Water and urea transport in human erythrocytes infected with the malaria parasite Plasmodium falciparum

Molecular and Biochemical Parasitology, 40 (1990) 269-278 269 Elsevier

MOLBIO 01330

Water and urea transport in human erythrocytes infected with the malaria parasite Plasmodium falciparum

M a r y A n n Z a n n e r 1, Wi l l i am R . G a l e y 2, J o s e p h V. Scale t t i 3, J e s p e r B r a h m 4 and D a v i d L. V a n d e r Jag t 1

Departments of 1Biochemistry, 2physiology, and 3Microbiology, University of New Mexico, School of Medicine, Albuquerque, NM, U.S.A., and 4Department of General Physiology and Biophysics, University of Copenhagen, The Panum Institute,

Copenhagen, Denmark

(Received 23 October 1989; accepted 18 December 1989)

The permeability properties of the human red cell membrane to various solutes are altered by malarial infection. In the present work we show that the permeability of the red cell membrane to water is also affected by the intraerythrocytic growth of the malaria parasite Plasmodium falciparum, whereas urea permeability appears unchanged. The data from infected cells show decreases in membrane surface area, cell volume, the osmotically active water fraction (W~n), and osmotic water permeability (Pf) as measured by stopped-flow spectroscopy. On the other hand, the data suggest an increase in diffusive water permeability (Pd) in infected cells with no change in urea permeability when measured by the continuous flow method. The decreased Pf/Pd ratio of infected cell membranes and its implications in the geometry of the red cell membrane water channel or pore are discussed.

Key words: Plasmodium falciparum; Red cell membrane; Water permeability; Urea permeability

Introduction

The growth of the malarial parasite Plasmo- dium falciparum in human red cells results in nu- merous biochemical, structural and functional changes in the host cell membrane. Previous studies have shown that the selective permeability of the red cell membrane is altered by malarial infection resulting in an increased transport of several solutes including amino acids [1-3], anions [4,5], cations [6,7], and polyols [8,9]. In recent years, Ginsburg et al. [9-11] and Kutner et al. [5] have provided evidence for the existence of pore-like permeation pathways in P. falciparum-infected human red cell membranes which

Correspondence address: David L. Vander Jagt, Department of Biochemistry, University of New Mexico, School of Med- icine, Albuquerque, NM, 87131, U.S.A.

Abbreviations: Pf, osmotic water permeability; Pd, diffusive water permeability; P . . . . urea permeability; W~n, osmotically active water fraction; Vis o isotonic red cell volume.

appear to facilitate the transport of various anions and non-electrolytes into and out of the cell. In light of the observed parasite-induced alterations in red cell membrane permeability properties, it was of interest to investigate the possibil- ity that the permeation pathways could contribute to an increased transport of water across infected human red cell membranes.

In normal red cells the major mechanism for net water transport through the membrane is the bulk flow of water through channels or pores. This ac- counts for 80-90% of the osmotic water flux, with the movement of water through the lipid bilayer accounting for about 10-20% of the total flux [12,13]. These conclusions were based on studies on artificial and normal biological membranes which showed that the ratio between the water permeability measured under an osmotic pressure gradient (Pf) and that measured under a diffusion gradient (Pd) is greater than unity [14,15]. Since the process of water transport across the red cell membrane is of considerable importance, es- pecially in the regulation of cell volume, the pres-

0166-6851/90/$03.50 t~) 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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ent study measured the transport of water across infected and uninfected red cell membranes under both isotonic and anisotonic conditions in or- der to determine if infection by P. falciparum al- ters the diffusive and/or the osmotic water permeability characteristics of the erythrocyte membrane. The transport of urea across infected and uninfected red cell membranes was also studied since it is a small non-electrolyte only slightly larger than water which should easily pass through the proposed permeation pathways.

Materials and Methods

Materials. Radioisotopes [3H]H20 and [14C]urea were purchased from Dupont NEN Research Products, Boston, MA. All other chemicals were purchased from Sigma Chemical Company, St. Louis, MO.

Preparation of erythrocytes. Blood was drawn from healthy donors by venipuncture into sterile heparinized tubes. After centrifugation, the plasma and buffy coat were removed and the packed cells were washed twice in sterile phos- phate-buffered saline (PBS), pH 7.4, 20°C. Sam- ples of the packed erythrocytes were used in the following procedures, and consisted of: (a) malaria-infected cells; (b) uninfected-incubated cells carried through the same experimental procedure as the infected cells; and (c) uninfected, fresh cells drawn on the day of the experiment.

