Protective effects of phosphodiesterase 2 inhibitor on depression- and anxiety-like behaviors: involvement of antioxidant and anti-apoptotic mechanisms.

Stress occurs in everyday life, but the relationship between stress and the onset or development of depression/anxiety remains unknown. Increasing evidence suggests that the impairment of antioxidant defense and the neuronal cell death are important in the process of emotional disorders. Chronic stress impairs the homeostasis of antioxidants/oxidation, which results in the aberrant stimulation of the cell cycle proteins where cGMP-PKG signaling is thought to have an inhibitory role. Phosphodiesterase 2 (PDE2) is linked to cGMP-PKG signaling and highly expressed in the limbic brain regions including hippocampus and amygdala, which may play important roles in the treatment of depression and anxiety. To address the possible effects of PDE2 inhibitors on depression-/anxiety-like behaviors and the underlying mechanisms, Bay 60-7550 (0.75, 1.5 and 3 mg/kg, i.p.) was administered 30 min before chronic stress. The results suggested that Bay 60-7550 not only restored the behavioral changes but also regulated Cu/Zn superoxide dismutase (SOD) levels differentially in hippocampus and amygdala, which were increased in the hippocampus while decreased in the amygdala. It was also significant that Bay 60-7550 regulated the abnormalities of pro- and anti-apoptotic components, such as Bax, Caspase 3 and Bcl-2, and the indicator of PKG signaling characterized by pVASP(ser239), in these two brain regions. The results suggested that Bay 60-7550 is able to alleviate oxidative stress and mediate part of the apoptotic machinery in neuronal cells possibly through SOD-cGMP/PKG-anti-apoptosis signaling and that inhibition of PDE2 may represent a novel therapeutic target for psychiatric disorders, such as depression and anxiety.

Anxiolytic effects of phosphodiesterase-2 inhibitors associated with increased cGMP signaling.

Phosphodiesterase (PDE)-2 is a component of the nitric-oxide synthase (NOS)/guanylyl cyclase signaling pathway in the brain. Given recent evidence that pharmacologically induced changes in NO-cGMP signaling can affect anxiety-related behaviors, the effects of the PDE2 inhibitors (2-(3,4-dimethoxybenzyl)-7-det-5-methylimidazo-[5,1-f][1,2,4]triazin-4(3H)-one) (Bay 60-7550) and 3-(8-methoxy-1-methyl-2-oxo-7-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-5-yl)benzamide (ND7001), as well as modulators of NO, were assessed on cGMP signaling in neurons and on the behavior of mice in the elevated plus-maze, hole-board, and open-field tests, well established procedures for the evaluation of anxiolytics. Bay 60-7550 (1 microM) and ND7001 (10 microM) increased basal and N-methyl-d-aspartate- or detanonoate-stimulated cGMP in primary cultures of rat cerebral cortical neurons; Bay 60-7550, but not ND7001, also increased cAMP. Increased cGMP signaling, either by administration of the PDE2 inhibitors Bay 60-7550 (0.5, 1, and 3 mg/kg) or ND7001 (1 mg/kg), or the NO donor detanonoate (0.5 mg/kg), antagonized the anxiogenic effects of restraint stress on behavior in the three tests. These drugs also produced anxiolytic effects on behavior in nonstressed mice in the elevated plus-maze and hole-board tests; these effects were antagonized by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (20 mg/kg). By contrast, the NOS inhibitor N(omega)-nitro-l-arginine methyl ester (50 mg/kg), which reduces cGMP signaling, produced anxiogenic effects similar to restraint stress. Overall, the present behavioral and neurochemical data suggest that PDE2 may be a novel pharmacological target for the development of drugs for the treatment of anxiety disorders.

Reversal of oxidative stress-induced anxiety by inhibition of phosphodiesterase-2 in mice.

