Adults

with

corrected

oesophageal

atresia

is

oesophageal

function

associated

with

complaints

and/or

quality

of

life

The

aim

of

this

study

was

to

evaluate

oesophageal

function

after

correction

of

oesophageal

atresia

in

adults

and

to

investigate

the

association

between

complaints

oesophageal

function

and

quality

of

life

-LRB-

QoL

-RRB-

Twenty-five

adults

were

included

who

participated

in

previous

follow-up

studies

during

which

complaints

of

dysphagia

and

gastro-oesophageal

reflux

-LRB-

GOR

-RRB-

results

of

upper

gastrointestinal

endoscopy

oesophageal

biopsies

and

QoL

had

been

collected

Manometry

was

performed

in

patients

h

pH-measurements

were

performed

in

patients

pH-values

-LRB-

sample

time

s

-RRB-

were

calculated

using

criteria

of

Johnson

and

DeMeester

Associations

were

tested

with

ANOVA

and

Ten

patients

-LRB-

-RRB-

reported

complaints

of

dysphagia

seven

-LRB-

-RRB-

of

GOR

The

amplitude

of

oesophageal

contractions

was

low

-LRB-

mmHg

-RRB-

in

four

patients

-LRB-

-RRB-

pH-measurements

showed

pathological

reflux

in

three

patients

-LRB-

-RRB-

Patients

reporting

dysphagia

more

often

had

disturbed

motility

-LRB-

P

-RRB-

and

lower

scores

on

the

domains

general

health

perceptions

-LRB-

-RRB-

-LRB-

P

-RRB-

standardised

physical

component

-LRB-

-RRB-

-LRB-

P

-RRB-

and

physical

well-being

-LRB-

GIQLI

-RRB-

-LRB-

-RRB-

No

other

associations

were

found

This

study

shows

a

high

percentage

of

oesophageal

motility

disturbances

and

a

moderate

percentage

of

GOR

after

correction

of

oesophageal

atresia

Patients

reporting

dysphagia

whom

more

often

had

disturbed

motility

seemed

to

be

affected

by

these

symptoms

in

their

QoL

Introduction

At

present

the

survival

rate

of

patients

with

oesophageal

atresia

-LRB-

OA

-RRB-

is

approximately

-LSB-

-RSB-

With

the

decreased

mortality

the

interest

in

morbidity

especially

the

long-term

results

after

correction

of

OA

has

increased

over

the

years

Several

long-term

follow-up

studies

have

shown

that

long

lasting

gastro-oesophageal

reflux

-LRB-

GOR

-RRB-

is

a

frequent

problem

after

correction

of

OA

although

intestinal

metaplasia

as

its

theoretical

consequence

is

rare

-LSB-

-RSB-

In

these

studies

GOR

has

been

either

diagnosed

by

upper

gastrointestinal

-LRB-

GI

-RRB-

endoscopy

with

biopsies

by

h

pH-measurements

or

by

both

showing

varying

degrees

of

GOR

Besides

pH-measurements

oesophageal

manometry

has

been

performed

in

several

studies

showing

oesophageal

motility

disturbances

in

most

patients

-LSB-

-RSB-

The

true

impact

of

this

finding

on

individual

daily

life

is

not

clear

In

our

centre

we

have

performed

several

long-term

follow-up

studies

in

a

relatively

large

group

of

patients

after

correction

of

OA

-LSB-

-RSB-

Patients

underwent

upper

GI

endoscopy

with

biopsies

and

quality

of

life

-LRB-

QoL

-RRB-

measurements

-LSB-

-RSB-

The

first

aim

of

this

study

was

to

evaluate

the

presence

of

GOR

and

oesophageal

motility

problems

in

a

group

of

our

adult

patients

treated

for

OA

The

second

aim

was

to

investigate

if

there

was

an

association

between

complaints

oesophageal

function

and

QoL-measurements

This

association

has

not

been

investigated

before

Because

of

the

influence

of

complaints

of

dysphagia

and

GOR

on

daily

life

we

hypothesised

that

patients

with

complaints

have

a

poorer

QoL

than

patients

without

complaints

Patients

and

methods

Twenty-five

patients

over

years

of

age

who

participated

in

previous

follow-up

studies

after

correction

of

OA

-LSB-

-RSB-

and

gave

informed

consent

to

participate

were

included

in

this

study

In

these

previous

studies

data

regarding

the

results

of

upper

GI

endoscopy

biopsies

of

the

distal

oesophagus

and

QoL

had

been

collected

From

the

QoL

study

we

used

the

results

of

the

Medical

Outcome

Study

Short-Form

Health

Survey

-LRB-

-RRB-

and

of

the

gastro-intestinal

quality

of

life

index

-LRB-

GIQLI

-RRB-

-LSB-

-RSB-

After

approval

of

the

study

protocol

by

the

Medical

Ethical

Committee

all

patients

received

a

written

invitation

to

participate

in

the

study

All

patients

who

gave

their

informed

consent

underwent

manometry

and

pH-measurements

and

were

asked

if

they

experienced

difficulties

swallowing

solid

foods

-LRB-

dysphagia

-RRB-

or

experienced

heartburn

or

retrosternal

pain

-LRB-

GOR-related

complaints

-RRB-

Oesophageal

manometry

was

performed

using

the

measurement

stationary

system

-LRB-

MMS

Enschede

The

Netherlands

-RRB-

with

software

version

The

pressure

was

measured

with

the

Unisensor

Microtip

catheter

type

with

three

pressure

transducers

on

a

cm

distance

from

each

other

The

lower

oesophageal

sphincter

basal

or

resting

pressure

-LRB-

LOSP

-RRB-

and

relaxation

after

swallowing

the

motility

in

the

oesophageal

body

after

at

least

six

wet

swallows

of

ml

water

and

the

upper

oesophageal

sphincter

pressure

-LRB-

UOSP

-RRB-

and

relaxation

were

calculated

The

amplitude

of

oesophageal

body

contractions

was

categorized

as

low

-LRB-

mm

Hg

-RRB-

moderate

-LRB-

mm

Hg

-RRB-

or

normal

-LRB-

mm

Hg

-RRB-

The

encountered

oesophageal

motility

disorders

were

classified

according

to

the

guidelines

proposed

by

Spechler

et

al.

