PLoS One. 2014 Oct 28;9(10):e110693.

β2-Adrenergic receptor-dependent attenuation of hypoxic pulmonary vasoconstriction prevents progression of pulmonary arterial hypertension in intermittent hypoxic rats.

Nagai H1, Kuwahira I2, Schwenke DO3, Tsuchimochi H4, Nara A5, Inagaki T4, Ogura S6, Fujii Y4, Umetani K7, Shimosawa T8, Yoshida K9, Pearson JT10, Uemura K11, Shirai M4.
  • 1Department of Forensic Medicine, Tokyo Medical and Dental University, Tokyo, Japan; Department of Forensic Medicine, The University of Tokyo, Tokyo, Japan; Department of Clinical Laboratory Medicine, The University of Tokyo, Tokyo, Japan.
  • 2Department of Pulmonary Medicine, Tokai University Tokyo Hospital, Tokyo, Japan.
  • 3Department of Physiology-Heart Otago, University of Otago, Dunedin, New Zealand.
  • 4Department of Cardiac Physiology, National Cerebral and Cardiovascular Center Research Institute, Osaka, Japan.
  • 5Department of Forensic Medicine, The University of Tokyo, Tokyo, Japan.
  • 6Department of Forensic Medicine, The University of Tokyo, Tokyo, Japan; Division of Laboratory Medicine, Department of Pathology and Microbiology, Faculty of Medicine, Nihon University School of Medicine, Tokyo, Japan.
  • 7Japan Synchrotron Radiation Research Institute, Hyogo, Japan.
  • 8Department of Clinical Laboratory Medicine, The University of Tokyo, Tokyo, Japan.
  • 9Department of Forensic Medicine, The University of Tokyo, Tokyo, Japan; Department of Forensic Medicine, Tokyo Medical University, Tokyo, Japan.
  • 10Monash Biomedical Imaging Facility and Department of Physiology, Monash University, Melbourne, Clayton, Victoria, Australia; Australian Synchrotron, Clayton, Victoria, Australia.
  • 11Department of Forensic Medicine, Tokyo Medical and Dental University, Tokyo, Japan.

 

Abstract

In sleep apnea syndrome (SAS), intermittent hypoxia (IH) induces repeated episodes of hypoxic pulmonary vasoconstriction (HPV) during sleep, which presumably contribute to pulmonary arterial hypertension (PAH). However, the prevalence of PAH was low and severity is mostly mild in SAS patients, and mild or no right ventricular hypertrophy (RVH) was reported in IH-exposed animals. The question then arises as to why PAH is not a universal finding in SAS if repeated hypoxia of sufficient duration causes cycling HPV. In the present study, rats underwent IH at a rate of 3 min cycles of 4-21% O2 for 8 h/d for 6 w. Assessment of diameter changes in small pulmonary arteries in response to acute hypoxia and drugs were performed using synchrotron radiation microangiography on anesthetized rats. In IH-rats, neither PAH nor RVH was observed and HPV was strongly reversed. Nadolol (a hydrophilic β(1, 2)-blocker) augmented the attenuated HPV to almost the same level as that in N-rats, but atenolol (a hydrophilic β1-blocker) had no effect on the HPV in IH. These β-blockers had almost no effect on the HPV in N-rats. Chronic administration of nadolol during 6 weeks of IH exposure induced PAH and RVH in IH-rats, but did not in N-rats. Meanwhile, atenolol had no effect on morphometric and hemodynamic changes in N and IH-rats. Protein expression of the β1-adrenergic receptor (AR) was down-regulated while that of β2AR was preserved in pulmonary arteries of IH-rats. Phosphorylation of p85 (chief component of phosphoinositide 3-kinase (PI3K)), protein kinase B (Akt), and endothelial nitric oxide synthase (eNOS) were abrogated by chronic administration of nadolol in the lung tissue of IH-rats. We conclude that IH-derived activation of β2AR in the pulmonary arteries attenuates the HPV, thereby preventing progression of IH-induced PAH. This protective effect may depend on the β2AR-Gi mediated PI3K/Akt/eNOS signaling pathway.

PMID: 25350545

 

Supplements:

Prior to our experiments, it was very difficult to expose rats to intermittent hypoxia (IH) in a stable manner. Generally, gas cylinders filled with nitrogen and air (or oxygen) have been used to expose animals to IH. Therefore, we used the biggest size of gas cylinders available (7,000 L) in our preliminary IH experiments. However, surprisingly, it only took 4 hours to empty the cylinders. Thus, we had to develop a new IH exposure system to enable us to perform IH experiments in a consistent manner. Within a year, we had successfully developed a new hypoxic system through trial and error. The most important feature of our IH system is that it contains a nitrogen generator and an air compressor (both of which were designed for industrial use), which make it possible to expose animals to a consistent level of IH for long periods. Thus, it is not necessary to exchange gas cylinders or pay attention to the residual volumes of gases during experiments involving IH exposure. In addition, the start and end points of each hypoxic exposure period (9:00 am to 5:00 pm, respectively, in this study) are controlled automatically using a programmable timer. Thus, rats can be subjected to prolonged IH exposure semi-automatically. As this IH system is ground-breaking and easy to use, we published a paper about it in the International Journal of Clinical and Experimental Physiology (1). Please refer to our paper if you are interested in IH experiments.

