Vol.

167,

March

No.

16,

2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

1990

413-418

ATRIAL NATRIURETIC PEX'TIDE REDUCESTHE BASAL LEVEL OF CYTOSOLIC F'REE Ca2+ IN GUINEA PIG CARDIAC MYOCYTES Masahi'ko

Teil

, Minor"

Akihiro 1Department

Hazama3,

of Molecular

and 31)epartment

1*, Toshinori

Horie

Yasunobu

2.Third

Diagnostics,

of Physiology, Sakyo-ku,

Received

.January

29,

Makita2,

Okada3,

Hiroshi

Department

Kyoto

University

Kyoto

606,

Suzukil,

Kawai 2

and Chuichi

of Internal

Faculty

Medicine

of Medicine,

Japan

1990

The cytosolic free Ca2+ concentration ([Ca2+].) was monitored in quiescent atria1 and ventricular myocytes isolated from'guinea-pig hearts by the furafluorescence ratio technique. Recombinant human atria1 natriuretic peptide (ANP) was found to reduce their basal [Ca2+]. level a dose-dependent manner. Dibutyryl-cGMP mimicked the effe:t of AN;: Neither the prior application of caffeine nor removal of extracellular N?: impaired the ANP effect. ANP had no inhibitory effect on voltage-gated Ca cur ents measured by a whole-cell patch clamp technique. The ANP-induced [Ca St Ii decrease was abolishe Thus, it is concluded that ANP reduces the basal the cGMP-mediated activation of the plasma myocytes. 0 1990 Rcademlc Press,

Inc.

The

cardiac

contractile release

atrium

but

also

atria1

is

of

particular

an endocrine

natriuretic

peptide

natriuretic

and

vasorelaxant

activity

free

Ca 2t level

accelerate

as a second

the

extracellular

([Ca2+li) for

the

messenger ANP level

has not first

for

yet

time,

[Ca2+]. 1 in mammalian

examined

evidence

that

(ANP),

(1).

the

which

Since

cytosolic

maneuvers

human

only

a and

diuretic, raising

the

Ca2+ appears

to

the

free

in the myocardium.

and ventricular

not

produce

possesses

(2-4),

However,

recombinant

is

cardiocytes

ANP secretion

secretion.

affects

been atria1

the

as it

Atria1

hormone

cytoplasmic act

interest

organ.

question whether 2t Ca concentration

This

study

ANP reduces

provides, the

basal

myocytes.

MAT5tIALSANDMETHODS Single atria1 and ventricular cells were enzymatically isolated from previously (5). To load with fura-2, adult guinea-pig hea ts, as described the myocytes (2 x 10 E cells) were suspended in a 2-ml of Tyrode's solution containing 10 uM fura-2/AM (Molecular Probes) and 5 % bovine serum for 30

*To whom reprint

requests

should

be addressed. 0006-291X/90

413

$1.50

Copyright 0 I990 by Academic Press, Inc. All rights of reproducrion in any form reserved.

Vol.

167,

No.

BIOCHEMICAL

2, 1990

AND

BIOPHYSICAL

RESEARCH

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min at 36'C. The cells were then washed once and stored on ice for at least 2 h before experiments. The cells were inoculated on the coverslip bottom of a small chamber (0.5 ml) placed on the stage of an inverted microscope (Nikon TMD). The bath was perfused with Tyrode's solution. The method of [Ca2+ii measurement was almost the same as described previously (6). The fluorescence images at excitation wavelengths of 340, 360 and 380 nm were collected from single fura-2-loaded myocytes at room temperature (about 25'C) and were digitized by an image processor (Hamamatsu Photonics). The fluorescence intensity ratio was calculated after background subtractio The 340 nm/380 nm ratio was found to be more sensitive to a small [Ca 9.+I. change than the 340 nm/ 360 nm, since the fluorescence int nsity at nm changed in mirror image with that at 380 nm. Thus, the 540 [Ca 5+ Ii was estimated as the mean value of the 340 nm/380 nm ratio in a given cardia In some experiments the furaratio was calibras;d into the [Ca ~+myocyteIi value using a sigmoidal standard curve determined in Ca EGTA buffer solutions containin 2 uM furafree acids (Molecular Probes) !2+ and various concentrations of Ca . voltagePatch-clamp whole-cell recordings (7) were made to measure gated Ca2+ currents in single ventricular myocytes incubated in Tyrode's To eliminate the contribution of both Nat and lowsolution at 33OC. To monitor threshold Ca2+ cusyents, a holding potential of -40 mV was used. high-threshold Ca currents, the cell was depolarized to t10 mV for 300 ms every 10 s. It is known that inward currents thus recorded consist both of transien exclusively due to the activation of high-threshold (Lty e) Ca5+c~;a~n~~s and of sustained currents, due to activation of the Preliminary Ca 5t channels as dell as delayed rectifier K+ channels. experiments showed that ANP did not affect the outward K+ currents. Tyrode's solution contained (in mM) 136.5 NaCl, 0.3 NaH2P04, 5.4 KCl, 1.8 CaC12, 0.5 MgC12, and 5 HEPES-NaOH (pH 7.4). A Nat-free solution was prepared by replacing NaCl in the Tyrode solution with equimolar N-methyl Dglucamine. Choline was not used because of its possible reactivity with the A high Kt solution acetylcholine receptor. was prepared by replacing NaCl in Tyrode's solution with KCl. During whole-cell recordings the cell was dialyzed with a patch pipette solution containing (in mM) 110 K-aspartate, 20 KCl, 5 MgCl 5 K2ATP, 5 Na2-creatine phosphate, 0.2 Na2GTP, 5 EGTA and 5 HEPES-KOH (pH 314). Since it is known that the amino acid sequence of ANP is almost identical throughout mammalian species, we employed recombinant ~1(l-28) human ANP, which was a gift from Suntory Ltd. Other drugs used were caffeine (Nakarai), N-methyl D-glucamine (Nakarai), sodium orthovanadate (Na3V04: Sigma) and dibutyryl cGMP (Sigma). Numerical data are given as mean + S.E. (number of cells). Statistical significance was evaluated by Student's paired t-test. RESULTS AND DISCUSSION Figure levels

