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Objective: We investigated whether a reduced activity in the Rho-A/Rho-kinase pathway could be involved in the impaired vascular reactivity observed in septic shock., Design: Ex vivo animal study., Setting: University research laboratory., Subjects: Male Wistar rats., Interventions: Rats received an intraperitoneal injection of lipopolysaccharide (LPS, 10 mg/kg) either 6 or 24 hours before the onset of our experiments. The effects of Y-27632 (a Rho-kinase inhibitor) were assessed in first-order mesenteric rings taken from these animals using wire myograph. The expression of Rho-A, Rho-kinases I and II, and the total and phosphorylated myosin phosphatase targeting subunit 1 (MYPT1) were assessed by Western blotting., Measurements and Main Results: The EC50 to Y-27632 was reduced from 2.10 microM (1.22-3.66 microM) (control) to 0.21 microM (0.09-0.44 microM), and 9.54 (0.82-110.30) nM in LPS-treated groups 6 and 24 hours, respectively. The increased potency of Y-27632 was partially reversed by endothelium removal at both 6 and 24 hours. Incubation of Nomega-nitro-l-arginine methyl ester hydrochloride or 1400W (a nonselective and an inducible nitric oxide synthase inhibitor, respectively) normalized the responses to Y-27632 seen 6 hours after LPS. However, 1400W had no effect, whereas Nomega-nitro-l-arginine methyl ester hydrochloride caused a partial reduction in the enhanced potency of Y-27632 found 24 hours after LPS. The soluble guanylate cyclase inhibitor oxadiazolo[4,3-alpha]quinoxalin-1-one was able to bring the Y-27632 response back to normal both 6 and 24 hours after LPS. Rho-A, Rho-kinase I, Rho-kinase II, and MYPT1 were increased in mesenteric arteries from endotoxemic rats, but the phosphorylated MYPT1 was significantly reduced. However, incubation with oxadiazolo[4,3-alpha]quinoxalin-1-one circumvented the inhibition of MYPT1 phosphorylation found in preparations from LPS-treated animals., Conclusions: Our findings revealed an impaired Rho-A/Rho-kinase-mediated phosphorylation of MYPT1 in vessels from endotoxemic animals in a cyclic guanosine monophosphate-dependent manner, suggesting that changes in mechanisms involved in calcium sensitization play a pivotal role in cardiovascular changes observed in septic shock.

Blockades of cytokine and oxygen radicals release are considered to be beneficial in reducing multiple organ injury and increasing the survival rate in sepsis/septic shock. Thus, we examined the protective efficacy of N-allylsecoboldine, a secoaporphine derivative with antioxidant and alpha1-adrenoceptor blocking activities, in rats treated with endotoxin (E. coli lipopolysaccharide, LPS). Pretreatment of LPS-treated rats with N-allylsecoboldine significantly attenuated the late-phase hypotension, hypoglycemia and incremental plasma tumor necrosis factor (TNF)-alpha. Overproduction of plasma nitrate in endotoxemia was not changed but the continuous decrease of urinary nitrate appeared to be partially ameliorated by N-allylsecoboldine. However, N-allylsecoboldine inhibited the inducible nitric oxide synthase (iNOS) protein expression in the renal cortex of endotoxemic rats. N-allylsecoboldine also improved the endotoxemia-induced organ injury as demonstrated from the conspicuous recovery of marker enzymes in the LPS-treated rats. Endotoxemia was associated with renal dysfunctions as indicated by decreases in renal blood flow, urinary potassium excretion, and renal nitrate clearance. However, pretreatment with N-allylsecoboldine showed significant alleviation of these renal dysfunctions. In addition, a lower dose of N-allylsecoboldine ameliorated the mortality of LPS-treated mice. This study demonstrates N-allylsecoboldine's ability to avail against acute renal failure and increase survival rate during endotoxemia. These beneficial effects may be attributed to the inhibition of iNOS expression, TNF-alpha production, and free radical scavenging activities. However, the role of alpha1-adrenoceptor antagonism for N-allylsecoboldine in sepsis remains unclear.

This study addressed the question of whether thaliporphine, a phenolic aporphine alkaloid obtained from Chinese herbs and possessing antioxidant and alpha-1 adrenoceptor antagonistic activity, has protective effects in endotoxaemic rats and we attempted to elucidate the mechanisms contributing to such protective effects. Injection of rats with endotoxin (E. coli lipopolysaccharide, LPS) induced severe hypotension and tachycardia as well as vascular hyporeactivity to noradrenaline. Pretreatment of LPS-treated rats with thaliporphine attenuated the delayed hypotension significantly whilst only a higher dose (1 mg/kg) of thaliporphine decreased LPS-induced tachycardia. LPS significantly increased nitric oxide (NO.) and superoxide anion (O(2).(-)) levels, a response that was reduced by pretreatment with 1 mg/kg thaliporphine. Endotoxaemia for 240 min resulted in a bell-shaped time course for the change of serum tumour necrosis factor-alpha (TNF-alpha) level with a peak at 60 min. Pretreatment of LPS-treated rats with 1 mg/kg thaliporphine significantly reduced the serum TNF-alpha level at 60 min. In addition, LPS caused a biphasic change in blood glucose and thaliporphine attenuated the late-phase decrease in blood glucose. Endotoxaemia induced multiple organ injury in the liver, kidney and heart, as indicated by increases of aspartate aminotransferase (GOT), alanine aminotransferase (GPT), creatinine (CRE), lactate dehydrogenase (LDH) and creatine phosphate kinase muscle-brain (CKMB) levels in serum. These increases of biochemical markers and inflammatory cell infiltration into injured tissues were reduced significantly by treatment with thaliporphine. In addition, thaliporphine increased the survival rate of LPS-treated mice dose-dependently. In conclusion, our results suggest that thaliporphine could be a novel agent for attenuating endotoxin-induced circulatory failure and multiple organ injury and may increase the survival rate. These beneficial effects of thaliporphine may be attributed to the suppression of TNF-alpha, NO. and O(2).(-) production.