Revisiting the mechanism of hypoxic pulmonary vasoconstriction using isolated perfused/ventilated mouse lung

Abstract

Hypoxic Pulmonary Vasoconstriction (HPV) is an important physiological mechanism of the lungs that matches perfusion to ventilation thus maximizing O₂ saturation of the venous blood within the lungs. This study emphasizes on principal pathways in the initiation and modulation of hypoxic pulmonary vasoconstriction with a primary focus on the role of Ca²⁺ signaling and Ca²⁺ influx pathways in hypoxic pulmonary vasoconstriction. We used an ex vivo model, isolated perfused/ventilated mouse lung to evaluate hypoxic pulmonary vasoconstriction. Alveolar hypoxia (utilizing a mini ventilator) rapidly and reversibly increased pulmonary arterial pressure due to hypoxic pulmonary vasoconstriction in the isolated perfused/ventilated lung. By applying specific inhibitors for different membrane receptors and ion channels through intrapulmonary perfusion solution in isolated lung, we were able to define the targeted receptors and channels that regulate hypoxic pulmonary vasoconstriction. We show that extracellular Ca²⁺ or Ca²⁺ influx through various Ca²⁺-permeable channels in the plasma membrane is required for hypoxic pulmonary vasoconstriction. Removal of extracellular Ca²⁺ abolished hypoxic pulmonary vasoconstriction, while blockade of L-type voltage-dependent Ca²⁺ channels (with nifedipine), non-selective cation channels (with 30 µM SKF-96365), and TRPC6/TRPV1 channels (with 1 µM SAR-7334 and 30 µM capsazepine, respectively) significantly and reversibly inhibited hypoxic pulmonary vasoconstriction. Furthermore, blockers of Ca²⁺-sensing receptors (by 30 µM NPS2143, an allosteric Ca²⁺-sensing receptors inhibitor) and Notch (by 30 µM DAPT, a γ-secretase inhibitor) also attenuated hypoxic pulmonary vasoconstriction. These data indicate that Ca²⁺ influx in pulmonary arterial smooth muscle cells through voltage-dependent, receptor-operated, and store-operated Ca²⁺ entry pathways all contribute to initiation of hypoxic pulmonary vasoconstriction. The extracellular Ca²⁺-mediated activation of Ca²⁺-sensing receptors and the cell–cell interaction via Notch ligands and receptors contribute to the regulation of hypoxic pulmonary vasoconstriction.

Introduction

Acute alveolar hypoxia causes pulmonary vasoconstriction, whereas acute hypoxemia causes systemic (e.g. coronary) vasodilation.¹ Hypoxic pulmonary vasoconstriction (HPV) is an important physiological mechanism for matching perfusion with ventilation, which ensures the maximal oxygenation of the venous blood in pulmonary artery (PA). HPV is a unique or intrinsic feature of the pulmonary vasculature.² Although HPV has been extensively studied,³ the exact cellular and molecular mechanisms still remain unclear. Pulmonary vasoconstriction, similar to systemic vasoconstriction, is caused by pulmonary vascular smooth muscle contraction.⁴ An increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) in pulmonary arterial smooth muscle cells (PASMCs) is a major trigger for pulmonary vasoconstriction. Removal or chelation of extracellular Ca²⁺ significantly inhibits agonist- and high K⁺-induced vasoconstriction in isolated PA rings,^5,6 indicating that Ca²⁺ influx through various Ca²⁺-permeable cation channels in the plasma membrane of PASMCs is required for pulmonary vasoconstriction.

One of the early proposed mechanisms of HPV is triggered by hypoxia-induced blockade of K⁺ channels in PASMCs, which induces membrane depolarization and subsequently the opening of voltage-dependent Ca²⁺ channels (VDCC) in the plasma membrane.^6,7 Ca²⁺ influx through VDCC results in a rise in [Ca²⁺]_cyt that triggers PASMC contraction and ultimately pulmonary vasoconstriction.⁸ Pharmacological blockade of VDCC using, for example, verapamil and nifedipine (Nif), significantly inhibits HPV but fails to abolish HPV,^5,9 while blockers of VDCC abolish the high K⁺-induced pulmonary vasoconstriction.^10–13 These observations suggest that Ca²⁺ influx through cation channels other than VDCC, such as receptor-operated Ca²⁺ channels (ROC) and store-operated Ca²⁺ channels (SOC), are also involved in initial increases in [Ca²⁺]_cyt in PASMC which trigger HPV.^9,14

There are six subtypes of VDCC based on functional characteristics and biophysical properties, including L-type, T-type, N-type, P-type, Q-type, and R-type VDCC.¹⁵ The high voltage-activated and slowly inactivating L-type VDCC have been substantially studied in vascular smooth muscle cells including PASMC. They are believed to play an important role in increasing [Ca²⁺]_cyt in PASMC during hypoxia.¹⁶ L-type VDCC is also highly expressed in other types of cells such as neuron, cardiomyocytes, skeletal muscle cells, fibroblast, and kidney cells.¹⁷ The low voltage-activated and rapidly inactivating T-type VDCC is implicated in the regulation of vascular smooth muscle cell proliferation,¹⁸ but their potential role in HPV is unclear. In addition to VDCC, there are multiple voltage-independent Ca²⁺-permeable channels that are responsible for agonist- and growth factor-induced increases in [Ca²⁺]_cyt in PASMC.^19–21 Activation of G protein-coupled receptor (GPCR), for instance, ROC formed by transient receptor potential (TRP) channels and SOC formed by Stromal interaction molecule (STIM) and Orai/TRP, are both involved in inducing increases in [Ca²⁺]_cyt required for stimulating cell contraction, migration, and proliferation.^22,23 Multiple GPCRs,²⁴ such as Ca²⁺-sensing receptors (CaSR),²⁰ muscarinic receptors (M₁),^25,26 and endothelin receptors (ET_A/ET_B)²⁷ and their ligands are implicated in the development and progression of pulmonary hypertension (PH). We have shown that CaSR contributes partially to acute HPV.²⁰ ET_A receptor mediates the HPV through inhibition of ATP-sensitive K⁺ channels in isolated rat lungs and intact animals.²⁸ Furthermore, endothelin-1 induces pulmonary vasoconstriction through ET_A receptor via phospholipase and inositol triphosphate (IP₃) and diacylglycerol (DAG) pathways.²⁹ There is substantial evidence that hypoxia stimulates many different signaling cascades and pathways to increase [Ca²⁺]_cyt in PASMC. It has been demonstrated that hypoxia induces Ca²⁺ release from the intracellular stores such as sarcoplasmic reticulum (SR).³⁰ Apart from Ca²⁺ influx through Ca²⁺-permeable cation channels and Ca²⁺ mobilization from intracellular stores, activation of Rho-kinase signaling may also be involved in HPV.^31,32

In this study, we used an isolated perfused/ventilated mouse lung model and pharmacological approaches to examine potential involvement of various membrane receptors and ion channels in HPV. The isolated perfused/ventilated lung preparation is a widely used ex vivo model to study mechanisms of HPV because it has no influence from central and peripheral nervous system and the systemic circulation, while the preparation includes the intact whole lungs that are ventilated by a mini-ventilator to emulate alveolar ventilation and superfused by an automatic pump to emulate pulmonary vascular perfusion.^5,33,34 Although we focused on using isolated perfused/ventilated lung preparation in this study, we have to note that other preparations and experimental models, such as intact animals, isolated pulmonary arterial rings, and isolated PASMC,^35–40 are all useful preparations for studying mechanisms of HPV.^3,41 The advantage of using the isolated perfused/ventilated lung to study HPV is that (i) it reflects the functional changes of the whole lung vasculature, (ii) it introduces alveolar hypoxia via ventilation to the vasculature, (iii) it allows to superfuse inhibitors into PA or the whole pulmonary vasculature via a perfusion pump to examine their effect; (iv) it shows very similar time-course and pharmacological properties shown in intact animals and human subjects; (v) it also minimizes the impact of other organs and nervous systems on HPV while maintaining the intact lung in a relatively physiological setting (e.g. consistently ventilated and perfused); and (vi) it allows us to examine whether genetic deletion or overexpression of certain genes affect HPV. Here, we aimed at utilizing the isolated perfused/perfused mouse lung model to revisit the mechanisms involved in HPV by focusing on Ca²⁺ signaling and its regulation.

