Neuropeptide K enhances glucocorticoid release by acting directly on the rat adrenal gland: the possible involvement of zona medullaris

BRAIN RESEARCH ELSEVIER

Brain Research 661 (1!)94) 91-96

Research report

Neuropeptide K enhances glucocorticoid release by acting directly on the rat adrenal gland: the possible involvement of zona medullaris G. Mazzocchi, L.K. Malendowicz, P.G. Andreis, V. Meneghelli, A. Markowska, A.S. Belloni, G.G. Nussdorfer * Department of Anatomy, Unicersity of Padua, Via Gabelli 65, 1-35121 Padua, ltalv Accepted 19 July 1994

Abstract

Neuropeptide K (NPK), a member of the kassinin-like tachykinin family, is contained in the rat hypothalamus and is known to stimulate pituitary ACTH release. The intraperitoneal bolus administration of NPK dose-dependently enhanced corticosteronc blood level not only in intact rats, but also in hypophysectomized/ACTH replaced animals. NPK did not affect corticosterone secretion of dispersed rat adrenocortical cells; however, it concentration-dependently raised basal corticostcrone production by decapsulated adrenal quarters (including both cortical and medullary tissues). Minimal and maximal effective concentrations were 10 ~ and 10 -8 M, respectively. 10 8 M NPK potentiated corticosterone response of adrenal quarters elicited by 10 12 M ACTH, but not that evoked by higher concentrations of ACTH. The direct corticosterone secrctagoguc effect of 10 ~ M NPK is annulled by 10-6 M a-helical-CRH or corticotropin-inhibiting peptide, competitive inhibitors of CRH and ACTH, respectively. In light of these findings, the hypothesis is advanced that NPK exerts a direct stimulatory action on adrenocortical secretion and that the mechanism underlying this effect of NPK may involve the activation of the intra-medullary C R H / A C T H system.

Keywords: Ncuropeptide K; Hypothalamo-pituitary-adrenal axis; Steroidogenesis; Adrenal cortex; Adrenal medulla; Rat

1. Introduction

Evidence is available that several m e m b e r s of the neuromedin (NM) family (e.g. NMB, N M N and NMU), which are widely distributed in the central nervous system, are able to regulate the activity of rat hypothalamo-pituitary-adrenal (HPA) axis [27]. N M K and N M L (neurokinin B and neurokinin A), belonging to the kassinin-like tachykinin group, are present in high concentrations in the rat hypothalamus [8,11,21,32-34,44, 49] and N M L is also contained in the anterior pituitary [8,12,19,32]. However, scarce attention has been paid to their possible effect on H P A axis: as far as we are aware, only Faria et al. [13] studied the action of N M L on C R H / A C T H release in rats. It is well known that the decapeptide N M L represents the fully processed form of neuropeptide K (NPK), a 36-amino acid peptide secreted in the rat

* Corresponding author. Fax: (39) 49 66 03 10. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights feserved SSD1 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 9 0 4 - X

hypothalamus [10,24,45]. Recently, Kalra and Kalra [20] showed that the intracerebroventricular administration of 0.5-2.5 nmol N P K markedly raises plasma corticosterone concentration in rats, being N M L much less effective. On these grounds, these investigators conclude that NPK may be the endogenous biologically active tachykinin normally activating H P A axis in rats, ,being N M L a sequential degradation product in the hypothalamus. Malendowicz [25] and Malendowicz and Markowska [27] have recently reviewed a large body of data indicating that several neuropeptides (e.g. neuropeptide Y, substance P, NMB, N M C and NMN), which are located in the hypothalamus and are able to stimulate A C T H release, are also contained in adrenal medulla and exert a direct secretagogue effect on adrenal cortex. Evidence is available that kassinin-like tachykinins may be included in this group of neuropeptides: (i) NMK, N M L and NPK are contained in chromaffin cells of bovine adrenal gland [9], and (ii) N M K and N M L enhance cortisol secretion by cultured bovine adrenocortical cells [50].

t)2

(; 'i4azzo((ki ct a/. / Brain Research riO1 (1994) ql -00

The

aim

of the

present

study was

to i n v e s t i g a t e

whether the mechanism underlying the glucocorticoid ( c o r t i c o s t e r o n e ) s e c r e t a g o g u e e f f e c t o f N P K in r a t s [2(i)] involves not only the stimulation of pituitary ACTH release, but also a direct effect on the adrenal gland.

