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Sphingosine kinase functionally links elevated transmural pressure and increased reactive oxygen species formation in resistance arteries

Matthias Keller^,1, Darcy Lidington^*,1, Lukas Vogel^*, Bernhard Friedrich Peter^*, Hae-Young Sohn, Patrick J. Pagano, Stuart Pitson, Sarah Spiegel^||, Ulrich Pohl^* and Steffen-Sebastian Bolz^*,2

^* Institute of Physiology,
Department of Cardiology, Ludwig-Maximilians-University, Munich, Germany;
Division of Hypertension and Vascular Research Division, Henry Ford Health System, Detroit, Michigan, USA;
Hanson Institute, Human Immunology, Institute of Medical and Veterinary Science, Adelaide, Australia; and
^|| Department of Biochemistry, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia, USA

²Correspondence: Institute of Physiology, Ludwig-Maximilians-University Munich, Schillerstrasse 44, Munich 80336, Germany. E-mail: bolz{at}lmu.de

SPECIFIC AIMS

Myogenic vasoconstriction is reported to require both sphingosine kinase 1 (Sk1) activation and the generation of reactive oxygen species (ROS). The aims of the present study were to: 1) establish a serial link between Sk1 and NAPDH oxidase; and 2) determine the functional consequences of pressure-induced ROS generation with respect to the myogenic response.

PRINCIPAL FINDINGS

1. Pressure-induced ROS generation is dependent on the activity of sphingosine kinase
Elevation of transmural pressure (TMP; from 45 to 110 mmHg) stimulated the translocation of a GFP-tagged Sk1 fusion protein from the cytosol to the plasma membrane, indicative of enzymatic activation (Fig. 1 A). Concurrently, production of ROS significantly increased in the vascular smooth muscle cells (VSMC) of isolated resistance arteries, as measured by 2,7-dichlorodihydro-fluorescein-diacetate (DCFH-DA) fluorescence (n=4; Fig. 1B ). The increase in ROS production was transient in nature, occurring within the first 2 min of elevated TMP; there was no additional ROS generation observed over a subsequent 8 min period (i.e., no significant increase in DCFH-DA fluorescence between 2 min and 10 min time points). To determine whether this ROS generation was dependent on the activity of Sk, arteries were transfected with either wild-type human Sk1 (hSk^wt) or its dominant-negative mutant (hSk^G82D). Overexpression of Sk1 (with hSk^wt) significantly enhanced pressure-induced ROS formation in resistance arteries (by 150±52%; n=4); basal ROS production was unaffected (Fig. 1C ). In contrast, expression of the dominant-negative mutant of Sk1 (hSk^G82D) abolished pressure-induced ROS generation (n=5; Fig. 1C ). Exogenous application of sphingosine-1-phosphate (S1P; 1 µmol/L), the enzymatic product of Sk1, stimulated ROS generation in both control and hSk^G82D-transfected resistance arteries (n=6; Fig. 1C ).

View larger version (44K): [in this window] [in a new window]   Figure 1. Pressure-induced ROS formation in resistance arteries is dependent on functional sphingosine kinase. A) Following elevation of transmural pressure (TMP; from 45–110 mmHg, 2 min), translocation of a GFP-Sk1 fusion protein from the cytosolic compartment to the plasma membrane was observed (arrows). The optical plane is positioned within the smooth muscle layer. The images are representative of 5 experiments. B) DCFH-DA-related fluorescence in VSMCs of resistance arteries increased following elevation of TMP (45–110 mmHg, 10 min). The confocal plane is positioned within the smooth muscle layer. C) Pressure-induced ROS formation (after 10 min elevated TMP) was significantly augmented in resistance arteries by overexpression of sphingosine kinase (with hSkwt) and abolished by expression of its dominant-negative hSkG82D mutant. Exogenous sphingosine-1-phosphate (S1P) significantly increased ROS production in control and hSkG82D-expressing resistance arteries. (*P<0.05 vs. respective treatment at 45 mmHg; #P<0.05 vs. control at 110 mmHg; n=4-5).

