induction of microvillous fusion and pinching-off by concanavalin a

Vol. 140, No. 2, 1986

October 30, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 766-772

INDUCTION OF MICROVILLOUS FUSION AND PINCHING-OFF BY CONCANAVALIN A

Toshiaki Nishihata Faculty of Pharmaceutical Sciences, Osaka University,

l-6 Yamadaoka, Suita Osaka 565, Japan

Paul R. Burton Dept. of Physiology and Cell Biology, Center for Biomedical

Research, University of Kansas, Lawrence, Kansas 66045

Larry J. Caldwell Pharmaceutical Research and Development Laboratories,

Teikoku Seiyaku Co., Ltd., 567 Sanbonmatsu, Ochi-cho, Okawa-gun, Kagawa 769-26, Japan

Received August 29, 1986

Concanavalin A (Con A) caused dramatic changes in the structure and function of rat colonic epithelium. The mophological effect was a pronounced, permanent alteration of the microvilli, including fusion and blebing. The functional change involved an increased permeability to passively transported hydrophilic marker compounds. The functional change was transient in nature. Since coadministration of glucose or mannose inhibited the effect of Con A, the mechanistic effect of Con A probably involves binding to saccharide components of the membrane surface. 0 1986 Academic Press, Inc.

Lectins have been isolated from a wide variety of organisms,

including bacteria, plants, and mammals. In plants the occurence

is quite common (1). Lectins often constitute the major protein

fraction of leguminous seeds. The poisonous nature of uncooked

beans is associated with lectins such as Con A. Lectins have

been used for special purposes such as carrier substances for

intraperitoneal injection. In these eases, lectins are coupled to

some enzyme or an anticancer agent (2,3). When injected, the

therapeutic agents are to some degree taken up into cells,

probably as the result of an endocytotic process which lectins

are known to stimulate (4,5). Although the effect of Con A have

been extensively studied with cell culture and with isolated

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Vol. 140. No. 2. 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

tissue preparations (6), relatively little information has been

forthcoming concerning the functional and structural impart in vivo.

In the present communication we examined the effect of C:-r: A

on the intestinal epithelium of the colon of the rat, both for

morphologic and for functional alteration of permeability.

Materials and Methods. All reagents used were of the best commercial grade available. Con A was obtained from Sigma Chemicals Inc. (St. Louis, USA).

Male Sprauge-Dawley rats, 200 to 250 g, were anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and placed on a 38 C surface. The colon was exposed by abdominal incision, and the administration of 0.25 ml test solution into the lumen of the ligated colon was performed according to the method in reference 7. The reason why a ligated colonic loop was employed in this study was to control exposure of the colonic epithelium to Con A and to determine the transport of test compound by measuring the remaining amount in the loop.

For the morphologic study and the stainability with trypan blue, 0.25 ml sodium phosphate buffer (0.1 M and pH7.0) containing 0.4 % trypan blue was administered into the lumen with Con A (100 pg/ml) or after rinsing out the Con A with 6 ml of buffer 15 minutes after treatment with Con A. Sample tissue was removed 15 min after administration of trypan blue. Morphologic change and trypan blue stainability were assayed by light- and electron-microscopy (7). Microvillar surface area was determined by measuring length and widith of each microvillus across 1 urn of epithelial cell surface, with the assumption that microvilli are cylindrical.

Absorption of marker compounds such as cefoxitin and sulfadimethoxine was determined by measuring the amount of compound remaining in the colonic loop, 15 min after administration of the test solution (0.1 M sulfadimethoxine and 0.05 M cefoxitin) at a volume of 0.25 ml per each colonic loop. Assay of sulfadimethoxin (8) and cefoxitin (9) was carried out by high performance liquid chromatography. Thus, absorption of test compounds was represented with an absorption clearance (CL) determined with the following equation. CL(ml/min/g-tissue) = [(A) - (R)]/[(C)x(period)x(wet weight 0 f tissue)]; where (A) and (R) represent the administered amounts and remaining amounts in the loop, (C) represents the initial concentration of compounds in the administered solution.

