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.

Sustained pulmonary vasoconstriction and excessive pulmonary vascular remodelling are two major causes of elevated pulmonary vascular resistance in patients with pulmonary arterial hypertension. The purpose of this study was to investigate whether chloroquine induced relaxation in the pulmonary artery (PA) and attenuates hypoxia-induced pulmonary hypertension (HPH).

Excessive pulmonary artery (PA) smooth muscle cell (PASMC) proliferation and migration are implicated in the development of pathogenic pulmonary vascular remodeling characterized by concentric arterial wall thickening and arteriole muscularization in patients with pulmonary arterial hypertension (PAH). Pulmonary artery smooth muscle cell contractile-to-proliferative phenotypical transition is a process that promotes pulmonary vascular remodeling. A rise in cytosolic Ca2+ concentration [(Ca2+) cyt ] in PASMCs is a trigger for pulmonary vasoconstriction and a stimulus for pulmonary vascular remodeling. Here, we report that the calcium homeostasis modulator (CALHM), a Ca2+ (and ATP) channel that is allosterically regulated by voltage and extracellular Ca2+, is upregulated during the PASMC contractile-to-proliferative phenotypical transition. Protein expression of CALHM1/2 in primary cultured PASMCs in media containing serum and growth factors (proliferative PASMC) was significantly greater than in freshly isolated PA (contractile PASMC) from the same rat. Upregulated CALHM1/2 in proliferative PASMCs were associated with an increased ratio of pAKT/AKT and pmTOR/mTOR and an increased expression of the cell proliferation marker PCNA, whereas serum starvation and rapamycin significantly downregulated CALHM1/2. Furthermore, CALHM1/2 were upregulated in freshly isolated PA from rats with monocrotaline (MCT)-induced PH and in primary cultured PASMC from patients with PAH in comparison to normal controls. Intraperitoneal injection of CGP 37157 (0.6 mg/kg, q8H), a non-selective blocker of CALHM channels, partially reversed established experimental PH. These data suggest that CALHM upregulation is involved in PASMC contractile-to-proliferative phenotypical transition. Ca2+ influx through upregulated CALHM1/2 may play an important role in the transition of sustained vasoconstriction to excessive vascular remodeling in PAH or precapillary PH. Calcium homeostasis modulator could potentially be a target to develop novel therapies for PAH.

Pulmonary vasoconstriction and vascular remodeling are two major causes for elevated pulmonary vascular resistance and pulmonary arterial pressure in patients with idiopathic pulmonary arterial hypertension (IPAH). An increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in pulmonary artery smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC proliferation, which causes pulmonary vascular remodeling. Store-operated Ca(2+) entry (SOCE), induced by depletion of stored Ca(2+) in the sarcoplasmic reticulum (SR), can increase [Ca(2+)](cyt) in PASMC independent of other means of Ca(2+) entry. Stromal interaction molecule (STIM) proteins, STIM1 and STIM2, were recently identified as both sensors for store depletion and signaling molecules to open store-operated Ca(2+) channels. We previously reported that SOCE was significantly enhanced in PASMC from IPAH patients compared to PASMC from normotensive control subjects. Enhanced SOCE plays an important role in the pathophysiological changes in PASMC associated with pulmonary arterial hypertension. In this study, we examine whether the expression level of STIM1 and STIM2 is altered in IPAH-PASMC compared to control PASMC and whether these putative changes in STIM1/2 expression level are responsible for enhanced SOCE and proliferation in IPAH-PASMC. Compared to control PASMC, the protein expression level of STIM2 was significantly increased whereas STIM1 protein expression was not significantly changed. In IPAH-PASMC, siRNA-mediated knockdown of STIM2 decreased SOCE and proliferation, while knockdown of STIM2 in control PASMC had no effect on either SOCE or proliferation. Overexpression of STIM2 in control PASMC failed to enhance SOCE or proliferation. These data indicate that enhanced protein expression of STIM2 is necessary, but not sufficient, for enhanced SOCE and proliferation of IPAH-PASMC.

Pulmonary hypertension is a chronic vascular disease characterized by pulmonary vasoconstriction and pulmonary arterial remodeling. Pulmonary arterial remodeling is mainly due to small pulmonary arterial wall thickening and lumen occlusion. Previous studies have described intravascular changes in lung sections using histopathology, but few were able to obtain a fine detailed image of the pulmonary vascular system. In this study, we used Microfil compounds to cast the pulmonary arteries in a rat model of monocrotaline-induced pulmonary hypertension. High-quality images that enabled quantification of distal pulmonary arterial branching based on the number of vessel bifurcations/junctions were demonstrated in this model. The branch and junction counts of distal pulmonary arteries significantly decreased in the monocrotaline group compared to the control group, and this effect was inversely proportional to the mean pulmonary artery pressure observed in each group. The patterns of pulmonary vasculature and the methods for pulmonary vessel casting are presented to provide a basis for future studies of pulmonary arterial remodeling due to pulmonary hypertension and other lung diseases that involve the remodeling of vasculature.