EPR studies on the kinetics of quenching singlet oxygen.

The specific reaction between singlet oxygen (1O2) and 2,2,6,6-tetra-methyl-piperidine (TEMP) was utilized to investigate the kinetics of singlet oxygen quenching. Rose bengal was used for the generation of singlet oxygen (1O2). The deuterium isotope effect (rho) was found to be 22.1. The rate constant (kT) of the reaction of TEMP with 1O2 in H2O, D2O and ethanol were found to be 9.0 x 10(5) M-1s-1, 1.5 x 10(6) M-1s-1 and 7.3 x 10(5) M-1s-1, respectively. The intercept of the plot of [TEMPO]-1 at different H2O/D2O ratios was found to be constant, indicating the lifetime of 3Sens is independent of solvent deuteration. The quenching of 1O2 by azide and the sensitizer itself were confirmed by the constant intercept on the kinetic plots. Thus, the quenching rate constants for azide anion in ethanol solution (96%) and for ground state rose bengal in 0.05 M phosphate buffer (pH 7.4), were found to be 8.36 x 10(8) M-1s-1 and 1.72 x 10(4) M-1s-1, respectively. These studies not only verify the specificity of the reaction between TEMP and 1O2 but also provide a sensitive and specific assay for 1O2.

The specificity and product of quenching singlet oxygen by 2,2,6,6-tetramethylpiperidine.

The specificity of 2,2,6,6-tetramethylpiperidine to singlet oxygen was shown using Rose Bengal as a singlet oxygen generator, and Xanthine-Xanthine Oxidase and KO2 as the sources for the superoxide radical. The highest concentration of produced-singlet oxygen occurred at 25% of O2 by Rose Bengal photosensitization. The linewidth of the EPR signal for photosensitized nitroxyl radical, increasing solvent polarity. Deuterated solvents enlarge the EPR signal intensity in a dose-dependent manner. No EPR signal increase was observed in xanthine-xanthine oxidase reaction or KO2 systems, indicating that TEMP does not react with the superoxide anion. Thus, reaction of TEMP with 1O2 is highly specific.

Functional properties of cells in rat trigeminal subnucleus interpolaris following local serotonergic deafferentation.

We have previously demonstrated increases in serotonin (5-HT) content and immunoreactivity within spinal trigeminal subnucleus interpolaris (SpVi) that are correlated with the functional changes observed in this subnucleus following adult infraorbital nerve (ION) transection. To assess the possible functional significance of this change, we have examined the influence of 5-HT afference upon the normal response properties of cells in SpVi. We employed local depletion of the transmitter, using 5,7-dihydroxtryptamine (5,7-DHT), in combination with extracellular single-cell recording. Chromatographic methods revealed a 97.6% depletion of 5-HT 24 hr after neurotoxin injection. Immunocytochemical procedures revealed depletion of 5-HT throughout SpVi. Physiological recordings were made from 403 SpVi cells in 5,7-DHT-injected rats and 387 cells in vehicle-injected rats. All recordings were made 19-27 hr after injection. Horseradish peroxidase (HRP) deposits from the recording electrode were used to mark recording tracks. 5-HT depletion did not influence receptive field (RF) location, size, or continuity, or the dynamic response characteristics of SpVi cells. It did, however, (1) alter the probability that certain types of somatosensory receptor surfaces would activate local-circuit neurons, and (2) influence the rate of firing of spontaneously active SpVi cells. There was a significant increase in the proportion of vibrissa-sensitive cells with infraorbital RF components, and a concurrent decrease in the proportion of guard-hair-sensitive cells. It therefore appears that 5-HT input to SpVi is necessary for some mechanoreceptive features of the normal functional organization of this area. These functional changes were interesting in that they were opposite to those found following adult ION transection, which increases 5-HT within SpVi. Thus, changes in 5-HT central afference to SpVi that follow ION damage may be responsible for at least one type of functional change observed following this peripheral lesion.

Generation of free radicals during photosensitization of hypocrellin A and their effects on cardiac membranes.

