103

J. Physiol. (1977), 265, pp. 103-118 With 6 text-figure8 Printed in Great Britain

EFFECTS OF CYTOCHALASIN B AND DIMETHYLSULPHOXIDE ON ISOSMOTIC FLUID TRANSPORT BY RABBIT GALL-BLADDER IN VITRO

BY 0. FREDERIKSEN AND P. P. LEYSSAC From the University Institute for Experimental Medicine, 71, Norre Alle', Dk-2100, Copenhagen, Denmark

(Received 12 May 1976) SUMMARY

1. Net fluid transport rate, transepithelial ohmic resistance and potential difference (p.d.), and unidirectional fluxes of Na+ were measured in rabbit gall-bladder preparations in vitro exposed on both sides to Ringer solutions of identical electrolyte composition. 2. Bilateral application of 1 % dimethylsulphoxide (DMSO), the solvent for cytochalasin B, rapidly and reversibly depressed net fluid transport rate by 15 % and increased the lumen positive p.d. by 1*5-2*0 mV. Resistance did not change significantly. These effects of DMSO were shown to be non-specific osmotic effects. 3. Cytochalasin B (10-5 M) applied bilaterally caused: (a) a progressive inhibition of net fluid transfer rate to 40-50 % of its control value within 60 min; the effect was partly reversible within 60 min and independent of the substrates glucose, glutamate and pyruvate; (b) a progressive depression of the mucosal-to-serosal Na+ flux within the first 30 min with no further change in the flux during the following 30 min of exposure to cytochalasin B; the effect was partly reversible within 70 min; (c) a rapid but moderate increase in the passive serosal-to-mucosal Na+ flux, which continued to increase gradually during the entire 60 min period of exposure to cytochalasin B; the effect was completely reversible within 70 min; (d) a prompt drop in ohmic resistance (30 %) and p.d. (40 %) with no further changes in these parameters during the following 60 min of exposure to cytochalasin B. The effect on resistance was partly reversible within 90 min; the effect on p.d. was completely reversible within 30 min. 4. The results are interpreted to indicate an early inhibitory action of cytochalasin B on the active transcellular pump mechanism and to

104 0. FREDERIKSEN AND P. P. LEYSSAC suggest a cytochalasin B-mediated progressive increase in cell membrane permeability to sodium resulting ultimately in a highly leaky epithelium. The results are compatible with the concept that a mechanochemical process is involved in isosmotic transcellular transport of fluid across lowresistance epithelia. INTRODUCTION

According to the conventional view isosmotic electrolyte and water transfer across low-resistance epithelia is transcellular and driven by active ion transport. Various models have been proposed in accordance with this general concept. Thus, the 'standing gradient-model' (Diamond & Bossert, 1967) postulates active sodium transport across the basolateral cell membrane into the lateral intercellular space creating a local gradient of osmotic activity, which may account for the passive transfer of water from the cell to the lateral space. A serious criticism of this model is the energy problem, since it would require an efficiency of oxygen consumption for sodium transport of at least 80-90 %, as emphasized by Bojesen & Leyssac (1965). This problem is overcome to some extent by the model proposed by Fr6mter, Rumrich & Ullrich (1973) for the renal proximal tubule, in which one third of the total sodium transport is driven by an active Na+ pump and one third is secondary to active transmembrane HCO3 transport. These two ion-pumps create an osmotic activity in the lateral intercellular space, which is the force responsible for paracellular water transfer across the junctional complex. The last third of the sodium transport is passive, due to solvent drag paracellularly. Several pieces of evidence are difficult to reconcile with the general concept underlying these models and their modifications. Thus, it has been shown in studies on isosmotic fluid transport by rabbit gall-bladder in vitro that marked changes in net sodium transport can occur acutely without changes in water transfer when sodium concentration (and osmolality) is changed abruptly and identically in the media bathing the serosal as well as the mucosal side; and suprabasal energy consumption was strictly correlated to fluid volume (water) transport rate independent of the actual amounts of sodium chloride transferred (Frederiksen & Leyssac, 1969). Cell volume regulation by kidney cortical cells depends on an ethacrynic acidsensitive pump distinct from the ouabain-sensitive Na+-K+ pump, and evidence has been provided suggesting that cell volume regulation involves a mechanochemical process (Kleinzeller & Knotkova, 1964). Similarly, transepithelial isosmotic solute and water transfer involves an ethacrynic acid-sensitive pump mechanism distinct from the ouabainsensitive Na+-K+ pump (Leyssac & Frederiksen, 1974a). For these reasons, and on the basis of recent structural evidence, Leyssac &

