248 Biochimica et Biophysica Acta, 717 (1982) 248-254 Elsevier Biomedical Press

BBA21168

PINOCYTOSIS OF POLY(a,/B-(N-2-HYDROXYETHYL))-DL-ASPARTAMIDE AND A TYRAMINE DERIVATIVE BY RAT VISCERAL YOLK SACS CULTURED IN VITRO

ABILITY OF PHENOLIC RESIDUES TO ENHANCE THE RATE OF PINOCYTIC CAPTURE OF A MACROMOLECULE

R. DUNCAN a, D. STARLING a F. RYP~,~EK b, j. DROBNIK b and J.B. LLOYD a,,

a Biochemistry Research Laboratory, Department of Biological Sciences, University of Keele, Keele, Staffordshire ST5 5BG (U.K.) and b Institute of Macromolecular Chemistry, Czechoslovak Academy of Sciences, 16206 Prague 6 (Czechoslovakia)

(Received November 27th, 1981)

Key words: Pinocytosis," Phenolic residue," Tyramine derivative," Poly(hydroxyethyOaspartamide; (Rat visceral yolk sac)

Incorporation of 20% tyramine residues into its structure greatly increased the rate of pinocytosis of poly(a,O-(N-2-hydroxyethyi))-DL-aspartamide 0PHEA) by rat visceral yolk sacs cultured in vitro. Both the parent macromolecule and the tyramine derivative (PHEA-tyramine) were captured by adsorptive pinocyto- sis, the higher affinity of the derivative for the yolk sac plasma membrane being responsible for its greater rate of capture. Using t2Sl-labelled PHEA-tyramine, the relationship between substrate concentration and rate of capture was determined, it was also shown that following internalization, the PHEA-tyramine linkage is resistant to intracellular hydrolysis. Fluorescence micrographs were consistent with capture of both substrates being by pinocytosis and illustrated the highly efficient concentration of the tyramine derivative by yolk sac endodermal cells.

Introduction

The affinity of a macromolecule for the plasma membrane greatly affects its rate of capture by pinocytosis. A macromolecule that binds strongly to the plasma membrane is efficiently con- centrated within the cell, whereas one that enters in a non-adsorptive fashion is captured much more slowly [1]. A number of receptor-mediated uptake processes have been described that permit the specific capture of particular macromolecules by particular c.ell types; currently the best understood are those governed by a carbohydrate recognition system (reviewed in Ref. 2). Rat hepatocyte mem-

* To whom correspondence should be addressed. Abbreviations: PHEA, poly( a,fl-( N-2-bYdroxyethyl)-D L- aspartamide; PVP, poly(vinylpyrollidone).

0304-4165/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

branes carry a unique receptor which facilitates the rapid uptake of asialoglycoproteins bearing terminal galactose residues. In contrast, uptake of mannose- or N-acetyl-glucosamine-terminated gly- coproteins is effected by receptors associated with Kupffer cells and alveolar macrophages. Fibrob- lasts possess a phosphomannose-recognising recep- tor involved in the adsorptive pinocytosis of lyso- somal enzymes.

It has been previously shown that a variety of 125I-labelled proteins are captured rapidly by ad- sorptive pinocytosis in the rat visceral yolk sac epithelium [3-5] and, although this adsorption does not appear particularly substrate-specific, its ef- fectiveness correlates well with the hydrophobicity of the molecule [5]. t25I-Labelled poly(vinylpyr- rolidone) (PVP) and [14C]sucrose enter this tissue non-adsorptively [6]. In this study we have demon-

PHEA:(~= -CH 2 -CH2-OH PHEA-tyromine: (~) = -CH2-CH2~-OH

~ O ~ ~ NH-CH2-CH2-OH ~ ' ~ "

- - - H O N ~-OHL J L ~ - ~

; ~,

Fig. 1. Chemical structure of PHEA and its tyramine deriva- tive. x =80.8 mol% and y = 19.2 rnol%.

strated that the introduction of p-hydroxyphenyl residues greatly increases the uptake by this tissue of a homopolymeric substrate. Poly(a,fl-(N-2-hy- droxyethyl))-DL-aspartamide (PHEA; Fig. 1) is a relatively inert macromolecule which has been ex- plored as a potential plasma expander [7] and also proposed as a possible drug carrier [8]. We have investigated pinocytic uptake of PHEA, and a PHEA derivative coupled to tyramine residues, by rat visceral yolk sacs cultured in vitro.

