Tohokn

J. exp.

Med ., 1976, 119, 201-209

Interrelationship

between

Sugar-Evoked

in

Potential

Difference

Transmural

Influxes

across

the

Mucosal

Increases and

Border

in

Sugar

the

Small

Intestine TAKESHI HOSHI, YUTCHI SUZUKI, TSUNETA KUSACHI* and YUTAKA IGARASHIt

Department of Physiology,Tohoku UniversitySchoolof Medicine, Sendai, 980

HOSHI,

T.,

Sugar-Evoked the

Mucosal

201-209 by

actively the

conductivity showed

both the

of

electrical

Ģ

sizes

of ĢPD's

PD's in

and

and

was

seen

shunt

which

sugar

transport;

Active

on and

changes

in

in

Na+

investigated al.

studies

have

dependent

varied

the

the

goldfish shown active

sugar-evoked ĢPD

parallel

and

relationship sugar

fluxes.

between

APP

It

to

influx;

transmural

of

D-glucose

and

the

presence

of

1970).

transmural

potential

small

(Smith that

of

1966) the

transport

of cannot

by in

the

of

these

of

hamsters

and

toads

be

taken

On as

a

the

between the

the

conduc coupling)

dominant

of

the

small

factor

paracellular

intestine

generates of

change

(Hoshi

and

is

medium

because

PD and

other

increase

medium

sugars

(Lyon

direct

correction

conductivity.

the

are the

correlation

intestine

(PD)

sugar-evoked ĢPD's sugars.

such simple

extracellular

such

intestine, estimated

one-to-one

small

difference

properties

intestines

of

electrical

D-galactose sodium

correlated, the

from

resistance

potential;

investi medium

parallelism

that

is the

medium

Transport

The

a

estimated

is concluded Js

was

sugar

No

(approximately

and

proportional

sugar

Curran

regardless

a

(3),

induced

closely

for

a close

Js

with

119

that

along

However,

flux

(Js's)

were

identical.

Na+

1976,

condition

(Km)

restored in

sugars

Js

between across

potential

difference

nearly

increase in

and

varied.

conductivity

stoichiometrical

transport.

in

1964),

current

directly

transport

Schultz

increase

sugar-induced

Na+

is

were

Ivied.,

the

concentration

medium

exp.

of the

regional

conductivity

Interrelationship and Sugar Influxes

transmural

Under APP

of

saturation

J. the

influxes

intestine.

measurements

for

in

unchanged,

medium

interrelation

dependent

1965;

the

the

flux

and

small

, Y.

Difference Tohoku

patterns

The

a fixed between

affecting

et

Js'S.

short-circuit

tivity,

istic

and when

half

IGARASHI

changes

(dPD's) pig

similar

the

and

Intestine.

between

guinea

of

T.

Potential

Small

remains

observed the

the

very

values

KUSACHI, Transmural

sugars

isolated

e.g.,

from

be

relationship

electrical

was

to

in

The

transported

in

and

Y., in

Border -

gated

SUZUKI, Increases

Crane Komatsu directly

known (Crane

character a concomitant

(ĢPD)

have

1966),

rats 1968).

related

hand,

there

is

measure

of

coupled

a

been (Barry These

to

Na+-

view

that

flows

Received for publication, March 10, 1976. Present address: Tokyo Research Laboratory,* Kowa Co. Ltd., Higashimurayama, Tokyo. Department of Pediatrics,t Tohoku University School of Medicine, Sendai 201

of

202

T. Hoshi

Na+

and/or

greatly

sugar,

modify

sugar

the

(Schultz actual

not

been

relationship

in

obtained

show

the

guinea

that

of

tion

between

that

among

where

APP

for

the

APP

basis

observed

of

by

small or

of

and

the

actual

epithelium flux

of

may Na+

sugar-evoked ĢPD's, study

in

or

a

influencing

proportional of

medium

conductivity

sugar

was

influx of

dominant

the

layer,

influencing

kinetic

sugar

of

border.

the

correla

This

the

active

of

a direct

indicates

resistance

the ĢPD-influx

properties

across

unchanged.

correction

restore

to

results

influxes

remains

only

was The

simple to

mucosal

epithelial

factor

some

a

this

interest

medium

sufficient

across

the

to

the

examine

interrelation.

varies,

conductivity

to

Particular

this

directly

however,

aimed

vitro.

conductivity

finding,

electrical

intestine

are

and

a

and

present

electrical

medium

is

this

fluxes

factors

parameters

shunt

the

The

the ĢPD's

resistance

paraceliular

sugar

detail. pig

when

situations

the ĢPD

between

factor

border

amplitude

On

the

parameters

between

in

dominant

mucosal

resistance

1967).

studied

a

in

al.

relationship

determine

the

electrical

interrelation

et

The has

since

et al.

of

the

relationship.

hexose

transport

were

measurements.

