cell volume regulation in rat thymocytes was investigated by using

Journal of Physiology (1993), 465, pp. 403-414 403With 7 figures

Printed in Great Britain

CELL VOLUME REGULATION IN RAT THYMOCYTES

BY A. ARRAZOLA, R. ROTA, P. HANNAERT, A. SOLER* AND R. P. GARAYFrom INSERM U2, Facultei de Medecine, Creteil, France and *Departamento

Fisiologia, Universidad de Granada, Granada, Spain

(Received 22 July 1991)

SUMMARY

1. DIOA (dihydroindenyl-oxy-alkanoic acid), a potent inhibitor of the K+-Cl- co-transport system, fully blocked regulatory volume decrease (RVD) in swelled ratthymocytes, with an IC50 of 2-2 + 0 5 x 10-5 mol 1-1 (mean + S.D., n = 4). Conversely,RVD was resistant to quinine, quinidine, apamin, cetiedil, amiloride, bumetanideand DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonate).

2. DIOA-sensitive RVD followed mono-exponential kinetics, with th (half-lifetime)of 1-3 min and maximal capacity (Cmax) of about 55% of the initial cell swelling.Cmax and the initial rate of RVD (V.) were both linear functions of the increase in cellvolume.

3. RVD was: (i) slightly increased by replacing external Cl- by NO3-, (ii) reversedby replacing external Na+ by K+ (in the presence of external Cl-) and (iii) inhibitedby cell K+ depletion. All these phenomena were blocked by DIOA (86 jmol 1-1).

4. Increased membrane potassium permeability by valinomycin was unable toaccelerate RVD or RVD reversal.

5. In the presence of DIOA, thymocytes responded like osmometers (the relativecell volume was a linear function of the reciprocal of the relative osmolality) in a largerange of osmolalities.

6. The results strongly suggest that RVD in rat thymocytes is mediated by theK+-Cl- co-transport system.

INTRODUCTION

We have previously found that rat thymocyte membranes have a 1: 1 K+-Cl- co-transport system (Soler, Rota, Hannaert, Cragoe & Garay, 1993). A similar co-transport system was well characterized in red blood cells, where it catalysesregulatory volume decrease (RVD; Kregenow, 1981; Lauf, 1988). However,Grinstein and co-workers (Grinstein, Dupre & Rothstein, 1982; Grinstein, Clarke,Rothstein & Gelfand, 1983; Grinstein, Rothstein, Saikadi & Gelfand, 1984; Grinstein& Dixon, 1989) reported that RVD in lymphoid cells was mediated by independentCl- and quinine-sensitive K+ channels. Therefore, we decided to investigate the roleof the K+-Cl- co-transport system in the RVD mechanism of rat thymocytes.

Cell volume regulation in rat thymocytes was investigated by using: (i) iontransport inhibitors, particularly DIOA, an inhibitor of the K+-Cl- co-transportMS 9576

A. ARRAZOLA AND OTHERS

system (Garay, Nazaret, Hannaert & Cragoe, 1988), quinine and DPAC 144 (5-nitro-2-(2-phenylethyl-amino)-benzoie acid), a new Cly channel inhibitor (Wangemann etal. 1986; for chemical structures see Fig. 1 in Soler et al. 1993; for IC50 see Table 1),(ii) ionophores and (iii) ion substitutions.

METHODS

Preparation of thymocytesRat thymocytes were prepared according to a previously described protocol (see Soler et al.

1993). Briefly, rats were rapidly decapitated and thymus from two to four male XWistar rats werehomogenized in Na'-K' Ringer medium at room temperature. The Na'-K' Ringer mediumcontained (mmol 1-1): 145 NaCl. 5 KCl. 10 3-(N79-morpholino)propanesulphonic acid (Mops)--tris(hydroxymethyl)aminomethane (Tris) buffer (pH 7 4 at room temperature), 1 MgCl2 and1 CaCl2. The cells were separated by aspiration with a pipette and washed three times with Na'-K'Ringer medium at room temperature.

