Swelling detection for volume regulation in the primitive eukaryote Giardia intestinalis: a common feature of volume detection in present-day eukaryotes

^a School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, N.S.W. 2052, Australia

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
It is increasingly evident that cell swelling is associated with the triggering of many biological processes, including progression of the cell cycle, hormonal response, and gene expression. However, the mechanism by which cell swelling is initially sensed and converted into intracellular signals is still ill-defined. We report here an early event in the detection of cell swelling and initiation of the volume regulatory response in Giardia intestinalis, an ancient representative of the eukaryotic kingdom. Giardial cell swelling, irrespective of the extent, was sensed at a cell volume of 1.06 x isosmotic volume (the threshold volume), at which the transition of the volume regulatory transport system from the `resting' to the `open' state occurred. Irreversible modification by p-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM) of reduced thiols affected the threshold volume, but in opposing manners: pCMB increased the threshold volume to 1.14 x and NEM decreased to 0.85 x isosmotic volume. The simple modification of the threshold volume by NEM caused a drastic reduction of giardial cell volume under isosmotic conditions, with a process strikingly similar to the opening of mitochondrial permeability transition pore, a causative event in stress-induced programmed cell death. Substantial evidence supports the hypothesis that modulation of the membrane thiol moieties at the threshold volume, causing the `all-or-nothing' type of swelling detection, represents the event linking cell swelling to the second messenger systems for volume regulation in present eukaryotes. Pathophysiological implications of alteration of the threshold volume are discussed.—Park, J.-H., Edwards, M. R., Schofield, P. J. Swelling detection for volume regulation in the primitive eukaryote Giardia intestinalis: a common feature of volume detection in present-day eukaryotes. FASEB J. 12, 571–579 (1998)

Key Words: NEM • RVD • giardiasis • giardial swelling-activated alanine transport • volume sensor

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
CELL SWELLING is a fundamental physiological challenge faced by all animal cells. It is induced by concentrative uptake of nutrients, catabolic cell function, or a decrease in ambient osmolarity (1–3). Interest in how a cell senses its volume has rapidly increased recently with substantial evidence that cell swelling can modulate various second messenger systems involved in important biological processes such as the progression of the cell cycle, hormonal response, oxidative stress, and gene expression (1, 4–8). For example, hyposmotic cell swelling activates a protein tyrosine kinase that, in turn, mediates activation of ERK and induction of the c-fos gene in cardiac myocytes (6). In the budding yeast, conditions leading to cell swelling, such as hyposmotic shock and increased levels of intracellular glycerol, cause activation of a pathway that blocks cell fusion and maintains cell integrity (7, 8).

Despite the wealth of knowledge about various cellular responses to cell swelling and the underlying second messenger systems, the earliest step for sensing cell swelling is still unknown. Swelling-activated (SA)² transport has provided a framework that will ultimately allow studies of the effect of cell swelling on the biological processes mentioned above. SA transports are not functionally active under physiological conditions; once stimulated, their activity in general relies on the extent of cell swelling (2, 9–11). Therefore, it is presumed that a cellular component that modulates its conformation rapidly and reversibly in accordance with the extent of cell swelling controls the activity of SA transports. This notion has led to the characterization of various signaling molecules that are potentially important in regulating SA transport (12–15). However, it has been a subject of debate as to which molecules act as a volume sensor that initially detects cell swelling, since they are measured in relation to changes in transport activity at different time courses or in the presence of an inhibitor. For example, the effect of macromolecular crowding (14, 15), modulation of protein kinase activities (11), and membrane deformation (16) have all been proposed as the primary volume sensor for KCl cotransport in erythrocytes. A volume sensor can unambiguously be selected by investigating its relation to a change in cell volume. However, this approach remains elusive due to difficulties in developing an appropriate methodology to monitor the continuous changes in cell volume with sufficient sensitivity to distinguish slight changes in cell volume during cell swelling and regulatory volume decrease (RVD).

Giardiasis, caused by the protozoan parasite Giardia intestinalis, has been recognized as the most frequently occurring water-borne disease worldwide (17, 18). The Giardia life cycle presents two morphologically distinct forms: trophozoites and cysts (17). Giardial transmission occurs through the ingestion of cysts and the disease is caused by the trophozoite forms. Besides its medical importance, Giardia is an excellent system with which to study the evolution of fundamental cellular processes, since it belongs to the earliest branch of the eukaryotic line of descent (19). This organism is anaerobic and lacks mitochondria and peroxisome organelles, suggesting that it may represent an anaerobic proto-eukaryote (17, 19). Maintenance of a constant cell volume in the face of osmotic stress is undoubtedly an evolutionarily ancient homeostatic process (3, 20). Giardia is, therefore, a potentially important and valuable resource for understanding ancestral physiological mechanisms of volume regulation present in eukaryotic organisms. Unlike higher eukaryotes, where taurine is ubiquitously present, in this organism alanine is the predominant amino acid pool and is accumulated up to 50 mM during growth (21). In response to hyposmotic stress and other conditions that lead to cell swelling, Giardia trophozoites increase unidirectional release of intracellular alanine via activation of a transport system termed GSAAT (giardial swelling-activated alanine transport), which is mainly responsible for giardial RVD (21, 22).

