Pathophysiology and management of Akathisia 70 years after the introduction of the chlorpromazine, the first antipsychotic.

OBJECTIVE: Akathisia is among the most troubling effects of psychiatric drugs as it is associated with significant distress on behalf of the patients, and it limits treatment adherence. Though it most commonly presents during treatment with antipsychotic drugs which block dopamine D2 receptors, Akathisia has also been reported during treatment with selective serotonin reuptake inhibitors (SSRIs), serotonin norepinephrine reuptake inhibitors (SNRIs), stimulants, mirtazapine, tetrabenazine and other drugs. MATERIALS AND METHODS: This article was designed as a narrative review on akathisia with a focus on its clinical presentation, pathophysiology and management. A PubMed search for akathisia was conducted which returned 8481 articles. RESULTS: Akathisia is experienced as severe restlessness commonly accompanied by dysphoria and purposeless movement which relieves subjective tension. It has been attributed to an imbalance between dopaminergic and noradrenergic neurotransmission in the basal ganglia. Acute akathisia commonly resolves upon treatment discontinuation but tardive and chronic akathisia may persist after the causative agent is withdrawn and prove resistant to pharmacological treatment. Even drugs which induce no other extrapyramidal side effects (such as clozapine, quetiapine, aripiprazole and cariprazine) may induce akathisia. A high index of suspicion should be maintained in patients with motor disabilities, drug-induced parkinsonism and those under mechanical restraint. Propranolol and low-dose mirtazapine are the most thoroughly studied pharmacological interventions for akathisia, though benzodiazepines, voltage-gated calcium channel blockers (gabapentin, pregabalin) and opioids may be effective. CONCLUSIONS: Pharmacological management may pose a challenge in chronic akathisia. Rotation between different pharmacological management strategies may be optimal in resistant cases. Discontinuation of the causative drug and use of b-blockers, mirtazapine, benzodiazepines or gabapentinoids for symptomatic relief is the basis of management.

Gas phase oxidants of cigarette smoke increase nitric oxide synthase and xanthine oxidase activities of rabbit brain synaptosomes.

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 microM) 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.

Inhibition of ultraviolet B-induced skin erythema by N-nitro-L-arginine and N-monomethyl-L-arginine.

Ultraviolet B (UVB)-irradiated human keratinocytes and human endothelial cells release nitrogen oxides, i.e. nitric oxide (NO). S-nitrosothiols, hydroxylamine (H2NOH) as well as ammonia (NH3) formed from L-arginine. Generation of these compounds was time and concentration-dependent and decreased by both N-monomethyl-L-arginine (L-NMMA) and N-nitro-L-arginine (L-NA). UVB radiation of the cells resulted in a concomitant increase of soluble guanylate cyclase (sGC) activity which was inhibited by L-NMMA and L-NA. S-nitrosothiols formed during the irradiation of the cells directly increased purified sGC activity by a mechanism characteristic of release of NO from a carried molecule. UVB-irradiated cells promptly increased thiobarbituric acid reacting substance (TBARS) (estimated as malondialdehyde. MDA) production which were inhibited by desferrioxamine. In in vivo experiments using guinea pigs subjected to UVB radiation, a Protection Factor (PF) of 2.25 +/- 0.75 was calculated when an emulsified cream formulation containing L-NMMA (1% w/w) and L-NA (1% w/w) was applied to their skin. In human volunteers subjected to UVB radiation, a dose-dependent increase of PF was observed. When an emulsified cream formulation containing L-NMMA (1% w/w) and L-NA (1% w/w) was applied to their skin the PF was 2.15 +/- 0.80: by increasing the concentration of L-NMMA (1% w/w) and L-NA (2% w/w) the PF was 4.25 +/- 1.25. The present results indicate that UVB radiation acts as a potent stimulator of human keratinocytes and endothelial cells to release nitrogen oxides that may diffuse out of the keratinocytes and endothelial cells, activating sGC in neighboring smooth muscle cells. This may be a major part of the integrated response of the skin leading to vasodilation and erythema.

Calmodulin-related changes in microsomal membrane fluidity during liver regeneration.

