Cardiac substances that influence blood-pressure II. Potent pressor activity in rat and rabbit atrial muscle

Vol.

129,

No.

June

14,

1985

2. 1985

BIOCHEMICAL

AND

BIOPHYSKAL

RESEARCH

COMMUNICATIONS Pages

CARDIAC SUBSTANCES TBAT INFLUENCE BLOOD-PRESSURE POTENT PRESSOR ACTIVITY IN RAT AND RABBIT ATRIAL

II.

J.J. Institute

Received

Nieuwenhuis

of Cellular University of

April

22,

and J.J.

Physiology, Pretoria,

472-478

MUSCLE

Theron

Department of Physiology, Pretoria, South Africa

1985

In addition to their natriuretic, diuretic and vasodilator activities, freshly prepared aqueous extracts of either rat or rabbit atria1 myocardium were shown to elicit significant increases in the blood-pressure of anaesthetized rats. Small aliquots (0.05 me1 intravenously administered caused a transient rise in mean arterial blood-pressure of up to 20 %. Slow infusion of 0.4 me right atria1 extract (corresponding to about one half of a rabbit right atria1 lobe) during 90 seconds caused the expected natriuresis and diuresis, together with a sustained elevation in arterial blood-pressure (ca 25 %) that returned to normal within 3 minutes. This potent pressor activity could not be detected in ventricular extracts. It was furthermore readily separable from the natriuretic peptides and catecholamines by ultrafiltration. The atria1 pressor factor is a small proteolytically unstable molecule (300 - 1000 dalton). 0 1985

Academic

Press,

Extracts

Inc.

from

ventricles,

contain

vasodilator

13-71,

Crude to

extracts

elicit

venous been

transient

both

to

the

of

the

mammalian

peptides (8,

atria1

9)

(l-4)

and

and

depressor into

adenine

with

as

which

ventricular

rats

nucleotide

in

arterial

(1,

2,

were

content

of

11). the

sometimes

reported upon

This crude

the

activities.

blood-pressure 10,

to

associated

(101 tissue

opposed

are

cardioinhibitory

responses

anaesthetized

heart,

activity heart

intrahas

tissue

ex-

rabbit

or

(11). We have

baboon

observed

atria,

degradation in

lobes

natriuretic

from

related

atria1

depressor

infusion

tracts

the

the

reported tomized

rats

increase

in

0006-291X/85 Copyright A# rights

under

labile

molecules,

arterial (131

time

prepared of

rat

some

ago

conditions

the

infusion,

of

a crude

extract

mean

arterial

$1.50 0 1985 by Academic Press, qf reproduction in any form

(see under of

that that

elicit

blood-pressure

that

(121

rat

extracts

minimize

an

immediate

also

11).

atrium,

472

from

ischemia and Very

anaesthesia,

blood-pressure.

Inc. reserved.

crude

into produced

and

biological

significant recently bilateral a small

increase it

has nephrec-

transient

been

Vol.

129,

No. 2, 1985

Although from

both

activity of

biologically rat

could

the

BIOCHEMICAL

ventricular

and not

unstable,

rabbit be

atria1 found

AND

we

have

myocardium, in

other

BIOPHYSICAL

succeeded a potent

types

of

rabbit

RESEARCH

in pressor muscular

COMMUNICATIONS

partially

isolating

factor.

