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Interferon-α2b (IFN-α2b) is being used intravesically for preventing recurrence and progression of superficial transitional cell carcinoma of the bladder. However, its mechanism of action when instilled intravesically is not yet elucidated. We monitored end products of nitric oxide (NO) in urine in 12 bladder cancer patients undergoing intravesical instillations of IFN-a2b. Urine end products of NO levels rose gradually after each instillation, reaching a peak value after the third instillation. Although the patients continued their treatment for 5 more weeks, no further alteration was observed. Inducible nitric oxide synthase (iNOS) expression was immunohistochemically evaluated in urinary bladder biopsy specimens before and after IFN-α2b instillations. It was shown that IFN-α2b induced urothelial iNOS expression, with subsequent oxidative stress. The peroxynitrite (ONOO–) formed from the combination of NO with superoxide (O2–) provides important clues in the role of ONOO– as a causative factor in the antineoplastic action of IFN-a2b.

In the present study we demonstrated that NO synthase and xanthine oxidase of synaptosomes isolated from rabbit brain cortex can be activated by the gas phase of cigarette smoke to produce nitric oxide and superoxide which react together to form peroxynitrite. Expose of synaptosomes, up to 3 hours, in the gas phase of cigarette smoke, a gradual increase in both nitric oxide and superoxide release that were inhibited by N-monomethyl-L-arginine (100 μM) and oxypurinol (1 mM), respectively, was observed. NO synthase and xanthine oxidase activities were increased approximately three fold after treatment of synaptosomes with the gas phase of cigarette smoke as compared with the gas phase deprived of oxidants. Synaptosomes treated with the gas phase of cigarette smoke dramatically increased 3-nitrotyrosine production (used as an index of peroxynitrite formation). Synaptosomes treated with the gas phase of cigarette smoke, promptly increased malondialdehyde production with subsequent decrease of synaptosomal plasma membrane fluidity estimated by fluorescence anisotropy of 1,4-(trimethyl-amino-phenyl)-6-phenyl-hexa-1,3,5-triene. Gas phase deprived of oxidants showed a small but not statistically significant (p > 0.05) effect on both malondialdehyde and membrane fluidity. In summary, the present results indicate that activation of NO synthase and xanthine oxidase of brain cells by oxidants contained in the gas phase of cigarette smoke lead to the formation of peroxynitrite a causative factor in neurotoxicity.

The association of [3H]-Met-enkephalin with synaptosomes isolated from rat brain cortex, when incubated for 30 min at 25°C follows a sigmoid path with a Hill coefficient h=1.25±0.04. Binding of Met-enkephalin into synaptosomes was saturable, with an apparent binding constant of 8.33±0.48 nM. At saturation, Met-enkephalin specific receptors corresponded to 65.5±7.2 nmol/mg synaptosomal protein. The Hill plot in combination with the biphasic nature of the curve to obtain the equilibrium constant, showed a moderate degree of positive cooperativity in the binding of Met-enkephalin into synaptosomes of at least one class of high affinity specific receptors. Met-enkephalin increased the lipid fluidity of synaptosomal membranes labelled with 1,6-diphenyl-1,3,5-hexatriene (DPH), as indicated by the steady-state fluorescence anisotropy [(ro/r)-1]-1. Arthenius-type plots of [(ro/r)-1]-1 indicated that the lipid separation of the synaptosomal membranes at 23.4±1.2°C was perturbed by Met-enkephalin such that the temperature was reduced to 15.8±0.8°C. Naloxone reversed the fluidizing effect of Met-enkephalin, consistent with the receptor-mediated modulation of membrane fluidity. Naloxone alone had no effect on membrane fluidity. NO release and cGMP production by NO-synthase (NOS) and soluble guanylate cyclase (sGC), both located in the soluble fraction of synaptosomes (synaptosol) were decreased by 82% and 80% respectively, after treatment of synaptosomes with Met-enkephalin (10-10–10-4 M). These effects were reversed by naloxone (10-4 M) which alone was ineffective in changing NO and cGMP production. We propose that Met-enkephalin achieved these effects through receptor mediated perturbations of membrane lipid structure and that inhibition of the L-Arg/NO/cGMP pathway in the brain may result in the antinociceptive effects of Met-enkephalin.

The binding of [14C]phenobarbital into synaptosomal plasma membranes of dog brain follows a sigmoid path. The “best fit” curve of this binding is the one described by the Hill equation (r2>0.93 and Hill coefficient,n=1.32). (Na+, K+)-stimulated ATPase and Ca2+-stimulated ATPase activities are modulated by phenobarbital. Arrhenius plots of (Na+, K+, Mg2+)-dependent ATPase revealed that phenobarbital (2mM) lowered the transition temperature and altered the Arrhenius activation energies of this enzyme. The allosteric inhibition by F- of the (Na+, K+)-stimulated ATPase was studied in control and phenobarbital-treated membranes. The lowering of the transition temperature and changes in Arrhenius activation energy about the transition temperature in combination with changes observed in the allosteric properties of the (Na+, K+)-stimulated ATPase by F-, produced by phenobarbital, would be expected if it is assumed that phenobarbital “fluidizes” synaptosomal plasma membranes.

