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We investigated the effect of altered extracellular pH, mitochondrial function, and ATP content on development of apoptosis in human pulmonary artery endothelial cells after treatment with staurosporine (STS). STS produced a concentration- and time-dependent increase in caspase-3 activity in pH 7.4 medium that reached a peak at 6 h. The increase in caspase activity was associated with significant DNA fragmentation. Fluorescent imaging of treated monolayers in pH 7.4 medium with Hoechst-33342-propidium iodide demonstrated a large percentage of apoptotic cells (~40%) with no evidence of necrosis. Caspase activity, DNA fragmentation, and percentage of apoptotic cells were reduced after STS treatment in acidic media (pH 7.0 and 6.6). The [Ca.sup.2+] chelator 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM inhibited STS-induced apoptosis, whereas the rise in intracellular [Ca.sup.2+] concentration in STS-treated cells in pH 7.4 medium was reduced in pH 7.0 medium. These results suggest that one mechanism for inhibitory effects of acidosis may be a pH-induced alteration in [Ca.sup.2+] signaling. Treatment with STS in the presence of oligomycin (10 [micro]M), an inhibitor of the mitochondrial [F.sub.0][F.sub.1]-ATPase, in glucose-free media abolished caspase activation and DNA fragmentation in association with severe ATP depletion (~2% of control cells). Imaging demonstrated a change in the mode of cell death from apoptosis to necrosis under these conditions. This change was linked to the level of ATP depletion, because STS treatment in the absence of glucose or the presence of oligomycin in media with glucose still leads to apoptosis in the presence of only moderate ATP depletion. These results demonstrate that pH, mitochondrial function, and ATP supply are important variables that regulate STS-induced apoptosis in human pulmonary artery endothelial cells. caspases; extracellular pH; mitochondria

Analysis of the effects of oxidant stress on the Na+/H+ antiport activity in monolayers exposed in the oxidant, tert-butyl hydroperoxide (t-BOOH), shows that oxidant stress attenuates Na+/H+ antiport activity in bovine pulmonary artery endothelial cells. Na+/H+ antiport regulates intracellular pH in these cells and t-BOOH decreases the rate of recovery from acidosis. The intracellular glutathione in these cells enable the maintenance of Na+/H+ antiport functions.

Cutaia, M., K. Tollefson, J. Kroczynski, N. Parks, and S. Rounds. Role of the Na/H antiport in pH-dependent cell death in pulmonary artery endothelial cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 278: L536-L544, 2000.--We investigated the role of intracellular pH ([pH.sub.i]) and Na/H exchange in cell death in human pulmonary artery endothelial cells (HPAEC) following a metabolic insult (inhibition-oxidative phosphorylation, glycolysis). Metabolic inhibition in medium at pH 7.4 decreased viability (0-15% live cells) over 6 h. Cell death was attenuated by maneuvers that decreased [pH.sub.i] and inhibited Na/H exchange (acidosis, Na/H antiport inhibitors). In contrast, cell death was potentiated by maneuvers that elevated [pH.sub.i] or increased Na/H exchange (monensin, phorbol ester treatment) before the insult. HPAEC demonstrated a biphasic [pH.sub.i] response following a metabolic insult. An initial decrease in [pH.sub.i] was followed by a return to baseline over 60 min. Maneuvers that protected HPAEC and inhibited Na/H exchange (acidosis, [Na.sup.+]-free medium, antiport inhibitors) altered this pattern, [pH.sub.i] decreased, but no recovery was observed, suggesting that the return of [pH.sub.i] to normal was mediated by antiport activation. Although Na/H antiport activity was reduced (55-60% of control) following a metabolic insult, the cells still demonstrated active Na/H exchange despite significant ATP depletion. Phorbol ester pretreatment, which potentiated cell death, increased Na/H antiport activity above the level observed in monolayers subjected to a metabolic insult alone. These results demonstrate that HPAEC undergo a pH-dependent loss of viability linked to active Na/H exchange following a metabolic insult. Potentiation of cell death with phorbol ester treatment suggests that this cell death pathway involves protein kinase C-mediated phosphorylation events. sodium/hydrogen exchange; intracellular pH; intracellular cytosolic calcium

Looks at the temporal pattern of hypoxemia in chronic obstructive pulmonary disease. Definition of the temporal profile of oxygen saturation during activities of daily living; Effectiveness of long-term oxygen therapy in improving outcomes in patients with advanced lung disease.

