385
B~ochlmlca et B~ophystca Acta, 419 (1976) 3 8 5 - - 3 9 0 © Elsevier S c m n t f f m P u b h ~ h m g C o m p a n y , A m s t e r d a m - - P r i n t e d m T h e N e t h e r l a n d s
BBA Report B B A 71 238
THE E F F E C T S OF REMOVAL OF SODIUM IONS FROM THE MUCOSAL SOLUTION ON SUGAR ABSORPTION BY RABBIT ILEUM
R J NAFTALIN and G D HOLMAN
Department of Physiology, Unwers~ty of Leicester, LE1 7RH (U K )* ( R e c e i v e d N o v e m b e r 3rd, 1 9 7 5 )
S u mmar y Net absorpt:on and accumulation of D-galactose, H-methyl D-glucose and low co n cen t r a t m ns of 3-O-methyl-D-glucose by sheets of rabbit ileum are observed even when Na ÷ m the mucosal solut:on :s replaced by chohne This mdmates that active sugar transport can occur m the dlrectmn opposite to the brush-border Na ÷ gradmnt
There is cons:derable disagreement concerning the effects of Na ÷ replacement m the solution bathing the mucosal surface of the small m t est m e on subsequent sugar absorption Perfuslon studms m VlVO [1 ] suggest that replacement o f NaC1 m the mucosal solutmn by e~ther lsotomc KCI or m an mto l has no effect on glucose absorptmn by human, dog or rat ileum With suspensions of chink mtestmal eplthehal cells, removal of Na ÷ from the external solut:on does n o t prevent active accumulatmn of D-galactose by cells whmh have been pr em c uba t e d with Na ÷ [2] On the ot her hand, several stud:es do suggest t hat the presence of a Na ÷ gradmnt across the brush-border f r o m mucosa-cell :s essential for act:ve sugar accumulatmn and absorption [3,
41 Rmaldo et al [5] who re-examined th~s question recently, measured transmural flux from mucosal-serosal solution and from serosal-mucosal solutmn of 20 mM 3-O-methyl D-glucose across an m v:tro preparat m n of rabbit ileum and concluded t hat when Na ÷ is umlaterally replaced by chohne m the mucosal solut:on, no slgmfmant act:ve sugar absorption occurs Since :t has been shown t ha t the a s y m m e t r y of brush-border sugar transport :s decreased at higher sugar concentrations [6] and t hat a s y m m e t r y of the brush-border sugar transport system is a f u n c t m n of sugar affinity for the brush-border, (Holman, G D and Naftahn, R J , m preparat:on) :t was *Present address of both authors Department of Physzology, Kings Collei~e, London, U K
Serosal solution Na + mequtx
140
140
0
140
140
0
140 140 0
140
140
0
Mucosai solution Na + meqm~
140
0
0
140
0
0
140 0 0
140
0
0
1 mM ~-methvl glucose 1 mM ~-methvl glucose 1 m M 1 3 -m e t h yl glucos(
2 mM galactose 2 mM galactose 2 mM galaetose
20 mM 3-0 methvl glucose 20 mM 3-0 meth:yl glucose 20 mM 3-0 meth,,l g~ucose 2 mM 3-0 methvl glucose 2 mM 3-O methyl glucose 2 mM 3-O methyl glucose
Sugar
7
5
7
5 5 5
6
6
5
4
5
5
n
~ 0 19
++
**~ ÷+
~
***
~ 0 0156"
0011+00016
0 072
0 347 + 0 026
0 553 * 0038 0101+0021 0 029 + 0005
0023+0003
0067+0019
0 315-+ 0 05
0334+0031
0 335 ± 0 38*
0 87
Jms (#tmol cm -2 h-I )-+SEM
+- 0 0 0 0 4
+ 0 0008 +0002 ~ 00054
±0004
±0002*
-+ 0 0 0 1 2
~ 0033
-+ 0 0 3 1
~ 0 035
00098-+00012
0 0048 ± 0 0011
0 0027
00068 001 0024
0021
0017
0009
027
0 17
0 206
Jsm (tzmol cm -2 h -~)±SEM
*
+ 0 67
I 05
4 89
12 43
3 85 155 0 48
032
088
2 73
030
~÷
+0213
0 613
= 0085
~*
± 0 50 + 0 2 0 s~ * 0 085'
~0057
+-008**
+ 0 56
+0033
0 507 + 0 053
1 63
R ±SEM
~ I 73 ' 0 264***
627*0626
13 1 1 ~ 1 3
11 7 8 + 0 69 551±098 **~ 2 92 ± 0 32 ~
235+014
392±027*** **~
z 13 ~*
: 0965"
+ 1 7
8 08 + 0 63
241
33 2
38 4
Ace (mM) 4SEM
0 0018
0067
0 54
0 55 009 0 005
00016
005
0 305
007
0 164
0 66
~ 0 00to
-0015
",)026
- 0 038 002*' + 0 001'
