RAPID COMMUNICATION ! COMMUNICATION RAPIDE

Phlorizin increases the permeability of intestinal rnucosal membrane to sodium

P. K. DINDA' AND I. T. BECK The <;ustroirrtestinul Diseuse Re.reurc-h Ilnlt, I)epartmerzt c$Medic.inr und L)epc~rtrnerzt of PhyLsio8ugy, Queerz'.~ Univrrsih.,

Kingsfon, Clnt. , C'anndf~ K7L 3H6

Received December 16, 1980

DINDA, P. K . , and BECK, I . T. 19$7. Phlorizin increases the permeability of intestinal rnucosal membrane to sodium. Can. J . Physiol. Pharmacol. 65: 579-586.

We reported previously that when jejunal transnlural glucose transport was inhibited by phlsrizin the ratio of Na:glucose transport increased from 2.0: 1 (in controls) to 3.3: 1 . To elucidate the mechanism of this increased ratio of Na:glucose transport, in the present study we have investigated the effect of phlorizin on Na uptake by brush border membrane vesicles and by everted sacs sf hamster jejunum. In experiments on membrane vesicles the following observations were made. The time course of Na uptakc showed that the control vesicles were in cornplete equilibriu~n with a Na-containing (100 m2a) medium between 30 and 90 min incubation. I11 these periods of incubation, the vesicles incubated with phlorizin presumably also equilibrated with the nledium. but lost their intravesicular Na during Millipore filtration and washing, and consequently the residual Na content was lower than that of controls. This effect of phlorizin was concentration dependent, and appeared to be unrelated to Na-coupled glucose transport, because it was also observed in thc absence of glucose. This loss c ~ f Na during Millipore tiltration and washing was also observed (i) when vesicles were equilibrated in a Na-containing solution in the absence of phlorizin and thenexposed to a similar solution containiilg phlorizin, or ( i i ) when vesicles were equilibrated in a Na-containing solution in the presence of phlorizin and then washcd repeatedly following Millipore filtration. Preincubation of vesicles for 10 niin in a Na- and glucose-free solution containing phlorizin followed by incubation for 30-90 s in solutions containing 1 mM glucose and various concentrations of Na (from 10 to 100 d) caused an increase in Na uptake from all concentrations of Na. After similar preincubation. when jejunal everted sacs wcre incubated for 15 s in a Na- and glucose-containing medium, Na uptake by the sacs increased. These findings suggest that phlorizin causes an increase in pernleability of mucosal membrane of the enterocyte to Na. This may cause a rapid dissipation of Na gradient and an increase in the ratio of Na:glucose transport. The dissipation of Na gradient may be an additional mechanism for phlcrrizin-induced inhibition of intestinal sugar transport.

DINDA, P. K., et BECK, I. T. 1987. Phlorizin increases the pernleabllity of intestinal rnucosal membrane to sodium. Can. J . Physiol. Phamlacol. 65 : 579-586.

Nous avons rapporte ant6rieuremcnt que lorsque le transport de glricose transillural jijunal etait inhibe par la phlori~ine, le rapport du transport Na:glucose augmmentait de 2.0: 1 (chez des t h o i n s ) i 3,3: 1 . Pour essayer d'expliqucr le m@canisme de cette augmentation du rapport du transport Na:glucose, nous avons examine, dans la presente etude. l'effet de la phlorizi~le sur la capture de Na par des vesicules de la membrane de bordure en brosse et par des sacs retournes de jkjunum de hamster. Dans des expkriences sur des vesicules, nous avons fait les observations suivantes. L'evolution ternporelle de la capture de Na rnontra que les v@sicules temoins Ctaient en equilibre parfait avec un milieu contenant du Na (100 mM) lors d'une incubation de 30 ii 90 min. Durant ces periodes d'incubation. les vCsicules incubees avec de la phlorizine s'kquilibrerent vraisemblablement aussi avec le milieu, rnais elles perdirent leur Na intrav6siculaire durant une filtratlon Millipore et un lavage; par consequent, la teneur en Na residuelle fut plus faible que celle des temoins. Cet effet de la phlorizhe etait fonction de la concentration et sembla ne pas etre relie au transport de glucose couple au Na, etant donnC qu9il fut observe aussi en 19absence de glucose. Cette perte de Na durant la filtration Millipore et He lavage fut aussi observee (i) lorsque les cellriles furent equilibrees dans une solution contenant du Na en I'absence de phlorizine, pour ensuite Ctre exposces ii une solution similaire contenant de la phlorizine, ou ( i i ) lorsque Ics vesicules furent equilibrkes dans une solution contenant du Na en prksencc de phlorizine, puis lavkes de maniere repetee apres la filtration Miflipore. La preincubation des v6sicules pendant 10 min dans une solution sans Na et sans glucose mais contenant de la phlorizine, suivie d'une incubation de 30 a 90 s dans des solutions contenant 1 mV de glucose et diverses concentrations de Na (de 10 21 100 mVl), provoqua une augmentation de capture de Na 2i toutes lcs concentrations de Na. Apres une prkincubation similaire, lorsque Bes sacs jejunaux retournes furent incubes pendant 15 s dans un milieu contcnant du Na et du glucose, la capture de Na par les sacs augmenta. Ces resultats sugghent que la phlorizine provoque unc augmentation de la 1m-nrn6abiliti de la membrane muqueusc de l'enterocyte au Na. Ceci peut provoquer une dissipation rapide du gradicnt de Na et rine augmentation du rapport du transport Na:glucose. La dissipation du gradient de Na peut Ctre un nrecanisrne sny~plementaire pour I'inhibition induite par la phlorizine du transport de sucre intehtinal.

