John S. Hothersall

Expression and Modulation of an NADPH Oxidase in Mammalian Astrocytes

Amyloid β peptides generate oxidative stress in hippocampal astrocytes through a mechanism sensitive to inhibitors of the NADPH oxidase [diphenylene iodonium (DPI) and apocynin]. Seeking evidence for the expression and function of the enzyme in primary hippocampal astrocytes, we confirmed the expression of the subunits of the phagocyte NADPH oxidase by Western blot analysis and by immunofluorescence and coexpression with the astrocyte-specific marker glial fibrillary acidic protein both in cultures and in vivo. Functional assays using lucigenin luminescence, dihydroethidine, or dicarboxyfluorescein fluorescence to measure the production of reactive oxygen species (ROS) demonstrated DPI and apocynin-sensitive ROS generation in response to the phorbol ester PMA and to raised [Ca2+]c after application of ionomycin or P2u receptor activation. Stimulation by PMA but not Ca2+ was inhibited by the protein kinase C (PKC) inhibitors staurosporine and hispidin. Responses were absent in transgenic mice lacking gp91phox. Expression of gp91phox and p67phox was increased in reactive astrocytes, which showed increased rates of both resting and stimulated ROS generation. NADPH oxidase activity was modulated by intracellular pH, suppressed by intracellular alkalinization, and enhanced by acidification. The protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone suppressed basal ROS generation but markedly increased PMA-stimulated ROS generation. This was independent of mitochondrial ROS production, because it was unaffected by mitochondrial depolarization with rotenone and oligomycin. Thus, the NADPH oxidase is expressed in astrocytes and is functional, activated by PKC and intracellular calcium, modulated by pHi, and upregulated by astrocyte activation. The astrocytic NADPH oxidase is likely to play important roles in CNS physiology and pathology.

Morphine peripheral analgesia depends on activation of the PI3Kγ/AKT/nNOS/NO/KATP signaling pathway

Morphine is one of the most prescribed and effective drugs used for the treatment of acute and chronic pain conditions. In addition to its central effects, morphine can also produce peripheral analgesia. However, the mechanisms underlying this peripheral action of morphine have not yet been fully elucidated. Here, we show that the peripheral antinociceptive effect of morphine is lost in neuronal nitric-oxide synthase null mice and that morphine induces the production of nitric oxide in primary nociceptive neurons. The activation of the nitric-oxide pathway by morphine was dependent on an initial stimulation of PI3Kγ/AKT protein kinase B (AKT) and culminated in increased activation of KATP channels. In the latter, this intracellular signaling pathway might cause a hyperpolarization of nociceptive neurons, and it is fundamental for the direct blockade of inflammatory pain by morphine. This understanding offers new targets for analgesic drug development.

Evidence for a cyclic GMP‐independent mechanism in the anti‐platelet action of S‐nitrosoglutathione