Based on our previous findings on the modifying effect of calmodulin (CaM) on the physiochemical properties of biomembrane, we have investigated the possible relationship between intracellular CaM content and endoplasmic reticulum (ER) membrane fluidity and function during liver regeneration. The degree of ER membrane fluidity was estimated by fluorescence polarization analysis with the 1,6-diphenyl-1,3,5-hexatriene probe. Microsomal guanylate cyclase (GC) was used as a functional parameter. The kinetics of the increase in the ER membrane fluidity during liver regeneration was strictly parallel to the CaM surge and was matched by an increase in GC activity. The stimulative effect of splenectomy on liver regeneration and its inhibition by Walker-256 tumor, inferred from the corresponding alterations of CaM levels, were mirrored by the modulation in GC activity. The fluidizing effect of CaM on ER membrane was concluded from the drop in thermotropic transition temperature from 28.3 +/- 1.6 degrees C in control membranes to 17.8 +/- 1.1 degrees C membranes from regenerating livers and to 19.8 +/- 1.2 degrees C in control membranes treated with CaM. Arrhenius plots of GC activity exhibited a transition temperature of 25.5 +/- 1.25 degrees C in controls, which shifted to 20.5 +/- 0.9 degrees C in ER membranes from regenerating livers and to 21.7 +/- 1.1 degrees C in control membranes treated with CaM. The Hill coefficient for the allosteric activation of the GC by Mn.GTP decreased from 1.49 +/- 0.16 in controls to 0.93 +/- 0.085 in membranes from regenerating cells and to 0.86 +/- 0.073 in CaM-treated membranes. Both effects of CaM were consistent with a fluidity increase in the enzyme's lipid microenvironment. The results of the present study suggest that an early key event in liver regeneration may be the CaM-induced modulation of ER membrane fluidity and function.

Increase of particulate nitric oxide synthase activity and peroxynitrite synthesis in UVB-irradiated keratinocyte membranes.

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.

Nitric oxide synthase and xanthine oxidase activities in the spermatic vein of patients with varicocele: a potential role for nitric oxide and peroxynitrite in sperm dysfunction.

PURPOSE: The 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 METHODS: Whole 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. RESULTS: Serum 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. CONCLUSIONS: Our data suggest a high oxidative stress due to the release of nitric oxide synthase and xanthine oxidase within the dilated spermatic vein. The 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.

Nitric oxide and peroxynitrite released by ultraviolet B-irradiated human endothelial cells are possibly involved in skin erythema and inflammation.

In this study we attempted to demonstrate whether endothelial cell nitric oxide synthase (eNOS) and xanthine oxidase (XO) could be activated to release nitric oxide (NO) and peroxynitrite (ONOO-) following exposure to ultraviolet B (UVB) radiation and to define whether this light-induced response could be involved in the pathogenesis of sunburn erythema and inflammation. Treatment of human endothelial cells with UVB (290-320 nm) radiation (up to 100 mJ/cm2) resulted in an increase of both NO and ONOO- release that was inhibited by NG-monomethyl-L-arginine (L-NMMA). Treatment of cell cytosol with various doses of UVB radiation (up to 20 mJ/cm2) resulted in a threefold increase of XO activity that was inhibited (approximately 90% by oxypurinol. In reconstitution experiments, when purified eNOS was added to purified XO, an almost fourfold increase in ONOO- production at 20 mj/cm2 UVB radiation was observed. UVB radiation (100 mg/cm2) decreased cell membrane fluidity, indicating changes in the physicochemical characteristics of the membranes. In in vivo experiments, when human volunteers were subjected to UVB light, a protection factor (PF) of 3.90 +/- 0.85 was calculated when an emulsified cream formulation containing nitro-L-arginine (L-NA; 2%) and L-NMMA (2%) was applied to their skin. The present studies indicate that UVB radiation acts as a potent stimulator of eNOS and XO in human endothelial cells. The cytotoxic effects of NO and ONOO- may be the main factors in the integrated response of the skin leading to vasodilatation, the first key event of erythema production and the inflammation process.

Tumour promoter tert-butyl-hydroperoxide induces peroxynitrite formation in human erythrocytes.

