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BOR - Papers in Press, published online ahead of print September 14, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.043034
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BIOLOGY OF REPRODUCTION 74, 88–94 (2006)
DOI: 10.1095/biolreprod.105.043034
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Sphingosine-1-Phosphate Acts via Rho-Associated Kinase and Nitric Oxide to Regulate Human Placental Vascular Tone1

Denise G. Hemmings 2 3, Nicola K. Hudson 4, Deborah Halliday 4, Maureen O'Hara 4, Philip N. Baker 4, Sandra T. Davidge 3, and Michael J. Taggart 45 

Department of Obstetrics and Gynecology, Perinatal Research Centre,3 University of Alberta, Edmonton, Alberta, Canada T6G 2S2 Maternal and Fetal Health Research Centre, Human Development Group4 Smooth Muscle Physiology Group, Cardiovascular Research5, University of Manchester, Manchester M13 9WL, United Kingdom

ABSTRACT

Sphingosine-1-phosphate (S1P), a bioactive lipid released from activated platelets, has been demonstrated in animal models to regulate vascular tone through receptor-mediated activation of Rho-associated kinase 1 and nitric oxide synthase 3. The role of S1P in regulation of human vascular tone (particularly during pregnancy, with its unique vascular adaptations and localized platelet activation) is unknown. We hypothesized that S1P would constrict small placental arteries through activation of Rho-associated kinases with modulation by nitric oxide. Reverse transcription-polymerase chain reaction of chorionic plate artery preparations detected mRNAs encoding all five receptors for S1P, and S1P induced dose-dependent vasoconstriction of both chorionic plate and stem villous isobarically mounted arteries, which at 10 µmol/L was 32.9% ± 3.86% (mean ± SEM) and 34.6% ± 7.01%, respectively. In stem villous arteries, S1P-induced vasoconstriction was enhanced significantly following inhibition of nitric oxide synthases with NG-nitro-L-arginine methyl ester (100 µmol/L, 52.6% ± 6.28%, P < 0.05). The S1P-induced vasoconstriction was reversed by Y27632, an inhibitor of Rho-associated kinases (10 µmol/L) in both chorionic plate (to 14.9% ± 4.95%) and stem villous arteries (to 2.71% ± 6.13%). The S1P added to alpha-toxin-permeabilized, isometrically mounted chorionic plate arteries bathed in submaximal Ca2+-activating solution induced Ca2+-sensitization of constriction, which was 47.7% ± 10.0% of that occurring to maximal Ca2+-activating solution. This was reduced by Y27632 to 18.4% ± 18.4%. Interestingly, S1P-induced vasoconstriction occurred in all isobarically mounted arteries but was inconsistent in isometrically mounted chorionic plate arteries. In summary, S1P-induced vasoconstriction in human placental arteries is mediated by increased Ca2+-sensitization through activation of Rho-associated kinases, and this vasoconstriction also is modulated by nitric oxide. Identification of these actions of S1P in the placental vasculature is important for understanding both normal and potentially abnormal vascular adaptations with pregnancy.

calcium, kinases, nitric oxide, placenta, pregnancy

INTRODUCTION

Sphingosine-1-phosphate (S1P) is a circulating, bioactive lipid that is released from activated platelets and that modulates vascular tone through receptor-mediated vasoconstrictor and vasodilator pathways, as demonstrated in a number of animal models [1]. The extent to which S1P mediates these vascular effects appears to be dependent on both species and vascular bed [2]. Although S1P has been implicated in human coronary artery disease [3], atherosclerosis [2], and abnormal regulation of vascular tone in aging [4], to our knowledge a role for S1P in vascular responses of intact human arteries has not been investigated directly. During pregnancy, regulation of vascular tone in both maternal and fetoplacental circulations is essential for normal fetal development [5], with increased tone or vascular resistance being associated with pregnancy-related disorders, such as intrauterine growth restriction and preeclampsia [6, 7]. A role for S1P in regulation of vascular tone in the human placenta with its unique vascularization and localized platelet activation is unknown.

Sphingosine-1-phosphate mediates its biological effects through as many as five receptors expressed on the cell surface, which originally were named EDG (Endothelial Differentiation Gene) but recently were renamed S1P1 to S1P5 (S1P1/EDG1, S1P2/EDG5, S1P3/EDG3, S1P4/EDG6, and S1P5/EDG8) [8, 9]. The S1P-specific receptors are expressed in the vascular endothelium (S1P1/EDG1 and S1P3/EDG3) and vascular smooth muscle cells (S1P1/EDG1, S1P2/EDG5, and S1P3/EDG3) in both animal and human cardiovascular tissues [1] and in whole placenta (S1P3/EDG3 and S1P5/EDG8) [8, 10, 11]. To our knowledge, however, expression of S1P-specific receptors has not yet been examined specifically in human placental arteries.

