The importance of adenosine in the pathogenesis of immediate hypersensitivity reactions is becoming increasingly clear. Adenosine, released from hypoxic lung tissue or activated mast cells, potentiates the stimulated release of granule-associated mast cell mediators in vitro. In vivo, inhaled adenosine induces bronchospasm in asthmatic subjects by a mechanism consistent with the augmentation of an ongoing, low-level release of preformed mast cell mediators, particularly histamine. Mast cell possess at least two types of cell surface adenosine receptors that singly or in combination result in the activation of phospholipase C and adenylate cyclase. Receptor occupation alone is not sufficient to induce mediator release, and the release of leukotrienes and prostaglandins is not enhanced by adenosine. The nature of these mast cell adenosine receptors, their individual signal transduction pathways, responses to regulatory stimuli, and the importance of each subtype in mast cell secretion are largely unknown. The scope of the project encompasses a thorough physiologic and pharmacologic characterization of two newly-cloned mast cell adenosine receptor subtypes. Receptors will be studied in a stable expression system such as transfected CHO cells as well as in anti-sense knock out experiments in transfected RBL cells. Antibodies to intracellular and extracellular domains of each receptor will be produced in order to quantitate receptor expression and examine changes in mast cell function in the presence of specific antibody. Regulation of adenosine receptor numbers will be studied at the mRNA and protein levels utilizing several agents previously shown to alter adenosine receptor expression in mast cells. Site-directed mutagenesis at selected phosphorylation sites may help to determine the kinases important in desensitization and down- regulation responses. Because adenosine is a naturally-occurring mediator and modulator of allergic responses, a clearer understanding of mast cell adenosine receptors will facilitate the development of therapeutic interventions useful in the treatment of asthma and allergic diseases.
