The main focus of this application is the intracellular pathways of A2b receptors. Compared to other adenosine receptors subtypes, the A2B have received little attention. However, recent evidence indicates that A2b receptors are involved in vasodilation, inhibition of vascular smooth muscle growth, regulation of intestinal secretion, and modulation of mast cell function. Our preliminary studies indicate that A2b receptors produce direct activation of human mat cells HMC-1 through pathways that appears to involve PLC activation through coupling with Gq proteins. These are novel observations. First this signaling pathway has not been previously associated with A2b receptors. Second, adenosine has only been shown to potentiate mast cells, but was not previously shown to produce direct activation of mast cells in vitro. Our studies, therefore, have the potential to better explain the observation that inhaled adenosine provokes bronchoconstriction in asthmatics in vivo. Our pilot studies also suggest that A2b receptors are present in human lung mast cells, and that their effects on mast cells obtained from asthmatics may be different from those obtained from normal subjects. We have broadened the focus of our original application to include studies that will confirm these new observation. Several changes have been made in the approach used to study the intracellular pathways of A2b receptors in HMC-1 cells. In particular, new and specific pharmacological probes are now available that will allow us to differentiate between the actions of A2b receptors from those of A2a or A3 receptors. Finally, we have added new studies designed to understand the structure-activity relationship of A2b antagonists. We are confident that are proposed studies will result in novel observations regarding A2b receptors and their regulation of mast cell function, with implications for asthma.
