using different concentrations in the inner medulla. In addition, (-)-sulpride D2 receptor probes than were used here, also failed to detect and LY171555, selective D2 receptor ligands, were ineffec- a positive hybridization signal with kidney RNA. Likewise, tive competitors for [3H]spiperone binding in the kidney. the oligonucleotide probes selected here for hybridization Likewise, although the [3H]spiperone binding in the inner were complementary to the putative transmembrane- medulla was stereoselective, with (+)-butaclamol more po- spanning regions (IV and VII) of the D2 receptor, regions that tent than the inactive stereoisomer (-)-butaclamol, the dis- are highly conserved among aminergic receptors. Thus, it is crimination between isomers was insignificant (3- to 4-fold) highly unlikely that the negative hybridization results were compared to the stereoselectivity shown by the central due to the kidney inner medulla DA receptor being an nervous system D2 receptor for butaclamol (10,000-fold) (26). alternatively spliced variant of the brain D2 receptor. A similar atypical pharmacological profile has been reported Relevant to this discussion is a recent report by Sokoloffet al. for [3H]spiperone binding in the renal cortex (17). (31), which was published while this manuscript was under Concern that [3H]spiperone might be binding to a non-DA review. These authors reported the cloning of a DA receptor receptor in the kidney led us to determine whether the protein from brain, which they have termed the D3 receptor. This that was being labeled in the inner medulla was the same size receptor is 52% homologous to the D2 receptor overall and 75% as the D2 receptor from brain and pituitary. Amlaiky and [125 homologous in the putative transmembrane-spanning regions. Caron (18) have reported that I]N3-NAPS, a photoaffinity The D3 receptor has a somewhat different pharmacological spiperone analogue, labeled a Mr ==94,000 peptide species profile compared to the D2 receptor and does not appear to be from striatal membranes and two peptides, Mr --120,000 and Mr -94,000, from the neurointermediate lobe ofthe pituitary. negatively linked to adenylyl cyclase, as is the D2 receptor (31). The from bovine had Interestingly, the D3 receptor may be expressed in the kidney, purified D2 receptor pituitary, however, since a weak signal was detected by the polymerase chain a Mr = 120,000 when labeled with [125I]N3-NAPS, suggesting that the smaller was a reaction (31). Examination of the competition binding data, Mr -=94,000 peptide species proteolytic the pharmacological profile of the D3 product ofthe Mr = 120,000 peptide species (19). Here, under however, indicates that conditions in which proteolysis was stringently controlled, receptor is much more similar to the brain D2 receptor than the we determined that the predominant specific incorporation of IMCD DA receptor. In addition, although quinpirole (LY171555) [125I]N3-NAPS was into a broad protein band ofMr ==120,000 was 100-fold more selective for the D3 receptor than the D2 in striatum, pituitary, and kidney inner medulla, with no receptor (31), it was the least potent ofany dopaminergic agonist labeling seen at Mr ==94,000. These results demonstrate that used here as a competitor for [3H]spiperone binding and it was the kidney inner medulla spiperone binding site is very also ineffective at stimulating PGE2 production from IMCD cells. similar in size to the D2 receptor from striatum and pituitary. In addition, the D2 receptor oligonucleotide probes used here for The D2 receptors from brain and pituitary have recently the RNA hybridization analyses were -71% complementary been cloned (21, 27-30). Using radiolabeled oligonucleotide (32/45 and 30/42 nucleotides) to the nucleotide sequence of the probes complementary to the rat brain D2 receptor, however, D3 receptor mRNA and may have been expected to hybridize we were unable to detect a positive hybridization signal from with kidney inner medulla RNA ifthe D3 receptor was expressed the kidney inner medulla. If the inner medulla DA receptor in the inner medulla. Taken together, these negative pieces of was actually the renal form of the brain D2 receptor, then evidence