METHODS

Guinea pigs of either sex weighing from 350 to 650 g were used. The entire length of the small intestine of the animal was divided into 10 segments starting from the level of Lig. Treitz. The portion supplied with blood by the superior and inferior ileal arteries was divided into 5 segments, and the portion supplied with blood by jejunal arteries was also divided into 5 segments. All segments were numbered from I to X starting from the uppermost jejunal segment. The animals were anesthetized with urethane (1 g/kg B.W., i.p.). After a midline incision of the abdominal wall, the terminal ileum was first pulled out and excised after ligating supplying arteries and both ends of the portion to be excised. The remaining intestine was put back into the abdominal cavity until the excision of the other segments. Usually, two or three segments were excised and examined at the same time. After finishing experiments with the first few segments, the second excision was performed to examine other segments. Sometimes, the upper segments were examined earlier than the lower in order to see time-dependent changes in both electrical and transport activities. During these procedures, the animals were kept warm with electric lumps. When pulsation of small arteries supplying blood to the intestine was very feeble or not visible at the occasion of the second or the third excision, the experiments were discontinued. In most eases, blood circulation was maintained undisturbed for more than 3 hr. The extirpation of the entire length of the intestine at once gave unsatisfactory results, because some of the segments had to be incubated in Ringer's solution for a few hr, and such a long incubation resulted in a marked decline in the transport function. The

excised

and

fixed

tip

of

the

intestine

by

was

the

the

inside

of

just

cm

below

tube,

was

immersed

in

the

tube

was

with

standand

solution, 220,

Na*

KHC03

concentration

The methods

length,

over

the

filled at

lower

2.5, was

of recording

rinsed

20

ml

standard same

A

low-Na+

had

KH2P04 varied,

of area.

the

The solution

in

1.5, of

solution,

outer

fenestrated everted

intestine

bubbled

with of

mannitol

PD were described

was

the

(in 1 .0, to

be

The

closed

,

and

, thus supported pure oxygen . The bathing

solution

MgSO4

everted

diameter.

area

composition

CaSO4 amount

standard

Ringer's

following

the transmural

the 5 mm

Temperature

and SO4--

0.25,

of

the

solution.

the

the

with

tube

margin

fenestrated

37•Ž. which

was

polyethylene

the

tied

constant

in

fenestrated

was

mannitol When

2-3

a multiply

tube,

regulated

the

segment,

over

was mM);

added

as

Na2S04

Tris-S04

in detail

solution used

10 was

the 25,

(pH

7.4).

changed.

in previous

papers

Sugar

Influx

and Transmural

PD

in Small Intestine

203

(Hoshi and Komatsu 1968, 1970). Short-circuit current was measured by passing DC current across the wall of the preparation . For this purpose, another type of the supporting tube was used. The fenestrated part of the tube was replaced by four stainless steel rods and the upper tube segment was replaced by a thicker (10 mm , I.D.) polyethylene tube. The everted intestine was fixed over this metal-supported portion . The reason for the use of metal rods was to get a homogeneous potential field inside the everted intestine . In this case, the area of the serosal surface in contact with the internal solution was 3.6 cm2. For passing current, non-polarizable electrodes made of Zn-ZnSO4 cells and bridges made of thin polyethylene tubes (2 mm, O.D.) filled with 2% agar-1 M Tris-S0, solution were employed. The tip of one of the bridges was placed inside the tube just above the portion covered by the intestine, and the tip of the PD-measuring bridge inside the tube was fixed around the center of the everted intestine and in the very vicinity of the serosal surface. The tip of the other bridge for passing current was placed at a remote portion in the external solution. The other PD-recording bridge was placed in the vicinity of the mucosal surface just outside the internal recording bridge. Fluid resistance between the PD-measuring electrodes was determined without the tissue before the experiments with the tissue. This was taken into consideration when short-circuit current was measured. Measurements intestine,

prepared

glucose

at

a

Preliminary

mM

the

same

ionic

sugars

about

hr.