-LSB-

-RSB-

Based

on

the

basal

LOSP

LOS

relaxation

peristaltic

wave

progression

and

distal

wave

amplitude

oesophageal

motility

disorders

were

classified

into

four

categories

-LRB-

-RRB-

inadequate

LOS

relaxation

-LRB-

-RRB-

uncoordinated

contraction

-LRB-

-RRB-

hypercontraction

and

-LRB-

-RRB-

hypocontraction

or

ineffective

oesophageal

motility

pH-measurements

were

performed

using

the

Comfortec

dual

channel

pH

probe

-LRB-

Sandhill

Scientific

-RRB-

which

was

positioned

with

the

pH

measurement

points

and

cm

above

the

manometrically

established

upper

border

of

the

LOS

The

position

of

the

probe

was

checked

by

X-ray

Ambulatory

pH

measurement

was

performed

during

h

using

the

GORD

pH-recorder

-LRB-

Sandhill

Scientific

-RRB-

with

a

sample

time

of

every

s

The

pH

values

were

calculated

using

the

criteria

of

Johnson

and

DeMeester

-LSB-

-RSB-

The

data

were

entered

into

a

database

and

analysis

was

performed

using

SPSS

-LRB-

Statistical

Package

of

the

Social

Sciences

-RRB-

for

Windows

We

tested

for

association

between

functional

results

and

QoL

by

applying

ANOVA

and

or

Fisher

's

exact

tests

Definitions

Before

testing

for

association

the

results

of

functional

tests

were

dichotomized

If

upper

GI

endoscopy

showed

a

normal

oesophagus

or

grade

I

oesophagitis

-LRB-

according

to

the

modified

system

of

Savary-Miller

-RRB-

-LSB-

-RSB-

this

was

scored

as

normal

Grade

II

oesophagitis

or

worse

was

scored

as

abnormal

If

the

biopsies

of

the

distal

oesophagus

showed

normal

oesophageal

epithelium

or

mild

reflux

oesophagitis

-LRB-

according

to

Ismael-Beigi

-RRB-

-LSB-

-RSB-

this

was

scored

as

normal

Moderate

oesophagitis

or

worse

was

scored

as

abnormal

As

all

patients

were

diagnosed

as

having

ineffective

oesophageal

motility

this

variable

could

not

be

used

Instead

we

used

the

amplitude

of

oesophageal

body

contractions

as

a

measure

of

outcome

of

manometry

because

decreased

amplitude

implies

a

defective

peristaltic

function

of

the

oesophagus

The

results

were

dichotomized

as

normal

or

abnormal

-LRB-

moderate

or

low

amplitude

-RRB-

The

results

of

pH-measurements

were

also

dichotomized

as

normal

or

abnormal

-LRB-

minor

or

pathological

reflux

-RRB-

Results

Patient

characteristics

Patient

characteristics

are

described

in

Table

All

patients

had

undergone

a

primary

end-to-end

anastomosis

for

Gross

type

C

OA

Two

patients

were

using

proton-pump

inhibitors

they

stopped

taking

their

medication

two

days

before

the

start

of

the

study

Table

characteristics

of

patients

participating

in

the

studyMean

-LRB-

range

-RRB-

or

n

-LRB-

-RRB-

Age

-LRB-

years

-RRB-

-LRB-

-RRB-

Gender

-LRB-

-RRB-

-LRB-

-RRB-

Concomitant

congenital

anomalies

-LRB-

-RRB-

-LRB-

-RRB-

Anorectal

-LRB-

-RRB-

Cardiac

-LRB-

-RRB-

Vertebral

-LRB-

-RRB-

Limb

-LRB-

-RRB-

Other

-LRB-

-RRB-

Anti-reflux

procedure

in

past

-LRB-

-RRB-

-LRB-

-RRB-

Anastomotic

stenosis

in

past

-LRB-

-RRB-

-LRB-

-RRB-

Current

educational

status

Primary

-LRB-

-RRB-

Basic

high

-LRB-

-RRB-

Advanced

high

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Unfortunately

it

was

impossible

to

perform

manometry

and

pH-measurements

in

four

patients

In

one

patient

it

was

impossible

to

introduce

the

catheter

through

the

nose

due

to

resistance

of

the

patient

who

decided

to

withdraw

from

the

study

In

three

out

of

four

patients

it

was

impossible

to

introduce

the

catheter

due

to

oesophageal

stricture

All

of

these

three

patients

had

been

treated

for

anastomotic

stricture

in

childhood

one

patient

had

undergone

anti-reflux

surgery

in

childhood

Two

of

these

three

patients

had

complaints

of

dysphagia

at

the

time

of

the

study

The

stricture

of

these

three

patients

was

treated

with

dilatation

Since

dilatations

may

influence

the

results

of

manometry

and

pH-measurements

no

measurements

were

performed

in

these

patients

Due

to

a

technical

failure

the

data

of

the

manometry

of

one

patient

could

not

be

retrieved

Dysphagia

was

reported

by

patients

-LRB-

-RRB-

GOR-related

symptoms

were

reported

by

patients

-LRB-

-RRB-

Manometry

The

data

of

patients

could

be

analyzed

The

upper

oesophageal

sphincter

-LRB-

UOS

-RRB-