The pathogenesis of several neuropsychiatric disorders, including anxiety and depression, has been linked to oxidative stress, in part via alterations in cyclic nucleotide signaling. Phosphodiesterase-2 (PDE2), which regulates cGMP and cAMP signaling, may affect anxiety-related behavior through reduction of oxidative stress. The present study evaluated the effects of oxidative stress on behavior and assessed the anxiolytic effects of the PDE2 inhibitor Bay 60-7550 [(2-(3,4-dimethoxybenzyl)-7-{(1R)-1-[(1R)-1-hydroxyethyl]-4-phenylbutyl}-5-methyl imidazo-[5,1-f][1,2,4]triazin-4(3H)-one)]. Treatment of mice with L-buthionine-(S,R)-sulfoximine (300 mg/kg), an inducer of oxidative stress, caused anxiety-like behavioral effects in elevated plusmaze, open-field, and hole-board tests through the NADPH oxidase pathway; these effects were antagonized by Bay 60-7550 (3 mg/kg) and apocynin (3 mg/kg), an inhibitor of NADPH oxidase. The Bay 60-7550-mediated decrease in oxidative stress (i.e., superoxide anion and reactive oxygen species generation in cultured neurons and total antioxidant capacity and lipid peroxides in amygdala and hypothalamus) and expression of NADPH oxidase subunits (i.e., p47 phox and gp91 phox expression in amygdala, hypothalamus, and cultured neurons) was associated with increased cGMP and phosphorylation of vasodilator-stimulated phosphoprotein at Ser239, suggesting an important role of cGMP-protein kinase G signaling in reduction of anxiety. Overall, the present results indicate that oxidative stress induces anxiety-like behavior in mice and that PDE2 inhibition reverses it through an increase in cGMP signaling. Thus, PDE2 may be a novel pharmacological target for treatment of anxiety in neuropsychiatric and neurodegenerative disorders that involve oxidative stress.

Oxidant--antioxidant imbalance in asthma: scientific evidence, epidemiological data and possible therapeutic options.

Prevalence of asthma has increased considerably in recent decades throughout the world especially in developed countries. Airway inflammation is thought to be prime cause for repeated episodes of airway obstruction in asthmatics. Several studies have shown that reactive oxygen species (ROS) play a key role in initiation as well as amplification of inflammation in asthmatic airways. Excessive ROS production in asthma leads to alteration in key enzymatic as well as nonenzymatic antioxidants such as glutathione, vitamins C and E, beta-carotene, uric acid, thioredoxin, superoxide dismutases, catalase, and glutathione peroxidases leading to oxidant-antioxidant imbalance in airways. Oxidant-antioxidant imbalance leads to pathophysiological effects associated with asthma such as vascular permeability, mucus hypersecretion, smooth muscle contraction, and epithelial shedding. Epidemiological data also support the scientific evidence of oxidant-antioxidant imbalance in asthmatics. Therefore, the supplementation of antioxidants to boost the endogenous antioxidants or scavenge excessive ROS production could be utilized to dampen/prevent the inflammatory response in asthma by restoring oxidant-antioxidant balance. This review summarizes the scientific and epidemiological evidence linking asthma with oxidant-antioxidant imbalance and possible antioxidant strategies that can be used therapeutically for better management of asthma.

Anxiogenic-like behavioral phenotype of mice deficient in phosphodiesterase 4B (PDE4B).

Phosphodiesterase-4 (PDE4), an enzyme that catalyzes the hydrolysis of cyclic AMP and plays a critical role in controlling its intracellular concentration, has been implicated in depression- and anxiety-like behaviors. However, the functions of the four PDE4 subfamilies (PDE4A, PDE4B, PDE4C, and PDE4D) remain largely unknown. In animal tests sensitive to anxiolytics, antidepressants, memory enhancers, or analgesics, we examined the behavioral phenotype of mice deficient in PDE4B (PDE4B-/-). Immunoblot analysis revealed loss of PDE4B expression in the cerebral cortex and amygdala of PDE4B-/- mice. The reduction of PDE4B expression was accompanied by decreases in PDE4 activity in the brain regions of PDE4B-/- mice. Compared to PDE4B+/+ littermates, PDE4B-/- mice displayed anxiogenic-like behavior, as evidenced by decreased head-dips and time spent in head-dipping in the holeboard test, reduced transitions and time on the light side in the light-dark transition test, and decreased initial exploration and rears in the open-field test. Consistent with anxiogenic-like behavior, PDE4B-/- mice displayed increased levels of plasma corticosterone. In addition, these mice also showed a modest increase in the proliferation of neuronal cells in the hippocampal dentate gyrus. In the forced-swim test, PDE4B-/- mice exhibited decreased immobility; however, this was not supported by the results from the tail-suspension test. PDE4B-/- mice did not display changes in memory, locomotor activity, or nociceptive responses. Taken together, these results suggest that the PDE4B subfamily is involved in signaling pathways that contribute to anxiogenic-like effects on behavior.