Materials and methods

Isolated perfused/ventilated mouse lung

C57BL/6 mice (approximately 25 g body weight, male, 8–10 weeks old) were used in this study, and the animal experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at The University of Arizona, Tucson, and University of California, San Diego. The background of trpc6^–/– (stock #37345) and notch3^–/– (stock # 010547) mice is C57Bl/6 mice and initial breeding pairs were obtained from Jackson’s Laboratory.^42,43

Mice were anesthetized by pentobarbital sodium (120 mg/kg) via intraperitoneal injection. After tracheostomy, isolated lungs were immediately ventilated with normoxic gas mixture of 21% O₂/5% CO₂ using a rodent ventilator (Minivent type 845, Harvard Apparatus, USA). The respiration rate was maintained at 80 breaths/min with a tidal volume of approximately 250 µl. Positive end expiratory pressure was maintained at 2 cmH₂O. End inspiratory pressure was measured by a pressure transducer (MPX type 399/2, Hugo Sachs Elektronik-Harvard Apparatus, Germany) connected to a tracheal catheter. The mice were placed in an isolated lung open perfusion system chamber (IL-1 Type 839, Harvard Apparatus, USA) with a heated water jacket at 37°C. After tracheal intubation, the chest was opened by median sternotomy and thymus and adipose tissue were carefully excised. Heparin (20 IU) was immediately injected into the right ventricle to prevent blood from coagulation.

A catheter was inserted into the main PA via the right ventricle, which was ligated together with ascending aorta using a 6-0 black silk suture. The PA catheter was connected with a pressure sensor (P75 Type 379, Hugo Sachs Elektronik-Harvard Apparatus, Germany) that was used to continuously measure pulmonary arterial pressure (PAP). Another catheter was inserted into the left atrium via a small incision of the left ventricle (LV) to allow perfusate to drain to reservoir. The pulmonary flow rate was set and maintained at 1 ml/min by a peristaltic pump (ISM 834, ISOMATEC, USA). The Powerlab data acquisition system (AD Instruments, CO, USA) was used to store and analyze the imaging data.

Physiological salt solution (PSS) or saline was occasionally applied to the isolated lungs to moisten the lung tissue. The lung vasculature was consistently superfused with PSS via a pump while the lung airway and alveoli were ventilated with normoxic or hypoxic gas. Raising extracellular [K⁺] from 4.7 mM to 40 mM in PSS causes membrane depolarization in PASMC and pulmonary vasoconstriction due to a shift of the equilibrium potential for K⁺ from –85 mV to −31 mV. Before experimentation, the isolated lungs were first superfused with the 40 mM K⁺-containing PSS (40 K), at least three times, to stabilize the basal PAP and the amplitudes of 40 K-induced increases in PAP. When the basal PAP was stabilized, the lungs were repetitively challenged by ventilation of hypoxic gas (1% O₂ in N₂, for 4 min) to induce an increase in PAP due to alveolar hypoxia-induced pulmonary vasoconstriction (HPV). In the interval of hypoxic challenges, the lungs were ventilated with normoxic gas (21% O₂ in N₂). Pharmacological effects of various ion channel blockers and membrane receptor inhibitors on HPV were examined by superfusion of PSS containing each of the inhibitors with prior treatment for up to 10 min before the lungs were ventilated with hypoxic gas. We do not have direct experimental data showing the optimal time for the maximal inhibition of inhibitors used in the study.

The following inhibitors and blockers were used in this study: (i) the blockers of VDCC, Nifedipine (Nif) (0.1 µM, for blocking L-type channels),⁵ TTA-A2 (30 µM, for blocking T-type channels),⁴⁴ and conotoxin (CoTX) (1 µM, for blocking P/Q type channels)⁴⁵; (ii) the inhibitors of Ca²⁺-activated chloride (Cl^–) channels, N-((4-methoxy)-2-naphthyl)-5-nitroanthranilic acid (MONNA or MON, 10 µM, for blocking TMEM16A/anoctamin-1 channels), and CaCCinh-A01 (10 µM, also for blocking TMEM16A/anoctamin-1 channels)⁴⁶; (iii) the inhibitors of non-selective cation channels and TRP channels, SKF-96365 (30 µM, for blocking TRP canonical (TRPC) channels), SAR-7334 (1 µM, for blocking TRPC6 channels),⁴⁷ capsazepine (CPZ) (30 µM, for blocking TRP vanilloid 1 (TRPV1) channels),⁴⁸ benzamil (Ben) (10 µM, for blocking TRP polycystic (TRPP) channels),⁴⁹ AM0902 (10 µM, for blocking TRPA channels),⁵⁰ and gadolinium (Gd³⁺, 10 µM for blocking TRP mucolipin (TRPML) channels)^51,52; (iv) the inhibitor of Notch signaling pathway, (2 S)-N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine 1,1-dimethylethyl ester (DAPT, 30 µM, for inhibiting γ-secretase)⁴³; and (v) the inhibitors of membrane receptors, NPS2143 (30 µM, for blocking extracellular calcium-sensing receptors/CaSR),^20,53 tropicamide (TRO) (10 µM, for blocking muscarinic receptors),⁵⁴ and BQ-123 (10 µM, for blocking endothelin receptor A).²⁸ The concentrations of various drugs used in this study were based on previously published literature. Table 1 lists all the inhibitors (and their targets) used in this study.

Table 1.

Inhibitors and drugs used in the study.

Inhibitors	Abbreviations	Target
Nifedipine	Nif	L-type of voltage-dependent Ca2+ channel
2 -(4-Cyclopropylphenyl)-N-[(1 R)-1-[5-(2,2,2-trifluoroethoxy)pyridin-2-yl]ethyl]acetamide (TTA-A2)	TTA-A2	T-type of voltage-dependent Ca2+ channel
Conotoxin	CoTX	P/Q-type of voltage-dependent Ca2+ channel
SKF-96365	SKF	Transient receptor potential (TRP)  canonical (C) channels
4-[[(1 R,2 R)-2-[(3 R)-3-amino-1-piperidinyl]-2,3-dihydro-1H-inden-1-yl]oxy]-3-chlorobenzonitrile dihydrochloride (SAR-7334)	SAR	TRPC6 channels
CPZ	CPZ	TRPV1 channels
Benzamil	Benzamil	TRPP channels
1-[[3-[2-(4-Chlorophenyl)ethyl]-1,2,4-oxadiazol-5-yl]methyl]-1,7-dihydro-7-methyl-6H-purin-6-one (AM 0902)	AM 0902	TRPA channels
Gadolinium	Gd3+	TRPML channels
2-Chloro-6-[(2 R)-3-[[1,1-dimethyl-2 -(2-naphthalenyl)ethyl]amino-2-hydroxypropoxy]benzonitrile hydrochloride (NPS 2143)	NPS	Calcium sensing receptor
TRO	TRO	Muscarinic M4 receptor
Cyclo (D-Trp-D-Asp-Pro-D-Val-Leu) (BQ-123)	BQ-123	Endothelin A receptor
N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl Ester (DAPT)	DAPT	γ-secretase (indirect inhibitor of Notch)
2-[(4-Methoxy-2-naphthalenyl)amino]-5-nitro-benzoic acid (MONNA)	MONNA	TMEM16A subunit of calcium  activated chloride channel
6 -(1,1-Dimethylethyl)-2-[(2-furanylcarbonyl)amino]-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylic acid (CaCCinh-A01)	A01	Ca2+-activated Cl– channel

