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Fluorescence study on the interaction of salicylate with rat small intestinal epithelial cells: Possible mechanism for the promoting effects of salicylate on drug absorption in vivo
Life Sciences, Vol. 37, pp. 523-530 Printed in the U.S.A.
Pergamon Press
FLUORESCENCE STUDY ON THE INTERACTION OF SALICYLATE WITH RAT SNALL INTESTINAL EPITHELIAL CELLS: POSSIBLE MECHANISM FOR THE PROMOTING EFFECTS OF SALICYLATE ON DRUG ABSORPTION IN VIVO Hiroshi Kajii, Toshiharu Horie, Masahiro Hayashi and Shoji Awazu* Department of Biopharmaceutics, Tokyo College of Pharmacy, 1432-I Horinouchi, Hachioji, Tokyo 192-03, Japan (Received in final form May 30, 1985) Summary The water-soluble drug, salicylate, was rapidly taken up by rat small intestinal epithelial cells. Salicylate, known to enhance the absorption of poorly absorbable drugs by rectum and small intestine, caused a significant decrease in the fluorescence polarization of 1,6-diphenyl-1 ,3,5-hexatriene (DPH) and a slight increase in the fluorescence polarization of 8-anilinol-naphthalene sulfonic acid (ANS) in the isolated rat small intestinal epithelial cell suspension. An increase in the membrane fluidity of epithelial cells may possibly contribute to the enhancement of drug absorption by salicylate. Salicylate in its ionized form at neutral pH is absorbed by the small intestine (I). Recently, non-surfactant adjuvants such as salicylate have been reported to accelerate rectal and small intestinal absorption of water-soluble compounds (2,3) and polypeptides (4). This enhancement of the permeability of various compounds by intestinal lumen by hydrophilic compounds is of considerable interest in view of the limited permeability of hydrophobic membranes toward hydrophilic compounds. To elucidate the absorption mechanism of salicylate and the accelerating mechanism by this and similar water-soluble compounds, an i__nnsit~ perfusion of gut lumen has been carried out (5,6). However an isolated epithelial cell system providing information directly on the interaction between hydrophobic membranes and hydrophilic compound has so far not been reported. We thus carried out a study on the uptake of ionized salicylate by isolated rat small intestinal epithelial cells and the perturbation of the cell membrane caused by salicylate, using fluorescence polarization spectroscopy. The results were found to provide some understanding of the enhanced permeability by salicylate observed in an in situ system. Materials and Methods Materials: 8-anilino-l-naphthalene sulfonic acid sodium salt (ANS), 1,6-diphenyl-1,3,5-hexatriene (DPH), fluorescein isothiocyanate dextran (FITC-dextran) (avg. mol. wt., 64,200), hyaluronidase (Type I-S) and bovine serum albumin (Fraction V) * To whom correspondence
should be addressed.
0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
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Membrane Fluidity on Intestinal Cells
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were p u r c h a s e d from Sigma Chemicals Co. (St. Louis, MO, U.S.A.). Sodium s a l i c y l a t e and t e t r a h y d r o f u r a n (THF) were obtained from Wako Pure Chemical I n d u s t r i e s Ltd. (Osaka, Japan). Silicon oil (KF961, 100) was obtained from S h i n - E t s u Chemical Co., Ltd. (Tokyo, Japan). All other c h e m i c a l s were of a n a l y t i c a l grade. P r e p a r a t i o n of rat small intestinal e p i t h e l i a l cells: Isolated rat small intestinal e p i t h e l i a l cells were prepared from male Wistar rats fasted o v e r n i g h t (190-230 g body weight) (Saitama Animals Lab., Saitama, Japan) a c c o r d i n g to a slightly modified method of Kimmich et al. (7). A 40 cm segment of a small intestine from the proximal end of the jejunum was used to isolate the e p i t h e l i a l cells. Cell v i a b i l i t y was estimated by trypan blue exclusion. All e x p e r i m e n t s were carried out on cell s u s p e n s i o n s of more than 80 % viability. The protein c o n c e n t r a t i o n in each cell suspension was d e t e r m i n e d by the method of Lowry et al. using bovine serum albumin as the standard (8). Uptake of s a l i c y l a t e by rat small intestinal e p i t h e l i a l cells: The c e n t r i f u g a l filtration method (9) was used to study the uptake of s a l i c y l a t e by isolated rat small intestinal e p i t h e l i a l cells from the rats. The cells were suspended in an incubation m e d i u m c o n t a i n i g 120 mM NaC1, 3 mM K2HPO~, I mM MgC1 6H~O, I mM C a C l ^ - 2 H p O and 10 mM Tris (hydroxymethyl) 2 a m ~ n o m e t h a n e , p~ 7.~. After p r e i n c u b a t i o n of 5 ml of the cell s u s p e n s i o n c o n t a i n i n g 3.1 mg p r o t e i n s / m l at 25°C for 5 min, the reaction was initiated by the addition of 5 ml of 2 mM salicylate solution p r e i n c u b a t e d at 25°C. One ml of the reaction mixture was placed on top of 0.5 ml silicone oil (density 1.02) in a polyethylene tube following a proper incubation time and then centrifuged for 15 sec in a Beckman m i c r o f u g e B (Beckman I n s t r u m e n t Inc., CA, U.S.A.). The reaction was terminated by separating the cells from the incubation medium. The cells were then suspended by the addition of I ml d i s t i l l e d water and 2 ml of ethanol were added to the cell suspension thus obtained. The cell s u s p e n s i o n was shaken v i g o r o u s l y for 30 min and c e n t r i f u g e d at 3,000 rpm for 10 min in a Kubota c e n t r i f u g e KN-70. One ml of d i s t i l l e d water, I ml of 6 N HC1 and 6 ml of ether were added to 0.5 ml of the supernatant. The mixture was shaken v i g o r o u s l y for 30 min and c e n t r i f u g e d at 3,000 rpm for 10 min. Four ml of 0.5 M borate buffer (pH 10) were then added to 5 ml of the s u p e r n a t a n t followed by vigorous shaking for 30 min and c e n t r i f u g a t i o n at 3,000 rpm for 10 min. F o l l o w i n g removal of the upper layer, the amount of s a l i c y l a t e in the lower layer was d e t e r m i n e d f l u o r o m e t r i c a l l y ( e x c i t a t i o n wavelength: 310 nm, emission wavelength: 400 nm). The volume of fluid adhering to the cells, d e t e r m i n e d using impermeable F I T C - d e x t r a n , was 5 . 