Asunto(s)
Becas , Humanos , Publicaciones Periódicas como Asunto , Pulmón/fisiología , Políticas Editoriales , Estados UnidosRESUMEN
Lung endothelium resides at the interface between the circulation and the underlying tissue, where it senses biochemical and mechanical properties of both the blood as it flows through the vascular circuit and the vessel wall. Endothelium performs the bidirectional signaling between the blood and tissue compartments that is necessary to maintain homeostasis while physically separating both, facilitating a tightly regulated exchange of water, solutes, cells, and signals. Disruption in endothelial function contributes to vascular disease, which can manifest in discrete vascular locations along the arterial-to-capillary-to-vein axis. While our understanding of mechanisms that contribute to endothelial cell injury and repair in acute and chronic vascular disease have advanced, pathophysiological mechanisms that underlie site-specific vascular disease remain incompletely understood. In an effort to improve the translatability of mechanistic studies of the endothelium, the American Thoracic Society convened a workshop to optimize rigor, reproducibility, and translation of discovery to advance our understanding of endothelial cell function in health and disease.
RESUMEN
Pulmonary hypertension (PH) arises from increased pulmonary vascular resistance due to contraction and remodeling of the pulmonary arteries. The structural changes include thickening of the smooth muscle layer from increased proliferation and resistance to apoptosis. The mechanisms underlying apoptosis resistance in PH are not fully understood. In cancer cells, high expression of aquaporin 1 (AQP1), a water channel, is associated with apoptosis resistance. We showed AQP1 protein was expressed in pulmonary arterial smooth muscle cells (PASMCs) and upregulated in preclinical PH models. In this study, we used PASMCs isolated from control male rats and the SU5416 plus hypoxia (SuHx) model to test the role of AQP1 in modulating susceptibility to apoptosis. We found the elevated level of AQP1 in PASMCs from SuHx rats was necessary for resistance to apoptosis and that apoptosis resistance could be conferred by increasing AQP1 in control PASMCs. In exploring the downstream pathways involved, we found AQP1 levels influence the expression of Bcl-2, with enhanced AQP1 levels corresponding to increased Bcl-2 expression, reducing the ratio of BAX to Bcl-2, consistent with apoptosis resistance. These results provide a mechanism by which AQP1 can regulate PASMC fate.
Asunto(s)
Apoptosis , Acuaporina 1 , Hipoxia , Indoles , Músculo Liso Vascular , Miocitos del Músculo Liso , Arteria Pulmonar , Pirroles , Animales , Acuaporina 1/metabolismo , Acuaporina 1/genética , Masculino , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Arteria Pulmonar/citología , Ratas , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Músculo Liso Vascular/citología , Pirroles/farmacología , Indoles/farmacología , Hipoxia/metabolismo , Ratas Sprague-Dawley , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/patología , Células Cultivadas , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Modelos Animales de EnfermedadRESUMEN
BACKGROUND: Conduit pulmonary arterial stiffening and the resultant increase in pulmonary vascular impedance has emerged as an important underlying driver of pulmonary arterial hypertension (PAH). Given that matrix deposition is central to vascular remodeling, we evaluated the role of the collagen crosslinking enzyme lysyl oxidase like 2 (LOXL2) in this study. METHODS AND RESULTS: Human pulmonary artery smooth muscle cells (PASMCs) subjected to hypoxia showed increased LOXL2 secretion. LOXL2 activity and expression were markedly higher in primary PASMCs isolated from pulmonary arteries of the rat Sugen5416 + hypoxia (SuHx) model of severe PH. Similarly, LOXL2 protein and mRNA levels were increased in pulmonary arteries (PA) and lungs of rats with PH (SuHx and monocrotaline (MCT) models). Pulmonary arteries (PAs) isolated from rats with PH exhibited hypercontractility to phenylephrine and attenuated vasorelaxation elicited by acetylcholine, indicating severe endothelial dysfunction. Tensile testing revealed a a significant increase in PA stiffness in PH. Treatment with PAT-1251, a novel small-molecule LOXL2 inhibitor, improved active and passive properties of the PA ex vivo. There was an improvement in right heart function as measured by right ventricular pressure volume loops in-vivo with PAT-1251. Importantly PAT-1251 treatment ameliorated PH, resulting in improved pulmonary artery pressures, right ventricular remodeling, and survival. CONCLUSION: Hypoxia induced LOXL2 activation is a causal mechanism in pulmonary artery stiffening in PH, as well as pulmonary artery mechanical and functional decline. LOXL2 inhibition with PAT-1251 could be a promising approach to improve pulmonary artery pressures, right ventricular elastance, cardiac relaxation, and survival in PAH.