Results and Discussion. After a brief exposure to Con A,

light-microscopic observations revealed minor epithelial dis-

continuity in the colon (Table I). However, transmission electron-

microscopy revealed many membraous vesicles around the microvillar

brush-border, which appeared to be the result of microvillar

blebing or "pinching-off". Fig. 1 shows a typical result for the

colonic epithelium. Additionaly, some of the damaged microvilli

appeared to be fused together '.s a result of Con A treatment.

Vol. 140, No. 2, 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table I. Effect of Con A (100 pg/ml) on rat colonic epithelium: surface disruption and stainability of trypan blue (TB) using the

in vivo rat colonic ligated loop

Treatment % disrupption TB stainability surface

buffer TB alone

Con A alone

0.91io.75 1.04t0.81 - [+I

Con A + glucose (0.3 M) Con A t mannose (0.3 M)

Con A + TB Con A t TB + glucose Con A + TB + mannose

7.96f6.38 ttt 1.09f0.97 1.27iO.84

pretreatment with Con A for 15 min (h: time after rinsing)

1h:buffer 7.14k6.23 1h:TB 8.8925.22 - (+I 2h:TB 6.11i4.23 - [+I 12h:buffer 6.94f6.17 12h:TB 7.02i6.94 - [+I

Notes to Table: The % disrupted surface refers to the length of measured surface which was discontinuous as a percentage of the total distance measured. TB stainability refers to the degree of stain with trypan blue of colonic epithelium. The coding is as follows: -, unstained; +, faintly stained; ++, moderately stained; +++, intensely stained. The parentheses indicate that the tissue from one of the five rats showed the indicated difference in staining from the other four samples. The brackets indicate that the tissue from two of the five rats showed the indicated difference in staining. In all other cases the tissue from all five rats were stained in the same manner.

There is some experimental evidence that surface carbohydra-

tes play a role in the functional and structural organization of

plasma membranes (2,10), but only in the cases of aggregation of

certain sponge cells have specific carbohydrate been shown to be

essential. The addition of 0.3 M D-glucose or 0.3 M D-mannose in

the administered solution inhibited the epithelial discontinuity

(Table I), and also inhibited the fusion and the pinching-off of

the microvilli (same appearance as with control in Fig. IA was

obtained). Thus, the fusion and pinching-off observed by Con A

alone, may occur by the same mecahnism of erythrocyte aggregation,

in which mannosyl- and glucosyl- groups at the membrane surface

are specific for Con A (11). The effect of Con A after binding to

the microvillous surface causes fusion and pinching-off of micro-

768


1. Fig. Electron micrograph of rat colonic epithelium treated with buffered saline (control, A) or with Con A (100 pg/ml, B) at pH7.0 for 15 min. In B, the membrane vesicles on the luminal side were formed from microvillous blebing or "pinching-off" (arrow). Some damaged microvilli were fused (double arrow). Abbreviations: b=membrane bound vesicle, g=glycocalyx, m=microvillus, v=membrane vesicle in the lumen. Mag x 81,000.

villi, rather than inducing internalization. This may be ;i

specific action of Con A displayed only microvillous surfaces.

The treatment of Con A did not result in endocytosis in the co

epithelium as it does in cell culture. Therefore, endocvtosis

Lon


probably is not one of the mechanisms for the increased permeabi-

lity reported below.

The observed morphological changes induced by Con A were

still apparent even 12 hours after rinsing the lumen of the

colonic loop with the buffer. One of the quantifiable changes so

induced was a reduction in surface area at the brush-border (Fig.

2). This surface reduction also did not correct itself within 12

hours, and therefore this change is considered to be permanent for

the purpose of this discussion. Essentially, the entire epi-

thelial surface will be replaced by new cells, within 1.5 days in

mice and 3 - 5 days in humans (12).

The colonic uptake of poorly absorbed marker compounds such

as trypan blue and cefoxitin was enhanced in the presence of Con

A (Table I and Fig. 2). However, upon rinsing the colonic lumen

with the buffer, the enhanced uptake was lost, ie. the barrier

properties of the mucosa were quickly restored. This restoration

of the barrier was seen despite the permanently disrupted appea-

rance of the brush border membrane (Table I and Fig. 2). These

results indicate that the enhanced uptake is possible only during

the membrane-damaging process such as fusion and pithing-off in

the presence of Con A, rather than cell death. When Con A is

removed from the system, the functionality is restored but not

the original cytoarchitecture. The addition of 0.3 M glucose or

mannose in the administered solution inhibited the increased

uptake of trypan blue (Table I) and cefoxitin (Fig. 2), which were

induced by Con A. These results indicate that the changes in

transport of test compounds is induced by the binding of Con A on

the colonic epithelial surface, which also induces the morpholo-

gic changes.