counter

fluid

the

by

that

used

for

each the

D-[3H] the

in

of

was

the

of

counted

to

determine

used

initial were

the

picked

torsion

HNO, in

up

having

samples

a

N

at influx

solution

on

0.1

were

linearly

solution

Ringer

weighed

I ml

fluids

mannitol

surface

cold

Na+

the

Then

paper,

sample

extraction

of

galactose

obtain

a

or

(0.1-0.2 ƒÊCi(ml).

preparations

with

everted

galactose

tracer

mM

incubation.

filter

The

presence

10 to

the sec

the

on

the in

adopted

10

ways. containing

[14C] in

incubation,

blotted

of

to

was

about

placing

LSC-601).

adhering

uptake incubated

for

following

a medium

respective

when

of

the

in

galactose

end

tubes,

in

with

min

rinsed

radioactivities

(Aloka

incubation

out

incubated

incubation

as

extracted The

10

At and

supporting

were

24

tion

border.

the

that

3-min

media

carried was

together

than

composition

from

The

more

Therefore,

incubation

detached

were above,

revealed for

mucosal

the

influx

concentration

time

Na+. the

from

described

desired

with

across

sugar

as

experiments

increased 150

of

were balance.

solution

a

liquid

the

for

scintilla

amount

of

the

preparations.

RESULTS

Regional differences along the length of the small intestine At

first,

under

fixed

(ĢPD)

was

tration

of

the

patterns

of ĢPD

significantly The Tat,

than

an

carnivorous transport segments

was from

observed omnivorous

by

at

function

mM.

As

regional

in

the

pattern

and

the

the

bathing

with in

Fig.

fluid

are

change

final was

the

twofold

concen added

colon,

there

to

influx

higher

and

galactose

segments

PD

the

and

about

all

which

caecum of

at

the ĢPD

duodenum, uptake

in The

D-galactose, 1,

both

out

difference.

in

no

did

show

not

the

genera differ

mannitol. of

animal

regional

(Barry

(Hoshi and

the

i.e.,

In

carried

regional

to

shown

difference,

observed,

were

the

measured

jejunum.

of

see

D-glucose was

of

were

to

influx

20

that

measurements

order adding

The

animal

(VI-X)

flux

in

mM. fluid

region

tion

and

examined 10

mucosal

similar ileal

electrical conditions

and the

employed

difference et

al.

1964)

Komatsu

generation in

and 1968).

of the

markedly

larger

subsequent

of

differs the

toad,

Because

an of

sugar-induced ĢPD, experiments.

from

the

those

of

the

insectvorous higher only

or sugar ileal

204

T. Hoshi

Fig.

1.

Regional

across

differences

the

the

right

line

in

ileal

mucosal the

the

in

rates

right

of

sizes

The

galactose

panel

segments.

the

border.

Cae,

the

mean

denote

PD

shows ĢPD's

from

the

Col

sugar-evoked

panel

uptake

indicates

Duo,

of

left

et al.

20

mM

value

the

change

induced galactose

of

rates

duodenum,

of

caecum

and by

sugar

10

mM

solution.

The

mannitol and

influx glucose, dotted

uptake

colon,

by

the

respectively.

Values of Km estimatedfrom electrical and flux measurements Both were both Fig.

the

simply cases,

a

both

the

points

2.

Double

Left relation

lines and

for the

tested.

panel

shows of

J,

to

glucose

plots

the sugar

and

influx

similar plots

difference

Glu.

sugar

concentration

Glucose

reciprocal

sugars

sugar

reciprocal

concentration.

cases,

of

and the

relationship,

double

sugar

identical

on

saturable

2 shows

against

Fig.

sugar-evoked ĢPD dependent

of ĢPD's Gal.

concentration.

and and

in

relation

of

to the

and

galactose

half

4PD's

to Each

the

as

mucosal bathing

and

compared

crossed

in

the

a function and

of the

concentration

value

is

mean •}S

each

seen. of

Js In

nearly between

concentration

, respectively. ([S]) , the .E.