Experimental mouse model of PH

C57BL/6 mice (approximately 25 g body weight, male, 8–10 weeks old) were used in this study, and the animal experimental protocol was approved by the IACUC at The University of Arizona, Tucson, and our University. C57Bl/6 J mice were exposed to normobaric hypoxia (10%) in a well-ventilated chamber for four weeks to induce PH. The hypoxia chamber had an oxygen sensor (ProOx P110-E702) which continuously monitored the oxygen levels. Following hypoxic exposure, mice were continuously anesthetized under inhaled isoflurane (1.5%). Right ventricle systolic pressure was measured by right heart catheterization using a pressure catheter (Millar Instruments, PVR1030, 1 F, 4 E, 3 mm, 4.5 cm, Colorado, USA) introduced via right jugular vein. Data were recorded and analyzed using Lab Chart Pro1.0 software (AD Instruments).

Lung angiography

Mice were anesthetized by intraperitoneal injection of pentobarbital sodium (120 mg/kg), and then heparin (20 IU) was injected immediately into the heart to prevent blood from clotting. A polyethylene (PE-20) tube was cannulated into the PA via the right ventricle; and phosphate-buffered saline was perfused through the PA using an automated pump (NE-300, Pump Systems, for 3 min at a speed of 0.05 ml/min). Then, 0.08 ml of microfil polymer (yellow) (FlowTech Inc., Carver, MA) was perfused into the PA at a speed of 0.05 ml/min. Then, the microfil polymer-filled lungs were kept at 4°C overnight. The next day, the lungs were dehydrated using different concentrations of ethanol: once in 50%, 70%, 80%, and 95% ethanol, and twice in 99.9% ethanol. After dehydration, the lungs were placed in methyl salicylate (Sigma Aldrich, USA) at room temperature on a shaker for overnight in order to show only the vasculature. Lungs were then photographed or imaged with a digital camera (MU1000, FMA050, Amscope, CA). The peripheral lung vascular image, covering the peripheral area of the lung, 1 mm width from the edge was selected with Photoshop CS software, and the branches on the images in Photoshop were outlined manually and later converted to binary images with NIH Image J 1.8v software for quantitative analysis. The total length of branches, the number of branches, and the number of junctions on the skeletonized images were obtained by Image J software and were normalized by the area selected within the peripheral regions of the lung.

Western blot

Lung tissues harvested from mice were homogenized with radioimmunoprecipitation (RIPA) buffer, followed by protein isolation. The samples were diluted with 6 × SDS-sample buffer (Boston BioProducts, USA), heated for 10 min at 95°C, and loaded on 10% SDS polyacrylamide gels. The protein samples were separated by electrophoresis and transferred to a 0.45 µm nitrocellulose membrane (BioRad, USA). The membrane was blocked with 5% bovine serum albumin (Sigma) in Tris-Buffered Saline with Tween 20 (TTBS) for an hour at room temperature and then incubated overnight at 4°C with anti-Notch3 (VMA00484, 1:1000, Bio-Rad) or anti-TRPC6 (bs-2393R, 1:1000, Bioss) primary monoclonal antibody. The membrane was washed with 1X Tris-Buffered Saline, 0.1% Tween® 20 Detergent (TBST) and then incubated for an hour at room temperature with the secondary anti-mouse or anti-rabbit IgG, Horseradish peroxidase (HRP)-linked antibody (1:5000; Cell Signaling). The membrane was subsequently developed after adding substrate (Thermo Fisher Scientific). All membranes were probed for Pan-Actin antibody (Cat# 4968S, 1:2000, Cell Signaling) or β-actin antibody (Cat# sc-47778, 1:1500, Santa Cruz Biotechnology) as internal controls. Band intensities on the membrane were quantified using Image J software.

Solutions and chemicals

The composition of PSS (perfusate) consisted of 120 mM NaCl, 4.3 mM KCl, 1.8 mM CaCl₂, 1.2 mM MgCl₂, 19 mM NaHCO₃, 1.1 mM KH₂PO₄, 10 mM glucose, and 20% fetal bovine serum (pH 7.4). To block endogenous prostaglandin synthesis, 3.1 µM sodium meclofenamate was added to the perfusate. High-K⁺ solution (or 40 mM K⁺ solution) was prepared by replacing NaCl with equimolar KCl (40 mM). Ca²⁺-free (0Ca) solution was prepared by replacing CaCl₂ with equimolar MgCl₂ with 1 mM EGTA added to chelate the residual Ca²⁺. Mg²⁺-free (0 Mg) solution was prepared by replacing MgCl₂ with equimolar NaCl. Nif, CPZ, AM-0902, CaCCinh, MONNA, Ben, TTA-A2, BQ-123, or TRO was dissolved in DMSO to make a stock solution and aliquoted for storage at –20°C. SKF-96335, SAR-7334, Gd³⁺, or CoTX was dissolved in water to make a stock solution and aliquoted for storage at –20°C. Aliquots were diluted into final PSS right before the time the inhibitor-containing PSS was perfused into the isolated lungs via the right ventricle.

Statistical analysis

The composite data are shown as mean ± standard error (SEM). Paired or unpaired Student’s t-test and one way analysis of variance (ANOVA) with Bonferroni multiple comparison test were used for statistical analysis. p Value <0.05 was considered as statistically significant.

Results

As shown in pulmonary angiogram, the mouse lungs are composed of a single large lobe on the left side (insert Fig. 1Aa) and four small lobes in the right side (insert Fig. 1Ab). The angiography images of the left and right lungs clearly demonstrate the vascular complexity and density of the pulmonary vascular tree. The highly organized branching pattern is shown from the left and right extrapulmonary arteries to peripheral pulmonary vasculature in all lobes of both sides (Insert Fig. 1Aa and Ab, upper panels). Inspection of the lung periphery region (1 mm width from the edge) at high magnification reveals large numbers of vascular branches and junctions (Insert Fig. 1Aa and Ab, lower panels). As shown in Fig. 1Ac, the total length of branches, the number of branches, and the number of junctions at a given area (1 mm²) are 8.5 ± 0.8 mm, 366.4 ± 42.6, and 161.1 ± 20.9 (n = 11), respectively, at the peripheral regions of the left lung (insert Fig. 1Ac).

Fig. 1.

