Pflfigers Archiv
Pfltigers Arch (1985) 404:293 - 299
EuropeanJournal
of Physiology 9 Springer-Verlag1985
Contraluminal sulfate transport in the proximal tubule of the rat kidney II. Specificity: sulfate-ester, sulfonates and amino sulfonates K. J. Ullrich, G. Rumrich, and S. Kl6ss Max-Plank-Institut ffir Biophysik, Kennedyallee 70, D-6000 Frankfurt/Main 70, Federal Republic of Germany
Abstract. In order to study the specificity for the contraluminal sulfate transport system the inhibitory potency of sulfate esters and sulfonate compounds on the 35SO42influx from the interstitium into cortical tubular cells in situ has been determined. The following was found: 1. From 10 sulfate monoesters tested 9 inhibited contraluminal sulfate influx with an app. Ki between 0.6 and 6 mmol/l; the two sulfate diesters tested, however, did not. 2. Out of 8 aliphatic sulfonate compounds only three, having a NH- or OHgroup in a suitable position, exerted a moderate inhibition (app. Ki ca. 2 - 6 mmol/1). 3. Amongst 14 benzene sulfonates tested only 2 compounds (5-nitrobenzene-sulfonate and 2-hydroxy-5-nitrobenzenesulfonate) inhibited with a Ki < 5 mmol/1. 4. Out of 10 naphthalene sulfonates tested 8 inhibited with a K~ < 5; the highest inhibition was seen with the NH-containing 8-anilinonaphthalene-l-sulfonate (ANS), but no inhibition with 2 compounds containing an amino group. 5. From the polycyclic sulfonates pyrene-3sulfonate and anthracene-l-sulfonate inhibited with a Ki of approximately 2 mmol/1, while no inhibition was seen with anthracene-2-sulfonate. 6. Out of 4 amino-sulfonates tested benzene-l-amino-sulfonate and a similar benzyl-analog inhibited with a K~ of 1 mmol/1 and smaller; cyclohexyl-1amino-sulfonate (cyclamate), however, inhibited only slightly (app. K~ of 6 mmol/1). The data indicate that sulfate monoesters are well accepted by the contraluminal sulfate transport system. The affinity of sulfonate compounds to this system depends on neighbouring OH-groups - N H groups, meta-positioned electronegative groups or a hydrophobic moiety in an appropriate position. Key words: Epithelial transport - Contraluminal cell membrane
Introduction In a previous publication we reported about some characteristics of the sulfate flux through the contraluminal cell side of the renal proximal tubule in situ [6]. It was shown that a sulfate countertransport takes place with a Km of 1.4 mmol/1 for sulfate influx from the interstitium into the cells. This sulfate flux was inhibited by thiosulfate (app. Ki 0.3 mmol/1), molybdate (0.8 mmol/1), oxalate (1.2 mmol/1) and bicarbonate (18 mmol/1). It is inhibited in the presence of Na + ions and, in Na+-free solutions, it is augmented by Offprint requests to: K. J. Ullrich to the above address
H § ions. In this and the following papers [ 7 - 9 ] we test a series of anions, i.e. sulfate, sulfonate and carboxylate compounds to learn more about structure, charge, electronic configuration and hydrophobicity of molecules which inhibit the contraluminal sulfate flux. In this paper data are presented which show that only sulfate monoester (4 O-atoms around the S-atom, 1 negative charge), not diester (4 O-atoms around the S-atom, not charged), inhibit contraluminal sulfate influx and that sulfonate (3 O-atoms around the S-atom, I negative charge) compounds inhibit readily if they have a suitable OH- or - N H - g r o u p near the sulfonate group or an electronegative group in the metaposition or a hydrophobic moiety in a proper position.
