Early (1 to 24h) plasma atrial natriuretic factor changes in the rat during antiorthostatic hypokinetic syspension

BIOCHEMICAL

Vol. 148, No. 2, 1987 October 29, 1987

AND BIOPHYSICAL

EARLY (1 to 24h) PLASMA ATRIAL

RESEARCH COMMUNICATIONS Pages 582-588

NATRIURETIC FACTOR CHANGES

IN THE RAT DURING ANTIORTHOSTATIC

HYPOKINETIC SUSPENSION.

Guillemette Gauquelin, Christophe Kazek, Anne-Marie Allevard, Rolande Garcin, Jacques Bonnod*, Jolanta Gutkowska-,

Lab.

and Claude

Gharib

Physiol. Fat. Med. Grange-Blanche 69373 LYON Cedex 08,

l IFFA-CREDO **Clinical Received

Marc cantin:

69210 L’Arbresle - France

Research Institute of Montreal H2W lR7 MONTREAL, Canada

September

14,

1987

ABSTRACT: Antiorthostatic hypokinetic suspension (AOH) in rat is currently used as an animal model for simulating weightlessness. This maneuver is responsible for a diuresis, a natriuresis and an increase in central venous pressure (CVP). Knowing the role of CVP in atria1 natriuretic factor (ANF) secretion, the aim of the study was to examine the early plasma ANF changes during AOH (angle 30-35’) using Morey’s model (tail suspension). The rats were divided into 4 groups : 24 population cage (PC), 24 isolated in separate cages (I), 24 were attached by the tail (Morey’s model) and remained in the horizontal position (attached horizontal : AH). At the end of this period of 7 d, 12 AH were suspended for 1, 2, 6 and 24 h (AOH) and sacrificed with the controls for plasma ANF determination. Our results show that the level of ANF is significantly (p<.O5) higher in AOH rats after 2 h of suspension (16.622 pg/ml vs 10.9+1.5). A significant increase is also observed between AOH and AH after 2h of suspengon (p < 0.05). Six hours after suspension ANF presents a sharp decline in AOH and no difference is observed between AOH and AH and I. Morey’s tail suspension model seems to be valid 0 1987 for the study of the early hormonal effects of simulated weightlessness for ANF. Academic

Press,

Inc.

In man, head-down many

bedrest

is currently

used

for simulating weightlessness because

of the physiological responses are similar to those

The head-down

suspension (antiorthostatic

observed

during space flight (1).

hypokinesia) is also currently

used in rat as a

model for simulating weightlessness (2). Indeed, body fluid compartmentalization, and electrolyte dependent.

metabolism, volume regulating

Head-down

of the body.

tilt

gravity

induces a marked fluid shift from the lower to the upper half

Increased pressure in both

atria (increased central venous pressure=CVP)

leads to hormonal modifications characterizing 0006-291X/87 Copyright All rights

hormones are in large part

water

$1.50 0 I987 by Academic Press. of reproduction in any ,fornr

Inc. reserved.

582

the Gauer-Henry reflex

: inhibition of

Vol.

148,

No. 2, 1987

secretion

AND

BIOPHYSICAL

of the renin-angiotensin-aldosterone

mammalian

atria1 myocytes

(4, 5) which

are known

ANF

is involved

blood

pressure

release

of

secrete

of potent

as atria1 natriuretic

(6, 7). For

these

are

modified

Another

problem

factors

of extracellular reasons, during

RESEARCH

natriuretic (ANF).

fluid

It

early

has

been

peptides

speculated

electrolyte whether

adaptative

was the choice of controls

(3). Moreover,

and diuretic

volume,

we investigated the

COMMUNICATIONS

and of vasopressin

system

a family

in the regulation

ANF

weightlessness. stress

BIOCHEMICAL

that

balance and

the synthesis

response

to

and

simulated

for such experiments

where

could play a major role.

MATERIALS Animal

and METHODS preparation

Male Wistar rats (230-250 g, IFFA-CREDO, les Oncins), kept at controlled room temperature under a 7 AM-7 PM light regimen, were fed regular pelleted rat chow and tap water “ad libitum” for 9 days prior to the experiment. On the first experimental day (adaptation period), all the animals were weighed and divided randomly and assigned to a predetermined treatment schedule, as shown in Table I. Rats (n=l2 par time point) were suspended in individual plastic cages using Morey’s tail suspension model (8) as modified by Roberts (9). A plastic hole-drilled disc was wrapped with adhesive tape to the tail and connected to a pulley by a plastic bar. The rats were capable of moving freely about the cage in any direction and had easy access to the food and water. These rats were maintained in the orthostatic position for seven days before the test period. 1, 2, 6 and 24 hours before the experiment, a head-down tilt of approximately 30-35” was applied (AOH). To separate the effects of orthostatic manipulations from the

