Experimental cystinuria: the cycloleucine model. I. Amino acid interactions in renal and intestinal epithelia.

Experimental Cystinuria: the Cycloleucine Model. I. Amino Acid Interactions in Renal and Intestinal Epithelia

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ANDRBG. CKAAN'A N D MICHEL, BERGERON 1Xpczrtc.rnent dephysiologie, Uni\'evs.itu'dc.Montvkal, Case postalc 6208, S ~ ~ c c r ~ r sAa.l MontrPul, u Q14c;hec f13C3T8 Received April 25, 1975 CRAAN, A. G . , and BERGEHON, M . 1975. Experimental cystinuria: the cycloleblcine model. I. Amino acid interactions in renal and intestinal epithelia. Can. J. Physiol. Pharmacol. 53, 1027-1036. The injection of cycloleucine (I-aminocyclopentanecarkoxylic acid (ACBC)) into rats groduces a hyperexcretion of dibasic amino acids and cystine, an aberration resembling cystinuria. This may constitute a model of experimental cystinu~ia,and the transport of amino acids involved in thi5 disease was studied with the techniques caf everted intestinal sacs (in vitvs) and micruinjections into renal tubules (in vivo). In everted sacsfrom normal rats, there was a decrease in transfer of L-cystine (0.03 mM), L-lysine (0.065 mlM) and r.-valine (0.065 mM) when and in acc~an~ulation ACPC was on the mucosal (luminal) side. Dibasic amino acids such as L-ariginine and L-lysine caused a similar inhibition of the transport of 6-cystine. However, when ACPC was on the serosal (antiluminal) side, a lesser effect was noted while arginine and lysine had no effect. Intestinal sacs from treated rats (ACPC, 300 mgikg x 3 days) transferred and accumaalated as much L-cystine as those from control rats. The interaction between cycloleucine and I--cystine was competitive at the luminal and non-competitive at the antiluminal side of the intestine. Cycloleucine inhibited L-lysine transport in a non-competitive fashion at either side of the intestine. r..-1,ysine also interacted in a non-competitive fashion with L-cystine transport at the lumina8 membrane. In proximal convoluted tubules, the presence caf L-arginine or ACBC caused a decrease in the transport of L-cystine and L-lysine. I,-Valine exerted no effect. Furthermore, L-lysine and ACPC did not impair the reabsorption of L-valine significantly. These res~altssuggest a functional heterogeneity between luminal and antiluminal membranes of renal and intestinal epithelia and the existence. at both membranes, of different transport sites for cystine and dibasic amino acids. CRA,~N A., G. et BERGERON, M. 1975. Experimental cystinuria: the cycloleucine model. I. Amino acid interactions in renal and intestinal epithelia. Can. J. Physiol. Pharmacol. 53, 1027-1036. L'injection de la cycloleucine (ACPC) a des rats reproduit une aminoacidurie touchant les acides amines dibasiques et la cystine, rappelant celle de la cystinurie. Nr~usavons utilise la technique des sacs intestinaux inverses (in vitro) et celle des microinjections intratubulaires dans le rein afin de mieux definir les rapports entre acides amines dans la cystinurie (in \viva). Au niveau de I'intestin, on note un effet inhibiteur de la cycloleucine sur le transfert et I'accumulation de la L-cystine (0.03 mM) de la L.-lysine(0.065 naM) et de la L-valine (0.065 mM). Cette inhibition est plus marquee du cBte luminal que du c6t6 antiluminal. Bar contre, la E,-arginineet la I.-lysine inhibent le transport intestinal de la L-cystime du cBte muqueux, rnais non du c6te sereux. Si I'ACPC est dsnnee de fason chronique (300 mgikg x 3 jours), on ne note aucune modification dans la cinetique de transport membranaire. 1,'interaction entre la cycloleucine et la cystine est de nature competitive la face luminale et non-competitive a la face antiluminale. L'interaction entre la cycloleucine et la lysine aux membranes luminales et antiluminales est de type noncompetitif. I1 en est de mime de I'interaction entre la lysine et la cystine a la face muqueuse de I'intestin. Au niveau du nephron, on observe, a la membrane luminale, une inhibition mutuelle eiltre la c.-lysine, la r.-arginine, la L-cystine et la cycloleucine. 'Toutefois, la L-valinene semble pas influencer le transport renal de la 1.-lysine et de la L-cystine. Ces resultats prouvent I'heterogeneite fonctionnelle des membranes luminales et antilurninales de I'epitheiium renal et intestinal et l'existence, a ces deux membranes, de differents transporteurs pour la cystine et les acides amines dibasiques.