Methods The experiments were performed in male Wistar rats (Winkelmann, Kirchborchen, FRG) of 2 0 0 - 2 5 0 g body weight fed an Altromin standard diet and tap water. The animals were anaesthetized by injecting Inactin (Byk Gulden, Konstanz, FRG) 120-150 mg/kg body weight intraperitoneally and placed on a heated operating table with a thermostat control set at 37~ An incision was made in the left flank and the kidney separated from the surrounding fascia. The capsule was stripped off. The kidney was immobilized in a plastic cup resting on cotton wool and covered by paraffin oil heated to 37 ~C. The renal artery and vein were isolated from the ureter so that they could be clamped. Unless stated otherwise the rats were infused with Na2SO4 solution (94 mmol/1 made up to isotonicity with NaC1). The prime dose was 60 mg NazSO4 per kg body weight, lasting infusion 1 mg NazSO4 per kg body weight and minute. This treatment elevated plasma sulfate concentration by ca. 1 mmol/1, i.e. from 0.7 mmol/1 to 1.8 mmol/1. The method of measuring contraluminal influx of 35SO2- from the interstitium into the cells and the mode of evaluating the transport parameters have been described previously [1]. Briefly, the renal artery and vein were clamped for each measurement of the disappearance rate of a 5SO] -. The proximal convoluted tubules then collapsed because the luminal fluid was reabsorbed, while glomerular filtration ceased. Immediately a thick blood capillary was impaled by an oil-filled sampling pipette (tip diameter about 6 gin). At a distance of 100-140 ~m from this glass capillary another blood vessel was punctured with a filling pipette (tip diameter about 7 gin). Through the filling pipette a rapid injection of an isotonic solution containing 10 gmol/1 3 s s o 2 - and 3H-inulin as extracellular space marker was made. After
294 Table 1. Effect of sulfatesters and sulfonates on contraluminal 4 s influx of 35SO 2- into cortical tubular cells (ACol.4 ~). The initial concentration of 35SO2- in the blood capillaries was 0.01 mmol/l. The decrease in this concentration within 4 s contact time is given as percentage and absolute value Substances added
Control Methylsulfate (k) Dimethylsulfate (e) Diethylsulfate (e) Pentylsulfate (k) Hexylsulfate (k) Octylsulfate (k) Decylsulfate (k) Dodecylsulfate (d) 2-Naphthylsulfate (e) Phenolphthaleindisulfate (m) Picosulfate; 4,4'-(2-pyridinylmethylene)diphenol bis (hydrogensulfate) (ester) (t) 4-Hydroxy-triamterenesulfate (ester): 2,4,7-triaminopteridine 6-(4-phenylsulfate) (*) Methanesulfonate (r) Ethanesulfonate (e) 2-Hydroxyethanesulfonate (isethionate) (v) 3-Hydroxypropane- 1-sulfonate (e) 2-Aminoethane sulfonate (taurine) (v) N-(4-azido-2-nitrophenyl)-2-aminoethane1-sulfonate (NAP-taurine) (pi) Tauroeholate (v) N-2-hydroxyethylpiperazineN'-2-ethanesulfonate (Hepes) (v) Benzenesulfonate (f) 2-Hydroxybenzenesulfonate (h) 4-Hydroxybenzenesulfonate (f) 5-Nitrobenzenesulfonate (e) 2-Hydroxy-5-nitrobenzenesulfonate(h) 2,4-Nitrobenzenesulfonate (e) Pyridine-5-sulfonate (f) 4-Hydroxypyridine-5-sulfonate (h) 2-Aminobenzenesulfonate (f) 4-Amino benzenesulfonate (f) 2,4-Aminobenzenesulfonate (j) 4-Thiocyanatobenzenesulfonate (*) 3,5-Isothiocyanato-4-methylbenzenesulfonate(*) 2-Aldehyd-5-chlorobenzenesulfonate (h) Naphthalene-l-sulfonate (m) 4-Amino-naphthaleneq-sulfonate (k) 4-Hydroxy-naphthalene-l-sulfonate (k) 4-Chloronaphthalene- 1-sulfonate (h) 5-Dimethylaminonaphthalene-:1-sulfonate (s) 8-Anilinonaphthalene-l-sulfonate(ANS) (f) 3,4-Naphthoquinone-l-sulfonate (k) Naphthalene-2-sulfonate (m) 5,6,7,8-Tetrahydronaphthalene-2-sulfonate (h) l-Hydroxynaphthalene-2-sulfonate (k) Diphenyl-4-sulfonate (h) Anthracene-2-sulfonate (h) Anthracene-l-sulfonate (h) Pyrene-3-sulfonate (h) Amidosulfonate (*) Benzene-aminosulfonate (p) 2-Hydroxybenzene-3-iodo-5-isopropylamino-ethanesulfonate (h) Cyclohexyl-aminosulfonate (cyclamate) (s)