7 9 ± 0 . 8 5 ~ I / m g protein (Mean±SE). All data for the uptake of salicylate were corrected on the basis of the amount of s a l i c y l a t e in this volume of fluid. F l u o r e s c e n c e m e a s u r e m e n t : F l u o r e s c e n c e m e a s u r e m e n t s were carried out using a Hitachi f l u o r e s c e n c e s p e c t r o p h o t o m e t e r 650-60. All f l u o r e s c e n c e m e a s u r e m e n t s were carried out at controlled t e m p e r a t u r e s , while stirring the epithelial cell suspension in a cuvett gently and c o n t i n u o u s l y by a magnetic stirrer equipped with a f l u o r e s c e n c e s p e c t r o p h o t o m e t e r to prevent s e d i m e n t a t i o n of the cells in the sample. E x c i t a t i o n and emission w a v e l e n g t h s for ANS f l u o r e s c e n c e m e a s u r e m e n t s were 380 nm and 480 rim, r e s p e c t i v e l y . The emission light was passed through a 430 nm cut-off fllter. A small volume of ANS solution was added to the cell suspension with a m i c r o s y r i n g e . F o l l o w i n g the complete mixing of the sample within a few seconds, the ANS f l u o r e s c e n c e was recorded and found to
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Membrane Fluidity on Intestinal Cells
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i n c r e a s e b i p h a s i c a l l y w i t h r e a c t i o n time ( F i g u r e I). A f l u o r e s c e n c e p o l a r i z a t i o n m e a s u r e m e n t of ANS in the cell s u s p e n s i o n was thus c a r r i e d out at 10 min f o l l o w i n g the a d d i t i o n of a small v o l u m e of ANS s o l u t i o n to the cell s u s p e n s i o n at 37°C, at w h i c h time the e q u i l i b r i u m s t a t e of ANS b e t w e e n the c e l l s and m e d i u m had been r e a c h e d .
100 -
-=
50
o/°~
/
0~0
---~0--0
Io--o-o--o
e,-.
z.__
,,-,z
0
u
0
i
l
I
I
5
i
,
i
v
Time, min
Fig.
I
10
'
i
,
i
I
15
I.
T i m e c o u r s e of f l u o r e s c e n c e i n c r e a s e and the e f f e c t of s a l i c y l a t e on ANS f l u o r e s c e n c e in i s o l a t e d rat small i n t e s t i n a l e p i t h e l i a l cells. Fluorescence measurements w e r e p e r f o r m e d at 2 5 ° C as d e s c r i b e d in m e t h o d s . A small v o l u m e of s a l i c y l a t e s o l u t i o n was a d d e d to a c u v e t t c o n t a i n i n g 20 ~ M ANS and 0 . 6 3 mg p r o t e i n s / m l at the time i n d i c a t e d by the arrow, after the f l u o r e s c e n c e i n t e n s i t y of ANS in the e p i t h e l i a l cell s u s p e n s i o n had r e a c h e d a p l a t e a u . The c o n c e n t r a t i o n of s a l i c y l a t e was 30 mM. More than 30 min w e r e r e q u i r e d to a t t a i n the e q u i l i b r i u m s t a t e of DPH l a b e l l i n g of the c e l l s at 37°C. The small i n t e s t i n a l e p i t h e l i a l cells, h o w e v e r , d e c r e a s e d in their v i a b i l i t y and a g g r e g a t e d w i t h time. To p r e v e n t this, the r e a c t i o n time for DPH l a b e l l i n g of the c e l l s was s h o r t e n e d . P r e p a r a t i o n of the e p i t h e l i a l c e l l s f r o m rat small i n t e s i n e was c a r r i e d out u s i n g a b u f f e r s o l u t i o n c o n t a i n i n g I ~ M DPH. The c e l l s thus p r e p a r e d w e r e r e a c t e d
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Membrane Fluidity on Intestinal Cells
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8
O
0
5
Time,min Fig.
10
15
2.
U p t a k e of s a l i c y l a t e by rat small i n t e s t i n a l e p i t h e l i a l cells. U p t a k e was s t u d i e d u n d e r s t a n d a r d c o n d i t i o n s d e s c r i b e d in m e t h o d s . The d a t a p o i n t s r e p r e s e n t mean v a l u e s o b t a i n e d from a s i n g l e e x p e r i m e n t ( t r i p l i c a t e m e a s u r e m e n t s at each s a m p l i n g time); bars r e p r e s e n t s t a n d a r d e r r o r . U p t a k e of s a l i c y l a t e was s t u d i e d in three rats and the r e s u l t s were r e p r o d u c i b l e . The c o n c e n t r a t i o n of s a l i c y l a t e was I mM. for a s e c o n d time w i t h 10 ~ M DPH on ice for 30 min. The DPH s o l u t i o n for l a b e l l i n g the c e l l s was p r e p a r e d by d i l u t i n g 2 mM DPH d i s s o l v e d in THF w i t h the b u f f e r and v i g o r o u s s t i r r i n g . All DPH l a b e l l i n g of the c e l l s was s h i e l d e d from light. The D P H - l a b e l e d c e l l s were e x p o s e d to e x c i t a t i o n l i g h t for less than 10 sec d u r i n g the f l u o r e s c e n c e p o l a r i z a t i o n m e a s u r e m e n t to a v o i d any r e v e r s i b l e p h o t o i s o m e r i z a t i o n of the DPH (10). E x c i t a t i o n and e m i s s i o n w a v e l e n g t h s in the f l u o r e s c e n c e p o l a r i z a t i o n m e a s u r e m e n t s were 380 nm and 480 nm for the A N S - l a b e l e d cells, and 380 nm and 455 nm for the D P H - l a b e l e d cells, r e s p e c t i v e l y , w i t h a 430 nm c u t - o f f filter for the e m i s s i o n side. S a l i c y l a t e in the m e d i u m had no i n f l u e n c e on the f l u o r e s c e n c e m e a s u r e m e n t of ANS and DPH at t h e s e e x c i t a t i o n and e m i s s i o n w a v e l e n g t h s .