Based largely on data from animal experiments, S1P-induced vasoconstriction has been suggested to occur primarily through S1P2/EDG5 and S1P3/EDG3 receptors on the vascular smooth muscle. Both S1P2/EDG5 and S1P3/EDG3 signal through the heterotrimeric G-proteins, Gi and Gq, to activate phospholipase C, leading to increased intracellular Ca2+, and also through G12/13, to activate Rho-associated kinase 1 (ROCK1) and increase Ca2+-sensitization of myofilament activation [1]. The S1P-induced vasodilation, on the other hand, may be mediated through the S1P1/EDG1 receptor on the endothelium via Gi to activate phosphoinositol-3-kinase and Akt, which leads to phosphorylation and activation of endothelial nitric oxide synthase 3 (NOS3) and release of a potent vasodilator, nitric oxide [12–14]. Activation of NOS3 also occurs through binding of the S1P3/EDG3 receptor by either S1P or phosphorylated FTY720, an S1P analog [15, 16]. A certain capacity for signaling overlap, however, exists between different S1P-specific receptors [15, 17]. Thus, the net vasoreactive effect of S1P will depend partly on the pattern of expression of S1P/EDG-receptor subtypes. Because the regulation of human placental vascular tone is essential for fetal development, it is important to understand the extent to which S1P mediates vasoactivity in this tissue.

Our hypothesis was that S1P will constrict small placental arteries through activation of Rho-associated kinases and increased Ca2+-sensitization and that constriction to S1P also will be modulated by nitric oxide. The objectives of the present study were to determine if small placental arteries from normal term pregnancies expressed S1P-specific receptors, to determine the vasoactive properties of S1P in chorionic plate and stem villous arteries, and to examine the roles of Rho-associated kinases and nitric oxide in these responses.

MATERIALS AND METHODS

Consumables

All chemicals were purchased from Sigma-Aldrich Company Ltd. except the following: NaCl, CaCl2, NaHCO3, KHPO4, K2EDTA, acetic acid, and methanol were purchased from BDH Laboratory Supplies; A23187, alpha-toxin (known throughout as {alpha}-toxin), and Y27632 from CN Biosciences; Trizol, reverse transcriptase, and PCR primers from Invitrogen; Taq DNA polymerase and oligonucleotides from Promega; and S1P from Biomol International L.P. A 10 mM stock solution of U46619 was made by dissolving in a 1:2 mixture of 100% ethanol and 1 mg/kg of sodium carbonate. Y27632 was made as a 10 mM stock dissolved in distilled water. {alpha}-Toxin was dissolved in distilled water as a stock solution of 2000 U/ml. One milligram of S1P was dissolved in 1–1.5 ml of warm (50°C) methanol. The solution was evaporated under nitrogen, and the powder redissolved in 0.01 M NaOH to a concentration of 2.64 mM. The S1P was diluted to final working concentrations in solutions containing 0.1% fatty acid-free BSA as a carrier.

Isolation of Human Placental Arteries

Small placental arteries (n = number of arteries throughout) were identified and isolated from both the chorionic plate and within the stem villous tree of human placentas (N = number of placentas throughout) that were obtained either by cesarean section (N = 10) or normal vaginal delivery (N = 18) at term (37–41 wk) following written informed consent in accordance with local ethical committee guidelines and the World Medical Association Declaration of Helsinki. Mode of delivery did not influence experimental results. The placental tissues were collected in Krebs solution (154 mmol/L of NaCl, 5.4 mmol/L of KCl, 1.2 mmol/L of MgSO4, 1.6 mmol/L of CaCl2, 5.5 mmol/L of glucose, and 10 mmol/L of MOPS) at pH 7.4, and small arteries were dissected and placed in physiological salt solution (PSS) bubbled with 95% air/5% CO2 (119 mmol/L of NaCl, 4.7 mmol/L of KCl, 1.2 mmol/L of MgSO4·7H2O, 25 mmol/L of NaHCO3, 1.17 mmol/L of KH2PO4, 0.03 mmol/L of K2EDTA, 5.5 mmol/L of glucose, and 1.6 mmol/L of CaCl2·2H2O) at pH 7.4. Some chorionic plate artery segments (total weight, 50–100 mg) were snap-frozen in liquid N2 and stored at –80°C until processed for RT-PCR (see below). For intact tissue contractile studies (see below), freshly isolated stem villous arteries (n = 16 arteries from nine placentas; lumen diameter at 40 mm Hg, 275 ± 27 µm [mean ± SEM]) or chorionic plate arteries (n = 8 arteries from four placentas; lumen diameter at 40 mm Hg, 284 ± 10 µm) were isobarically mounted between two glass cannulae on a pressure myograph system (Living Systems Instrumentation), tied off, and equilibrated at a pressure of 40 mm Hg for 30 min at 37°C. Intraluminal pressures between 20 and 40 mm Hg approximate that found in the fetoplacental circulation [18]. In another set of experiments, chorionic plate arteries were mounted on two 40-µm wires in chambers of a Danish Myotech wire myograph containing 5–7 ml of PSS for either intact (338 ± 36 µm; N = 7, n = 28) or permeabilization studies (344 ± 22 µm; N = 8, n = 16). Placental arteries were normalized by applying a vessel stretch, which was equivalent to a tension of 0.9 of L5.1kPa, where L5.1kPa is the vessel diameter at which the active effective pressure is 5.1 kPa, equivalent to 20 mm Hg, and equilibrated in PSS for 20 min at 37°C (25°C for permeabilized arteries to prevent tissue deterioration because of proteolytic enzyme activity) before experimentation.