responded

normally

to

swallowing

in

all

patients

Mean

UOS

pressure

was

mm

Hg

Oesophageal

contractions

were

observed

in

all

patients

One

or

more

propulsive

contractions

were

observed

in

patients

All

patients

showed

one

or

more

non-transmitted

contractions

Retrograde

contractions

were

observed

in

patients

Mean

minimum

oesophageal

body

amplitude

pressure

was

mm

Hg

Mean

maximum

oesophageal

body

amplitude

pressure

was

mm

Hg

The

amplitude

of

oesophageal

body

contractions

was

low

-LRB-

mm

Hg

-RRB-

in

patients

-LRB-

-RRB-

moderate

-LRB-

mm

Hg

-RRB-

in

-LRB-

-RRB-

and

normal

-LRB-

mm

Hg

-RRB-

in

-LRB-

-RRB-

In

all

patients

the

LOS

showed

complete

relaxation

upon

swallowing

Mean

LOSP

was

mm

Hg

According

to

the

guidelines

of

Spechler

et

al.

all

patients

were

classified

as

having

ineffective

oesophageal

motility

-LSB-

-RSB-

The

LOS

pressure

was

normal

and

LOS

relaxation

was

complete

in

all

patients

Wave

progression

varied

from

normal

to

absent

progression

and

the

distal

wave

amplitude

was

low

in

of

wet

swallows

-LRB-

data

not

shown

-RRB-

pH-measurements

The

data

of

patients

could

be

analyzed

The

results

are

described

in

Table

pH-measurements

showed

a

normal

pattern

in

patients

-LRB-

-RRB-

minor

reflux

in

-LRB-

-RRB-

and

pathological

reflux

in

-LRB-

-RRB-

In

both

of

the

patients

who

were

taking

proton-pump

inhibitors

pH-measurements

showed

pathological

reflux

Table

Results

of

h

pH-measurements

in

patientsProximal

mean

SDDistal

mean

SDTotal

time

pH

Upright

time

pH

Supine

time

pH

SD

standard

deviation

Associations

The

association

between

symptoms

and

results

of

endoscopy

oesophageal

biopsies

manometry

pH-measurements

and

QoL

is

shown

in

Table

Patients

reporting

dysphagia

more

often

had

disturbed

motility

-LRB-

P

-RRB-

These

patients

also

had

significantly

lower

scores

on

the

domains

general

health

perceptions

-LRB-

P

-RRB-

and

standardised

physical

component

-LRB-

P

-RRB-

of

the

and

on

the

domain

physical

well-being

-LRB-

P

-RRB-

of

the

GIQLI

Table

between

symptoms

and

results

of

endoscopy

oesophageal

biopsies

manometry

pH-measurements

and

quality

of

lifeComplaintsDysphagiaGORYesNoYesNoEndoscopy

biopsies

scales

Physical

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Physical

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Emotional

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Bodily

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

General

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Social

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Mental

-LRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

component

summary

scales

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

GIQLI

scales

Physical

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

GI

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Social

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Emotional

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Total

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Data

are

shown

as

n

or

mean

-LRB-

standard

deviation

-RRB-

GOR

gastro-oesophageal

reflux

Medical

Outcome

Study

Short

Form

Health

Survey

PCS

physical

component

summary

MCS

mental

component

summary

GIQLI

gastro-intestinal

quality

of

life

index

P

P

P

P

No

association

was

found

between

complaints

of

GOR

and

quality

of

life

nor

between

the

results

of

endoscopy

and

the

results

of

pH-measurement

and/or

manometry

nor

between

the

results

of

oesophageal

biopsies

and

the

results

of

pH-measurement

and/or

manometry

-LRB-

data

not

shown

-RRB-

Problems

with

the

initial

surgical

repair

in

childhood

-LRB-

i.e.

anastomotic

leak

or

stricture

needing

dilatation

-RRB-

did

not

influence

the

findings

in

the

current

study

Discussion

The

results

of

the

present

study

confirm

that

oesophageal

motility

disturbances

are

frequently

present

after

correction

of

OA

Low

or

moderate

amplitude

of

oesophageal

body

contractions

were

found

in

patients

-LRB-

-RRB-

all

patients

showed

one

or

more

non-transmitted

contractions

and

retrograde

contractions

were

observed

in

patients

-LRB-

-RRB-

All

patients

met

the

manometric

features

of

ineffective

oesophageal

motility

as

described

by

Spechler

et

al.

