Zinc Transport by Isolated, Vascularly Perfused Rat Intestine and

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17 Zinc Transport by Isolated, Vascularly Perfused Rat Intestine and Intestinal Brush Border Vesicles M I C H A E L P. M E N A R D , P A U L O E S T R E I C H E R R O B E R T J. COUSINS

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Rutgers University, Department of Nutrition, New Brunswick, NJ 08903

Studies of zinc transport with isolated, vascularly perfused rat intestine and brush border vescicles fail to support a role for a unique binding ligand i n zinc absorption. Evidence suggests that a physiological pH, none of the ligands tested significantly enhanced zinc uptake or transfer by the perfused intestine. Uptake studies with brush border membrane vescicles also support these findings. In vescicles from both zinc normal and zinc depleted rats, zinc transfer does not require energy. With vescicles from normal rats, the Jmax i s 5.4 nmoles per m i n , with a Km of 0.41 mM. The Km i s not changed, but the Jmax increases to 12.0 nmoles per min with vescicles from zinc depleted rats. Electrophoretic data suggests a change i n brush border membrane protein composition occurs during zinc deficiency. These proteins could influence zinc uptake during a reduction i n dietary zinc supply. The b i o a v a i l a b i l i t y of a mineral nutrient i s influenced by a plethora of factors. Composition of the diet can influence the interaction between specific dietary components and metals. The physical characteristics of these interactions will determine how much mineral i s available for absorption. These interactions are undoubtedly influenced by the acid conditions of the gastric secretions and the hydrolytic a c t i v i t y of the intestine. In order to understand how these factors might influence the absorption of a nutrient metal such as zinc, i t i s f i r s t necessary to understand the mechanism of zinc absorption and the systemic factors which regulate i t . Current address: University of Florida, Department of Food Science and Human Nutrition, Gainesville, FL 32611 J

0097-6156/83/0210-0233$06.00/0 © 1983 American Chemical Society Inglett; Nutritional Bioavailability of Zinc ACS Symposium Series; American Chemical Society: Washington, DC, 1983.


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Twenty years ago C o t z i a s £t a l . (1) provided the f i r s t evidence for homeostatic control of zinc absorption. Subsequent reports from numerous l a b o r a t o r i e s have r e i n f o r c e d that hypothesis. Studies w i t h both ruminants (2) and r a t s (3,4) have shown z i n c a b s o r p t i o n i s depressed by high l e v e l s of dietary zinc. Conversely, z i n c d e f i c i e n c y r e s u l t s i n an increase i n z i n c a b s o r p t i o n ( 5 ) , w i t h maximal a b s o r p t i o n o c c u r r i n g w i t h i n s e v e r a l days of d e p l e t i o n ( 6 ) . The mechanisms which account f o r t h i s homeostatic c o n t r o l have not been f u l l y delineated. The mechanism of z i n c absorption involves s e v e r a l d i s t i n c t phenomena. These include 1) uptake and t r a n s f e r of z i n c at the l u m i n a l membrane, 2) i n t r a c e l l u l a r i n t e r a c t i o n between the i n t r a c e l l u l a r z i n c pool and newly absorbed z i n c , 3) t r a n s f e r of z i n c at the b a s o l a t e r a l membrane to the p o r t a l c i r c u l a t i o n and 4) t r a n s f e r of z i n c i n a s e r o s a l to mucosal d i r e c t i o n ( 7 ) . The c o n t r i b u t i o n s of any number of these components and t h e i r i n t e r a c t i o n s a l s o i n f l u e n c e the extent of z i n c a b s o r p t i o n and thus account f o r the wide range of b i o a v a i l a b i l i t y of z i n c i n v a r i o u s foods ( 8 ) . While numerous s t u d i e s on the i n f l u e n c e of metabolic c o n d i t i o n s on z i n c a b s o r p t i o n have been reported, there i s at present no concensus on the s p e c i f i c s of t h i s process. E a r l i e r studies by Pearson et^ a l . ( 9 ) . Sahagian et a l . (10) and Oberleas et a l . (11) provided no consistent evidence f o r an energy requirement f o r the a b s o r p t i o n of z i n c . These s t u d i e s c o n f l i c t , however, w i t h more recent e f f o r t s which support the concept of a requirement f o r a c t i v e transport i n some phase of absorption. Kowarski e_t a l . found that 2,4 dinitrophenol, an i n h i b i t o r of ATP p r o d u c t i o n , caused a decrease i n z i n c transport by r a t j e j u n a l segments suggesting an energy r e q u i r i n g process was involved (12). S i m i l a r l y , Schwarz and Kirchgessner provided evidence t h a t , i n both zinc-depleted and p a i r - f e d r a t s , z i n c uptake i n i n t e s t i n a l segments was i n h i b i t e d by t h i s compound (13). Since an energy requirement has been defined i n l i v e r parenchymal c e l l s (14), i t seems p l a u s i b l e that an energy requirement may e x i s t f o r z i n c a b s o r p t i o n by i n t e s t i n a l c e l l s . However, due to the f u n c t i o n a l p o l a r i t y of the mucosal c e l l , the energy requirement may be at e i t h e r the brush border membrane or the b a s o l a t e r a l membrane. Definitive proof requires i s o l a t i o n of the i n d i v i d u a l membranes f o r separate study. Uptake of z i n c at the brush border membrane i s undoubtedly i n f l u e n c e d by a s s o c i a t i o n of the metal w i t h v a r i o u s compounds i n the i n t e s t i n a l lumen. A point of contention, however, has been the hypothesis that a unique z i n c - b i n d i n g l i g a n d i s a r e q u i s i t e f o r z i n c absorption. Attempts to i d e n t i f y such a compound and e s t a b l i s h a f u n c t i o n have yet to be c o n v i n c i n g l y adduced. Nevertheless, a v a r i e t y of compounds have been

