Low temperature-induced cell surface membrane vesicle shedding is associated with DNA fragmentation

Temperature shift conditions of 0 degree to 22 degrees C or 0 degree to 37 degrees C induce the formation and shedding of membrane vesicles (MV) from P815 tumor cell surfaces. When the MV shedding process takes place at 22 degrees C it occurs without changes in cell surface membrane permeability, whereas at 37 degrees C, changes in permeability to 51Cr and trypan blue do occur, thus mimicking the lymphocyte-mediated lytic process of tumor cells [1]. The present studies demonstrate that nuclear DNA fragmentation also occurs in both 0 degree to 22 degrees C and 0 degree to 37 degrees C temperature shifts but cell surface membrane permeability to DNA fragments occurs only in the latter condition, i.e., 0 degree to 37 degrees C. The microtubule-stabilizing agent deuterium oxide (D2O) inhibited the MV shedding process, the changes in membrane permeability, and DNA fragmentation. When P815 cells which had been induced to shed MV by the 0 degree to 22 degrees C temperature shift were labeled with 51Cr and used as targets for alloimmune lymphocytes, they were found to be as susceptible to T-cell lysis as control P815 cells. This result indicates that the lytic effect of alloimmune T lymphocytes can be exerted at the target cell surface membrane level independently of nuclear DNA fragmentation.     

Effects of cyclic nucleotides on the shedding of tumor cell surface membranes

Mastocytoma P815 cells can be induced to shed membrane vesicles (MV) from their surfaces by low temperature [24], a condition known to disrupt microtubules. In the present study we have investigated the effects of various nucleotides on this tumor cell surface behaviour. Our data show that the cyclic nucleotides cAMP and cGMP significantly inhibit the low temperature-induced shedding of MV from the tumor cell surfaces. GTP and the phosphodiesterase inhibitor theophylline exhibited similar inhibitory effects. These data are interpreted in terms of nucleotide-induced stabilization of cytoskeletal components. Transmission (TEM) and scanning electron microscopy (SEM) of cells shedding MV revealed that these structures originated from blebs on the cell surface.     

Oxygen consumption by tumor cells during membrane vesicle shedding

The incubation of mastocytoma P815 cells at low temperature (0 degrees C/1-2 hr), with a subsequent shift to greater than or equal to 20 degrees C results in the formation and shedding of membrane vesicles from the tumor cell surfaces. This process, when occurring at physiologic temperature (37 degrees C), mimics the morphological and membrane permeability changes occurring during T-lymphocyte mediated cytolysis of tumor cells. The latter is an oxygen dependent event, but it is not known whether this requirement is at the effector T cell or at the tumor cell level. The present study investigated the oxygen consumption rates of mastocytoma P815 cells induced to shed membrane vesicles by a temperature shift (0 degrees C/1-2 hrs----greater than or equal to 20 degrees C). Results showed that cells undergoing the membrane vesicle shedding process had significantly higher oxygen requirements than control non-shedding cells. Inhibition of the shedding process with deuterium oxide and hexylene glycol, reduced the oxygen consumption rates of low temperature treated cells to the level of control cells. The oxygen consumption rates of the latter were unaffected by these microtubule stabilizing agents. These data indicate that the oxygen required for immune T-cell mediated lysis of tumor cells may be at the target tumor cell level.     

A possible role for K channels in tumor cell injury : Membrane vesicle shedding and nuclear DNA fragmentation

Mastocytoma P815 tumor cells subjected to low temperature (O degrees C/l h) and shifted to 22 degrees or 37 degrees C undergo morphological, physiological and biochemical changes which are analogous to those induced by immune effector cells, i.e., changes in cell-surface morphology and membrane permeability, elevated O2 consumption rates and nuclear DNA fragmentation [18-21]. Utilizing this low-temperature shift method for the induction of cell injury, we investigated the possible role of K+ channels in this process. Results show that the two classical K+ channel blockers, tetraethylammonium (TEA) and 4-aminopyridine (4-AP), inhibited the low temperature-induced cell-surface membrane vesicle shedding as well as the nuclear DNA-fragmentation process. These results indicate that K+ channel function is required for tumor-cell injury as manifested by nuclear DNA fragmentation and cell-surface membrane vesicle (MV) shedding.     