-LSB-

-RSB-

The

manometric

findings

in

the

present

study

are

comparable

to

those

described

in

other

studies

-LSB-

-RSB-

Based

on

pH-measurements

the

prevalence

of

GOR

in

the

current

patient

group

is

lower

than

expected

pH-measurements

showed

minor

or

pathological

reflux

in

patients

-LRB-

-RRB-

of

these

patients

had

undergone

anti-reflux

surgery

in

the

past

In

other

studies

the

prevalence

of

GOR

based

on

pH-measurements

varies

from

to

-LSB-

-RSB-

Unfortunately

the

criteria

used

for

diagnosing

GOR

and

the

age

of

the

patient

groups

studied

also

vary

between

studies

The

question

is

what

the

influence

is

of

the

disturbed

oesophageal

motility

and

GOR

found

in

previous

studies

on

the

daily

life

of

adults

with

corrected

OA

This

is

the

first

study

in

which

complaints

and

QoL

after

correction

of

OA

have

been

combined

with

long-term

studies

of

oesophageal

function

endoscopy

oesophageal

biopsies

manometry

and

pH-measurements

Patients

reporting

dysphagia

more

often

had

disturbed

motility

and

showed

significantly

lower

scores

on

the

domains

general

health

perceptions

and

standardised

physical

component

of

the

and

on

the

domain

physical

well-being

of

the

GIQLI

However

GOR-related

complaints

were

not

associated

with

disturbed

oesophageal

function

and

did

not

influence

QoL

It

is

important

to

consider

that

this

group

of

patients

has

grown

up

with

these

symptoms

and

may

probably

have

gotten

used

to

it

The

fact

that

complaints

of

dysphagia

affect

the

QoL

and

GOR-related

complaints

do

not

affect

QoL

may

be

explained

by

the

influence

of

these

complaints

on

daily

activities

such

as

eating

Motility

problems

after

correction

of

OA

were

first

reported

by

Haight

-LSB-

-RSB-

in

The

main

cause

of

the

abnormal

oesophageal

motility

after

correction

of

OA

is

not

clear

Some

studies

propose

a

congenital

nervous

abnormality

as

the

cause

of

motility

disturbances

In

the

foetal

rat

model

for

OA

abnormalities

were

found

in

the

course

and

branching

pattern

of

the

vagal

nerves

-LSB-

-RSB-

However

an

acquired

cause

is

also

suggested

i.e.

surgical

damage

to

vagal

fibres

that

innervate

the

oesophagus

-LSB-

-RSB-

Abnormal

oesophageal

motility

can

cause

symptoms

of

dysphagia

It

can

also

worsen

the

effects

of

GOR

since

malfunction

of

the

peristaltic

pump

will

result

in

a

delayed

clearance

with

a

longer

period

of

stasis

of

refluxed

material

in

the

oesophagus

In

conclusion

this

study

shows

a

high

percentage

of

oesophageal

motility

disturbances

and

a

moderate

percentage

of

GOR

after

correction

of

OA

Only

patients

reporting

dysphagia

who

more

often

had

disturbed

motility

appeared

to

be

affected

by

these

symptoms

in

their

QoL

O O O B I O O O O O O O O B I I O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

