This study describes a novel mechanism of regulation of the high-affinity Na ؉ -dependent adenosine transporter (CNT2) via the activation of A 1 adenosine receptors (A 1 R). This regulation is mediated by the activation of ATP-sensitive K ؉ (K ATP ) channels. The high-affinity Na ؉ -dependent adenosine transporter CNT2 and A 1 R are coexpressed in the basolateral domain of the rat hepatocyte plasma membrane and are colocalized in the rat hepatoma cell line FAO. The transient increase in CNT2-mediated transport activity triggered by (؊)-N 6 -(2-phenylisopropyl)adenosine is fully inhibited by K ATP channel blockers and mimicked by a K ATP channel opener. A 1 R agonist activation of CNT2 occurs in both hepatocytes and FAO cells, which express Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B mRNA channel subunits. With the available antibodies against Kir6.X, SUR2A, and SUR2B, it is shown that all of these proteins colocalize with CNT2 and A 1 R in defined plasma membrane domains of FAO cells. The extent of the purinergic modulation of CNT2 is affected by the glucose concentration, a finding which indicates that glycemia and glucose metabolism may affect this cross-regulation among A 1 R, CNT2, and K ATP channels. These results also suggest that the activation of K ATP channels under metabolic stress can be mediated by the activation of A 1 R. Cell protection under these circumstances may be achieved by potentiation of the uptake of adenosine and its further metabolization to ATP. Mediation of purinergic responses and a connection between the intracellular energy status and the need for an exogenous adenosine supply are novel roles for K ATP channels.Adenosine is an autocoid that acts physiologically on specific cell surface G protein-coupled receptors, four of which (A 1 , A 2A , A 2B , and A 3 ) have been cloned and described pharmacologically (18,23,39). A 1 adenosine receptors (A 1 R) mediate a broad range of signaling responses by coupling to Gi and Go proteins (18,23,33). The activation of A 1 R regulates several membrane and intracellular proteins, such as adenylate cyclase, phospholipase C, Ca 2ϩ channels (23, 39), and K ϩ channels (3,26,35). mRNAs encoding the four adenosine receptors have been detected in liver cells (12). In isolated rat hepatocytes, the activation of A 1 R triggered Ca 2ϩ -mediated glycogenolysis, the activation of A 2 adenosine receptors stimulated cyclic AMP (cAMP)-mediated gluconeogenesis, and the activation of A 3 adenosine receptors increased cytosolic Ca 2ϩ and decreased cAMP levels, with minor changes in glycogen metabolism (20). In hepatocyte plasma membranes, the stimulation of adenylate cyclase activity and the inhibition of low-K m cAMP phosphodiesterase activity are coordinately regulated by glucagon, and A 1 R can inhibit glucagon-stimulated cAMP accumulation by blocking the effect of glucagon on phosphodiesterase activity (40). Moreover, a signal pathway involving A 2a adenosine receptors, Gi proteins, phospholipase C, protein kinase C (PKC) ␦, PKC ε, and p38 mitogen-activated prote...