Materials and Methods

Chemicals. 125i.Labelled PVP and [125i]iodide (preparations IM.33P and IMS.30) were from the Amersham International, U.K. Tissue culture medium 199 was from GIBCO, Bio-Cult Ltd., Paisley, Scotland and inactivated calf serum (pre- paration CS07) was from Wellcome Reagents, Be- ckenham, Kent. 2,4-Dinitrophenol was from Sigma (London) Chemical Co., Poole, Dorset. Aquamount GT GURR was from Hopkins and Williams and OCT compound from Miles Labs Ltd, Slough. Tyramine was from Fluka AG Buchs, Switzerland.

Preparation of fluorescence-labelled PHEA poly- mers. Polysuccinimide was fluorescence-labelled with N-(2-aminoethyl-carbonyl)-6-aminofluo- rescein [8]. PHEA was prepared by total aminoly- sis of the fluorescence-labelled polysuccinimide with 2-aminoethanol in dimethylformamide [9]. PHEA-tyramine was prepared by partial aminoly- sis of the fluorescence-labelled polysuccinimide with tyramine, followed by reaction of the remain- ing succinimide residues with 2-aminoethanol [9,10].

249

Fig. 1 shows the chemical structure of PHEA and PHEA-tyramine. Both polymers contained ap- prox. 0.5 mol% of fluorescence label. The mean molecular weights were 11000 (PHEA) and 8900 (PHEA-tyramine).

Preparation of 1251-labelled PHEA-tyramine. PHEA-tyramine (20 nag) was dissolved in 9ml 0.05 M phosphate buffer (pH 8.0) and Na [125 I]iodide (1 mCi) was added. After stirring for at least lmin at 4°C, chloramine T (4ml of a 1 mg/ml solution) was added and after 8 min the reaction was stopped with sodium metabisulphite (3 ml of a 2 mg/ml solution). Sodium iodide (100 mg) was added before dialysis against 1% sodium chloride to remove all the [125I]iodide from the preparation. Paper electrophoresis (barbitone buffer pH 8.6, 400 V, 35 min) was used to assess the amount of contaminating [125I]iodide in the preparation.

Uptake of ~ 25I-labelled PHEA-tyramine. The up- take of 125I-labelled PHEA-tyramine was mea- sured using a method previously described for the uptake of l:SI-labelled PVP Ill]. Uptake was de- fined as the volume of culture medium (pl) whose contained substrate was captured per mg of yolk sac protein per h. Uptake values were corrected to compensate for the effect of substrate depletion during the incubation period, before calculation of uptake values [12]. In certain experiments 2,4-di- nitrophenol was added, simultaneously with the substrate, to the culture medium at a concentra- tion (50/xg/ml) previously shown [13] to inhibit pinocytosis in the yolk sac system.

In some experiments yolk sacs were incubated in the absence of serum, as previously described [4]. The uptake of 125I-labelled PHEA-tyramine in this system was measured in the presence of vari- ous concentrations of unlabelled PHEA-tyramine.

Uptake of 125I-labelled PVP was measured [l 1] in the presence of PHEA or of non-radioactively labelled PHEA-tyramine (100 /~g/ml). In these experiments PHEA or PHEA-tyramine was intro- duced simultaneously with 125I-labelled PVP.

Uptake of PHEA and PHEA-tyramine as as- sessed by fluorimetric assay. Rat visceral yolk sacs were incubated singly in Erlenmeyer flasks con- taining l0 ml medium 199 supplemented with 10% calf serum. PHEA or PHEA-tyramine (10/~g/ml) was added to each flask at the beginning of the

50C

experiment and incubations were carried out at 37°C for periods up to 7 h. At each sampling time, yolk sacs were removed, washed three times (2 min) in ice-cold saline and homogenized individually in 1 ml distilled water with l0 passes of a motor- driven Teflon pestle using a Tri-R homogenizer at setting 2. Trichloroacetic acid (0.5 ml of a 10% solution) was added to each tube and the precipi- tate pelleted by spinning a MSE Coolspin at 3100 r.p.m, for 20 min at 4°C. Supernatant (1 ml) was withdrawn from each tube and adjusted to greater than pH 8.0 by addition of 1 N sodium hydroxide (0.23 ml). The fluorescence was measured using a Perkin-Elmer Model 3000 fluorimeter, excitation wavelength 492 nm, emission wavelength 516 nm.

In certain experiments monoiodoacetate was present in the culture medium at a concentration (10 #g/ml) previously shown [13] to inhibit pino- cytosis in the yolk sac. Monoiodoacetate was used in these experiments, since 2,4-dinitrophenol would interfere with the fluorimetric, assay.

