Actu Physiol Scund 1990, 139, 393-404

Prostaglandin E, binding sites in human renal tissue: characterization and localization by radioligand binding and autoradiography I>.-O.E R I K S S O N , B. L A R S S O N , H. H E D L U N D " and K.-E. A N D E R S S O N Departments of Clinical Pharmacology and * Urology, University Hospital of Lund, Sweden ERIKSSON, L.-O., LARSSON, B., HEDLUND, H. & ANDERSON,K.-E. 1990. Prostaglandin E, binding sites in human renal tissue : characterization and localization by radioligand binding and autoradiography. Acta Physiul Scund 139,393-404. Received 30 December 1988, accepted 13 October 1989. ISSN 0001-6772. Departments of Clinical Pharmacology and Urology, University Hospital of Lund, Sweden. The prostaglandin E, (PGE,) binding site in human kidney was characterized in membrane preparations from cortex, outer medulla and inner medulla using radioligand binding techniques. The localization of the binding sites for ['HH]PGE, was visualized autoradiographically. In the membrane suspensions, the highest, level of specific [3H]PGE, binding was detected in the outer medulla (Bmax= 335+28 fmol mg-l protein) followed by the inner medulla (Bma,= 258kz1 fmol mg-' protein) and the cortex (B,,,,, = 143& 22 fmol mg..' protein). T h e binding was of high affinity with K,) values between 3.7 and 6.2 IIM in the various regions. Unlabelled prostaglandins competed for the ['HIPGE, binding sites in the following rank order of potency: PGE, x PGE, > PGFZ1% PGA, > PGB, > PGI, z PGD,. Autoradiographs revealed that a high density of [3H]PGE, (2 nM) binding sites were located on the distal tubule, particularly on the thick ascending limbs of Henle. Lower densities of [3H]PGE, binding sites were found on the medullary collecting ducts and possibly on the thin loops of Henle. In contrast, no specific ['HH]PGE, binding could be found on the proximal tubule, glomeruli or on blood vessels. This distribution is in accordance with the assumed site of action for the salt and water regulatory function of PGE,.

Key words : autoradiography, human, kidney, prostaglandin E,, receptor binding

Accumulating evidence suggests that prostaglandins (PGs) play a role at numerous sites of importance for renal function. Cells from various renal structures such as the tubules, the glomeruli and the medullary interstitium as well as the renal blood vessels have the capacity to synthesize and release PGs, which influence the regulation of renal blood flow, glomerular filtration rate, renin release, and renal electrolyte and water excretion (Schlondorff & Ardaillou 1986). The main effect of PGE, in the kidney is to augment salt and water excretion in both animal and man through a direct effect on the tubular Correspondence : L.-0. Eriksson, Department of Clinical Pharmacology, Lund University Hospital, S-221 85 Lund, Sweden.

membrane and/or by increasing the renal blood flow (Johnston et al. 1967, Shea-Donohue et al. 1979, Stokes 1979, Fadem et al. 1982). The thick ascending limbs of Henle, the collecting ducts, the glomeruli and the renal arterioles may hence be important target structures for endogenously synthesized PGE,. The biological activity of PGE, is assumed to be exerted via specific receptors on the various target tissues. Using radioligand binding techniques, the renal PGE, receptors have previously been studied in rabbit (Attallah & Lee 1973) and rat (Oien et al. 1979, Limas & Limas 1984, Eriksen et al. 1987). We have combined radioligand membrane binding and autoradiography in a study of the rat renal [3H]PGE, binding site (Eriksson et al. 1990). I n the present study the

393 14

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same technique was applied on h u m a n kidney to identify a [3H]PGE, binding site and t o examine its characteristics a n d regional distribution.