We have recently developed a light-scattering technique that provides the necessary sensitivity and reproducibility for real-time detection of slight changes in giardial cell volumes (22). Using this and other techniques, we report here that Giardia did not sense the extent of cell swelling nor the gradual changes in cell volume during RVD, but recognized a certain cell volume (the threshold volume) for subsequent volume regulation. The threshold volume, which was only 6% greater than isosmotic volume, was a key factor in controlling GSAAT activity. The threshold volume was modulated by thiol reagents, indicating that the volume signal is transduced at the threshold volume via modulation of reduced thiols.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Parasite
Giardia intestinalis trophozoites (Portland-1 strain) were grown anaerobically in TYI-S-33 growth medium supplemented with 10% fetal calf serum. The trophozoites in late log phase were harvested by centrifugation (700 g) and resuspended at a cell density of 7–9 x 10⁷ cells/ml in isosmotic phosphate-buffered saline (PBS) (1.8 mM KH₂PO₄/5 mM K₂HPO₄/150 mM NaCl pH 7.4=308 mosmol/kg). The protein content was determined by the method of Lowry et al. (23), using bovine serum albumin as a standard.

Experimental design
All experiments were carried out at 25°C. Cell swelling or shrinkage was induced osmotically by exposure of stock giardial suspensions to the mixture of PBS and distilled water or a volume of 1.5 M NaCl to give a desired osmolarity. Medium osmolarity was measured using a ONE-TEN Osmometer (Fiske). Osmolarity of the growth medium for our cells was 308 mosmol/kg, and is defined as isosmolarity. In experiments in which cells were exposed to high concentrations of compounds, medium isosmolarity was maintained by reducing the NaCl content correspondingly.

The light-scattering technique used to measure changes in giardial cell volume has previously been described in detail (22). Briefly, an absorbance (A) change in giardial suspensions was monitored at 550 nm and sampled at various time intervals ranging from every 2 to 10 s with a spectrophotometer connected to a microcomputer system. The A₅₅₀ values were converted to the b values that are directly proportional to cell volumes, using the double reciprocal approximation with giardial protein concentration (P) given by 1/A₅₅₀ = 0.273 + b/P. The b values were then used to estimate relative cell volume with the equation: b_t/b_tc = (1/A_550t-0.273) P_t/(1/A_550tc-0.273) P_tc, where subscript t refers to the parameter at a particular time after hyposmotic challenge and tc to that observed simultaneously under isosmotic conditions. The ratio of b_t/b_tc is equal to relative cell volume. Light-scattering data shown in this paper are highly reproducible (SEM<0.01) and are representative of at least 15 observations. The slope of RVD was estimated from the straight line fitted to changes in relative volume and is expressed as 1/h.

Uptake of 2-aminoisobutyric acid (AIB) began with the addition of 0.1 ml stock cell suspensions to 0.14 ml PBS of varying osmolarities, containing the final 1 mM [1-¹⁴C]AIB (0.1 µCi/ml). The efflux studies were performed in cells preloaded with 1 mM L-[2,3-³H]alanine (2.5 µCi/ml), as described (21). At predetermined intervals, duplicate 0.1 ml aliquots were removed, layered over 0.4 ml oil (a blend of dibutyl phthalate and iso-octyl phthalate (4:1, 1.03 g/ml)), and centrifuged at 16,000 g for 30 s. The supernatant was aspirated, the aqueous layer and surface of the oil were washed gently three times, and the oil and remaining water were aspirated. The cell pellet was lysed with 0.4 ml 1% (v/v) Triton X-100, deproteinized with 0.75 ml 5.8% (w/v) trichloroacetic acid, and centrifuged at 16,000 g for 5 min. A 1 ml supernatant was then added to scintillant (0.5% (w/v) 2,5-diphenylosazole in 1:2 (v/v) Triton X-100:toluene) and the radioactivity was counted. The intracellular radioactivity was expressed as nmol/(mg cell protein) for the uptake studies and as a percentage of that measured in cells with normal volumes for the efflux studies.

Flux studies were also performed in the presence of inhibitors. The preincubation time and concentration dependencies of the effect of p-chloromercuribenzoate (pCMB) on hyposmotically activated alanine release (at 214 mosmol/kg) showed that the optimal conditions involve a 10 min preincubation of cells with 0.2 mM pCMB at isosmotic PBS (data not shown), and these conditions were used for the inhibition studies. Without preincubation, pCMB of up to 0.5 mM did not affect hyposmotically activated alanine release, indicating that its effect originates from covalent binding of pCMB to thiols. The step of washing to remove the unreacted reagents after preincubation was unnecessary and thus omitted.

For amino acid analysis, 0.25 ml stock cell suspensions were added to 0.75 ml isosmotic or hyposmotic PBS. At predetermined intervals, duplicate 0.4 ml aliquots were removed and centrifuged directly through oil into 0.1 ml 20% (w/v) sulphosalicylic acid and 0.02 ml 5 mM ß-alanine. The mixture containing the cell pellet was assayed for the intracellular amino acids by using a Beckman system 6300 amino acid analyzer (21).

