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Isolation of gill cells
Hochachka and Mommsen (eds.), Biochemistry and molecular biology of fishes, vol. 3 © 1994 Elsevier Science B.V. All rights reserved.
C H A P T E R 21
Isolation of gill cells PIETER M . V E R B O S T , G E R T FLIK AND H A R O L D C O O K * Department of Animal Physiology, Faculty of Science, University of Nijmegen, Toernooiveld, 6525 ED Nijmegen, The Netherlands, and *Department ofZoology, University of Alberta, Edmonton, Alberta, T6G 2H7 Canada
I. II.
Introduction Cell isolation procedures 1. Density or differential centrifugation 2. Flow cytometry 3. Immunodissection/cell culture III. References
/.
Introduction
The gills of fish constitute a multifunctional and complex organ where blood vessels and branchial epithelium meet. The branchial epithelium consists of various cell types that fulfil respiratory, osmoregulatory and excretory functions. The predomi nant cell types are pavement cells, mucus cells and ionocytes ("chloride cells") that 1415 all have their specific location in the gill s t r u c t u r e . The main feature of the filament epithelium (cells surrounding the filament) is the presence of ionocytes 14 (ion transporting cells) in freshwater as well as in seawater species . Ionocytes are mainly located in the interlamellar region of the filament epithelium and on the trailing edge of the filaments. This may be visualized in situ with confocal laser scanning microscopy after staining of the ionocytes with the fluorescent dye DASPEI (Fig. 1). DASPEI, 2-P-dimethyl-aminostyrylpyridylethyl-iodide, is a vi 2 tal stain for mitochondria with sufficient specificity for the metabolically active 18 ionocytes . The lamellar epithelium lining the lamellae is very thin and this is 1 1 1 4 where most of the respiratory exchanges across the branchial epithelium o c c u r . The respiratory cell is the predominant cell in this epithelium which is in fact the same cell type as the pavement cell in the filament epithelium. It is well established that the lamellar epithelium stems from nondifferentiated cells of the filament epithelium which might explain why ionocytes are not strictly confined to the filament epithelium but may also be found in the lamellar epithelium. The mitochondria-rich ionocyte is most likely the site of active transepithelial 5 6 71617,28 ion transport in both freshwater and seawater fishes ' ' . However, a pure preparation of ionocytes in sufficient amounts for in vitro studies has never been obtained. Typical problems in the gill cell isolation are the high degree of cell
240
P.M. Verbost, G. Flik and H. Cook
Fig. 1. Vital stain (DASPEI) of the mitochondria-rich ionocytes of a gill filament of freshwater tilapia. Note the high density of ionocytes on the trailing edge. Photograph obtained by confocal fluorescence microscopy showing a composition of 10 indepent scan collected over 100 μπ\ in depth ( x 2 0 0 ) . Bar indicates 100 μπι.
aggregation (mucus and D N A from disrupted cells may contribute to cell clus tering) and the small difference in size and density between ionocytes and other cell types. Some species, i.e. eel (Anguilla sp.), pinfish (Lagodon rhomboides), de velop considerably more and larger ionocytes in seawater which allows separation of ionocytes on the basis of cell size (see below). In tilapia (Oreochromis sp.), however, there is hardly any increase in size or number of ionocytes after trans fer to seawater 2 3. In freshwater, the intercellular junctions between ionocytes and their adjacent cells are much tighter than in seawater which may mean that gill cells from seawater fish are easier dispersed than cells from specimens adapted to freshwater. In this chapter a method of gill cell isolation is reviewed which has proved useful for studying aspects of ion transport functions. The method described is easy, can be employed for gills of both freshwater and seawater adapted fish, and produces a suspension of viable gill cells ready for further purification or biochemical assays. In addition methods for further isolation of ionocytes are presented. The techniques and the rationale of the methodology will be addressed.
Isolation of gill cells
241
IL Cell isolation procedures The branchial epithelium is scraped free from the underlying cartilaginous filaments using the edge of a glass slide. The epithelial blend produced by this mechanical procedure includes intact cells, cell clusters, cell debris, connective tissue fragments, and small blood vessels. First the red blood cells are removed from this mixture (even after thorough gill perfusion we found 5 0 - 7 0 % of the scraped cells to be red blood cells in trout and tilapia preparations); concomitantly cell clusters are fractionated into separate cells. Remaining cell clusters are removed by filtration (see below). Incubation of the scrapings for 20 min at room temperature in lysis-medium (9 21 parts 0.17 Μ NH 4 C1, 1 part 0.17 Μ Tris/HCl pH 7.4; from Yust et al ) results in blood cell lysis and tissue fractionation. Incubation in lysis-medium results in 2 + 2+ lysis of red blood cells specifically. An incubation in C a - and M g - f r e e medium (i.e. balanced salt solution without addition of calcium and magnesium and