Removal of extracellular Ca²⁺ decreases the basal pulmonary arterial pressure (PAP) and abolishes the acute hypoxia-induced increase in PAP in isolated perfused/ventilated mouse lungs. (A) Representative lung angiograph of the left lung (a) and right lung lobes (b) from a C57/BL6 mouse. Summarized data (c, means ± SE) showing the total branch length, number of branch, and number of junctions of the left lung vasculature per square millimeter of area (n = 11 mouse lungs). (B) Schematic diagram (a) of the isolated perfused/ventilated lung preparation and representative records (b) of pulmonary arterial pressure (PAP) in the lungs ventilated with hypoxic gas (1% O₂ in N₂) by tracheal intubation (upper insert) or perfused with high-K⁺ solution through a right ventricular/pulmonary arterial catheter (lower insert). (C) Representative record (a) of PAP before, during, and after ventilation with hypoxic gas (Hyp, (1% O₂ in N₂ for 4 min) when the lung was perfused with physiological salt solution (PSS) with or without (Ca²⁺-free) 1.8 mM extracellular Ca²⁺. Summarized data (means ± SE, n = 6 mouse lungs) showing the basal PAP and the acute hypoxia-induced increases in PAP before (Cont), during (0Ca), and after (Rec) the lungs are superfused with Ca²⁺-free (0Ca) PSS. ***p < 0.001, *p < 0.05 vs. Cont (blue) and Rec (dark red) bars.

For measuring PAP in the open perfusion isolated perfused/ventilated lung, we used (i) a mini pump to consistently superfuse PSS into PA via right ventricle and (ii) a mini ventilator to ventilate room air (normoxic control, 21% O₂) into the airway and alveoli (insert Fig. 1Ba). PAP was measured by a pressure transducer and recorded by Power Lab (AD Instruments) via a catheter connected to the perfusion tube (Fig. 1Ba). By ventilating hypoxic gas mixture (1% O₂, 5% CO₂ in N2), we were able to observe a significant increase in PAP due to HPV (Fig. 1Bb, upper inset). By perfusing high-K⁺ PSS, we were able to observe an increase in PAP due to 40 mM K⁺-induced pulmonary vasoconstriction (Fig. 1Bb, lower inset). By perfusing PSS containing a vasoconstrictive agonist, we were able to observe an increase in PAP due to agonist-induced pulmonary vasoconstriction (data not shown). The amplitude of PAP increase during four minutes of alveolar hypoxia was at the range of 3–5 mmHg (insert Fig. 1C), while the amplitude of 40 mM K⁺-induced increase in PAP was around 6–9 mmHg.

HPV is dependent on extracellular Ca²⁺ influx

Removal of extracellular Ca²⁺ in the perfusate (Ca²⁺-free) or PSS significantly decreased the basal PAP and abolished the alveolar hypoxia-induced increase in PAP due to HPV (insert Fig. 1Ca). Upon restoration of extracellular [Ca²⁺] to 1.8 mM, the basal PAP returned to control level (insert Fig. 1Cb) and the hypoxia-induced increase in PAP (insert Fig. 1Cc) was also fully recovered (insert Fig. 1Ca–c). These results indicate that extracellular Ca²⁺ is not only necessary for maintaining basal PAP, but also required for alveolar hypoxia-induced increase in PAP due to HPV. The 20% decrease in basal PAP and the 90% inhibition of HPV when the pulmonary vasculature was superfused with Ca²⁺-free PSS indicate that Ca²⁺ influx through Ca²⁺-permeable cation channels in PASMC plays an important role in the regulation of pulmonary vascular reactivity and HPV.

HPV is dependent on Ca²⁺ influx through L-type of VDCC

In the next set of experiment, we aimed to identify specific VDCC that contribute to HPV in mouse lung using selective blockers for different types of VDCC. Superfusion of Nif (0.1 µM), a L-type VDCC blocker, significantly and reversibly inhibited alveolar hypoxia-induced increases in PAP due to HPV (insert Fig. 2a). Intrapulmonary perfusion of TTA-A2 (30 µM), a specific blocker of T-type VDCC slightly decreased the basal PAP but had negligible effect on alveolar hypoxia-induced increase in PAP (insert Fig. 2b). CoTX (1 µM), by selectively blocking P/Q-type VDCC, had no effect on the basal PAP and the amplitude of alveolar hypoxia-induced increase in PAP (insert Fig. 2c). These results indicate that Ca²⁺ influx through, at least, L-type VDCC is involved in alveolar hypoxia-induced increase in PAP due to HPV.

Fig. 2.

Blockade of L-type of voltage-dependent Ca²⁺ channels (VDCC) significantly inhibits hypoxia-induced increase in pulmonary arterial pressure (PAP) in isolated perfused/ventilated mouse lungs. (a–c) Representative records (left panels) showing changes of pulmonary arterial pressure (PAP) induced by ventilation of hypoxic gas (1% O₂ for 4 min) before, during, and after perfusion of nifedipine (Nif, 0.1 µM, a L-type VDCC blocker, (a), TTA-A2 (30 µM, a T-type VDCC blocker, (b) or ϖ-Conotoxin (CoTX, 1 µM, an N, P/Q-type VDCC blocker, (c). Summarized data (means ± SE, right panels, n = 6 mouse lungs) showing the acute hypoxia-induced increases in PAP before (Control), during, and after (Recovery) perfusion with PSS containing Nif (a), TTA-A2 (b), or CoTX (c). ***p < 0.001 vs Control (c, blue) bars and Recovery (R, dark red) bars.

HPV is dependent on Ca²⁺ influx through TRP-formed non-selective cation channels

TRP channels have been demonstrated to form ROC^55,56 and SOC.^57,58 To determine specific TRP channels involved in HPV in mouse lung, we used selective blockers for different TRP isoforms in the next set of pharmacological experiments. As shown in Fig. 3, intrapulmonary arterial superfusion of SKF-96365 (SKF, 30 µM) (insert Fig. 3a), a blocker of TRPC channels,⁵⁹ and SAR-7334 (SAR, 1 µM) (insert Fig. 3b), a specific blocker of TRPC6 channels,⁴⁷ had no obvious effect on the basal PAP, but significantly and reversibly inhibited alveolar hypoxia-induced increases in PAP. Blockade of TRPV1 channel by CPZ (30 µM) also exerted significant and reversible inhibitory effect on alveolar hypoxia-induced increases in PAP or HPV (insert Fig. 3c), whereas selective blockers for TRPP channels, Ben (10 µM), TRPA channels, AM0902 (AM, 10 µM) and TRPML channels, Gd³⁺ (10 µM) negligibly affected HPV or alveolar hypoxia-induced increases in PAP (insert Fig. 3d–f). Consistent with the pharmacological experiments using TRPC6 blocker SAR-7334 (insert Fig. 3b) and TRPC blocker SKF-96365 (insert Fig. 3a), genetic deletion of the TRPC6 gene (insert Fig. 3g) significantly inhibited HPV in isolated mouse lungs (insert Fig. 3h). The amplitude of alveolar hypoxia-induced increase in PAP in isolated perfused/ventilated lungs from trpc6^–/– mice was similar to that in isolated lungs from wild-type (WT) mice when the lungs were superfused with PSS containing the TRPC blocker SKF-96365 or the TRPC6 blocker SAR-7334 (insert Fig. 3g and Fig. 3a and b). These results led us to conclude that TRPC channels (especially the TRPC6 channel) and TRPV channels (e.g. TRPV1) are involved in acute hypoxia-induced Ca²⁺ influx and increases in [Ca²⁺]_cyt in PASMC that triggers HPV.

Fig. 3.

