527
605th MEETING, STRATHCLYDE
Non-specific adsorptive pinocytosis
JOHN B. LLOYD and K E N N E T H E. WILLIAMS
Biochemistry Research Laboratory, Department of Biological
Sciences, University of Keele. Keele. Staffs. ST5 SBG, U.K.
Unlike phagocytosis, which is typically a substrate-induced
phenomenon, pinocytosis appears to be a constitutive
activity in most animal cell types (see Pratten et al., 1980;
Steinman et al., 1983). Pinocytic vesicles are constantly
being formed from invaginations of the plasma membrane,
irrespective of the availability of substrates; hence the
analogies of a paternoster-style elevator (Lloyd, 1980) or a n
escalator (Steinman et al., 1983) have been used. Although
the rate of pinosome formation is usually unaffected by substrate, constitutive pinocytosis is eminently capable of substrate selection. Substrates that enter cells by this route can
do so either non-adsorptively (in the fluid phase) or
adsorbed to the plasma membrane that is being internalized. Several substrates may be captured by the same
pinocytic invagination, the rate of uptake reflecting in each
case the concentration of the molecule in the ambient fluid,
its strength of binding to the membrane binding sites, and
the abundance of these sites on those regions of the plasma
membrane that are internalized.
In addition to this ubiquitous cellular phenomenon, there
are reports of pinocytic events that are highly substratespecific: either the pinocytic invaginations form only in
response to a ligand attaching to a cell-surface receptor or
the pinosomes contain exclusively one receptor-bound substrate, excluding other substrates, or both. In many cases of
substrate uptake by pinocytosis it is not yet known whether
capture is by the constitutive mode or by a substrateinduced event. Here we suggest applying the principle
known as Occam's razor. It is clearly absurd to postulate a
separate class of event for every substrate, natural or
synthetic, found to be captured by pinocytosis. It would be
wise therefore to assume uptake of a substrate to be by
constitutive pinocytosis until evidence to the contrary is
found.
I t has been the traditional understanding for some 2
decades that substances captured by pinocytosis are routed
to the lysosomes. This view is now seen as over-simple:
some pinosomes avoid the lysosomes and fuse again with
the plasma membrane, releasing their contents to the same
or to another extracellular compartment. Nevertheless,
pinocytosis followed by delivery to the lysosomes is a major
route in most cells, and one seen by many investigators to
have interesting therapeutic potential in two fields. First,
the lysosomal storage diseases, unlike most inborn errors of
metabolism, are uniquely amenable to 'replacement therapy' with exogenous enzymes. Secondly, chemotherapeutic
agents attached to macromolecules can enter cells only by
pinocytosis, offering the potential for tissue-specific action
if tissue-specific uptake can be achieved. Both of these
novel approaches to therapy depend upon effective and if
possible, cell-specific pinocytic uptake. There is therefore
much current interest ir? identifying those features of a
macromolecule that lead to its avid uptake by cells.
The publication recently of two excellent reviews on the
mechanisms and substrate specificity of pinocytosis (Besterman & Low, 1983: Steinman et a/., 1983) obviate the
necessity of a lengthy account here. The remainder of the
present paper concerns some important aspects of methodology and reports recent investigations on non-specific
pinocytosis in the authors' laboratories.
E.vporimentu1 approaches
The measurement of pinocytic uptake is most satisfactory
with cells in culture, where one may be confident that all the
VOl.
12
cells are exposed to the same concentration of substrate
(Williams, 1981). The most convenient substrates are radiolabelled macromolecules, which cannot enter cells otherwise than by endocytosis. Macromolecules that can be
radiolabelled and that are not digested within the cell after
pinocytic uptake are ideal marker substrates. Uptake of
lysosomally degraded macromolecules can be quantified by
measuring both the cell accumulation and the appearance of
digestion products in the culture medium; in such cases it is
essential to demonstrate that digestion products arise only
from digestion subsequent to pinocytosis (see Livesey &
Williams, 1979; Pratten & Lloyd, 1982).
Experimentally measured uptake rates are most usefully
expressed as clearance rates. A widely accepted unit is the
Endocytic Index (Williams et al., 1975a), defined as the
volume of culture medium whose substrate content has been
captured by a defined amount of living tissue in a given
time. Normalization of data in this way permits a direct
comparison between rates of uptake either of different substrates or by different cell types (see Pratten er al., 1980).
The rate of clearance of a substrate will only equal the rate
of its endocytosis if there is no substrate returned to the
medium; Besterrnan et al. (1981) describe a careful investigation of this aspect of the calculation.
Identification of substrates that enter pinocytic vesicles
entirely in the fluid phase is not without difficulty. A widely
employed indicator of fluid-phase uptake is an Endocytic
Index independent of substrate concentration. There are
however pitfalls in using this criterion (Williams et al.,
19756). Another indicator relies on essentially circumstantial evidence. In the rat yolk-sac it was found (Roberts
et al., 1977) that [14C]sucrose, '251-labelled polyvinylall
pyrrolidone and some batches of colloidal [ 198A~]gold
had an Endocytic Index of approx. 2pl/mg of tissue protein
per h. It was concluded that this value represented the rate
of fluid uptake by the cells of this tissue, since it seemed
inconceivable that three such different substrates should
coincidentally have exactly the same affinity for membrane
binding sites.
