Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1981 Dec 1;91(3):716–727. doi: 10.1083/jcb.91.3.716

Exocytosis of pinocytosed fluid in cultured cells: kinetic evidence for rapid turnover and compartmentation

PMCID: PMC2112800  PMID: 7328118

Abstract

The uptake and fate of pinocytosed fluid were investigated in monolayers of pulmonary alveolar macrophages and fetal lung fibroblasts using the fluid-phase marker, [14C]sucrose. Initial experiments revealed that cellular accumulation of chromatographically repurified [14C]sucrose was not linear with incubation time. Deviation from linearity was shown to be due to constant exocytosis of accumulating marker. Chromatographic analysis revealed that the cells were unable to metabolize sucrose and were releasing it intact by a process that was temperature-sensitive but not dependent on extracellular calcium and magnesium. A detailed analysis of the kinetics of exocytosis was undertaken by preloading cells with [14C]sucrose for various lengths of time and then monitoring the appearance of radioactivity into isotope- free medium. Results indicated that modeling the process of fluid-phase pinocytosis and subsequent exocytosis required at least two intracellular compartments in series, one compartment being of small size and turning over very rapidly (t1/2 = 5 min in macrophages, 6--8 min in fibroblasts) and the other compartment being apparently larger in size and turning over very slowly (t1/2 = 180 min in macrophages, 430--620 min in fibroblasts). Computer-simulation based on this model confirmed that the kinetics of efflux faithfully reflected the kinetics of influx and that the rate of efflux completely accounted for the deviation from linearity of accumulation kinetics. Moreover, the sizes of the compartments and magnitude of the intercompartment fluxes were such that the majority of fluid internalized in pinocytic vesicles was rapidly returned to the extracellular space via exocytosis. This result provides direct experimental evidence for a process previously thought necessary based solely on morphological and theoretical considerations. Furthermore, the turnover of pinocytosed fluid was so dynamic that accumulation deviated from linearity even within the first few minutes of incubation. We were able to show that the kinetics of exocytosis allowed calculation of the actual pinocytic rate, a rate that was nearly 50% greater than the apparent initial rate obtained from the slope of the uptake curve over the first 10 min.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BREWER D. B., HEATH D. Lysosomes and vacuolation of the liver cell. Nature. 1963 Jun 8;198:1015–1016. doi: 10.1038/1981015b0. [DOI] [PubMed] [Google Scholar]
  2. Becker G., Ashwood-Smith M. J. Endocytosis in Chinese hamster fibroblasts. Inhibition by glucose. Exp Cell Res. 1973 Dec;82(2):310–314. doi: 10.1016/0014-4827(73)90346-7. [DOI] [PubMed] [Google Scholar]
  3. Berlin R. D., Oliver J. M. Surface functions during mitosis. II. Quantitation of pinocytosis and kinetic characterization of the mitotic cycle with a new fluorescence technique. J Cell Biol. 1980 Jun;85(3):660–671. doi: 10.1083/jcb.85.3.660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berlin R. D., Oliver J. M., Walter R. J. Surface functions during Mitosis I: phagocytosis, pinocytosis and mobility of surface-bound Con A. Cell. 1978 Oct;15(2):327–341. doi: 10.1016/0092-8674(78)90002-8. [DOI] [PubMed] [Google Scholar]
