S1P₂ receptor-dependent Rho-kinase activation mediates vasoconstriction in the murine pulmonary circulation induced by sphingosine 1-phosphate

Abstract

Vasoactive properties of sphingosine 1-phosphate (S1P) have been demonstrated by many investigators to vary in systemic vascular beds. These variations appear to reflect differential S1P receptor expression in the vasculature of these tissues. Although S1P has been demonstrated to enhance endothelial barrier function, induce airway hyperresponsiveness, and modulate immune responses in the lung, the pulmonary vasomotor effects of S1P remain poorly defined. In the present study, we sought to define the vasoregulatory effects of S1P in the pulmonary vasculature and to elucidate the underlying mechanisms operative in effecting the response in the intact lung. S1P (10 μM) increased pulmonary vascular resistance (PVR) by 36% in the isolated perfused mouse lung. S1P-induced vasoconstriction was reduced by 64% by concomitant administration of the Rho-kinase inhibitor Y27632 (10 μM). Similarly, the S1P response was attenuated by >50% after S1P₂ receptor antagonism (JTE-013; 10 μM) and in S1P₂ receptor null mice. In contrast, S1P₃ receptor antagonism (VPC23019; 10 μM) had no effect on the contractile response to S1P. Furthermore, we confirmed the role of Rho-kinase as an important regulator of basal vasomotor tone in the isolated perfused mouse lung. These results suggest that S1P is capable of altering pulmonary vascular tone in vivo and may play an important role in the modulation of pulmonary vascular tone both in the normal lung and under pathological conditions.

sphingosine 1-phosphate (S1P) has emerged as an important signaling lipid in recent years. Through activation of a family of five cell surface receptors (S1P₁–SIP₅) expressed differentially in nearly all tissues, S1P participates in a wide variety of physiological processes including angiogenesis, cell survival, inflammatory cell trafficking, cytokine production, cell motility and migration, cytoskeletal reorganization, endothelial barrier regulation, and the control of vasomotor tone (7, 10–12, 15, 17, 21, 25–27, 34, 43). In the lung, S1P and small molecule analogs of S1P are the focus of considerable interest in the pathogenesis and pharmacotherapy of acute lung injury, asthma, and possibly chronic obstructive pulmonary disease (COPD) (8, 27, 33, 34, 36, 37, 43, 44, 46). This is in part due to S1P-induced endothelial barrier enhancement (10, 27, 34, 43, 44), induction of airway hyperresponsiveness (36, 37), modulation of mast cell recruitment and cytokine production (33), and regulation of apoptosis (8, 46).

In the systemic circulation, differential expression of three S1P receptors (S1P₁, S1P₂, and S1P₃) on both the vascular endothelium and smooth muscle leads to regionally variable effects of S1P on vasomotor tone (3, 6, 40). For example, both S1P₁ and S1P₃ receptor signaling have been linked to vasoconstriction in renal and mesenteric resistance vessels (3, 7, 48), whereas S1P elicits potent vasoconstriction in isolated cerebral arteries via an S1P₃ pathway-dependent mechanism (39, 40). In contrast, S1P relaxes precontracted aortic rings via an S1P₃ receptor-dependent mechanism (29). Similar disparities in vasomotor function persist in what is known about the effects of S1P on the pulmonary vasculature. Two studies have demonstrated that S1P increases tension in isolated conduit pulmonary arterial segments, lending evidence that S1P exerts a vasoconstrictive response in the pulmonary vasculature (16, 45). Another study demonstrated that S1P activates endothelial nitric oxide synthase (eNOS) downstream of S1P₁ receptor activation in cultured bovine microvascular endothelial cells, lending evidence that S1P drives vasorelaxation in the pulmonary vasculature (28). Given the inherent regional differences of S1P in vasomotor control in the systemic circulation and the contradictory data from prior studies of the vasomotor effects of S1P on the pulmonary vasculature, the effects of S1P on vasomotor tone in the intact pulmonary vasculature cannot be inferred from existing evidence.

The purpose of the current study, therefore, was to examine the vasomotor effects of S1P in the intact murine pulmonary circulation and to identify the S1P receptor and proximal signaling events that account for S1P vasoconstrictor effects in the intact lung. We originally hypothesized that S1P₃ was critical, since S1P₃ contributes to S1P-mediated vasoconstriction in the cerebral vasculature (39, 40), has been implicated in vasomotor control in the aorta (29), and is coupled to Rho-kinase (20, 21), an important mediator of pulmonary vascular tone (9) and constriction in response to a variety of stimuli (2, 9, 18, 22, 35). We ultimately revealed that S1P induces pulmonary vasoconstriction in a Rho-kinase-sensitive (e.g., Y27632) fashion via the S1P₂ receptor. These observations are important for the development of small-molecule pharmacological agents that will selectively act in a therapeutic fashion on the pulmonary circulation in disease states such as pulmonary arterial hypertension or acute lung injury.

