The objective of this application is to provide the applicant with an in-depth training experience that will result in her becoming an independent and funded investigator at the end of this work. Our long-range goal is to study ion transport modulation in red cells and other tissues in an over-stimulated environment that may lead to additional pathology. Dehydration of hemoglobin S-containing erythrocytes favors the formation of dense cells due to increased hemoglobin S polymerization in sickle cell disease. The preliminary results indicate that the potent vasoactive peptide endothelin-1 induces dehydration of sickle erythrocytes. In addition, it was found that endothelin-1 induced activation of Ca(2+)- activated K(+) channel (Gardos channel) via an endothelin receptor type B- mediated process in normal and sickle erythrocytes. The central hypothesis of this application is that the elevated systemic and local concentrations of endothelin-1 contributes to the pathogenesis of vaso-occlusive episodes by induced dehydration via the Gardos channel. This hypothesis will be tested by defining the regulatory mechanism(s) by which endothelin-1 induces Gardos channel activation and red cell dehydration in sickle cell disease. To this end, the functional properties of the Gardos channel will be evaluated in mouse and human erythrocytes containing Hb A and S by following these specific aims: 1) Establish the mechanism(s) by which endothelin-1 activates Gardos channel in sickle erythrocytes: to test the hypothesis that endothelin-1 induced a signaling pathway that changes the Ca(2+) affinity constant of the Gardos channel via a protein kinase C-mediated mechanism(s) in normal and sickle erythrocytes. 2) Identify the mechanism(s) by which endothelin-1 induces dehydration in sickle erythrocytes: to define the physiological pathway(s) of endothelin-1 induced dense red cell formation in normal and sickle cells and its regulatory mechanism(s) by specific blockers of endothelin-1 receptors. Erythrocytes from mice transgenic for hemoglobin S will be studied in vitro and in vivo to assess the role of endothelin receptors in sickle cell dehydration by using endothelin-1 selective receptor antagonists. It is the expectation that this work will determine the mechanism(s) identified are expected to provide critical information on pathological activation of the Gardos channel in sickle erythrocytes. Finally, it is expected that these data will provide preliminary results for the subsequent planned submission of an R01 application. The research proposed in the application is significant because it will provide us with a more comprehensive knowledge of erythrocyte volume regulatory mechanism(s). These, in turn, will help to develop a novel therapeutic strategy to reduce sickle cell formation, and decrease the occurrence of vaso-occlusive episodes. It is additionally significant, because it will provide the means for the applicant to establish an independent research career and to become competitive for a tenure-track assistant professional position.
