Neuropeptide-Y acutely stimulates rat zona glomerulosa in vivo

Neuropeptides (1987) 9, 257-262 C; Longman Group UK Lid 1987

NEUROPEPTIDE-Y

ACUTELY STIMULATES RAT ZONA GLOMERULOSA

IN VIVO

Giuseppina

Mazzocchi

Department

of Anatomy, Via Gabelli 65, I-35100 Padova, Italy

(Reprint requests

and Gastone G. Nussdorfer

to GGN) ABSTRACT

Neuropeptide-Y (but

acutely enhanced the plasma concentration

(NPY)

not that of corticosterone)

hypothalamo-hypophyseal axis and renin-angiotensin interrupted. dose of NPY

of aldosterone

in both normal animals and in rats

whose

system were pharmacologically

The maximal response was obtained with a dose of 150 pg/kg. raised the

capsular adrenal

activity of llB-hydroxylase

(zona glomerulosa),

and

This

18-hydroxylase in the

but not that of llR-hydroxylase

in the

inner adrenocortical layers (zonae fasciculata and reticularis). These findings seem

to indicate that NPY is specifically

stimulation

late

of the

steps of the

and directly involved in the acute

secretory

activity of the

rat zona

glomerulosa. INTRODUCTION Numerous

lines of evidence indicate that many regulatory peptides, which are contained in adrenal chromaffin cells (for review, see l), are involved in the

positive or negative modulation

of the secretory

glomerulosa.

These adrenoglomerulotropic

VIP (5) and

somatostatin

peptides

activity

of the rat zona

include enkephalins

(Z-4),

(6-9).

Neuropeptide-Y (NPY) is contained in the adrenal medulla of various animal species (l,lO,ll), and it seemed worthwhile to examine whether this substance is able to affect the activity of the zona

glomerulosa

as well. METHODS

Treatment of animals. group of animals California)

Adult male rats of Wistar strain (200 g) were used.

was subcutaneously

with dexamethasone

infused

A

(Alzet osmotic pumps; Palo Alto,

(lO,ug/kg/h; Decadron, Merk, Milan, Italy) plus

ACTH (0.01 IU/kg/h; Sigma, St. Louis, Missouri) and captopril (0.5 mg/kg/h; Capoten, Squibb, Milan, Italy) plus angiotensin II (10 pg/kg/h; Sigma) for 24 h. The dosage was that indicated by Nussdorfer and Mazzocchi (5). Another group of rats was infused for 24 h with normal saline. Both groups of animals

257

NEUR.

D

were divided into nine subgroups 30 min before the sacrifice, 200, 250 and 300 pg/kg;

(of six rats each). Eight subgroups received,

an i.p. injection of NPY (25, 50, 75, 100, 150,

Sigma)

dissolved

in 0.2 ml saline. The ninth subgroup

was given only the vehicle and served as a control. Another 24 rats, those used in the previous experiment,

were infused with

akin to dexamethasone/ACTH

and captopril/angiotensin

II as described above. One half of the animals received an i.p. injection of 150 pg/kg NPY 30 min before the sacrifice, and the other half was given only the aqueous vehicle. Their adrenal glands were used for the enzymatic dosages. All the rats were killed by decapitation between 10:00 and 11:00 a.m. Hormonal dosages. corticosterone

Trunk blood was collected

were extracted and purified

from each rat. Aldosterone

aldosterone was determined by radioimmuno-assay, Diagnostic

Products

(Los Angeles, California),

assayed by competitive Enzymatic dosages.

protein binding

and

(12). The plasma concentration using kits purchased

of

from

and that of corticosterone

was

(13).

The adrenals of each rat were quickly removed and cleaned of

adhering fat, and the zona glomerulosa was separated by incising and stripping the

Decapsulated

connective capsule.

Capsular

(zona glomerulosa)

reticularis)

adrenals were halved and demedullated.

and decapsulated

were weighed and homogenized

pH 7.4) buffer containing

1mM

adrenals

(zonae fasciculata

in 0.25 M sucrose/tris

EDTA. IlB-Hydroxylase

(180H) activities were evaluated on a mitochondrial

and

HCl (50 mM,

(11ROH) and 18-hydroxylase fraction

obtained

by

differential centrifugation at 10,000 g for 15 min (14), by measuring the rates of conversion

of

to corticosterone Statistical

exogenous

ll-deoxycorticosterone

and aldosterone,

analysis.

respectively

The statistical

and corticosterone

(Sigma)

(15).

significance

of results was assessed by

ANOVA and Duncan's multiple range test.