Data analysis
Data are analyzed by paired or unpaired Student's t test. P < 0.01 was considered a statistically significant difference. A lag period observed from the flux studies or cell volume changes was estimated from the fitted line of the data to a nonlinear least squares iteration according to the Marquardt algorithm, using the equation: Y(t) = At + A/B[exp(-Bt)-1], where Y(t) is a relative cell volume or intracellular AIB content at time t, and A and B are fitted constants. Most data obtained from the flux experiments are presented as means ± SEM for n observations.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The `all-or-nothing' type of swelling detection at the threshold volume for the transient activation of GSAAT
In response to the decrease in medium osmolarity greater than 40 mosmol from isosmolarity (308 mosmol/kg), Giardia trophozoites swelled ( Fig. 1a). Maximal cell volume was a linear function of osmolarity shift and a line fitted to the data intersected to the X axis at close to isosmotic volume ( Fig. 1a, inset), indicating that cellular solutes were not changed during the swelling period. At the 40 mosmol decrease from isosmolarity ( Fig. 1a), RVD did not follow cell swelling. However, RVD was observed at decreases greater than 60 mosmol. Under these conditions, lowering the medium osmolarity had no effect on the slope of RVD but significantly increased the time required for the completion of RVD. Uptake of AIB, a marker for GSAAT activity (21, 22), was measured under these medium osmolarities ( Fig. 1b). Uptake was negligible at the 40 mosmol decrease from isosmolarity, consistent with the lack of RVD under these conditions. At decreases greater than 60 mosmol, uptake was observed after a lag period of 1.3 ± 0.3 min (n=4). However, osmolarity changes did not affect either the lag period or the uptake rate, which varied between 1.2 ± 0.3 and 1.3 ± 0.4 min (n=4) or between 0.38 ± 0.03 and 0.42 ± 0.02 nmol/(mg protein)/min (n=4), respectively.

View larger version (30K): [in this window] [in a new window]   Figure 1. The relations of giardial cell volume changes to GSAAT activity. a) Cell volume changes were measured after exposure, at time zero, to 308 (+), 270 (—), 248 (), 214 (), 180 (), or 157 () mosmol/kg. The traces included in the lines were used to estimate the slope of RVD. Inset shows maximal cell volume at a given osmolarity change ( ). Values are means ± SEM (n=21). b) Uptake of AIB was measured after Giardia trophozoites were exposed to 270 (), 248 (), 214 (), 180 (), or 157 () mosmol/kg. Values are averages of four separate determinations. For most data points, the standard error was smaller than the symbol size; error bars have been omitted for clarity. c) At a given osmolarity, maximal cell volume was estimated from cell swelling curves, similar to Fig. 1a, and the final concentration of AIB taken up was estimated from the plateau of AIB uptake. Values are means ± SEM (n=6). Where not shown, errors were smaller than the symbols. d) At 0 (), 2 (), 4 (), or 8 () min after cells were hyposmotically swollen at 214 mosmol/kg (), AIB uptake was initiated by the addition of 1 mM [14C]AIB. Values are means ± SEM (n5).

Since the rates of RVD and AIB uptake were fixed at varying medium osmolarities, the relatedness of RVD to AIB uptake could be predicted by comparing maximal cell volume to the amount of AIB taken up at a given osmolarity. As shown in Fig. 1c, the total amount of AIB taken up increased in direct proportion to maximal cell volume. The extrapolation of a line fitted to the data intercepted the X axis at an 1.060 ± 0.005 x isosmotic volume (IV) (n=12), indicating that AIB uptake will not occur at a cell volume of less than 1.06 x IV. This value fits well with the data shown in Fig. 1a, b. Lack of RVD observed at the 40 mosmol decrease from isosmolarity is due to insufficient cell swelling to activate GSAAT. In addition, RVD plateaued coincidentally at volumes similar to this value. Thus, a cell volume of 1.06 x IV represents the threshold volume at which cell swelling is sensed for subsequent GSAAT activation.

If the threshold volume is the only factor regulating GSAAT, its activity should remain unchanged at cell volumes above the threshold volume. To investigate this possibility, we measured AIB uptake at various cell volumes ( Fig. 1d). At 2 or 4 min after hyposmotic challenge, cell volume was approximately 1.16 or 1.11 x IV, respectively. However, despite these volume differences, the uptake rates remained similar to that measured at the initiation of cell swelling [the rate (nmol/(mg protein)/min)=0.40±0.01 at 0 min, 0.41±0.02 at 2 min, and 0.36±0.07 at 4 min, n4]. This finding indicates strongly that Giardia senses cell swelling at the threshold volume via an `all-or-nothing' manner for transient activation of GSAAT.

pCMB inhibits GSAAT by increasing the threshold volume
In normal Giardia the 60 mosmol decrease from isosmolarity was sufficient to reach cell volumes above the threshold volume, causing transient activation of GSAAT ( Fig. 1a, b). However, in pCMB-treated cells, RVD was completely abolished at this osmolarity shift ( Fig. 2a). Further decreases in medium osmolarity characterized RVD, which completed at cell volumes of approximately 1.14 x IV, well above the maximal cell swelling induced by the 60 mosmol decrease from isosmolarity. These findings indicate that inhibition of GSAAT by pCMB results from an increase in threshold volume. We also found the reduced slope of RVD in pCMB-treated cells [the slope of RVD (1/h)=-0.72±0.12 vs. -2.35±0.15 in untreated cells, n12; P<0.001] and determined that this was due to the inhibition of flux shown in Fig. 2b. This finding suggests that the pCMB effect on GSAAT results in part from the direct inhibition of transport activity.