Extraction of RNA and RT-PCR

Total RNA was prepared using Trizol and treated with DNaseI to remove any contaminating genomic DNA, and the first-strand cDNA was synthesized using reverse transcriptase. Previously published oligonucleotide primers were used for S1P1/EDG1 (predicted product size, 481 bp), S1P3/EDG3 (506 bp) [19], S1P2/EDG5 (315 bp), S1P4/EDG6 (420 bp) [20], and ß-actin (546 bp) [21]. Oligonucleotide sequences for S1P5/EDG8 were constructed based on their human nucleotide sequences (http://www.genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi; 352 bp). The primers used were as follows: human S1P1/EDG1, 5'-CCA CAA CGG GAG CAA TAA CT-3' and 5'-GTA AAT GAT GGG GTT GGT GC-3'; human S1P2/EDG5, 5'-CAT TGC CAA GGT CAA GCT GT-3' and 5'-ACG ATG GTG ACC GTC TTG AG-3'; human S1P3/EDG3, 5'-TCA GGG AGG GCA GTA TGT TC-3' and 5'-CTG AGC CTT GAA GAG GAT GG-3'; human S1P4/EDG6, 5'-ACG GGA GGG CCT GCT CTT CA-3' and 5'-AAG GCC AGC AGG ATC ATC AG-3'; human S1P5/EDG8, 5'-AAG GCC TAC GTG CTC TTC TG-3' and 5'-GCG TGT AGA TGA TGG GGT TC-3'; human ß-Actin, 5'-GGG ACC TGA CTG ACT ACC TC-3' and 5'-ACT CGT CAT ACT CCT GCT TG-3'. The PCR amplification was performed using Taq DNA polymerase and specific primers in a programmable PCR machine. The PCR protocols were optimized as follows: human S1P1/EDG1 and S1P3/EDG3, initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 45 sec, annealing at 50°C for 30 sec, and extension at 72°C for 1 min, and then a final extension for 10 min at 72°C; human S1P4/EDG6 and human ß-actin, initial denaturation at 95°C for 2 min and 30 sec, followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min, and then a final extension for 5 min at 72°C; human S1P2/EDG5, initial denaturation at 94°C for 2 min, followed by 40 cycles of denaturation at 94°C for 15 sec, annealing at 55°C for 30 sec, and extension at 72°C for 1 min, and a final extension at 72°C for 5 min; human S1P5/EDG8, initial denaturation at 95°C for 2 min, followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C for 1 min, and then a final extension for 5 min at 72°C. The RT-PCR products were visualized on a 1.5% agarose gel electrophoresis and their nucleotide sequences confirmed as the appropriate S1P genes following gel extraction and sequencing using primers in both the forward and reverse directions and analysis through the BLAST database. Two negative controls were performed; each reaction was done in the absence of reverse transcriptase and specific primers. The positive control was human heart cDNA (Becton Dickinson).

Intact Tissue Contractile Studies on the Pressure Myograph

Studies on intact chorionic plate or stem villous placental arteries were conducted in a single- or dual-chamber pressure myograph system in N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]-buffered PSS (Hepes-PSS; 142 mmol/L of NaCl, 4.7 mmol/L of KCl, 1.17 mmol/L of MgSO4, 1.18 mmol/L of KH2PO4, 1.56 mmol/L of CaCl2, 10 mmol/L of Hepes, and 5.5 mmol/L of glucose) at pH 7.4 to ensure that pH was maintained on addition of S1P. After equilibration, a control, 5-min exposure to high-K+ solution (140 mmol/L of KCl-containing Hepes-PSS isosmotically substituted for NaCl) was performed to test for contractile viability. Arteries were then exposed to incremental doses of S1P (1.0 nmol/L to 20 µmol/L) for 10 min at each concentration. In some studies, parallel arteries were preincubated with 100 µmol/L of the nitric oxide synthase-inhibitor NG-nitro-L-arginine methyl ester (L-NAME; Calbiochem) for 20 min before addition of S1P. In studies examining the role of Rho-associated kinases in intact arteries mounted on the pressure myograph system, incremental doses of S1P (0.1 nmol/L to 10 µmol/L) were added for 10 min at each concentration. After stabilization at 10 µmol/L of S1P, the arteries were then exposed to incremental doses (0.1–10 µmol/L) of Y27632, an inhibitor of both ROCK1 and ROCK2 [22, 23] for 10 min at each concentration. At the end of the experiment, the passive lumen diameter of the arteries were measured at 40 mm Hg after incubation for 10 min in EGTA-Ca2+-free PSS (142 mM NaCl, 4.7 mM KCl, 1.17 mM MgSO4, 1.18 mM KH2PO4, 10 mM Hepes, and 2 mM EGTA) and papaverine (0.1 mM; Sigma). Arterial diameters were assessed on the pressure myograph system using either a digital filar eyepiece (Lasico) on a compound microscope as previously described [4, 24] or were visualized with a video camera and measured digitally using a video dimension analyzer and computer software (WinDaq/Lite, Dataq Instruments, Inc.).