2. Pressure-induced ROS are generated primarily by NADPH oxidase
NAPDH oxidase is proposed in this study to be the source of pressure-induced ROS formation. Employing arteries transfected with hSk^wt, NADPH oxidase was inhibited with either a specific inhibitor peptide (gp91ds-tat) or by co-transfection with a dominant-negative Rac construct (N17Rac; Rac is a regulatory subunit of the NADPH oxidase complex). A prerequisite for the use of gp91ds as a specific inhibitor of NADPH oxidase is the expression of the NADPH oxidase subunit gp91^phox, which we confirmed in hamster gracilis muscle resistance arteries by Western blot analysis (positive staining for gp91^phox was observed in all 3 resistance artery lysates tested). Inhibition of NADPH oxidase with gp91ds-tat significantly reduced basal production of ROS (by 45±15%) and prevented pressure-induced ROS formation within the first 2 min of elevated TMP (n=5). Inhibition of NADPH oxidase by co-transfection of N17Rac yielded similar observations. For both inhibition strategies, ROS formation was significantly elevated after 10 min of increased TMP, although the absolute ROS production remained substantially lower than in hSk^wt-expressing control arteries. Thus, while NADPH is clearly the primary source of pressure-induced ROS formation, other pressure-sensitive sources of ROS appear to exist. Stimulation of resistance arteries expressing N17Rac with 1 µmol/L exogenous S1P failed to induce a significant increase in DCFH-DA fluorescence, consistent with the proposed link between Sk1 and NADPH oxidase and the hypothesis that NADPH oxidase is the primary source of pressure-induced ROS formation.

3. Pressure-induced ROS modulate myogenic vasoconstriction by increasing calcium sensitivity
We determined the functional consequences of NADPH oxidase inhibition with respect to myogenic vasoconstriction and vasomotor responses. The specific inhibitor peptide gp91ds, which effectively blocked pressure-induced ROS formation, significantly reduced myogenic vasoconstriction and the Ca²⁺ sensitivity of the VSMC contractile apparatus (Fig. 2 ). Basal Ca²⁺ and pressure-induced Ca²⁺ entry were unaffected (Fig. 2) , as were the vasomotor responses to 0.3 µmol/L noradrenaline and 3 µmol/L acetylcholine.

View larger version (14K): [in this window] [in a new window]   Figure 2. Functional effects of NADPH oxidase inhibition with gp91ds. A) Treatment of vessels with gp91ds (Chariot delivery) prevented pressure-induced ROS formation (n=4). B) Pressure-induced vasoconstriction in resistance arteries was significantly reduced following delivery of gp91ds (n=5). C) The kinetics and amplitude of the pressure-induced increase in smooth muscle Ca2+i were preserved following transfection of gp91ds. Because the gp91ds treatment protocol resulted in an artifactual reduction in the Fura-2 ratio (see Results for details), direct comparison of pressure-induced Ca2+ in the pre-gp91ds and gp91ds treatment groups required normalization to resting Ca2+ (n=5) D) Resting tone was not affected following transfection of gp91ds, however, constriction of depolarized resistance arteries (120 mmol/L K+) in response to increasing concentrations of extracellular Ca2+ was moderately attenuated (n=5). *Significant difference vs. control/pre-gp91ds.

Inhibition of NAPDH oxidase by transfection of N17Rac or with the chemical inhibitor diphenyleneiodonium (DPI; 1 µmol/L) both abolished myogenic vasoconstriction and reduced Ca²⁺ sensitivity, but also affected several other functional parameters. Basal Ca²⁺ and resting tone were reduced, the kinetic of the pressure-induced Ca²⁺ entry was altered and the response to noradrenaline (but not acetylcholine) was attenuated. Despite the additional effects of these inhibitors, the vessels maintained their ability to constrict, and the dose-response curves for Ca²⁺-independent vasoconstriction in response to exogenous S1P remained unaltered.