Effect of cholesterol on the valinomycin-mediated uptake of rubidium into erythrocytes and phospholipid vesicles

Human erythrocytes have been treated with lipid vesicles in order to alter the cholesterol content of the cell membrane. Erythrocytes have been produced with cholesterol concentrations between 33 and 66 mol% of total lipid. The rate of valinomycin-mediated uptake of rubidium into the red cells at 37°C was lowered by increasing the cholesterol concentration of the cell membrane. Cholesterol increased the permeability to valinomycin at 20°C of small (less than 50 nm), unilamellar egg phosphatidylcholine vesicles formed by sonication. Cholesterol decreased the permeability to valinomycin at 20°C of large (up to 200 nm) unilamellar egg phosphatidylcholine vesicles formed by freezethaw plus brief sonication. It is concluded that cholesterol increases the permeability of small membrane vesicles to hydrophobic penetrating substances while above the transition temperature but has the opposite effect on large membrane vesicles and on the membranes of even larger cells.     

Membrane alterations in winter rye and Brassica napus cells during lethal freezing and plasmolysis

Abstract Cells fixed during freezing or plasmolysis were used to study membrane alterations in hardened and non-hardened Brassica napus suspension-cultured cells and rye leaf mesophyll cells. The plasmalemma in non-hardened rye mesophyll cells formed multilamellar vesicles during lethal freezing at high subzero temperatures (–5°C). These vesicles became highly condensed at lower subzero temperatures (–10°C). Conversely, cold-hardened rye mesophyll cells did not undergo membrane alterations at these temperatures. The results from plasmolysis of B. napus and rye mesophyll cells hardened by ABA at 25 °C and low temperature (2°C), respectively, verify the cell response to lethal freezing. Again there was a continuum of responses with 1 kmol m^?3 balanced salt causing multilamellar protrusions. Appression of the plasmalemma against the tonoplast to form multilamellar vesicles and the invagination of these vesicles into the tonoplast were also observed in rye cells undergoing lethal plasmolysis. Increasing the plasmolysing solution to 3 kmol m^?3 occasionally caused the formation of multilamellar vesicles on the cell surface of hardened rye mesophyll cells.     

Enrichment and localization of ganglioside G(D3) and caveolin-1 in shed tumor cell membrane vesicles

Tumor cell ganglioside shedding has been implicated in the process of tumor formation. Previously, we identified three forms of tumor ganglioside shedding: micelles, monomers and membrane vesicles. Here, we have explored the membrane vesicle form of ganglioside shedding, using a newly identified human ovarian carcinoma cell line, CABA I. These cells synthesize and express a spectrum of gangliosides, including the disialoganglioside, G(D3). Immunostaining using the monoclonal antibody R24 confirmed G(D3) expression and its presence in the plasma membrane of these cells. Cellular gangliosides were detected in the culture supernatant by HPTLC autoradiography, confirming an active shedding rate of 3% of cellular gangliosides/24 h. CABA I cell membranes also express caveolin-1, a characteristic protein marker for caveolae, which was detected by flow cytometric analysis and by Western blotting in both the cell membranes and the isolated membrane vesicles. To further define the expression of G(D3) and caveolin-1, we used immunogold electron microscopy. This revealed localization of G(D3) in small clusters in the plasma membrane as well as enrichment and localization of ganglioside G(D3) and caveolin-1 in shed membrane vesicles, with 58-78% of vesicles carrying both G(D3) and caveolin-1. Together, these results suggest that membrane vesicle shedding originates in plasma membrane domains enriched in gangliosides and caveolin-1.     

Undifferentiated and differentiated L6 myoblast plasma membranes. I: Comparison of the morphology of plasma membrane vesiculation and the factors influencing the vesiculation process

Monolayer cultures of a variety of cell lines can be induced to shed plasma membrane vesicles by exposure to 25 mM formaldehyde--2 mM dithiothreitol. An analysis of the process of membrane shedding can provide a method to probe the general characteristics of the plasma membrane in different cell populations. We have, therefore, analyzed membrane shedding in L6 myoblasts at different stages of differentiation. The results show that the morphology of membrane shedding is comparable in undifferentiated or differentiated L6 myoblasts, as are the factors that influence the process of membrane shedding. L6 myoblasts at various stages of differentiation have similar requirements for metabolic energy, physiological temperature, pH, and monovalent and divalent cations.     

