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Characterization of glycerophosphocholine phosphodiesterase activity and phosphatidylcholine biosynthesis in cultured retinal microcapillary pericytes. Effect of adenosine and endothelin-1

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Lipids

Abstract

In pericytes from bovine retina, the enzyme glycerophosphocholine phosphodiesterase, catalyzing the hydrolysis of sn-glycero-3-phosphocholine to glycero-3-phosphate and choline, has been characterized with respect to pH optimum, metal ion dependence, K m, inhibitors, and subcellular localization. In these cells, the natural substrate sn-glycero-3-phosphocholine was present at relatively high concentration (6.4±1.2 nmol/mg protein), and the EDTA-sensitive phosphodiesterase activity was also found to be markedly high (9.80±1.5 nmol/min/mg protein) compared to the estimated in liver and brain (1–3 nmol/min/mg protein) or in renal epithelial cell culture (0.27 nmol/min/mg protein). The reaction conditions were in general agreement with those found earlier in brain and other tissues. The majority of the enzyme specific activity was located in the plasma membrane, whereas a minor part was present in the microsomal fraction. The physiological significance of the high catabolic phosphodiesterase activity in these cells may be related to the tranfer, followed by deacylation, of lysophosphatidylcholine from the bloodstream to nervous tissue. In addition, capillary pericytes in culture were able to incorporate 3H-choline rapidly into choline-containing soluble phosphorylated intermediates and into phosphatidylcholine. To find a positive and negative effector on phosphatidylcholine formation, adenosine, an important intercellular mediator in the retina in response to alterations in oxygen delivery, and endothelin-1, a potent paracrine mediator present at the blood-brain and blood-retina barrier, were tested. The cells cultured for 1 or 24 h in a medium containing adenosine at concentrations of 10−6 and 10−4 M showed significant reduction in 3H-choline incorporation compared to control cultures, whereas endothelin-1, at a concentration of 10 and 100 nM, caused stimulation of phosphatidylcholine biosynthesis. These findings provide evidence that both agonists may modulate phosphatidylcholine metabolism in pericytes.

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Abbreviations

CDP-Cho:

cycidine diphosphocholine

CT:

CTP:phosphocholine cytidylyltransferase

EC:

endothelial cells

ET-1:

endothelin 1

γ-GPT:

γ-glutamyltranspeptidase

GroPCho:

sn-glycero-3-phosphocholine

GroPChoPDE:

glycerophosphocholine phosphodiesterase

lyso-PtdCho:

lysophosphtidylcholine

PCho:

phosphorylcholine

PLA2 :

phospholipase A2

PtdCho:

phosphatidylcholine

SN:

supernatant

References

  1. Alberghina, M., Infarinato, S., Anfuso, C.D., and Lupo, G. (1994) 1-Acyl-2-lysophosphatidycholine Transport Across the Rat Blood-Retina and Blood-Brain Barrier, FEBS Lett 351, 181–185.

    Article  PubMed  CAS  Google Scholar 

  2. Sipione, S., Lupo, G., Anfuso, C.D., Albanese, V., and Alberghina, M. (1996) Phosphatidylcholine Synthesis-Related Enzyme Activities of Bovine Brain Microvessels Exhibit Susceptibility to Peroxidation, FEBS Lett 384, 19–24.

    Article  PubMed  CAS  Google Scholar 

  3. Baldwin, J.J., and Cornatzer, W.E. (1968) Rat Kidney Glycerylphosphorylcholine Diesterase, Biochim. Biophys. Acta 164, 195–204.

    PubMed  CAS  Google Scholar 

  4. Zablocki, K., Miller, S.P.F., Garcia-Perez, A., and Burg, M.G. (1991) Accumulation of Glycerophosphocholine (GPC) by Renal Cells: Somotic Regulation of GPC: Choline Phosphodiesterase, Proc. Natl. Acad. Sci. USA 88, 7820–7824.

    Article  PubMed  CAS  Google Scholar 

  5. Abra, R.M., and Quinn, P.J. (1976) Some Characteristics of sn-Glycero-3-Phosphocholine Diesterase from Rat Brain, Biochim. Biophys. Acta 431, 631–639.

    PubMed  CAS  Google Scholar 

  6. Spanner, S., and Ansell, G.B. (1982) Activation of Glycerophosphocholine Phosphodiesterase in Rat Forebrain by Ca2+, Biochem. J 62, 689–693.

    Google Scholar 

  7. Kanfer, J.N., and McCartney, D.G. (1988) Developmental and Regional Quantitation of Glycerophosphorylcholine Phosphodiesterase Activities in Rat Brain, Neurochem. Res. 13, 803–806.