RESUMEN
Numerous studies have demonstrated that endostatin (ES), a potent angiostatic peptide derived from collagen type XVIII α 1 chain and encoded by COL18A1, is elevated in pulmonary arterial hypertension (PAH). It is important to note that elevated ES has consistently been associated with altered hemodynamics, poor functional status, and adverse outcomes in adult and pediatric PAH. This study used serum samples from patients with Group I PAH and plasma and tissue samples derived from the Sugen/hypoxia rat pulmonary hypertension model to define associations between COL18A1/ES and disease development, including hemodynamics, right ventricle (RV) remodeling, and RV dysfunction. Using cardiac magnetic resonance imaging and advanced hemodynamic assessments with pressure-volume loops in patients with PAH to assess RV-pulmonary arterial coupling, we observed a strong relationship between circulating ES levels and metrics of RV structure and function. Specifically, RV mass and the ventricular mass index were positively associated with ES, whereas RV ejection fraction and RV-pulmonary arterial coupling were inversely associated with ES levels. Our animal data demonstrate that the development of pulmonary hypertension is associated with increased COL18A1/ES in the heart as well as the lungs. Disease-associated increases in COL18A1 mRNA and protein were most pronounced in the RV compared with the left ventricle and lung. COL18A1 expression in the RV was strongly associated with disease-associated changes in RV mass, fibrosis, and myocardial capillary density. These findings indicate that COL18A1/ES increases early in disease development in the RV and implicates COL18A1/ES in pathologic RV dysfunction in PAH.
Asunto(s)
Endostatinas , Disfunción Ventricular Derecha , Remodelación Ventricular , Animales , Endostatinas/metabolismo , Humanos , Masculino , Femenino , Disfunción Ventricular Derecha/metabolismo , Disfunción Ventricular Derecha/fisiopatología , Ratas , Hipertensión Arterial Pulmonar/metabolismo , Hipertensión Arterial Pulmonar/fisiopatología , Hipertensión Arterial Pulmonar/patología , Ratas Sprague-Dawley , Colágeno Tipo XVIII/metabolismo , Colágeno Tipo XVIII/genética , Persona de Mediana Edad , Adulto , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/fisiopatología , Hipertensión Pulmonar/patología , Progresión de la Enfermedad , Modelos Animales de Enfermedad , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/fisiopatología , Ventrículos Cardíacos/patologíaRESUMEN
Pulmonary hypertension (PH) is a condition in which remodeling of the pulmonary vasculature leads to hypertrophy of the muscular vascular wall and extension of muscle into nonmuscular arteries. These pathological changes are predominantly due to the abnormal proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs), enhanced cellular functions that have been linked to increases in the cell membrane protein aquaporin 1 (AQP1). However, the mechanisms underlying the increased AQP1 abundance have not been fully elucidated. Here we present data that establishes a novel interaction between AQP1 and the proteolytic enzyme caspase-3. In silico analysis of the AQP1 protein reveals two caspase-3 cleavage sites on its C-terminal tail, proximal to known ubiquitin sites. Using biotin proximity ligase techniques, we establish that AQP1 and caspase-3 interact in both human embryonic kidney (HEK) 293A cells and rat PASMCs. Furthermore, we demonstrate that AQP1 levels increase and decrease with enhanced caspase-3 activity and inhibition, respectively. Ultimately, further work characterizing this interaction could provide the foundation for novel PH therapeutics.NEW & NOTEWORTHY Pulmonary arterial smooth muscle cells (PASMCs) are integral to pulmonary vascular remodeling, a characteristic of pulmonary arterial hypertension (PAH). PASMCs isolated from robust animal models of disease demonstrate enhanced proliferation and migration, pathological functions associated with increased abundance of the membrane protein aquaporin 1 (AQP1). We present evidence of a novel interaction between the proteolytic enzyme caspase-3 and AQP1, which may control AQP1 abundance. These data suggest a potential new target for novel PAH therapies.
