Uptake of tomato lectin by the adult rat small intestine in vitro

Abstract

T h e phenomenon of macromolecular uptake in the adult intestine has now generally been accepted. Most authors agree that the degree of absorption is nutritionally insignificant, but that the penetration of the intestinal epithelium by antigenic macromolecules may be o f immunological importance [ 1, 21. This absorption of intact macromolecules is thought to occur predominantly by transcellular endocytosis and may be involved in the pathogenesis of food allergies and inflammatory bowel diseases. Recently. however, interest has centred on the uptake of intact proteins in the gut as a potential route for the therapeutic administration of biologically active peptide drugs and vaccines. We have been using an improved everted gut sac system [3] to study the uptake of a non-toxic lectin from tomatoes, a dietary glycoprotein of molecular weight, 7 1 kDa. as it has been shown to be taken up intact by rat and human intestine it1 vivo, to bind to the intestine without causing any deleterious effects [4] and to have potential for slowing gastrointestinal transit [ 51. Quantification and characterization of the uptake of this lectin will be useful in evaluating its potential as an adjunct to drug delivery in the gastrointestinal tract. Everted gut sacs were prepared from the small intestines of adult male Wistar rats and incubated for times up to 2 h in 1 0 ml of tissue culture medium 100 at 37°C in the presence of 1'51-labelled tomato lectin ( 2 pg/ml). Sacs were removed from the flasks at intervals. blotted dry and the serosal fluid collected. Sacs were then washed four times in ice-cold saline and dissolved in 1 M-NaOH. Samples of the external medium, serosal fluid and dissolved tissue were taken for gamma counting. T h e protein content of the sacs was determined by the method of Lowry et af . [ 61 as modified by Peterson [ 71. Uptake of tomato lectin was compared with two controls, I '51-labelled polyvinylpyrrolidone (PVP), an inert polymer, often used as a marker for fluid-phase endocytosis, and 1'51labelled bovine serum albumin (BSA), a degradable protein of similar molecular mass to the lectin. Uptakes were calculated as nanograms of substrate associated with gut tissue or in the serosal fluid per milligram of gut protein. T h e uptake of tomato lectin is shown in Fig. 1(u). At 37C, the uptake into gut tissue increased linearly with time at a rate of 26 ng/h per mg o f protein. Passage into the serosal fluid was also linear, but much slower, the rate being 1.7 ng/h per mg o f protein. Fig. 1( h ) shows uptake of the two controls. T h e rate of uptake of lectin by the tissue was 11 times higher than BSA and 20 times higher than PVP, but transfer to the serosal fluid was only 4.3 and 5 times greater than BSA and PVP, respectively. This showed that the proportion of lectin transferred across the mucosa into the serosa was less than either control, and indicated accumulation of lectin within the enterocytes. To determine the mechanism( s) of lectin uptake, incubations were carried out using low temperature as an inhibitor of endocytosis (Fig. l a ) . These results showed that lectin uptake into the tissue was reduced, but not negated at 4°C. Rather than true uptake, this persistent tissue association at low temperature represented binding of the lectin to the mucosa and was indicative of an absorptive component in lectin uptake at 37°C. This conclusion was confirmed by