49Biochem. J. (1978) 172,49-56Printed in Great Britain

Effect of Parathyrin on the Transport Properties of IsolatedRenal Brush-Border Vesicles

By CARLA EVERS, HEINI MURER and ROLF KINNE*Max-Planck-Institut fur Biophysik, Kennedyallee 70, 6000 Frankfurt (Main) 70, Germany

(Received 4 November 1977)

The transport properties- of brush-border membrane vesicles isolated by a calcium-precipitation method from the renal cortex of normal and parathyrin (parathyroidhormone)-treated rats were studied by a rapid-filtration technique. Parathyrin eliciteda dose-dependent decrease in the Na+-dependent phosphate uptake by the brush-bordermembrane vesicles, but the uptake of D-glucose, Na+ and mannitol was not affected. Amaximum inhibition of 30% was observed after the application of 30 U.S.P. unitsintramuscularly 1 h before the animals were killed. Intravenous infusion of dibutyrylcyclic AMP (0.5-1.5mg) also decreased the phosphate uptake by the brush-bordervesicles. Both dibutyryl cyclic AMP and parathyrin were ineffective when added in vitroto brush-border membrane vesicles isolated from normal rats. These data suggest thatparathyrin exerts its action on the phosphate reabsorption in the renal proximal tubuleby affecting the Na+/phosphate co-transport system in the brush-border membrane.The effects of parathyrin on Na+ and glucose transport, however, seem to be due toalterations to the driving forces for transport and not to the brush-border transportsystems.

Parathyrin administration causes phosphaturia byinhibiting phosphate transport in the proximal aswell as in the distal tubule (Agus et al., 1971, 1973;Amiel et al., 1970; Brunette et al., 1973; Gekle,1971; Knox et al., 1976).With respect to the biochemical events, it seems

that the action of parathyrin is initiated at the contra-luminal cell side (Shlatz et al., 1975) by activationof an adenylate cyclase (Nelson et al., 1970), and thatthe appearance of cyclic AMP in the tubular fluidprecedes the effect of parathyrin on the phosphatereabsorption (Chase & Aurbach, 1967; Nelson et al.,1970). Butlen & Jard (1972) provided further evidencethat the presence of cyclic AMP in the tubular lumenmight be essential for the action of parathyrin on thephosphate reabsorption. The further reactions whichin turn are influenced by cyclic AMP are, however,unknown.From a biophysical viewpoint the alterations of

phosphate transport by parathyrin can be broughtabout either by changes in the properties of thetransport system, leaving the driving forces un-altered, or by changes in the driving forces withoutan alteration of the transport system itself, or by acombination of both processes. The following studieswere undertaken to distinguish between these possi-bilities. For this purpose, the transport properties ofrenal brush-border membrane vesicles isolated from

Abbreviation used: Hepes, 4-(2-hydroxyethyl)-l-piper-azine-ethanesulphonic acid.

* To whom reprint requests should be addressed.

Vol. 172

proximal tubules of normal and hormone-treatedanimals were compared. The results demonstratethat parathyrin administration specifically decreasesthe maximum velocity of the Na+/phosphate co-transport system in the brush-border membrane.This finding indicates that parathyrin exerts its actionon the phosphate transport by an alteration to themembrane-bound transport system rather than by achange in the driving forces.

Materials and Methods

Isolation ofbrush-border membranes

Male Wistar rats (160-180g body wt.; ten for eachexperiment) were killed by a blow to the neck andtheir kidneys were placed as quickly as possible intoice-cold mannitol buffer (10mM-mannitol/2mm-Tris/HCl, pH 7.1). They were decapsulated and thin slicesof the renal cortex (approx. 1-2mm thick) wereprepared. Brush-border membrane vesicles wereisolated by the calcium-precipitation method origin-ally described by Booth & Kenny (1974) as modifiedby Evers et al. (1978). In short, the renal cortex ishomogenized in mannitol buffer (the most criticalperiod for the maintenance of the altered state of themembrane seems to be the time between the deathof the animal and the homogenization of the tissuein mannitol buffer; after that the parathyrin-induceddecrease in phosphate transport is quite stable), thenCaCl2 is added to the suspension to a final concentra-