Blockade of transient receptor potential (TRP) channels significantly inhibits the acute hypoxia-induced increase in pulmonary arterial pressure (PAP) in isolated perfused/ventilated mouse lungs. (a–f) Representative records (left panels) showing changes of PAP induced by ventilation of hypoxic gas (1% O₂ in N₂ for 4 min) before, during, and after perfusion of SKF 96365 (SKF, 30 µM, a TRPC channel blocker, a), SAR-7334 (SAR, 1 µM, a TRPC6 channel blocker, b), capsazepine (CPZ, 30 µM, a TRPV1 channel blocker, c), benzamil (Ben, 10 µM, a TRPP3 channel blocker, d), AM0902 (AM, 10 µM, a TRPA channel blocker, e), or gadolinium (Gd³⁺, 10 µM, a TRPML channel blocker, f). Summarized data (means ± SE, right panels, n = 6 mouse lungs) showing the hypoxia-induced increases in PAP before (Control), during, and after (Recovery) perfusion with PSS containing SKF, SAR, CPZ, Ben, AM, or Gd³⁺, respectively. **p < 0.01, *p < 0.05 vs. control (Cont, blue) bars. (g) Representative Western blot image (left panel) and summarized data (means ± SE, right panel, n = 3 independent experiments from five mice) of TRPC6 in whole lung tissues from wild-type (WT) and Trpc6 knock-out (trpc6^–/–) mice. ***p < 0.001 vs. WT. (h) Representative records (left panels) showing changes of PAP when the lungs from WT and trpc6^–/– mice are ventilated with normoxic (21% O₂ in N₂) and hypoxic (1% O₂ in N₂, for 4 min) gas. Summarized data (means ± SE, n = 5 mouse lungs) showing basal PAP (middle panel) and acute hypoxia-induced rises in PAP (right panel) in isolated lungs from WT (blue) and trpc6^–/– (red) mice. ***p < 0.001 vs WT.

ND: not detectable.

Regulation of HPV by CaSR, a GPCR

CaSR, ET_A and M₄ receptors are three GPCRs expressed in PASMC, which are activated by extracellular Ca²⁺,²⁰ endothelin-1 (ET-1)⁶⁰ and acetylcholine (ACh),⁶¹ respectively. It has been demonstrated that activation of these receptors induce Ca²⁺ influx through ROC and SOC, thus increasing [Ca²⁺]_cyt in PASMCs.²⁰ As shown in Fig. 4, intrapulmonary arterial perfusion of NPS2143 (30 µM), an allosteric blocker of CaSR, significantly and reversibly inhibited alveolar hypoxia-induced increase in PAP (insert Fig. 4a). However, neither TRO (10 µM), a specific M4 receptor blocker, nor BQ-123 (10 µM), a selective ET_A receptor blocker, exerted any effect on alveolar hypoxia-induced increase in PAP or HPV (insert Fig. 4b and c). These results indicate that activation of CaSR is involved in mediating or modulating acute alveolar hypoxia-induced pulmonary vasoconstriction by priming ROC and SOC.²⁰

Fig. 4.

Blockade of calcium sensing receptor (CaSR) significantly inhibits hypoxia-induced increase in pulmonary arterial pressure (PAP) in isolated perfused/ventilated mouse lungs. (a–c) Representative records (left panels) showing changes of PAP induced by ventilation of hypoxic gas (1% O₂ in N₂ for 4 min) before, during, and after perfusion of NPS 2143 (NPS, 30 µM, an allosteric CaSR antagonist, a), tropicamide (TRO, 10 µM, a muscarinic receptor antagonist, b), or BQ-123 (BQ, 10 µM, an endothelin receptor A antagonist, c). Summarized data (means ± SE, right panels, n = 5 mouse lungs) showing the hypoxia-induced increases in PAP before (Control, c), during, and after (Recovery, R) perfusion with PSS containing NPS, Tro, or BQ, respectively. ***p < 0.001 vs Control (C, blue) bars and Recovery (R, dark red) bars.

Regulation of HPV by notch signaling pathway

Notch signaling has been implicated in lung vascular development,⁶² upregulated Notch ligands (e.g. Jaged-1), and Notch receptors (e.g. Notch1 and Notch3) have been linked to concentric pulmonary vascular remodeling and occlusive intimal lesions in patients with PAH.^42,43,63 Acute superfusion of DAPT (30 µM), a γ-secretase inhibitor that blocks Notch signaling in signal-receiving cells,⁴³ significantly and reversibly diminished the amplitude of alveolar hypoxia-induced increase in PAP due to HPV (insert Fig. 5a). The 50% reduction of acute HPV by DAPT (insert Fig. 5a, right panel) implied that rapid cleavage of Notch receptors or formation of Notch intracellular domain (NICD) was involved in HPV by, directly or indirectly, modulating hypoxia-induced increase in [Ca²⁺]_cyt in PASMC. Furthermore, in isolated perfused/ventilated lungs from notch3^–/– mice (insert Fig. 5b), the amplitude of alveolar hypoxia-induced increase in PAP was approximately 45% less than that in isolated lungs from the WT littermates (insert Fig. 5c). These data indicate that Notch3, a Notch receptor that is predominantly expressed in vascular smooth muscle cells, might be involved in regulation of HPV.

Fig. 5.

Inhibition of Notch signaling attenuates hypoxia-induced increase in pulmonary arterial pressure (PAP) in isolated perfused/ventilated mouse lungs. (a) Representative records (left panel) showing changes of PAP induced by ventilation of hypoxic gas (1% O₂ in N₂ for 4 min) before, during, and after perfusion of DAPT (30 µM, a γ-secretase inhibitor). Summarized data (means ± SE, right panel, n = 5 mouse lungs) showing the hypoxia-induced increases in PAP before (Cont), during (DAPT), and after (Rec) perfusion with PSS containing DAPT. (b) Representative Western blot images (left panel) and summarized data (means ± SE, right panel, n = 3 independent experiments from five mice) showing Notch3 expression levels in lung tissues isolated from WT and notch3^–/– mice. ***p < 0.001 vs. WT. (c) Representative records (left panels) of PAP before, during, and after ventilation of hypoxic gas (1% O₂ in N₂ for 4 min) in WT and Notch3 knock-out (notch3^–/–) mice. Summarized data (means ± SE, n = 5 mouse lungs, right panel) showing acute hypoxia-induced increase in PAP in WT and notch3^–/– mice. ***p < 0.001 vs. WT (c).

ND: not detectable.

HPV is not affected by Ca²⁺-activated Cl^– channel activity

Intracellular or cytosolic Cl^– concentration ([Cl^–]_cyt) is very high in smooth muscle cells⁶⁴; the estimated [Cl^–]_cyt in vascular smooth muscle cells is in the range of 30–50 mM.^65–67 Due to the high [Cl^–]_cyt, the equilibrium potential of Cl^– is thus less negative than the resting membrane potential in smooth muscle cells like PASMC. Accordingly, activation of Cl^– channel, such as Ca²⁺-activated Cl^– (Cl_Ca) channels, in PASMC would result in inward currents (or Cl^– efflux) and therefore membrane depolarization, which may subsequently activate VDCC, induce Ca²⁺ influx, and increase [Ca²⁺]_cyt in PASMC. Intrapulmonary arterial superfusion of CaCCinh-A01 (A01, 10 µM), a specific blocker of Cl_Ca channels, and MONNA (MO, 10 µM), a specific blocker of TMEM16A which forms Cl_Ca channel, had no effect on HPV or alveolar hypoxia-induced increases in PAP (insert Fig. 6a and b). These results indicate that activation of Ca²⁺-activated Cl^– channels, such as TMEM16A, are not involved in the alveolar hypoxia-induced pulmonary vasoconstriction. The Ca²⁺-induced membrane depolarization due to activation of Cl_Ca channels during hypoxia may be compromised by the Ca²⁺-induced membrane repolarization or hyperpolarization due to activation of Ca²⁺-activated K⁺ channels in PASMC.⁶⁸

Fig. 6.