CYTOCHALASIN B ISOSMOTIC FLUID TRANSPORT 105 Frederiksen (1974a) proposed a mechanochemical pump mechanism for transcellular isosmotic fluid transport. The structural basis is, firstly, the presence in brush-border and terminal web of proximal tubule cells and intestinal epithelium of organized and polarized actin-like filaments (heavy meromyosin-reacting) and other muscle proteins including myosin, tropomyosin and c-actinin (Z-band protein) (Rostgaard & Thuneberg, 1972; Mooseker & Tilney, 1975). Motility of microvilli in isolated brush-borders has been shown directly (Thuneberg & Rostgaard, 1969) and its calcium dependency demonstrated (Mooseker, 1974). Secondly, a system of so-called 'scalloped sacs' and distended endoplasmic reticulum has been disclosed in the transporting outermost living cell layer of the frog skin epithelium by applying a modest hydrostatic pressure to the inside of the skin. The number of these 'vacuoles' correlated linearly with the short-circuit current suggesting that this organelle is directly involved in sodium transport (Vouete, M0llgard & Ussing, 1975). A similar sacculotubular organelle, extending from the microvilli crypts to the lateral cell membrane, along which it distends, is highly developed in the plexus chorioideus, an isosmotic transporting epithelium (M0llghrd & Saunders, 1975). Thus, it constitutes a likely pathway for transcellular transfer of isosmotic fluid volumes propelled by contractile mechanical pump activity. The present study was designed to test the prediction that substances such as cytochalasins, which are known to interfere with cell motility (Wessels, Spooner, Ash, Bradley, Luduena, Taylor, Wrenn & Yamada, 1971; Carter, 1972), among other effects, should inhibit net fluid transfer rate if the pump mechanism involves a mechanochemical element. The results confirmed this prediction. METHODS

Gall-bladders of white female rabbits weighing 2-5-3-0 kg were removed and prepared for gravimetric measurement of net fluid transport rate as previously described (Diamond, 1964; Frederiksen & Leyssac, 1969). The composition of the control Ringer solution (in m-mole/l.) was: Na+, 114-7; K+, 7 0; Ca2+, 2-0; Mg2+, 1-2; CC, 102-0; HC03, 17-5; S042-, 1-2; H2P042-, 1-2; monoglutamate, 5 0; glucose, 11.0. The pH was adjusted to 7 3-7 4 by equilibration with 96% 02 and 4% CO2 at 370 C. All experiments were carried out at 370 C and the bladders were bathed both inside and outside with the same Ringer solution, unless otherwise stated in the text. For one of the cytochalasin B series, sodium pyruvate was added to the control Ringer to give a final concentration of 2-5 mm. For another series a Ringer solution was prepared without addition of glutamate and glucose. Cytochalasin B was dissolved in freshly distilled dimethylsulphoxide (DMSO); a stock solution containing cytochalasin B, 1-0 mg/ml. DMSO was prepared and kept in the refrigerator until use. The stock solution was added to the Ringer solutions

106

0. FREDERIKSEN AND P. P. LEYSSAC

(inside as well as outside medium) to give a final concentration of cytochalasin B of 5 jg/ml. and 0-5 % DMSO. For the cytochalasin series, D.MSO was added to the Ringer solutions used in the control periods, giving a final concentration of 0-5%. Freshly distilled DMSO was always used. When using the gall-bladder sac preparation for measurements of net fluid transport the protocol was the same as that described in a previous paper (Leyssac, Bukhave & Frederiksen, 1974). The changes in net transport rate were not corrected for the spontaneous decrease in transport rate with time observed in the previous series (Leyssac et al. 1974). Unless direct records from an individual experiment are presented, the results are expressed as the percentage of control net transfer rate and are given as means + S.E. of mean. MeasurementM of electrical potential and ohmic resistance. Transepithelial potential difference (p.d.) and ohmic resistance were measured across gall-bladders cut open and mounted in an Ussing chamber as sheets separating two half-chambers (10-0 cm3) with an exposed area of 0-9 cm2, as previously described in detail (Leyssac et al. 1974). DMSO was added either to the mucosal, serosal, or both media in microlitre volumes to give a final concentration of 1-0%, except for the cytochalasin series, in which mucosal as well as serosal control media contained 0-5 % DMSO; when changing from control to experimental solutions both half-chambers were emptied and the solutions rapidly replaced by otherwise identical Ringer solutions containing cytochalasin B (5 ,sg/ml.) and 0-5 % DMSO. Flux mea8urement8. In preliminary flux studies, the gall-bladder sac preparation was used as described in detail previously (Leyssac et al. 1974). Briefly, net fluid transfer rate was measured under control conditions (including 0-5 % DMSO) until a steady level had been obtained. The bladder was then emptied, re-filled with control Ringer containing 22Na 2-4 /sc/ml. and weighed. After flushing the outside of the bladder sac with non-radioactive Ringer it was immediately transferred to a beaker containing 25-0ml. control Ringer (+0-5% DMSO). Samples of 100l #. outside (serosal) solution were obtained every 30 sec during the flux period for the counting of radioactivity. At the end of the measurement the gall-bladder was weighed and emptied. The specific activities both of the solution originally instilled in the bladder and of that re-aspirated at the end of the period were determined for calculation of the mean specific activity. The bladder was then washed, re-filled with and bathed in non-radioactive control Ringer for continued measurement of net fluid transport in order to ascertain that a significant change had not occurred. The bladder was subsequently emptied, re-filled with and transferred to the test solution containing cytochalasin B (5 ,ug/ml.) but otherwise identical in composition to that used in the control period. After measurements of net fluid transport in the presence of cytochalasin B for various periods of time, as stated in the text, the flux measurement was repeated (22Na added to the cytochalasin B-containing Ringer). Mucosal-toserosal 22Na flux was obtained by plotting the sample radioactivity versus time; the flux rate was taken as the slope of the last linear part of the curve and the mucosalto-serosal sodium flux (/zequiv/hr) was calculated from the flux of radioactivity, the mean specific activity and the mean volume of the serosal (outside) bathing medium. Net sodium flux was calculated from the measured net fluid transport rate and the estimated sodium concentration of the absorbate, assuming fluid absorption isosmotic with the luminal solution. The unidirectional seromal-to-mucosal flux was calculated as the difference between the mucosal-to-serosal sodium flux and the net sodium flux. When 22Na fluxes were measured across the gall-bladder wall mounted as a sheet in an Ussing chamber, as described above for measurements of p.d. and the resistance

