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Cardiovascular Research 2005 68(1):56-64; doi:10.1016/j.cardiores.2005.05.013
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Copyright © 2005, European Society of Cardiology

Sphingosylphosphorylcholine-induced vasoconstriction of pulmonary artery: Activation of non-store-operated Ca2+ entry

Gavin D. Thomas, Vladimir A. Snetkov, Rupal Patel, Richard M. Leach, Philip I. Aaronson and Jeremy P.T. Ward*

Department of Asthma, Allergy and Respiratory Science, GKT School of Medicine, King's College London, Guy's Campus, London SE1 9RT, UK

* Corresponding author. c/o Cardiovascular Biology and Medicine, 2nd floor New Hunt's House, King's College London, Guy's Hospital Campus, London SE1 1UL. Tel.: +44 20 7848 6695; fax: +44 20 7403 8640. Email address: Jeremy.ward{at}kcl.ac.uk

Received 12 February 2005; revised 12 May 2005; accepted 17 May 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Acknowledgments
 References
 
Objective: Sphingosylphosphorylcholine (SPC) is an important lipid mediator that has been implicated in vascular disease. As it has not been studied in the pulmonary circulation, we examined its mechanisms of action in rat small intrapulmonary arteries (IPA).

Methods: IPA were mounted on a myograph for recording tension and intracellular Ca2+ concentration ([Ca2+]i). Ca2+ sensitisation was examined in {alpha}-toxin permeabilized IPA, and by Western blot analysis of MYPT1 phosphorylation.

Results: SPC induced a slow but powerful vasoconstriction in IPA associated with an elevation in [Ca2+]i, with an EC50 for vasoconstriction of 12 ± 2 µM. Removal of extracellular Ca2+ increased the EC50 to 76 ± 33 µM (p<0.01) and abolished the rise in [Ca2+]i. Endothelial denudation or inhibition of NO synthase with L-NAME enhanced vasoconstriction. Treatment with pertussis toxin or the PLC inhibitor U731223 had no effect on SPC-induced vasoconstriction. The Rho kinase inhibitor Y27632 reduced SPC-induced vasoconstriction by ~70% and abolished both SPC-induced Ca2+ sensitisation in permeabilized IPA and the associated increase in MYPT1 phosphorylation; Ca2+ sensitisation was substantially inhibited by GDPβS. La3+ and 2-APB, at concentrations previously shown to block capacitative Ca2+ entry in IPA, suppressed SPC-induced vasoconstriction to the same extent as removal of extracellular Ca2+; residual tension was abolished by Y27632. Diltiazem was relatively ineffective. 2-APB also abolished the SPC-induced rise in [Ca2+]i. However, treatment with thapsigargin to empty intracellular stores had no effect on the elevation of [Ca2+]i induced by SPC.

Conclusion: We present evidence that SPC is a powerful vasoconstrictor of IPA and the novel finding that SPC-induced vasoconstriction in IPA is dependent on activation of a Ca2+ entry pathway with a similar sensitivity to La3+ and 2-APB as capacitative Ca2+ entry, although its activation is not dependent on emptying of PLC/IP3 or thapsigargin-sensitive intracellular stores.

KEYWORDS Pulmonary artery; Lysophospholipids; Sphingosylphosphorylcholine; Capacitative Ca2+ entry

Abbreviations: 2-APB, 2-aminoethoxydiphenylborane • CCE, Capacitative Ca2+ entry • IPA, intrapulmonary arteries • L-NAME, N{omega}-nitro-L-arginine methyl ester • LPA, lysophosphatidic acid • MYPT1, myosin phosphatase targeting subunit • PLC, phospholipase C • PTX, pertussis toxin • PSS, physiological salt solution • SPC, Sphingosylphosphorylcholine • S1P, Sphingosine-1-phosphate

This article is referred to in the Editorial by R. Schubert (pages 5–7) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Acknowledgments
 References
 
Sphingosylphosphorylcholine (SPC) belongs to a group of membrane-derived lysophospholipids which includes sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA). Lysophospholipids are important cardiovascular mediators with multifarious effects, including modulation of intracellular [Ca2+], mitogenesis and differentiation [1–3]. They are present in plasma at high nanomolar concentrations, released from activated platelets [4,5], and are increased in inflammation and atherosclerosis [6]. SPC is formed by N-deacylation of sphingomyelin, an abundant membrane lipid, whereas S1P is produced from sphingosine by sphingosine kinase [3].

Various lysophospholipid G-protein-coupled receptors (GPCR) have been identified, including the S1P (S1P1–5) and LPA (LPA1–4) families [7]. Although SPC is a low affinity ligand for S1P receptors, this cannot account for all its actions and specific receptors have been proposed [1,7]. Lysophospholipids also have direct intracellular actions [1,8], and it has been proposed that sphingosine kinase and S1P may parallel the phospholipase C (PLC) and inositol trisphosphate (IP3) pathway and mediate agonist-induced Ca2+ release [8,9].