mmol/1
Accl,4s of 35SO~
app. Ki retool/1
P against control
1.7 > 10 > 25 2.2 5.5 2.2 1.3 1.1 4.3 0.56
< 0.001 NS NS < 0.001 < 0.01 < 0.001 < 0.001 <0.001 <0.01 < 0.00:1
%
pmol/1
5 5 5 5 5 5 5 5 5 5
45.15 -+ 1.4 26.8 -+ 2.5 41.0 -+ 2.3 44.5 +_ 1.9 30.0 -+ 2.8 35.5 • 3.2 30.0 _+3.8 23.7 -+ 3.6 22.4 -+3.5 36.0 -+1.7 14.5 -+ 2.0
4.5 _+ 0.14 2.7 -+ 2.5 4.:1 _+ 0.23 4.5 -+ 0.19 3.0 -I- 0.28 3.6 -+ 0.32 3.0• 2.4 _+ 0.36 2.2-+0.35 3.6-+0.17 :1.5 -+ 0.20
5
33.2 -+3.3
3.3_+0.33
3.1
46.8 47.6 46.9 38.5 37.9 45.6
-+3.1 -+ 2.5 -+ 2.7 • _+2.8 -+ 2.0
4.7-+0.31 4.8 -+ 0.25 4.7 +_ 0.27 3.8_+0.14 3.8 -+ 0.28 4.6 _+ 0.2
>25 > 25 > 25 6.3 5.7 > 25
NS NS NS <0.05 < 0.05 NS
29.7 +1.3 43.5 -+1.9
3.0-+0.13 4.4_+0.19
2.2 >25
< 0.001 NS
10 10 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 1 5 5 5 5 5 5 5 5 5 5
44.1 43.2 41.9 47.2 33.8 15.8 40.4 36.6 39.8 47.4 40.4 39.5 39.2 37.1 38.1 36.3 41.6 36.2 36.1 46.4 12.5 34.9 33.8 32.7 30.2 38.0 42.5 29.7 24.5 46.2 21.7
4.4-+ 0.27 4.3__0.12 4.2 -+ 0.28 4.7-+0.11 3.4_+0.23 1.6-+0.32 4.0-+ 0.23 3.7-+0.19 4.0-+0.18 4.7 _ 0.22 4.0-+ 0.18 3.9-+ 0.34 3.9 _+ 0.27 3.7+0.24 3.8 +_ 0.33 3.6 -+ 0.28 4.2 -+ 0.28 3.6 _+ 0.42 3.6-+0.45 4.6 _+ 0.2 1.3 _+0.25 3.5_+0.16 3.4-+0.15 3.3_+0.16 3.0_+ 0.29 3.8 • 0.22 4.3 -+ 0.19 3.0 _+ 0.22 2.4 -+ 0.34 4.6 + 0.26 2.2-/-0.24
> 25 >25 > 14 >25 3.3 0.64 > 9 4.6 7.9 > 25 > 9 > 7 7.0 5.0 5.8 4.5 > 12 4.4 4.3 > 25 0.09 3.8 3.3 2.9 2.3 5.7 > 17 2.2 1.4 > 25 :1.1
NS NS NS NS <0.001 <0.001 NS <0.01 <0.05 NS NS NS < 0.05 <0.01 < 0.05 < 0.001 NS < 0.05 <0.05 NS <0.001 < 0.001 <0.001 <0.001 < 0.001 < 0.05 NS < 0.001 < 0.001 NS <0.001
5 5
8.4 -+ 2.6 38.3 -+2.1
0.8 -+ 0.26 3.8-+0.21
0.28 6.0
< 0.001 <0.05
5 150 5 5 5 5 5 5
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<0.001
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4 s, as checked by an acoustic signal, the test solution was withdrawn by the sampling pipette. The 35SO~- and 3H-inulin counts were measured in a Nuclear Chicago scintillation counter with Picofluor 15 (Packard, Frankfurt, FRG) as scintillation fluid. The standard solution which was injected into the capillaries contained in mmol/l: 150 Na +, 4 K +, 154 gluconate. All substances added replaced an equivalent amount of gluconate so that the osmolality remained constant. The solutions were gassed with pure O2 and the pH was set to 7.4. Further specifications are given in the legends. The substances which were tested for their ability to inhibit contraluminal 35802- influx were added to the capillary perfusate in a concentration of 5 mmol/1 so that an app. Ki of up to 10 retool/1 could be detected. The calculation of the apparent Ki values was performed by a computer program which is described in [I], assuming competitive inhibition and a Km of sulfate being 1.4 mmol/1 [6]. All values are given as mean 4- SEM. 35SO42in a specific activity of 1,400 Ci/mmol was obtained from New England Nuclear, Dreieich, FRG. The source of the inhibitors used is indicated in Table 1, where (s) = Sigma, M/inchen, F R G , (f)= Fluka G m b H , Neu-Ulm, FRG, (m) = Merck, Darmstadt, F R G (e) = EGA-Chemie,
Effect of sulfate esters (5 mmol/1) on 4 s contraluminal influx of 35SO42- into renal cortical tubular cells. Starting concentration was 10 pmol/1. The decrease of this concentration within 4 s contact time (Ace1.4s) was 4.5 gmol/1 under control conditions. Each bar represents the mean + SEM of 7 - 9 samples from 2 animals. * P < 0.05, 9* P < 0.01, ***P < 0.0t