Introduction Cycloleucine, or 1-aminocyclopentanecarboxylic acid (ACPC), is known to produce a hyperexcretion of &basic amino acids and cystine in humans and rats (Brown 1967; Goyer 'Boursier du Ministkre de 19Education du QuCbec.

et al. 1969; Lemonnier et al. 1971). This

urinary aberration is reminiscent of the syndrome of cystinuria, and the administration Of this n0n-metabolizable amino acid to rats may constitute a model for the disease and help elucidate its prithophysiology. Studies on the intestine have been useful in


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CAN. J. PHYSIBL. PHARMACOL. VOL. 54, 1975

describing cystinuria. Genetically distinct forms of this syndrome have been identified as dependent upon how transport defects found in the kidney are expressed in the intestine (Rosenberg et a%. 1966; Morin et ak. 1971). In the Iight of these findings, the present work is an attempt to compare the behavior of the two epithelia vis-2-vis the amino acids predominantly found in the urine of cystinuric patients. Dibasic an~inoacids and cystine were reported to share the same transport mechanism in both renal and intestinal cells (Dent and Rose 1951 ; Dent et a&. 1954; Wilson 1962; Fox et ul. 1964; Wiseman 1968; Young and Frecdman 1971; Mannck 1972). Few explanatioils have been given to account for the mediation by a hypothetical dibasic amino acid carrier of the transport of cystine, a sulfur-containing neutral amino acid. A link was shown between amino acid structures and their transport mechanism, suggesting that cystine satisfies the structural requirements for the dibasic amino acid transport systems (Christensen and Gullen 1971; Silbernagl and Deetjen 1972b). But in more generalized terms, Reiser and Christiansen ( 1972) have suggested the possibility of overlaps between transport nnechanisms of neutral and dibasic amino acids. These interactions were shown to exist to some extent by Webber et al. (1961, 1962, 1963). More specifically, a similar inhibition between ACPC, dibasic amino acids and some neutral amino acids was demonstrated in renal cortex slices ( Holtzapple et al. 1970), although amino acids of identical groups may differ in their interactions with other amino acids (Robinson and Felber 1964). Our results show different modes of interaction among the dibasic amino acids and cystine. The characteristics of cycloleucine inhibition on cellular transport vary not only between intestinal and renal epithelia but also between luminal and antiluminal membranes of these epithelia. Methods Intestinal Transport Female Sprague-Bawley rats weighing 150-280 g were decapitated after a 20-h fast. The small intestine was dissected free and flushed thoroughly with a Hgrebs-Ringer bicarbonate solution until clean. Bt was immediately turned inside out, placed in a chilled Krebs-Ringer bicarbonate solution gassed with 95%

O2 : 5 % COz and divided into sacs 4 cm long. Six to eight consecutive sacs were chosen from the jejunum and the proximal ileum (Wilson and Wiseman 1954). Each everted sac was ligated at both ends and filled with a Krebs-Ringer bicarbonate buffer containing a labeled amino acid and inulin, making up a serosal volume of 0.4 ml. It was then incubated in a 25-rnl Erlen~neyerflask containing 10 ml of the same buffer, labeled inulin (New England Nuclear) and amino acids such as L-lysine, L-valine, cycloleucine (New England Nuclear) and L-cystine (Schwarz/R4ann Corporation) . V n l a b e l e d L-amino acids (Schwartz/ Mann and Calbiochem) were added to bring the solutions to physiological concentrations. The presence sf inulin was necessary to estimate the extracellular space, which represented on the average 14.7% of the total tissue wet weight. This value was obtained by sgectrophotometric dosage and liquid scintillation analysis with 3H- or j4C-labeled inulin. A similar value has previously been found for the same tissue (Esposito and Csaky 1974). The flask was gassed with 95% 0, : 5% COz and placed in a Bubnoff incubator at 37 "C. Incubation was for 30 min with 128 strokes per minute. The radioactivity of the intestinal sacs and of serosal and mucosal solutions was determined in a Packard Tri-Carb liquid scintillation spectrometer. Intestinal segments were diluted in a tissue and gel solubilizer (Protosol, New England Nuclear) before the addition of liquid scintillation solution (Aquasol, New England Nuclear), pH adjustment and counting. Corrections were made for background, quenching and reciprocal interference of '*C and 'H channels.