oesophageal atresia
manometry
quality of life


Purinergic inhibition of Na + , K + , Cl − cotransport in C11-MDCK cells : Role of stress-activated protein kinases Previously , we observed that sustained activation of P2Y1 leads to inhibition of Na + , K + , Cl − cotransport -LRB- NKCC -RRB- in C11 cells resembling intercalated cells from collecting ducts of the Madin-Darby canine kidney . This study examined the role of stress-activated protein kinases -LRB- SAPK -RRB- in NKCC inhibition triggered by purinergic receptors . Treatment of C11 cells with ATP led to sustained phosphorylation of SAPK such as JNK and p38 . Activation of these kinases also occurred in anisomycin-treated cells . Surprisingly , we observed that compounds SP600125 and SB202190 , known as potent inhibitors of JNK and p38 in cell-free systems , activated rather than inhibited phosphorylation of the kinases in C11 cells . Importantly , similarly to ATP , all the above-listed activators of JNK and p38 phosphorylation inhibited NKCC . Thus , our results suggest that activation of JNK and/or p38 contributes to NKCC suppression detected in intercalated-like cells from distal tubules after their exposure to P2Y1 agonists . Na + , K + , Cl − cotransporter -LRB- NKCC -RRB- , providing electroneutral symport of monovalent ions in the stoichiometry of 1Na + : 1K + : 2Cl − and selectively inhibited by bumetanide and other high-ceiling diuretics , belongs to the superfamily of Cl − - coupled monovalent cation cotransporters . Two isoforms of this carrier have been cloned from vertebrate cDNA libraries . A ubiquitous NKCC1 isoform is expressed in all types of cells studied so far -LSB- 1 -RSB- , including the basolateral membrane of epithelial cells derived from collecting ducts of the Madin-Darby canine kidney -LRB- MDCK -RRB- -LSB- 2 -RSB- . This isoform contributes to cell volume regulation , adjustment of -LSB- Cl − -RSB- i above the values predicted by Nernst equilibrium potential and transcellular movement of salt and in the secretory epithelium -LSB- 1 , 3 -RSB- . Unlike NKCC1 , three alternatively spliced variants of the NKCC2 isoform were found exclusively on the apical membrane of renal epithelial cells from the macula densa and thick ascending limb of Henle 's loop . In the thick ascending limb , NKCC2 plays a key role in bulk salt reabsorption -LSB- 3 -RSB- , whereas in the macula densa this carrier is involved in sensing extracellular Cl − concentration and regulating renal function via tubuloglomerular feedback -LSB- 4 -RSB- . Because of compensatory reactions occurring in proximal segments via tubuloglomerular feedback , collecting ducts are considered a major target for regulation of water/salt and acid-base homeostasis by hormones and neurotransmitters , including vasopressin , bradykinin , atrial natriuretic peptide , prostanoids , mineralocorticoids , and catecholamines -LSB- 5 -RSB- . Under certain circumstances , extracellular nucleotides , such as ATP , UTP , and ADP , can also regulate renal epithelium function by activating two receptor subtypes . P2Y receptors are coupled to heterotrimeric G proteins and expressed in all types of renal cells studied so far , whereas P2X receptors correspond to ligand-gated cation channels , and their expression in the kidney is mainly limited to mesangial and vascular smooth muscle cells -LSB- 6 -- 10 -RSB- . The renal ion transport systems affected by P2Y receptors have been explored mainly in MDCK cells where extracellular ATP leads to transient Cl − secretion -LSB- 11 -RSB- . Two populations of cells have been isolated from commercially available stocks of MDCK cells : C7 - and C11-MDCK cells -LSB- 12 -RSB- . C7 cells have high transepithelial electrical resistance -LRB- Rte -RRB- , are peanut lectin negative , maintain pHi at 7.39 , and have large K + conductance , thus resembling principal cells from the collecting ducts . C11 cells , on the other hand , resemble intercalated cells ; they have low Rte , are peanut lectin positive , maintain pHi at 7.16 , and have large Cl − and H + conductances -LSB- 12 -RSB- . We were the first to report that transient activation of basolateral NKCC in ATP-treated C7 cells is evoked by elevation of -LSB- Ca2 + -RSB- i -LSB- 13 -RSB- and contributes to transepithelial Cl − secretion -LSB- 14 -RSB- . We also noted that in contrast to C7 and other cells studied so far , sustained application of ATP to C11 cells resulted in sharp inhibition of this carrier -LSB- 15 -RSB- that was absent under the addition of other modulators of transepithelial ion fluxes -LSB- 16 -RSB- . Several hypotheses might be proposed to explain the cell type-specific impact of extracellular nucleotides on NKCC activity . First , NKCC inhibition is mediated by the P2 receptor isoform whose expression is limited to intercalated cells . However , in a recent study , we established that NKCC inhibition in ATP-treated C11 cells is caused by activation of P2Y1 receptors -LSB- 13 -RSB- whose expression has been detected in most of the cell types studied so far , including C7 cells -LSB- 17 -RSB- . Second , in intercalated cells , sustained activation of P2Y1 receptors leads to inhibition of the cell type-specific NKCC isoform . This hypothesis contradicts data showing that prolonged exposure of C11 cell monolayers to ATP abolished the increment of bumetanide-sensitive 86Rb uptake across the basolateral membrane by transfection with human NKCC1 -LSB- 18 -RSB- . Third , delayed NKCC1 inhibition seen in ATP-treated C11 cells is mediated by the de novo expression of intercalated cell-specific inhibitor -LRB- s -RRB- of this carrier . However , the inhibitory action of ATP on NKCC activity in C11 monolayers was preserved in the presence of inhibitors of RNA and protein synthesis -LSB- 18 -RSB- . Fourth , NKCC1 inhibition is caused by the intercalated cell-specific signaling cascade that couples P2Y1 activation with NKCC1 inhibition . In renal epithelial cells , P2Y receptors trigger diverse signaling , including transient elevation of -LSB- Ca2 + -RSB- i , cAMP production , activation of phospholipase C -LRB- PLC -RRB- , phospholipase