Effects of adrenomedullin on angiotensin II stimulated atrial natriuretic peptide and arginine vasopressin secretion in healthy humans

Abstract

Aims

Adrenomedullin is a newly described peptide that has widespread tissue distribution. Its presence in cardiovascular (including vascular endothelial cells, smooth muscle cells, and cardiac atria and ventricles) and renal tissues, together with its vasodilatory and natriuretic properties, suggest a role in blood pressure regulation and fluid and electrolyte balance.

Methods

Nine normal volunteers were studied to determine whether or not adrenomedullin influenced plasma atrial natriuretic peptide and arginine vasopressin concentrations during systemic angiotensin II infusion.

Results

A significant (P = 0.02) augmentation of atrial natriuretic peptide concentrations, but no suppression of arginine vasopressin concentrations, was found with coinfusion of adrenomedullin and angiotensin II when compared with vehicle and angiotensin II.

Conclusions

Despite its vasodilator and natriuretic action, adrenomedullin significantly augmented angiotensin II-stimulated plasma atrial natriuretic peptide concentrations in healthy humans. This provides further evidence of a synergistic interaction between adrenomedullin and atrial natriuretic peptide and suggests that adrenomedullin may have a role in fluid and electrolyte balance and blood pressure regulation.

Introduction

Atrial natriuretic peptide (ANP) and arginine vasopressin (AVP) are hormones involved in sodium and water balance and blood pressure regulation. In vivo and in vitro studies suggest that there may be an interaction between adrenomedullin and these regulatory peptides. In one study, in an ovine model of pacing-induced heart failure, ADM infusion (10 and 100 ng kg⁻¹ min⁻¹) led to an increase in the plasma concentration of ANP [1]. In another study, in humans, ANP infusion resulted in a four fold increase in the plasma concentration of ADM [2].

Central administration of ADM inhibits the secretion of AVP caused by hyperosmolality and hypovolaemia in rats [3].

A rise in plasma ANP [4] and AVP [5] concentrations has been previously found in healthy humans following intravenous angiotensin II infusion. We have investigated the effect of ADM on angiotensin II stimulated ANP and AVP concentrations in healthy human volunteers.

Methods

Subjects

Nine healthy volunteers (eight male and one female) aged 29–31(mean 29.6) years were studied on two separate occasions separated by at least 1 week. Each volunteer refrained from caffeine on the day of the study and alcohol for the previous 24 h. The protocol had been approved by the hospital committee on medical ethics and each volunteer gave written informed consent. The investigation conformed with the principles outlined in the Declaration of Helsinki.

Protocol

Subjects were studied in a quiet clinical laboratory, sitting upright with their legs horizontal. At the start of each study intravenous cannulae were sited in each forearm. After 35 min of rest, an infusion of either ADM (Peptides International, Lousville, Kentucky) [3 pmol kg⁻¹ min⁻¹] or placebo was commenced and continued for 60 min. After a further 15 min an infusion of angiotensin II 10 ng kg⁻¹ min⁻¹ (Clinalfa, Laufelfingen, Switzerland) was started and continued for 45 min. During preparation ADM was initially diluted in 3 ml Haemaccel (Behring) before addition to 60 ml of 0.9% sodium chloride for infusion at 1 ml min⁻¹.

Blood samples

Blood samples were taken at (Figure 1):

Figure 1.

Timing of blood sampling.

35 min – baseline, after rest

50 min – after 15 min of ADM and before the start of angiotensin II infusion

80 min – after 30 min of angiotensin II

95 min – at the end of both the ADM/placebo and angiotensin II infusions.

Samples for measurement of ADM and ANP were collected in tubes containing K-EDTA and aprotinin (2000 U). Samples for measurement of AVP were collected in lithium heparin tubes. All were immediately placed on ice and plasma separated within 10 min. Plasma was stored at −20 ° C until assayed.

Hormone assays

ADM was measured using a commercially available radioimmunoassay kit (Phoenix Inc., Affiniti Research, Exeter, UK) [6]. Plasma ANP and AVP were measured by radioimmunoassay as previously described [7, 8].

Statistical analysis

Data are presented as means and s.e.mean. Paired Student's t-tests were used to compare the change (δ) in plasma concentration of ANP, AVP and ADM between the start (50 min) and end of angiotensin II infusion (95 min).

Results

Plasma ADM concentration

Mean(± s.e. mean) plasma ADM was 8.7(± 1.2) pmol l⁻¹ at baseline and rose by 2.1(± 0.5) pmol l⁻¹ on the placebo/angiotensin II day. Mean plasma ADM was 9.5(± 0.4) pmol l⁻¹ at baseline and rose by 3.6(± 0.7) pmol l⁻¹ on the ADM/angiotensin II day.