Background: Food insecurity is a major public health concern in the United States, particularly for pregnant and postpartum individuals. In 2020, ∼13.8 million (10.5%) U.S. households experienced food insecurity. However, the association between food security and pregnancy outcomes in the United States is poorly understood., Purpose: The purpose of this review was to critically appraise the state of the evidence related to food insecurity as a determinant of health within the context of pregnancy in the United States. We also explored the relationship between food insecurity and pregnancy outcomes., Methods: PubMed, CINAHL, Web of Science, and Food and Nutrition Science databases were used. The inclusion criteria were peer-reviewed studies about food (in)security, position articles from professional organizations, and policy articles about pregnancy outcomes and breastfeeding practices. Studies conducted outside of the United States and those without an adequate definition of food (in)security were excluded. Neonatal health outcomes were also excluded. Included articles were critically appraised with the STROBE and Critical Appraisal Skills Program checklists., Results: Nineteen studies met the inclusion criteria. Inconsistencies exist in defining and measuring household food (in)security. Pregnant and postpartum people experienced several adverse physiological and psychological outcomes that impact pregnancy compared with those who do not. Intersections between neighborhood conditions and other economic hardships were identified. Findings regarding the impact of food insecurity on breastfeeding behaviors were mixed, but generally food insecurity was not associated with poor breastfeeding outcomes in adjusted models., Conclusion: Inconsistencies in definitions and measures of food security limit definitive conclusions. There is a need for standardizing definitions and measures of food insecurity, as well as a heightened awareness and policy change to alleviate experiences of food insecurity., Competing Interests: No competing financial interests exist., (© Veronica C. Pasha et al., 2024; Published by Mary Ann Liebert, Inc.)

Background: The COVID-19 pandemic had its first peak in the United States between April and July of 2020, with incidence and prevalence rates of the virus the greatest in the northeastern coast of the country. At the time of study implementation, there were few studies capturing the perspectives of nurses working the frontlines of the pandemic in any setting as research output in the United States focused largely on treating the disease., Objective: The purpose of this study was to capture the perspectives of nurses in the United States working the frontlines of the COVID-19 pandemic's first wave. We were specifically interested in examining the impact of the pandemic on nurses' roles, professional relationships, and the organizational cultures of their employers., Design: We conducted an online qualitative study with a pragmatic design to capture the perspectives of nurses working during the first wave of the United States COVID-19 pandemic. Through social networking recruitment, frontline nurses from across the country were invited to participate. Participants provided long form, text-based responses to four questions designed to capture their experiences. A combination of Latent Dirichlet Allocation--a natural language processing technique--along with traditional summative content analysis techniques were used to analyze the data., Setting: The United States during the COVID-19 pandemic's first wave between May and July of 2020., Results: A total of 318 nurses participated from 29 out of 50 states, with 242 fully completing all questions. Findings suggested that the place of work mattered significantly in terms of the frontline working experience. It influenced role changes, risk assumption, interprofessional teamwork experiences, and ultimately, likelihood to leave their jobs or the profession altogether. Organizational culture and its influence on pandemic response implementation was a critical feature of their experiences., Conclusions: Findings suggest that organizational performance during the pandemic may be reflected in nursing workforce retention as the risk for workforce attrition appears high. It was also clear from the reports that nurses appear to have assumed higher occupational risks during the pandemic when compared to other providers. The 2020 data from this study also offered a number of signals about potential threats to the stability and sustainability of the US nursing workforce that are now manifesting. The findings underscore the importance of conducting health workforce research during a crisis in order to discern the signals of future problems or for long-term crisis response., Tweetable Abstract: Healthcare leaders made the difference for nurses during the pandemic. How many nurses leave their employer in the next year will tell you who was good, who wasn't., Competing Interests: Declaration of Competing Interest The authors report no conflicts of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Endobronchial Ultrasound-guided Transbronchial Needle Aspiration (EBUS-TBNA) has revolutionized the diagnostic approach to mediastinal diseases. Lesions located lateral to the pulmonary artery (trans-PA, Station 5), or in the lumen of the PA (intra-PA) are in the 'blind-spot' of EBUS., Objectives: We describe a case series where EBUS guided trans-pulmonary or intra-pulmonary aspiration (EBUS-TIPNA) was used for diagnosis., Methods: Retrospective analysis of 10 patients who had EBUS-TIPNA over 3 years in 2 centres. The inclusion criteria included patients with station 5 lesions, or intrapulmonary artery lesions, where no other option was possible., Results: The study included 4 males and 6 females, mean age 52 years, with 7 trans-PA and 3 intra-PA lesions. Adequacy was seen in 10/10, and a definitive diagnosis was made in 9/10 patients. There were no procedure-related complications., Conclusion: EBUS-TIPNA can be done as a safe and successful procedure and adds to the armamentarium of Convex Probe-EBUS (CP-EBUS), in carefully selected patients., (© 2018 John Wiley & Sons Ltd.)