Vol. 37, No. 6, 1985
The
Membrane Fluidity on Intestinal Cells
fluorescence
polarization, Ii,
-
P,
is defined
527
as
I~
P = I, + Ia where Ill and I~ are the fluorescence intensities parallel and p e r p e n d i c u l a r to the excitation polarization plane, r e s p e c t i v e l y (11). Results Ionized salicylate uptake by isolated rat small intestinal epithelial cells was studied at 25°C. This uptake was rapid, and e q u i l i b r i u m was complete within 3 min, as evident from Figure 2. The interaction between ANS and the epithelial cells showed the biphasic behavior, adsorption and uptake, depicted in Figure I. The following explanation seems applicable. Adsorption of ANS to the epithelial cells was quickly completed. The equilibrium state of ANS uptake by the cells was attained within 10 min. The addition of a small volume of salicylate solution to the cell suspension labeled with ANS then caused the ANS fluorescence to decrease instantly, indicating the rapid interaction between salicylmte and epithelial cell membranes. The uptake profile of salicylate by epithelial cells, considered to reflect the rapid phase appearing in less than 10 sec and the slow phase reaching e q u i l i b r i u m within 3 min, suggests adsorption to and uptake of salicylate into the cells, respectively. Such a profile has also been reported by Schwenk et al. (12) and Schwartz et al. (13) in the uptake of b r o m o s u l f o p h t h a l e i n and taurocholic acid by hepatocytes. To study the effects of salicylate on isolated rat small intestinal epithelial cells, the fluorescence polarization of two different types of fluorescent probes, ANS and DPH-labeled epithelial cells, was measured. ANS and DPH in the m e m b r a n e s are reported to be near the surface and in the interior of the membrane, r e s p e c t i v e l y (14). Thirty mM salicylate which caused e n h a n c e m e n t of w a t e r - s o l u b l e drug absorption in situ (2,3,4) increased the ANS fluorescence polarization slightly but significantly (Table I). The DPH fluorescence polarization decreased s i g n i f i c a n t l y at this c o n c e n t r a r t i o n (Table I). Salicylate thus causes membrane perturbation whose nature varies according to the domain of the epithelial cell membrane. This perturbation was not as extensive as the m e m b r a n e damage detectecd by trypan blue exclusion, since the change in the cell viability by the presence of 30 mM salicylate was less than 4 %. Discussion Two possible transport pathways, paracellular and transcellular routes, have been presented for inorganic and organic ions in the small intestine (15). However, the transport routes for ionized salicylate in vivo have not been revealed, though salicylate is absorbed from the small intestine (I). The data in Figure 2 show salicylate to be rapidly taken up by rat small intestinal epithelial cells in a manner comparable to that in human red blood cells (16). Thus, a t r a n s c e l l u l a r as well as p a r a c e l l u l a r route may both be importantly involved in in vivo salicylate absorption. The rapid interaction between salicylate and epithelial cells detected by the fluorescence of ANS-labeled cells in Figure I indicates the adsorption of salicylate to
528
Membrane Fluidity on Intestinal Cells
TABLE
Vol. 37, No. 6, 1985
I
Effects of S a l i c y l a t e on F l u o r e s c e n c e P o l a r i z a t i o n of ANS and D P H - l a b e l e d Rat Small I n t e s t i n a l E p i t h e l i a l Cells Adjuvant
Fluorescence Polarization of ANS
control
0.272 ± 0.001
30 mM SA
0.282 ± 0.001"*
(9) (10)
Fluorescence Polarization of DPH 0.281
± 0.001
0.259 ± 0.001"*
(15) (15)
F l u o r e s c e n c e p o l a r i z a t i o n values are given as mean values ± standard error. The number of d e t e r m i n a t i o n s appears in brackets. ** F l u o r e s c e n c e p o l a r i z a t i o n s of ANS and DPH differed significantly (P < 0.01) from those of the control (in the absence of SA). The e p i t h e l i a l cell v i a b i l i t y (%) tested by trypan blue exclusion was 84.3 ~ 4.2 in the absence of SA and 83.3 ± 3.1 in the presence of 30 mM SA for the ANS f l u o r e s c e n c e p o l a r i z a t i o n experiments, and 92.1 ± 1.8 in the absence of SA and 88.6 ± 3.5 in the presence of 30 mM SA for the DPH f l u o r e s c e n c e p o l a r i z a t i o n e x p e r i m e n t s (Means ± SE, n=4). Cell c o n c e n t r a t i o n s were 1.0 -1.4 mg proteins/ml. F l u o r e s c e n c e p o l a r i z a t i o n m e a s u r e m e n t s were carried out at 37°C.
