School of Biochemistry and Molecular Biology, University of New South Wales, Sydney, NSW 2052, Australia1
Division of Biochemistry and Molecular Biology, Australian National University, Canberra, ACT 0200, Australia2
Author for correspondence: Giancarlo A. Biagini. Tel: +61 2 9385 2043. Fax: +61 2 9385 1483. e-mail: G.Biagini{at}unsw.edu.au
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: anaerobic protozoa, osmoregulation, volume regulation, membrane transport, alanine
Abbreviations: [1-14C]AIB, 2-amino[1-14C]isobutyrate; DIDS, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid disodium salt; NEM, N-ethylmaleimide; NPPB, 5-nitro-2-(3-phenylpropylamino)benzoic acid; RVD, regulatory volume decrease
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Free-living Hexamita species have been observed in low-O2 marine and freshwater environments (Fenchel et al., 1995 ; Fenchel & Finlay, 1995
). In addition, parasitic species of Hexamita (which may have free-living life stages) have been observed in a variety of organisms, both invertebrate (e.g. shellfish; Kulda & Nohýnková, 1978
) and vertebrate (e.g. fish; Buchmann et al., 1995
; Ferguson, 1979
); in this context, Hexamita spp. are of substantial economic significance.
In adapting to the variety of habitats in which it is found, Hexamita must cope with extreme variations in the osmolality of its environment. Such variations pose a challenge to cell volume. In many protozoa, osmoregulation and cell volume control is achieved via the operation of a contractile vacuole. However, Hexamita reportedly lacks this organelle (Brugerolle, 1974 ). Other protozoa rely on the modulation of intracellular levels of osmotically active solutes (osmolytes) via the operation of volume-sensitive membrane transport pathways. For example, the closely related diplomonad Giardia intestinalis possesses a large pool of free amino acids (Knodler et al., 1994
). Upon exposure to hypo-osmotic stress, G. intestinalis has been shown to release amino acids, principally alanine (present at approx. 50 mM; Knodler et al., 1994
), via swelling-activated transport pathways (Park et al., 1995
, 1997
). The efflux of amino acids, perhaps together with other intracellular solutes, results in a net loss of water from the swollen cells and thereby allows G. intestinalis to undergo a regulatory volume decrease (RVD), back to its initial cell volume (Park et al., 1995
, 1997
, 1998
). The use of alanine as the principal organic osmolyte has also been observed in a range of other protozoa including Crithidia luciliae (Bursell et al., 1996
), Acanthamoeba castellanii (Geoffrion & Larochelle, 1984
) and Leishmania major (Darling et al., 1990
; Vieira et al., 1996
).
In this study we have investigated the ability of H. inflata to regulate its volume in aniso-osmotic media and have characterized the mechanisms involved.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell volume measurements.
Total cell volumes were measured using a Coulter Multisizer fitted with a 100 µm aperture tube. Signals from the Multisizer were collected on Multisizer AccuComp software. Results are expressed as the mean of the cell volume distribution from approximately 4000 cells for each time point. Intracellular water space was measured as described by Knodler et al. (1994) , with the exception that [1-14C]mannitol was used rather than [1-14C]inulin to estimate the extracellular water volume in the cell pellets.
Amino acid analysis.
Intracellular amino acid analysis was performed as described by Knodler et al. (1994) , with ß-alanine as the internal standard, on a Beckman 6300 amino acid analyser.
2-Amino[1-14C]isobutyrate influx.
Volume-sensitive amino acid transport was investigated using 2-amino[1-14C]isobutyrate ([1-14C]AIB), a non-metabolizable analogue of alanine. [1-14C]AIB influx measurements were carried out essentially as described by Park et al. (1995 , 1998
). H. inflata cell suspensions (typically 24x107 cells ml-1) in PBS of varying osmolality were incubated with [1-14C]AIB [specific activity 0·5 µCi mol-1 (37 kBq mol-1)]. At specific time intervals aliquots of the suspension were overlaid onto oil (a mixture of dibutyl phthalate and diiso-octyl phthalate, 4:1, v/v; 1·03 g ml-1) in a microcentrifuge tube and centrifuged at 10000 g for 20 s, sedimenting the cells below the oil. Cell pellets were lysed with 0·1% (v/v) Triton X-100, precipitated with 6% (w/v) trichloroacetic acid and centrifuged at 10000 g for 30 s. For scintillation counting, samples (1 ml) were mixed with scintillant (9 ml) containing 0·5% (w/v) 2,5-diphenyloxazole in Triton X-100/toluene (1:2, v/v).