Cultivation of parasites. P. falciparum, Colom- bian strain FCB1 (clone NC-I), was grown in human erythrocytes at 37°C [16]. Culture dishes contained 2-3% erythrocytes in RPMI-1640 medium supplemented with 5 mM glutamine, 35 mM 4-(2-hydroxyethyl)-l-piperazine-ethanesulfonic acid (Hepes), 24 mM sodium bicarbonate, gen- tamicin, 33 mg 1-1 and 10% (v:v) horse serum (Hyclone), pH 7.2. When parasitemia reached 8-10%, the parasitized cells were concentrated to >95% by flotation on Plasmagel [17] to provide a trophozoite-rich sample of erythrocytes.

Determination of osmotic water permeability (t f). Pf was determined using the stopped-flow method previously described by Galey [18]. In this method

osmotic volume changes induced by mixing cells with hypertonic solutions are followed by changes in light scattering by the cells.

The resulting experimental cell volume versus time curve was fitted to the predicted Pf derived from the integration of the following equation:

dv/dt = PfAVw [Ci (Vo - b')(Vt - b') - C,n] (1)

where A=cell surface area in cm2; Vw=:partial molar volume of water (approx. 18 cm 3 mol-1; Ci--osmolality before mixing of solution in which cells are suspended; Cm=osmolality after mixing of solution in which cells are suspended; Vo=cell volume before mixing at time=0; Vt--cell volume at time t; b'--red cell volume osmotically inactive in cm 3 and is equal to (1-Weff)V. War is the fraction of the cellular water volume which is osmotically active [19]. The experimental data curve was fitted to a Runga-Kutta integration [20] of the dv/dt equation by estimating the Pf in a Fibbon- acci search program. The minimum least squares fit of the experimental curve to the integrated theoretical curve was taken to be the value of Pf for a given set of data.

Determination of the osmotically active water fraction (WefO. The isotonic volume Viso is made up of an osmotically inactive volume consisting of intracellular molecules and bound water and an osmotically active fraction [21]. When the osmolality of the extracellular medium is altered, the cell responds with a change in its volume of osmotically active water to obtain osmotic equilib- rium. The osmotically active fraction of the cell volume is known as the Well. The War of non-infected and infected red cells was determined using the hematocrit method for calculation of red cell volume under anisotonic conditions as described by Savitz et al. [21]. A plot of Hcto/Hctiso versus "rriso/'rr o yielded a straight line with a slope of Weff and an intercept of 1-Wef f or b, where HCto=hematocrit at each of the various osmolalities; Hcti~o=hematocrit at isotonicity; -rro=varlous solution osmolalities and ~riso=isotonic osmolality. Giemsa-stained cell samples at all osmolalities showed intact red cells with or without parasites.

The value of Waf is also used in the determi-

nation of the total cell volume at isotonicity (Viso) as shown in the following equation:

V o = Vi~ o [1 - Wen +Weff ('a'i~dXro) ] (2)

where Vo=volume at various osmolalities [21].

Determination of surface area. Samples of malaria-infected and uninfected red blood cells were suspended in a series of buffers of progressively decreasing osmolality. These samples were then fixed with 2.5% glutaraldehyde in cacodylate buffer (0.075 M), pH 7.2. Aliquots (0.1 ml) of fixed cell suspensions were placed on poly-(L-lys- ine) (1 mg/ml) coated coverslips and incubated for 1 h. After treatment with 1% osmium tetroxide for 30 min the cells were dehydrated through graded concentrations of ethanol (50-100%), air- dried and coated with gold. A comparison with critical-point freeze-dried preparations showed no improvement in ultrastructural details over the air- dried preparations. Specimens were observed and photographed utilizing a Hitachi $800 scanning electron microscope equipped with a field emis- sion source. Cell diameters of the spherically swollen cells at the lowest pre-lytic osmolality were measured for all cell samples and the corresponding cell surface areas were calculated.

Determination of cell solvent water. Solvent water content of malaria-infected and uninfected red cells was determined by weighing known volumes of packed cell samples before and after drying to constant weight. In all calculations of volume, the extracellular medium trapped between the packed cells was assumed to be about 2% [22]. The cellular solvent volume was then calculated by di- viding the water content by the number of cells in the sample which was determined by Coulter counting.