In the present study we demonstrated that human erythrocytes under normal conditions release small amounts of peroxynitrite (ONOO-) that can be considerably increased by the tumour promoter t-butylhydroperoxide (t-BHP), with a subsequent increase in lipid peroxidation and inhibition of Ca2+ pump ATPase activity. By causing oxidative stress in human erythrocytes with t-BHP, ONOO- release was increased approximately ten fold. N-monomethyl-L-arginine (L-NMMA) inhibited ONOO- release by approximately 90% while D-NMMA had no effect. The interaction of t-BHP with hemoglobin (Hb) and methemoglobin (MetHb) caused the production of superoxide (O2-) and hydrogen peroxide (H2O2). The differential direct effect of t-BHP on Hb and MetHb was investigated by taking their spectra in the presence or absence of cytochrome C. Erythrocyte membranes treated either with t-BHP or with ONOO- were subjected to oxidative stress with a subsequent increase in malondialdehyde (MDA) production and decrease in membrane fluidity, as estimated by the fluorescence polarization of 1,6,diphenyl-1,3,5-hexatriene (DPH). Ca2+ pump ATPase activity was decreased in t-BHP and/or ONOO-(-)treated erythrocytes, indicating that the subsequent intracellular calcium increase promoted ONOO- production by activating the Ca(2+)-calmodulin-dependent NO-synthase activity. These results suggest that hemoglobin oxidation by the tumour promoters play a key role in oxidative damage to erythrocytes and that the t-BHP/ Hb redox system could be a useful tool for investigating the tumour promoting efficacy of organic hydroperoxides.

Alterations of nitric oxide synthase and xanthine oxidase activities of human keratinocytes by ultraviolet B radiation. Potential role for peroxynitrite in skin inflammation.

In the present study, we demonstrated that NO synthase (cNOS) and xanthine oxidase (XO) of human keratinocytes can be activated to release NO, superoxide (O2-) and peroxynitrite (ONOO-) following exposure to ultraviolet B (UVB) radiation. We defined that this photo induced response may be involved in the pathogenesis of sunburn erythema and inflammation. Treatment of human keratinocytes with UVB (290-320 nm) radiation (up to 200 mJ/cm2) resulted in a dose-dependent increase in NO and ONOO- release that was inhibited by N-monomethyl-L-arginine (L-NMMA). NO and ONOO- release from keratinocytes was accompanied by an increase in intracellular cGMP levels. Treatment of human keratinocyte cytosol with various doses of UVB (up to 100 mJ/cm2) resulted in an increase in XO activity that was inhibited by oxypurinol. UVB radiation (up to 100 mJ/cm2) of keratinocytes resulted in a 15-fold increase in S-nitrosothiol formation, which directly increased purified soluble guanylate cyclase (sGC) activity by a mechanism characteristic of release of NO from a carrier molecule. In reconstitution experiments, when UVB-irradiated (20 mJ/cm2) purified cNOS isolated from keratinocyte cytosol was combined with UVB-irradiated (20 mJ/cm2) purified XO, a 4-fold increase in ONOO- production, as compared to nonirradiated enzymes, was observed. ONOO- synthesized by NO and O2- following UVB radiation of cNOS and XO was inhibited by oxypurinol (100 microM). UVB radiation of keratinocyte cytosol resulted in an increase in oxygen free radical production, consistent with the increased production of ONOO- by UVB-irradiated keratinocyte cytosol. In in vivo experiments, when experimental animals were subjected to UVB radiation, a protection factor (PF) of 6.5 +/- 1.8 was calculated when an emulsified cream formulation containing nitro-L-arginine (L-NA) (2%) and L-NMMA (2%) was applied to their skin. The present study indicates that UVB radiation acts as a potent stimulator of cNOS and XO activities in human keratinocytes. NO and ONOO- may exert cytotoxic effects in keratinocytes themselves, as well as in their neighboring endothelial and smooth muscle cells. This may be a major part of the integrated response leading to erythema production and the inflammation process.

NO synthase and xanthine oxidase activities of rabbit brain synaptosomes: peroxynitrite formation as a causative factor of neurotoxicity.