2. Materials and methods 2. l. In Lit,o experiments

Adult male Wistar rats, weighing 200_+20 g, were purchased from Charles-River (Como, Italy). A number of animals was hypophysectomized via the parapharyngeal approach, and the completeness of the operation was checked as previously detailed [1]. Starting from the 4th post-operative day, hypophysectomized rats were subcutaneously (s.c.) infused for 7 days with ACTH (human; Peninsula, Merseyside, UK) dissolved in 0.9% NaCI, at a rate (0.1 pmol/min) that was previously shown to maintain a normal blood level of ACTH and after 5 days to completely reverse the adrenal atrophy induced by bypopbysectomy [28]. The infusion was performed by implanting Alzet osmotic pumps Mod. 2001 (Alza, Palo Alto, CA), which release at a constant rate for a week. A number of rats was sham-hypophysectomized, and s.c. infused with the saline vehicle. On the 7th day of infusion, sham-operated and hypophysectomized/ACTH-replaced rats were divided into equal groups (n = 6), which were intraperitoneally injected with increasing doses of NPK (Peninsula) (from 10 12 to 10-s mol/rat) dissolved in 0.2 ml saline, 60 rain before the sacrifice. The animals were decapitated between /)9.30 and 10.30 h, their trunk blood was collected, and plasma separated and stored at -20°C. ACTt] and corticosterone were extracted from plasma and purified [40,42], and their concentration was measured by specific RIA. ACTH (Rat)-RIA kit (Peninsula): sensitivity, 15 pg/tube. Cross-reactivity: ACTH (rat, human), 100%; ACTH~_24, 0.01%; other ACTH fragments and pituitary hormones, 0%. Intra- and inter-assay variations were 7.3% and 9.4%, respectively. CORTX-RIA kit (Eurogenetix, Milan, Italy): sensitivity, 25 pg/tube. Cross-reactivity: corticosterone and cortisol. 100%; DOC and progesterone, 2%; other steroids, less than 0.001%. Intra- and inter-assay variations were 5.5% and 7.1%, respectively.

tion was carried out l'or 9() rain in a shaking baih at 37'(i ir~ an atmosphere of 95% O~ and 5% ('O~. Corticosteronc production ~as assayed by nlcasuviug its conccJ~tration in the incubatkm media. The hormone was extracl~d and purified by HPLC as previously detailed [36], and its conccntraficm measured by RIA (see above). [ntra- anti inter-assay ~ariati~ms x~crt: 7.2% and 8.6c~, respectively. 2.3. Statis'tics

Data were expressed as means_+ S.E., and their statistical comparison was performed by ANOVA, followed by the Multiple Range Test of Duncan.

3. Results 3.1. I n v i v o e x p e r i m e n t s

NPK bolus injection did not change ACTH plasma c o n c e n t r a t i o n in h y p o p h y s e c t o m i z e d / A C T H - r e p l a c e d r a t s , b u t it d o s e - d e p e n d e n t l y i n c r e a s e d it in s h a m - o p e r a t e d a n i m a l s . T h e r i s e w a s a l r e a d y s i g n i f i c a n t at a d o s e o f 100 p m o l / r a t ( 3 5 % ) a n d r e a c h e d its m a x i m u m ( 5 5 % ) at a dose of 1 nmol/rat (Fig. 1). C o n v e r s e l y , N P K e n h a n c e d c o r t i c o s t e r o n e p l a s m a c o n c e n t r a t i o n in b o t h sham-operated and hypophysectomized/ACTH-rep l a c e d r a t s , b e i n g t h e first g r o u p o f a n i m a l s m o r e s e n s i t i v e t o t h e n e u r o p e p t i d e t h a n t h e s e c o n d o n e . In t h e s h a m - o p e r a t e d r a t s t h e e f f e c t w a s a l r e a d y signific a n t a t a d o s e o f 10 p m o l / r a t ( 2 2 % ) a n d a t t a i n e d its m a x i m u m ( 4 0 % ) a t a d o s e o f 100 p m o l / r a t , w h i l e in hypophysectomized/ACTH-replaced a n i m a l s a signifi-

40

fiasma ACTH concentration(pM)

35intact rats

2,2. In citro experiments

Intact rats akin to those employed in in vivo experiments were decapitated, and their adrenal glands promptly removed and freed of pericapsular fat. The left glands were gently decapsulated to separate zona glomerulosa, and then bisected; adrenal halves were then enucleated to eliminate zona medullaris. Dispersed inner (zonae fasciculata/reticularis) adrenocortical cells were obtained by collagenase/DNase I digestion and mechanical disaggregation [43]. Dispersed cells obtained from 4 adrenals were pooled to obtain a single cell suspension, and 6 cell preparations for each incubation experiment were employed. The right adrenals were decapsulated and cut into slices containing cortex and medulla [1]. Both kinds of preparations were placed in Medium 199 (DIFCO, Detroit, MI) and potassium-free Krebs-Ringer bicarbonate buffer with 0.2% glucose (2:1, v/v), containing 5 mg/ml human serum albumin. Dispersed cells and adrenal slices were incubated (3 >( 105 cells or 6-8 mg/ml), in replicates of 6 each, with NPK (from 10- 12 to 10 6 M). Adrenal slices were incubated, in replicates of 8 each, with ACTH (10 12 lO-iO or 10 - s M), 10 - 6 M a-helical-CRH (a-CRH; Peninsula) or i0 0 M corticotropin-inhibiting peptide (CIP; Peninsula), in the presence or absence of 10 -8 M NPK. The incuba-