86Rb+ and [1-14C]AIB efflux.
The involvement of K+ in the volume regulatory response was investigated using 86Rb+ as a congener for K+. Cells were harvested by gentle centrifugation (650 g) and preloaded with 86Rb by incubation with 86RbCl [12 µCi ml-1 (3774 kBq ml-1)] for 2·5 h in fresh culture medium. After the pre-incubation the cells were washed (three times) to remove extracellular 86Rb+ and resuspended in PBS. Efflux time-courses commenced with the addition of an aliquot of the 86Rb+-loaded cells into either iso-osmotic PBS or hypo-osmotic (diluted with H2O) PBS of varying osmolality. At predetermined time intervals aliquots were taken and centrifuged through the oil mix. Supernatant and pellet fractions were processed as described above.
For dual-labelling experiments, designed to monitor concomitantly the efflux of [1-14C]AIB and 86Rb+, cells were loaded with [1-14C]AIB by subjecting them to a hypo-osmotic shock (150 mosmol kg-1) in the presence of [1-14C]AIB (as described by Park et al., 1998 ). The [1-14C]AIB-loaded cells cells were then washed in PBS, loaded with 86Rb+ (12 µCi ml-1; 2·5 h), and subjected to the efflux protocol described above. The concentrations of [1-14C]AIB and 86Rb+ in the dual-label samples were estimated by measuring 14C and 86Rb radioactivity over different ranges and subtracting the contribution of 86Rb to the c.p.m. in the 14C range.
Suspension of cells in media having an osmolality of 150 mosmol kg-1 caused a rapid lysis of a small proportion (14±2%) of the cells (as estimated by total cell counts and confirmed in initial efflux experiments). The efflux time course data were corrected accordingly.
Estimation of intracellular K+ concentration.
The intracellular K+ concentration of H. inflata under the growth conditions used in this study was estimated from the equilibrium distribution of 86Rb+. The validity of this approach rests on the assumption that the equilibrium distribution of 86Rb+ between the intra- and extracellular solutions is the same as that for K+. Cells were incubated with 86RbCl (approx. 0·2 µCi ml-1) overnight in culture medium. The amount of 86Rb+ taken up by the cells was determined by centrifuging the cells through the oil mix and processing the cell pellet as described above. From the measured intracellular 86Rb+ content, the intracellular water space (measured as above) and the extracellular 86Rb+ concentration, it was possible to determine the transmembrane 86Rb+ distribution ratio ([86Rb+]i/[86Rb+]o, where [86Rb+]i and [86Rb+]o are the intra- and extracellular 86Rb+ concentrations, respectively). The concentration of K+ in the medium ([K+]o) was determined by flame photometry (Radiometer), and the intracellular K+ concentration ([K+]i) calculated using the equation [K+]i=[K+]ox([86Rb+]i/[86Rb+]o).
Effect of inhibitors on 86Rb+ and [1-14C]AIB efflux.
A number of inhibitors of ion and organic osmolyte channels were tested for their ability to inhibit [1-14C]AIB and 86Rb+ efflux. These included BaCl2 (2 mM), quinine (1 mM), tamoxifen (10 µM), 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid disodium salt (DIDS, 500 µM), niflumate (200 µM), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 µM) and N-ethylmaleimide (NEM, 200 µM). Cells were preloaded with AIB and 86Rb+ as described above and incubated for 15 min with the inhibitors before being subjected to a hypo-osmotic shock (150 mosmol kg-1) for 5 min. The inhibitor concentration was maintained pre- and post-osmotic shock. Cells were then processed as described above. Experiments were performed in triplicate and repeated at least twice.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An increase in the extracellular osmolality, from 300 mosmol kg -1 to 350400 mosmol kg-1, achieved by the addition of appropriate amounts of mannitol, choline chloride, NaCl or KCl, resulted in a marked reduction of cell volume within 12 min. In all cases the cells failed to show a volume recovery and remained shrunken for up to 30 min (Fig. 1).
