Action of AT1 subtype angiotensin II receptors of the medial preoptic area on gonadotropins and prolactin release

Neuropeptides (1998) 32 (1), 51-55 © HarcourtBraceand CompanyLtd 1998

A c t i o n of A T 1 s u b t y p e angiotensin II r e c e p t o r s of the medial preoptic area on gonadotropins and prolactin release R. C. M. Dornelles, C. R. Franci Faculdade de Medicina de Ribeir~.o Preto, Departamento de Fisiologia, Universidade de S~.o Paulo, Brazil

Summary. This study determined the effect of the selective angiotensin II (A II) AT 1 receptor subtype antagonist Iosartan in the medial preoptic area (MPOA) of ovariectomized rats, treated with estrogen or untreated, on the release of gonadotropins (LH and FSH) and prolactin (PRL). The MPOA is sensitive to the action of A II and contains cell bodies of neurons producing luteinizing hormone-releasing hormone and a large density of estradiol receptors. Plasma FSH was not altered in any situation. However, Iosartan blocked and estradiol facilitated the stimulating and inhibitory effects of A II microinjection into the MPOA on LH and PRL secretion, respectively. The results indicate that these effects are mediated by AT1 receptors in the MPOA and that estradiol may modulate them. On the other hand, Iosartan itself reduced LH secretion in ovariectomized rats, indicating that the increase in the secretion of this hormone, after removal of the negative feedback caused by estradiol, is due, at least in part, to the action of A II on AT~ receptors of the MPOA.

INTRODUCTION

Angiotensin II (AII) is an octapeptide which plays a role in cardiovascular control, homeostasis and pituitary hormone release? The demonstration of the existence of a cerebral renin-angiotensin system 2 and the increased concentration of A II receptors in the central nervous system (CNS) and pituitary 3 indicate that A I I may exert a neuroendocrine action and may be involved in the control of pituitary function. Two AII receptor subtypes have been characterized in peripheral tissues ¢~ and in the brain. 6-9 The AT~ receptors are sensitive to blockade by losartan (DUP-753) and the AT2 receptors s are sensitive to blockade by CGP 42112 and PD 12317Z The medial preoptic area (MPOA) is involved in the control of the cyclic release of gonadotropins. This area contains a large number of estrogen (E2) receptors, Received 2 July 1997 Accepted 30 October 1997 Correspondence to: C. R. Franci, Departamento de Fisiologia, Faculdade de Medicina de Ribeir~o Preto, Av. Bandeirantes, 3900 - Campus USP 14049-900, Ribeir~.o Preto- SP, Brazil. Tel: 55-16-6023022, Fax: 55-16-6330017; e-mail: [email protected]

luteinizing hormone-releasing hormone (LHRH) cells ~° and A II receptors al and shows an increased cell discharge frequency upon AII administration. 12 Over the last few years, it has been shown 13-16that A II acts by regulating the hypothalamo-pituitary-gonadal axis and cyclic reproductive activity in animals. Intracerebroventricular (icy) microinjection of A II increases the release of luteinizing hormone (LH) in rats during proestrus and in ovariectomized rats (OVX) treated with E2 and progesterone (P4). Furthermore, in rats with a regular estrous cycle, saralasin (a peptidergic A II blocker) or enalapril (an A I I converting enzyme inhibitor) inhibits the pre-ovulatory LH surge and blocks ovulation. Several lines of evidence support the existence of a dual control of prolactin (PRL) secretion by A I I in rats, one as an inhibitor through the CNS and the other as a stimulator at the adenohypophyseal level, as central administration of A I I inhibits PRL secretion I3,17 while hypophyseal administration stimulates it. ~7-~9 Different studies have shown that the action of AII on the control of LH and PRL secretion is E2 dependent. The purpose of the present study was to determine the action of AT~ receptors in the MPOA on LH, FSH and PRL secretion as a function of estradiol. 51

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MATERIALS AND METHODS

Radioimmunoassay (RIA)

Animals

The radioimmunoassay (RIA) procedure for measuring plasma LH, FSH and PRL has been described previously.22.23 The lowest detectable amount of LH RP 3 standard was 0.04ng/ml, the inter-assay coefficient of variation was 16.8%, and the intra-assay coefficient of variation was 3.44%. Plasma FSH data are expressed in terms of the RP 2 standard and the lowest detectable amount was 0.19 ng/ml, the inter-assay coefficient of variation was 10.2%, and the intra-assay coefficient of variation was 2.5%. The lowest detectable amount of PRL RP3 standard was 0.19 ng/ml, the inter-assay coefficient of variation was 12.16%, and the intra-assay coefficient of variation was 2.33%.