Renal Transport - ~MicropupacdureStudy in Viva The in vivo technique of microinjections was adapted from Bergeron and Morel (1969) and Bergeron and Vadeboncoeur ( 197 1) . Animals were anesthetized with Nembutal (Abkott, 50 mg/kg), a tracheotomy was made, and a polyethylene, catheter inserted into the right jugular vein. Mannitol diluted to 5 % in isotonic saline was infused at the rate of 66.1 ml/min with a constant-infusion pump. The left kidney was exposed by the technique of Gottschalk and Myle (1956). Microinjections were made through the renal capsule under stereo~nicroscopic control into a selected superficial convolution with the aid of a De Fonbrune micromanipulator. Several of these were successively made at the same point with different amino acid concentrations taken at random. Each injection lasted 466-50 s, and up to 20 microinjections (14 nl) could be made into four to six proximal tubules in each experiment. With the addition of lissamine green to the injectate, the injection rate was kept as constant as possible by visual control, and retrograde flow could be avoided. The proxinnal and the distal %I a series of experiments, a Krebs-Ringer-Tris buffer gassed with Oa was used as the incubating solution for greater pH stability, and the substitution of this buffer for the 95% O2 : 5% @O2gassed bicarbonate solution did not affect the results.

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CRAAN AND BERGERON: EXPERIMENTAL CYSTINURIA

TABLE1. EkTect of cycloleucine (ACPC) on transfer and accunlulation of ~ - [ ~ ~ S ] c y s t i n e " S/M

Inhibition,


-

Colltrol ACPC in m~acosalfluid Control ACPC in serosal fluid

1.61 + 0 . 12 (13) I . 1 2 k 0 . 0 3 (15) 1 . 7 4 k 0 . 0 9 (11) 1.48k0.06 (12)

ICjEC

Inhibition, %

-

-

24.6* 14-9**

5.78+(9.50 (12) 3 . 4 4 k 0 . 4 0 (15) 5 . 5 2 k 0 . 3 5 (10) 4 . 3 6 k 0 . 2 7 (11)

-

40.5** 21 .O***

-

~Transfer(S/M) and accumulation (ICiEC) representTistribution ratios. ~ r a n s f e ris the r a t i o 7 ~ - c y s t i n econcentration in the serosal solution to its concentration in the mucosal solution; accumulation is the ratio of intracellular to extracellular concentrations after incubation for 30 min of everted intestinal sacs. Values are rneans SE followed by number of experiments in parentheses. Initial concentrations: unlabeled m M ; cycloleucine (ACPC), 5 mM. cystine. 0.03 mM; [35S]cystine, 3 ?; *D < 0.005.

+

TABLE2.

Ef'fect of cycloleucine (ACPC) on transfer and accumulation of L-[3M]lysine" S/M

Control ACPC in mucosal fluid Control ACPC in serosal fluid

1.33 (9) 1 .OO (10) 1 .55 (9) 1.33 (11)

Inhibition, Y, -

24.8k4.2* -

14.2k2.5"

ICjEC 4.11 (10) 2.67 (12) 4.19 (8) 3.21 (11)

Is~hibition, -

35.024.2* -

23.4+3.6*

"Initial concentrations: unlabeled lysine, 0.065 K I M ; ['Hllysine, 10-5 m M ; cycloleucine, 5 rnM. See 'Table 1 for further explanations. * p < 0.01.

transit time of the dye was noted, and compared for st~ccessiveinjections in a given tubule. Labeled inulin was Lased as an indicator of the validity of each microinjection since this polysaccharide is found in its totality in the collected urine. Only inicroinjections that showed an inulin excretion of more than 85% of the injected solrntion were counted and used in the results. Urine was collected directly into vials containing 10 ml of liquid scintillation cocktail (Aquasol, New England Nuclear). Each injection was followed by two 5-min urine collections. The 14C and "H content was determined in a Packard Tri-Carb liquid scintillation spectrometer, as described above. Thus, knowing the amount of the radioactive amino acids injected and excreted, the results can be expressed as percentages of absorption. The degree of competition is better assessed by comparing, for a given tubule, the absorption of radioactivity in the experimental injections (excess unlabeled amino acid) with the absorption in the control injection (NaCl). (See Dubord and Bergeron (1974) for further details.)