Inglett; Nutritional Bioavailability of Zinc ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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suggested as having a unique r e l a t i o n s h i p to z i n c absorption. Included are N,N,N -trimethyl-l,2-ethanediamine (15), a peptide (16), p r o s t a g l a n d i n (17), c i t r i c a c i d (18), p i c o l i n i c a c i d (19), or amino a c i d residues ( 2 0 ) . This subject has been reviewed'recently by Cousins (21). Recent s t u d i e s by Cousins £t a l . (22) have f a i l e d to provide evidence f o r a s p e c i f i c moiety involved i n the absorption process. They have shown that the low molecular weight z i n c b i n d i n g l i g a n d s observed by v a r i o u s i n v e s t i g a t o r s can be explained as a r t i f a c t s of the experimental protocol used. Intestinal preparations appear to be susceptable t o p r o t e o l y t i c a c t i v i t y which can a l t e r z i n c b i n d i n g p r o f i l e s i n mucosal c y t o s o l . This r e s u l t s from atypical binding between intracellular components and degradation products. Similarly, Cherian noted rapid degradation i n post-mitochondrial supernatants of mucosal c y t o s o l which had been heated b r i e f l y (23). Further s t u d i e s by Cousins e_t a l . have shown that e l e v a t i o n of the i n t e s t i n a l z i n c content to very high l e v e l s i n v i t r o a l s o y i e l d s anomolous b i n d i n g c h a r a c t e r i s t i c s . Apparently, exogenous z i n c s a t u r a t e s higher molecular weight b i n d i n g s i t e s and a t y p i c a l b i n d i n g to lower molecular weight species r e s u l t s (22). Some of these f i n d i n g s have been confirmed by Lônnerdal et a l . ( 2 4 ) . The second step i n zinc absorption i n v o l v e s the i n t r a c e l l u l a r i n t e r a c t i o n of z i n c w i t h v a r i o u s compounds which may enhance or impede absorptive processes. I n 1969, Starcher noted that r a d i o a c t i v e copper, given o r a l l y , a s s o c i a t e d w i t h a low molecular weight p r o t e i n (25). Subsequently, t h i s mucosal p r o t e i n was i s o l a t e d and c h a r a c t e r i z e d by Richards and Cousins, who c l a s s i f i e d i t as a m e t a l l o t h i o n e i n (26), and who f u r t h e r showed that i t was induced i n response to z i n c a d m i n i s t r a t i o n (5). The appearance of t h i s m e t a l l o t h i o n e i n , w i t h p r o p e r t i e s s i m i l a r t o those described f o r both r a t (27) and human (28) l i v e r m e t a l l o t h i o n e i n , appears to be r e l a t e d to changes i n both d i e t a r y z i n c s t a t u s and plasma z i n c l e v e l s ( 5 ) . The s y n t h e s i s of mucosal m e t a l l o t h i o n e i n has been shown to be under t r a n s c r i p t i o n a l c o n t r o l (29,30). Menard e_t a l . reported that dietary zinc administration resulted i n enhancement of metallothionein mRNA t r a n s c r i p t i o n and i t s subsequent t r a n s l a t i o n , to y i e l d nascent m e t a l l o t h i o n e i n p o l y p e p t i d e s ( 3 1 ) . The i n t e s t i n a l m e t a l l o t h i o n e i n appearance was c o r r e l a t e d to both an increase i n mucosal z i n c content p r i m a r i l y a s s o c i a t e d w i t h the p r o t e i n and w i t h a decrease i n serum z i n c l e v e l s . I n a d d i t i o n , Smith e_t a l . , using the i s o l a t e d , v a s c u l a r l y perfused i n t e s t i n a l system, reported an inverse r e l a t i o n s h i p between the s y n t h e s i s of m e t a l l o t h i o n e i n and z i n c t r a n s f e r to the p o r t a l system, confirming e a r l i e r s t u d i e s (32). Factors i n v o l v e d i n z i n c t r a n s f e r across the b a s o l a t e r a l membrane to the c i r c u l a t i o n have not been w e l l c h a r a c t e r i z e d . However, Smith and Cousins have suggested that the t r a n s f e r of z i n c across the s e r o s a l membrane may be the r a t e l i m i t i n g step f