Column chromatography. Samples of PHEA- tyramine and of the 125I-labelled PHEA-tyramine preparation were applied to a Sephadex G-75 col- umn and eluted with 0.1 M NaEHPO 4. In the case of PHEA-tyramine the resultant fractions were assayed for fluorescence; with 125I-labelled PHEA-tyramine the fractions were assayed for radioactivity.

Fluorescence microscopy. Visceral yolk sacs were incubated with PHEA-tyramine (100 #g/ml) at 37°C for 5 h. Each yolk sac was washed three times (2min) in ice-cold saline (1%), mounted in OCT compound on a chuck, and frozen in liquid nitrogen. Sections (6#m) were cut using a Slee cryostat (type HR) at - 20°C and placed on slides. After mounting in Aquamount, they were ex- amined using a Leiz Orthoplan fluorescence mi- croscope. Photographs were taken using a Leitz Orthomat camera and Kodak Ectochrome (ASA 200) film uprated to ASA 800 during development.

Results

PHEA-tyramine was radioactively labelled very efficiently, 83% of the 125I present in the reaction mixture becoming attached to the polymer. No free [usI]iodide was detectable in the preparation after dialysis, nor was there any measurable re-

Ve 125I-

3oc 1 ; 6

~ 2oc "°'°~'q' b 4 o

u ~ 10C 2

o _ 1

20 40 60 80 100 Fraction No

Fig. 2. Elution from Sephadex G-75 of the unlabelled PHEA- tyramine preparation ( O - - - - O), the 125I-labelled PHEA- tyramine preparation (@ @), and uSI-labelled PHEA- tyramine in medium 199 after incubation with 10 yolk sacs for 6.5 h (m m).

lease during storage at 4°C or incubation at 37°C. Sephadex G-75 chromatography of the PHEA. tyramine before and after labelling with ~25I showed preferential labelling of the higher molecu-

4 0 0

"8

3 0 0 O.

10C

0

/ -8- --8 .... ~- - 4} - - o _ _ _8_ _

3 4 5 Time (h)

250

Fig. 3. Uptake of t25I-labetled PHEA-tyramine by yolk sacs cultured in medium 199 containing 10% calf serum in the presence of 2,4-dinitrophenol (50 #g/ml) ( O - - - - - O ) and with no addition (@ 0). Each point represents data derived from a single yolk sac.

lar weight material, resulting in a radioactively labelled preparation with a similar degree of poly- dispersity, but a slightly higher mean molecular weight, compared with the original sample (Fig. 2).

x25I-labelled PHEA-tyramine was captured avidly by yolk sacs. Accumulation was linear with time over a 6.5 h incubation period and was effec- tively abolished by the presence of 2,4-di- nitrophenol (50 #g/ml) (Fig. 3).

The presence of PHEA or unlabelled PHEA- tyramine (100/~g/ml) in cultures with 125 I-labelled PVP as substrate did not alter its rate of capture, the mean rates of uptake being 2.07, 2.28 and 2.25 # l /mg protein per h with no addition, and in the presence of PHEA and PHEA-tyramine, respec- tively.

In order to detect degradation of pinocytosed 125I-labelled PHEA-tyramine, 10 visceral yolk sacs were incubated together in an Erlenmeyer flask containing 10 ml medium 199, supplemented with 10% calf serum and 125I-labelled PHEA-tyramine (60 #g/ml), for 6.5 h at 37°C. Samples (1 ml) of culture medium were applied to a Sephadex G-75 column and eluted with 0.1 M Na2HPO 4 buffer. Fractions were then assayed for radioactivity and the resultant elution profile is showh in Fig. 2. In this experiment 90% of the 125I-labelled PHEA- tyramine had been captured by the yolk sacs, and the elution profile of the residual radioactivity

80

6O

4oi . c i

8 c

2c

o

h / 0.___.8.----0"----8-

1 2 3 4 5 6 7 Time (h)

Fig. 4. Uptake of PHEA (O O) and PHEA-tyramine (O O) by yolk sacs cultured in medium 199 containing 100% calf serum. Each point represents data derived from a single yolk sac.

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displays two interesting features. First, that this polymer is not hydrolysed intracellularly to yield 125I-labelled degradation products small enough to diffuse back into the culture medium. This ob- servation demonstrates that the rate of accumula- tion of radioactivity by the tissue represents the total rate of uptake of substrate. Secondly, the size distribution of the remaining 125I-labelled PHEA- tyramine indicates preferential uptake of higher molecular weight material. Although [125 I]iodide is a major component of the residual radioactivity, it accounts for only 3.7% of the total radioactivity originally present in the sample of 125I-labelled PHEA-tyramine.