*

30-

25-

20-

,s10

hymh, o,ni,U

I NPK (.log mot/rat)

|

I

|

I

I

|

0

12

11

I0

9

8

Fig. 1. Effects of NPK bolus administration on plasma ACTH concentration in intact (sham-hypophysectomized) and hypopbysectomized/ACTH-replaced rats. Values are means_+ S.E. (n = 6). * P < 0.01 from the respective basal (no NPK administered)group.

G. Mazzocchi et al. /Brain Research 661 (1994) 91-96

400

1200

93

corticosteroneproduction (pmol/mg. h)

plasma corticosterone concentration (nM) O

1000-

350-

© 300-

800-

250-

600-

200-

400-

controls

<~ NPK lO-eM

<~> intact rats 150-

hypophysectomized ACTH-replacedrats

100

,

o

1'2

,

NPK (-log tool/rat) , ,

11

1'0

0

ACTH (-log M) 0

0

Fig. 2. Effect of NPK bolus administration on plasma corticosterone concentration in intact (sham-hypophysectomized) and hypophysectomized/ACTH-replaced rats. Values are means+_ S.E. (n ~ 6). + P < 0.05 and * P < 0.01 from the respective basal (no NPK administered) group. c a n t rise ( 2 0 - 2 2 % ) w a s o b s e r v e d o n l y w h e n 100 p m o l N P K w e r e i n j e c t e d (Fig. 2). 3.2. In L,itro experiments N P K did n o t s i g n i f i c a n t l y a f f e c t c o r t i c o s t e r o n e p r o d u c t i o n by d i s p e r s e d a d r e n o c o r t i c a l cells (Fig. 3). C o n -

300

200-

corticosteroneproduction (pmol/lOScells~ or mg~.h)

I

I

l

I

0

12

10

8

Fig. 4. Effect of l0 -~ M NPK on ACTH-stimulated corticosterone production by decapsulated adrenal quarters. Values are means+ S,E. (n = 8). + P < 0.05 from the respective control group.

v e r s e l y , it r a i s e d c o r t i c o s t e r o n e s e c r e t i o n o f d e c a p s u l a t e d a d r e n a l slices, b e i n g t h e m i n i m a l a n d m a x i m a l e f f e c t i v e c o n c e n t r a t i o n s 10 -~ M ( 6 3 % ) a n d 10 - 8 M (2-fold), r e s p e c t i v e l y (Fig. 3). A s e x p e c t e d , A C T H c o n c e n t r a t i o n - d e p e n d e n t l y inc r e a s e d c o r t i c o s t e r o n e s e c r e t i o n o f a d r e n a l slices: 1 0 - , 2 M, 2.4-fold; 10 1(1 M, 5.4-fold; a n d 10 s M, 8.6-fold. 10 - 8 M N P K i n d u c e d a 2-fold rise in b a s a l c o r t i c o s terone secretion, and evoked a 50%-rise of that stimul a t e d by 10 -12 M A C T H ; t h e n e u r o p e p t i d e did n o t

250 250-

corticosteroneproduction (pmol/mg. h)

*

200" 200150150-

o 100-

100-

50-

a

1® dispersedcells ~ decapsulatedquarte~I

50-

[ [ ] a-CRH ,~, CIP ]

NPK I-log M) NPK (-log M) 0

I

I

o

12

1;

l

I

l

0

6

7

Fig. 3. Effect of NPK on corticosterone production by dispersed inner adrenocortical cells and decapsulated adrenal quarters. Values are means _+S.E. (n = 6). * P < 0.01 from the respective basal (no NPK added) group.

0

I

I

0 8 Fig. 5. Effect o f l 0 6 M a-CRHand 10 6 M CIP on basal and 10 s M NPK-stimulated corticosterone production by decapsulated adrenal quarters. Values are means+_S.E. (n = 8), * P < 0.01 from basal (no NPK added) group; ~ P < 0.01 from the respective control group.

g4

(;. Mazzo~chi ~,t aL / Brain Research OO[ (1994) g] 90

apparently affect corticosterone responses to 10 j'~ M or 1() '~ M ACTH (Fig. 4). ~-CRH and CIP (10 ~' M) did not change basal corticosterone secretion of adrenal slices, but they completely annulled either l0 s M CRH and 10 s M ACTH-evoked increases (data not shown) or 10 -s M NPK-induced 2-fold rise in corticosterone output (Fig. 5).