e p i t h e l i a l cell membranes. This is also reflected in the biphasic behavior of salicylate uptake by e p i t h e l i a l cells in Figure 2. This direct and rapid i n t e r a c t i o n between cell m e m b r a n e s and s a l i c y l a t e is a key point in the e l u c i d a t i o n of the m e c h a n i s m by which drug a b s o r p t i o n is enhanced by salicylate. Two d i f f e r e n t f l u o r e s c e n t probes were used to study the effects of salicylate on e p i t h e l i a l cell membranes: ANS reported to monitor regions near the m e m b r a n e surface and DPH by which the features of a m e m b r a n e interior can be u n d e r s t o o d (13). A slight increase in ANS f l u o r e s c e n c e p o l a r i z a t i o n was observed in the presence of 30 mM salicylate (Figure I). This implies that the motion of m o l e c u l e s in the exterior of cell m e m b r a n e s is slightly restricted under such a condition which, incidentally, caused a decrease in DPH f l u o r e s c e n c e p o l a r i z a t i o n (Table I). Thus, the presence of s a l i c y l a t e causes the interior of a cell m e m b r a n e to become more fluid. Cell membrane p e r t u r b a t i o n s detected by ANS and DPH f l u o r e s c e n c e p o l a r i z a t i o n occurred i m m e d i a t e l y following the addition of s a l i c y l a t e were i n d e p e n d e n t of time (data not shown). N i s h i h a t a et al. reported that the effects of salicylate on the rectum in situ could be e l i m i n a t e d by washing the rectum with buffer but not those of sodium lauryl sulfate (2). The presence of 30 mM salicylate did not give rise to the s i g n i f i c a n t change in the v i a b i l i t y of rat small intestinal epithelial cells, as detected by trypan blue exclusion (Table I). However, the v i a b i l i t y of rat small intestinal epithelial cells decreased to 28 in the presence of 0 . 0 1 % sodium lauryl sulfate. This is c o n s i s t e n t with the results of N i s h i h a t a et al. (2), which indicated sodium lauryl sulfate to cause i r r e v e r s i b l e changes in the rectum. Also, this decrease suggests that the changes in ANS and DPH f l u o r e s c e n c e p o l a r i z a t i o n s in the cells following the addition of salicylate are caused by some m e c h a n i s m whose mode of action differs from that of a detergent. N i s h i h a t a et al. also observed the h e m o l y s i s induced by salicylate and the transport inhibition of salicylate by chemical m o d i f i c a t i o n of proteins in the human red blood cells. They suggest that the m e m b r a n e protein
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Membrane Fluidity on Intestinal Cells
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fraction is responsible for the enhancLng effects of saLicylate on the uptake of compounds (16). Salicylate also shows antihemolytic activity at a lower c o n c e n t r a t i o n of salicylate (17). Membrane proteins are important to the protection by certain drugs for osmotic hemolysis of red blood cells (18). The interaction between salicylate and membrane proteins of epithelial cells may thus contribute t) the slight increase in the fluorescence polarization of ANS in the cell membranes. Doubtless, the rapid adsorption of salicylate to the epithelia] cell membranes triggers such a ~nembrane perturbation, which would Ln turn induce perturbation between membrane lipids and proteins. Such a membrane perturbation o c c u r r i n g near the surface of membrane can be transferred to the interior of the me~nbrane causLng its interior to become more fluid, as was shown to be the case by DPH fluorescence polarization. This enhances the p e r m e a b i l i t y of the compound in epithelial cells. Consequently, the effect of salicylate in promoting the absorption of w a t e r - s o l u b l e and poorly absorbable drugs in vivo is likely enhanced as a result of the membrane perturbations. The present study has d e m o n s t r a t e d for the first time ~nembrane p e r t u r b a t i o n s arising directly from an adjuvant in isolated intestinal epithelial cells, as detected by fluorescence polarization. Acknowledgement The authors wish to thank Mr. for their technical assistances.
Toru
Kimura
and Miss
Kyoko
Mine
References I 2 3 4 5 6 7 8. 9. 10. 11. 12. 13. 14. 15.
H.Kunze, G . R e h b o c k and W.Vogt, N a u n y n - S c h n i e d e b e r g ' s Arch. Pharmacol. 273, 331-340 (1972) T.Nishihata, J . H . R y t t L n g and T.Higuchi, J. Pharm. Sci. 71, 865-868 (1982) T.Nishihata, H.Takahagi and T.Higuchi, J . Pharm. Pharmacol. 35, 124-125 (1983) T.Nishihata, J.H.Ryttlng, T.Higuchi and L.Caldwell, J. Pharm. Pharmacol. 33, 334-335 (1981) H . O c h s e n f a h r t and D.Winne, N a u n y n - S c h m t e d e b e r g ' s Arch. Pharmacol. 281, 197-217 (19"74) A.Karino, M.Hayashi, T.Horie, S.Awazu, H.Minami and M.Hanano, J. Pharm. Dyn. 5, 410-417 (1982) G.A.Kimmich, BiOchemistry, ~, 3659-3668 (1970) O.H.Lowry, N.J.Rosebrough, A.L.Farr and R.J.Randail, J. Biol. Chem. 193, 265-275 (1951) H.Baur, S . K a s p e r e k and E.Pfaff, H o p p e - S e y l e r ' s Z. Physiol. Chem. 356, 827-838 (1975) M . S h i n i t z k y a~id Y.Barenholz, J. Biol. Chem. 24__99, 2652-2657 (1974) G.Weber, Adv. Protein Chem. ~, 415-459 (1953) M.Schwenk, R.Burr, L . S c h w a r z and E.Pfaff, Eur. J. Biochem. 64, 189-197 (1976) L.S.Schwarz, R.Burr, M.Schwenk, E.Pfaff and H.Greim, Eur. J. Biochem. 55, 617-623 (1975) F.Podo and J.K.Blasie, Proc. Natl. Acad. Sci. 74, 1032-1036 (1977) P.G.Ruifrok and W.E.Mol, Biochem. Pharmacol. 32, 637-640 (1983)