Asunto(s)
Acuaporina 1 , Caspasa 3 , Músculo Liso Vascular , Miocitos del Músculo Liso , Arteria Pulmonar , Animales , Humanos , Masculino , Ratas , Acuaporina 1/metabolismo , Acuaporina 1/genética , Caspasa 3/metabolismo , Proliferación Celular , Células HEK293 , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/patología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Ratas Sprague-DawleyRESUMEN
Pulmonary arterial hypertension (PAH) is a morbid disease characterized by significant lung endothelial cell (EC) dysfunction. Prior work has shown that microvascular endothelial cells (MVECs) isolated from animals with experimental PAH and patients with PAH exhibit significant abnormalities in metabolism and calcium signaling. With regards to metabolism, we and others have shown evidence of increased aerobic glycolysis and evidence of increased utilization of alternate fuel sources (such as fatty acids) in PAH EC. In the realm of calcium signaling, our prior work linked increased activity of the transient receptor potential vanilloid-4 (TRPV4) channel to increased proliferation of MVECs isolated from the Sugen/Hypoxia rat model of PAH (SuHx-MVECs). However, the relationship between metabolic shifts and calcium abnormalities was not clear. Specifically, whether shifts in metabolism were responsible for increasing TRPV4 channel activity in SuHx-MVECs was not known. In this study, using human data, serum samples from SuHx rats, and SuHx-MVECs, we describe the consequences of increased MVEC fatty acid oxidation in PAH. In human samples, we observed an increase in long-chain fatty acid levels that was associated with PAH severity. Next, using SuHx rats and SuHx-MVECs, we observed increased intracellular levels of lipids. We also show that increasing intracellular lipid content increases TRPV4 activity, whereas inhibiting fatty acid oxidation normalizes basal calcium levels in SuHx-MVECs. By exploring the fate of fatty acid-derived carbons, we observed that the metabolite linking increased intracellular lipids to TRPV4 activity was ß-hydroxybutyrate (BOHB), a product of fatty acid oxidation. Finally, we show that BOHB supplementation alone is sufficient to sensitize the TRPV4 channel in rat and mouse MVECs. Returning to humans, we observe a transpulmonary BOHB gradient in human patients with PAH. Thus, we establish a link between fatty acid oxidation, BOHB production, and TRPV4 activity in MVECs in PAH. These data provide new insight into metabolic regulation of calcium signaling in lung MVECs in PAH.NEW & NOTEWORTHY In this paper, we explore the link between metabolism and intracellular calcium levels in microvascular endothelial cells (MVECs) in pulmonary arterial hypertension (PAH). We show that fatty acid oxidation promotes sensitivity of the transient receptor potential vanilloid-4 (TRPV4) calcium channel in MVECs isolated from a rodent model of PAH.
Asunto(s)
Antineoplásicos , Hipertensión Arterial Pulmonar , Animales , Humanos , Ratones , Ratas , Calcio/metabolismo , Células Endoteliales/metabolismo , Hipertensión Pulmonar Primaria Familiar/metabolismo , Ácidos Grasos/metabolismo , Lípidos , Pulmón/metabolismo , Hipertensión Arterial Pulmonar/metabolismo , Canales Catiónicos TRPV/metabolismoRESUMEN
Background: Conduit pulmonary arterial stiffening and the resultant increase in pulmonary vascular impedance has emerged as an important underlying driver of pulmonary arterial hypertension (PAH). Given that matrix deposition is central to vascular remodeling, we evaluated the role of the collagen crosslinking enzyme lysyl oxidase like 2 (LOXL2) in this study. Methods and Results: Human pulmonary artery smooth muscle cells (PASMCs) subjected to hypoxia showed increased LOXL2 secretion. LOXL2 activity and expression were markedly higher in primary PASMCs isolated from pulmonary arteries of the rat Sugen 5416 + hypoxia (SuHx) model of severe PH. Similarly, LOXL2 protein and mRNA levels were increased in pulmonary arteries (PA) and lungs of rats with PH (SuHx and monocrotaline (MCT) models). Pulmonary arteries (PAs) isolated from rats with PH exhibited hypercontractility to phenylephrine and attenuated vasorelaxation elicited by acetylcholine, indicating severe endothelial dysfunction. Tensile testing revealed a a significant increase in PA stiffness in PH. Treatment with PAT-1251, a novel small-molecule LOXL2 inhibitor, improved active and passive properties of the PA ex vivo. There was an improvement in right heart function as measured by right ventricular pressure volume loops in-vivo with