Since it is not possible to 125I-label PHEA, comparison of pinocytic capture of PHEA and PHEA-tyramine was achieved by measurement of fluorescence.

Fig. 4 illustrates their uptake over a 6.5 h in- cubation period. It can be seen that the PHEA- tyramine derivative is accumulated considerably faster than PHEA. The non-linearity of accumula- tion of PHEA-tyramine, which can be attributed to a decrease of the conceniration of this substrate during the culture period, owing to its rapid accu- mulation in the tissue, precludes an accurate calcu- lation of initial rates of uptake. However, over the initial 1-3 h period the rate of uptake of PHEA- tyramine was consistently between 10 and 12 times (11.42, 10.71, 10.68) higher than that of PHEA. Accumulation of fluorescence label by the yolk sacs, when incubated with either substrate, was inhibited by inclusion of monoiodoacetate (10 /xg/ml) (results not shown).

The rate of uptake of 125I-labelled PHEA- tyramine (6 #g/ml) was considerably greater when measured in the absence of 10% calf serum, the rate of capture (mean- + S.E.) measured in three separate experiments being 280.11 -+ 33.58 # l /mg protein per h. The mean (-+S.E.) value measured for 12SI-labelled PVP in matched control experi- ments in the absence of serum was 4.26-+ 1.16 /xl/mg protein per h. Fig. 5 shows the uptake, in the absence of serum, of 125I-labelled PHEA- tyramine (1.6 #g/ml) with addition of unlabelled PHEA-tyramine (10-100 #g/ml). Elevation of the total concentration of PHEA-tyramine in the cul- ture medium led to a decreased rate of uptake for the 125I-labelled PHEA-tyramine. Uptake values

252

300[ °

250

2oo

UJ 150

T!

0 25 50 75 I O0 q 2 5 Concentration of PHEA-tyrQmJne (#g lml )

Fig. 5. The effect of substrate concentration on the rate of uptake of 125 I-labelled PHEA-tyramine by yolk sacs cultured in the absence of serum. Each point represents data derived from either two or three separate experiments (mean -+ S.E.).

for 125I-labelled PHEA-tyramine in terms of #g of substrate captured/mg yolk sac protein per h were used to produce a Lineweaver-Burk plot, yielding K m of 71.43 / ,g /ml and Vma x of 20.65 /~g/mg protein per h. Using a molecular weight for PHEA-tyramine of 8900, these values become 6.4 #M and 1.88 nmols /mg protein per h, respec- tively.

Fluorescence microscopy of yolk sacs incubated with PHEA-tyramine showed the tissue to be

heavily labelled with fluorescent polymer, the greatest concentration of fluorochrome being in the apical region of the cells of the visceral endo- derm (Fig. 6). There was also a definite band of fluorescence visible along the other surface of the tissue; this represented material either bound to, or internalized by, the flattened mesothelial cells that cover that area. At higher power it was possi- ble to visualize fluorescence concentrated within very small vesicular structures in the apical regions of endodermal cells, which presumably represent pinosomes and secondary lysosomes. Incubation of yolk sacs with PHEA also produced tissue that was fluorescence-labelled, but the concentration of fluorochrome was much less, as demonstrated by the increased exposure time necessary to obtain the image shown in Fig. 6. Apart from the lower intensity of fluorescence, the pattern of labelling was identical. When examined under ultraviolet light, sections from yolk sacs incubated in culture medium alone showed some evidence of fluores- cence, particularly in the apical regions of the endodermal cells. This fluorescence probably originates from certain of the constituents of medium 199 pinocytosed during incubation. Al- though the background fluorescence may contrib-

Fig. 6. Fluorescence micrographs of yolk sacs after incubation in culture medium at 37°C for 5 h with (a) no addition, (b) PHEA (200 /~g/ml), or (c) PHEA-tyramine (100 /~g/ml). Although both polymers contained the same proportion of fluorescence label, the tyramine derivative has an approx. 50% lower specific fluorescence; consequently PHEA-tyramine was used at twice the concentration of PHEA in order to compensate for this difference. Material obtained after incubation with PHEA-tyramine was photographed with an exposure time of 10 s, whereas the other specimens were exposed for 30 s.

ute a significant proportion of the fluorescence seen with PHEA, it is only a very small fraction of the total fluorescence seen after exposure to PHEA-tyramine.