This

tissue,

inclusive

myocardium. MATERIALS

AND

METHODS

Only glass-distilled, deionized water were used. Pronase-CB" was obtained from Calbiochem-Behring (USA). L-norepinephrine bitartrate and the adenine nucleotides AMP, ADP and ATP were from Sigma (USA). All other chemicals were analytical reagent grade from E. Merck (Germany). Isotonic saline consisted of 0.9% aqueous sodium chloride. Ultrafiltration membranes, supplied by the Amicon Corporation (USA), were prepared as suggested by the manufacturer. The Ultropac Lichrosorb RP-18 (5 pm) liquid chromatography column was supplied by LKB (Sweden). Male New Zealand white rabbits and male Sprague-Dawley rats were kept in small cages and fed a balanced diet and tap water ad libitum. Preparation of the crude tissue extracts: Fifty rats, weighing between 300 and 400 g were terminated by decapitation and the hearts immediatly excised. Both atria and the apical halves of the ventricles were dissected out, rinsed in cold saline and blotted dry. The atria and ventricles were separately pooled and placed in liquid nitrogen. The tissues were crushed while frozen, quickly weighed and half of each sample homogenized in ice cold water (1 gin 4 me; rotating teflon pestle in a glass tube). The other halves were homogenized The extracts were in cold 5 mM sodium phosphate-buffered saline, pH 7.2 (PBS). heated in a boiling water bath for 2 min, centrifuged (12000x g, 30 min, 4°C) and placed on ice. Each rabbit, weighing from 2 to 4 kg, was terminated by a single blow at the apical half of the ventricle and the back of the head. Working rapidly, the left and right atria1 myocardium were dissected. Samples were also taken from the tn. femoris biceps (white skeletal muscle). The samples were rinsed in cold saline, blotted dry, separately wrapped in aluminium foil and placed in liquid nitrogen. The process was repeated for a total of 10 animals. From each individual sample was prepared a crude extract in water (1 g in 4 me) as above.

The injection) effect extract

effects of each were determined on natriuresis/diuresis (0.4 ml administered

sample on mean arterial blood-pressure within the hour (see below and were determined for a pooled during 90 secl.

Table rabbit

(0.05 1).

me

per

The right

atria1

Ultrafiltration: The rabbit myocardial crude extracts obtained above were pooled according to origen (designated RA, LA and V), and 4 mt of each (corresponding to 1 g of fresh tissue) was filtered sequentially through a YM2 (retentivity 1000 dalton) and a YCO5 (retentivity > 300 dalton) ultrafiltration membrane. Each retentate was resuspended in 2 me water and its biological activity evaluated (see below and Table 11. The low molecular weight (< 300) pressor activity: The final ultrafiltrates obtained from the rabbit right and left atrium and ventricle (respectively designated RA-5F, LA-SF and V-SF), each elicited a pressor effect qualitatively similar to L-norepinephrine (14). Using the bitartrate salt of the latter, a dose-pressor response curve was constructed (l-100 uM in saline), and the The norepinephrine activities in norepinephrine-equivalent units determined. content of RA-5F was also measured by high performance liquid chromatography Treatment of a small portion of RA-5F with activated aluminium oxide (15). (16) abolished the activity, catecholamines being effectively absorbed at neutral pH (16).

473

Vol.

129,

BIOCHEMICAL

No. 2. 1985

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

The YCOS-retentate obtained from The 300 - 1000 dalton pressor activity: eliciting a pressor response, was treated with the rabbit right atrium (RA-5R), A small sample (0.02 me) was mixed with an a proteolytic enzyme a* follows. equal volume of 0.2M Tris (hydroxymethyl)aminomethane - hydrochloric acid buffer, One half of the mixture, to which was added 0.025 me water served as pH 7.6. control. The remaining half was treated with 0.025 me Pronase-CB solution After incubation for six hours at 38'C, the solutions were filtered (1 mq/me). through a PM 10 membrane (retentivity 10 000 daltonl and retested for their effects on arterial blood-pressure. Determination of the adenosine nucleotide content of the rabbit muscle extracts: Four samples from each tissue type were selected at random (before their biological activities were determined). The concentrations of 5'-ATP, 5'-ADP and 5'-AMP in each sample were measured by reversed-phase high pressure liquid chromatography (17) using the lichrosorb RP-18 column and 1% methanol The flow rate was 1 me/min in 0.05 M ammonium phosphate (pH 6.0) as eluent. and detection by absorbance at 254 nm. Biological activities: The biological activities of the samples and fractions described above, in terms of their effects on natriuresis/diuresis and on arterial blood-pressure were studied as reported earlier (1, 11). When necessary, test solutions were first made isotonic by addition of the appropriate amount of 3.6% aqueous sodium choride solution.