Once the fluid-phase pinocytic rate has been established
for a particular experimental system, it is reasonable to conclude that any macromolecule entering with a higher Endocytic Index does so by adsorptive endocytosis. This
inference is strengthened if the Endocytic Index is found to
decrease with increasing substrate concentration, indicating saturability of binding sites. However, two further
pieces of experimental evidence are needed to establish
adsorptive endocytosis. First, there is the possibility that the
substrate enters solely in the fluid phase but also exerts a
pharmacological action on the tissue, increasing the rate of
pinosome formation and in consequence increasing the
basal rate of fluid entry. This is often an implausible
explanation, since radiolabelled macromolecules used as
probes for pinocytosis are commonly used at very low
concentrations, but it may be simply excluded by measuring
the Endocytic Index of a known fluid-phase substrate in the
presence of the substance in question in non-radioactive
form and in the concentration at which it is used as a pinocytic substrate. Secondly, it is important to distinguish
between genuine pinocytic uptake and mere adherence of
the substrate to the cell surface. Often the kinetics of substrate accumulation provide the necessary evidence. Pinocytic uptake of all non-degradable (non-toxic) substrates in
any defined system should follow the same time-course,
typically constant accumulation over a lengthy period. Thus
the kinetics of uptake of the substrate under investigation
may be compared with those of a known pinocytic substrate. In contrast, the extent of surface binding without
528
pinocytosis typically reaches a maximum quite rapidly,
when binding sites are fully occupied. Surprisingly, however, there are exceptions to this rule: Duncan et al. (1979)
demonstrated a slow progressive, but non-pinmytic, binding of a polycation-colloidal [ *g8A~]gold
complex by rat
yolk-sacs. Another valuable method of distinguishing
pinocytic from non-pinocytic uptake is the use of inhibitors.
A range of inhibitors of pinocytosis is known, including low
temperature, fluoride, 2,4-dinitrophenol, rotenone, cytochalasin B, EGTA, NH,Cl and colchicine (Duncan &
Lloyd, 1978; Pratten & Lloyd, 1979; Livesey & Williams,
1979; Livesey et al., 1980). Comparison of the effects of
inhibitors on the uptake of an established pinocytic substrate and of the macromolecule being investigated can
provide strong indications concerning the latter’s mode of
uptake.
Properties of adsorptive substrates
Investigations in this laboratory over some 10 years have
examined pinocytosis in the 17.5-day rat visceral yolk-sac
cultured in vitro. Although pinocytosis in this tissue has an
important physiological role (see Freeman et al., 1981), our
choice of experimental system rests upon its great stability
and the reproducibility of the data it yields. To our
knowledge it is unrivalled in these respects. Another system
we use extensively is the rat peritoneal macrophage cultured
in vitro. The two techniques were first described by Williams
et al. (1975~1,b ) and by Pratten et al. (1977).
Our initial finding, using the rat yolk-sac system,
concerned the uptake of bovine serum albumin (Moore et
al., 1977). It was found that the rate of uptake could be
increased by graded increases in the extent of denaturation
of the protein. The modified species did not alter the rate of
vesicle formation, and so it was concluded that denaturation
of bovine serum albumin increased its affinity for the yolksac cell surface and so increased its uptake by non-specific
‘constitutive’ pinocytosis. Further experiments by Ibbotson
& Williams (1979) confirmed this interpretation for formaldehyde-denatured bovine serum albumin and indicated a
dissociation constant for this species and the plasma
membrane of approx. 1p ~ .
Several chemical treatments, i.e. with formaldehyde, urea
or decreased pH, all increased the binding affinity of bovine
serum albumin. This suggested that exposure of hydrophobic residues or the masking of (cationic) lysine residues
was responsible for increasing affinity. However, subsequent experiments with polypeptides other than albumin
(Livesey & Williams, 1981) gave two surprising results.
First, the native forms of bovine insulin, egg-white
lysozyme and bovine ribonuclease all entered the yolk sac
mainly by adsorptive pinocytosis; and secondly, treatment
with formaldehyde, urea or low pH either had no effect or
decreased the rate of uptake. Livesey & Williams (1981)
tentatively conclude from these data that the yolk-sac
surface contains two classes of binding sites-for hydrophobic and for cationic ligands. Further data on the uptake
of porcine lactate dehydrogenase H4 and M4 (Kooistra &
Williams, 1981) were concordant with this conclusion.
Parallel data using rat peritoneal macrophages suggested
similar characteristics for these cells (Kooistra et al., 1981).
Using competitive-binding studies, Livesey & Williams
(1982) provided strong evidence that the two classes of
binding sites on the yolk-sac surface are functionally
independent. Polypeptides binding because of their hydrophobic character include insulin and formaldehyde-treated
albumin; those binding because of cationic properties
include calcitonin, ribonuclease and lysozyme.
BIOCHEMICAL SOCIETY TRANSACTIONS
Polypeptides are complex molecules, and it is difficult to
identify the precise effects on their structures of a modifier
such as urea or formaldehyde. In contrast, synthetic homopolymers are simpler and can be tailor-made to contain
graded amounts of a desired substituent. We have recently
shown that the substitution of a poly(DL-aspartamide) with
(hydrophobic) phenolic residues increased its rate of pinocytosis approx. 10-fold (Duncan et al., 1982). In subsequent
work (R. Duncan, H. C. Cable, P. Rejmanova, J . Kopecek
& J. B. Lloyd, unpublished work) a polymethacrylamide has
been substituted with 1-20mol% tyrosinamide. Up to
approximately lOmol%, uptake was predominantly by
fluid-phase pinocytosis. Beyond 1Omol% adsorptive uptake
supervened. In the rat peritoneal macrophage we have used
synthetic polymers to demonstrate the effect of additional
cationic character on the uptake of polyvinylpyrrolidone
(Pratten et al., 1982). Polyvinylpyrrolidone enters the cells
by fluid-phase pinocytosis, but the incorporation of 8%
vinylamine residues changes it into an adsorptive substrate.
The authors’ research alluded to in this paper has been
generously supported by the Science and Engineering Research
Council, the Cancer Research Campaign and the Medical
Research Council. We thank our several collaborators whose
contributions are acknowledged by name throughout the text and
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