We have measured the ability of a range of NO donor compounds to stimulate cyclic GMP accumulation and inhibit collagen‐induced aggregation of human washed platelets. In addition, the rate of spontaneous release of NO from each donor has been measured spectrophotometrically by the oxidation of oxyhaemoglobin to methaemoglobin. The NO donors used were five s‐nitrosothiol compounds: S‐nitrosoglutathione (GSNO), S‐nitrosocysteine (cysNO), S‐nitroso‐N‐acetyl‐DL‐penicillamine (SNAP), S‐nitroso‐N‐acetyl‐cysteine (SNAC), S‐nitrosohomocysteine (homocysNO), and two non‐nitrosothiol compounds: diethylamine NONOate (DEANO) and sodium nitroprusside (SNP). Using 10 μM of each donor compound, mean ±s.e.mean rate of NO release ranged from 0.04±0.001 nmol min−1 (for SNP) to 3.15±0.29 nmol min−1 (for cysNO); cyclic GMP accumulation ranged from 0.43±0.05 pmol per 108 platelets (for SNP) to 2.67±0.31 pmol per 108 platelets (for cysNO), and inhibition of platelet aggregation ranged from 40±6.4% (for SNP) to 90±3.8% (for SNAC). There was a significant positive correlation between the rate of NO release and the ability of the different NO donors to stimulate intra‐platelet cyclic GMP accumulation (r=0.83; P=0.02). However, no significant correlation was observed between the rate of NO release and the inhibition of platelet aggregation by the different NO donors (r=−0.17), nor was there a significant correlation between cyclic GMP accumulation and inhibition of aggregation by the different NO donor compounds (r=0.34). Comparison of the dose‐response curves obtained with GSNO, DEANO and 8‐bromo cyclic GMP showed DEANO to be the most potent stimulator of intraplatelet cyclic GMP accumulation (P<0.001 vs both GSNO and 8‐bromo cyclic GMP), but GSNO to be the most potent inhibitor of platelet aggregation (P<0.01 vs DEANO, and P<0.001 vs 8‐bromo cyclic GMP). The rate of NO release from GSNO, and its ability both to stimulate intra‐platelet cyclic GMP accumulation and to inhibit platelet aggregation, were all significantly diminished by the copper (I) (Cu+) chelating agent bathocuproine disulphonic acid (BCS). In contrast, BCS had no effect on either the rate of NO release, or the anti‐platelet action of the non‐nitrosothiol compound DEANO. Cyclic GMP accumulation in response to GSNO (10−9–10−5M) was undetectable following treatment of platelets with ODQ (100 μM), a selective inhibitor of soluble guanylate cyclase. Despite this abolition of guanylate cyclase stimulation, GSNO retained some ability to inhibit aggregation, indicating the presence of a cyclic GMP‐independent component in its anti‐platelet action. However, this component was abolished following treatment of platelets with a combination of both ODQ and BCS, suggesting that Cu+ ions were required for the cyclic GMP‐independent pathway to operate. The cyclic GMP‐independent action of GSNO, observed in ODQ‐treated platelets, could not be explained by an increase in intra‐platelet cyclic AMP. The impermeable thiol modifying agent p‐chloromercuriphenylsulphonic acid (CMPS) produced a concentration‐dependent inhibition of aggregation of ODQ‐treated platelets, accompanied by a progressive loss of detectable platelet surface thiol groups. Additional treatment with GSNO failed to increase the degree of aggregation inhibition, suggesting that a common pathway of thiol modification might be utilized by both GSNO and CMPS to elicit cyclic GMP‐independent inhibition of platelet aggregation. We conclude that NO donor compounds mediate inhibition of platelet aggregation by both cyclic GMP‐dependent and ‐independent pathways. Cyclic GMP generation is related to the rate of spontaneous release of NO from the donor compound, but transfer of the NO signal to the cyclic GMP‐independent pathway may depend upon a cellular system which involves both copper (I) (Cu+) ions and surface membrane thiol groups. The potent anti‐platelet action of GSNO results from its ability to exploit this cyclic GMP‐independent mechanism.

Copper chelation-induced reduction of the biological activity of S-nitrosothiols.

1 The effect of copper on the activity of the S‐nitrosothiol compounds S‐nitrosocysteine (cysNO) and S‐nitrosoglutathione (GSNO) was investigated, using the specific copper chelator bathocuproine sul‐phonate (BCS), and human washed platelets as target cells. 2 Chelation of trace copper with BCS (10 μm) in washed platelet suspensions reduced the inhibition of thrombin‐induced platelet aggregation by GSNO; however, BCS had no significant effect on the anti‐aggregatory action of cysNO. BCS inhibited cyclic GMP generation in response to both cysNO and GSNO. 3 The effect of BCS was rapid (within 30 s), and could be abolished by increasing the platelet concentration to 500 times 109 1−1. 4 In BCS‐treated platelet suspensions, the addition of Cu2+ ions (0.37–2.37 μm) led to a restoration of both guanylate cyclase activation and platelet aggregation inhibition by GSNO. 5 The anti‐aggregatory activity of GSNO was reduced in a concentration‐dependent manner by the copper (I)‐specific chelators BCS and neocuproine, and to a smaller extent by desferal. No effect was observed with the copper (II) specific chelator, cuprizone, the iron‐specific chelator, bathophenanthroline sulphonate, or the broader‐specificity copper chelator, d‐penicillamine. 6 In both BCS‐treated and ‐untreated platelet suspensions, cys NO was more potent than GSNO as a stimulator of guanylate cyclase. In BCS‐treated platelet suspensions there was no significant difference between the anti‐aggregatory potency of cysNO and GSNO; however, in untreated suspensions, GSNO was significantly more potent than cysNO. Thus, when copper was available, GSNO produced a greater inhibition of aggregation than cysNO, despite being a less potent activator of guanylate cyclase. 7 The breakdown of cysNO and GSNO was measured spectrophotometrically by decrease in absor‐bance at 334 nm. In Tyrode buffer, cysNO (10 μm) broke down at a rate of 3.3 μm min−1. BCS (10 μm) reduced this to 0.5 μm min−1. GSNO, however, was stable, showing no fall in absorbance over a period of 7 min even in the absence of BCS. 8 We conclude that copper is required for the activity of both cysNO and GSNO, although its influence on anti‐aggregatory activity is only evident with GSNO. The stimulatory effect of copper is unlikely to be explained solely by catalysis of S‐nitrosothiol breakdown. The enhancement by copper of th anti‐aggregatory activity of GSNO, relative to cysNO, suggests that copper may be required for biological activity of GSNO which is independent of guanylate cyclase stimulation.