|
Swelling-activated amino acid release
Analysis of the free intracellular amino acid pool of H. inflata revealed alanine to be the major component, present at a concentration of 69 mM under iso-osmotic conditions (Table 1). Leucine, glycine, valine and glutamate were also found at high (>2 mM) concentrations. The concentration of proline fluctuated between samples to a greater extent than that of any of the other amino acids, as reflected in the relatively high standard deviation. The total free amino acid pool of H. inflata, under iso-osmotic conditions, was estimated to be approximately 95 mM.
|
Swelling-activated transport of [1-14C]AIB and 86Rb+
The pathway(s) involved in swelling-activated amino acid release from H. inflata were investigated in more detail by measuring the transmembrane flux of [1-14C]AIB, a structural non-metabolizable analogue of alanine. [1-14C]AIB did not enter the cells under iso-osmotic conditions. However, on decreasing the osmolality, there was a slight (approx. 2 min) lag, after which [1-14C]AIB was taken up (Fig. 2a). The rate of uptake and final intracellular [1-14C]AIB concentration were dependent upon the magnitude of the osmotic stress to which the cells were subjected, with the highest rate of uptake observed at the lowest osmolality tested. The elevated rate of uptake was maintained for 46 min, after which it decreased. A plot of the amount of AIB taken up by cells after 6 min exposure to solutions of varying osmolalities shows a sigmoidal dependence on the extracellular osmolality (Fig. 2b
).
|
|
Intracellular K+ concentration
To gain some insight into the extent to which the swelling-activated efflux of K+ contributed to the RVD response of H. inflata following a hypo-osmotic challenge, the intracellular K+ content was estimated from the equilibrium distribution of 86Rb+. Cells preincubated overnight in iso-osmotic growth medium containing 86Rb+ accumulated the radioisotope to a concentration some 2·5 times that in the extracellular solution, yielding an estimate for intracellular K+ concentration of 84±13 mM (SEM, n=5).
Effect of inhibitors on the swelling-activated efflux of [1-14C]AIB and 86Rb+
A range of reagents shown previously to inhibit the swelling-activated transport of solutes from various cell types were tested for their effects on the efflux of [1-14C]AIB and 86Rb+ from H. inflata after hypo-osmotic challenge (150 mosmol kg-1). The thiol reagent NEM and the anion-transport inhibitor niflumic acid both lysed the cells at concentrations down to 200 µM and their effect on efflux could therefore not be evaluated. The K+-channel blockers BaCl2 (2 mM) and quinine (1 mM) and the anion-channel blockers DIDS (500 µM), NPPB (100 µM) and tamoxifen ( 10 µM) were all without significant inhibitory effect.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Increasing the extracellular osmolality by the addition of various solutes caused H. inflata to shrink and it failed to show any volume recovery over a 30 min period. Failure to regulate cell volume in response to osmotic shrinkage is not uncommon amongst the cells of higher eukaryotes (Hallow & Knauf, 1994 ), and has also been observed in other protozoa (e.g. Giardia; Park et al., 1995
). It could be argued that cell shrinkage is unlikely to be life-threatening, at least in the short-term, and H. inflata does not expend resources on mounting a rapid volume-regulatory response under these conditions.
Osmotic swelling poses a more serious challenge for a cell as, unless it is countered, the cell is at risk of bursting. We have demonstrated that H. inflata can regulate its volume in response to a hypo-osmotic challenge and that it does so through the loss of both amino acids and K+.