  5. Bowers B., Olszewski T. E., Hyde J. Morphometric analysis of volumes and surface areas in membrane compartments during endocytosis in Acanthamoeba. J Cell Biol. 1981 Mar;88(3):509–515. doi: 10.1083/jcb.88.3.509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bowers B., Olszewski T. E. Pinocytosis in Acanthamoeba castellanii. Kinetics and morphology. J Cell Biol. 1972 Jun;53(3):681–694. doi: 10.1083/jcb.53.3.681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cohn Z. A., Benson B. The in vitro differentiation of mononuclear phagocytes. 3. The reversibility of granule and hydrolytic enzyme formation and the turnover of granule constituents. J Exp Med. 1965 Sep 1;122(3):455–466. doi: 10.1084/jem.122.3.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cohn Z. A., Ehrenreich B. A. The uptake, storage, and intracellular hydrolysis of carbohydrates by macrophages. J Exp Med. 1969 Jan 1;129(1):201–225. doi: 10.1084/jem.129.1.201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Curry D. L., Bennett L. L., Grodsky G. M. Dynamics of insulin secretion by the perfused rat pancreas. Endocrinology. 1968 Sep;83(3):572–584. doi: 10.1210/endo-83-3-572. [DOI] [PubMed] [Google Scholar]
  10. DOUGLAS W. W., POISNER A. M. STIMULUS-SECRETION COUPLING IN A NEUROSECRETORY ORGAN: THE ROLE OF CALCIUM IN THE RELEASE OF VASOPRESSIN FROM THE NEUROHYPOPHYSIS. J Physiol. 1964 Jul;172:1–18. doi: 10.1113/jphysiol.1964.sp007399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Davies P. F., Ross R. Mediation of pinocytosis in cultured arterial smooth muscle and endothelial cells by platelet-derived growth factor. J Cell Biol. 1978 Dec;79(3):663–671. doi: 10.1083/jcb.79.3.663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Douglas W. W. The mechanism of release of catecholamines from the adrenal medulla. Pharmacol Rev. 1966 Mar;18(1):471–480. [PubMed] [Google Scholar]
  13. Duncan R., Pratten M. K. Membrane economics in endocytic systems. J Theor Biol. 1977 Jun 21;66(4):727–735. doi: 10.1016/0022-5193(77)90241-7. [DOI] [PubMed] [Google Scholar]
  14. Fell H. B., Dingle J. T. Endocytosis of sugars in embryonic skeletal tissues in organ culture. I. General introduction and histological effects. J Cell Sci. 1969 Jan;4(1):89–103. doi: 10.1242/jcs.4.1.89. [DOI] [PubMed] [Google Scholar]
  15. Hokin L. E. Effects of calcium omission on acetylcholine-stimulated amylase secretion and phospholipid synthesis in pigeon pancreas slices. Biochim Biophys Acta. 1966 Jan 25;115(1):219–221. doi: 10.1016/0304-4165(66)90066-3. [DOI] [PubMed] [Google Scholar]
  16. Houghton B. A., Stidworthy G. H. A growth history comparison of the human diploid cells WI-38 and IMR-90: proliferative capacity and cell sizing analysis. In Vitro. 1979 Sep;15(9):697–702. doi: 10.1007/BF02618249. [DOI] [PubMed] [Google Scholar]
  17. Kaplan J., Nielsen M. Pinocytic activity of rabbit alveolar macrophages in vitro. J Reticuloendothel Soc. 1978 Dec;24(6):673–685. [PubMed] [Google Scholar]
  18. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  19. Lee G. T., Engelhardt D. L. Protein metabolism during growth of Vero Cells. J Cell Physiol. 1977 Aug;92(2):293–301. doi: 10.1002/jcp.1040920218. [DOI] [PubMed] [Google Scholar]
  20. Low R. B. Protein biosynthesis by the pulmonary alveolar macrophage: conditions of assay and the effects of cigarette smoke extracts. Am Rev Respir Dis. 1974 Oct;110(4):466–477. doi: 10.1164/arrd.1974.110.4.466. [DOI] [PubMed] [Google Scholar]
  21. Muller W. A., Steinman R. M., Cohn Z. A. The membrane proteins of the vacuolar system. II. Bidirectional flow between secondary lysosomes and plasma membrane. J Cell Biol. 1980 Jul;86(1):304–314. doi: 10.1083/jcb.86.1.304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Novikoff A. B. The endoplasmic reticulum: a cytochemist's view (a review). Proc Natl Acad Sci U S A. 1976 Aug;73(8):2781–2787. doi: 10.1073/pnas.73.8.2781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ose L., Ose T., Reinertsen R., Berg T. Fluid endocytosis in isolated rat parenchymal and non-parenchymal liver cells. Exp Cell Res. 1980 Mar;126(1):109–119. doi: 10.1016/0014-4827(80)90475-9. [DOI] [PubMed] [Google Scholar]
