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We recently demonstrated that activation of the pulmonary sensory neurons plays a critical role in prevention of endotoxin-induced shock by releasing calcitonin gene-related peptide (CGRP) in rats. CGRP increased the endothelial production of prostacyclin (PGI(2)) in the lungs, thereby preventing endotoxin-induced shock response by inhibiting tumor necrosis factor-alpha (TNF-alpha) production. Since antithrombin (AT) enhances sensory neuron activation, we hypothesized that AT might reduce endotoxin-induced hypotension by enhancing the activation of pulmonary sensory neurons in rats. We examined this possibility using a rat model of endotoxin shock. AT-induced effects including reduction of hypotension (n = 5) and inhibition of induction of iNOS (n = 4 or 5) and TNF- alpha (n = 5) in the lungs of endotoxin-treated animals were completely reversed by pretreatment with capsazepine (CPZ) (n = 4 or 5), a vanilloid receptor antagonist, or CGRP(8-37), a CGRP receptor antagonist (n = 4 or 5). AT enhanced endotoxin-induced increases in lung tissue levels of CGRP (n = 4), but this effect of AT was not seen in animals pretreated with CPZ (n = 4). CGRP produced therapeutic effects (n = 5) similar to those induced by AT, and such therapeutic effects were completely abrogated by pretreatment with indomethacin (n = 4). AT increased CGRP release from cultured dorsal root ganglion neurons only in the presence of anandamide (n = 5), and AT-induced increase in CGRP release was not observed in the presence KT5720, an inhibitor of protein kinase A (n = 5). AT markedly increased intracellular levels of cAMP in the presence of anandamide (n = 5). These results strongly suggested that AT might reduce endotoxin-induced hypotension in rats by enhancing activation of sensory neurons via activation of protein kinase A.

Objective: We attempted to determine whether activation of the sensory neuron contributes to reduction of endotoxin-induced hypotension by inhibiting tumor necrosis factor (TNF)-alpha production via calcitonin gene-related peptide (CGRP) release in rats., Design: Prospective, randomized, controlled study., Setting: Research laboratory at a university medical center., Subjects: Wistar rats weighing 220-280 g., Interventions: Mean arterial blood pressure was measured in rats administered endotoxin intravenously. Animals were pretreated with capsazepine (a vanilloid receptor antagonist), CGRP(8-37) (a CGRP receptor antagonist), and indomethacin before endotoxin administration. Levels of CGRP, 6-keto-prostaglandin F1alpha, TNF-alpha, and cytokine-induced neutrophil chemoattractant (CINC) were measured by enzyme immunoassay methods. The concentration of NO2/NO3 was measured using the Griess reagent. Tissue levels of messenger RNA of the inducible form of nitric oxide synthase (iNOS) and TNF-alpha were determined by reverse transcription polymerase chain reaction., Measurements and Main Results: Both lung levels of CGRP and plasma levels of 6-keto-prostaglandin F1alpha were increased after intravenous administration of endotoxin (5 mg/kg), peaking at 90 mins after endotoxin administration. Increases in plasma levels of 6-keto-prostaglandin F1alpha at 90 mins after endotoxin administration (766 +/- 134 pg/mL) were inhibited by pretreatment with capsazepine (373 +/- 44 pg/mL, p < .05), CGRP(8-37) (406 +/- 64 pg/mL, p < .05), and indomethacin (154 +/- 40 pg/mL, p < .05). Although none of the pretreatments affected a series of endotoxin-induced responses, including increases in lung tissue levels of TNF-alpha, CINC, and iNOS and the resultant hypotension in animals given 5 mg/kg endotoxin, such pretreatments enhanced these pathologic responses in animals given a smaller dose of endotoxin (1 mg/kg) to the same extent as those induced by 5 mg/kg of endotoxin, suggesting that shock responses induced by 5 mg/kg endotoxin are maximum responses and activation of sensory neurons in endotoxin-treated rats is essentially a reparative response., Conclusion: Activation of sensory neurons might contribute to reduction of endotoxin-induced hypotension by releasing CGRP, which is capable of promoting endothelial production of prostacyclin.

