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Free radicals have been implicated in cerebral ischemia reperfusion (IR) injury. Massive production of nitric oxide and superoxide results in continuous formation of peroxynitrite even several hours after IR insult. This can produce DNA strand nicks, hydroxylation and/or nitration of cytosolic components of neuron, leading to neuronal death. Peroxynitrite decomposition catalysts 5,10,15,20-tetrakis(N-methyl-4'-pyridyl)porphyrinato iron (III) (FeTMPyP) and 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III) (FeTPPS) have been demonstrated to protect neurons in in vitro cultures; however, their neuroprotective efficacy in cerebral IR injury has not been explored. In the present study, we investigated the efficacy and the therapeutic time window of FeTMPyP and FeTPPS in focal cerebral ischemia (FCI). FCI was induced according to the middle cerebral artery occlusion (MCAO) method. After 2 h of MCAO and 70 h of reperfusion, the extent of neurological deficits, infarct and edema volume were measured in Sprague-Dawley rats. FeTMPyP and FeTPPS were administered at different time points 2, 6, 9 and 12 h post MCAO. FeTMPyP and FeTPPS (3 mg kg(-1), i.v.) treatment at 2 and 6 h post MCAO produced significant reduction in infarct volume, edema volume and neurological deficits. However, treatment at latter time points did not produce significant neuroprotection. Significant reduction of peroxynitrite in blood and nitrotyrosine in brain sections was observed on FeTMPyP and FeTPPS treatment. As delayed treatment of FeTMPyP and FeTPPS produced neuroprotection, we tested whether treatment had any influence over the apoptotic neuronal death. DNA fragmentation and in situ nick end-labeling assays showed that FeTMPyP and FeTPPS treatment reduced IR injury-induced DNA fragmentation. In conclusion, peroxynitrite decomposition catalysts (FeTMPyP and FeTPPS) produced prominent neuroprotection even if administered 6 h post MCAO and the neuroprotective effect is at least in part due to the reduction of peroxynitrite and apoptosis.

1. The influence of AII on contractile dysfunction, regulation of the tyrosine kinase-dependent signaling molecule extracellular signal-regulated kinase (ERK), and natriuretic peptide gene expression were examined in the noninfarcted left ventricle (NILV) and right ventricle (RV) during the early phase of remodeling post-myocardial infarct (MI) in the rat. The selective AT(1) receptor antagonist irbesartan was administered10 h following coronary artery ligation, and rats were killed either at 4-day or 2-week post-MI. 2. At 4 days post-MI, left ventricular systolic pressure (LVSP: sham=125+/-12, MI=91+/-4 mmHg) was decreased, whereas left ventricular end-diastolic pressure (LVEDP: sham=9+/-2, MI=17+/-2 mm Hg), right ventricular systolic (RVSP: sham=26+/-1, MI=34+/-2 mm Hg), and end-diastolic pressures (RVEDP: sham=3+/-0.5, MI=7+/-1 mm Hg) were increased. ERK phosphorylation was significantly elevated in the NILV and RV. 3. Irbesartan (40 mg x kg(-1)/day(-1)) administration did not improve left ventricular function, or suppress increased ERK phosphorylation in the 4-day post-MI rat. By contrast, irbesartan therapy normalized RVSP (MI+irbesartan=25+/-1 mm Hg), RVEDP (MI+irbesartan=3+/-0.3 mm Hg), and reduced ERK1 (MI=3.0+/-0.6, MI+irbesartan=2.0+/-0.3-fold increase), and ERK2 (MI=3.8+/-0.8, MI+irbesartan=2.2+/-0.5-fold increase) phosphorylation. 4. In 2-week post-MI rats, biventricular dysfunction was associated with increased prepro-ANP, and prepro-BNP mRNA expression. Irbesartan therapy normalized RVSP, attenuated RVEDP, and abrogated natriuretic peptide mRNA expression (prepro-ANP; MI=9+/-2, MI+irbesartan=2+/-1-fold increase, prepro-BNP; MI=6+/-2, MI+irbesartan=1+/-1-fold increase), whereas both transcripts remained elevated in the NILV despite the partial attenuation of LVEDP. 5. These data suggest that the therapeutic benefit of irbesartan treatment during the early phase of remodeling post-MI was associated with the preferential amelioration of RV contractile function and phenotype.