Mitochondrial superoxide production during oxalate-mediated oxidative stress in renal epithelial cells

Crystals of calcium oxalate monohydrate (COM) in the renal tubule form the basis of most kidney stones. Tubular dysfunction resulting from COM-cell interactions occurs by mechanism(s) that are incompletely understood. We examined the production of reactive oxygen intermediates (ROI) by proximal (LLC-PK1) and distal (MDCK) tubular epithelial cells after treatment with COM (25-250 microg/ml) to determine whether ROI, specifically superoxide (O(2)(*-)), production was activated, and whether it was sufficient to induce oxidative stress. Employing inhibitors of cytosolic and mitochondrial systems, the source of ROI production was investigated. In addition, intracellular glutathione (total and oxidized), energy status (ATP), and NADH were measured. COM treatment for 1-24 h increased O(2)(*-) production 3-6-fold as measured by both lucigenin chemiluminescence in permeabilized cells and dihydrorhodamine fluorescence in intact cells. Using selective inhibitors we found no evidence of cytosolic production. The use of mitochondrial probes, substrates, and inhibitors indicated that increased O(2)(*-) production originated from mitochondria. Treatment with COM decreased glutathione (total and redox state), indicating a sustained oxidative insult. An increase in NADH in COM-treated cells suggested this cofactor could be responsible for elevating O(2)(*-) generation. In conclusion, COM increased mitochondrial O(2)(*-) production by epithelial cells, with a subsequent depletion of antioxidant status. These changes may contribute to the reported cellular transformations during the development of renal calculi.

Role of a copper (I)-dependent enzyme in the anti-platelet action of S-nitrosoglutathione.

1 S‐nitrosoglutathione (GSNO) is a potent and selective anti‐platelet agent, despite the fact that its spontaneous rate of release of nitric oxide (NO) is very slow. Our aim was to investigate the mechanism of the anti‐aggregatory action of GSNO. 2 The biological action of GSNO could be mediated by NO released from S‐nitrosocystylglycine, following enzymatic cleavage of GSNO by γ‐glutamyl transpeptidase. The anti‐aggregatory potency of GSNO was not, however, altered by treatment of target platelets with the γ‐glutamyl transpeptidase inhibitor acivicin (1 mM). γ‐Glutamyl transpeptidase is not, therefore, involved in mediating the action of GSNO. 3 The rate of breakdown of S‐nitrosoalbumin was increased from 0.19 ± 0.086 nmol min−1 to 1.52 ± 0.24 nmol min−1 (mean±s.e.mean) in the presence of cysteine (P < 0.05, n = 4). Inhibition of platelet aggregation by S‐nitrosoalbumin was also significantly increased by cysteine (P < 0.05, n = 4), suggesting that the biological activity of S‐nitrosoalbumin is mediated by exchange of NO from the protein carrier to form the unstable compound cysNO. Breakdown of GSNO showed a non‐significant acceleration in the presence of cysteine, from 0.56 ± 0.22 to 1.77 ± 0.27 nmol min−1 (mean±s.e.mean) (P = 0.064, n = 4), and its ability to inhibit platelet aggregation was not enhanced by cysteine. This indicates that the anti‐platelet action of GSNO is not dependent upon transnitrosation to form cysNO. 4 Platelets pretreated with the copper (I)‐specific chelator bathocuproine disulphonic acid (BCS), then resuspended in BCS‐free buffer, showed resistance to the inhibitory effect of GSNO. These findings suggest that BCS impedes the action of GSNO by binding to structures on the platelet, rather than by chelating free copper in solution. 5 Release of NO from GSNO was catalysed enzymatically by ultrasonicated platelet suspensions. This enzyme had an apparent Km for GSNO of 12.4 ± 2.64 μm and a Vmax of 0.21 ± 0.03 nmol min−1 per 108 platelets (mean±s.e.mean, n = 5). It was inhibited by BCS, but not by the iron chelator bathophenathroline disulphonic acid, nor by acivicin. 6 We conclude that the stable S‐nitrosothiol compound GSNO may exert its anti‐platelet action via enzymatic, rather than spontaneous release of NO. This is mediated by a copper‐dependent mechanism. The potency and platelet‐selectivity of GSNO may result from targeted NO release at the platelet surface.