Effect of ADM on haemodynamic response to angiotensin II

Mean(± s.e. mean) systolic and diastolic blood pressures rose by 20(± 4) [95% CI 12,28] and 19(± 4) [95% CI 11,27] mmHg, respectively, from a baseline of 129(± 5)/68(± 4) mmHg on the placebo/angiotensin II day. Mean(± s.e. mean) systolic and diastolic blood pressures rose by 27(± 4) [95% CI 21,35] and 21(± 4) [95% CI 13,29] mmHg, respectively, from a baseline of 117(± 6)/69(± 4) mmHg on the placebo/angiotensin II day. The rise in blood pressure with angiotensin II infusion was not statistically different between the two study days.

Effect of ADM on the angiotensin II mediated increase in plasma ANP concentration

Data from two of the nine subjects were incomplete and therefore excluded from analysis. As previously reported, systemic [4] intravenous infusion of angiotensin II results in an increase in plasma ANP concentration (Figure 2). This increase in ANP was augmented by the coadministration of ADM, most notably at the 95 min time point. The plasma ANP concentration rose by an average of 15(± 7) [95% CI 2,28] pmol l⁻¹ from a baseline mean of 7(± 1) pmol l⁻¹ on the placebo angiotensin II infusion day and by 26(± 9) [95% CI 8,44] pmol l⁻¹ from a baseline of 8(± 2) pmol l⁻¹ on the ADM/angiotensin II infusion day. This difference was statistically significant (P = 0.02). A greater rise in ANP was seen on the ADM coinfusion day in all seven subjects.

Figure 2.

Effect of ADM on angiotensin II stimulated ANP secretion. ▴ ADM/angiotensin II, ▪ placebo/angiotensin II.

Effect of ADM on the angiotensin II mediated increase in plasma AVP concentration

The data from all nine subjects were complete. As previously reported, systemic intravenous infusion of angiotensin II caused an increase in plasma AVP concentration [5]. This increase in AVP concentration was not attenuated during coadministration of ADM (Figure 3). The plasma AVP concentration rose by an average of 1.2(± 0.2) [95% CI 0.8,1.6] pg ml⁻¹ from a baseline mean of 1.3(± 0.3) pg ml⁻¹ on the placebo angiotensin II infusion day and by an average of 1.0(± 0.2) [95% CI 0.6,1.4] pg ml⁻¹ from a baseline of 1.1(± 0.2) pg ml⁻¹ on the ADM/angiotensin II infusion day.

Figure 3.

Effect of ADM on angiotensin II stimulated AVP secretion. ▴ ADM/angiotensin II, ▪ placebo/angiotensin II.

Discussion

We have found that systemic administration of ADM augments the increase in plasma ANP concentration induced by intravenous infusion of angiotensin II in healthy humans. This effect is seen with small increments in plasma ADM concentration within the pathophysiological range [6]. No inhibition of angiotensin II stimulated AVP by ADM was found.

Our findings are novel and add to the existing literature linking these peptide hormones. There are two previous in vivo infusion studies of the relationship between ADM and ANP. In the first, in an ovine model of heart failure, a rise in plasma ANP concentration was found following completion of infusion of ADM [1]. Conversely, in the second, a systemic infusion study of ANP in healthy human volunteers found that ANP caused a rise in plasma ADM concentration [2]. The authors of this study suggest that ADM may mediate some of the actions of ANP. Our findings and those in the ovine model of heart failure suggest that this stimulatory relationship may be reciprocal. Our results and those of Rademaker et al. [1] are also surprising, in two ways. Firstly, by acting as a vasodilator (and presumably reducing atrial stretch) ADM might be expected to decrease rather than increase plasma ANP concentrations. Secondly, two in vitro studies, suggest that ADM inhibits ANP secretion from isolated rat atria [9] and neonatal cardiomyocyte cultures [10]. Why then have the in vivo studies shown the opposite effect? One possibility is that ADM may, in some way, reduce the clearance of ANP. There are no reports in the literature examining the effects of ADM on ANP clearance. It seems unlikely that ADM binds to ANP clearance receptors. ADM may, however, be a substrate for neutral endopeptidase (NEP) which also metabolizes ANP [11]. It is not known, however, whether the affinity of NEP for ADM is greater than for ANP.

No inhibition of angiotensin II stimulated AVP was found in the current study. This is in contrast to previous evidence in conscious rats [3]. This study, however, investigated the action of ADM administered intracerebroventricularly, as opposed to into a peripheral vein. It is possible that ADM acts centrally, but not peripherally, to inhibit angiotensin II stimulated AVP secretion and systemic administration has no effect because ADM does not cross the blood brain barrier.

In summary we have found for the first time that small increments in plasma ADM concentration augment the rise in angiotensin II stimulated ANP concentrations. This is further evidence that ADM may play an important role in fluid and electrolyte balance and blood pressure regulation.