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Background
Under hypoxia, exaggerated compensatory responses may lead to acute mountain sickness. The excessive vasodilatory effect of nitric oxide (NO) can lower the hypoxic pulmonary vasoconstriction (HPV) and peripheral blood pressure. While NO is catalyzed by various nitric oxide synthase (NOS) isoforms, the regulatory roles of these types in the hemodynamics of pulmonary and systemic circulation in living hypoxic animals remain unclear. Therefore, this study aims to investigate the regulatory effects of different NOS isoforms on pulmonary and systemic circulation in hypoxic rats by employing selective NOS inhibitors and continuously monitoring hemodynamic parameters of both pulmonary and systemic circulation.
Methods
Forty healthy male Sprague–Dawley (SD) rats were randomly divided into four groups: Control group (
Results
Under normoxia, mean arterial pressure and total peripheral vascular resistance were increased, and ascending aortic blood flow and serum NO concentration were decreased in the L‐NAME and AG groups. During hypoxia, pulmonary arterial pressure and pulmonary vascular resistance were significantly increased in the L‐NAME and AG groups.
Conclusions
This compensatory mechanism activated by inducible NOS and endothelial NOS effectively counteracts the pulmonary hemodynamic changes induced by hypoxic stress. It plays a crucial role in alleviating hypoxia‐induced pulmonary arterial hypertension.
INTRODUCTION
When people rapidly ascend to high altitude areas (typically above 2500 m), the partial pressure of oxygen in the blood decreases. However, when the compensatory responses to hypoxia are insufficient to counteract the effects of hypoxia, a series of acute altitude-related illnesses can occur. Uneven hypoxic pulmonary vasoconstriction (HPV) is an adaptive vascular response to hypoxia in which the pulmonary blood vessels constrict. It primarily occurs in the pulmonary arterioles, leading to an increase in pulmonary arterial pressure. HPV plays a crucial role in redirecting blood flow from poorly ventilated or hypoxic lung regions to better-ventilated regions, thereby optimizing oxygen delivery.1 In addition to reducing altitude and administering oxygen, common treatments2 for HAPE include phosphodiesterase (PDE) inhibitors such as Sildenafil (Viagra), Tadalafil (Cialis), and Aminophylline (with PDE inhibition function), which can inhibit PDE, thereby reducing the degradation of cGMP. Nitric oxide (NO) induces cGMP through guanylate cyclase enzyme activation to exert vasodilatory effects. As a major vasodilator in the body, NO plays a significant role in dilating the pulmonary vasculature during hypoxia.3,4 A clinical study5 has shown an increase in exhaled NO levels in children with acute asthma, and inhaled NO6 can improve oxygen saturation and reduce inflammatory cytokine storms in COVID-19 patients7 and ameliorate acute respiratory distress syndrome.8 NO is produced by L-arginine catalyzed by nitric oxide synthase (NOS) in vivo. Endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and neuronal nitric oxide synthase (nNOS) have been identified as being responsible for the production of NO.9,10 eNOS is mainly distributed in vascular endothelial cells and is involved in regulating vascular tone, vascular permeability, leukocyte adhesion, and platelet activation. iNOS is widely distributed in all tissues and cells of the body and is mainly activated by hypoxia, cytokines, lipopolysaccharides, etc. nNOS is mainly distributed in the nervous system, and the NO it produces acts primarily in the central nervous system.
A previous study11 found that the body can exert protective effects on the cardiovascular system by upregulating NOS under hypoxia, and another study found12 that NO primarily contributes to vasodilation when generated by eNOS under normoxia in isolated lungs. Asymmetric dimethylarginine (ADMA), which competitively binds NOS to reduce NO production, along with brain natriuretic peptide (BNP), serotonin (5-HT), and others, have become susceptibility markers for assessing high altitude pulmonary edema (HAPE)13 However, there has been limited in-depth research on the source of NO within the entire organism in live experimental animals. In this study, we specifically address this gap by investigating the effects of NO produced by different NOS on both pulmonary circulation and systemic circulation during hypoxia at a systemic level. This focus is of immense significance for effectively preventing and treating high-altitude pulmonary edema and providing protection against acute hypoxic exposure.