Background: Prolonged air leak (PAL) is a life-threatening condition that can present either as bronchopleural fistula, or alveolar-pleural fistula (APF). Although numerous bronchoscopic treatments are described, they are either expensive, not widely available in the developing world or have limited success. We describe our experience with a novel customized endobronchial silicone blocker (CESB) for PAL treatment., Methods: This is a retrospective study of PAL patients who underwent CESB placement. The air leak was localized using a balloon occlusion test. The CESB was uniquely designed by molding silicone stent pieces into a conical shape, deployed with rigid bronchoscopy into the appropriate segment, and reinforced with cyanoacrylate glue to prevent migration. In patients with APF, pleurodesis was performed after leak resolution to prevent recurrence. Following this, the CESB was removed after 6 weeks., Results: Forty-nine CESBs were placed in 31 patients (25 male individuals, 6 female individuals) with mean age of 49.7±19.7 years. The PALs included APF (n=16), bronchopleural fistula (n=14), and airway-mediastinal fistula (n=1). The average diameter of the CESB used was 7.9±2.9 mm. There was resolution of the PAL in 26 of 31 patients (84%). The CESB migrated in 5 patients with no adverse events. Pleurodesis was performed in 13 of 16 patients with APF, to prevent recurrence. No other significant complications were observed., Conclusions: CESBs represent a safe, effective, and innovative approach in the management of PAL. They should be considered in patients who are not surgical candidates, fail surgery, or those who have a recurrence following surgery.

Background: Current histopathological classification and TNM staging have limited accuracy in predicting survival and stratifying patients for appropriate treatment. The goal of the study is to determine whether the expression pattern of functionally important regulatory proteins can add additional values for more accurate classification and prognostication of non-small lung cancer (NSCLC)., Methods: The expression of 108 proteins and phosphoproteins in 30 paired NSCLC samples were assessed using Protein Pathway Array (PPA). The differentially expressed proteins were further confirmed using a tissue microarray (TMA) containing 94 NSCLC samples and were correlated with clinical data and survival., Results: Twelve of 108 proteins (p-CREB(Ser133), p-ERK1/2(Thr202/Tyr204), Cyclin B1, p-PDK1(Ser241), CDK4, CDK2, HSP90, CDC2p34, β-catenin, EGFR, XIAP and PCNA) were selected to build the predictor to classify normal and tumor samples with 97% accuracy. Five proteins (CDC2p34, HSP90, XIAP, CDK4 and CREB) were confirmed to be differentially expressed between NSCLC (n=94) and benign lung tumor (n=19). Over-expression of CDK4 and HSP90 in tumors correlated with a favorable overall survival in all NSCLC patients and the over-expression of p-CREB(Ser133) and CREB in NSCLC correlated with a favorable survival in smokers and those with squamous cell carcinoma, respectively. Finally, the four proteins (CDK4, HSP90, p-CREB and CREB) were used to calculate the risk score of each individual patient with NSCLC to predict survival., Conclusion: In summary, our data demonstrated a broad disturbance of functionally important regulatory proteins in NSCLC and some of these can be selected as clinically useful biomarkers for diagnosis, classification and prognosis.