View larger version (42K): [in this window] [in a new window]   Figure 2. The effect of pCMB on cell volume change and GSAAT activity under varying medium hyposmolarities. a) Cells were preincubated for 10 min in isosmotic PBS in the presence (symbols) or absence (solid lines) of 0.2 mM pCMB. At the time marked by an arrowhead, medium was made hyposmotic [248 (), 214 (), or 180 () mosmol/kg]. Traces included in the lines were used to estimate the slope of RVD. b) [3H]Alanine-loaded cells were preincubated in isosmotic PBS for 10 min in the presence (open symbols) or absence (filled symbols) of 0.2 mM pCMB. Medium was then made hyposmotic [248 (), 214 (), or 180 () mosmol/kg] and intracellular radioactivity was measured. Values are averages of four separate determinations. Error bars have been omitted for clarity. c) Inhibition: the loaded cells were incubated for 10 min in isosmotic PBS with or without 0.2 mM pCMB. Reactivation: half of the cells in each group were incubated with 5 mM DTT in isosmotic PBS for 5 min; the remaining half were incubated with PBS only. At the end of the second incubation period, groups of cells were then incubated for 5 min at 214 mosmol/kg. Values are expressed as percentages of the radioactivity released from the untreated control cell and are means ± SEM (n=4).

pCMB is not absolutely specific for thiols, but also reacts with other functional groups such as carboxyl, amino, and tyrosyl, producing stable derivatives of benzoic acid (24). The product of pCMB binding to a thiol, mercaptide, however, is labile and the reaction is reversed in the presence of excess thiols (25). This property was used to investigate whether inhibition of GSAAT by pCMB was primarily through the thiol binding. As shown in Fig. 2c, GSAAT inhibition by pCMB was completely reversed by the addition of dithiothreitol (DTT). As a control, treatment of cells with DTT alone had no effect on GSAAT activity.

NEM activates GSAAT by lowering the threshold volume, resulting in cell shrinkage under isosmotic conditions
Figure 3a shows the effect of N-ethylmaleimide (NEM) on giardial cell volume. It was found that cells begin to shrink under isosmotic conditions (1/h=-2.45±0.12, n=11). NEM-induced cell shrinkage was preceded by a lag period of 1.7 ± 0.3 min (n=12). The subsequent flux studies in the NEM-treated cells showed a drastic increase in the unidirectional release of intracellular alanine ( Fig. 3b) and uptake of AIB ( Fig. 3c) during cell shrinkage. Uptake nearly ceased at 14 min after NEM treatment, indicating the NEM effect is transient. We also analyzed the effect of NEM on intracellular amino acids ( Fig. 3d). In NEM-treated cells, the total amino acid pool was significantly decreased. This decrease was due mainly to the selective reduction of the intracellular neutral amino acids alanine, glycine, and valine, excluding the possibility of membrane damage by NEM leading to an increase in membrane permeability in a nonspecific manner. The pattern of these changes corresponded well to that observed in hyposmotically swollen cells (21). Overall, these findings indicate that NEM, like cell swelling, can induce the transient activation of GSAAT, which causes cell shrinkage under isosmotic conditions.

View larger version (29K): [in this window] [in a new window]   Figure 3. The effect of NEM on giardial cell volume, alanine release, AIB uptake, and intracellular amino acids under isosmotic conditions. a) At time zero, cells were exposed to isosmotic PBS containing 0.1 mM NEM (). For the effect of pCMB on NEM-induced cell shrinkage (), cells were incubated with 0.2 mM pCMB for 10 min prior to exposure to 0.1 mM NEM. b) Intracellular [3H]alanine was measured in the loaded cells treated with 0.1 mM NEM. Values are means ± SEM (n=3). c) AIB uptake was initiated by the addition of 1 mM [14C]AIB at time zero or 14 min after NEM treatment. Values are means ± SEM (n=3). d) Intracellular amino acids were analyzed in NEM-treated cells at the predetermined intervals and expressed as nmol/(µl cell volume), based on the calculation that 1 mg of cell protein represents 4 µl of intracellular water space (21). The seven amino acids shown represent 89% of the total amino acid pool under isosmotic conditions. Values are averages of two separate determinations.

To investigate whether NEM and cell swelling activate GSAAT via the same pathway, we measured the effect of NEM on the cells that were preexposed to hyposmotic medium for a selected time period ( Fig. 4a). NEM treatment at 0.5, 4, or 8 min represents the cells at the swelling, RVD, or post-RVD period, respectively. When the cells at the post-RVD period were treated with NEM, they proceeded to shrink after a lag period. When added to the cells undergoing cell swelling or RVD, NEM did not alter the slope of RVD, but increased the duration of RVD, resulting in RVD being complete at cell volumes below the isosmotic volume. These findings suggest that NEM activates GSAAT not by affecting transport activity, but by altering the sensor that detects cell swelling.

View larger version (37K): [in this window] [in a new window]   Figure 4. The effect of medium osmolarity on NEM-induced cell shrinkage. a) At time zero, cells were exposed to hyposmotic medium of 214 mosmol/kg (), to which 0.1 mM NEM was added at the times marked by arrowheads. The time intervals were 0.5 (), 4 (), and 8 (X) min. b) Cells were initially exposed to isosmotic PBS () or to hyperosmotic medium [340 (), 370 (), or 400 () mosmol/kg], to which 0.1 mM NEM was added at the times marked by arrowheads.