METHODS

Animal protocols were approved by the University of Pittsburgh Animal Care and Use Committee.

Animal husbandry.

Eight-week-old C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME) and allowed to acclimate for at least 48 h in the animal facility before being used in experiments. During the acclimation period, mice were provided free access to water and chow. S1P₂ receptor-deficient (S1P₂^−/−) breeding pairs (C57/SvJ129 background) and littermate controls (S1P₂^+/+) were generously provided by Dr. Joe G. N. Garcia (University of Illinois at Chicago, Chicago, IL) as approved by the originator of the strain, Dr. Richard Proia (National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD) (23).

Reagents.

S1P (product S9666; Sigma, St. Louis, MO) and the putative S1P₂ antagonist JTE-013 (product 2392; Tocris Bioscience, Ellisville, MO) were dissolved in methanol (final concentration 10 mM). The Rho-kinase inhibitor Y27632 was purchased in aqueous solution from Calbiochem (product 688001; 5 mM). VPC23019, a putative S1P₃ antagonist, was purchased from Avanti Polar Lipids (product 857360P; Alabaster, AL), solubilized in 1:20 solution of 1 N HCl and DMSO according to the manufacturer's instructions, and diluted to 1 mM in aqueous solution supplemented with 3% (wt/vol) low-endotoxin bovine serum albumin (A9430; Sigma). All stock solutions were stored at −20°C and diluted to working concentration in perfusion buffer at the time of experimentation.

Isolated mouse lung perfusion buffer.

Premixed Krebs-Henseleit modified buffer (K3753; Sigma) was dissolved in reverse-osmosis deionized water (18.2 MΩ) according to the supplier's instructions and supplemented with the following compounds: 2.54 mM CaCl₂·2H₂O, 25.0 mM NaHCO₃, and 23.0 mM NaCl. The pH was adjusted to 7.1 with HCl, lyophilized low-endotoxin bovine serum albumin (A9430; Sigma) was added to a final concentration of 4% (wt/vol), and the buffer was sterile-filtered and stored at 4°C (final pH 7.4).

Isolated perfused mouse lung.

All studies were conducted using a commercially available isolated perfused mouse lung apparatus (IL-1; Harvard Apparatus, Boston, MA). Mice were anesthetized with intraperitoneal pentobarbital sodium (90 mg/kg). Tracheostomy was performed for initiation of mechanical ventilation (Harvard Apparatus Minivent; tidal volume 175 μl, rate 90–100 breaths/min, PEEP 3 cmH₂O, room air). An incision was made from the sternal notch to the xiphoid process and extended bilaterally along the inferior borders of the rib cage to the posterior abdominal wall. The diaphragm was incised and detached circumferentially to the midline of the posterior thoracic/abdominal wall. The thoracic cavity was opened via midline sternotomy, exposing the heart and lungs, and heparin (100 IU/100 μl) was slowly injected into the right ventricle. The pulmonary artery and left atrium were cannulated in situ via the corresponding cardiac ventricles, and catheters were secured in place using 6-0 silk suture. Inspired gas was switched to room air + 5% CO₂ to maintain pH 7.35–7.40 during perfusion studies. The pulmonary circulation was perfused in a nonrecirculating manner with perfusion buffer at constant flow (0.5 ml/min) delivered by a computer-controlled peristaltic pump (Reglo; Harvard Apparatus). Temperature was maintained at 37°C within a water-jacketed perfusion chamber, and humidity was preserved with cellophane wrap sealed over the preparation. Pulmonary artery (P_PA), left atrium (P_LA), and airway pressures (P_AW) were continuously sampled at 100 Hz by an analog-to-digital converter (National Instruments, Austin, TX) and were displayed and recorded using customized computer software (MATLAB; The Mathworks, Natick, MA) running on a laptop personal computer (Inspiron; Dell, Round Rock, TX).

Pharmacological interventions.

Immediately following cannulation, the pulmonary circulation was perfused for a 25-min acclimation period. Mice not achieving a stable PVR during the acclimation period were rejected from further study. Perfusate reservoir selection was automated using a computer-controlled valve actuator (Hamilton, Reno, NV) allowing for precise control of timing of pharmacological interventions.

For dose-response studies, following the acclimation period, buffer solution containing S1P [1–100 μM, n = 3 (1, 5, 20, and 100 μM) and n = 13 (10 μM)] was introduced for 20 min, followed by washout with fresh perfusion buffer (Fig. 1A). In a subset of animals (n = 7), S1P (10 μM) was reintroduced for 20 min after a 25-min washout period, followed a third time by washout and restimulation (Fig. 1B).

Fig. 1.

Protocol timeline. Depicted is the timeline of pharmacological intervention in studies involving sphingosine 1-phosphate (S1P) alone (A and B) or S1P in the presence of an inhibitor (C).

For inhibitor studies, following the acclimation period, lungs were perfused with inhibitor [Y27632, 1 μM (n = 4), 10 μM (n = 5), or 20 μM (n = 4); VPC23019, 10 μM (n = 5); JTE-013, 10 μM (n = 5); or nitro-l-arginine methyl ester (l-NAME), 100 μM (n = 4)] for 20 min before introduction of S1P. S1P (10 μM) was then introduced in the presence of the inhibitor for 20 min, followed by washout with fresh buffer. After the 20-min washout period, S1P (10 μM) was reintroduced for 20 min absent the inhibitor, followed by washout with fresh buffer (Fig. 1C). To test for specificity of the S1P₂-dependent vasoconstrictor response, in a separate set of experiments, control and S1P₂^−/− mouse lungs were perfused with endothelin-1 (ET-1; 100 nM) for 20 min, followed by washout with fresh Krebs buffer. Separate control experiments were conducted with vehicle for each pharmacological agent to exclude vehicle-induced vasoactive responses (data not shown).

Data processing and analysis.

After data collection, the continuous P_PA data were transformed by a custom-designed digital filter to remove the transient pressure artifact introduced by positive-pressure ventilation (Fig. 2). Briefly, the nonregressive, zero-phase shift digital filter exploits the change in distribution of a short segment of continuous P_PA data from unimodal to bimodal, introduced by positive-pressure ventilation. A symmetrical, moving square window (100 data points, 1 s of data) is used to sample the raw P_PA continuous data. A pressure histogram (100 bins) is then generated from the sampled raw P_PA data and fit to a polynomial function. The mean (peak) of the first distribution (lowest pressure) is recorded, and the window is advanced by a single data point. After advancing through the entire raw P_PA dataset in an iterative manner, we saved the output of the filter in a separate file. Pilot experiments in our laboratory have shown that the output of the filter reliably and accurately tracks the baseline P_PA when positive-pressure ventilation is discontinued (Fig. 2B) and provides a means to substantially increase the signal-to-noise ratio and improve the ability to parametrically curve fit small amplitude changes in P_PA. From the filtered signal, locally averaged P_PA and P_LA data were extracted, and onset and offset responses to S1P were cut using custom-designed data analysis software. The cut data were imported into MATLAB, and S1P onset responses were analyzed using the built-in curve-fitting toolbox (Fig. 2, C and D).

Fig. 2.

Signal processing technique and curve fitting procedure. A: the sequence of data collection, signal processing, and curve fitting. B: raw airway pressure (P_AW; solid trace, top), raw pulmonary arterial pressure (P_PA; shaded trace), and the filtered P_PA (solid trace, bottom) in the isolated perfused mouse lung being mechanically ventilated or statically inflated with the ventilator turned off. Of note is how the cyclic nature of the raw P_PA tracing approximates that in the airway and the accuracy with which the filtered P_PA signal approximates the basal P_PA signal in the presence of mechanical ventilation and the raw P_PA signal in the absence of mechanical ventilation. C: the closeness of fit of the filtered P_PA signal superimposed on the raw P_PA response (unfiltered) to stimulation with 10 μM S1P. D: the filtered P_PA response to 10 μM S1P stimulation with the location of cursor placement for data extraction for offline nonparametric curve fit analysis (shown in E). The linear portion of the response is fit to a line described by the function f(t) = P2∗t + b, where P2 represents the slope of the line (mmHg/s) and b represents the y-intercept of the line (E). Subsequently, the onset of the S1P response is fit to an equation describing the summation of exponential and linear components, f(t) = P1 + (P2∗t) + [P3∗(1 − e^{−1∗P4∗t})], where t is time, P1 represents the baseline P_PA (mmHg), P2 is the slope previously defined (mmHg/s), P3 represents the magnitude of the exponential component (mmHg), and P4 represents the time constant of the exponential phase (1/s; E).

Statistical analysis.

