Geclosporin

To clarify the interaction between arginase and nitric oxide (NO) production in the kidney with normal and high glucose levels, renal cortical slices from male Sprague-Dawley rats were incubated in Hank's solution containing various concentrations of L-norvaline (Nval; an arginase inhibitor), 500 U/mL superoxide dismutase, and either 5 mmol/L (normal) or 20 mmol/L (high) glucose (n = 5 per group). Incubation with Nval increased renal cortical NOX (nitrite + nitrate) production dose-dependently, indicating competition between arginase and NO synthase (NOS) for the substrate (L-arginine). In the basal condition without Nval, high glucose also increased NO(X) production to a rate 3 times that observed during incubation with normal glucose (P < .01). This effect of high glucose was not altered by Nval. Rather, the effects of high glucose and Nval were additive, indicating that the activity of NOS per se is enhanced by high glucose. Direct assay of arginase and NOS activities confirmed stimulation of both enzymes under the high glucose condition (P < .05, P < .01, v normal glucose, respectively). However, high glucose did not change the amount of L-arginine present in renal cortical slices. These data reveal that arginase competes with NOS for L-arginine in the renal cortex, and that high glucose increases the activity of both enzymes without affecting the amount of substrate. These results suggest that increased NOS activity, rather than altered substrate availability, may be the principal factor underlying increased NO synthesis in diabetic kidneys.

Lysine tagging of hydrophobic peptides of parent sequence KKAAALAAAAALAAWAALAAAKKKK‐NH2 has been shown to facilitate their synthesis and purification through water solubilization, yet not impact on the intrinsic properties of the hydrophobic core sequence with respect to its insertion into membranes in an α‐helical conformation. However, due to their positively charged character, such peptides often become bound to phospholipid head groups in membrane surfaces, which inhibits their transbilayer insertion and/or prevents their transport across cellular bilayers. We sought to develop more neutral peptides of membrane‐permeable character by replacing most Lys residues with uncharged peptoid [N‐(R)glycyl] residues, which might similarly confer water solubility while retaining membrane‐interactive properties of the hydrophobic core. Several “peptoid‐tagged” derivatives of the parent peptide were prepared with varying peptoid content, with five of the six Lys residues replaced with peptoids Nala and/or Nval. Conformations of these peptides measured by circular dichroism spectroscopy demonstrated that these water‐soluble peptides retain the α‐helix structure in micelles (lysophosphatidylcholine and sodium dodecyl sulfate) notwithstanding the known helix‐breaking capacity of the peptoid tags. Blue shifts in Trp fluorescence spectra and quenching experiments with acrylamide confirmed that peptoid‐tagged peptides insert spontaneously into micellar membranes. Results suggest that upon introduction of uncharged tags, the interaction between the membrane and the peptides is dominated by the hydrophobicity of the peptide core rather than the electrostatic interactions between the Lys and the head groups of the lipids. The overall findings indicate that peptoid residues are effective surrogates for Lys as uncharged water‐solubilizing tags and, as such, provide a potentially valuable feature of design of membrane‐interactive peptides. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004