Blockade of Ca²⁺-activated Cl^– (Cl_Ca) channels fails to inhibit hypoxia-induced rise in pulmonary arterial pressure (PAP) in isolated perfused/ventilated mouse lungs. (a and b) Representative records (left panels) showing changes of PAP induced by ventilation of hypoxic gas (1% O₂ in N₂ for 4 min) before, during, and after perfusion of MONNA (MON, 10 µM, a TMEM16A/anoctamin-1 blocker, a), CaCCinh-A01 (A01, 10 µM, a Cl_Ca channel blocker, b). Summarized data (means ± SE, right panels, n = 5 mouse lungs) showing the hypoxia-induced increases in PAP before (Control), during, and after (Recovery) perfusion with PSS containing MON (a) or A01 (b).

Chronic hypoxic exposure of mice inhibits acute alveolar hypoxia-induced pulmonary vasoconstriction

Early studies showed that chronic exposure of mice (and rats) to hypoxia enhances pulmonary vasoconstrictive reactivity in response to various agonists,¹² while humans living in high altitude have blunted response to acute hypoxia.⁶⁹ In rats, it has been reported that acute HPV was significantly inhibited in chronically hypoxic rats.^32,70–72 In this study, we examined whether chronic hypoxia-mediated structural changes in the pulmonary vasculature affects acute HPV. Mice were first exposed to hypoxia for four weeks, which led to significant increases in (a) right ventricular systolic pressure (Fig. 7a), a surrogate measurement of pulmonary arterial systolic pressure, (b) right ventricular contractility (i.e. RV-± dP/dt_max) (Fig. 7b), and (c) Fulton Index, the ratio of the weight of LV to the weight of LV and septum (S) (RV/(LV + S)) (Fig. 7c) in comparison to normoxic controls. The heart rate in normoxic control mice (507 ± 24 beats/min, n = 11) and chronically hypoxic mice (488 ± 28 beats/min, n = 8) was not changed significantly (p = 0.611). Following chronic hypoxic exposure, the lung vasculature underwent significant changes revealed by angiography (Fig. 7d and e). The total length of vascular branches, the number of branches, and the number of junctions between vascular branches were all significantly decreased in the lungs from chronically hypoxic mice compared to normoxic controls (Fig. 7d and e). These data show that chronic hypoxia resulted in significant pulmonary vascular remodeling.

Fig. 7.

Acute hypoxia-induced pulmonary vasoconstriction is inhibited in isolated perfused/ventilated from mice with chronic hypoxia-mediated pulmonary hypertension. (a): Representative records showing right ventricular pressure (RVP, left panels) and summarized data (means ± SE, n = 5–8, right panel) showing right ventricular systolic pressure (RVSP), a surrogate measurement of pulmonary arterial systolic pressure, in normoxic control mice (Nor), and chronically hypoxic mice (for four weeks, Hyp-4w). ***p < 0.001 vs. Nor. (b): Representative record showing right ventricle (RV) ± dP/dt (RV- ± dP/dt, left panels) and summarized data (means ± SE, n = 5–8, right panel) showing the maximal RV ± dP/dt (RV ± dP/dt_max) values in Nor and Hyp-4w mice. ***p < 0.001 vs. Nor. (c): Fulton index, the ratio of the weight of right ventricle (RV) to the weight of left ventricle (LV), and septum (S) (RV/(LV + S)) of the heart isolated from in Nor and Hyp-4w mice. (d): Representative angiographic images (8 × or 30 × in magnification) showing the pulmonary vascular structure in a Nor control mouse (left) and a Hyp-4w mouse (right). The 30 × image in right shows a peripheral region of the whole lung (8 × image). (e): Summarized data (means ± SE, n = 9–10) showing the total length of pulmonary vascular branches (left), the number of branches (middle), and the number of junctions of the vascular branches (right) in Nor control mice (blue) and chronically hypoxic (Hyp-4w) mice (red). **p < 0.01, ***p < 0.001 vs. normoxic control. (f): Representative record showing changes of pulmonary arterial pressure (PAP) before, during, and after acute ventilation of hypoxic gas mixture (Hyp, 1% O₂ in N₂, for 4 min in each challenge) in isolated perfused/ventilated lungs from a normoxic (Nor) control mouse (blue) and a chronically hypoxic (Hyp-4w) mouse (red). The averaged data (means ± SE, n = 8–10 mice) showing the increase in PAP induced by a series of consecutive hypoxia challenges. (g): Summarized data (means ± SE, n = 8–10) showing the basal PAP (left) and acute alveolar hypoxia-induced increases in PAP (right) in isolated perfused/ventilated lungs from Nor mice (blue) and Hyp-4w mice (red). **p < 0.01, ***p < 0.001 vs. Nor.

Then, we examined and compared the basal PAP, determined by measuring the basal pulmonary vascular pressure under the constant flow rate of perfusion, and the amplitude of acute (5 min) alveolar hypoxia-induced increase in PAP in isolated perfused/ventilated lungs from normoxic control mice and chronically hypoxic mice. As shown in Fig. 7f and g, when the perfusion rate was maintained at the same level, the basal PAP in the isolated perfused/ventilated lungs from chronically hypoxic lungs was significantly higher than that in the isolated lungs from normoxic control mice (Fig. 7f). The increased basal PAP in the isolated perfused/ventilated lungs from chronically hypoxic mice was due apparently to the chronic hypoxia-induced structural changes in the pulmonary vasculature (Fig. 7d), including concentric pulmonary arterial wall thickening due to medial hypertrophy. In the isolated perfused/ventilated lungs from chronically hypoxic mice, acute alveolar hypoxia (for 5 min) was still able to induce a decent increase in PAP (Fig. 7f). The amplitude of acute alveolar hypoxia-induced PAP increases in isolated lungs of chronically hypoxic mice, however, was significantly diminished in comparison to the lungs from normoxic control mice (Fig. 7f and g). These data indicate that chronic hypoxia-mediated pulmonary vascular remodeling or structural changes diminished the pulmonary vascular reactivity in response to acute alveolar hypoxia. The chronic hypoxia-mediated pulmonary vascular remodeling may enhance the pulmonary vascular reactivity to other agonists.¹²

Discussion

In this study, we used the isolated perfused/ventilated mouse lung model, previously optimized by our laboratory,⁵ to revisit the Ca²⁺ signaling mechanisms involved in acute HPV. Specifically, we focused on the potential roles of voltage-dependent/independent Ca²⁺ and Cl^– channels as well as GPCRs and Notch receptors. The ex vivo experiments indicate that HPV is primarily dependent of Ca²⁺ influx through various voltage-dependent and -independent Ca²⁺ channels in PASMC; the process is regulated or modulated by Notch and CaSR signaling cascades. Removal of extracellular Ca²⁺ abolished HPV, while blockade of voltage-dependent Ca²⁺ entry through L-type VDCC and receptor-operated Ca²⁺ entry through TRP channels significantly and reversibly inhibited HPV. These data also indicate that the mechanism of HPV or acute hypoxia-induced Ca²⁺ influx is not due to a single pathway³; multiple ion channels and signaling cascades are involved to ensure HPV. Since there is no appropriate morphometric technique to evaluate the whole pulmonary vascular tree quantitatively, the isolated perfused/ventilated lung is an excellent ex vivo model to study the mechanisms involved in HPV. The use of knockout (KO) mice provides more convincing data on the role of different proteins and genes played in the initiation and regulation of HPV.