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16. T.Nish~hats, T°Higuchi and AoKamada, Life Sci. 34, 427-436 (1984) 17. A.D.Inglot and E.Wolna, Biochem. Pharmacol. 17, 269-279 (1968) 18. T.Horie, Y.Sugiyama, S.Awazu and M.Hanano, J. Pharm. Dyn. ~, 116-122 (1981)
Pergamon Press
FLUORESCENCE STUDY ON THE INTERACTION OF SALICYLATE WITH RAT SNALL INTESTINAL EPITHELIAL CELLS: POSSIBLE MECHANISM FOR THE PROMOTING EFFECTS OF SALICYLATE ON DRUG ABSORPTION IN VIVO Hiroshi Kajii, Toshiharu Horie, Masahiro Hayashi and Shoji Awazu* Department of Biopharmaceutics, Tokyo College of Pharmacy, 1432-I Horinouchi, Hachioji, Tokyo 192-03, Japan (Received in final form May 30, 1985) Summary The water-soluble drug, salicylate, was rapidly taken up by rat small intestinal epithelial cells. Salicylate, known to enhance the absorption of poorly absorbable drugs by rectum and small intestine, caused a significant decrease in the fluorescence polarization of 1,6-diphenyl-1 ,3,5-hexatriene (DPH) and a slight increase in the fluorescence polarization of 8-anilinol-naphthalene sulfonic acid (ANS) in the isolated rat small intestinal epithelial cell suspension. An increase in the membrane fluidity of epithelial cells may possibly contribute to the enhancement of drug absorption by salicylate. Salicylate in its ionized form at neutral pH is absorbed by the small intestine (I). Recently, non-surfactant adjuvants such as salicylate have been reported to accelerate rectal and small intestinal absorption of water-soluble compounds (2,3) and polypeptides (4). This enhancement of the permeability of various compounds by intestinal lumen by hydrophilic compounds is of considerable interest in view of the limited permeability of hydrophobic membranes toward hydrophilic compounds. To elucidate the absorption mechanism of salicylate and the accelerating mechanism by this and similar water-soluble compounds, an i__nnsit~ perfusion of gut lumen has been carried out (5,6). However an isolated epithelial cell system providing information directly on the interaction between hydrophobic membranes and hydrophilic compound has so far not been reported. We thus carried out a study on the uptake of ionized salicylate by isolated rat small intestinal epithelial cells and the perturbation of the cell membrane caused by salicylate, using fluorescence polarization spectroscopy. The results were found to provide some understanding of the enhanced permeability by salicylate observed in an in situ system. Materials and Methods Materials: 8-anilino-l-naphthalene sulfonic acid sodium salt (ANS), 1,6-diphenyl-1,3,5-hexatriene (DPH), fluorescein isothiocyanate dextran (FITC-dextran) (avg. mol. wt., 64,200), hyaluronidase (Type I-S) and bovine serum albumin (Fraction V) * To whom correspondence
should be addressed.
0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
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Membrane Fluidity on Intestinal Cells
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were p u r c h a s e d from Sigma Chemicals Co. (St. Louis, MO, U.S.A.). Sodium s a l i c y l a t e and t e t r a h y d r o f u r a n (THF) were obtained from Wako Pure Chemical I n d u s t r i e s Ltd. (Osaka, Japan). Silicon oil (KF961, 100) was obtained from S h i n - E t s u Chemical Co., Ltd. (Tokyo, Japan). All other c h e m i c a l s were of a n a l y t i c a l grade. P r e p a r a t i o n of rat small intestinal e p i t h e l i a l cells: Isolated rat small intestinal e p i t h e l i a l cells were prepared from male Wistar rats fasted o v e r n i g h t (190-230 g body weight) (Saitama Animals Lab., Saitama, Japan) a c c o r d i n g to a slightly modified method of Kimmich et al. (7). A 40 cm segment of a small intestine from the proximal end of the jejunum was used to isolate the e p i t h e l i a l cells. Cell v i a b i l i t y was estimated by trypan blue exclusion. All e x p e r i m e n t s were carried out on cell s u s p e n s i o n s of more than 80 % viability. The protein c o n c e n t r a t i o n in each cell suspension was d e t e r m i n e d by the method of Lowry et al. using bovine serum albumin as the standard (8). Uptake of s a l i c y l a t e by rat small intestinal e p i t h e l i a l cells: The c e n t r i f u g a l filtration method (9) was used to study the uptake of s a l i c y l a t e by isolated rat small intestinal e p i t h e l i a l cells from the rats. The cells were suspended in an incubation m e d i u m c o n t a i n i g 120 mM NaC1, 3 mM K2HPO~, I mM MgC1 6H~O, I mM C a C l ^ - 2 H p O and 10 mM Tris (hydroxymethyl) 2 a m ~ n o m e t h a n e , p~ 7.~. After p r e i n c u b a t i o n of 5 ml of the cell s u s p e n s i o n c o n t a i n i n g 3.1 mg p r o t e i n s / m l at 25°C for 5 min, the reaction was initiated by the addition of 5 ml of 2 mM salicylate solution p r e i n c u b a t e d at 25°C. One ml of the reaction mixture was placed on top of 0.5 ml silicone oil (density 1.02) in a polyethylene tube following a proper incubation time and then centrifuged for 15 sec in a Beckman m i c r o f u g e B (Beckman I n s t r u m e n t Inc., CA, U.S.A.). The reaction was terminated by separating the cells from the incubation medium. The cells were then suspended by the addition of I ml d i s t i l l e d water and 2 ml of ethanol were added to the cell suspension thus obtained. The cell s u s p e n s i o n was shaken v i g o r o u s l y for 30 min and c e n t r i f u g e d at 3,000 rpm for 10 min in a Kubota c e n t r i f u g e KN-70. One ml of d i s t i l l e d water, I ml of 6 N HC1 and 6 ml of ether were added to 0.5 ml of the supernatant. The mixture was shaken v i g o r o u s l y for 30 min and c e n t r i f u g e d at 3,000 rpm for 10 min. Four ml of 0.5 M borate buffer (pH 10) were then added to 5 ml of the s u p e r n a t a n t followed by vigorous shaking for 30 min and c e n t r i f u g a t i o n at 3,000 rpm for 10 min. F o l l o w i n g removal of the upper layer, the amount of s a l i c y l a t e in the lower layer was d e t e r m i n e d f l u o r o m e t r i c a l l y ( e x c i t a t i o n wavelength: 310 nm, emission wavelength: 400 nm). The volume of fluid adhering to the cells, d e t e r m i n e d using impermeable F I T C - d e x t r a n , was 5 . 