Bacterial lipopolysaccharide (LPS) activates the immune system and promotes inflammation via Toll-like receptor (TLR) 4, which regulates the synthesis and release of tumor necrosis factor (TNF)-␣ and other inflammatory cytokines. Previous studies have shown that the nucleoside adenosine suppresses LPS-stimulated TNF-␣ release in human UB939 macrophages by activating an adenosine A 3 receptor (A 3 AR) subtype on these cells. In this study, we examined the mechanism(s) underlying A 3 AR-dependent inhibition of TNF-␣ release in a mouse (RAW 264.7) cell line. Treatment of RAW 264.7 cells with LPS (3 g/ml) increased TNF-␣ release, which was reduced in a dose-dependent manner by adenosine analogs N 6 -(3-iodobenzyl)-adenosine-5Ј-N-methyluronamide (IB-MECA) and R-phenylisopropyladenosine and reversed by selective A 3 AR blockade. The increase in TNF-␣ release was preceded by an increase in intracellular Ca 2ϩ levels. Inhibition of intracellular Ca 2ϩ release by IB-MECA, a selective agonist of the A 3 AR, or with BAPTA-AM, an intracellular Ca 2ϩ chelator, reduced LPSstimulated TNF-␣ release. Activation of the A 3 AR or inhibition of intracellular Ca 2ϩ release also reduced LPS-stimulated nuclear factor-B (NF-B) activation and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Similar inhibition by A 3 AR was observed for LPS-stimulated inducible nitric-oxide synthase. These data support the contention that inhibition of LPS-stimulated release of inflammatory molecules, such as TNF-␣ and NO via the A 3 AR, involves suppression of intracellular Ca 2ϩ signaling, leading to suppression of NF-B and ERK1/2 pathways.

The expression of inducible nitric-oxide synthase (iNOS) in vascular smooth muscle cells leads to prolonged vasorelaxation in vivo and contributes to the profound vasodilation induced by bacterial lipopolysaccharide (LPS) in septic shock. This induction of iNOS depends, in large part, on activation of nuclear factor (NF)-B. Hypertonicity regulates the activity of NF-B in different cell lines; as such, we propose that it should also regulate the expression of iNOS. Thus, the goal of this study was to determine whether hypertonicity regulates iNOS expression and function in smooth muscle cells and to elucidate the mechanism(s) underlying this process. Treatment of hamster ductus deferens (DDT 1 MF-2) cells and porcine aortic smooth muscle cells with either mannitol (50 mM) or NaCl (50 mM) reduced LPS-stimulated iNOS expression and nitric oxide release. Both of these agents also reduced the activation of NF-B induced by LPS, tumor necrosis factor-␣ and interleukin-1␤ in smooth muscle cells. This inhibitory action was caused by suppression of IB-␣ phosphorylation, a prerequisite for ubiquitination and degradation of this protein, and showed additivity with N-benzoyloxycarbonyl (Z)-Leu-Leuleucinal (MG-132), an inhibitor of proteasomal degradation of IB-␣. Furthermore, exposure to mannitol inhibited the activity of IB kinase, an enzyme involved in phosphorylation of IB-␣. Mannitol was unable to affect the induction of iNOS produced by overexpression of RelA in DDT 1 MF-2 cells, suggesting that this agent does not have additional downstream inhibitory actions on this activated NF-B subunit. Taken together, these data suggest that these hypertonic solutions may prove useful as anti-inflammatory agents, especially against conditions associated with increased NF-B activity.

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A male worker entered an underground manure pit and lost consciousness. His coworker tried to rescue him and also lost consciousness in the pit. Emergency service arrived in minutes and removed both of them from the pit. Both men suffered anoxic brain injury and died in the hospital. Cases from Iowa Fatality Assessment and Control Evaluation Program Data Base program were reviewed. Occupational mortality rate was found to be 7.4 per 10,000 per year for occupational deaths related to agriculture compared to 3.1 per 10,000 per year for deaths not related to agriculture. In most of the cases associated with sewage or livestock waste handling, victims died of asphyxiation. While manure pit injury is rare, it has an extremely high fatality rate. The most effective strategy to decrease mortality is active prevention.

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