[Ca²⁺]_cyt in PASMC can be increased by Ca²⁺ influx through various cation channels in the plasma membrane and Ca²⁺ release or mobilization from individual intracellular stores.⁷³ In PASMC, there are at least three classes of Ca²⁺-permeable channels responsible for Ca²⁺ influx: (a) VDCC which are opened by membrane depolarization, (b) ROC which are opened by DAG upon receptor activation, and (c) SOC which are opened by a reduction of [Ca²⁺] level in the SR due to active or passive depletion of stored Ca²⁺.^12,74,75 Activity of Na⁺ pump (or Na⁺/K⁺ ATPase) and K⁺ channel play a vital role in maintaining and regulating the resting membrane potential (E_m) in PASMC. Acute hypoxia has been demonstrated to inhibit K⁺ channels,⁷⁶ which subsequently causes membrane depolarization and opening of VDCC thereby increasing [Ca²⁺]_cyt in PASMC.^7,77–80 The acute hypoxia-mediated increase in [Ca²⁺]_cyt by membrane depolarization is believed to be, at least, mediated by Ca²⁺ influx through L-type VDCC formed by the pore-forming subunits Ca_V1.1 (α_1S), Ca_V1.2 (α_1C), Ca_V1.3 (α_1D), and/or Ca_V1.4 (α_1F).⁶⁶ The results from this study show that only blockade of L-type of VDCC inhibits HPV, whereas the blockers for T-, N-, P-, and Q-type of VDCC seem to have little effect on HPV. Although Nif is a dihydropyridine Ca²⁺ channel blocker that selectively blocks L-type VDCC,^81–83 Nif and other dihydropyridine VDCC blockers have been shown to inhibit the adenosine A_2B receptor, a GPCR in colonic tissues and cells.⁸⁴ The inhibitory effect of Nif on adenosine A_2B receptors has been implicated having therapeutic potential for diarrhea and related diseases.⁸⁴ In addition, it has been shown that T-type of VDCC is involved in hypoxia-induced PH in rats⁸⁵; but more experiments are needed to define the role of different types of VDCC in the initiation and regulation of HPV.

Acute hypoxia-mediated increase in [Ca²⁺]_cyt may also result from Ca²⁺ influx through ROC formed by the TRP channels. Indeed, blockade of non-selective cation channels formed by TRP channels significantly inhibited HPV, while isolated perfused/ventilated lungs from trpc6^–/– mice exhibit significantly reduced amplitude of HPV in comparison to WT littermates (see Fig. 3g).⁸⁶ TRPC6 channel is an important ROC in smooth muscle cells^87–89 and is functionally coupled to CaSR, a GPCR, to mediate CaSR-associated Ca²⁺ influx in PASMC.^20,90 The data from this study imply that CaSR-associated Ca²⁺ influx through ROC formed by TRP channels (e.g. TRPC6) is also an important pathway for hypoxia-induced increase of [Ca²⁺]_cyt in PASMC and HPV. One of the concerns with regard to the pharmacological experiments using different TRPC inhibitors is lack of potent and selective inhibitor for TRPC6. SKF-96365 is a non-selective blocker of TRPC6 and other TRPC channels (e.g. TRPC3 and TRPC7).⁹¹ Furthermore, it has been reported that SKF-96365 also blocks T-type of VDCC; the SKF-96365 seems to induce more potent blockade effect on VDCC than on TRPC3. SKF-96365 has been shown to block other subtypes of VDCC (including L-type, N-type, and P/Q-type).⁹² Another TRPC6 antagonist, SAR-7334, can also bind to TRPC3 and TRPC7.⁴⁷ Recently, another potent, selective and orally available TRPC6 blocker, BI749327, has been shown to have promising therapeutic effects on renal and cardiac fibrosis.⁹³ Apart from TRPC channels, TRPV channels (e.g. TRPV4) have also been implicated in HPV.⁹⁴ Our data in this study show that blockade of TRPV1 channels with CPZ reversibly inhibit acute HPV. However, CPZ may also non-specifically bind to nicotine ACh receptors and TRPM8 channels.⁹⁵ Furthermore, TRPV1 and TRPV4 also contribute to capsaicin- and serotonin-induced pulmonary vasoconstriction, respectively,⁴⁸ while TRPV4 is also implicated in chronic hypoxia-induced PH.⁹⁴

Our ex vivo data from this study indicate that CaSR receptors are involved in or required for HPV. Activation of CaSR increases IP₃ and DAG. IP₃ activates IP₃R in the SR and induces Ca²⁺ mobilization, while DAG activates ROC in the plasma membrane and induces receptor operated calcium entry (ROCE). Both IP₃-mediated Ca²⁺ mobilization and DAG-mediated Ca²⁺ influx contribute to increasing [Ca²⁺]_cyt in PASMC that is required for causing pulmonary vasoconstriction.^96–98 Therefore, ROCE induced by DAG-mediated activation of ROC (formed by TRPC6 and TRPV1, for example) and SOCE induced by the IP₃R–STIM interaction and IP₃-induced active store depletion (upon activation of membrane receptors like CaSR in the plasma membrane) are all involved in triggering HPV. Interestingly, it has been reported that in rabbit portal vein smooth muscle cells, TRPC6/7 channels can be activated independent of DAG by eliminating the inhibitory response of phosphatidylinositol 4,5-bis phosphate.⁹⁹ The results from this study also indicate a potential role of Notch activation in HPV. Inhibition of γ-secretase with DAPT significantly attenuates HPV, while notch3^–/– mice exhibit significantly reduced amplitude of HPV (Fig. 5). These data suggest that NICD may participate in regulating HPV. These data confirmed our previously published results and we further showed that hypoxia activated Notch signaling, which enhances SOCE via direct interaction with TRPC6 leading to HPV and development of chronic hypoxia-induced PH. Thus, Notch signaling is involved in regulation of cytosolic Ca²⁺.⁴³ It is, however, unknown whether and how NICD is involved in enhancing CaSR-mediated ROCE/SOCE and/or IP3R/STIM interaction in PASMC.¹⁰⁰ Recently, it was shown that DAPT, a γ-secretase inhibitor that inhibits Notch signaling, also attenuated pulmonary fibrosis in mice through inhibition of pericyte proliferation and transition.¹⁰¹

