Regulation of endothelial intracellular adenosine via adenosine kinase epigenetically modulates vascular inflammation.

The molecular mechanisms underlying vascular inflammation and associated inflammatory vascular diseases are not well defined. Here we show that endothelial intracellular adenosine and its key regulator adenosine kinase (ADK) play important roles in vascular inflammation. Pro-inflammatory stimuli lead to endothelial inflammation by increasing endothelial ADK expression, reducing the level of intracellular adenosine in endothelial cells, and activating the transmethylation pathway through increasing the association of ADK with S-adenosylhomocysteine (SAH) hydrolase (SAHH). Increasing intracellular adenosine by genetic ADK knockdown or exogenous adenosine reduces activation of the transmethylation pathway and attenuates the endothelial inflammatory response. In addition, loss of endothelial ADK in mice leads to reduced atherosclerosis and affords protection against ischemia/reperfusion injury of the cerebral cortex. Taken together, these results demonstrate that intracellular adenosine, which is controlled by the key molecular regulator ADK, influences endothelial inflammation and vascular inflammatory diseases.The molecular mechanisms underlying vascular inflammation are unclear. Here the authors show that pro-inflammatory stimuli lead to endothelial inflammation by increasing adenosine kinase expression, and that its knockdown in endothelial cells inhibits atherosclerosis and cerebral ischemic injury in mice.

An Adenosine-Mediated Glial-Neuronal Circuit for Homeostatic Sleep.

Sleep homeostasis reflects a centrally mediated drive for sleep, which increases during waking and resolves during subsequent sleep. Here we demonstrate that mice deficient for glial adenosine kinase (AdK), the primary metabolizing enzyme for adenosine (Ado), exhibit enhanced expression of this homeostatic drive by three independent measures: (1) increased rebound of slow-wave activity; (2) increased consolidation of slow-wave sleep; and (3) increased time constant of slow-wave activity decay during an average slow-wave sleep episode, proposed and validated here as a new index for homeostatic sleep drive. Conversely, mice deficient for the neuronal adenosine A1 receptor exhibit significantly decreased sleep drive as judged by these same indices. Neuronal knock-out of AdK did not influence homeostatic sleep need. Together, these findings implicate a glial-neuronal circuit mediated by intercellular Ado, controlling expression of homeostatic sleep drive. Because AdK is tightly regulated by glial metabolic state, our findings suggest a functional link between cellular metabolism and sleep homeostasis. SIGNIFICANCE STATEMENT: The work presented here provides evidence for an adenosine-mediated regulation of sleep in response to waking (i.e., homeostatic sleep need), requiring activation of neuronal adenosine A1 receptors and controlled by glial adenosine kinase. Adenosine kinase acts as a highly sensitive and important metabolic sensor of the glial ATP/ADP and AMP ratio directly controlling intracellular adenosine concentration. Glial equilibrative adenosine transporters reflect the intracellular concentration to the extracellular milieu to activate neuronal adenosine receptors. Thus, adenosine mediates a glial-neuronal circuit linking glial metabolic state to neural-expressed sleep homeostasis. This indicates a metabolically related function(s) for this glial-neuronal circuit in the buildup and resolution of our need to sleep and suggests potential therapeutic targets more directly related to sleep function.

Diabetes-induced decrease of adenosine kinase expression impairs the proliferation potential of diabetic rat T lymphocytes.

The proliferative response of T lymphocytes is a crucial step in cell-mediated immunity. This study was undertaken to investigate the mechanisms leading to the impaired proliferative response of diabetic T lymphocytes. T cells that had been isolated from the spleen of normal rats and cultured in medium containing 20 mm glucose and no insulin displayed the same degree of proliferative impairment as cells isolated from diabetic rats. The rate of T-cell proliferation, when induced with concanavalin A or anti-CD3 and anti-CD28 antibodies, was not affected by the inhibition of nucleoside transporters. T cells cultured at high glucose concentrations in the absence of insulin displayed decreased expression of adenosine kinase, and released measurable extracellular quantities of adenosine. Under resting conditions, the level of cAMP was 5.9-fold higher in these cells compared to cells grown in low glucose and in the presence of insulin. Experiments with specific adenosine receptor agonists and antagonists showed that adenosine-induced suppression of diabetic T cell proliferation was mediated by the A2A adenosine receptor, but not by the A2B receptor. Treatment of diabetic T cells with 10 microm H-89, a specific protein kinase A inhibitor, restored T-cell proliferation. These results show that suppressed proliferation of diabetic T lymphocytes is evoked by the decreased expression of adenosine kinase, leading to the outflow of adenosine from the cell. Extracellular adenosine then stimulates the A2A receptor and induces cAMP production, leading to the activation of protein kinase A, and suppression of T-cell proliferation.

Isolation and immunological properties of adenosine kinase.

Bovine liver adenosine kinase is a 43 kDa protein that catalyzes the transfer of phosphate from GTP or ATP to adenosine. Its immunological properties were compared to other GTP-binding proteins of approximately 40 kDa, in particular those involved in signal transduction, such as Gs and Gi, the stimulatory and inhibitory regulatory proteins of adenylyl cyclase, Gt, from the visual excitation system, and Go, a similar protein of unknown function. Antibodies elicited in rabbits against adenosine kinase did not significantly cross-react with other guanyl nucleotide-binding proteins. Antibodies against the other GTP-binding proteins did not react with adenosine kinase. Thus these GTP-binding proteins do not exhibit immunological cross-reactivity.

Immunological studies with different classes of mutants affected at the adenosine kinase locus in CHO cells.

Adenosine kinase (AK) from CHO cells has been purified to homogeneity and specific antibodies to it have been raised in rabbits. Using this antibody, the presence of a specific cross-reacting protein (CRP) in cell extracts of different classes of mutants resistant to purine nucleoside analogs which are affected in AK has been investigated by the immunoblotting technique. Results of our studies show that 31 of the 32 independently selected class A AK- mutants (obtained at high frequency in presence of adenosine analogs toyocamycin, tubercidin, 6-methylmercaptopurine riboside, or pyrazofurin and containing no measurable activity of AK in cell extracts) contained similar amounts of a specific CRP as seen in the parental AK+ cells. The CRP in the parental and different mutant cell lines has the same relative molecular mass as purified AK. Similar results were obtained with two mutants each of the class B and C type (selected in presence of C-nucleosides formycin A and formycin B), which are also affected in AK but show novel properties. The presence of equivalent amounts of the CRP in the vast majority of the class A mutants strongly indicates that the high frequency of those mutants in CHO cells is not a result of an epigenetic or deletion type of event, but that such mutants may contain missense types of mutations at a presumed "mutational hot spot" within the structural gene for adenosine kinase.