C. EVERS, H. MURER AND R. KINNE

tion of 10mM. The aggregated mitochondria, lyso-somes, endoplasmic reticulum and basolateral plasmamembranes are removed by a low-speed (12min at5)0g) centrifugation. The brush-border membranesremain in the supernatant and are then sedimentedby centrifugation for 12 min at 15000g. The calcium-precipitation step is repeated once and the finalsediment is suspended in buffer (lOOmM-mannitol/20mM-Hepes/Tris, pH7.4) and used for transportstudies. The brush-border membrane vesicles ob-tained by this procedure are predominantly orientedright-side out asjudged from freeze-fracture electron-microscopy and by immunological techniques(Haase et al., 1978).

Treatment ofanimals before death

Normally a crude parathyrin preparation (P-20;Lilly, Indianapolis, IN, U.S.A.) was injected (30U.S.P. units/animal) intramuscularly 1 h before theanimals were killed. For dose-response experiments,a highly purified hormone preparation (Inolex, ParkForest, South Illinois, U.S.A.) was used. In someexperiments [arginine]vasopressin (0.1 nmol/animal;Ferring, Malmo, Sweden) instead of parathyrin wasinjected intramuscularly 1 h before the animals werekilled. The hormone preparations were always dis-solved in water containing 0.2% phenol. Animalsused for control experiments received only an equalamount (0.3ml/animal) of phenol solution in water.Dibutyryl cyclic AMP (0.5 or 1 mg) was injected intothe jugular vein of animals kept in Inactin (thiobuta-barbital sodium; Byk-Gulden, Konstanz, Germany)narcosis 20 min before removal of the kidneys.The cyclic nucleotide was dissolved in 0.2mlof 150OmM-mannitol/100 mM-NaCI/2.6mM-KCl/3 mm-CaC12. Control animals kept under anaesthesia re-ceived 0.2ml of the same solution without thecyclic nucleotide.

Determination ofprotein and enzymes

Protein was determined, after precipitation of themembranes with ice-cold 10% (w/v) trichloroaceticacid, by the procedure of Lowry et al. (1951), withbovine serum albumin as standard. Alkaline phos-phatase (EC 3.1.3.1) and Na+ + K+-stimulatedadeno-sine triphosphatase (EC 3.6.1.3) were determined asdescribed by Berner & Kinne (1976); acid phosphat-ase (EC 3.1.3.2) was measured with a test kit (Merck,Darmstadt, Germany, no. 3378). These enzymeactivities were measured semi-automatically with theLKB reaction-rate analyser 8600 at 37°C. Succinatedehydrogenase (EC 1.3.99.1) was determined by themethod of Gibbs & Reimers (1965). Glucose 6-phosphatase (EC 3.1.3.9) was measured as describedby Bode et al. (1974). Maltase (EC 3.2.1.20) wasmeasured by a modification of the method of Sacktor(1968).

Transport studies

Uptake of labelled substrates by the isolatedmembrane vesicles was measured by a Millipore-filtration technique as described previously(Hoffmannet al., 1976; Hopfer et al., 1973; Kinne et al., 1975a).The exact compositions of the incubation media aregiven in the legends to the Figures.

Materials

All chemical reagents were of the highest chemicalpurity available. Radioactive isotopes were pur-chased from New England Nuclear (Boston, MA,U.S.A.).

Results

Biochemical characteristics of the brush-border mem-brane vesicles isolated from normal and parathyrin-treated rats

As shown in Table 1 the administration of para-thyrin to normal rats does not influence the specificactivities, enrichment or recovery of brush-bordermarker enzymes alkaline phosphatase and maltasein the final membrane preparation. Similarly thecontamination with basolateral plasma membranesand with other intracellular organelles is not altered.Therefore it can be concluded that the hormone doesnot alter the behaviour of the brush-border vesiclesduring isolation and that membrane fractions ofidentical purity are obtained from control andhormone-treated animals.