0002~'
z 002 ~
-+ 0 0 5
* 0015
+ 0 025'+
' 0 17
Jnet (pmol cm -2 h -1)
~'
F l u x e s o f s u g a r s ~ e r e m e a s u r e d a t 3 0 , 6 0 a n d 9 0 m m a f t e r a d d i t m n o f t h e r a d m i s o t o p e as p r e ~ m u s l v d e s c r i b e d [ 6 9 ] T h e R i n g e r s o l u t m n s ~ e r e g a s s e d c o n t i n u o u s l ~ ~ l t h 9 5 % O ~ , 5% C O . a n d ~ e r e m a i n t a i n e d a t 3 7 ~ C M - S f l u x e s ~ e r e m e a s u r e d ~ l t h ~ H - l a b e l l e d s u g a r , s - m f l u x e s w i t h ~ 4 C - l a b e l l e d s u g a r s l d e n t w a l s u g a ~ c o n c e n t r a t l o n s ~ e r e p l a c e d in m u e o s a l a n d serosal c h a m b e r s Following incubation, the tissues ~ere washed m ice-cold chohne chloride Ringer and then extracted for 4 h in 0 1 M HNO~ The ~atm R was obtained from the ratm of dpm, ~H/dpm ~4C-labelled sugar extracted from the tissues Tissue sugar concentratmn zs c a l c u l a t e d o n t h e b a s i s t h a t ~t ~s p r e s e n t e n t * r e l v ~ l t h i n t h e c e l l s p a c e , c e l l w a t e r ~s o b t a i n e d f r o m t h e w e t - d r y w e i g h t o f t*ssue l e s s ] 0 % fo* t h e e x t r a c e l l u l a r t l m d [ 9 , 1 4 ] Ringer solutmncontams 1 4 0 m M NaC1 1 0 m M K H C O ~ 0 4 m M K H 2 P O , , 2 4 m M K 2 H P O ~ , 1 2 C a C L , 1 2 m M M g C 1 , m c h o h n e / R m g e r , chohne chloude issubstltuted f o r NaC1 3 - 0 M e t h y l g l u c o s e a n d f l - m e t h v l g l u c o s e ~ e r e o b t a i n e d f r o m K o c h L i g h t L t d ~H, ~ C ' - l a b e l l e d D - g a l a c t o s e ~ C - l a b e l l e d 3 - O m e t h y l g l u c o s e a n d 3 H - l a b e l l e d g l u c o s e ( u s e d as s t a r t i n g m a t e r i a l f o r s v n t h e m s o f l a b e l l e d ~ - m e t h v l g l u c o s e ) w e r e all o b t a i n e d f r o m t h e R a d m - C h e m i c a l C e n t r e A m e r s h a m ~H-labelled 3-0 methv! glucose and ~aC-labelled S-methyl glucose were obtained from Ne~ England Nuclear Ltd all the othe~ ohemieals ~ere Analar grade The rabbits used ~ere 2--3 kg Ne~ Zealand white rabbits ,~hleh were killed by intravenous inlect*on of Nembutal T h e t e r m i n a l i l e u m wa~ d ~ s s e e t e d I m m e d m t e b a n d s t u p p e d o f ~ts s e r o s a a n d o u t e r m u s c l e l a y e r s b e f o r e m o u n t i n g as a f l a t s h e e t m t h e f l u x c h a m b e r s S t a t i s t i c s t h e s l g m f i ( a n c e lex e ls ~ e l e c a l c u l a t e d u s i n g S t u d e n t s t t e s t ( u n p n r e d m e a n s s o l u t i o n ) * = P ~ 0 0 5 ** = P % 0 0 2 ' *' =P < 0 0011ndxeate the significance of the differencebet~eenthe figure and that m the row above
TABLE I
387
TABLE
Ib
T h e N a + c o n c e n t r a t i o n xn t h e m u c o s a l i n c u b a t i o n b a t h w a s m e a s u r e d f o n o w m g 9 0 m m i n c u b a t i o n [ N a +] w a s e s t i m a t e d b y f l a m e p h o t o m e t r y T i s s u e [ N a +] w a s c a l c u l a t e d f r o m t h e H N O a e x t r a c t s F r o m t h e m u e o s a l [ N a +] , n e t s - m N a + f l u x w a s c a l c u l a t e d a n d f o u n d t o b e c o n s t a n t f o r all s u g a r s t e s t e d a n d f o r all [ s u g a r ] t e s t e d Initial concentration Mucosal solution 0 0
of Na+mequlv
Concentration of Na + meqmv m mucosal solutmn following 90 mln mcubatmn
Serosal solutmn
n
0 140
16 25
095-+ 0073 3 4 4 -+ 0 1 7 T t s s u e [ N a +] m e q u l v / l l t r e cell w a t e r after 90 mln incubation
0 0 140
0 140 140
Calculated net S-M Na + flux
16 26 13
455-+04 2 2 3 7 -+ 0 9 2 4 4 61 + 2 4 7