Measurement of cell volumeWashed fresh thymocytes were resuspended at room temperature in Na+-K' Ringer medium,

containing 10 mmol I1- glucose, at a thymocrit of 3-88 (v/v). The osmolality of the suspendingmedium was adjusted to 308 + 5 mosmol kg-'. The cell suspension was preincubated for 30 min at37 'C. After this period the cells were diluted in different incubation media at 37 'C (final thymocritwas 0-03-0 1 %). Changes in cell volume were followed in a Coulter counter ZM (Coulter Electronics,Luton, UK). For each incubation media, calibration of volumes measured by the Coulter counterwas performed by using standard spherical latex particles (Coultronic France S.A., Andilly,France) in the range of 11 a to 333 yim3.

The effect of anisotonicity and ion substitutionIn experiments with anisotonic media, the incubation media contained a Na'-K' Ringer media

of the following composition (mmol 1-1): (140+x) NaCl, 5 KCl, 1 MgCl2. 1 CaCI2. 10 Mops-Tris(pH 7-4 at 37 'C) and 10 glucose; where x varied from - 140 to + 180 mmol 1-'. The osmolality ofthe solutions was measured by using a Knauer semi-micro-osmometer (Oberursel, FRG).To study the effect of the external ion composition on cell volume regulation, Cl- content in the

hypotonic media was substituted mole by mole with N03-. In other experiments, Na' content in

the hypotonic media was replaced mole by mole with K+ (for further details see legends to figures).To study the effect of internal K+ on cell volume regulation. Na+-loaded-K+-depleted cells were

prepared by using a previously published method (Soler et al. 1993).

Ion transport inhibitors and ionophoresDIOA and the other compounds used in the study have been described previously (see Soler et

al. 1993). DIOA was provided by E. Cragoe Jr (Nacogdoches, TX, USA). DPAC 144 was

synthesized by G. Moinet and Th. Imbert (Lab. Anphar Rolland, Chilly Mazarin, France).Bumetanide was a gift from Leo Laboratories (Vernouillet. France). Cetiedil was obtained fromInnothera Laboratories (Arcueil, France). Valinomycin, gramicidin D and the other compoundswere either from Sigma or Merck (distributed through Coger, Paris, France). The free acid (or base)form of the ion transport inhibitors was neutralized with Tris base (or with Mops).To study the effect of ion transport inhibitors and ionophores on cell volume regulation in rat

thymocytes, the compounds were added to the incubation media from freshly prepared,concentrated stock solutions in water or dimethyl sulphoxide (DMSO), provided that the finalconcentrations of these solvents had no effect per se on cell volume (final DMSO concentration inthe flux media was always lower than 0-1 %). In particular, DIOA was tested from stock solutionscontaining 50-500 mmol 1-1 of compounds in DMSO. All drugs were tested in concentra-tion-response curves. Table 1 shows IC50 values for the inhibitory action of compounds on fluxescatalyzed by different ion transport systems in human and rat red cells (red cell fluxes measuredby previously published methods; Garay, Hannaert. Nazaret & Cragoe, 1986; Garay et al. 1988).

404

CELL VOLUME REGULATION

RESULTS

Regulatory volume decrease in rat thymocytesFigure 1 (left side, upper panel) shows the cell volume distribution of a population

of fresh thymocytes incubated in isotonic Na+-K+ Ringer medium at 37 'C. It canbe seen that cell volume distribution was unimodal, although slightly shifted to the

TABLE 1. Inhibitory activity of compounds on K+ and/or Cl- transport systems

IC.50 (mol l-l)

K+-Cl- Na+-K+-Cl- Cl--HCO3- Ca2+-dependent Na+-K+Compound co-transport co-transport exchanger K+ channels pump

