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. 1994 Dec 1;127(5):1217–1232. doi: 10.1083/jcb.127.5.1217

Filipin-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules

PMCID: PMC2120262  PMID: 7525606

Abstract

Caveolae or noncoated plasmalemmal vesicles found in a variety of cells have been implicated in a number of important cellular functions including endocytosis, transcytosis, and potocytosis. Their function in transport across endothelium has been especially controversial, at least in part because there has not been any way to selectively inhibit this putative pathway. We now show that the ability of sterol binding agents such as filipin to disassemble endothelial noncoated but not coated plasmalemmal vesicles selectively inhibits caveolae-mediated intracellular and transcellular transport of select macromolecules in endothelium. Filipin significantly reduces the transcellular transport of insulin and albumin across cultured endothelial cell monolayers. Rat lung microvascular permeability to albumin in situ is significantly decreased after filipin perfusion. Conversely, paracellular transport of the small solute inulin is not inhibited in vitro or in situ. In addition, we show that caveolae mediate the scavenger endocytosis of conformationally modified albumins for delivery to endosomes and lysosomes for degradation. This intracellular transport is inhibited by filipin both in vitro and in situ. Other sterol binding agents including nystatin and digitonin also inhibit this degradative process. Conversely, the endocytosis and degradation of activated alpha 2- macroglobulin, a known ligand of the clathrin-dependent pathway, is not affected. Interestingly, filipin appears to inhibit insulin uptake by endothelium for transcytosis, a caveolae-mediated process, but not endocytosis for degradation, apparently mediated by the clathrin-coated pathway. Such selective inhibition of caveolae not only provides critical evidence for the role of caveolae in the intracellular and transcellular transport of select macromolecules in endothelium but also may be useful for distinguishing transport mediated by coated versus noncoated vesicles.