PAT-1251. Importantly PAT-1251 treatment ameliorated PH, resulting in improved pulmonary artery pressures, right ventricular remodeling, and survival. Conclusion: Hypoxia induced LOXL2 activation is a causal mechanism in pulmonary artery stiffening in PH, as well as pulmonary artery mechanical and functional decline. LOXL2 inhibition with PAT-1251 is a promising approach to improve pulmonary artery pressures, right ventricular elastance, cardiac relaxation, and survival in PAH. New & Noteworthy: Pulmonary arterial stiffening contributes to the progression of PAH and the deterioration of right heart function. This study shows that LOXL2 is upregulated in rat models of PH. LOXL2 inhibition halts pulmonary vascular remodeling and improves PA contractility, endothelial function and improves PA pressure, resulting in prolonged survival. Thus, LOXL2 is an important mediator of PA remodeling and stiffening in PH and a promising target to improve PA pressures and survival in PH.
RESUMEN
We have previously identified mitogen-activated protein kinase-activated protein kinase 2 (MK2) is required for caspase-3 nuclear translocation in the execution of apoptosis; however, little is known of the underlying mechanisms. Therefore, we sought to determine the role of kinase and nonkinase functions of MK2 in promoting nuclear translocation of caspase-3. We identified two non-small cell lung cancer cell lines for use in these experiments based on low MK2 expression. Wild-type, enzymatic and cellular localization mutant MK2 constructs were expressed using adenoviral infection. Cell death was evaluated by flow cytometry. In addition, cell lysates were harvested for protein analyses. Phosphorylation of caspase-3 was determined using two-dimensional gel electrophoresis followed by immunoblotting and in vitro kinase assay. Association between MK2 and caspase-3 was evaluated using proximity-based biotin ligation assays and co-immunoprecipitation. Overexpression of MK2 resulted in nuclear translocation of caspase-3 and caspase-3-mediated apoptosis. MK2 directly phosphorylates caspase-3; however, phosphorylation status of caspase-3 or MK2-dependent phosphorylation of caspase-3 did not alter caspase-3 activity. The enzymatic function of MK2 was dispensable in nuclear translocation of caspase-3. MK2 and caspase-3 associated together and a nonenzymatic function of MK2, chaperoned nuclear trafficking, is required for caspase-3-mediated apoptosis. Taken together, our results demonstrate a nonenzymatic role for MK2 in the nuclear translocation of caspase-3. Furthermore, MK2 may function as a molecular switch in regulating the transition between the cytosolic and nuclear functions of caspase-3.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Apoptosis , Caspasa 3/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Non-small cell lung cancers (NSCLCs) demonstrate intrinsic resistance to cell death, even after chemotherapy. Previous work suggested defective nuclear translocation of active caspase-3 in observed resistance to cell death. We have identified mitogen-activated protein kinase-activated protein kinase 2 (MK2; encoded by the gene MAPKAPK2) is required for caspase-3 nuclear translocation in the execution of apoptosis in endothelial cells. The objective was to determine MK2 expression in NSCLCs and the association between MK2 and clinical outcomes in patients with NSCLC. Clinical and MK2 mRNA data were extracted from two demographically distinct NSCLC clinical cohorts, North American (The Cancer Genome Atlas, TCGA) and East Asian (EA). Tumor responses following first round of chemotherapy were dichotomized as clinical response (complete response, partial response, and stable disease) or progression of disease. Multivariable survival analyses were performed using Cox proportional hazard ratios and Kaplan-Meier curves. NSCLC exhibited lower MK2 expression than SCLC cell lines. In patients, lower tumor MK2 transcript levels were observed in those presenting with late-stage NSCLC. Higher MK2 expression was associated with clinical response following initial chemotherapy and independently associated with improved 2-yr survival in two distinct cohorts, 0.52 (0.28-0.98) and 0.1 (0.01-0.81), TCGA and EA, respectively, even after adjusting for common oncogenic driver mutations. Survival benefit of higher MK2 expression was unique to lung adenocarcinoma when comparing across various cancers. This study implicates MK2 in apoptosis resistance in NSCLC and suggests prognostic value of MK2 transcript levels in patients with lung adenocarcinoma.