MATERIA1,S AND M E T H O D S Tissue preparation. Human kidneys were obtained from seven patients (Table I ) undergoing nephrectomy because of a renal cell carcinoma. Following resection, the kidneys were immediately bisected and placed in ice-cold Tris-HC1 buffer ( 1 0 mM) containing NaCl ( I 50 mM) and indomethacin ( 3 0 PM), p H 7.6, at 22 "C (buffer A). All subsequent procedures of membrane preparation %ere conducted at 4 "C. The kidneys were dissected free of tumour, connective tissue and fat. The remaining tissue was cut into its defined anatomical regions under a dissection microscope. Only those specimens of cortex, outer medulla and inner medulla which appeared free of disease were used for further preparations. The tissue for membrane preparation was stored frozen at -80 "C until used. Tissue specimens for macro- and microautoradiography were frozen in isopentane at -40 "C for 20 s and stored frozen at -80 "C until sections were made (see below). Membrane preparation. T h e tissue was cut into small pieces and homogenized carefully in 60 ml icecold buffer A with a Polytron P T 10/35 homogenizer (Kinematica) for 10-15 s. T h e rough homogenate was further homogenized by the use of glass homogenizers with Teflon pestles. The honiogenates were centrifuged at 700 g for 1 5 min at 4 "C and the supernatant was stored on ice while the pellet was suspended in fresh ice-cold buffer A, homogenized and recentrifuged at the same speed. The combined supernatants were centrifuged at 40,000 g for 30 min at 4 "C. The final pellet was suspended in ice-cold buffer A containing 2 mM MgCI, (buffer B). T h e final protein concentration was adjusted to approximately 5 mg ml-', corresponding to approximately 300 pg protein per incubation tube. Protein concentration was determined by the method of Lowry et al. (1951). Rudioligand binding. The radioligand binding studies on membranes and on slide-mounted sections from the human kidney were performed essentially as previously described for the rat kidney (Eriksson et al. '990). Membrune binding. In membrane saturation experiments, aliquots (60PI) of membrane suspensions were incubated with 20 p1 of various concentrations of [3H]PGE, (giving final concentrations of 0.25-1 5 nM), 20 ,ul buffer A or 20 pl unlabelled PGE, (10 PM, final concentration). The association of ['HIPGE, to and dissociation from its binding site were studied in preliminary experiments in the same system using I nM of the radiolabelled ligand. Similarly, inhibition

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Prostuglandin E , receptors in human kidney of ['lI]PGE, binding ( I nM) was studied using different PGs of various concentrations. After incubation to steady state at 37 "C, the process was terminated by adding 1 . 5 ml of ice-cold Tris-HC1 ( 5 0 mM) to the incubation mixture followed by rapid filtration under reduced pressure through Whatman G F / C glass fibre filters. T h e filters were rapidly washed three times with 5-ml portions of ice-cold Tris-HCI (jo mM). The radioactivity retained on the filters was subsequently determined in a Beckman liquid scintillation counter (model 1,s 1801). Non-specific binding of [311]PGEZwas defined as the radioactivity bound to membranes in the presence of a high concentration of unlabelled PGE, (10 PM). Specific hinding of ["IIIPGE, was defined as total binding minus non-specific binding. Microautorudiography. Frozen I 2-14 p m sections of each anatomical region were cut at - 15 "C on a cryostat (Bright Instrument Co. Ltd, Huntingdon, England), mounted onto chrome-alum/gelatinecoated slides and placed under vacuum at 4 "C overnight. Binding sites for [WIPGE, were labelled in zitro by covering the sections with zoopl of a solution containing 2 nM ['HIPGE,, diluted in buffer A, in the presence or absence of I O ~ MPGE,. The sections were subsequently placed in a humidity chamber and incubated to steady state at 37 "C. Some sections were incubated in buffer A and used for control of positive chemography. Following incubation, the slides were washed three times, 20 s each, in 50 mM Tris-HC1 at 4 "C, rinsed in ice-cold distilled water and dried under a stream of cold air. Coverslips which had previously been coated with photographic emulsion (Ilford K z emulsion) were placed over the sections and fixed to one end of the slide with adhesive tape. The emulsion was held in contact with the section by binder clips. The sections were subsequently stored dessicated in light-proof' boxes at 4 ° C for 8-10 weeks. After exposure, the coverslip was partially separated from the slide so that the emulsion could be developed and the section histologically fixed and stained with Mayer's haematoxylin and counter-stained with o. I o/A erythrocin. Some sections were stained for alkaline phosphatase with naphthol-AS-TR phosphate reagent containing fast red T R salt and counter-stained with Mayer's haematoxylin (Hack & Helmy 1974). The sections were mounted in Kaiser's glycerin-gelatin (Merck, Darmstadt, FRG) under dark-field illumination and viewed under bright-field and dark-field illumination. Macroautorudiograph,y. T h e frozen kidney was embedded in an aqueous gel of carboxymethylcellulose in a metal frame. The embedded specimen was subsequently frozen in n-hexan cooled with dry ice ( - 70 "C). Sections (20 p i ) were cut sagittally right through the kidney using a heavy sledge microtome (LKB PMV 450) operating at -20 "C. All sections were obtained by applying an adhesive tape (3M no.