Results Intestinal Transport in Normal Rats Table 1 shows the values of mucosal-serosal transfer and intracellular accumulation of L-cystine with respect to the cffect of cycloleucine. From the mucosal side of the intestine, cycloleucine ( 5 m,M) exerted an inhibition of 24.6 a/o on the transfer and 40.5% on the aecurnulation of L-[35S]cystine ( 3 x 18-"M). The transfer

and accumulation sf L-[xS]cystine decreased by 14.9 and 21 .O% , respectivcly (Table I ). Cycloleucine depressed the intestinal transport of I>-["Hllysine (lo-') m.41) and ra-[:3rjS]cystine by nearly the same magnitude of values (Table 2 ) ; and as in the case sf 1,-cystine, serosal inhibition of L-lysine by cycloleucine was less than its mucosal inhibition. L-Valine ( 6 x 10-"*41) transport also dccreased in the presence of cycloleucine ( 5 mM) on either the mucosal or the serosal side of the intestine (Table 3 ) . Thc accumulation of this neutral amino acid was less affected by cycoleucine on the mucosal side than was that of cystine or lysine. The effect of L-arginine (2.4 mM) on the transport of 1,-cystine by everted intestinal sacs is shown in Table 4. When L-arginine was placed in the mucosal solution, the transfer of L-cystine decreased 15.4% from control values and the accumulatioar diminished by 25.4%. However, from the serosal side, L-arginine had no effect. Similar results were found when we substituted L-lysine for L-arginine (Table 5). Intestinal Transport in Cycloleucine-Treated Rats Injections of eycloleucine (300 and 688 mg/'kg) were administered daily for 3 days to 12 Sprague-Dawley rats. They were listless,

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CAN. J. PHYSIBL. PHARMACBL. kYOL.53, 1975

TABLE3. Efkct of cycloleucine (ACPC) on transfer and accumulation of ~-[~M]vaiine"


S/M Control ACPC in nnucosal fluid Control ACPC in serosal fluid

3 . 6 8 k 0 . 2 9 (17) 2.94k0.22 (19) 3 . 9 0 k 0 . 2 4 (11) 3 . 3 9 k 0 . 1 5 (15)

'Initial concentrations: u~llabeledvaline, 0.065 m M ; ["haline, tions. * p < 0.001. **p 'p 00.1. ***p < 0.05. ****p < 0.1.

TABLE 4.

Inhibition, j7, 20. I*** -

13. I**** 6x

10.73k 8.65 7.78k0.46 13.41+0.58 10.09f 0.41

Inhibition, "j, (20) (23) (11) (13)

-

27.5** -

25.9*

m M ; cycloleucine (ACPC), 5 m M . See Table 1 for filrther explana-

Effect of L-arginine on transfer and accumulation of ~-[~~S]cystiale" S/M

Control Arginine in mucssal fluid A~gininein serosal fluid

IC/EC

Inhibition,

15.4k3.0* 1.7*3.6**

1.17 (10) 0.99 (12) 1.15 (14)

"Initial concentrations: unlabeled cystia~e,0.03 m M ; [35S]cystine,3 x tions. *p < 0.001. **No significant difference from control values.

