Chronic aminoguanidine attenuates renal dysfunction and injury in aging rats.

We have previously shown that aging is associated with increased lipid peroxidation, reductions in renal function, and increased glomerular sclerosis. The mechanism(s) responsible for these age-related changes are not clear. The purpose of the present studies was to determine if there was an increase in inducible nitric oxide synthase (iNOS) with aging, and if so, whether inhibition of iNOS would prevent aging injury by preventing free radical-mediated lipid peroxidation. iNOS protein expression in the kidney increased by approximately 90% by 24 months. Inhibition of iNOS by aminoguanidine (0.1% in drinking water) for 9 months, beginning at 13 months of age, reduced blood pressure, improved glomerular filtration rate by 70%, and renal plasma flow by 40%, whereas glomerular sclerosis was considerably reduced. Renal F2-isoprostanes and malondialdehyde levels, markers of oxidative stress and lipid peroxidation, were not reduced by aminoguanidine. Aminoguanidine also did not attenuate immunostaining for advanced glycosylation end products (AGE) in the kidneys. These findings suggest that aminoguanidine attenuates aging renal dysfunction by inhibiting a pathophysiologic function of iNOS that is independent of free radical-mediated lipid peroxidation or significant effects on AGE deposition.

Gender differences in the renal nitric oxide (NO) system: dissociation between expression of endothelial NO synthase and renal hemodynamic response to NO synthase inhibition.

Many studies have shown that nitric oxide (NO) production is higher in the systemic vasculature of females than males and is stimulated during pregnancy, a high estrogen state. The present study was performed in rats to determine whether females had a greater expression of endothelial NO synthase (eNOS) in kidneys than did males; whether there were gender differences in the excretion of NO metabolites, nitrate/nitrite; and whether there were gender differences in the renal hemodynamic response to NO synthase inhibition. The renal levels of eNOS mRNA (as measured by ribonuclease protection assays) and protein (as measured by Western blot) were 80% higher in kidneys from females than from males (P < .001). Urinary excretion of NO metabolites, nitrate/nitrite, were not different between males and females. To inhibit eNOS, rats were treated with nitro-L-arginine methyl ester (L-NAME, 3 to 4 mg/kg/day) for 2 weeks. Although there were no differences in basal renal hemodynamics between males and females, when factored for kidney weight, chronic L-NAME increased renal vascular resistance by 130% in males but by only 60% in females, and decreased renal plasma flow by 40% in males but had no effect in females. These data show that although the renal levels of eNOS mRNA and protein are higher in females than in males, the renal vasculature of males is more responsive to NO synthase inhibition. The data suggest that the renal vasculature of males may be more dependent on NO than is the renal vasculature in females.

Angiotensin II stimulates synthesis of endothelial nitric oxide synthase.

Previous studies have suggested that NO may play an important role in protecting the renal vessels from angiotensin II (ANGII)-mediated vasoconstriction. One possible mechanism for this interaction is that ANGII could stimulate NO production in the kidney by increasing endothelial NO synthase (NOS III). The present studies were performed in rats to determine whether acute or chronic elevations in ANGII are associated with enhanced renal NOS III mRNA or protein synthesis. In both acute and chronic studies captopril (20 microg/kg/min) was given I.V. to inhibit endogenous ANGII production. Acute suprarenal infusion of ANGII (8 ng/kg/min) for 110 minutes had no effect on arterial pressure but decreased GFR and renal plasma flow by 20% and 30%, respectively, and increased renal vascular resistance by 70%. Acute ANGII increased renal NOS III mRNA by 70% (as determined by ribonuclease protection assay), but had no effect on renal NOS III protein concentration (as detected by Western blot analyses). In contrast, chronic infusion of ANGII (5 ng/kg/min) for 10 days, increased arterial pressure by 30% and tended to reduce GFR and renal plasma flow. Chronic ANGII had no effect on renal NOS III mRNA levels, but increased NOS III protein by 90%. These data suggest that ANGII can stimulate NOS III synthesis and suggest that this may be one of the mechanisms whereby AngII may enhance NO production.

Interaction of tubulin and microtubule proteins with vanadate oligomers.