Experiments were performed on aduk female Wistar rats (180 g) which had been kept in a light-, humidity- and temperature-controlled environment (lights from 7.00 a.m. to ZOO p.m., 22 + 2°C). Rats had free access to water and chow. Surgery and instrumentation Before each surgery the animals were anesthetized with I ml/100 g tribromoethanol (Aldrich Chem. Comp. Inc.) in 2.5% saline, administered intraperitoneally (i.p.). Animals were ovariectomized (OVX) and 14 days later, a unilateral stainless steel cannula (16.5 mm in length) was implanted into the MPOA (coordinates: AP 2.2.; L + 0.8; V -7.9), 20 using a Kopf stereotaxic instrument. A guide cannula was held in place by two skull screws and dental cement (Simplex Dental of Brazil, S.A.) and provided with a mandril to prevent obstruction. After surgery the animals were returned to individual cages. One week after stereotaxic surgery the animals were either unprimed (OVV group) or primed (OVBE group) with estradiol benzoate (50 ~g/ml corn oil, injected s.c.) for 3 days before the experiment. An intra-atrial catheter of silicone tubing (Dow Coming, 0.020 x 0.037 diameter) was inserted through the jugular vein on the day before the experiment by the technique described previously. 2~ On the day of the experiment an extension of polyethylene tubing (PE-50) filled with heparin-0.9% NaC1 (1:40 ml) was attached to the distal end of the jugular cannula.

Histological analysis The brains were removed at the end of the experiments and fixed with 10% formalin for histological analysis to confirm the position of the cannula in the MPOA. Figure 1 shows the placement of the cannula in the MPOA. Only the animals with the cannula implanted into this area were considered for hormone secretion analysis. Data analysis Data were analysed statistically using computer software (SAS) that performs analysis of variance for repeated measure, followed by the Tukey new multiple range test. Results are expressed as mean + SEM. Differences were considered significant when P < 0.05. RESULTS

Drug administration and blood withdrawal Microinjection of saline (0.15 M), A II (Sigma; 100 pmoles) and losartan (2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'(1H-tetrazol-5-yl)biphenyl-4-1) methyl]-imidazole, potassium salt; Du Pont; 100 pmoles) were performed according to the following procedure: microinjection was made in a volume of 1 gl using a 10-gl Hamilton syringe connected by polyethylene tubing (PE 10) to a Mizzy needle in the stainless steel cannula. The tubing and needle had been previously filled with the solution to be injected. The m.i. into MPOA were carried out lOmin (NaC1 or losartan) and 20 min (NaC1 or A II) after basal bleeding. Heparinized blood samples (0.8m l) were collected from the external jugular vein cannula at the following intervals: -20 (basal bleeding), 0, 10, 20, 30 and 60 min, while the animal was freely moving in the cage. The volume of all samples was replaced immediately after each bleeding with an equivalent volume of saline. Plasma was separated by centrifugation at 4°C and stored frozen until the day of assay. Neuropeptides (1998) 32(1), 51-55

Plasma FSH did not alter after microinjection of NaC1/NaC1 and NaC1/A II or losartan/NaC1 and losartan/A II into the MPOA of unprimed (Fig. 2A) or primed animals (Fig. 2B). There was a significant increase in plasma LH 60 min after microinjection of AII into the MPOA of animals of the unprimed group (Fig. 3A). Losartan, with or without AII, reduced plasma LH at 20 and 30 min after injection and blocked the increase in plasma LH induced by A II at 60 min. In the animals of the primed group (Fig. 3B), the stimulating effect of A II started at 20 rain and was maintained up to 30 and 60 min after microinjection. This effect was blocked by losartan. However, losartan itself did not alter LH secretion. Plasma PRL in animals of the unprimed group was significantly reduced only at 60 min after microinjection of AII into the MPOA (Fig. 4A). However, in the animals of the primed group (Fig. 4B), A I I significantly reduced PRL release at 10 min. The inhibitory action of A I I was blocked by losartan in both groups. © Harcourt Brace and Company Ltd 1998

AT 1 receptors in MPOA and LH, FSH and PRL

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Fig. 1 Illustration of the placement of the cannula in the medial preoptic area. On the left, a schematic drawing illustrates a cerebral hemisphere where the medial preoptic area is represented by the abbreviation MPOA. On the right, as a complementary hemisphere, a histological section embedded in paraffin and stained with Nissl is shown. The arrow indicates the placement of the cannula tip in the MPOA. 40

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Fig. 2 Effect of: - l - NaCI + NaCI, - 0 - NaCI + A II, --Ell.. Iosartan + NaCI and ..©.- Iosartan + A II administered into the MPOA of unprimed ovariectomized (A) and estrogen-primed ovariectomized rats (B) on FSH secretion. Values are means + SEM. The number of animals in the groups ranged from 10 to 26.