[Traduit par la revue)

Introduction we found that a concentration of phlorizin, which inhibited both Phlorizin is known to be a competitive inhibitor of intestinal

sugar uptake (Alvarado 1 967; Alvarado and Crane 1962, 1963). which is reported to be stoichiometrically related to Na influx across the mucosal ~nembrane (Kaunitz et al. 1982; Kaunitz and Wright 1984; Kirninich and Randles 1980). In a previous study

'Author to whom correspondence may be sent at the following atldress: Div. of C;astroentcrology, Hotel Dieu Hospital, Kingston. Ont., Canada K7L 3N6.

intestinal glucose transport andglucose-dependent transmural potential difference by approximately 40%. had no effect on the net transport of Na (Dinda et al. 1975). This was manifested by a difference in the ratio of Na:glucose transport. In the absence of phlorizin the ratio was 2.0 1 , while in the presence of phlorizin the ratio increased to 3 -3 1. This increase in the transport of Na in relation to that of glucose was difficult to interpret. However, we tentatively suggested that the phlorizin-induced inhibition of glucose-linked electrogenic Na transport was probably accom-

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

WA

ST

AT

E U

NIV

LIB

RA

RIE

S on

11/

13/1

4Fo

r pe

rson

al u

se o

nly.

580 CAN. J . PHYSIOL. PHARMrZCOL. VOL. 65, 1987

panied by an increase in nonelectrogenic transport of this ion (Dinda e t a l . 1975). The present study was undertake11 to ex- amine whether phlorizin caused an incrcase in nonelectrsgenic Na transport, and if so , how this effect was mediated. For this investigation, we studied thc effect sf pbIorizin on Na uptake by the jejuraal brush border membrane (BBM) vesicles and also b y everted sacs of the jejunum. The fcdlowing is a detailed outline of these experiments.

Methods Msrteria1.s

All labelled cornpounds ("N~cI , [ '~] inul in , n-[ '~]mannitol, n - ( ' ~ ] ~ l u c o s e ) were purchased from New England Nuclear, Morat- real, Que.. Canada. N-2-Wydroxyethylpipera~ine-N'-2-ethanesulf~~r1ic iacid (HEPES), tris(hydroxyrlaethy~)a11lino~11et~~ar~e hydrogen chloride (Tris-HCl), phloriizin. and u-mannitol were obtained from Signaa Chem- ical Co., St. Louis, MO. Phlori~in was recrystallized in our labc~ra- tory. Other reagents obtained froni various sources were of highest purity available.

Anirnuis Slx- to eight-wcek-old hamsters were used. They had easy access to

food and water. On the day of experiment, the anlmal wah killed by decapitation, and the small intestme was excised and flushed with ice-cold Krebs-Ringer bicarbonate solution (KRBS composition: Na' , 143.5; K I , 5.9; C1-, 128; c a 2 I , 2.5; H2P04 , 1.2; ~ g " , 1.2: SO4" , 1.2; HC03-, 25.0; pH 7.4). Two groups of experiments were carried out.

C;rotdp I: eflect of phlorizira on Ma ctnd glttcose upbake by brush border rnernhrarer ve.sicies

BBM vesicles were prepared from hamster je~lanum employing a rnodifred method of Hopfer et al. (1973). The details of the method and the purity of the preparation were similar to those described by us previously (Dinda and Beck 198 1 , 1982; Dinda et al. 19791, except that in the present study EDTA was conlpletely omitted from the isolation buffer, Based on sucrase activity, the purity of the BBM was 32 + 2 (naean f SE, n = 32) fold.

For the measurement of Na uptake, the final membrane preparation was suspended in 10 nuV1 HEPES-Tris buffer containing 206) tnU D-mannitol and 0.1 nuV1 MgS04. Na uptake by the BBM vesicles was measured by a Millipore filtration technique.' The method was based on that described by SigPist-Nelson et al. (1975). In this group, eight series of experiments were carried out. Speclfic details of the method for each series are given in the legends to the figures. The general outline of the naethod is as follows: aliquots (usually 0.025 or 0.050 EL) of the menabrane suspension were prewar~iaed (i.e., preincubated at 25°C for 10 min in the presence or in the absence of phlorizin, and the uptake was initiated by the addition of an equal volume of grewarmed (25°C) incubatic~n solution. The solute concentratioaa of the incubation solution was such that the addition of this solution to an equal volume of' membrane suspension resulted in the following composition of the cornplete reaction mixture: 10-100 m4f NaCl, tracer amounts of 2 2 ~ a C l , 100-280 11~44 D-mannitol, 0.1 mhl MgSO,, 10 mhl HEPES- Tris (pH 7.4j, appropriate amounts of BBM vesicles with or without 1 nlM glucose, and 0.05-0.75 rM phlorizin. In all experiments the osrncplality of the final reactiorl mixture was maintained at 290 rnosmoli kg. The incubation was carried out at 25°C for variable lengths of tirne. The uptake was terminated by the addition of 4.0 m% ice-cold "stop solution" to the reaction tube. The composition of the stop solution was BO IT&! HEPES-Tris (pH 7.4). 500 ~Taaaf n-niannitol, tracer amounts of u-['~]rnannitc~l and 10 mV MgS04. The BBM vesicles were collected rapidly over a Millipore filter (HA025, pore size 0.45 pm]. Unless otherwise stated. the vesicles over the Millipore filter were washed with

"n this paper, the expressions "Na uptake by the vesicles'' and "'Na content of the vesicles" are used to denote the residual Na content of the vesicles after Millipare filtration and subsequent washing irrespective of the length of incubation.

a 4.0-nL wash solution which had a composition sinmilar to that o f thc stop solution, except that it did not contain 11-['~lmannitol. 'I'he MiBli- pore filter. together with the vesicles. was transfened to a counting vial. The 2 2 ~ a and % of the sample were assayed using a liquid scintiIlation spectrometer (LKB, model 12 15 Rackbeta) as described previously (Binda and Beck 1981). The % counts of the sample were used to correct for the 2 ' ~ a adhering to the vesicles and the MilBipore filter owing to insufficient washing (Hopfer and Ciroseclose I980; Sigrist-Nelson et al. 1975 j.