The association of [125I-]calmodulin with rat brain synaptosomal plasma membranes, when incubated for 1 h at 25° in the presence or in absence of 20 µM Ca2+, follows a sigmoid path with a Hill coefficient h=1.79±0.12 and h=1.72±0.11, respectively. The total association of calmodulin with the membrane increased approx. 60%–80% at all the range of calmodulin concentrations used in the presence of 20 µM Ca2+. A three fold increase of guanylate cyclase activity was shown in the presence of low concentrations of calmodulin (up to 10 mM); higher concentrations (up to 40 mM) however, led to a progressive inhibition of the enzyme activity with respect to maximal stimulation. Calmodulin increased the lipid fluidity of synaptosomal plasma membranes labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH), as indicated by the steady-state fluorescence anisotropy [(ro/r)-1]-1. Arrhenius-type plots of [(ro/r)-1]-1 indicated that the lipid separation of the membrane at 22.7±1.2° was perturbed by calmodulin such that the temperature was reduced to 16.3±0.9° and 15.5±0.8° in the absence or in the presence of 20 µM Ca2+. Arrhenius plots of guanylate cyclase and acetylcholinesterase activities exhibited brak points at 25.7±1.4° and 22.3±1.0° in control synaptosomal plasma membranes, respectively. The break point for the guanylate cyclase was reduced to 16.3±0.9° in calmodulin treated synaptosomal plasma membranes whereas that of acetylcholinesterase remained unaffected (21.1±0.9°). The allosteric properties of guanylate cyclase by Mn-GTP (as reflected by changes in the Hill coefficient) were modulated by calmodulin while those of acetylcholinesterase by fluoride (F-) were not altered. We propose that calmodulin achieves these effects through asymmetric perturbations of the membrane lipid structure and that increase in membrane fluidity of the inner leaflet of the membrane induced by calmodulin may be an early key event to the process of neurotransmitter release.

PurposeThe oxidative and reductive stresses within the varicocele veins were estimated. Nitric oxide synthase and xanthine oxidase activities, as well as nitric oxide, S-nitrosothiols and superoxide release within the spermatic vein in patients with varicocele, and the role of the noxious oxidant peroxynitrite formed from nitric oxide and superoxide in sperm dysfunction were determined.Materials and MethodsWhole blood samples were drawn from a peripheral vein and a dilated varicocele vein before ligation. Nitric oxide synthase, xanthine oxidase, nitric oxide and peroxynitrite were measured by novel spectro-fluorophotometric methods. S-nitrosothiols were estimated by a luminol-chemiluminescence method. Serum and red blood cell antioxidant capacity was determined by a chemiluminescence reaction.ResultsSerum nitric oxide synthase and xanthine oxidase activities, as well as nitric oxide, peroxynitrite and S-nitrosothiol levels were greater in the spermatic vein compared to the peripheral vein. Serum antioxidant capacity was greater in varicocele veins compared to peripheral veins. In contrast, the antioxidant capacity of red blood cells was less in the varicocele veins, which was consistent with an increased rate of peroxynitrite production.ConclusionsOur data suggest a high oxidative stress due to the release of nitric oxide synthase and xanthine oxidase within the dilated spermatic vein. This reaction resulted in dramatic formation of nitric oxide, peroxynitrite and S-nitrosothiols, which are biologically active. Formation of peroxynitrite from the reaction of nitric oxide with superoxide could be a causative factor for impaired sperm function in patients with varicocele.

Here we demonstrate that human keratinocytes possess a Ca2+/calmodulin-dependent particulate NO synthase that can be activated to release NO after exposure to UVB radiation. UVB irradiation (up to 20 mJ/cm2) of human keratinocyte plasma membranes resulted in a dose-dependent increase in NO and l-[3H]citrulline production that was inhibited by approx. 90% in the presence of N-monomethyl-l-arginine (l-NMMA). In time-course experiments with UVB-irradiated plasma membranes the changes in NO production were followed by analogous changes in soluble guanylate cyclase (sGC) activity. In reconstitution experiments, when particulate NO synthase was added to purified sGC isolated from keratinocyte cytosol, a 4-fold increase in cGMP was observed; the cGMP was increased by NO synthesized after UVB irradiation (up to 20 mJ/cm2) of particulate NO synthase. A 5-fold increase in superoxide (O2-) and a 7-fold increase in NO formation followed by an 8-fold increase in peroxynitrite (ONOO-) production by UVB (20 mJ/cm2)-irradiated keratinocyte microsomes was observed. UVB radiation (20 mJ/cm2) decreased plasma membrane lipid fluidity as indicated by steady-state fluorescence anisotropy. Membrane fluidity changes were prevented by l-NMMA. Changes in Arrhenius plots of particulate NO synthase in combination with changes in its allosteric properties induced by UVB radiation are consistent with a decreased fluidity of the lipid microenvironment of the enzyme. The present studies provide important new clues to the role of NO and ONOO- released by UVB-irradiated human keratinocytes in skin erythema and inflammation.