In the present study we demonstrated that synaptosomes isolated from rabbit brain cortex contain NO synthase and xanthine oxidase that can be activated by ultraviolet B radiation and Ca2+ accumulation to produce nitric oxide and superoxide which react together to form peroxynitrite. Irradiation of synaptosomes with ultraviolet B (up to 100 mJ/cm2), or increase the intrasynaptosomal calcium concentration using various doses (up to 100 mu M) of the calcium ionophore A 23187, a gradual increase in both nitric oxide and peroxynitrite release that was inhibited by N-monomethyl-L-arginine (100 mu M) was observed. The rate of nitric oxide release and cyclic GMP production by NO synthase and soluble guanylate cyclase, both located in the soluble fraction of synaptosomes (synaptosol), were increased approximately eight fold after treatment of synaptosomes with Ultraviolet B radiation (100 mJ/cm2). In reconstitution experiments, when purified NO synthase isolated from synaptosol was added to xanthine oxidase, in the presence of the appropriate cofactors and substrates, a ten fold increase in peroxynitrite production at various doses (up to 20 mJ/cm2) of UVB radiation was observed. Ultraviolet B irradiated synaptosomes 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. Desferrioxamine (100 mu M) tested in Ultraviolet B-irradiated synaptosomes showed a decrease (approximately 80%) in malondialdehyde production with subsequent restoration of the membrane fluidity to that of non-irradiated (control) synaptosomes. Ca(2+)-stimulated ATPase activity was decreased after Ultraviolet B (100 mJ/cm2) radiation of synaptosomes indicating that the subsequent increase of intrasynaptosomal calcium promoted peroxynitrite production by a calmodulin-dependent increase of NO synthase and xanthine oxidase activities. Furthermore, it was shown that UVB-irradiated synaptosomes were subjected to higher oxidative stress by exogenous peroxynitrite (100 mu M) compared to non-irradiated (control) synaptosomes. In summary, the present results indicate that activation of NO synthase and xanthine oxidase of brain cells lead to the formation of peroxynitrite providing important clues in the role of peroxynitrite as a causative factor in neurotoxicity.

Nitric oxide and peroxynitrite production by human erythrocytes: a causative factor of toxic anemia in breast cancer patients.

In the present study we demonstrated that human erythrocytes possess a NO synthase (NOS) that can be activated by oxidative stress and Ca2+ accumulation to produce nitric oxide (NO), and that this activation could be involved in the pathogenesis of toxic anaemia in breast cancer patients. By causing oxidative stress in human erythrocytes with hydrogen peroxide (H2O2) (100 microM), or by increasing the intracellular calcium concentration using various doses (up to 100 microM) of the calcium ionophore A23187, a gradual increase in both NO and peroxynitrite (ONOO-) release that was inhibited by N-monomethyl-L-arginine (L-NMMA) (1mM) was observed. Time-dependent experiments using hemolysates showed a linear rise of NO production which was elevated by 60% in the presence of superoxide dismutase (SOD) (100 U). NOS isolated from hemolysates was constitutively expressed and was dependent on NADPH, Ca2+/calmodulin, tetrahydrobiopterin and flavins. In reconstitution experiments, when purified NOS, isolated from erythrocytes, was added to purified soluble guanylate cyclase (sGC), isolated from endothelial cells, in the presence of the appropriate cofactors and substrates, a linear increase in cGMP production at various concentrations (up to 50 microM) of H2O2 was observed. Furthermore, it was shown that erythrocytes from breast cancer patients were subjected to higher oxidative stress by ONOO- (100 microM), with a consequential increase of membrane rigidity, than erythrocytes from healthy individuals. Such mechanic changes may result in shortening of the lifespan of erythrocytes, a feature of toxic anemia in cancer patients.

Nitric oxide, peroxynitrite and nitroso-compounds formation by ultraviolet A (UVA) irradiated human squamous cell carcinoma: potential role of nitric oxide in cancer prognosis.