Student's t-tests or one-way ANOVA were used to analyze data between groups where appropriate. Post hoc Bonferroni tests were conducted when the ANOVA indicated significance. Data are means ± SE unless otherwise indicated. Statistical significance was defined at P < 0.05.

RESULTS

S1P induces vasoconstriction in the pulmonary circulation in a dose-dependent manner.

S1P introduction into the perfusion buffer (1–100 μM) induced a dose-dependent vasoconstriction in the isolated mouse lung (Fig. 3) that is characterized by a rapid onset that saturates (i.e., reaches a plateau phase) quickly (t_1/2 = 20–40 s), followed by a gradual linear increase in P_PA that is sustained in the presence of S1P for up to 4 h (data not shown). Examination of the P_PA recording with and without dynamic ventilation shows that ventilation introduced a substantial (1–2 mmHg) transitory artifact (Fig. 2B), thereby decreasing the signal-to-noise ratio of the S1P response (Fig. 2C, unfiltered P_PA recording) and impairing quantification of the vasoconstrictor response, particularly when the magnitude of the response was low, as shown with low concentrations of S1P (Fig. 3A). To remove this artifact, increase the signal-to-noise ratio of the S1P-induced vasoconstrictor response, and maximize the statistical power of parametric curve-fitting methodology, we filtered the P_PA signal using a custom-designed digital filter to remove the ventilator artifact (Fig. 2, B and C). The close fit of filtered data to the raw signal from a representative P_PA response to 10 μM S1P demonstrates the accuracy of the filter (Fig. 2C). Extracting the filtered signal at the onset and offset of S1P infusion reveals the complex vasoconstrictor response of the intact pulmonary vasculature to S1P (Fig. 2D). The response is best fit by an equation that combines a slowly developing linear response following a rapid onset exponential component:

$$\text{f}(t)=\text{P}1+(\text{P}2\ast t)+\left[\text{P}3\ast (1-e^{-\text{P}4\ast t})\right]$$

where t represents time (s), P1 defines baseline P_PA (mmHg), P2 represents the slope of the linear phase of the response (mmHg/s), P3 quantifies the amplitude of the exponential response (mmHg), and P4 represents the time constant of the exponential phase (1/s; Fig. 2E, R² ≥ 0.95 for all [S1P] >1 μM). Using this approach, we observed that both the magnitude of the exponential component (P3) and the slope of the linear component (P2) of the curve fit increased with increasing S1P concentration (Fig. 3B). The total S1P-induced vasoconstrictor response, calculated as the sum of the exponential and linear percent increase in PVR over baseline at 20 min of S1P exposure, also exhibited dose dependence (Fig. 3B). Both the exponential component and the total S1P-induced vasoconstrictor response fit well to a sigmoidal curve with K_d ≈ 21 μM (R² = 0.99, Fig. 3B). Although the linear component generally increased with increasing concentrations of S1P (Fig. 3B), it did not fit to a standard sigmoidal dose-response curve at the doses administered. Repetitive stimulation of the isolated lung with 10 μM S1P in 20-min increments separated by 25-min washout periods revealed that the exponential component of the response remained stable with repeated exposures, whereas the minimal linear component observed in the initial 10 μM S1P response generally increased with sequential stimulation (Table 1). Given that we used a nonsaturating dose of S1P (10 μM) in all of our pharmacological inhibition studies, that the linear component accounted for <10% of the total increase in PVR at this dose, and that the exponential component is saturable, comparisons between inhibitor responses were drawn between the exponential components exclusively.

Fig. 3.

S1P induces reversible and dose-dependent vasoconstriction in the pulmonary circulation. A: representative raw P_PA tracings in response to increasing doses of S1P, arranged in a waterfall presentation. The ordinate represents change in P_PA from baseline (ΔP_PA; each tick mark = 2 mmHg). B: the dose dependence of the S1P vasoconstrictor response, measured by an increase in pulmonary vascular resistance (PVR) calculated 20 min after S1P stimulation (K_{d exp} = 20.7 μM). Both the exponential and linear components of the vascular response increase with concentration of S1P ([S1P]).

Table 1.