Steinheim, F R G , (r) = Riedel-de HaEn, Seelze, F R G , (h) = Dr. K. H. Lang, Hoechst AG, Frankfurt/Main, FRG, (k) = Kodak, Rochester, NY, USA, ( v ) = Serva, Heidelberg, F R G ( t ) = Thomae, Biberach, FRG, ( d ) = Bio-Rack, Richmond, CA, USA, (i) = ICN, Plainview, NY, USA, (p) = Pfisel, Frankfurt, FRG, (pi) = Pierce, Rockford, IL, USA. (*) 4-thiocyanatobenzene-sulfonate and 3,5-isothiocyanato-4-methylbenzene sulfonate were kindly provided to us by Prof. K. F. Schnell, Regensburg, F R G , amino sulfonate by Dr. F. Fahrenholz, Frankfurt/Main, and 4-hydroxytriamterenesulfate by Prof. E. Mutschler, Frankfurt/ Main, FRG.
Results
After 4 s contact time the 35SO~- concentration in the stopped peritubular perfusate was decreased from 10 pmol/1 to 5.5 pmol/1, i.e. by 45% under control conditions (Table 1). As can be seen from Fig. I and Table 1 this decrease in the contraluminal 35SO42- concentration (Acel,4s) is smaller in the presence of 5 mmol/1 of sulfate monoesters, but not with
296 sulfate diesters. A n exception a m o n g s t the sulfate m o n o esters is p-hydroxy-triamterene-sulfate-ester, which exerted no inhibition (Fig. 1 b). This indicates that at least one negative charge has to remain on the sulfate to interact with the contraluminal sulfate t r a n s p o r t system. The aliphatic sulfonate c o m p o u n d s methane sulfate and ethane sulfonate do not inhibit the contraluminal ~5S O ~ - influx (Fig. 2). The same holds for aminoethanesulfonate (taurine) and taurocholate. However, if the taurine molecule is N-substituted as in N A P - t a u r i n e a m o d e r a t e inhibition was seen (app. K~ 2.2 mmol/1). F u r t h e r m o r e , molecules which contain an O H group as 2-hydroxyethanesulfonate and 3-hydroxy-prop a n e - / - s u l f o n a t e show slight inhibition (app. K~ 6.0 mmol/1, Table 1). As shown in Fig. 3 a, b the a r o m a t i c benzene sulfonates do not or only slightly inhibit contraluminal 35SO~- influx. There are, however, some exceptions, namely the benzene sulfonate with a nitro group in meta-position (app. K~ 3.3mmol/1) a n d especially if this c o m p o u n d has an additional O H group (2-hydroxy-5-nitro-benzene-l-sulfonate, app. Ki 0.64 mmol/1). The situation is quite different with naphthalene sulfonates (Fig. 4). Out o f the ten naphthalene sulfonate c o m p o u n d s tested, seven inhibit contraluminal 35SO]influx with an app. K~ between 2 and 5 mmol/1, but one,
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Effect of benzene sulfonates (5 mmol/1) on 4 s contraluminal influx of 35SO~- into renal cortical tubular cells. Starting concentration was I0 gmol/1. The decrease of this concentration within 4 s contact time (Acc~.4s) was 4.5 Ixmol/1under control conditions. Each bar represents the mean -t- SEM of 8 - 9 samples from 2 animals. * P < 0.05, ** P < 0.01, 9** P < 0.001
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Fig. 4a, b. Effect of naphthalene sulfonates (--I-- = 5 mmol/1; ""iii. . . . i mmol/1) on 4 s contraluminal influx of 35SO42-into renal cortical tubular cells. Starting concentration was 10 gmol/1. The decrease of this concentration within 4 s contact time (Acd.4s) was 4.5 gmol/1 under control conditions. Each bar represents the mean + SEM of 8 - 1 6 samples from 2 - 4 animals. *P < 0.05, ***P < 0.001
8-anilinonaphthalene-l-sulfonate, exerted highest inhibition (app. Ki 0.09 mmol/1). Only two compounds, which have an amino group in the C4-position or a dimethylamino-group in the C5-position, have no effect. One might conclude from these data that 1) at an appropriate hydrophobicity a SOs alone is sufficient for interaction with the contraluminal SO 2- transport site and 2) a neighbouring NH-group enhances the interaction and 3) amino side groups prevent the interaction. Diphenylsulfonate interacts with a similar affinity as naphthalene-l-sulfonate (Table 1). The affinity rises with the 3-ring anthracene-l-sulfonate (app. Ki 2.2 mmol/1) and the 4-ring pyrene-3-sulfonate (app. Ki 1.4 mmol/1) (Fig. 5). Interestingly, however, 5 mmol/1 anthracene-2-sulfonate did not inhibit at all. This indicates that rising hydrophobicity of sulfonate compounds augments the affinity for the contraluminal sulfate transport system. The hydrophobic moiety, however, has to be in a proper position in relation to the sulfonate group. Because we observed with cyclamate a small inhibition of contraluminal 35SO~- influx, we tested other amino
