Mitochondrial ATP synthase inhibition and nitric oxide are involved in muscle weakness that occurs in acute exposure of rats to monocrotophos

Organophosphate poisoning in the context of self-harm is a common medical emergency in Asia. Prolonged muscle weakness is an important but poorly understood cause of morbidity and mortality of the poisoning. This study examined mitochondrial function and its modulation by nitric oxide in muscle weakness of rats exposed to an acute, oral (0.8LD50) dose of monocrotophos. Muscle mitochondrial ATP synthase activity was inhibited in the rat in acute exposure to monocrotophos while respiration per se was not affected. This was accompanied by decreased mitochondrial uptake of calcium and increased levels of nitric oxide. Reactive cysteine groups of ATP synthase subunits were reduced in number, which may contribute to decreased enzyme activity. The decrease in ATP synthase activity and reactive cysteine groups of ATP synthase subunits was prevented by treatment of animals with the nitric oxide synthase inhibitor, L-NG Nitroarginine methyl ester, at 12 mg/kg body weight for 9 days in drinking water, prior to monocrotophos exposure. This indicated a role for nitric oxide in the process. The alterations in mitochondrial calcium uptake may influence cytosolic calcium levels and contribute to muscle weakness of acute organophosphate exposure

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.

Signalling mechanisms in contraction-mediated stimulation of intracellular NO production in cat ventricular myocytes

In this study we sought to determine whether contractile activity has a role as a signalling mechanism in the activation of intracellular nitric oxide (NOi) production induced by electrical stimulation of cat ventricular myocytes. Field stimulation (FS) of single ventricular myocytes elicited frequency-dependent increases in NOi that were blocked by the calmodulin (CaM) inhibitor 10 μm W-7 and partially inhibited by the phosphatidylinositol 3′-kinase (PI-(3)K) inhibitor 10 μm LY294002. Increasing extracellular [Ca2+] caused a concentration-dependent increase in FS-induced NOi that was partially inhibited by LY294002. The negative inotropic agents BDM (5 mm) or blebbistatin (10 μm) decreased cell shortening and NOi production without concomitant changes in L-type Ca2+ current (ICa,L) or [Ca2+]i transients. The positive inotropic agents EMD 57033 or CGP 48506 (1 μm) increased cell shortening and NOi production without concomitant changes in ICa,L or [Ca2+]i transients. FS-induced NOi production was decreased in myocytes infected (100 multiplicity of viral infection (MOI); 24 h) with a replication-deficient adenovirus expressing a dominant-negative mutant of protein kinase B (Akt) compared with cells infected with a control adenovirus expressing β-galactosidase. FS-induced NOi was partially inhibited by either endothelial (eNOS) or neuronal nitric oxide synthase (nNOS) inhibitors and completely blocked by simultaneous exposure to both. FS-induced [Ca2+]i transients were increased by the nNOS inhibitor nNOS-I (0.24 μm), decreased by the eNOS inhibitor L-NIO (1 μm) and unchanged by exposure to both inhibitors. We conclude that in cat ventricular myocytes, FS-induced NOi production requires both Ca2+-dependent CaM signalling and Ca2+-independent PI-(3)K–Akt signalling activated by contractile activity. FS activates NOi production from both eNOS and nNOS, and each source of NOi exerts opposing effects on [Ca2+]i transient amplitude. These findings are important for understanding the regulation of NOi signalling in the normal and mechanically failing heart

Phenylephrine acts via IP(3)-dependent intracellular NO release to stimulate L-type Ca(2+) current in cat atrial myocytes

This study determined the effects of α(1)-adrenergic receptor (α(1)-AR) stimulation by phenylephrine (PE) on L-type Ca(2+) current (I(Ca,L)) in cat atrial myocytes. PE (10 μm) reversibly increased I(Ca,L) (51.3%; n = 40) and shifted peak I(Ca,L) activation voltage by −10 mV. PE-induced stimulation of I(Ca,L) was blocked by each of 1 μm prazocin, 10 μml-NIO, 10 μm W-7, 10 μm ODQ, 2 μm H-89 or 10 μm LY294002, and was unaffected by 10 μm chelerythrine or incubating cells in pertussis toxin (PTX). PE-induced stimulation of I(Ca,L) also was inhibited by each of 10 μm ryanodine or 5 μm thapsigargin, by blocking IP(3) receptors with 2 μm 2-APB or 10 μm xestospongin C or by intracellular dialysis of heparin. In field-stimulated cells, PE increased intracellular NO (NO(i)) production. PE-induced NO(i) release was inhibited by each of 1 μm prazocin, 10 μml-NIO, 10 μm W-7, 10 μm LY294002, 2 μm H-89, 10 μm ryanodine, 5 μm thapsigargin, 2 μm 2-APB or 10 μm xestospongin C, and unchanged by PTX. PE (10 μm) increased phosphorylation of Akt, which was inhibited by LY294002. Confocal microscopy showed that PE stimulated NO(i) release from subsarcolemmal sites and this was prevented by 2 mm methyl-β-cyclodextrin, an agent that disrupts caveolae formation. PE also increased local, subsarcolemmal SR Ca(2+) release via IP(3)-dependent signalling. Electron micrographs of atrial myocytes show peripheral SR cisternae in close proximity to clusters of caveolae. We conclude that in cat atrial myocytes PE acts via α(1)-ARs coupled to PTX-insensitive G-protein to release NO(i), which in turn stimulates I(Ca,L). PE-induced NO(i) release requires stimulation of both PI-3K/Akt and IP(3)-dependent Ca(2+) signalling. NO stimulates I(Ca,L) via cGMP-mediated cAMP-dependent PKA signalling. IP(3)-dependent Ca(2+) signalling may enhance local SR Ca(2+) release required to activate Ca(2+)-dependent eNOS/NO(i) production from subsarcolemmal caveolae sites