Calculation of permeability coefficients Pd and Purea" The permeability coefficient P (cm s -1) is determined from the equation:

P = kV/A (3)

where V/A is the ratio of intracellular solvent water to cell membrane area. Cell volumes and

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surface areas of all cell samples were calculated as described above.

Determination of the rate constants for efflux of water and urea. Washed red cells were resus- pended to a hematocrit of approximately 60% and radioactively labeled urea ([14C]urea) and water ([3H]H20) were added to give a concentration of 0.2-1 txCi m1-1 of each tracer in the suspension. The rate of tracer efflux from cell samples labeled with radioactive water and urea was determined at room temperature by the continuous flow method as described by Brahm [13].

Results

Osmotic water permeability (P~) The osmotically active water fraction: Weft. The determination of osmotic water permeability or P~ depends on the rate of red cell volume change created by alteration of the extracellular osmotic environment. When the osmolality of the extracellular medium is altered, the cell responds with a change in its volume of osmotically active water, or effective water fraction (Wen). The value of Wef f is included in Eqn. 1 which fits the cell volume vs. time curve and is used to calculate Pf. Therefore it was necessary to determine if infected red cells had the same effective water volume as normal cells. The data in Table I show a significant decrease in the infected cell Weee when compared to incubated-uninfected cells (P<0.001). The infected cells have a Wef t of 0.27---0.02 vs. 0.65---0.03 and 0.62---0.03 for the uninfected fresh cells and uninfected incubated cells respectively. The numbers in Table I represent the average Wen values of eight samples from two separate experiments with two different donors. Although the uninfected WCf~ values in Table I are somewhat higher than the 0.58 reported in the literature for normal cells [18,21], all values were reproduci- ble, with the plots yielding correlation coefficients of 0.98 or better. The results in Table I indicate that the infected red cells have less than half of the osmotically active water fraction of uninfected cells.

Analysis of the experimental data derived from the stopped-flow method requires that the relationship between red cell volume and light scat-

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T A B L E I

Determinat ion of osmotic water permeabili ty of uninfected and malaria-infected red cells

Cells Diameter (ixm) Area Wen Visa (× 10 -12 cm 3) Cell count ( x 106 ~1-1) Pc (× 10-2 X 10 -6 cm 2 cm s - l )

Fresh uninfected 7.06-+0.04 1.56 0.65-+0.03 117 7.80-+0.38 2.61-+0.08 Incubated unin- 6.96---0.03 1.52 0.62-+0.03 114 7.84-+0.46 2.60-+0.08 fected Malaria infected 6.06-+0.06 1.15 0.27-+0.02 80 11.18-+0.43 0.99-+0.03

Plus and minus values represent the s tandard error of the mean for N values of each determination; N = 9 for cell count; N = 8 for Wef f and N = 9 for Pf. Calculations for area and Viso and are described in the text.

tering be known. Galey [18] has shown that a linear relationship exists between volume and light scattering for normal uninfected red cells. Our results indicate that a linear relationship also exists between red cell volume and light scattering for red cells infected with P. falciparum (Fig. 1). As can be seen, there is an inverse relationship between cell volume and the amount of light scattered by suspensions of infected and uninfected cells, with the cell volumes at various ton- icities (Vo) calculated from Eqn. 2.

Cell surface area. Determinat ion of the osmotic water permeability, Pf, requires that the cell membrane area be known. The membrane surface area was estimated by measuring the diameters of swollen infected and uninfected cells at their lowest pre-lytic osmolality of 160 mOsm 1-1. Examples of swollen uninfected and infected cells are shown in Fig. 2. Calculation of surface area was made from the measured mean diameters of

5000 / [ • malaria-infected l • uninfected

i--- ...J i.d ¢.9r~ r 4000

2000

~J n- 2000

1000 70 80 go 100 1 I0 120 130

RBC VOLUME

Fig. 1. Resul ts of a typical exper iment exhibiting the inverse linear relationship between red cell volume and 90 ° light scattering at 535 n m for uninfected and malaria-infected red cells.

Details of exper iment described in text.

7.06---0.04 i~m, 6.96---0.03 ~m, and 6.06___0.06 I~m for fresh-uninfected, incubated-uninfected and malaria-infected cells respectively. The infected mean cell diameter was significantly less than that of infected cells (P<0.01) and yielded a 24% decrease in surface area with a value of 1.15 × 10 - 6

cm 2 (Table I). Our calculated surface area values of 1.56 × 10 -6 cm 2 and 1.52 x 10 -6 cm 2 for fresh- uninfected cells and incubated-uninfected cells respectively, are in good agreement with the published value of 1.42 x 10 -6 cm 2 for normal red blood cell surface area [23-25].