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i n z i n c a b s o r p t i o n and uptake to the c i r c u l a t i o n (33). A s i m i l a r suggestion was made by Kowarski et_ a l . (12). Zinc transport at the b a s o l a t e r a l membrane appears to be a l i n e a r f u n c t i o n of the lumen z i n c content and d i r e c t l y r e l a t e d to the mucosal z i n c lumen z i n c content and d i r e c t l y r e l a t e d to the mucosal z i n c concentration. Zinc t r a n s f e r to the c i r c u l a t i o n requires albumin as the c a r r i e r p r o t e i n (23). T r a n s f e r r i n does not appear to be s i g n i f i c a n t l y involved with p o r t a l t r a n s f e r of z i n c , although t h i s has been proposed p r e v i o u s l y by Evans and Winter (34). The nature and s i g n i f i c a n c e of endogenous z i n c s e c r e t i o n i n t o the i n t e s t i n e i s not w e l l understood. Secretion could occur as t r a n s c e l l u l a r f l u x i n the s e r o s a l to mucosal d i r e c t i o n (12,35) and/or as pancreatic and other glandular secretions (36). Evidence i n support of r o l e s of the pancreas and l i v e r i n c o n t r i b u t i n g to t h i s p o o l of z i n c i s c o n f l i c t i n g (37,38). In any event, endogenous s e c r e t i o n has been shown to be d i r e c t l y r e l a t e d to z i n c intake at or above the d i e t a r y requirement (39). However, the p h y s i o l o g i c a l s i g n i f i c a n c e of t h i s phenomena remains to be defined. In recent experiments, we have employed two approaches to examine s p e c i f i c aspects of z i n c absorption. The i s o l a t e d , v a s c u l a r l y perfused r a t i n t e s t i n e has been employed to examine a b s o r p t i o n at the organ l e v e l . I s o l a t e d brush border membranes from r a t i n t e s t i n e have been used to study the t r a n s f e r of z i n c across the i n t e s t i n a l surface. The i s o l a t e d , v a s c u l a r l y perfused r a t i n t e s t i n e system has been used to i n v e s t i g a t e the i n f l u e n c e of v a r i o u s z i n c - b i n d i n g ligands on z i n c absorption. With t h i s approach the f u n c t i o n a l i n t e g r i t y of the i n t e s t i n e w i t h respect to s e v e r a l m i n e r a l s , i n c l u d i n g calcium, i r o n and z i n c uptake, and subsequent t r a n s f e r of these minerals to t h e i r r e s p e c t i v e serum transport p r o t e i n s i s maintained (32,33). The i n t e s t i n a l p e r f u s i o n system allows the simultaneous measurement of both mucosal z i n c uptake (retention) and transfer to the portal circulation ( a b s o r p t i o n ) , and thus provides d e t a i l e d information on the nature of the mechanisms of both uptake from the lumen and t r a n s f e r to albumin i n the p o r t a l c i r c u l a t i o n . Male r a t s used f o r the perfusion experiments were maintained under e s t a b l i s h e d c o n d i t i o n s and were f a s t e d 16-24 hours p r i o r to surgery as p r e v i o u s l y described (35). In the f i r s t s e r i e s of s t u d i e s the luminal perfusate was at pH 4.2. This perfusate c o n s i s t e d of M199 t i s s u e c u l t u r e medium which contained a v a r i e t y of amino a c i d s , v i t a m i n s and minerals plus glucose. The p e r f u s a t e was supplemented (at 110 umolar) w i t h L-histidine HC1, L-cysteine, L-methionine, L-tryptophan, 2 - p i c o l i n i c a c i d , c i t r i c a c i d , or reduced g l u t a t h i o n e . The mixture was infused i n t o the lumen at 0.39 ml per min f o r 20 min and 0.10 ml per min f o r the f i n a l 40 min of the experiments. The small i n t e s t i n e was then removed and mucosal