Dedicated linear endobronchial ultrasound (EBUS) is an established modality in sampling central pulmonary lesions, mediastinal and hilar lymph nodes to diagnose and stage lung malignancies as well as evaluate a number of inflammatory conditions. In addition, power Doppler modality of EBUS allows for vascular evaluation. We describe a case where EBUS visualized a submucosal lesion within a vascular malformation, which was eventually proven to be a carcinoid tumor. Computed tomographic scan of the chest failed to differentiate the 2 structures and bronchoscopy showed no endobronchial component. This case underscores the added diagnostic capabilities of endobronchial Doppler ultrasonography. We suggest considering EBUS in investigating central opacities associated with vascular features within the lung parenchyma when technically feasible.

Sphingomyelin (SM), a major sphingolipid in the lipid raft microdomains of the cell membrane, is synthesized by plasma membrane-bound sphingomyelin synthase 2 (SMS2). SMS2 is required for the maintenance of plasma membrane microdomain fluidity and receptor-mediated responses to inflammation in macrophages. However, the exact mechanism of SMS2 activation in endothelial barrier disruption and lung injury is not fully understood. To define the role of SMS activation in lung injury, we hypothesized that the inhibition of SM synthesis may provide protection against acute lung injury (ALI) by preserving endothelial barrier function. Using SMS2-silencing RNA (siRNA) treatment in human pulmonary endothelial cells (HPAECs) and tricyclodecan-9-yl-xanthogenate (D609), a competitive inhibitor of SMS, and phosphatidylcholine-specific phospholipase C in a murine model of bacterial LPS injury, we studied the role of sphingomyelin synthesis in ALI. Results show that pretreating mice with D609 significantly attenuated LPS-induced lung injury, as measured by a significant decrease in wet to dry ratio, bronchoalveolar lavage fluid cell and protein counts, and myeloperoxidase activity in lung tissue. Similarly, LPS-induced endothelial barrier disruption was significantly reduced in HPAECs pretreated with D609 or SMS2 siRNA, as demonstrated by an increase in paracellular integrity on an FITC-dextran assay, by the inhibition of LPS-induced stress fibers, and by the formation of cortical actin rings and lamellipodia at the periphery. These results indicate that D609 attenuates LPS-mediated endothelial barrier dysfunction and lung injury in mice through inhibition of SMS, suggesting a novel and essential role of SMS inhibition in modulating endothelial barrier integrity via actin cytoskeletal activation, with a potential therapeutic role in ALI.

Endobronchial stents have been used occasionally to treat benign conditions such as tracheobronchomalacia (TBM). This report describes a unique case of a patient with crescentic TBM in whom Dynamic Y stent was placed on 2 separate occasions to control symptoms and resulted in identical posterior wall stent fractures within a year of stent placement, both times. A silicone Y stent was substituted for the dynamic stent, and it has been effective in controlling symptoms for 9 months without complications. A literature review of cases of fractured Dynamic Y stents is made and factors affecting the choice of stent type for crescentic TBM are explored.