In vascular smooth muscle cells, intracellular [Cl^–] is very high so the equilibrium potential for Cl^– is less negative than the resting membrane potential. Activation of Cl^– channels under resting conditions would thus cause Cl^– efflux and membrane depolarization.¹⁰² Ca²⁺-activated Cl^– (Cl_Ca) channels, formed by TMEM16A, are expressed in arterial smooth muscle cells and exert an important role in the regulation of smooth muscle excitation–contraction coupling and vascular tone.^103–105 It has been demonstrated that Cl_Ca channels or TMEM16A are upregulated in PASMC of chronically hypoxic rats and monocrotaline-treated rats,^106,107 while increased activity of Cl_Ca channels (formed by TMEM16A) is an important contributor to the changes in electromechanical coupling of PA and membrane depolarization in PASMC from animals with experimental PH.¹⁰⁷ In the current study, however, intrapulmonary superfusion of the Ca²⁺-activated Cl^– channel blockers, MONNA and A01, had little effect on HPV. Though, MONNA was claimed to be a selective blocker of TMEM16 Ca²⁺-activated Cl^– channels, its selectivity has been challenged by a study which showed that MONNA induced dose-dependent relaxation in rat mesenteric arteries in the absence of Cl^– gradient.¹⁰⁸ Recently, it was reported that chronic administration of benzbromarone attenuated pulmonary vascular remodeling in two different experimental models of PH through inhibition of TMEM16A.¹⁰⁹ Inhibition of cystic fibrosis transmembrane conductance regulator (CFTR), a Cl^– channel that is also permeable to HCO₃^–,^110,111 significantly attenuated HPV.¹¹² In PASMC, hypoxia causes CFTR to interact with TRPC6. Inhibition of CFTR attenuates hypoxia-induced TRPC6 translocation to caveolae and sphingosine-1-phosphate-mediated Ca²⁺ mobilization in PASMC. These data indicate that sphingolipid-mediated interaction of CFTR, a Cl^–/HCO3^– channel, and TRPC6, a non-selective cation channel, in PASMC plays an important role in HPV.¹¹²

It is clear from our data that Ca²⁺ influx through various Ca²⁺-permeable cation channels is involved in the initiation of HPV; however, Ca²⁺-independent activation of Rho kinase and enhancement of Ca²⁺ sensitivity of contractile proteins have also been implicated in the development of HPV.¹¹³ Hypoxia seems to be able to activate Rho kinase in both PASMCs and endothelial cells.^38,113,114 Rho kinase appears to play an important role in mediating both the acute and chronic effects of hypoxia on pulmonary circulation.¹¹⁵

Alveolar hypoxia induces pulmonary vasoconstriction to match the perfusion with ventilation ensuring maximal oxygenation of the venous blood in PA. Persistent hypoxia, however, causes sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling and subsequently PH.^116,117 It has been demonstrated that chronic alveolar hypoxia downregulates Kv channels and upregulates TRP channels in PASMC causing pulmonary vasoconstriction and vascular remodeling.^118,119 In 1978, McMurtry and colleagues reported that lungs from chronically hypoxic rats have decreased acute hypoxia-mediated pulmonary vasoconstriction.⁷² In mice, we observed the same results that chronic exposure of mice to hypoxia for four weeks resulted in PH characterized by significant pulmonary vascular remodeling; however, the acute HPV was significantly inhibited in isolated perfused/ventilated lungs from chronically hypoxic mice (Fig. 7). These data imply that acute hypoxia induces pulmonary vasoconstriction by mechanisms that may be shared by chronic hypoxia to induce pulmonary vasoconstriction and vascular remodeling. The proposed mechanisms include impaired lung vascular endothelial function,⁷⁰ mitochondrial dysfunction^120–122 and metabolic shift,^123,124 functional and transcriptional changes of ion channels and membrane receptors,³ and Ca²⁺-sensitive intracellular signaling proteins and transcription factors.¹²⁵

Here, we used an ex vivo mouse model, isolated perfused/ventilated lung preparation, for a series of comprehensive pharmacological experiments to define the Ca²⁺ signaling mechanisms involved in acute hypoxia-induced pulmonary vasoconstriction. The isolated perfused/ventilated lung model we used in this study has both strengths and drawbacks, but we believe the benefits outweigh the drawbacks. To investigate the precise mechanisms of HPV, investigators have used intact animals, isolated lungs, isolated pulmonary arteries, and freshly-dissociated and primary cultured PASMC and endothelial cells.^41,79,126 Studies in vessels or arterial rings provide important information about, for example, two phases of HPV and the involvement of contractile proteins and the involvement of contractile proteins and Ca²⁺-sensitive and -insensitive signaling proteins. However, results obtained from freshly-dissociated or primary cultured cells are very different from the vessels or the whole vasculature in isolated lungs or the intact animals. One of the advantages of using the isolated perfused lung to study HPV is that it minimizes the impact of systemic organs¹²⁷ while maintaining the intact lungs and allowing a more physiological setting for transport of solutes across capillary membrane and exchange of O₂ and CO₂ across the blood–air barrier.¹²⁸ Although acute hypoxia causes vasoconstriction in isolated vessels or rings, the kinetics of HPV response in vessels or rings is different from HPV in humans and intact animals. In isolated perfused/ventilated lungs, the kinetics of HPV is similar to that in intact animals and humans. Overall, the advantage of using the isolated perfused/ventilated lung to study HPV is that (i) it reflects the functional changes of the whole lung vasculature, (ii) it introduces alveolar hypoxia via ventilation to the vasculature (instead of using hypoxemic solution to perfuse into the vessels), (iii) it allows us to superfuse inhibitors via perfusion pump into PA or the pulmonary vasculature to examine their effect; (iv) it shows very similar time-course shown in intact animals and healthy subjects; (v) it minimizes the impact of other organs and nervous systems on HPV while maintaining the intact lung in a relatively physiological setting (e.g. the preparation is consistently ventilated through the airway and alveoli, and perfused through the pulmonary arteries, capillaries, and vens); and (vi) it allows us to examine whether genetic deletion (e.g. KO mice) or overexpression (e.g. transgenic mice) of specific genes affects HPV.

The data from our study indicate that extracellular Ca²⁺, or Ca²⁺ influx through various Ca²⁺-permeable channels in the plasma membrane, is required for HPV. Removal of extracellular Ca²⁺ abolished HPV, while blockade of L-type VDCC (with Nif), non-selective cation channels (with SKF) and TRP channels (with SAR and CPZ) significantly and reversibly inhibited HPV. Furthermore, blockers of CaSR and Notch receptors also attenuated HPV. These results led us to conclude that Ca²⁺ influx through L-type voltage-gated Ca²⁺ channels and TRPC6-formed ROC plays an important role in the initiation of pulmonary vasoconstriction during alveolar hypoxia; however, contribution of Ca²⁺ channels in the plasma membrane and cation channels in the intracellular organelles to the regulation of HPV cannot be ruled out. From our study, we have confirmed our and other investigators’ data on the critical role of Ca²⁺ signaling in HPV. We believe that present pharmacological study utilizing various inhibitors of ion channels and membrane receptors in isolated perfused/ventilated mouse lung model provides data giving us a comprehensive overview on the involvement of ion channels and membrane receptors in HPV. Furthermore, the results of our study could serve as a template for selecting inhibitors for the future research.

In this study, we also included mouse angiography images showing left and right lungs from normoxic control and chronically hypoxic C57Bl/6 J mice with detailed quantification (i.e. the total length of lung vascular branches, the number of vascular branches, and the number of junctions among branches). The mouse lung angiography data can be used as a template to further study chronic hypoxia-mediated pulmonary vascular remodeling in WT mice and various KO and transgenic mice. The mouse lung angiography technique is a simple and economic method in comparison to the expensive micro-CT imaging approach.¹²⁹

In summary, the data from this study indicate that Ca²⁺ influx through voltage-gated, receptor-operated, and SOC in the plasma membrane of PASMC plays an important role in the initiation of HPV, while the extracellular Ca²⁺-mediated activation of CaSR and the cell–cell interaction via Notch ligands and receptors contribute to the regulation of HPV.