7 9 ± 0 . 8 5 ~ I / m g protein (Mean±SE). All data for the uptake of salicylate were corrected on the basis of the amount of s a l i c y l a t e in this volume of fluid. F l u o r e s c e n c e m e a s u r e m e n t : F l u o r e s c e n c e m e a s u r e m e n t s were carried out using a Hitachi f l u o r e s c e n c e s p e c t r o p h o t o m e t e r 650-60. All f l u o r e s c e n c e m e a s u r e m e n t s were carried out at controlled t e m p e r a t u r e s , while stirring the epithelial cell suspension in a cuvett gently and c o n t i n u o u s l y by a magnetic stirrer equipped with a f l u o r e s c e n c e s p e c t r o p h o t o m e t e r to prevent s e d i m e n t a t i o n of the cells in the sample. E x c i t a t i o n and emission w a v e l e n g t h s for ANS f l u o r e s c e n c e m e a s u r e m e n t s were 380 nm and 480 rim, r e s p e c t i v e l y . The emission light was passed through a 430 nm cut-off fllter. A small volume of ANS solution was added to the cell suspension with a m i c r o s y r i n g e . F o l l o w i n g the complete mixing of the sample within a few seconds, the ANS f l u o r e s c e n c e was recorded and found to
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Membrane Fluidity on Intestinal Cells
525
i n c r e a s e b i p h a s i c a l l y w i t h r e a c t i o n time ( F i g u r e I). A f l u o r e s c e n c e p o l a r i z a t i o n m e a s u r e m e n t of ANS in the cell s u s p e n s i o n was thus c a r r i e d out at 10 min f o l l o w i n g the a d d i t i o n of a small v o l u m e of ANS s o l u t i o n to the cell s u s p e n s i o n at 37°C, at w h i c h time the e q u i l i b r i u m s t a t e of ANS b e t w e e n the c e l l s and m e d i u m had been r e a c h e d .
100 -
-=
50
o/°~
/
0~0
---~0--0
Io--o-o--o
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z.__
,,-,z
0
u
0
i
l
I
I
5
i
,
i
v
Time, min
Fig.
I
10
'
i
,
i
I
15
I.
T i m e c o u r s e of f l u o r e s c e n c e i n c r e a s e and the e f f e c t of s a l i c y l a t e on ANS f l u o r e s c e n c e in i s o l a t e d rat small i n t e s t i n a l e p i t h e l i a l cells. Fluorescence measurements w e r e p e r f o r m e d at 2 5 ° C as d e s c r i b e d in m e t h o d s . A small v o l u m e of s a l i c y l a t e s o l u t i o n was a d d e d to a c u v e t t c o n t a i n i n g 20 ~ M ANS and 0 . 6 3 mg p r o t e i n s / m l at the time i n d i c a t e d by the arrow, after the f l u o r e s c e n c e i n t e n s i t y of ANS in the e p i t h e l i a l cell s u s p e n s i o n had r e a c h e d a p l a t e a u . The c o n c e n t r a t i o n of s a l i c y l a t e was 30 mM. More than 30 min w e r e r e q u i r e d to a t t a i n the e q u i l i b r i u m s t a t e of DPH l a b e l l i n g of the c e l l s at 37°C. The small i n t e s t i n a l e p i t h e l i a l cells, h o w e v e r , d e c r e a s e d in their v i a b i l i t y and a g g r e g a t e d w i t h time. To p r e v e n t this, the r e a c t i o n time for DPH l a b e l l i n g of the c e l l s was s h o r t e n e d . P r e p a r a t i o n of the e p i t h e l i a l c e l l s f r o m rat small i n t e s i n e was c a r r i e d out u s i n g a b u f f e r s o l u t i o n c o n t a i n i n g I ~ M DPH. The c e l l s thus p r e p a r e d w e r e r e a c t e d
526
Membrane Fluidity on Intestinal Cells
Vol. 37, No. 6, 1985
8
O
0
5
Time,min Fig.
10
15
2.
U p t a k e of s a l i c y l a t e by rat small i n t e s t i n a l e p i t h e l i a l cells. U p t a k e was s t u d i e d u n d e r s t a n d a r d c o n d i t i o n s d e s c r i b e d in m e t h o d s . The d a t a p o i n t s r e p r e s e n t mean v a l u e s o b t a i n e d from a s i n g l e e x p e r i m e n t ( t r i p l i c a t e m e a s u r e m e n t s at each s a m p l i n g time); bars r e p r e s e n t s t a n d a r d e r r o r . U p t a k e of s a l i c y l a t e was s t u d i e d in three rats and the r e s u l t s were r e p r o d u c i b l e . The c o n c e n t r a t i o n of s a l i c y l a t e was I mM. for a s e c o n d time w i t h 10 ~ M DPH on ice for 30 min. The DPH s o l u t i o n for l a b e l l i n g the c e l l s was p r e p a r e d by d i l u t i n g 2 mM DPH d i s s o l v e d in THF w i t h the b u f f e r and v i g o r o u s s t i r r i n g . All DPH l a b e l l i n g of the c e l l s was s h i e l d e d from light. The D P H - l a b e l e d c e l l s were e x p o s e d to e x c i t a t i o n l i g h t for less than 10 sec d u r i n g the f l u o r e s c e n c e p o l a r i z a t i o n m e a s u r e m e n t to a v o i d any r e v e r s i b l e p h o t o i s o m e r i z a t i o n of the DPH (10). E x c i t a t i o n and e m i s s i o n w a v e l e n g t h s in the f l u o r e s c e n c e p o l a r i z a t i o n m e a s u r e m e n t s were 380 nm and 480 nm for the A N S - l a b e l e d cells, and 380 nm and 455 nm for the D P H - l a b e l e d cells, r e s p e c t i v e l y , w i t h a 430 nm c u t - o f f filter for the e m i s s i o n side. S a l i c y l a t e in the m e d i u m had no i n f l u e n c e on the f l u o r e s c e n c e m e a s u r e m e n t of ANS and DPH at t h e s e e x c i t a t i o n and e m i s s i o n w a v e l e n g t h s .