5 81 # t m o l . c m - 2 - h - 1
considered worthwhile e xa m m m g the effects of mucosal deprivation of Na* on the absorption o f 3-O-methyl glucose at b o t h high and low concentrations and on D--galactose and H-methyl-D-glucose (methyl/3-D-glucopyranoslde) transport (both these last sugars have higher afhmtms for the brush-border than 3-O-methyl glucose [7]) Table I shows t hat removal of Na + f r om the mucosal solutmn alone causes a slgmhcant reduction m m-s flux of 3-O-methyl glucose, at b o t h 2 and 20 mM, 1 mM m e t h y l glucose and 2 mM D-galactose It can also be seen that ratios o f 3H/14C-labelled sugar entering the tissue from the mucosal and serosal solutions respectively are all reduced by replacement of mucosal Na ÷ by chDhne Ex c e pt in the case of 20 mM 3-O-methyl glucose removal of mucosal Na ÷ increases the serosal-mucosal sugar fluxes Removal of Na ÷ from b o t h mucosal and serosal solution causes a furthel slgnlfmant decrease m Jms and net flux m all conditions except with 20 mM 3-O-methyl glucose and reductions in tissue accumulation and the specific activity ratio occur in all conditions Additionally, there is a f ur t her increase m serosal-mucosal fluxes of 14C-labelled sugars in all cases shown in Table I when Na ÷ is removed from b o t h serosal and mucosal solutions, compared with the effect of unilateral removal o f Na ÷ f r o m the mucosal solution The similarities between these results and those report ed by Rmaldo et al [ 5], are that no changes m Jms, net flux or Jsm of 20 mM 3-O-methyl glucose are observed when Na ÷ is removed f r om b o t h mucosal and serosal solutions, compared with fluxes observed when the Na ÷ m the mucosal solution is unilaterally replaced However, significant reductions m Jms, Jnet, tissue accumulatmn and specffm actlwty ratio of 3H/14C-labelled sugars are observed m all condltmns shown in Table I except with 20 mM 3-O-methyl glucose It can be seen (Table II) that no measurable effect of serosal Na ÷ on 20 mM 3-O-methyl glucose t~ansport is observed because the brush-border transport system is partially saturated and f u r t h e r m o r e the optimal brush-border permeablhty a s y m m e t r y towards 3-O-methyl glucose ~s considerably less than for galactose or ~3-methyl glucose The results indicate that, except with 20 mM 3-O-methyl glucose, when
Serosal solution Na+ mequlv
140
140
0
140
140
0
140 140 0
140
140
0
Mucosal solution Na+ mequl~,
140
0
0
140
0
0
140 0 0
140
0
0
3-Omethvl glucose 3-0 methyl glucose 3-0 methyl glucose
1 mM~-methyl glucose lmMfl-methyl glucose lmMfl-methvl glucose
2mMgalactose 2mMgalactose 2mMgalactose
2 mM
2 mM
2mM
20mM3-O-methyl glucose 2 0 m M 3-O m e t h y l glucose 20mM 3-O-methyl glucose
Sugar
7
5
7
5 5 5
6
6
5
4
5
--5-
n
0022
0096
0 38
0 289 0058 0020
00147-+
0041
0 17
0021
0 021
0064
+0002**
+0021"**
-+ 0 0 2 7
-+ 0 0 1 9 ± 0 011"~* +00038*
0002"
± 001"**
-+ 0 0 2 3
+- 0 0 0 2 2
-+ 0 0 0 2 6 *
+ 002
00063
-+ 0 0 0 2 6 *
-+ 0 0 0 1 "
+ 00005
0012
+00016**
00046+00012
00028
00027-+ 00003 0 0048+- 00009" 0012 -+00017**
0 0136 + 0003
0008
0 0042-+ 00006
0015
0 0076 + 00012
00156-+ +- 0 0 0 2 2
00064
± 0009
0007
001
0 0028
0019-+00047
0013-+00014
0030-+
0050 + 0 002 0 0 3 1 +- 0 0 0 3 1 " * 0034-+0005
0042
0034-+
0058-+
0 062-+ 0 007**
0030
0040-+
**
e
+ 0005"*
± 0 004
± 0007
0007
+ 0003
0025±0005
0019+-00026
0031
*
0078-+ 0 0063 0 0 3 6 +- 0 0 0 2 5 * * 4 0045-+0008
0048
0044-+
0 0 7 3 -+ 0 0 1 4
0071
0042
0 044 ± 0006
P32
1 4
2 76
4 1
195
242
167 5
113 3 16 3 168
i 08
5 1
40 5
P2a
P2"~
P t ~.