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c e l l s harvested w i t h a g l a s s s l i d e . The mucosa was homogenized and centrifuged. Aliquots of the post-mitochondrial supernatant and the^çortal e f f l u e n t from the v a s c u l a r p e r f u s i o n were analyzed f o r Zn content by gamma ray spectrometry. A second s e r i e s of experiments was conducted i n the same manner as above, but the pH of the luminal perfusate was adjusted to pH 6.6. The i n f l u e n c e of 110 uM EDTA was a l s o i n v e s t i g a t e d at t h i s pH. In a t h i r d s e r i e s of experiments, the perfusate was supplemented w i t h h i s t i d i n e , tryptophan, 2 - p i c o l i n a t e , or c i t r a t e at 550 umolar. When the pH of the luminal perfusate was at 4.2, only methionine had a s i g n i f i c a n t (P < .05) e f f e c t on the amount of z i n c appearing i n the p o r t a l c i r c u l a t i o n . However, h i s t i d i n e and g l u t a t h i o n e both s i g n i f i c a n t l y (P < .05) enhanced the amount of z i n c sequestered i n the i n t e s t i n a l mucosal c e l l s . As shown i n Figure 1, when the luminal perfusate was adjusted to pH 6.6 to b e t t e r simulate the i n t e s t i n a l environment, each of the compounds added to the perfusate appeared to decrease the amount of z i n c t r a n s f e r r e d to the portal circulation. H i s t i d i n e , cysteine, tryptophan and glutathione decreased transfer significantly. In marked c o n t r a s t , however, EDTA was found to s i g n i f i c a n t l y enhance t r a n s f e r to the p o r t a l supply. The r e t e n t i o n of z i n c by mucosal cells was s i g n i f i c a n t l y decreased by adding h i s t i d i n e , tryptophan, 2 - p i c o l i n i c a c i d and g l u t a t h i o n e , as seen i n Figure 2. EDTA d i d not a l t e r z i n c r e t e n t i o n i n the mucosal c e l l s . Increasing the amount of h i s t i d i n e , 2 - p i c o l i n a t e and c i t r i c a c i d i n the lumen perfusate to a c o n c e n t r a t i o n of 550 uM had no e f f e c t on e i t h e r mucosal r e t e n t i o n or transmucosal f l u x of zinc. The same concentration of tryptophan, however, s i g n i f i c a n t l y decreased the amount of z i n c taken up and transferred. The r e s u l t s presented by these experiments f a i l to support the concept that a s i n g l e low-molecular weight, n a t u r a l l y o c c u r r i n g species (ligand) uniquely enhances z i n c absorption. The d i f f e r e n t compounds had v a r i o u s e f f e c t s on both mucosal uptake and r e t e n t i o n and p o r t a l t r a n s f e r of z i n c that were dependent upon both luminal pH and c o n c e n t r a t i o n of the l i g a n d . In recent s t u d i e s , Hurley e£ a l . reported no e f f e c t of v a r i o u s l i g a n d s on z i n c concentrations i n r a t dams and pups (40). Moreover, these p e r f u s i o n data r e a f f i r m the f i n d i n g that EDTA enhances z i n c absorption (41,42). The uptake of z i n c at the luminal surface and eventual t r a n s f e r to the c i r c u l a t i o n i s l i k e l y due to many f a c t o r s a c t i n g together to i n f l u e n c e z i n c absorption. The a f f i n i t y w i t h which z i n c i s bound by a p a r t i c u l a r d i e t a r y component may i n f l u e n c e i t s a v a i l a b i l i t y f o r uptake at the brush border membrane system. A l t e r a t i o n s i n z i n c uptake noted under c o n d i t i o n s of d i f f e r e n t lumenal pH may be due to the changing degree of i o n i z a t i o n of v a r i o u s l i g a n d s , rendering the z i n c more or l e s s a v a i l a b l e f o r transport across the membrane.