Transport properties of brush-border membranevesicles isolatedfrom normal and hormone-treated rats

Fig. 1 gives an example of the phosphate uptakeby renal brush-border vesicles isolated from theproximal tubules of normal and parathyrin-treatedrats. Both vesicle preparations show a very rapidinitial uptake phase which leads to an intravesicularaccumulation of phosphate inside the vesicles (over-shoot). However, as detailed quantitatively in Table2, in brush-border vesicles isolated from parathyrin-treated rats the phosphate uptake during the first 20sand the overshoot are decreased by about 30 %.During the present study it was observed that the

phosphate uptake varied considerably from prepara-tion to preparation. As shown in Fig. 2, after 20s ofincubation uptake as low as 120% and as high as250% of the equilibrium value could be observed.The high variability of the phosphate uptake (com-pared with the glucose uptake) might reflect the factthat phosphate transport in the proximal tubule issubjected to regulation by a variety of factors (hor-monal and nutritional) that are difficult to control.In a given population of animals, however, the

1978

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EFFECT OF PARATHYRIN ON RENAL PHOSPHATE TRANSPORT

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Fig. 1. Effect of parathyrini on the phosphate uptake byisolated renal brush-border membrane vesicles

Control rats (o) received 0.3ml of 0.2%. phenolintramuscularly 1 h before death; parathyrin-treatedrats (e) received 0.3ml of Parathormone (Eli Lilly,100 U.S.P. units/ml). Phosphate uptake was studiedin lOOmM-mannitol / 20mM-Hepes / Tris (pH7.4) /0.1 mM-phosphate/l0OmM-NaCl medium. The resultsare expressed as percentage of the uptake observedafter 60min (equilibrium), which amounted to

00 o 0.357nmol/mg of protein for the control rats and° 0.322 for the parathyrin-treated rats. Results from

one typical experiment are given.

phosphate uptake by brush-border vesicles isolated. ay after treatment with parathyrin was always lower

C.6 6 e,; rr° ^ > than uptake by the membranes isolated from thenon-treated animals, and seemed to be linearlyrelated to the transport observed in the controlmembranes (Fig. 2). This means that the percentageinhibition was quite constant, although the absolute

m~ N - - decrease varied greatly. Therefore paired data

°~ t-~obtained from the same population of animals at thesame time and under identical conditions are alwaysgiven. Representative experiments are shown whichcould be repeated at least three times with different,animal populations yielding qualitatively similar

results.

@ . Table 3 shows that the decrease in phosphate

o O:3.uptake can be elicited by different preparations ofCA parathyrin (highly purified hormone preparationO< v: provokes similar alterations to those with crude

preparations), and that [arginine]vasopressin, an-

<U _ other peptide hormone acting on the kidney but atD blX different sites on the nephron, does not seem to

E 70 E influence the characteristics of the phosphate trans-

o port.

<o .o The effect of parathyrin on the phosphate uptakeWL1; -; > v is also dependent on the concentration of parathyrin

Vol. 172

51

C. EVERS, H. MURER AND R. KINNE

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Fig. 2. Correlation between initial uptake rates of Pi inrenal brush-border vesicles isolated from control animals

andfrom parathyrin-treated animalsThe experimental conditions were identical withthose given in Fig. 1. The solid line represents theregression line calculated from all experimentalpoints by the least-squares method (r = regressioncoefficient). The dashed lines indicate 0, 25 and 50%4inhibition of uptake.

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administered to the animals (see Fig. 3a); however,addition of parathyrin to the vesicles after isolationof membranes from normal animals does not changethe transport properties (Fig. 3b).

Table 2 shows in addition that parathyrin underthe conditions of these experiments specificallydecreases the Na+-dependent phosphate uptake bythe brush-border vesicles. The uptake of phosphatein the presence of choline instead of Na+ is notaffected by the hormone. Similarly parathyrin seemsnot to affect significantly the uptake of D-glucoseand 22Na by the membrane vesicles, or the amount ofphosphate, glucose and Na+ found in the vesiclesafter 2h of incubation in the Na+-containing medium.Therefore it can be concluded that both preparationscontain the same intravesicular volume of 3.3,1 permg of protein. Since the rate of mannitol entry (whichmonitors unspecific permeability properties of themembrane) into the two vesicle preparations is alsoidentical, one can further assume that the surface/volume ratio and therefore the size of the transportingvesicles is not different (Evers et al., 1978).