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Selected References

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  1. Anderson R. G., Kamen B. A., Rothberg K. G., Lacey S. W. Potocytosis: sequestration and transport of small molecules by caveolae. Science. 1992 Jan 24;255(5043):410–411. doi: 10.1126/science.1310359. [DOI] [PubMed] [Google Scholar]
  2. Bamezai A., Goldmacher V. S., Rock K. L. Internalization of glycosyl-phosphatidylinositol (GPI)-anchored lymphocyte proteins. II. GPI-anchored and transmembrane molecules internalize through distinct pathways. Eur J Immunol. 1992 Jan;22(1):15–21. doi: 10.1002/eji.1830220104. [DOI] [PubMed] [Google Scholar]
  3. Bolard J. How do the polyene macrolide antibiotics affect the cellular membrane properties? Biochim Biophys Acta. 1986 Dec 22;864(3-4):257–304. doi: 10.1016/0304-4157(86)90002-x. [DOI] [PubMed] [Google Scholar]
  4. Bretscher M. S., Thomson J. N., Pearse B. M. Coated pits act as molecular filters. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4156–4159. doi: 10.1073/pnas.77.7.4156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brown M. S., Goldstein J. L. Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis. Annu Rev Biochem. 1983;52:223–261. doi: 10.1146/annurev.bi.52.070183.001255. [DOI] [PubMed] [Google Scholar]
  6. Bumbasirević V., Pappas G. D., Becker R. P. Endocytosis of serum albumin-gold conjugates by microvascular endothelial cells in rat adrenal gland: regional differences between cortex and medulla. J Submicrosc Cytol Pathol. 1990 Jan;22(1):135–145. [PubMed] [Google Scholar]
  7. Bundgaard M. Vesicular transport in capillary endothelium: does it occur? Fed Proc. 1983 May 15;42(8):2425–2430. [PubMed] [Google Scholar]
  8. Davis E. C., Shivers R. R. Ordered distribution of membrane-associated dense plaques in intact quail gizzard smooth muscle cells revealed by freeze-fracture following treatment with cholesterol probes. Anat Rec. 1992 Mar;232(3):385–392. doi: 10.1002/ar.1092320308. [DOI] [PubMed] [Google Scholar]
  9. De Bruyn P. P., Michelson S., Bankston P. W. In-vivo endocytosis by bristle-coated pits and intracellular transport of endogenous albumin in the endothelium of the sinuses of liver and bone marrow. Cell Tissue Res. 1985;240(1):1–7. doi: 10.1007/BF00217551. [DOI] [PubMed] [Google Scholar]
  10. Dickson R. B., Willingham M. C., Pastan I. alpha 2-macroglobulin adsorbed to colloidal gold: a new probe in the study of receptor-mediated endocytosis. J Cell Biol. 1981 Apr;89(1):29–34. doi: 10.1083/jcb.89.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Elias P. M., Goerke J., Friend D. S., Brown B. E. Freeze-fracture identification of sterol-digitonin complexes in cell and liposome membranes. J Cell Biol. 1978 Aug;78(2):577–596. doi: 10.1083/jcb.78.2.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Faustmann P. M., Teutrine S., Krause D., Dermietzel R. Subarachnoidal macrophages share a common epitope with resident non-cerebral macrophages and show receptor-mediated endocytosis of albumin-gold and IgG-gold complexes. J Neuroimmunol. 1991 Dec;35(1-3):79–88. doi: 10.1016/0165-5728(91)90163-2. [DOI] [PubMed] [Google Scholar]
  13. Frøkjaer-Jensen J. Three-dimensional organization of plasmalemmal vesicles in endothelial cells. An analysis by serial sectioning of frog mesenteric capillaries. J Ultrastruct Res. 1980 Oct;73(1):9–20. doi: 10.1016/0022-5320(80)90111-2. [DOI] [PubMed] [Google Scholar]
  14. Galis Z., Ghitescu L., Simionescu M. Fatty acids binding to albumin increases its uptake and transcytosis by the lung capillary endothelium. Eur J Cell Biol. 1988 Dec;47(2):358–365. [PubMed] [Google Scholar]
  15. Geoffroy J. S., Becker R. P. Endocytosis by endothelial phagocytes: uptake of bovine serum albumin-gold conjugates in bone marrow. J Ultrastruct Res. 1984 Dec;89(3):223–239. doi: 10.1016/s0022-5320(84)80039-8. [DOI] [PubMed] [Google Scholar]
  16. Ghinea N., Eskenasy M., Simionescu M., Simionescu N. Endothelial albumin binding proteins are membrane-associated components exposed on the cell surface. J Biol Chem. 1989 Mar 25;264(9):4755–4758. [PubMed] [Google Scholar]
  17. Ghinea N., Fixman A., Alexandru D., Popov D., Hasu M., Ghitescu L., Eskenasy M., Simionescu M., Simionescu N. Identification of albumin-binding proteins in capillary endothelial cells. J Cell Biol. 1988 Jul;107(1):231–239. doi: 10.1083/jcb.107.1.231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ghitescu L., Bendayan M. Transendothelial transport of serum albumin: a quantitative immunocytochemical study. J Cell Biol. 1992 May;117(4):745–755. doi: 10.1083/jcb.117.4.745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ghitescu L., Fixman A., Simionescu M., Simionescu N. Specific binding sites for albumin restricted to plasmalemmal vesicles of continuous capillary endothelium: receptor-mediated transcytosis. J Cell Biol. 1986 Apr;102(4):1304–1311. doi: 10.1083/jcb.102.4.1304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Goldstein J. L., Brown M. S., Anderson R. G., Russell D. W., Schneider W. J. Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Annu Rev Cell Biol. 1985;1:1–39. doi: 10.1146/annurev.cb.01.110185.000245. [DOI] [PubMed] [Google Scholar]