1. Bi-directional GABA-transport was studied by performing uptake and superfusion experiments in human embryonic kidney 293 cells stably expressing the rat GABA transporter rGAT-1. 2. K(M) and V(max) values for [(3)H]-GABA uptake were 11.7+/-1.8 microM and 403+/-55 pmol min(-1) 10(-6) cells (n=9), respectively. 3. Kinetic analysis of outward transport was performed by pre-labelling the cells with increasing concentrations of [(3)H]-GABA and triggering outward transport with 333 microM GABA. Approximate apparent K(M) and V(max) values were 12 mM and 50 pmol min(-1) 10(-6) cells, respectively. 4. GABA re-uptake inhibitors (RI; e.g. tiagabine), as well as, substrates of the rGAT-1 (e.g. GABA, nipecotic acid) concentration dependently decreased [(3)H]-GABA uptake and increased efflux of [(3)H]-GABA from pre-labelled cells. The IC(50) values for inhibiting uptake and the EC(50) values for increasing efflux were significantly correlated (r(2)=0.99). 5. On superfusion, RI antagonized the efflux-enhancing effect of the substrates. The effect of the latter was markedly augmented in the presence of ouabain (100 microM), whereas the effect of RI remained unchanged. The most likely explanation for the release enhancing effect of RI is interruption of ongoing re-uptake. 6. The structural GABA-analogue 2,4-diamino-n-butyric acid (DABA) exhibited a bell-shaped concentration response curve on [(3)H]-GABA efflux with the maximum at 1 mM, and displayed a deviation from the sigmoidal inhibition curve in uptake experiments in the same concentration range. At concentrations below 1 mM, DABA inhibited [(3)H]-GABA uptake non-competitively, while at 1 mM and above the inhibition of uptake followed a competitive manner. 7. The results provide information of GABA inward and outward transport, and document a complex interaction of the rGAT-1 with its substrate DABA.

A number of phenanthrolines and benzoquinolines were examined for their ability to activate epithelial chloride secretion by measuring short circuit current (SCC) using the mouse colon epithelium. 1,10 phenanthroline stimulated electrogenic chloride secretion with an EC50 of 612±10 μ and a Hill slope of 4.9±0.3. A similar pharmacology was demonstrated by both 1,7 and 4,7 phenanthrolines, 7,8 benzoquinoline and phenanthridine. Evidence that the increase in SCC caused by 1,10 phenanthroline was due to chloride secretion is based upon (a) inhibition of the current by furosemide, (b) failure of cystic fibrosis (CF) colons to respond and (c) an associated net flux of 36Cl−. 1,10 Phenanthroline affected neither the generation of cyclic AMP or the concentration of intracellular Ca2+ in colonic epithelial cells. 1,10 phenanthroline affected the chloride conductance of the apical membrane, as shown by an increase in chloride current in ‘apical membrane only' preparations in the presence of an apical to basolateral chloride gradient. The increase in chloride current was inhibited by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and was not present in CF colons. Additionally, 1,10 phenanthroline activated basolateral K+ channels, both Ca2+- and cyclic AMP-sensitive channels, as shown by inhibitor studies with charybdotoxin (ChTX) and XE991, and after the apical membrane was permeabilized with nystatin. The phenanthrolines and benzoquinolines described here, with dual actions affecting CFTR and basolateral K+ channels, may constitute useful lead compounds for adjunct therapy in CF. Keywords: CFTR, cystic fibrosis, chloride channel openers, phenanthrolines, 7,8 benzoquinoline, phenanthridine, potassium channels, cyclic AMP, intracellular Ca2+ Introduction Increased interest in agents affecting epithelial chloride channels arose after it was shown that the cystic fibrosis transmembrane conductance regulator (CFTR) is also a chloride channel in many epithelial membranes (Anderson et al., 1991). Not only have blockers of CFTR been described but considerable interest has also focused on CFTR chloride channel openers (Schultz et al., 1999). The most common mutation in cystic fibrosis (CF) is ΔF508, in which phenylalanine is absent from position 508. This mutant protein (ΔF508CFTR) can function as a chloride channel (Drumm et al., 1991), however only a minute fraction of this protein is transported to the apical domain of epithelial cells (Ward & Kopito, 1994). Instead the quality control mechanisms of the cell cause the mutant protein to be degraded in the proteasome. Therefore attempts are being made to increase the delivery of ΔF508 CFTR to the membrane (Brodsky, 2001; Fischer et al., 2001; Fuller & Cuthbert, 2000). Thus agents which open CFTR channels are of interest, particularly so if they also activate ΔF508 CFTR. The present classes of CFTR openers are as follows; phosphatase inhibitors (Becq et al., 1998), xanthines, particularly CPX (Guay-Broder et al., 1995), benzimidazolones such as EBIO (Devor et al., 1996; Cuthbert et al., 1999), flavones, for example genistein (Illek et al., 1995), benzoxazolones like chlorzoxazone (Singh et al., 2000) and finally the MPB compounds described by Becq et al. (1999). The purpose of this study was to further investigate the actions of 1,10 phenanthroline on chloride secretion in the mouse colon following the observations made by Duszyk et al. (1999) that 1,10 phenanthroline, a powerful inhibitor of matrix metalloproteases (MMPs) activated a chloride conductance, measured as whole cell current, in human airway epithelial cells, Calu-3. No effect was found in airway cells lacking CFTR, namely A549 cells. Furthermore, an antibody to MMP2 caused a similar effect in Calu-3 cells as did 1,10 phenanthroline, suggesting metalloproteinase inhibition was responsible. A further aim was to explore structure activity relations in the phenanthroline isomers and to ask if both heterocyclic nitrogens in the tricyclic aromatic molecules were necessary for the effect. From various experimental approaches we conclude that the phenanthrolines and related structures constitute a new class of agents that stimulate chloride secretion.