Chronic Obstructive Pulmonary Disease (COPD) frequently has a significant impact on patients' everyday activity. Because of this, accurate measurement of daily activity is of particular interest. Although accelerometers are an objective means of measuring daily activity, these devices sense vibrations and erroneously score motor vehicle travel (MVT) as moderate physical activity. It is the objective of this study to develop a new method to analyze accelerometry data that would accurately classify MVT as non-acceleration, or sitting/standing. As sitting/standing has a different pattern of count-to-count variability than walking, we hypothesized that a rolling standard deviation (RSD), which is a measurement of volatility in the data, would more accurately classify periods of MVT than analysis based on activity counts alone. Twenty-two subjects with COPD were studied. A training set of 15% of the dataset was used to establish an RSD-threshold during MVT based on the upper 95%-confidence interval. The accuracy of the RSD thresholds were tested and presented as sensitivity, specificity and receiver operating curves. Results demonstrated high sensitivity and specificity suggesting that the RSD not only accurately classified MVT, but had a low rate of misclassification. The RSD analysis scored more MVT as sitting/standing than assessment by VMU alone. The accuracy of accelerometers to define the profile of daily activity in sedentary populations, such as those with COPD, is greatly improved.

Background: The frequency of oxygen desaturation during daily activities in chronic obstructive pulmonary disease (COPD) is poorly defined. The BODE index predicts survival in COPD. The purpose of this study was to determine the relationship between BODE scores and oxygen saturation during daily activities., Methods: Seventy-eight patients with COPD (FEV(1) = 37%) underwent ambulatory oximetry and activity monitoring. We defined four activity categories: Walking, Slow-Intermittent-Walking (SIW), Active-Not-Walking (ANW), and Rest. We quantified oxygen desaturation during activity using a desaturation index (DSI = % time oxygen saturation <90%). BODE scores were categorized into three groups: group I (0-3), II (4-6), and III (7-10)., Results: The percentage of patients demonstrating oxygen desaturation (DSI ≥ 10%) during each activity was 55% for Walking, 35% for SIW, 15% for ANW, and 28% for Rest. There was a strong association between BODE score and desaturation for Walking and SIW. During Walking, 21, 44, and 86% of patients in BODE groups I, II, and III, respectively, demonstrated desaturation. The DSI for Walking and SIW was increased in patients in BODE groups II and III compared to group I (P < 0.006, P < 0.007, respectively). BODE score was also linked to long-term oxygen therapy (LTOT) usage; the majority of patients not on LTOT (89%) had a BODE score <7. The majority of patients on LTOT (84%) demonstrated desaturation during Walking, but 42% of patients not on LTOT also demonstrated desaturation. In this subgroup of patients not on LTOT, all patients with a BODE score ≥ 7 demonstrated desaturation during Walking., Conclusions: The link between the BODE index and oxygen desaturation during daily activities suggests that desaturation is linked to disease severity. Our data suggest that patients with a BODE score ≥ 7 should be evaluated for desaturation during daily activities. Use of the BODE index to screen for exertional desaturation may have value as a tool that can lead to the earlier identification of patients who may be candidates for LTOT.

Sphingomyelin synthase (SMS) catalyzes the synthesis of sphingomyelin (SM) and is required for maintenance of plasma membrane microdomain fluidity. Of the two isoforms of mammalian SMS, SMS1 is mostly present in the trans-Golgi apparatus, whereas SMS2 is predominantly found at the plasma membrane. SMS2 has a role in receptor mediated response to inflammation in macrophages, however, the role of SMS2 in vascular permeability, pulmonary edema, and lung injury have not been investigated. To define the role of SMS activation in lung injury, we utilized a lipopolysaccharide (LPS)-induced lung edema model. SMS activity was measured and correlated with the severity of lung injury. Within 4 h of LPS treatment, SMS activity was increased significantly and remained upregulated up to 24 h. Comparison of LPS-induced lung injury in SMS2 knockout (SMS2(-/-)) and wild-type littermate control mice showed that inflammation, cytokine induction, and lung injury were significantly inhibited in SMS2(-/-) mice. Our results suggest that a deficiency of SMS2 can diminish the extent of pulmonary edema and lung injury. Furthermore, we show that depletion of SMS2 was sufficient to decrease MAP kinase-JNK activation, severity of LPS-induced pulmonary neutrophil influx, and inflammation, suggesting a novel role of SMS2 activation in lung injury.

