Chloride channel inhibition blocks the protection of ischemic preconditioning and hypo-osmotic stress in rabbit ventricular myocardium.

The objective of this study was to examine the role of chloride (Cl-) channels in the myocardial protection of ischemic preconditioning (IP). Isolated rabbit ventricular myocytes were preconditioned with 10-minute simulated ischemia (SI) and 20-minute simulated reperfusion (SR) or not preconditioned (control). The myocytes then received 180-minute SI or 45-minute SI/120-minute SR. Indanyloxyacetic acid 94 (IAA-94, 10 micromol/L) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 1 micromol/L) was administered before IP or before SI or SI/SR to inhibit Cl- channels. Electrophysiological studies indicate that these drugs, at the concentrations used, selectively abolished Cl- currents activated under hypo-osmotic conditions (215 versus 290 mOsm). IP significantly (P<0.001) reduced the percentage of dead myocytes after 60-minute (30.8+/-1.3%, mean+/-SEM), 90-minute (35.3+/-1.3%), and 120-minute (39.2+/-1.7%) SI compared with controls (44.7+/-1.6%, 54.5+/-1.3%, and 58.9+/-1.8%, respectively) and after 45-minute SI/120-minute SR (36.3+/-0.6%) compared with control (56.6+/-2.2%). Hypo-osmotic stress also produced protection similar to IP. IAA-94 or NPPB abolished the protection of both IP and hypo-osmotic stress. In buffer-perfused rabbit hearts preconditioned with three 5-minute ischemia/10-minute reperfusion cycles given before the 40-minute long ischemia and 60-minute reperfusion, IP significantly (P<0.0001) reduced infarct size (IP+vehicle, 4.7+/-0.9%, versus control+vehicle, 26.6+/-3.3%; mean+/-SEM). Again, IAA-94 or NPPB abolished the protection of IP. Our results implicate Cl- channels in the IP protection of the myocardium against ischemic/reperfusion injury and demonstrate that hypo-osmotic stress is capable of preconditioning cardiomyocytes.

Ischemic preconditioning: differences in protection and susceptibility to blockade with single-cycle versus multicycle transient ischemia.

BACKGROUND: We compared ischemic preconditioning (IP) induced with a single cycle of transient ischemia and reperfusion with that induced by multiple cycles in terms of (1) efficacy of protection against myocardial necrosis and (2) susceptibility to pharmacological blockade by inhibition of protein kinase C (PKC) or elevation of cAMP. METHODS AND RESULTS: All rabbits were subjected to 30 minutes of regional ischemia and 90 minutes of reperfusion in vivo. IP was induced with either one or three cycles of 5-minute transient ischemia and 10-minute reperfusion given before the 30-minute ischemia. Drug-treated hearts received a bolus dose of one of the following just before the 30-minute ischemia: (1) the PKC inhibitor chelerythrine (3.8 mg/kg), (2) the PKC inhibitor polymyxin B (10 mg/kg), or (3) the cAMP-increasing agent NKH477 (45 microg/kg). IP induced with either one or three cycles of transient ischemia and reperfusion significantly protected the heart against infarction, although the extent of protection was significantly greater with three-cycle IP. Chelerythrine, polymyxin B, or NKH477 alone did not alter infarct size in control hearts, nor did they increase infarct size in hearts preconditioned with three-cycle IP. In contrast, when IP was induced with only a single cycle, all three of these drugs significantly increased infarct size above that of the untreated one-cycle IP group. However, infarct size in all three of these drug-treated one-cycle IP groups was still significantly lower than that in the corresponding drug-treated controls, indicating a partial block of IP. CONCLUSIONS: Three-cycle IP provided more effective protection against myocardial necrosis than one-cycle IP and was less susceptible to blockade by inhibitors of PKC or an agent that increases cAMP levels. However, single-cycle IP was only partially blocked by either inhibition of PKC or stimulation of cAMP production. Neither activation of the PKC pathway nor reduced formation of cAMP alone fully accounted for the necrosis protection by IP even when induced with only a single cycle of transient ischemia.

Clinical laboratory investigation of the Sanofi ACCESS CK-MB procedure and comparison to electrophoresis and Abbott IMx.

This evaluation was undertaken to verify the application protocol for the CK-MB assay on the ACCESS Immunoassay Analyzer (Sanofi Diagnostics Pasteur, Chaska, MN). The results show that the ACCESS CK-MB assay total imprecision was 6.8% to 9.1%. Analytical linearity of the ACCESS CK-MB assay was excellent in the range of < 1-214 micrograms/L. A comparison of the ACCESS CK-MB assay with the IMx (Abbott Laboratories, Abbott Park, IL) method shows good correlation r = 0.990 (n = 108). Linear regression analysis yielded Y = 1.36X-0.3, Sx/y = 7.2. ACCESS CK-MB values also correlated well with CK-MB by electrophoresis with r = 0.968 (n = 132). The linear regression equation for this comparison was Y = 1.08X + 1.4, Sx/y = 14.1. The expected non-myocardial infarction range of CK-MB determined by the ACCESS system was 1.3-9.4 micrograms/L (mean = 4.0, n = 58). The ACCESS CK-MB assay would appear to be rapid, precise and clinically useful.

Oxidation of spin trap 5,5-dimethyl-1-pyrroline-1-oxide in an electron paramagnetic resonance study of the reaction of methemoglobin with hydrogen peroxide.