P21
PI:
c m - h -1 i S E M
T h e u m d l r e c t l o n a l f l u x e s w e r e c a l c u l a t e d a c c o r d i n g t o t h e f o l l o w i n g r e l a t l o n s h a p PO = J1j/C1 I t is a s s u m e d t h a t t h e f l u x e s a r e a t s t e a d y - s t a t e a n d t h a t t h e t m s u e beha~eskmetlcallvasasmglecompartment Onthlsbaslsltcanbocalculated thatJ12=J~t R +JL3, J21 =J~I(I + R),J2~ =J13(1+ 1/R) andJ32 =J,~ + JI~/R [ q ] w h e r e c o m p a r t m e n t s 1, 2 a n d 3 a r e t h e m u c o s a l , t a s s u e a n d s e r o s a l f l u i d r e s p e c t i v e l y
T A B L E I1
o~ 0¢
389 the normal Na ÷ gradmnt is reversed, slgmfmant net sugar transport against the direction of the Na ÷ gradmnt remains. The evidence for this statement is based on t w o independently measured variables, net flux and tissue accumulation, o f three dxfferent n o n - m e t a b o h z e d sugars [8,9,10] Table II shows the calculated umdlrectlonal permeablhtms o b t a m e d f r o m the observed data according to the equations derived previously [9] The data show, cont r a r y to the predmtlons of the Na ÷ gradmnt hypothesis, that the exit p e r m e a b l h t y of 3-O-methyl glucose,/~-methyl glucose and galactose are all increased when mtracellular [Na ÷] is lowered, first by replacing mucosal Na ÷ and increased stdl f ur t her by removmg b o t h mucosal and serosal Na +. The p e r m e a b l h t y ratio of ~-methyl glucose PI2/P2~ is 24 when the distribution of Na ÷ between the tissue water and mucosal solution is approxi m at el y 10 The Na ÷ gradmnt hypothesis predicts that m steady-state, the distribution ratm o f sugars, or the p e r m e a b l h t y ratm P12/P2~, should n o t exceed the d l s t n b u t m n ratm of [Na ÷] (mucosal)/[Na +] (cell) [11] Here it is observed that that the brush-border umdlrectlonal pe r m e a b l ht y ratm to ~-methyl glucose exceeds the predicted ratm by at least two orders of magnitude when the [Na +] m the mucosal solutmn is replaced by chohne We observe m Table Ib that alteratmn of the sugar c o n c e n t r a t m n , or t y p e does n o t affect s-m Na ÷ m ove m ent , y e t m some cases the um dl rect m nal permeability ratms of sugars across the brush-border are so high (Table II) that m order to satisfy the requirements of the Na ÷ gradmnt hypothesis the [Na +] m the mucosal unstlrred layer would have to be higher than is, m fact, present m the serosal solutmn If this were the case, then, according to the Na ÷ gradmnt hypothesis, 20 mM 3-O-methyl glucose should be accumulated, since 20 mM 3-O-methyl glucose is not accumulated when Na + is unilaterally removed f r o m the mucosal solutmn, and smce it is unhkel y that the [Na ÷] m the brush-border regmn will be very much higher than is present within the cell fluid, it can be referred t hat latent reversal of the imposed Na ÷ gradmnt across the brush-border following unilateral removal of Na+from the solutmn bathing the mucosal surface is an improbable explanatmn of the results presented here It may be concluded f r om these results that although the presence of Na + m the mucosal solutmn accelerates sugar influx across the brush border, it is n o t essentml for net sugar absorptmn and accumulatmn by the small intestine on the ot her hand, Na ÷ is reqmred to maintain the activity of the tissue Na + p u m p The reciprocal rise and fall of sugar entry and exit permeability on actlvatmn of the tissue Na+pump by mcreasmg cell Na ÷ is consistent with the view that actively transported sugars cross the brush-border by convect~ve-dfffusmn the force generating this convective flow may arise from osmotm pressure gradients across the lateral-based border These are formed due to the actmn of the Na + pum p whmh deposits h y p e r t o n m NaC1 m the extra-cellular space [6,12,13,14] The authors wish to t hank the M R C for fmancml assistance
390
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14