RESULTS Fig. 1A shows that NPY, at a dose less than 100 pg/kg,

did not evoke any signi

ficant change in the plasma level of aldosterone in saline-infused

rats. Doses

induced a maximum increase of about 19-20% (P
higher than 100 pg/kg

75 pg/kg and 100 ug/kg

NPY

caused a

23% (PC 0.05) and 30% (P< 0.01)

rise,

A maximal response (36%, P< 0.01) was observed with 150 pg/kg respectively. any apparent enhancement of the the dose did not yield NPY. Further rising aldosterone

response. No changes were noted in the blood level of corticosterone

in either of the groups of animals

(Fig. 1).

administration of 150 ,ug/kg NPY significantly (P (0.01) raised the activity of 1lROH and 180H in dexamethasone/ACTHand capsular adrenal captopril/angiotensin II-infused rats (Fig. 2). No significant changes were

The acute

observed in the 1lROH activity of inner adrenal layers (Fig. 2).

258

%40

30

30

+

+ 20

20

10

10,

0

0,

Effect of various doses of NPY on the plasma concentrations of in saline-infused (A) and aldosterone (O----O) and corticosterone ( M) dexamethasone/ACTH- and captopril/angiotensin II-infused rats (B). +, PcO.05; * , P< 0.01. Results are expressed as percent of the basal values, which are

Figure 1.

shown in Table 1.

DISCUSSION The present findings suggest that NPY affects rat zona glomerulosa

by stimula

ting the late steps of aldosterone

synthesis (i.e. 1lROH and 180H activities). This effect of NPY seems to be direct. In fact, we can exclude the possibility that this peptide acts by increasing ACTH release and/or angiotensin production, since it stimulates hypophyseal

aldosterone

by the simultaneous

administration

doses of ACTH/angiotensin The

secretion also in animals whose

axis and renin-angiotensin

lower response

pharmacologically

hypothalamosystem were pharmacologically interrupted

of dexamethasone/captopril

to NPY of saline-infused animals, in comparison to the manipulated rats, may be only apparent. It is conceivable

that it is due to the fact that NPY, in saline-infused glomerulosa

and maintenance

II (5).

whose aldosterone

production

is already

rats, stimulates

a zona

enhanced by the stress

caused by the i.p. injection, as it can be seen by comparing the basal plasma levels of aldosterone and corticosterone in both groups of rats (Table 1). Our dose-response curves indicate that NPY, at a dose of 150 pg/kg, is able to evoke a maximal secretory response of the zona glomerulosa. Since 98% of corticosterone is produced by inner adrenal layers in Rodentia (161, our

259

660

1

.I E .

*

: . 8

600

400

1 *

.E E .

.B

1000 .f

$er

E .

r”

$

. JI r 600

*.a:.* .::: -::: .::: .::: *::: *::: *::: *:;: .::: *::: .::: -:::

750

...’ . . . . ..I . . . . ..I . . . ...’ .::. .::. .::.

400

‘...‘.’ :::: .::: . . . . ..I

A

500-

B

Figure 2.

Effect of 150 pg/kg NPY on the activities of 1lBOH (A) and 180H (B) in the capsular adrenals, and of 1lBOH in the inner adrenocortical layers (C) of dexamethasone/ACTH- and captopril/angiotensin II-infused rats. Standard errors are indicated.

*, PC 0.01.

results indicate that the specific target of NPY is the zona glomerulosa. Also the absence of effect of NPY on the activi-cy of 1lBOH of inner adrenal layers confirm the specificity which may

reflect

of the adrenoglomerulotropic

action of this peptide, lack a cells

the fact that zona fasciculata-reticularis

sufficient number of specific receptors for NPY. The existence of high-affinity specific receptors for NPY in the rat adrenal cortex is currently being checked by autoradiography

in our laboratory.

Table 1. Plasma levels of aldosterone

and corticosterone

30 min after an i.p.

injection of 0.2 ml saline. Aldosterone (ng/dl) Saline-infused

28.8 + 3.1

rats

Dexamethasone/ACTH- and captopril/angiotensin IIinfused rats Values are group means * SD.

19.5 L 2.5*

*, P< 0.01

260

Corticosteronc

oJg/dl) 13.6 + 2.0 8.1 & 1.3"

the adrenoglomerulotropic

The physiological

meaning of

to be elucidated;

There are indications

(18) (17) and nor-adrenaline co-released with catecholamines cats (20).

Some morphological to the

action of NPY has yet

that NPY is co-stored with adrenaline and that it is in the chromaffin granules,

during physical

exercise in humans

(19) and i1

data suggest the existence of a venous return

from the

medulla

grounds,

it seems possible to hypothesize

ou-ter portion of the adrenal cortex (21). On these in the that NPY may be involved

paracrine control of the zona glomerulosa

by the zona medullaris.

REFERENCES 1. Kondo, H. (1985). neuropeptides

Immunohistochemical

analysis

in the adrenal gland. Archivium

of the localization

Histologicum

of

Japonicum 48:

453-481. 2. Racz, K., Glaz, E., Kiss, R., Lada, G., Varga, S., Di Glena, K., Medzihradsky,

K., Lichtwald, K. and Vecsei, P. (1980). Adrenal cortex, a

newly recognized and Biophysical 3. Bevilacqua,

peripheral

site of action of enkephalins.

Research Communications

Biochemical

97:1346-1353.