Isotonic red cell volume (Visa). Isotonic red cell volume (Visa) is also a factor in the calculation of Pf. The cell volume at isotonicity (Vi~o) for uninfected incubated cells was calculated from Eqn. 2 incorporating the measured diameter approxi- mated at pre-lytic osmolality to estimate 1/o. This resulted in a Viso value of 114 × 10 -12 cm 3 for the uninfected incubated cells (Table I), which is in close agreement with the published value for red cell volume of 104 × 10 -12 cm 3 [26]. From our coulter determinations we found the number of infected cells per unit volume of packed cells to be significantly greater than uninfected cells (P<0.01) as shown in Table I. From these data we can see that the infected cells are about 70% as large as uninfected cells, assuming extracellular space is the same as for normal uninfected packed cells. Since infected cells have volumes which are 70% of normal cells as described above, a Viso o f 80 X 10 -12 c m 3 (70% of 114 × 10 -12 C:TI 3)

was estimated for the infected cells (Table I).

Calculation of Pf. The incorporation of the cor- rect values for cell volume, surface area and Wen into Eqn. 1 results in a significant decrease in the

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Fig. 2. Scanning electron micrographs of P. falciparum-infected and uninfected human red cells at isotonic and pre-lytic osmolalities. (a) Uninfected red cells, 300 mOsm; (b) Malaria-infected red cells, 300 mOsm; (c) Uninfected red cell, 160 mOsm; (d)

Malaria-infected red cell, 160 mOsm. Bar, 2.0 p,m.

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TABLE If

Determination of permeability coefficients for diffusive water (P0) and urea (Pur~a)

Diffusive water

Fresh uninfected Incubated uninfected Malaria-infected

k (s -1) ed a k (S - l) ed a k (s -1) ed a

Donor 1 48.50 2.02 45.60 1.95 81.40 3.21 Donor 2 103.00 4.27 105.30 4.51 118.20 4.66

Urea

Fresh uninfected Incubated uninfected Malaria-infected

k (s -1) Purea a k (S -1) Purea a k (s 1) pu~eaa

Donor 1 11.40 0.47 12.80 0.54 12.30 0.49 Donor 2 13.00 0.53 11.30 0.48 11.70 0.46

apermeability coefficients (x 10 -3 cm s -1) calculated from V/A ratios of 4.15, 4.28 and 3.94 x 10 -5 cm for fresh-uninfected, incubated-uninfected and malaria-infected red cells, respectively.

osmotic water permeability of red cells infected with P. falciparum as compared to uninfected cells (P<0.001) as shown in Table I. The results of nine determinations from several donors yield mean Pf values of 2.61---0.06 × 10 -2 cm s - I , 2.60---0.04 x 10 -2 cm s -1, and 0.99---0.03 × 10 -2 cm s -1 for uninfected-fresh cells, uninfected-incubated cells and malaria-infected red cells respectively.

Urea and diffusive water permeability The permeability coefficient, P, in the equa-

tion P=kV/A, represents the true permeability of the membrane to a solute and incorporates the solvent water volume (V) of the red cell which has been determined to be 64.70 × 10 -12 cm 3 under isotonic conditions [13]. Since our data for malaria-infected red cells show a 30% decrease in isotonic volume (Table I), we must assume that the solvent water volume of infected red cells is also decreased by 30%. This results in a solvent water volume of 45.29 x 10 -12 cm 3 and a V/A ratio of 3.94 x 10 -5 cm for malaria-infected cells (Table II). We also found that the amount of water in a given packed cell volume is approximately the same for infected cells and uninfected cells. This was determined by the fact that the wet weight minus dry weight is 0.69 g water m1-1 packed cells and 0.71 g water m1-1 packed cells for infected cells and uninfected cells respectively. This supports the assumption that the amount of cell water is reduced in each infected cell since the number of infected cells is increased

per unit volume as compared to uninfected cells. Inasmuch as urea is a small non-electrolyte, it

was of interest to determine if the membrane transport of this molecule is increased by malarial infection via the proposed permeation pathways. Permeability coefficients for u r e a (/°urea) transport from uninfected and infected cells were calculated from the V/A ratios described above and the rate constants of tracer effiux (Table II). There appears to be little difference in urea permeability between infected and uninfected cells, or between the individual donors (Table II). However, the data from two separate donors suggest an increase in diffusive water permeability of malaria-infected cells as compared to uninfected cells, with donor 1 exhibiting a larger increase in Pa than donor 2. The reasons for these differences between donors are unclear. This is partic- ularly interesting since both the diffusive water permeability and urea permeability determinations were done simultaneously.