CYTOCHALASIN B ISOSMOTIC FLUID TRANSPORT

107

the following procedure was used. The transepithelial resistance and p.d. were recorded throughout the entire experiment. When stable values were obtained, 20 /tc of carrier-free 22Na was added to either the serosal or the mucosal control bathing medium (including 0-5% DMSO) giving a final radioactivity of 2 ,uc/ml. Samples (250 ju) were then collected from the opposite bathing medium and immediately replaced by 250 #1. non-radioactive Ringer. Samples were obtained every 4 min for the counting of radioactivity throughout a 45 min control period. The half-chambers were then emptied and re-filled with otherwise identical solutions containing cytochalasin B, 5 jug/ml. (+ 0 5% DMSO). An aliquot of 22Na (20 juc) was added to the same side as in the control period and samples were again collected from the opposite side at 4 min intervals for 60 min. Finally the flux measurement was repeated in a recovery period of about 90 min with the bladder wall bathed with control Ringer. Separate gall-bladders were used for measurement of fluxes in each direction. The unidirectional 22Na flux was obtained from the slope of the increase in radioactivity with time. Linearity of the slope, i.e. steady-state flux, was obtained within a few minutes. The sodium flux (n-equiv x min' x cm-2) was calculated from the 22Na flux, the specific activity on the 'hot' side, the volume of the bathing medium and the exposed area of the bladder wall. No corrections for back-flux of 22Na were made, since the ratio of radioactivity on the two sides generally did not exceed 1:100. Samples were counted in a y-well scintillation counter (Selektronik, Copenhagen) to a significance level better than 2 %. 22Na was obtained as carrier-free 22NaCl (Amersham, Bucks) and cytochalasin B was a generous gift from S. B. Carter, Imperial Chemical Industries Ltd, Alderley Park, Cheshire). RESULTS

Effects of DMSO Dimethylsulphoxide was used as solvent for the present test substance, cytochalasin B. In order first to test the possible effects of this solvent on the in vitro fluid absorption rate and the electrical parameters, the following control experiments were carried out. In pilot experiments it was observed that 0-5 % DMSO applied bilaterally, i.e. a concentration equal to that used for testing cytochalasin B effects, changed the electrical p.d. moderately without any significant change in net fluid transport rate. In order to investigate this effect in further detail the concentration of DMSO was increased to 1 %. Effect on net fluid transfer rate. Addition of DMSO bilaterally (serosal as well as mucosal side) to the control Ringer solution, giving a final concentration of 1 % DMSO, caused an immediate 15-20 % reduction in net fluid transport rate which remained stable at this new level in the continued presence of DMSO for 80 min. The effect was completely and immediately reversible, as seen from Fig. 1, suggesting a passive, possibly osmotic, effect of DMSO. The osmotic activity of 1 % DMSO equals about 140 m-osmole/kg. In order to test this suggestion further 1 % DMSO was added unilaterally first to the medium bathing the mucosal side and subsequently to that

108 0. FREDERICKSEN AND P. P. LEYSSAC bathing the serosal side. As seen from the record of a representative experiment (Fig. 2), DMSO on the mucosal side depressed net fluid absorption rate markedly. The rate of transport rapidly returned to the control value upon return to control medium. Subsequent application of 1 % DMSO to the serosal medium immediately and reversibly augmented net fluid transfer rate, consistent with an osmotic effect. 1 % DMSO bilaterally

120 ,o I-

0

60

n 40 20

.

,

I

I

,

60 120 Time (min)

0

180

Fig. 1. Effect of 1 % dimethylsulphoxide (DMSO) added to both mucosal and serosal medium on net fluid absorption rate relative to the steadystate control value. Means + s.E. of mean (n = 4). Mucosal

Serosal 1 %DMSO

1 %DMSO

25 C

E- 20 E- A -* 10 15 __ C

L.