Discussion

~25 I-labelled PHEA-tyramine was captured very rapidly by the rat visceral yolk sac, its rate of uptake, when measured in the presence of calf serum, being some 40 times that measured for the non-adsorptive substrate 125I-labelled PVP. This high rate of uptake indicates either that PHEA- tyramine is an adsorptive substrate or that PHEA-tyramine stimulates the rate of formation of pinocytic vesicles at the plasma membrane. The inability of unlabelled PHEA-tyramine (100 /~g/ml) to increase the rate of uptake of 1251- labelled PVP, when both were present in the same incubation, eliminates the second alternative and suggests that the rapid clearance of 125I-labelled PHEA-tyramine is due to its high affinity for the plasma membrane. The complete inhibition of up- take of ~25I-labelled PHEA-tyramine by a con- centration of 2,4-dinitrophenol known to inhibit pinocytosis in the yolk sac system confirms that this substrate is truly pinocytosed and is not sim- ply accumulating progressively on the exposed surfaces of the tissue. The rapid uptake of 125I- labelled PHEA-tyramine was not followed by in- tracellular degradation of the polymer, a result compatible with previous reports [14] of its limited biodegradability.

To investigate whether the tyramine residues play any part in determining the high affinity of 125I-labelled PHEA-tyramine for the yolk sac plasma membrane, it was necessary to compare its rate of capture with that of the parent PHEA polymer. Although the fluorescence properties of macromolecules have been used before [15] to quantify rates of pinocytosis, we found the technique, although adequate, less satisfactory for precise quantitation than our radioactive assay. However, the data obtained showed clearly that PHEA-tyramine was captured 10-11 times faster than PHEA. Incubations with monoidoacetate present confirmed that neither polymer can enter except by pinocytosis. Fluorescence microscopy revealed a distribution of fluorochrome indicative

253

of uptake by pinocytosis and confirmed that PHEA-tyramine was captured more rapidly than PHEA.

It has been previously shown that omission of serum from the culture medium leads to an in- crease in the rate of capture of adsorptive sub- strates [3]. Uptake of 125I-labelled PHEA-tyramine (6 #g/ml) was considerably faster in the absence of serum than in its presence and indeed, in the absence of serum, the rate of uptake was some 66 times greater than that of the fluid-phase marker ~25I-labelled PVP. Increasing the substrate con- centration, by addition of unlabelled PHEA- tyramine to the culture medium, decreased the rate of uptake of 125I-labelled PHEA tyramine, an ob- servation explicable on the basis of competition for a limited number of membrane receptors. An alternative explanation, that PHEA-tyramine in high concentration inhibits pinocytic vesicle for- mation, is ruled out by our observation that PHEA-tyramine (100 #g/ml) has no effect on the rate of capture of ~25I-labelled PVP.

Michaelis-Menten analysis of the effect of sub- strate concentration yielded a K m of 6.4/~M and Vm, x of 1.88 nmols/mg (based on the average molecular weight of 8900) for the pinocytic cap- ture of 125I-labelled PHEA-tyramine. Assuming the g m approximates to the dissociation constant of the substrate-binding site complex, the affinity of PHEA-tyramine for the yolk sac plasma mem- brane is somewhat lower than that of lysosomal enzymes for the fibroblast plasma membrane [16,17] and of 125I-labelled asiolo-orosomucoid for the hepatocyte [18] both of these having dissocia- tion constants in the nM range. The value mea- sured for I25I-labelled PHEA-tyramine in the yolk sac is somewhat higher than, but comparable with values reported for other substrates in this tissue [4,5].

Synthetic macromolecules such as PHEA have been proposed as potential carriers of chem- otherapeutic agents [19,20], their intended role being to confine uptake of drug by cells to the pinocytic route and thus afford the possibility for directing drug action to the specific cell type where it is required. Efficiency of targeting such a drug- carrier complex depends on the rate of pinocytosis in the offending cell type, the size of the carrier complex [21] and also the magnitude and specific-

254

ity of its affinity for the plasma membrane. It is known that incorporation of certain sugar residues into liposome membranes can significantly alter the body distribution after administration in vivo [22]. Here we have shown that inclusion of tyra- mine residues into the structure of a polyasparta- mide dramatically increased its rate of capture by the visceral yolk sac. Experiments reported elsewhere [23] have shown that, following their in vivo administration, certain derivatives of PHEA, including PHEA-tyramine, accumulate in the kid- ney more rapidly and to a greater extent than the parent polymer. Therefore, it is possible that the pinocytosis-enhancing properties of the tyramine residue are to some extent, cell-specific.

Acknowledgements

We thank the Cancer Research Campaign for financial support and the British Council for sup- porting the collaboration under their Academic Links with Eastern Europe scheme. Thanks also to Mr. F. Brian and his colleagues for preparation of material for fluorescence microscopy.

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