Intact and Permeabilized Placental Arteries on the Wire Myograph

As many as four arterial segments isolated from each placenta were run in parallel in adjacent organ chambers. All vessels responded to high-K+ solution with a maintained constriction. In some experiments, the response to S1P in intact, nonpermeabilized chorionic plate arteries mounted on the wire myograph was conducted. The following permeabilization techniques are based on the results of previous studies [25, 26]. The PSS-equilibrated chorionic plate arteries were exposed to a mock intracellular relaxing solution of pCa 9, where pCa = log10[Ca2+] ([Ca2+]i = 0.001 µM; no active tension; 10 mmol/L of sodium creatine phosphate, 5.2 mmol/L of Na2ATP, 7.3 mmol/L of magnesium methane sulfonate, 74 mmol/L of potassium methane sulfonate, 1 mmol/L of K2EGTA, and 30 mmol/L of PIPES; pH 7.1 with KOH) and equilibrated for 10–15 min. This relaxing solution was then aspirated, and a 25-µl droplet of submaximal activating solution of pCa 6.7 ([Ca2+]i = 0.2 µM; induces submaximal active tension) containing 500 U/ml of {alpha}-toxin plus 10 µmol/L of A23187 was carefully pipetted directly onto the vessel. {alpha}-Toxin permeabilization was assumed to be complete when the ensuing constriction either had reached a plateau or, in cases of low constriction after permeabilization, after 30 min. The artery was returned to relaxing solution, and the subsequent constriction to activating solution of pCa 4.5 ([Ca2+]i = 31.5 µM; induces maximal active tension) was monitored. On return to relaxing solution, agonist-induced Ca2+-sensitization of constriction was then examined as follows: Vessels were exposed, in turn, to the submaximal activating solution of pCa 6.7, solution of pCa 6.7 plus 10 µmol/L of GTP (GTP/pCa 6.7), and GTP/pCa 6.7 solution plus 10 µmol/L of S1P or 1 µmol/L of U46619. Once the constriction had reached a plateau, the effect of Y27632 (10 µmol/L) on S1P or U46619-induced Ca2+-sensitization was examined. To confirm that agonist-induced constriction was maintained at a plateau over the same time period as Y27632 addition, constriction in a parallel time-control vessel was monitored simultaneously in the presence of S1P or U46619 alone. The Ca2+-sensitization of constriction was calculated as the change in tension observed by addition of S1P or U46619 to GTP/pCa 6.7 solution as a percentage of the change in tension observed on previous exposure to pCa 4.5 solution. Activating solutions of pCa 6.7 and pCa 4.5 contained 10 mmol/L of EGTA and were prepared by addition of the appropriate amount of CaEGTA.

Calculations

Percentage constriction in Figures 2 and 3 were calculated as follows: ((D1 – D2)/D1) x 100, where D1 is the arterial lumen diameter with or without L-NAME treatment before S1P addition and D2 is the arterial diameter after S1P addition. Basal tone was calculated as follows: ((D3 – D1)/D3) x 100, where D3 is the passive arterial lumen diameter in calcium-free Hepes-PSS at 40 mm Hg and D1 is the arterial lumen diameter in Hepes-PSS at 40 mm Hg. Basal tone was calculated in parallel arteries treated with or without L-NAME. No significant differences were found in lumen diameter before and after L-NAME treatment in the same arteries (data not shown).


