Effect of intermedin/adrenomedullin2 on the pulmonary vascular bed in hypoxia-induced pulmonary hypertensive rats

doi:10.1016/j.lfs.2017.11.031

Life Sciences

Volume 192, 1 January 2018, Pages 62-67

https://doi.org/10.1016/j.lfs.2017.11.031 Get rights and content

Abstract

Aims

This study aimed to investigate the effect and mechanism of action of intermedin/adrenomedullin₂ (IMD/AM₂) on the pulmonary vascular bed in pulmonary hypertensive rats.

Materials and methods

Male Sprague-Dawley rats were exposed to hypobaric hypoxia for 3 weeks to induce pulmonary hypertension (PHT). The development of PHT was confirmed by histopathological analyses and measurement of hematocrit, basal perfusion pressure, and right ventricle hypertrophy. Subsequently, the effect of IMD/AM₂ in pulmonary hypertensive rats was assessed with both, isolated organ bath and isolated lung perfusion studies.

Key findings

In the PHT group, the basal perfusion pressure and % hematocrit were increased, and right ventricle hypertrophy occurred after 3 weeks of hypoxia exposure. Increased medial wall thickness was also observed in the pulmonary artery with histopathological analysis. In the PHT, the nitric oxide-mediated vasodilation caused by IMD/AM₂ in the pulmonary vascular bed and this was as potent as the control group. Acetylcholine responses were also protected in pulmonary hypertensive rats.

Significance

Our results showed for the first time in in vitro studies that IMD/AM₂ administration causes potent, concentration-dependent vasodilation in the main and resistance pulmonary arteries of rats with PHT. Based on these results, IMD/AM₂ might be considered as a future therapeutic target for PHT treatment.

Introduction

Pulmonary hypertension (PHT) is a rare and severe disease, characterized by elevated pulmonary arterial pressure. It progresses rapidly, causing serious consequences, such as right ventricular failure and death. Though PHT pathobiology is still not well-understood, vasoconstriction, pulmonary vessel wall remodeling, and thrombosis are known to result in elevated resistance to the pulmonary vascular system. Many endogenous mediators, growth factors, and exogenous stimuli play important roles in these pathological changes. Adjunctive and symptomatic therapies targeting these factors are applied for PHT treatment. Although new drugs such as prostanoids, endothelin receptor antagonists, and phosphodiesterase-5 inhibitors have recently provided significant improvements in hemodynamic parameters, there is still no definitive treatment for the disease [1], [2], [3], [4], [5].

The members of the calcitonin gene-related peptide (CGRP) family, especially CGRP and adrenomedullin, demonstrate potent activity in cardiovascular systems. They are both cardioprotective and effective in cardiac diseases such as congestive heart failure and myocardial ischemia [6]. Based on the cardioprotective and potent vasorelaxant action of these peptides, their effect on PHT has been tested in many studies. Endogenous CGRP levels were reduced in hypoxia-induced PHT; hence, CGRP infusions prevented hypoxia-induced PHT [7], [8]. Adrenomedullin reduced the pulmonary arterial pressure in pulmonary hypertensive rats [9], [10], [11]. Additionally, intravenous administration or inhalation of adrenomedullin decreased the pulmonary arterial pressure and vascular resistance in PHT patients [12].

Intermedin/adrenomedullin₂ (IMD/AM₂) is the latest member of the CGRP family; similar to other members of the family, IMD/AM₂ has a significant effect on the cardiovascular system [13], [14]. Intraperitoneal IMD/AM₂ injections attenuate myocardial injury and protect the heart against ischemia-reperfusion injury [15]. Another study showed that low dose administration of IMD/AM₂ reduces pulmonary ischemia/reperfusion injury in lungs of mice [16]. IMD/AM₂ is also a potent vasorelaxant in several vascular beds such as rat aorta, mesenteric and porcine coronary arteries, and reduces arterial blood pressure [14], [17], [18], [19], [20], [21]. IMD/AM₂ was suggested as a putative drug candidate for cardiometabolic diseases based on its positive effect on hemodynamic and cardiac functions [22]. Our previous studies reported that IMD/AM₂ provides a marked vasorelaxation in the isolated main pulmonary artery (PA); additionally, intra-arterial (i.a.) bolus injections of IMD/AM₂ decreased the perfusion pressure of the pulmonary vascular bed in isolated rat lungs [23]. In recent years, several studies have been reported the effect of CGRP and adrenomedullin on PHT. However, no study investigated the effect of IMD/AM₂ on pulmonary vascular bed with PHT in vitro. In this study, we determined the role and mechanism of action of IMD/AM₂ on the isolated main PA and perfused lungs of rats with hypoxia-induced PHT.

Section snippets

Animals

Male Sprague-Dawley rats (300–350 g) were used in all the experiments. The rats were housed in a room with a 12 h/12 h light/dark cycle at a constant temperature (22 ± 1 °C), and were provided access to food and water ad libitum. All experiments were approved by the Hacettepe University Animal Experimentations Local Ethics Board (2015/2–3). All procedures involving animals were conducted in accordance with the EU Directive 2010/63/EU.

