QUANTITATIVE STUDIES OF PINOCYTOSIS I. Kinetics of Uptake of [~sI]Polyvinylpyrrolidone by Rat Yolk Sac Cultured In Vitro

A method is described for the in vitro culture of 17.5-day rat visceral yolk sac. Tissue survival was good as judged by light and electron microscopy. The rate of pinocytic uptake of ~25I-labeled polyvinylpyrrolidone by the tissue was constant both within and between experiments. Within the concentrat ion range 0 .15-24 ~tg/ml, the a2~I-labeled polyvinylpyrrolidone neither stimulated nor inhibited pinocytosis. The system offers many advantages in the quantitative study of the physical basis of pinocytosis. Many important questions concerning pinocytosis remain unanswered. For example, do pinocyticaily ingested solutes enter chiefly in free solution, or adsorbed to the plasma membrane? By what means and to what extent can pinocytosis be stimulated and inhibited? Such questions can be answered only by experiments in which pinocytosis of substances is studied quantitatively. Unfortunately, although theoretical analyses of the pinocytic process have been made (l, 2), the experimental data available are inadequate to test their validity. Some data, such as those gathered on the clearance of macromolecules from the bloodstream of intact animals or from organ perfusates, are difficult to interpret because of the many unmeasurable physiological variables inherent in such systems. Studies with isolated cells or unicellular organisms cultured in vitro, although attractive because potentially simpler to interpret, have in general proved disappointing since the rate of pinocytic uptake has frequently been observed to change markedly within the course of a single experiment and the reproducibility of experiments has been poor. This is true of Gosselin's (1) data on the uptake of colloidal gold by rabbit peritoneal macrophages, regarded by Jacques (2) as the most complete analysis available, and of the data of Chapman-Andresen (3, 4) on uptake of several solutes by Amoeba species. Some authors have approached the quantitation of pinocytosis by measuring morphological parameters such as the number of pinocytic vesicles visible in a ~zell. In this way, Cohn (5, 6) has produced evidence on the stimulation of pinocytosis in macrophages by a range of chemicals, and Chapman-Andresen (3, 4) has published similarly on Amoeba. But, as Ryser (7) and Jacques (2) point out, changes in the rate of pinocytosis of a solute do not necessarily parallel changes in morphological parameters. In this and the accompanying paper we present an experimental method that appears to have considerable advantages over previously described methods for studying quantitative aspects of pinocytosis. The tissue used is the visceral yolk sac of THE JOURNAL OF CELL BIOLOGY . VOLUME 64, 1975 . pages 113-122 113 on July 8, 2017 jcb.rress.org D ow nladed fom the 17.5-day p regnan t rat , ma in t a ined in organ culture. S o m e of the work has been repor ted briefly e l sewhere (8, 9, 10). M A T E R I A L S A N D M E T H O D S Organ Culture of Yolk Sac Wistar rats, aged 3-6 too, from an inbred laboratory colony were brother-sister mated overnight. If sperm were detected in the vagina next morning, pregnancy was timed from midnight of the night of the mating. At 17.5 days, rats were killed by cervical dislocation, conceptuses removed under sterile conditions, and yolk sacs dissected free from fetus, amnion, and placenta under culture medium, which consisted of 9 vol of medium 199 mixed with ! vol of inactivated calf serum (both obtained from WeUcome Reagents, Beckenham, Kent, U.K.; preparations TC 20 and CS 07, respectively). The yolk sacs were opened out by small incisions from the cut edge originally adjacent to the chorioallantoic placenta (see Fig. 1), rinsed in three changes of sterile culture medium, and incubated in sterile Erlenmeyer flasks (50 ml) containing 9.0 ml of culture medium. The air in each flask was displaced by a mixture of oxygen and carbon dioxide (95:5) and the vessel stoppered with a sterile silicone-rubber bung. Flasks were placed in a Unitemp water bath (Baird & Tatlock [London] Ltd., Romford, Essex, U.K.) maintained at 37.0 ~ • 0.2~ and the shaker attachment was regulated to a stroke of 3.4 cm at a frequency of 100 • 5 strokes per min. After 15-20 min, 30 /~g of 12Sl-labeled polyvinylpyrrolidone ([125]PVP; mean tool wt 30,000 40,000; preparation IM 33P from RadiochemAMNION ~ -PLACENTA FIGURE 1 Dissection of a 17.5-day rat yolk sac for organ culture. ical Centre, Amersham, Bucks., U.K.) was added to each flask as a solution in 1.0 ml of culture medium. The flasks were then regassed, stoppered, and replaced in the water bath. In experiments to determine the uptake of [ '~q]PVP, yolk sacs were removed after a period of incubation, agitated three times for 2 min in changes of ice-cold 0.9% NaCI (30 ml) to remove extracellular substrate, and stored at 2 0 ~ until assay. Before assay, each yolk sac was homogenized in water (2 ml) with a Virtis 45 homogenizer (Techmation Ltd., Edgware, Middx., U.K.) at top speed, and the suspension was diluted to 5.0 ml with water. Portions of the yolk sac homogenates (I.0 ml) and of the corresponding culture media ( 1.0 ml), each in a 3-ml disposable tube, were assayed for radioactivity with a gamma spectrometer (Packard Instruments Ltd., Caversham, Berks., U.K.). The protein content of each yolk sac was determined by the method of Lowry et al. (1 I) with bovine serum albumin (Sigma [London] Chemical Company Limited, London SWS; type I1) as reference protein. In experiments to measure the release of [~z~I]PVP from cultured yolk sac, three yolk sacs were incubated separately for 6 h in the presence of [~51]PVP (40 ~g/ml), and each was transferred into a 10-ml portion of sterile, substrate-free culture medium and incubated for 2 min to remove extracellular substrate. This process was repeated twice more before the yolk sacs were incubated separately for a further 6 h in 10.0 ml of substrate-free culture medium. In another such experiment, 1% wt/vol PVP of average mol wt 40,000 (PVP-40; Sigma [London] Chemical Co. Ltd.) was added to the substrate-free culture medium used in both the wash and the reincubation. in both series of experiments, samples of culture media (two, each of 1.0 ml) were withdrawn in sterile pipettes and replaced by an equal volume of the appropriate fresh culture medium which had been warmed to 37~ and gassed. The activity in the tissue itself at the end of the 12-h overall culture period was assayed as described above, and the total activity released into the culture medium over the second 6-h period was calculated by the following formula: Tn = IOC,~,=, ,+ 2Z~=0. C, where T~ is the radioactivity (counts per minute) released up to the time of the n th sampling, and C~ the counts per minute per milliliter of culture medium of the i th sample after correcting for background.