Abstract-Blood flow to contracting skeletal muscle is tightly coupled to the oxygenation state of hemoglobin. To investigate if ATP could be a signal by which the erythrocyte contributes to the regulation of skeletal muscle blood flow and oxygen (O 2 ) delivery, we measured circulating ATP in 8 young subjects during incremental one-legged knee-extensor exercise under conditions of normoxia, hypoxia, hyperoxia, and Key Words: skeletal muscle blood flow Ⅲ erythrocytes Ⅲ oxygen sensor Ⅲ oxygen delivery T he signals regulating the rapid adjustments of skeletal muscle blood flow to exercise and altered inspiratory O 2 fraction have been the focus of more than a century of research. Recently, the idea has been advanced that optimal local circulatory adjustments to alterations in blood oxygenation and exercise require the existence of extracellular and cellular O 2 sensors coupled to signal-transduction systems, which are capable of evoking the appropriate vascular responses in contracting and resting muscle. 1-9 The extensive available evidence demonstrating that alterations in circulating O 2 are inversely related to changes in exercising skeletal muscle blood flow provides indirect support for this notion. 10 -20 To assess the primary extracellular site of O 2 sensing, this laboratory's initial approach was to elucidate whether alterations in skeletal muscle blood flow to exercise in humans were associated with either the amount of O 2 dissolved in circulating plasma or the amount of O 2 bound to hemoglobin. 16,19 Using normoxia, hypoxia, anemia, anemiaϩhypoxia, COϩnormoxia, and COϩhyperoxia as interventions, we found compelling evidence indicating that elevations in exercising muscle blood flow and vascular conductance are independent of pronounced alterations in PaO 2 (40 to 540 mm Hg) but are closely linked to the reductions in arterial oxyhemoglobin. 16,19 Our findings suggest that the main vascular O 2 sensor locus is located in the erythrocyte itself, rather than in the PO 2 sensitive regions of the endothelium or vascular smooth muscle.The red blood cell itself might be involved in the regulation of local blood flow and O 2 delivery by signaling O 2 availability in the erythrocyte. This theory is consistent with the findings from two independent groups of researchers who have demonstrated that red blood cells release ATP 3 and NO 4,5 in response to a fall in hemoglobin O 2 saturation. The release of NO from the S-nitrosohemoglobin molecule with the lowering in oxyhemoglobin is thought to induce the diffusion of the NO group to the vascular endothelium where it stimulates vessel relaxation. 4 -5 ATP in turn can induce vasodilatation by binding to P 2y -purinergic receptors located on the vascular endothelial cells to release NO-and/or endothelium-derived hyperpolarization factors, which diffuse to the vascular smooth muscle and result in vasodilatation. 3 The vasodilator potency of ATP is clearly supported by Original