Hydrogen sulfide improves neutrophil migration and survival in sepsis via K+ATP channel activation.

RATIONALE Recovering the neutrophil migration to the infectious focus improves survival in severe sepsis. Recently, we demonstrated that the cystathionine gamma-lyase (CSE)/hydrogen sulfide (H(2)S) pathway increased neutrophil recruitment to inflammatory focus during sterile inflammation. OBJECTIVES To evaluate if H(2)S administration increases neutrophil migration to infectious focus and survival of mice. METHODS Sepsis was induced by cecal ligation and puncture (CLP). MEASUREMENTS AND MAIN RESULTS The pretreatments of mice with H(2)S donors (NaHS or Lawessons reagent) improved leukocyte rolling/adhesion in the mesenteric microcirculation as well as neutrophil migration. Consequently, bacteremia levels were reduced, hypotension and lung lesions were prevented, and the survival rate increased from approximately 13% to approximately 80%. Even when treatment was delayed (6 h after CLP), a highly significant reduction in mortality compared with untreated mice was observed. Moreover, H(2)S pretreatment prevented the down-regulation of CXCR2 and l-selectin and the up-regulation of CD11b and G protein-coupled receptor kinase 2 in neutrophils during sepsis. H(2)S also prevented the reduction of intercellular adhesion molecule-1 expression in the endothelium of the mesenteric microcirculation in severe sepsis. Confirming the critical role of H(2)S on sepsis outcome, pretreatment with dl-propargylglycine (a CSE inhibitor) inhibited neutrophil migration to the infectious focus, enhanced lung lesions, and induced high mortality in mice subjected to nonsevere sepsis (from 0 to approximately 80%). The beneficial effects of H(2)S were blocked by glibenclamide (a ATP-dependent K(+) channel blocker). CONCLUSIONS These results showed that H(2)S restores neutrophil migration to the infectious focus and improves survival outcome in severe sepsis by an ATP-dependent K(+) channel-dependent mechanism.

Hydrogen sulfide augments neutrophil migration through enhancement of adhesion molecule expression and prevention of CXCR2 internalization: role of ATP-sensitive potassium channels.