Co

I

14; O~~~~~~~~I

II

l1

30

60

.

90

120

180 150 Time (min)

210

240

270

Fig. 2. Record of an experiment showing the effects on absolute net fluid transfer rate following unilateral addition and subsequent removal of 1 % DMSO to the mucosal side and thereafter to the serosal side.

CYTOCHALASIN B ISOSMOTIC FLUID TRANSPORT 109 Effect on transepithelial electrical p.d. and ohmic resistance. Data from a typical experiment are given in Fig. 3. It is seen that the application of 1 % DMSO unilaterally to the mucosal side caused an immediate increase in the lumen positive p.d. from 3 to 8 mV accompanied by a very slight decrease in transmural resistance. The changes were rapidly reversible upon return to the control medium. Conversely, when 1 % DMSO was Mucosal Serosal 1 % DMSO 1 % DMSO

Mucosal+serosal 1 % DMSO

80 60-

~-40 _I !

~~I

X20

i

I

I

8

E 6

;2

30

60

a

90

t'>I

120 150 Time (min)

180

210

240

Fig. 3. Records of an experiment showing the effects on ohmic resistance, RT (open circles) and transepithelial potential difference, Vfm-Vir (filled circles) following addition of 1 % DMSO first to the mucosal medium, subsequently to the serosal medium, and finally to both sides simultaneously.

applied unilaterally to the serosal side, the p.d. decreased from 3 to 1 mV while resistance did not change significantly. Finally, it appears from Fig. 3 that the recorded changes in p.d. following simultaneous addition of DMSO to both sides are consistent with a summation of the two opposite effects obtained by unilateral application of the solvent to the mucosal and serosal side, respectively; after a transient marked increase (to 8 mV) the p.d. stabilized within 10 min at 5 mV (lumen positive), a value 1-5-2-0 mV higher than that recorded in the control medium. Resistance did not change significantly. Effect of cytochala8in B Effect on isosmotic net fluid transfer rate. The inhibitory effect on fluid transport of cytochalasin B, 5 ,tg/ml. (10-5 M), applied to both serosal and

0. FREDERIKSEN AND P. P. LE YSSAC mucosal sides of gall-bladders following a control incubation period in otherwise identical media containing 0-5 % DMSO without cytochalasin B is shown in Fig. 4. Addition of cytochalasin B to control Ringer solution (containing glucose and glutamate, filled circles) caused an immediate inhibition of net fluid transport rate, which progressively fell to 50 % of the control value within 110

160

Control

Cytochalasin B I

140 In

120 0 IC

0 %I

100 _

0

1-

0 oU

0. 0.

80 F

C

-

z5

60 _

._

z

40 _

_+

AlI

I3 201-

0

-

I

I

60 30 Time (min)

I

I

90

Il

120

Fig. 4. Effect of cytochalasin B (5,ug/ml. in both mucosal and serosal medium) on net fluid absorption rate relative to the preceding steady-state control value. Filled circles represent data from six gall-bladders bathed in control Ringer (containing glucose and glutamate); the final steadystate value was obtained after 160-200 min of exposure to cytochalasin B. Filled triangles are data from six gall-bladders bathed in Ringer solutions containing glucose, glutamate and pyruvate. Open circles represent data from six gall-bladders pre-incubated in control Ringer until stable absorption rates were obtained (within 60-80 min); glucose and glutamate were then removed and a new but lower steady-state value was obtained defined as 100 %); subsequently cytochalasin B was added in the continued absence of substrates. The arrows indicate the time of removal of cytochalasin B from the media. Means ± S.E. of mean.

CYTOCHALASIN B ISOSMOTIC FLUID TRANSPORT 111 60 min, reaching an almost stable value equal to 30-40 % of the control value after exposure to cytochalasin B for more than 2 hr. Additional experiments (not given in Fig. 4) showed an inhibition of about 90 % after exposure to cytochalasin B, 10 jug/ml. for 2 hr or more, while a cytochalasin B concentration of 1 jug/ml. (2 x 10-6 M) had hardly any inhibitory effect on fluid transport. Addition of pyruvate (2.5 mM) to the control Ringer solution (filled triangles, Fig. 4) did not significantly influence the inhibitory effect of cytochalasin B, 5 jtg/ml. Removal of the substrates, glutamate and glucose, from the medium in the control period (open circles, Fig. 4) reduced the transport capacity from an average value of about 135 % down to the relative value designated 100 %, representing the new steady-state level obtained in the absence of substrates. Subsequent addition of cytochalasin B (5 jug/ml.) in the continued absence of substrates caused a further inhibition of net fluid transport to about 40 % within 60 min. As is apparent from Fig. 4, the relative depression of net fluid transfer rate by cytochalasin B was not significantly influenced by the substrates glucose, glutamate or pyruvate. Further, the cytochalasin B effect was partly reversible, as seen from the slow increase in net trasport rate following removal of cytochalasin B (marked with arrows, Fig. 4) from both substrate-free and substrate-containing media. Effects on unidirectional fluxes of Na+. In preliminary experiments (mentioned in the discussion to the paper presented at the Sixth Alfred Benzon Symposium; Leyssac & Frederiksen (1974b)) the effects of cytochalasin B on the unidirectional Na+ fluxes were studied on the gall-bladder sac preparation. The results indicated that exposure to cytochalasin B (5 jug/ ml.) bilaterally for 120 min or more caused an increase in both the directly measured mucosal-to-serosal Na+ flux and in the calculated serosal-tomucosal Na+ flux, the latter effect being more pronounced than the former, thus accounting for the measured 60 % inhibition of net transfer rate. These effects seem to suggest an increase in the permeability of the epithelium. However, in two experiments mucosal-to-serosal Na+ flux had decreased when measured after exposure to cytochalasin B for only 30 min, suggesting that an early cytochalasin B effect might be an inhibition of the active transport accompanied by a slight increase in the passive serosal-to-mucosal flux. It was, therefore, decided to extend these observations by measuring the unidirectional fluxes of sodium across the gallbladder wall mounted as a membrane-sheet in an Ussing chamber. This preparation allows both undirectional fluxes to be measured directly and continuously during the control period and after the addition of cytochalasin B to both sides. In addition the p.d. and resistance can be recorded simultaneously. The results of the flux measurements are given in Fig. 5. In all three