S a l t T m a n , D A , R e c t o r F C a n d F o r d t r a n , J S ( 1 9 7 2 ) J Chn In~cst 51, 870 8 8 5 K l m m m h , G A ( 1 9 7 0 ) B l o c h e m l s t r v 9, 3 6 6 9 - - 3 6 7 7 G o l d n e r , A M , S c h u l t z a n d C u r r a n . P F ( 1 9 6 9 ) J G e n P h ) s l o l 53, 3 6 2 - - 3 8 3 Crane, R K ( 1 9 6 5 ) F e d Proc 24, 1 0 0 0 - - 1 0 0 6 R m a l d o , J E , J e n m n g s . B L , Frlzzell, R A a n d S c h u l t z , S G ( 1 9 7 5 ) A m J Ph:~slol 2 2 8 , 854--860 Naftahn, R J and Holman, G D (1974) Blochlm Blophys Acta 3 7 3 , 4 5 3 - - 4 7 0 Holman, G D and Naftahn. R J Blochlm Blophys Acta, submitted L a n d a u , B R . Bernsteln, L a n d Wilson, T H ( 1 9 6 2 ) A m J P h y m o l 203, 2 3 7 - - 2 4 6 N a f t a h n , R a n d C u r r a n , P F ( 1 9 7 4 ) J M e m b r a n e Blol 16, 2 5 7 - - 2 7 8 Gsaky, T Z a n d Thale, M J ( 1 9 6 0 ) J Physlol ( L o n d o n ) 151, 5 9 - - 0 5 S c h u l t z , S G a n d C u r r a n . P 1~ ( 1 9 7 0 ) Physlol R e v 50, 6 3 7 - - 7 1 8 D i a m o n d , J M a n d Bossert, W H ( 1 9 6 7 ) J G e n Physlol 50, 2 0 6 1 - - 2 0 8 3 H o l m a n , G D a n d N a f t a h n , R J ( 1 9 7 5 ) B l o c h l m B l o p h y s A c t a 382, 2 3 0 - - 2 4 5 N a f t a h n , R J a n d S i m m o n s , N L , J P h y m o l , m t h e press
B~ochlmlca et B~ophystca Acta, 419 (1976) 3 8 5 - - 3 9 0 © Elsevier S c m n t f f m P u b h ~ h m g C o m p a n y , A m s t e r d a m - - P r i n t e d m T h e N e t h e r l a n d s
BBA Report B B A 71 238
THE E F F E C T S OF REMOVAL OF SODIUM IONS FROM THE MUCOSAL SOLUTION ON SUGAR ABSORPTION BY RABBIT ILEUM
R J NAFTALIN and G D HOLMAN
Department of Physiology, Unwers~ty of Leicester, LE1 7RH (U K )* ( R e c e i v e d N o v e m b e r 3rd, 1 9 7 5 )
S u mmar y Net absorpt:on and accumulation of D-galactose, H-methyl D-glucose and low co n cen t r a t m ns of 3-O-methyl-D-glucose by sheets of rabbit ileum are observed even when Na ÷ m the mucosal solut:on :s replaced by chohne This mdmates that active sugar transport can occur m the dlrectmn opposite to the brush-border Na ÷ gradmnt
There is cons:derable disagreement concerning the effects of Na ÷ replacement m the solution bathing the mucosal surface of the small m t est m e on subsequent sugar absorption Perfuslon studms m VlVO [1 ] suggest that replacement o f NaC1 m the mucosal solutmn by e~ther lsotomc KCI or m an mto l has no effect on glucose absorptmn by human, dog or rat ileum With suspensions of chink mtestmal eplthehal cells, removal of Na ÷ from the external solut:on does n o t prevent active accumulatmn of D-galactose by cells whmh have been pr em c uba t e d with Na ÷ [2] On the ot her hand, several stud:es do suggest t hat the presence of a Na ÷ gradmnt across the brush-border f r o m mucosa-cell :s essential for act:ve sugar accumulatmn and absorption [3,
41 Rmaldo et al [5] who re-examined th~s question recently, measured transmural flux from mucosal-serosal solution and from serosal-mucosal solutmn of 20 mM 3-O-methyl D-glucose across an m v:tro preparat m n of rabbit ileum and concluded t hat when Na ÷ is umlaterally replaced by chohne m the mucosal solut:on, no slgmfmant act:ve sugar absorption occurs Since :t has been shown t ha t the a s y m m e t r y of brush-border sugar transport :s decreased at higher sugar concentrations [6] and t hat a s y m m e t r y of the brush-border sugar transport system is a f u n c t m n of sugar affinity for the brush-border, (Holman, G D and Naftahn, R J , m preparat:on) :t was *Present address of both authors Department of Physzology, Kings Collei~e, London, U K
Serosal solution Na + mequtx
140
140
0
140
140
0
140 140 0
140
140
0
Mucosai solution Na + meqm~
140
0
0
140
0
0
140 0 0
140
0
0
1 mM ~-methvl glucose 1 mM ~-methvl glucose 1 m M 1 3 -m e t h yl glucos(
2 mM galactose 2 mM galactose 2 mM galaetose
20 mM 3-0 methvl glucose 20 mM 3-0 meth:yl glucose 20 mM 3-0 meth,,l g~ucose 2 mM 3-0 methvl glucose 2 mM 3-O methyl glucose 2 mM 3-O methyl glucose
Sugar
7
5
7
5 5 5
6
6
5
4
5
5
n