In

case. at

(Km)

mucosal

D-galactose

sugar

was values

ordinate

concentration

border fluid.

kinetics,

of ĢPD

were

saturation

Js'S

across mucosal

Michaelis-Menten

denote D-glucose of

the

amplitudes

galactose

the

(Js) in

The right

the

TABLE

1.

The by

and

the

(number

galactose

were

values

of ĢPDmax,

incubated

maximum

sugars

values electrical

•} s.E.

of

Influx

both

were

glucose

Sugar

listed

estimated

in ĢPDmax insignificant

in

the

standard

of

experiments)

the

Jmax

PD in Small Intestine

and

at

presented

in

the

1.

were

PD

There

and

for

the

the

no

and

measurements

given

flux

as

data.

(JmaX)

Km

differences

are

values for

both

between Small

galactose

mean

The

and

(p>0.5).

and

estimated Preparations

are

influx

significant

glucose

galactose

ileum.

Values

PD

205

and pig

37•Ž.

maximum

flux

between

glucose

Guinea

medium

(ĢPDmax),

values

Km

measurements.

similarly

Table

from and

flux

change in

Jmax

and

was

PD

and Transmural

Km

differences

slso

statistically

(p>0.4).

Relationship between sugar-dependent increase in short-circuit current and sugar influx

(Js)

Fig.

3

and

the

shows

concentration

in

experiments.

Js

Fig.

seen

the

increase

Effects

solution values

of

of and

of

indicate •}S.E.

GIs,

the

of

Na

flux

Na+

galactose are

Na+

expressed

Na+

in was

both

and

medium the

solution that

range in

medium increments

mucosal

is

over

the

3.

effect

is increased,

parallel and

the

It

concentration are

the

galactose-induced

Js

fixed

at

curves

for

Js

on

ionic

galactose

increase fluxes

are

this

short

(smoles-min-1•g-1

Na+

Moreover, approximately

influx in

Galactose series

increase

against

tested.

influxes

(ĢIsc).

throughout

and ĢIsc

from ĢIsc,

concentration

galactose

progressively

concentrations

(5 mM)-induced

on current

5 mM

and ĢIsc,

calculated

as

concentration short-circuit

(Js) circuit wet

current wt.).

Na+

concentration the the

from

of

as

5 mM

value

of

same

at

galactose

(GIs).

The

Vertical

bars

206

T. Hoshi

any Na+ concentration, suggesting in the influx mechanism.

et al.

one-to-one

coupling

between

galactose

and

Na+

Effect of medium Na+ concentration on the sugar-evokedJPD Fig.

4 shows

the

evoked •}ƒ¢PD. to

40

in

the

mM.

Above

Na+

a

changed.

ratio

of

solution

(6.92

ductivity,

to

restore

For

mmho/cm) that

the

was

of ĢPD

direct

it

at

a

of

the

Na+

were

corrected The

certain

Na+

solution

correlation

was

for

of ĢPD

for

the

made

medium

in

150

the mM

Na+-

(con This

conductivity PD

by

standard

(=17.6/6.92).

sugar-evoked

the

multiplied of

in

mannitol,

conductivity,

that

2.46

20

concentration

was

recorded

mM)-

increase

by

was

to

of

with

medium

correction

factor

(10 from

Na+

concentration

a

glucose

replaced

when

employed

by

between

the

concentration decreased

varied

obtained.

correction

Na+

gradually

medium

multiplied

on

of

example, ĢPD's

were a simple

range

experiments, the

of ĢPD's

curve

conductivity

indicates

sugar

these

sizes Js

concentration

the

range,

of

mmho/cm).

17.0

finding

the

size

specific

in

concentration

the

the

Na+

largest

In

with

way;

medium

conductivity

When

parallel

following a

this

electrical

line

of was

concentration.

therefore, was

effect

The ĢPD

is enough

change

and

the

influx.

Fig.

4.

The

Closed

effect

correction

those

medium

Na+

indicate

for

relationship as

of

circles

the

medium

galactose in

Fig.

on

uncorrected,

conductivity

between presented

concentration

values

(see

influx

(Js)

the

glucose

open

the

text).

and

The

medium

(10

circles dotted

Na+

mM)-induced ĢPD

the

values curve

concentration

after indicates

(the

same

. the the data

3).

