Abstract 12708: Oxidative Stress and Extracellular Signal-regulated Protein Kinase (ERK) are Responsible for Impaired Desensitization and Irreversible Activation of Thrombin Receptor in Vascular Smooth Muscle, Which Underlie Cerebral Vasospasm
Background: Cerebral vasospasm is a critical determinant in the prognosis of subarachnoid hemorrhage (SAH). We clarified the increased expression and impaired desensitization of thrombin receptor PAR1 to increase vascular contractility after SAH using a rabbit model. The impaired PAR1 desensitization is attributable to oxidative stress and causes irreversible contraction after thrombin stimulation, which is a characteristic of cerebral vasospasm. Elucidating the molecular mechanism underlying the impairment of PAR1 desensitization is thought to be a key step for developing new therapeutic strategies for vasospasm.
Objective: The mechanism underlying impaired PAR1 desensitization was investigated using cultured smooth muscle cells, with a focus on the role of oxidative stress and the related protein kinases.
Methods and Results: In the cells at confluence on culture day 8, PAR1AP induced a transient [Ca2+]i elevation, followed by a sustained elevation at 65% of the peak level, as monitored with fura-2 fluorometry. After removing PAR1AP, [Ca2+]i returned to the pre-stimulation level. The second stimulation induced 80% of the first response. Thrombin induced a sustained [Ca2+]i elevation, which irreversibly persisted even after terminating thrombin stimulation. Serotonin induced a sustained [Ca2+]i elevation, and the second stimulation induced 99% of the first response. In the cells treated with diphenyleneiodonium chloride (DPI), a NOX inhibitor, from day 1 to day 8, PAR1AP and thrombin induced a transient [Ca2+]i elevation, which spontaneously returned to the pre-stimulation level. The second stimulation induced only a residual response. Treatment with vitamin C, tempol or N-acetyl-L-cysteine had no effect. The treatment with ERK inhibitor, PD184352 or PD0325901, 15 min before and during PAR1 stimulation exhibited the similar effect to that seen with DPI. The inhibitors of JNK, p38 kinase or Rho kinase had no effect. Either DPI or ERK inhibitors had little effect on the responses to serotonin.
Conclusion: Oxidative stress and an ERK pathway contribute to the impaired desensitization and irreversible activation of PAR1. Targeting oxidative stress and ERK could be a novel strategy to normalize the increased vascular reactivity after SAH.
- © 2013 by American Heart Association, Inc.