Michael P. Gordge

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.

Inhibition of NADPH supply by 6-aminonicotinamide: effect on glutathione, nitric oxide and superoxide in J774 cells.

We have examined the integrity of J774 cell nitric oxide (NO) production and glutathione maintenance, whilst NADPH supply was compromised by inhibition of the pentose pathway with 6‐aminonicotinamide. In resting cells 6‐phosphogluconate accumulation began after 4 h and glutathione depletion after 24 h of 6‐aminonicotinamide treatment. Cellular activation by lipopolysaccharide/interferon‐λ decreased glutathione by ∼50% and synchronous 6‐aminonicotinamide treatment exacerbated this to 31.2% of control (P<0.05). In activated cells NO− 2 production was inhibited by 60% with 6‐aminonicotinamide (P<0.01), and superoxide production by 50% (P<0.01) in zymosan‐activated cells. NADPH production via the pentose pathway is therefore important to sustain macrophage NO production whilst maintaining protective levels of glutathione.

Megakaryocyte apoptosis: sorting out the signals

British Journal of Pharmacology (2005) 145, 271–273. doi:10.1038/sj.bjp.0706202

S-nitrosothiols as selective antithrombotic agents – possible mechanisms

S‐nitrosothiols have a number of potential clinical applications, among which their use as antithrombotic agents has been emphasized. This is largely because of their well‐documented platelet inhibitory effects, which show a degree of platelet selectivity, although the mechanism of this remains undefined. Recent progress in understanding how nitric oxide (NO)‐related signalling is delivered into cells from stable S‐nitrosothiol compounds has revealed a variety of pathways, in particular denitrosation by enzymes located at the cell surface, and transport of intact S‐nitrosocysteine via the amino acid transporter system‐L (L‐AT). Differences in the role of these pathways in platelets and vascular cells may in part explain the reported platelet‐selective action. In addition, emerging evidence that S‐nitrosothiols regulate key targets on the exofacial surfaces of cells involved in the thrombotic process (for example, protein disulphide isomerase, integrins and tissue factor) suggests novel antithrombotic actions, which may not even require transmembrane delivery of NO.

Fabrication of a novel poly(3-hydroxyoctanoate) / nanoscale bioactive glass composite film with potential as a multifunctional wound dressing

Fabrication of a composite scaffold of nanobioglass (n‐BG) 45S5 and poly(3‐hydroxyocatnoate), P(3HO) was studied for the first time with the aim of developing a novel, multifunctional wound dressing. The incorporation of n‐BG accelerated blood clotting time and its incorporation in the polymer matrix enhanced the wettability, surface roughness and bio‐compatibility of the scaffold.

Cell surface thiol isomerases may explain the platelet-selective action of S-nitrosoglutathione

S-nitrosoglutathione (GSNO) at low concentration inhibits platelet aggregation without causing vasodilation, suggesting platelet-selective nitric oxide delivery. The mechanism of this selectivity is unknown, but may involve cell surface thiol isomerases, in particular protein disulphide isomerase (csPDI) (EC 5.3.4.1). We have now compared csPDI expression and activity on platelets, endothelial cells and vascular smooth muscle cells, and the dependence on thiol reductase activity of these cell types for NO uptake from GSNO. csPDI expression was measured by flow cytometry and its reductase activity using the pseudosubstrate dieosin glutathione disulphide. This activity assay was adapted and validated for 96-well plate format. Flow cytometry revealed csPDI on all three cell types, but percentage positivity of expression was higher on platelets than on vascular cells. Consistent with this, thiol isomerase-related reductase activity was higher on platelets (P < 0.01), and cellular activation (with either phorbol myristate acetate or ionomycin) increased csPDI activity on both platelets and smooth muscle cells, but not on endothelium. Intracellular NO delivery from GSNO was greater in platelets than in vascular cells (P < 0.002), and was more sensitive to thiol isomerase inhibition using phenylarsine oxide (P < 0.05). Increased surface thiol isomerase activity on platelets, compared with cells of the vascular wall, may explain the platelet-selective actions of GSNO and help define its antithrombotic potential.

Highly elastomeric poly(3-hydroxyoctanoate) based natural polymer composite for enhanced keratinocyte regeneration

ABSTRACT A novel nanocomposite material combining the biocompatible, elastomeric, natural, biodegradable homopolymer poly(3-hydroxyoctanoate) (P(3HO)) with hemostatic and antibacterial bioactive glass nanoparticles (n-BG) was developed as a matrix for skin related applications. P(3HO) is a unique member of the family of natural polyhydroxyalkanoate biopolymers. The P(3HO)/n-BG composite films were fabricated using the solvent casting method. Microstructural studies revealed n-BG particles both embedded in the matrix and deposited on the surface, which introduced nanotopography and increased its hydrophilicity. The composite exhibited an increase in the Young’s modulus when compared to the control, yet maintained flexible elastomeric properties. These changes in the surface topography and chemistry of the composite system led to an increase of protein adsorption and cytocompatibility for the seeded human keratinocyte cell line. The results from this study demonstrated that the fabricated P(3HO)/n-BG composite system is a promising novel matrix material with potential applications in skin tissue engineering and wound healing. GRAPHICAL ABSTRACT