A simple calculation enables a semi-quantitative assessment of the relative contribution of amino acids and K+ loss to the overall RVD. In the absence of any RVD mechanisms operating (i.e. if the cells behaved as perfect osmometers) a 50% reduction of the osmolality should have resulted in a doubling of the cell water volume, from 214 fl to 428 fl. As is evident from Fig. 1, 7 min after the reduction of the osmolality from 300 to 150 mosmol kg-1 the total cell volume was approximately 312 fl and the cell water volume therefore approximately 264 fl (assuming that the contribution of cell solids to the total volume was unaffected by osmolality), some 164 fl less than the volume predicted had the cell lacked the capacity to volume regulate. From Table 1
, during 7 min in hypo-osmotic medium each cell lost 10·5 fmol of amino acids. Over the same period each lost approximately 6 fmol of K+ (approx. 30% of the total intracellular K+ content; from Fig. 3
). If it is assumed that the loss of amino acids and K+ was accompanied by sufficient water to produce a solution of osmolality equivalent to that of the extracellular solution, and that the osmotic coefficients of the amino acids and K+ are 1 and 0·95, respectively, then it can be calculated that the loss of amino acids from the cell resulted in the net loss of approximately 70 fl of water, and the loss of K+ resulted in the net loss of approximately 40 fl of water. The loss of K+ from the cell must necessarily have been accompanied by the loss of an equivalent amount of a counter-balancing ion; and assuming that this was either Cl- or another monovalent anion, this accounts for a further 40 fl of water loss.
In summary, 7 min after the suspension of H. inflata in a medium of half the original osmolality, the parasite had an intracellular water volume some 164 fl less than that predicted had the cells been unable to undergo RVD. Of this 164 fl, approximately 70 fl may be attributed to the loss of amino acids and 80 fl to the loss of K+ together with a charge-balancing counter-anion, accounting almost fully for the observed RVD.
The volume-regulatory efflux of amino acids has been described in a number of different protozoa, including Giardia (Park et al., 1995 ), Crithidia (Bursell et al., 1996
), Acanthamoeba (Geoffrion & Larochelle, 1984
) and Leishmania (Darling et al., 1990
; Vieira et al., 1996
). In all cases, as with H. inflata, the predominant intracellular amino acid that was lost in the greatest quantity during RVD was alanine. In Leishmania, hypo-osmotic challenge was reported to result in increased 86Rb+ efflux; however, it was concluded that this made only a minor (<4%) contribution to the RVD (Blum, 1992
). This contrasts with the situation in H. inflata, in which the efflux of K+, together with its counter-anion, contributes approximately half of the total RVD. We have observed a similar substantial contribution by K+ to the total RVD in Giardia pertaining to hypo-osmotic challenge (S. Maroulis & M. R. Edwards, unpublished). To our knowledge, these observations of the diplomonads Hexamita and Giardia represent the first reports of the loss of K+ making a significant contribution to the RVD response in a protozoon.
The strategy of releasing a combination of organic and inorganic solutes following cell swelling is common in cells from higher eukaryotes. Cells from vertebrates typically contain high cytosolic concentrations of the sulfonic amino acid taurine and/or polyols such as myo-inositol and sorbitol and these are released in response to cell swelling via broad-specificity osmolyte channels (Kirk, 1997 ). Such cells commonly also possess one or more swelling-activated K+ and Cl- transport pathways (channels and/or cotransporters) which mediate the volume-regulatory efflux of KCl (Hallow & Knauf, 1994
).
The finding that the volume-regulatory efflux in H. inflata was not inhibited by reagents that inhibit swelling-activated K+ and anion channels in vertebrate cells is consistent with the pathway(s) operating in H. inflata being quite different from their vertebrate counterparts. The observation that osmotic swelling induces an increase in both the unidirectional influx (Fig. 2) and efflux (Fig. 3
) of the alanine analogue AIB is consistent with the pathway being bi-directional, with the net flux of amino acids from the cell being determined simply by the large outward concentration gradient for these solutes. If a single pathway mediates the efflux of all of the amino acids lost from the cell, it is clearly one with a broad substrate specificity, though the relative loss of the different amino acids from the cell does suggest a degree of selectivity, with the pathway having an apparent preference for neutral amino acids (Table 1
). The volume-regulatory loss of amino acids from other protozoa has been attributed to a swelling-activated channel (Vieira et al., 1996
; Bursell et al., 1996
; Park et al., 1998
) and the data are consistent with a similar pathway operating in H. inflata. Whether it is this same pathway that mediates the volume-regulatory efflux of K+ remains to be established.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Biagini, G. A., McIntyre, P. S., Finlay, B. J. & Lloyd, D. (1998). Carbohydrate and amino acid fermentation in the free-living primitive protozoon Hexamita sp.Appl Environ Microbiol 64, 203-207.