    Article  PubMed  CAS  Google Scholar 

  8. Kanfer, J.N., and McCartney, D.G. (1989) Glycerophosphorylcholine Phosphocholine Phosphodiesterase Activity of Rat Brain Myelin, J. Neurosci. Res. 24, 231–240.

    Article  PubMed  CAS  Google Scholar 

  9. Mitra, J., and Chowdhury, M. (1991) Purification and Characterization of Rat Uterine Glycerophosphorylcholine Diesterase and Its Tissue-Specific Induction by 17β-Estradiol, Endocrinology 129, 1147–1154.

    Article  PubMed  CAS  Google Scholar 

  10. Hirschi, K.K., and D’Amore, P.A. (1996) Pericytes in the Microvasculature, Cardiovasc. Res. 32, 687–698.

    Article  PubMed  CAS  Google Scholar 

  11. Li, Q., and Puro, D.G. (2001) Adenosine Activates ATP-Sensitive K+ Currents in Pericytes of Rat Retinal Microvessels: Role of A1 and A2a Receptors, Brain Res. 907, 93–99.

    Article  PubMed  CAS  Google Scholar 

  12. Bursell, S.E., Clermont, A.C., Oren, B., and King, G.L. (1995) The in vivo Effect of Endothelins on Retinal Circulation in Non-diabetic and Diabetic Rats, Invest. Ophthalmol. Vis. Sci. 36, 596–607.

    PubMed  CAS  Google Scholar 

  13. Dehouck, M.P., Vigne, P., Torpier, G., Breittmayer, J.P., Cechelli, R., and Frelin, C. (1997) Endothelin-1 as a Mediator of Endothelial Cell-Pericyte Interactions in Bovine Brain Capillaries, J. Cerebr. Blood Flow Metab. 17, 464–469.

    CAS  Google Scholar 

  14. Kawamura, H., Oku, H., Li, Q., Sakagami, K., and Puro, D.G. (2002) Endothelin-Induced Changes in the Physiology of Retinal Pericytes, Invest. Ophthalmol. Vis. Sci. 43, 882–888.

    PubMed  Google Scholar 

  15. McGinty, A., Scholfield, C.N., Liu, W.H., Anderson, P., Hoey, D.E., and Trimble, E.R. (1999) Effect of Glucose on Endothelin-1-Induced Calcium Transient in Cultured Bovine Retinal Pericytes, J. Biol. Chem. 274, 25250–25253.

    Article  PubMed  CAS  Google Scholar 

  16. Cook, H.W., Palmer, F.B.S.T., Byers, D.M., and Spence, M.W. (1988) Isolation of Plasma Membranes from Cultured Glioma Cells and Application to Evaluation of Membrane Sphingomyelin Turnover, Anal. Biochem. 174, 552–560.

    Article  PubMed  CAS  Google Scholar 

  17. Alberghina, M., Buonacera, P., Agodi, A., and Giuffrida Stella, A.M. (1988) Occurrence of Phospholipase A1-A2 and Lysophosphatidylcholine Acyltransferase activities in Axolemma-Enriched Fraction of Brain Stem, Optic Pathway, and Craniospinal Nerves of the Rabbit, J. Neurosci. Res. 19, 79–87.

    Article  PubMed  CAS  Google Scholar 

  18. Takayama, M., Itoh, S., Nagasaki, T., and Tanumizu, I. (1977) A New Enzymatic Method for Determination of Serum Choline-Containing Phospholipids, Clin. Chim. Acta 79, 93–98.

    Article  PubMed  CAS  Google Scholar 

  19. Williams, S.K., Gillis, J.E., Matthews, M.A., Wagner, R.C., and Bitensky, M.W. (1980) Isolation and Characterization of Brain Endothelial Cells: Morphology and Enzyme Activity, J. Neurochem. 35, 374–381.

    Article  PubMed  CAS  Google Scholar 

  20. Barak, A.J., and Tuma, D.J. (1978) Determination of Free Choline and Phosphorylcholine in Rat Liver, Lipids 14, 304–308.

    Google Scholar 

  21. Orlowski, M., and Meister, A. (1963) γ-Glutamyl-p-nitroanilide: A New Convenient Substrate for Determination and Study of l-and d-γ-Glutamyltranspeptidase Activities, Biochim. Biophys. Acta 73, 679–681.

    Article  PubMed  CAS  Google Scholar 

  22. Folch, J., Lees, M., and Sloane Stanley, G.H. (1957) A Simple Method for the Isolation and Purification of Total Lipids from Animal Tissues, J. Biol. Chem. 226, 497–509.

