244s
Biochemical Society Transactions ( 1 992) 20
Nucleoside transporters in human placenta L. FELIPE BARROS., N1Cf BEAUMONT', SIMON M. JARVIS#, JAMES D. YOUNG , PETER J.F. HENDERSON+, DAVID L. YUDILEVICH*, CHRIS THRASIVOULOU' and STEPHEN A. BALDWINO *University of Chile, Casilla 70055 Santiago 7, Chile; 'Royal Free Hospital School of Medicine (Universit of London), Rowland Hill Street, London NW3 2PF, U.K.; !University of Kent, Canterbury, Kent C T 2 7NJ, U.K.; University of Alberta, Edmonton, Canada T6G 2H7; 'University of Cambridge, Cambridge CB2 IQW, U.K. T h e mechanisms by which small molecules and ions pass across t h e syncytiotrophoblast a r e of g r e a t interest, given t h a t this tissue forms t h e primary barrier between t h e m a t e r n a l and f e t a l circulations in t h e human placenta. Many d i f f e r e n t transport systems have been d e t e c t e d and characterized in t h e trophoblast. For example, w e recently reported t h e identification of t h e erythrocyte-type glucose transporter (GLUTI) a s t h e major hexose transporter present in placenta, and using immunological techniques showed t h a t this protein is present a t both surfaces of t h e syncytiotrophoblast, but not in t h e capillary endothelium [l]. Similarly, in a previous study we reported t h e presence of broad-specificity, nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporters in both brush-border and basal membrane vesicles derived f r o m t h e m a t e r n a l and f e t a l surfaces of t h e human placental syncytiotrophoblast respectively 121. In t h e present study w e have used polyclonal antibodies raised against t h e NBMPR-sensitive nucleoside transporter from human erythrocytes to examine t h e relationships between t h e placental and e r y t h r o c y t e proteins, and to investigate further t h e distribution of nucleoside transporters within t h e placenta. A polyclonal antiserum was raised against t h e purified human e r y t h r o c y t e nucleoside transporter a s previously described 131. This antiserum contained antibodies which recognised t h e purified protein on Western blots. However, i t was important to exclude t h e possibility t h a t i t also contained antibodies capable of recognising t h e human erythrocyte glucose transporter, because both proteins m i g r a t e a s broad bands of identical apparent M, (55000) on SDS/polyacrylamide gels [41. Furthermore, purification of t h e nucleoside transporter entails its separation f r o m a large excess of glucose transporter 141. Confirmation of t h e specificity of t h e antiserum was provided by Western blotting: t h e antibodies were found not to recognise blots of t h e human glucose transporter when this was produced, f r e e of contamination with nucleoside transporter, a s a recombinant protein in insect cells. Syncytiotrophoblast brush-border and basal membranes were isolated f r o m fresh t e r m human placenta a s previously described 121. Contamination with erythrocyte membranes was negligible, a s shown by t h e virtual absence of acetylcholinesterase activity. On Western blots of t h e brush-border membranes t h e anti-nucleoside transporter antibodies recognised a protein t h a t migrated a s a broad band of apparent M, 55000, essentially identical in its mobility t o t h e human e r y t h r o c y t e transporter [3,4J. Surprisingly, t h e antibodies did not recognise any proteins on blots of basal membranes, despite t h e presence of equal concentrations of NBMPR-binding s i t e s in t h e t w o placental membrane fractions. This situation c a n be contrasted with our previous finding t h a t both fractions contain t h e s a m e glucose transporter isoform (GLUTl) [ I ] . In order to confirm these findings, and to investigate t h e
t h e possible presence of nucleoside transporters a t o t h e r locations in t h e placenta, c r y o s t a t sections of perfusionfixed (4% paraformaldehyde) placenta and umbilical cord were prepared for examination by confocal immunoepifluorescence microscopy. Sections w e r e blocked by incubation for 90 min with 5% normal swine serum, followed by overnight incubation with pre-immune s e r u m o r antinucleoside transporter serum, diluted 1:50 in phosphatebuffered saline. A f t e r washing, bound antibody was d e t e c t e d by incubation for 90 min with a fluorescein conjugate of swine anti-rabbit IgC, which had been pre-absorbed with an homogenate of fixed human placental tissue. Sections w e r e then washed and mounted in Citifluor anti-fade medium for examination using a Bio-Rad MRCGOO confocal laser scanning microscope. Pre-immune s e r u m showed no staining of e i t h e r chorionic tissue sections or sections of umbilical cord. However, t h e anti-nucleoside transporter antibodies showed strong staining of both t h e brush-border s u r f a c e of t h e syncytiotrophoblast and of f e t a l capillary endothelial cells in cross-sections of chorionic villi. No staining of t h e basal s u r f a c e of t h e trophoblast was d e t e c t a b l e , an observation which paralleled t h e lack of staining of basal membranes on Western blots. This lack of staining could not have resulted from damage to or loss of t h e basal membranes during section preparation because, in parallel experiments, these membranes w e r e found t o b e strongly stained by antibodies against t h e glucose transporter [ 11. In t h e umbilical cord, intense staining of t h e endothelium by anti-nucleoside transporter antibodies was observed in t h e umbilical vein but not in t h e umbilical a r t e r y , although both endothelia w e r e strongly stained by antibodies raised against human von Willebrand factor. Interestingly, we w e r e unable t o d e t e c t t h e GLUT1 glucose transporter isoform in t h e endothelial cells of e i t h e r t h e chorionic villus or t h e umbilical cord using confocal epi-immunofluorescence. In conclusion, t h e results of both Western blotting and immunofluorescence studies suggest t h a t a t least t w o isoforms of NBMPR-sensitive nucleoside transporter a r e present in human placenta, only o n e of which is recognised by t h e anti-erythrocyte transporter antibodies. The d e t e c t i o n of an abundance of t h e immunologically cross-reactive isoform in t h e umbilical cord vein endothelium is consistent with our previous observation t h a t cultured human umbilical vein endothelial cells (HUVEC) contain a high concentration of NBMPR-binding sites [5]. Its physiological significance is unclear, b u t t h e transporters may b e important for c l e a r a n c e of circulating adenosine, which is known t o be a vasoconstrictor in t h e human placental vascular bed IS]. We thank t h e C a n c e r Research Campaign, t h e Wellcome Trust, t h e Medical Research Council and t h e British Diabetic Association for support. Ethics C o m m i t t e e approval was obtained. 1.