Loss of membrane phospholipid asymmetry in platelets and red cells may be associated with calcium-induced shedding of plasma membrane and inhibition of aminophospholipid translocase

Influx of calcium in platelets and red cells produces formation of vesicles shed from the plasma membrane. The time course of the shedding process closely correlates with the ability of both cells to stimulate prothrombinase activity when used as a source of phospholipid in the prothrombinase assay. This reflects increased surface exposure of phosphatidylserine, presumably resulting from a loss in membrane asymmetry. Evidence is presented that the shed vesicles have a random phospholipid distribution, while the remnant cells show a progressive loss of membrane phospholipid asymmetry when more shedding occurs. Removal of intracellular calcium produces a decrease of procoagulant activity of the remnant cells but not of that of the shed vesicles. This is consistent with reactivation of aminophospholipid translocase activity, being first inhibited by intracellular calcium and subsequently reactivated upon calcium removal. Involvement of aminophospholipid translocase is further supported by the observation that reversibility of procoagulant activity is also dependent on metabolic ATP and reduced sulfhydryl groups. The finding that this reversibility process is not apparent in shed vesicles may be ascribed to the absence of translocase or to a lack of ATP. These data support and extend the suggestion made by Sims et al. [1989) J. Biol. Chem. 264, 17049-17057) that membrane fusion, which is required for shedding to occur, produces transient flip-flop sites for membrane phospholipids. Furthermore, the present results indicate that scrambling of membrane phospholipids can only occur provided that aminophospholipid translocase is inactive.     

Shedding of membrane vesicles mediates fibroblast growth factor-2 release from cells

Fibroblast growth factor-2 (FGF-2), a polypeptide with regulatory activity on cell growth and differentiation, lacks a conventional secretory signal sequence, and its mechanism of release from cells remains unclear. We characterized the role of extracellular vesicle shedding in FGF-2 release. Viable cells released membrane vesicles in the presence of serum. However, in serum-free medium vesicle shedding was dramatically down-regulated, and the cells did not release FGF-2 activity into their conditioned medium. Addition of serum to serum-starved cells rapidly induced intracellular FGF-2 clustering under the plasma membrane and into granules that colocalized with patches of the cell membrane with typical features of shed vesicle membranes. Shed vesicles carried three FGF-2 isoforms (18, 22, 24 kDa). Addition of vesicles to endothelial cells stimulated chemotaxis and urokinase plasminogen activator production, which were blocked by anti-FGF-2 antibodies. Treatment of intact vesicles with 2.0 m NaCl or heparinase, which release FGF-2 from membrane-bound proteoglycans, did not abolish their stimulatory effect on endothelial cells, indicating that FGF-2 is carried inside vesicles. The comparison of the stimulatory effects of shed vesicles and vesicle-free conditioned medium showed that vesicles represent a major reservoir of FGF-2. Thus, FGF-2 can be released from cells through vesicle shedding.     

Glycocalyceal bodies in a human rectal carcinoma cell line and their interstitial collagenolytic activities.

In co-cultivation on a membrane of connective tissue matrix (CTM) obtained from human dura mater, human adenocarcinoma cells (RCM-1) degraded CTM. Morphologically, the destruction of CTM was associated with the shedding of membrane vesicles from the cells. Transmission electron microscopy, using ruthenium red (RR), showed that the shed vesicles were composed of various-sized membrane bound vesicles (MV). A large majority were small glycocalyceal bodies (G-bodies) measuring 20-120 nm in diameter, composed of an amorphous matrix of moderate electron-density surrounded by an RR-positive, trilaminar membrane. G-bodies were separated from medium-sized and large MVs by ultracentrifugation. Ultrastructural observation of the isolated collagen fibrils from CTM co-cultured with RCM-1 cells, showed G-bodies attached to degraded collagen fibrils with characteristic transverse notches along their axes. The lesions occurred as microerosions in the apolar region between the e and d bands of collagen fibrils. Collagenolytic activity in serum-free RCM-1 conditioned medium was localized in the G-body and MV fractions (80% and 20% of the total activity, respectively, when tested against 3H-labeled type I collagen). No activity was detected in the supernatant. The activity in G-bodies was also confirmed by ultrastructural analysis using reconstituted native type I collagen fibrils. The results suggest that RCM-1 cells release interstitial collagenase as a component of G-bodies which facilitates local breakdown of connective tissue during the process of invasion.     