METHODS
Animals
To exclude the physiological changes caused by estrogen and considering the characteristic occurrence of high-altitude pulmonary edema (predominantly in young males), male rats were selected as the experimental subjects because they demonstrate a more stable and consistent set of physiological features than females under equivalent conditions. Forty healthy adult male Sprague–Dawley rats weighing 351.90 ± 6.04 g were raised in a free environment with a room temperature of 23°C, a 12-h light–dark cycle, and unrestricted access to food and water. The animals were provided by Jiangsu Huachuang Xinuo Pharmaceutical Technology Co., Ltd (License number: SCXK(Su)2020-0009, NO.202239305; Taizhou, China). Rats were randomly divided into four groups, 10 rats per group. The non-selective NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) was used for the L-NAME group, while NG-nitro-D-arginine methyl ester (D-NAME), which is structurally similar but lacks NOS inhibitory activity, was used as a control for the Control group. We are using D-NAME in the control group to eliminate the vehicle's influence and remove the impact of chemical elements in the drug. The iNOS-specific inhibitor aminoguanidine (AG) was used for the AG group, and the nNOS-specific inhibitor 7-nitroindazole (7-NI) was used for the 7-NI group. The inhibitory effect on eNOS was deduced by subtracting the inhibition induced by AG (an iNOS inhibitor) and 7-NI (an nNOS inhibitor) from the overall inhibition caused by L-NAME. This approach allowed us to isolate and quantify the specific impact on eNOS activity within our experimental framework. Each group was subdivided into normoxic and hypoxic conduction.
Reagents and materials
All drugs were purchased from Sigma Company (Louis, MO, USA). In this experiment, D-NAME (10 mg/kg, 100 μL), L-NAME (10 mg/kg, 100 μL),14,15 and AG (100 mg/kg, 100 μL)16 were prepared using physiological saline and administered via the femoral vein, while 7-NI (30 mg/kg, 200 μL)17,18 was prepared as a suspension in peanut oil (No. 47119, Sigma, Louis, MO, USA) and administered intraperitoneally. The dose of these drugs was 0.1 mL (300 g of body weight). Heparin saline solution was prepared by dissolving heparin powder (BS145, Biosharp, Hefei, China) in physiological saline to a concentration of 1000 U/mL for storage. The working concentration was further diluted to 50 U/mL. Pentobarbital sodium (071025, Terui, Nanjing, China) was prepared as a sterile 2.5% solution in physiological saline for anesthesia. All drugs used were prepared immediately before use.
Hypoxic gas containing 15% oxygen is prepared by mixing nitrogen (N2) and air (2200 m a.sl., 582 mmHg, Xining, China). This concentration was confirmed to be effective in previous studies performed by our research group.19
The experimental polyethylene catheters were all non-toxic medical catheters (Natsume Manufacturing, Tokyo, Japan).
Surgery
The surgical procedure was conducted according to a previously established protocol.20 The details are as follows. After anesthesia with 2.5% pentobarbital sodium (50 mg/kg, i.p.), rats were fixed in a supine position on an animal temperature control pad set at 37°C (Physitemp, Clifton, NJ, USA) to maintain the rectal temperature at 37 ± 0.3°C. A midline cervical incision and a left inguinal incision were made to insert catheters into the vessels (right external jugular vein, left common carotid artery, and left femoral vein). Polyethylene catheters were inserted into the right external jugular vein (catheter ID 0.80 mm, OD 1.00 mm) to measure central venous pressure (CVP), the left common carotid artery (catheter ID 0.50 mm, OD 0.80 mm) to measure mean arterial pressure (MAP), and the left femoral vein (catheter ID 0.80 mm, OD 1.00 mm) for intravenous administration of drugs and saline (4 mL/kg/h) to supplement lost fluids.
A tracheal tube (ID 2.00 mm, OD 3.0 mm) was inserted into the trachea for mechanical ventilation and measurement of airway pressure (Paw). Mechanical ventilation was set at a respiratory rate of 70 breaths/min and a tidal volume of 7 mL/kg, while positive end-expiratory pressure of 2.5 cmH2O was provided to prevent alveolar collapse. A midline incision was made on the chest, and electrocautery was used for hemostasis. A pulse-wave Doppler ultrasound probe (MC2PSS, Transonic Systems, Ithaca, NY, USA) was placed on the ascending aorta to dynamically measure ascending aortic blood flow (ABF). A polyethylene catheter (ID 0.50 mm, OD 0.80 mm) was inserted into the right ventricle and advanced into the pulmonary artery to monitor mean pulmonary artery pressure (PAP), and another polyethylene catheter (ID 0.35 mm, OD 0.60 mm) was inserted into the left atrium to measure left atrial pressure (Pla). The wound was covered with a transparent, clear plastic sheet to minimize moisture loss. The average duration of the entire surgical procedure was maintained within 20–30 min.
Experimental data acquisition
Each catheter was connected to a pressure sensor (positioned at the level of the left atrium of the heart) to monitor real-time dynamic changes in MAP, PAP, CVP, Pla and Paw. ABF was measured with an ultrasound probe. All data were recorded at a frequency of 200 Hz using the PowerLab biological data acquisition system (AD Instruments, Castle Hill, Australia). The PowerLab software system calculated heart rate (HR) based on the waveform of MAP. Data collection of experimental measurements began after a 10-min stabilization period following the completion of surgery. Data collection consisted of two phases: first, monitoring of hemodynamic parameters under normoxic conditions for 10 min after administration of different inhibitors drugs (D-NAME, L-NAME, AG, 7-NI); second, after administering the drugs for 20 minutes, hemodynamic changes were monitored for 5 min under acute hypoxia via a ventilator. Pulmonary vascular resistance (PVR) was calculated using the formula PVR = (PAP-Pla)/ABF. Total peripheral vascular resistance (TVR) was calculated using the formula TVR = (MAP-CVP)/ABF.20,21
Sample collection and blood gas analysis
Fifty microlitres of arterial blood was collected from the left common carotid artery for blood gas analysis at different time points. After successful completion of the surgery, 100 μL of peripheral blood was collected via the external jugular vein at different time points.
Measurement of nitric oxide (
Nitric oxide (NO) detection was performed strictly according to the instructions for the NO detection kit (A012-1, Jiancheng, Nanjing, China). Peripheral blood NO levels were measured by the nitrate reductase method, using the total concentration of nitrate (NO3−) and nitrite (NO2−) in serum as a representative measure of NO content.
Statistics
All results are presented as mean ± SE. Statistical analysis was performed with SPSS software (version 27.0, SAS Institute Inc., Cary, NC, USA). Two-way analysis of variance (ANOVA) with Bonferroni's multiple comparison test was used for comparisons between multiple groups. A p value less than 0.05 was considered statistically significant.
RESULTS
Blood gas analysis and Hct before and after thoracotomy
To observe the basic physiological parameters of rats, we compared the peripheral blood Hct and blood gas analysis before and after surgery. The results showed that the physiological parameters of rats in each group remained stable after the surgical interventions (Table 1). There was no significant difference in blood gas analysis and Hct before and after thoracotomy (p > 0.05), and no hyperventilation or hypoventilation.
TABLE 1 Blood gas analysis and hematocrit (Hct) comparison before and after thoracotomy.
| pH | PaO2 (mmHg) | Hct (%) | ||||
| Before | After | Before | After | Before | After | |
| Control | 7.40 (0.01) | 7.41 (0.01) | 99.90 (1.69) | 98.5 (1.38) | 49.93 (1.43) | 46.32 (1.27) |
| L-NAME | 7.41 (0.01) | 7.39 (0.01) | 101.10 (1.96) | 95.34 (2.02) | 50.93 (0.48) | 49.93 (0.50) |
| AG | 7.39 (0.01) | 7.39 (0.01) | 100.39 (1.09) | 98.03 (1.07) | 51.12 (0.68) | 50.17 (0.78) |
| 7-NI | 7.39 (0.01) | 7.40 (0.01) | 100.27 (0.87) | 99.32 (0.86) | 50.96 (0.68) | 49.29 (0.52) |
Acute hypoxic stress on arterial blood gas analysis in rats under the effects of different
To observe the effects of hypoxic stress on acid–base imbalance and the degree of hypoxia in rats under the effect of different NOS inhibitors, we detected blood gas analysis before drug administration, 10 minutes after drug administration, and 5 minutes after hypoxic stimulation. The results are summarized in Figure 1. There was no significant change in arterial blood gas results before and after administering the different drugs. After acute hypoxic stimulation, PaO2 decreased most in the L-NAME group, followed by the AG group. The PaO2 change in the 7-NI group was similar to the Control group. Arterial blood pH was slightly decreased but not significantly. No significant changes in PaCO2 were observed.
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Serum
To observe the effects of different NOS inhibitors and the influence of hypoxic stress on serum NO levels, we measured NO levels in peripheral blood before and after administering drugs under normoxic and hypoxic conditions. After administration of L-NAME and AG, the NO levels in the peripheral blood of rats decreased (p < 0.05), while the changes in the 7-NI and Control groups were insignificant. Under hypoxia, NO levels increased to varying degrees in each group, with statistically significant increases in the Control group, AG group, and 7-NI group but not in the L-NAME group. Both the Control group and the 7-NI group showed higher values compared to the AG group and L-NAME group (Figure 2).