TABLE EXPERIMNTAL

Adaptation

I SCHEDULE

Test

Period

period 1 day

Day

-9

0

Population

cage

Isolated

(I)

Attached tail

(PC)

by

12

per

cage

-7

5 per I,

0

cage

12

#I

9,

1

0

12

”

”

1 per

cage

(AH)

,*

11

11

1 per

cage

(AH)

and

ANF

(I)

Ih

2h

6h

24h

*

*

*

*

x

*

*

*

*

l

*

* AH

*

*

*

the

(horizontal)

AH

Suspended (30°)

*Blood for

AOH

was

drawn

under

AOH for

each

point).

penthobarbital

anesthesia

583

measured

(n

= 6 for

PC,

I and

AH and

* AOH

n = 12

BIOCHEMICAL

Vol. 148, No. 2, 1987

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

effects of hypokinesia, a group of rats was suspended by the same method as the antiorthostatic rats, except that these rats were maintained in a horizontal position. These are identified as orthostatic rats (AH: Attached Horizontally). Another control group consisted of physically unrestrained rats housed individually in plastic cages and were classified as isolated rats (I). A third group of controls were housed five in a standard cage and they are identified as (PC : Population Cage). Six rats of each group were killed at the same time as the antiorthostatic hypokinetic. Consequently, our study on the effects of head-down tilt was based first on comparisons between the antiorthostatic hypokinesic and the orthostatic hypokinesic rats and secondly on comparison of the isolated animals and those housed five to a cage. During the adaptation period, the rats were weighed daily. Preparation of blood samples At 1, 2, 6 and 24 h after head-down suspension, the experimental rats and their controls were anesthetized with pentobarbital sodium (60 mg/kg body weight, i.p.). Blood was coyected by aorta puncture. One ml, taken separately was processed for plasma Na+, K , proteins and osmolality analysis. The hematocrit was also measured. Three ml of -9ood were placed 9 ice-chilled plastic tubes containing the protease jyhibitors EDTA (10 M), pepstatin (lo- M), and phenylmethylsulfonyl fluoride (PMSF, 3.10 M) for plasma IR-ANF (99-l 26). Plasma was obtained by centrifugation at 1500 g for 10 min at 4°C and stored at -7O“C until assayed. ANF was extracted from plasma by means of Vycor glass beads (Corning Glass Works Corning, WK) and measured by a specific radioimmunoassay (RIA) (10). The heart was removed immediately after sacrifice the right and left atria were dissected separately and atria1 ANF concentrations were measured by RIA (11). Atria1 protein content was assessed by a modification of Bradford’s methods (12). Plasma and atria1 IR-ANF concentrations were determined twice, and the mean of the two values was recorded. The data were evaluated by two-way analysis of variance with repeated measures to lobally test the time effect, the group effect and the group interaction over time fF-test). If a significant effect was detected, Newmann et Keul’s test was used to determine which treatment means were significantly different. Results are expressed as means -+ SEM.

RESULTS Table II depicts the hematocrit

plasma osmolality,

sodium, potassium and protein

values at 4 times of the test period in each of the four experimental groups. During all the

test

period,

no difference

osmolality and hematocrit. an initial

was found

in plasma sodium, potassium, proteins,

During the first two days of the adaptation period, there was

slight loss of weight for the orthostatic

hypokinetic

and isolated rats. Then

the growth rate remained similar in all groups (data not shown). Figure

1

illustrates

the

plasma ANF

concentrations (middle panel) and left four periods after was significantly

levels

(upper panel), right

atria1

atria1 ANF concentrations (lower panel) for the

suspension for antiorthostatic higher than for antiorthostatic

rats and controls. Plasma ANF in PC hypokinetic,

orthostatic

hypokinetic

isolated rats (p
ANF

and isolated rats. After

difference

two hours of suspension, plasma ANF

was signicantly higher in antiorthostatic

584

or

hypokinetic

rats than after

one

Vol.

148,

BIOCHEMICAL

No. 2, 1987

AND

TABLE EFFECT

OF SIMULATED

Na+

139+1

AH

13920.5

i

139+0.5 141+0.7 -

latter

721 pg/ml decline,

OSMOLALITY

6h

24h

140+0.5 14021

13722

14221

138+2-

13823

14022

137+1 -

13723

14121

141+0.7 -

13821

(%) 47.7+1.4 49.4+1.2 -

49.3+0.6 -

4820.7

AH

47.9+0.9 47.622

48

48.520.5

i

48.921

49.320.9

49

46.5+1.2 48.8~1

50.9+0.5 -

PC

51

50.6+0.5 -

~0.4

Osmolality

(mOsm/kg

20.7

0.5

H20)