DISCUSSION The present resuks show that losartan and estradiol facilitate the stimulating and inhibitory effects of A I I microinjection into the MPOA on LH and PRL secretion, respectively. However, blockade of the AT~ receptors of the MPOA in ovariectomized rats reduced LH, but not FSH or PRL secretion. This effect of losartan on LH secretion was not observed in ovariectomized rats treated with E2. The exogenous administration of A I I by the icy route to ovariectomized rats together with the replacement of sex steroid stimulates LH release. ~3,24,2~A I I does not exert its action directly in the anterior pituitary, but presumably © Harcourt Brace and Company Ltd 1998

modifies LHRH secretion. 26 Endogenous A I I m a y be involved in the control of LH secretion, since the blockade of the A I I receptor (saralasin} or inhibition of A I I synthesis (enalapril) by icv microinjection on the day of proestrus has been shown to reduce LH release and inhibit ovulation. ~4 During proestrus there is an increase in A I I release in the anterior hypothalamic-medial preoptic area 27 compared to diestrus I. Furthermore, the administration of A II-specific antibodies into the third ventricle of female OVX rats reduced plasma LH and the LHRH level in the median eminence and MPOA. 28,29 A II increases the discharge frequency in most MPOA neurons. This fact suggests a direct mechanism of action Neuropeptides (1998) 32(1), 51-55

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Fig. 4 Effect of --E- NaCI + NaCI, - 0 - NaCI + A II, ..[~.. Iosartan + NaCI and ..O.. Iosartan + A II administered into the MPOA of unprimed ovariectomized (A) and estrogen-primed ovariectomized rats (B) on PRL secretion. Values are means + SEM. * P < 0.05 versus NaCI + NaCI; a p < 0.05 Iosartan + A II versus NaCI + A II. The number of animals in the groups ranged from 10 to 18.

of A I I on neuronal elements in this region. 12 Therefore, the increase in LH secretion after ovariectomy m a y be due, at least in part, to the action of A I I on AT~ receptors in the MPOA, where it would stimulate the activity of LHRH neurons. There is no information in the literature about the possible action of A I I on FSH secretion. In the present study, no alteration in the plasma FSH was observed after the administration of A II or losartan into the MPOA in ovariectomized rats treated with estradiol or untreated. This represents additional evidence that the control of FSH release is independent of LH. Neuropeptides (1998) 32(1), 51-55

The present resuks show that AII microinjected into the MPOA reduces PRL secretion through AT 1 receptors and this effect is potentiated by estrogen therapy. Immunoneutralization of All through icy administration of specific antiserum in ovariectomized rats indicated that endogenous cerebral A I I m a y inhibit PRL secretion? TM Other investigators reported a central inhibitory action of AII on PRL secretion, but only in the presence of estrogenY The inhibitory action of A I I on PRL secretion m a y be due, at least in part, to the stimulation of the dopaminergic system since this effect was blocked by a dopaminergic antagonist. ~3 Another possible mechanism of this © Harcourt Brace and Company Ltd 1998

AT 1 receptors in MPOA and LH, FSH and PRL

i n h i b i t o r y a c t i o n m a y b e t h e r e l e a s e of a p e p t i d e associa t e d w i t h L H R H (GAP) w h i c h i n h i b i t s PRL s e c r e t i o n . 32 T h e d i s t r i b u t i o n of cells i m m u n o r e a c t i v e to p r o - L H R H 40-53 (GAP) w a s i d e n t i c a l to t h a t o b s e r v e d for LHRH. 33 T h e c o - r e l e a s i n g of L H R H a n d GAP m a y r e g u l a t e LH a n d PRL s e c r e t i o n in a n i n v e r s e r e l a t i o n s h i p u n d e r s o m e physiological circumstances. In s u m m a r y , t h e s t i m u l a t i n g o r i n h i b i t o r y a c t i o n of A I I in t h e M P O A o n LH a n d PRL s e c r e t i o n , r e s p e c t i v e l y , o c c u r s t h r o u g h AT 1 r e c e p t o r s a n d is f a c i l i t a t e d b y g o n a d a l steroids. T h e i n c r e a s e in LH s e c r e t i o n a f t e r o v a r i e c t o m y m a y b e d u e , at l e a s t in part, to t h e a c t i o n of A II o n AT 1 r e c e p t o r s in t h e MPOA.

ACKNOWLEDG EMENTS W e t h a n k S6nia A p a r e c i d a Z a n o n Baptista for t e c h n i c a l s u p p o r t a n d Dr E u c l i d e s Braga M a l h e i r o s for p e r f o r m i n g t h e statistical analysis. G r a n t s f r o m FAPESP a n d CAPES.

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Neuropeptides (1998) 32(1), 51-55