SPC is a powerful vasoconstrictor of all arteries so far examined; although more efficacious than S1P it acts over the same concentration range [10–14], lending support for a specific SPC receptor. Recent reviews have concluded that SPC-induced vasoconstriction is mediated via pertussis toxin (PTX) sensitive Gi-proteins, IP3-induced Ca2+ release, and Rho kinase-mediated Ca2+ sensitisation, with Ca2+ entry via voltage-dependent L-type channels (see Refs. [2,3]). However Ca2+ sensitisation may be the major or sole mechanism in cerebral and coronary arteries [12,13], possibly mediated via a GTP-independent pathway [13,15].

There is little information concerning the effects of lysophospholipids on Ca2+ channels, although Itagaki et al. have reported that both S1P and LPA activate voltage-independent Ca2+ entry in human neutrophils and HL60 cells, the former via a store-independent action on channels underlying capacitative Ca2+ entry (CCE) [16,17]. SPC is reported not to activate CCE in rat mesenteric artery [10].

In the pulmonary circulation Rho kinase-mediated Ca2+ sensitisation and voltage-independent Ca2+ entry pathways such as CCE play particularly important roles in agonist-induced vasoconstriction, hypoxic pulmonary vasoconstriction, and the pathogenesis of pulmonary hypertension (Reviewed in Refs. [18,19]). Moreover, lung disease is commonly associated with inflammation and microemboli, factors that might be expected to increase lysophospholipids, and levels of S1P are reported to be ~8 fold greater in lung than elsewhere [20]. Lysophospholipids such as SPC could therefore potentially play a role in the increased pulmonary vascular resistance associated with pulmonary hypertension, as proposed for vasospasm associated with ischaemic heart and cerebrovascular disease [12,13]. However, as far as we are aware there have been no studies concerning lysophospholipids in the pulmonary circulation.

We therefore examined the effects of SPC on rat small intrapulmonary arteries (IPA). We report the novel findings that SPC-induced vasoconstriction of IPA was insensitive to PTX, inhibition of PLC, or emptying of Ca2+ stores with thapsigargin, but was associated with activation of voltage-independent Ca2+ entry via a La3+ and 2-APB-sensitive pathway, coupled with GTP-dependent and Rho kinase-mediated Ca2+ sensitisation.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 5. Conclusions
 Acknowledgments
 References
 
2.1 Tissue preparation
Male Wistar rats (200–300 g) were killed by cervical dislocation; the investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The lungs were removed and small (~300 µm i.d.) intrapulmonary arteries (IPA) mounted in a myograph (Danish MyoTechnology, Denmark), containing physiological salt solution (PSS) gassed with 95% air/5% CO2 (pH 7.4) at 37 °C, before being normalised as previously described [21]. IPA were equilibrated with three 2 min exposures to 80 mM K+ PSS (KPSS; isotonic replacement of NaCl by KCl). Endothelial denudation was achieved by rubbing the artery lumen with a human hair, and confirmed by loss of relaxation to acetylcholine. Concentration–response relationships were obtained by cumulative addition of SPC. Experiments using La3+ were performed in HEPES PSS to prevent precipitation. To investigate the role of Gi-proteins, IPA were incubated with PSS containing 400 ng/ml PTX or vehicle (water) for 2 h [22,23].

2.2 Simultaneous force and [Ca2+]i recording
IPA were incubated for 1.5 h at 20 °C in PSS with 4 µM Fura PE-3/AM. Temperature was increased to 37 °C for 30 min to promote intracellular cleavage to Fura PE-3 before washing with PSS. The myograph was mounted on an inverted microscope (Nikon Diaphot, Nikon Ltd.) combined with a microfluorimeter (Cairn Ltd., Faversham, U.K.). Force was recorded simultaneously with the ratio of emission intensities at >510 nm from excitation wavelengths of 340 and 380 nm (F340/380); changes in [Ca2+]i are expressed as the change in F340/380 elicited by SPC as a proportion of the change in F340/380 elicited by KPSS in the same preparation (%KPSS–F340/380) [21]. Autofluorescence did not change under the conditions used, and had an extremely small (<1%) effect on %KPSS–F340/380.

2.3 {alpha}-Toxin permeabilization of IPA
Isometric force was recorded in{alpha}-toxin-permeabilized arteries, as described previously [24,25]. Briefly, IPA were mounted as above, but incubated at 26 °C. Arteries were equilibrated in Ca2+-free relaxing solution containing 1 mM EGTA, and permeabilized by incubation at pCa 6.5 with 60 µg/ml {alpha}-toxin until the resulting vasoconstriction reached a plateau. IPA were then re-equilibrated with solution containing 10 mM EGTA and sub-maximal vasoconstriction elicited by reducing the pCa to ~6.8 by adjusting the K2EGTA/CaEGTA ratio. Experiments were performed in the presence of 10 µM cyclopiazonic acid (CPA) to obviate effects of Ca2+ release.