A2 -LRB- PLA2 -RRB- , prostaglandin synthase , protein kinase C -LRB- PKC -RRB- , and protein kinase A -LRB- PKA -RRB- -LSB- 19 , 20 -RSB- . We did not observe any effect of extracellular Ca2 + and intracellular Ca2 + chelator on NKCC inhibition in C11 cells -LSB- 15 , 21 -RSB- . The inhibitory action of ATP is also preserved in the presence of inhibitors of PLC , PLA2 , and prostaglandin synthase -LRB- U73122 , AACOCF3 , and indomethacin , respectively -RRB- as well as under activation of adenylate cyclase with forskolin and isoproterenol and inhibition of PKA with H-89 -LSB- 15 , 21 -RSB- . In C11 cells , NKCC inhibition was also revealed in the presence of the PKC activator 4β-phorbol-12-myristate-13-acetate -LRB- PMA -RRB- . However , the same action of PMA was also documented in C7 cells -LSB- 15 -RSB- . Moreover , suppression of NKCC in ATP-treated cells was not affected in the presence of PKC inhibitors -LRB- staurosporine and calphostin C -RRB- and after downregulation of this enzyme by chronic exposure to PMA -LSB- 15 , 21 -RSB- . Viewed collectively , these data strongly suggest that none of the above-listed intermediates of the intracellular signaling cascade is involved in the purinergic inhibition of NKCC -LSB- 15 , 21 -RSB- . In contrast to the above-listed signals documented in both principal - and intercalated-like cells , activation of P2Y receptors resulted in phosphorylation of members of the superfamily of mitogen-activated protein kinases -LRB- MAPK -RRB- , such as the extracellular signal-activated protein kinases Erk1 and Erk2 and the Jun N-terminal kinase JNK1 . Compound PD98059 , a potent Erk inhibitor , strongly suppressed Erk1 and Erk2 phosphorylation in ATP-treated C11 cells , but did not affect the inhibitory action of ATP on NKCC activity -LSB- 15 , 21 -RSB- . Considering these results , we designed the present investigation to examine the involvement of JNK and p38 , a member of the stress-activated protein kinases -LRB- SAPK -RRB- , in P2Y-induced NKCC inhibition . Methods Cell culture C11-MDCK cells were obtained from Dr. M. Gekle -LRB- University of Würzburg , Germany -RRB- and cultured in DMEM supplemented with 2.5 g/l sodium bicarbonate , 2 g/l hydroxyethylpiperazine ethanesulfonic acid -LRB- HEPES -RRB- , 100 U/ml penicillin , 100 μg/ml streptomycin , and 10 % fetal bovine serum -LRB- Gibco Laboratories , Burlington , ON , Canada -RRB- . The cells were passaged upon reaching subconfluent density by treatment in Ca2 + - and Mg2 + - free Dulbecco 's phosphate-buffered saline -LRB- PBS -RRB- with 0.1 % trypsin from Sigma -LRB- St. Louis , MO , USA -RRB- , then scraped from the flasks with a rubber policeman . Dispersed cells were counted and inoculated at 1.25 × 103 cells/cm2 . Before experimentation , the cells were subjected to 24-h serum deprivation in the presence of 0.1 % bovine serum albumin -LRB- BSA -RRB- . NKCC measurement Serum-deprived cells seeded in 24-well plates were washed twice with 2 ml of PBS and incubated for 30 min at 37 °C in 1 ml of Cl − - depleted medium A containing 140 mM Na gluconate , 5 mM K gluconate , 1 mM MgSO4 , 1 mM CaCl2 , 5 mM D-glucose , and 20 mM HEPES-Tris -LRB- pH 7.4 -RRB- . After 30 min of Cl − depletion , ATP and other test compounds were added at concentrations indicated in the figure legends . In a major part of the experiments , preincubation with ATP was limited to 30 min . Then , Cl − - depleted medium was replaced with 0.25 ml of medium B containing 140 mM NaCl , 5 mM KCl , 1 mM MgCl2 , 1 mM CaCl2 , 5 mM D-glucose , 20 mM HEPES-Tris -LRB- pH 7.4 -RRB- , ~ 1 μCi/ml 86Rb , and 50 μM ouabain ± 10 μM bumetanide . In 5 min , 86Rb uptake was terminated by the addition of 2 ml of ice-cold medium C containing 100 mM MgCl2 and 10 mM HEPES-Tris buffer -LRB- pH 7.4 -RRB- . The cells were then transferred on ice , washed 4 times with 2 ml of ice-cold medium C , and lysed with 1 ml of a 1 % SDS/4 mM ethylenediaminetetraacetate -LRB- EDTA -RRB- mixture . The radioactivity of the cell lysate was measured with a liquid scintillation analyzer . The rate of 86Rb -LRB- K + -RRB- influx was calculated as V = A/amt where A was the radioactivity in the sample -LRB- cpm -RRB- , a was the specific radioactivity of 86Rb -LRB- K + -RRB- -LRB- cpm/nmol -RRB- in the incubation medium , m was the protein content in the sample measured by modified Lowry 's method -LRB- mg -RRB- , and t was the incubation time -LRB- min -RRB- . NKCC activity was estimated as the rate of ouabain-resistant , bumetanide-sensitive 86Rb influx . Western blotting C11 cells grown in 6-well plates were incubated for 24 h in DMEM containing 0.1 % BSA and stimulated with ATP in Cl − - depleted medium A. Then , the cells were washed twice with ice-cold PBS and lysed in 200 μl of lysis buffer containing 20 mM Tris-HCl -LRB- pH 7.5 -RRB- , 150 mM NaCl , 10 % glycerol , 1 % Triton X-100 , 0.1 % SDS , 2 mM EDTA , 2 mM ethyleneglycoltetraacetic acid -LRB- EGTA -RRB- , 0.25 % deoxycholate , 1 mM phenylmethylsulfonyl fluoride , 1 μg/ml leupeptin , 1 μg/ml aprotinin , 200 μM Na-orthovanadate , and 1 mM NaF . The lysed cells were scraped and centrifuged at 13,000 rpm for 5 min , and an equal volume of clear lysates containing 20 μg of protein was treated for 5 min at 95 °C and applied on 10 % polyacrylamide gel , followed by electrophoresis and transfer to Immobilon-P membranes -LRB- Millipore Corp. , Bedford , MA , USA -RRB- . The membranes were washed with PBS containing 0.05 % Tween 20 -LRB- PBS-Tween -RRB- and 0.5 % skim milk , and incubated overnight at 4 °C with antibodies . After incubation , the membranes were washed 3 times with PBS-Tween , incubated for 1 h with horseradish peroxidase-conjugated antibody -LRB- Santa Cruz Biotechnology , Santa Cruz , CA , USA -RRB- , washed with PBS-Tween , and the protein bands were visualized with an enhanced chemiluminescence detection kit -LRB- Santa Cruz Biotechnology , Santa Cruz , CA , USA -RRB- before exposure to X-ray film . Relative protein content was quantified by the NIH image program . Chemicals ATP , ouabain , bumetanide , forskolin , and PMA were obtained from