Human red cellsDIOA 10-5 Inactive 10oo 6 x 10-5 InactiveDPAC 144 5x 10-5 10-4 lo-5 3x 10-4 7x10-4Bumetanide Inactive 6 x 10-' 3 x 10-4 Inactive InactiveDIDS Inactive Inactive 3 x 10-7 PI InactiveAmiloride Inactive Inactive Inactive Inactive InactiveEIPA Inactive 8x 10-4 105 5 x 10-4 2 x 10-4Quinidine Inactive Inactive Inactive 10-4 InactiveQuinine Inactive 10-3 Inactive 2 x 10-4 Inactive

Rat red cellsDIOA 105 Inactive 8 x 10-6 6 x 10-5 n.m.Bumetanide n.m. 5 x 10s n.m. n.m. n.m.DIDS n.m. n.m. 10-7 n.m. n.m.

Ion fluxes were measured by using previously published methods (Garay et at. 1986, 1988). SomeIC50 values given in this table are from Garay et al. 1986, 1988. For cetiedil see Berkowitz &Orringer, 1982. Apamin is inactive in red cells (for other cells see Burgess, Claret & Jenkinson,1981; Hugues et al. 1982; Traore et al. 1986). EIPA increased red cell K+ leak at concentrationshigher than 5 x 10-5 mol l-l. n.m., not measured. PI = partial inhibitor (DIDS inhibited about50% of Ca2+-dependent K+ efflux at concentrations higher than 10-6 mol I-'). DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulphonate; EIPA, ethyl-isopropyl-amiloride; DIOA, [(dihydro-indenyl)oxy]alkanoic acid.

right. For the sake of simplicity we measured the mode of this unimodal distribution.Under basal conditions, cell volume distribution had a mode of 128-3±541 pm3(mean+ S.D. of 31 experiments).Figure 1 shows the rapid increase in cell volume that took place in hypotonic

media (192 + 5 mosmol kg-') and the regulatory volume decrease (RVD) thatfollowed 20-30 min thereafter.

Pharmacological properties of RVDFigure 2 shows the effect of ion transport inhibitors on thymocyte RVD (see

structures of compounds in Soler et al. 1993 and ion flux IC50 in Table 1). It can beseen that DIOA was an efficient RVD inhibitor, with an IC50 of 2-2 + 0 5 x 10-5 mol I`(mean+ S.D., n = 4).DPAC 144 was previously reported to inhibit renal Cl- channels with an IC50 of

8 x 10-8 mol V-l (Wangemann et al. 1986). Figure 2 shows that at these phar-14 PHY 465

405


macological concentrations, DPAC 144 was unable to modify RVD in ratthymocytes. Indeed, 2-3 orders of magnitude higher DPAC 144 concentrations wererequired to inhibit RVD (Fig. 2, see Discussion).

Generally speaking, RVD was resistant to inhibitors of Ca2+-sensitive K+ channels.Thus, a very slight RVD inhibition (< 10%) was found with quinine (tested

Isotonicity50 +86,u DIOA

0

50 Hypotonicity

C 1 :

.0

50 _ ,I.

0.

0 0 150-300__150_3000.

~50 -

0r 15

50-

30 I

0 150 300 0 150 300

Cell volume (Pum3)Fig. 1. Regulatory volume decrease (RVD) in rat thymocytes. Hypotonic shock(192 mosmol kg-') induced a rapid cell swelling, which was partially reduced in about30 min. DIOA fully blocked RVD. Note that fresh thymocytes exhibited unimodal cellvolume distribution, slightly shifted to the right (left side, upper panel).

between 5 x 10-6 and 2 x 10-4 mol 1-1). Cetiedil also induced slight RVD inhibition( 25% at 2 x 10-4 mol 1-1). Apamin (up to 10-6 mol l-l) was unable to significantlymodify RVD. Quinidine (10-4 to 10-3 mol 1-1), the most potent of this series ofcompounds, only induced 25-35% of RVD inhibition (Fig. 2).