When pCMB-treated cells were treated with NEM under isosmotic conditions ( Fig. 3a), cell shrinkage was inhibited and plateaued at cell volumes greater than those observed in untreated cells (0.95±0.02 xIV, n=18; P<0.001). Since pCMB inhibited GSAAT by increasing the threshold volume ( Fig. 2a), it was conceivable that NEM may activate GSAAT by lowering the threshold volume to below the isosmotic volume. Figure 4b shows the relationships between NEM-induced cell shrinkage and ambient hyperosmolarity. Plus (+) symbols show cell shrinkages induced by an increase in medium osmolarity of up to 130% isosmolarity (the post-cell shrinkage=0.96, 0.92, or 0.85xIV at 340, 370, or 400 mosmol/kg, respectively). At the cell volume of 0.96 or 0.92 x IV, NEM treatment induced cell shrinkage, with a pattern similar to that observed under isosmotic conditions. However, these initial volume decreases significantly reduced the extent of NEM-induced cell shrinkage that completed coincidentally at approximately 0.85 x IV. In cells at this volume, NEM-induced cell shrinkage was completely abolished. These findings indicate that GSAAT activation by NEM is caused primarily by lowering the threshold volume where the volume sensor is present.

The location of pCMB-and NEM-sensitive thiols
To investigate the location of pCMB- and NEM-sensitive thiols, two other thiol reagents—p-chloromercuribenzene sulfonate (pCMBS) and 3-(N-maleimidopropinyl)biocytin (MPB)—were tested for their effect on isosmotic or perturbed cell volume at 214 mosmol/kg ( Table 1). pCMBS is a structural analog of pCMB, but in pCMBS the weakly acidic carboxyl function of pCMB (pK 4) is replaced by a strongly acidic sulfonic acid (pK 1.5), yielding a water-soluble agent (26). Since octanol/water partition coefficients of pCMB and pCMBS are 1.95 x 10^-2 and 5.46 x 10^-3, respectively (27), pCMB is thought to be more hydrophobic than pCMBS and interacts easily with thiol groups within the membrane. MPB shares with NEM a functional maleimide group, but due to its bulk size and the possession of an ionizable carboxyl group, it is hydrophilic and membrane impermeant (28). pCMBS showed an effect similar to that of pCMB on giardial cell volume; it had no effect on either isosmotic cell volume or maximal cell swelling, but decreased RVD. However, direct comparison of inhibition of RVD by both inhibitors showed that at 0.2 mM pCMBS was a less potent inhibitor of GSAAT than pCMB (P<0.01). Despite possessing a functional group structure identical to that of NEM, at concentrations of up to 0.5 mM, MPB had no effect on isosmotic cell volume or on cell swelling followed by RVD.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Here we demonstrate that Giardia intestinalis, an archezoan `biological fossil' of eukaryotic evolution, senses a certain cell volume—a threshold volume—for volume regulation. The threshold volume was only 6% greater than isosmotic cell volume, which was undistinguishable when using intracellular water space measurement (a method often applied for monitoring cell volume changes) due to the high inherent standard error (21). Two lines of evidence, albeit indirect, indicate that detection of cell swelling at a threshold volume may represent a common feature regulating SA transport in present-day eukaryotes. First, in most eukaryotes cell volume does not fully recover after RVD, but remains larger than the original volume (2, 3, 29), indicating the involvement of a cell volume other than isosmotic volume for inactivation of SA transports. Second, due to the lack of a rigid cell wall, eukaryotic cells behave in an osmometric fashion: the relationship between the rate of cell swelling and the osmotic gradient is linear (22). This property of cell volume has been used routinely to determine the membrane permeability of water (30). However, activation of SA transports is, in most cases, a nonlinear, sigmoidal function of an increase in cell volume with a threshold at close to the isosmotic volume, suggesting the presence of threshold volume (9–11, 14, 31).

The diagram below illustrates a model integrating a variety of observations in the literature with our findings and demonstrates how eukaryotic cells may sense swelling and activate SA transports. Cell swelling is sensed at the threshold volume, allowing the transition of transport from a `resting' (R) to an `open' (A) state. The resultant A state becomes independent of a volume signal. As indicated above, this phase may represent an evolutionarily conserved part of transport-mediated volume regulation in eukaryotes. For transport with activity related linearly to the extent of osmotic shifts, the A state is further subjected to up-regulation by something other than volume per se, probably [as suggested by others (11, 12, 14, 15)] by attendant changes in cellular impermeants. As a result, the A' state mimics the graded response to the extent of cell swelling. Recent studies of SA transports have focused on the detection of regulators involved in this graded response phase and have determined a variety of cytoplasmic factors that control SA transport in a cell type- and transport-specific manner (1–3). An important implication of the proposed model is that activation/inactivation of transport (R to A conversion) is an event distinct from regulation of the activated transport (A to A' conversion). A' state transports, such as KCl cotransport, would thereby require two signals—one volume-related and the other probably not related—for the graded response to cell swelling. The requirement of these two signals for fully functional KCl cotransport has recently been reported (32).

