Richard A. Oeckler

Stretch Enhances Contraction of Bovine Coronary Arteries via an NAD(P)H Oxidase–Mediated Activation of the Extracellular Signal–Regulated Kinase Mitogen-Activated Protein Kinase Cascade

Abstract— This study examines the effects of an increase in passive stretch in endothelium-removed bovine coronary artery on oxidant-induced changes in force generation. Increasing passive stretch on the arterial segments from 5 to 20 g for 20 minutes caused a subsequent increase (P <0.05) in force generation to 30 mmol/L KCl or 0.1 &mgr;mol/L serotonin compared with the prestretch control response. Also associated with the passive stretch were increases in superoxide detection by lucigenin and a selective increase in extracellular signal–regulated kinase (ERK) mitogen-activated protein (MAP) kinase phosphorylation measured by Western analysis. The stretch-induced increase in force generation was eliminated by inhibition of the ERK pathway by the MEK inhibitor PD98059 but not by inhibitors of the p38 MAP kinase pathway (SB202190) or c-Jun N-terminal protein kinase pathway (SP200169). Additionally, stretch-induced increases in both ERK phosphorylation and force generation were attenuated by inhibition of tyrosine kinases (genistein), src (PP2), and specific sites on the epidermal growth factor receptor (EGFR) (AG1478). Probes for oxidant signaling, including NAD(P)H oxidase inhibitors (diphenyliodonium and apocynin) or enhancement of peroxide consumption (ebselen) but not inhibition of xanthine oxidase (allopurinol), attenuated the effects of stretch on both ERK phosphorylation and force generation. Furthermore, stretch caused an increase in EGFR phosphorylation and cytosolic to membrane translocation of the p47phox NAD(P)H oxidase subunit. Hydrogen peroxide also elicited contraction through EGFR phosphorylation and ERK. In summary, stretch seems to enhance force generation via ERK signaling through an EGFR/src-dependent mechanism activated by peroxide derived from a stretch-mediated activation of the NAD(P)H oxidase, a response that may contribute to hypertensive alterations in vascular reactivity.

Superoxide in the Vascular System

Oxidant production and regulation is becoming increasingly important in the study of vascular signaling mechanisms, and recent reviews have characterized some of the possible roles for known downstream products of superoxide formation. In this review, we will examine current research in the field, with a special emphasis on the role of the superoxide molecule itself and its place amongst the slightly better understood roles of peroxide and peroxynitrite. The regulatory roles of oxidant species are wide-ranging, and their involvement in processes ranging from intracellular and receptor signaling mechanisms that regulate endothelial mediator release and vascular contractile function to processes that control cellular growth and apoptosis has been implied. Cellular sources of superoxide production and metabolism and the chemical interaction of oxidant species with specific components of cellular signaling mechanisms are considered important factors which determine physiological responses that control vascular function.

Determinants of Plasma Membrane Wounding by Deforming Stress

Once excess liquid gains access to air spaces of an injured lung, the act of breathing creates and destroys foam and thereby contributes to the wounding of epithelial cells by interfacial stress. Since cells are not elastic continua, but rather complex network structures composed of solid as well as liquid elements, we hypothesize that plasma membrane (PM) wounding is preceded by a phase separation, which results in blebbing. We postulate that interventions such as a hypertonic treatment increase adhesive PM-cytoskeletal (CSK) interactions, thereby preventing blebbing as well as PM wounds. We formed PM tethers in alveolar epithelial cells and fibroblasts and measured their retractive force as readout of PM-CSK adhesive interactions using optical tweezers. A 50-mOsm increase in media osmolarity consistently increased the tether retractive force in epithelial cells but lowered it in fibroblasts. The osmo-response was abolished by pretreatment with latrunculin, cytochalasin D, and calcium chelation. Epithelial cells and fibroblasts were exposed to interfacial stress in a microchannel, and the fraction of wounded cells were measured. Interventions that increased PM-CSK adhesive interactions prevented blebbing and were cytoprotective regardless of cell type. Finally, we exposed ex vivo perfused rat lungs to injurious mechanical ventilation and showed that hypertonic conditioning reduced the number of wounded subpleural alveolus resident cells to baseline levels. Our observations support the hypothesis that PM-CSK adhesive interactions are important determinants of the cellular response to deforming stress and pave the way for preclinical efficacy trials of hypertonic treatment in experimental models of acute lung injury.

Integration of Pulmonary Mechanics in a Personalized Approach to Mechanical Ventilation

The concept of safe ventilation continues to evolve. Initially, a low tidal volume (VT) strategy,[1][1] then an open lung strategy,[2][2],[3][3] and now as mortality improvements have stagnated and promising targets such as PEEP and plateau pressure yield unsatisfactory and conflicting results,[4][4