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810, Minnesota Mining and Manufacturing Co.) to the section surface of the frozen specimen before cutting according to Ullberg (1954). The section attached to the adhesive tape was freeze-dried at - 20 "C for 48 h. Binding sites for PGE, were labelled in citro in a similar manner as described above for the glass-mounted sections with minor modifications. Sections were used for developing autoradiograms of total and non-specific binding, as well as for evaluation of positive chemography (buffer-incubated sections). In addition, controls were included for radioligand and buffer-incubated tapes without any sections. Autoradiograms were made by apposition of the sections to [ 3H]Ultrofilm (LKB, Bromma, Sweden). Following exposure for 10 weeks the film was developed as previously described (Eriksson et al. 1990). The autoradiograms were analysed with a computer image analyser (IBAS 2 , Kontron Bildanalyse GmbH, Munich, FRG) in order to generate pseudocolour images of PGE, binding areas. Data analyses. Results are expressed as means SEM of n experiments. Regression lines were calculated by the least-squares method. The dissociation constant ( K J and the,maximum number of binding sites (BmJ were calculated from plots according to Scatchard (1949). T o assess for the possibility of two separate populations of binding sites, the data were analysed by the LIGAND computer program (Munson & Rodbard 1980) and Hill plots (Bennett & Yamaniura 1985). In the competition studies, the concentration of the tested compound that produced a 507;decrement of the specific binding of the radiolabelled ligand (ICJ was calculated graphically and transformed to inhibition constants ( K , ) using the method described by Cheng & Prusoff (1973). Statistical comparisons of data between anatomical regions were made by one way analysis of variance (one-way ANOVA) and by Student's t-test. Differences were considered statistically significant if P < 0.05. Drugs, chemicals and equipment. ['H]prostaglandin E, ([3€I]PGE,), 160-zoo Ci mmol- I, was obtained from New England Nuclear, Boston, MA, USA. Unlabelled PGs were all purchased from Sigma, St Louis, MO, USA. Indomethacin and Sc 42867 were kindly supplied respectively by Merck Sharp & Dohme, Rahway, NJ, USA, and Searle Chicago, IL, USA. Naphthol-AS-TR phosphate, fast red T R salt, haematoxylin and erythrocin were obtained from Sigma, St Louis, MO, USA. Kaiser's glycerin-gelatin was obtained from Merck, Darmstadt, FRG. ['HIUltrofilm and Ilford Kz emulsion were respectively obtained from LKB, Bromma, Sweden, and Ilford Ltd, Mobberly, England. GF/C filters were obtained from Whatman International Ltd, Maidstone, England. All routine chemicals were either purchased from Sigma, St Louis, MO, USA, or from Merck, Darmstadt, FRG. 14.2

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L.-0. Eriksson et al. 2 . Dissociation constants ( K J and maximum number of binding sites (B,,,,J from saturation studies with ['HIPGE, in different regions of the human kidney