Isolated perfused mouse lung vasoconstrictor responses to S1P

Intervention	n	Total PVR, %increase	Exponential Component, %increase	Linear Component, %increase	Baseline PVR, mmHg•ml−1•min
S1P (C57BL/6J)
Control	13	41.0 ± 4.8	36.3 ± 4.5	2.94 ± 2.6	6.9 ± 0.5
2nd Stimulation	7	36.0 ± 7.8	27.9 ± 6.4	6.4 ± 4.7	8.6 ± 0.9
3rd Stimulation	7	45.7 ± 8.5	24.9 ± 4.3	17.4 ± 5.3*	10.1 ± 0.9
Y27632 + S1P
1 μM	4	21.6 ± 4.3	26.1 ± 4.2	−4.6 ± 3.2	5.1 ± 1.7
10 μM	5	17.9 ± 2.9*	13.2 ± 2.5*	4.8 ± 1.6	7.9 ± 1.0
20 μM	4	12.4 ± 1.2*	10.4 ± 2.6*	2.0 ± 1.9	3.3 ± 0.3
VPC23019 + S1P	5	37.4 ± 1.6	38.0 ± 4.6	−0.6 ± 3.8	5.8 ± 0.4
JTE-013 + S1P	5	21.7 ± 3.2*	18.8 ± 2.6*	2.9 ± 1.3	6.1 ± 0.5
S1P (C57BL6/SvJ129)
S1P2+/+	5	52.3 ± 5.7	27.5 ± 2.9	24.8 ± 3.9	5.4 ± 0.5
S1P2+/−	5	27.0 ± 8.2*	20.7 ± 3.4	8.1 ± 8.6*	5.8 ± 0.5
S1P2−/−	5	14.0 ± 5.0*	8.9 ± 2.9*	5.1 ± 3.3*	5.2 ± 1.0

Values are means ± SE; n = no. of experiments. For all interventions, sphingosine 1-phosphate (S1P) concentration was 10 μM; inhibitor concentrations were 10 μM except for Y27632, the Rho-kinase inhibitor, as indicated. VPC23019, S1P₃ inhibitor; JTE-013, S1P₂ inhibitor. Total pulmonary vascular resistance (PVR) and the percent increase for both the exponential and linear components were measured 20 min after S1P introduction; baseline PVR was measured immediately before S1P introduction.

^*P < 0.05 compared with appropriate S1P control.

S1P requires Rho-kinase activation to effect vasoconstriction in the pulmonary circulation.

Preperfusion of the isolated mouse lung with buffer containing the Rho-kinase inhibitor Y27632 attenuated the pulmonary vascular response to 10 μM S1P in a dose-dependent manner (PVR increase from baseline: 36.3 ± 4.5%, S1P alone; 26.1 ± 4.2%, S1P + 1 μM Y27632; 13.2 ± 2.5%, S1P + 10 μM Y27632; 10.4 ± 2.6%, S1P + 20 μM Y27632; P = 0.002 by 1-way ANOVA; Fig. 4, A and C). In addition, perfusion with Y27632 significantly reduced baseline PVR in the isolated perfused mouse lung (−39.3 ± 10.4%, 1 μM Y27632 compared with acclimation baseline, P < 0.05 by paired t-test; −17.2 ± 3.0%, 10 μM Y27632 compared with acclimation baseline, P < 0.0005; and −39.7 ± 3.8%, 20 μM Y27632 compared with acclimation baseline, P < 0.0005; Fig. 4, A and B).

Fig. 4.

Rho-kinase inhibition reduces baseline PVR and blocks S1P-induced vasoconstriction in the pulmonary circulation. A: representative filtered P_PA tracings from lungs exposed to 10 μM S1P in the absence or presence of the Rho-kinase inhibitor Y27632 in increasing concentrations (1, 10, and 20 μM as indicated by horizontal bars). Tracings were normalized to acclimation baseline to demonstrate relative differences. B: the reduction in baseline PVR resultant from Y27632 introduction in increasing concentrations (n = 4–5 each). *P < 0.05; †P < 0.0005 compared with acclimation baseline (by paired t-test). PVR reductions represent the difference between the PVR immediately before Y27632 introduction and that immediately before S1P introduction. C: the increase in PVR in response to 10 μM S1P in the absence (n = 13) or presence of Y27632 in increasing concentrations (1 μM, n = 4; 10 μM, n = 5; and 20 μM, n = 4). *P = 0.002 compared with S1P alone (by 1-way ANOVA). PVR increases depicted represent the magnitude of the exponential component of the response.

S1P-induced pulmonary vasoconstriction is dependent on S1P₂ cell surface receptor activation.