.~

112 0. FREDERIKSEN AND P. P. LEYSSAC experiments it appears that the mucosal-to-serosal Na+ flux (including the active component of the transport) was consistently depressed within the first 30 min to a value about 30 % less than the control level. It then remained stable for the following 30 min. This effect was partly reversible. In three separate experiments, the passive serosal-to-mucosal Na+ flux continued to increase moderately during the entire 60 min period of exposure to cytochalasin B. This effect was almost completely reversible within 70 min. Cytochalasin B

(5 /Ig/ml) 800

-

-

C4

2

.~400

ii|

U~

0*~~~~

z4

200

0'

-3

06000

90

1005

-a--

-30

0

30

90 60 Time (min)

120

150

Fig. 5. Effects of cytochalasin B (5/cg/ml. in both mucosal and serosal medium) on the unidirectional fluxes of Na+ (JN.+). The data represent the absolute values recorded in six separate experiments (three for mucosal-to-serosal (M -+ S) and three for serosal-to-mucosal (S -+ M) flux measurements.

Thus, cytochalasin B (5 ,ug/ml.) depressed the flux ratio markedly. Net sodium flux, calculated from these data, was inhibited about 70 % within 60 min, in fair agreement with the 50-60 % inhibition of net fluid transport measured directly in six separate experiments utilizing the sac preparation (cf. Fig. 4). Effects on transepithelial electrical p.d. and resistance. Cytochalasin B (5,ug/ml.) resulted in a prompt 30 % reduction of the resistance accompanied by an equally rapid drop in p.d. from 5 mV (in the presence of 0.5 % DMSO) to 3 mV (lumen positive). No further change occurred in either p.d. or resistance during the following 60 min exposure to the drug.

CYTOCHALASIN B ISOSMOTIC FLUID TRANSPORT 113 After re-introduction of control media, the p.d. returned to its control value within 30 min while the effect of cytochalasin B on resistance was only partly and slowly reversible within the 90 min recovery period. Cytochalasin B

(5 pg/ml.)

~~40 E 30 x

20 1-

cc 10

0 -

30

0

30

60

90

1

1

120

150

120

150

6 I-,

E

4 E

20

1

-30

0

30

60 Time (min)

90

Fig. 6. Effects of cytochalasin B (5 #ag/ml. in both mucosal and serosal medium) on ohmic resistance, RT (open circles) and transepithelial potential difference, ?m - 0I. (filled circles). Means + S.E. of mean (n = 8). DISCUSSION

The results obtained with 0 5-1 0 % DMSO make it likely that DMSO has no effect on net isosmotic fluid transport other than a non-specific osmotic effect. Thus, increasing the osmolality significantly on the mucosal side by addition of a poorly permeable solute should increase the passive serosal-to-mucosal water flux, thereby reducing net water flux. Since the permeability of the gall-bladder epithelium is cation-selective (Wright & Diamond, 1968; Barry, Diamond & Wright, 1971), this passive water flux would create a lumen positive streaming potential, in agreement with the present data with 1 % DMSO (see Fig. 3). Conversely, increasing serosal medium osmolality should cause the opposite effect, as also observed (Fig. 3). Because in vitro preparations of rabbit gall-bladder with identical solutions bathing both sides maintain absorption of a fluid approximately