Asunto(s)
Adenocarcinoma del Pulmón , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Caspasa 3/uso terapéutico , Células Endoteliales , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genéticaRESUMEN
Exposure to hypoxia, due to high altitude or chronic lung disease, leads to structural changes in the pulmonary vascular wall, including hyperplasia and migration of pulmonary arterial smooth muscle cells (PASMCs). Previous studies showed that hypoxia upregulates the expression of Na+/H+ exchanger isoform 1 (NHE1) and that inhibition or loss of NHE1 prevents hypoxia-induced PASMC migration and proliferation. The exact mechanism by which NHE1 controls PASMC function has not been fully delineated. In fibroblasts, NHE1 has been shown to act as a membrane anchor for actin filaments, via binding of the adaptor protein, ezrin. Thus, in this study, we tested the role of ezrin and NHE1/actin interactions in controlling PASMC function. Using rat PASMCs exposed to in vitro hypoxia (4% O2, 24 h) we found that hypoxic exposure increased phosphorylation (activation) of ezrin, and promoted interactions between NHE1, phosphorylated ezrin and smooth muscle specific α-actin (SMA) as measured via immunoprecipitation and co-localization. Overexpression of wild-type human NHE1 in the absence of hypoxia was sufficient to induce PASMC migration and proliferation, whereas inhibiting ezrin phosphorylation with NSC668394 suppressed NHE1/SMA co-localization and migration in hypoxic PASMCs. Finally, overexpressing a version of human NHE1 in which amino acids were mutated to prevent NHE1/ezrin/SMA interactions was unable to increase PASMC migration and proliferation despite exhibiting normal Na+/H+ exchange activity. From these results, we conclude that hypoxic exposure increases ezrin phosphorylation in PASMCs, leading to enhanced ezrin/NHE1/SMA interaction. We further speculate that these interactions promote anchoring of the actin cytoskeleton to the membrane to facilitate the changes in cell movement and shape required for migration and proliferation.
RESUMEN
Dysregulated metabolism characterizes both animal and human forms of pulmonary hypertension (PH). Enzymes involved in fatty acid metabolism have previously not been assessed in human pulmonary arteries affected by pulmonary arterial hypertension (PAH), and how inhibition of fatty acid oxidation (FAO) may attenuate PH remains unclear. Fatty acid metabolism gene transcription was quantified in laser-dissected pulmonary arteries from 10 explanted lungs with advanced PAH (5 idiopathic, 5 associated with systemic sclerosis), and 5 donors without lung diseases. Effects of oxfenicine, a FAO inhibitor, on female Sugen 5416-chronic hypoxia (SuHx) rats were studied in vivo using right heart catheterization, and ex vivo using perfused lungs and pulmonary artery ring segments. The impact of pharmacologic (oxfenicine) and genetic (carnitine palmitoyltransferase 1a heterozygosity) FAO suppression was additionally probed in mouse models of Schistosoma and hypoxia-induced PH. Potential mechanisms underlying FAO-induced PH pathogenesis were examined by quantifying ATP and mitochondrial mass in oxfenicine-treated SuHx pulmonary arterial cells, and by assessing pulmonary arterial macrophage infiltration with immunohistochemistry. We found upregulated pulmonary arterial transcription of 26 and 13 FAO genes in idiopathic and systemic sclerosis-associated PAH, respectively. In addition to promoting de-remodeling of pulmonary arteries in SuHx rats, oxfenicine attenuated endothelin-1-induced vasoconstriction. FAO inhibition also conferred modest benefit in the two mouse models of PH. Oxfenicine increased mitochondrial mass in cultured rat pulmonary arterial cells, and decreased the density of perivascular macrophage infiltration in pulmonary arteries of treated SuHx rats. In summary, FAO inhibition attenuated experimental PH, and may be beneficial in human PAH.