The P~/Pd ratio. Bulk flow through a channel, dri- ven by a pressure difference, is represented by the Poiseuille equation where flow is proportional to the pore radius raised to the fourth power (r4). Diffusive flow is proportional to the square of the pore radius (r 2) since it is related to the area available for diffusion (,rrr2). The ratio Pf/Pd re- lates to a geometric property of the pore, and there are two interpretations of this relationship in the literature at present. In the analyses of

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TABLE III

PflPd ratios of infected and uninfected cells and possible interpretations

Ceils Pe/Pd ratio S Equivalent pore radius b Water molecules per (x 10 -a cm) single-file channel c

Fresh-uninfected Donor 1 12.9 11.7 13 Donor 2 6.1 7.2 6

Incubated-uninfected Donor 1 13.3 11.9 13 Donor 2 5.7 6.9 6

Malaria-infected Donor 1 3.1 4.2 3 Donor 2 2.1 2.8 2

apf and Pd values are from Tables I and II and both are in units of x 10 -2 cm s -1. bThe equivalent pore radii were calculated from equations of Solomon [33]. CAccording to the interpretations of Levitt [29] and Rosenberg and Finkelstein [30].

Renkin [27] and Solomon [28], the Pf/Pd ratio is interpreted to be directly proport ional to the square of the pore radius of some equivalent pore. On the other hand, in small channels such as the gramicidin channel where water permeates by single-file diffusion, the P(Pd ratio has been shown to be equal to the number of water molecules aligned single file in the pore [29-32].

Whichever interpretat ion is used, the fundamental relationship between the physical parameters of the Pf/Pd ratio can be used to evaluate the characteristics of the membrane pores. The data f rom Tables I and II were used to calculate the Pe/Pd ratios listed in Table III. As can be seen, the malaria-infected cells have a decreased ratio as compared to uninfected cells regardless of the differences between the donors, with values of 2.1 and 3.1 as compared to 12.9, 6.1 and 13.3, 5.7 for fresh-uninfected and incubated-uninfected cells respectively. The equivalent pore radii were calculated f rom the Pf/Po ratios according to Solo- mon [28] and the data show a decrease in the pore radius of infected cell membranes as compared to non-infected cell membranes (Table III) . The values of the pore radii of uninfected cells f rom donor 2 are in good agreement with the equivalent pore radius of 6.5---0.6 x 10 -8 cm calculated for normal red cells by Solomon [33]. When the Pf/Po ratio is interpreted in this manner , the data indicate that the size of the water channel is decreased in malaria-infected cell membranes .

If we interpret the Pe/P d ratio to relate to the number of water molecules diffusing through the

pore single-file, the infected cells show a decrease with values of 2 and 3 molecules as compared to 6 and 13 for the uninfected cells (Table III). These results indicate a decrease in the thickness of the membrane and/or in the length of the channel. It should be noted that the estimated values of 6 and 13 water molecules diffusing through the pore in a single-file fashion in uninfected cell membranes are in good agreement with the 5 to 9 water molecules per water channel reported by Levitt [29], Rosenberg and Finklestein [30], Brahm [34] and Macey [35].

Discussion

The development of the malaria parasite, P. falciparum, in red cells requires that the red cell membrane play a major role in maintaining a physiologically and biochemically stable intraerythrocytic environment by providing routes for the access of metaboli tes and removal of catabolites [2]. Various transport mechanisms in the malaria-infected cell have been studied, and it appears that the alteration of intrinsic red cell membrane propert ies is a prominent modification induced by the parasite to accommodate to its needs. It has been shown that infected cells uti- lize f rom 10 to 50 times more glucose than uninfected cells [2,36,37]. The uptake of exogenous amino acids is also accelerated in malaria-infected cells, and these increases have been attrib- uted to both changes in diffusion and cart ier-me- diated transport propert ies of the red cell

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membrane [1,38]. In addition, increases in intracellular sodium and calcium have been reported in malaria-infected cells [6,7].