114 0. FREDERIKSEN AND P. P. LE YSSAC equal to a sodium chloride solution isosmotic with the bathing medium over a range of osmolarities from 80-600 m-osmole (Diamond, 1964; Whitlock & Wheeler, 1964), then increasing osmolality equally on both sides should result in the transfer into the lateral intercellular space of an isosmotic sodium chloride solution, i.e. a solution with higher sodium chloride concentration than that in the medium where the poorly permeant solute contributes to the total osmolality. A sodium chloride concentration difference across the tight junction should therefore be created; and since a major part of the passive ion fluxes across low resistance epithelia probably takes place paracellularly across the junctional complex, the site of very low ohmic resistance (Fr6mter, 1972), a lumen positive diffusion potential would result (cf. Whitlock & Wheeler, 1964; Machen & Diamond, 1969). Such a change would account for the measured increase in p.d. following addition of 1 % DMSO bilaterally (Fig. 3); and possibly also for the modest drop in net fluid transfer rate (Fig. 1) because of the increased passive back-flux of sodium. It should be mentioned that the changes observed in the p.d. after unilateral addition of 1 % DMSO, corresponding to an increase in osmolality of about 140 m-osmole/kg, equal those obtained by addition of only 50 mm sucrose to a similar HCO3--Ringer (0. Frederiksen, unpublished), indicating a considerably lower reflexion coefficient of DMSO than of sucrose. Cytochalasin B at concentrations of 10-5 to 10-6 M inhibits cell movements, e.g. cytokinesis, pinocytotic, phagocytotic and at least some exocytotic processes (Wessels et al. 1971; Carter, 1972; Copeland 1973): i.e. processes involving membrane or particle motility, or both, possibly linked to a contractile mechanism. Active transmembrane hexose transport is inhibited by cytochalasin B at concentrations of 1O-7 to 10-8 M (Mizel & Wilson, 1972; Kletzien & Perdue, 1973; Taverna & Langdon, 1973). Some effects of cytochalasin B, such as inhibition of transmitter discharge from cholinergic nerves and of histamine secretion from mast cells, seem to be due to blockade of sugar transport resulting in substrate depletion (Douglas, 1974). A similar metabolic effect of cytochalasin B is excluded as a possible cause of the inhibition of net fluid transport by rabbit gall-bladder. Firstly, it has been shown that, unlike other gallbladder epithelia, rabbit gall-bladder epithelium is incapable of active glucose uptake across the brush-border (Mirkovitch, Sepulveda, Menge & Robinson, 1975); secondly, the present data demonstrate that the effect is independent of the access to the substrates glucose, glutamate and pyruvate. Cytochalasin B at a concentration of 10 ,ug/ml. (2 x 10-5) may disintegrate, or disorganize, or do both to the actin-like microfilament systems in living cells (cf. for example, Wessels et al. 1971; Baudnin, Stock, Vincent

CYTOCHALASIN B ISOSMOTIC FLUID TRANSPORT 115 & Grenier, 1975). However, microfilament systems supposed to be involved in force production may be left apparently unchanged as far as filament ultrastructure is concerned in spite of disturbed motility (Goldman, 1972), suggesting that motility may be inhibited by cytochalasin B without demonstrable microfilament disorganization (Carter, 1972; Copeland, 1973). However, it should be mentioned also that actin function may be altered by cytochalasin B without any demonstrable ultrastructural changes of the actin filaments. This possibility is suggested by in vitro experiments with muscle actin, myosin and actomyosin. Forer, Emmersen & Behnke (1972) found no ultrastructural changes in F-actin exposed to cytochalasin B (1 mM) but functional inhibition of the F-actin could be demonstrated (Spudich & Lin, 1972). Nevertheless, it still remains a fact that no direct evidence has been obtained indicating that the effects of cytochalasin B on motile processes are due to a direct effect on the membrane-associated actomyosin; the bulk of evidence, though, suggests that such an action is a likely possibility. However, even a non-actomyosin mediated membrane effect may interfere indirectly with an actomyosin mediated contractile system by changing membrane properties, e.g. ion permeability, possibly involved in the triggering of a contractionrelaxation cycle. The early inhibition of the active sodium transport, indicated by the inhibition of the mucosal-to-serosal Na+ flux, suggests an effect of cytochalasin B presumably on a trans-cellular component of the fluid transfer process; and the slow action is compatible with an intracellular site of action. Intracellular binding sites for the congeneric compound cytochalasin D have also been shown directly (Tannenbaum, Tannenbaum & Goldman, 1975). Consistent with this suggestion is the observation that luminal perfusion of the rat submaxillary salivary duct with 10 jug/ml. of cytochalasin B depressed both net absorption of sodium and net secretion of potassium with a similar half-time of about 30 min without changing the transductal p.d. (Schneyer, 1974). But, in addition, cytochalasin B has effects on several aspects of epithelial passive permeability: (1) The continuous, slow rise in the passive serosal-to-mucosal Na+ flux in spite of unaltered electrical resistance for 60 min or more and the late increase in both of the unidirectional fluxes suggest an increase in cell membrane sodium (or ion) permeability. This effect contributes to the inhibition of net fluid transport and is responsible ultimately for a complete leakiness of the epithelium. (2) The immediate drop in the transmural p.d. and resistance recorded after addition of cytochalasin B indicates an increased permeability of the junctional complex of the gall-bladder, which accounts for about 95 % of the total conductance (Fr6mter, 1972). The marked difference in time

