We propose to test the hypothesis that adenosine (ADO) is a major cause of free flow exercise hyperemia. A major unknown is the concentration of ADO in the interstitial space, to which vascular smooth muscle is exposed. Tissue adenosine content measurements are difficult to interpret because of lack of knowledge of compartmentation of adenosine between intra- and extracellular sites. Adenosine measurements in venous plasma cannot be interpreted with certainty because of a lack of knowledge of the nature of capillary transport of adenosine. We plan to characterize capillary transport of ADO and then use this information along with measurements of arterial and venous plasma ADO concentration ([ADO]) to calculate interstitial ADO concentration ([ADO]ISF). With regard to capillary transport, we will test our working hypothesis that ADO moves across capillary endothelium by a combination of simple diffusion through water filled channels and facilitated transport via the nucleoside carrier of the endothelial cell membrane. This will be done using autoradiography for localization of tracer adenosine and indicator diffusion experiments to determine if capillary ADO transport exhibits inhibition by dipyridamole, saturation kinetics and counter transport. Based on the results of these experiments, we will develop a technique for measurement of capillary transport of ADO in each exercise experiment so that [ADO]ISF can be calculated from arterial and venous concentrations. Then we will observe changes in muscle blood flow and [ADO]ISF during free flow exercise and during dose response curves to exogenous and endogenous ADO. We will study the same variables during infusion of a transport inhibitor, an adenosine receptor antagonist, and an ecto-5 feet nucleotidase inhibitor. We will then see if changes in [ADO]ISF can quantitatively account for the observed changes in blood flow in each of these circumstances. This will provide a strong test of the adenosine hypothesis. In addition, these experiments will provide basic information about capillary transport of adenosine which should be of use to investigators studying other aspects of adenine nucleotide and nucleoside homeostasis.
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