Naloxone enhances angiotensin II-induced increase in serum luteinizing hormone concentrations in normal women

Regulator), Peptides, 51 (1994) 161-167 © 1994 Elsevier Science B.V. All rights reserved 0167-0115/94/$07.00

161

REGPEP 01646

Naloxone enhances angiotensin II-induced increase in serum luteinizing hormone concentrations in normal women Vittorio Coiro a,,, Corrado Cigarini c, Riccardo Volpi a, Luigi Capretti d, Alberto B a c c h i - M o d e n a b and Paolo Chiodera a "Department of lnternal Medicine, University of Parma, Via Gramsci 14, 43100 Parma (Italy), bDepartment of Obstetrics and Gynecology, University of Parma, Parma (Italy), CDivision of Obstetrics and Gynecology, Hospital of Reggio Emilia, Reggio Emilia (Italy) and d Division of Internal Medicine, Hospital of Codogno, Codogno (Italy) (Received 17 August 1993; revised version received 20 January 1994; accepted 1 February 1994)

Key words." Naloxone; Angiotensin II; Luteinizing hormone

Summary The present study was carried out in order to establish whether the stimulatory effect of angiotensin II (ANG II) on serum concentration of LH in women is under control by endogenous opioid peptides. For this purpose, the effect of ANG II (infusion for 60 min of successively increasing doses of 4, 8 and 16 ng/kg/min; each dose for 20 min) on serum LH levels was evaluated during administration of saline or the opioid antagonist naloxone (4 mg in an i.v. bolus followed by 10 mg over 2 h) in 7 normal women in both follicular and luteal phase. In all subjects, tests with saline alone (control test) or naloxone alone were performed. No significant changes in LH levels were observed in any test performed in the follicular phase and in the control test performed in the luteal phase. In contrast, in the luteal phase, both ANG II and naloxone induced significant increments in LH levels (23~o increase vs. baseline by ANG II and 54~o by naloxone). When naloxone and A N G II were given together stimulated LH increase was greatly enhanced. The mean peak was 154~o higher than baseline and was higher than the sum of the individual peaks produced by naloxone and ANG II alone. These findings suggest inhibition by endogenous opioids of the stimulating action of ANG II on LH secretion during the luteal phase.

* Corresponding author.

162 Introduction

The finding that a systemic administration of angiotensin II (ANG II) stimulates LH release in women during the luteal phase [ 1] led to hypothesize a possible role for A N G II in regulation of cyclic reproductive activity in humans. At present, the neuroendocrine mechanisms underlying A N G II action are still uncertain. In recent years, attention has been focused on the involvement of endogenous opioids, particularly beta endorphin [2] as inhibitory modulators of LH secretion [3,4]. Since A N G II has been found able to play a role at hypothalamic-pituitary level in the regulation of opioid peptide secretion [5-7], we hypothesized a possible interaction between A N G II and endogenous opioids in the control of LH secretion. In order to clarify this issue, the LH response to the administration of A N G II in normal women during luteal phase was evaluated either in the presence or absence of the opioid antagonist naloxone. Control experiments with naloxone alone were performed in the same subjects.

menstrual cycles). The same tests were repeated on luteal phase (22nd day of the same menstrual cycles). In both follicular and luteal phase, tests were carried out in random order and followed a similar procedure. Experimental procedure

At 08.00 h a 21 gauge cannula was inserted into the left antecubital vein and a double lumen indwelling catheter was placed into the right antecubital vein of subjects lying in the recumbent position after an overnight fast and rest in bed. The cannula was used for blood sampling, whereas the catheter served for the administration of A N G II, naloxone and/or normal saline (NaC1 0.9~o). ANG H test

A basal blood sample was taken 30 min after needle insertions (time 0) and was followed by a 60 min infusion of A N G II (Asp-1, Ile-5 angiotensin II Hypertensin, Ciba Geigy Italy, dissolved in 50 ml of normal saline) in successively increasing doses of 4, 8 and 16 ng/kg/min, each dose for 20 min. Further blood samples were collected 30, 60, 90, 120, 150 and 180 min after the beginning of A N G II infusion.

Materials and Methods ANG H plus naloxone test

Seven healthy women (aged 25-31 years) participated in this study after giving their informed consent. None of the subjects had clinical or laboratory evidence of endocrine, hepatic or renal disease. All women were within 10~o of their ideal body weight and had a history of regular menstrual cycles of normal duration (26 to 30 days). No drugs were taken by any subject for at least 3 weeks before the experimental days. Basal body temperature and plasma levels of ovarian steroids were evaluated dally and served as criteria to determine the precise period of the menstrual cycle. Each subject was tested four times (control, A N G II, naloxone and A N G II plus naloxone test) on follicular phase (5th day of four following regular

This test was performed as the previously described A N G II test, except for the injection of an i.v. bolus of 4 mg naloxone (Narcan, Crinos, Italy) in 10 ml of normal saline at time 0, just before A N G II infusion. Naloxone injection was followed by the infusion over 2 h of 10 mg naloxone diluted in 100 ml of normal saline. In the A N G II test an equal volume of normal saline (i.e., 110 ml) was given instead of naloxone. Naloxone test

This test was performed as the previously described A N G II plus naloxone test, except for the infusion of 50 ml normal saline instead of ANG II.