Table 4 shows the effect of parathyrin on thekinetic parameters of the phosphate-transport systemin the brush-border membrane. Both at 100mm- and40mM-NaCl the hormone mainly seems to affect the

1978

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EFFECT OF PARATHYRIN ON RENAL PHOSPHATE TRANSPORT

Table 3. Effects ofparathyroid gland extract (Lilly), highly purified parathyrin (Inolex) and [arginine]vasopressin (Ferring)injection on phosphate uptake by isolated brush-border vesicles

The experiments were carried out as indicated in the legend to Fig. 1. The values are expressed as percentage ofequilibrium uptake. For the experiments with parathyroid extract the mean values+ S.D. of 11 experiments areindicated. For purified parathyrin three independent experiments are given. For [arginine]vasopressin two inde-pendent experiments are given.

Hormone preparationParathyroid extract

(Lilly, P-20)

Parathyrin (Inolex)

[Arginine]vasopressin

Control (n = 11)

Hormone injection (n = 11) (30 U.S.P. units/animal)ControlHormone injection (10 U.S.P. units/animal)ControlHormone injection (0.1 nmol/animal)

Uptake after 20s(% of equilibrium value)

193.5 +45.20.01>P>0.005132.7+ 34.8

170 187 276135 136 178

310 290300 280

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Fig. 3. Effect ofparathyrin on phosphate uptake by isolatedbrush-border membrane vesicles; comparison ofapplicationofparathyrin in vivo (a) versus the effect ofparathyrin on

isolated membrane vesicles in vitro (b)The experiments in vivo (a) were carried out as indi-cated in the legend to Fig. 1 and the results are

expressed as percentage of phosphate uptake bybrush-border vesicles isolated from control rats.Results from one typical experiment are given. Forthe experiments in vitro (b), brush-border membranevesicles were isolated from normal rats and para-thyrin at doses indicated in the Figure was added tothe incubation media. The results are expressed aspercentage of phosphate uptake after 20s (o) andafter 1 min (e) in the incubation media containing no

parathyrin. For experiments in vivo and in vitrohighly purified hormone (Inolex) was used. Theincubation media were as indicated in the legend toFig. 1. -log [M] is the negative logarithm of the con-centration of parathyrin in the incubation medium.

Vol. 172

Vmax. value of the system, but the affinity of thephosphate/Na+ co-transport system to phosphateseems to be unchanged. Similarly the sensitivity ofthe phosphate/Na+ co-transport to Na+ is apparentlyunaffected by parathyrin treatment of the animals(Fig. 4).

Effect of cyclic AMP on the transport properties ofisolated brush-border vesicles

Since cyclic AMP is thought to be the mediatorof the action of parathyrin on the phosphate trans-port in renal cells, the effect of cyclic AMP on thetransport properties of the vesicles was investigated.In a first set of experiments, dibutyryl cyclic AMPwas infused into the animals; the membranes wereisolated after infusion and phosphate transport wasstudied in the absence of cyclic AMP in the incuba-tion medium. As shown in Fig. 5, under these con-ditions a decrease in the phosphate transport by thebrush-border vesicles (even higher in magnitude thanthe parathyrin effect) could be observed. The effectivecyclic AMP concentration can be estimated to beapprox. 3,11M at the time of removal of the kidney,if the same clearance and distribution volume as inman (Broadus et al., 1970) is assumed. Addition ofcyclic AMP in vitro to brush-border membranevesicles isolated from control rats did not altersignificantly the transport properties of the vesicles(Fig. 5).

Discussion

Nature of alteration in phosphate uptake by isolatedbrush-border vesicles

The brush-border membrane contains a Na+/phosphate co-transport system which at pH 7.4facilitates the electroneutral transfer of secondary

53

C. EVERS, H. MURER AND R. KINNE

Table 4. Effect ofparathyrin on kinetic parameters ofphosphate uptake by brush-border membranesThe incubation media contained lOOmM-D-mannitol, 20mM-Hepes/Tris, pH7.4, KH232P04 and lOOmM-NaCI or40mM-NaCl and 60mM-choline chloride. The phosphate concentration of the incubation media was varied from 0.1to 1.OmM. Apparent Km and apparent Vmax. values were calculated from regression lines obtained by least-squaresanalysis in Eadie-Hofstee plots (regression coefficients = 0.96-0.99). Five different concentrations of phosphate wereused and the transport experiments were performed in triplicate.

