Charles A. Downs

β-Adrenergic agonists differentially regulate highly selective and nonselective epithelial sodium channels to promote alveolar fluid clearance in vivo

β-Adrenergic receptors (β-AR) increase epithelial sodium channel (ENaC) activity to promote lung fluid clearance. However, the effect of selective β-AR agonist on highly selective cation (HSC) channels or nonselective cation (NSC) channels in alveolar type 1 (T1) and type 2 (T2) cells is unknown. We hypothesized that stimulation with β(1)-AR agonist (denopamine) or β(2)-AR agonist (terbutaline) would increase HSC and/or NSC channel activity in alveolar epithelial cells. We performed single-channel measurements from T1 and T2 cells accessed from rat lung slices. Terbutaline (20 μM) increased HSC ENaC activity (open probability, NP(o)) in T1 (from 0.96 ± 0.61 to 1.25 ± 0.71, n = 5, P <0.05) and T2 cells (from 0.28 ± 0.14 to 1.0 ± 0.30, n = 8, P = 0.02). Denopamine (20 μM) increased NSC NP(o) in T1 cells (from 0.34 ± 0.09 to 0.63 ± 0.14, n = 7, P = 0.02) and in T2 cells (from 0.47 ± 0.09 to 0.68 ± 0.10, P = 0.004). In vivo X-ray imaging of lung fluid clearance and ICI 118,551 selective inhibition of β(2)-ARs confirmed patch-clamp findings. cAMP concentrations increased following treatment with denopamine or terbutaline (n = 3, P < 0.002). The effects of systemic (intraperitoneal, IP) and local (intratracheal, IT) modes of delivery on lung fluid clearance were assessed. IT delivery of denopamine promoted alveolar flooding, whereas IP delivery promoted delayed fluid clearance. In summary, β-AR agonists differentially regulate HSC and NSC in T1 and T2 cells to promote lung fluid clearance in vivo, and the mode of drug delivery is critical for maximizing β-AR agonist efficacy.

Stress and Inflammation: A Biobehavioral Approach for Nursing Research:

Despite known advantages, the use of biobehavioral approaches in nursing research remains limited. The purposes of this article are to (1) present applications of stress and inflammation in various health conditions as examples of biobehavioral concepts and (2) stimulate similar applications of biobehavioral concepts in future nursing research. Under a biobehavioral conceptual framework, studies on stress and selective inflammatory biomarkers in cardiovascular, cancer, and pulmonary health are reviewed and summarized. Inflammation underlies many diseases, and stress is a significant source of increased inflammation. Biobehavioral concepts of stress and inflammation are highly relevant to nursing research concerned with health-related issues. Diverse biobehavioral concepts are readily applicable and should be utilized in nursing research with children and adults. To stimulate further biobehavioral research, more training and resources for nurse scientists, more unified conceptual definitions and biobehavioral conceptual frameworks, rigorous and expanded methodologies, and more collaboration are essential.

Receptor for Advanced Glycation End-Products Regulates Lung Fluid Balance via Protein Kinase C–gp91phox Signaling to Epithelial Sodium Channels

The receptor for advanced glycation end-products (RAGE), a multiligand member of the Ig family, may play a crucial role in the regulation of lung fluid balance. We quantified soluble RAGE (sRAGE), a decoy isoform, and advanced glycation end-products (AGEs) from the bronchoalveolar lavage fluid of smokers and nonsmokers, and tested the hypothesis that AGEs regulate lung fluid balance through protein kinase C (PKC)-gp91(phox) signaling to the epithelial sodium channel (ENaC). Human bronchoalveolar lavage samples from smokers showed increased AGEs (9.02 ± 3.03 μg versus 2.48 ± 0.53 μg), lower sRAGE (1,205 ± 292 pg/ml versus 1,910 ± 263 pg/ml), and lower volume(s) of epithelial lining fluid (97 ± 14 ml versus 133 ± 17 ml). sRAGE levels did not predict ELF volumes in nonsmokers; however, in smokers, higher volumes of ELF were predicted with higher levels of sRAGE. Single-channel patch clamp analysis of rat alveolar epithelial type 1 cells showed that AGEs increased ENaC activity measured as the product of the number of channels (N) and the open probability (Po) (NPo) from 0.19 ± 0.08 to 0.83 ± 0.22 (P = 0.017) and the subsequent addition of 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-N-oxyl decreased ENaC NPo to 0.15 ± 0.07 (P = 0.01). In type 2 cells, human AGEs increased ENaC NPo from 0.12 ± 0.05 to 0.53 ± 0.16 (P = 0.025) and the addition of 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-N-oxyl decreased ENaC NPo to 0.10 ± 0.03 (P = 0.013). Using molecular and biochemical techniques, we observed that inhibition of RAGE and PKC activity attenuated AGE-induced activation of ENaC. AGEs induced phosphorylation of p47(phox) and increased gp91(phox)-dependent reactive oxygen species production, a response that was abrogated with RAGE or PKC inhibition. Finally, tracheal instillation of AGEs promoted clearance of lung fluid, whereas concomitant inhibition of RAGE, PKC, and gp91(phox) abrogated the response.