  21. Hansen S. H., Sandvig K., van Deurs B. The preendosomal compartment comprises distinct coated and noncoated endocytic vesicle populations. J Cell Biol. 1991 May;113(4):731–741. doi: 10.1083/jcb.113.4.731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Huet C., Ash J. F., Singer S. J. The antibody-induced clustering and endocytosis of HLA antigens on cultured human fibroblasts. Cell. 1980 Sep;21(2):429–438. doi: 10.1016/0092-8674(80)90479-1. [DOI] [PubMed] [Google Scholar]
  23. Jefferies W. A., Brandon M. R., Hunt S. V., Williams A. F., Gatter K. C., Mason D. Y. Transferrin receptor on endothelium of brain capillaries. Nature. 1984 Nov 8;312(5990):162–163. doi: 10.1038/312162a0. [DOI] [PubMed] [Google Scholar]
  24. Kartenbeck J., Stukenbrok H., Helenius A. Endocytosis of simian virus 40 into the endoplasmic reticulum. J Cell Biol. 1989 Dec;109(6 Pt 1):2721–2729. doi: 10.1083/jcb.109.6.2721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Keller G. A., Siegel M. W., Caras I. W. Endocytosis of glycophospholipid-anchored and transmembrane forms of CD4 by different endocytic pathways. EMBO J. 1992 Mar;11(3):863–874. doi: 10.1002/j.1460-2075.1992.tb05124.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. King G. L., Johnson S. M. Receptor-mediated transport of insulin across endothelial cells. Science. 1985 Mar 29;227(4694):1583–1586. doi: 10.1126/science.3883490. [DOI] [PubMed] [Google Scholar]
  27. Milici A. J., Watrous N. E., Stukenbrok H., Palade G. E. Transcytosis of albumin in capillary endothelium. J Cell Biol. 1987 Dec;105(6 Pt 1):2603–2612. doi: 10.1083/jcb.105.6.2603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mommaas-Kienhuis A. M., Nagelkerke J. F., Vermeer B. J., Daems W. T., van Berkel T. J. Visualization of the interaction of native and modified low density lipoproteins with isolated rat liver cells. Eur J Cell Biol. 1985 Jul;38(1):42–50. [PubMed] [Google Scholar]
  29. Montesano R., Roth J., Robert A., Orci L. Non-coated membrane invaginations are involved in binding and internalization of cholera and tetanus toxins. Nature. 1982 Apr 15;296(5858):651–653. doi: 10.1038/296651a0. [DOI] [PubMed] [Google Scholar]
  30. Oliver C. Endocytic pathways at the lateral and basal cell surfaces of exocrine acinar cells. J Cell Biol. 1982 Oct;95(1):154–161. doi: 10.1083/jcb.95.1.154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Palade G. E., Bruns R. R. Structural modulations of plasmalemmal vesicles. J Cell Biol. 1968 Jun;37(3):633–649. doi: 10.1083/jcb.37.3.633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pitas R. E., Boyles J., Mahley R. W., Bissell D. M. Uptake of chemically modified low density lipoproteins in vivo is mediated by specific endothelial cells. J Cell Biol. 1985 Jan;100(1):103–117. doi: 10.1083/jcb.100.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Predescu D., Simionescu M., Simionescu N., Palade G. E. Binding and transcytosis of glycoalbumin by the microvascular endothelium of the murine myocardium: evidence that glycoalbumin behaves as a bifunctional ligand. J Cell Biol. 1988 Nov;107(5):1729–1738. doi: 10.1083/jcb.107.5.1729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rabkin R., Tsao T., Elliot S. J., Striker L. J., Striker G. E. Insulin uptake and processing by cultured mouse glomerular endothelial cells. Am J Physiol. 1993 Aug;265(2 Pt 1):C453–C459. doi: 10.1152/ajpcell.1993.265.2.C453. [DOI] [PubMed] [Google Scholar]
  35. Raposo G., Dunia I., Delavier-Klutchko C., Kaveri S., Strosberg A. D., Benedetti E. L. Internalization of beta-adrenergic receptor in A431 cells involves non-coated vesicles. Eur J Cell Biol. 1989 Dec;50(2):340–352. [PubMed] [Google Scholar]
  36. Roberts R. L., Sandra A. Receptor-mediated endocytosis of insulin by cultured endothelial cells. Tissue Cell. 1992;24(5):603–611. doi: 10.1016/0040-8166(92)90031-2. [DOI] [PubMed] [Google Scholar]
  37. Rothberg K. G., Heuser J. E., Donzell W. C., Ying Y. S., Glenney J. R., Anderson R. G. Caveolin, a protein component of caveolae membrane coats. Cell. 1992 Feb 21;68(4):673–682. doi: 10.1016/0092-8674(92)90143-z. [DOI] [PubMed] [Google Scholar]
  38. Rothberg K. G., Ying Y. S., Kamen B. A., Anderson R. G. Cholesterol controls the clustering of the glycophospholipid-anchored membrane receptor for 5-methyltetrahydrofolate. J Cell Biol. 1990 Dec;111(6 Pt 2):2931–2938. doi: 10.1083/jcb.111.6.2931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schnitzer J. E., Bravo J. High affinity binding, endocytosis, and degradation of conformationally modified albumins. Potential role of gp30 and gp18 as novel scavenger receptors. J Biol Chem. 1993 Apr 5;268(10):7562–7570. [PubMed] [Google Scholar]
