Abstract-Extracellular adenosine production by the GPI-anchored Ecto-5Ј-Nucleotidase (Ecto-5Ј-Nu) plays an important role in the cardiovascular system, notably in defense against hypoxia. It has been previously suggested that HMG-CoA reductase inhibitors (HRIs) could potentiate the hypoxic stimulation of Ecto-5ЈNu in myocardial ischemia. In order to elucidate the mechanism of Ecto-5Ј-Nu stimulation by HRIs, Ecto-5Ј-Nu activity and expression were determined in an aortic endothelial cell line (SVAREC) incubated with lovastatin. Lovastatin enhanced Ecto-5Ј-Nu activity in a dose-dependent manner. This increase was not supported by de novo synthesis of the enzyme because neither the mRNA content nor the total amount of the protein were modified by lovastatin. By contrast, lovastatin enhanced cell surface expression of Ecto-5Ј-Nu and decreased endocytosis of Ecto-5Ј-Nu, as evidenced by immunostaining. This effect appeared unrelated to modifications of cholesterol content or Ecto-5Ј-Nu association with detergent-resistant membranes. The effect of lovastatin was reversed by mevalonate, the substrate of HMG-CoA reductase, by its isoprenoid derivative, geranyl-geranyl pyrophosphate, and by cytotoxic necrotizing factor, an activator of Rho-GTPases. Stimulation of Ecto-5Ј-Nu by lovastatin enhanced the inhibition of platelet aggregation induced by endothelial cells. In conclusion, lovastatin enhances Ecto-5Ј-Nu activity and membrane expression in endothelial cells. This effect seems independent of lowering cholesterol content but could be supported by an inhibition of Ecto-5Ј-Nu endocytosis through a decrease of Rho-GTPases isoprenylation. This benefit is already apparent at the early step of endothelial dysfunction, which precedes the development of atherosclerosis. 4,5 Some of the effects of these cholesterol-lowering drugs, particularly those observed in short-term exposure, could be supported by mechanisms independent of lowering of plasma LDL concentration. 1,6 Induction of adenosine production in the extracellular space by the GPI-anchored Ecto-5Ј-Nu could be one of these mechanisms. Indeed, the production of adenosine by Ecto-5Ј-Nu is implicated in the regulation of endothelial functions 7-9 and in defense against hypoxia. 9 -12 Ecto-5Ј-Nu activity is increased by tissular hypoxia, notably in cardiomyocytes and endothelial cells. 13,14 The enzyme has been implicated in myocardial preconditioning, 13,15 and it was recently shown that HRIs potentiate the stimulation of Ecto-5Ј-Nu activity induced by ischemia in cardiomyocytes. 16 However, it is not known whether HRIs also enhance Ecto-5Ј-Nu activity in endothelium, a main site of adenosine production, 17 and the mechanisms whereby HRIs enhance Ecto-5Ј-Nu have not been studied. Some hypotheses can be put forward. First, it has been shown that HRIs can modulate the activity of enzymes, such as endothelial nitric oxide synthase, at the transcriptional level. 6 On the other hand, HRIs can induce posttranslational events, which influence cell surface expression of proteins....

Abstract-Extracellular adenosine production by the glycosyl-phosphatidyl-inositol-anchored Ecto-5Ј-Nucleotidase plays an important role in the defense against hypoxia, particularly in the intravascular space. The present study was designed in order to elucidate the mechanisms underlying hypoxia-induced stimulation of Ecto-5Ј-Nucleotidase in endothelial cells. For this purpose, aortic endothelial cells (SVARECs) were submitted to hypoxic gas mixture. Hypoxia (0% O 2 for 18 hours) induced a 2-fold increase of Ecto-5Ј-Nucleotidase activity (V max 19.78Ϯ0.53 versus 8.82Ϯ1.12 nmol/mg protein per min), whereas mRNA abundance and total amount of the protein were unmodified. By contrast, hypoxia enhanced cell surface expression of Ecto-5Ј-Nucleotidase, as evidenced both by biotinylation and immunostaining. This effect was accompanied by a decrease of Ecto-5Ј-Nucleotidase endocytosis, without modification of Ecto-5Ј-Nucleotidase association with detergent-resistant membranes. Finally, whereas cholesterol content was unmodified, hypoxia induced a time-dependent increase of saturated fatty acids in SVARECs, which was reversed by reoxygenation, in parallel to Ecto-5Ј-Nucleotidase stimulation. Incubation of normoxic cells with palmitic acid enhanced Ecto-5Ј-Nucleotidase activity and cell surface expression. In conclusion, hypoxia enhances cell surface expression of Ecto-5Ј-Nucleotidase in endothelial cells. This effect could be supported by a decrease of Ecto-5Ј-Nucleotidase endocytosis through modification of plasma membrane fatty acid composition.

In the cochlea, endolymph is a K-rich and Na-poor fluid. The purpose of the present study was to check the presence and to assess the role of epithelial Na channel (ENaC) in this organ. alpha-, beta-, and gamma-ENaC subunit mRNA, and proteins were detected in rat cochlea by RT-PCR and Western blot. alpha-ENaC subunit mRNA was localized by in situ hybridization in both epithelial (stria vascularis, spiral prominence, spiral limbus) and nonepithelial structures (spiral ligament, spiral ganglion). The alpha-ENaC-positive tissues were also positive for beta-subunit mRNA (except spiral ganglion) or for gamma-subunit mRNA (spiral limbus, spiral ligament, and spiral ganglion), but the signals of beta- and gamma-subunits were weaker than those observed for alpha-subunit. In vivo, the endocochlear potential was recorded in guinea pigs under normoxic and hypoxic conditions after endolymphatic perfusion of ENaC inhibitors (amiloride, benzamil) dissolved either in K-rich or Na-rich solutions. ENaC inhibitors altered the endocochlear potential when Na-rich but not when K-rich solutions were perfused. In conclusion, ENaC subunits are expressed in epithelial and nonepithelial cochlear structures. One of its functions is probably to maintain the low concentration of Na in endolymph.