Ovarian hormones in man: their effects on resting vascular tone, angiotensin converting enzyme activity and angiotensin II-induced vasoconstriction

Abstract

Aims

Oestrogens in women have been shown to cause vasodilation which may reflect alterations in the activity of vascular angiotensin converting enzyme (ACE) and/or sensitivity to angiotensin II. The aim of this study was to assess the effect of ovarian hormones on vascular tone, vascular ACE activity and vasoconstriction to angiotensin II in males.

Methods

Eight volunteers were randomised in a crossover design to oestradiol, medroxy-progesterone, and placebo. Vasoconstriction to angiotensin I and angiotensin II was assessed by forearm plethysmography.

Results

Although baseline forearm flow was increased with oestradiol, suggesting generalized vasodilation, there were no changes in the vasoconstrictor responses to angiotensin I or angiotensin II. Medroxy-progesterone affected neither baseline flow nor vasoconstrictor responses. The results expressed as percentage reduction in flow (mean ± s.d.) were: angiotensin I 48 pmol ml⁻¹: placebo −48 ± 14%; oestradiol −42 ± 16%; medroxyprogesterone −43 ± 8% and for angiotensin II 16 pmol ml⁻¹: placebo −42 ± 10%; oestradiol −39 ± 11%; medroxyprogesterone −46 ± 13%.

Conclusions

Acute administration of oestradiol caused vasodilation in males, the effect was not due to alterations in vascular ACE activity or to altered sensitivity to angiotensin II.

Introduction

Premenopausal women have a lower prevalence of atherosclerotic vascular disease than men or postmenopausal women, this is believed to relate to the protective vascular effects of ovarian hormones [1].

Oestrogens produce vasodilation, potentially via multiple mechanisms such as the release of nitric oxide from the endothelium [2]. One possible mechanism is inhibition of angiotensin converting enzyme. Proudler et al. showed that both oestrogens and progesterone as hormone replacement therapy (HRT), can reduce serum angiotensin converting enzyme (ACE) activity in postmenopausal women [3, 4].

This study was carried out to investigate the effect of acute ovarian hormones on forearm blood flow, plasma ACE activity, and tissue ACE activity in addition to the vasoconstrictor response to angiotensin II. Tissue ACE activity was measured by the differential vasoconstriction to angiotensin I against angiotensin II as angiotensin I requires conversion to angiotensin II for biological effect.

We chose to use normal male volunteers rather than females because their oestrogen and progesterone status could be more easily manipulated without bias from endogenous hormones. It is known that oestrogen receptors are present on male endothelial cells [5], and intravenous oestrogens, given acutely, improve coronary blood flow in men in response to acetylcholine [6] and the cold pressor test [7].

Methods

This study was of a double-blind, randomised crossover design. Informed consent was obtained to the study protocol, which had been previously approved by the Tayside Committee for Medical Research Ethics and this investigation conformed with the principles outlined in the Declaration of Helsinki.

Eight healthy male volunteers, aged 20–35 years, with no history or clinical evidence of cardiovascular disease were studied on three separate occasions 1 week apart. All patients had fasted and abstained from cigarettes for a minimum of 8 h.

Randomised medication was taken orally as two doses, the first 12 h and the second 2 h before the study. Doses were as follows; 2 mg oestradiol valerate (Climaval, Sandoz), 20 mg medroxyprogesterone acetate (Provera, Upjohn), or placebo. Blood was taken on arrival for ACE activity, oestradiol, progesterone, and testosterone.

Blood for hormones was spun at 3000 rev min⁻¹ for 15 min, the serum was collected and stored at −20 °C until analysed using radio-immunoassay (Sorin Biomedica, Italy). Serum ACE activity was analysed by automated analysers (Technicon Axon, Bayer Diagnostics, Basingstoke UK).

Forearm plethysmography was performed using a standard protocol, which has been previously described [8]. A 27G needle was inserted into the nondominant brachial artery. After 30 min rest infusing 0.9% saline, baseline strain gauge measurements were taken. The average ratio of three measurements from both arms was taken as the baseline ratio of forearm blood flow.

Angiotensin I was then infused at three concentrations, 12 pmol min⁻¹, 24 pmol min⁻¹, and 48 pmol min⁻¹ which were expected to produce 25%, 35% and 50% reduction in blood flow under normal conditions. Each concentration was infused over 7 min, with readings taken over the last two minutes. Following the third dose, 0.9% saline was infused until the blood flow ratio had returned to baseline. Angiotensin II was then infused at 4 pmol min⁻¹, 8 pmol min⁻¹, and 16 pmol min⁻¹, which we expected to produce similar reductions in flow. An identical protocol was followed for each study.

The peptides were dissolved in 0.9% saline, and the infusion rate was kept constant at 1 ml min⁻¹ throughout the study period (Grasby 310 syringe pump, Grasby Medical). All peptides were supplied by Calbiochem/Novobiochem, Nottingham, England. Discussion with the company established that the peptide activity was stable over 24 h after reconstitution, but solutions were made up freshly within 45 min of each study.

Statistical analysis

Comparisons between the biochemical parameters were performed using Student's t-test. For statistical analysis, the ratio of flow measurements from the infused arm over those from the control arm was used, a standard way of expressing forearm flow data [9]. The reductions in flow to angiotensin I and II were compared on each study day by calculating the area under the curve, and comparing these using paired Student's t-tests. All results are expressed as mean ± s.d. or mean and confidence intervals (95% CI), and statistical significance was accepted for P values < 0.05.