without 2+ Ca -chelators) facilitates cell isolation. One may consider to add chelating agents 2+ 2+ (EDTA, citrate) to capture C a and M g and by doing so prevent cell aggregation and inhibit endogenous protease activity. Also, collagenase and hyaluronidase may be used to facilitate tissue disruption. However, in our experience, the use of chelating agents either alone or in combination with lytic enzymes produces a cell population with less stable characteristics (autolysis and cell aggregation). Moreover, the use of lytic enzymes may damage cell surface receptors and is expensive. Following red cell lysis, cells are suspended at the beginning and resuspended at the end of this incubation period by passing them through a 10 ml pipet (3 mm bore diameter). With 2.5 g tissue scrapings per 10 ml lysis-medium (around 7 3.25 χ 10 cells per 10 ml) lysis is optimal. The cells are now sieved through 100 μπι mesh nylon gauze to remove persistent cell clusters. The resulting suspension consists primarily of single cells and subcellular debris and is centrifuged at 150 g for 5 min at 4°C in a swing-out rotor (BS4402/A rotor, Heraeus). The pelleted cells 2 + 2+ - 1 are washed in C a - and M g - f r e e balanced salt solution (containing in mmol L : 160 NaCl, 8.5 KCl, 5 N a H C 0 3 , 0.5 N a H 2 P 0 4 , 1.5 N a 2 H P 0 4 , 5 glucose, pH 7.4, - 1 osmolarity 280 mOsm L ) and finally resuspended in the required assay medium. The red cell content of this preparation is less than 2%. Table 1 shows the result of the lysis method for two marker enzymes for + + 4 1 2 2 0 ionocytes, succinate dehydrogenase (SDH) and N a / K - A T P a s e ' . The removal + + of red cells (which contain nondetectable S D H and N a / K - A T P a s e activity) results in a 3.1-fold increase in the ionocyte density of the cell suspension. This is close to the 3.4 times purification of ionocytes from seawater adapted pinfish after density 10 centrifugation as reported by Hootman and Philpott . Our results imply that the centrifugation method merely removes the blood cells from the original cell suspension and this was further confirmed when we separated tilapia gill cells on continuous and discontinuous Percoll gradients (the "ionocyte band", at 1.09-1.10 g -1 + + m l density, was enriched 2.9 to 4.2 times in N a / K - A T P a s e activity; unpublished observations). Figure 2 shows that this isolation method including the blood cell
242
P.M. Verbost, G Flik and H. Cook
Fig. 2. Electron micrographs of gill cells isolated with the "red cell lysis method" described in the text. A. Mature ionocyte showing normal mitochondria; the black edge around the nucleus signals the beginning of apoptosis ( χ 17,000). Β. Ionocyte showing the tubular system and mitochondria typical for cells of this stage ( χ 14,000). C. Two ionocytes showing severe (left) and early (right) apoptosis. The cell with severe apoptosis shows typical condensation of the tubular system ( χ 18,000). D . Segment of cytoplasm of a ionocyte, showing normal and some apoptotic mitochondria ( χ 45,000).
243
Isolation of gill cells TABLE 1 +
Succinate dehydrogenase ( S D H ) and N a + / K - A T P a s e activity in gill cell suspensions of freshwater tilapia before and after lysis of red blood cells Marker enzyme Succinate dehydrogenase +
Na+/K -ATPase a
b
a
η
Before lysis mean ± SEM
After lysis mean ± SEM
Enrichment (fold)
7
0.86 ± 0.09
2.71 ± 0.72
3.2
2.41 ± 0.56
6.53 ± 1.07
6 1
1
b
Activity in extinction units (A4oonm) h mg protein. Activity in μπιοί P, h of enzyme activity after lysis over activity before lysis.
0
3.1 1
mg
1
c
p r o t e i n . Ratio
lysis does not affect the ultrastructural integrity in the majority of the isolated cells. Most ionocytes show normal mitochondria and possess the tubular system that is characteristic for the mature stages of these cells. The tubular system has + 12 been shown to be the site of N a / K + - A T P a s e s . We also observed some ionocytes 25,26 in different phases of apoptosis, i.e. cell death by intrinsic f a c t o r s . Figure 3 illustrates the viability of this preparation as indicated by biochemical parameters. The cellular content of the second messenger cAMP may be manipulated by the adenylyl cyclase activator forskolin and by teleocalcin, an inhibitor of the adenylyl 22 cyclase from the corpuscles of Stannius , respectively. The cell viability as assessed 9 by Trypan Blue exclusion indicated that 95 to 98% of the gill cells were intact, independent of whether they were isolated from freshwater or from seawater gills. A cell suspension from a seawater adapted tilapia as well of freshwater adapted tilapia typically contains 10 to 13% ionocytes based on staining with DASPEI. Three strategies will be described now to further purify ionocytes.
cAMP content in dispersed FW tilapia gill cells 180
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control
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5.0
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forek.
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Ω. Fig. 3. c A M P content of dispersed gill cells from freshwater adapted tilapia after treatment with forskolin or teleocalcin. Incubation was for 10 min at 37°C. Bars represent means of 3 experiments ±SD.