IC/EC

Inhibition, Y,

3.35 (10) 2.50 (1 1) 3.23 (14)

25.4 *4.2* 3.58_+3.$**

-

m M ; arginine, 2.4 m M . See Table 1 for further explana-

TABLE5. EfTect of L-Iysine on transfer and accumulation of ~ - [ ~ ~ S ] c y s t i n e "

S/M Control Lysine in mucosal fluid Control Lysine in serosal fluid

1.32 (9) 1 .on (9) 1.35 (8j 1 .24 (7)

Inhibition, -

23.5i-2.2" 6 . 7 + 3.2***

"Initial concentrations: unlabeled cystine, 0.03 m M ; [35S]cystine,3 x 10- " M ; tions. *p < 0.001. * * p i0.005. ***No significant difference from control values.

ate less than normal, and a continuous loss of weight ensued (Fig. 1 ) . On the 4th day, they were killed 2 h after a fourth injection of cycloleucine. Urine pH was 6.0 and urinary abnormalities included a light ketonuria and a proteinuria (30-100 mg/100 mH). The intestines of these rats were removed and everted. The transfer and intracellular accumulation of e-cystine did not change significantly from those of control rats (Table 6 ) . Some of the everted sacs from pre-treated rats were incubated in media containing cycloBeucine ( 5 mM) either on the ltsrninal or the antijuminal side. Hn the case of cycloleucine in the luminal ss%ution,the transfer sf L-cystine decreased by 36.6% from control values and the accumulation by 52.7% (Table 7 ) . From the serosal side of the intestine of pse-treated rats, cycloleucine exerted an inhibitidn that was

IC/EC 4.31 (8) 3.07 (8) 2.92 (7) 2.84 (7)

Inhibition, -

28.8&5.3** -

2.7&3.8***

lysine, 2.4 m M . See Table 1 for further explana-

less important than on the mucosal side, a reminder of the mucosal-serosal difference demonstrated in non-treated rats.

Kinetic Studies The observed luminal-antilurninal difference with respect to cycloleucine inhibition of the intestinal transport of neutral and dibasic amino acids gave us little information as to the nature of their interactions. Figures 2-6 represent Lineweaver-Burk plots of the reciprocal of the rate sf accumulation versus the reciprocal of the amino acid substrate concentration. From the rnamcssal side of the intestine, cycloleucine ( 5 mM) inhibited the intracellular accumulation sf L - [ ~ ~ c y s t i nine a classically competitive fashion, causing a twofold increase of the dissociation constant of the substrate-

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C R 4 4 N 4NII ESERGER(1N: EXPERIMENTAL CYSTINUKIA


i n normal e and cycloleucine-treated rats TABLE6. Transfer and accumulation of ~ - [ ~ ~ S ] c y s tin

Normal g "day- ' )" ACPG treatment (300 n ~ kgACPC treatment (600 mg kg-' day -I)' -

S/M

IC'/EG

1.71 k 0 . 0 5 (16) 1 . 7 3 k 0 . 0 5 (23)" I . 4 8 + 0 . 1 3 (6)*

6 . 8 4 k 0 . 4 0 (18) 6 . 3 7 k 0 . 4 0 (23)* 6 . 5 5 t 0 . 4 8 (6)*

-

B Z n r;itro experiments followed a 3-day treatnaent with cycloleucine (ACPC) administered intsapesitoneally. See Table 1 for further explanations. *No significant difference froin normal rats.

in cycloleucine-treated ratsu TABLE, 7. Effect of cycloleucine (ACPC) on transfer and accumulation of ~-[~%S]cystine S/M

1.83kO.08 (13) 1.16t0 (13) 1 . 6 0 + 0 . 0 4 (10) 1 . 4 8 k 0 . 0 4 (6)

Control ACPC in mucosal fluid Control ACPC in serosal fluid

Inhibition, 'r,

ICiEC

-

7 . l O t 0 . 6 1 (13) 3 . 3 6 k 0 . 1 6 (13) 5 . 4 3 5 0 . 2 9 (10) 4.39kO.32 (4)

36.6* -

12.5-

Inhibition, -

52.7* -

19.2**

#See Table 1 for explanations. * p < 0.001. **p < 0.05.

I 0

I 2

I 4

I 6

I

8

DA"5

FIG. 1. Weight variations in female SpragueDawley rats. Each point is the mean r SE. Bay 0 started at 1500 hours. All animals were weighed at 0900 and 1500 hours every day. Treated rats received regular injections of cycloleucine (38 mg kg-"lay-') at 1500 hours. Oscillations in the weight s f the control group reflect circadian rhythm in the pattern of eating. The rhythm was lost in the treated group. After day 2, all animals were subjected to an 18-h fast, with free access to water, during which a parallel loss of weight was observed in both groups.