Table

Compartment 0

1, 0

\ 50

100

150

Cortex Outer medulla Inner medulla

3.7 f o . 7 6.2 f I .3

5.0 f0.4

protein)

n

143 f22 335 28 258 f2 1

4 4 4

Values represent meanf SEM of n different experiments performed in triplicate using separate membrane preparations. between the regions studied. Steady state was however reached within 20-40 min. Addition of 10 ,UM unlabelled PGE, was found to reverse the specific binding in a time-dependent manner. T h e binding of [3H]PGE, to cortex, outer and I 0 100 200 300 inner medulla was found tp be saturable with increasing concentrations of the radioligand. T h e data obtained were analysed by Scatchard plots (Fig. I ) . As can be seen in Table 2, binding sites along the nephron appear to have the same affinity for PGE,, although the K,, value in cortex is somewhat lower than those in the outer and inner medulla (one-way ANOVA, P = 0.19). In contrast, the numbers of binding sites were different along the nephron (one-way ANOVA, I 0 100 200 300 P < 0.001). Outer medulla had the highest number of binding sites followed by inner %-PGE, bound frnol rng-' Fig. I . Transformation of the binding data, obtained medulla and cortex (Table 2). T h e Scatchard plots in Fig. I appeared in saturation studies with ['HIPGE, in membranes from cortex ( 0 )(Y = -o.gg), outer medulla (0)somewhat curved, especially in the outer and (Y = -0.97) and inner medulla (A) (r = -0.96) of inner medulla, indicating the possible existence the human kidney, according to Scatchard. The K,, of more than one population of [3H]PGE, and B,,,axvalues for cortex, outer medulla and inner binding sites in these regions. A two-site binding medulla were 3.5 n M and 136 fmol mg-', 5.6 n M and model was therefore tested by applying the same 323 fmol mg-' and 4.8 nM and 255 fmol mg respec- data to the LIGAND program. T h e fitted curve for tively. Each point represents the average of four the two-site model did not give a better fit to the separate experiments performed in triplicate. data than the one-site model. A one-site model was also supported by results from Hill plots performed on the data given in Fig. I , which also revealed slopes close to unity. Taken together, RESULTS only a single binding site for [3H]PGE,, in concentrations u p to approximately 1 5 nM, could Radioligund membrane binding be detected in the human kidney under our assay I n preliminary experiments it was found that the conditions. time of incubation to reach a steady state of T h e specificity of the [3H]PGE, binding site binding at I n M of ['HIPGE, varied somewhat was examined in competitive binding experi-

Fig. 3. Computer-generated pseudocoloured images of ["H]Ultrolilm autoradiographs of 31labelled PGE, binding sites in the human kidney. Red areas represent the highest receptor density and blue areas low or undetectable receptor densities. (a) Section with total binding showing highest density of binding sites in the outer medulla (OM) followed by inner medulla (IM) and cortex (C). (b) T h e pattern of ["HlPGE, binding was abolished by the addition of 1 0 PM unlabelled PGE, in the incubation medium.

Prostaglandin E , receptors in human kidney

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Fig. 2. Inhibition of specific r3H]PGE, binding to membranes from the outer medulla of the human kidney by various prostaglandins: PGE, (a),PGE, (O), PGF,, (A), PGA, (Q), PGB, PGI, (H),PGD, (+). Membrane suspensions were incubated with I nM of [3H]PGE, in the presence of the indicated concentrations of different prostaglandins. The amounts of [3H]PGE, binding inhibited by the various concentrations of the competitors were calculated as per cent of specific binding. Values represent meanf SEM of 4-5 separate experiments performed in duplicate. Inhibition constants (Ki) are given in Table 3 .