Colonic absorption of sulfadimethoxine, which is ordinarily

well absorbed, was decreased in the presence of Con A, and

770


(Al

25 iB)

.z c) a

.rl x E12.5

:

L

I _---

Q

: OI”

----g

4 5r x

Time, after administration of Con A, h

Fig. 2. Effect of Con A (100 pg/ml) on the surface area (A) of ~110~1s brush-border membrane of rat colonic epithelial cells, and on the absorption of cefoxitin (B) and sulfadimethoxine (C) from colinic lumen, using an in vivo intestinal loop method. Symbols in the figure are as follows: circles, treatment with Con A alone (100 pg/ml); triangles, Con A + D-glucose (0.3M); squares, Con A + mannose (0.3 M).

Treatment periods of Con A without or with sugar was 15 min as shown with m , and then Con A was rinsed out with 6 ml of the buffer. Absorption of cefoxitin and sulfadimethoxine were determined for 15 min after the administration, as shown within figure. Closed symbols represent the results just after the treartment of Con A (A) and represent the simultaneous administration of cefoxitin and sulfadimethoxine with Con A. Open symbols after pretreatment with Con A represents the results after rinsing out Con A.

Each value represents the mean k S.D. (nz&). a;p<O.Ol versus the result without Con A treatment.

remained compromised even 12 hours after rinsing (Fig. 2). Ii. in

generally assumed that absorption of lipophilic compounds such 2s

sulfadimethoxine involves passive diffusion across the lipid I~ayer

of the brush border membrane (13). The present result is easily

rationalized in view of the apparent permanent reduction in

absorptive surface area after Con A treatment (Fig. 2).

771


In conclusion, treatment of the colon with Con A was seen to

dramatically alter both structure and function of the absorptive

epithelium. Although structural changes were considered to be

rather permanent, the functional aspects of the epithelium were

largely restored after rinsing away the Con A. Further, since

the fusion and pithing-off of the epithelial microvilli after ad-

ministration of Con A was inhibited by a simultaneous administra-

tion of high concentrations of glucose or mannose, it is

considered that the morphological changes induced by Con A occurs

via the same mechanism as with erythrocyte aggregation.

References

1.

2.

;:

2: 7.

8.

9.

10.

11.

12.

13.

Allen N. K. and Brillantinel B. A. (1965) J. Immunol. 102:1295-1299 Barker, S. A., Giblin, A. G., Gray, G. J., and Brown, W. H., :;:92) Carbohydrate Res 36:23-33

Edlso;, T. and Holrori, K. (1974) Nature 265:81-82 P. J. and Cohn, Z. A. (1974) J. Exptl. Med. 1~+0:136&-

1386 Goldman, R. (1974) FEBS Lett 46:203-208 Nicolson, G. L. (1974) Int. Rev. Cytol. 39:89-190 Sithigorngul, P., Burton, P., Nishihata, T., and Caldwell, L., (1983) Life Sci. 33:1025-1032 Nishihata, T., Yata, N., and Kamada, A., (1978) Chem. Pharm. Bull. 26:3353-3363 Nishihata, T., Takahagi, H., Yamamoto, M., Tomida, H., Rytting, J. H., and Higuchi, T., (198.4) J. Pharm. Sci. 73:109-112 Burgger, M. M. and Goldberg, A. R. (1967) Proc. Natl. Acad. Sci., USA 57:359-366 Goldstein, I. J. (1976) RConcanavalin A as a Tool", eds, H. Bittiger and H. P. Scnebli, Wiley, London, p.55-65 Rogers, A. W., (1983) I'Cells and Tissues", Academic Press,New York and London, p. 198 Shanker, C. S. (1962) Pharmacol. Rev. 14:501-509

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induction of microvillous fusion and pinching-off by concanavalin a

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