244 1. Density or differential
P.M. Verbost, G. Flik and H. Cook
centrifugation
Centrifugation steps will only be useful when the density or size of the ionocytes differs from that of the other cell types. However, this procedure will therefore be limited to certain species (as discussed above) and to a subfraction of the ionocytes because only the fully grown ones will be distinct enough. Furthermore, the tendency of the gill cells to stick together may hamper separation; cell clusters - 1 disturb the gradient. The use of bovine serum albumin (0.1 mg m l ) and DNase (50 - 1 μ g m l ) in our hands does not significantly alleviate cell gluing that develops within 30 to 60 min when the cells are left on ice and even faster at higher temperatures. One can use large volumes of medium to prevent cell clustering (pers. observation), but this is not a favourable solution in centrifugation procedures, especially not in density centrifugation where small volumes need to be loaded on the gradient. Operating as quickly as feasible in volumes as large as possible seems the best guarantee for success. We predict that at least in analytical studies this procedure — performed after the lysis method — may yield highly purified ionocyte samples. 2. Flow cytometry After loading gill cells with DASPEI they can be separated on a flow cytometer. The area of the DASPEI-fluorescence signals and the right angle scatter are recorded in list mode using a flow cytometer 50H (Ortho Instruments, Westwood, CA) equipped with a 5 W Argon ion laser (Fig. 4). The 488 nm line of the laser is used for excitation and a bandpass filter 515-530 nm (for green) for detection of fluorescence. The flow cytometer selects cells with a certain level of fluorescence and expresses them as a percentage of the total number of cells. The labeled gill cells from tilapia are not homogeneous but appear to exist of two distinct populations (Fig. 4). Although it is tempting to hypothesize that these groups represent different developmental stages of the ionocyte, more microscopical studies are necessary. An advantage of this method is that the cells can be kept in rather large -1 volumes (around 1 mg m l cell protein) which prevents or delays cell clustering. A disadvantage is the low yield; at a maximum collecting speed of 1500 to 2000 ionocytes per min (flow rate around 300 cells per s) it takes 4 h to collect 250,000 cells because of all the obligatory operation steps with the flow cytometer. This yield is usually too low to allow subsequent enzyme assays. However, for histochemical purposes or to produce an immunogen, this procedure yielding a cell population + consisting for 99.5% of ionocytes, should be considered. The purification in N a / + K -ATPase activity of this preparation is 2.9 to 3.2 times. This is much lower than the purification of 7.5 times predicted on the basis of the microscopical results (from 13% ionocytes before flow cytometry to 99.5% ionocytes after) and + + of the doctrine that N a / K - A T P a s e resides in the ionocytes. This discrepancy could be an indication that not all mitochondria rich cells (preferentially stained by + + DASPEI) carry N a / K - A T P a s e . For mitochondria-rich cells in frog skin epithelium 19 it has recently been shown that only 50% of the cells were ouabain sensitive . If a similar situation exists in the fish gill this would explain the discrepancy in
245
Isolation of gill cells
Fig. 4. Bivariate fluorescence analysis of gill cells from freshwater tilapia labeled with DASPEI (for 15 min with 4 μΐ of a saturated DASPEI solution in phoshate buffer per 100 μΐ cell suspension, followed by a wash step). Plots show the right angle scatter ( R A S ) on the x-axis and the fluorescence ( 5 1 5 - 5 3 0 nm) on the j>-axis, where each dot represents o n e cell. The RAS gives an indication of the cell diameter. A. Total population. B. The window isolated from A, 10.5% of the population in A, with almost exclusively fluorescent cells. C. Isolated window after longer data acquisition then in Β clearly showing two different groups of labeled cells. One group of smaller cells and higher fluorescence and a group of larger cells with lower fluorescence.
enrichment of fluorescent cells and N a + / K + - A T P a s e activity. In this case, the Na+/ K + -ATPase activity is defined as the ouabain-inhibitable fraction of the measured ATP-hydrolysis. 3. Immunodissectionlcell
culture
Recently an indirect immunoaffinity method for isolating large and highly purified populations of hormonally responsive epithelial cells has been developed for mam malian kidney c e l l s 1 , 2 1. The mammalian nephron is comparable in morphological and functional complexity to the fish gill. A detailed localization of the action of hormones or the presence of transport enzymes requires a separation of the different cell types. The outline of the procedure is as follows. Four weeks old female BALB/c mice are initially immunized intraperitoneally with 10 7 freshly pre pared (isolated) cells and boosted after 3 weeks intravenously on three successive days with 5 χ 10 6 cells 3. The monoclonal antibodies produced are incubated with a mixed gill cell suspension (as harvested with the lysis method). After washing the cells, the suspension is added to goat anti-mouse IgX coated bacterial dishes (where X is the Ig subclass of the monoclonal antibody). After 15 min incubation the dishes are washed carefully. Subsequently, the adherent cells are scraped off the dishes and collected by centrifugation (for further details see ref. 3). Immunodissected cells from rabbit collecting duct were successfully brought into primary culture on collagen coated permeable filters and formed physiologically functional monolayers 3. The two bottlenecks in the development of this method for gill cells (ionocytes) are the production of monoclonal antibodies and the provision of a sterile cell mixture for the immunodissection and subsequent culturing. In our attempts to
246
P.M. Verbost, G. Flik and H. Cook
raise antibodies against ionocytes we ended up with a few mucus-specific-antibodies. Apparently, mucus due to its glycoproteinaceous character is very antigenic and not easily removed from the cells during isolation. The other problem is that the cells used for a primary culture have to be sterile. In contrast to mammalian kidney cells the gill cells are not sterile from the beginning of the isolation and despite performance of the purification procedures under sterile conditions bacterial infections are to fear. It is possible, however, to culture pavement cells as shown by at least three groups in Europe (M. Avella and B. Lahlou, France; P. Part, Sweden; H. Witters, Belgium; all pers. communications) and one group in 13 Taiwan studying heat shock proteins in cultured pavement cells of carp (Cyprinus carpio). Thus the indirect immunodissection technique combined with the culture of isolated cells may provide an elegant method to facilitate further studies of the biochemical and cell physiological events underlying solute transport in the complex gills.