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Figure 2.

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Zn retention by mucosal cytosol following intestinal perfusion as Figure 1. *Significantly different from control at Ρ < 0.05.



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In a d d i t i o n to f a c t o r s i n f l u e n c i n g luminal uptake of z i n c , t r a n s f e r across the b a s o l a t e r a l membrane has been shown to be dependent on the concentration of albumin i n the p o r t a l c i r c u l a t i o n (33). These i n v e s t i g a t i o n s suggest that metabolic f a c t o r s which a f f e c t the albumin concentration i n the plasma may a l s o a f f e c t the r a t e of p o r t a l z i n c t r a n s f e r . I t should be noted that EDTA d i d not enhance z i n c accumulation w i t h i n the mucosal c e l l s yet i t increased t r a n s f e r to the vascular perfusate. These r e s u l t s suggest that b a s o l a t e r a l membrane transport of z i n c i s enhanced by EDTA. We have proposed (35), as has Davies (38), that b a s o l a t e r a l transport to the c i r c u l a t i o n i s the r a t e l i m i t i n g phase of z i n c absorption. Since EDTA and z i n c might be transported as a complex (42), the l a t t e r may transverse t h i s b a r r i e r more e a s i l y and thus increase z i n c absorption. In a d d i t i o n to the v a s c u l a r p e r f u s i o n system s t u d i e s , we have employed brush border membrane v e s i c l e s , i s o l a t e d from r a t mucosa, to determine more c l o s e l y the parameters of mucosal z i n c t r a n s p o r t (43). These v e s i c l e preparations represent the best means c u r r e n t l y a v a i l a b l e to d e l i n e a t e the c h a r a c t e r i s t i c s of z i n c t r a n s f e r i n t o mucosal c e l l s . The technique permits i s o l a t i o n of the m i c r o v i l l o u s membrane f r e e of other c e l l u l a r contaminants, as determined by e s t a b l i s h e d procedures (44). For these experiments, brush border v e s i c l e s were obtained by a m o d i f i c a t i o n of the p r e c i p i t a t i o n technique of K e s s l e r et a l . (45). B r i e f l y , the small i n t e s t i n e was perfused v i a the mesenteric a r t e r y w i t h i c e c o l d 0.9% s a l i n e , the lumen r i n s e d w i t h i c e c o l d s a l i n e , and the mucosa scraped o f f w i t h a glass s l i d e . The mucosal t i s s u e was suspended i n a 50mM D-mannitol/10 mM Hepes/Tris b u f f e r (pH 6.7), and homogenized with a blender f o r 5 min. Calcium c h l o r i d e was added to a f i n a l concentration of 10 mM, and the r e s u l t i n g mixture was c e n t r i f u g e d at 3000 xg f o r 15 min at 4°C The supernatant was r e c e n t r i f u g e d at 42,000 xg f o r 20 min, and the p e l l e t d i s s o l v e d i n the same b u f f e r c o n t a i n i n g 10 mM EGTA, adjusted to pH 6.7. This mixture was c e n t r i f u g e d again at 42,000xg f o r 20 min and the p e l l e t resuspended i n the o r i g i n a l b u f f e r and r e c e n t r i f u g e d to r i n s e the v e s i c l e s . The p e l l e t was then suspended i n the appropriate incubation buffer, homogenized with a Potter-Elvehjem homogenizer, and r e c e n t r i f u g e d . The final p e l l e t was then suspended w i t h a 25 gauge needle i n the appropriate buffer f o r the uptake s t u d i e s . The above p r e p a r a t i v e steps were c a r r i e d out at 4°C. Energy requirements f o r z i n c transport were i n v e s t i g a t e d by measuring z i n c uptake under the i n f l u e n c e of sodium and ATP e q u i l i b r a t i o n . None was found, suggesting that an energy requirement, i f one e x i s t s as has been proposed (12,13), must be at the b a s o l a t e r a l membrane. Once the requirements f o r optimal z i n c uptake by brush border v e s i c l e s were d e f i n e d , the parameters of z i n c uptake