  24. Phaire-Washington L., Wang E., Silverstein S. C. Phorbol myristate acetate stimulates pinocytosis and membrane spreading in mouse peritoneal macrophages. J Cell Biol. 1980 Aug;86(2):634–640. doi: 10.1083/jcb.86.2.634. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pratten M. K., Williams K. E., Lloyd J. B. A quantitative study of pinocytosis and intracellular proteolysis in rat peritoneal macrophages. Biochem J. 1977 Dec 15;168(3):365–372. doi: 10.1042/bj1680365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Quintart J., Leroy-Houyet M. A., Trouet A., Baudhuin P. Endocytosis and chloroquine accumulation during the cell cycle of hepatoma cells in culture. J Cell Biol. 1979 Sep;82(3):644–653. doi: 10.1083/jcb.82.3.644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Roberts A. V., Williams K. E., Lloyd J. B. The pinocytosis of 125I-labelled poly(vinylpyrrolidone), [14C]sucrose and colloidal [198Au]gold by rat yolk sac cultured in vitro. Biochem J. 1977 Nov 15;168(2):239–244. doi: 10.1042/bj1680239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schneider Y. J., Tulkens P., de Duve C., Trouet A. Fate of plasma membrane during endocytosis. I. Uptake and processing of anti-plasma membrane and control immunoglobulins by cultured fibroblasts. J Cell Biol. 1979 Aug;82(2):449–465. doi: 10.1083/jcb.82.2.449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schneider Y. J., Tulkens P., de Duve C., Trouet A. Fate of plasma membrane during endocytosis. II. Evidence for recycling (shuttle) of plasma membrane constituents. J Cell Biol. 1979 Aug;82(2):466–474. doi: 10.1083/jcb.82.2.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Steinman R. M., Brodie S. E., Cohn Z. A. Membrane flow during pinocytosis. A stereologic analysis. J Cell Biol. 1976 Mar;68(3):665–687. doi: 10.1083/jcb.68.3.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Steinman R. M., Cohn Z. A. The interaction of soluble horseradish peroxidase with mouse peritoneal macrophages in vitro. J Cell Biol. 1972 Oct;55(1):186–204. doi: 10.1083/jcb.55.1.186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Steinman R. M., Silver J. M., Cohn Z. A. Pinocytosis in fibroblasts. Quantitative studies in vitro. J Cell Biol. 1974 Dec;63(3):949–969. doi: 10.1083/jcb.63.3.949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tartakoff A. M., Vassalli P. Plasma cell immunoglobulin secretion: arrest is accompanied by alterations of the golgi complex. J Exp Med. 1977 Nov 1;146(5):1332–1345. doi: 10.1084/jem.146.5.1332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tartakoff A., Vassalli P., Détraz M. Comparative studies of intracellular transport of secretory proteins. J Cell Biol. 1978 Dec;79(3):694–707. doi: 10.1083/jcb.79.3.694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Thilo L., Vogel G. Kinetics of membrane internalization and recycling during pinocytosis in Dictyostelium discoideum. Proc Natl Acad Sci U S A. 1980 Feb;77(2):1015–1019. doi: 10.1073/pnas.77.2.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wagner R., Rosenberg M., Estensen R. Endocytosis in Chang liver cells. Quantitation by sucrose- 3 H uptake and inhibition by cytochalasin B. J Cell Biol. 1971 Sep;50(3):804–817. doi: 10.1083/jcb.50.3.804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Williams K. E., Kidston E. M., Beck F., Lloyd J. B. Quantitative studies of pinocytosis. I. Kinetics of uptake of (125I)polyvinylpyrrolidone by rat yolk sac cultured in vitro. J Cell Biol. 1975 Jan;64(1):113–122. doi: 10.1083/jcb.64.1.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. van Furth R. Origin and kinetics of monocytes and macrophages. Semin Hematol. 1970 Apr;7(2):125–141. [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES