This long term goal of our laboratories is to understand the kinetic mechanism that enable normal tissues to mount an appropriately timed and appropriately limited response to stress or crisis. Local release of adenine nucleotides and adenosine, and metabolism of these highly pharmacoactive compounds by cell surface enzymes are major components of the homeostatic response in many physiologic and pathophysiologic situations. We propose to evaluate the capacities of ventricular myocytes isolated from rabbit heart, and microvascular endothelial cells cultured from pig and rabbit heart, to produce extracellular adenosine and adenine nucleotides, to regulate extracellular nucleotide hydrolysis, and to respond to ATP, ADP, and adenosine.
The specific aims are: 1) Determine whether the metabolic and transport capabilities of each cell type sustain net adenosine efflux sufficient to account for the extracellular adenosine observed during coronary ischemia. 2) Determine whether hypoxia causes ATP release from myocytes and coronary microvascular endothelial cells. 3) Determine how the kinetic properties of the extracellular reaction sequence, ATP leads to ADP leads to AMP leads to adenosine, regulate the relative concentrations of these compounds at the surfaces of both cell types, and develop kinetic models which can predict the entire time course of the reaction sequence when a bolus of one or more of the components is introduced. 4) Examine cellular responses to ATP, ADP, and adenosine, and where possible, relate the rate and extent of these responses to the shifting extracellular agonist concentrations driven by release and extracellular metabolism. Stimulation (endothelial cells) and inhibition (myocytes) of cAMP synthesis and efflux will be used as cellular end-points for response to adenosine. Prostaglandin synthesis and changes in intracellular calcium concentration will be measured as indices of response to ATP and ADP. 5) Characterize the export of cAMP from each of the cell types, and determine the extent to which this process contributes to regulation of cellular cAMP turnover. Pharmacologically active concentrations of adenine nucleotides or adenosine occur extracellularly during neurotransmission, thrombus formation, and coronary ischemia. The experiments proposed here should provide important information for understanding the regulation of the rate and extent of coronary response during these events.
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