Amiloride, bumetanide and DIDS were very poor RVD inhibitors (Fig. 2).

Investigation ofRVD by using DIOAThe above results clearly showed that DIOA was a potent RVD inhibitor in rat

thymocytes (Fig. 2). In order to minimize potential toxic effects and obtain full RVD

406


0 106 10-5 104

Compound concentration (M)

407

10-3

Fig. 2. Pharmacological properties of RVD in rat thymocytes. RVD was measured after15 min incubation in hypotonic media (192 + 5 mosmol kg-'). Values represent means

of 3-4 experiments for each compound. DIOA inhibited RVD with an IC50(22 + 0-5 x 10-5 mol 1-l) similar to that inhibiting K+-Cl- co-transport in rat thymocytes(Soler et al. 1993) and red blood cells (Table 1). DPAC 144 inhibited RVD, but at muchhigher concentrations than those required to inhibit renal Cl- channels (8 x 10-8 mol 1-l,Wangemann et al. 1986) and very similar to those inhibiting K+-Cl- co-transport inhuman erythrocytes (Table 1). Other tested compounds were inactive or poorly active on

RVD.

195

z3. 180a

0)

D 1650

EE) 1 50

0

> 135(1u120

Hypotonic medium + DIOA1 .4 A

v S ~~~~~CiL- f a max0 5 10 15

Time (min)

Hypotonic medium

Isotonic medium

0 ti 10 20 30 40 50

Time (min)

Fig. 3. Kinetic analysis of DIOA-sensitive RVD (hypotonic media at 192 + 5 mosmolkg-'). Inset: single exponential component for RVD. V(t) represents the differencebetween thymocyte volume at time t and that at equilibrium. RVD can be described bymeans of two kinetic parameters: (i) Cmax = maximal RVD and (ii) tp, the time whereV(t) = Cmax/2.

14-2

0-0

0cc


inhibition, we selected a DIOA concentration of 86 /imol 1-1 (Fig. 2). Figure 1 (right-hand side) clearly showed that 86 ,tmol 1I1 DIOA fully blocked RVD.

Figure 3 shows the time course ofRVD in hypotonic media (192 + 5 mosmol). Theinset shows that it can be analysed as a single exponential component. Therefore,

Ideal /60 full regulation /

He45/E / / Hypotonic media

15 Hypertonic media

15 30 45 60 75 90

Initial change in cell volume (Mm3)Fig. 4. Maximal RVD and RVI capacities (Cmax) as a function of the initial change in cellvolume (given as absolute values). RVD was a linear function of cell swelling with a slopeof about 055, indicating that rat thymocytes can reduce by about 55% any increase incell volume (in a large range of osmolalities). RVI showed low capacity. The same resultswere obtained in two other sets of experiments.

RVD can be described by means of two parameters: Cmax, the maximal RVDcapacity and ti, the time where RVD equals Cmax/2 (Fig. 3).

2

For the sake of simplicity we transformed tL into VI, the initial rate of RVD, byusing the following equation:

VO =-n2C0(m1)tiI

In eleven experiments performed at 192 + 5 mosmol, we obtained Cmax =32-7 + 3-8 /tm3 and V. = 14-4 + 6-9 ,m3 min- (mean + S.D.).RVD was investigated as a function of the degree of cell swelling. Figure 4 shows

that Cmax was a linear function of the increase in cell volume with a slope of about0 55. This indicates that rat thymocytes can reduce the initial cell swelling by about55%, and this independently of the absolute value of the initial increase in cellvolume. VO was also a linear function of the increase in cell volume with a slope ofabout 0-19 min- (data not shown).

