Factors modifying cholesterol uptake by intestinal rings

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BBA 73 043

Factors modifying cholesterol uptake by intestinal rings The role of the intestinal plasma membrane in determining intestinal absorption of cholesterol was investigated in jejunal slices from hamsters. The linearitv of uptake with concentration of micellar cholesterol, and low Q10 suggested uptake by simple diffusion 1. Requirement of metabolic energy, membrane integrity and cholesterol exchange were then investigated for evidence of carrier-mediated processes. Sodium taurodeoxycholate (Maybridge Chemical Co., Tintagel, England), cholesterol (Nutritional Biochemicals Corp., Cleveland, Ohio), i-monoolein (The Hormel Institute, Austin, Minn.) and [4-14C]- and [3Hlcholesterol (New England Nuclear Corp., Boston, Mass.) were 98 % pure or better, verified by thin-layer chromatography. Enzymes and metabolic inhibitors were obtained commercially. Micellar solutions (5.4 mM monoolein, o.25 mM cholesterol, 6.o mM sodium taurodeoxyeholate, KrebsRinger phosphate buffer, p H 6.3) were prepared with a Branson Sonifier Model SI25, 7.5 A, 5 rain. Jejunal rings were mixed from hamster pairs of one sex fasted 24 h. Rings (lOO-2oo rag) were incubated in I ml micellar solution, with IO/,moles glucose in a Dubnoff metabolic shaker, at 37 °, for 3o rain, in lOO % O~. The fluid was decanted at termination. The rings were rinsed with chilled bile salt buffer. Experimental modifications were: omission of glucose; comparison of N 2 for O 2 gas phases using a double hood; substitution of buffer ions; addition of test substances, 2oo and 5oo ~g. Rings in modified media were incubated simultaneously with rings under control conditions. Results of experiments performed in duplicate 3 times yielded means and standard errors. Lipids were extracted from homogenized rings and fluids before and after incubation. Lipid classes were separated by thin-layer silicic acid chromatography. The radioactivity of cholesterol was assayed by liquid scintillation counting z. Mean recovery of radioactivity from tissues and media after incubation was 94% of the activity recovered from initial incubation solutions, entirely as free sterol. Uptake was calculated as m~moles cholesterol taken up per IOO mg intestinal rings. The mean uptake -~ S.E. for ~2 pairs of incubations of intestine with o.25 mM cholesterol (25o m/~moles) was 41.6 ~ 1.1o6 m/~moles (range 35.2-49.2)/lOO nag per 3o rain, averaging 16.6 % of the sterol incubated (Table I). Metabolic activi@. When glucose was omitted, cholesterol uptake diminished by~ 19 %. In an atmosphere of N2, intestinal slices took up o~oO/~oless sterol than in O,,. When sodium azide was added, uptake of cholesterol decreased by 38 %. Iodoacetamide increased cholesterol uptake by 57 °.0Membra~w effects. Phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4) addition to the incubation medium decreased sterol uptake 5o %. With 2 mM lysolecithin added to i mM cholesterol micellar solutions, sterol uptake after 8 rain was IOI mtmmles/Ioo mg (control, 2o2 m~moles) and at 3o min it was 136 m~moles (control, 224 m~moles). Inclusion of amphotericin B in incubations doubled cholesterol uptake. Hypocholesteremic drugs. With nicotinic acid, sterol uptake increased by 5o %. Modificatiom where sterol ,uptake approximated simultaneous control values (range 30.6-49.9 m~moles). Replacing buffer Na + by K+; addition of 2. 7 mM Ca 2+ and 1. 3 mM Mg2+; NaF, KCN, N-ethyhnaleimide; dimethylsulfoxide I.O ,o or 2. 5 ,'o O'

O/

•

Biochim. Biophys. Acta, i5o (I968) 727-7-'9

728

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TABIA c 1 CHOLESTFROL

l." P T A I'~ F,

Cholesterol uptake is expressed in mpmoles • S.l 4. per Ioo mg {incubation, 3° rain with 2,50 m/mloles cholesterol). Values m parentheses are the number of paired incubations. I ~ o.oot for all experimental values compared to control using Student'sltest. Control value: 41.6 : ~.ol (~-').

