isozymes (CA I. C A 11. C A Ill and CA IW in an umbilical vein endothelial cell line (EA-hv926). ISABELLE MAHIEU~, ANAND SAGGAR-MALIK~. ETIENNE ...
308s Biochemical Society Transactions (1995) 23 Localisation a n d characterisation of c a r b onic a n h y d r a s e isozymes ( C A I. C A 11. C A Ill and C A IW in a n umbilical vein endothelial cell line (EA-hv926)
ISABELLE M A H I E U ~ , A N A N D S A G G A R - M A L I K ~ . ETIENNE HOLLANDEJ AND NICK CARTER1 1Medical Genetics Unit and 2Blood Pressure Unit, St. George's Hospital Medical School, London SW17 ORE U.K. 3Department of Cell Biology, Paul Sabatier University, Toulouse, France. Carbonic anhydrases I and I1 (CA I and C A 11) are present within endothelial ( I ) and red blood cells (2) but are regarded as being functionally absent from blood plasma (2). However, it appears that extracellular C A can participate directly in plasma C 0 2 HC03--H+ reactions. Evidence for such 'intravascular' C A activity in rabbit, guinea pig lung and rat (3.4). rabbit liver (3) and rabbit and cat hind limb (3.5) has been documented in numerous reports. Results of histological, biochemical and physiological studies of C A in the human, cat and rat lung suggest that intravascular C A activity is due to an extracellular C A localised on the luminal surface of vascular endothelial cells (6.7.8) and probably represents the high activity, membrane bound C A IV isozyme. Thc aim of the present work was to study the precise distribution and expression of C A 1, 11, I11 and IV in an umbilical vein endothelial cell line, EA-hy926, using immunocytochemistrymd immunoblotting techniques. This permanently established cell line was derived by fusing human umbilical vein endothelial cells with the permanent human pulmonary cell line A549 (9). Cells were routinely grown at 37°C in Dulbecco's modified Eagle medium (GIBCO) supplemented with 15% heat inactivated foetal bovine serum, 2%J HAT and 1% glutamine. At early confluency, the monolayer constituted a mixture of cells with diffuse limits and clusters of polygonal, well defined cells (Fig.lA). At late confluency, all the cells in the monolayer became polygonal (Fig.2A). Doubling time of cell cultures was approximately 19h. For immunocytochemistry, the monolayers were fixed in situ with 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer for 30 mins and washed 3 times in the same buffer. Cells were then incubated for 1h at room temperature with rabbit anti-human C A I. C A 11 and C A I11 and anti-bovine C A IV antibodies, followed by a 1h incubation with a second peroxydase-conjugated anti-rabbit antibody. Peroxidase activity was Evealed by diaminobenzidine. Three independently prepared antisera to highly purified Cterminal, N-terminal and active site amino acid sequences of the C A IV protein were also used. Controls incubated with preimmune serum as opposed to specific anti-carbonic anhydrase did not stain. C A I, 11, 111 and IV expression was evaluated using Western blotting. Samples were electrophoresed on a 10% S D S discontinuous polyacrylamide gel. then transferred onto a nitrocellulose membrane (Hybond C, Amersham International) and incubated with the antibodies described above. The resulting complex was visualised using a biotin-avidin alkaline phosphacasc system. Immunocytochemical techniques showed that C A I, C A 11, and C A 111 were highly expressed in umbilical vein endothelial cells. Diffuse staining of the cytoplasm suggested that the CAs could be associated with cytoskeleton components (Fig 1C.D.E). However, even at higher magnification, light microscopy yielded few clues as to-specific cellular localisations. C A IV was demonstrated in cell membrane as early as the sccond day in culture (Fig IF). On the fifth day, all the cells in the monolayer wen: stained (Fig. IG). The staining was weak, consistent with the results of Henry et al. ( I ) who showed that only 2%) of C A in the lung was chemically hound to cell membranes. After Western b~oltingC A I. 11, and III were visualised as 29 kD proteins and C A IV as a single immunorcactive band of 55 kD (Fig.H). After incubation of memhranc pellets with Triton X- I00 a second, well-defined 45 k D hand appeared, suggesting an association between C A IV and a lipid or a glycolopid membrane fraction (Fig. I). Separate experiments failed to demonstrate C A existence in the pulmonary cell linc A 549 (data not shown).
Given that EA-hy926 cells were maintained in culturc in the absence of a source of CA, it would appear that they arc able to synthesise these enzymes and are. therefore, a good model to study C A functions in endothelial cells. It is conceivable that umbilical vein enzymcs. whilst having no role in placental gas exchange, may relate in some way to other processes such as homeostatic regulation. Indeed. C A involvement in blood pH regulation has heen demonstrated in the lung whcrc i t accelerates the release of carbon dioxide from plasma hicarbonate and so diminishes C 0 2 - H C 0 3 - - H + disequilibrium generated hy transcapillary CO2 exchange. In the placenta Pailurc to ic-cstahlish equilibrium within the capillary system would lead to continued reequilibration in the postcapillary vasculaturc including the umbilical vein. In the latter context, C A 1, I1 and Ill would then represent constitutive enzymes, implicated. in 'house-kecping' functions such as intracellular pH regulation.
Bibliography : 1. Henry R.P., Dodgson. S.J., Forstcr R.E.. and Storey B.T. (1986) J. Appl. Physiol. 60. 638-645 2. Maren T.H. (1967) Physiol. Rev. 47.597-781 3. O'Brasky J.E. and Crandall E.D. (1980) J. Appl. Physiol. 49. 21 1-217 4. Crandall E.D. and O'Brasky J.E. (1978) J. Clin. Invest. 62, 6 18-622 5. Effros R.M. and Weissman M.L. ( 1979) J. ADDI. .. Physiol. 47. 1090-1098 6. Lonnerholm G. and Wistrand P. (1982) Pediatr. Res. 16. 40741 1 7. Hansson M.A., Nye P.C.G. and Torrance R.W. (1981) 319. 93- 109 8. Lonnerholm G. (1980) Acta Physiol. Scand. 108. 197- I99 9. Edgell C.J., Mc Donald C.C. and Graham J.B. (1983) Proc. Nat. Acad. Sci. USA 80,3734-3737
We gratefully acknowledgc financial support from the Association for Spina Bifida and Hydrocephalus.
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(A) 4 day-old and (B) 7 day-old EA-hy926 cells i n culturc. Bar: 5Opm.Cytoplasmic localisation of C A I (C). C A I1 (D). C A Ill (El and membrane localisation of C A 1V (F) in 2 day-old cells. Membrane localisation of C A IV in 5 day-old cells (G). Bar:Spm. (H) C A I (I), C A I1 (2). C A 111 (3) and C A IV (4) characterization by immunob~olting.(I) C A IV characterization after incuhation with Triton X-100 using anti-bovine C A IV antihody ( I ) and antisera to C terminal (2). N terminal (3) and active site (4) synthetic pcptidcs.