116 0. FREDERIKSEN AND P. P. LEYSSAC constants between this latter effect and the measured changes in net fluid transfer rate (with a half-time of about 30 min), in the active mucosal-toserosal Na+ flux, and the slow rise in the passive serosal-to-mucosal Na+ flux excludes, however, that this permeability effect on the paracellular shunt-path is a major cause, if any at all, of the inhibition of net fluid transfer. The present data cannot exclude nor substantiate that the inhibitory effect of cytochalasin B on the active transcellular pump might be indirect, mediated by the increase in cell membrane permeability to sodium, since indirect (Frederiksen & Leyssac, 1969) as well as direct evidence (Cremaschi, H6nin & Calvi, 1971) has indicated that transepithelial isosmotic fluid transport across rabbit gall-bladder is inhibited by an increased intracellular sodium concentration. This effect itself creates a problem in view of the conventional concept since it would appear paradoxical that an active Na+ (or NaCl) ion pump responsible for the extrusion of Na+ (or NaCl) out of the cell, is inhibited by an elevation of the intracellular sodium concentration. Summarizing, the present results have shown that 10-5 to 10-6 M of cytochalasin B causes a progressive and reversible inhibition of net isosmotic fluid transfer across the gall-bladder epithelium. This effect is due both to inhibition of the active, transcellular pump mechanism and to an increase in the cellular permeability. Previous physiological evidence has suggested a mechanochemical process for the transcellular transfer of isosmotic volumes across low-resistance epithelia. A highly organized actomyosin-system is present in the microvillus and terminal web of these cells with additional actin filaments extending throughout the entire cytoplasma; and a sacculotubular organelle extends from the apical to the lateral cell membrane. Similar concentrations of cytochalasin B reversibly inhibit cell motility in a large variety of non-muscle cells. Thus, the present observations are not contradictory to the concept that the contractile system is directly involved in the propulsion of fluid through the cell via a distinct subcellular tubular organelle. According to this concept a basic actomyosin mediated process might underlie transport in general, whether it be transport of the organism relative to the surroundings (cell movements) or transport of the surroundings (the bathing medium) relative to the organism (transcellular fluid transport). However, the present evidence is indirect and an alternative interpretation is not excluded. Thus, further studies are highly warranted before this question can be definitely answered. The present study was supported by grants from the Danish Medical Research Council. The skilful technical assistance of Anni Salomonsson, Pia Svaneborg and COnnio Teindrup is acknowledged.

CYTOCHALASIN B ISOSMOTIC FLUID TRANSPORT

117

REFERENCES BARRY, P. H., DIAMOND, J. M. & WRIGHT, E. M. (1971). The mechanism of cation permeation in rabbit gallbladder. Dilution potentials and biionic potentials. J. Membrane Biol. 4, 358-394. BAUDUIN, H., STOCK, C., VINCENT, D. & GRENIER, F. (1975). Microfilamentous system and secretion of enzymes in the exocrine pancreas. Effect of Cytochalasin B. J. cell Biol. 66, 165-181. BojEsoN, E. & LEYsSAC, P. P. (1965). The kidney cortex slice technique as a model for sodium transport in vivo. Acta physiol. 8cand. 65, 20-32. CARTER, S. B. (1972). The cytochalasins as research tools in cytology. Endeavour 31, 77-82. COPELAND, M. (1973). The cellular response to cytochalasin B: a critical overview. Cytologia, 39, 709-727. CREMASCHI, D., HANIN, S. & CALVI, M. (1971). Inhibition of NaCl-NaHCO3 pump by high levels of Na+ salts in rabbit gall bladder epithelial cells. Atti Accad. naz. lincei Rc. 50, 216-220. DIAMOND, J. M. (1964). Transport of salt and water in rabbit and guinea pig gallbladder. J. gen. Physiol. 48, 1-14. DIAMOND, J. M. & BOSSERT, W. H. (1967). Standing-gradient osmotic flow. A mechanism for coupling of water and solute transport in epithelia. J. gen. Physiol. 50, 2061-2083. DOUGLAS, W. W. (1974). Involvement of calcium in exocytosis and the exocytosisvesiculation sequence. Biochem. Soc. Symp. 39, 1-28. FORER, A., EMMERSEN, J. & BEHNKE, 0. (1972). Cytochalasin B: does it affect actin-like filaments? Science, N.Y. 175, 774-776. FREDERIKSEN, 0. & LEYSSAC, P. P. (1969). Transcellular transport of isosmotic volumes by the rabbit gall-bladder in vitro. J. Physiol. 201, 201-224. FROMTER, E. (1972). The route of passive ion movement through the epithelium of Necturus gallbladder. J. Membrane Biol. 8, 259-301. FROMTER, E., RUMRICH, G. & ULLRICH, K. J. (1973). Phenomenologic description of Na+, Cl- and HCO3- absorption from proximal tubules of the rat kidney. Pflugers Arch. ge8. Phy8iol. 343, 189-220. GOLDMAN, R. D. (1972). The effects of cytochalasin B on the microfilaments of baby hamster kidney (BHK-21) cells. J. cell Biol. 52, 246-254. KLEINZELLER, A. & KNOTKOVA, A. (1964). The effect of oubain on the electrolyte and water transport in kidney cortex and liver slices. J. Physiol. 175, 172-192. KLETZIEN, R. F. & PERDUE, J. F. (1973). The inhibition of sugar transport in chick embryo fibroblasts by cytochalasin B. J. biol. Chem. 248, 711-719. LEYSSAC, P. P., BUKHAVE, K. & FREDERIKSEN, 0. (1974). Inhibitory effect of prostaglandins on isosmotic fluid transport by rabbit gall-bladder in vitro, and its modification by blockade of endogenous PGE-biosynthesis with indomethacin. Acta physiol. scand. 92, 496-507. LEYSSAC, P. P. & FREDERIKSEN, 0. (1974a). An alternative model for isosmotic water transport. In Secretory Mechani8m8 of Exocrine Glands, ed. THORN, N. A. & PETERSEN, 0. H., pp. 432-448. Copenhagen: Munksgaard. LEYSSAC. P. P. & FREDERIKSEN, 0. (1974b). Discussion. In Secretory Mechani8ms of Exocrine Glands, ed. THORN, N. A. & PETERSEN, 0. H., P. 445. Copenhagen: Munksgaard. MACHEN, T. E. & DIAMOND, J. M. (1969). An estimate of the salt concentration in the lateral intercellular spaces of rabbit gall-bladder during maximal fluid transport. J. Membrane Biol. 1, 194-213.