sulfonates (Fig. 6) and found that benzene-l-aminosulfonate with an apparent Ki of 1.1 nmlol/1, has a good and 2-hydroxybenzene-3-iodo-5-isopropyl- l-aminoethane sulfonate with an app. Ki of 0.3 mmol/1 has a strong inhibitory potency. From this, one may conclude that a hydrogen-bond forming - N H - g r o u p in ortho-position to the SOs might be sufficient to promote interaction with the contraluminal sulfate transport mechanism. Amidosulfonate, however, does not inhibit, supposedly because it behaves as zwitter ion.
Discussion
One of the main characteristics of an enzyme or transporter is its substrate specificity. The iiaformation concerning the possible molecular structure of the enzyme or transporter might be great, if it has a high substrate specificity. At low specificity, however, the information might be limited, but
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Fig. 6. Effect of aminosulfonates (5 retool/l) on 4 s contraluminal influx of 3SSO~- into renal cortical tubular ceils. Starting concentration was 10 ~tmol/l. The decrease of this concentration within 4 s contact time (Ac~,4,) was 4.5 p,mol/lunder control conditions. Each bar represents the mean _ SEM of 7 - 9 samples from 2 animals. 9 P < 0.05, ***P < 0.001
nevertheless valuable. With this in mind we started a systematic study of the contraluminal sulfate transport of the proximal renal tubule using series of sulfate-esters, sulfonates, amino-sulfonates (present study), disulfonates, di- and tricarboxylates, sulfocarboxylates [7], salicylates and dihydroxybenzoates [8], phenolphthaleins, sulfonphthaleins, sulfamoyl compounds and diphenylamino carboxylates [9]. For our first goal, to get information about the interaction of possible substrates with the transporter, it was irrelevant whether the substrate was transported itself or whether it interacted with the transport site without being transported. This question anyhow could be answered by our in situ methods only, if the respective substances, labeled with high specific activity, would be available. Otherwise it requires countertransport studies with contraluminal membrane vesicles [2]. Furthermore, it was not intended in this
study to evaluate the type of inhibition. Still, however, we wanted to give a quantitative figure of the degree of inhibition and we calculated apparent Ki values, assuming competitive inhibition [1]. This approach proved already to be very valuable in elucidating the specificity of the contraluminal transport system for dicarboxylates [5]. Compared to monocarboxylates [4] and dicarboxylates [5] complete series of analogs (sulfate-ester, sulfonate etc.) are not commercially available. Thus, some key substances were synthetized and others given to us by Dr. H. J. Lang from the Hoechst AG, FRG. Nevertheless there remain gaps which make some evidences rather circumstantial. If one considers the molecular structure and the transport function of the band 3 protein in the red cells, which is the best investigated anion transport system at the moment [3], it appears that the basolateral sulfate transport system in the proximal renal tubule seems to be different from it: the red cell system does not accomplish net sulfate