In a series of experiments the effect of plalorizin on glucose iaptake by BBM vesicles was examined. The raaethods employed for this uptake measmenlent were sirmailar to those described by us previously (Ilincia and Beck 1981). A11 uptake measurements were made in triplicate and all experiments were carried out inamediately after the isolation of the membranes (within 15 xninj. Unless otherwise stated, uptake is ex- pressed per milligrams protein, which was determined by the mcthod of Lowry et al. (1951). Results are presented as net uptake. This was calculated by subtracting the adherent " ~ a (nonspecific Na cc~intent) frona the total 2 2 ~ a uptake. Hn same experinaents the RBM vesicles were examined by electron microscopy, the method for which was similar to that described by us previously (Dinda and Beck 198 I ).

Group 11: ejfkct ofphlorizin orz "NU uptake bv ever1c.d sac-s o f h a r ~ ~ ~ t c ~ r jejer num

A 3-crn long piece of jejunurma. beginning frona the liganient of Treitz, was everted and divided into two equal segments, thc proximal and the distal. Two everted sacs were prepared, and each was filled with 0.25 mE MWBS. Paired experinmetits were performecf using these two sacs, of which one served as ccpntrol and thc other as experimental. In altersaate experiments, the proximal scgnaent served as the control and the distal segment as experimental; while in others, this arrange- ment was reversed. The control sac was preincukated for 85 nlin in a glucose- and Na-frec buffer (composition: 10 1miM HEPES-T'ris, pH 7.4, 300 11z1tI mannitol, 0.1 mM MgS04) and them incubated f i~ r 15 id, in KRBS containing 1 0 rnM glucose aamd tracer mounts of '"a and [%]inulin. The 2 2 ~ a served as the probe ~m-nolecule, while the j3~] inu l in was used for the co~aectiorm of 2%a content of the fluid adhering to the ~iaucosal surface. The experimental sac was preincubated and incubated slmilarly, except that the preincubation as well as the incubation solution for this sac contained 0.50 mill phlorizin. All incubation and preincubation teinperatures were 37°C. After thc incubation, time sac was taken directly on a filter paper (Whatman No. 1) and the rnucosal surface was blotted gently. The solution inside tlac sac was drained. taking care that the scrosal tluid did not contaminate the fluid adhering on the mucosal surface. The tissue from both ends, which were tied to rnake the sac, was discarded. The intestine was then opened along the nlesenteric line and the scrosal side was blotted. The tissue was divicted into two paits of approximately equal sizes. One part was dried at 105°C to constant weight to determine the dl-y-to-wet weight ratio. The other part was extracted for 48 h in 3.0 snL, water at 4°C as cisscribed by us previously (Dinda et al. 1972, 1975). An alicluot of this extract arad an aliquot of suitably diluted incubation sc~lutiola were assaycd for ['~linulin and 2 2 ~ a using a liquid scintillation spectrometer. -The uptake of ' ' ~ a per grlam of dry tissue was calculated and cnrrcctcd for the ' 2 ~ a content of the incubation solution adhering to the tissue. The methods for these calculaticms were similar to those desca-ibccl hy us previously (Dinda et al. 1972. 1997, 1975).

Results are expressed as the mean ? SE of the number of cxpcri- nlents indicated in the Results section. 'Ihe significance of rnearl diffkr- ences was determined by Student ' a t-test as applicable to pair' ccsmpari.- sons as dcscribed by Snedecor anti Cochran ( 1980). Bn some instances sorrelation and regression anaalyscs wrrc pet-fc>s~aaed iind the regrcssioils were compared by the method applicabls to Inore than two rcgrcssionx (Zar B 974).

Resuits Group I : efscce ofphlorizin on Na and glut-osr upauke Eqt..jqjunul

WBM ve,sirles Eight series s f cxperiments were carried (-jut using BBM

vesicles. The results of each serics are described below.

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

WA

ST

AT

E U

NIV

LIB

RA

RIE

S on

11/

13/1

4Fo

r pe

rson

al u

se o

nly.

R A P ~ D COMMUNICAT~ON 1 COMMUNICATION RAPIDE 58 1

7 -- -r - -- 4 0 tT0 8 0

TIME (mln)

PHL

PHL

PHL

FIG. 1. Time course of action of phlorizin (0.25,0.50, and 0.75 mM PHL) on Na content of BBM vesicles. Aliquots of vesicle suspension (vesicles suspended in 10 mM HEPES-Tris, pH 7.4, 200 mM man- nitol, 0.1 mM MgS04) were prewarmed for 10 rnin at 25OC before the incubations wereinitiated. he composition of the complete incubation mixture was 100 mM * * N ~ C I ; 100 mM mannitol; 0.1 mM MgS04; 1 rnM glucose; 10 mM HEPES-Tris (pH 7.4); appropriate amounts of BBM vesicles; and 0 (control), 0.25, 0.50, or 0.75 mM phlorizin. In each experiment, the Na content in the absence and in the presence of the three concentrations of phlorizin was measured in the same run using the same membrane preparation. For the measurement of Na content of the vesicles, a Millipore filtration technique was employed. The results are mean + SE of eight experiments. Values significantly higher than controls are indicated by asterisks.

Series l a The purpose of these experiments was to investigate the time

course of action of phlorizin on the Na content of BBM vesicles in the presence of 1 mM glucose. The incubations were carried out for 15 and 30 s and 1 , 2 , 3 ,5 , 10,30,60, and 90 min. Other details were the same as described under Methods. The effect of 0.25, 0.50, and 0.75 mM phlorizin was investigated. None of these phlorizin concentrations had any effect on the nonspecific Na content at any of the incubation periods employed. Conse- quently, the pattern of phlorizin-induced changes in total and net Na content was similar. Figure 1 shows that the net Na content of the control BBM vesicles increased progressively with the incubation time for the first 30 min, and between 30 and 90 min remained stable, suggesting that from 30 min the vesicles were in complete equilibrium with the medium. In the presence of 0.25,0.50, or 0.75 mM phlorizin in the incubation medium, the Na content of the vesicles was not different from that of the controls during the first 5 min, except that at 5 rnin there was a slight but significant increase in Na content of the vesicles incubated with 0.50 mM phlorizin. After this, however, the residual Na content of the phlorizin-treated vesicles was always lower than that of controls. This decreased Na content (Y) was dependent on the phlorizin concentration of the medium (X). After 90 min incubation, the correlation (r) and regression coefficient, i.e., slope (6) of this concentration-dependent de- crease in the net Na content of the vesicles were as follows: r = -0.80 ( p < 0.001) and b = -96 t lO(SE) (p < 0.001); n = 32.