The possibility that methemoglobin (metHb) may function as a biological Fenton reagent to produce hydroxyl radical from hydrogen peroxide is investigated by electron paramagnetic resonance (EPR) spin-trapping techniques. The spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) gives a nine-line EPR spectrum and no hydroxyl radical or superoxide spin adduct signals for the metHb/H2O2 system. From the known hyperfine splitting constants, the spectrum is assigned to 5,5-dimethylpyrrolidone-2(2)-oxyl-(1) (DMPOX), an oxidized derivative of DMPO. The likely involvement of the peroxidase activity of metHb in this reaction is suggested by the oxidation of DMPO to DMPOX by horseradish peroxidase as well. Furthermore, peroxidase inhibitors prevent the formation of DMPOX. Spectrophotometric assays confirm the peroxidase activity of metHb toward typical phenolic and nonphenolic substrates under the conditions used for the EPR experiments. The visible absorption spectra indicate the formation of a ferrylHb intermediate and its reduction by DMPO. Glutathione and ascorbic acid compete with DMPO as electron donors in the reaction to form thiyl and ascorbate radicals. Neither hydroxyl radical nor any other signal is observed when N-tert-butyl-alpha-phenylnitrone (PBN) is used as the spin trap in the metHb/H2O2 system. It is concluded that methemoglobin-bound iron may not catalyze the Fenton reaction forming hydroxyl radical, but can oxidize a variety of substrates, including DMPO, in a peroxidase-type reaction.

Inhibition of superoxide-generating NADPH oxidase of human neutrophils by lazaroids (21-aminosteroids and 2-methylaminochromans).

Lazaroids (21-aminosteroids and 2-methylaminochromans) are a new series of drugs designed and demonstrated to protect against tissue damage after trauma and/or ischemia. It has been suggested that the protective effects of lazaroids are derived from their potent actions to inhibit iron-dependent lipid peroxidation, but whether this is sufficient to explain their therapeutic effects is unknown. In an effort to better understand their mechanism of action, these drugs were tested for other modes of antioxidant activity such as scavenging superoxide and hydroxyl radicals and inhibition of production of oxygen free radicals by human neutrophils stimulated with phorbol myristate acetate. Using an ESR spin-trapping technique, with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for superoxide and hydroxyl radicals, we found that the lazaroids U74500A and U78518F are, at best, weak scavengers of superoxide radicals whereas U78518F is a strong scavenger of hydroxyl radicals. In addition, lazaroids were found to be strong inhibitors (60-80% inhibition at 50 microM) of the superoxide-generating NADPH oxidase of human neutrophils. Inhibition of NADPH oxidase by lazaroids in cell-free systems suggested the action to be on the activated enzyme rather than on the process of activation. This may represent an important mode action of lazaroids and suggests their potential use in ischemic/inflammatory conditions involving oxygen free radical production by activated phagocytic cells.

Superoxide dismutase (SOD)-catalase conjugates. Role of hydrogen peroxide and the Fenton reaction in SOD toxicity.

Superoxide dismutase (SOD) has been championed as an effective antioxidant for the treatment of ischemia-reperfusion injury in a wide variety of tissues. Unfortunately a bell-shaped dose-response curve has been observed, whereby SOD at higher concentrations loses its effectiveness and may even enhance the extent of reperfusion injury. Using the xanthine/xanthine oxidase reaction to generate superoxide radicals, we have attempted to examine the role of the Fenton reaction in SOD toxicity observing that high SOD levels along with micromolar concentrations of Fe2+ greatly increased the production of the highly toxic hydroxyl radical. The production of superoxide radicals and their conversion to hydroxyl radicals were measured by using the spin-trapping agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and electron spin resonance (ESR). Attempts to counter the toxicity of SOD involved the covalent conjugation of SOD to catalase in an effort to lower the available free H2O2 and thus minimize the extent of the Fenton reaction. The conjugate was tested in both the xanthine/xanthine oxidase system and a rat heart model of ischemia-reperfusion. In the xanthine/xanthine oxidase model, the combination of SOD and Fe2+ results in an enhanced production of hydroxyl radicals which is inhibited by the inclusion of catalase. In reperfused ischemic hearts, working at levels of free SOD which were either toxic or failed to give any protection against reperfusion injury, an equivalent amount of SOD conjugated to catalase resulted in an 80% return to normal mechanical function of the reperfused hearts. We attribute the toxicity of free SOD in hearts subjected to ischemia-reperfusion injury to the production of hydroxyl radicals as a result of the increased Fenton reaction. This reaction is inhibited by the presence of catalase conjugated to SOD.

Electron spin resonance study on the permeability of superoxide radicals in lipid bilayers and biological membranes.

The permeability of lipid bilayers and biological membranes to superoxide free radicals was examined by using superoxide dismutase (SOD)-loaded lipid vesicles and SOD-loaded erythrocyte ghosts. After exposing SOD lipid vesicles and SOD ghosts to enzymatically produced superoxide radicals and using spin-trapping and electron spin resonance (ESR) techniques, we found that SOD entrapped within erythrocyte ghosts effectively scavenges external O2.- while SOD inside the lipid bilayers has no effect. These results confirm that O2.- is able to cross through a biological plasma membrane but not across a pure lipid bilayer. The data provide instruction as to how and where anti-oxidant therapy is to be approached relative to the site of oxygen free radical production.

Superoxide dismutase: improving its pharmacological properties by conjugation with human serum albumin.

Superoxide Dismutase has been reported to offer important pharmacological advantages in modifying oxygen toxicity as a result of its ability to scavenge oxygen free radicals. This has proven most exciting in reducing damage associated with post-reperfusion damage following myocardial ischemia. Unfortunately Superoxide Dismutase has a circulation life time of only a few minutes making the exact time of its administration crucial and somewhat impractical. We report here on the production of SOD-Albumin conjugates which have important advantages over free SOD in terms of stability, extended circulation time and reduced immunogenicity.