Lack of effect of synthetic atrial natriuretic factor on rubidium uptake by human erythrocytes

Vol., 130, August

No. 3. 1985 15,

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1985

LACK

OF ON

Natividad

EFFECT OF RUBIDIUM

Hernando,* Fernandez-Cruzf

* Renal

Physi

May

22,

SYNTHETIC UPTAKE Carlos

opathol

ogy

+Hosp i tal Medicine, Received

1066-1071

Pages

BY

ATRIAL HUMAN

Ca.ramelo,f; and Josh

San

Alberta Lopez-Nouoa*

M.

Fundac

Laboratory, Madrid,

Cl inico Complutense

NATRIURETIC ERYTHROCYTES

FACTOR

Tejedor,*

i on

Arturo

Jimenez

Diaz,

Spain Carl Uniuersi

Depar tmen Madrid,

us, ty,

t of Spain

1985

Summary. The effect of synthetic atria1 natriuretic fa.ctor on the ouabain-sensi tive and the furosemide-sensi tive rubidium uptake by human erythrocytes has been studied. This pept i de wi th potent diuretic and natriuretic effects did not affect any rubidium uptake sys.tem at concentrations of 10 -7 and lgmg M. These results do no t supper t that the natriuretic effect is based on the i nh i bi t i on of act i ve transport systems in the renal tubules. D 1985 Academic Press, Inc.

There atria of

i s

have

the

f 1u i d

1 ived

[2-81.

acids,

bu t

from

24

The

mechan

obscure

to

33

hemodynamic In i nves.t

acids

of

i gated

i E a

to

of show

also

s

further huma.n

0006-291X/85 $1.50 Copyright 0 1985 by Academic Press, Inc. Ail rights of reproduction in any form reserved.

that

tn

the

caus.es

regul

1 ong

mol

thesi ecu1

i c

ev i dence

poi

the

mechan

i sm

the

effect

s.UCh

n0t the

zed

by

seweral

Iof

152

e

ion

propert

on

r a. t s

of

port

ides

i a.1 bu t

into

iANF)

N-ter-minal i uret

ion

atr

ha.ue

factor s.yn

at

rat

injected

a. n d

i a.n

si 9n i f i ca.nt

muscle

pept

mammal

that

when

natr-

these of

effects

on

the

of most

mediated order-

rat

action

though

uresi

es

s. e y CIe n c e d

fragments

i sm

qranul _

natriuretic

the

i ng

observed

ventricular

a.trial

in

amino

al

di

purified,

several

been

at r- i a 1

of

the

s.upport

pa.rticipate ha.s

and

ANF

,

it

homogena.tes

i s.01 ated,

groups

we

pu r i f i ed

eu i dence

directly

Thus ,

Recently been

e

to

natriures.is

wh i 1 e

effects.

derabl ty

ume. or

I1 921,

has

consi

capa.ci

vol

homogenates sh or t

now

am i n 0 ra.nging

i es

the

rite

,

t0

rat

C-i-81

.

k i dney

is

d i s. t a 1

or

C9-111. define erythrocvtes

1066

of

act

ion

of

a

of

ANF

,

.z y n t h e t / c

Vol.

130,

ANF

BIOCHEMICAL

No. 3, 1985

(8-33

proper

aminoacids,

ooabain sensitive + + Na , K -ATPase pump

of

BIOPHYSICAL

Sharp

Merck,

on

t i es

measure

AND

and

RESEARCH

Dohme)

radioactive

tiue

Rb

uptake,

a measure

activity,

that

Our

results

demonstrate

the

studied

transport

systems

of

the

sinthetic in

natrioretic uptake,

and Na

on

a

f urosemi

de-

t i nward

-K

ANF human

with

rubidium

+ sensi

COMMUNICATIONS

does

not

cotranspor

t ,

affect

any

of

erythrocytes.