Series 1 b Similar experiments were carried out to examine whether the

phlorizin-induced changes in Na content of vesicles in the ab- sence of glucose were similar to those described above in the presence of glucose. Accordingly, we studied the time course of

action of phlorizin under the same experimental conditions as described above, but with the omission of glucose from the reaction mixture. The results were comparable to those illus- trated in Fig. 1 and after 90 rnin incubation, the correlation and regression of the net Na content versus the concentration of phlorizin used were (r = -0.90 ( p < 0.001) and b = - 101 2 9(SE) ( p < 0.001), n = 32) also similar to those found in experiments illustrated in Fig. 1.

Series 2 Experiments were carried out to determine the magnitude of

the phlorizin-induced inhibition of glucose uptake under the conditions of the experiments of series 1. In the absence of phlorizin (controls), the peak of glucose uptake (overshoot) occurred at 30 s. At this time, the net glucose uptake by the control vesicles was 0.77 2 0.07 and by those incubated with 0.05,0.10,0.25,and0.50mMphlorizinwas0.43 *0.05,0.32 t 0.03,O. 18 2 0.02, and 0.09 )- 0.0 1 nmollmg protein (mean 2 SE, n = 16).

Series 3 These experiments were carried out to examine the effect of

phlorizin on Na content of BBM vesicles, after the vesicles were fully equilibrated in a Na-containing medium under control conditions. Aliquots of BBM vesicles were incubated in the absence of glucose as described for control vesicles in the experiments of series 1 b. After 80 rnin incubation, the reaction in four tubes was terminated and the Na content of the vesicles was measured. This provided the base-line values. The remain- ing tubes were divided into two batches: 0.05-mL control solu- tion (containing no phlorizin) was added to the tubes of the first batch and the same volume of solution containing phlorizin to the tubes of the second batch. After these additions, the incuba- tion of these tubes was continued for 5 s to 60 min. The two added solutions had a composition which was (except for the presence or absence of phlorizin) similar to each other, and except for the presence of 1 mM glucose, was also similar to the reaction medium to which they were added. The solutions added to the tubes of the second batch contained adequate phlorizin to give a concentration of 0.50 mM in the final reaction mixture. The effect of these additions on the Na content of the vesicles as a percentage of the corresponding control at each time period is shown in Fig. 2. As can be seen, after addition of phlorizin the residual Na content of the vesicles decreased progressively with time, and after 30 rnin these vesicles contained virtually no Na.

Series 4 The objective of these experiments was to investigate the

effect of increasing number of washings (following Millipore filtration) on the residual Na content of the vesicles incubated in the absence (control) and in the presence of phlorizin. Aliquots of BBM suspensions were incubated with the control solution or with solutions containing 0.25 or 0.50 mM phlorizin. After 90 min, the reaction was terminated and the content of the tubes was filtered by Millipore filtration. Filters from each group in duplicate or triplicate were washed 1, 2, 3, or 4 times using 4.0 mL of wash solution each time. Figure 3 shows that with the usual single wash, phlorizin caused a dose-dependent decrease in ,residual Na content of the vesicles (compare the first bar of panels A, B, and C). Repeated washing up to 4 times had no significant effect on the residual Na content of the control vesicles (compare 1-4 washes in panel A), but caused a progres- sive loss of Na from the vesicles incubated with phlorizin (compare 1-4 washes in panels B and C). The Na loss owing to repeated washing was also dependent on the phlorizin concen-

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

WA

ST

AT

E U

NIV

LIB

RA

RIE

S on

11/

13/1

4Fo

r pe

rson

al u

se o

nly.

582 CAN. J . PHYSIOL. PHARMACOL. VOL. 65, 1987

"50, ADDITION OF PHLORIZIN IEXTRAMSICULAR SOLUTION MAINTAINED AT 100 rnM No)

B +!

PHLORIZIN, 0.5 mM

2 0 35 6 5 TIME (min)

FIG. 2. Changes in 2 2 ~ a content of 22~a- loaded vesicles (i .e. , equili- brated in 100 mM 2 2 ~ a ~ 1 ) after incubation with a phlorizin-containing (0.5 rnM) sblution for different periods of time followed by Millipore filtration and washing. Vesicles were preincubated for 80 min in l ( jmM HEPES-Tris (pH 7.4) containing 100 mM 2 2 ~ a ~ l , 100 mM mannitol, and 0.1 mM MgS04. After this, the vesicles were exposed to a control (which was the same as the preincubation solution except for the presence of glucose which gave a final concentration of 1 mM) or to a phlorizin-containing solution (which, except for the presence of phlor- izin, had the same composition as that of the control solution). Other details were the same as described in legend of Fig. 1. Results are expressed as percent of controls and are mean + SE of nine experi- ments.

@) CONTROL

(B) 0.25 mM PHLORIZIN

(C) 0.50 rnM PHLORlZlN

NUMBER OF WASHES

FIG. 3. Effect of increasing numbers of washing (following Milli- pore filtration) on Na content of control and phlorizin-treated vesicles. Vesicles were incubated in the absence (control) and in the presence of 0.25 or 0.50 mM phlorizin under. the same conditions described in Fig. 1. After 90 min incubation, the vesicles were collected over Milli- pore filters and washed 1 , 2 , 3 , or 4 times using a 4.0-mL wash solution each time. Other details were the same as in Fig. I . Results are mean +- SE of 10 experiments.

tration of the medium. The slopes (b) of the regression of Na content of the vesicles versus the number of washes shown in panel A (control), B (0.25 mM phlorizin), and C (0.50 mM phlorizin) of Fig. 3 wcre all significant. Comparison of the slopes indicated that the slope was significantly higher ( p < 0.01) for the vesicles incubated with 0.25 or 0.50 mM phlorizin than that for the controls. The difference in slope between the two concentrations of phlorizin (panel B vs. C) approached significance ( p < 0.1 > 0.05).

