Role of RhoA/Rho-Kinase and Nitric Oxide in Vascular Reactivity in Rats with Endotoxaemia

• Main Authors: Mei-Hui Liao, 廖美惠
• Other Authors: Chin-Chen Wu
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/57592168979906513098

博士 === 國防醫學院 === 醫學科學研究所 === 101 === Smooth muscle contractility is dually regulated by cytoplasmic Ca2+ concentration and Ca2+ sensitivity. The pathway of RhoA/Rho-kinase (RhoA/ROK) is the major cellular target for regulating Ca2+ sensitivity of agonist-induced contraction (including α1-adrenergic agonist). The activation of RhoA leads to stimulation of ROK that can phosphorylate and subsequently inactivate myosin light chain phosphatase, favoring myosin light chain phosphorylation, actin-myosin interaction and cell contraction. Previous studies have shown that protein kinase G (PKG) inhibits RhoA-induced Ca2+ sensitization by phosphorylating Ser188 of RhoA. In addition, the RhoA/ROK pathway is inhibited by cyclic guanosine monophosphate (cGMP) dependent mechanism after lipopolysaccharide (LPS) in small mesenteric arteries. These indicate that cGMP/PKG pathway regulates the RhoA/ROK activity leading to vasodilation. Administration of rats with LPS induces a biphasic hypotension (an early hypotension and a delayed hypotension) and causes a sustained attenuation to vasoconstrictor agents. The early hypotension induced by LPS is proposed to be mainly associated with the release of endogenous bradykinin (BK), which activates endothelial nitric oxide synthase (eNOS), resulting in vasodilation. In addition, excessive NO production by inducible NOS (iNOS) participates in LPS-induced delayed hypotension and aortic hyporesponsiveness to noradrenaline (NA). However, there is no in vivo evidence to explore the effect of RhoA/ROK in animals with endotoxaemia and the interaction of RhoA/ROK and NO in the regulation of vascular reactivity at different time-point in endotoxaemia in vivo and ex vivo experiments. In this study, we tried to examine (i) the role of RhoA/ROK in the regulation of vascular reactivity and (ii) the relationship between RhoA/ROK and NO at different time-point of endotoxaemia. Male Wistar rats were intravenously infused for 10 min with saline or E. coli endotoxin (LPS, 10 mg/kg) and divided into five groups: (i) Control, sacrificed 6 h after saline infusion; (ii) LPS1h, sacrificed 1 h after LPS infusion; (iii) LPS2h, sacrificed 2 h after LPS infusion; (iv) LPS4h, sacrificed 4 h after LPS infusion; and (v) LPS6h, sacrificed 6 h after LPS infusion. LPS1h and LPS2h were regarded as early endotoxaemia, whereas LPS4h and LPS6h were regarded as late endotoxaemia. We recorded the changes of haemodynamics, biochemical variables, pressor response to NA, RhoA activity, NO levels as well as BK levels. In addition, we evaluated vascular reactivity ex vivo. Our results indicated that LPS produced a biphasic hypotension and sustained vascular hyporeactivity to NA in vivo. Interestingly, this hyporeactivity did not occur in ex vivo during early endotoxaemia. This could be due to increases of aortic RhoA activity and myosin phosphatase targeting subunit 1 phosphorylation. In addition, pressor response to NA and vascular reactivity in early endotoxaemia were inhibited by ROK inhibitor, Y27632. Furthermore, plasma BK was increased at 10 min and aortic endothelial NO synthase expression was increased at 1 h after LPS. In late endotoxaemia, the hypotension and vascular hyporeactivity was associated with aortic inducible NO synthase expression and an increased serum NO level. In the present study, our results demonstrate that an increased RhoA activity may compensate vascular reactivity in early endotoxaemia ex vivo, while in late endotoxaemia, the large production of NO by iNOS inhibiting RhoA activity leads to vascular hyporeactivity in vivo and ex vivo. Therefore, both RhoA/ROK and iNOS/NO pathways play important roles in regulation of vascular reactivity and blood pressure in endotoxaemic rats. We propose that a combination of using NO/cGMP pathway inhibitors and specific RhoA activators could be a novel therapeutic strategy in the vascular hyporeactivity associated with endotoxaemic shock. Different animal models have been developed to study the pathophysiology and treatment of sepsis. Polymicrobial sepsis induced by cecal ligation and puncture (CLP) is a commonly used model because it is similar to the progression and characteristics of human sepsis. CLP animals have circulatory failure, multiple organ dysfunction syndrome, metabolic acidosis, and electrolyte imbalance, as seen in patients with severe sepsis. Over the past year, this laboratory has focused on the treatment of sepsis, attempting to develop new therapeutic drugs to improve the survival of animals induced by CLP. Indded, after therapeutic interventions, the alterations of these parameters were attenuated along with the improvement of survival rate. After therapeutic interventions, the alterations of these parameters were attenuated along with the improvement of survival rate. I observed that if some of these parameters were dramatically changed in CLP-induced sepsis, rats survived less than 18 h. It seems that these parameters could predict the early death of animals in this model. However, the relationships between mortality and haemodynamic or serum parameters in the CLP model have not yet been studied. Thus, the aim of this study was to examine which possible biomarkers were associated with mortality in the CLP-induced sepsis model. Animals were divided into three groups: (1) sham-operated (SOP); (2) survivors (at 18 h) of CLP; and (3) non-survivors (died between 9 h and 18 h) of CLP. The changes of haemodynamics, biochemical variables, blood gas, and electrolytes were monitored during the 18-h observation. Compared with survivors, non-survivors showed significant difference in (1) blood glucose; (2) lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, creatinine, and blood urea nitrogen in serum; and (3) base excess, HCO3-, PaCO2, potassium, and calcium in whole blood at 9 h after CLP. However, after receiver operating characteristic (ROC) curve and multifactor dimensionality reduction analysis, the union of HCO3- plus blood glucose served as the best biomarker of early death in rats with CLP-induced sepsis. Thus, these parameters could guide experimental procedures for making right interventions when utilizing CLP as a sepsis model in rats.