163 Control test

Control test

This test followed an identical procedure as the abovedescribed tests, except for the administration of 160 ml normal saline instead of naloxone and A N G II. Systolic/diastolic blood pressure was monitored with a mercury sphygmomanometer at each sampling time during all tests.

The administration of normal saline did not change the serum concentrations of LH at any time duririg tests (Fig. 1A and B).

Assays

Serum L H levels were measured in all samples with a specific RIA [8], using commercial kits. The lower limit of sensitivity was 1.5 mIU/ml. Intra- and inter-assay coefficients of variation were 6~o and 9~o, respectively. Plasma 17-/~-estradiol (E2) [9] and progesterone (P) [10] concentrations were measured in basal samples (time 0) by specific RIA, using commercial kits. All samples from the same subject were measured in duplicate in the same assay. Hematocrit and sodium concentrations were measured in all specimens. Hematocrit was measured by Drummond microhematocrit (Drummond Scientific, Broomal, CA) and serum sodium concentrations were evaluated by flame photometry.

Statistics

Results were analyzed with the Wilcoxon's matched pair rank sum test and analysis of variance, as appropriate. Values are expressed as mean+ S.E.

A N G H test

The infusion of increasing amounts of A N G II in the follicular phase did not produce any significant change in LH levels (Fig. 1B). In contrast, when administered in luteal phase, the infusion of A N G II induced a significant increase in serum LH levels, that reached plateau values 23 ~o higher than baseline at 60 min (Fig. 1A). The LH response to A N G II lasted for at least 1 h after the end of A N G II infusion (P<0.02, at 60 and 180 min vs. baseline; P < 0 . 0 5 at 90 and 120 min; NS at 150 min vs. baseline) (Fig. 1A). LH levels during A N G II test were significantly higher than during control test (F= 14.17; P < 0.02). Naloxone test

The administration of naloxone in the follicular phase did not modify the serum levels of LH (Fig. 1B), whereas it induced a striking LH response in the luteal phase (Fig. 1A). When tested on the 22nd day of the cycle naloxone induced a 54~o increase in serum LH concentrations, which remained constant during naloxone infusion and slowly declined thereafter (P<0.01 at 30, 60, 90 and 120 min vs. baseline; P < 0 . 0 5 at 150 min vs. baseline). LH levels during naloxone test were significantly higher than during control test (F=21.40, P < 0 . 0 1 ) (Fig. 1A).

Results

A N G H plus naloxone test

The mean plasma levels of E2 were 65.2_+ 2.8 pg/ml (mean + S.E. of 7 women. For each woman, a mean value was calculated from those obtained at time O of the 4 tests) in the follicular phase and 79.3 + 4.8 pg/ml in the luteal phase. P concentrations in the plasma were 1.2 + 0.2 ng/ml in the follicular phase and 9.7 + 0.8 ng/ml in the luteal phase.

When A N G II and naloxone were given together in the follicular phase, LH level did not change (Fig. 1B). In contrast a striking LH increase was observed in the luteal phase ( P < 0.01 vs. baseline at all examined points) (Fig. 1A). Plateau values were 154~o higher than baseline, were reached at 30 rain and lasted until the end of naloxone infusion (120 min). Thereafter, LH levels slowly declined during

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Fig. 1. Effects of saline, Ang I1, naloxone or the combination of Ang I1 plus naloxone on serum LH levels during luteal (A) and follicu;. lar (B) phase. Each point represents the mean + S.E. of seven observations. Statistical differences between curves: follicular phase none; luteal phase: Ang II vs. control (saline) test, F = 14,17, P<0.02; naloxone vs. control test, F = 21.40, P<0.01; Ang II plus naloxone vs. naloxone test, F = 26.40, P<0.01; Ang II plus naloxone vs. Ang II, F = 28,74, P<0.01.

165 the next hour. LH levels during the A N G II plus naloxone test were significantly higher than during naloxone test (F= 26.40, P < 0.01) and during A N G II test (F= 28.74, P < 0 . 0 1 ) (Fig. 1A). The administration of A N G II induced a significant increase in systolic/diastolic blood pressure (ANG II test: luteal phase baseline: 105/70_+ 4/2 mmHg; 30 rain: 130/80 + 3/1 (P<0.01 vs. baseline); 60 min: 140/85 + 4.2 ( P < 0.01); 90 min: 130/80 + 5/3 ( P < 0.01 vs. baseline). Similar values were observed at corresponding times in all tests where A N G II was given, both in follicular and luteal phase and regardless of the presence of naloxone. None of the subjects experienced side effects after A N G II and/or naloxone treatments. Hematocrit (time 0 of control test, luteal phase: 44.2+ 1.7~o) and serum sodium concentrations (luteal phase: 140.7 + 0.7 mEq/1) were similar at all points of all tests both in follicular and luteal phase regardless of pharmacological treatments.