Series 5

-e CONTROL * I -o- 0.lOmM PHLORIZIN

* T

INCUBATION TIME ( s

FIG. 4. Effect of preincubation with 0.1 mM phlorizin on Na uptake by the vesicles during subsequent incubation with glucose and Na. Aliquots of vesicle suspension (which was glucose and Na free) were preincubated for 10 min in the absence and in the presence of 0.1 rnM phlorizin. The vesicles were then incubated with a glucose- and Na- containing solution. The composition of the final incubation medium was 100 mM " N ~ C I , 100 mM mannitol, 0.1 mM MgS04, 1 mM glucose without (control) or with 0.1 mM phlorizin. Incubation times are shown in the abscissa and Na content of the vesicles in the ordinate. Results are mean 2 SE of 15 experiments. Values significantly differ- ent from the controls are indicated by asterisks. **, p < 0.01.

of phlorizin on 2 2 ~ a uptake during early incubation periods. Aliquots of BBM suspensions (i.e., vesicles suspended in 10 mM HEPES-Tris, pH 7.4, containing 200 mM mannitol and 0.1 mM MgS04) were preincubated for 10 min without (control) or with 0.1 mM phlorizin (experimental). The preincubated aliquots of BBM vesicles were then incubated for 10-90 s in a solution containing 1 mM glucose and 100 mM 2 2 ~ a ~ l without (control) or with 0.1 mM phlorizin (experimental). Figure 4 shows that at all times of incubation, Na uptake by the vesicles preincubated and incubated with 0.1 mM phlorizin was significantly higher (p < 0.01) than that of the controls. Similar observations were also made when the incubations were carried out in the absence of glucose (data not presented).

Series 6 These experiments were carried out to examine the effect of

phlorizin (0.1 mM) on Na uptake from incubation solutions containing various concentrations of Na. These experiments were similar to those described under series 5 , except that following 10 min preincubation the incubations were carried out for 8 s only, and that the Na concentration of the final incubation was 10, 20, or 50 mM. Figure 5 shows that Na uptake by the phlorizin-treated vesicles was significantly higher than that by the controls with all concentrations of Na in the incubation solution.

Series 7 In these experiments, we examined whether the increased Na

uptake in the presence of phlorizin was concentration depen- dent, and if so, how did it relate to the time of exposure of the

The purpose of these experiments was to investigate the effect vesicles to phlorizin. Two sets of experiments were performed.

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

WA

ST

AT

E U

NIV

LIB

RA

RIE

S on

11/

13/1

4Fo

r pe

rson

al u

se o

nly.

RAPID COMMUNICATION 1 COMMUNICATION RAPIDE

No CONCENTRATION FIG. 5. Effect of preincubation with 0. I mM phlorizin on Na uptake

by the vesicles during subsequent incubation with glucose and various concentrations of Na. These experiments were similar to those de- scribed in Fig.4, except that the ' 2 ~ a ~ l concentration of the final incubation medium was 10, 20, or 50 mM and that the concentration of mannitol was increased appropriately to adjust the osmolality. Incuba- tion periods were 8 s. Results are mean + SE of 20 experiments. Values significantly different from the controls are indicated by asterisks. ***, p < 0.001.

In both sets, the vesicles were preincubated and incubated as described for the experiments of series 5. In the first set, the preincubation was carried out without (control) or with O.1,0.2, 0.3, 0.4, or 0.50 mM phlorizin for 3 min, followed by incuba- tion with a glucose- and Na-containing solution for 30 s. Experi- ments of the second set were in all respects similar to those of the first set except that the length of the preincubation was increased to 15 min. Figure 6A demonstrates that after 3 rnin preincuba- tion, there was a progressive increase in Na uptake with the increase in phlorizin concentration of the medium. As compared with controls, all increases were statistically significant except for that caused by 0.1 mM phlorizin. In contrast, when the preincubation period was extended to 15 min, the residual Na content of vesicles exposed to 0.1 or 0 .2 mM concentration did not change, but of those exposed to 0.3, 0.4, or 0.50 mM phlorizin decreased significantly (p < 0.01 ) (Fig. 6B).

Series 8 These experiments were carried out to examine whether

phlorizin causes lysis or reduction in size of the vesicles. BBM vesicles were incubated in the absence (control) and in the presence of 0.75 mM phlorizin. After the incubation, the vesi- cles were processed for transmission electron microscopy. Ex-

3 man PREINCUBATION 15 min PRElNCUBATlON

FIG. 6. Effect of preincubation with 0 (control), 0.1, 0.2, 0.3, 0.4, and 0.5 mM phlorizin (PHL) for 3 rnin (A) and 15 rnin (B) on residual Na content during subsequent incubation with a glucose- and Na- containing solution. The preincubation and the incubation were carried out as described in Fig. 4. Results are mean -t SE of eight experiments. Values significantly different from the controls are indicated by aster- isks. * , p c0 .025 ; * * , p < 0.010; * * * , p < 0.005.

amination of the electron micrographs by two observers, who were not aware of the treatment, did not reveal any evidence of lysis or reduction in size.

Group 11: eflect of phlorizin on 2 2 ~ a uptake by everted jejunal sacs

Twenty paired experiments were carried out. As described in the Method section, 2 2 ~ a uptake in the absence of phlorizin was measured using one of these sacs (control) and in the presence of 0.50 mM phlorizin employing the other (experimental). The total 2 2 ~ a content of the control sacs was (mean +SE) 3.84 k 0.16 pmo1.g t i ssue1-15 s-'. Of this, 1.84 + 0.09 pmol consti- tuted 2 2 ~ a content of the adherent incubation solution and the remaining 2.0 5 0.13 pmol was the net 2 2 ~ a content (i.e., net 2 2 ~ a uptake). The corresponding values for the phlorizin- treated sacs were 4.72 + 0.30, 2.00 -+ 0.1 1, and 2.72 5 0.22, respectively. The difference between the two groups of sacs was significant for the total 2 2 ~ a content ( p < 0.025) and also for the net 2 2 ~ a content ( p < 0.05), but not for the 2 2 ~ a content of the adhering incubation solution.

