Reduced function of the RhoA/Rho‐Kinase pathway contributes for the vascular refractoriness to phenylephrine in septic mice

Abstract

Hypotension and reduced vascular responses to vasoconstrictors are two hallmarks of septic shock. Activation of the RhoA/Rho‐Kinase pathway plays an important role in the vascular effects of ligands able to increase blood pressure. We have investigated the hypothesis that a reduced functionality of this pathway plays a role in the hyporesponsiveness to alpha‐1 adrenergic agonists in experimental sepsis induced by bacterial lipopolysaccharide (LPS) or Klebsiella pneumonia (KP). Male Swiss mice (3–4 months; n = 6 each group) were used in this study. For the LPS model, animals received an intraperitoneal injection of LPS (011‐B4; 7.5 mg/kg), or sterile saline (0.1 ml/10 g). For the KP model, the mice were anesthetized with ketamine/xylazine (80 and 20 mg/kg i.p.) and were inoculated with a sub‐lethal dose of KP (4 × 108 CFU), or sterile saline (0.05 ml), which were administered directly into their trachea. At 6 h or 18 h after LPS injection, and 6 h or 24 h after inoculation of KP, the animals were anesthetized with ketamine/xylazine (100 and 20 mg/kg, i.m.), and had their femoral vein and carotid artery cannulated for intravenous administration of drugs and direct measurement of the mean arterial pressure (MAP), respectively. After stabilization of MAP all groups received the selective Rho‐kinase inhibitor Y‐27632 (0.1 mg/kg, i.v.), or vehicle (10 μl/10 g, i.v.), followed by bolus infusions of phenylephrine (PE, 3, 10 and 30 nmol/kg). Each subsequent drug was administered only when the MAP returned to basal values. After the recording of MAP, blood samples were collected and used for Griess reaction, the animals were euthanized with anesthetic overdose and had the aorta collected and subjected to Western blotting detection of Rho‐A, ROCK I and ROCK II. Animals from both LPS and KP groups presented systemic hypotension, reduced reactivity to PE, and increased plasma levels of nitrate+nitrite, as detected at early (6 h) or late (18 h and 24 h, respectively) periods. Administration of 0.1 mg/kg Y‐27632 was unable to alter PE‐increased MAP in control animals, but resulted in further reduction in the hypertensive effect of PE at 18 h after LPS, as well as at 6 h and 24 h after inoculation of KP. For instance, the increase in MAP induced by 30 nmol/kg PE before and after Y‐2763, respectively, was 46.1 ± 4.8 and 38.5 ± 5.5 mmHg in control animals, and 27.5 ± 5.2 and 10.7 ± 2.3 mmHg at 18 h after LPS injection. In spite of the lack of significant changes in the levels of RhoA, ROCK I and ROCK II in the aortas from KP‐treated animals, the Western blotting analysis revealed that both RhoA and Rock II were significantly increased at 6 h after LPS, compared with samples from control animals. However, the levels of RhoA were reduced by 53% in the LPS 18 h groups. Our results reveal that the pressor effect of PE is more sensitivity to inhibition of ROCK in animals subjected to experimental models of sepsis, and suggest that changes in the expression or function of components of the Rho‐A/Rho‐ kinase pathway may contribute to the cardiovascular failure that takes place in septic shock.Support or Funding InformationFAPESC, SC, Brazil (TR2012000367) and CNPq, Brazil (448738/2014)