AOH

30321

30121

30321.5

2985.5

AH

298+3_

301+1.5 -

301.823

i

298+229821

30322

30121.5

29721

PC

300+2.5 -

301+2_

29921.5

29821.5

K+ (mmol/l)

AOH

5+0.2

5~0.2

420.2

4.520.3

AH

5+1 5~0.2

4+0.2 5+0.3 -

4

i

4+0.2 4+0.3 -

4

-+O.l LO.2

PC

5+0.2 -

420.2

420.2

4

LO.3

(g/l)

AOH

6321

AH

i

60+1 6323

PC

6222

being

suspension,

(p < 0.05).

orthostatic

Na+,

AOH

Proteins

of

ON SERU4

Hematocrit

Serum

hour

AND SERW

2h

AOH

Serum

COMMUNICATIONS

(mmol/l) 1 h

PC

RESEARCH

II

WEIGHTLESSNESS

HEMATOCRIT

Serum

BIOPHYSICAL

respectively). and no difference

5922

5922

62+1 -

66+5

65+3 65+4 -

6522

6221

593

66~3

16.6 + 2 pg/ml

A significant

and isolated

61+2 62+3 -

rats

increase at 2 hours

Six hours between

after the

in the former was

also noted

of suspension suspension,

three

and

groups

this

10.9

+

1.5

pg/ml

in the

between

the antiorthostatic,

(16.6+2pg/ml,

7.521 pg/ml

parameter

was found.

presented

and

a sharp

Vol. 148, No. 2, 1987

BIOCHEMICAL

AND BIOPHYSICAL

q Pc ml

RESEARCH COMMUNICATIONS

q AH q AOH

.1 Figure

1.

Effect right

hypokinetic

24 of suspension (upper (lower

panel), panel).

vs I and AH vs AOH at 1 hour

concentration

rats after

6 hours

of simulate weightlessness on plasma ANF (middle panel) and left atria1 concentration

* p t 0.05 + p < 0.05

The atria1 ANF

2

was higher in the right

2 h of suspension compared to

atrium

of antiorthostatic

of suspension. No difference

1 h

was found later.

During the all stages of the test period, no difference

atrium was noted

between

in the left

groups.

the

DISCUSSION Experimental

model

Various techniques have

been

used

to

suspend

the

rat

to

weightlessness. All

simulate

of the methods used to produce head-down suspension in rats attempted to reproduce the physiological effects of simulated weightlessness, more particularly of

blood

Consequently,

volume.

The

problem

in these studies was to choose a correct

our conclusions on the effects

comparisons of the antiorthostatic

a cephalad shift

of weightlessness are

based

control.

primarily

on

hypokinetic rats and orthostatic hypokinetic rats. But 586

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 148, No. 2, 1987

our data on the rats that were those isolated rats

in plastic

are smaller

housed five in a standard

cages. The baseline values in orthostatic

than those reported

appear

to be due to the method

animals

received

a standard Whether

cage are very

A relationship

potential

housed together

of weightlessness

between

RNA

ANF

(mRNA)

participation

acute

simulated

factor

during volume

and Kirsh urine flow

of ANF

synthesis

(maximum

at

ANF

increase

in

plasma

level

IR-ANF

and this is the first

investigation,

disappeared pressure central

of

changes

hours.

suspension

It has been demonstrated

changes

after

animals.

hours

was observed

of

suspension,

suspension.

in the rat occurred

within

of ANF

evaluation

(16)

examine

the

the effects

of

Gauer, Reevers in a increase

in the

simulation

at 9”

was

rapid

very

suspension

levels.

early

in plasma ANF

this

In our after

difference

central

They found that

the first

an

compared

in the rat.

but

in

studies

in the right atrium

have reported

(45’ tilt)

and

We recorded

of this phenomenon

of

in water

to pretilt

the increase

the head-down

587

elevation

of ANF.

Interestingly,

et al (22)

the release

results

had returned

two

one hour

and they decreased that

plasma

study an elevation

to

Shellock

in the rat during

venous pressure

to head-down

plasma

of a natriuretic

(18, 19). Henry,

ANF

a higher ANF concentration

six

The secretion

induced by simulation

This

observation

compared

we studied

(20) in a head-down

1 hr plasma

or isolated

suspension

after

rats.

of ANF -

(17). To further

that an atria1 distension

concentration

was

hours

in baseline

and changes

to fluid volume,

in human.

and by

hypokinesic

two

all

housed five in

(I 51, morphological

pre-proANF

The early changes

to orthostatic

present

(because

and secretion

atrium

had long been postulated

during an immersion

plasma

transient,

for rat

We have also reported

30 minutes)

demonstrated

on synthesis

by bioassay

to demonstrate

documented.

in

do not

(13) in anesthetized

elevations

on plasma and atria1 ANF.

and sodium excretion.

an increase

or anesthesia

by Horky

in the

in adjustments

expansion

the first

rat is not well

RIA

from

or isolated

(13, 14). These results

caused

simulation

analysis

weightlessness

were

hypokinetic

Our baseline levels in rats

to those reported

balance has been demonstrated

and messenger

very different

to be clarified.