would suggest that the brain D3 receptor and the kidney there are two possibilities that could account for the negative inner medulla DA receptor are not identical. Downloaded by guest on September 28, 2021 3174 Physiology/Pharmacology: Huo et al. Proc. Natl. Acad. Sci. USA 88 (1991) Thus, the most likely explanation for the lack of hybrid- the natriuretic/diuretic effects of DA, whether formed in- ization seen here with D2 receptor probes and kidney inner trarenally or administered exogenously. medulla RNA is that the kidney [3H]spiperone binding site represents a DA receptor encoded by a different gene than We would like to thank Drs. Jack Peter Green, Ravi Iyengar, and the brain D2 receptor. The marked differences in the phar- Pramod Srivastava for helpful discussions and Dr. George Prell for assistance with the statistical analysis. This work was supported by macology of the [3H]spiperone binding in the kidney inner the American Heart Association (Grant 881190), the Irma T. Hirschl medulla compared to the striatal D2 receptor suggests that the Foundation, and the National Institutes of Health (Grant HL42485). two receptors are distinct, whereas the fact that both bind D.P.H. is an American Heart Association Established Investigator. [3H]spiperone and are a similar size suggests that the kidney inner medulla DA receptor is D2-like. Thus, on the basis of 1. Goldberg, L. I. (1972) Pharmacol. Rev. 24, 1-29. 2. Lokhandwala, M. F. & Barrett, R. J. (1982) J. Auton. Pharmacol. 3, our findings, we speculate that the kidney inner medulla DA 189-215. receptor represents a newly discovered DA receptor sub- 3. Wasserman, K., Huss, R. & Kullman, R. (1980) Naunyn-Schmiedebergs type, designated here as DA2K. This is consistent with recent Arch. Pharmacol. 312, 77-83. 4. McGrath, B., Bode, K., Luxford, A., Howden, B. & Jablonski, P. (1985) speculation as to the existence of additional peripheral DA Clin. Exp. Pharmacol. Physiol. 12, 343-352. receptor subtypes (32). There is some precedence in support 5. Hegde, S. S., Jadhav, A. L. & Lokhandwala, M. F. (1989) Hypertension of multiple D2/DA2 receptor genes, as three distinct a2- 13, 828-834. 6. Frederickson, E. D., Bradley, T. & Goldberg, L. I. (1985) Am. J. adrenergic receptor genes with a heterogenous pattern of Physiol. 249, F236-F240. tissue expression have been cloned (33). 7. Felder, R. A., Felder, C. C., Eisner, G. M. & Jose, P. A. (1989) Am. J. We further determined that the DA2K receptor in the inner Physiol. 257, F315-F327. medulla was functional, since bromocriptine and DA, but not 8. Kebabian, J. W. & Calne, D. B. (1979) Nature (London) 277, 93-96. 9. Goldberg, L. I. & Kohli, J. D. (1979) Commun. Psychopharmacol. 3, LY171555, stimulated PGE2 production in IMCD cells, and 447-456. that the DA-mediated stimulation could be blocked selec- 10. Hegde, S. S., Ricci, A., Amenta, F. & Lokhandwala, M. F. (1989) J. tively by the DA2 antagonist domperidone. Pharmacol. Exp. Ther. 251, 1237-1245. Additional sup- 11. Huo, T. & Healy, D. P. (1989) Am. J. Physiol. 257, F414-F423. port that the DA-stimulated PGE2 production was via the 12. Stier, C. T., Cowden, E. A. & Allison, M. E. (1982) J. Pharmacol. Exp. DA2K receptor comes from the demonstration that [3H]dom- Ther. 220, 366-370. peridone binds to IMCD cells in vitro with a pharmacological 13. Seri, I. & Aperia, A. (1988) Am. J. Physiol. 254, F196-F201. 14. Felder, C. C., McKelvey, A. M., Gitler, M. S., Eisner, G. M. & Jose, profile similar to the profile for [3H]spiperone binding to P. A. (1989) Kidney Int. 36, 183-193. kidney inner medulla membranes (T.H. and D.P.H., unpub- 15. Felder, R. A., Nakamura, K. T., Robillard, J. E., Kanadjian, M. & Jose, lished data). The DA stimulation of PGE2 production in P. A. (1988) Pediatr. Nephrol. (NY) 2, 156-162. 16. Galbusera, M., Garattini, S., Remuzzi, G. & Mennini, T. (1988) Kidney IMCD cells is consistent with a report by Yoshimura et al. Int. 33, 1073-1077. (24) in which they showed that DA and the DA2 agonist 17. Hietala, J., Syvalahti, E. & Roytta, M. (1988) J. Receptor Res. 8, bromocriptine increased urinary excretion of PGE2. PGE2 is 753-771. 18. Amlaiky, N. & Caron, M. G. (1986) J. Neurochem. 