Blum, J. J. (1992). Effect of osmolarity on 86Rb+ uptake and release by Leishmania donovani.J Cell Physiol 152, 111-117.[Medline]
Brugerolle, G. (1974). Contribution a létude cytologique et phylétique des diplozaires (zoomastigophorea, diplozoa, Dangeard 1910).Protistologica 1, 83-90.
Buchmann, K., Uldal, A. & Lyholt, H. C. K. (1995). Parasite infections in Danish trout farms.Acta Vet Scand 36, 283-298.[Medline]
Bursell, J. D. H., Kirk, J., Hall, S. T., Gero, A. M. & Kirk, K. (1996). Volume-regulatory amino acid release from the protozoan parasite Crithidia luciliae.J Membr Biol 154, 131-141.[Medline]
Darling, T. N., Burrows, C. M. & Blum, J. J. (1990). Rapid shape change and release of ninhydrin-positive substances by Leishmania major promastigotes in response to hypoosmotic stress.J Protozool 37, 493-499.[Medline]
Fenchel, T. & Finlay, B. J. (1995). Ecology and Evolution in Anoxic Worlds. Oxford: Oxford University Press.
Fenchel, T., Bernard, C., Esteban, G., Finlay, B. J., Hansen, P. J. & Iversen, N. (1995). Microbial diversity and activity in a Danish fjord with anoxic deep water.Ophelia 43, 45-100.
Ferguson, H. W. (1979). Scanning and transmission electron microscopic observations of Hexamita salmonis (Moore, 1923) related to mortalities in rainbow trout fry Salmo gairdneri Richardson.J Fish Dis 2, 57-67.
Geoffrion, Y. & Larochelle, J. (1984). The free amino acid contribution to osmotic regulation in Acanthamoeba castelanii.Can J Zool 62, 1954-1959.
Hallow, K. R. & Knauf, P. A. (1994). Principles of cell volume regulation. In Cellular and Molecular Physiology of Cell Volume Regulation, pp. 3-29. Edited by K. Strange. Boca Raton, FL: CRC Press.
Kirk, K. (1997). Swelling activated organic osmolyte channels.J Membr Biol 158, 1-16.[Medline]
Knodler, L. A., Edwards, M. R. & Schofield, P. J. (1994). The intracellular amino acid pools of Giardia intestinalis, Trichomonas vaginalis and Crithidia luciliae. Exp Parasitol 79, 117-125.[Medline]
Kulda, J. & Nohýnková, E. (1978). Flagellates of the human intestine and of the intestines of other species. In Parasitic Protozoa, pp. 2-127. Edited by J. P. Kreier. New York: Academic Press.
Leipe, D. D., Gunderson, J. H., Nerad, T. A. & Sogin, M. L. (1993). Small subunit ribosomal RNA of Hexamita inflata and the quest for the first branch in the eukaryotic tree.Mol Biochem Parasitol 59, 41-48.[Medline]
van Keulen, H., Guttel, R. R., Gates, M. A., Campbell, S. R, Erlansden, S. L., Jarrol, E. L., Kulda, J. & Meyer, E. A. (1993). Unique phylogenetic position of diplomonadida based on the complete small subunit ribosomal RNA sequence of Giardia ardeae, G. muris, G. duodenalis and Hexamita sp.FASEB J 7, 223-231.
Park, J. H., Schofield, P. J. & Edwards, M. R. (1995). The role of alanine in the acute response of Giardia intestinalis to hypo-osmotic shock.Microbiology 141, 2455-2462.
Park, J. H., Schofield, P. J. & Edwards, M. R. (1997). Giardia intestinalis: volume recovery in response to cell swelling.Exp Parasitol 86, 19-28.[Medline]
Park, J. H., Edwards, M. R. & Schofield, P. J. (1998). 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.
Vieira, L. L., Lafuente, E., Gamarro, F. & Cabantchik, Z. I. (1996). An amino acid channel activated by hypotonically induced swelling of Leishmania major promastigotes.Biochem J 319, 691-697.[Medline]
Received 17 August 1999;
accepted 9 November 1999.