118

0. FREDERIKSEN AND P. P. LE YSSAC

MIRKOVITCH, V., SEPULVEDA, F. V., MENGE, M. & ROBINSON, J. W. L. (1975). Active amino-acid and sugar uptake by gall bladder epithelium in dog, guineapig and man. Pfliuger8 Arch. ge8. Physiol. 355, 319-330. MIZEL, S. B. & WILSON, L. (1972). Inhibition of the transport of several hexoses in mammalian cells by cytochalasin B. J. biol. Chem. 247, 4102-4105. M0LLGARD, K. & SAUNDERS, N. R. (1975). Complex tight junctions of epithelial and of endothelial cells in early foetal brain. J. Neurocytol. 4, 453-468. MOOSEKER, M. S. (1974). Brush border motility: microvillar contraction in isolated brush border model. J. cell Biol. 63, 231 a. MOOSEKER, M. S. & TILNEY, L. G. (1975). Organization of an actin filament-membrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells. J. cell Biol. 67, 725-743. ROSTGAARD, J. & THUNEBERG, L. (1972). Electron microscopical observations of the brush border of proximal tubule cells of mammalian kidney. Z. Zellfor8ch. mikrosk. Anat. 132, 473-496. SCHNEYER, L. H. (1974). DifferentiaJ effects of cytochalasin B on Na and K transport in a perfused salivary duct. Am. J. Phy8iol. 227, 606-612. SPUDICH, J. A. & LIN, S. (1972). Cytochalasin B, its interaction with actin and actomyosin from muscle. Proc. natn. Acad. Sci. U.S.A. 69, 442-446. TANNENBAUM, J., TANNENBAUM, S. W. & GOLDMAN, G. C. (1975). Subcellular localization of binding sites for cytochalasin D: evidence from activation energies. Biochim. biophys. Acta 413, 322-327. TAVERNA, R. D. & LANGDON, R. G. (1973). Reversible association of cytochalasin B with the human erythrocyte membrane: inhibition of glucose transport and the stoichiometry of cytochalasin binding. Biochim. biophy8. Acta 323, 207-219. THUNEBERG, L. & ROSTGAARD, J. (1 969). Motility of microvilli. A film demonstration. J. Ultrastruct. Res. 29, 578a. VoUTE, C. L., M0LLGARD, K. & USSING, H. H. (1975). Quantitative relationship between active sodium transport, expansion of endoplasmic reticulum and specialized vacuoles ('scalloped sacs') in the outermost living cell layer of the frog skin epithelium (Rana temporaria). J. Membrane Biol. 21, 273-289. WESSELS, N. K., SPOONER, B. S., ASH, J. F., BRADLEY, M. O., LUDUENA, M. A., TAYLOR, E. L., WRENN, J. T. & YAMADA, K. M. (1971). Microfilaments in cellular and developmental processes. Science, N. Y. 171, 135-143. WHITLOCK, R. T. & WHEELER, H. 0. (1964). Coupled transport of solute and water across rabbit gallbaldder epithelium. J. cdin. Invest. 43, 2249-2265. WRIGHT, E. M. & DIAMOND, J. M. (1 968). Effects of pH and polyvalent cations on the selective permeability of gall-bladder epithelium to monovalent ions. Biochim. biophys. Acta 163, 57-74.