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Changes in hemodynamic parameters under the effects of different drugs
After drug administration in each group, it was observed that MAP and △MAP significantly increased in the L-NAME group. Specifically, MAP and △MAP were notably higher in the L-NAME group than in the Control and 7-NI groups, and the △MAP increase in the L-NAME group was higher than in the AG group (Figure 3). A gradual decrease in ABF was observed in both the L-NAME and AG groups compared to the baseline (0 points). Furthermore, △ABF in the L-NAME and AG groups exhibited a significant decrease compared to the Control and 7-NI groups (Figure 3). No statistically significant increase was observed in PAP and △PAP in any group (p > 0.05) (Figure 3). Compared to the baseline, TVR and △TVR gradually increased in both the L-NAME and AG groups. TVR and △TVR in the L-NAME and AG groups were higher than those in the Control and 7-NI groups (Figure 4). Similarly, PVR and △PVR in the L-NAME and AG groups notably increased compared to the baseline after administration. △PVR in the L-NAME and AG groups remained higher than in the Control and 7-NI groups (Figure 4). Likewise, CVP, Pla, HR, and Paw remained relatively stable with no significant changes (Figure 5).
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Changes in hemodynamic parameters during hypoxia with different drug effects
When the rats were exposed to hypoxia (15% O2), both MAP and △MAP in all groups significantly decreased compared to the level at the start of hypoxia (Figure 3). PAP and △PAP in the L-NAME and AG groups showed a significant increase compared to the level at the start of hypoxia (Figure 3). ABF and △ABF in the L-NAME and AG groups decreased to varying degrees compared to the level at the start of hypoxia, with significant reductions in the L-NAME and AG groups (Figure 3). TVR and △TVR in all groups decreased to different degrees during hypoxia, but the declines were insignificant compared to the level at the start of hypoxia (Figure 4). PVR and △PVR increased in all groups to different degrees during hypoxia, with the order for greatest to least degree of increase being L-NAME, AG, 7-NI, and Control groups (Figure 4). PVR in the L-NAME and AG groups significantly increased compared to the Control group, and PVR and △PVR in L-NAME were significantly higher than the 7-NI group (Figure 4). Additionally, PVR in the L-NAME group was significantly higher than in the AG group (Figure 4). CVP increased slightly but not statistically (Figure 5). No significant changes were observed in Pla, HR, and Paw (Figure 5).
DISCUSSION
We found that endogenous NO produced by eNOS and iNOS plays a crucial role in regulating systemic circulation under normoxia. Additionally, the endogenous NO generated by these enzymes also significantly impacts the regulation of pulmonary circulation under hypoxia. However, the regulatory effect of NO produced by nNOS on both pulmonary and systemic circulation under normoxia or hypoxia appears less pronounced.
To eliminate confounding factors in the experiment, the Labchart biological signal acquisition system was employed for real-time and dynamic monitoring of physiological parameters in rats. The hemodynamic parameters such as CVP, MAP, PAP, Pla, and ABF were monitored at a frequency of 200 Hz. A positive end-expiratory pressure of 2.5 cm water column was applied to prevent lung collapse. Arterial blood gas analysis and peripheral blood hematocrit measurement were performed before and after thoracotomy to assess the physiological stability of the rats. Acute hypoxic stress modeling utilized ventilation with a gas mixture of 15% O2 and 85% N2, a concentration validated in previous studies by our research group,19 ensuring effective visualization of hemodynamic changes during the rapid transition from normoxia to hypoxia.
In this experiment, after the application of L-NAME, endogenous NO production decreased (Figure 2), and MAP and TVR increased, ABF gradually decreased (Figures 3 and 4). After applying AG to inhibit iNOS, endogenous NO and ABF decreased, and TVR increased. After treatment with the nNOS inhibitor 7-NI and the non-NOS inhibitor D-NAME, the changes in NO production and hemodynamic parameters were insignificant. TVR, △TVR, and △PVR increased in the L-NAME and AG groups, associated with a fall in ABF.22 We observed that after the administration of different drugs the most significant changes in hemodynamic parameters occurred in the L-NAME group, followed by the AG group. The effect of 7-NI on hemodynamic parameters was minimal and can be considered negligible. This suggests that the administration of L-NAME (non-selective NOS inhibitor) and AG (iNOS inhibitor) affects the hemodynamics of rats. It can be inferred that the NO produced by iNOS and eNOS participates in maintaining the hemodynamic stability of the systemic circulation under normoxic conditions. A report that inhibition of iNOS by AG can increase blood pressure in rats23 is consistent with our findings. Furthermore, NO produced by NOS had a negligible effect on blood gas analysis under normoxia.