Growth temperature and OprF porin affect cell surface physicochemical properties and adhesive capacities of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> MF37

Pseudomonads adapt to various ecological niches by forming biofilms, which first requires bacterial adhesion on surfaces. We studied the influence of growth temperature on surface physicochemical properties of Pseudomonas fluorescens MF37 and on its adhesive capacities onto inert surfaces. It presented a global hydrophilic character, measured by microbial adhesion to solvent (MATS), and showed a cell surface more hydrophilic at 8 and 28°C than at 17°C. Moreover, P. fluorescens MF37 was more adhesive at 17°C. This critical temperature thus should be carefully taken into account in food safety. Adhesion onto inert surfaces is thus influenced by the growth temperature, which modifies the bacteria cell wall properties through changes in the outer membrane components. Therefore, we studied the effect of the loss of OprF, the major outer membrane protein, known to act as an adhesin (root, and endothelial cells). The OprF-deficient mutant was able to adhere to surfaces, but showed the same physicochemical and adhesion properties on abiotic surfaces whatever the growth temperature. OprF is thus not essential in this adhesion process. However, we suggest that OprF is involved in the bacterial environmental temperature sensing by P. fluorescens.This work was supported by a grant from the Région Bretagne (Doctoral fellowship to G.H.).     

Hexose and amino acid transport by chicken embryo fibroblasts infected with temperature-sensitive mutant of rous sarcoma virus: Comparison of transport properties of whole cells and membrane vesicles

The effect of transformation on hexose and amino acid transport has been studied using whole cells and membrane vesicles of chicken embryo fibroblasts infected with the temperature-sensitive mutant of the Rous sarcoma virus, TS-68. In whole cells, TS-68-infected chicken embryo fibroblasts cultured at the permissive temperature (37°C) had a 2-fold higher rate of 2-deoxy-d-glucose uptake than the same cells cultured at the non-permissive temperature (41°C). However, both the non-transformed and transformed cells had comparable rates of α-aminoisobutyric acid transport. Membrane vesicles, isolated from TS-68-infected chicken embryo fibroblasts cultured at 41°C or 37°C, displayed carrier-mediated, intravesicular uptake of d-glucose and α-aminoisobutyric acid. Membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 37°C had an approx. 50% greater initial rate of stereospecific hexose uptake than the membrane vesicles from fibroblasts cultured at 41°C. The two types of membrane vesicle had similar uptake rates of α-aminoisobutyric acid. The results of hexose and amino acid uptake by the membrane vesicles correlated well with those observed with the whole cells. K_m values for stereospecific d-glucose uptake by the membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 41 and 37°C were similar, but the V value was greater for the membrane vesicles from TS-68-infected cells cultured at 37°C. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicle. These findings suggest that the membrane vesicles retained many of the features of hexose and amino acid transport observed in whole cells, and that the increased rate of hexose transport seen in the virallytransformed chicken embryo fibroblasts was due to an increase in the number or availability of hexose carriers.     

Plasma membrane vesiculation: a cellular response to injury.

The shedding of plasma membrane vesicles has been shown to result from exposure of monolayer cell cultures to formaldehyde and other sulfhydryl blocking agents. Incubation of cells in concentrations of these agents as low as 5 to 10 mM for intervals as brief as fifteen minutes is effective (Scott, 1976). Plasma membrane vesiculation has been shown to be an energy-dependent process that requires Ca++ and physiological temperature. Following plasma membrane vesiculation, cell monolayers appear intact by phase microscopy and show only slight evidence of cell injury by electron microscopy. In view of these observations, the question has been raised whether plasma membrane vesiculation is compatible with continued cell growth and metabolism. The experiments described in this paper were designed to answer these questions. We pulse exposed 3T3 mouse embryo cells to concentrations of formaldehyde, between 2.5 and 250 mM, for intervals 15, 30 or 60 min. Cell momolayers were then washed in a variety of different media in an attempt to reverse the effect of formaldehyde on cells. Cell monolayers were thereafter assayed for the shedding of plasma membrane vesicles and for their ability to transport 2-deoxy-D-glucose. Cells were also replated in serum-containing medium and their ability to grow was assayed over a seven day interval. The results show an inverse relationship between the shedding of plasma membrane vesicles and the ability of the cells to transport nutrients and to grow. We interpret these data to suggest that the process of plasma membrane vesiculation results from a form of cell injury which blocks cellular metabolism and growth.     