transport, while the contraluminal proximal tubular system does [2]. Both systems, however, show a broad substrate specificity and interact with the same molecules of widely varying structure. In fact, if the affinity of the same substrates for both systems are found to be different, this does not prove that both systems are different, but they may be similar or identical and apparent differences are caused by recruitment or modifier sites. This again might be clarified if contraluminal vesicles with uniform sidedness are available. From the data presented in this paper it became clear that sulfate monoesters do interact with the contraluminal sulfate transport system, but sulfate diesters do not. An exception was 4-hydroxy-triamterenesulfate-ester, where the NH2-groups on the pteridine skeleton might prevent interaction, as was also observed with amino-naphthalenesulfonates (Fig. 4). Since simple sulfonate compounds do not interact with the contraluminal sulfate transport system, the question arises whether additional groups can promote interaction. This is indeed the case, if the sulfonates have a neighbouring OH-group and/or an electronegative group in the meta-position. This can be seen by the differences between ethane-sulfonate and 2-hydroxy-ethane sulfonate (Fig. 2) and between benzenesulfonate, 5-nitro-benzene1-sulfonate and 2-hydroxy- 5-nitrobenzene- 1-sulfonate (Fig. 3 a). The enhancing effect of an electronegative group (SOj, C O O - , SO2NH2, NO2, C1) in the meta-position and/ or that of an OH-group in the ortho-position can also be seen in our studies with disulfonates, dicarboxylates and sulfocarboxylates [7], and with salicylate analogs [8]. A strong enhancing influence on the interaction of sulfonates with the contraluminal sulfate carrier can also be attributed to a neighbouring - N H - g r o u p , as can be seen by the difference between taurine and NAP-taurine (Fig. 2), between naphthalene sulfonate and 8-anilino-naphthalene-lsulfonate (Fig. 4a), between benzene sulfonate or amidosulfonate and benzene-l-amino-sulfonate (Figs.3a, 6), but also between 3-carboxy-4-aminobenzene-l-sulfonate (app. Ki 4.7 mmol/1) and 3-carboxy-4-ethylaminobenzene-l-sulfonate (app. Ki 0.4 mmol/1) [7]. One might consider that the neighbouring OH- or - N H - g r o u p functions as a hydrogen donor in forming a hydrogen bond. In addition the introduction of a hydrophobic moiety (ethyl-, or benzene group) to the nitrogen atom might be important. It has to be stressed, however, that a neighbouring OH-group is not always effective as can be seen in 2-hydroxybenzene-sulfonate compared to benzene-sulfonate (Fig. 2) or 1-hydroxy-
299 Table 2. Effect of SH-reagents on contraluminal 4 s influx of 35SO,~- into cortical tubular cells (Ac~,4s). The initial concentration of 35SO2 -
in the blood capillaries was 0.01 mmol/1. The decrease in this concentration within 4 s contact time is given as percentage and absolute value Substances added
mmol/1
Accl.4s of 35SO2%
Control 4-Chloromercurybenzenesulfonate (e) 4-Hydroxymercurybenzenesulfonate (e) Mercurybichloride (m) l-[3-(Chloromercuri)-2-methoxypropyl]-urea (chlormerodrin)
naphthalene-2-sulfonate (Fig. 4) compared to naphthalene2-sulfonate or in pyridine-4-hydroxy-5-sulfonate compared to pyridine-5-sulfonate (Fig. 3 a). Most of the benzene sulfonate analogs have a Ki higher than 5.0, while the corresponding naphthalene sulfonates have an app. Ki between 3 and 4 (Table 1). Since the app. Ki decreases in the sequence benzenesulfonate ( > 2 5 mmol/1) > naphthalene-l-sulfonate (4.6 mmol/1) > anthracene-1sulfonate (2.2 mmol/1) > pyrene-3-sulfonate (1.4 mmol/1), one might suggest that a hydrophobic moiety is able to promote interaction of a sulfonate group with the contraluminal