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Table 3. Inhibition constants (K,) for various prostaglandins to the [3H]PGE, binding sites in membranes from the outer medulla of the human kidney ~

~

Compound

Log K, (M)

Mean K, (MI

PGE, PGE, PGF,, PGA, PGB, PGI, PGD,

-8.37k0.07 -8.37f0.15 -6.65k0.05 -6.58fo.18 - 6.38 0.03 -5.63f0.08 - 5.55 & 0.02

4.3 x 4.3 x

IO-~ IO-~

’

2.2 X 10 2 . 6 10-’ ~

4.2x 2.3 x 2.8 X

10-’ IO-~ 10-6

n

5 5 4 4 4 4 4

Values are given as mean fSEM for log K,. Sc 42867 and furosemide, in high concentrations (IO-’ M), failed to displace [‘HIPGE, ( < 100/0) from its binding sites in membranes from outer medulla ( n = I).

ments with various PGs using membranes from the outer medulla. The PGs tested competed for the [3H]PGE, binding sites in the following rank order of potency (Fig. 2):

P G E , z P G E , > PGF,, z PGA, > PGB, > P G 1 , z PGD,. Possibly it is more correct to regard the displacement obtained by PGI, as being caused by 6-keto PGF,,, since PGI, is known to be hydrolysed with a half-life between 3.5 and 10.5 min at 25 “C and pH 7.48 (Cho & Allen 1978). No additional inhibition of [“HIPGE, was obtained when any of these unlabelled PGs were added in high concentrations together with 10,UM PGE, as compared to separate applications of I O ~ MPGE,, suggesting that these substances compete for the same binding sites as PGE,. The inhibition constants ( K J of these compounds are given in Table 3.

Autoradiography The macroautoradiographic localization of the [3H]PGE, binding sites in the human kidney is shown in Fig. 3(a). Binding was observed in all three anatomical regions, but there was a regional variation. The highest density was observed in the outer medullary zone followed by the inner medulla. A binding of lower density was detected in the cortex. In all cases these patterns were

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Fig. 4. The bright-field micrograph (a) shows a section of cortex of the human kidney with glomeruli (G), proximal tubule (PT) and distal tubule (DT). The corresponding dark-field micrograph (b) shows low concentrations of silver grains over glomeruli and proximal tubules but high concentration over distal tubules. The bright-field micrograph (c) shows a distal tubule surrounded by proximal tubules in the cortex. A high concentration of grains is found only on the distal tubule in the corresponding dark-field micrograph (d). The bright-field micrograph (e) shows a blood-vessel in cortex with no specific localization of silver grains in the corresponding dark-field micrograph (f) ( x zoo). abolished by r o p ~unlabelled PGE, (Fig. 3 b). Autoradiographs from the tape surface and from buffer-treated sections were all negative. Microautoradiographs, obtained with the coverslip technique, of the cortex showed that

grains were distributed in highest density over distal tubules, which were fewer in number and whose cytoplasm stains less deeply with erythrocin than those of the proximal tubules. In addition, the dye exclusion test with alkaline

Prostaglandin E , receptors in human kidney

Fig. 5 . Binding of ["HJPGE, to the outer medulla of the human kidney. The bright-field micrograph (a) shows structures with relatively large tubular cells whose cytoplasm stains with erythrocin (some marked with arrows), most likely representing the thick portion of the ascending limbs of Henle. Total binding is shown in the corresponding dark-field micrograph (b), which demonstrates the high density of binding to this segment of the nephron. The bright-field micrograph (c) shows a collecting duct in outer medulla, characterized by its lightly erythrocinstained cells of considerable height, with detectable but low silver grain density (see arrow) on the corresponding dark-field picture (d) ( x zoo).

Fig. 6. Localization of' ['HIPGE, binding to human renal inner medulla. (a) Bright-field view of an area in the papilla showing collecting ducts (CD). T h e corresponding dark-field micrograph (b) shows low but a significant amount of specific silver grains on the collecting ducts in the papilla (x

100).