Ultraviolet A (UVA) irradiated human squamous cell carcinoma (SCC-13) releases nitrogen oxides, i.e. nitric oxide (NO), peroxynitrite (ONOO-), nitrosocompounds, ammonia (NH3) and hydroxylamine (H2NOH) formed from L-arginine. Formation and/or release of these nitrogen oxides was time and concentration-dependently stimulated by UVA and decreased by N-monomethyl-L-arginine (L-NMMA), a compound that inhibits NO synthase activity. UVA irradiation of SCC-13 cells resulted in concomitant increase in soluble guanylate cyclase (sGC) which was inhibited by L-NMMA. The increased NO and ONOO- production evoked by dibutyryl cGMP and 3-isobutyl-l-methyl-xanthine (IBMX) represents an additional positive feedback mechanism that could serve to maintain NO and ONOO- release for extended periods following UVA radiation. Using an in vitro chemical model system, it was demonstrated that oxidation of NH3 to NO by hydroxyl radical (.OH) at physiological pH is chemically feasible. UVA irradiated SCC-13 cells induced a luminol-enhanced chemiluminescence signal that reaches a peak within 1 min. The modulation of this signal by ebselen is consistent with a rate-determining step corresponding to the disproportionation of a luminol-superoxide (O2-) complex. UVA irradiated SCC-13 cells promptly increased malondialdehyde (MDA) production with subsequent decrease of plasma membrane fluidity. Desferrioxamine tested in UVA irradiated SCC-13 cells showed a concentration dependent decrease in MDA production with subsequent restoration of the membrane fluidity to the normal level. Furthermore, it was shown that squamous cell carcinoma possesses higher NO synthase and sGC activity as compared to normal keratinocytes. Such an increase in NO production may be directly related to the poor prognosis of squamous cell carcinoma.

Modulation of particulate nitric oxide synthase activity and peroxynitrite synthesis in cholesterol enriched endothelial cell membranes.

Endothelium-derived relaxing factor/nitric oxide (EDRF/NO) is produced by the vascular wall and is a key modulator of vascular tone and blood pressure. Since reduced EDRF/NO release from the endothelium is a major key event in the development of atherosclerosis, we investigated the effect of cholesterol on endothelial cell particulate (membrane-bound) NO synthase activity. Low concentrations (up to 0.2 mM) of liposomal cholesterol progressively activated plasma membrane-bound NO synthase. Increasing cholesterol concentration above that which maximally stimulated enzyme activity produced a progressive inhibition with respect to the control value. In time course experiments using endothelial cell plasma membranes enriched with cholesterol, changes in NO production were followed by analogous changes in soluble guanylate cyclase activity (sGC). N-Monomethyl-L-arginine (L-NMMA) (1 mM) inhibited particulate NO synthase activity at all cholesterol concentrations used with subsequent decreases in cGMP production. Egg lecithin liposomes (free of cholesterol) had no effect on NO synthase activity. A three-fold increase in superoxide (O2-) and a 2.5-fold increase in NO formation followed by an eight-fold increase in peroxynitrite (ONOO-) production by cholesterol-treated microsomes isolated from endothelial cells was observed, one which rose further up to eight-fold in the presence of superoxide dismutase (SOD) (10 U/mL). Cholesterol had no effect on Lubrol-PX solubilized membrane-bound NO synthase or on cytosolic (soluble) NO synthase activities of endothelial cells. Cholesterol modulated lipid fluidity of plasma membranes labelled with 1,6-diphenyl-1,3,5-hexatriene (DPH) as indicated by the steady state fluorescence anisotropy [(ro/r)-1]-1. Arrhenius plots of [(ro/r)-1]-1 indicated that the lipid phase separation of the membranes at 26.2 +/- 1.5 degrees was elevated to 34.4 +/- 1.9 degrees in cholesterol-enriched membranes, consistent with a general decrease in membrane fluidity. Cholesterol-enriched plasma membranes treated with egg lecithin liposomes showed a lipid phase separation at 27.5 +/- 1.6 degrees, indicating the reversible effect of cholesterol on membrane lipid fluidity. Arrhenius plots of NO synthase activity exhibited break point at 26.9 +/- 1.8 degrees which rose to 35.6 +/- 2.1 degrees in 0.5 mM cholesterol-treated plasma membranes and decreased to 21.5 +/- 1.4 degrees in plasma membranes treated with 0.2 mM cholesterol. The allosteric properties of plasma membrane-bound NO synthase inhibited by Mn2+ (as reflected by changes in the Hill coefficient) were changed by cholesterol, consistent with modulations of the fluidity of the lipid microenvironment of the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)

Release by ultraviolet B (u.v.B) radiation of nitric oxide (NO) from human keratinocytes: a potential role for nitric oxide in erythema production.