S1P₂ and S1P₃ cell surface receptors couple to the G protein, G_12/13, which signals through Rho-kinase to inhibit MLC phosphatase activity, thus promoting actin stress fiber formation and contraction of pulmonary vascular endothelial and smooth muscle cells (20, 21). S1P₃ receptor antagonism with VPC23019 (10 μM) did not significantly alter PVR in the isolated perfused mouse lung at baseline or in response to 10 μM S1P stimulation (PVR increase from baseline, 38.0 ± 4.6%, S1P + VPC23019 vs. 36.3 ± 4.5%, S1P alone; P = 0.1; Fig. 5). In contrast, JTE-013, a putative S1P₂ receptor antagonist, significantly attenuated the S1P vasoconstrictor response in the intact mouse lung (PVR increase from baseline, 18.8 ± 2.6%, S1P + 10 μM JTE-013 vs. 36.3 ± 4.5%, S1P alone; P = 0.002; Fig. 5). Because it is well known that JTE-013 is not specific for the S1P₂ receptor and may affect vasoconstrictor response to ET-1 and the thromboxane analog U46619 (39), we utilized a more precise genetic approach in which the S1P₂ receptor was deleted by whole body gene ablation. The total vasoconstrictor response to 10 μM S1P was similar between C57BL/6J and S1P₂^+/+ mixed background control mice, although the linear component comprised a greater proportion of the response in C57/SvJ129 compared with C57BL/6J mouse lungs (Table 1). The 10 μM S1P vasoconstrictor response was nearly completely abolished in S1P₂^−/− mice (8.9 ± 2.9% PVR increase from baseline, P = 0.002 compared with wild type, n = 5; Fig. 6) and attenuated in mice heterozygous for S1P₂ receptor expression (20.7 ± 3.4%, P = 0.16, n = 5; Table 1). Previous authors have demonstrated altered systemic vasoreactivity in S1P₂^−/− mice (24, 39) and have suggested that these mice express increased eNOS activity (42) or dedifferentiated vascular smooth muscle (13). Therefore, we examined the vasoconstrictive properties of ET-1 and S1P in the presence of l-NAME, a competitive inhibitor of eNOS activity, in isolated S1P₂ null mouse lungs. ET-1 induced potent vasoconstriction in control and S1P₂^−/− mouse lungs (4.7 ± 0.2- vs. 6.0 ± 0.9-fold increase, respectively; P = 0.19, n = 4). Furthermore, the presence of l-NAME (100 μM) in the perfusion buffer did not reverse the inhibition of S1P-induced vasoconstriction observed in S1P₂ null mouse lungs (12.8 ± 4.5% PVR increase from baseline, P = 0.47 compared with S1P₂^−/−, n = 4; Fig. 6).

Fig. 5.

S1P₂ receptor antagonism attenuates S1P-induced pulmonary vasoconstriction. A: representative filtered P_PA tracings from lungs exposed to 10 μM S1P alone or in the presence of the S1P₃ inhibitor VPC23019 (10 μM) or the S1P₂ receptor inhibitor JTE-013 (10 μM). B: the increase in PVR from baseline in response to 10 μM S1P alone (n = 13), 10 μM S1P in the presence of 10 μM VPC23019 (n = 5), or 10 μM S1P in the presence of 10 μM JTE-013 (n = 5). *P = 0.03 compared with S1P controls. PVR increases depicted represent the magnitude of the exponential component of the response.

Fig. 6.

S1P-induced vasoconstriction requires the S1P₂ cell surface receptor. A: representative filtered P_PA tracings from S1P₂^+/+ C57/SvJ129 mouse lungs exposed to 10 μM S1P, S1P₂^−/− mouse lungs exposed to 10 μM S1P, or S1P₂^−/− mouse lungs exposed to 10 μM S1P in the presence of 100 μM nitro-l-arginine methyl ester (l-NAME). B: the increase in PVR in response to 10 μM S1P in S1P₂^+/+ mouse lungs (n = 5), 10 μM S1P in S1P₂^−/− mouse lungs (n = 5), or 10 μM S1P in the presence of 100 μM l-NAME in S1P₂^−/− mouse lungs (n = 4). *P = 0.004 compared with S1P₂^+/+ control (by 1-way ANOVA). PVR increases depicted represent the magnitude of the exponential component of the response.

DISCUSSION

In this study, we have used an isolated perfused mouse lung model coupled with signal analysis and parametric curve fitting techniques to examine the vasoactive properties of S1P in the intact pulmonary vasculature. Our data demonstrate that exogenous S1P induces dose-dependent vasoconstriction in the lung via the S1P₂ cell surface receptor and a Rho-kinase-mediated signal transduction pathway. Furthermore, our study verifies the role of Rho-kinase as a regulator of basal vasomotor tone in the isolated perfused mouse lung. These results suggest that S1P is capable of modulating pulmonary vascular tone in vivo and may play an important role in the regulation of pulmonary vascular tone both in the normal lung and under pathological conditions. For example, it is possible that S1P pharmacology participates in the vasoreactive response of the pulmonary circulation to hypoxia, since sphingosine kinase is known to be upregulated in hypoxia (1), and downstream signaling cascades (e.g., Rho-kinase) play a role in the hypoxic pulmonary vasoconstrictor response (9).

