Mechanisms associated to impaired activity of cardiac P-type ATPases in endothelial nitric oxide synthase knockout mice

The effect of long-lasting in vivo restriction of nitric oxide (NO) bioavailability on cardiac and renal P-type ATPases critical for intracellular ion homeostasis is controversial. Previous work has shown in eNOS knockout (eNOS−/−) mice hearts that Na+/K+- and Ca2+-ATPase activities were depressed but the underlying mechanisms are still unclear. The goal of this study was to characterize potential alterations responsible for impaired enzyme activity in eNOS−/− mice. Na+/K+-ATPase activity from crude preparations of adult male eNOS−/− mice hearts and kidneys was reduced compared with wild-type animals (32 %, p < 0.05 and 16 %, p < 0.0001, respectively). Immunoblot analysis showed that although the expression of the predominant (or exclusive, for the kidney) Na+/K+-ATPase á1 isoform was not significantly changed, there was an important downregulation of the less abundant á2 isoform in the heart (57 %, p < 0.0001). In addition, although cardiac Ca2+-ATPase activity was unaltered, the expression of sarco/endoplasmic reticulum Ca2+-ATPase 2 protein in eNOS−/− mice was very high (290 % compared with wild-type animals, p < 0.0001) without any significant change in phospholamban expression. Consistent with these findings, the content of cardiac and renal free sulfhydryl groups, essential for the catalytic function of such ATPases, was decreased (23 %, p < 0.01 and 35 %, p < 0.05, respectively). Altogether, the present results suggest that the absence of eNOS promotes a compartmentalized altered redox balance that affects the activity and expression of ion transport ATPases (AU)

Mechanisms associated to impaired activity of cardiac P-type ATPases in endothelial nitric oxide synthase knockout mice.

The effect of long-lasting in vivo restriction of nitric oxide (NO) bioavailability on cardiac and renal P-type ATPases critical for intracellular ion homeostasis is controversial. Previous work has shown in eNOS knockout (eNOS(-/-)) mice hearts that Na(+)/K(+)- and Ca(2+)-ATPase activities were depressed but the underlying mechanisms are still unclear. The goal of this study was to characterize potential alterations responsible for impaired enzyme activity in eNOS(-/-) mice. Na(+)/K(+)-ATPase activity from crude preparations of adult male eNOS(-/-) mice hearts and kidneys was reduced compared with wild-type animals (32 %, p < 0.05 and 16 %, p < 0.0001, respectively). Immunoblot analysis showed that although the expression of the predominant (or exclusive, for the kidney) Na(+)/K(+)-ATPase α1 isoform was not significantly changed, there was an important downregulation of the less abundant α2 isoform in the heart (57 %, p < 0.0001). In addition, although cardiac Ca(2+)-ATPase activity was unaltered, the expression of sarco/endoplasmic reticulum Ca(2+)-ATPase 2 protein in eNOS(-/-) mice was very high (290 % compared with wild-type animals, p < 0.0001) without any significant change in phospholamban expression. Consistent with these findings, the content of cardiac and renal free sulfhydryl groups, essential for the catalytic function of such ATPases, was decreased (23 %, p < 0.01 and 35 %, p < 0.05, respectively). Altogether, the present results suggest that the absence of eNOS promotes a compartmentalized altered redox balance that affects the activity and expression of ion transport ATPases.

Anti-inflammatory effects of LASSBio-998, a new drug candidate designed to be a p38 MAPK inhibitor, in an experimental model of acute lung inflammation.

We investigated the effects of LASSBio-998 (L-998), a compound designed to be a p38 MAPK (mitogen-activated protein kinase) inhibitor, on lipopolysaccharide (LPS)-induced acute lung inflammation in vivo. BALB/c mice were challenged with aerosolized LPS inhalation (0.5 mg/ml) 4 h after oral administration of L-998. Three hours after LPS inhalation, bronchoalveolar lavage fluid was obtained to measure the levels of the proinflammatory cytokines TNF-α (tumor necrosis factor-α) and IL-1 (interleukin-1) and the chemokines MCP-1 (monocyte chemoattractant protein-1) and KC (keratinocyte chemoattractant). In addition, neutrophil infiltration and p38 MAPK phosphorylation was measured. L-998 inhibited LPS-induced production of TNF-α and IL-1ß and did not alter KC and MCP-1 levels. Furthermore, L-998 also significantly decreased neutrophil accumulation in lung tissues. As expected, L-998 diminished p38 MAPK phosphorylation and reduced acute lung inflammation. Inhibition of p38 MAPK phosphorylation by L-998 was also demonstrated in LPS-challenged murine C57BL/6 peritoneal macrophages in vitro, with concentration-dependent effects. L-998 suppressed LPS-induced lung inflammation, most likely by inhibition of the cytokine-p38 MAPK pathway, and we postulate that L-998 could be a clinically relevant anti-inflammatory drug candidate.

Aspirin and indomethacin reduce lung inflammation of mice exposed to cigarette smoke.

Neutrophil accumulation response to cigarette smoke (CS) in humans and animal models is believed to play an important role in pathogenesis of many tobacco-related lung diseases. Here we evaluated the lung anti-inflammatory effect of aspirin and indomethacin in mice exposed to CS. C57BL/6 mice were exposed to four cigarettes per day during 4 days and were treated i.p. with aspirin or indomethacin, administered each day 1h before CS exposure. Twenty four hours after the last exposure, cells and inflammatory mediators were assessed in bronchoalveolar lavage (BAL) fluid and the lungs used for evaluation of lipid peroxidation, p38 mitogen-activated protein kinase (MAPK) phosphorylation and nuclear transcription factor kappaB (NF-kappaB) activation. Exposure to CS resulted in a marked lung neutrophilia. Moreover, the levels of oxidative stress-related lipid peroxidation, prostaglandin E(2) (PGE(2)), interleukin 1beta (IL-1beta), monocyte chemotactic protein 1 (MCP-1), and activated NF-kappaB and p38 MAPK were greatly increased in CS group. Aspirin or indomethacin treatment led to a significant reduction of neutrophil influx, but only aspirin resulted in dramatic decrease of inflammatory mediators. Moreover, both drugs reduced lung p38 MAPK and NF-kappaB activation induced by CS. These results demonstrate that short-term CS exposure has profound airway inflammatory effects counteracted by the anti-inflammatory agents aspirin and indomethacin, probably through COX-dependent and -independent mechanisms.