Discussion To investigate whether phlorizin influences nonelectrogenic

Na transport across the intestine, in the experiments of group I we studied the effect of this agent on Na transport by BBM vesicles (as determined from Na content of the vesicles). BBM was used because (a) it is now well established that transport characteristics of BBM vesicles reflect the absorptive processes of the mucosal membrane of the intestine in vivo (Murer and Kinne 1980), and (b) phlorizin is known to interfere with Na- coupled glucose transport across the brush border membrane of the enterocyte. Na transport by the vesicles was measured using a Millipore filtration technique, which has been used widely in the literature for the measurement of uptake of various solutes by the BBM. In fact, except for the recently described voltage- sensitive dye technique (which measures electrogenic transport processes only by monitoring transmembrane potential differ- ence), Millipore filtration is the only technique available for transport studies using membrane vesicles (Stevens et al. 1984).

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

WA

ST

AT

E U

NIV

LIB

RA

RIE

S on

11/

13/1

4Fo

r pe

rson

al u

se o

nly.

584 CAN. J. PHYSIOL. PHARMACOL. VOL. 65, 1987

In the first series of experiments on BBM vesicles (group I), increase in permeability of the brush border membrane of the we studied the time course of action of 0 .25 ,0 .50 , and 0.75 mM enterocyte to Na. phlorizin on Na uptake. In these experiments we made three important observations, which appear to be consistent with a phlorizin-induced increased permeability of the vesicles to Na. First, none of the concentrations of phlorizin had any effect on Na content of the vesicles during the first 30-s incubation (Fig. l ) , when Na-coupled glucose uptake by the vesicles was depressed by this glycoside (series 2, group I). Therefore, in the presence of phlorizin the uptake of Na in excess of the amount that could be accounted for Na-coupled glucose transport was probably the result of an increased nonelectrogenic Na uptake. However, in our experiments there was no additional driving force that could stimulate one of the nonelectrogenic Na trans- port mechanisms described in the literature (e .g. , Na-Cl co- transport (Fan et al. 1983) and Na-H exchange (Cassano et al. 1984; Gunther and Wright 1983)). Thus, it appears the in- creased Na uptake (in relation to Na-coupled glucose transport) was probably the result of an enhanced passive diffusion of Na into these vesicles. This increased passive diffusion of Na appears to be independent of Na-coupled glucose transport, because the effects of phlorizin in the absence of glucose were similar to those observed in the presence of glucose. Secondly, at 5 min incubation, there was a slight but significant increase in Na content of the vesicles incubated with 0.50 mM phlorizin, but not in those incubated with the other two concentrations of this agent (Fig. 1). This transient increase in Na content in the presence of 0.50 mM phlorizin is consistent with an enhanced passive permeation of Na. It is possible that the other two concentrations of phlorizin also produced a similar transient increase in Na uptake, but after different periods of incubation, and that these periods did not coincide with the ones we em- ployed. Thirdly, between 30 and 90 min incubation, when the control vesicles maintained a stable Na content ( i .e . , in equilib- rium with the medium). the ~hlorizin-treated vesicles demon- strated a concentration-depen&t decrease in residual Na con- tent (Fig. 1). This was unexpected, because if the effect of phlorizin was to inhibit Na-dependent glucose transport only, it should have depressed Na content of the vesicles during the early incubation periods. In the latter periods of incubation, the Na content of the phlorizin-treated vesicles should have been similar to that of controls. This, however, did not occur. There- fore, one may wonder whether phlorizin caused a reduction in the size of the vesicles (similar to osmotic shrinkage) or lysis of some, but not all, vesicles. This, however, does not appear to be the situation, because our electron microscopic studies did not give any indication of such changes in the phlorizin-treated vesicles (series 8 , group I). Furthermore, phlorizin has been used extensively to characterize transport processes of intestinal BBM vesicles (Ferguson et al. 198 1; Lemaire et al. 1982; Toggenburger et al. 1978), and there is no evidence to suggest this glycoside (especially in concentrations used in the present study) causes reduction in size or lysis of vesicles. In view of this, we believe that between 30 and 9 0 min incubation the Na content of the phlorizin-treated vesicles was similar to that of the controls, but these vesicles became so leaky that they lost some of their intravesicular Na during Millipore filtration and washing. Consequently, the residual Na content of the phlor- izin-treated vesicles was lower than that of the controls. All subsequent experiments were designed to substantiate that phlorizin-treated vesicles lost their intravesicular Na during Millipore filtration and washing, and that phlorizin caused an

Evidence for loss of Na from phlorizin-treated vesicles during Millipore filtration and washing

In the next series of experiments, we examined whether the loss of intravesicular Na also occurred when Na-loaded vesicles (i .e. , equilibrated in 100 mM Na in the absence of phlorizin) were exposed to phlorizin before Millipore filtration and washing. After exposure to phlorizin, the vesicles were found to lose Na progressively with the time of exposure, and after 30 min the vesicles contained virtually no Na (Fig. 2). Theoretically this loss of Na could have occurred either during the incubation in the presence of phlorizin or during the Millipore filtration. If it did occur during the incubation, then the loss of Na had to be the result of active extrusion from the intravesicular fluid (because at the time of the addition of phlorizin the intra- and extra- vesicular fluids had similar Na concentrations). In these experi- -

ments, however, conditions necessary for active extrusion of Na were not present. Therefore, active extrusion of Na from the vesicles could not have taken place. Accordingly, it is likely the loss of intravesicular Na occurred during Millipore filtration and washing. If this is so, it could only happen as a result of a phlorizin-induced permeability of the vesicles to Na.