FIG.2. Lineweaver-Burk representation of the influence of cycloleucine (ACPC, 5 m M ) on e-cystine transport across lrmn~inalmembranes of the intestine. Each point is the average value from 4 experiments. S, cystine concentration; v, velocity of uptake. There is a chaasge in the K , but not in the V.

carrier complex, corresponding to a decrement sf the affinity of L-cystine for its carrier (Fig. 2 ) . For L-cystine entry from the serssal side, cyclsleucine inhibition was of the non-conlpetitive type, whereby the V sf L-cystine accumulation decreased slightly (Fig. 3 ) . When the K , displacement caused by cyclsleucine on the rnercosa (Fig. 2) is compared with the V


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CAN. J . PHYSIOI,. PHARMACOL. VOL. 53, 1975

L-[W]Valine was also injected into proximal convoluted tubules in the presence of different amino acids. Reabsorption sf it in the presence of cycloleucine 182.5 m M ) represented 97.2 % of control reabsorption. In tlae presence of L-lysine 112.5 mM), it was of the order sf 95 5%. Doubling the concentration of these unlabeled amino acids did not change the re-

-,.

L lys~rle4 5rnM ACPC

FIG. 3. Lineweaver-Burk representation of the influence of cycloleucine (ACPC, 5 m M ) on L-cystine transport across antiluminal membranes of the intestine. Each point is the average value from 4 experiments. S, cystine concentration; v, velocity of uptake. There is a slight change in the $.' but not in the K,.

displacement of the non-competitive inhibition (Fig. 3 ) , it is obvious that cycloleucine inhibition of L-cystine accumulation is not only more pronounced at the n~ucosal membrane (Table % ) but is also of a different nature from that of the serosal side. The presence of cycloleucine (ACPC) on either side of the intestine caused a non-competitive inhibition of thc intracellular accumulation of ~ - [ . ~ ~ C ] t y s(Figs. i n e 4 and 5 ) . A comparison of the V displacement seen in Fig. 4 with that of Fig. 5 shows a more pronounced cdfect of cycloleucine on the luminal membrane of the intestine, as already seen in Table 2. As suggested by the data of Tables 4 and 5, the dibasic amino acids had no effect on the serosal side; their inhibition of L-cystine transport from the lun~inalside seems to follow a non-competitive pattern (Fig. ti), which is in contrast to the effect of ACPC at this very membrane. Renal Tubular Transport Figure 7 shows the reabsarption of L-[WJIysine and L-["qcystine from the luminal fluid of proximal tubules. Reabsorption of both amino acids was depressed by the addition of an excess of unlabeled L-arginine or cycloleucine. A similar concentration of unlabeled L-valine added to the injection solution showed no inhibition of the labeled amino acids.

FIG.4. kineweaver-Burk representation s f the influence of cycloleucine (ACPC, 5 m M ) on L-lysine transport across BuminaI membranes of the intestine. Each point is the average value from 4 experiments. S, cystine concentration; v, velocity of uptake. There is a change in the V, but not in the K,,,.

FIG. 5. Lineweaver-Burk representation of the influence of cycloleucine (ACPC, 5 m M ) on I,-lysine transport across antilurninaI membranes of the intestine. Each point is the average value from 4 experiments. S, Bysine concentration; v, velocity of uptake. There is a change in the V, but not in the K,.

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CRAAN A N D BERGERON: EXPERIMENTAL CYSTHNURIA ACPC

% OF CONTROL

n Ls,-

1 l 2 5rnM


I

FIG.6. kineweaver-Burk representation s f the influence of L-lysine (5 mM) on L-cystine transport across luminal membranes of the intestine. Each point is the average value from 4 experiments. S, cystine concentration; v, velocity of uptake. There is a change in the V , but not in the K,,,.