In recent years a number of studies have proposed that pore-like pathways in malaria-infected cells provide transmembrane routes for various solutes, some of which are normally impermea- ble to uninfected red cells [5,9,10]. The number of such pores at the trophozoite stage of infection has been reported to be 8-16 per cell, each with an estimated radius of 7A [10,11]. These conclusions were based on the results of solute-induced hemolysis experiments and size exclusion data, methods which have been recently questioned by Sherman [39]. On the other hand, Ginsburg and Stein [40] have suggested that the permeability characteristics of the infected cell membrane do not fit a pore model, but follow the mode of non- Stokesian diffusion. The validity of the non- Stokesian biophysical model has been challenged by Silfen et al. [41] on the grounds that the model is based on transport data acquired through sub- strate-induced hemolysis experiments which they believe do not reflect the true permeability of the membrane to the solute.

To our knowledge there have been no studies concerning the permeability of the infected cell membrane to water. Regarding the pore-like characteristics of the proposed permeation pathways, and the fact that the majority of water flux occurs through membrane channels or pores, it seemed possible that the transport of water across infected cell membranes would be increased as compared to uninfected cells. Another stimulus for this investigation was the belief that water permeability measurements of malaria-infected cells as compared to uninfected cells would result in further insight concerning the routes of water and solutes through the red cell membrane and perhaps characteristics of the membrane itself.

We have taken a fundamental approach to the concepts of channels based on the principles of hydrodynamics initially used by Renkin [27] and subsequently applied to pores in the red cell membrane by Paganelli and Solomon [14] and Solomon [28]. This approach is based on the difference between the bulk flow (P0 and diffusive flow (Pd) of water through membrane channels or pores, and the interpretation of the ratio of these

two parameters. The decrease in osmotic water permeability (P0 observed in malaria-infected cells (Table I) indicates a net loss of water permeability resulting from bulk flow through membrane channels in these cells. However, the data in Table II suggest an increase in diffusive water permeability in infected cells which could relate to changes in the infected cell membrane matrix as suggested by Ginsburg and Stein [42]. When comparing the Pf/Pd ratios of infected cells to uninfected cells, the data show a significant decrease in the ratio and in the corresponding equivalent pore radius of infected cells (Table III). Both of these results indicate that malarial infection decreases the size of the red cell membrane water channel or pore and thus decreases the bulk flow of water through the infected cell membrane. Alternatively, malarial infection of the red cell can be said to decrease the thickness of the membrane or the length of the water channel if the Pf/Pd ratio is interpreted according to the single-file theory [29,30]. Regardless of interpretation, a decrease in bulk water transport across the infected cell membrane in vivo could result in the inability of the infected cell to traverse small ves- sels by not decreasing its cell water volume.

The transport of urea across red cell membranes is thought to follow asymmetric carrier kinetics [43--45]. On the other hand, Solomon et al. [46] have proposed that urea, anions and water share a common path through the integral membrane protein Band 3. Nevertheless, urea, a small nonelectrolyte, is a useful probe for membrane channels since it has a radius of about 2A. There- fore, urea should traverse the 7A pathway with ease, and one would expect the urea permeability of infected cell membranes to be increased. How- ever, the data show this not to be the case, since there are little differences in urea permeability between uninfected and infected cells (Table II). Since the malaria-infected cells do show changes in both osmotic and diffusive water permeabili- ties with no corresponding change in urea permeability, the data suggest independent pathways for the transport of urea and water across red cell membranes.

In summary, we conclude the following: (1) The data do not support the existence of 7~

pores in the malaria-infected cell membranes as

proposed by Ginsburg et al. [10,11]. If pores of this size were available to contribute to the transport of water and urea the results should show increases in osmotic water permeability (Pf), the P~/Pd ratio and urea permeability in infected cells. However, the data show decreases in Pf and the Pf/Pa ratio, and no significant change in urea permeability in these cells.

(2) The results of the urea and water permeability measurements do not exhibit parallel changes in infected cells and thus the data suggest independent pathways for urea and water transport.

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Acknowledgements

This work was supported by NIH Grant AI21214 to D.V.J. and NATO Research Grant 334/87 to J.B. and W.R.G.J . Brahm was the re- cipient of an Alfred Benzon Senior Investigator Fellowship. The authors kindly acknowledge the following for providing excellent technical assist- ance: Thomas Sisneros, Naomi Campos, Lucy Hunsaker, Betty Richter and Judi Davis.

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Water and urea transport in human erythrocytes infected with the malaria parasite Plasmodium falciparum