Involvement of nitric oxide (NO) in the regulation of stress susceptibility and adaptation in rats.

The present study evaluated the regulatory role of nitric oxide (NO) in stress susceptibility and adaptation in rats. Acute restraint stress (RS x1) reduced the number of entries and time spent in the open arms in the elevated plus maze (EPM) test and raised plasma corticosterone levels. RS (x1)-induced neurobehavioral suppression and raised corticosterone levels were attenuated by pretreatment with the NO precursor, L-arginine (500 and 1000 mg/kg)and unaffected or further aggravated by NO synthase inhibitor, L-NAME or 7-nitroindazole (10 and 50 mg/kg). Biochemical assay of plasma and brain homogenates showed that these RS - induced behavioral and neuroendocrinal changes were associated with lowered levels of plasma and brain total nitrates/nitrites (NOx). L-Arginine attenuated the RS-induced suppression of NOx levels in plasma and brain, whereas, the NO synthase inhibitors tended to produce reverse effects. In the experiments involving repeated stress i.e. RS (x5), exposure resulted in attenuation/reversal of (a) neurobehavioral suppression in the EPM test and (b) lowered brain NOx, that was seen after RS (x1). The RS (x5)-induced changes in EPM parameters and brain Nox were further potentiated after L-arginine pretreatment, whereas, the NO synthase inhibitors were less effective. Rats were screened as high and low emotional in the open-field test, and high emotional rats showed greater(a) behavioral suppression in the EPM, (b) corticosterone responses (c) brain NOx suppression, and (d) cold-restraint stress (CRS) induced gastric mucosal lesions as compared to their low emotional counterparts. L-Arginine pretreatment was more effective in modulating the above RS induced stress responses/markers in the high emotional group of rats. Our data suggest that NO plays a differential role during exposure to acute and repeated stress situations, and that the relationship between stress and emotionality status may be under the regulatory influence of NO.

Immobilization stress causes extra-cellular oxidant-antioxidant imbalance in rats: restoration by L-NAME and vitamin E.

Stress has been shown to be associated with altered homeostasis that may lead to oxidant-antioxidant imbalance. Non-enzymatic antioxidants are important regulators of reactive oxygen species produced in extra-cellular milieu and represent the first line of defense against them. Extra-cellular non-enzymatic antioxidants may be disturbed by the production of superoxide and nitric oxide and this has not been studied in stressful situation previously. In the present study, effects of immobilization stress (IS), both acute (IS x 1) and repeated (IS x 7) were assessed on extra-cellular total antioxidant capacity measured as plasma ferric reducing antioxidant power (FRAP) and protein sulfhydryls, and oxidative stress measured as leukocyte superoxide generation, plasma nitric oxide production (total nitrates and nitrites, NOx) and lipid peroxides in rats. Effects of pretreatment with nitric oxide synthase (NOS) inhibitors and vitamin E were also studied on these biochemical parameters. The results showed that both IS x 1 and IS x 7 resulted in extra-cellular oxidant-antioxidant imbalance as oxidant generation was increased and non-enzymatic antioxidants were depleted. Pretreatment either with NOS inhibitors or vitamin E restored stress-induced extracellular oxidant-antioxidant imbalance implying their potential role as antioxidants. Our data suggest that there is extra-cellular oxidant-antioxidant imbalance in the stressed rats, with greater magnitude of severity in repeated stress paradigm. Augmentation of antioxidant defenses might be beneficial in long-term stress.

Pharmacological and biochemical studies on the possible role of nitric oxide in stress adaptation in rats.