The urgent need for efficacious drugs to treat chronic hepatitis C virus (HCV) infection requires a concerted effort to develop inhibitors specific for virally encoded enzymes. We demonstrate that 2-C-methyl ribonucleosides are efficient chain-terminating inhibitors of HCV genome replication. Characterization of drug-resistant HCV replicons defined a single S282T mutation within the active site of the viral polymerase that conferred loss of sensitivity to structurally related compounds in both replicon and isolated polymerase assays. Biochemical analyses demonstrated that resistance at the level of the enzyme results from a combination of reduced affinity of the mutant polymerase for the drug and an increased ability to extend the incorporated nucleoside analog. Importantly, the combination of these agents with interferon-␣ results in synergistic inhibition of HCV genome replication in cell culture. Furthermore, 2-C-methyl-substituted ribonucleosides also inhibited replication of genetically related viruses such as bovine diarrhea virus, yellow fever, and West African Nile viruses. These observations, together with the finding that 2-C-methyl-guanosine in particular has a favorable pharmacological profile, suggest that this class of compounds may have broad utility in the treatment of HCV and other flavivirus infections. Hepatitis C virus (HCV)1 is the most common blood-borne infection and a major cause of chronic liver disease and liver transplantation in industrialized countries. The prevalence of HCV infection is estimated to be ϳ5-fold greater than HIV infection and ranges from 1-5% in most developed countries (1). Current therapy is both poorly tolerated and has limited efficacy, with less than 50% response rates among patients infected with the most prevalent virus genotype (1b) (1). Currently approved drugs for the treatment of hepatitis C are interferon-␣ and ribavirin, neither of which appears to act directly on the virus, and their antiviral effects appear to be mediated by multiple, indirect mechanisms. Therefore, there is a need for more efficient and better tolerated anti-HCV agents.The success of antiviral therapies based on chemotherapeutic agents targeting viral polymerases has prompted intense efforts to develop inhibitors of HCV NS5B, the virally encoded RNA-dependent RNA polymerase (RdRp). Studies with HIV reverse transcriptase validate the clinical utility of two distinct classes of viral polymerase inhibitors, nucleoside and non-nucleoside inhibitors. Nucleoside inhibitors function as competitive substrate analogs that prevent RNA chain elongation when incorporated by the viral enzyme, resulting in premature chain termination (2, 3). HIV reverse transcriptase non-nucleoside inhibitors bind to a site residing outside the enzyme active site and inhibit catalysis by an allosteric mechanism (4, 5). Several putative allosteric binding sites on the surface of HCV NS5B have been suggested based on recent structural studies (6 -8), and several chemical classes of NS5B non-nucleoside inhibitors have ...

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is essential for the replication of viral RNA and thus constitutes a valid target for the chemotherapeutic intervention of HCV infection. In this report, we describe the identification of 2-substituted nucleosides as inhibitors of HCV replication. The 5-triphosphates of 2-C-methyladenosine and 2-O-methylcytidine are found to inhibit NS5B-catalyzed RNA synthesis in vitro, in a manner that is competitive with substrate nucleoside triphosphate. NS5B is able to incorporate either nucleotide analog into RNA as determined with gel-based incorporation assays but is impaired in its ability to extend the incorporated analog by addition of the next nucleotide. In a subgenomic replicon cell line, 2-C-methyladenosine and 2-O-methylcytidine inhibit HCV RNA replication. The 5-triphosphates of both nucleosides are detected intracellularly following addition of the nucleosides to the media. However, significantly higher concentrations of 2-C-methyladenosine triphosphate than 2-O-methylcytidine triphosphate are detected, consistent with the greater potency of 2-C-methyladenosine in the replicon assay, despite similar inhibition of NS5B by the triphosphates in the in vitro enzyme assays. Thus, the 2-modifications of natural substrate nucleosides transform these molecules into potent inhibitors of HCV replication. Hepatitis C virus (HCV)1 infection is the leading cause of sporadic, post-transfusion, non-A non-B hepatitis (1, 2). One hundred seventy million people worldwide are thought to be infected with hepatitis C virus of which an estimated 4 million reside in the United States (3). Approximately 80% of infected individuals progress to chronic infection. Long term chronic HCV infection can lead to liver cirrhosis and to hepatocellular carcinoma (4 -6). Currently, the recommended therapy is treatment with a combination of interferon ␣2b and ribavirin, which results in a sustained viral response in 40% of patients (7,8). Investigational therapies using a combination of pegylated interferon and ribavirin have lead to an sustained viral response in 54% of patients, but the response rate (42%) of patients harboring HCV genotype 1 is lower (9, 10). Consequently, additional therapies for HCV infection are needed.Antiviral chemotherapies based on administration of analogs of deoxynucleosides have been widely successful as treatment for HIV, herpes virus, and hepatitis B infection (11,12). Intracellular phosphorylation of the nucleoside analog to the triphosphate creates the active form of the inhibitor that then serves as a substrate for the viral polymerase. Generally, incorporation of the nucleotide analog at the 3Ј-end of the replicating viral DNA causes termination of DNA synthesis, owing to the lack of the 3Ј-hydroxyl required for extension. These successes suggest that an investigation of ribonucleoside analogs as inhibitors of HCV replication would be worthwhile.The HCV NS5B protein, the RNA-dependent polymerase responsible for the synthesis of the viral RNA geno...

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