Ion substitutionsThe next series of experiments was designed to investigate DIOA-sensitive RVD

as a function of the external and internal ionic compositions. Figure 5 shows theeffect of external K+ and Cl- on RVD. It can be seen that RVD was reversed by

408

CELL VOLUME REGULATION 409

190 _

KCI

° 170 -

0 ~~~~~~~~KNO3o Do 150 DIQA0)E

> 130 NaCI

0 ~~~~~~0Q o Q ~~~~~~NaNO,110 _

0 10 20 30

Time (min)Fig. 5. Cell volume changes in hypotonic media (192 mosmol) as a function of the externalK+ and Cl- concentrations. It can be seen that RVD was: (i) slightly increased in NO3-media and (ii) reversed by replacing external NaCl with KCl (note that a small RVDreversal exists in KNO3 media). All these cell volume changes were inhibited by86 #mol 1-1 DIOA.

TABLE 2. DIOA-sensitive RVD as a function of internal K+ contentInternal K+ content VO Cmax(mmol (1 cells)-1) (/tm3 min') (#m3)116-5+3-9t 14-1+3-1 31-6+3-868-3+6-9 6-8+2-3* 24-0+331**37-0+ 7-7 2-9+ 1-4* 152 + 7-1*

Values are given as means+ S.D. (n = 3). Internal K+ was replaced by equivalent amounts ofNa+by using the nystatin technique (Soler et al. 1993). RVD was measured in hypotonic Na+-K+ Ringermedia (183 + 5 mosmol kg-'). For all cell samples, exchangeable Cl- content was between 23 and30 mmol (1 cells)-'.*P < 0-05, **P < 0-01 (Student's t test).t Fresh cells.

replacing external Na+ by K+, and that this reversal required the presence of externalCl- (note, however, that a small RVD reversal exists in KNO3 media). Moreover,replacement of external Cl- by NO3- slightly increased the rate of RVD (Fig. 5).

Table 2 shows the effect of internal K+ on RVD. It can be seen that cell K+depletion induced a statistically significant RVD inhibition (for both V. and Cmax).

IonophoresRVD and RVD reversal were investigated as a function of membrane potassium

permeability. Potassium permeability was increased by using ionophores.Gramicidin enhanced not only potassium but also sodium permeability (thymocyte

sodium content increased by about 40% after 40 min incubation with 0-5 Itmol 1` ofgramicidin). Therefore, we decided to use valinomycin, a highly selective potassiumionophore (Pressman, 1976).


140

Isotonic media

'E 130 -

.$_C

E 120 Hypertonic media

0

E

-@110 -

Hypertonic media: ~~~~~~~~+DIOA

0 15 30 45 60

Time (min)Fig. 6. Regulatory volume increase (RVI) in rat thymocytes. It can be seen that RVI

was much less important than RVD and fully blocked by DIOA.

TABLE 3. Effect of valinomycin on cell volume changes in hypotonic media

VO CmaxCondition (1um3 min') (#m3)

RVDControl 15-0+2-8 (4) 33-9+341 (4)Valinomycin, 2 fmol 1-1 5-5+ 3-5 (4)* 28-9+3-5 (4)Valinomycin, 10 #mol l-' 6 9+ 3 4 (4)** 30*7 + 8 4 (4)

RVD reversalControl 10-9+3-6 (3) 33-6+8 1 (3)Valinomycin, 2 #smol 1-1 6-9 + 2-7 (3) 26-5+ 8-1 (3)Valinomycin, 10t mol 1-1 5z4+34 (3) 33-8+11-4 (3)

Values are given as mean + S.D. The number ofexperiments is indicated in parentheses. *P = 0057,P = 0059 (Student's t test). Volume changes were measured in hypotonic media at

183 + 5 mosmol kg-'. For RVD reversal, NaCl was replaced mole by mole with KC1.

Valinomycin concentrations higher than 0-5 ytmol 11 strongly stimulated Rb+influx, reaching maximal flux stimulation at 5-10 jtmol -1. Moreover, maximalhyperpolarizing effects were previously found at 2 ptmol 11 valinomycin (Soler et al.1993). Therefore, we used such a concentration range to test the effect of valinomycinon RVD and RVD reversal.