Sigma -LRB- St. Louis , MO , USA -RRB- ; anisomycin , cell-permeable inhibitors of JNK and p38 kinases -LRB- compounds SP600125 and SB202190 , respectively -RRB- and their negative controls -LSB- N1-methyl-1,9-pyrazoloanthrone -LRB- MPA -RRB- and compound SB202474 , respectively -RSB- were purchased from Calbiochem -LRB- La Jolla , CA , USA -RRB- , and 86RbCl from Dupont -LRB- Boston , MA , USA -RRB- . Anti-phospho-SAPK/JNK -LRB- Thr183/Tyr185 -RRB- and anti-phospho-p38 -LRB- Thr180/tyr182 -RRB- antibodies were provided by Cell Signaling Technology Inc. -LRB- Hornby , ON , Canada -RRB- . Salts , D-glucose , and buffers were obtained from Sigma -LRB- St. Louis , MO , USA -RRB- and Anachemia -LRB- Montreal , QC , Canada -RRB- . Statistics The data were analyzed by Student 's t-test or the t-test for dependent samples , as appropriate . Significance was defined as p < 0.05 . Results In a previous study , we observed that 1-h preincubation of C11 cells in Cl − - depleted medium abolished the transient activation of NKCC by ATP but sharply increased baseline NKCC activity , allowing more precise estimation of the inhibitory action of P2Y-induced signaling -LSB- 13 -RSB- . Considering these results , Cl − - depleted cells were used in the present experiments . Figure 1 shows that NKCC inhibition in Cl − - depleted cells occurred after a 10-min lag phase with maximal attenuation of the carrier 's activity in 30 -- 40 min of ATP addition . Fig. 1Time dependence of NKCC inhibition in C11 cells triggered by the addition of 100 μM ATP Incubation for 2 min with ATP was sufficient to induce full-scale phosphorylation of JNK1 -LRB- p46 -RRB- and p38 kinases -LRB- Fig. 2 -RRB- . Phosphorylation of JNK2 in ATP-treated cells was observed after a 5-min lag phase , and its time course was negatively correlated with NKCC inhibition . In contrast to sustained JNK phosphorylation , p38 phosphorylation was partially normalized in 40 min of ATP addition . Activation of adenylate cyclase with forskolin did not affect SAPK phosphorylation . Very modest p38 and JNK phosphorylation was detected in 20 min of PMA addition . These results show that neither PKA nor PKC is involved in SAPK phosphorylation by ATP , which is consistent with the lack of involvement of these serine-threonine kinases in inhibition of NKCC in ATP-treated C11 cells -LSB- 15 , 16 , 21 -RSB- . Fig. 2Representative blots showing the time-dependent action of 100 μM ATP , 10 μM forskolin , and 0.1 μM PMA on SAPK phosphorylation in C11 cells Consistent with numerous previous observations , anisomycin sharply elevated SAPK phosphorylation -LRB- Fig. 3 -RRB- . Figure 4a reveals that the action of anisomycin on JNK phosphorylation was lower than that of ATP ; in contrast , anisomycin was a more potent activator of p38 phosphorylation than ATP . However , these differences were not statistically significant . We observed that 40-min exposure to anisomycin resulted in the inhibition of NKCC to about the same level as was detected in ATP-treated cells -LRB- Fig. 4b -RRB- . Fig. 3Representative blots revealing the effects of SP600125 -LRB- a -RRB- , MPA -LRB- b -RRB- , SB202190 -LRB- c -RRB- , and SB202474 -LRB- d -RRB- on JNK and p38 phosphorylation in C11 cells under control conditions and in the presence of 100 μM ATP or 0.1 μM anisomycin . ATP and anisomycin were added during the last 40 min of preincubation of cells in Cl − - depleted medium . Compounds SP600125 , MPA , SB202190 , and SB202474 were added at the indicated concentrations 30 min before ATP or anisomycinFig . 4Effect of ATP and anisomycin on JNK and p38 phosphorylation -LRB- a -RRB- and NKCC -LRB- b -RRB- in C11 cells . 100 μM ATP and 0.1 μM anisomycin were added during the last 40 min of preincubation of cells in Cl − - depleted medium . Phosphoprotein content and NKCC activity in the absence of ATP and anisomycin were taken as 1.0 and 100 % , respectively . Mean values from three -LRB- a -RRB- or four -LRB- b -RRB- independent experiments are shown With cell-free systems , it was demonstrated that several newly synthesized cell-permeable compounds inhibited purified or recombinant JNK and p38 without significant action on other protein kinases . Thus , compound SP600125 inhibited recombinant JNK1-3 with an IC50 of 0.05 -- 0.20 μM , whereas half-maximal inhibition of other members of the MAPK superfamily , such as Erk and p38 as well as PKA and cyclin-dependent kinases , was observed at concentrations higher than 5 μM -LSB- 22 , 23 -RSB- . It should be underlined that SP600125 was a much less potent inhibitor of JNK in intact cells . Indeed , inhibition of JNK in the anisomycin-treated KB-3 carcinoma cell line was detected after 1-h preincubation with this compound at concentrations higher than 3 μM -LSB- 23 -RSB- . To decrease JNK phosphorylation by two - to threefold in PMA-treated Jurkat cells , SP600125 should be added at a concentration of 50 μM -LSB- 22 -RSB- . Surprisingly , we observed that at a concentration of 20 μM SP600125 and SB202190 , i.e. , potent inhibitors of p38 SAPK in cell-free systems , increased JNK and p38 phosphorylation to the level comparable with their phosphorylation in the presence of ATP and anisomycin -LRB- Fig. 3a and c -RRB- . Moreover , like other potent activators of JNK and p38 phosphorylation , such as ATP and anisomycin , both SP600125 and SB202190 sharply decreased NKCC activity in C11 cells -LRB- Fig. 5 -RRB- . In contrast , neither JNK/p38 phosphorylation -LRB- Fig. 3b and d -RRB- nor NKCC activity in the presence and absence of ATP -LRB- data not shown -RRB- was affected by the inactive structural analogues of SP600125 and SB202190 , compounds MPA and SB202474 , respectively . Fig. 5Effect of ATP , SP600125 , and SB202190 on NKCC activity in C11 cells ; 100 μM ATP was added during the last 40 min of preincubation of cells in Cl − - depleted medium . Compounds SP600125 and SB202190 were added at 20 μM concentration 30 min before ATP . NKCC activity in control cells was taken as 100 % . Mean ± SE values from experiments performed in quadruplicate are shown Increased phospho-JNK and phospho-p38 