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phosphatase staining revealed that only distal tubules were labelled. Little or no accumulation of silver grains was observed over the proximal tubular cells (whose cytoplasm stains deeply with erythrocin), cortical collecting ducts (which were distinct because of their lightly erythrocinstained cells; not shown), glomeruli or blood vessels (Fig. 4a-f). In the outer medulla, almost all specific binding was associated with the distal tubule, most likely representing the thick ascending limbs of Henle. Specific labelling could also be found over collecting ducts in this region although the grains were relatively sparse (Fig. 5 a-d). In the inner medulla, the collecting ducts were found to have a significant amount of specific binding, although the density was lower than expected from results of the membrane radioligand binding studies (Fig. 6a and b). The histological identification of the thin loops of Henle was somewhat difficult. However, it is possible that this part of the nephron has binding sites for r3H]PGE,, since labelling was observed over some structures which appeared to be the thin loops of Henle. Generally, the bindirig pattern of [3H]PGE, was totally abolished on autoradiographs of adjacent sections incubated with [3H]PGE, in the presence of excess of unlabelled PGE, (10p ~ ) . DISCUSSION In the present study the [3H]PGE, binding sites in human kidney were characterized and localized by radioligand membrane binding and autoradiography. Our results show that [3H]PGE, bound concentration-dependently to membranes obtained from renal cortex, outer medulla and inner medulla. T h e binding was time-dependent, reversible and saturable. This is what would be expected from a radiolabelled receptor. In the displacement studies it was found that PGE, and PGE, were equipotent in inhibiting [3H]PGE, binding to human kidney outer medullary membranes. The other PGs tested were all found to be much less potent in inhibiting the [3H]PGE, binding. This is consistent with the assumption that [3H]PGE, receptors were labelled under our assay conditions. The results of the saturation experiments were analysed by Scatchard plots. Since some of the Scatchard plots appeared curved, especially in the outer and inner medulla, the possible existence of more than one population of binding

sites was considered. Mathematical modelling analysis of the saturation data using the LIGAND program revealed, however, monophasic binding of [3H]PGE,, indicating one population of binding sites. This was also supported by the results of Hill plots. It is possible that the concentration of the radioligand used was not high enough (0.25-15 nM) to label a site with lower affinity, which then could be distinguished from a high-affinity site by the LIGAND program. However, in the displacement studies high concentrations of unlabelled PGE, were used, and the LIGAND program could still not distinguish between two separate binding sites for [3H]PGE,. I n addition, a Hill plot performed on the same data revealed a slope close to unity. These data do not support the assumption that two separate populations of [3H]PGE, binding sites were present. It is possible, however, that there exist two different high-affinity sites for [3H]PGE, that cannot be separated with the tests used for multiple rsites. For example, [3H]PGE, may bind to high-affinity receptors for both PGE, and PGE,. This could possibly occur since the results from the competition studies showed that there is a high degree of 'cross-reactivity ' between the two PGs (Fig. 2, Table 3 ) . The binding sites labelled in the present study have a dissociation constant between 3.7 and 6.2 nM in the studied regions of the human kidney. The affinity in outer medulla and inner medulla was very similar, whereas there was a tendency towards higher binding affinities (lower K , values) in the cortex. The differences in K , values were, however, not statistically significant. The density of receptors, estimated from Scatchard plots, revealed a statistically significant difference between the three anatomical regions ( P < 0.001). The highest density of binding sites for [3H]PGE, was found in outer medulla (335 fmol mg-' protein) followed by inner medulla (258 fmol mg-l protein) and cortex (I43 fmol mg-' protein). This order of receptor densities in the human kidney was different from that previously found in the rat kidney (Eriksson et al. 1990).In the rat, the [3H]PGE, binding sites were most abundant in the outer medulla followed by the cortex, while almost no binding sites were found in the inner medulla. The high density of binding sites in the human renal medulla fits well with the fact that the mammalian medulla is the major site of PGE,

