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THE EARLY DAYS of PINOCYTOSIS by CICILY CHAPMAN-ANDRESEN

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THE EARLY DAYS of PINOCYTOSIS by CICILY CHAPMAN-ANDRESEN Carlsberg Res. Commun. Vol. 49, p. 179-186, 1984 THE EARLY DAYS OF PINOCYTOSIS by CICILY CHAPMAN-ANDRESEN Institute of Cell Biology and Anatomy, Universitetsparken 15, DK-2100 Copenhagen O. Keywords: Pinocytosis, amoebae 1. INTRODUCTION photographs and descriptions of this process, The earliest suggestion of "cell drinking" showing that during active movements at the cell appears to be the medically orientated theory surface, droplets of the fluid medium were put forward by the American physician engulfed; these droplets were at first of irregular MELTZER in 1904 (51). This hypothesis, for shape, but became spherical as they passed into which there was no experimental evidence, was the interior of the cell, sometimes fusing with clearly influenced by METCHNIKOF~S (52) de- other droplets, and rapidly decreasing in size. It scription and definition ofphagocytosis in 1883. was in this classical paper that the term pinocy- If cells can eat, why should they not also drink? tosis first appeared, at the suggestion of Lewis' MELTZER'S theory sought to explain the forma- colleague, DAVID M. ROBINSON; the designa- tion of oedema by the transport of fluid though tion was derived from the Greek, rltvetv to drink, ceils, which "sipped" fluid from the surrounding and rvxo~ cell. Later LEWIS (47) observed the medium, a process which this author called same phenomenon in cultured malignant cells. "potocytosis'. Apart from an observation by Thus the term pinocytosis appeared in the litera- EDWARDS (27) in 1925 on food cup formation ture 50 years after METCHNIKOFFhad described induced by neutral inorganic salt solutions in and defined phagocytosis. amoebae, there were no further published data The observations of MAST and DOYLE (49) in or theories on fluid uptake until 1930. In this 1934 on the uptake of fluid by freshwater amoe- year, a short editorial in the Journal of the bae, a process for which these authors also used American Medical Association by MAIN- Lewis' term pinocytosis, differed in two respects WARING (,48) under the title "Hydrophago- from LEWIS' descriptions of the process in mac- cytosis" brought to the attention of physicians rophages; in the macrophages droplets were the cine films of the movements of rat macro- engulfed directly at the cell surface, while the phages in culture which LEWIS had demon- amoebae formed channels on rather immobile strated at meetings in the United States. pseudopodia, at the base of which vacuoles were MAINWARING pointed out that "the Lewis phe- pinched off from the channels. The other differ- nomenon", during which the macrophages cap- ence between pinocytosis in these two cell types tured and swallowed plasma from the surround- was that while the macrophages continually ing medium, could be of importance in cellular formed droplets from the nutrient medium, the nutrition, and in "mechanical filtration of puri- amoebae required a "stimulus", in the form of fication of body fluids", as well as "disposal of diluted sea water or egg albumin, before pinocy- souble antigens and .. synthesis of specific anti- tosis could take place: it should be noted here bodies". Then in 1931 LEWIS (46) published that while the macrophage medium was a nu- Springer-Verlag 0105-1938/84/0049/0179/$01.60 C. CHAPMAN-ANDRESEN: Early days of pinocytosis Figure 1. a and b. Pinocytosis in rat omental macrophages, a. Redrawn from MAST (46), the first paper on pinocytosis, b. Phase contrast photograph (CHAPMAN-ANDRESEN).C and d. Pinocytosis in fresh water amoebae. c. Drawing from MAST and DOYLE (49), the first paper on this subject, d. Phase contrast photograph (CHAPMAN-ANDRESEN). trient medium, the amoeba medium, which was details of the morphology of the process (see a low concentration of inorganic salts, could not Figure 1). be considered as nutrient. Higher concentra- Meanwhile, since METCHNIKOFF'S first stu- tions of salts and of albumin, a protein, could dies and his further exploration ofphagocytosis however be nutritional importance, hence the through many animal groups from amoeba to early emphasis on this aspect of the significance man, considerable data had been accumulated ofpinocytosis. These early observations on mac- from in vivo and in vitro observations on a wide rophages and amoebae were all made on living variety of cells, which phagocytosed living or cells without the aid of phase contrast or other dead bacteria, organic or inorganic particles of accessory optics, but they give clear and full different types. The significance of phagocy- 180 Carlsberg Res. Commun. Vol. 49, p. 179-186, 1984 C. CHAPMAN-ANDRESEN:Early days of pinocytosis tosis-enhancing factors from normal and sensi- After confirmation of the need for protein or tized serum was realized, and the influence of salt (8, 24) in the medium before pinocytosis many factors, such as temperature, salts, pH and could take