NaCl(mM)

1004010040

Km(mM)

0.360.320.300.28

Vmax.(nmol/20s per mg

of protein)1.560.650.960.27

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Fig. 4. Effect ofparathyrin on Na+ activation ofphosphateuptake

The experiments were carried out as indicated inFig. 1. The phosphate concentration was 0.1 mm, andthe NaCl concentration was varied from 25 to100mM as indicated in the Figure (sodium was re-placed by potassium). The results of one typicalexperiment are expressed as percentage of the Na+-stimulated uptake of phosphate at lOOmM-NaCl.Control rats: o, uptake after 20s; *, uptake after1min; parathyrin-treated animals: O, uptake after20s; *, uptake after 1 min.

phosphate and two Na+ ions (Hoffmann et al., 1976).In such a co-transport system, both the chemicalconcentration difference of phosphate and the chemi-cal concentration difference of Na+ across the vesiclemembrane act as predominant driving forces forthe phosphate uptake. Therefore a decrease in phos-phate transport (measured in the presence of a Na+and phosphate gradient) as observed after para-thyrin treatment does not necessarily imply that thephosphate-transport system itself is altered but couldalso be due to a decrease in the driving forces. Sucha decrease could be brought about by an increase inthe Na+ permeability of the brush-border membrane.

Thereby the Na+ gradient across the vesicle membranewhich is present at the beginning of the transportexperiment would be dissipated faster, and conse-quently a lower initial rate ofphosphate uptake wouldbe observed. This seems unlikely, however, since theuptake of 22Na by the brush-border vesicles is notaltered by parathyrin and another Na+-drivenco-transport system, the D-glucose/Na+ co-transport,is also not changed by the hormone. Therefore weare inclined to conclude that the phosphate/Na+ co-transport system itself is affected by the hormone.This conclusion is supported by the results of theexperiments performed to study the properties of thephosphate-transport system. The hormone-induceddecrease of the apparent Vmax. might be explained bya decrease in the number of transport sites or by alower mobility of a hypothetical carrier system for Pi.The biochemical nature of the parathyrin- and

cyclic AMP-elicited change of the phosphate-transport system has yet to be clarified. Our datasuggest that the mere presence of cyclic AMP at theluminal membrane is not sufficient to provoke achange in the phosphate-transport system, but thatadditional cellular processes are required. Some ofour own preliminary experiments (results not shown)indicate that cyclic AMP even in the presence ofATP is unable to decrease the phosphate transportby the vesicles, although cyclic AMP-dependentprotein kinases have been found to be concentratedin renal brush-border membranes (Kinne et al.,1975b). This might simply be due to the fact that thebrush-border vesicles used in this study are orientedright-side out (Haase et al., 1978), i.e. the formercytoplasmic side of the membrane is at the inside ofthe vesicles, and thus the catalytic centre of proteinkinase might not be accessible for ATP.

There is another noteworthy aspect of the studiespresented above concerning a possible relationshipbetween the phosphate-transport system and thealkaline phosphatase present in the brush-bordermembrane. After the application of parathyrin thealkaline phosphatase activity in the homogenate and

1978

RatsControl

+Parathyrin

54

EFFECT OF PARATHYRIN ON RENAL PHOSPHATE TRANSPORT 55

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0

50 o v so51 0 0 L'/I

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Dibutyryl cyclic -log 1M]AMP injected (mg)

Fig. 5. Effect of cyclic AMP on phosphate uptake by isolated brush-border vesicles; comparison ofapplication ofcyclic AMPin vivo (a) versus the effect ofcyclic AMP on isolated membrane vesicles in vitro (b)

Dibutyryl cyclicAMP was injected in vivo (a) as described in the Materials and Methods section and uptake experimentswere carried out as indicated in the legend to Fig. 1. The results are expressed as percentage of uptake of P1 after 20s(o) and after 1 min (e) observed in brush-border vesicles isolated from animals that received no cyclic AMP. For theexperiments in vitro (b) brush-border membrane vesicles were isolated from normal rats (no injection) and dibutyrylcyclic AMP in amounts indicated in the Figure was added to the incubation media. The results are expressed aspercentage of uptake of P1 in the incubation media containing no cyclic AMP. -log [M] is the negative logarithmof the cyclic AMP concentration (M) in the incubation medium.

in the isolated brush-border membranes remainedconstant, although the phosphate transport wasdecreased by about 30%. Thus it seems very unlikelythat alkaline phosphatase and the phosphate-transport system are functionally inter-related.