Prostaglandin E , receptors in human kidney synthesis in the kidney (Angglrd & Oliw 1981). It is known from previous studies that the most important sodium- and water-regulating segments of the nephron, i.e. the thick ascending limbs of Henle’s loop and the collecting ducts, synthesize PGE, (Bohman 1977, Smith & Bell 1978, Jackson et al. 1980, Schlondorff et al. 1982). Macroautoradiographs of the distribution of r3H]PGE, binding in the human kidney are in close agreement with the results obtained from the radioligand binding in membrane suspensions. The highest binding was found in the outer medulla followed by the inner medulla and the cortex. This distribution is also different from that of the rat kidney where no binding could be detected in the inner medulla (Eriksson et al. 1990). Microautoradiographs revealed a high density of binding sites of [3H]PGE, on the thick ascending limbs of Henle. Binding sites of lower density were found on the collecting ducts, and possibly there were some binding sites on the thin loops of Henle. This supports the suggestion that the natriuretic and water diuretic actions of PGE, are mediated by receptors on these segments of the nephron. I n contrast to the findings in the human kidney, microautoradiographs of the rat kidney (Eriksson et al. 1990) revealed that the [3H]PGE, binding sites were almost exclusively located on the distal tubules (most likely on the thick ascending limbs of Henle) and not on collecting ducts. No [3H]PGE, binding sites were found on the human proximal tubules, which is in accordance with the results from our study in the rat (Eriksson et al. 1990) and from in vivo micropuncture studies in rat and dog showing that neither intravascular nor intratubular infusions of exogenous PGE, affected proximal tubule salt and water reabsorption (Kokko 1981). As in the rat kidney (Eriksson et al. 1990), no [3H]PGE, binding sites were found on glomeruli or on blood vessels in the human kidney. This observation contrasts with the finding that PGE, has a direct receptor-mediated vasodilating effect on the vasculature in the kidney (Lifschitz 1981, Edwards 1985) and a relaxing effect on the smooth muscle-like mesangial cells (ScharSchmidt & Dunn 1983, Scharschmidt et al. 1983). The evidence that PGE, may dilate the renal vasculature derives from studies where PGE, was administered directly into the renal artery of the dog (Vander 1968, Fulgraff et al.

401

1974), the rabbit (Larsson & Anggird 1973) and the rat (Haylor & Towers 1982). The lowest dose of PGE, that has been given directly into the renal artery of the dog has been 5-6 times higher than the circulating plasma concentration of PGE, (Lifschitz 1981). Interestingly, this ‘low’ dose induced only small changes in the renal blood flow (RBF). I n contrast, higher doses of PGE, increased the RBF. The lack of [3H]PGE, binding sites on blood vessels, found in the present investigation, and the findings of PGE,mediated effects on renal vascular tissue seem contradictory. However, there may be several explanations : (a) There may be different receptors on the epithelial cells of the nephron and on the smooth muscle cells of blood vessels and glomeruli. Smooth muscle cells in blood vessels and glomeruli may, for example, have low-affinity PGE, receptors. These receptors will then only be labelled to a minor extent and may possibly not be detectable on autoradiographs in the present study because a low concentration (2 nM) of the radiolabelled ligand was used for localization of the receptors. In favour of this explanation is the studies from two independent laboratories which reported K D values of 12 nM and 14 nM for [3H]PGE, binding sites on isolated rat glomeruli (Friedlander et al. 1983, Chaudari & Kirschenbaum 1985). In addition, Freidlander et al. (1983) performed Scatchard transformations of data from competitive inhibition of [3H]PGE, binding to isolated rat glomeruli by increasing concentrations of unlabelled PGE, and found K,, values between 10 and I O O ~ M . The possible existence of a site with lower affinity is also supported by the fact that a significant vasodilatation and increase in RBF occur only when high doses of PGE, are administered intrarenally to dogs (Lifschitz 1981). Furthermore, previous reports in man suggest that basal production of PGE, contributes only to a minor extent in the regulation of normal renal haemodynamics and glomerular functions (Haylor 1980), but becomes important during states of ischaemia (Kimberly et al. 1978, Abe et al. 1981,Blum et al. 1983), hypovolaemia (Arisz et al. 1976, Walshe & Venuto 1979, Arroyo et al. 1983) and sodium depletion (Muther et al. 1981) when the renal synthesis of PGE, is enhanced. (b) The method employed to detect PGE, receptors may have certain limits. For example

