Renal function is highly dependent on endothelium-derived nitric oxide (NO). Several renal disorders have been linked to impaired NO bioavailability. The enzyme that is responsible for the synthesis of NO within the renal endothelium is endothelial NO synthase (eNOS). eNOS-mediated NO generation is a highly regulated cellular event, which is induced by calcium-mobilizing agonists and fluid shear stress. eNOS activity is regulated at the transcriptional level but also by a variety of modifications, such as acylation and phosphorylation, by its cellular localization, and by protein-protein interactions. The present review focuses on the complex regulation of eNOS within the endothelial cell.

The insulin-responsive glucose transporter GLUT4 plays an essential role in glucose homeostasis. A novel assay was used to study GLUT4 trafficking in 3T3-L1 fibroblasts/preadipocytes and adipocytes. Whereas insulin stimulated GLUT4 translocation to the plasma membrane in both cell types, in nonstimulated fibroblasts GLUT4 readily cycled between endosomes and the plasma membrane, while this was not the case in adipocytes. This efficient retention in basal adipocytes was mediated in part by a C-terminal targeting motif in GLUT4. Insulin caused a sevenfold increase in the amount of GLUT4 molecules present in a trafficking cycle that included the plasma membrane. Strikingly, the magnitude of this increase correlated with the insulin dose, indicating that the insulin-induced appearance of GLUT4 at the plasma membrane cannot be explained solely by a kinetic change in the recycling of a fixed intracellular GLUT4 pool. These data are consistent with a model in which GLUT4 is present in a storage compartment, from where it is released in a graded or quantal manner upon insulin stimulation and in which released GLUT4 continuously cycles between intracellular compartments and the cell surface independently of the nonreleased pool.

—5-Methyltetrahydrofolate (MTHF), the active form of folic acid, has been reported to restore NO status in hypercholesterolemic patients. The mechanism of this effect remains to be established. We assessed the effects of L- and D-MTHF on tetrahydrobiopterin (BH ₄ )–free and partially BH ₄ -repleted endothelial NO synthase (eNOS). Superoxide production of eNOS and the rate constants for trapping of superoxide by MTHF were determined with electron paramagnetic resonance using 5-diethoxyphosphoryl-5-methyl-1-pyrroline- N -oxide (DEPMPO) as spin trap for superoxide. NO production was measured with [ ³ H]arginine–citrulline conversion or nitrite assay. The rate constants for scavenging of superoxide by L- and D-MTHF were similar, 1.4×10 ⁴ ms ^–1 . In BH ₄ -free eNOS, L- and D-MTHF have no effect on enzymatic activity. In contrast, in partially BH ₄ -repleted eNOS, we observe a 2-fold effect of MTHF on the enzymatic activity. First, superoxide production is reduced. Second, NO production is enhanced. In cultured endothelial cells, a similar enhancement of NO production is induced by MTHF. In the present study, we show direct effects of MTHF on the enzymatic activity of NO synthase both in recombinant eNOS as well as in cultured endothelial cells, which provides a plausible explanation for the previously reported positive effects of MTHF on NO status in vivo.

In addition to its role in selective protein degradation, the conjugation of ubiquitin to proteins has also been implicated in the internalization of plasma membrane proteins, including the α‐factor receptor Ste2p, uracil permease Fur4p, epithelial sodium channel ENaC and the growth hormone receptor (GHR). Binding of GH to its receptor induces receptor dimerization, resulting in the activation of signal transduction pathways and an increase of GHR ubiquitination. Previously, we have shown that the ubiquitin conjugation system mediates GH‐induced GHR internalization. Here, we present evidence that a specific domain of the GHR regulates receptor endocytosis via the ubiquitin conjugation system. This ubiquitin‐dependent endocytosis (UbE) motif consists of the amino acid sequence DSWVEFIELD and is homologous to sequences in other proteins, several of which are known to be ubiquitinated. In addition, we show that GH internalization by a truncated GHR is independent of the presence of lysine residues in the cytosolic domain of this receptor, while internalization still depends on an intact ubiquitin conjugation system. Thus, GHR internalization requires the recruitment of the ubiquitin conjugation system to the GHR UbE motif rather than the conjugation of ubiquitin to the GHR itself.

The ubiquitin conjugation system is involved in ligand-induced endocytosis of the growth hormone receptor (GHR) via a cytosolic 10-amino acid ubiquitin-dependent endocytosis motif. Herein, we demonstrate that the proteasome is also involved in growth hormone receptor down-regulation. Ligand-induced degradation was blocked in the presence of specific proteasomal inhibitors. In addition, growth hormone (GH) internalization was inhibited, whereas the transferrin receptor cycle remained unaffected. A truncated GHR entered the cells independent of proteasome action. In addition, we show that GH internalization is independent of the presence of lysine residues in the cytosolic domain of the receptor, whereas its internalization can still be inhibited by proteasomal inhibitors. Thus, GHR internalization requires proteasome action in addition to an active ubiquitin conjugation system, but ubiquitination of the GHR itself seems not to be required.