RESULTS The ficant

rat

atria1

pressor

extracts

that

extracts,

reactions elicited

whether in

the

strong

AND

DISCUSSION

prepared

anaesthetized depressor

in

PBS

rat,

in

effects

or

water,

elicited

contrast

(Table

1).

to Extracts

siqni-

the

ventricle of

rabbit

Table 1. Effects of crude and ultrafiltered extracts of rat and rabbit myocardial tissue on rat mean arterial blood-pressure. expressed as the percentage change after intravenous injection of 0.05 me into an anaesthetized rat Activity,

A%

Rat

Crude

extract

Retentate, > 1 kdal Ultrafiltration, < .3 kdal Retentate, .3 - 1 kdal Proteolysis,

(5F) (5R3)

Rabbit

V

A

-18

+12

S -25

V k 6a

-23

t 7

LA -6

_' 16

RA +15

i

NS

NS

NS

NS

+9

+7

+8

+8

+11

-25

control + Pronase-CB"

-12

t14 +16 +3

Ventricle (V), left (LA) and right (RA) atrium (A) and skeletal muscle (Sl. Rabbit muscle extracts were prepared in water, those from rates in PBS. Extracts of rabbit heart in water gave comparable results (A -21% and +lO% for ventricle and atrium respectively). Pre-injection blood-pressures, 115 f 15 mmHg. a Mean change + SEM for 10 rabbits before the samples were pooled. 474

7

Vol.

129,

heart

ventricle

were as

No. 2, 1985

and

therefore the

The

are

in

of

reported

the

left

1.

arterial

greatly

right

on

half

of

a right

and,

contrary

and

significant

to

previous

long

diastolic

pressures

infusion,

as

infusion

was both

the

also

elevated

to

returned

the

to

This

same

normal

(1)

immediate increase

was and

Upon

within

about

systolic

extent.

the

natriuresis an

the

of of

caused

set),

mean

sample

and

blood-pressure.

rabbit

the

equivalent

10,

(90

of

cessation

3 min

without

atria1

of

atrium-specific

between

extracts

is,

cardiocytes

immunoreactive

atria1 the

left

probably

due

to

interest

to

note

AMP than

the

of

nucleotides conditions

the

were that

their that

the

determined down

for

a number

atria1 than

often

of

content rabbit

while

.005),

should

the

also

similar be

in

our

crude

ATP

and

ADP.

lobe its

475

of

crude

right

dissimilar of

animal

species

contain

counterpart

It

2).

were

that,

in

extracts

which

were

The

response

(Table

stressed

more (19).

significantly

values

(18).

contain

a depressor (11)

and

populations

apparently left

elicited

nucleotide

atria

It

(20).

since

factor

adenine

(PC

surprising

right

extracts

the

effects

reported

natriuretic atria1

break

not

were of

ventricles dog

blood-pressure

perhaps,

granules

Interestingly,

that

the

of

less reported

the

is is

present prepared

5'-

in

the study,

under

far

for

extracts

extracts

A pooled

diuresis

maintained

prepared

muscle

raised

expected

arterial

blood-pressure

difference

left

case

% in

intro-

blood-pressure

ventricular

consistently

131,

without were

arterial

The

containing

(l-5,

as

individual

degrees.

the

prevent

tachyphylaxes.

The

fact

caused

25

being

the

apparent

of

and

effects.

hand,

me,

reports

mean

skeletal

varying

(0.04

lobe)

rise as

other in

extracts atria1

sustained

the

lobes)

to

muscle

onrat

their

right

activities

blood-pressure,

in

albeit

atria1

crude

and

skeletal

extracts

arterial

varied

myocardium,

white

COMMUNICATIONS

designed

biological

from

the

left

being

labile

crude

While

blood-pressure,

rabbit

the

depressed

atrium

atria1

highly

RESEARCH

the

conditions

Extracts of

Table

BIOPHYSICAL

between

the

water, of

effects

consistently

right

of

in

substances.

comparison.

AND

(distinguishing

degradation

foreign

extracts

atria

prepared

possible

ducing

BIOCHEMICAL

Vol.