47, 196-204. the most abundant renal prostaglandin in the rat and the 19. Senogles, S. E., Amlaiky, N., Falardeau, P. & Caron, M. G. (1988) J. highest levels are produced in the inner medulla (25). PGE2 Biol. Chem. 263, 18996-19002. is known to be a potent physiological antagonist of the 20. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, 156-159. 21. Monsma, F. J., Jr., McVittie, L. D., Gerfen, C. R., Mahan, L. C. & hydroosmotic effects ofvasopressin in the collecting duct and Sibley, D. R. (1989) Nature (London) 342, 926-929. also directly inhibits sodium reabsorption in the distal neph- 22. Sato, M. & Dunn, M. J. (1984) Am. J. Physiol. 247, F423-F433. ron (25). The demonstration that DA stimulates PGE2 pro- 23. Leysen, J. E., Niemegeers, C. J. E., Tolenaere, J. P. & Laduron, P. M. duction, together with the (1978) Nature (London) 272, 168-171. known physiological properties of 24. Yoshimura, M., Takashina, R., Takahasi, H. & Ijichi, H. (1987) Agents DA and PGE2 as vasodilators and inhibitors of sodium Actions 22, 93-99. reabsorption, suggests that these agents may act in concert to 25. Dunn, M. J. (1983) in Renal Endocrinology, ed. Dunn, M. J. (Williams & modulate water and electrolyte excretion in the inner me- Wilkins, Baltimore), pp. 1-74. 26. Seeman, P. (1981) Pharmacol. Rev. 32, 229-313. dulla. Attempts to block DA-mediated renal responses by 27. Bunzow, J. R., Van Tol, H. H. M., Grandy, D. K., Albert, P., Salon, J., inhibition of prostaglandin synthesis, however, have led to Christie, M., Machida, C. A., Neve, K. A. & Civelli, 0. (1988) Nature conflicting results (34) but should be reexamined in light of (London) 336, 783-787. the present findings. 28. Grandy, D. K., Marchionni, M. A., Makam, H., Stofko, R. E., Alfano, M., Frothingham, L., Fischer, J. B., Burke-Howie, K. J., Bunzow, The presence of DA2K receptors in the distal nephron J. R., Server, A. C. & Civelli, 0. (1989) Proc. Natl. Acad. Sci. USA 86, raises new possibilities regarding the action of DA within the 9762-9766. kidney. DA is known to be produced intrarenally by uptake 29. Giros, B., Sokoloff, P., Martres, M. P., Riou, J. F., Emorine, L. J. & and decarboxylation of L-dopa by the proximal tubules in Schwartz, J. C. (1989) Nature (London) 342, 923-926. 30. Chio, C. L., Hess, G. F., Graham, R. S. & Huff, R. M. (1990) Nature proportion to sodium load (35-37). Indeed, the major portion (London) 343, 266-269. of urinary DA excreted appears to be derived from the 31. Sokoloff, P., Giros, B., Martres, M. P., Bouthenet, M. L. & Schwartz, intrarenal conversion of plasma L-dopa (38). The local syn- J. C. (1990) Nature (London) 347, 146-151. thesis ofDA together with the presence ofrenal DA receptors 32. Anderson, P. H., Gingrich, J. A., Bates, M. D., Dearry, A., Falardeau, P., Senogles, S. E. & Caron, M. G. (1990) Trends Pharmacol. Sci. 11, has led to the concept that DA may be an intrarenal natri- 231-236. uretic substance (5, 35, 39). It is possible to speculate that 33. Lorenz, W., Lomasney, J. W., Collins, S., Regan, J. W., Caron, M. G. DA, produced by the proximal tubules under conditions of & Lefkowitz, R. J. (1990) Mol. Pharmacol. 38, 599-603. increased sodium load, could filter down and bind to DA2K 34. Manoogian, C., Nadler, J., Ehrlich, L. & Horton, R. (1988) J. Lab. Clin. Endocrin. Metab. 66, 678-683. receptors on IMCD cells. Activation of DA2K receptors could 35. Lee, M. R. (1982) Clin. Sci. 62, 439-448. then inhibit vasopressin activity either directly, as has been 36. Chan, Y. L. (1976) J. Pharmacol. Exp. Ther. 199, 17-24. shown for DA in rabbit cortical collecting ducts (40), or 37. Baines, A. D. & Chan, W. (1980) Life Sci. 26, 253-259. indirectly through release of PGE2. A reduction in vasopres- 38. Zimlichman, R., Levinson, P. D., Kelly, G., Stull, R., Keiser, H. R. & Goldstein, D. S. (1988) Clin. Sci. 75, 515-520. sin activity would result in a decrease in water reabsorption 39. Ball, S. G. & Lee, M. R. (1977) Br. J. Clin. Pharmacol. 4, 115-119. by the collecting ducts. Thus, the IMCD DA2K receptor may 40. Muto, S., Tabei, K., Asano, Y. & Imai, M. (1985) Eur. J. Pharmacol. 114, be topologically situated to play a significant role in mediating 393-397. Downloaded by guest on September 28, 2021