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. 2009 Mar 6;284(10):6348-60.
doi: 10.1074/jbc.M807040200. Epub 2008 Dec 17.

Thromboxane A2-induced bi-directional regulation of cerebral arterial tone

Ronald L Neppl  1 , Lubomir T Lubomirov, Ko Momotani, Gabriele Pfitzer, Masumi Eto, Avril V Somlyo
Affiliations

Thromboxane A2-induced bi-directional regulation of cerebral arterial tone

Ronald L Neppl et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
Effects of thromboxane A2 and cGMP on rat MCA contractility. A, representative force trace from endothelium-denuded, nonpermeabilized MCA stimulated with 154 mm K+ and 300 nm U-46619 with and without 25 μm 8-Br-cGMP pretreatment. Contractile force induced by 300 nm U-46619 is reversed by treatment with 10 μm Y-27632 (inset). B, representative force trace from endothelium-denuded, nonpermeabilized MCA stimulated with 154 mm K+ and 25 μm 8-Br-cGMP following maximal sustained 300 nm U-46619 contraction. C, summary of the effects of 8-Br-cGMP added pre- or post-stimulation with U-46619. The effects of 8-Br-cGMP on contractile force are normalized to that of the average maximal U-46619 contraction (dashed line). Error bars represent mean ± S.D.; *, p < 0.05 with respect to control, n = 4 independent experiments.
FIGURE 2.
FIGURE 2.
U-46619-induced RhoA activation is inhibited with 8-Br-cGMP pretreatment. A, representative RhoA Western blots following Rhotekin pulldown of stimulated intact cerebral vasculature. Increased RhoA in pulldown fraction with respect to whole homogenate (WH) are observed following U-46619 treatment. B, quantification of changes in RhoA activation following U-46619 alone, and in the presence of (following pretreatment) 8-Br-cGMP. Rabbit cerebral vessels were used for the rhotekin pulldown assay because of protein requirements. Error bars represent mean ± S.D; *, p < 0.05 with respect to control, n = 3 independent experiments.
FIGURE 3.
FIGURE 3.
8-Br-cGMP pretreatment inhibits maximal TXA2R-mediated myosin RLC20 phosphorylation without reducing MLCP inhibitory phosphorylation of MYPT1. A, representative Western blots of U-46619- and 8-Br-cGMP-induced changes in the phosphorylation states of MYPT1, CPI-17, and RLC20 in the intact cerebral vasculature. PDBu, phorbol 12,13-dibutyrate. B, pretreatment with 8-Br-cGMP has no significant effect on U-46619-induced phosphorylation of MYPT1 at both Thr-696 and Thr-853, while significantly reducing maximal U-46619-induced myosin RLC20 phosphorylation with respect to U-46619 alone. Error bars represent mean ± S.E.; *, p < 0.05 with respect to control, n = 5 independent experiments.
FIGURE 4.
FIGURE 4.
TXA2R activation, not 8-Br-cGMP stimulation, leads to increased MYPT1 phosphorylation at Ser-695 in the intact cerebral vasculature. A, representative Western blots depicting increased MYPT1 phosphorylation at Ser-695 in response to U-46619 stimulation with and without 8-Br-cGMP pretreatment in the intact cerebral vasculature and pulmonary artery. Stimulation with either 25 μm 8-Br-cGMP or 10 μm 6-Bnz-cAMP alone does not result in increased Ser-695 phosphorylation. The absence of changes in basal MYPT1 Ser-695 phosphorylation following U-46619 stimulation in intact rat pulmonary artery (Pulm. A.) indicate that the TXA2R activation-induced increase in MYPT1 Ser-695 phosphorylation is specific to the cerebral vasculature. Endothelium-denuded pulmonary artery, both control and U-46619-stimulated, has lower MYPT1 Ser-695 phosphorylation levels than their intact endothelial counterparts. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B, membrane permeabilization abrogates basal and U-46619-induced Ser-695 phosphorylation in the cerebral vasculature. Stimulation with either 6-Bnz-cAMP or 8-Br-cGMP, unlike U-46619, results in increased MYPT1 Ser-695 phosphorylation with respect to pCa 6.2 control. C, quantification of changes in MYPT1 Ser-695 phosphorylation in both the intact and permeabilized cerebral vasculature. Error bars represent mean ± S.E.; *, p < 0.05 with respect to control, n = 4 independent experiments.