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under optimal c o n d i t i o n s were determined f o r v e s i c l e s from both z i n c normal and 4 day d e f i c i e n t r a t s . Rats were maintained on c o n t r o l d i e t s w i t h f i n a l z i n c concentrations of e i t h e r 1 or 50 ppm f o r periods up to 4 days. Experiments were i n i t i a t e d by adding z i n c i n e x t r a v e s i c u l a r (luminal) concentrations v a r y i n g from 34 uM to 450 uM to approximately 100 ug of v e s i c u l a r p r o t e i n i n the appropriate b u f f e r . E x a c t l y one minute l a t e r , the r e a c t i o n was terminated and R e s i d e s c o l l e c t e d on a M i l l i p o r e f i l t e r f o r measurement of Zn t r a n s f e r . The e f f e c t of some postulated z i n c binding ligands on the uptake of z i n c i n v e s i c l e s was determined by adding e i t h e r glutathione, c i t r a t e or 2 - p i c o l i n a t e at an extravesicular c o n c e n t r a t i o n of 384 uM to the appropriate incubation b u f f e r c o n t a i n i n g 192 uM z i n c . Uptake was determined at v a r i o u s times up to 60 minutes. In order to determine whether the e f f e c t of the l i g a n d s on z i n c uptake was s p e c i f i c , or due to a general e f f e c t on the membrane, the i n f l u e n c e of the l i g a n d s on D-glucose uptake was analyzed. The l i g a n d s , i n the above concentrations, were added to a l i q u o t s of v e s i c l e s c o n t a i n i n g 200 uM D-glucose, and uptake at v a r i o u s times up to 60 minutes were determined, as described above. The r e s u l t s of these experiments a l s o f a i l to support a requirement f o r a c t i v e transport at the brush border membrane. Uptake at ImM and 192 uM z i n c concentrations showed no evidence of e i t h e r a Na or ATP requirement. Studies i n v o l v i n g the i n f l u e n c e of a 4 day z i n c d e p l e t i o n on the same parameters y i e l d e d s i m i l a r r e s u l t s . R e s u l t s from these s t u d i e s support the f i n d i n g s of Davies (38), who suggested two p o s s i b l e pathways f o r z i n c absorption at d i f f e r e n t l u m i n a l z i n c concentrations. Our r e s u l t s at 192 uM z i n c show a r a p i d uptake of z i n c to s a t u r a t i o n l e v e l s , w h i l e at higher (ImM) concentrations z i n c uptake continued to increase throughout the experiment. These r e s u l t s are i l l u s t r a t e d i n Figure 3. When 1 minute z i n c uptake was measured at e x t r a v e s i c u l a r concentrations of from 34 uM to 450 uM, uptake increased i n a c u r v i l i n e a r fashion towards a maximal rate of z i n c uptake. Uptake at concentrations from 1 mM to 3 mM increased l i n e a r l y with z i n c concentration. When the uptake k i n e t i c s were presented as an Eadie-Hofstee p l o t , e x t r a v e s i c u l a r z i n c concentrations of 450 uM and below y i e l d e d a s t r a i g h t l i n e c h a r a c t e r i s t i c of c a r r i e r mediated uptake, as seen i n Figure 4. C a l c u l a t i o n s show that w i t h v e s i c l e s from c o n t r o l r a t s , z i n c uptake occurs w i t h a Km of 0.38 mM and a Jmax of 5.4 nmoles per min. Values obtained w i t h v e s i c l e s from 4 day z i n c d e f i c i e n t r a t s showed no s i g n i f i c a n t v a r i a t i o n i n Km, (0.44 mM), but the Jmax increased s i g n i f i c a n t l y to 12.0 nmoles per min. S t a t i s t i c a l analyses of the k i n e t i c data were performed (46). These data suggest that w h i l e half-maximal uptake was not a l t e r e d i n d i e t a r y z i n c d e p l e t i o n , the uptake c a p a c i t y of the brush border membrane increased d r a m a t i c a l l y . F i n a l l y , to i n v e s t i g a t e the e f f e c t of z i n c d e p l e t i o n on