If the above hypothesis is correct, repeated washing, instead of a usual single wash, should cause a gradual decrease in Na content from the phlorizin-treated vesicles, but not from the controls. We examined whether this is the case (group I, series 4), and found that washing up to 4 times caused a progressive decrease in Na content of the vesicles (Fig. 3). These findings are consistent with a dose-dependent increase in leakiness of the membrane to Na. These experiments also demonstrate the in- adequacy of the Millipore filtration technique for accurate measurement of solute uptake by membrane vesicles when the permeability of the latter to the solute in question is increased.

Further evidence for increased permeubility of mucosul mem- brane to Na

The above findings suggest, but do not prove unequivocally, that phlorizin increases the permeability of the vesicles to Na. To provide direct evidence of increased passive diffusion, it is necessary to demonstrate that Na uptake by the vesicles is enhanced in the presence of phlorizin. Since the phlorizin- treated vesicles lose their Na during Millipore filtration and washing, we realized that by employing this technique it would not be possible to demonstrate the increased uptake quantitative- ly. The loss of Na from the vesicles, however, appears to depend not only on the phlorizin concentration of the medium (Figs. 1 and 3), but also on the time of exposure of the vesicles to this agent (Fig. 2). In view of this, if an appropriate phlorizin con- centration and an appropriate time of exposure are selected, even using the Millipore technique, it may be possible to dem- onstrate an increased uptake qualitatively, if not quantitatively. Indeed as shown in Fig. 4 , preincubation with 0.1 mM phlorizin in a Na- and glucose-free solution for 10 min, followed by incubation in a solution containing 100 mM Na and 1 mM glucose for 10, 15 ,30 ,60 , and 90 s caused a significant increase in Na uptake by the vesicles at all times of incubation. A similar increased uptake was also observed with other Na concentra- tions of the incubation solutions (Fig. 5) . It is to be noted that higher concentrations of phlorizin produce the same effect, if the preincubation or the incubation is carried out for a shorter period of time (see Fig. 6A). Thus, an increase in Na uptake can

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

WA

ST

AT

E U

NIV

LIB

RA

RIE

S on

11/

13/1

4Fo

r pe

rson

al u

se o

nly.

RAPID COMMUNICATION I COMMI:NICATION RAPIDE 585

be demonstmted with any concentration of phlorizin if the time transport by phlorizin analogs. Arch. Biochem. Biophys. 117: 248- of preincubation and thetirne of incubation-are adjusted appro- priately. On the other hand, the loss of Na from the vesicles during Milliptare filtration and washing may exceed the in- creased uptake when thc vesicles become too leaky owing to prolonged preincubation with a high concentration of phlorizin (Fig. 6B).

To ascertain the phlorizin-induced increased Na permeability of the BBM vesicles was not related to the methodology em- ployed (vesicle preparation, uptake measurement, etc .), we also investigated the effect of phlorizin on Na uptake by everted jejunal sacs. These experiments also showed a higher Na uptake by the phlorizin-treated sacs than that by the controls (Group 11). These findings on everted sacs and those on BBM vesiclcs suggest phlorizin iricreases the pernleability of mucosal mem- brane to Na. Such an increase in Na permeation and a concom- itant inhibition of glucose uptake should cause an increased ratio of Na:glucose transport, without affecting the stoichiometry of carrier-mediated cotransport csf Na and glucose. The intestinal BBM is known to contain a P-glucosidase which catalyses the hydrolysis of phlorizin to phloretin and glucose (Diedrich 1968; hlaIathi and Crane 1969). Phloretin is also an inhibitor of intestinal sugar transport, but its effect is 100 times less than that of phlorizin, and the inhibition is uncompetitive (Estep and Goldner 1974). The present study does not differentiate whether the increased permeability of the BBM to Ka was caused by phlorizin or by its aglycon, phloretin.

Although phlorizin is known to inhibit intestinal sugar trans- port by competing with sugar molecules for a common binding site on the mucosal membrane (Alvarado and Crane 1962; Diedrich 19661, there is no report in which the effect of this agent on intestinal Na transport has been investigated. The obsenation of the present study that phlorizin increases Na permeation is likely to cause a rapid dissipation of Na gradierlt across the brush border membrane. Since the Na gradient is the driving force for sugar transport (Crane 1968; Schultz and Curran 1969), its fall or abolition by phlorizin could be a second rncchanism by which this agent inhibits intestinal absorption of sugars.

Acknowledgments We wish to thank Dr. @. P. Morris for his assistance in

electron microscopic studies. This work was supported by grant No. MT.4254 from the Medical Research Council of Canada.

A~,VARADO, F. 1967. Hypothesis for the interaction of phlorizin and phloretin with membrane carriers for sugar. Biochim. Biophys. Acta. 135: 483-495.

ALVARADO. F., and CRANE, K. K. 1962. Phlori~in as a connpetitive inhibitor of the active transport of sugars by hamster small intestine, in virro. Biochim. Biophys. Acta. 56: 170- 172.

1963. Studfes on the rnechanisni of intestinal absorption of sugars. VII. Phenylglycosidc transport and its possible relationship to phlorizin inhibitiisn of the active transport of sugars by the small intestine. Biochi~n. Biophys. Acta. 93: 116-135.

CASSANO. G. , STIEGER, B., and M ~ R ~ R . H. 1984. Na/H- and Cll OH-exchange in rat je.junal and rat proximal tubular brush border membrane vesicles. Pfluegers Arch. 400: 309-317.

CRANE, R. K . 1968. Absorption of sugars. Irz Handbook of phpsiolo- gy. Sect.6. Alimentary canal. Vol.111. Intestinal absorption. Edited by C. F. Code and W. Hcidel. American Physiological Society, Washington, DC. pp. 1323- 135 1.

~ ~ I E I ~ R I C H , D. F. 1966. 6:ompetitive inhibition of intestinal glucosc

256. 1968. Is phloretin thc sugar transport inhibitor in intestine?

Arch. Biochem. Biophys. 127: 803-812. DINDA, P. K.. and BECK, 1. T. 198 1. Ethanol-induced inhibition of

glucose transport across the isolated brush-border membrane of hamsterjejunum. Dig. Dis. Sci. 26: 23-32.