Acute Effects of Cigarette Smoke Extract on Alveolar Epithelial Sodium Channel Activity and Lung Fluid Clearance

Cigarette smoke contains high levels of reactive species. Moreover, cigarette smoke can induce cellular production of oxidants. The purpose of this study was to determine the effect of cigarette smoke extract (CSE)-derived oxidants on epithelial sodium channel (ENaC) activity in alveolar type 1 (T1) and type 2 (T2) cells and to measure corresponding rates of fluid clearance in mice receiving a tracheal instillation of CSE. Single-channel patch clamp analysis of T1 and T2 cells demonstrate that CSE exposure increases ENaC activity (NPo), measured as the product of the number of channels (N) and a channels open probability (Po), from 0.17 ± 0.07 to 0.34 ± 0.10 (n = 9; P = 0.04) in T1 cells. In T2 cells, CSE increased NPo from 0.08 ± 0.03 to 0.35 ± 0.10 (n = 9; P = 0.02). In both cell types, addition of tetramethylpiperidine and glutathione attenuated CSE-induced increases in ENaC NPo. Biotinylation and cycloheximide chase assays indicate that CSE-derived ROS increases channel activity, in part, by maintaining cell surface expression of the α-ENaC subunit. In vivo studies show that tracheal instillation of CSE promoted alveolar fluid clearance after 105 minutes compared with vehicle control (n = 10/group; P < 0.05).

Age-related differences in cigarette smoke extract-induced H2O2 production by lung endothelial cells

Cigarette smoke causes oxidative stress in the lung resulting in injury and disease. The purpose of this study was to determine if there were age-related differences in cigarette smoke extract (CSE)-induced production of reactive species in single and co-cultures of alveolar epithelial type I (AT I) cells and microvascular endothelial cells harvested from the lungs (MVECLs) of neonatal, young and old male Fischer 344 rats. Cultures of AT I cells and MVECLs grown separately (single culture) and together (co-culture) were exposed to CSE (1, 10, 50, 100%). Cultures were assayed for the production of intracellular reactive oxygen species (ROS), hydroxyl radical (OH), peroxynitrite (ONOO(-)), nitric oxide (NO) and extracellular hydrogen peroxide (H(2)O(2)). Single and co-cultures of AT I cells and MVECLs from all three ages produced minimal intracellular ROS in response to CSE. All ages of MVECLs produced H(2)O(2) in response to CSE, but young MVECLs produced significantly less H(2)O(2) compared to neonatal and old MVECLs. Interestingly, when grown as a co-culture with age-matched AT I cells, neonatal and old MVECLs demonstrated ~50% reduction in H(2)O(2) production in response to CSE. However, H(2)O(2) production in young MVECLs grown as a co-culture with young AT I cells did not change with CSE exposure. To begin investigating for a potential mechanism to explain the reduction in H(2)O(2) production in the co-cultures, we evaluated single and co-cultures for extracellular total antioxidant capacity. We also performed gene expression profiling specific to oxidant and anti-oxidant pathways. The total antioxidant capacity of the AT I cell supernatant was ~5 times greater than that of the MVECLs, and when grown as a co-culture and exposed to CSE (≥ 10%), the total antioxidant capacity of the supernatant was reduced by ~50%. There were no age-related differences in total antioxidant capacity of the cell supernatants. Gene expression profiling found eight genes to be significantly up-regulated or down-regulated. This is the first study to describe age-related differences in MVECLs exposed to CSE.

Functional assessment of chronic obstructive pulmonary disease.

Purpose: To describe available methods for assessing functional capacity in persons with chronic obstructive pulmonary disease (COPD). Data sources: An extensive literature review is used to provide pertinent information. Conclusions: COPD disease affects millions of Americans and is physically and psychologically distressing. The hallmark of chronic obstructive pulmonary disease is irreversible airflow limitation and dyspnea. Dyspnea is a major contributor to decreased exercise capacity and functional status in this population. Understanding the methods to complete a functional assessment is important for all practitioners caring for this population. Implications for practice: This paper provides an overview of current methods used to assess functional status, including pulmonary function testing, exercise testing, and anthropomorphic and self-report measurements. In addition, there is discussion of the indications and contraindications for exercise testing in chronic obstructive pulmonary disease and the clinical significance of performing a global composite of functional ability.Purpose: To describe available methods for assessing functional capacity in persons with chronic obstructive pulmonary disease (COPD). Data sources: An extensive literature review is used to provide pertinent information. Conclusions: COPD disease affects millions of Americans and is physically and psychologically distressing. The hallmark of chronic obstructive pulmonary disease is irreversible airflow limitation and dyspnea. Dyspnea is a major contributor to decreased exercise capacity and functional status in this population. Understanding the methods to complete a functional assessment is important for all practitioners caring for this population. Implications for practice: This paper provides an overview of current methods used to assess functional status, including pulmonary function testing, exercise testing, and anthropomorphic and self‐report measurements. In addition, there is discussion of the indications and contraindications for exercise testing in chronic obstructive pulmonary disease and the clinical significance of performing a global composite of functional ability. Disclosure The author reports no competing interests. To obtain CE credit for this activity, go to and click on the CE Center. Locate the listing for this article and complete the post‐test. Follow the instructions to print your CE certificate.