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Figure 3. Zinc uptake by brush border membrane vesicles as a function of tim under optimal (control) conditions at extravesicular zinc concentrations of 1 (Φ)αηά 0.192 mM (Ο).

Vo/S

Figure 4. Eadie-Hofstee plot of 1 min zinc uptake by brush border membrane vesicles from control (+Zn) and 4-day zinc deficient (—Zn) rats.



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the p r o t e i n composition of the brush border membrane, v e s i c l e s were obtained from c o n t r o l , 4 day z i n c d e f i c i e n t and 8 day z i n c deficient rats. Equivalent concentrations of v e s i c u l a r proteins were subject to SDS-polyacrylamide gel electrophoresis. As seen i n Figure 5, z i n c d e f i c i e n c y r e s u l t e d in d e f i n i t e changes i n brush border membrane protein composition. In z i n c d e p l e t i o n s t u d i e s , a general decrease i n protein synthesis i s believed to occur. However, when equivalent amounts of p r o t e i n were subject to e l e c t r o p h o r e s i s , there was a r e l a t i v e increase i n the synthesis of a t l e a s t two proteins. These had m i g r a t i o n s e q u i v a l e n t to p r o t e i n s of approximately 45,000 and 63,000 d a l t o n s . The r o l e of these p r o t e i n s i n z i n c uptake has not been e s t a b l i s h e d . However, since t h e i r appearance c o i n c i d e s w i t h a s i g n i f i c a n t increase i n Jmax, questions as to t h e i r f u n c t i o n i n the membrane can be raised. I t should be pointed out that the i n t e n s i t y of the 45,000 d a l t o n p r o t e i n band a l s o increased i n p a i r - f e d animals, although not to the extent observed i n the z i n c depleted r a t s . The need f o r f u r t h e r s t u d i e s on the r e l a t i o n of these p r o t e i n s to z i n c uptake i s apparent. Brush border membrane v e s i c l e s a l l o w the determination of z i n c uptake under p r e c i s e l y c o n t r o l l e d c o n d i t i o n s . When the e f f e c t of v a r i o u s z i n c b i n d i n g l i g a n d s on z i n c uptake was measured, v a r y i n g r e s u l t s were obtained. Although z i n c uptake w i t h p i c o l i n a t e and c i t r a t e a t 60 min of incubation was s i g n i f i c a n t l y lower than c o n t r o l v a l u e s , i n i t i a l r a t e s of z i n c uptake d i d not vary s i g n i f i c a n t l y . These r e s u l t s support previous s t u d i e s wherein these compounds d i d not a l t e r z i n c a b s o r p t i o n (47). Moreover, when the e f f e c t of these l i g a n d s on D-glucose uptake was t e s t e d , no d i f f e r e n c e was noted. Therefore, i t appears that the response noted above i s s p e c i f i c for z i n c uptake, p o s s i b l y r e l a t i n g to the a f f i n i t y of the ligands f o r zinc. The s t u d i e s reported here using the i s o l a t e d , v a s c u l a r l y perfused r a t i n t e s t i n e system and i s o l a t e d brush border membrane v e s i c l e s f a i l t o support a r o l e f o r a s p e c i f i c zinc-binding ligand involved i n z i n c uptake i n the r a t . Rather, the extent of z i n c uptake i n v o l v e s the i n t e r a c t i o n of several phenomena, including both extracellular and i n t r a c e l l u l a r reactions. I t appears that the major pathway of z i n c uptake under normal d i e t a r y c o n d i t i o n s i n v o l v e s the t r a n s f e r of z i n c from v a r i o u s d i e t a r y components to a c a r r i e r mediated transport system a t the brush border membrane. The net a b s o r p t i o n of z i n c from the lumen could involve a competition between v a r i o u s d i e t a r y components, z i n c binding l i g a n d s and the membrane c a r r i e r f o r z i n c . Thus, i n some cases, those compounds i n the lumen w i t h a higher a f f i n i t y f o r z i n c than the membrane component w i l l be l e s s l i k e l y to permit t r a n s f e r of z i n c t o the c a r r i e r , w h i l e compounds w i t h a lower a f f i n i t y f o r z i n c w i l l increase the amount of z i n c made