Short term effects

Epstein

of blood collection,

similar

of the rats

ANF levels remains

electrolyte

by some investigators

the same dose of anesthesic).

the stress

cage were

venous the peak

60 min of exposure

or rose (20’) during the next 7 h. directly

correlates

with

changes in

BIOCHEMICAL

Vol. 148, No. 2, 1987

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

right atria1 pressure in water immersed rats (23). Volume expansion, by enhancing venous return, could elevate right atria1 pressure and increase the concentration of right atria1 ANF. In summary, the changes in the synthesis and secretion of ANF are comparable to those observed during bed rest and water immersion in humans. The antiorthostatic hypokinetic

rat therefore

appears to be a good model for the study of the hormonal

system during simulated weightlessness. ACKNOWLEDGMENTS This work was supported by grants from CNES (87.1242), DRET (87086), UER de Biologie Humaine (Universitd Claude Bernard) and Centre J. Cartier. We thank J. Carew for correction of the English. REFERENCES 1. Greenleaf, J.E. (1984) 3. Appl. Physiol.:Respirat. Environ. Exercise 57, 619-633 2. Deavers, D.R., Musacchia, X.J. and Meininger, GA. (1980) J. Appl. Physiol. 49, 576-582 3. Gauer, O.H., Henry, J.P. and Behn, C. (1970) Ann. Rev. Physiol. 32, 547-595 4. de Bold, A.J., Borenstein, H.B., Veress, A.T. and Sonnenberg, H. (1981) Life Sci. 28, 89-94 5. Garcia, R., Cantin, M., Thibault, C., Ong, H. and Genes& J. (1981) Experientia 38, 1071-1073 6. de Bold, A.J. (1985) Science 230, 767-770 7. Cantin, M. and Genest, J. (1985) Endocrine Rev. 6, 107-l 27 8. Morey, E.R. (1979) Biosc ience 29, 168-l 72 9. Roberts, W.E. (1984) Bone Mineralisation Workshop. Brussels Jan. ESA SP.203, July 1984 10. Gutkowska, J., Horky, K., Schiffrin, E.L., Thibault, G., Garcia, R., De Lean, A., Hamet, P., Tremblay, J., Anand-Srivostava, M.B., Januszewicz, P., Genest, J. and Cantin, M. (1986) Fed. Proc. 45, 2101-2105 11. Gutkowska, J., Thibault, G., Januszewicz, P., Cantin, M. and Genest, J. (1984) Biochem. Biophys. Res. Comm. 122, 593-601 12. Spector, T. (1978) Annal. Biochem.86, 142-146 13. Horky, K., Gutkowska, J., Garcia, R., Thibault, G., Genest, J. and Cantin, M. (1985) Biochem. Biophys. Res. Comm. 129, 651-657 14. Long, R.E., Tholken, H., Ganten, D., Luft, F.C., Ruskoaho, H. and Unger, T. (1985) Nature (Lond.) 314, 264-266 15. Thibault, G., Garcia, R., Cantin, M. and Cenest, J. (1983) Hypertension 5 (suppl. I), 1.75-1.80 16. Marie, J.P., Guillemot, H. and Hatt, P.Y. (1976) Pathol. Biol. 24, 549-555 17. Nakayama, K., Girkuso, H., Hirobi, T., Inagama, S. and Nakanishi, 5. (1984) Nature 310, 699-70 1 18. Maack, T., Camargo, M.J.F., Kleinert, H.D., Laragh, J.H. and Atlas, S.A. (1985) Kidney Intern. 27, 607-615 19. Palluk, R., Gaida, W. and Hoelke W. (1985) Life Sci. 36, 1416-l 425 20. Gharib, C., Gauquelin, G., Geelen, G., Cantin, M., Gutkowska, J., Mauroux, J.L. and Guell, A. (1985) The Physiologist 28 (suppl.), S.30-S.33 21. Epstein, M., Loutzenhiser, R., Friedlead, E., Aceto, R.M., Camargo, M.J.F. and Atlas, S.A. (1987) J. Clin. Invest. 79, 738-745 22. Shellock, F.G., Swan, H.J.C. and Rubin, S.A. (1985) Aviat. Space Environ. Med. 56, 791-795 23. Katsube, N., Schwartz, D. and Needleman, P. (1985) Biochem. Biophys. Res. Comm. 133. 937-944 588