Thromboxane A2-induced Bi-directional Regulation of Cerebral Arterial Tone

Ronald L Neppl,Lubomir T Lubomirov,Ko Momotani,Gabriele Pfitzer,Masumi Eto,Avril V Somlyo

doi:10.1074/jbc.m807040200

Abstract

Myosin light chain phosphatase plays a critical role in modulating smooth muscle contraction in response to a variety of physiologic stimuli. A downstream target of the RhoA/Rho-kinase and nitric oxide (NO)/cGMP/cyclic GMP-dependent kinase (cGKI) pathways, myosin light chain phosphatase activity reflects the sum of both calcium sensitization and desensitization pathways through phosphorylation and dephosphorylation of the myosin phosphatase targeting subunit (MYPT1). As cerebral blood flow is highly spatio-temporally modulated under normal physiologic conditions, severe perturbations in normal cerebral blood flow, such as in cerebral vasospasm, can induce neurological deficits. In nonpermeabilized cerebral vessels stimulated with U-46619, a stable mimetic of endogenous thromboxane A2 implicated in the etiology of cerebral vasospasm, we observed significant increases in contractile force, RhoA activation, regulatory light chain phosphorylation, as well as phosphorylation of MYPT1 at Thr-696, Thr-853, and surprisingly Ser-695. Inhibition of nitric oxide signaling completely abrogated basal MYPT1 Ser-695 phosphorylation and significantly increased and potentiated U-46619-induced MYPT1 Thr-853 phosphorylation and contractile force, indicating that NO/cGMP/cGKI signaling maintains basal vascular tone through active inhibition of calcium sensitization. Surprisingly, a fall in Ser-695 phosphorylation did not result in an increase in phosphorylation of the Thr-696 site. Although activation of cGKI with exogenous cyclic nucleotides inhibited thromboxane A2-induced MYPT1 membrane association, RhoA activation, contractile force, and regulatory light chain phosphorylation, the anticipated decreases in MYPT1 phosphorylation at Thr-696/Thr-853 were not observed, indicating that the vasorelaxant effects of cGKI are not through dephosphorylation of MYPT1. Thus, thromboxane A2 signaling within the intact cerebral vasculature induces "buffered" vasoconstrictions, in which both the RhoA/Rho-kinase calcium-sensitizing and the NO/cGMP/cGKI calcium-desensitizing pathways are activated.

Highlights

Physiologic control of cerebral circulation is modulated metabolically via PO2 and PCO2, as well as via eicosanoids, endothelin, and nitric oxide (NO).2 Disruptions in the blood-brain barrier either by traumatic head injury or subarachnoid hemorrhage can cause prolonged and severe perturbations to normal cerebral blood flow
Effects of Thromboxane A2 and cGMP Signaling on Middle Cerebral Artery Contractility—To assess the general effects of TXA2 receptors (TXA2R) stimulation and cGMP signaling in the intact cerebral vasculature, we examined the effects of the stable thromboxane A2 mimetic U-46619 and 8-Br-cGMP on the contractility of the intact middle cerebral artery (MCA, Fig. 1)
The maximal contractile force induced by 300 nM U-46619 (10 min) is reversible upon addition of the Rho-kinase inhibitor Y-27632 (Fig. 1A, inset), not completely, suggesting a component of TXA2R-mediated contractile force is independent of Rho-kinase

Summary

Introduction

Physiologic control of cerebral circulation is modulated metabolically via PO2 and PCO2, as well as via eicosanoids, endothelin, and nitric oxide (NO). Disruptions in the blood-brain barrier either by traumatic head injury or subarachnoid hemorrhage can cause prolonged and severe perturbations to normal cerebral blood flow. Cyclic nucleotide-induced relaxation of vascular smooth muscle occurs through several potential downstream signaling pathways initiating Ca2ϩ desensitization, which include phospho-inhibition of RhoA1⁄7GTP at Ser-188 by cyclic GMPdependent kinase (cGKI) [14, 15], cGKI phosphorylation of telokin (16 –18), and inhibition of RhoA activation through protein kinase A (PKA)-dependent phosphorylation of G␣13 [19]. The nitric oxide donor SNP was shown to decrease CPI-17 phosphorylation coincident in time with the rise in [cGMP] and MLCP activity in porcine carotid arteries [31] It remains uncertain whether activation of cGKI in cerebrovascular smooth muscle inhibits RhoA activation and MYPT1 phosphorylation as in cell culture systems [32], or whether cyclic nucleotide-mediated phosphorylation of telokin, such as in phasic smooth muscles or dephosphorylation of CPI-17, contributes to activation of MLCP in the cerebral vasculature, which is a focus of this study

Methods

Results

Conclusion