Table 3 shows that valinomycin had modest effects on both RVD and RVDreversal: (i) V. was reduced, but this effect was non-statistically significant for RVDreversal and only borderline for RVD and (ii) Cmax was unchanged by valinomycin(for both RVD and RVD reversal).

Isotonic mediaIncubation of thymocytes in isotonic Na+-K+ Ringer media containing DIOA

(86 #amol 1-1) induced a small increase in cell volume (3-05 ± 0-90 jm3, mean + S.D.,n = 8, after 1-2 min of incubation with DIOA).

410


Regulatory volume increase

Generally speaking, regulatory volume increase (RVI) in rat thymocytes wasmuch less important than RVD. Figure 6 shows the time course of RVI.Interestingly, DIGA fully inhibited this phenomenon (Fig. 6). Conversely, RVI was

240

E 210 2.0E' 1.5

0 ~~~~~~~~~0-~10E

150 . .E 5 0.00 ____ ____-0 1 2 3 4> 120 1/Relative osmolality

Q90

0 100 200 300 400 500 600 700 800Osmolality (mosmol kg-')

Fig. 7. Osmotic behaviour of DIOA-treated thymocytes (cell volume as a function of theexternal osmolality). The inset shows that, for a large range of osmolalities, the relativecell volume was a linear function of the reciprocal of the relative osmolality.

resistant to: amiloride (25 x 10-4 moll-'), DIDS (2-5 x 10-4mol -1), bumetanide(10-4 mol l-1) and hydrochlorothiazide (10- mol l-'). EIPA (ethyl-isopropyl-amil-oride) at 10-4 mol 1-1 was able to inhibit about 72% of RVI.

Similarly to RVD, RVI could also be analysed as a single exponential componentwith two parameters, Cmax and V.. Figure 4 shows Cmax for RVI as a function of thedegree of cell shrinkage. It can be seen that thymocytes were able to fully reversesmall cell volume decreases. However, Cmax saturates at moderate cell shrinkage andthe capacity for regulating large decreases in cell volume was lower than that forregulating RVD (Fig. 4). The same was observed for VJ, which reached maximalvalues of 1-1P5 ,gm3 min'.

Osmotic behaviour of DIOA-treated thymocytesThe use of DIOA allowed us to see how thymocytes respond to changes in tonicity

(at 37 TC) when cell volume regulation is blocked. Figure 7 shows cell volume as afunction of the external osmolality. The inset shows that, for a large range ofosmolalities, the relative cell volume was a linear function of the reciprocal of therelative osmolality (see also Grinstein et al. 1984).

DISCUSSION

Swollen rat thymocytes exhibited fast and important regulatory volume decrease(RVD), which reduced cell swelling by about 55% with a half-time of 1-3 min.

Several observations strongly suggested that RVD in rat thymocytes wasmediated by the K+-Cl- co-transport system. First, DIGA potently inhibited RVD,

411


with an IC50 (2 x 10-5 mol 11) close to that found for inhibiting K+-Cl- co-transportfluxes in rat thymocytes (8 x 10t mol l-l, Soler et al. 1993), human red blood cells(1o-5 mol l-1, Garay et al. 1988) and rat erythrocytes (10-s mol l-l, Table 1).

Second, the percentage cell volume decrease (with initial rate of about 716% min-',calculated from RVD kinetics) coincided with the percentage of cell K+ extrusion bythe co-transport system (about 6-5% min-', calculated from flux data in Soler et al.1993).

Third, the behaviour of RVD with respect to ion substitutions was that expectedfor a K+-CL- co-transport system, i.e. (i) replacement of external Cl- by N03-slightly increased RVD, suggesting the disappearance of a small inward K+-Cl- co-transport, (ii) RVD was reversed (cell volume increased) by replacing external Na+by K+, suggesting activation of inward K+-Cl- co-transport, (iii) RVD reversalrequired the presence of external Cl-, as expected for a coupled inward K+-Cl- co-transport (however, a small RVD reversal was observed in KNO3 media; see alsoSoler et al. 1993) and (iv) RVD was reduced by cell K+ depletion as expected for acoupled outward K+-Cl- co-transport.