This report also provides evidence that, whatever the molecular identity of the volume sensor, modulation of the associated thiol moieties represents the mechanism by which a volume signal is transduced at the threshold volume. In Giardia, irreversible modification of the reduced thiols by pCMB increased the threshold volume from 1.06 to 1.14 x IV, with sensitivity depending on the hydrophobicity of thiol reagents (pCMB>pCMBSMPB, Fig. 2a and Table 1). In contrast, NEM treatment decreased the threshold volume to 0.85 x IV ( Fig. 3a, Fig. 4b). The NEM covalent reaction required for GSAAT activation was complete in less than 10 s, during which time our amino acid analysis data showed no significant changes in the intracellular level of cysteine, the major low-molecular weight thiol in Giardia (33), which might otherwise occur by sequestering the intracellular NEM (data not shown). Thus, these findings indicate that the target thiols for NEM, like the pCMB-sensitive thiols, reside at the plasma membrane. The inability of pCMB to react with NEM-sensitive thiols is not readily explicable. One possible explanation is that steric constraints prevent pCMB from reaching the stimulatory thiols. Alternatively, it is possible that NEM may participate in the reactions that are chemically distinct from pCMB binding to a thiol, since the electron acts as a leaving group. The functional importance of reduced thiols in ligand translocation has been implicated in a variety of transporters; their sequestering by thiol reagents inhibits transport activity. However, activation by thiol reagents is frequently observed in SA transports (34–36). Activation by pCMB has been reported in SA taurine release in astrocytes (34). KCl cotransport in erythrocytes from a variety of sources is activated by NEM treatment under isosmotic conditions (11, 35). Similar to GSAAT, this cotransport requires a lag period prior to activation either by cell swelling or NEM treatment under isosmotic conditions. These observations support the hypothesis that modulation of the reduced thiols in the volume sensor may commonly be the hitherto missing link between cell swelling and the underlying messenger systems.

The simple modification of the threshold volume by NEM caused a drastic effect on giardial cell volume, as cell shrinkage occurred under isosmotic conditions. This mode of regulation by NEM of giardial volume sensing is strikingly similar in nature to that of the mitochondrial voltage-dependent permeability transition pore, in which opening of the pore is preceded by a lag period, proceeds via an `all-or-nothing' manner, and NEM increases the periods of the opening state by shifting its gating potential to higher values (37, 38). Recent reports indicate that the pore opening, followed by mitochondrial swelling, is a causative event in cell death due to oxidative stress and other causes (38). Bcl-2 and Bcl-X_L have also been shown to exert their antiapoptotic function by preventing mitochondrial swelling (39, 40). Since GSAAT activation by NEM caused a drastic reduction of cell volume that is also a fundamental feature of apoptosis, it is possible that an intracellular component showing this NEM-mimetic effect on the sensor may play a crucial role in the control of programmed cell death.

There is now substantial evidence that SA transport of organic solutes, such as taurine and polyol, in vertebrate cells occurs via an anion channel, collectively termed VSOAC (20). However, unlike the vertebrate counterpart, GSAAT is insensitive to chloride channel blockers including furosemide and niflumate (21), indicating the apparent difference in transport proteins for both systems. Since volume regulation is one of the parameters that determines cell life or death in a given habitat, GSAAT has the potential to provide new targets for the development of chemotherapeutics against giardiasis. Likewise, this may provide a new site of chemotherapy against a range of protozoan parasite infections, since the release of intercellular alanine is commonly responsible for volume regulation in other protozoan parasites, including Leishmania and Crithidia (31, 41). In addition, the finding that the threshold volume can be modulated and induce the activation of SA transport under physiological conditions has an important pathophysiological implication. Giardia-induced diarrhea involves transient calcium accumulation in infected intestinal cells, followed by the net efflux of electrolytes and the obliged water (42, 43). This process is similar to the response of uninfected host cells to hyposmotic stress via activation of the SA channel (44, 45). Striking similarities are also reported between the VSOAC in erythrocytes and the pathway responsible for the enhanced permeability of malaria-infected erythrocytes (46); the possible role of band 3, a candidate for VSOAC (47, 48), in this process has been suggested (49). Thus, it is highly probable that these parasite-induced pathways may result from an alteration by parasites of the threshold volume for SA transports in the host cells rather than from de novo synthesis, followed by membrane targeting, or from directly affecting the host SA transporters.

	ACKNOWLEDGMENTS

 
We thank George Grossman for technical support. This work was supported by grants from the DEET (to J.-H.P.) and ARC (to P.J.S. and M.R.E.) in Australia.

	FOOTNOTES

 
¹ Correspondence: Seattle Biomedical Research Institute, 4 Nickerson St., Seattle, WA 98109, USA. E-mail: parkjh{at}u.washington.edu

² Abbreviations: SA, swelling-activated; GSAAT, giardial swelling-activated alanine transport; RVD, regulatory volume decrease; AIB, 2-aminoisobutyric acid; PBS, phosphate-buffered saline; IV, isosmotic volume; pCMB, p-chloromercuribenzoate; NEM, N-ethylmaleimide; DTT, dithiothreitol; pCMBS, p-chloromercuribenzene sulfonate; MPB, 3-(N-maleimidopropinyl)biocytin.