Figure 2
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  		FIG. 2. S1P-induced vasoconstriction of human chorionic plate placental arteries (A) and stem villous arteries (B) was enhanced by L-NAME only in stem villous arteries. Decreased lumen diameter of placental arteries mounted on a pressure myograph were measured in response to cumulative doses of S1P (1.0 nmol/L to 20 µmol/L) in the presence or absence of L-NAME, an inhibitor of nitric oxide synthases (100 µmol/L) in parallel vessels and reported as the mean ± SEM. A significant difference between curves in B was assessed by two-way ANOVA with the Fisher LSD post hoc test and is denoted by an asterisk (P < 0.01)


Figure 3
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  		FIG. 3. S1P-induced vasoconstriction in human placental arteries was reversed by inhibition of Rho-associated kinases. A) A representative tracing of a stem villous artery mounted on a pressure myograph and constricted to cumulative doses of S1P (0.1 nmol/L to 10 µmol/L) followed by relaxation induced by cumulative doses of Y27632, an inhibitor of Rho-associated kinases (0.1–10 µmol/L). Vessel contractile viability initially was assessed by constriction to 140 mmol/L of KCl. Bar indicates a timescale of 10 min. All tracings were summarized for both chorionic plate arteries (B; n = 4) or stem villous arteries (C; n = 3) at 10 µmol/L of S1P and 10 µmol/L of S1P plus 10 µmol/L of Y27632 and shown as the mean ± SEM. An asterisk denotes significance (P < 0.05) using a paired t-test

Statistics

All measurements are reported as the mean ± SEM, with n = number of samples. A paired t-test and one-way or two-way ANOVA with the Fisher least-significant-difference (LSD) multiple-comparison post hoc test were used to analyze significant differences as described in the figure legends and Results. Significance was accepted at P < 0.05. In Figure 2B, the S1P concentration at which the lumen diameter initially was significantly reduced compared to the lumen diameter in the absence of S1P (threshold concentration) was assessed using a one-way ANOVA with the Fisher LSD method of multiple comparisons. This analysis was repeated for arteries pretreated with L-NAME.

RESULTS

S1P-Specific Receptor Expression in Placental arteries

Using RT-PCR in isolated chorionic plate arteries from four different placentas, we showed consistent mRNA expression for S1P3/EDG3 and S1P5/EDG8 (four of four preparations were positive) but more variable expression of mRNA encoding S1P1/EDG1 (three of four preparations), S1P2/EDG5 (two of four preparations), and S1P4/EDG6 (two of four preparations) (Fig. 1). Each preparation showed similar signals for mRNA encoding ß-actin.


Figure 1
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  		FIG. 1. S1P-specific receptor expression in human chorionic plate artery preparations. S1P-specific receptor transcripts for S1P1/EDG1 (481 bp), S1P2/EDG5 (315 bp), S1P3/EDG3 (506 bp), S1P4/EDG6 (420 bp), and S1P5/EDG8 (352 bp) receptors were assessed by RT-PCR in each of 4 placental artery preparations (1–4). ß-Actin transcripts (546 bp) were assessed as an internal arterial control. For each transcript, the band corresponding to the appropriate number of bp is identified with an arrow. Negative control experiments were done in the absence of reverse transcriptase (RT) or in the absence of primers (–ve). Human heart cDNA was used as a positive (+ve) control

S1P-Induced Vasoconstriction of Placental Arteries and Modulation by Nitric Oxide

The placental vascular response to increasing doses of S1P initially was examined using isobarically mounted arteries. The S1P induced vasoconstriction of both chorionic plate (Fig. 2A) and stem villous (Fig. 2B) arteries in a dose-dependent manner. At 10 µmol/L, the maximum constriction was 32.9% ± 3.86% and 34.6% ± 7.01% for chorionic plate (Fig. 2A) and stem villous (Fig. 2B) arteries, respectively.

The role of nitric oxide in modulation of S1P-induced vasoconstriction also was examined in a parallel set of either chorionic plate (Fig. 2A) or stem villous (Fig. 2B) isobarically mounted arteries. No difference was observed in vasoconstriction of chorionic plate arteries in the presence or absence of L-NAME, an inhibitor of nitric oxide synthases (Fig. 2A). In stem villous arteries, however, preincubation with L-NAME resulted in an enhanced sensitivity to S1P, with significant constriction initially occurring at a concentration of 0.5 µmol/L compared to 5 µmol/L in the absence of L-NAME. In addition, significantly enhanced responses in the presence of L-NAME were observed at higher concentrations of S1P (20 µmol/L), with a maximum constriction of 68.2% ± 6.29% (compared to 45.2% ± 7.51% in the absence of L-NAME, P < 0.01) (Fig. 2B). No significant differences were found in the basal tone of either chorionic plate (9.39% ± 4.18% in the presence of L-NAME vs. 10.2% ± 2.32% in the absence of L-NAME) or stem villous (4.37% ± 8.13% vs. 3.72% ± 0.96%) arteries at 40 mm Hg before the addition of S1P.