    PubMed  CAS  Google Scholar 

  23. Lupo, G., Anfuso, C.D., Ragusa, N., Strosznajder, R.P., Walski, M., and Alberghina, M. (2001) t-BuOOH and oxLDL Enhance Phospholipid Hydrolysis in Lipopolysaccharide-Stimulated Retinal Pericytes, Biochim. Acta 1557, 143–155.

    Google Scholar 

  24. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) Protein Measurement with the Folin Phenol Reagent, J. Biol. Chem. 193, 265–275.

    PubMed  CAS  Google Scholar 

  25. Jamil, H., and Vance, D.E. (1991) Substrate Specificity of CTP:phosphocholine Cytidyltransferase, Biochim. Biophys. Acta 1086, 335–339.

    PubMed  CAS  Google Scholar 

  26. Schor, A.M., Allen, T.D., Canfied, A.E., Sloan, P., and Schor, S.L. (1990) Pericytes Derived from the Retinal Microvasculature Undergo Calification in vitro, J. Cell Sci. 97, 449–461.

    PubMed  Google Scholar 

  27. Frey, A., Mackelein, B., Weiler-Guttler, H., Mockel, B., Flach, R., and Gassen, H.G. (1991) Pericytes of the Brain Microvasculature Express γ-Glutamyltranspeptidase, Eur. J. Biochem. 202, 421–429.

    Article  PubMed  CAS  Google Scholar 

  28. Risau, W., Dingler, A., Albrecht, U., Dehouck, M.P., and Cecchelli, R. (1992) Blood-Brain Barrier Pericytes Are the Main Source of γ-Glutamyltranspeptidase Activity in Brain Capillaries, J. Neurochem. 58, 667–672.

    Article  PubMed  CAS  Google Scholar 

  29. Yavin, E. (1976) Regulation of Phospholipid Metabolism in Differentiating Cells from Rat Brain Cerebral Hemispheres in Culture, J. Biol. Chem. 251, 1392–1397.

    PubMed  CAS  Google Scholar 

  30. Belloni-Olivi, L., Bressler, J.P., and Goldstein, G.W. (1992) Retinal Microvessels Express Less γ-Glutamyltranspeptidase than Brain Microvessels, Curr. Eye Res 11, 203–211.

    PubMed  CAS  Google Scholar 

  31. Webster, G.R., Marples, E.A., and Thompson, R.H.S (1957) Glycerylphosphorylcholine Diesterase Activity of Nervous Tissues, Biochem. J. 65, 374–377.

    PubMed  CAS  Google Scholar 

  32. Anfuso, C.D., Lupo, G., and Alberghina, M. (1999) Amyloid β but Not Bradykinin Induces Phosphatidylcholine Hydrolysis in Immortalized Rat Brain Endothelial Cells, Neurosci. Lett. 271, 151–154.

    Article  PubMed  CAS  Google Scholar 

  33. Stitt, A.W., Anderson, H.R., Gardiner, T.A., Bailie, J.R., and Archer, D.B. (1994) Receptor-Mediated Endocytosis and Intracellular Trafficking of Insulin and Low-Density Lipoprotein by Retinal Vascular Endothelial Cells, Invest. Ophthalmol. Vis. Sci. 35, 3384–3392.

    PubMed  CAS  Google Scholar 

  34. Alberghina, M., and Gould, R.M. (1990) Levels of Choline Intermediates in the Visual System Structures and in Peripheral Nerve of the Rat: Comparison with Neural Tissues of a Lower Vertebrate (Mustelus canis) and an Invertebrate (Loligo pealei), Neurochem. Int. 17, 599–604.

    Article  CAS  Google Scholar 

  35. Fox, I.H., and Kelley, W.N. (1978) The Role of Adenosine and 2′-Deoxy Adenosine in Mammalian Cells, Annu. Rev. Biochem. 47, 655–686.

    Article  PubMed  CAS  Google Scholar 

  36. Meininger, C.J., Schelling, M.E., and Granger, H.J. (1988) Adenosine and Hypoxia Stimulate Proliferation and Migration of Endothelial Cells, Am. J. Physiol. 255, H554–H562.

    PubMed  CAS  Google Scholar 

  37. Ethier, M.F., Chander, V., and Dobson, J.G. (1993) Adenosine Stimulates Proliferation and Migration of Endothelial Cells, Am. J. Physiol. 265, H131–H138.

    PubMed  CAS  Google Scholar 

  38. Jackson, J.A., and Carlson, E.C. (1992) Inhibition of Bovine Retinal Microvascular Pericyte Proliferation in vitro by Adenosine, Am. J. Physiol. 263, H634–H640.