1. The mechanism of human sunburn is poorly understood but its characteristic features include the development of erythema. In this study we attempted to determine whether human keratinocytes possess a nitric oxide (NO) synthase (NOS), if this enzyme could be activated to release NO following exposure to ultraviolet B (u.v.B) and to define whether this photo-induced response could be involved in the pathogenesis of sunburn erythema. 2. Treatment of human keratinocytes with various doses of u.v.B (290-320 nm) radiation (up to 100 mJ cm-2) resulted in a dose-dependent release of NO and cyclic GMP production that was reduced by NG-monomethyl-L-arginine (L-NMMA). 3. u.v.B irradiation of keratinocyte cytosol at varying doses (up to 50 mJ cm-2), resulted in a gradual rise in NO production, with a concomitant increase in soluble guanylate cyclase activity (sGC). 4. NOS isolated from the keratinocyte cytosol was constitutively expressed and was dependent on NADPH, Ca2+/calmodulin, tetrahydrobiopterin and flavins. 5. In reconstitution experiments, when purified NOS was added to purified sGC, both isolated from keratinocyte cytosol, a four fold increase in cyclic GMP was observed. The GMP was increased by NO synthesized following u.v.B radiation (up to 20 mJ cm-2) of NOS. 6. In in vivo experiments, guinea-pigs were subjected to u.v.B light. A Protection Factor (PF) of 8.71 +/- 2.85 was calculated when an emulsified cream formulation containing L-NMMA (2%) was applied to their skin. 7. The present results indicate that u.v.B radiation acts as a potent stimulator of NOS in keratinocytes. NO is lipophilic and may diffuse out of the keratinocytes, activating sGC in endothelial cells and neighbouring smooth muscle cells. This may be a major part of the integrated response of the skin leading to vasodilatation and erythema.

Met-enkephalin receptor-mediated increase of membrane fluidity modulates nitric oxide (NO) and cGMP production in rat brain synaptosomes.

The association of [3H]-Met-enkephalin with synaptosomes isolated from rat brain cortex, when incubated for 30 min at 25 degrees 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. Arrhenius-type plots of [(ro/r)-1]-1 indicated that the lipid separation of the synaptosomal membranes at 23.4 +/- 1.2 degrees C was perturbed by Met-enkephalin such that the temperature was reduced to 15.8 +/- 0.8 degrees 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Scavenging effects of hemoglobin and related heme containing compounds on nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke. A new method for protection against the dangerous cigarette constituents.

The present study refers to the utilization of hemoglobin and related heme containing substances in scavenging noxious compounds contained in the gas phase of cigarette smoke (e.g. nitric oxide (NO), nitrogen oxides (NOx), hydrogen peroxide (H2O2), carbon monoxide (CO), aldehydes, trace elements and carcinogenic nitrosocompounds) which were up to today insufficiently retained by conventional cigarette filters. Hemoglobin impregnated conventional cigarette filters were capable of withholding the above noxious components of the cigarette smoke up to 90%. Similar results were also obtained when solid hemoglobin was sandwiched between two common filters so that all cigarette smoke drawn through the filter comes into contact with the active groups of the hemoglobin molecules (Fe3+, Fe2+, -SH, -NH2). The present study also shows that noxious oxidants contained in cigarette smoke can be retained and neutralized by appropriate scavengers like: a) substances which contain stereospecifically bound iron, b) substances which contain porphyrin ring with iron (e.g. protoporphyrin), c) substances which contain porphyrin ring that does not necessarily contain iron, d) substances which contain porphyrin ring complexed with other metals (e.g. Cu2+, Mg2+). We have also demonstrated that rat alveolar macrophages challenged by cigarette smoke release both superoxide (O2-) and NO the interaction of which resulted in the formation of peroxynitrite (ONOO-). Alveolar macrophages continue to release NO/ONOO- for 30 min following two or three puffs of smoke. Similar results were also obtained in experiments with human volunteers. It was shown that during cigarette smoking the ratio of NO/ONOO- in the inhaled smoke was 1:0.5 while in the exhaled smoke was 1:9, due to secondary redox reactions taking place in the lung resulting in the ONOO- formation. When smokers inhaled cigarette smoke passed through a conventional filter containing hemoglobin, a 70% reduction of both NO and ONOO- in their exhaled cigarette smoke was observed. All findings prove conclusively that, alveolar macrophages exposed to cigarette smoke evoke a dramatic increase of NO, NOx, ONOO- and H2O2 inside the lung. These substances stimulate by a positive feed back mechanism the alveolar macrophages and perhaps even endothelium of the alveolar vessels, to produce more oxidants resulting in lung damage.