Figure 5. SDS-acrylamide gel electrophoresis of brush border membrane proteins from rats of adequate zinc status (0), following a 4-day zinc depletion (4), and following an 8-day zinc depletion (8). Proteins migrating with approximate molecular weights of 63,000 and 45,000 daltons are more apparent after 4 and 8 days of dietary zinc restriction.



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a v a i l a b l e f o r t r a n s p o r t . Some z i n c - c h e l a t e complexes could be absorbed i n t a c t although there i s l i t t l e data on t h i s aspect of z i n c t r a n s p o r t . D i g e s t i v e a c t i o n could a l t e r these i n t e r a c t i o n s and i n f l u e n c e the a v a i l a b i l i t y of z i n c f o r the membrane carrier. Evidence f o r a s i m i l a r membrane c a r r i e r f o r z i n c i n humans has been suggested from studies of z i n c uptake i n a c r o d e r m a t i t i s enteropathica. I n 1979, using mucosal samples obtained from a c r o d e r m a t i t i s p a t i e n t s , Atherton e t a l . reported t h a t , a t lower lumenal z i n c c o n c e n t r a t i o n s , brush border z i n c uptake was i n h i b i t e d i n these p a t i e n t s , an e f f e c t overcome a t higher z i n c concentrations (48). As a r e s u l t of these s t u d i e s , they suggested the p o s s i b i l i t y of a membrane a s s o c i a t e d c a r r i e r f o r z i n c i n the brush border membrane. I n the a c r o d e r m a t i t i s enteropathica p a t i e n t s , t h i s c a r r i e r could be a l t e r e d o r absent. Their hypothesis i s supported by f i n d i n g s i n r a t s by both Davies (38) and by t h i s l a b o r a t o r y (43). Based upon these and e a r l i e r s t u d i e s , z i n c absorption may be suggested t o occur i n the f o l l o w i n g steps. Under normal d i e t a r y c o n d i t i o n s where an adequate supply o f z i n c i s provided, i n i t i a l uptake o f z i n c t o the mucosal membrane appears t o depend on i t s net a v a i l a b i l i t y f o r uptake t o and t r a n s p o r t by a membrane a s s o c i a t e d z i n c c a r r i e r . This a v a i l a b i l i t y i s , i n t u r n , dependent on the z i n c b i n d i n g a f f i n i t y of b i n d i n g l i g a n d s d and the membrane c a r r i e r . Zinc i s transported i n t o the mucosal c e l l , where i t may e i t h e r enter into i n t r a c e l l u l a r reactions, including binding to proteins, newly synthesized t h i o n e i n polypeptides (31) and/or b i n d i n g t o i n t r a c e l l u l a r membrane components. The net f l u x of these r e a c t i o n s determines the a v a i l a b i l i t y o f z i n c f o r t r a n s f e r a t the b a s o l a t e r a l membrane t o albumin i n the p o r t a l c i r c u l a t i o n . The albumin content of the plasma may a l s o r e g u l a t e t h i s f i n a l phase o f z i n c absorption (33). Given the e x i s t e n c e of ATPase i n the b a s o l a t e r a l membrane, the t r a n s p o r t of z i n c across t h i s membrane may proceed by a c t i v e transport. Several i n v e s t i g a t o r s have suggested z i n c absorption i n v o l v e s a c t i v e t r a n s p o r t (12,13,39). However, f u r t h e r s t u d i e s are needed t o determine mechanisms of z i n c t r a n s p o r t across t h i s membrane system.

Acknowledgments The work from the author's l a b o r a t o r y (RJC) discussed i n t h i s review was supported by USPH, NIH Grant No. AM 18555 from the N a t i o n a l I n s t i t u t e of A r t h r i t i s , Diabetes, D i g e s t i v e and Kidney Diseases.


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Zinc Transport by Isolated, Vascularly Perfused Rat Intestine and

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