My N. Helms - US grants

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] The alveolar epithelium in normal lungs is comprised of two morphologically distinct types of cells (type 1 and type 2) that are responsible for maintaining lung fluid balance. Tight regulation of alveolar fluid clearance is essential for maintaining a dry breathing space, and hence, proper gas exchange. It has been established that net ion transport through amiloride-sensitive epithelial sodium channels (ENaC), located on the apical surface of alveolar epithelial cells, play a critical role in fluid clearance in normal lung. However, the specific mechanisms regulating ENaC function are not completely understood. Within the alveoli, complex regulatory mechanisms must also be in place to balance the redox state of type 1 and type 2 cells, since inspired oxygen is converted to superoxide anions (O2-). Excessive O2- production, caused by high oxygen tensions, can lead to tissue damage and cell death, whereas insufficient oxygenation can result in anything from fatigue to life threatening conditions. In vivo, superoxides react quickly and irreversibly with nitric oxide (NO) to form peroxynitrite. We hypothesize that endogenous 02- binding to NO limits nitric oxide inhibition of ENaC function, thereby enhancing alveolar fluid clearance. Indeed, we have preliminary data suggesting that nitric oxide-unresponsive AT1 cells may have elevated levels of O2-, and that increasing O2- enhances Na transport. To investigate the role of redox signaling in alveolar fluid clearance, and more specifically, ENaC function, I will utilize single channel patch clamp analysis to examine ion transport in lung slice preparations, bio-molecular techniques to examine redox signaling in pneumocytes, and perform whole lung studies in vivo. My first aim, performed during the mentored phase, directly examines the role of O2- in alveolar fluid clearance. Successful completion of this aim will naturally transition into aims 2 and 3, which utilizes several protocols that will be established in the mentored phase, as well as incorporate new approaches to studying type 1 and type 2 cells. The second aim will determine the role of NO in lung function, and lastly, the third aim examines the putative reciprocal relationship between O2- and NO regulation of lung fluid balance. The studies proposed have real clinical relevance, and the potential for a very productive independent research career. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): By bringing together investigators with varied expertise in epithelial transport and cell signaling regulation of ion channels/transporters with clinicians focused on disease processes, the Epithelial Physiology and Cell Biology Workshop stimulates collaborations and catalyzes scientific progress , as has been exemplified by the successes of the previous meetings. This workshop is held annually (since 2004) in Telluride, Colorado with logistical meeting support provided by the Telluride Science Research Center. The cutting edge nature of the topics to be presented is reflected in the co-organizers, Drs. John Cuppoletti, My N. Helms, Thomas Kleyman, Peter Snyder, and a diverse range of speakers. Topics will include (but is not limited to) the role of free radicals in ion transport, molecular modeling of sodium channels based on the X-ray crystal structure of acid sensing ion channels, and the potential use of prostone based compounds to target chloride channels. Participation by junior researchers in this workshop will also be enhanced through a proposed Research Recognition Travel Award. PUBLIC HEALTH RELEVANCE: The annual Epithelial Physiology and Cell Biology Workshop, held in conjunction with the Telluride Science Research Center, sets new direction for scientific research. The Principal Investigator, together with Drs. Kleyman, Cuppoletti, and Snyder requests R13 funds that will defray the cost of travel for trainees who wish to attend this scientific workshop.

DESCRIPTION (provided by applicant): Bringing together investigators with varied expertise in epithelial transport and cell signaling regulation of ion channels/transporters with clinicians focused on disease process, the Epithelial Physiology and Cell biology Workshop stimulates collaborations and catalyzes scientific progress, as has been exemplified by the successes of the previous meetings. This workshop is held annually (since 2004) in Telluride, Colorado with logistical meeting support provided by the Telluride Science Research Center. The cutting edge nature of topics to be presented is reflected in the co-organizers involved, Drs. John Cuppoletti, My N. Helms, Thomas Kleyman, Peter Snyder, and a diverse range of speakers. Topics will include (but is not limited to) the role of free radicals in ion transport, molecular modeling of sodium channels based on the X-ray crystal structure of acid sensing ion channels, and the potential use of prostone based compounds to target chloride channels. Participation by junior researchers in this workshop will also be enhanced through a proposed Research Recognition Travel Award.

Project Summary/Abstract Epithelial sodium channels (ENaC) serve as the rate limiting step in net salt and water removal from the airspace by generating osmotic gradients for subsequent water transport. In the lung, the regulation of ENaC is fine-tuned so that a precise volume of water continually lines the airway epithelium, which keeps the lungs appropriately moist for effective gas exchange. In some patients with lung injury, it is not clear why lung ENaC fails to function. Normal ENaC activity is critically important in preterm infants, since they are born with immature lung, low levels of ENaC protein, and often require oxygen supplementation. In this study, we look at whether oxygen supplementation (hyperoxia) confounds lung injury by turning off ENaC. We use a term mouse model of hyperoxia induced oxidative stress in our studies. Our rationale is that under high oxygen, the antioxidant glutathione is oxidized to its disulfide form, termed GSSG. Our preliminary data indicates that GSSG can alter ENaC function through direct post translational modification of conserved Cys thiols on ENaC, albeit the modified Cys have not been mapped. Whether GSSG modifies ENaC under a biologically relevant model of hyperoxia induced lung injury has not been evaluated. Our goal is to delineate biologically relevant post-translational modification of lung ENaC by GSSG and determine whether lowering lung GSSG levels can improve health outcomes for preterm infants requiring oxygen supplementation.