Asunto(s)
Hipertensión Pulmonar , Hipertensión Arterial Pulmonar , Esclerodermia Sistémica , Animales , Modelos Animales de Enfermedad , Ácidos Grasos/metabolismo , Femenino , Humanos , Hipertensión Pulmonar/patología , Hipoxia/metabolismo , Ratones , Arteria Pulmonar/metabolismo , Ratas , Esclerodermia Sistémica/patología , Remodelación VascularRESUMEN
BACKGROUND: Obstructive sleep apnea, characterized by sleep fragmentation and chronic intermittent hypoxia (CIH), is a risk factor for Alzheimer's disease (AD) progression. Recent epidemiological studies point to CIH as the best predictor of developing cognitive decline and AD in older adults with obstructive sleep apnea. However, the precise underlying mechanisms remain unknown. This study was undertaken to evaluate the effect of CIH on pathological human tau seeding, propagation, and accumulation; cognition; synaptic plasticity; neuronal network excitability; and gene expression profiles in a P301S human mutant tau mouse model of AD and related tauopathies. METHODS: We exposed 4- to 4.5-month-old male P301S and wild-type mice to an 8-week CIH protocol (6-min cycle: 21% O2 to 8% O2 to 21% O2, 80 cycles per 8 hours during daytime) and assessed its effect on tau pathology and various AD-related phenotypic and molecular signatures. Age- and sex-matched P301S and wild-type mice were reared in normoxia (21% O2) as experimental controls. RESULTS: CIH significantly enhanced pathological human tau seeding and spread across connected brain circuitry in P301S mice; it also increased phosphorylated tau load. CIH also exacerbated memory and synaptic plasticity deficits in P301S mice. However, CIH had no effect on seizure susceptibility and network hyperexcitability in these mice. Finally, CIH exacerbated AD-related pathogenic molecular signaling in P301S mice. CONCLUSIONS: CIH-induced increase in pathologic human tau seeding and spread and exacerbation of other AD-related impairments provide new insights into the role of CIH and obstructive sleep apnea in AD pathogenesis.
Asunto(s)
Enfermedad de Alzheimer , Tauopatías , Animales , Modelos Animales de Enfermedad , Hipoxia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Plasticidad NeuronalRESUMEN
Upon sensing a reduction in local oxygen partial pressure, pulmonary vessels constrict, a phenomenon known as hypoxic pulmonary vasoconstriction. Excessive hypoxic pulmonary vasoconstriction can occur with ascent to high altitude and is a contributing factor to the development of high-altitude pulmonary edema. The carbonic anhydrase inhibitor, acetazolamide, attenuates hypoxic pulmonary vasoconstriction through stimulation of alveolar ventilation via modulation of acid-base homeostasis and by direct effects on pulmonary vascular smooth muscle. In pulmonary arterial smooth muscle cells (PASMCs), acetazolamide prevents hypoxia-induced increases in intracellular calcium concentration ([Ca2+]i), although the exact mechanism by which this occurs is unknown. In this study, we explored the effect of acetazolamide on various calcium-handling pathways in PASMCs. Using fluorescent microscopy, we tested whether acetazolamide directly inhibited store-operated calcium entry or calcium release from the sarcoplasmic reticulum, two well-documented sources of hypoxia-induced increases in [Ca2+]i in PASMCs. Acetazolamide had no effect on calcium entry stimulated by store-depletion, nor on calcium release from the sarcoplasmic reticulum induced by either phenylephrine to activate inositol triphosphate receptors or caffeine to activate ryanodine receptors. In contrast, acetazolamide completely prevented Ca2+-release from the sarcoplasmic reticulum induced by hypoxia (4% O2). Since these results suggest the acetazolamide interferes with a mechanism upstream of the inositol triphosphate and ryanodine receptors, we also determined whether acetazolamide might prevent hypoxia-induced changes in reactive oxygen species production. Using roGFP, a ratiometric reactive oxygen species-sensitive fluorescent probe, we found that hypoxia caused a significant increase in reactive oxygen species in PASMCs that was prevented by 100 µM acetazolamide. Together, these results suggest that acetazolamide prevents hypoxia-induced changes in [Ca2+]i by attenuating reactive oxygen species production and subsequent activation of Ca2+-release from sarcoplasmic reticulum stores.