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the ‘vascular PGE, receptor subtype’ may require a ‘co-factor’ which is missing in our assay. (c) Alternatively, the [3H]PGE, binding sites in the renal vasculature were occupied by endogenously produced PGE,. The influence of endogenously produced PGE, on [3H]PGE, binding is, however, uncertain, as judged from previous studies on the rat (Limas & Limas 1984, Eriksen et al. 1987). In these studies Eriksen et al. (1987) found an increase of [3H]PGE, binding sites in the inner medulla, thick ascending loop of Henle and in glomerular and medullary vessels after 4 days of indomethacin treatment compared to controls without indomethacin. It was, however, unclear whether an up-regulation of the [3HjPGE, binding sites occurred after 4 days of indomethacin treatment or whether a ‘pseudo’ downregulation by endogenously produced PGE, was the reason for the lower number of binding sites observed in the absence of indomethacin. I n contrast, Limas & Limas (1984) did not find an increased number of [3H]PGE, binding sites in outer medullary membranes after 4 days of aspirin treatment. Vascular tissue, collecting ducts, the thick ascending limbs of Henle, and medullary interstitial cells are all known to synthesize PGE, (Schlondorff & Ardaillou 1986). Since vascular tissue, collecting ducts, and medullary interstitial cells contain more cyclo-oxygenase than does the thick ascending limb of Henle, it cannot be excluded that the number of [3H]PGE, binding sites was reduced as a consequence of occupation of these sites by endogenously produced PGE,. In such a case, this PGE, would presumably reflect the PGE, level before extirpation of the kidneys, since indomethacin (30 ,LLM)was present in the tissue collection buffer and throughout the entire assay procedure. Thus, the reasons for the discordance between the physiological effect of PGE, on the renal vasculature and the lack of [3H]PGE, binding sites on renal blood vessels in the present study remain obscure. The PGE, synthesis has been reported to be greater in the inner medulla than in the outer medulla (Larsson & Anggdrd 1973, Sraer et al. 1983). This is not in line with the view that PGE, exerts its natriuretic effect mainly via PGE, receptors on the thick ascending limb of Henle, which is most abundant in the outer medulla, but suggests that the site of production

of PGE, in the medulla is different from its site of natriuretic action. However, the high density of [3H]PGE, binding sites on the thick ascending limb of Henle is in agreement with the assumed site of action for the natriuretic effect of PGE,. In summary, this study demonstrates [3H]PGE, binding in the human kidney which is time-dependent, reversible and saturable. This is what would be expected for a ligand-receptor interaction. In addition, the rank order ofpotency of some PGs was that expected for an interaction with a PGE, receptor. The binding sites were more abundant on the thick ascending limbs of Henle, followed by the collecting ducts. This distribution is in agreement with the assumption that those structures are the main targets for the salt and water regulatory effects of PGE, (Stokes & Kokko 1977, Stokes 1979). Hence, the [3HjPGE, binding sites studied under the present assay condition are most likely binding sites representing receptors that mediate the functional effects of PGE,. This is, to the best of our knowledge, the first time a human renal PGE, receptor has been characterized and localized. The authors are greatly indebted to Mr Stefan Seth for technical assistance with computer-generated images, Dr Anders Neil for help with data processing with the LIGAND program and to Dr Hans Henriksson for his advice on the histological evaluation. The skilful technical assistance of Ms Christina Olsson is gratefully acknowledged. The writers wish to thank Ms Anette Persson for her excellent secretarial assistance. This study was supported by funds from Merck, Sharp & Dohme (Sweden) AB and from the faculty of medicine, University of Lund, Sweden.

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