S1P induces complex changes in pulmonary vascular tone in the intact lung.

Investigation in the intact lung preserves the physiological interaction of the endothelium and smooth muscle, which together regulate vascular tone in vivo. In addition, the isolated lung model preserves the endothelial heterogeneity of the pulmonary circulation, where substantial structural and functional differences exist between conduit and microvascular endothelial cells (19), and incorporates the effects of periodic mechanical forces that are imparted on the lung during the respiratory cycle and may affect vascular tone. Thus, unlike the use of isolated vascular rings or cultured cell-based models, the isolated perfused mouse lung enables the assessment of the net integrated vasomotor response to pharmacological stimulation across the entire pulmonary vasculature and allows for exploitation of transgenic and knockout technologies in the exploration of vasomotor regulatory mechanisms. The signal processing and parametric curve fitting methodologies we applied to the P_PA response elicited by S1P in the intact lung enabled us to resolve two distinct components of vasoconstriction: 1) a rapid-onset exponential phase that reached a maximal level of vasoconstriction within minutes of exposure; and 2) a slowly developing linear phase that resulted in the steady increase of vascular tone that did not saturate during a 4-h perfusion with S1P-containing buffer. Because of limitations of the model and differential expression of S1P receptors on endothelial and smooth muscle cells within the pulmonary vasculature, further work is necessary to determine the segment(s) of the pulmonary vasculature involved in the response, the cell type(s) that contribute to S1P-mediated vasoconstriction in the lung, the physiological significance of and differential mechanisms underlying the distinct components of the response, and the potential variability in the pulmonary vascular response to S1P and downstream signaling mechanisms in different species. Furthermore, whether the in vivo pulmonary vascular response to S1P is temporized by activation of eNOS or is manifest as vasorelaxation in preconstricted vessels, as suggested by previous investigators using reductionist models (16, 28, 45), remains to be determined. Despite these limitations, our data enable us to conclude that the net whole organ response to S1P in the pulmonary vasculature is a complex, dynamic vasoconstriction with both fast and slow components primarily mediated through the S1P₂ cell surface receptor and the downstream signaling molecule Rho-kinase.

Rho-kinase signaling is important for maintenance of basal vascular tone and for vasoconstriction induced by S1P in the pulmonary circulation.

Rho-kinase activity has been implicated in the maintenance of pulmonary vascular tone (9) and in the pulmonary vascular response to various stimuli including acute and chronic hypoxia (9, 18), reactive oxygen species (22, 35), angiotensin II (2), and ET-1 (49). Furthermore, Rho-kinase inhibition has been implicated as potential therapy for pulmonary arterial hypertension (14, 30, 31). Our study confirms the previously reported dose-dependent effect of Rho-kinase inhibition on basal pulmonary arterial tone and highlights the central role of Rho-kinase in pulmonary vasoregulation, adding S1P to the list of compounds requiring its activation to induce vasoconstriction in the pulmonary circuit.

S1P₂ is the predominant cell surface receptor responsible for S1P-induced vasoconstriction in the pulmonary circulation.

Numerous studies have demonstrated vasoactive effects of S1P in systemic vascular beds. The authors of these studies have implicated different signaling pathways responsible for these effects as being reflective of differential receptor expression in various tissues, although each has implicated a single receptor subtype responsible for the S1P effect in the given tissue. Salomone et al. (40) identified S1P₃-dependent vasoconstriction in cerebral arteries from rats compared with the lack of an effect in coronary or femoral arteries from the same animals. Bischoff and colleagues (3, 4) demonstrated potent S1P-induced renal and mesenteric vascular constriction in rats that were pertussis toxin sensitive, thus implicating G_i protein dependence, predominantly a feature of S1P₁ receptor signaling rather than S1P₂ or S1P₃.