Received for publication November 21, 1997. Accepted for publication January 7, 1998.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Häussinger, D., and Lang, F. (1991) Cell volume in the regulation of hepatic function: a mechanism for metabolic control. Biochim. Biophys. Acta 1071, 331–350[Medline]
2. Sarkadi, B., and Parker, J. C. (1991) Activation of ion transport pathways by changes in cell volume. Biochim. Biophys. Acta 1071, 407–427[Medline]
3. Chamberlin, M. E., and Strange, K. (1989) Anisosmotic cell volume regulation: a comparative view. Am. J. Physiol. 257, C159–C173[Abstract/Free Full Text]
4. Krause, U., Rider, M. H., and Hue, L. (1996) Protein kinase signaling pathway triggered by cell swelling and involved in the activation of glycogen synthase and acetyl-CoA carboxylase in isolated rat hepatocytes. J. Biol. Chem. 271, 16668–16673[Abstract/Free Full Text]
5. Waldegger, S., Barth, P., Raber, G., and Lang, F. (1997) Cloning and characterization of a putative human serine/threonine protein kinase transcriptionally modified during anisotonic and isotonic alterations of cell volume. Proc. Natl. Acad. Sci. USA 94, 4440–4445[Abstract/Free Full Text]
6. Sadoshima, J., Qiu, Z., Morgan, J. P., and Izumo, S. (1996) Tyrosine kinase activation is an immediate and essential step in hypotonic cell swelling-induced ERK activation and c-fos gene expression in cardiac myocytes. EMBO J. 15, 5535–5546[Medline]
7. Philips, J., and Herskowitz, I. (1997) Osmotic balance regulates cell fusion during mating in Saccharomyces cerevisiae. J. Cell Biol. 138, 961–974[Abstract/Free Full Text]
8. Davenport, K. R., Sohaskey, M., Kamada, Y., Levin, D. E., and Gustin, M. C. (1995) A second osmosensing signal transduction pathway in yeast. Hypotonic shock activates the PKC1 protein kinase-regulated cell integrity pathway. J. Biol. Chem. 270, 30157–30161[Abstract/Free Full Text]
9. Kirk, K., Ellory, J. C., and Young, J. D. (1992) Transport of organic substrates via a volume-activated channel. J. Biol. Chem. 267, 23475–23478[Abstract/Free Full Text]
10. Jackson, P. S., and Strange, K. (1993) Volume-sensitive anion channels mediate swelling-activated inositol and taurine efflux. Am. J. Physiol. 265, C1489–C1500[Abstract/Free Full Text]
11. Starke, L. C., and Jennings, M. L. (1993) K-Cl cotransport in rabbit red cells: further evidence for regulation by protein phosphatase type 1. Am. J. Physiol. 264, C118–C124[Abstract/Free Full Text]
12. Tilly, B. C., van den Berghe, N., Tertoolen, L. G. J., Edixhoven, M. J., and de Jonge, H. R. (1993) Protein tyrosine phosphorylation is involved in osmoregulation of ionic conductances. J. Biol. Chem. 268, 19919–19922[Abstract/Free Full Text]
13. Schwiebert, E. M., Millis, J. W., and Stanton, B. A. (1994) Actin-based cytoskeleton regulates a chloride channel and cell volume in a renal cortical collecting duct cell line. J. Biol. Chem. 269, 7081–7089[Abstract/Free Full Text]
14. Parker, J. C. (1993) In defense of cell volume? Am. J. Physiol. 265, C1191–C1200[Abstract/Free Full Text]
15. Minton, A. P., Colclasure, G. C., and Parker, J. C. (1992) Model for the role of macromolecular crowding in regulation of cellular volume. Proc. Natl. Acad. Sci. USA 89, 10504–10506[Abstract/Free Full Text]
16. Kracke, G. R., and Dunham, P. B. (1992) Volume-sensitive K-Cl cotransport in inside-out vesicles made from erythrocytes membranes from sheep of the low-K phenotype. Proc. Natl. Acad. Sci. USA 87, 8575–8579[Abstract/Free Full Text]
17. Adam, R. D. (1991) The biology of Giardia spp. Microbiol. Rev. 55, 706–732[Abstract/Free Full Text]
18. Farthing, M. J. G. (1992) . New perspectives in giardiasis. J. Med. Microbiol. 37, 1–2[Medline]
19. Sogin, M. L., Gunderson, J. H., Elwood, H. J., Alonso, R. A., and Peattie, D. A. (1989) Phylogenic meaning of the kingdom concept: an unusual ribosomal RNA from Giardia lamblia. Science 243, 75–77[Abstract/Free Full Text]
20. Strange, K., Emma, F., and Jackson, P. S. (1996) Cellular and molecular physiology of volume-sensitive anion channels. Am. J. Physiol. 270, C711–C730[Abstract/Free Full Text]
21. Park, J. H., Schofield, P. J., and Edwards, M. R. (1995) The role of alanine in the acute response of Giardia intestinalis to hypo-osmotic shock. Microbiology 141, 2455–2462
22. Park, J. H., Schofield, P. J., and Edwards, M. R. (1997) Giardia intestinalis: Volume recovery in response to cell swelling. Exp. Parasitol. 86, 19–28[Medline]
23. Lowry, O. H., Rosebrough, N. H., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275[Free Full Text]
24. Rothstein, A. (1970) In Current Topics in Membranes and Transport (Bronner, F., and Kleinzeller, A., eds) pp. 135–176, Academic Press, New York
25. Peerce, B. E., Cedilote, M., and Clarke, R. D. (1993) Role of carboxyl and sulfhydryl residues on rabbit small intestinal brush-border membrane Na⁺-glucose cotransporter. Am. J. Physiol. 264, G294–G299[Abstract/Free Full Text]
26. Yan, R.-T., and Maloney, P. C. (1993) Identification of a residue in the translocation pathway of a membrane carrier. Cell 75, 37–44[Medline]
