Hypoxic Pulmonary Vasoconstriction in Isolated Mouse Lungs

Abstract

BackgroundHypoxic pulmonary vasoconstriction (HPV) is a physiological mechanism of lung to ensure maximal gas exchange and blood oxygenation. Persistent and global alveolar hypoxia leads to sustained vasoconstriction and vascular remodeling causing pulmonary hypertension. It has been demonstrated that acute HPV is initiated by an increase in cytosolic [Ca2+] ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMC) and modulated by factors released from endothelium. Acute HPV is well reserved in isolated perfused/ventilated lungs, whereas HPV in isolated pulmonary arterial rings shows different pattern in comparison to HPV in intact animals and humans. In this study, we aimed to use isolated perfused/ventilated mouse lungs to further study potential mechanisms involved in development of HPV.MethodsC57BL/6 mice (male, 8–10 weeks old) were anesthetized by pentobarbital sodium (120 mg/kg). After tracheostomy, isolated lungs were immediately ventilated with normoxic gas (21% O2/5% CO2) using a rodent ventilator (80 breaths/min with a tidal volume of 250 μl). The mice were then placed in an isolated lung system chamber with a heated water jacket at 37°C. After tracheal intubation, the chest was opened and a catheter was inserted into the main PA via the right ventricle to measure PAP. The lung vasculature was consistently superfused with physiological salt solution (PSS) via a pump while lung airway and alveoli were ventilated with normoxic or hypoxic gas. Before experimentation, the isolated lung was first superfused with the 40 mM K+‐containing PSS (40K) to stabilize the basal PAP. The lung was then repetitively challenged by ventilation of hypoxic gas (1% O2 in N2, for 4 min) to induce an increase in PAP (due to HPV). Pharmacological effects of various inhibitors on HPV were examined by superfusion of PSS containing the inhibitors while the lung was ventilated with hypoxic gas.ResultsOur results show that HPV was dependent on Ca2+ influx through a) nifedipine‐sensitive L‐type voltage‐dependent Ca2+ channels; b) SKF‐sensitive non‐selective cation channels; and c) TRP channels like TRPC6, TRPV1 and TRPP. HPV in isolated perfused lung was also significantly inhibited by blocking Ca2+‐sensing receptors (CaSR) and Notch receptors and by extracellular Mg2+ and Mn2+. Interestingly, blockade of Ca2+‐activated Cl− channels and inhibition of muscarinic receptors and endothelin receptors had little effect on HPV. Furthermore, inhibition of nitric oxide synthase (eNOS) potentiated HPV.ConclusionAn increase in [Ca2+]cyt due to Ca2+ influx through various Ca2+‐permeable cation channels in PASMC influx is required for the initiation of acute HPV. Both voltage‐gated Ca2+ channels (e.g., CaV1.2) and receptor‐operated Ca2+ channels (TRPC6/V1/P) are involved in mediating Ca2+ influx to induce HPV. G protein‐coupled receptor/CaSR‐mediated synthesis of intracellular signaling molecules (e.g., diacylglycerol and inositol 1, 4, 5‐trisphosphate) and Notch‐associated cell‐to‐cell interaction also play important roles in HPV. These data indicate that multiple mechanisms are involved in the initiation and regulation of HPV.Support or Funding InformationNIH NIHLBI HL135807