Despite the presence of multiple S1P receptor subtypes in the lung (specifically S1P₁, S1P₂, and S1P₃), the fact that the majority of the S1P-induced vasoconstrictor response in the isolated perfused mouse lung can be very accurately modeled by a single exponential equation suggests that the majority of S1P-induced vasoconstriction in the lung is mediated through a single receptor subtype. The importance of Rho-kinase in mediating the S1P response in the pulmonary vasculature lead us to explore the roles of the S1P₂ and S1P₃ cell surface receptors, because these receptors are known to signal through Rho-kinase downstream of G_12–13 activation (20, 21). Our pharmacological studies using S1P receptor antagonists and S1P₂ receptor null mice support the notion that a single S1P receptor, S1P₂, is responsible for the majority of S1P-induced vasoconstriction in the lung. In contrast to published reports demonstrating S1P₃-dependent vasoconstriction in the cerebral vasculature (39, 40), S1P₃ receptor inhibition with VPC23019 failed to inhibit S1P-induced vasoconstriction in the intact mouse lung (Fig. 5). On the other hand, S1P₂ receptor inhibition with JTE-013 markedly reduced pulmonary vasoreactivity to S1P (Fig. 5). Since JTE-013 remains the only commercially available S1P₂ antagonist, and a recent report demonstrated that JTE-013 nonspecifically inhibits vasoconstrictors in the cerebral vasculature, we also used lungs from S1P₂ null mice to investigate the role of the S1P₂ receptor in S1P-mediated vasoconstriction in the pulmonary circulation (39). In S1P₂ null mouse lungs, S1P-induced vasoconstriction was reduced by over 65% (Fig. 6 and Table 1). Several previous studies have shown that S1P₂ receptor null mice may exhibit blunted vasoconstrictor responses due to increased eNOS activity (42) or dedifferentiation of vascular smooth muscle (13, 47). We found that inhibition of eNOS with a saturating concentration of l-NAME had no effect on the S1P vasoconstrictor response in isolated S1P₂ null lungs (Fig. 6), demonstrating that the attenuation in vasoconstriction observed in these lungs is not likely due to increased eNOS-derived NO production. Additional studies, in which we demonstrated that ET-1 induced robust vasoconstriction in the S1P₂ null mouse lung, argue against the possibility that the attenuated S1P-induced vasoconstriction in S1P₂ null mouse lung is due to a generalized impairment of the vascular smooth muscle apparatus. Collectively, the data from our experiments with JTE-013 in wild-type mice and the studies in S1P₂ receptor null mice support the role of the S1P₂ receptor as the primary cell surface receptor mediating S1P-dependent vasoconstriction in the murine pulmonary vasculature.

S1P₂ receptor signaling, a heretofore poorly defined pathway, is increasingly recognized as being important in angiogenesis, endothelial barrier function, and the regulation of systemic vasomotor tone (24, 39, 41). Our experiments now introduce a potentially important vasoregulatory role for the S1P₂ cell surface receptor in the pulmonary circulation. Furthermore, the graded response to S1P observed in S1P₂-deficient mice compared with heterozygotes for S1P₂ receptor expression suggests a potentially dose-dependent effect of S1P₂ receptor signaling in the regulation of pulmonary vascular tone (Table 1). Further investigation is warranted to examine S1P₂ receptor signaling in more detail as it pertains to vasomotor control in the lung.

Conclusions.

With these experiments, we have clearly demonstrated a complex constrictor response of the pulmonary vasculature to exogenous S1P administration that is dependent on Rho-kinase activation downstream of the cell surface receptor S1P₂. Because sphingosine kinase isoforms have been demonstrated to be upregulated in hypoxia (1) and Rho-kinase signaling has been implicated in hypoxic pulmonary vasoconstriction (9), it is possible that S1P pharmacology participates in the vasoreactive response of the pulmonary circulation to hypoxia. In addition, the literature supports a role for endogenous pulmonary vasoconstrictors (e.g., thromboxane receptor agonists, ET-1, and Rho-kinase) in the pathophysiology of pulmonary arterial hypertension (5, 32, 38). S1P is produced endogenously by activated platelets, endothelial cells, and other cell types and signals through Rho-kinase to invoke vasoconstriction in the lung. The potential role S1P production in the pulmonary circulation may play in the pathogenesis of pulmonary hypertension remains to be investigated. Finally, S1P has been demonstrated to reduce lung edema formation and to improve gas exchange in animal models of inflammatory acute lung injury (27, 34, 43). Regional alterations in vasomotor tone, potentially involving S1P signaling, may play a significant role in maintenance of gas exchange in this devastating syndrome. Further experimentation is necessary and indicated to determine the role of endogenous S1P signaling in the control of vasomotor tone in the lung, to elucidate the role of vasomotor regulation in the evolution and resolution of acute lung injury, and to further examine potential therapeutic implications of S1P receptor pharmacology for diseases such as pulmonary arterial hypertension and acute lung injury.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants K08 HL083101 (to B. J. McVerry) and R37 HL65697 (to B. R. Pitt).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).