Modi/icatio~ of incubatiou ~,ixl,rc

Cholesterol uptake (m/t**whs A S.I']. /?g1" lOO

No glucose ioo% N e Sodium azide lodoacetamide Ph(}spholipaseA Amphotericin B Amphotericin B Nicotinic acid

33.3 :: I.OI -'9.1 1.2o 25.8 3.5 ° (}5.3 : .t.2T 21.8 • L7[ 8{~.o ; 5.oo 73 .2 4 .6o (}-'.4 {}.S8

{2oo/,g) (2oot~g) (2oo!~g) (2oo Pg) (5oo/~g) (2ooyg)

1fig)

(3} (3) (3) (3) (3) {3) (3) (3)

v i t a m i n A" n e o m y c i n , c e t r i m i d e , e t h a n o l ; t)hospholipase C ( p h o s p h a t i d y l c h o l i n e c h o l i n e p h o s p h o h y d r o l a s e , EC 3.I.4.3), t)hospholipase D ( p h o s p h a t i d y l c h o l i n e phosp h a t i d o h y d r o l a s e , E C 3.1.4.4). Exchange between cholesterol previously taken up aud sterol in the medium. Int e s t i n a l rings w e r e i n c u b a t e d 8 rain in 4 b e a k e r s w i t h • ml o.25 m M lnicellar chole s t e r o l c o n t a i n i n g z24, 850 c o u n t s / r a i n aH c h o l e s t e r o l a n d rinsed. Tissues f r o m 2 b e a k e r s w e r e t r a n s f e r r e d i m m e d i a t e l y to b e a k e r s c o n t a i n i n g i 7 5 o o c o u n t s / n f i n [4-14C]cholesterol and i n c u b a t e d an a d d i t i o n a l 8 rain. R a d i o a c t i v i t y was l n e a s u r e d in tissues i n c u b a t e d in Jail)cholesterol w i t h o u t a n d w i t h s u b s e q u e n t t r a n s f e r to ~HC c h o l e s t e r o l s o l u t i o n s (Table II). R i n g s t o o k u p labeled cholesterol f r o m b o t h m e d i a lint r e l e a s e d a b o u t half t h e [aHicholester()l t a k e n up in t h e first 8 rain i n t o the second i n c u b a t i o n m e d i u m in t h e s u c c e e d i n g period, d e m o n s t r a t i n g e x c h a n g e . TABLI'2 11 ]:;XCHAN(;G

OF

C I t O L l z 2 S T F . R O L B]£,T\V]*;IBN T I S S U ] : ;

l ncubalio,

First with 7al I]cholesterol Second with 14C :cholesterol

AND

I',IEI)IUM

Up/abe W label Co,,ts~'miJz per ~oo mZ

%

n C

:%r

1~C

aH

o 1(}t2

,'¢ r 3 1 3£S I

o IO.5

{;..5 3. r

I n h i b i t i o n of e n e r g y - y i e l d i n g r e a c t i o n s h a d s m a l l influence on cholesterol u p t a k e i n t e s t i n a l slices. K i n e t i c d a t a did n o t s u p p o r t cholesterol a c c u m u l a t i o n " u p h i l l " a g a i n s t a g r a d i e n t , or a c t i v e t r a n s p o r t 1. S e g m e n t s p e r f u s e d in vitro t o o k u p c h o l e s t e r o l e q u a l l y f r o m m u c o s a or serosa, w i t h no a s y m m e t r i c t r a n s p o r t 6. T h e s e d a t a i n d i c a t e t h a t m i c e l l a r c h o l e s t e r o l is t a k e n up b y t h e i n t e s t i n e b y diffusion. S i m i l a r conclusions w e r e r e a c h e d for f a t t y a c i d u p t a k e a , 4. i n c o n t r a s t , u n d e r a n a e r o b i c conditions, a n d u s i n g m e t a b o l i c i n h i b i t o r s i n t e s t i n a l a b s o r p t i o n of m o s t sugars a n d anfino acids was d e p r e s s e d p r o f o u n d l y i m p l y i n g " a c t i v e " t r a n s p o r t a. t) 3,