place, it soon became clear that not all hormones was studied. Reviews such as those of salts or all proteins were equally effective MUDD et al. (53) and of BERRY and SPIES (1) "inducers", as the solutes which were responsi- summarize the extensive research in this field up ble for the initiation of the process were now to 1949. It can thus be seen that phagocytosis had called. Using a quantitative method for estimat- early proved to be a subject of interest to medical ing the intensity of pinocytosis, based on the physiologists, and was soon attributed a role in mean number of channels formed under stan- certain diseases and in normal body repair dard conditions, the inducing capacity of a range functions. However, the concept ofpinocytosis, of water soluble proteins of differing iso-ionic during the first two decades after its first descrip- point was explored. The data showed that under tion, was either ignored, rejected as pathological conditions where the predominant charge of the in mammalian cells e.g. by BESSlS (2), or consi- protein was positive, pinocytosis was induced dered as a normal cellular process only in cul- (9). In general, non-toxic inorganic salts were tured cells and in Protozoa. effective inclucers at approximately neutral pH, although calcium salts were an exception: non- toxic basic dyes were also good inducers. 2. STUDIES ON PINOCYTOSIS AT Further experiments with these different CARLSBERG LABORATORIUM types of inducers confirmed the importance of The above introduction shows briefly the the interaction between cell surface and inducer background against which studies on pinocy- for the initiation ofpinocytosis; e.g. the presence tosis were started at Carlsberg Laboratorium, of fluorescein-labelled protein or basic dyes first at the Cytochemical Department, and from could be visualised on the cell surface. This first 1956 at the Physiological Department under phase of surface binding was independent of HEINZ HOLTER, who had again spotted an temperature (9) but was influenced by condi- important field in cell biology, which could be tions in the medium. Changes in the pH of the developed with the material and techniques medium and in its ionic strength, however, available at Carlsberg. influenced different types of inducers in differ- Cultures of the fresh water amoebae Amoeba ent ways, indicating that surface binding was proteus and Chaos carolinense (then known as several types, showing varying degrees of labil- C. chaos or Pelomyxa carolinensis) had been ity. These observations led to the division of kept in the laboratory for some years, for bioche- inducers into three types, according to the fac- mical, physiological and cytological studies, of-. tors which led to the release of the inducer from ten using microtechniques, which permitted the cell surface, or in modem terminology various types of measurements to be made on "enhance the receptor-ligand dissociation". single amoebae; studies with radioactive tracers Inorganic salts, as well as neutral salts of amino were also in progress. HEINZ HOLTER, together acids were effective as inducers only when the with a newly arrived guest from U.S.A., JOHN M. active solutes were present in the medium: on MARSHALL JR., then commenced studies on transfer to a non-inducing medium pinocytosis pinocytosis in fresh water amoebae. In these first ceased immediately. Surface-bound protein was experiments, the uptake of a protein, fluorescein not released on transfer of the amoebae to a labelled gamma globulin, was explored by mea- non-inducing medium of the same pH as that of surements of fluorescence in single amoebae. the inducing solution, while immediate release This (43) was the first of a series of studies on occurred if the pH of the non-protein medium different aspects of pinocytosis which issued differed (19). For basic dyes, the conditions were from HOLTER'Slaboratory during the next two similar to those shown by protein inducers. decades, the results of his collaboration with Finally, in a small group of compounds, so far guests from Denmark and abroad and with the comprising the copper phthalocyanin dye alcian laboratory's own co-workers. blue, and rhuthenium red, the dye, once bound Carlsberg Res. Commun. Vol. 49, p. 179-186, 1984 181 C. CHAPMAN-ANDRESEN: Early days of pinocytosis the uptake and fate of material ingested by pinocytosis. Further methods available at that time for measuring uptake was the use of radio- active compounds as tracers; rather few were then available compared with the vast range now commercially produced. In the first experi- t] b c ments, glucose, labelled with C-14, was used, as carbohydrates had been shown to be non-induc- ing (17), and hence might give a measure of the volume of fluid ingested. Non-radioactive albu- min was used to induce pinocytosis, and the fate ?& of the ingested material was followed by direct measurements of radio activity and by autora- diography. The glucose was metabolised by the d e amoebae, thus showing that it penetrated the Figure 2.
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