77/. References 1. Allen, M.A., A. Nakao, W.K. Sonnenburg, Μ. Burnatowska-Hledin, W.S. Spielman and W.L. Smith. Immunodissection of cortical and medullary thick ascending limb cells from rabbit kidney. Am. J. Physiol. 255: F704-F710, 1988. 2. Bereiter-Hahn, J. Dimethylaminostyrylmethylpyridiniumiodine (DASPMI) as a fluorescent probe for mitochondria in situ. Biochim. Biophys. Acta 423: 1-14, 1976. 2+ 3. Bindeis, R.J.M., A. Hartog, J. Timmermans, and C.H. van Os. Active C a transport in primary cultures of rabbit kidney CCD: stimulation by 1,25-dihydroxyvitamin D3 and PTH. Am. J. Physiol. 261: F799-F807, 1991. 4. Epstein, F.H., P. Silva, and G. Kormanik. Role of Na/K-ATPase in chloride cell function. Am. J. Physiol. 238: R 2 4 6 - R 2 5 0 , 1980. 2+ 5. Flik, G., S.E. Wendelaar Bonga, and J.C. Fenwick. Active C a transport in plasma membranes of branchial epithelium of the North-American eel, Anguilla anguilla LeSueur. Biol. Cell 55: 2 6 5 - 2 7 2 , 1985. 2+ 6. Flik, G., J.H. Rijs, and S.E. Wendelaar Bonga. Evidence for high affinity C a - A T P a s e activity 2+ transport in membrane preparations of the gill epithelium of the cichlid fish and ATP-driven C a Oreochromis mossambicus. J. Exp. Biol. 119: 335-347, 1985. 7. Foskett, J.K., and C. Scheffey. The chloride cell: definitive identification as the salt secretory cell in teleosts. Science 215: 164-166, 1982. 8. Foskett, J.K., H.A. Bern, T.E. Machen, and M. Conner. Chloride cells and the hormonal control of teleost fish osmoregulation./. Exp. Biol. 106: 2 5 5 - 2 8 1 , 1983. 9. Grinstein, S. and W. Furuya. Receptor-mediated activation of electropermeabilized neutrophils. /. Biol. Chem. 263: 1779-1783, 1988. 10. Hootman, S.R. and C.W. Philpott. Rapid isolation of chloride cells from pinfish giW.Anat. Ree. 190: 6 8 7 - 7 0 2 , 1978. 11. Hughes, G.M. Fish respiratory homeostasis. Symp. Soc. Exp. Biol. 18: 8 1 - 1 0 7 , 1964. 12. Karnaky Jr., K.J., L.B. Kinter, W.B. Kinter, and C.E. Stirling. Teleost chloride cell. I. Autoradio + + graphic localization of gill N a / K - A T P a s e in killifish Fundulus heteroclitus adapted to low and high salinity environments./. Cell Biol. 70: 157-177, 1976. 13. Ku, C.C. and S.N. Chen. Heat shock proteins in cultured gill cells of common carp CypHnus carpio L. Bull. Inst. Zool. Acad. Sinica (Taipei). 30: 319-330, 1991. 14. Laurent, P. Gill internal morphology. In: Fish Physiology, Vol. XA, W.S. Hoar and D.J. Randall (eds.), Academic Press, New York, pp. 7 3 - 1 8 3 , 1984. 15. Payan, P., J.P. Girard, and N. Mayer-Gostan. Branchial ion movements in teleosts: the roles of respiratory and chloride cells. In: Fish Physiology, Vol. XB, W.S. Hoar and D.J. Randall (eds.), Academic Press, New York, pp. 3 9 - 6 3 , 1984. 16. Perry, S.F., and C M . Wood. Kinetics of branchial calcium uptake in the rainbow trout: effects of
Isolation of gill cells
247
acclimation to various external calcium levels./. Exp. Biol. 116: 4 1 1 - 4 3 4 , 1985. 17. Perry, S.F, and G. Flik. Characterization of branchial transepithelial calcium fluxes in freshwater trout, Salmo gairdneri. Am. J. Physiol. 254: R 4 9 1 - R 4 9 8 , 1988. 18. Perry, S.F, and P.J. Walsh. Metabolism of isolated fish gill cells: contribution of epithelial chloride c e l l s . / . Exp. Biol. 144: 5 0 7 - 5 2 0 , 1989. 19. Rick, R. Intracellular ion concentrations in the isolated frog skin epithelium: evidence for different types of mitochondria-rich c e l l s . / . Membr. Biol. 127: 227-236, 1992. 20. Sargent, J.R., A.J. Thomson, and M. Bornacin. Activities and localization of succinic dehydrogenase + + and N a / K -activated adenosine triphosphatase in the gills of fresh water and sea water eels (Anguilla anguilla). Comp. Biochem. Physiol. 51B: 6 5 - 7 9 , 1975. 21. Stanton, R.C., D.L. Mendrick, H.G. Rennke, and J.L. Seifter. U s e of monoclonal antibodies to culture rat proximal cells. Am. J. Physiol. 251: C780-C786, 1986. 22. Verbost, P.M., A. Butkus, P. Willems, and S.E. Wendelaar Bonga. Indications for two bioactive principles in the corpuscles of Stannius,/. Exp. Biol. Ml: 2 4 3 - 2 5 2 , 1993. 23. Verbost, P.M., Th.J.M. Schoenmakers, G. Flik, and S.E. Wendelaar Bonga. Kinetics of ATP+ 2+ transport in basolateral membranes from gills of freshwater- and and N a - g r a d i e n t driven C a seawater-adapted tilapia. / Exp. Biol. 186: 9 5 - 1 0 8 , 1994. 24. Wendelaar Bonga, S.E. and C.J.M. van der Meij. Degeneration and death, by apoptosis and necrosis, of the pavement and chloride cells in the gills of the teleost Oreochromis mossambicus. Cell Tissue Res. 255: 2 3 5 - 2 4 3 , 1989. 25. Wendelaar Bonga, S.E., G. Flik, P.H.M. Balm, and J.C.A. van der Meij. The ultrastructure of chloride cells in the gills of the teleost Oreochromis mossambicus during exposure to acidified water. Cell Tissue Res. 259: 5 7 5 - 5 8 5 , 1990. 26. Wyllie, A.H., J.F.R. Kerr, and A.R. Currie. Cell death: the significance of apoptosis. Int. Rev. Cyt. 68: 2 5 1 - 3 0 6 , 1980. 27. Yust, I., R.W. Smith, J.R. Wunderlich, and D.L. Mann. Temporary inhibition of antibody-dependent, cell-mediated cytotoxicity by pretreatment of human attacking cells with ammonium chloride. /. Immunol. 116: 1170-1172, 1976. 28. Zadunaisky, J.A. The chloride cell: the active transport of chloride and the paracellular pathways. In: Fish Physiology, Vol. X B , W S . Hoar and D.J. Randall (eds.), Academic Press, New York, pp. 129-176, 1984.
C H A P T E R 21
Isolation of gill cells PIETER M . V E R B O S T , G E R T FLIK AND H A R O L D C O O K * Department of Animal Physiology, Faculty of Science, University of Nijmegen, Toernooiveld, 6525 ED Nijmegen, The Netherlands, and *Department ofZoology, University of Alberta, Edmonton, Alberta, T6G 2H7 Canada
I. II.
Introduction Cell isolation procedures 1. Density or differential centrifugation 2. Flow cytometry 3. Immunodissection/cell culture III. References
/.
Introduction
The gills of fish constitute a multifunctional and complex organ where blood vessels and branchial epithelium meet. The branchial epithelium consists of various cell types that fulfil respiratory, osmoregulatory and excretory functions. The predomi nant cell types are pavement cells, mucus cells and ionocytes ("chloride cells") that 1415 all have their specific location in the gill s t r u c t u r e . The main feature of the filament epithelium (cells surrounding the filament) is the presence of ionocytes 14 (ion transporting cells) in freshwater as well as in seawater species . Ionocytes are mainly located in the interlamellar region of the filament epithelium and on the trailing edge of the filaments. This may be visualized in situ with confocal laser scanning microscopy after staining of the ionocytes with the fluorescent dye DASPEI (Fig. 1). DASPEI, 2-P-dimethyl-aminostyrylpyridylethyl-iodide, is a vi 2 tal stain for mitochondria with sufficient specificity for the metabolically active 18 ionocytes . The lamellar epithelium lining the lamellae is very thin and this is 1 1 1 4 where most of the respiratory exchanges across the branchial epithelium o c c u r . The respiratory cell is the predominant cell in this epithelium which is in fact the same cell type as the pavement cell in the filament epithelium. It is well established that the lamellar epithelium stems from nondifferentiated cells of the filament epithelium which might explain why ionocytes are not strictly confined to the filament epithelium but may also be found in the lamellar epithelium. The mitochondria-rich ionocyte is most likely the site of active transepithelial 5 6 71617,28 ion transport in both freshwater and seawater fishes ' ' . However, a pure preparation of ionocytes in sufficient amounts for in vitro studies has never been obtained. Typical problems in the gill cell isolation are the high degree of cell
240
P.M. Verbost, G. Flik and H. Cook
Fig. 1. Vital stain (DASPEI) of the mitochondria-rich ionocytes of a gill filament of freshwater tilapia. Note the high density of ionocytes on the trailing edge. Photograph obtained by confocal fluorescence microscopy showing a composition of 10 indepent scan collected over 100 μπ\ in depth ( x 2 0 0 ) . Bar indicates 100 μπι.
aggregation (mucus and D N A from disrupted cells may contribute to cell clus tering) and the small difference in size and density between ionocytes and other cell types. Some species, i.e. eel (Anguilla sp.), pinfish (Lagodon rhomboides), de velop considerably more and larger ionocytes in seawater which allows separation of ionocytes on the basis of cell size (see below). In tilapia (Oreochromis sp.), however, there is hardly any increase in size or number of ionocytes after trans fer to seawater 2 3. In freshwater, the intercellular junctions between ionocytes and their adjacent cells are much tighter than in seawater which may mean that gill cells from seawater fish are easier dispersed than cells from specimens adapted to freshwater. In this chapter a method of gill cell isolation is reviewed which has proved useful for studying aspects of ion transport functions. The method described is easy, can be employed for gills of both freshwater and seawater adapted fish, and produces a suspension of viable gill cells ready for further purification or biochemical assays. In addition methods for further isolation of ionocytes are presented. The techniques and the rationale of the methodology will be addressed.
Isolation of gill cells
241
IL Cell isolation procedures The branchial epithelium is scraped free from the underlying cartilaginous filaments using the edge of a glass slide. The epithelial blend produced by this mechanical procedure includes intact cells, cell clusters, cell debris, connective tissue fragments, and small blood vessels. First the red blood cells are removed from this mixture (even after thorough gill perfusion we found 5 0 - 7 0 % of the scraped cells to be red blood cells in trout and tilapia preparations); concomitantly cell clusters are fractionated into separate cells. Remaining cell clusters are removed by filtration (see below). Incubation of the scrapings for 20 min at room temperature in lysis-medium (9 21 parts 0.17 Μ NH 4 C1, 1 part 0.17 Μ Tris/HCl pH 7.4; from Yust et al ) results in blood cell lysis and tissue fractionation. Incubation in lysis-medium results in 2 + 2+ lysis of red blood cells specifically. An incubation in C a - and M g - f r e e medium (i.e. balanced salt solution without addition of calcium and magnesium and without 2+ Ca -chelators) facilitates cell isolation. One may consider to add chelating agents 2+ 2+ (EDTA, citrate) to capture C a and M g and by doing so prevent cell aggregation and inhibit endogenous protease activity. Also, collagenase and hyaluronidase may be used to facilitate tissue disruption. However, in our experience, the use of chelating agents either alone or in combination with lytic enzymes produces a cell population with less stable characteristics (autolysis and cell aggregation). Moreover, the use of lytic enzymes may damage cell surface receptors and is expensive. Following red cell lysis, cells are suspended at the beginning and resuspended at the end of this incubation period by passing them through a 10 ml pipet (3 mm bore diameter). With 2.5 g tissue scrapings per 10 ml lysis-medium (around 7 3.25 χ 10 cells per 10 ml) lysis is optimal. The cells are now sieved through 100 μπι mesh nylon gauze to remove persistent cell clusters. The resulting suspension consists primarily of single cells and subcellular debris and is centrifuged at 150 g for 5 min at 4°C in a swing-out rotor (BS4402/A rotor, Heraeus). The pelleted cells 2 + 2+ - 1 are washed in C a - and M g - f r e e balanced salt solution (containing in mmol L : 160 NaCl, 8.5 KCl, 5 N a H C 0 3 , 0.5 N a H 2 P 0 4 , 1.5 N a 2 H P 0 4 , 5 glucose, pH 7.4, - 1 osmolarity 280 mOsm L ) and finally resuspended in the required assay medium. The red cell content of this preparation is less than 2%. Table 1 shows the result of the lysis method for two marker enzymes for + + 4 1 2 2 0 ionocytes, succinate dehydrogenase (SDH) and N a / K - A T P a s e ' . The removal + + of red cells (which contain nondetectable S D H and N a / K - A T P a s e activity) results in a 3.1-fold increase in the ionocyte density of the cell suspension. This is close to the 3.4 times purification of ionocytes from seawater adapted pinfish after density 10 centrifugation as reported by Hootman and Philpott . Our results imply that the centrifugation method merely removes the blood cells from the original cell suspension and this was further confirmed when we separated tilapia gill cells on continuous and discontinuous Percoll gradients (the "ionocyte band", at 1.09-1.10 g -1 + + m l density, was enriched 2.9 to 4.2 times in N a / K - A T P a s e activity; unpublished observations). Figure 2 shows that this isolation method including the blood cell
242
P.M. Verbost, G Flik and H. Cook
Fig. 2. Electron micrographs of gill cells isolated with the "red cell lysis method" described in the text. A. Mature ionocyte showing normal mitochondria; the black edge around the nucleus signals the beginning of apoptosis ( χ 17,000). Β. Ionocyte showing the tubular system and mitochondria typical for cells of this stage ( χ 14,000). C. Two ionocytes showing severe (left) and early (right) apoptosis. The cell with severe apoptosis shows typical condensation of the tubular system ( χ 18,000). D . Segment of cytoplasm of a ionocyte, showing normal and some apoptotic mitochondria ( χ 45,000).
243
Isolation of gill cells TABLE 1 +
Succinate dehydrogenase ( S D H ) and N a + / K - A T P a s e activity in gill cell suspensions of freshwater tilapia before and after lysis of red blood cells Marker enzyme Succinate dehydrogenase +
Na+/K -ATPase a
b
a
η
Before lysis mean ± SEM
After lysis mean ± SEM
Enrichment (fold)
7
0.86 ± 0.09
2.71 ± 0.72
3.2
2.41 ± 0.56
6.53 ± 1.07
6 1
1
b
Activity in extinction units (A4oonm) h mg protein. Activity in μπιοί P, h of enzyme activity after lysis over activity before lysis.
0
3.1 1
mg
1
c
p r o t e i n . Ratio
lysis does not affect the ultrastructural integrity in the majority of the isolated cells. Most ionocytes show normal mitochondria and possess the tubular system that is characteristic for the mature stages of these cells. The tubular system has + 12 been shown to be the site of N a / K + - A T P a s e s . We also observed some ionocytes 25,26 in different phases of apoptosis, i.e. cell death by intrinsic f a c t o r s . Figure 3 illustrates the viability of this preparation as indicated by biochemical parameters. The cellular content of the second messenger cAMP may be manipulated by the adenylyl cyclase activator forskolin and by teleocalcin, an inhibitor of the adenylyl 22 cyclase from the corpuscles of Stannius , respectively. The cell viability as assessed 9 by Trypan Blue exclusion indicated that 95 to 98% of the gill cells were intact, independent of whether they were isolated from freshwater or from seawater gills. A cell suspension from a seawater adapted tilapia as well of freshwater adapted tilapia typically contains 10 to 13% ionocytes based on staining with DASPEI. Three strategies will be described now to further purify ionocytes.
cAMP content in dispersed FW tilapia gill cells 180
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244 1. Density or differential
P.M. Verbost, G. Flik and H. Cook
centrifugation
Centrifugation steps will only be useful when the density or size of the ionocytes differs from that of the other cell types. However, this procedure will therefore be limited to certain species (as discussed above) and to a subfraction of the ionocytes because only the fully grown ones will be distinct enough. Furthermore, the tendency of the gill cells to stick together may hamper separation; cell clusters - 1 disturb the gradient. The use of bovine serum albumin (0.1 mg m l ) and DNase (50 - 1 μ g m l ) in our hands does not significantly alleviate cell gluing that develops within 30 to 60 min when the cells are left on ice and even faster at higher temperatures. One can use large volumes of medium to prevent cell clustering (pers. observation), but this is not a favourable solution in centrifugation procedures, especially not in density centrifugation where small volumes need to be loaded on the gradient. Operating as quickly as feasible in volumes as large as possible seems the best guarantee for success. We predict that at least in analytical studies this procedure — performed after the lysis method — may yield highly purified ionocyte samples. 