Beyond the PhD: Putting the Right Tools in Your Research Toolbox

Postdoctoral training is vital to a successful career for nurse researchers with a biological or biobehavioral focus. Such training provides structured time to devote to gaining substantive knowledge, expanding one’s biological-methods repertoire, and writing grants. However, for unknown reasons, relatively few nurses pursue postdoctoral training. A few plausible explanations include a near critical shortage of nursing faculty coupled with an aging population in need of health care, a lack of available mentoring for predoctoral students to pursue postdoctoral training, and the difficulty of navigating the process of finding and choosing the right match for a postdoctoral experience. The purposes of this article are to provide a rationale for choosing postdoctoral training, review common fellowship opportunities, and discuss the process of finding and choosing the right match for postdoctoral training. The authors provide two prospective plans for postdoctoral training and include a plan for staying on track during the postdoctoral experience.

Formaldehyde exposure during pregnancy.

Background:Pregnancy is a particularly vulnerable time for exposure to indoor air pollutants, such as formaldehyde (FA), which is linked to spontaneous abortion, congenital malformations, and premature birth. Purpose:To determine personal exposure to FA during pregnancy, and to identify the relationship between FA exposure levels and potential residential sources of FA. Study Design and Methods:The study sample consisted of 140 pregnant women recruited from obstetrical clinics in Huntsville, Alabama. Formaldehyde exposure was measured by FA vapor monitor badges. Questionnaires were administered to participants to identify potential residential sources of FA. Urine cotinine, a surrogate for tobacco smoke exposure, was also used as an indicator of a possible source of residential exposure to FA. Results:The mean level of FA exposure by vapor monitor badge was 0.04 parts per million (ppm) (SD = 0.06; range 0.003-0.54 ppm). Minimum risk levels of 0.03 and higher were found in 36.4% of participants. Exposure levels of FA were higher in spring than winter (p < 0.001). Exposure levels of FA were correlated with indoor temperature of dwellings (p < 0.02), installation of new carpet within last 5 years (p < 0.04), and use of nail polish (p < 0.01). No relationship was found between FA exposure and urine cotinine levels. Clinical Implications:Formaldehyde exposure may increase at various times in the lives of women; however, it is of particular concern during pregnancy because of perinatal risk to the exposed fetus.

Hydrogen Peroxide Stimulates Exosomal Cathepsin B Regulation of the Receptor for Advanced Glycation End-Products (RAGE)

Exosomes are nano‐sized vesicles that are secreted into the extracellular environment. These vesicles contain various biological effector molecules that can regulate intracellular signaling pathways in recipient cells. The aim of this study was to examine a correlation between exosomal cathepsin B activity and the receptor for advanced glycation end‐products (RAGE). Type 1 alveolar epithelial (R3/1) cells were treated with or without hydrogen peroxide and exosomes isolated from the cell conditioned media were characterized by NanoSight analysis. Lipidomic and proteomic analysis showed exosomes released from R3/1 cells exposed to oxidative stress induced by hydrogen peroxide or vehicle differ in their lipid and protein content, respectively. Cathepsin B activity was detected in exosomes isolated from hydrogen peroxide treated cells. The mRNA and protein expression of RAGE increased in cultured R3/1 cells treated with exosomes containing active cathepsin B while depletion of exosomal cathepsin B attenuated RAGE mRNA and protein expression. These results suggest exosomal cathepsin B regulates RAGE in type 1 alveolar cells under conditions of oxidative stress. J. Cell. Biochem. 119: 599–606, 2018.

Immunological methods for nursing research: from cells to systems.

Scientists and clinicians frequently use immunological methods (IMs) to investigate complex biological phenomena. Commonly used IMs include immunocytochemistry (IC), enzyme-linked immunosorbent assays (ELISA) and flow cytometry. Each of these methodologies exploits a common principle in IMs —the binding of an antibody to its antigen. Scientists continue to develop new methodologies, such as high-throughput immunohistochemistry (IHC) and in vivo imaging techniques, which exploit antibody—antigen binding, to more accurately answer complex research questions involving single cells up to whole organ systems. The purpose of this paper is to discuss established and evolving IMs and to illustrate the application of these methods to nursing research.