  40. Schnitzer J. E., Carley W. W., Palade G. E. Albumin interacts specifically with a 60-kDa microvascular endothelial glycoprotein. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6773–6777. doi: 10.1073/pnas.85.18.6773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Schnitzer J. E., Carley W. W., Palade G. E. Specific albumin binding to microvascular endothelium in culture. Am J Physiol. 1988 Mar;254(3 Pt 2):H425–H437. doi: 10.1152/ajpheart.1988.254.3.H425. [DOI] [PubMed] [Google Scholar]
  42. Schnitzer J. E., Oh P. Albondin-mediated capillary permeability to albumin. Differential role of receptors in endothelial transcytosis and endocytosis of native and modified albumins. J Biol Chem. 1994 Feb 25;269(8):6072–6082. [PubMed] [Google Scholar]
  43. Schnitzer J. E., Oh P. Antibodies to SPARC inhibit albumin binding to SPARC, gp60, and microvascular endothelium. Am J Physiol. 1992 Dec;263(6 Pt 2):H1872–H1879. doi: 10.1152/ajpheart.1992.263.6.H1872. [DOI] [PubMed] [Google Scholar]
  44. Schnitzer J. E., Pinney E. Quantitation of specific binding of orosomucoid to cultured microvascular endothelium: role in capillary permeability. Am J Physiol. 1992 Jul;263(1 Pt 2):H48–H55. doi: 10.1152/ajpheart.1992.263.1.H48. [DOI] [PubMed] [Google Scholar]
  45. Schnitzer J. E., Siflinger-Birnboim A., Del Vecchio P. J., Malik A. B. Segmental differentiation of permeability, protein glycosylation, and morphology of cultured bovine lung vascular endothelium. Biochem Biophys Res Commun. 1994 Feb 28;199(1):11–19. doi: 10.1006/bbrc.1994.1185. [DOI] [PubMed] [Google Scholar]
  46. Schnitzer J. E., Sung A., Horvat R., Bravo J. Preferential interaction of albumin-binding proteins, gp30 and gp18, with conformationally modified albumins. Presence in many cells and tissues with a possible role in catabolism. J Biol Chem. 1992 Dec 5;267(34):24544–24553. [PubMed] [Google Scholar]
  47. Schnitzer J. E. gp60 is an albumin-binding glycoprotein expressed by continuous endothelium involved in albumin transcytosis. Am J Physiol. 1992 Jan;262(1 Pt 2):H246–H254. doi: 10.1152/ajpheart.1992.262.1.H246. [DOI] [PubMed] [Google Scholar]
  48. Severs N. J., Simons H. L. Caveolar bands and the effects of sterol-binding agents in vascular smooth muscle plasma membrane. Single and double labeling with filipin and tomatin in the aorta, pulmonary artery, and vena cava. Lab Invest. 1986 Sep;55(3):295–307. [PubMed] [Google Scholar]
  49. Silverstein S. C., Steinman R. M., Cohn Z. A. Endocytosis. Annu Rev Biochem. 1977;46:669–722. doi: 10.1146/annurev.bi.46.070177.003321. [DOI] [PubMed] [Google Scholar]
  50. Sparrow C. P., Parthasarathy S., Steinberg D. A macrophage receptor that recognizes oxidized low density lipoprotein but not acetylated low density lipoprotein. J Biol Chem. 1989 Feb 15;264(5):2599–2604. [PubMed] [Google Scholar]
  51. Steinberg D., Parthasarathy S., Carew T. E., Khoo J. C., Witztum J. L. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med. 1989 Apr 6;320(14):915–924. doi: 10.1056/NEJM198904063201407. [DOI] [PubMed] [Google Scholar]
  52. Söllner T., Whiteheart S. W., Brunner M., Erdjument-Bromage H., Geromanos S., Tempst P., Rothman J. E. SNAP receptors implicated in vesicle targeting and fusion. Nature. 1993 Mar 25;362(6418):318–324. doi: 10.1038/362318a0. [DOI] [PubMed] [Google Scholar]
  53. Tran D., Carpentier J. L., Sawano F., Gorden P., Orci L. Ligands internalized through coated or noncoated invaginations follow a common intracellular pathway. Proc Natl Acad Sci U S A. 1987 Nov;84(22):7957–7961. doi: 10.1073/pnas.84.22.7957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Van Deurs B., Nilausen K. Pinocytosis in mouse L-fibroblasts: ultrastructural evidence for a direct membrane shuttle between the plasma membrane and the lysosomal compartment. J Cell Biol. 1982 Aug;94(2):279–286. doi: 10.1083/jcb.94.2.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Villaschi S., Johns L., Cirigliano M., Pietra G. G. Binding and uptake of native and glycosylated albumin-gold complexes in perfused rat lungs. Microvasc Res. 1986 Sep;32(2):190–199. doi: 10.1016/0026-2862(86)90053-1. [DOI] [PubMed] [Google Scholar]
  56. Wagner R. C., Chen S. C. Transcapillary transport of solute by the endothelial vesicular system: evidence from thin serial section analysis. Microvasc Res. 1991 Sep;42(2):139–150. doi: 10.1016/0026-2862(91)90082-m. [DOI] [PubMed] [Google Scholar]
  57. Wagner R. C., Robinson C. S., Cross P. J., Devenny J. J. Endocytosis and exocytosis of transferrin by isolated capillary endothelium. Microvasc Res. 1983 May;25(3):387–396. doi: 10.1016/0026-2862(83)90028-6. [DOI] [PubMed] [Google Scholar]
  58. YAMADA E. The fine structure of the gall bladder epithelium of the mouse. J Biophys Biochem Cytol. 1955 Sep 25;1(5):445–458. doi: 10.1083/jcb.1.5.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. van Deurs B., Hansen S. H., Petersen O. W., Melby E. L., Sandvig K. Endocytosis, intracellular transport and transcytosis of the toxic protein ricin by a polarized epithelium. Eur J Cell Biol. 1990 Feb;51(1):96–109. [PubMed] [Google Scholar]

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