4. Discussion

Our present in vivo findings obtained in sham-operated rats agree with those of Kalra and Kalra [20], who reported that the intravenous injection of 2.5 nmol NPK markedly increases plasma corticosterone level within 10-60 rain. These results indicate that circulating NPK acts on median eminence and pituitary, two regions located outside the blood-brain barrier, to increase A C T H release. However, our experiments demonstrate that the NPK-induced rise in the blood level of corticosterone is not exclusively due to the stimulating effect of the neuropeptide on pituitary corticotropes, since it also occurs in hypophysectomized rats in the absence of any relevant change in the plasma concentration of ACTH. Taken together these data strongly suggest that NPK may be included in that family of neuropeptides exerting a direct stimulatory effect on adrenal glands (see Introduction). This contention appears to be in keeping with the demonstration that substance P, an endecapeptide structurally related to NPK and NML and with the same distribution in the HPA axis [9,18,22], exerts a direct modulatory action on aldosterone secretion by rat adrenal gland both in vivo and in vitro [16,35,37,50]. Our in vitro experiments provide clearcut evidence that the direct corticosterone secretagogue effect of NPK requires the integrity of the adrenal tissue, since it manifests itself in adrenal slices but not when dispersed adrenocortical cells are employed. Parenthetically, it must be noted that a good accord exists between the in vitro and in vivo minimal and maximal effective concentrations of NPK. In fact, given that the peritoneal absorption rate of peptides in rats is very high and assuming 10-12 ml of blood per rat [38], it may be calculated that the bolus intraperitoneal injection of the minimal (10 - ~ mol) and maximal (10 -~ mol) doses of NPK produce blood concentrations of the peptide of about 10 - 9 and 10 -v M, respectively. Several investigations indicate that the integrity of adrenal tissue is frequently needed for several neuropeptides may exert their steroidogenic action [46,47]. This may be due to the fact that, as it has been recently demonstrated for oxytocin [48], the integrity of cell-tocell contacts may allow the spreading of the secretagogue signal from responsive to unresponsive cells.

However, this does not seem to be the case as far as NPK is concerned, since preliminary data (not shown) indicate that this neuropeptide does not elicit any appreciable secretory response by cnucleated (i.c. demedullated) adrenal quarters. Compelling evidence suggests that in rats adrenal medulla, by secreting catecholamines and many regulatory peptides, exerts a paracrine control of the cortical function [6,15,17,26,30,39], the morphologic background of which may be the presence of abundant interdigitations between medullary and cortical tissues occurring in the adrenals of this species [4,7,14]. Hence, it seems reasonable to think that medullary chromaffin cells play a pivotal role in the mediation of the direct glucocorticoid secretagogue effect of NPK. Our present results confirm this contention and throw light on the mechanism underlying this effect of NPK. Evidence is available of the existence of a C R H / A C T H system in the rat adrenal medulla, which may affect the cortex function in a paracrine manner [2,3,5,28] and whose activity is enhanced when hypothalamo-pituitary C R H / A C T H system is suppressed by hypophysectomy [29]. It has also been shown that interleukin-I/3 exerts a direct secretagogue effect on the rat adrenal cortex by stimulating such a local intramedullary system [1,31]. In light of these considerations it appears conceivable that NPK, in addition to stimulate the central branch of C R H / A C T H system, may also activate the intramedullary peripheral one. Our present findings lend strong support to this contention, inasmuch as the NPK-induced corticosterone response of adrenal slices is completely blocked by both a - C R H and CIP, two competitive inhibitors of CRH [41] and A C T H [23], respectively. This hypothesis may also easily explain why 10 -s M NPK potentiates corticosterone response of adrenal slices induced by low concentrations of ACTH, but not those evoked by higher ones. In fact, it has been calculated that the maximal CRH- or interleukin-1/3-evoked stimulation of medullary A C T H release may produce intra-adrenal concentrations of A C T H of about 1()- '~ M [31]. In conclusion, our investigation demontrates that NPK is a potent stimulator of rat HPA axis, that acts both on the central and peripheral (intramedullary) branch of the C R H / A C T H system. Since NPK is contained in adrenal medulla [9], it may be hypothesized that this neuropeptide may be involved in the regulation of the complex mechanisms whereby adrenal medulla paracrinally controls steroid secretion.

References

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[37]

[38]

[39]

[40]

[41]

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