levels in SP600125 - and SB202190-treated C11 cells , revealed in our study , are in contrast with the inhibitory action on these SAPK in cell-free systems -LSB- 22 , 23 -RSB- . Indeed , the Calbiochem catalogue notes that SP600125 and SB202190 inhibit purified JNK and p38 with IC50 of 50 and 16 nM , respectively . It should be underlined that both compounds are ATP-competitive kinase inhibitors , and the data reported were obtained at an ATP concentration of 5 μM , i.e. , 1,000-fold lower than the intracellular ATP concentration . For this reason , in experiments with intact cells , these inhibitors are used at much higher concentrations where diverse side effects can not be excluded . For more details , see -LSB- 24 -RSB- . Discussion The data obtained in our study show that NKCC inhibition in ATP-treated C11 cells is accompanied by activation of JNK and/or p38 . We also report here that activation of these SAPK with three distinct stimuli , such as actinomycin , SP600125 , and SB202190 , results in NKCC inhibition . Additional experiments should be performed to establish the causal relationship between SAPK activation and NKCC inhibition . It should also be noted that the modest inhibitory action of ATP on NKCC was preserved in the presence of the above-listed activators of SAPK . This observation suggests that the signaling cascade is not limited to the activation of these kinases . Recent studies have demonstrated that the with no lysine -LRB- K -RRB- kinase member WNK4 downregulates NKCC1 via the STE20-related kinase PASK interacting with the conserved domain in the NKCC1 N-terminus -LSB- 25 -- 27 -RSB- . The role of WNK kinases in ATP-induced NKCC1 inhibition limited to intercalated cells deserves further investigation . Two questions should be answered to evaluate the role of purinergic-dependent ion transporters in the regulation of renal function in vivo . First , are concentrations of ATP and other extracellular nucleosides sufficient for activation of P2Y receptors on the apical and basolateral surfaces of collecting duct epithelia ? Second , what is the physiological consequence of purinergic regulation of NKCC and other ion transporters in principal and intercalated cells comprising collecting ducts ? In peripheral blood , ectonucleotidases maintain circulating levels of ATP < 10 nM -LSB- 28 -RSB- , a concentration at which renal P2 receptors with ID50 -LRB- ATP -RRB- > 1 μM -LSB- 29 -RSB- can not be activated . However , nucleosides can act in paracrine and autocrine ways , reaching high extracellular concentrations after sympathetic -LSB- 30 -RSB- and cholinergic -LSB- 31 -RSB- stimulation or exposure to shear -LSB- 32 , 33 -RSB- , osmotic -LSB- 34 -- 36 -RSB- , and ischemic stresses -LSB- 33 , 37 -- 39 -RSB- . Sympathetic innervation is probably the major source of nucleosides for basolateral purinergic receptors -LSB- 38 -RSB- whereas shear and osmotic stresses , occurring in proximal tubules and the juxtaglomerular apparatus , contribute to ATP release through the apical membrane in tubular fluid -LSB- 20 , 40 -RSB- . Using chimeric Staphylococcus aureus protein A-luciferase bound to endogenous antigens on the human airway epithelium , it was shown that hypotonic shock leads to elevation of ATP concentration in surface fluid from ~ 0.001 to 1 μM -LSB- 41 -RSB- . Released ATP is rapidly metabolized by ectoenzymes to adenosine . Importantly , modest elevation of UTP -LSB- 33 -RSB- and adenosine -LSB- 42 -RSB- sharply increased ATP release from MDCK cells , suggesting a positive feedback loop . It is well documented that osmolality of tubular fluid in collecting ducts varies from ~ 400 to 1,500 mOsm -LSB- 43 -RSB- . Baseline concentrations of ATP and other P2Y agonists in collecting duct tubular fluid and their modulation by osmotic perturbations remain unknown . Both C7 and C11 cells , resembling principal and intercalated cells , are highly abundant with P2Y1 and P2Y2 receptors -LSB- 13 , 17 -RSB- . In these cells , P2Y1 and P2Y2 receptors have been shown to mainly reside on the basolateral and apical membranes , respectively -LSB- 44 -RSB- . Using monolayers of C7 cells , it was noted that basolateral P2Y1 receptors activate Cl − secretion via PLA2-PKA-mediated activation of Cl − channels -LSB- 17 -RSB- . Our studies demonstrated that P2Y2 receptors contribute to Cl − secretion via Ca2 + - calmodulin-mediated activation of NKCC1 -LSB- 14 -RSB- -LRB- Fig. 6a -RRB- . Glanville and coworkers reported that in mouse collecting ducts , basolateral NKCC possesses the same affinity for K + and -LRB- K0 .5 ~ 1.5 mM -RRB- -LSB- 45 -RSB- . Because bumetanide markedly decreased the acidification rate in NH4Cl-treated cells , they proposed a key role for NKCC1 in H + secretion and handling by intercalated cells . With intercalated-like C11 cells , we observed that apical P2Y2 receptors transiently activated NKCC1 via Ca2 + - calmodulin-mediated signaling , whereas sustained activation of basolateral P2Y1 receptors inhibited this carrier -LSB- 13 -RSB- . The present data suggest that NKCC1 inhibition in intercalated cells evoked by P2Y1 agonists is caused by activation of SAPK -LRB- Fig. 6b -RRB- . Further studies should be performed to examine the relative contribution of purinergic signaling in the final adjustment of salt reabsorption and acid-base balance by principal and intercalated cells in vivo as well as the pathophysiological implications of these regulatory pathways . Fig. 6Possible mechanisms of purinergic signaling in the regulation of Cl − secretion and acid-base homeostasis by principal -LRB- a -RRB- and intercalated -LRB- b -RRB- cells from collecting ducts in vivo . 1 Cl − channels , 2 H + - ATPase , 3 anion exchange , PLC phospholipase C , IP3 inositol 1,4,5-triphosphate , COX cyclooxygenase , PGE1 prostaglandin E1 , CAMK -LRB- Ca2 + + CaM -RRB- - dependent protein kinase , CA carbonic anhydrase , a.m. and b.m. apical and basolateral membranes , respectively . For other abbreviations and more details , see text

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

cl− cotransport
k+
na+
stress-activated protein kinases