Chemicals

Rat intermedin (IMD/AM_{2 (17–47)}) was purchased from Bachem Labs

Development of chronic hypoxia-induced pulmonary hypertension

Secondary PHT was developed in rats after 3 weeks of hypobaric hypoxia exposure. Basal perfusion pressure of these rats were increased by approximately two-fold compared to that in the control group (*p < 0.05; Fig. 1A). Right ventricular hypertrophy occurred in the PHT group. (*p < 0.05; Fig. 1B). The increased hematocrit level was found to be statistically significant compared to that of the control group (*p < 0.05; Fig. 1C).

A statistically significant increase in muscle content was detected in the

Discussion

IMD/AM₂ has potent vasodilator effect on pulmonary vascular bed. Until now only a few studies address the role of IMD/AM₂ in the pulmonary vascular system in PHT. In present study, chronic hypoxia caused pathophysiological changes such as polycythemia and right ventricular hypertrophy in the pulmonary system leads to the development of PHT. These changes did not affect the vasorelaxation of IMD/AM₂ and our study was the first in vitro study to demonstrate that IMD/AM₂ was still a potent

Conclusions

IMD/AM₂ is an important vasorelaxant in the pulmonary vascular system and protects this effect on chronic hypoxia-induced PHT. Considering these results, along with its stabilizing effect on microvasculature and beneficial role in vascular remodeling in the pulmonary system, IMD/AM₂ is an important target for the development of PHT treatment strategies.

Acknowledgements

This study was supported by the Hacettepe University Scientific Research Projects Coordination Unit (Project numbers: THD-2016-9281).

Conflict of interest

The authors declare that there are no conflicts of interest.

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Adrenomedullin 2/intermedin (AM2/IMD) is a member of calcitonin related gene peptide family and an important nitric oxide mediated vasorelaxant in various vascular beds. However, the mechanism of post receptor-interaction is not clear and may differ depending on tissue type and species. In this study, we aimed to investigate the exact mechanism and the role of BK_Ca and calcium channels on the vasorelaxant effect of AM2/IMD in rat PA. Changes in the AM2/IMD-mediated vasorelaxation were evaluated in the presence of various inhibitors. CGRP_(8-37) (10⁻⁶ M), L-NAME (10⁻⁴ M), ODQ (10⁻⁵ M), SQ22536 (10⁻⁴ M), H89 (10⁻⁶ M), TEA (10⁻² M), iberiotoxin (3 × 10⁻⁷ M), and verapamil (10⁻⁵ M), all partly or completely inhibited the vasorelaxation. The relaxation was also abolished by removal of the endothelium, or in KCl precontracted PAs. AM2/IMD did not elicit vasorelaxation in the Ca²⁺-free conditions. However, the vasorelaxation was not inhibited with AM_(22-52) (10⁻⁶ M), 4-AP (3 × 10⁻³ M), glibenclamide (10⁻⁵ M), apamin (3 × 10⁻⁷ M), TRAM-34 (10⁻⁵ M), and La⁺³ (10⁻⁴ M). AM2/IMD −induced changes in intracellular calcium levels and isometric force were monitored simultaneously in fura-2-loaded, endothelium-intact PAs. The AM2/IMD-induced increase in intracellular Ca²⁺ concentration was inhibited in the presence of iberiotoxin and verapamil, whereas no change was observed with La³⁺ incubation. Our data suggest that the cAMP/PKA pathway is one of the important pathways AM2/IMD-induced vasorelaxation. AM2/IMD acts through activation of endothelial BK_Ca and subsequently causes hyperpolarization of the endothelial cell membrane. The hyperpolarization induces Ca²⁺ influx, which leads to NO production and subsequent vasorelaxation.

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Effect of intermedin/adrenomedullin2 on the pulmonary vascular bed in hypoxia-induced pulmonary hypertensive rats

Abstract

Aims

Materials and methods

Key findings

Significance

Introduction

Section snippets

Animals

Chemicals

Development of chronic hypoxia-induced pulmonary hypertension

Discussion

Conclusions

Acknowledgements

Conflict of interest

Clin. Chest Med.

Mol. Ther.

Peptides

Peptides

Peptides

J. Biol. Chem.

Cardiovasc. Diabetol.

Peptides

Peptides

Biochem. Biophys. Res. Commun.

FEBS Lett.

Peptides

Peptides

Pulm. Pharmacol. Ther.

The pharmacological treatment of pulmonary arterial hypertension

Pharmacol. Rev.

Pulmonary arterial hypertension

Circulation

Pulmonary hypertension due to common respiratory conditions: classification, evaluation and management strategies

J. Clin. Med.

Advances in therapeutic interventions for patients with pulmonary arterial hypertension

Circulation

Vascular actions of calcitonin gene-related peptide and adrenomedullin

Physiol. Rev.

Effect of intermedin/adrenomedullin₂ on the pulmonary vascular bed in hypoxia-induced pulmonary hypertensive rats