Heart failure is a complex clinical phenomenon. Among a variety of functional defects, a common one is the constriction of arterioles and pre- capillary resistance vessels. The tone of these vessels is controlled by local regulatory factors generated in the heart muscle cells (cardiac myocytes), in the cells lining the blood vessels (vascular endothelial cells), as well as by hormonal and neural factors. Adenosine is a key regulator which is released by cardiac myocytes in response to hypoxia and increased work load and by vascular endothelial cells. Adenosine causes relaxation of vascular smooth muscle; this results in an increased diameter of the blood vessels and an increased blood supply to the heart muscle. Adenosine exerts its effects via external cell surface receptors, called A1 and A2. The predominant adenosine receptors on cardiac myocytes are A1, and on vascular smooth muscle and endothelial cells are A2. When adenosine occupies these receptors, they respond by causing a decrease (A1 receptors) or an increase (A2 receptors) in the levels of cyclic AMP in the cell. Cyclic AMP acts as a second messenger which affects many aspects of cellular regulation. Adenosine also acts on intracellular receptors. These receptors are poorly understood. Upon occupancy by adenosine, they also reduce the concentration of cyclic AMP, but they probably work via other mechanisms too. Recently adenosine has started to be used in clinical medicine, because of its beneficial effects in certain arrhythmias, and it appears to be preferable for this purpose to some older drugs. We propose to study factors that regulate the production of adenosine. Two intracellular 5'-nucleotidases which hydrolyze nucleotides to nucleosides in the heart cell have been identified. One of these, which we call N-I, prefers AMP as substrate and is responsible for producing the adenosine that is released for heart cells to act as an intercellular signal. The other 5'-nucleotidase, N-II, prefers IMP as substrate and produces inosine. Both are regulated enzymes. N-I and N-II also occur in other cells and organs, in different relative amounts that remain to be determined. Although its normal function is not clear, N-II is of great interest because it can act as a phosphotransferase. It is the only enzyme known to phosphorylate 2',3'-dideoxy analogs of nucleosides, a family of drugs that is useful in the amelioration of AIDS and other viral diseases. A better understanding of the regulation of the phosphotransferase activity of N-II may lead to an improved efficacy of dideoxy nucleoside drugs. We have isolated two adenosine-binding proteins that have no known enzymatic activities and that do not use up adenosine by unknown reactions. We believe that they may be regulatory proteins, but their targets remain unknown. The effects of over-expressing these proteins on protein phosphorylation and purine nucleotide metabolism will be investigated. The long term objective of this proposal is to provide a better understanding of the biochemical mechanisms that regulate the blood supply to the heart, of the function of the two cytosolic 5'-nucleotidases in health and disease, and of the function of the intracellular adenosine- binding proteins.
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