Differences in lipid fluidity among isolated plasma membranes of normal and leukemic lymphocytes and membranes exfoliated from their cell surface

The fluorescence polarization technique with 1,6-diphenyl 1,3,5-hexatriene as a probe was used to determine the lipid microviscosity, $\text{η}$, of isolated plasma membranes of mouse thymus-derived ascitic leukemia (GRSL) cells and of extracellular membraneous vesicles exfoliated from these cells and occurring in the ascites fluid. For comparison, $\text{η}$ was also determined in isolated plasma cell supernatants.For isolated plasma membranes of thymocytes and GRSL cells $\text{η}$ values at 25° C amounted to 4.67 and 3.28 P, respectively, which were higher than the microviscosities of the corresponding intact cells, 3.24 and 1.73 P, respectively.Microviscosities inextracellular membranes of thymocytes and GRSL cells were 5.96 and 5.83 P, respectively. The fluidity difference between these membranes and plasma membranes was most pronounced for the leukemic cells and was thereby correlated with a large difference in cholesterol/phospholipid molar ratio (1.19 for extracellular membranes and 0.37 for plasma membranes). It is proposed that extracellular membraneous vesicles are shed from the surface of GRSL cells similar to the budding process of viruses, that is by selection of the most rigid parts of the host cell membrane.Liposomes of total lipid extracts of plasma membranes and extracellular membranes of both cell types exhibited about the same microviscosity as the corresponding intact membranes, indicating virtually no contribution of (glyco)-protein to the lipid fluidity as measured by the fluorescence polarization technique. For both cell types $\text{η}$ (25° C) values of liposomes consisting of membrane phospholipids varied between 1.5 and 1.9 P, much lower than the values for total lipids, indicating a significant rigidizing effect of cholesterol in each type of membrane.     

Receptors for IgM on rat spleen cells

Ox red blood cells (ORBCs) sensitized with rat IgM antibodies formed antibody-coated red blood cell rosettes (EAR) around 9% of rat spleen cells freshly prepared at 4 °C, while an enhancement of the rosette-forming cell (RFC) frequency to 18–20% was observed after having exposed the spleen cells to a temperature shift from 4 to 37 °C. Although the temperature shift was found to increase the RFC frequency, a shedding of IgM-Fc receptors IgM-FcR into the medium was also detected (IgM-FcR-I). Regeneration of shed receptors within 5 hr has been proved, and the sheading-regeneration cycle could be repeated several times. IgM-Fc receptors detectable after the temperature shift (IgM-FcR-II) are neither shed nor detectable on freshly prepared spleen cells. This latter type of receptor is expressed only after incubating the cells at 37 °C for an optimal period of 8–10 hr. Both type I and II IgM-FcRs were detected on T lymphocytes as well as B lymphocytes; therefore they do not mark subpopulation. Both receptors are sensitive to trypsin and the activity of both requires Ca²⁺ ions. Shed receptors are able to inhibit EAR formation by cells carrying either IgM-FcR-I or IgM-FcR-II. They are sensitive to higher temperatures and agglutinate ORBCs sensitized by IgM antibodies in the presence of Ca²⁺ ions. EAR-inhibiting capacity was detected in two fractions obtained by gel chromatography of the supernatant after shedding. The elution volume of one of the active fractions corresponds to 130,000 daltons (D), and that of the other to approximately 50,000 D.     