l~iochim, l~wHm,s. Acta, i5 o (1oo8) 7-'7--7z9

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729

Phospholipase A releases hydrophobic segments of the liquid crystalline bimolecular lipid leaflet of the cell membrane, and decreases ceil permeability. Lysolecithin, the product of phospholipase A action on lecithin, decreased sterol absorption. Neither phospholipases C nor D, affecting hydrophilic groups of phospholipids influenced sterol uptake. Lysolecithin may affect the membrane, the micellar dispersion of cholesterol, or its adsorptionL Where intestine was incubated with lysolecithin in similar concentration, it was taken up and acylated indicating viability of the preparation s. Amphotericin B disrupts lipid structures containing sterols with a free hydroxyl group 9 when in contact with the sterol-containing surface. Interaction with membrane sterols affecting sterol orientation increases cell permeability ~°. Conformational changes induced may secondarily affect binding affinity and transport velocity of sterols. The cholesterol in the intestinal plasma membrane is large relative to phospholipid, with a ratio of I. I I (ref. ~I) resembling that for the erythrocyte. Exchange of cholesterol between intestinal membrane and medium in the present study,, and between erythrocyte membranes and serum lipoproteins 1~, indicates processes differing from those in culture lymphocytes which showed no exchange of cholesterol between cells and medium la. In that system, cholesterol was taken up by adsorption. Cholesterol is absorbed from a micellar phase of intestinal content containing conjugated bile salts, fatty acids, monoglycerides and lecithin (or lysolecithin). F a t t y acids, monoglycerides and cholesterol m a y be absorbed by diffusion fronl a molecular phase, rather than a micellar phase ~4. The mechanism of entry of individual micellar components such as cholesterol into intestinal epithelial cells is as yet unknown and warrants further investigation at the cell, organ and whole animal level. This work was supported in part by U.S. Public Health Service Grants H E 10742 and T I AM 5347. Miss LORETTA BOGAN provided excellent teclmical assistance.

Department of Medicine, State Universit3, of New York Downstate Medical Center, 13rook@n, N . Y .

E L A I N E B. lgELDMAN

;12o 3 (U.S.A.)

E. ]7}. FELDMAN,P'ederatioJt Pro&, 26 (1967) 47 I. E. B. FELDMaN AND B. BOR6STROM, Biochim. Biophys. Acta, I25 (I966) 148. J, JOHNSTON AND 13. BORGSTRO~a, Biochim. Biophys. Acta, 84 (1964) 412. D. PORTE, JR. AND C. ENTENMAN, Am. J. Physiol., 2o8 (*965) 6o 7. T. H. \VILSON, IntestiJ,al Absorption, \V. B. Saunders, Philadelphia, 1962. g. B. FELDMAN, C/in. Res., 15 (I967) 459G. H. ROTHBLAT AND D. I{RITCHE.VSKY,Circulation, 36 (1967) 35. A. NILSSON AND B. BORGSTROM, Biochim. Biophys. Acta, 137 (1967) 240. S. C. NIysKv, Arch. Biochem. Biophys., lO2 (1963) i8o. N. S. LICHTF,NST~IN AND A. LEAF, J. Clin. lnvesl., 44 0965) 1328. J. B. I;INFaN, R. COL~YMANAND \V. A, (~REEN, A'1I*L N . Y . Acad. Sci., 137 (1960) 414. J. S. HAGERM:\N .XND R. G. GOULD, Proc. Soc. Exptl. Biol. Med., 78 (1951) 329. (;. H. ROTHBLAT, [{. H. HARTZI~;LL, JR., H. MIALHI~ AND D. I"~RITCHEVSKY,Biochim. Biophys. Acta, 116 {1960) 13314 \V. J. SIM>IONDS, A. I:. HOFM.~.NN AIVD E. THEODOR, J. Cli~z. 1Jzvest., 46 (1967) 874.

I 2 3 4 5 6 7 8 9 io ii 12 13

Received February I2th, 1968 Biochim. Biophys. Acta, 15o (1968) 727 729