S1P-Induced Vasoconstriction in Placental Arteries Was Dependent on Rho-Associated Kinases

To investigate further the placental vascular response to S1P, we examined the role of Rho-associated kinases using Y27632, a well-characterized inhibitor [22, 23]. Vasoconstriction in either chorionic plate or stem villous isobarically mounted arteries induced by increasing doses of S1P was reversed on addition of increasing doses of Y27632, as depicted in a representative tracing of a stem villous artery (Fig. 3A). Constriction at 10 µmol/L of S1P was similar in both chorionic plate and stem villous arteries. At the maximum dose of Y27632 (10 µmol/L), S1P-induced constrictions were reduced to 14.9% ± 4.95% in chorionic plate (Fig. 3A) and 2.71% ± 6.12% in stem villous (Fig. 3B) arteries.

S1P Induced Ca2+-Sensitization of Vasoconstriction Through Rho-Associated Kinases

The above experiments suggested that S1P-induced vasoconstriction of human placental arteries was mediated through activation of Rho-associated kinases and, therefore, may involve Ca2+-sensitization of the myofilaments. This mechanistic possibility was investigated further using arteries permeabilized with {alpha}-toxin. Chorionic plate arteries were isometrically mounted, thus allowing both endothelial and vascular smooth muscle cells to be permeabilized with {alpha}-toxin and the ionophore A23187. The main benefits of this preparation are inhibition of endothelial-mediated vasoactive substances and that the Ca2+ concentration of the bathing solution surrounding the myofilaments can be kept constant. The latter situation allows for the study of S1P-induced changes in Ca2+-sensitization of constriction. After permeabilization, the arteries were placed in a low-calcium solution (pCa 9), in which the vessels do not develop tension. Addition of a high-calcium activating solution (pCa 4.5) induced constriction, which increased the tension measurement. After washout of pCa 4.5 with pCa 9 solution, the vessels were then placed in a submaximal calcium solution (pCa 6.7) in the presence of GTP, in which the vessels only attained a small increase in tension. On addition of 10 µmol/L of S1P (Fig. 4) or 1 µmol/L of U46619 (data not shown), a further substantial increase in tension was found, demonstrating that S1P increased the sensitivity of the arteries to this submaximal calcium concentration, with constriction maintained over time (Fig. 4A). This Ca2+-sensitization by S1P was calculated as 47.7% ± 10.0% of the maximal constriction induced by pCa 4.5 and only occurred in arteries from three of eight placentas tested. Y27632 reduced this Ca2+-sensitization in the S1P-constricted arteries to 18.4% ± 18.4% (n = 3) (Fig. 4B). Arteries from all placentas in this series of experiments (N = 8), including those unresponsive to S1P, all responded with maintained constrictions to the thromboxane mimetic U46619 (Ca2+-sensitization of 64.2% ± 8.30% of pCa 4.5 constriction) and were all reduced by Y27632 (to Ca2+-sensitization of 19.0% ± 11.8% of pCa 4.5 constriction).


Figure 4
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  		FIG. 4. S1P induced constriction through Rho-associated kinases-dependent Ca2+-sensitization in permeabilized human chorionic plate arteries. Shown are representative tracings of {alpha}-toxin and A23187 permeabilized placental arteries mounted on a wire myograph, illustrating the pronounced Ca2+-sensitization of constriction by S1P (10 µmol/L) in a submaximal calcium solution pCa 6.7/GTP (A andB) and its attenuation by Y27632 (B; 10 µmol/L). A time control (A) shows maintenance of the S1P-induced constriction over time. Constriction induced by incubation of the artery in an activating calcium concentration (pCa 4.5) is shown for comparison purposes. The duration of treatment with either S1P alone (A) or with S1P and Y27632 (B) are denoted by horizontal bars above each figure. The small bar indicates a timescale of 10 min

S1P-Induced Vasoconstriction in Isometrically Mounted Intact Arteries

Because isobarically mounted chorionic plate arteries from all tested placentas responded to S1P with vasoconstriction, we sought to determine if the lack of response in permeabilized arteries of several placentas was caused by the permeabilization technique itself or a difference in responsiveness to S1P of isobarically or isometrically mounted arteries. A further set of experiments therefore was conducted with intact, nonpermeabilized, isometrically mounted chorionic plate arteries. Similar to the permeabilization studies, we found that only a proportion of isometrically mounted arteries (in this case, from five of seven placentas) constricted in response to S1P, and in some cases, this response was weak. Arteries from all the placentas in this series of experiments, including those unresponsive to S1P, responded with maintained constrictions to high-K+ solution. In the S1P-responsive arteries, the constriction to 10 µmol/L was 19.4% ± 9.70% the high-K+ solution. As in the isobarically mounted chorionic plate arteries (Fig. 2A), no significant alteration was observed in the dose-responsiveness of isometrically mounted chorionic plate arteries to S1P in the presence of L-NAME. Maximum S1P-induced constrictions (at 40 µmol/L, 46.4% ± 20.5% of high K+) were reduced significantly by Y27632 (to 0.05% ± 0.05% of high K+).