Finally, blockade of the Na+-K+-Cl- co-transport system, Na+-H+ exchanger,Cl--HCO3- exchanger or Ca2+-sensitive K+ channels had little or no effect on RVD.Moreover, RVD was resistant to pharmacological concentrations of DPAC 144, i.e.those previously found to inhibit Cl- channels in basolateral membranes of Henle'sloop cells (IC50 = 8 x 1o-8 mol l-1, Wangemann et al. 1986). This compound was ableto inhibit RVD at high concentrations (JC50, 3 x 1O- mol l-', Fig. 2), close to thatrequired to inhibit the K+-Cl- co-transport system in human red blood cells(5 x 10-5 mol t-1, Table 1).The above results contrasted with those of Grinstein and co-workers (1982, 1983,

1984, 1989) suggesting that RVD in lymphoid cells was mediated by independent K+and Cl- channels. Those authors postulated that: (i) cell swelling opens Cl- channels,and the membrane becomes depolarized approaching the Cl- equilibrium potential,(ii) potassium permeability thus becomes the rate-limiting step for the RVDmechanism and (iii) an elevation in cytosolic calcium, triggered by cell swelling,opens Ca2+-sensitive K+ channels and accelerates RVD.

In a previous study (Soler et al. 1993) we found that hypotonic shock was unableto increase cytosolic free calcium content, depolarize thymocyte membranes(membrane potential measured after 5 min of the initiation of RVD) or activateCa2+-sensitive K+ channels.

In the present study, we were unable to confirm the RVD sensitivity to Ca2+-sensitive K+ channel blockers (Grinstein et al. 1982, 1984). For instance, Grinstein etal. (1982) reported that quinine fully blocked RVD in human peripheral bloodlymphocytes with an IC50 of about 5 x 10-5 mol 1-1. Here we found that quinine wasalmost ineffective on thymocyte RVD, even at concentrations of 2 x 10-4 mol-1 (seeResults) Moreover, Grinstein et al. (1984) found that cetiedil was a very potentinhibitor of RVD-dependent potassium efflux (IC50 = 2 x 10-6 mol 1-1). In our

experiments, cetiedil was a very poor RVD inhibitor, even at concentrations of2 x 1W-' mol 1-1 (see Results). Moreover, quinidine, a more potent inhibitor of Ca2+-sensitive K+ channels than quinine (Table 1), blocked not more than 1/3 of RVD,and this at high concentrations (higher than those required to inhibit Ca2+-sensitive

412

CELL VOLU-ME REGULATION

K+ channels in red blood cells, Table 1). Finally, apamin, an excellent blocker ofCa2+-sensitive K+ channels in several kind of cells (Burgess, Claret & Jenkinson,1981; Hughes, Romey, Duval. Vincent & Lazdunski. 1982; Traore, Cognard,Poitreau & Raymond. 1986) was unable to modify RVD in rat thymocytes.A further discrepancy with Grinstein et al. (1984) concerns the effect of potassium

ionophores. These authors found that gramicidin accelerates cell volume changes inhypotonic media, and therefore concluded that potassium permeability was the rate-limiting step for RVD (Grinstein et al. 1984; see also Grinstein and co-workers; 1982,1983, 1989). In preliminary experiments we confirmed the RVD acceleration bygramicidin, but we found that this ionophore markedly increased sodiumpermeability (see Results; for gramicidin selectivity see Pressman, 1976). Therefore,we decided to repeat this type of experiment by using valinomycin, a highly selectivepotassium ionophore (Pressman, 1976). In contrast with gramicidin, valinomycinwas not able to accelerate RVD or RVD reversal.One permanent preoccupation of our study was to conciliate our findings with