Mechanism ofaction of parathyrin on transport pro-cesses in the proximal tubule

Since it has been demonstrated that the activephosphate reabsorption in rat proximal tubule isNa+-dependent (Baumann et al., 1975b), it could beconcluded that this transport involves a coupling ofphosphate and Na+ flux across the brush border viaa Na+/phosphate co-transport system. This systemis inhibited after parathyrin administration to therats. The observed change of 30% in vitro in the rateof phosphate uptake of the vesicles agrees quite wellwith the degree of inhibition of phosphate transportobserved in micropuncture experiments in vivo (Aguset al., 1971; K. J. Ullrich, G. Rumrich, S. Kloss,unpublished work). This indicates that in theseexperiments in vivo and in ours in vitro, the same rate-limiting factors existed; other factors such as de-creased phosphate permeability of the peritubularcell membrane, or changes in the driving forces forthe Na+-phosphate co-transport (which include theelectrochemical potential difference of Na+ andphosphate across the luminal membrane) are appar-ently not crucial for the parathyrin-cyclic AMPaction on phosphate transport.The latter factors and perhaps even effects on the

paracellular pathways of transport might be involvedin the inhibitory effect of parathyrin and cyclic AMP

on iso-osmotic volume reabsorption and glucosetransport in the proximal tubule (Baumann et al.,1975a), since our data provide no evidence for aninhibition of transport systems mediating the trans-cellular transport of Na+ and D-glucose. The per-meability of the luminal membrane for 22Na, and theactivity of the Na++K+-stimulated adenosine tri-phosphatase in the homogenate of the renal cortex,apparently remain unchanged. Similarly no alterationof the glucose/Na+ co-transport system in the brush-border membrane as studied under the conditions invitro could be detected.

We thank Professor Dr. K. J. Ullrich for valuablediscussions during the performance of the experimentsand the preparation of the manuscript. We also thankMrs. F. Papavassiliou and Mr. G. Rumrich for assistancein the dibutyryl cyclic AMP infusion experiments.

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Amiel, C., Kuntzinger, H. & Richet, G. (1970) PflugersArch. 317, 93-109

Baumann, K., Chan, Y. L., Bode, F., Papavassiliou, F. &Wagner, M. (1975a) in Biochemical Aspects of RenalFunction, Current Problems in Clinical Biochemistry(Angielski, S. & Dubach, U. C., eds.), vol. 4, pp. 223-228, Hans Huber Publishers, Bern

Baumann, K., de Rouffignac, C., Roinel, N., Rumrich, G.& Ulirich, K. J. (1975b) Pflugers Arch. 356, 287-297

Berner, W. & Kinne, R. (1976) PflugersArch. 361, 269-277

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56 C. EVERS, H. MURER AND R. KINNE

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Hopfer, U., Nelson, K., Perrotto, J. & Isselbacher, K. J.(1973) J. Biol. Chem. 248, 25-32

Kinne, R., Murer, H., Kinne-Saffran, E., Thees, M. &Sachs, G. (1975a) J. Membr. Biol. 21, 375-395

Kinne, R., Shlatz, L. J., Kinne-Saffran, E. & Schwartz,I. L. (1975b) J. Membr. Biol. 24, 145-159

Knox, F. G., Haas, J. A. & Lechene, C. P. (1976) KidneyInt. 10, 216-220

Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall,R. J. (1951) J. Biol. Chem. 193, 265-275

Nelson, G. L., Chase, L. R. & Aurbach, G. D. (1970)Endocrinology 86, 511-518

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Shlatz, L. J., Schwartz, I. L., Kinne-Saffran, E. & Kinne,R. (1975) J. Membr. Biol. 24, 131-144

1978