DISCUSSION

Sphingosine-1-phosphate is a bioactive lipid with many receptor-mediated biological activities, including a novel role in vascular tone regulation (for review, see [1]). The S1P-mediated vascular constriction is variable in the animal vascular beds examined to date, including basilar [27], coronary [28], cerebral [29, 30], as well as renal and mesenteric [31]; at low concentrations [14] and in conduit arteries such as the aorta [15], S1P may induce relaxation. The involvement of this modulator of vascular tone in human pregnancy, in which the importance of maternal and fetoplacental vascular tone are evident [7], is unknown. We show that S1P contributes to vascular tone in small, fetal-derived placental arteries through the Ca2+-sensitization activities of Rho-associated kinases and, potentially, through modulation by nitric oxide. To our knowledge, this is the first report demonstrating a vascular response in intact human arteries to S1P.

Arteries from the chorionic plate and stem villous both exhibit constriction to S1P. Interestingly, however, whereas the chorionic plate arteries mounted under isobaric conditions were consistently reactive, isometrically mounted arteries showed more variability in responsiveness. Even so, all were reactive to the thromboxane mimetic U46619 or high K+ stimulation, as described previously [26]. Vessel size is unlikely to be responsible for this variability, because no significant differences were found in lumen diameters for intact chorionic plate arteries on either system. Direct access of S1P to the vessel lumen on the wire myography system possibly may have facilitated activation of NOS3, resulting in enhanced nitric oxide production and the apparent loss of constriction in some of these vessels. Preincubation with L-NAME in chorionic plate arteries, however, did not modulate the constrictor response, as suggested previously by the lack of response to endothelium-dependent vasodilators, such as bradykinin [32, 33]. Well-characterized differences exist in contractile responsiveness of arteries mounted under isobaric or isometric conditions [34, 35]. These include an alteration in vasoconstrictor responsiveness that most likely also underlies the differences in response to S1P observed between isobaric and isometric preparations in the present study.

Although the placental artery preparations in the present study were consistently collected in such a way that they contained both large and small arteries in approximately the same proportion, variability exists in the expression of mRNA encoding S1P-specific receptors. Half the preparations expressed mRNA encoding S1P1/EDG1 receptors known to signal to NOS3, whereas half and all of the preparations expressed mRNA encoding S1P2/EDG5 and S1P3/EDG3 receptors, respectively, which are known to be involved in constriction events in other vessels. The expression of mRNA encoding S1P5/EDG8 receptors was surprising, and the functional significance of this in placental vessels remains to be resolved. The variability of receptor expression in placental arteries caused by size or location requires further investigation, to be resolved on development of sufficiently specific antibodies for examination of placental tissues by immunohistochemistry and Western blotting (current commercially available antibodies are not sufficient for this task). In addition, whereas the mode of delivery does not appear to be important in the in vitro response to S1P observed here, it is possible that in vivo, other factors that alter S1P efficacy or S1P-specific receptor expression may play accessory roles; examples include differentiation [36], fluid shear stress [37], and cytokines, such as tumor necrosis factor {alpha} [38] and vascular endothelial growth factor [39].

The concentration of S1P at which significant vasoconstriction in placental arteries first occurs is at least twofold higher than that reported in normal plasma (0.2–0.5 µM [40]). Although this suggests that vascular constriction may occur only when circulating S1P levels are elevated, the placental vascular bed is unique with potentially elevated local levels of S1P through increased blood flow and platelet activation. On the other hand, the fetoplacental vasculature is protected from maternal circulating factors by the syncytiotrophoblast layer. Thus, sources of S1P affecting the fetal vasculature likely include only the syncytiotrophoblast or cytotrophoblast cells, placental stromal cells, and fetal circulatory cells.

Sphingosine-1-phosphate activates NOS3, with ensuing release of the vasodilator nitric oxide in a number of vascular and cell culture systems [12–14]. Preincubation with L-NAME, an inhibitor of nitric oxide synthases that in other vascular beds successfully ameliorates nitric oxide-attributable relaxation, resulted in enhanced sensitivity to S1P-induced constriction of stem villous, but not chorionic plate, arteries. In the absence of a sigmoidal curve, we were unable to calculate EC50 values to compare the sensitivity of each curve to S1P-induced constriction. We found, however, that stem villous arteries treated with L-NAME showed constriction to S1P that was initially significant at a lower dose than arteries without L-NAME treatment and also showed greater constriction at the maximum S1P dose administered, suggesting an increased sensitivity to S1P-induced vasoconstriction. Interestingly, although a prominent agonist-mediated, nitric oxide-dependent vasodilation has not been a general feature reported for chorionic plate arteries [18, 32, 33] with the exception of calcitonin gene-related peptide [41], a number of reports have demonstrated L-NAME sensitivity in stem villous arteries [42–45]. Vasoactive differences between chorionic plate and stem villous arteries are not limited to nitric oxide but also have been documented in response to other substances [46, 47].