FIG.8. Reabsorption of L-["Hlvaline by renal proximal tubule epithelium in normal rats. A solution (14 nl) of this labeIed amino acid (2 m M ) was injected through the renal capsule into proximal tubules in the presence of 0.45% NaCl (control) or an excess of another amino acid (12.5 and 25 m M ) . The values for amino acid reabsorption were calcnlated by subtracting the amount excreted from the amount injected and expressed as a percentage of the control injection in the same tubule.

with the tubular transport of L-valine at the apical membrane of the nephron (Fig. 8).

Discussion Diflerences in Membrane Function Our results show a net competitive inhibition of L-cystine entry by cycloleucine at the luminal surface sf the intestine, while at the antilumind membrane, cycloleucine inhibition was of a lesser magnitude and followed a non-competitive type of kinetics. These findings confirm FIG.7. Reabsorption of L-[3H]lysine and L-[~%S]the membrane heterogeneity of transport epicystine by renal proximal tubule epithelium in thelia, as previously demonstrated in the kidney normal rats. A soltutican (14 nl) of these labeled amino acids (0.27 maif) was injected through the (Silverman et a/. 1970a, 1970h; Bergeron and renal capsule into proximal tubules in the presence Vadeboncseur 197I ; Foulkes 197 1; Dubord of 0.45% NaC1 (control) o r an excess of another and Bergeron 1974; Scriver and Bergeron amino acid (12.5 mA.4). The values for amino acid 1974). reabsorption were calculated by subtracting the As in the intestine, luminal entry of L-lysine amount excreted from $he amount injected and expresed as a percentage of the control injection in and L-cystine was depressed in renal tubules the same tubule. in the presence of cycloleucine. Using kidney cortex slices, Holtzapple et nl. (1978) also absorption value of L-[3H]valine. These values demonstrated that cycloleucine inhibits the upwere not statistically different from control re- take of L-lysixne and a-aminoisobutyrie acid in absorption values, and they indicate an absence a competitive fashion. Since the use of cortex of interference sf cycloleucine and L-lysine slices accounts for uptake from only the anti-


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CAN. J. PHYSIOL. PHARMACOL. VOL. 53, 1975

luminal surface, probably due to an in viivo occlusion of tubular lumina, we must regard cycloleucine inhibition in this preparation as an antiluminal phenomenon (Wedden and Weiner 1973). These data suggest different transport characteristics in the intestine and thc kidney, at least at the antiluminal site. This difference was seen also in the transport characteristics of L-valine. Cycloleucine inhibited the transport of this neutral amino acid from either the mucosal or the scrosal side of the intestine. However, its reabsorption remained unaffected at thc apical membrane sf the renal proximal tubule, while inhibition was demonstrated at the antiluminal membrane (Holtzapple et al. 1970).

A INTESTINAL CELL

AC PC 1

8 RENAL TUBULAR CELL LUMEN

AC PC

I

L .-

Tran.~porrInteracrions between Anzine Acids in Expc.rinlentak Cystinuricz ~ x p l a ntions a for cystinuria have varied from the proposal of Dent and Rose (195 1) of a common renal transport system for dibasic ainino acids and cystine to more recent evidence for the parallel occurrence of common and separate carriers (Roseraberg et a/. 1962, 1967a; Fox et al. 1964; Perheentupa and Visakorpi 11965; Brodehl et a&. 1966; Msrin el al. B 97 1; Silbernagl and Deetjen 1 9 7 2 ~ ) . Since Milne et a!. ( 2 961 ) suggested an intestinal defect in cystinuria, the general consensus is that cystine, lysine, arginine and srnithine are transported across the intestinal brush border by a common mechanism (Asatoor et al. 1962; Thier et al. 1964, 1945). The specificity of the latter was determined by the failure of glycine or cysteine to compete with dibasic amino acids for intestinal transport (Thier el a!. 1964; Wosenberg et al. 1947h). This transport system appears to be absent in cystinuric paticnts (Roscnberg et a&. 19661, but these amino acids can still be absorbed in the form of sligopeptides from the intestinal lumen (HelBier et a/. 1970). Similar amino acid distribution ratios obtained from our in vitro studies in normal and cycloleucine-treated rats suggest that no intracellular structure is affected in cystinuric intestine. Therefore the defect would seem to reside at the membrane level rather than bc the result of cytotoxicity due t s cycloleucine. Besides, we could not detect any morphological changes in renal cells of cycloleucine-treated rats on clec-