1982. Effect of ethanol on pcptidases of hamster jejunal brush- border rnembrane. Am. J . Physiol. 242: G442-G447.

D~NDA, P. K. , BECK. M., and BECK, I. T. 1972. Effect of changes in the osmolality of the lurninal fluid on intracellular concentration of solutes in the hamster jejunum. Can. J . Physiol. Pharmacol. 50: 72-82.

1975. Isoosmotic transport of fluid across the hamster small intestine in the presence of phloriziim-induced inhibition of sugar transport. Can. J. Physiol. Pharmacol. 53: 375-382.

1977. Sortie observations on the determination of extracellular fluid volunie of jejunal tissue using [.3~]inulin and [14~] inu l in . Calm. J . Physiol. Phannacol. 55: 389-393.

DINDA, P. K . , BECK, I. T . , BECK, M., and MCELI~IC;OTT, T. F. 1975. The effect of ethanol on sodiurn dependent glucose transport in the small intestine. Gastroenterology, 68: 15 17- 1526.

L ~ I N D A , 9 . K. , HURST, R. Q . , and BECK, I . T. 1979. Effect of ethanol on disaccharidases of hamster jejunal brush border membrane. Am. J . Physiol. 237: E68-E76.

ESTEP, J . A., and GOIDNER, .4. M. 1974. Effect of phlorizin on galactose influx in rabbit intestine. Biochim. Biophys. Acta, 367: 371-374.

FAN, C. C., FAUST, R. G. , and PCIWEI~I~, D. W. 1983. Coupled sodium-chloride transport by rabbit ileal brush-border membrane vesicles. Am. J . Physiol. 244: G375-G385.

FERGUSON, D. R., MATTHEWS, E. K . , O'CONNOK, M. D. L.. and S c ~ w z , A. D. 1981. The intc~action of phenformin and phlorizin with brush border membrane vesicles, phospholipid liposomes, and phospholipid liposonles containing brush border membrane protein. Biochem. Phar~nacol. 30: 161 3- 16 19.

GUNTHER, R. D., and WRIGHT, E. M. 1983. iVat, Lic , and C1. transport by brush border membranes from rabbit jejunum. J . Menibr. Biol. 74: 85-94.

HOPFER, U . , and GROSECI,OSE, R. 1980. The mechanism of ~ a ' -

dependent D-glucose transport. J . Biol. Chem. 255: 4453-4462. HOPFER, bj . , NEI,SON. K . , PERRCITTC), J . , and ISSELBACHEK. K. J .

1973. Glucose transport in isolated brush border membrane from rat small intestine. J. Biol. Chem. 248: 25-32.

KAUNITZ, J . D., and WRIGHT, E. M. 1984. Kinetics of sodium D- glucose cotransport in bovine intestinal brush border vesicles. J . Membr. Biol. 79: 41 -5 1.

KAUNITZ, J . D., GUNI'HER, R., and \VR~GHT, E. M. 8982. Involve- ment of multiple sodium ions in intestinal D-glucose transport. Proc. Natl. Acad. Sci. U.S.A. 79: 2315-2318.

KIMMICH, G. A., and RANDLES, J . 1980. Evidence for an intestinal ~a + :sugar transport coupling stoichiometry of 2.0. Biochirn. Bio- phys. Acta, 596: 439-444.

LEMAIRE, J . , MAESTRAC'CI, D., LAPRADE, R.. and SAUVE, R. 1982. Mechanism of ncomycin stirnulation of D-glucose uptake in rabbit intestinal brush border membrane. Blochim. Biophys. Acta, 686: 119-129.

LOWRY, 0. H . , ROSEBRCIUGH, N. J . , FARR, A. L., and RANDAIL, K. J . 195 1. Protein measurement with the Folin phenol reagent. J . Biol. Chem. 193: 265-275.

h/l~n.~.ricnl, P., and CRANE, R. K. 1969. Phlorizin hydrolase: a P- glucosidase of hamster intestinal brush bordcr membrane. Biocliim. Biophys. Acta, 173: 245-256.

MURER, H., and KINNE, K. 1980. The use of isolated membrane vesicles to study epithelial transport processes. J . Membr. Biol. 55: 81-95.

ScHur,'rz, S. G . , and CUKKAN, P. F. 1969. The role of sodium in

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

WA

ST

AT

E U

NIV

LIB

RA

RIE

S on

11/

13/1

4Fo

r pe

rson

al u

se o

nly.

586 CAN. J . PHYSHOL. PHARMACOL. VOI,. 65, I987

non-electrolyte transport across atminiaa cell membranes. Physiolo- TO(>C~ENRUW(~ER, G. , KESSLER. M., ROTHSTEIN, A., SEMENZA, G . , gist. 12: 437-452. and TANNENRAUM, C. 1978. Si~niIarity in effects of ~ a ' gradients

SIGMIST-NELSON, K. MUREK, H. , and HQPFER, U . 1975. Active ala- and mernbrane potentials on D-glucose transport by, and phlorizin nine transport in isolated B~rush border membranes. J . Biol. Chen~. binding to, vesicles derived fronn brush borders of rabbit intestinal 250: 5674-5680. mucosal cells. J. Menmbr. Biol. 40: 269-290.

SNEDECOR, G. Mr., and COCHRAN, W. G. 1980. Statistical methods. ZAR, J. M. 1974. Biostatistical analysis. Prentice-Hall. Inc., Englc- 7th ed. Iowa State Ul~iversity Press, Amcs, HA. wood Cliffs, N3. pp. 228-25 I .

STEVENS. B. R. , KAUNITZ, J . , and WRIC~HT, E. M. 1984. Intestinal transpofl of amino acids and sugars: advances using membrane vesicles. Annu. Rev. Physiol. 46: 417-433.

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

WA

ST

AT

E U

NIV

LIB

RA

RIE

S on

11/

13/1

4Fo

r pe

rson

al u

se o

nly.