those of Grinstein and co-workers (1982, 1983, 1984, 1989). We realized that most ofthese studies were performed in peripheral blood lymphocytes and that differences inRVD were found between B and T lymphocytes. On the other hand, we knew fromprevious studies in red cells that the K+-Cl- co-transport system dramaticallydecreases following red cell maturation (see, for instance, Lauf, 1988). Therefore, onepossible interpretation for the above discrepancies is that in human peripheral bloodlymphocytes, the RVD mechanism is different from that in rat thymocytes. Thisdeserves further investigation.

It appears therefore that, similarly to red blood cells, the underlying mechanismof RND) in rat thymocytes is a swelling-induced, net KCl extrusion through a DIOA-sensitive K+-Cl- co-transport system. It is important to mention that rat thymocyteshave much higher K+-Cl- co-transport fluxes than human red blood cells. Thus,thymocytes may rapidly regulate acute cell swelling, in contrast with humanerythrocytes where RVD has a half-time of about 5 h (calculated from Garay et al.1988).

It has been previously observed that, in the cold, lymphocytes behave likeosmometers, i.e. following the Boyle-van't Hoff relationship (linearity of 1/7T vs.volume, where 7T is osmotic pressure) (Roti-Roti & Rothstein, 1973; Grinstein et al.1984). Interestingly, we found here the same results at 37 0C, when thymocytes areincubated in the presence of DIOA. This further confirmed that the K+sl- co-transport system is an important cell volume regulator in rat thymocytes.

In isotonic media, DIOA induced a small thymocyte cell swelling. This agrees withour previous flux data showing that under physiological conditions the K+<l- co-transport system is able to extrude significant amounts of intracellular K+ and Cl-(Soler et al. 1993).Rat thymocytes exhibited a very small regulatory volume increase (RVI). This

was resistant to amiloride, DIDS and bumetanide, thus excluding the involvementof the Na+-H' exchanger, the Cl--HCO3- exchanger and the Na'-K+sl- co-transport system. The results obtained with EIPA on RVI deserve a comment. Highconcentrations of this compound (10-4 mol 1-l) were able to partially inhibitthymnocyte RVI. However, the lack of RVI inhibition with amiloride suggested a

413


non-specific effect of EIPA on a transport pathway different from the Na+-H+exchanger. Indeed, Table 1 shows that EIPA has many side-effects on other iontransport systems.One surprising result was that DIOA fully inhibited RVI, suggesting the

involvement of the K+-Cl- co-transport in the RVI mechanism (see also Soler et al.1993). Such a potential role deserves further investigation.In conclusion, our results strongly suggest that the K+-Cl- co-transport system is

responsible for the regulatory volume decrease (RVD) of swelled rat thymocytes.

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BURGESS, G. M., CLARET, M. & JENKINSON, D. H. (1981). Effects of quinine and apamine on thecalcium-dependent potassium permeability of mammalian hepatocytes and red cells. Journal ofPhysiology 317, 67-90.

GARAY, R. P., HANNAERT, P., NAZARET, C. & CRAGOE, E. J. JR (1986). The significance of therelative effects of loop diuretics and anti-brain edema agents on the Na+, K+, C1 cotransportsystem and the Cl-/NaCO3- anion exchanger. Naunyn-Schmiedeberg's Archives of Pharmacology334, 202-209.

GARAY, R. P., NAZARET, C., HANNAERT, P. & CRAGOE, E. J. JR (1988). Demonstration of a [K+,Cl-]-cotransport system in human red cells by its sensitivity to [(dihydroindenyl)oxy]alkanoicacids: Regulation of cell swelling and distinction from the bumetanide-sensitive [Na+, K+, Cl-]-cotransport system. Molecular Pharmacology 33, 696-701.

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cell volume regulation in rat thymocytes was investigated by using

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