Results

There was no evidence of side-effects from the treatments.

Biochemical parameters

Table 1 shows that neither oestradiol, nor medroxy-progesterone had any effect on circulating ACE activity.

Table 1.

Biochemical parameters, baseline blood flow, and differences in blood flow ratios due to angiotensin I and II infusions, following placebo, oestradiol, and medroxyprogesterone (n = 8, mean ± s.d.).

		Placebo	Oestradiol	Medroxy-progesterone
Serum ACE (units l−1)		42.8 ± 23	38.2 ± 14	48 ± 20
Oestradiol (pmol l−1)		77 ± 14	164 ± 22**	76 ± 16
Progesterone (nmol l−1)		1.8 ± 0.2	1.6 ± 0.3	1.8 ± 0.3
Testosterone (nmol l−1)		28 ± 1.9	26 ± 1.3	26 ± 2.5
Mean forearm blood flow (ml/100 ml forearm min−1
Baseline		1.5 ± 0.3	2.0 ± 0.6*	1.6 ± 0.6
Mean difference in absolute blood flow (95% CI)		−0.54 (−1.01; −0.06)		−0.18 (−0.57; 0.19)
Mean difference in blood flow ratios to AI and AII (95%CI)
Angiotensin I	12 pmol min−1	−0.07 (−0.17, 0.03)		−0.04 (−0.17, 0.09)
	24 pmol min−1	−0.02 (−0.15, 0.11)		0.05 (−0.06, 0.17)
	48 pmol min−1	−0.05 (−0.22, 0.12)		−0.04 (−0.14, 0.06)
Angiotensin II	4 pmol min−1	−0.03 (−0.14, 0.08)		−0.08 (−0.18, 0.03)
	8 pmol min−1	0.03 (−0.09, 0.14)		0.02 (−0.05, 0.09)
	16 pmol min−1	−0.02 (−0.09, 0.06)		0.06 (0, 0.11)

^**P < 0.0001 vs placebo

^*P < 0.03 vs placebo.

Table 1 shows the levels of oestradiol, progesterone and testosterone. These samples were taken 2 h after the second dose of treatment. Testosterone levels were unaffected. Oestradiol rose in all volunteers to levels comparable with the menstrual phase in premenopausal females. Progesterone was also measured, but medroxy-progesterone does not cross react with the assay.

Blood flow results

At baseline oral oestradiol produced significant vasodilation compared with placebo in the subjects (Table 1).

Figure 1 shows the fall in blood flow ratios i.e vasoconstriction in the infused arm, on each day. There were no differences noted between either treatment and placebo. 95% confidence intervals for differences are noted in Table 1.

Figure 1.

Ratio of blood flow in infused arm/control arm during infusions of a) angiotensin I and b) angiotensin II, comparing oestradiol (▴), and medroxy-progesterone (•) treatment compared with placebo (▪).

Discussion

This study has shown that in normal male volunteers, short-term dosing with oestradiol causes vasodilation, but that neither oral oestradiol nor medroxyprogesterone had any effect on plasma ACE, vascular ACE activity or vasoconstriction to angiotensin II. There has been great interest in the mechanism behind the acute vasodilatory effect of oestradiol, and its ability to potentiate the effects of vasodilators in women [2] and men [6, 7].

In contrast to the data presented here, Proudler et al. [3, 4] showed reductions in circulating ACE activity in postmenopausal women in response to HRT. However our study looked specifically at the acute effects of each hormone independently rather than combination of the two over 6 months as an HRT preparation.

The data presented show that even in young men, oestradiol acutely causes vasodilation. In contrast to our findings, Mabe et al. [10] found in men that norethisterone, but not mestranol could increase the dose of angiotensin II required to cause a 20 mmHg increase in diastolic blood pressure. They used a combined protocol with 6 days of norethisterone followed by 6 days of mestranol, with no washout phase, and hence their protocol examined more chronic hormonal effects.

It is possible that other non-ACE pathways for angiotensin I conversion exist in the normal forearm. However enalaprilat infused into the forearm almost completely abolishes vasoconstriction to angiotensin I, and acutely has no effect on blood flow [11], making significant non ACE conversion unlikely in the normal forearm.

A limitation of this study is the relatively small sample size. A previous study from our group with similar numbers demonstrated vascular ACE inhibition by C-type natriuretic peptide [12]. The present study, had 65% power to show a 30% fall in vasoconstriction to angiotensin I, i.e. from −48% to −32%, at 0.05 significance [13]. In a similar experimental model C-type natriuretic peptide caused a 60% fall in the vasoconstriction to angiotensin I [12]. Therefore although the sample size was small it is unlikely that we failed to detect any important inhibition of vascular ACE by female ovarian hormones.

As the main end points in this study are negative, it is important to assess adherence with treatment. All eight subjects demonstrated 60–100% increases in serum oestradiol on the oestradiol day, suggesting good compliance.

In conclusion, despite oestradiol inducing vasodilation, neither oestradiol nor medroxy-progesterone affected vascular ACE activity or vasoconstrictor responses to angiotensin II. Therefore neither inhibition of ACE activity nor reduction of sensitivity to angiotensin II is involved in oestradiol-induced vasodilation.