In this study, we have addressed the role of H2S in modulating neutrophil migration in either innate (LPS-challenged naive mice) or adaptive (methylated BSA (mBSA)-challenged immunized mice) immune responses. Treatment of mice with H2S synthesis inhibitors, dl-propargylglycine (PAG) or β-cyanoalanine, reduced neutrophil migration induced by LPS or methylated BSA (mBSA) into the peritoneal cavity and by mBSA into the femur/tibial joint of immunized mice. This effect was associated with decreased leukocyte rolling, adhesion, and P-selectin and ICAM-1 expression on endothelium. Predictably, treatment of animals with the H2S donors, NaHS or Lawesson’s reagent, enhanced these parameters. Moreover, the NaHS enhancement of neutrophil migration was not observed in ICAM-1-deficient mice. Neither PAG nor NaHS treatment changed LPS-induced CD18 expression on neutrophils, nor did the LPS- and mBSA-induced release of neutrophil chemoattractant mediators TNF-α, keratinocyte-derived chemokine, and LTB4. Furthermore, in vitro MIP-2-induced neutrophil chemotaxis was inhibited by PAG and enhanced by NaHS treatments. Accordingly, MIP-2-induced CXCR2 internalization was enhanced by PAG and inhibited by NaHS treatments. Moreover, NaHS prevented MIP-2-induced CXCR2 desensitization. The PAG and NaHS effects correlated, respectively, with the enhancement and inhibition of MIP-2-induced G protein-coupled receptor kinase 2 expression. The effects of NaHS on neutrophil migration both in vivo and in vitro, together with CXCR2 internalization and G protein-coupled receptor kinase 2 expression were prevented by the ATP-sensitive potassium (KATP+) channel blocker, glybenclamide. Conversely, diazoxide, a KATP+ channel opener, increased neutrophil migration in vivo. Together, our data suggest that during the inflammatory response, H2S augments neutrophil adhesion and locomotion, by a mechanism dependent on KATP+ channels.

Alternative pathways of glucose utilization in brain. Changes in the pattern of glucose utilization in brain during development and the effect of phenazine methosulfate on the integration of metabolic routes.

Abstract The activities of alternative pathways of glucose metabolism in developing rat brain were evaluated by measurement of the yields of 14CO2 from glucose labeled with 14C on carbons 1, 2, 3 + 4, 6 and uniformly labeled glucose, from the detritiation of [2-3H]glucose and from the incorporation of 14C from specifically labeled glucose into lipids by brain slices from cerebral hemispheres and cerebellum. The glycolytic route and tricarboxylic acid cycle (14CO2 yield from carbons 3, 4, and 6 of glucose) increased during development. The flux through the glutamate-γ-aminobutyric route (14CO2 yield from carbon 2-carbon 6 of glucose) also showed an increase with development. In contrast, the proportion of glucose metabolized via the pentose phosphate pathway was markedly decreased as development progressed. The artificial electron acceptor, phenazine methosulfate, was used as a probe to investigate the effect of alterations in the redox state of NADP + NADPH couple on a number of NADP-linked systems in developing brain. Phenazine methosulfate produced a massive (20- to 50-fold) stimulation of the pentose phosphate pathway, in contrast, the incorporation of glucose carbon into fatty acids and flux through the glutamate-γ-aminobutyrate shunt were sharply decreased. The effects of phenazine methosulfate on the incorporation of glucose into glyceride glycerol, on the flux of glucose through the pyruvate dehydrogenase reaction and tricarboxylic acid cycle, all processes linked to the NAD + NADH couple, appeared to be minimal in the brain at the stages of development studied, i.e., 1, 5, 10, 20 days, and in the adult rat. The significance of the massive reserve potential of the pentose phosphate pathway in the developing brain is discussed.

The multifaceted photocytotoxic profile of Hypericin

Photodynamic therapy (PDT) is an established anticancer treatment employing a phototoxin (photosensitizer), visible light and oxygen. The latter is photochemically converted into reactive oxygen species, which are highly toxic to the cells. Hypericin, a natural pigment of hypericum plants, is prominent among photosensitizers. The unique perylenequinone structure of hypericin is responsible for its intriguing multifaceted photochemical cytotoxicity. The diverse photodynamic action of hypericin targets a range of subcellular organelles most importantly the mitochondria and the endoplasmic reticulum (ER)-Golgi complex. Hypericin exerts its phototoxicity through intricate mechanisms, implicating key proteins, vital enzymes, organelle membranes and changes in cellular homeostasis. This, depending on drug and light administration conditions, leads to cell death, which occurs mainly by the induction of apoptosis and/or necrosis. Cell photosensitization with hypericin is also associated with the stimulation of macroautophagy, which may promote cell demise when the apoptotic machinery is defective. Herein, we aim to integrate the most important findings with regard to hypericin photocytotoxicity, into a unified scenario, detailing its potential in cancer photomedicine.