sulfate transport system. Again there seem to exist conditions which prevent the interaction of a sulfonate comp o u n d containing a hydrophobic moiety with the sulfate transport system, namely with 4-amino-naphthalene-lsulfonate and 5-dimethylamino-naphthalene-l-sulfonate (Fig. 4) or anthracene-l-sulfonate (Fig. 5). In the two previous compounds the amino group might prevent interaction; in the latter case the hydrophobic moiety might have an unfavourable position in relation to the sulfonate group. Both mercury compounds, which we tested, 4-chloromercury-benzene-sulfonate and 4-hydroxymercury-benzene-sulfonate inhibited contraluminal sulfate transport (Table 2). To exclude whether these compounds inhibit because of their SH-blocking ability, we tested also 5 m m o l / l chlormerodrin (1-[3-(chlormercury)-2-methoxypropyl]-urea) and 0.1 mmol/1 HgCI2. Both substances inhibit the contraluminal sulfate transport, too. Thus, SHgroups seem to be located at or near the sulfate binding site or at least seem to play a role in the functional state of the contraluminal sulfate transport system. Furthermore, it would be very interesting to know whether these compounds are transported into the cell or interact only with the transport system without being transported. It is hoped that countertransport experiments with vesicles solve these questions.
P against control
~mol/1
5 5 0.1
45.15 • 32.90 • 22.80 • 38.20 •
1.4 1.6 2.9 3.2
5
39.50 • 1.6
4.5 • 3.3 • 2.3 • 3.8 •
0.14 0.16 0.29 0.32
4.0 • 0.16
0.001 0.001 0.05 0.05
Furthermore we thank Prof. H. Fasold and Dr. F. Fahrenholz, Frankfurt/Main, and Prof. Wollenberg, Homburg/Saar, for valuable suggestions.
References
Acknowledgements. We thank Dr. H. J. Lang for suggesting and
1. Fritzsch G, Haase W, Rumrich G, Fasold H, Ullrich KJ (1984) A stopped flow capillary perfusion method to evaluate contraluminal transport parameters of methylsuccinate from interstitium into renal proximal tubular cells. Pfliigers Arch 400: 250- 256 2. LSw I, Friedrich T, Burckhardt G (1984) Properties of an anion exchanger in rat renal basolateral membrane vesicles. Am J Physiol 246 : F 3 3 4 - F342 3. Passow H (1985) Molecular aspects of the band 3 proteinmediated anion transport across the red blood cell membrane. Rev Physiol Biochem Pharmacol. Springer, Berlin Heidelberg New York Tokyo (in press) 4. Ullrich KJ, Rumrich G, K16ss S (1982) Reabsorption of monocarboxylic acids in the proximal tubule of the rat kidney. II. Specificity for aliphatic compounds. Pflfigers Arch 395:220226 5. Ullrich KJ, Rumrich G, K16ss S (1984) Secretion and contraluminal uptake of dicarboxylic acids in the proximal convolution of rat kidney. Pflfigers Arch 400: 241 - 249 6. Ullrich KJ, Rumrich G, K16ss S (1984) Contraluminal sulfate transport in the proximal tubule of the rat kidney. I. Kinetics, effect of K +, Na +, Ca 2+, H + and anions. Pflfigers Arch 402: 264-- 271 7. Ullrich KJ, Rumrich G, K16ss S (1985) Contraluminal sulfate transport in the proximal tubule of the rat kidney. III. Specificity: disulfonates, di- and tri-carboxylates and sulfocarboxylates. Pflfigers Arch 404: 300- 306 8. Ullrich KJ, Rumrich G, K16ss S (1985) Contraluminal sulfate transport in the proximal tubule of the rat kidney. IV. Specificity: salicylate analogs. Pflfigers Arch 404: 307 - 310. 9. Ullrich KJ, Rumrich G, Kl6ss S (1985) Contraluminal sulfate transport in the proximal tubule of the rat kidney. V. Specificity: phenolphthaleins, sulfonphthaleins and other sulfo dyes, sulfamoyl-compounds and diphenylamine-2-carboxylates. Pfl/igers Arch 404:311-318
providing us with many compounds (Table 1) without which some conclusions drawn in this paper would not have been possible.
Received January 7, 1985/Accepted April 16, 1985