Hypocrellin A (HA), a peryloquinone derivative, has recently been isolated from a fungus Hypocrella bambusae. This lipid soluble pigment, in combination with phototherapy, has been used to treat many skin diseases including the keloids caused by scalding and burns. We have studied the effects of photosensitized HA on biomembranes using pig heart microsomes. Photosensitization of HA was found to peroxidize the membrane lipids in the cardiac microsomes. The photodamage imposed by HA depended not only on the concentration of HA but also on the time of irradiation and pH of the system. Superoxide dismutase (SOD), ascorbic acid, beta-carotene and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) inhibited the lipid peroxidation approximately 50, approximately 50, approximately 30 and approximately 97%, respectively. Spin trapping in combination with EPR spectroscopic techniques was used to identify the reactive free radicals during the photoreaction. Formation of superoxide anion radical, (O2-.), was identified by the SOD-inhibitable DMPO-O2- EPR spectrum. Both SOD and ascorbic acid inhibited the EPR signal intensity in a dose-dependent manner with rate constants of 6.78 x 10(8) M-1 s-1 and 1.82 x 10(4) M-1 s-1, respectively. The lifetime of O2-., under these conditions, was found to be 1.1 s. Photoirradiation of HA yielded a HA free radical with a g = 2.002 which was not suppressed by SOD but in the presence of reductants such as ascorbic acid and catechol the septum was completely suppressed. The increase of the EPR signal intensity and malondialdehyde formation with increasing pH may be due, in part, to the production of predominant *HA- species at high pH which would be more reactive with oxygen to yield O2-.. These results indicate that the lipid peroxidation of the cardiac membranes observed during photooxidation of HA may arise, in part, from the interaction of membrane lipids with reactive species of oxygen and HA free radical produced during the photo-irradiation.

Time course of serotonergic afferent plasticity within rat spinal trigeminal nucleus following infraorbital nerve transection.

High-performance liquid chromatography with electrochemical detection (HPLC-ED) and immunocytochemistry were used to examine the time course of serotonergic afferent plasticity within trigeminal subnucleus interpolaris (SpVi) following infraorbital nerve (ION) transection in adult rats. Biochemical analysis was also performed in trigeminal subnucleus caudalis (SpVc) to examine the possibility of transient lesion-induced changes in this region. No significant changes in serotonin (5-HT) or 5-hydroxyindoleacetic acid (5-HIAA) concentration, or in density of 5-HT-immunoreactive (5-HTIR) axonal varicosities were observed in either subnucleus on the lesioned side, up to 51 days following ION cut. However, at 76-79 days post-lesion, a significant increase in 5-HT concentration was again demonstrated within SpVi.

Orofacial pain sensitivity in adult rats following neonatal infraorbital nerve transection.

A modification of the formalin test was used to assess orofacial pain sensitivity in adult rats that received infraorbital nerve transection at birth. Normal and neonatally lesioned adult animals received an injection of either 5% formalin or saline vehicle into the whiskerpad and the duration of whiskerpad rubbing was observed for 45 min. Normal rats given formalin exhibited the previously reported biphasic pattern of rubbing. Neonatally lesioned rats given formalin did not exhibit this pattern, and were indistinguishable from either of the saline control groups.

Vasoactive intestinal peptide, a singlet oxygen quencher.

The neuropeptide vasoactive intestinal peptide (VIP), a highly basic 28-amino acid peptide, has a widespread distribution in the body. The functional specificity of this peptide not only includes its potent vasodilatory activity, but also its role in protecting lungs against acute injury, in preventing T-lymphocyte proliferation and in modulating immune function. We have investigated the possible antioxidant properties of VIP and found that VIP does not have significant O2-, OH., or H2O2 scavenging ability. However, VIP was found to inhibit, in a dose-dependent manner, the 1O2-dependent 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO) formation. 1O2 was produced in photosensitizing systems using rose bengal or methylene blue as sensitizers and was detected as TEMP-1O2 product (TEMPO) by electron paramagnetic resonance (EPR) spectroscopic techniques. The formation of TEMPO signal was strongly inhibited by known singlet quenchers, e.g. beta-carotene, histidine as well as azide, but not by catalase (20 micrograms/ml) which removes H2O2 and mannitol (6 mM) or ethanol (5.9 mM) which remove OH.. Superoxide dismutase (2.5 micrograms/ml) inhibited the photoreaction up to 20% by removing O2- and most probably by blocking the secondary charge transfer pathway of 1O2 formation. These results suggest that the formation of nitroxide radical by 1O2 attack on TEMP may be used as a simple and specific assay for 1O2, and VIP can serve as an effective 1O2 scavenger/quencher, thus it may modulate the oxidative tissue injury caused by this reactive species of oxygen.