FIGURE 5.
FIGURE 5.
In vivo MYPT1 Ser-695 phosphorylation is dependent upon NOS signaling. A, treatment with the NOS inhibitor l-NAME completely abrogates both basal and U-46619-induced MYPT1 phosphorylation at Ser-695 in the intact cerebral vasculature. NOS inhibition results in increased MYPT1 phosphorylation at both Thr-696 and Thr-853. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B, quantification of changes in MYPT1 Thr-696 (left panel) and Thr-853 (right panel) phosphorylation. Bars represent mean ± S.E.; *, p < 0.05 with respect to control, n = 4. Significant differences between experimental conditions are indicated. C, quantification of changes in cyclic nucleotide concentrations in response to l-NAME, U-46619, SNP, and forskolin. Intact cerebral vessels were treated with 100 μm l-NAME and 25 μm SNP for 60 and 30 min, respectively, or with 20 μm forskolin for 20 min. 300 nm U-46619 was added in the presence of l-NAME for the last 10 min. Significant changes in cGMP (left panel) were observed following stimulation with U-46619 (22%, Experiment 1) and SNP (28%) with respect to control. A separate experiment was performed under identical condition as Experiment 1 in which U-46619 increased cGMP by ∼86%. Treatment with forskolin significantly increased cAMP (right panel) >300-fold as compared with control. Results represent mean ± S.D.; *, p < 0.05 with respect to control, n = 3. D, l-NAME treatment augments U-46619 contractile response. Helical strips with an intact endothelium were cut from the MCA. Force induced by l-NAME treatment alone indicates the presence of an intact endothelium. l-NAME treatment significantly enhanced U-46619-induced contractile force. Results represent mean ± S.D.; *. p < 0.05 with respect to control, n = 3.
FIGURE 6.
FIGURE 6.
Immunofluorescent localization of MYPT1 and phospho-MYPT1 subpopulations in the intact cerebral vasculature. A, profound change in spatial localization of MYPT1 following 10 min of stimulation with U-46619 indicating MYPT1 accumulation at the plasma membrane. B, spatial localization of the phospho-MYPT1 Thr-696 and Thr-853 subpopulations. Changes in the spatial localization of phospho-MYPT1 Thr-853 following U-46619 stimulation are readily observable with respect to control; however, no such changes are detected in the phospho-MYPT1 Thr-696 subpopulation. C, spatial redistribution of MYPT1 following 10 min of U-46619 stimulation (A) is abrogated in the presence of and following 10 min of 8-Br-cGMP pretreatment. Treatment with 8-Br-cGMP alone does not result in any noticeable changes in either the intensity or localization of MYPT1. The images are representative of data from 8 to 12 independent experiments acquired under identical laser and power gain settings. Scale bar, 10 μm.
FIGURE 7.
FIGURE 7.
8-Br-cGMP inhibits U-46619-induced MYPT1 association with the detergent-soluble membrane fraction. A, representative Western blots of the subcellular distribution of MYPT1 following U-46619 stimulation with and without 8-Br-cGMP pretreatment. Membranes reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and vimentin indicate minimal contamination of the detergent-soluble (Det. Soluble) fraction from either the cytosolic or pellet (detergent-insoluble) fractions. B, quantification of changes in MYPT1 subcellular distribution indicates U-46619 stimulation significantly increases MYPT1 association with the detergent-soluble fraction. Pretreatment with 8-Br-cGMP completely inhibits the U-46619-induced association of MYPT1 with the detergent-soluble fraction. Rabbit cerebral vessels were used for the fractionation assay because of protein requirements. Error bars represent mean ± S.D.; *, p < 0.05 with respect to control, n = 5.
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