Although urinary trypsin inhibitor (UTI) has been shown to inhibit tumor necrosis factor (TNF)-alpha- production, the detailed mechanism(s) remains unclear. This study was undertaken to elucidate the molecular mechanism(s) underlying this inhibitory effect in monocytes in vitro and in rats given lipopolysaccharide (LPS). TNF-alpha production by monocytes stimulated with LPS (100 ng/ml) was inhibited by UTI at concentrations higher than 100 U/ml. Expression of early growth response factor-1 (Egr-1) and phosphorylation of extracellular signal-regulated protein kinases 1/2 in monocytes stimulated with LPS were inhibited by UTI. UTI (50,000 U/kg i.v.) inhibited LPS (5 mg/kg i.v.)-induced increases in lung tissue levels of Egr-1, TNF-alpha mRNA, and TNF-alpha in rats. UTI inhibited LPS-induced hypotension by inhibiting pulmonary induction of inducible nitric oxide synthase (iNOS). We previously demonstrated that anti-TNF-alpha antibody and aminoguanidine, a selective inhibitor of iNOS, reduced LPS-induced hypotension in this animal model. Furthermore, we also reported that reduction of LPS-induced coagulation abnormalities in rats did not affect inflammatory responses and hypotension in this animal model. Taken together, these observations strongly suggested that UTI inhibited LPS-induced production of TNF-alpha by inhibiting activation of the extracellular signal-regulated protein kinases 1/2-Egr-1 pathway in monocytes, which might at least partly contribute to reduction of hypotension through inhibition of iNOS induction in rats given LPS.

Antithrombin (AT) prevents Escherichia coli-induced hypotension in animal models of sepsis, and it further reduces the mortality of patients with septic shock. In the present study, we examined whether AT may prevent the endotoxin (ET)-induced hypotension by promoting the endothelial release of prostacyclin (PGI(2)) in rats. Intravenous administration of AT (250 U/kg) prevented both hypotension and the increases in plasma levels of NO(2)(-)/NO(3)(-) in rats given ET. Lung expression of messenger RNA (mRNA) for tumor necrosis factor-alpha (TNF-alpha) was transiently increased after ET administration, followed by the increases in lung tissue levels of TNF-alpha. Both the lung activity of the inducible form of nitric oxide synthase (iNOS) and the lung expression of iNOS mRNA in animals administered ET were gradually increased after the TNF-alpha mRNA expression had peaked. Administration of AT significantly inhibited these increases. Neither DEGR-F.Xa, a selective inhibitor of thrombin generation, nor Trp(49)-modified AT, which is not capable of promoting the endothelial release of PGI(2), showed any effects on these changes induced by ET. Administration of antirat TNF-alpha antibody produced effects similar to those induced by AT. Indomethacin pretreatment abrogated the effects induced by AT. Iloprost, a stable derivative of PGI(2), produced effects similar to those of AT. These findings suggested that AT prevents the ET-induced hypotension by inhibiting the induction of iNOS through inhibiting TNF-alpha production. These effects of AT could be mediated by the promotion of endothelial release of PGI(2) and might at least partly explain the therapeutic effects for septic shock.

Although urinary trypsin inhibitor (UTI) has been shown to inhibit tumor necrosis factor (TNF)-1-production, the detailed mechanism(s) remains unclear. This study was undertaken to elucidate the molecular mechanism(s) underlying this inhibitory effect in monocytes in vitro and in rats given lipopolysaccharide (LPS). TNF-α production by monocytes stimulated with LPS (100 ng/ml) was inhibited by UTI at concentrations higher than 100 U/ml. Expression of early growth response factor-1 (Egr-1) and phosphorylation of extracellular signal-regulated protein kinases 1/2 in monocytes stimulated with LPS were inhibited by UTI. ETI (50,000 U/kg iv) inhibited LPS (5 mg/kg iv)-induced increases in lung tissue levels of Egr-1, TNF-α mRNA, and TNF-α in rats. UTI inhibited LPS induced hypotension by inhibiting pulmonary induction of inducible nitric oxide synthase (iNOS). We previously demonstrated that anti-TNF-α antibody and aminoguanidine, a selective inhibitor of iNOS, reduced LPS-induced hypotension in this animal model. Furthermore, we also repotted that reduction of LPS-induced coagulation abnormalities in rats did not affect inflammatory responses and hypotension in this animal model. Taken together, these observations strongly suggested that UTI inhibited LPS-induced production of TNF-α by inhibiting activation of the extracellular signal regulated protein kinases 1/2-Egr-1 pathway in monocytes, which might at least partly contribute to reduction of hypotension through inhibition of iNOS induction in rats given LPS. [ABSTRACT FROM AUTHOR]