2. 3. 4.
5. 6.
Barros, L.F., Baldwin, S.A., Jarvis, S.M., Cowen, 'I., Thrasivoulou, C., Beaumont, N. & Yudilevich, D.L. (1992) J . Physiol. 446, 345P Barros, L.F., Bustamante, J.C., Yudilevich, D.L. & Jarvis, S.M. (1991) J . Membr. Biol. 119, 151-161 Kwong, F.Y.P., Fincharn, H.E., Young, J.U. & Baldwin, S.A. (1989) Biochem. Soc. Trans. 17, 74:%-744 Kwong, F.Y.P., Davies, A., Tse, C.M., Young, J.D., Henderson, P.J.F. & Baldwin, S.A. (1988) I3iochvrn. J . 255, 243-249 Sobrevia, L., Morgan, D.M.L., Jarvis, S.M. & Yudilevich, D.L. (1991) J. Physiol. (1-ond). 438, 277P Slegel, P., Kitagawa, H. & Maguire, M.H. (1988)Anal. Biochem. 171, 124-134
Biochemical Society Transactions ( 1 992) 20
Nucleoside transporters in human placenta L. FELIPE BARROS., N1Cf BEAUMONT', SIMON M. JARVIS#, JAMES D. YOUNG , PETER J.F. HENDERSON+, DAVID L. YUDILEVICH*, CHRIS THRASIVOULOU' and STEPHEN A. BALDWINO *University of Chile, Casilla 70055 Santiago 7, Chile; 'Royal Free Hospital School of Medicine (Universit of London), Rowland Hill Street, London NW3 2PF, U.K.; !University of Kent, Canterbury, Kent C T 2 7NJ, U.K.; University of Alberta, Edmonton, Canada T6G 2H7; 'University of Cambridge, Cambridge CB2 IQW, U.K. T h e mechanisms by which small molecules and ions pass across t h e syncytiotrophoblast a r e of g r e a t interest, given t h a t this tissue forms t h e primary barrier between t h e m a t e r n a l and f e t a l circulations in t h e human placenta. Many d i f f e r e n t transport systems have been d e t e c t e d and characterized in t h e trophoblast. For example, w e recently reported t h e identification of t h e erythrocyte-type glucose transporter (GLUTI) a s t h e major hexose transporter present in placenta, and using immunological techniques showed t h a t this protein is present a t both surfaces of t h e syncytiotrophoblast, but not in t h e capillary endothelium [l]. Similarly, in a previous study we reported t h e presence of broad-specificity, nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporters in both brush-border and basal membrane vesicles derived f r o m t h e m a t e r n a l and f e t a l surfaces of t h e human placental syncytiotrophoblast respectively 121. In t h e present study w e have used polyclonal antibodies raised against t h e NBMPR-sensitive nucleoside transporter from human erythrocytes to examine t h e relationships between t h e placental and e r y t h r o c y t e proteins, and to investigate further t h e distribution of nucleoside transporters within t h e placenta. A polyclonal antiserum was raised against t h e purified human e r y t h r o c y t e nucleoside transporter a s previously described 131. This antiserum contained antibodies which recognised t h e purified protein on Western blots. However, i t was important to exclude t h e possibility t h a t i t also contained antibodies capable of recognising t h e human erythrocyte glucose transporter, because both proteins m i g r a t e a s broad bands of identical apparent M, (55000) on SDS/polyacrylamide gels [41. Furthermore, purification of t h e nucleoside transporter entails its separation f r o m a large excess of glucose transporter 141. Confirmation of t h e specificity of t h e antiserum was provided by Western blotting: t h e antibodies were found not to recognise blots of t h e human glucose transporter when this was produced, f r e e of contamination with nucleoside transporter, a s a recombinant protein in insect cells. Syncytiotrophoblast brush-border and basal membranes were isolated f r o m fresh t e r m human placenta a s previously described 121. Contamination with erythrocyte membranes was negligible, a s shown by t h e virtual absence of acetylcholinesterase activity. On Western blots of t h e brush-border membranes t h e anti-nucleoside transporter antibodies recognised a protein t h a t migrated a s a broad band of apparent M, 55000, essentially identical in its mobility t o t h e human e r y t h r o c y t e transporter [3,4J. Surprisingly, t h e antibodies did not recognise any proteins on blots of basal membranes, despite t h e presence of equal concentrations of NBMPR-binding s i t e s in t h e t w o placental membrane fractions. This situation c a n be contrasted with our previous finding t h a t both fractions contain t h e s a m e glucose transporter isoform (GLUTl) [ I ] . In order to confirm these findings, and to investigate t h e