Strong evidence exists for NOS3 expression and physiological influences of nitric oxide in placental vascular function [45, 48, 49]. In addition, nitric oxide is important in fetoplacental artery dilation in response to flow [43]. Taken together, these data suggest that nitric oxide elicited through other means, such as S1P, also may regulate placental arterial tone (at least in stem villous arteries). Our data showing increased constriction in the presence of L-NAME without evidence of differences in basal nitric oxide production is consistent with S1P-induced production of nitric oxide and modulation of placental vascular tone.

Vascular smooth muscle contractions occur through increased intracellular Ca2+ and/or increased Ca2+-sensitization of the contractile filaments by activation of Rho-associated kinases, both of which occur in response to extracellular S1P in animal tissues [1]. Because the importance of activation of Rho-associated kinases in mediating constriction in the vasculature has been shown to be variable depending on the agonist [50], it is important to examine specifically the magnitude of involvement in S1P-mediated vasoconstriction. The significant presence of message for S1P-specific receptors known to signal through Rho-associated kinases (S1P2/EDG5 and S1P3/EDG3), the dose-dependent relaxation to Y27632 (an effective inhibitor of both ROCK1 and ROCK2), and the considerable involvement of Ca2+-sensitization in the response to S1P demonstrate the importance of this signaling pathway for tone development in human placental arteries. We previously showed expression of ROCK2 in placental arteries isolated from normal term placentas and, using Y27632, demonstrated a similar role for activation of Rho-associated kinases in agonist-induced constriction of these vessels with a thromboxane mimetic, U46619 [26]. The reduction of S1P-induced Ca2+-sensitization of tone by Y27632 in permeabilized arteries was almost complete. This suggests that a major route of S1P-dependent constriction of human placental arteries is activation of Rho-associated kinases, leading to Ca2+-sensitization. These results are consistent with a recent report demonstrating that 5-hydroxytryptamine-induced constriction of human umbilical arteries also involves activation of ROCK2 [51]. Interestingly, nitric oxide can induce relaxation by decreasing Ca2+-sensitization through activation of myosin light-chain phosphatase and, potentially, through inhibition of ROCK activity [52, 53].

Altered regulation of vascular tone by Rho-associated kinases may be involved in cardiovascular diseases, such as hypertension or vasospasm, and disturbances in this signaling pathway also may induce endothelial dysfunction [54]. Interestingly, it has been shown recently that ROCK2 expression is increased in the placental syncytiotrophoblast from women with preeclampsia, a hypertensive disorder of pregnancy [55]. Currently, however, it is unknown whether this increased expression also occurs in the placental vasculature of women with preeclampsia. In addition, the potential importance of S1P in vascular function has been inferred from studies examining its role in situations of vascular dysfunction, including aging [4], atherosclerosis [2] and coronary artery disease [3]. Moreover, when sphingosine kinase is overexpressed (potentially leading to elevated S1P levels) in vascular smooth muscle cells, an increase occurs in vascular tone and myogenic responses of intact microvascular arteries that is dependent on the Rho-associated kinase signaling pathways [56]. In the present study, it is particularly interesting that each placental artery preparation examined expressed mRNA for S1P3/EDG3 receptors, because these receptors have been implicated in mediating S1P agonist-induced hypertension [57]. Therefore, it will be intriguing to establish if altered S1P-mediated vascular tone is involved in pregnancy-related disorders in which maternal and fetoplacental vascular tone is elevated.

In summary, we show that S1P induces vasoconstriction in human vasculature, specifically in the placental bed, through a Rho-associated kinase-dependent Ca2+-sensitization mechanism. This vasoconstriction is modulated in stem villous arteries by nitric oxide, likely through S1P-mediated activation of NOS3. The balance between these two opposing functional responses to S1P likely influences tone regulation in this specialized vascular bed, and dysregulation of this equilibrium may contribute to the etiology of pregnancy-related disorders.

FOOTNOTES

1 Supported by Ardana Bioscience and the Canadian Institutes of Health Research (CIHR). D.G.H. is supported jointly by the Heart and Stroke Foundation of Canada and CIHR. S.T.D. is supported jointly by a Canada Research Chair in Women's Cardiovascular Health and as a Senior Scholar of the Alberta Heritage Foundation for Medical Research. Back

2 Correspondence: Denise G. Hemmings, Department of Obstetrics and Gynecology, Perinatal Research Centre, 232 HMRC, University of Alberta, Edmonton, AB, Canada T6G 2S2. FAX: 780 492 1308; denise.hemmings{at}ualberta.ca Back

Received: 20 April 2005.

First decision: 14 May 2005.

Accepted: 12 September 2005.

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