The involvement of nitric oxide (NO) in stress adaptation was evaluated in rats using the elevated plus maze test. Repeated restraint stress RS(x 5) for 5 days resulted in an increase in the percentage number of entries and percentage time spent when compared to a single restraint stress RS(x 1) exposure. In the repeated RS treatment groups, the nitric oxide donor, L-arginine (500 and 1000 mg/kg, i.p.) slightly increased the elevated plus maze test parameters when compared to the corresponding vehicle-treated group. The nitric oxide synthase (NOS) inhibitors, N-nitro-L-arginine methyl ester (L-NAME, 10 and 50 mg/kg, i.p.) and 7-nitroindazole (10 and 50 mg/kg, i.p.) produced differential responses in both the parameters with L-NAME exhibiting greater reduction in open arm entries and open arm time, whereas 7-nitroindazole produced only small differences in both the elevated plus maze parameters. Biochemical data showed that repeated restraint stress resulted in higher levels of brain nitrates and nitrites (NOx) as compared to that of single restraint stress exposure. Further, in L-arginine (1000 mg/kg, i.p.)-treated rats, brain NOx was lowest in the single restraint stress group, followed by repeated restraint stress and (no restraint stress) controls. The results are suggestive of the role of nitric oxide in stress adaptation and this may be due to the effects of restraint stress on brain NOS activity.

Increased oxidative stress and altered levels of antioxidants in asthma.

BACKGROUND: Reactive oxygen species might play an important role in the modulation of airway inflammation. There is evidence of an oxidant-antioxidant imbalance in asthma. Although several oxidants and antioxidants are likely to be involved, alterations in only limited parameters have been studied in isolation. OBJECTIVE: We investigated changes in a wide range of oxidants and antioxidants to create a comprehensive picture of oxidant-antioxidant imbalance. METHODS: In the peripheral blood of 38 patients with bronchial asthma and 23 control subjects, oxidative stress was measured in terms of superoxide anion generation by leukocytes, lipid peroxidation products, total nitrates and nitrites, total protein carbonyls, and total protein sulfhydrils in plasma. Antioxidant status was evaluated by measuring red blood cell superoxide dismutase and catalase activity, total blood glutathione, and glutathione peroxidase activity in red blood cells and leukocytes and total antioxidant capacity in plasma. RESULTS: Asthmatic patients showed increased superoxide generation from leukocytes, increased total nitrites and nitrates, increased protein carbonyls, and increased lipid peroxidation products and decreased protein sulfhydrils in plasma, indicating increased oxidative stress. They also showed increased superoxide dismutase activity in red blood cells and increased total blood glutathione and decreased glutathione peroxidase activity in red blood cells and leukocytes. Red blood cell catalase activity and the total antioxidant capacity of plasma were not altered. CONCLUSION: There are alterations in a wide array of oxidants and antioxidants, with balance shifting toward increased oxidative stress in asthma. Therapeutic augmentation of the antioxidant defenses might be beneficial.

Modulation of stress-induced neurobehavioral changes by nitric oxide in rats.

The involvement of nitric oxide (NO) in stress-induced neurobehavioral changes in rats was evaluated using the elevated plus maze and open field tests. Restraint stress (1 h) reduced both the number of entries and time spent in open arms, with both expressed as percent of controls (no restraint stress), and these changes were reversed with diazepam (1 mg/kg) and the NO precursor, L-arginine (500 and 1000 mg/kg) pretreatment. The nitric oxide synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME) (50 mg/kg), aggravated restraint stress effects in the elevated plus maze test, whereas the lower dose (10 mg/kg) of the drug attenuated the same. In the open field test, the restraint stress-induced (a) increased entry latency and (b) decreased ambulation and rearing were reversed by diazepam and L-arginine and L-NAME (10 mg/kg), whereas L-NAME (50 mg/kg) aggravated restraint stress effects. The neuronal nitric oxide synthase inhibitor, 7-nitroindazole (10 and 50 mg/kg), did not influence these restraint stress-induced behavioral changes to any significant extent. Biochemical data showed that L-NAME (10 and 50 mg/kg.) induced opposite effects on the total brain nitrate/nitrite content during restraint stress. The results indicate a possible involvement of NO in stress-induced neurobehavioral effects.