2. Flow cytometry After loading gill cells with DASPEI they can be separated on a flow cytometer. The area of the DASPEI-fluorescence signals and the right angle scatter are recorded in list mode using a flow cytometer 50H (Ortho Instruments, Westwood, CA) equipped with a 5 W Argon ion laser (Fig. 4). The 488 nm line of the laser is used for excitation and a bandpass filter 515-530 nm (for green) for detection of fluorescence. The flow cytometer selects cells with a certain level of fluorescence and expresses them as a percentage of the total number of cells. The labeled gill cells from tilapia are not homogeneous but appear to exist of two distinct populations (Fig. 4). Although it is tempting to hypothesize that these groups represent different developmental stages of the ionocyte, more microscopical studies are necessary. An advantage of this method is that the cells can be kept in rather large -1 volumes (around 1 mg m l cell protein) which prevents or delays cell clustering. A disadvantage is the low yield; at a maximum collecting speed of 1500 to 2000 ionocytes per min (flow rate around 300 cells per s) it takes 4 h to collect 250,000 cells because of all the obligatory operation steps with the flow cytometer. This yield is usually too low to allow subsequent enzyme assays. However, for histochemical purposes or to produce an immunogen, this procedure yielding a cell population + consisting for 99.5% of ionocytes, should be considered. The purification in N a / + K -ATPase activity of this preparation is 2.9 to 3.2 times. This is much lower than the purification of 7.5 times predicted on the basis of the microscopical results (from 13% ionocytes before flow cytometry to 99.5% ionocytes after) and + + of the doctrine that N a / K - A T P a s e resides in the ionocytes. This discrepancy could be an indication that not all mitochondria rich cells (preferentially stained by + + DASPEI) carry N a / K - A T P a s e . For mitochondria-rich cells in frog skin epithelium 19 it has recently been shown that only 50% of the cells were ouabain sensitive . If a similar situation exists in the fish gill this would explain the discrepancy in
245
Isolation of gill cells
Fig. 4. Bivariate fluorescence analysis of gill cells from freshwater tilapia labeled with DASPEI (for 15 min with 4 μΐ of a saturated DASPEI solution in phoshate buffer per 100 μΐ cell suspension, followed by a wash step). Plots show the right angle scatter ( R A S ) on the x-axis and the fluorescence ( 5 1 5 - 5 3 0 nm) on the j>-axis, where each dot represents o n e cell. The RAS gives an indication of the cell diameter. A. Total population. B. The window isolated from A, 10.5% of the population in A, with almost exclusively fluorescent cells. C. Isolated window after longer data acquisition then in Β clearly showing two different groups of labeled cells. One group of smaller cells and higher fluorescence and a group of larger cells with lower fluorescence.
enrichment of fluorescent cells and N a + / K + - A T P a s e activity. In this case, the Na+/ K + -ATPase activity is defined as the ouabain-inhibitable fraction of the measured ATP-hydrolysis. 3. Immunodissectionlcell
culture
Recently an indirect immunoaffinity method for isolating large and highly purified populations of hormonally responsive epithelial cells has been developed for mam malian kidney c e l l s 1 , 2 1. The mammalian nephron is comparable in morphological and functional complexity to the fish gill. A detailed localization of the action of hormones or the presence of transport enzymes requires a separation of the different cell types. The outline of the procedure is as follows. Four weeks old female BALB/c mice are initially immunized intraperitoneally with 10 7 freshly pre pared (isolated) cells and boosted after 3 weeks intravenously on three successive days with 5 χ 10 6 cells 3. The monoclonal antibodies produced are incubated with a mixed gill cell suspension (as harvested with the lysis method). After washing the cells, the suspension is added to goat anti-mouse IgX coated bacterial dishes (where X is the Ig subclass of the monoclonal antibody). After 15 min incubation the dishes are washed carefully. Subsequently, the adherent cells are scraped off the dishes and collected by centrifugation (for further details see ref. 3). Immunodissected cells from rabbit collecting duct were successfully brought into primary culture on collagen coated permeable filters and formed physiologically functional monolayers 3. The two bottlenecks in the development of this method for gill cells (ionocytes) are the production of monoclonal antibodies and the provision of a sterile cell mixture for the immunodissection and subsequent culturing. In our attempts to
246
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raise antibodies against ionocytes we ended up with a few mucus-specific-antibodies. Apparently, mucus due to its glycoproteinaceous character is very antigenic and not easily removed from the cells during isolation. The other problem is that the cells used for a primary culture have to be sterile. In contrast to mammalian kidney cells the gill cells are not sterile from the beginning of the isolation and despite performance of the purification procedures under sterile conditions bacterial infections are to fear. It is possible, however, to culture pavement cells as shown by at least three groups in Europe (M. Avella and B. Lahlou, France; P. Part, Sweden; H. Witters, Belgium; all pers. communications) and one group in 13 Taiwan studying heat shock proteins in cultured pavement cells of carp (Cyprinus carpio). Thus the indirect immunodissection technique combined with the culture of isolated cells may provide an elegant method to facilitate further studies of the biochemical and cell physiological events underlying solute transport in the complex gills.