1 depicts

in

single

reversibly

reduced

dependency

of

the

This

the

receptor

preparation

over

dose-response and (see

messenger,

membrane-permeable

of ANP effects

over

In

2 in Ref.

8).

well activation It

is

mediates the ANP-dependent cGMP analog, dibutyryl 414

on basal

In both

rod-shaped maximum with

therefore [Ca 2t Ii

rat

at

for

at

about

low8

ANP binding that

reduction. also

dose-

myocytes,

cardiac

likely

ANP

to saturate

effect

those in

cGMP,

the

ventricular appeared

[CaZtli

myocytes,

2 summarizes

The effect a half

coincides cyclase

cells.

Figure

10 -9 M .

10m7 M, giving curve

(B)

[Ca2+li.

on [Ca2+li.

guanylate Fig.

results

and ventricular

basal

at doses

ANP concentrations M.

(A)

ANP effects

ANP was effective

second

representative

atria1

reduced

to

sarcolemmal cGMP, as a In fact, the

basal

a

Vol.

167, No. 2, 1990

BIOCHEMICAL

Atrial

A

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

cell

ANP

(O.lrrM)

.

1

-l-

2 14 Ventricular

2 I B

cells

ANP

. 1

(0.5uM).

--.-

/

2 L--J--c

db-cGMP

( 1 mM 1

.

1

5

0 Time

Figure (A)

and nm), ratio (3)

1. Effects of ANP and dibutyryl cGMF' (db-cGMP) on [&'+I. in atria1 arldentricular (B, C) myocytes. The abscissa indicates timk in minute the ordinate the ratio of t e furafluorescence excited at 340 and 380 which is indicative of [Ca9 +li. Percent reduction in the fluorescence was 20 + 8 % (3) in atria1 cells treated with 0.1 uM ANP or 25 + 9 % in ventricular cells exposed to 1 mM db-cGMP.

[Ca2+li

in

(Fig.

ventricular

depletion

in

from

Fig.

the

involvement

Even

in

though

the

effect

was not

fully

reversible

involved

in

sensitive

ANP reduced

the

basal

[Ca2+li

even

after

the

[Ca2+li

channel

the

of

currents

pulses

in

frog in

j.nhibited to inhibit

on the it

3B,

by the the

of the

that not

application

sarcolemmal

Thus, out.

3C,

ANP

myocytes. may not

reduction

in

could

be

voltagetake

obtained

the place

by both

voltage-gated

Ca2+ channel by ANP: ANP did not

impaired

by

of

a depolarizing

high-threshold

upon

(L-type)

depolarizing not

shown),

command as found

(10).

prior

of the

Fig.

the membrane

induced

myocytes

(9).

membrane

by 1 nM ANP (data

ANP effect

membrane

ventricular

of evidence

amplitude

myocytes

suggested

in

depolarization

were

ventricular

in

ANP-induced

e 1 evation

the

plasma is

the

The

shown

that

two pieces

suppressed

ventricular Fig.

in

indicated

3D). in

unlikely

Ca2+

can be ruled

as

sarcolemmal

is

membrane

[Ca2+li

K+ (Fig.

Therefore, (11). ANP-induced down-regulation activation

[Ca2+li

by depolarizations

it

through

basal

Also,

of

by 10 mM caffeine ANP effect

Na+,

Furthermore,

recorded

As shown known

extracellular

(-110 mV) was not

previously

the

techniques

degree

concentration

(SR)

by SR in

no appreciable

myocytes. induced

affect

reticulum

participates

and patch-clamp

activities

largely

of

ANP effect. since

quiescent

Ca2+

sarcoplasmic Ca 2+ uptake

absence

Ca2+

fura-

3A,

decreased the 2t Na+-Ca exchangers

Therefore,

basal

of

the

consistently

in

myocytes,

1C). As shown

the

10

(min)

in reducing

the

basal

[Ca2+]i

was

of 40 UM orthovanadate, which is 2+ pump in a variety of cell species Ca

that

the

basal

major

[Ca2+li is 2+ Ca pump.

415

mechanism

responsible

an increased

for

Ca2+ efflux

Vol.

167,

No.

BIOCHEMICAL

2, 1990

200

AND

BIOPHYSICAL

RESEARCH

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* P