M., Vago, T., Raggi, U., Scorza, D., Proverbio, M., Malacco,

E. and Norbiato,

G. (1982). In vitro steroidogenic

a stable analog of methionine-enkephalin. the control of aldosterone Investigations

production.

properties

Opiate-dopamine

of FK33824,

interaction

in

Journal of Endocrinological

5:277-280.

4. Robba, C., Mazzocchi, administration glomerulosa

G. and Nussdorfer G.G. (1986). Effects of chronic of a methionine-enkephalin analogue on the zona

of the rat adrenal cortex. Research in Experimental Medicine

186:173-178. 5. Nussdorfer,

G.G. and Mazzocchi,

(VIP) stimulates

aldosterone

G. (1987). Vasoactive intestinal peptide secretion by rat adrenal glands in vivo.

Journal of Steroid Biochemistry 6. Aguilera,

26, in press.

G., Harwood, J.P. and Catt, K.J. (1981). Somatostatin modulates II in adrenal glomerulosa zone. Nature 292:

the effects of angiotensin 262-263.

7. Boscaro, M., Scaroni, C., Edwards, C.R.W. and Mantero, F. (1982). Inhibitory effect of somatostatin on the aldosterone response to angiotensin II: in vitro studies. Journal of Endocrinological Investigations 5:173-178. 8. Rebuffat, P., Robba, C., Mazzocchi,

G. and Nussdorfer,

G.G. (1984).

Inhibitory effect of somatostatin on the growth and steroidogenic capacity of rat adrenal zona glomerulosa. Journal of Steroid Biochemistry 21:387-390. 9. Mazzocchi,

G., Robba, C., Rebuffat, P., Gottardo, G. and Nussdorfer, G. on the zona glomerulosa of rats treated

G. (1985). Effect of somatostatin

261

with angiotensin concentrations.

II

or

captopril:

stereology

Journal of Steroid Biochemistry

and

plasma

hormone

23:353-356.

10. Majane, E.A., Alho, H., Kataoka, Y., Lee, C.H. and Yang, H.Y.T. Neuropeptide

(1985).

Y in bovine adrenal glands: distribution and characterization.

Endocrinology

117:1162-1168.

11. Lundberg, J.M., Hockfelt, T., Hemsen, A., Theodorsson-Norheim, E., Pernow, J * , Hamberger, B. and Goldstein, M. (1986). Neuropeptide Y-like immunore activity in adrenaline of pheochromocytoma

cells of adrenal medulla and in tumors and plasma

patients. Regulatory

Peptides 13:169-182.

12. Sippell, W.G., Bidlingmaier, F., Becker, H., Briinig, T., Dorr, M., Golder, W ., Holmann, G. and Knorr, D. (1978). Simultaneous radioimmunoassay of plasma aldosterone,

corticosterone,

ll-deoxycorticosterone,

17-hydroxyprogesterone,

11-deoxycortisol,

of Steroid Biochemistry

9:63-74.

progesterone,

cortisol and cortisone. Journal

13. Spat, A. and Jozan, S. (1972). Competitive protein binding assay of Journal of Steroid Biochemistry 3:755-759. corticosterone. 14. Nussdorfer,

G.G. and Mazzocchi, G. (1983). Long-term effects of ACTH on cells: a coupled stereological and enzymological study.

rat adrenocortical

Journal of Steroid Biochemistry

19:1753-1756.

15. Kramer, R.E., Gallant, S. and Brownie, A.C. (1979). The role of cytochrome P450 in the action of sodium depletion on aldosterone biosynthesis in rats. Journal of Biological

Chemistry 254:3953-3958.

G.G. (1986). Cytophysiology

16. Nussdorfer,

of the adrenal cortex. Interna-

tional Review of Cytology 98:1-405. 17. Kuramoto, H., Kondo, H. and Fujita, T. (1986). Neuropeptide tyrosine (NPY)-like immunoreactivity in adrenal chromaffin cells and intraadrenal nerve fibers. Anatomical

la.

Varndell,

I.M., Polak, J.M., Allen, J.H., Terenghi, G. and Bloom, S.R.

(1984). Neuropeptide containing

Record 214:321-328.

tyrosine

(NPY) immunoreactivity

cells and nerves of the mammalian

in norepinephrine

adrenal gland. Endocrinology

114:1460-1462. 19. Lundberg, J.M., Martinsson, A., Hemsen, A., Theodorsson-Norheim, E., Svedenberg, J., Ekblom, B. and Hiemdahl, P. (1985). Corelease of neuropeptide Y and catecholamines during physical exercise in man. Biochemical and Biophysical

Research Communications

133:30-36.

20. Lundberg, J.M., Fried, G. and Pernow, J. (1986). Co-release of neuropeptide Y and catecholamines upon adrenal activation in the cat. Acta Physiologica

Scandinava

120:231-238.

21. Gonzalo-Sanz, L.M. and Insausti, R. (1976). The structure of the suprarenal venous system and its possible functional significance. Acta Anatomica

95:309-318.

Date Received 262

26/z/87

Date Accepted o/3/87