129,

No. 2, 1985

Table (AABP)

BIOCHEMICAL

2. Adenine of crude

Tissue

nucleotide aqueous

-9

atrium

Ventricle

muscle

Skeletal aMean f b Compared between

The extended

the

rabbit

and

without

delay.

retentive

for The

all adenine

Low

molecular For

be

(Table

1). was

ted

(14)

not

shown),

the

confirmed

as

aliquots allowing

< 10

32 + 13

c 10

67 t 30

71 k 45 < 10

r 500

a

atria1 the

the

lost

of

myocardial

from

in

YCOS-membrane

procedures the

activities excess

1000 the

of

(general

dalton natriuretic

3600

dalton

retentivity

blood-pressure

could

atrium, pointed

the

can

be

out characterized

pressor

activity: myocardial

follows.

estimate

as

that

norepinephrine

x 10

to

The

oriyen

Firstly, l-100

ascribed

above.

were

that

YC05-membrane,

two

be distin-

476

apparent

presence

of

activities

pressor

contained might

the

in

pressor

studied,

M L-norepinephrine

of

observed

follows.

a dose-response -6

the

only have

activities small

been curve solutions

norepinephrine

(2). > 300

1). by

upon

(11).

type

through

are

be

ultrafiltration

extract

masses

the

may

temperatures to

to

pressor

ultrafiltrates

rough

reduced

subjected

right

difference

labile,

at

affecting

of

of

significant

according

samples

molecular

right

final

possibility

pooled

retained

as

indicate

even

were

(Table

extracts

The

using

71 + 29

1290

biologically

extracts,

fractions

C-z 300)

three

t-test

being

using

(11)

by

b

84 f 51

Student's .005).

<

the

distinguished

elicited

t 7

whose

the

mass

all

500 2 150

activities

nucleotides could

_+ 5

separated

muscle

excepting

that

of

three

depressor

cases

-28

three

separation

heart

b

37 + 11

ATP weight

wet

b

89 t 46

? 4

extracts

(YM2)

least

ADP nmole/g

+16

crude

peptides

at

guised

the

Passage

molecular

blood-pressure myocardial muscle

AMP

activity,

resultant

vasoactive

dalton),

agent

the

membrane

After

were

pressor of

tissue,

arterial and

COMMUNKATIONS

N = 4.

with ventricle, the means (P

storage

Therefore,

RESEARCH

and effect on rabbit skeletal

of

f 4a

-30

SEM,

atria1

BIOPHYSICAL

AABP %

atrium

Right

and

content extracts

extracted

Left

AND

molecules

the

causative

was

construc(results concen-

Vol.

129,

No. 2, 1985

trations

to

with

those

be made:

sor

to

phrine

is

the

The

of

l),

the

appears

bation

pressor

to

be

of

the

presence

indicating

that

the

a peptidic

nature.

In

conclusion,

and

in

rabbit

atrium-specific atriotonin

activity

and

22).

(16),

activated

aluminium

abolished

the

by

confirmed

nmole/g

pres-

reversed-

that

fresh

bioassay

norepine-

rabbit

right

atrium)

system.

mass

'C

and

pH

of

the

proleolytic

structure

of

7.6.

be

granule

is

still

establish

its

from

rabbit

the

between more

of

the

for

which

the

conjecture.

Work

dalton.

is

incu-

activity

is

at

least

in

of

the

part,

the

of

rat

trivial

name to

progress

de-

l),

relationship in

The

under

myocardium

any

muscle

(Table

we propose but

chemical

1000 hours

is,

atria1

atrium-specific,

atria1

Pronase-CB"

substance

in

and six

most

a signifi-

right

than

enzyme active

retained

300

However,

factor,

to

dalton)

for

identified

pressor

< 300

extracted

stable

appears

to

norepinephrine

rat

molecular

we have

It

the

relatively

38

a peptidic

atriotonin.

for

(4.0

favourably

with

pH

retentivity

the

conditions

stroyed

(general

establishing

activity

in

RA-5F

(151,

amounts

compares 21,

neutral

chromatography

COMMUNICATIONS

activity:

YC05-membrane

amount

(15, of

RA-5F

RESEARCH

weight

tissue

aliquot

of

response

pressor

wet

at

sufficient

pressor

BIOPHYSICAL

nmole/g heart

analysis

in

Atrium-specific

1-5

a small

liquid

present

AND

catecholamines

performance

elicit

(Table

of

Finally,

high

of

mammalian

absorb

activity.

cant

for

treatment

known

phase

to

values

reported

Secondly, oxide,

BIOCHEMICAL

the

to

isolate

from

the

structure.