Discussion

During luteal phase the regulation of LH secretion undergoes profound neuroendocrine changes, because it becomes sensitive not only to naloxone, but also to A N G II stimulation. These phenomena probably result from the combined effects of estrogens and progesterone, because it is wellknown that neither naloxone nor- A N G II are able to stimulate LH secretion during the follicular phase, when only ovarian estrogen production occurs. In addition, in the present study we have found that during the follicular phase neither the concomitant administration of A N G II and naloxone is able to stimulate LH secretion. The development of the sensitivity of LH to naloxone during the luteal phase suggests the establishment of an inhibitory control on LH secretion by endogenous opioids. In the same period, LH develops its sensitivity to A N G II stimulation, probably because of activation of A N G II expressing neurons

by ovarian steroids [ 11]. In fact, a significant increase in A N G II concentration at hypothalamic level has been reported by Phillips et al. after treatment with progesterone of estrogen-primed ovariectomized rats [ 11 ]. These findings support the hypothesis that A N G II becomes an important stimulatory regulator of LH secretion at a time of the menstrual cycle, when high circulating levels of both estrogen and progesterone occur [1]. The data presented here suggest a possible link between endogenous opioids and A N G II in the control of LH during the luteal phase. In fact, the LH releasing effect of A N G II was greatly enhanced by naloxone, suggesting a modulation by endogenous opioids o f A N G II action. In other words, only in the absence of an inhibitory opioid tone, A N G II was able to express its total LH releasing activity. Both naloxone and A N G II are supposed to stimulate L H - R H release at hypothalamic level [ 12-17], since none of the two substances stimulate LH release from the pituitary in vitro [ 12,18,19]. Naloxone easily crosses the blood brain barrier (BBB) [20], whereas A N G II is devoid of this capacity [21]. However, A N G II binding to circumventricular organs [22] and possible A N G II effects on the median eminence (which is located outside to BBB), make conceivable the effect of systemically administered A N G II on hypothalamic L H - R H release [23,24]. At present, it is unknown whether endogenous opioids and A N G II impinge upon the same neurotransmitter(s). However, a suggestive answer to this question might be hypothesized by taking into account previous studies in rats. In this species, catecholamines are activated by estrogens and progesterone and become important mediators of the stimulatory feed-back actions of the ovarian hormones on LH [25]. There is considerable evidence that A N G II stimulates L H - R H secretion by facilitating the release of norepinephrine in the preoptic area [26]. Both A N G II and norepinephrine require the presence of ovarian steroids to stimulate LH release. In fact, in ovariectomized rats the intraventricular administration of either A N G II or norepi-

166 n e p h r i n e inhibits i n s t e a d o f stimulating L H s e c r e t i o n [17,26]. H o w e v e r , in the s a m e e x p e r i m e n t a l m o d e l either A N G II or n o r e p i n e p h r i n e a d m i n i s t r a t i o n p r o d u c e d significant s t i m u l a t i o n o f L H release, w h e n given to o v a r i e c t o m i z e d rats p r e - t r e a t e d with estrogen and p r o g e s t e r o n e [17,26]. O n the o t h e r h a n d , n a l o x o n e - s e n s i t i v e e n d o g e n o u s o p i o i d s inhibit L H secretion in e s t r o g e n and p r o g e s t e r o n e - t r e a t e d o v a r i e c t o m i z e d rats by r e d u c i n g hyp o t h a l a m i c c a t e c h o l a m i n e r g i c activity [27]. T a k e n together

these

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e s t r o g e n / p r o g e s t e r o n e rich milieu o f the luteal p h a s e c a t e c h o l a m i n e s are a c t i v a t e d by steroids a n d thus d e v e l o p their s t i m u l a t o r y action o n L H secretion. In this c o n d i t i o n , A N G

II a n d e n d o g e n o u s

opioids

might exert their o p p o s i t e effects o n L H secretion (i.e., s t i m u l a t o r y for A N G II a n d inhibitory for o p i o ids) by m o d u l a t i n g c a t h e c o l a m i n e r g i c n e u r o t r a n s m i s sion. F u r t h e r studies are n e e d e d to s u b s t a n t i a t e this hypothesis.

Acknowledgements V.C. was s u p p o r t e d in p a r t by M i n i s t e r o U n i v e r sitfi R i c e r c a Scientifica T e c n o l o g i c a ( R o m a , Italy).

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