The role of raft domains in human red blood cell transmembrane signaling and membrane cholesterol transport

In addition to transporting oxygen and carbon dioxide, red blood cells (RBCs) actually regulate blood flow in the smallest arteries. The RBCs accomplish this by releasing adenosine triphosphate (ATP) in areas of reduced blood flow. RBCs can detect reduced oxygen concentration, reduced pH, and, as well, the shear stress of deformation. Reduced oxygen concentration, reduced pH and physical deformation are the "signals" which cause red cells to elicit local production of nitric oxide, which can increase regional blood flow. Thus, in addition to carrying out complex transport functions, the RBCs play a critical role in the regulation of blood flow.; Forcing RBCs through filters is the method most commonly used to study deformation-induced ATP release. The shear stress imposed on RBCs by passing them through filters has been estimated to be almost three times higher than the time-average physiological shear stress (24 dyn•cm-2) that they encounter in the body. In this thesis, a method based on centrifugation was developed to generate those physiological levels of shear stress actually found in the circulation: This approach permitted us to study the ATP release pathway in vitro. Several key molecular components of this pathway, including the inhibitory G proteins (Gi), various forms of the enzyme known as adenylyl cyclase (AC) and also the cyclic AMP dependent protein kinases, were studied. Their membrane abundance was measured both in the specialized "lipid raft" and "non-raft" membrane domains. The subunits of the Gi were found to migrate from membrane non-raft domains into raft domains in the presence of specific activator molecules. AC was localized in the membrane raft regardless of the presence of activators. Based on these findings, we suggest that special membrane raft domains function to isolate and concentrate the specialized components of membrane signaling systems. These signaling components appear to interact in response to the molecule mastoparan. This isolation and concentration of signaling components may have advantages and even be essential for the highly specific rapid responses of RBC to specific molecular and mechanical signals.; In addition to the essential role that membrane lipid rafts play in signal transduction, they also facilitate the movement of cholesterol into the RBC membrane. Bovine serum albumin (BSA) was found to increase the rate of incorporation of free cholesterol into RBC plasma membranes. Lipid rafts may serve as the membrane regions where most of the BSA-facilitated cholesterol incorporation takes place, suggesting that lipid rafts also play an important role in cholesterol transport. Clearly this pathway may have a fundamental role in the development of hardening of the arteries or atherosclerotic change.