t h e possible presence of nucleoside transporters a t o t h e r locations in t h e placenta, c r y o s t a t sections of perfusionfixed (4% paraformaldehyde) placenta and umbilical cord were prepared for examination by confocal immunoepifluorescence microscopy. Sections w e r e blocked by incubation for 90 min with 5% normal swine serum, followed by overnight incubation with pre-immune s e r u m o r antinucleoside transporter serum, diluted 1:50 in phosphatebuffered saline. A f t e r washing, bound antibody was d e t e c t e d by incubation for 90 min with a fluorescein conjugate of swine anti-rabbit IgC, which had been pre-absorbed with an homogenate of fixed human placental tissue. Sections w e r e then washed and mounted in Citifluor anti-fade medium for examination using a Bio-Rad MRCGOO confocal laser scanning microscope. Pre-immune s e r u m showed no staining of e i t h e r chorionic tissue sections or sections of umbilical cord. However, t h e anti-nucleoside transporter antibodies showed strong staining of both t h e brush-border s u r f a c e of t h e syncytiotrophoblast and of f e t a l capillary endothelial cells in cross-sections of chorionic villi. No staining of t h e basal s u r f a c e of t h e trophoblast was d e t e c t a b l e , an observation which paralleled t h e lack of staining of basal membranes on Western blots. This lack of staining could not have resulted from damage to or loss of t h e basal membranes during section preparation because, in parallel experiments, these membranes w e r e found t o b e strongly stained by antibodies against t h e glucose transporter [ 11. In t h e umbilical cord, intense staining of t h e endothelium by anti-nucleoside transporter antibodies was observed in t h e umbilical vein but not in t h e umbilical a r t e r y , although both endothelia w e r e strongly stained by antibodies raised against human von Willebrand factor. Interestingly, we w e r e unable t o d e t e c t t h e GLUT1 glucose transporter isoform in t h e endothelial cells of e i t h e r t h e chorionic villus or t h e umbilical cord using confocal epi-immunofluorescence. In conclusion, t h e results of both Western blotting and immunofluorescence studies suggest t h a t a t least t w o isoforms of NBMPR-sensitive nucleoside transporter a r e present in human placenta, only o n e of which is recognised by t h e anti-erythrocyte transporter antibodies. The d e t e c t i o n of an abundance of t h e immunologically cross-reactive isoform in t h e umbilical cord vein endothelium is consistent with our previous observation t h a t cultured human umbilical vein endothelial cells (HUVEC) contain a high concentration of NBMPR-binding sites [5]. Its physiological significance is unclear, b u t t h e transporters may b e important for c l e a r a n c e of circulating adenosine, which is known t o be a vasoconstrictor in t h e human placental vascular bed IS]. We thank t h e C a n c e r Research Campaign, t h e Wellcome Trust, t h e Medical Research Council and t h e British Diabetic Association for support. Ethics C o m m i t t e e approval was obtained. 1.
2. 3. 4.
5. 6.
Barros, L.F., Baldwin, S.A., Jarvis, S.M., Cowen, 'I., Thrasivoulou, C., Beaumont, N. & Yudilevich, D.L. (1992) J . Physiol. 446, 345P Barros, L.F., Bustamante, J.C., Yudilevich, D.L. & Jarvis, S.M. (1991) J . Membr. Biol. 119, 151-161 Kwong, F.Y.P., Fincharn, H.E., Young, J.U. & Baldwin, S.A. (1989) Biochem. Soc. Trans. 17, 74:%-744 Kwong, F.Y.P., Davies, A., Tse, C.M., Young, J.D., Henderson, P.J.F. & Baldwin, S.A. (1988) I3iochvrn. J . 255, 243-249 Sobrevia, L., Morgan, D.M.L., Jarvis, S.M. & Yudilevich, D.L. (1991) J. Physiol. (1-ond). 438, 277P Slegel, P., Kitagawa, H. & Maguire, M.H. (1988)Anal. Biochem. 171, 124-134