In general, sympathoadrenergic activation is considered to be one of the most important pathogenic factors in various symptoms of sleep apnea syndrome (SAS) (2). Thus, beta adrenergic receptor (AR) blockers are administered as an antihypertensive therapy to patients with SAS. In the present study, we first examined whether chronic IH exposure induces systemic arterial hypertension. Blood pressure was measured using the tail-cuff method every week during a 6-week period of IH exposure. As a result, it was found that systolic blood pressure was increased after one week’s IH exposure, and the hypertension persisted throughout the 6-week study period (Figure 1). In addition, the urine catecholamine levels of rats that had been exposed to IH for 6 weeks were significantly increased. These findings indicate that the sympathoadrenergic system activation-induced systemic hypertension observed in SAS patients can be reproduced in rats by experimental IH stimulation.

Next, we evaluated the effects of sympathoadrenergic activation on pulmonary arterial blood pressure. Pulmonary arterial hypertension (PAH) is observed in roughly 20–40% SAS patients (3). It is known that acute exposure to hypoxic gas induces hypoxic pulmonary vasoconstriction (HPV). Thus, repeated HPV during sleeping might contribute to inducing PAH. In addition, it is also known that activation of the sympathoadrenergic system results in the dilation of the pulmonary arteries. Therefore, it is possible that IH-induced sympathoadrenergic activation attenuates HPV, and hence, helps to prevent PAH progressing. The present study indicates that HPV is strongly attenuated by IH stimulation via beta2AR in small pulmonary arteries. Furthermore, the chronic inhibition of beta2AR induced prolonged increases in pulmonary arterial pressure without causing medial wall hypertrophy. On the other hand, beta1AR in the lungs did not contribute to attenuating HPV. These results demonstrate that beta2AR in the lungs plays an important role in attenuating HPV and suggest that the chief pathogenesis of IH-induced PAH is hypercontraction of the pulmonary arteries.

In a previous study involving the direct measurement of sympathetic signals, we demonstrated that the activity of the pulmonary sympathetic nervous system was significantly increased in IH rats (4). Nevertheless, the protein expression level of beta1AR was not increased in the whole brain, and the direct administration of a beta1AR blocker into the cerebral ventricle attenuated the hypoxia-induced increase in pulmonary sympathetic activity and exacerbated the magnitude of the HPV. However, these phenomena were not observed after the blockade of beta2AR in the brain. These results suggest that beta1AR, but not beta2AR, in the brain plays an important role in attenuating HPV.

Taken together, these findings indicate that beta1AR in the brain and beta2AR in the lungs contribute to attenuating HPV. It remains unclear whether beta1AR in the brain and beta2AR in the lungs cooperate with each other to attenuate HPV; however, it was revealed that both beta1 and 2AR play pivotal roles in preserving the pulmonary circulation during prolonged IH, and it was suggested that sympathoadrenergic activity helps to prevent the progression of PAH. We hope that our research aids IH studies and helps to elucidate the pathophysiology of PAH in SAS.

 

fig.1

Figure 1 The changes in systolic blood pressure seen during 6 weeks of IH exposure

Blood pressure was measured every week using the tail-cuff method in normoxic rats (N rats) and intermittent hypoxic rats (IH rats) (n=10 each). Systolic blood pressure (SBP) was increased after a week of IH exposure, and similarly high SBP values were seen throughout the 6-week IH exposure period. Statistically significant differences in SBP were detected between the groups after 4 weeks’ IH exposure.

*Significant difference between the N and IH rats (*P<0.05).

 

References

  1. Nagai H, Tsuchimochi H, Yoshida K-i, Shirai M, Kuwahira I. A novel system including an N2 gas generator and an air compressor for inducing intermittent or chronic hypoxia. International Journal of Clinical and Experimental Physiology. 2014;1(4):307-10.
  2. Dempsey JA, Veasey SC, Morgan BJ, O’Donnell CP. Pathophysiology of sleep apnea. Physiological reviews. 2010;90(1):47-112.
  3. Sylvester JT, Shimoda LA, Aaronson PI, Ward JP. Hypoxic pulmonary vasoconstriction. Physiological reviews. 2012;92(1):367-520.
  4. Shirai M, Tsuchimochi H, Nagai H, Gray E, Pearson JT, Sonobe T, et al. Pulmonary vascular tone is dependent on the central modulation of sympathetic nerve activity following chronic intermittent hypoxia. Basic Res Cardiol. 2014;109(5):432.

 

 

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