    PubMed  CAS  Google Scholar 

  39. Dubey, R.K., Gillespie, D.G., Mi, Z., Suzuki, F., and Jackson, E.K. (1996) Smooth Muscle Cell-Derived Adenosine Inhibits Cell Growth, Hypertension 27, 766–773.

    PubMed  CAS  Google Scholar 

  40. Tinton, S., and Buc-Calderon, P. (1995) Homocystein Enhances the Inhibitory Effect of Extracellular Adenosine on the Synthesis of Proteins in Isolated Rat Hepatocytes, Biochem. J. 310, 893–896.

    PubMed  CAS  Google Scholar 

  41. Dawicki, D.D., Chatterjee, D., Wyche, J., and Rounds, S. (1997) Extracellular ATP and Atlenosine Cause Apoptosis of Pulmonary Artery Endothelial Cells, Am. J. Physiol. 273, L485–L494.

    PubMed  CAS  Google Scholar 

  42. Mlodzik, K., Loffing, J., Le Hir, M., and Kaissling, B. (1995) Ecto-5′-Nucleotidase Is Expressed by Pericytes and Fibroblasts in the Rat Heart, Histochem. Cell Biol. 103, 227–236.

    Article  PubMed  CAS  Google Scholar 

  43. Maccumber, M.W., Ross, C.A., and Snyder, S.H. (1990) Endothelin in Brain: Receptors, Mitogenesis, and Biosynthesis in Glial Cells, Proc. Natl. Acad. Sci. Usa 87, 2359–2363.

    Article  PubMed  CAS  Google Scholar 

  44. Takahashi, K., Brooks, R.A., Kanse, S.M., Ghatei, M.A., Kohner, E.M., and Bloom, S.R. (1989) Production of Endothelin-1 by Cultured Bovine Retinal Endothelial Cells and Presence of Endothelin Receptors on Associated Pericytes, Diabetes 38, 1200–1202.

    PubMed  CAS  Google Scholar 

  45. Yoshimoto, S., Ishizaki, Y., Kurihara, H., Sasaki, T., Yoshizumi, M., Yanagisawa, M., Yazaki, Y., Masaki, T., Takakura, K., and Murota, S. (1990) Cerebral Microvessel Endothelium Is Producing Endothelin, Brain Res. 508, 283–285.

    Article  PubMed  CAS  Google Scholar 

  46. Lee, T.S., Hu, K.Q., Chao, T., and King, K.Q. (1989) Characterization of Endothelin Receptors and Effects of Endothelin on Diacylglycerol and Protein Kinase C in Retinal Capillary Pericytes, Diabetes 38, 1643–1646.

    PubMed  CAS  Google Scholar 

  47. Chakravarthy, U., Gardiner, T.A., Anderson, P., Archer, D.B., and Trimble, E.R. (1992) The Effect of Endothelin-1 on the Retinal Microvascular Pericyte, Microvasc. Res. 43, 241–254.

    Article  PubMed  CAS  Google Scholar 

  48. Yamagishi, S., Hsu, C., Kobayashi, K., and Yamamoto, H. (1993) Endothelin-1 Mediates Endothelial Cell-Dependent Proliferation of Vascular Pericytes, Biochem. Biophys. Res. Comm. 191, 840–846.

    Article  PubMed  CAS  Google Scholar 

  49. Baldi, E., Musial, A., and Kester, M. (1994) Endothelin Stimulates Phosphatidylcholine Hydrolysis Through Both PLC and PLD Pathways in Mesangial Cells, Am. J. Physiol. 266, F957–F965.

    PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Biochemistry, Faculty of Medicine, University of Catania, Viale Andrea Doria 6, 95125, Catania, Italy

    Carmelina D. Anfuso, Simonetta Sipione, Gabriella Lupo, Nicolò Ragusa & Mario Alberghina

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  1. Carmelina D. Anfuso
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  2. Simonetta Sipione
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  3. Gabriella Lupo
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  4. Nicolò Ragusa
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  5. Mario Alberghina
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Correspondence to Mario Alberghina.

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Anfuso, C.D., Sipione, S., Lupo, G. et al. Characterization of glycerophosphocholine phosphodiesterase activity and phosphatidylcholine biosynthesis in cultured retinal microcapillary pericytes. Effect of adenosine and endothelin-1. Lipids 38, 45–52 (2003). https://doi.org/10.1007/s11745-003-1030-z

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  • DOI: https://doi.org/10.1007/s11745-003-1030-z

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