METHODS Natriuretic effect. Natriuretic effect was studied in consc i ous rats instrumented as preuiously pub1 ished C123. In brief, after anesthes i a, catheters were placed into a 1 ow dose nembutal femoral ue in and artery and in the urinary bladder, and conducted subcutaneous1 y to the dorsum of the neck and then exteriorized. Glomerular f i 1 trat i on rate (GFR) was measured by standard C ! HI min inul in c 1 earance techn i ques c121. After 2, 20, basal cl earance periods, ANF (0.1 ug/kg body weight in 0.25 ml of O.p% NaCl> was injected intravenously and 3, 20 min clearance periods performed. Urine was co1 lected in prewe i ghed plastic tubes containing mineral oil. Sodi urn, potassi urn and chloride were measured with a selective electrodes device (Astra 4, Beckmann>. +

+

Rb transport and ATPase measurement. Rb uptake was measured by a minor modification of the technique described by Bernstein and Israel C131. In brief, hepar in-treated blood was fresh1 y obtai ned from heal thy adul t donors by venous puncture. Plasma and buffy coat were separated by centrifugation and erythrocytes were washed three times with 10 volumes of ice-cold, i soosmot i c Kfree Krebs-Hensel e i t solution (140 mM NaCl, 1 mM CaCl2 , ;5 mM NaCCBH,, 5 mM dextrose). Then, 200 pl of the red ccl Is (packed ccl 1 volume of 97 & 1 %) were added to tubes containing 2.8 ml of K-free , Krebs-Hensel e i t sol ut i on, 1, 2, 4, 5, or 7 mM RbCl and about 300,000 cpm of 86Rb. Paired series of tubes contained al so ouabai n cl.2 ml’l) or furosemide (1 mM>.In two complete series of tubes ANF was added to achieve final concentrations of 10” and 10-g tfl. After 30 min incubation at 37 OC in a shalt i ng water ba t i-l , ccl 15 9.1e r e washed Z times. wi tti ice-c,~ld zgo r&f ‘S Ii C Le.,:z ‘5 e ) cczrrta i n i rgg 0 . 2 ml-: ou aba i i-1 * Exdi oact ccl 1 s i.*ias counted in 5 gamma counter. Erythroc,+te membranes were obtained k..ja, K ,ATPase prelu act i 1.~ i t:*, meas.ur ed ac An crJmp.we

-3. 1 y c. j 5. 0 f -_ dt tterences.

Ij a r ; jr, ot

c e ‘Z. <;g.j I>!<,~& ;+ *, r mea.ns

i151.

RESULTS

1067

i 1%~i t:ti

i ous! StiJdent

present

in

D:.+ i,:..pstnn y rjes,cr t

j E i bed test

ts; g,zrj

red :?,r,,:. [ 147 .

i,dere

IJE,~~

.3 r, L’

t,:

Vol. 130, No. 3, 1985

TABLE

1.

BIOCHEMICAL

EFFECT

OF

SYNTHETIC

BASAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ATRIcSL FLNCTI ON

ATRIAL

NATRIURETIC

O-20

min

f. 7.1

UNaV umol

GFR ml/mi

i

0.7

28.3

/m i n

0.6

i

0.1

4.1

i

1.2

RENFIL

INJECTION

min

40-60

min

/m i n

1.1

f

0.2

2.6

i

0.3

0.5

i

0.1

0.3

i

0.1

9.9

f.

1 .3

4.2

f

0.5

1.2

*

0.5

0.4

L 0.1

1.6

f

0.8

1.0

2

0.2

0.7

L 0.1

0.4

i

0.1

*

*

* n

Abbrev potasc, +: pertod

ON

*

7.2

in

UKU umol

FACTOR

FACTUR

20-40

U.V u 1 /m

NATRIURETIC

s

9r ations urn excre gnificant

U.U : urine t i on, GFR: difference

volume; glomerular

UN’aV, filtration 0.01) with

Sodium

excretion; rate. respect to

UKV the

basal

:

Vol.