~ 0 19
++
**~ ÷+
~
***
~ 0 0156"
0011+00016
0 072
0 347 + 0 026
0 553 * 0038 0101+0021 0 029 + 0005
0023+0003
0067+0019
0 315-+ 0 05
0334+0031
0 335 ± 0 38*
0 87
Jms (#tmol cm -2 h-I )-+SEM
+- 0 0 0 0 4
+ 0 0008 +0002 ~ 00054
±0004
±0002*
-+ 0 0 0 1 2
~ 0033
-+ 0 0 3 1
~ 0 035
00098-+00012
0 0048 ± 0 0011
0 0027
00068 001 0024
0021
0017
0009
027
0 17
0 206
Jsm (tzmol cm -2 h -~)±SEM
*
+ 0 67
I 05
4 89
12 43
3 85 155 0 48
032
088
2 73
030
~÷
+0213
0 613
= 0085
~*
± 0 50 + 0 2 0 s~ * 0 085'
~0057
+-008**
+ 0 56
+0033
0 507 + 0 053
1 63
R ±SEM
~ I 73 ' 0 264***
627*0626
13 1 1 ~ 1 3
11 7 8 + 0 69 551±098 **~ 2 92 ± 0 32 ~
235+014
392±027*** **~
z 13 ~*
: 0965"
+ 1 7
8 08 + 0 63
241
33 2
38 4
Ace (mM) 4SEM
0 0018
0067
0 54
0 55 009 0 005
00016
005
0 305
007
0 164
0 66
~ 0 00to
-0015
",)026
- 0 038 002*' + 0 001'
0002~'
z 002 ~
-+ 0 0 5
* 0015
+ 0 025'+
' 0 17
Jnet (pmol cm -2 h -1)
~'
F l u x e s o f s u g a r s ~ e r e m e a s u r e d a t 3 0 , 6 0 a n d 9 0 m m a f t e r a d d i t m n o f t h e r a d m i s o t o p e as p r e ~ m u s l v d e s c r i b e d [ 6 9 ] T h e R i n g e r s o l u t m n s ~ e r e g a s s e d c o n t i n u o u s l ~ ~ l t h 9 5 % O ~ , 5% C O . a n d ~ e r e m a i n t a i n e d a t 3 7 ~ C M - S f l u x e s ~ e r e m e a s u r e d ~ l t h ~ H - l a b e l l e d s u g a r , s - m f l u x e s w i t h ~ 4 C - l a b e l l e d s u g a r s l d e n t w a l s u g a ~ c o n c e n t r a t l o n s ~ e r e p l a c e d in m u e o s a l a n d serosal c h a m b e r s Following incubation, the tissues ~ere washed m ice-cold chohne chloride Ringer and then extracted for 4 h in 0 1 M HNO~ The ~atm R was obtained from the ratm of dpm, ~H/dpm ~4C-labelled sugar extracted from the tissues Tissue sugar concentratmn zs c a l c u l a t e d o n t h e b a s i s t h a t ~t ~s p r e s e n t e n t * r e l v ~ l t h i n t h e c e l l s p a c e , c e l l w a t e r ~s o b t a i n e d f r o m t h e w e t - d r y w e i g h t o f t*ssue l e s s ] 0 % fo* t h e e x t r a c e l l u l a r t l m d [ 9 , 1 4 ] Ringer solutmncontams 1 4 0 m M NaC1 1 0 m M K H C O ~ 0 4 m M K H 2 P O , , 2 4 m M K 2 H P O ~ , 1 2 C a C L , 1 2 m M M g C 1 , m c h o h n e / R m g e r , chohne chloude issubstltuted f o r NaC1 3 - 0 M e t h y l g l u c o s e a n d f l - m e t h v l g l u c o s e ~ e r e o b t a i n e d f r o m K o c h L i g h t L t d ~H, ~ C ' - l a b e l l e d D - g a l a c t o s e ~ C - l a b e l l e d 3 - O m e t h y l g l u c o s e a n d 3 H - l a b e l l e d g l u c o s e ( u s e d as s t a r t i n g m a t e r i a l f o r s v n t h e m s o f l a b e l l e d ~ - m e t h v l g l u c o s e ) w e r e all o b t a i n e d f r o m t h e R a d m - C h e m i c a l C e n t r e A m e r s h a m ~H-labelled 3-0 methv! glucose and ~aC-labelled S-methyl glucose were obtained from Ne~ England Nuclear Ltd all the othe~ ohemieals ~ere Analar grade The rabbits used ~ere 2--3 kg Ne~ Zealand white rabbits ,~hleh were killed by intravenous inlect*on of Nembutal T h e t e r m i n a l i l e u m wa~ d ~ s s e e t e d I m m e d m t e b a n d s t u p p e d o f ~ts s e r o s a a n d o u t e r m u s c l e l a y e r s b e f o r e m o u n t i n g as a f l a t s h e e t m t h e f l u x c h a m b e r s S t a t i s t i c s t h e s l g m f i ( a n c e lex e ls ~ e l e c a l c u l a t e d u s i n g S t u d e n t s t t e s t ( u n p n r e d m e a n s s o l u t i o n ) * = P ~ 0 0 5 ** = P % 0 0 2 ' *' =P < 0 0011ndxeate the significance of the differencebet~eenthe figure and that m the row above
TABLE I
387
TABLE
Ib
T h e N a + c o n c e n t r a t i o n xn t h e m u c o s a l i n c u b a t i o n b a t h w a s m e a s u r e d f o n o w m g 9 0 m m i n c u b a t i o n [ N a +] w a s e s t i m a t e d b y f l a m e p h o t o m e t r y T i s s u e [ N a +] w a s c a l c u l a t e d f r o m t h e H N O a e x t r a c t s F r o m t h e m u e o s a l [ N a +] , n e t s - m N a + f l u x w a s c a l c u l a t e d a n d f o u n d t o b e c o n s t a n t f o r all s u g a r s t e s t e d a n d f o r all [ s u g a r ] t e s t e d Initial concentration Mucosal solution 0 0