ACKNOWLEDGEMENTS The South

authors

African

schagne

and

Research

gratefully Medical

W.S.

Centre,

acknowledge Research

Council

Joubert.

They

Pretoria,

for

also

the and

assistance

technical

wish

providing

financial

to

assistance

thank

the

experimental

H.A.

by Grove

B.C.

Labu-

Animal

animals.

REFRRENCES 1. 2.

De Bold, A.J., Borenstein, H.B., Veress, Life Sci. 28, 89-94. Trippodo, N-C., Macphee, A.A., Cole, F.E., Proc. Sot. Exp. Biol. Med. 170, 502-508. 477

A.T. and

and

Sonnenberg,

Blakesley,

M. H.L.

(1981) (1982)

Vol.

3.

4.

5. 6. 7.

8. 9.

10. 11. 12. 13. 14. 15. 16. 17.

18. 19.

20. 21. 22.

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No.

2, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Atlas, S.A., Kleinert, H.D., Camargo, M.J., Januszewics, A., Sealy, J-E., Laragh, J.H., Schilling, J.W., Lewicki. J.A., Johnson, L.K., and Maack, T. (1984) Nature 309, 717-719. Geller, D.M., Currie, M.G., Wakitani, K., Cole, B-R., Adams, S.P., X.F., Siegel, N.R., Eubanks, S.R., Gallupi, G-R., and Needleman, P. Fok, (1984) Biochem. Biophys. Res. Commun. 120, 333-338. Currie, M.G., Geller, D.M., Cole, B.R., Boylan, J.G., YuSheng, W., Holmberg, S.W., and Needleman, P. (19831 Science 221, 71-73. Winquist, R.J., Faison, E-P., and Nutt, R.F. (1984) Eur. J. Pharmacol. 102, 169-173. Garcia, R., Thibault, G., Nutt, R.F., Cantin, M., and Genest, J. (19841 Biochem. Biophys. Res. Commun. 119, 685-688. Tang, J.. Webber, R.J., Chang, D., Chang, J.K., Kiang, J., and Wei, E.T. (1984) Regul. Peptides9, 53-59. Seymour, A.A., Blaine, E-H., Mazack, E.K., Smith, S.G., Stabilito, I.I., Haley, A.B., Napier, M.A., Whinnery, M.A., and Nutt, R.F. (1985) Life Sci. 36, 33-44. Ackermann, U., Irizarwa, T.G., Milojevic, S., and Sonnenberg, H. (1984) Can. J. Physiol. Pharmacol. 62, 819-826. Nieuwenhuis, J.J., Labushagne, B.C., Theron,. J.J., and Biagio, R.P. (19841 Biochem. Biophys. Res. Commun. 125, 1074-1081. J-J., Biagio, R.P., Meyer, B.J., and Viljoen, C.C. (1980) Theron, Geneeskunde 22, 255-258. M.B., Clough, D.L., Chen, J.S., Link, W.T., and Haddy, F.J. Pamnani, (1984) Proc. Sot. Exp. Biol. Med. 176, 123-131. Nielsen, G.D. (1977) Acta Pharmacol. Toxicol. 40, 75-86. Holmgren, S., Abrahamsson, T., Almgren, O., and Eriksson, B.M. (1981) Cardiovasc Res. 15, 680-689. Chang, C.C. (19641 Int. J. Neuropharmacol. 3, 643-649. Anderson, F.S., and Murphy, R.C. (1976) J. Chromatogr. 121, 251-262. Timm-Kennedy, M., El-Khatib, E., Huet, M., and Yunge, L. Cantin, M., (1979) Anat. Rec. 193, 55-70. Cantin, M., Gutkowska, J., Thibault, G., Milne, R.W., Ledoux, S., MinLi, S., Chapeau, C., Garcia, R., Hamet, P., and Genest, J. (1984) Histochemistry 80, 113-127. Thomas, R.A., Rubio, R., and Berne, R.M. (1975) J. Molec. Cell. Cardiol. 7, 115-123. K., andTorchiana, M.L. (1963) Acta Physiol. Angelakos, E.T., Fuxe, Stand. 59, 184-192. Petch, M.C., and Nayler, W.G. (1979) Brit. Heart J. 41, 340-344.

478