CONCLUSIONS AND SIGNIFICANCE

Myogenic vasoconstriction is a critical element involved in the regulation of tissue perfusion and systemic blood pressure. Although it was discovered almost a century ago, a comprehensive understanding of the mechanisms controlling myogenic activity remains elusive. Previous reports have identified ROS generation and Sk1 activity as necessary regulatory elements for this response. The present study presents the first evidence linking Sk1/S1P signaling and elevated ROS generation following increased TMP; it provides mechanistic insight into the functional role of ROS as a modulator of Ca²⁺ sensitivity and hence myogenic vasoconstriction.

Increased ROS generation is a rapid and relatively short-lived response to elevated TMP, displaying the steepest increase in production within the first 2 min. The sensitivity of pressure-induced ROS formation to the genetic manipulation of Sk1 activity strongly implicates Sk1 as the upstream mediator linking increased TMP to NADPH oxidase activation (Fig. 3 ). Consistent with these observations, exogenous S1P stimulated a robust NADPH oxidase-dependent increase in ROS generation. Having established a functional link between Sk1 activity and NADPH oxidase-mediated ROS production in response to increased TMP, we sought to determine whether the generated ROS contribute to the described previously pleiotropic effects of Sk1 in resistance arteries.

View larger version (46K): [in this window] [in a new window]   Figure 3. Schematic representation of proposed pathways. Increased transmural pressure activates stretch-sensing mechanisms that putatively lead to calcium influx and activation of Sk1. Sk1 converts sphingosine (Sph) to sphingosine-1-phosphate (S1P). Extracellular S1P acts as a receptor ligand and activates several signaling pathways, including RhoA/Rho kinase and Rac. RhoA/Rho kinase increase in apparent Ca2+ sensitivity by inhibiting myosin light chain phosphatase (MLCP). The activation of Rac is associated with increased formation of O2– via NADPH oxidase. This pathway also modulates apparent Ca2+ sensitivity, albeit to a lesser degree than RhoA/Rho kinase. Current literature suggests that O2– affects the RhoA/Rho kinase pathway at different levels (thin dotted lines).

A full, pressure-induced myogenic vasoconstriction requires the generation of ROS, which we demonstrated to increase the Ca²⁺ sensitivity of the smooth muscle contractile apparatus. The Ca²⁺ desensitizing effect of ROS inhibition is clearly modulatory, since it can be nullified via RhoA/Rho kinase activation by exogenous S1P (i.e., in DPI-treated or N17Rac-expressing resistance vessels). In accordance, the myogenic response was not completely inhibited by gp91ds peptide, despite the ablation of pressure-induced ROS generation. This indicates that NADPH-derived ROS are not required for the initiation of myogenic vasoconstriction, but that ROS production modifies and ultimately amplifies an already initiated myogenic response. Pressure-induced Ca²⁺ entry was not affected by the gp91ds peptide, indicating that it is independent of ROS generation and is either an upstream signal or a parallel one. In contrast both DPI and N17Rac affected basal Ca²⁺ and pressure-induced Ca²⁺ entry (independent of NADPH oxidase, based on the observation that these parameters are not affected by the gp91ds peptide). Thus, it is not surprising that these inhibitors completely abolished myogenic vasoconstriction.

Our results suggest a mandatory role for Sk1/S1P in mediating pressure-induced ROS formation. The initial elevation in ROS is entirely dependent on the activation of NADPH oxidase, suggesting that O₂^– is the initial ROS signaling molecule. Under normal physiological conditions, this newly described relationship contributes to the regulation of myogenic reactivity, a critical element for the regulation of tissue perfusion that also feeds back to systemic blood pressure. This relationship (between Sk1/S1P and NADPH oxidase/O₂^–) could also possess an important role under pathophysiological conditions where ROS production is elevated (e.g., atherosclerosis, hypertension and endothelial dysfunction).

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4075fje;

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