ACPC

BLOOD

ACPC

FIG.9. Model for transport interactions between cycloleucine (ACPC) , cystine ( C ) , lysine (L) , ornithine ( 8 ) and arginine ( A ) at luminal and antiluminal membranes of intestinal and renal epithelial cells. Blocks represent transport systems, and indentations on their surfaces are specific transport sites. The model is supported by daea obtained from varisras authors and our data (see text). (- - - -) Competitive inhibition, (------- ) non-competitive inhibition, ( . . . . .I inhibition not defined as yet.

tromicrographs previously done in this laboratory (Bergeron, unpublished data). Therefore ultrastructural changes could not account for the cystinuria caused by cycloleucine. Data obtained with the cycloleucine model are summarized in Fig. 9. At the luminal surface of the intestine ( 9 A ) , cycloleucine compctes with cystine for the site of attachment that is specific for cystine and located on a cystine - dibasic amino acid carrier; simultaneously. it could create an allosteric effect on the lysine site. This is reflected by a competitive interaction between cycloleucine and cystine and a non-competitive interaction between ACPC and lysine (Figs. 2 and 4). At the antiluminal side, cyclsleucine binds either the cysti~re-or the lysine-transport protein at sites that are different from those utilized for their respective transport. This explains modification in the V rather than in the K,, (Figs. 3 and 5 ) . Experiments with L-valine suggest the existence at both mucosaI and serosal membranes of neutral amino acid carriers that can react


CRAAN AND BEWCERON: EXPERIMENTAI, CYSTINURIA

with cycisleucine (Table 3 ) . Such carriers seem to be independent sf transport processes for cystine and dibasic amino acids (Wilson and Scriver 1967; Segal and Smith 1969). Figure 9B shows a mosaic of transport pathways at both membranes of the kidney. There exists a transport system that is specific for cystine-cysteine ( C ) , a second system for dibasic amino acids (LOA) and a third that is shared by cystine-cysteine and dibasic amino acids (LOAC) , all three rnecl~anismsbeing independent of transport processes of neutral amino acids. At the antilurninal sur%ace, all dibasic amino acids are taken up from the peritubular fluid, presumably by a common mechanism (LOB) that is not shared by cystine. This model is consistent with data obtained from various investigations (Rosenberg et wH. 1962, 1967a; Fox et ak. 1964; Berheentupa and Visakorpi 1965; Brodehl et ak. 1966; Morin et ak. 1971; Silbernagl and Deetjen 197%); it also prok7ides an explanation for isolated hypercystinuria (Brsdehl et al. 1966) and hypcrdibasicaminoacidb~ria (Whelan and Scriver 1968; Oyanagi et ak. 1970; Simell and Perheentupa B974), and may fit the genotypic variations already described in human cystinuria (Wssenberg et al. 1966; Morin et ak. 1971). This work was supported by a grant (hIT-2862) from the Medical Research Council of Canada and the Banting Research Foundation. T h e authors would like to thank Mrs. C . Schwab, L. Alle Ando and Miss C. Sabourin for technical assistance, E. Wupnik for the photography and Miss J. Manson for reviewing the manuscript. A S A T O ~ RA., M., LACEY,B. W . , LONDON,14. R., and MILNE,M. I). 1962. Amino acid metabolism in cystinuria. Clin. Ssi. 23,285-304. BERGERON, M., and MORE^ , F. 1969. Amino acid transport in rat renal tubules. Am. J. Physiol. 216, 1139-1 149. BERGERON, M . , and V~r>reoNcoEuR,M. 1971. Microinjections of 1.-leaacine into tubules and peritalbular capiilaries of the rat. 11. The malleic acid model. Nephron, $, 367-374. BRODEHH., J.. GELIISSFN, K. and KOWALEWSKI, S. 1966. Isolated cystinuria (without Bysine-ornithine-argininuriaj in a Fdmily with hypocalcemic tetany. Third International Congress of Nephrology, Washington, D.C. p. 165. (Abstr.). BROWN,13. R . 1967. Aminoacidkaria resulting from cyclofeucine administration in man. Science, 157 (3787), 432434. CEPRISTENSEN. H. N.. and CUI,I

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