During acute hypoxic stress, the serum NO levels in the Control, AG, and 7-NI groups demonstrated the largest elevations (Figure 2), consistent with similar previous studies.24,25 Under acute hypoxic stress, the MAP of each group decreased to varying degrees (Figure 3), consistent with some previous research findings26 but in contrast to others.27,28 The possible reasons for this phenomenon can be explained as follows. (a) During acute hypoxia, pulmonary vasoconstriction leads to an increase in PVR and a decrease in the amount of blood returning from the pulmonary circulation to the left atrium, resulting in a decrease in ABF and a decrease in MAP (Figure 3). (b) Then more NO is produced to dilate the pulmonary arterioles constricted under hypoxia, and at the same time, the NO acts on the peripheral vasculature, causing peripheral vasodilation. Under hypoxic stress, PAP and PVR increased, with the L-NAME group exhibiting the most significant increase, followed by the AG group (Figure 4). The serum NO level in the L-NAME group showed no significant increase, while the AG group exhibited slightly higher NO levels than the L-NAME group (Figure 2). These data indicate that pulmonary circulation is mainly regulated by endogenous NO produced by both iNOS and eNOS. Acute hypoxic stress can activate NOS,25,29,30 and post-translational modifications of eNOS can increase NO release.31 Studies focusing on hypoxia12 have indicated that iNOS and eNOS play important roles in the basal regulation of vascular tone and indicated an increase in NOS expression during chronic hypoxia.32,33 However, some research has reported that endogenous NO release changes under chronic hypoxia.34,35 In our study, the role of different NOS under acute hypoxia was a complementary finding to their role under chronic hypoxia.
We must acknowledge some limitations associated with the different drug formulations and administration methods. Particularly, in the case of the nNOS inhibitor 7-NI, its poor solubility posed a challenge. We opted for a peanut oil formulation and administered it via intraperitoneal injection to address this issue. This approach aimed to ensure the complete dissolution of the drug and maintain a stable dosage while allowing observation of drug efficacy within a relatively short time frame.36 However, these variations in treatment approach might have influenced the experimental outcomes.
CONCLUSIONS
In conclusion, our results indicate that both iNOS and eNOS jointly regulate systemic circulation in normoxic rats. The regulatory effect of endogenous NO produced by iNOS and eNOS does not significantly affect the regulation of pulmonary circulation under normoxia, but during acute hypoxic stress, the body counteracts the increase in PAP induced by hypoxia primarily through the production of endogenous NO by activating iNOS and eNOS. In other words, the NO produced by iNOS and eNOS plays a protective role in pulmonary circulation under hypoxia. This compensatory reaction activated by iNOS and eNOS during hypoxic stress is vital to maintaining normal life activities.
AUTHOR CONTRIBUTIONS
Huan Zhang conducted the animal experiments and wrote the manuscript. Yu Zhang conducted the animal experiments and designed the study. Xiaojun Wang and Jie Liu analyzed the data. Wei Zhang designed the study and wrote the manuscript.
ACKNOWLEDGMENTS
We want to express our gratitude to the ‘Research Centre for High Altitude Medicine of Qinghai University’ for providing the experimental platform. Also, we would like to thank Dr. Andrea Mendoza for language assistance.
FUNDING INFORMATION
This work was supported by the National Natural Science Foundation of China (grant numbers 81560301 and 81160012), the Natural Science Foundation of Qinghai Province (grant number 2022-ZJ-905), and ‘2022 Qinghai Province Kunlun Talents High-end Innovation and Entrepreneurship Talents’ Outstanding Talent Project.
CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.
DATA AVAILABILITY STATEMENT
Available upon request through correspondence.
ETHICS APPROVAL STATEMENT
All animal care and experimental procedures were conducted in strict accordance with the U.K. Animals (Scientific Procedures) Act 1986, the ARRIVE guidelines 2.0, and the China Practice for the Care and Use of Laboratory Animals. The study was approved by the China Zoological Society (approval number: GB 14923-2010) and the Experimental Committee of Qinghai University. All possible measures were taken throughout the experiments to minimize animal pain and suffering.
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