Ultraviolet-irradiated human endothelial cells elaborate nitric oxide that may evoke vasodilatory response.

Human skin absorbs solar ultraviolet radiation which evokes vasodilation by a mechanism that is unknown. In this work, we show that ultraviolet (290-320 nm) irradiation of cultured human endothelial cells with doses (60 mJ/cm2) evoked nitric oxide (NO) release that was sustained for more than 60 min. In addition to augmenting NO, cyclic GMP production by human endothelial cells was also increased in a time-dependent manner. Ultraviolet stimulation of NO production was also observed with the enzyme NO synthase purified from the cytosol of human endothelial cells. These results indicate that ultraviolet radiation stimulates NO release, which stimulates cyclic GMP production by vascular smooth muscle, which results in a relaxation response.

Evoked effects of cholesterol binding on integral proteins and lipid fluidity of dog brain synaptosomal plasma membranes.

Binding of cholesterol into dog brain synaptosomal plasma membranes (SPM) within the limits of concentration used (0.5-5 microM) follows an exponential curve described by the general formula y = a.ebx. This curve, which represents the total binding (specific and nonspecific), acquires sigmoid character in the presence of 100 microM cholesterol glucoside, with a Hill coefficient of h = 2.98 +/- 0.18. The specific activity of the Na+/K+-transporting ATPase and Ca2+-transporting ATPase rose after a 2-h preincubation of SPM with cholesterol (up to 5 microM) or its glucoside (up to 50 microM) to at least 50% above their original values. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) increased with cholesterol glucoside (50 microM) incorporation. Cholesterol (5 microM) had no effect on the DPH fluorescence polarization. Arrhenius plots of Na+/K+-transporting ATPase activity exhibited a break point at 23.2 +/- 1.1 degrees C in control SPM, which was elevated to 29.5 +/- 1.4 degrees C in SPM treated with cholesterol glucoside (50 microM) and abolished in SPM treated with cholesterol (5 microM). The allosteric properties of SPM-bound Na+/K+-transporting ATPase inhibited by F- and Ca2+-transporting ATPase inhibited by Na+ (as reflected by changes in the Hill coefficient) were modulated by cholesterol. It could be stated that cholesterol glucoside (50 microM) produced an increased packing of the bulk lipids, while cholesterol (5 microM) increased the fluidity of the lipid microenvironment of both Na+/K+-transporting ATPase and Ca2+-transporting ATPase.

Evaluation of membrane fluidity effects and enzyme activities alterations in adriamycin neurotoxicity.

Adriamycin (ADR) increased the lipid fluidity of dog brain synaptosomal plasma membranes (SPM) 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 phase separation of the membrane at 23.3 +/- 1.2 degrees was perturbed by ADR such that the temperature was reduced to 16.2 +/- 1.1 degrees. Arrhenius plots of (Na+ + K+)-stimulated ATPase activity exhibited a break point at 22.8 +/- 1.1 degrees in control SPM which was reduced to 15.8 +/- 1.0 degrees in ADR treated SPM, suggesting differences in the interaction of (Na+ + K+)-stimulated ATPase with lipids between ADR treated and untreated SPM. (Na+ + K+)-stimulated ATPase and Ca2+-stimulated ATPase activities were increased at a concentration range 10(-18)-10(-15) M of ADR; higher concentrations (up to 10(-7) M), however, led to a progressive inhibition of the enzyme activities. The allosteric properties of SPM-bound (Na+ + K+)-stimulated ATPase by fluoride (F-) (as reflected by changes in the Hill coefficient) were modulated by ADR whereas those of SPM-bound acetylcholinesterase remained unaffected. We propose that ADR achieves these effects through asymmetric perturbations of the membrane lipid structure and that changes in membrane fluidity may be an early key event in ADR induced neurotoxicity.