77/. References 1. Allen, M.A., A. Nakao, W.K. Sonnenburg, Μ. Burnatowska-Hledin, W.S. Spielman and W.L. Smith. Immunodissection of cortical and medullary thick ascending limb cells from rabbit kidney. Am. J. Physiol. 255: F704-F710, 1988. 2. Bereiter-Hahn, J. Dimethylaminostyrylmethylpyridiniumiodine (DASPMI) as a fluorescent probe for mitochondria in situ. Biochim. Biophys. Acta 423: 1-14, 1976. 2+ 3. Bindeis, R.J.M., A. Hartog, J. Timmermans, and C.H. van Os. Active C a transport in primary cultures of rabbit kidney CCD: stimulation by 1,25-dihydroxyvitamin D3 and PTH. Am. J. Physiol. 261: F799-F807, 1991. 4. Epstein, F.H., P. Silva, and G. Kormanik. Role of Na/K-ATPase in chloride cell function. Am. J. Physiol. 238: R 2 4 6 - R 2 5 0 , 1980. 2+ 5. Flik, G., S.E. Wendelaar Bonga, and J.C. Fenwick. Active C a transport in plasma membranes of branchial epithelium of the North-American eel, Anguilla anguilla LeSueur. Biol. Cell 55: 2 6 5 - 2 7 2 , 1985. 2+ 6. Flik, G., J.H. Rijs, and S.E. Wendelaar Bonga. Evidence for high affinity C a - A T P a s e activity 2+ transport in membrane preparations of the gill epithelium of the cichlid fish and ATP-driven C a Oreochromis mossambicus. J. Exp. Biol. 119: 335-347, 1985. 7. Foskett, J.K., and C. Scheffey. The chloride cell: definitive identification as the salt secretory cell in teleosts. Science 215: 164-166, 1982. 8. Foskett, J.K., H.A. Bern, T.E. Machen, and M. Conner. Chloride cells and the hormonal control of teleost fish osmoregulation./. Exp. Biol. 106: 2 5 5 - 2 8 1 , 1983. 9. Grinstein, S. and W. Furuya. Receptor-mediated activation of electropermeabilized neutrophils. /. Biol. Chem. 263: 1779-1783, 1988. 10. Hootman, S.R. and C.W. Philpott. Rapid isolation of chloride cells from pinfish giW.Anat. Ree. 190: 6 8 7 - 7 0 2 , 1978. 11. Hughes, G.M. Fish respiratory homeostasis. Symp. Soc. Exp. Biol. 18: 8 1 - 1 0 7 , 1964. 12. Karnaky Jr., K.J., L.B. Kinter, W.B. Kinter, and C.E. Stirling. Teleost chloride cell. I. Autoradio + + graphic localization of gill N a / K - A T P a s e in killifish Fundulus heteroclitus adapted to low and high salinity environments./. Cell Biol. 70: 157-177, 1976. 13. Ku, C.C. and S.N. Chen. Heat shock proteins in cultured gill cells of common carp CypHnus carpio L. Bull. Inst. Zool. Acad. Sinica (Taipei). 30: 319-330, 1991. 14. Laurent, P. Gill internal morphology. In: Fish Physiology, Vol. XA, W.S. Hoar and D.J. Randall (eds.), Academic Press, New York, pp. 7 3 - 1 8 3 , 1984. 15. Payan, P., J.P. Girard, and N. Mayer-Gostan. Branchial ion movements in teleosts: the roles of respiratory and chloride cells. In: Fish Physiology, Vol. XB, W.S. Hoar and D.J. Randall (eds.), Academic Press, New York, pp. 3 9 - 6 3 , 1984. 16. Perry, S.F., and C M . Wood. Kinetics of branchial calcium uptake in the rainbow trout: effects of
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acclimation to various external calcium levels./. Exp. Biol. 116: 4 1 1 - 4 3 4 , 1985. 17. Perry, S.F, and G. Flik. Characterization of branchial transepithelial calcium fluxes in freshwater trout, Salmo gairdneri. Am. J. Physiol. 254: R 4 9 1 - R 4 9 8 , 1988. 18. Perry, S.F, and P.J. Walsh. Metabolism of isolated fish gill cells: contribution of epithelial chloride c e l l s . / . Exp. Biol. 144: 5 0 7 - 5 2 0 , 1989. 19. Rick, R. Intracellular ion concentrations in the isolated frog skin epithelium: evidence for different types of mitochondria-rich c e l l s . / . Membr. Biol. 127: 227-236, 1992. 20. Sargent, J.R., A.J. Thomson, and M. Bornacin. Activities and localization of succinic dehydrogenase + + and N a / K -activated adenosine triphosphatase in the gills of fresh water and sea water eels (Anguilla anguilla). Comp. Biochem. Physiol. 51B: 6 5 - 7 9 , 1975. 21. Stanton, R.C., D.L. Mendrick, H.G. Rennke, and J.L. Seifter. U s e of monoclonal antibodies to culture rat proximal cells. Am. J. Physiol. 251: C780-C786, 1986. 22. Verbost, P.M., A. Butkus, P. Willems, and S.E. Wendelaar Bonga. Indications for two bioactive principles in the corpuscles of Stannius,/. Exp. Biol. Ml: 2 4 3 - 2 5 2 , 1993. 23. Verbost, P.M., Th.J.M. Schoenmakers, G. Flik, and S.E. Wendelaar Bonga. Kinetics of ATP+ 2+ transport in basolateral membranes from gills of freshwater- and and N a - g r a d i e n t driven C a seawater-adapted tilapia. / Exp. Biol. 186: 9 5 - 1 0 8 , 1994. 24. Wendelaar Bonga, S.E. and C.J.M. van der Meij. Degeneration and death, by apoptosis and necrosis, of the pavement and chloride cells in the gills of the teleost Oreochromis mossambicus. Cell Tissue Res. 255: 2 3 5 - 2 4 3 , 1989. 25. Wendelaar Bonga, S.E., G. Flik, P.H.M. Balm, and J.C.A. van der Meij. The ultrastructure of chloride cells in the gills of the teleost Oreochromis mossambicus during exposure to acidified water. Cell Tissue Res. 259: 5 7 5 - 5 8 5 , 1990. 26. Wyllie, A.H., J.F.R. Kerr, and A.R. Currie. Cell death: the significance of apoptosis. Int. Rev. Cyt. 68: 2 5 1 - 3 0 6 , 1980. 27. Yust, I., R.W. Smith, J.R. Wunderlich, and D.L. Mann. Temporary inhibition of antibody-dependent, cell-mediated cytotoxicity by pretreatment of human attacking cells with ammonium chloride. /. Immunol. 116: 1170-1172, 1976. 28. Zadunaisky, J.A. The chloride cell: the active transport of chloride and the paracellular pathways. In: Fish Physiology, Vol. X B , W S . Hoar and D.J. Randall (eds.), Academic Press, New York, pp. 129-176, 1984.