Vol. 37, No. 6, 1985
The
Membrane Fluidity on Intestinal Cells
fluorescence
polarization, Ii,
-
P,
is defined
527
as
I~
P = I, + Ia where Ill and I~ are the fluorescence intensities parallel and p e r p e n d i c u l a r to the excitation polarization plane, r e s p e c t i v e l y (11). Results Ionized salicylate uptake by isolated rat small intestinal epithelial cells was studied at 25°C. This uptake was rapid, and e q u i l i b r i u m was complete within 3 min, as evident from Figure 2. The interaction between ANS and the epithelial cells showed the biphasic behavior, adsorption and uptake, depicted in Figure I. The following explanation seems applicable. Adsorption of ANS to the epithelial cells was quickly completed. The equilibrium state of ANS uptake by the cells was attained within 10 min. The addition of a small volume of salicylate solution to the cell suspension labeled with ANS then caused the ANS fluorescence to decrease instantly, indicating the rapid interaction between salicylmte and epithelial cell membranes. The uptake profile of salicylate by epithelial cells, considered to reflect the rapid phase appearing in less than 10 sec and the slow phase reaching e q u i l i b r i u m within 3 min, suggests adsorption to and uptake of salicylate into the cells, respectively. Such a profile has also been reported by Schwenk et al. (12) and Schwartz et al. (13) in the uptake of b r o m o s u l f o p h t h a l e i n and taurocholic acid by hepatocytes. To study the effects of salicylate on isolated rat small intestinal epithelial cells, the fluorescence polarization of two different types of fluorescent probes, ANS and DPH-labeled epithelial cells, was measured. ANS and DPH in the m e m b r a n e s are reported to be near the surface and in the interior of the membrane, r e s p e c t i v e l y (14). Thirty mM salicylate which caused e n h a n c e m e n t of w a t e r - s o l u b l e drug absorption in situ (2,3,4) increased the ANS fluorescence polarization slightly but significantly (Table I). The DPH fluorescence polarization decreased s i g n i f i c a n t l y at this c o n c e n t r a r t i o n (Table I). Salicylate thus causes membrane perturbation whose nature varies according to the domain of the epithelial cell membrane. This perturbation was not as extensive as the m e m b r a n e damage detectecd by trypan blue exclusion, since the change in the cell viability by the presence of 30 mM salicylate was less than 4 %. Discussion Two possible transport pathways, paracellular and transcellular routes, have been presented for inorganic and organic ions in the small intestine (15). However, the transport routes for ionized salicylate in vivo have not been revealed, though salicylate is absorbed from the small intestine (I). The data in Figure 2 show salicylate to be rapidly taken up by rat small intestinal epithelial cells in a manner comparable to that in human red blood cells (16). Thus, a t r a n s c e l l u l a r as well as p a r a c e l l u l a r route may both be importantly involved in in vivo salicylate absorption. The rapid interaction between salicylate and epithelial cells detected by the fluorescence of ANS-labeled cells in Figure I indicates the adsorption of salicylate to
528
Membrane Fluidity on Intestinal Cells
TABLE
Vol. 37, No. 6, 1985
I
Effects of S a l i c y l a t e on F l u o r e s c e n c e P o l a r i z a t i o n of ANS and D P H - l a b e l e d Rat Small I n t e s t i n a l E p i t h e l i a l Cells Adjuvant
Fluorescence Polarization of ANS
control
0.272 ± 0.001
30 mM SA
0.282 ± 0.001"*
(9) (10)
Fluorescence Polarization of DPH 0.281
± 0.001
0.259 ± 0.001"*
(15) (15)
F l u o r e s c e n c e p o l a r i z a t i o n values are given as mean values ± standard error. The number of d e t e r m i n a t i o n s appears in brackets. ** F l u o r e s c e n c e p o l a r i z a t i o n s of ANS and DPH differed significantly (P < 0.01) from those of the control (in the absence of SA). The e p i t h e l i a l cell v i a b i l i t y (%) tested by trypan blue exclusion was 84.3 ~ 4.2 in the absence of SA and 83.3 ± 3.1 in the presence of 30 mM SA for the ANS f l u o r e s c e n c e p o l a r i z a t i o n experiments, and 92.1 ± 1.8 in the absence of SA and 88.6 ± 3.5 in the presence of 30 mM SA for the DPH f l u o r e s c e n c e p o l a r i z a t i o n e x p e r i m e n t s (Means ± SE, n=4). Cell c o n c e n t r a t i o n s were 1.0 -1.4 mg proteins/ml. F l u o r e s c e n c e p o l a r i z a t i o n m e a s u r e m e n t s were carried out at 37°C.