130,

8lOCHEMlCAL

No. 3, 1985

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

[Rb! , mM Figure

The

1. Effect of synthetic atria1 natriuretic factor on Rb uptake human at several Rb by errthrocrtes concentrations. Pane I A: total Rb uptake . Pane 1 8: ouabain-sensitive Rb uptake. Panel C:, furosemide-sensi tive Rb uptake. 0: control experiments. 0 : experiments in presence of ANF 10-7 M. 0 :exper iments in presence of ANF 10-9 M. Dara are shown as average f SEM of 4 experiments, each by dupl icate.

mecha.n

studi

i sms

es of

distal

phenomenon

a

the

1.

studies use

these

suggest

cause

C17,18

of

i uret

decrease

in

natriuresis or

d i screpanc

the

or

a.re

lack can

of be

a

the

ea.r.

E om e

in

a

tubular the

fact

extract

different

as

suggest

definite

based

ci

reabsorption

others

atria1

in

not

tubular

whereas

purified

peptides,

effects

proximal

i es

uncompletely

synthetic

i c

Cl.51

19-111

These used

natr

more effect

that

some

whereas doses

or

by

the

a

others routes

of

administration. To

our

knowledge,

our

resul

ts

-9 that

ANF

at

doses

of

10

and

10

+ sensi

tiue

Rb

uptake,

which

demonstrate

first

reflects

M does + +

not

affect

Na

pump

activity

,K

ouaba

sensitive

sodium

uptake,

which 1069

reflects

Na

in

c131, +

furosemide

time

-7

+ ,K

i nwar

d

Vol.

130,

No. 3, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

+ cotransport,

and

which er

in

part

c131.

i

et

and

furosemide

represents

i throcytes

Pamnan

ouabain

al

passive

These

Cf91

insensitive

resul

which

Rb

inflow

ts

reported

of

are

compatible

that

crude

uptake

Rb wi

,

into

th

atria1

the

those

of

extract

did +

not

affect

by

ouabain

tail

arteries.

that

ANF

fai

dependent

led

ATPase

with

our to

to

a

the

inhibit

has

studi

i urn

es

failed

to

transport

and

medu

11 ary

possi

bi

that

Some

we

no

uptake

studies this

will

be

potent

egnents.

Sharp

&

and

required

of

,K

-ATPase

by

to

dilucidate

Inigo

Alvarez

inhi

not

of

ANF

on that for

t ion

and the

support diuresis

renal bi

ia

flow

and the

the1

by

tubules. of

erythrocyte

+

authors

for work

do

in

epi

responsible medullary

natriuresis

,161,

synthetic the

Further

ANF.

mechanism

of

action

of

agent.

The

Dohme, This

the

Na

natriuretic

Acknowl

factor.

and

in

t

support

be

results

evidence

+ rubidium

experiments

mechanisms

found

ated effect

increases

i nduces

c9-ii

i sol

be

postulated

diuresis

could

must

cotranspor been

inhibitory

Our

agreement

ANF

or

wi th

other

C181.

of

has

and

direct

through

transport

summary

5

effect

ANF

active

In

a

washout

1 i ty

inhibiting

experiments

effect

In

+ K

extract was + + Na ,K -ATPase

renal

ATPase

tubular

and

i al

effect

than

i uresi

i c

and

up take

demonstrate + Na and .

atr

diuretic

natr

diuresis

urea

that

cardiac

and

detect

hemodynam

natriuresis

of

c171.

i ntrarenal

membranes

reported

direct

data

ouabain-sensitive

different

for

far

been

Rb

our

erythrocyte

activity

a

sensitive

transport,

the

natriuretic

responsible

so

ouabain

to

of

Although

micropuncture

sod

to

mechanism

inhibition.

have

addition

the

Thus

due

In

it

affect

C19,XJl.

nor

activity

results

unable

a5

insensitive

has

acknowledge

supplying been de

Toledo

the supported Foundat 1070

Dr. synthetic in

Bean,

by

Merck,

natriuretic

atria1 part

ion.

A.G.

grants

from

FIG

Vol.

130,

BIOCHEMICAL

No. 3, 1985

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

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