Kinetic properties of active hexose transport in the guinia pig intestine The can

be

sizes of the

results taken

are

of as

corrected

electrogenic

a

the direct

for

above measure

medium

active

hexose

observations of

sugar

conductivity. transport

indicate influx

across

Based were

on

that

the

the

brush

this

investigated

finding by

sugar-evoked ĢPD border , kinetic recording

when

their

properties the ĢPD's

Sugar

Fig.

5.

The

relationship

concentration plots.

at

various

were where

At

tion

a

and

data fixed namely,

resulted

the

were Na+ the

active

herbivorous

sugar animal

amplitude

different were

Na+

for

on

in ĢPDmax

similar

medium

(Vmax)

to

the

the guinea

of

were

also

mixed-type

ileum

of

the

the

mucosal

the

recorded ĢPD's

summarized

in

Fig.

5,

way. to

The

and

A similar across

sizes

results

line.

and

Lineweaver-Burk

conductivity.

conformed

a straight

observed.

dPD

Lineweaver-Burk's

data

207

concentrations.

The

to all

Intestine

glucose-induced

Na+

The

according

fell

transport

the

concentrations.

plotted

was

of

corrected

concentration, data

PD in Small

medium

conductivity.

in a decrease kinetics

the at

of ĢPD's

medium

a

mixed-type in

values

for

the

relation,

glucose

galactose

corrected

and Transmural

between

of The

Influx

a

Michaelis-Menten

reduction in

an

increase

kinetics rabbit

of

Na+ in

type concentra

Km,

has

been

(Goldner

et

thus

the

observed al.

1969),

pig.

DISCUSSION

Electrical events associated with active sugar transport in the small intestine can be regarded as phenomena related to cotransport of sugars and Na+ (Lyon and Crane 1966; Schultz et al. 1967; Hoshi and Komatsu 1970). There is good evidence for that the coupled transport of sugars and Na+ takes place at the brush border membrane (Goldner et al. 1969; Hoshi and Komatsu 1970; Maruyama and Hoshi 1972). Charge separation due to the coupled and preferential transport of Na+ with sugar may be responsible for the generation of an electromotive force (EMF) at the luminal membrane. The strength of the EMF would be proportional to coupled fluxes of Na+ and sugars, since a good parallelism is seen between the increases in short-circuit current and sugar influxes. Therefore, it is easily explain ed that the PD change across the intestinal wall due to such an EMF is directly proportional to sugar influx across the mucosal border when the resistance

208

T. Hoshi

parameters

of the cell layer

The epithelial cell of low-resistance paracellular 1971; Frizzell and Schultz be directly proportional

et al.

are unchanged.

the small intestine is known to be short-circuited by a shunt (Rose and Schultz 1971; White and Armstrong 1972). The conductance of the shunt has been shown to to electrical conductivity of the incubation medium

(Frizzell and Schultz 1972). Therefore, an EMF generated within the cell in association with sugar transport is also short-circuited by the low-resistance paracellular shunt. Such a situation explains why a simple correction for medium conductivity of the sugar-evoked JPD enables us directly to correlate it with sugar influx across the mucosal border. There may be other possible factors influencing the relationship between sugar evoked JPD and mucosal sugar influx. For example, changes in the cell membrane resistances can influence this relationship. However, as compared with the dominant effect of the shunt resistance, the influence of changes in membrane resistance seems to be of minor Debnam

and

relationship

rate

rent

Km's

they

showed

of

epithelial

cells

resistance

in

however, must

such

a

is quite

either

in

vitro

to

record

the

malabsorption. can

also

tion

as

In provide

sugar

of

mechanism,

and

paracellular

shunt

the

the out

two is

directly

2)

the

dominant

resistance

things,

proportional

coupling

of

factor which

is

man

and

sugar affecting

proportional

of

and

the

Na-r

requires

the

the ĢPD-flux to

medium

(1974)

PD

assess change

transport

func

.

However,

further

sugar

generated

in

al. to

the

1970)

influx

sugar intestine

et

of

and

EMF

sugar

by

small

attempt

sugar

still

the ĢPD

to

an

Komatsu

change

1)

the

sugar

compared.

