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-1β 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.
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.
The regulatory function of α 1B -adrenoceptors in mammalian heart homeostasis is controversial. The objective of the present study was to characterize the expression/activity of key proteins implicated in cardiac calcium handling (Na + /K + -ATPase and Ca 2+ -ATPases) and growth (ERK1/2, JNK1/2 and p38) in mice with cardiac-selective overexpression of constitutively active mutant α 1B -adrenoceptor (CAMα 1B -AR), which present a mild cardiac hypertrophy phenotype. Immunoblot assays showed that myocardial plasma membrane Ca 2+ -ATPase (PMCA) expression was increased by 30% in CAMα 1B -AR mice (N = 6, P < 0.05), although there was no change in sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA2) expression. Moreover, total Ca 2+ -ATPase activity was not modified, but a significant increase in the activity of the thapsigargin-resistant (PMCA) to thapsigargin-sensitive (SERCA) ratio was detected. Neither Na + /K + -ATPase activity nor the expression of α 1 and α 2 subunit isoforms was changed in CAMα 1B -AR mouse hearts. Moreover, immunoblot assays did not provide evidence for an enhanced activation of the three mitogen-activated protein kinases studied in this stage of hypertrophy. Therefore, these findings indicate that chronic cardiac α 1B -AR activation in vivo led to mild hypertrophy devoid of significant signs of adaptive modifications concerning primary intracellular calcium control and growth-related proteins, suggesting a minor pathophysiological role of this adrenergic receptor in mouse heart at this stage of development.
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