The long range goals of this investigation are to: 1) gain a clearer understanding of the local regulation of blood flow, with particular emphasis on the role played by adenosine, 2) determine the factors involved in the formation, release and metabolism of adenosine and, 3) determine the effects of adensine on parenchymal cell function. To achieve these goals, studies will be performed at the cellular level, in the in situ and isolated perfused heart and in the brain and the following will be determined 1) whether adenosine stimulation of endothelial cells grown on microbeads release a substance that relaxes vascular smooth muscle (VSM using endothelial-denuded arterial rings and co-cultured VSM cells, 2) in cardiac myocytes, to what extent adenosine is formed in the cytosol, whether the intracellular concentration can be increased with nucleoside transport blockers and whether intracellular formation is enhanced by a reduction in pH, 3) the relative activi- ties of S-adenoslhomocysteine hydrolase, nucleoside phosphorylase and adenosine deaminase in cardiac myocytes and endothelial cells, 4) the type of adenosine receptor on cultured VSM by noting the relative potencies of several A1 and A2 adenosine agonists and the effect on cAMP and cGMP contents, 5) the permeability of endothelial cell monolayers to adenosine with H-adenosine and whether this process is affected by adenosine receptor agonists, 6) by use of selective agonists and measurements of cAMP & cGMP, whether the hypoxia-induced increase in glycolytic flux is mediated by Al or A2 adenosine receptors in cultured endothelial cells & cardiac myoctyes. Since knowledge of the interstitial fluid (ISF) concentration of adenosine is critical for evaluation of the nucleoside's role in blood flow regulation, a thin porous nylon disc has been devised for application to the epicardium to reflect the ISF levels of adenosine. This technique will be used to determine the regional ISF adenosine concentration with concomitant flow measurements in the in situ dog heart & isolated perfused heart during a variety of interventions known to alter the 02 supply/02 demand ratio. In ISF adenosine concentration in a region of low 02 supply can be simultaneously compared with that in a region of high 02 supply. In addition, the accuracy of myocardial transudates as a measure of ISF adenosine can be determined and further evaluation of the adenosine hypotheses can be achieved by measuring ISF adenosine during homocysteine, alkylxanthine, & adenosine deaminase administration. Another series of experiments will utilize the microdialysis technique in the brain to determine whether adenosine mediates the regional increase in blood flow in response to auditory & visual stimuli. Since adenosine is now known to mediate a number of physiological processes and is also efficacious in the treatment of certain cardiae arrhythmias, a better understanding of its metabolism and mechanisms of action are pertinent to human health.