Malignant primary tracheal tumors are extremely rare. The most common malignant primary tracheal tumors are squamous cell carcinoma and adenoid cystic carcinomas. In this brief report, we describe a patient who presented with a primary papillary squamous cell carcinoma in-situ at multiple areas in the trachea with a significant airway obstruction. Our case was successfully managed using a combination of electrocautery and argon photocoagulation for endotracheal ablation of the tumor and adjuvant external beam radiotherapy.

Chronic obstructive pulmonary disease (COPD) is associated with significant morbidity and mortality. Its possible association with obstructive sleep apnea is a major cause of concern for clinicians. As the prevalence of both COPD and sleep apnea continues to rise, further investigation of this interaction is needed. In addition, COPD patients are at risk for hypoventilation during sleep due to the underlying respiratory dysfunction. In this study, 13 COPD subjects and 13 non-COPD control subjects were compared for the presence and severity of obstructive sleep apnea and nocturnal hypoventilation. All 26 subjects had presented to a sleep clinic and showed no signs of daytime hypoxemia. After matching for BMI and age, COPD subjects had a similar prevalence of sleep apnea with a lower degree of severity compared to the control subjects. However, less severe events, such as RERA, occurred at similar rates between the two groups. There was no significant difference between groups in the magnitude of oxyhemoglobin desaturation during sleep. Interestingly, severity and presence of nocturnal hypoxemia correlated with that of sleep apnea in the control group, but not in the COPD subjects. In conclusion, COPD without daytime hypoxemia was not a risk factor for sleep apnea or nocturnal hypoventilation in this study.

Post-obstructive pneumonia is the result of airway obstruction, commonly due to lung cancer. The majority of patients with lung cancer are non-operable and incurable at initial presentation. The clinical course of these patients is steadily downhill with complicating events such as post-obstructive pneumonia, respiratory failure, pleural effusion, and severe debilitation. Non-invasive conventional options for post-obstructive pneumonia include broad-spectrum antibiotics, combined with attempts at relieving the obstruction using radiation therapy. There is scant literature on the natural course of post-obstructive pneumonia, disease resolution, and optimal treatment. With the recent surge in technologic advances in interventional pulmonology, multiple airway recanalizaton options are now available. In this paper, we describe the causes of post-obstructive pneumonia, and the role of interventional pulmonary procedures in -establishing an open airway.

Tuberculosis (TB) persists as a worldwide health crisis. Nearly one third of the world's population is infected with TB and it remains a critical public health issue. In the past two decades, international efforts have focused on improving identification of those infected with TB as well as supporting molecular microbiologists in developing better diagnostic techniques. Furthermore, implementation of programs such as directly observed therapy have assisted more patients in receiving and completing therapy. This review aims to identify some of the more relevant findings in the field of TB over the past few years with a special emphasis on TB in urban communities.

Background: The benefits of long-term oxygen supplementation in COPD patients with hypoxemia are well established. The standard approach to prescribing oxygen uses a static assessment of oxygen requirements in a hospital or clinic setting. The assumption behind this approach is that patients will maintain a "therapeutic" hemoglobin oxygen saturation (SpO2) in the outpatient setting. We questioned the validity of this assumption, and hypothesized that many patients may demonstrate significant oxygen desaturation during normal activities of daily living. STUDY DESIGN, METHODS, AND MEASUREMENTS: We determined if oxygen supplementation maintained a therapeutic SpO2 level in patients with COPD (n = 27), using the technique of ambulatory oximetry monitoring (AOM). AOM consisted of using a portable oximeter to monitor SpO2, pulse rate, and patient activity while patients were engaged in normal activities of daily living over an extended time period (approximately 18 h). The portable oximeter collected and stored these data every 15 s over the monitored time period. Each AOM recording was manually scored for desaturation events and other key variables, including average SpO2 over the monitoring period, the average number of desaturation events per hour, and the percentage of monitored time deleted secondary to artifacts., Setting: University-affiliated Veterans Affairs Medical Center., Patients: All subjects were patients with stable COPD with no recent history of hospitalization or exacerbation of their lung disease., Results: This cohort of patients demonstrated a surprising frequency of desaturation below the recommended target SpO2 value (90%), which averaged approximately 25% of AOM recording time. There was wide variability among patients in the percentage of time SpO2 was below the target value (range, 3 to 67% of AOM recording time). Motion artifact on the AOM recordings was not a major problem; an average of 8% of the recording time was deleted secondary to artifacts in this patient cohort., Conclusions: The results demonstrate that AOM is feasible and accurate with an acceptable level of motion artifact. These results also suggest that the standard approach for prescribing oxygen may lead to subtherapeutic SpO2 values in the outpatient setting. AOM holds promise as a tool to monitor the adequacy of oxygen prescriptions in the outpatient setting in patients with lung disease.