Proteomic analysis of microvesicles derived from human colorectal cancer cells

Microvesicles (MV) are membrane vesicles secreted from the plasma and endosomal membrane compartment by various cell types such as hematopoietic, epithelial, and tumor cells. Actively growing tumor cells shed MV, and the rate of shedding increases in malignant tumors. Although recent progress in this area has revealed that tumor-derived MV play multiple roles in tumor growth and metastasis via immune escape, tumor invasion, and angiogenesis, the mechanism of vesicle formation and the biological roles of tumor-derived MV are not understood. Here, we report the first global proteomic analysis of highly purified MV from human colorectal cancer cells. Using 1D SDS gel electrophoresis and nano-LC-MS/MS analyses, we identified a total of 547 microvesicular proteins from three independent experiments with high confidence; 416 proteins were identified at least in two trials, including 181 as yet unreported proteins. We identified 49 proteins involved in the biogenesis of MV, including annexins, ADP-ribosylation factors, and Rab proteins. We also identified 28 proteins that may function in tumorigenesis via promotion of migration, invasion, and growth of tumor cells, immune modulation, metastasis, and angiogenesis. Taken together with previously reported results, our observations suggest that tumor-derived MV may act as communicasomes, that is, extracellular organelles that play diverse roles in intercellular communication. This information will help elucidate the biogenesis and functions of tumor-derived MV, and aid in the development of effective vaccines for various cancers, including colorectal cancer.     

Isolation and characterization of large (0.5–1.0 μm) cytoskeleton-free vesicles from human and rabbit erythrocytes

Large (0.5–1.0 μm) cytoskeleton-free vesicles were obtained, by ‘budding’, from fresh human and rabbit erythrocytes incubated at 45°C and titrated with EDTA and CaCl₂. This process occurs without hemolysis. The isolated vesicles maintain their cytoplasmic integrity and normal membrane orientation, and are resistant to hemolysis over the pH range 5.0–11.0 and temperature range 4–50°C. The only membrane proteins detected in vesicles from human erythrocytes were band 3 region polypeptides and bands PAS-1, PAS-2 and PAS-3. Vesicles obtained from rabbit erythrocytes were similarly simple. Because of their size and stability these vesicles are amenable to both kinetic and quantitative analysis using the same experimental techniques employed in studies of synthetic lipid membranes. The results obtained in this study indicate that these vesicles are essentially markedly simplified biological cells, and thus may be useful as a biologically relevant model membrane system for examining the molecular interactions which occur within, across and between cell membranes.     

Recovery of human neutrophils from complement attack: removal of the membrane attack complex by endocytosis and exocytosis

Nucleated cells can resist lysis by and recover from complement attack even after formation of the potentially cytolytic membrane attack complex on the cell surface. We have found that human neutrophils resist complement lysis by the physical removal of membrane attack complexes by both endocytic and exocytic process. The latter mechanism predominates, vesiculation being detectable within 60 sec of initiating the complement cascade. Sixty-five percent of the formed complexes are removed on plasma membrane vesicles, although only 2% of the cell surface is lost. Ultrastructural examination revealed that these vesicles were covered with ring-like "classical" complement lesions. Analysis of these vesicles by gel electrophoresis indicated that C9 was present exclusively in the form of a sodium dodecyl sulfate-resistant, high m.w. complex. In contrast, the 35% of C9 that remained associated with the cells was found to be inaccessible to a C9-specific monoclonal antibody, and was partly degraded, suggesting internalization of the membrane attack complex and proteolysis of some C9 molecules. The molar ratio of C9 to C8 was 12 to 1 on shed vesicles and on recovered cells.     

Uptake of reconstituted Na,K-ATPase vesicles by isolated lymphocytes measured by FACS, confocal microscopy and spectrofluorometry

Na,K-ATPase (EC, 3.6.1.37, Na,K-ATPase) is a fundamental vital membrane transport and receptor system which, after biosynthesis, is exported to the plasma membrane in inside-out vesicles. Na,K-ATPase can be extracted form the natural membrane and inserted into artificially formed phosphatidylcholine vesicles (liposomes). The ultrastructure of the reconstituted vesicles has been fully described. In the present work, the Na,K-ATPase-vesicles were labeled with fluorescent tracers either in their water or membrane phase, incubated with freshly isolated human lymphocytes, and the resulting cellular fluorescence measured with fluorescence activated cell sorting (FACS), confocal microscopy and spectrofluorometry. The FACS data show that all lymphocytes take up Na,K-ATPase-vesicles in a dose-and temperature-dependent fashion. Three-dimensional analysis of the fluorescence by confocal microscopy reveals that the fluorescence is contained within the cells. Quantitative determination by spectrofluorometry indicates that depending on the vesicle/cell ratio, a single lymphocyte takes up 650 to 36,500 vesicles within 30 min at 37°C together with up to about 200,000 renal Na,K-ATPase molecules.