e p i t h e l i a l cell membranes. This is also reflected in the biphasic behavior of salicylate uptake by e p i t h e l i a l cells in Figure 2. This direct and rapid i n t e r a c t i o n between cell m e m b r a n e s and s a l i c y l a t e is a key point in the e l u c i d a t i o n of the m e c h a n i s m by which drug a b s o r p t i o n is enhanced by salicylate. Two d i f f e r e n t f l u o r e s c e n t probes were used to study the effects of salicylate on e p i t h e l i a l cell membranes: ANS reported to monitor regions near the m e m b r a n e surface and DPH by which the features of a m e m b r a n e interior can be u n d e r s t o o d (13). A slight increase in ANS f l u o r e s c e n c e p o l a r i z a t i o n was observed in the presence of 30 mM salicylate (Figure I). This implies that the motion of m o l e c u l e s in the exterior of cell m e m b r a n e s is slightly restricted under such a condition which, incidentally, caused a decrease in DPH f l u o r e s c e n c e p o l a r i z a t i o n (Table I). Thus, the presence of s a l i c y l a t e causes the interior of a cell m e m b r a n e to become more fluid. Cell membrane p e r t u r b a t i o n s detected by ANS and DPH f l u o r e s c e n c e p o l a r i z a t i o n occurred i m m e d i a t e l y following the addition of s a l i c y l a t e were i n d e p e n d e n t of time (data not shown). N i s h i h a t a et al. reported that the effects of salicylate on the rectum in situ could be e l i m i n a t e d by washing the rectum with buffer but not those of sodium lauryl sulfate (2). The presence of 30 mM salicylate did not give rise to the s i g n i f i c a n t change in the v i a b i l i t y of rat small intestinal epithelial cells, as detected by trypan blue exclusion (Table I). However, the v i a b i l i t y of rat small intestinal epithelial cells decreased to 28 in the presence of 0 . 0 1 % sodium lauryl sulfate. This is c o n s i s t e n t with the results of N i s h i h a t a et al. (2), which indicated sodium lauryl sulfate to cause i r r e v e r s i b l e changes in the rectum. Also, this decrease suggests that the changes in ANS and DPH f l u o r e s c e n c e p o l a r i z a t i o n s in the cells following the addition of salicylate are caused by some m e c h a n i s m whose mode of action differs from that of a detergent. N i s h i h a t a et al. also observed the h e m o l y s i s induced by salicylate and the transport inhibition of salicylate by chemical m o d i f i c a t i o n of proteins in the human red blood cells. They suggest that the m e m b r a n e protein
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Membrane Fluidity on Intestinal Cells
529
fraction is responsible for the enhancLng effects of saLicylate on the uptake of compounds (16). Salicylate also shows antihemolytic activity at a lower c o n c e n t r a t i o n of salicylate (17). Membrane proteins are important to the protection by certain drugs for osmotic hemolysis of red blood cells (18). The interaction between salicylate and membrane proteins of epithelial cells may thus contribute t) the slight increase in the fluorescence polarization of ANS in the cell membranes. Doubtless, the rapid adsorption of salicylate to the epithelia] cell membranes triggers such a ~nembrane perturbation, which would Ln turn induce perturbation between membrane lipids and proteins. Such a membrane perturbation o c c u r r i n g near the surface of membrane can be transferred to the interior of the me~nbrane causLng its interior to become more fluid, as was shown to be the case by DPH fluorescence polarization. This enhances the p e r m e a b i l i t y of the compound in epithelial cells. Consequently, the effect of salicylate in promoting the absorption of w a t e r - s o l u b l e and poorly absorbable drugs in vivo is likely enhanced as a result of the membrane perturbations. The present study has d e m o n s t r a t e d for the first time ~nembrane p e r t u r b a t i o n s arising directly from an adjuvant in isolated intestinal epithelial cells, as detected by fluorescence polarization. Acknowledgement The authors wish to thank Mr. for their technical assistances.
Toru
Kimura
and Miss
Kyoko
Mine
References I 2 3 4 5 6 7 8. 9. 10. 11. 12. 13. 14. 15.
H.Kunze, G . R e h b o c k and W.Vogt, N a u n y n - S c h n i e d e b e r g ' s Arch. Pharmacol. 273, 331-340 (1972) T.Nishihata, J . H . R y t t L n g and T.Higuchi, J. Pharm. Sci. 71, 865-868 (1982) T.Nishihata, H.Takahagi and T.Higuchi, J . Pharm. Pharmacol. 35, 124-125 (1983) T.Nishihata, J.H.Ryttlng, T.Higuchi and L.Caldwell, J. Pharm. Pharmacol. 33, 334-335 (1981) H . O c h s e n f a h r t and D.Winne, N a u n y n - S c h m t e d e b e r g ' s Arch. Pharmacol. 281, 197-217 (19"74) A.Karino, M.Hayashi, T.Horie, S.Awazu, H.Minami and M.Hanano, J. Pharm. Dyn. 5, 410-417 (1982) G.A.Kimmich, BiOchemistry, ~, 3659-3668 (1970) O.H.Lowry, N.J.Rosebrough, A.L.Farr and R.J.Randail, J. Biol. Chem. 193, 265-275 (1951) H.Baur, S . K a s p e r e k and E.Pfaff, H o p p e - S e y l e r ' s Z. Physiol. Chem. 356, 827-838 (1975) M . S h i n i t z k y a~id Y.Barenholz, J. Biol. Chem. 24__99, 2652-2657 (1974) G.Weber, Adv. Protein Chem. ~, 415-459 (1953) M.Schwenk, R.Burr, L . S c h w a r z and E.Pfaff, Eur. J. Biochem. 64, 189-197 (1976) L.S.Schwarz, R.Burr, M.Schwenk, E.Pfaff and H.Greim, Eur. J. Biochem. 55, 617-623 (1975) F.Podo and J.K.Blasie, Proc. Natl. Acad. Sci. 74, 1032-1036 (1977) P.G.Ruifrok and W.E.Mol, Biochem. Pharmacol. 32, 637-640 (1983)
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16. T.Nish~hats, T°Higuchi and AoKamada, Life Sci. 34, 427-436 (1984) 17. A.D.Inglot and E.Wolna, Biochem. Pharmacol. 17, 269-279 (1968) 18. T.Horie, Y.Sugiyama, S.Awazu and M.Hanano, J. Pharm. Dyn. ~, 116-122 (1981)