of Na+mequlv
Concentration of Na + meqmv m mucosal solutmn following 90 mln mcubatmn
Serosal solutmn
n
0 140
16 25
095-+ 0073 3 4 4 -+ 0 1 7 T t s s u e [ N a +] m e q u l v / l l t r e cell w a t e r after 90 mln incubation
0 0 140
0 140 140
Calculated net S-M Na + flux
16 26 13
455-+04 2 2 3 7 -+ 0 9 2 4 4 61 + 2 4 7
5 81 # t m o l . c m - 2 - h - 1
considered worthwhile e xa m m m g the effects of mucosal deprivation of Na* on the absorption o f 3-O-methyl glucose at b o t h high and low concentrations and on D--galactose and H-methyl-D-glucose (methyl/3-D-glucopyranoslde) transport (both these last sugars have higher afhmtms for the brush-border than 3-O-methyl glucose [7]) Table I shows t hat removal of Na + f r om the mucosal solutmn alone causes a slgmhcant reduction m m-s flux of 3-O-methyl glucose, at b o t h 2 and 20 mM, 1 mM m e t h y l glucose and 2 mM D-galactose It can also be seen that ratios o f 3H/14C-labelled sugar entering the tissue from the mucosal and serosal solutions respectively are all reduced by replacement of mucosal Na ÷ by chDhne Ex c e pt in the case of 20 mM 3-O-methyl glucose removal of mucosal Na ÷ increases the serosal-mucosal sugar fluxes Removal of Na ÷ from b o t h mucosal and serosal solution causes a furthel slgnlfmant decrease m Jms and net flux m all conditions except with 20 mM 3-O-methyl glucose and reductions in tissue accumulation and the specific activity ratio occur in all conditions Additionally, there is a f ur t her increase m serosal-mucosal fluxes of 14C-labelled sugars in all cases shown in Table I when Na ÷ is removed from b o t h serosal and mucosal solutions, compared with the effect of unilateral removal o f Na ÷ f r o m the mucosal solution The similarities between these results and those report ed by Rmaldo et al [ 5], are that no changes m Jms, net flux or Jsm of 20 mM 3-O-methyl glucose are observed when Na ÷ is removed f r om b o t h mucosal and serosal solutions, compared with fluxes observed when the Na ÷ m the mucosal solution is unilaterally replaced However, significant reductions m Jms, Jnet, tissue accumulatmn and specffm actlwty ratio of 3H/14C-labelled sugars are observed m all condltmns shown in Table I except with 20 mM 3-O-methyl glucose It can be seen (Table II) that no measurable effect of serosal Na ÷ on 20 mM 3-O-methyl glucose t~ansport is observed because the brush-border transport system is partially saturated and f u r t h e r m o r e the optimal brush-border permeablhty a s y m m e t r y towards 3-O-methyl glucose ~s considerably less than for galactose or ~3-methyl glucose The results indicate that, except with 20 mM 3-O-methyl glucose, when
Serosal solution Na+ mequlv
140
140
0
140
140
0
140 140 0
140
140
0
Mucosal solution Na+ mequl~,
140
0
0
140
0
0
140 0 0
140
0
0
3-Omethvl glucose 3-0 methyl glucose 3-0 methyl glucose
1 mM~-methyl glucose lmMfl-methyl glucose lmMfl-methvl glucose
2mMgalactose 2mMgalactose 2mMgalactose
2 mM
2 mM
2mM
20mM3-O-methyl glucose 2 0 m M 3-O m e t h y l glucose 20mM 3-O-methyl glucose
Sugar
7
5
7
5 5 5
6
6
5
4
5
--5-
n
0022
0096
0 38
0 289 0058 0020
00147-+
0041
0 17
0021
0 021
0064
+0002**
+0021"**
-+ 0 0 2 7
-+ 0 0 1 9 ± 0 011"~* +00038*
0002"
± 001"**
-+ 0 0 2 3
+- 0 0 0 2 2
-+ 0 0 0 2 6 *
+ 002
00063
-+ 0 0 0 2 6 *
-+ 0 0 0 1 "
+ 00005
0012
+00016**
00046+00012
00028
00027-+ 00003 0 0048+- 00009" 0012 -+00017**
0 0136 + 0003
0008
0 0042-+ 00006
0015
0 0076 + 00012
00156-+ +- 0 0 0 2 2
00064
± 0009
0007
001
0 0028
0019-+00047
0013-+00014
0030-+
0050 + 0 002 0 0 3 1 +- 0 0 0 3 1 " * 0034-+0005
0042
0034-+
0058-+
0 062-+ 0 007**
0030
0040-+
**
e
+ 0005"*
± 0 004
± 0007
0007
+ 0003
0025±0005
0019+-00026
0031
*
0078-+ 0 0063 0 0 3 6 +- 0 0 0 2 5 * * 4 0045-+0008
0048
0044-+
0 0 7 3 -+ 0 0 1 4
0071
0042
0 044 ± 0006
P32
1 4
2 76
4 1
195
242
167 5
113 3 16 3 168
i 08
5 1
40 5
P2a
P2"~
P t ~.