27. Mannuzzu, L. M., Moronne, M. M., and Macey, R. I. (1993) Estimate of the number of urea transport sites in erythrocyte ghosts using a hydrophobic mercurial. J. Membr. Biol. 133, 85–97[Medline]
28. Zhen, R.-G., Kim, E. J., and Rea, P. A. (1994) Localization of cytosolically oriented maleimide-reactive domain of vaculoar H⁺-pyrophosphatase. J. Biol. Chem. 269, 23342–23350[Abstract/Free Full Text]
29. Dall'Asta, V., Rossi, P. A., Bussolati, O., and Gazzola, G. C. (1994) Regulatory volume decrease of cultured human fibroblasts involves changes in intracellular amino-acid pool. Biochim. Biophys. Acta 1220, 139–145[Medline]
30. van Os, C. H., Deen, P. M. T., and Dempster, J. A. (1994) Aquaporins: water selective channels in biological membranes. Molecular structure and tissue distribution. Biochim. Biophys. Acta 1197, 291–309[Medline]
31. Bursell, J. D. H., Kirk, J., Hall, S. T., Gero, A. M., and Kirk, K. (1996) Volume-regulatory amino acid release from the protozoan parasite Crithidia luciliae. J. Membr. Biol. 154, 131–141[Medline]
32. Kelley, S. J., and Dunham, P. B. (1996) Mechanism of swelling activation of K-Cl cotransport in inside-out vesicles of LK sheep erythrocyte membranes. Am. J. Physiol. 270, C1122–C1130[Abstract/Free Full Text]
33. Brown, D. M., Upcroft, J. A., and Upcroft, P. (1993) Cysteine is the major low-molecular weight thiol in Giardia duodenalis. Mol. Biochem. Parasitol. 61, 155–158[Medline]
34. Martínez, A., Muñoz-Clares, R. A., Guerra, G. Morán, J., and Pasantes-Morales, H. (1994) Sulfhydryl groups essential for the volume-sensitive release of taurine from astrocytes. Neurosci. Lett. 176, 239–242[Medline]
35. Lauf, P. K., Bauer, J., Adragna, N. C., Fujise, H., Zade-Oppen, A. M. M., Ryu, K. H., and Delpire, E. (1992) Erythrocyte K-Cl cotransport: properties and regulation. Am. J. Physiol. 263, C917–C932[Abstract/Free Full Text]
36. Koo, S.-P., Higgins, C. F., and Booth, I. R. (1991) Regulation of compatible solute accumulation in Salmonella typhimurium: evidence for a glycine betaine efflux system. J. Gen. Microbiol. 137, 2617–2625[Medline]
37. Bernardi, P., Vassanelli, S., Veronese, P., Colonna, R., Szabo, I., and Zoratti, M. (1992) Modulation of the mitochondrial permeability transition pore. Effect of protons and divalent cations. J. Biol. Chem. 267, 2934–2939[Abstract/Free Full Text]
38. Petronilli, V., Cstantini, P., Scorrano, L., Colonna, R., Passamonti, S., and Bernardi, P. (1994) The voltage sensor of the mitochondrial permeability transition pore is tuned by the oxidation-reduction state of vicinal thiols. J. Biol. Chem. 269, 16638–16642[Abstract/Free Full Text]
39. Zamzami, N., Susin, S. A., Marcetti, P., Hirsch, T., Gomez-Monterrey, I., Castedo, M., and Kroemer, G. (1996) Mitochondrial control of nuclear apoptosis. J. Exp. Med. 183, 1533–1544[Abstract/Free Full Text]
40. Vander Heiden, M. G., Chandel, N. S., Williamson, E. K., and Schumacker, P. T. (1997) Bcl-X_L regulates the membrane potential and volume homeostasis of mitochondria. Cell 91, 627–637[Medline]
41. Darling, T. N., Burrows, C. M., and Blum, J. J. (1990) Rapid shape change and release of ninhydrin-positive substances by Leishmania major promastigotes in response to hypo-osmotic stress. J. Protozool. 37, 493–499[Medline]
42. Gorowara, S., Ganguly, N. K., Mahajan, R. C., and Walia, B. N. (1992) Study on the mechanism of Giardia lamblia induced diarrhoea in mice. Biochim. Biophys. Acta 1138, 122–126[Medline]
43. Gorowara, S., Ganguly, N. K., Mahajan, R. C., and Walia, B. N. (1994) Involvement of intracellular calcium stores in Giardia lamblia induced diarrhoea in mice. FEMS Microbiol. Lett. 120, 231–236[Medline]
44. Hazama, A., and Okada, Y. (1990) Biphasic rises in cytosolic free Ca²⁺ in association with activation of K⁺ and Cl^- conductance during the regulatory volume decrease in cultured human epithelial cells. Pfluegers Arch. 416, 710–714[Medline]
45. Tilly, B. C., Edixhoven, M. J., van den Berghe, N., Bot, A. G., and de Jonge, H. R. (1994) Ca(²⁺)-mobilizing hormones potentiate hypotonicity-induced activation of ionic conductances in intestine 407 cells. Am. J. Physiol. 267, C1271–C1278[Abstract/Free Full Text]
46. Kirk, K., and Horner, H. A. (1995) Novel anion dependence of induced cation transport in malaria-infected erythrocytes. J. Biol. Chem. 270, 24270–24275[Abstract/Free Full Text]
47. Fievet, B., Gabillat, N., Borgese, F., and Motais, R. (1995) Expression of band 3 anion exchanger induces chloride current and taurine transport: structure-function analysis. EMBO J. 14, 5158–5169[Medline]
48. Goldstein, L., and Brill, S. R. (1991) Volume-activated taurine efflux from skate erythrocytes: possible band 3 involvement. Am. J. Physiol. 260, R1014–R1020[Abstract/Free Full Text]
49. Schofield, A. E., Reardon, D. M., and Tanner, M. J. A. (1992) Defective anion transport activity of the abnormal band 3 in hereditary ovalocytic red blood cells. Nature (London) 355, 836–837[Medline]