Little is known about the effects of prolonged hypoxic exposure on membrane ion transport activity. The Na+/H+ antiport is an ion transport site that regulates intracellular pH in mammalian cells. We determined the effect of prolonged hypoxic exposure on human pulmonary arterial endothelial cell antiport activity, gene expression, and localization. Monolayers were incubated under hypoxic or normoxic conditions for 72 h. Antiport activity was determined as the rate of recovery from intracellular acidosis. Antiport isoform identification and gene expression were determined with RT-PCR and Northern and Western blots. Antiport localization and F-actin cytoskeleton organization were defined with immunofluorescent staining. Prolonged hypoxic exposure decreased antiport activity, with no change in cell viability compared with normoxic control cells. One antiport isoform [Na+/H+ exchanger isoform (NHE) 1] that was localized to the basolateral cell surface was present in human pulmonary arterial endothelial cells. Hypoxic exposure had no effect on NHE1 mRNA transcript expression, but NHE1 protein expression was upregulated. Immunofluorescent staining demonstrated a significant alteration of the F-actin cytoskeleton after hypoxic exposure but no change in NHE1 localization. These results demonstrate that the decrease in NHE1 activity after prolonged hypoxic exposure is not related to altered gene expression. The change in NHE1 activity may have important consequences for vascular function.

Apoptosis may be important in the exacerbation of endothelial cell injury or limitation of endothelial cell proliferation. We have found that extracellular ATP (exATP) and adenosine cause endothelial apoptosis and that the development of apoptosis is linked to intracellular metabolism of adenosine [Dawicki, D. D., D. Chatterjee, J. Wyche, and S. Rounds. Am. J. Physiol. 273 (Lung Cell Mol. Physiol. 17): L485-L494, 1997]. In the present study, we investigated the mechanism of this effect. We found that exATP, adenosine, and the S-adenosyl-L-homocysteine (SAH) hydrolase inhibitor MDL-28842 caused apoptosis and decreased the ratio of S-adenosyl-L-methionine to SAH compared with untreated control cells. Using release of soluble [3H]thymidine as a measure of DNA fragmentation, we found that the effect of adenosine on soluble DNA release was potentiated by coincubation with homocysteine. These results suggest that the mechanism of exATP- and adenosine-induced endothelial cell apoptosis involves inhibition of SAH hydrolase. exATP-induced apoptosis was enhanced by an inhibitor of adenosine deaminase, whereas exogenous adenosine-induced apoptosis was partially inhibited by an adenosine deaminase inhibitor. These results suggest that adenosine deaminase may also be involved in the mechanism of adenosine-induced endothelial cell apoptosis. Adenosine and MDL-28842 caused intracellular acidosis as assessed with the fluorescent probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. The cell-permeant base chloroquine prevented adenosine-induced acidosis but not apoptosis. Thus, although intracellular acidosis is associated with adenosine-induced apoptosis, it is not necessary for this effect. We speculate that exATP- and adenosine-induced endothelial cell apoptosis may be due to an inhibition of methyltransferase(s) activity. Purine-induced endothelial cell apoptosis may be important in limiting endothelial cell proliferation after vascular injury.