P21
PI:
c m - h -1 i S E M
T h e u m d l r e c t l o n a l f l u x e s w e r e c a l c u l a t e d a c c o r d i n g t o t h e f o l l o w i n g r e l a t l o n s h a p PO = J1j/C1 I t is a s s u m e d t h a t t h e f l u x e s a r e a t s t e a d y - s t a t e a n d t h a t t h e t m s u e beha~eskmetlcallvasasmglecompartment Onthlsbaslsltcanbocalculated thatJ12=J~t R +JL3, J21 =J~I(I + R),J2~ =J13(1+ 1/R) andJ32 =J,~ + JI~/R [ q ] w h e r e c o m p a r t m e n t s 1, 2 a n d 3 a r e t h e m u c o s a l , t a s s u e a n d s e r o s a l f l u i d r e s p e c t i v e l y
T A B L E I1
o~ 0¢
389 the normal Na ÷ gradmnt is reversed, slgmfmant net sugar transport against the direction of the Na ÷ gradmnt remains. The evidence for this statement is based on t w o independently measured variables, net flux and tissue accumulation, o f three dxfferent n o n - m e t a b o h z e d sugars [8,9,10] Table II shows the calculated umdlrectlonal permeablhtms o b t a m e d f r o m the observed data according to the equations derived previously [9] The data show, cont r a r y to the predmtlons of the Na ÷ gradmnt hypothesis, that the exit p e r m e a b l h t y of 3-O-methyl glucose,/~-methyl glucose and galactose are all increased when mtracellular [Na ÷] is lowered, first by replacing mucosal Na ÷ and increased stdl f ur t her by removmg b o t h mucosal and serosal Na +. The p e r m e a b l h t y ratio of ~-methyl glucose PI2/P2~ is 24 when the distribution of Na ÷ between the tissue water and mucosal solution is approxi m at el y 10 The Na ÷ gradmnt hypothesis predicts that m steady-state, the distribution ratm o f sugars, or the p e r m e a b l h t y ratm P12/P2~, should n o t exceed the d l s t n b u t m n ratm of [Na ÷] (mucosal)/[Na +] (cell) [11] Here it is observed that that the brush-border umdlrectlonal pe r m e a b l ht y ratm to ~-methyl glucose exceeds the predicted ratm by at least two orders of magnitude when the [Na +] m the mucosal solutmn is replaced by chohne We observe m Table Ib that alteratmn of the sugar c o n c e n t r a t m n , or t y p e does n o t affect s-m Na ÷ m ove m ent , y e t m some cases the um dl rect m nal permeability ratms of sugars across the brush-border are so high (Table II) that m order to satisfy the requirements of the Na ÷ gradmnt hypothesis the [Na +] m the mucosal unstlrred layer would have to be higher than is, m fact, present m the serosal solutmn If this were the case, then, according to the Na ÷ gradmnt hypothesis, 20 mM 3-O-methyl glucose should be accumulated, since 20 mM 3-O-methyl glucose is not accumulated when Na + is unilaterally removed f r o m the mucosal solutmn, and smce it is unhkel y that the [Na ÷] m the brush-border regmn will be very much higher than is present within the cell fluid, it can be referred t hat latent reversal of the imposed Na ÷ gradmnt across the brush-border following unilateral removal of Na+from the solutmn bathing the mucosal surface is an improbable explanatmn of the results presented here It may be concluded f r om these results that although the presence of Na + m the mucosal solutmn accelerates sugar influx across the brush border, it is n o t essentml for net sugar absorptmn and accumulatmn by the small intestine on the ot her hand, Na ÷ is reqmred to maintain the activity of the tissue Na + p u m p The reciprocal rise and fall of sugar entry and exit permeability on actlvatmn of the tissue Na+pump by mcreasmg cell Na ÷ is consistent with the view that actively transported sugars cross the brush-border by convect~ve-dfffusmn the force generating this convective flow may arise from osmotm pressure gradients across the lateral-based border These are formed due to the actmn of the Na + pum p whmh deposits h y p e r t o n m NaC1 m the extra-cellular space [6,12,13,14] The authors wish to t hank the M R C for fmancml assistance
390
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14
S a l t T m a n , D A , R e c t o r F C a n d F o r d t r a n , J S ( 1 9 7 2 ) J Chn In~cst 51, 870 8 8 5 K l m m m h , G A ( 1 9 7 0 ) B l o c h e m l s t r v 9, 3 6 6 9 - - 3 6 7 7 G o l d n e r , A M , S c h u l t z a n d C u r r a n . P F ( 1 9 6 9 ) J G e n P h ) s l o l 53, 3 6 2 - - 3 8 3 Crane, R K ( 1 9 6 5 ) F e d Proc 24, 1 0 0 0 - - 1 0 0 6 R m a l d o , J E , J e n m n g s . B L , Frlzzell, R A a n d S c h u l t z , S G ( 1 9 7 5 ) A m J Ph:~slol 2 2 8 , 854--860 Naftahn, R J and Holman, G D (1974) Blochlm Blophys Acta 3 7 3 , 4 5 3 - - 4 7 0 Holman, G D and Naftahn. R J Blochlm Blophys Acta, submitted L a n d a u , B R . Bernsteln, L a n d Wilson, T H ( 1 9 6 2 ) A m J P h y m o l 203, 2 3 7 - - 2 4 6 N a f t a h n , R a n d C u r r a n , P F ( 1 9 7 4 ) J M e m b r a n e Blol 16, 2 5 7 - - 2 7 8 Gsaky, T Z a n d Thale, M J ( 1 9 6 0 ) J Physlol ( L o n d o n ) 151, 5 9 - - 0 5 S c h u l t z , S G a n d C u r r a n . P 1~ ( 1 9 7 0 ) Physlol R e v 50, 6 3 7 - - 7 1 8 D i a m o n d , J M a n d Bossert, W H ( 1 9 6 7 ) J G e n Physlol 50, 2 0 6 1 - - 2 0 8 3 H o l m a n , G D a n d N a f t a h n , R J ( 1 9 7 5 ) B l o c h l m B l o p h y s A c t a 382, 2 3 0 - - 2 4 5 N a f t a h n , R J a n d S i m m o n s , N L , J P h y m o l , m t h e press