Microtubule assembly is known to be regulated by the phosphorylation of microtubule-associated proteins (MAPs), and is thus sensitive to phosphatase inhibitors. We have investigated the direct interaction between phosphatase inhibitors (vanadate, sodium fluoride, and okadaic acid) and microtubule proteins. Vanadate self-assembles into oligomers, primarily dimer, tetramer, and decamer in 0.1 M Pipes, pH 6.9. Oligomer concentrations and their direct binding to tubulin and MAPs were determined by 51V NMR. The assembly of microtubule protein (MTP) is strongly inhibited by decavanadate binding to MAPs and only weakly inhibited by tetravanadate binding to MAPs. Decavanadate will inhibit both MAP2 and tau-induced assembly. Decavanadate binds to MAP2 at 26 sites [Ka > or = (1.0-1.3) x 10(5) M-1]. The mechanism appears to involve competitive binding to MAPs, presumably at or near the microtubule binding domains, and reduced affinity for microtubules. The assembly of MAP-free, phosphocellulose-purified tubulin (PC-tubulin) is only weakly inhibited by decavanadate, although decavanadate binds to tubulin at four independent sites (Ka > or = 1.0 x 10(5) M-1). Monomeric vanadate, a strong phosphatase inhibitor, does not interact with tubulin or MAPs, and thus does not bind to the exchangeable nucleotide binding site on tubulin. Sodium fluoride stimulates both PC-tubulin and MTP assembly by a nonspecific effect, probably involving water structure formation. Wyman analysis suggests an absence of direct or specific binding to tubulin (d ln K/d ln [NaF] = 0.214). NaCl is nearly as effective in promoting assembly of PC-tubulin, but inhibits MTP assembly.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple sites for subtilisin cleavage of tubulin: effects of divalent cations.

Limited digestion of pig brain GDP-tubulin by subtilisin was carried out in the presence of Mg2+, Mn2+, Ca2+, Zn2+, or Be2+. Isoelectric focusing, followed by SDS-PAGE, revealed characteristic divalent cation-dependent changes in the alpha- and beta-tubulin cleavage patterns. Previous studies revealed that the beta-cleavage pattern is different for heterodimers and microtubules [Lobert and Correia, 1992: Arch. Biochem. Biophys. 296: 152-160]. Divalent cation effects on subtilisin digestion of tubulin indicate different classes of divalent cation binding sites. Western blot analysis locates the proteolytic zone at residue 430 or higher in both subunits for all conditions. Turbidity and electron microscopy reveal that GDP-tubulin cleaved by subtilisin in the presence of Mg2+, Ca2+, or Mn2+ forms sheets of rings. Mn2+ induces ring formation in uncleaved GDP-tubulin. Isotype-depleted tubulin was generated by the removal of class III beta-tubulin using immunoaffinity chromatography. Subtilisin digestion of the depleted fraction and the purified class III beta-tubulin demonstrates that cleavage occurs at three to four distinct sites. Thus, subtilisin-digested tubulin is more heterogeneous than was previously reported and the cleavage sites depend on solution conditions, divalent cations, and the state of assembly. This has important implications for experiments that utilize subtilisin-digested tubulin for studying microtubule-associated protein binding.

Myelin protein expression in dissociated superior cervical ganglia and dorsal root ganglia cultures.

Schwann cells of the adult rat superior cervical ganglia (SCG) synthesize negligible levels of the major myelin glycoprotein, P0, in vivo. This suggests that the sympathetic axons of the SCG are deficient in one of more components involved in the regulation of myelin protein expression. Here we have compared the ability of neurites from neonatal rat SCG and embryonic rat dorsal root ganglia (DRG) to induce Schwann cell expression of myelin proteins after growth in culture using a serum-free medium. Steady-state P0 mRNA levels in the SCG and DRG culture paradigms were determined with a sensitive polymerase chain reaction (PCR) assay that amplified cDNA produced by reverse transcription of mRNA. This semiquantitative assay showed a linear response to increasing amounts of P0 and actin mRNA and required substantially less cellular RNA than typical hybridization techniques. Using the PCR assay, we found that SCG cultures contained significantly lower amounts of P0 mRNA than did DRG cultures. To further confirm that SCG cultures have negligible expression of myelin proteins, immunoblot analyses were done to examine the steady-state levels of both P0 and myelin basic protein. While nonmyelinating DRG cultures had readily detectable amounts of these myelin-specific proteins, neither could be demonstrated in the SCG cultures. The data indicate that SCG neurites lack one or more signals needed to induce myelin protein expression. Employing SCG and DRG cultures in comparative biochemical studies should prove useful in identifying the axonal molecule(s) involved in the regulation of myelin protein expression.