Read in

in

conditions.

induced in

the

changes

with

records

aspects

(Hoshi

between

important

one-to-one

in

PD

interrelation

changes

to

in

were

simplicity.

dynamic

sugar-evoked

complex

function

instantaneous

paper

changes

conditions

PD

behaved prior

sugar-induced ĢPD

the

appa

Vmax,

experimental

different

the

However,

starved

some

their

the

change

the

previous

of

taransport

because

a

that

technical

PD

concerning

in

structural

transport

its

experiments,

information

point

of

glucose-induced

in

on

chemically

between

absorption,

resulted

of

sugar

of

partly

of

vivo

and

agreement

or

likely

in

measurements.

rate

under

recording

because

vitro

is

good

fasted

when

assessing

vivo

concerning results

border

for

interpretation

knowledge

it occurred

reservation

limitation,

demonstrated

proper

present

in

been

correlation

some

useful or

maximum

rats

absorption

chemical

the

have that

with

found

had

experiments,

in

sugar

and

semistarvation

may

made

Despite

tivity.

their

They

rats or

indicates

be

transport

with

fasting

observations with

electrical and

when

parameters

This,

both

the ĢPDmax

As

made

associated

absorption.

from

that

recently

change

sugar

differently

experiments.

a

(1975)

PD

obtained

somewhat

tried

Levin

between

determined

flux

significance.

flux in

across

basic .

The

association the

mucosal

electrogenic

influx

relationship

is

electrical

conduc

the

Sugar

Influx

and Transmural

PD in Small

Intestine

209

References

1) Barry, R.P.C., Dikstein, S., Matthews, J., Smyth, D.H. & Wright, E.M. (1964) Electrical potential associated with intestinal sugar transfer. J. Physiol. (Loud.), 171, 316-338. 2) Crane, R.K. (1965) Na-t-dependent transport in the intestine and other animal tissues. Fed. Proc., 24, 1000-1005. 3) Debnam, E.S. & Levin, R.J. (1975) Effects of fasting and semistarvation on the kinetics of active and passive sugar absorption across the small intestine in vivo. J. Physiol. (Lout.), 252, 681-700. 4) Frizzell, R.A. & Schultz, S.G. (1972) Ionic conductances of extracellular shunt pathway in rabbit ileum. Influence of shunt on transmural sodium transport and electrical potential differences. J. gen. Physiol., 59, 318-346. 5) Goldner, A.M., Schultz, S.G. & Curran, P.F. (1969) Sodium and sugar fluxes across the mucosal border of rabbit ileum. J. gen. Physiol., 53, 362-383. 6) Hoshi, T. & Komatsu, Y. (1968) Sugar-evoked potential in isolated toad intestine. Jap. J. Physiol., 18, 508-519. 7) Hoshi, T. & Komatsu, Y. (1970) Effects of anoxia and metabolic inhibitors on the sugar-evoked potential and demonstration of sugar-outflow potential in toad intestine. Tohoku J. exp. Med., 100, 47-50. 8) Lyon, I. & Crane, R.K. (1966) Studies on transmural potential in vitro in relation to intestinal absorption. 1. Apparent Michaelis constants for Na+-dependent sugar transport. Biochim. biophys. Arta (Arnst.), 112, 278-291. 9) Maruyama, T. & Hoshi, T. (1972) The effect of D-glucose on the electrical potential profile across the proximal tubule of newt kidney. Biochim. biophys. Acta (Amst.), 282, 214-225. 10) Read, N.W., Holdsworth, C.D. & Levin, R.J. (1974) Electrical measurement of intestinal absorption of glucose in man. Lancet, 2, 624-627. 11) Rose, R.C. & Schultz, S.G. (1971) Studies on the electrical potential profile across rabbit ileum. Effects of sugars and amino acids on transmural and transmucosal electrical potential differences. J. gen. Physiol., 57, 639-664. 12) Schultz, S.G. & Curran, P.F. (1970) Coupled transport of sodium and organic solute. Physiol. Rev., 50, 637-718. 13) Schultz, S.G., Curran, P.F. & Wright, E.M. (1967) Interpretation of hexose dependent electrical potential differences in small intestine. Nature (Lout.), 214, 509-510. 14) Smith, M.W. (1966) Sodium-glucose interactions in the goldfish intestine. J. Physiol. (Lord.), 182, 559-573. 15) White, J.E. & Armstrong, W. McD. (1971) Effect of transported solutes on membrane potentials in bullfrog small intestine. Amer. J. Physiol., 221, 194-201.