2.4 Western blotting
IPA were incubated for 60 min with and without SPC (30 µM). They were then snap frozen in liquid nitrogen and homogenized in SDS buffer containing protease and phosphatase inhibitors (Sigma, U.K.). Samples were centrifuged and loaded onto 4–12% NUPAGE Bis–Tris gels, electrophoretically separated and transferred to nitrocellulose membranes in 25 mM Tris, 192 mM glycine and 20% methanol using a mini Trans-blot unit. Membranes were washed in Tris-buffered saline (TBS; 20 mM Tris–HCl, pH 7.5 and 500 mM NaCl) and blocked with 5% skimmed milk in TBS before probing with phospho-MYPT1 antibodies (Upstate, NY, USA) at 1:1000 dilution in 1% milk in TBS containing 0.1% Tween overnight at 4 °C. The membranes were then probed with goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:10,000 dilution; Chemicon Int., CA, USA) for 1 h at room temperature and exposed to West Femto chemiluminescent substrate (Pierce Biotechnology, IL, U.S.A.). Band intensity was quantified using ImageJ software. For total MYPT1, membranes were stripped for 1 h at room temperature and re-probed with pan-MYPT1 antibodies (Upstate, NY, U.S.A.). Bands were visualized using West Pico chemiluminescent substrate.

2.5 Solutions and drugs
Physiological salt solution (PSS) contained (in millimole): NaCl 118; NaHCO3; 24; KCl 4; CaCl2 1.8; MgSO4 1; NaH2PO4 0.434; glucose 5.56. HEPES buffered PSS contained (in millimole): NaCl 140, KCl 4, CaCl2 1.8, MgCl2 1, HEPES 10, glucose 5.56. For nominally Ca2+-free PSS CaCl2 was omitted. All chemicals were obtained from Sigma-Aldrich Ltd. (Poole, UK) or Calbiochem (Notts., UK), except 2-APB (2-aminoethoxydiphenylborane; Tocris Cookson Ltd., Bristol, UK). Lysophospholipids were dissolved in 2:1 chloroform:methanol, and stored at –80 °C under argon. Before use, solvent was evaporated and the residue dissolved in water.

2.6 Calculations, curve fitting and statistical analysis
Developed force is presented in terms of the response to the final 2 min exposure to KPSS, and expressed as %KPSS. Values for EC50 (IC50), maximum response and Hill coefficient were derived from fitting to the Hill equation (Sigmaplot, SPSS Inc., Chicago, USA). Results are shown as mean ± S.E.M., and compared using paired or unpaired Student's t test, or ANOVA with Holm–Sidak post hoc as appropriate (SigmaStat, SPSS Inc., Chicago, USA).


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 5. Conclusions
 Acknowledgments
 References
 
3.1 SPC-mediated vasoconstriction of rat small IPA
SPC caused a concentration-dependent, slowly developing vasoconstriction in IPA that took 30–50 min to reach a plateau; time to reach plateau was independent of concentration. In contrast, the associated elevation in [Ca2+]i required ~10 min to stabilise (Fig. 1). The concentration–force relationship was steep, with a Hill coefficient of 2.4 ± 0.9, maximum vasoconstriction of 85.9 ± 8.6% KPSS, and EC50 of 12.4 ± 2.3 µM (n = 8, Fig. 2A). SPC-induced vasoconstriction was not easily reversed. In experiments where force and [Ca2+]i were measured simultaneously, 10 µM SPC elicited a steady-state rise in force of 20 ± 0.7% of the response to KPSS and in [Ca2+]i of 16 ± 2% KPSS–F340/380 (n = 3); at 30 µM the elevations were 71 ± 3% KPSS and 34 ± 3% KPSS–F340/380 respectively (n = 7).


Figure 1
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  		Fig. 1 A trace typical of 6 others showing the effect of 30 µM SPC on force and [Ca2+]i (expressed as F340/380) in a rat IPA. The response to depolarisation with KPSS is shown for comparison.

 

Figure 2
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  		Fig. 2 Panel A shows the SPC concentration–response relationship for IPA (filled circles), in the absence of extracellular Ca2+ (open squares), the presence of 100 µM L-NAME (open circles) or following removal of endothelium (filled squares). Symbols are mean ± S.E.M. of 4–13 experiments. Panel B shows equivalent responses to S1P (filled triangles) and LPA (open diamonds); n = 4–5 experiments.

 
Removal of extracellular Ca2+ caused a rightward shift in the SPC concentration–response relationship and made it less steep, such that vasoconstriction at 30 µM SPC was significantly smaller (p<0.001; Fig. 2A). It also essentially abolished the SPC-induced elevation of [Ca2+]i (see Section 3.3 and Fig. 3B).


Figure 3
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  		Fig. 3 Panel A shows the effect of PTX (open circles) or vehicle (filled circles) on SPC-induced vasoconstriction of IPA. Symbols represent mean ± S.E.M. of 4 experiments. Panel B shows typical traces of the effect of SPC (30 µM) and PGF2{alpha} (0.1 µM) on [Ca2+]i in IPA, in the presence and absence of extracellular Ca2+. Traces typical of at least 5 others.

 
The nitric oxide synthase inhibitor L-NAME (100 µM) increased maximum vasoconstriction to SPC (111 ± 7% KPSS, n = 13, p<0.05), but did not alter the EC50 (10.8 ± 1.4 µM) (Fig. 2A); L-NAME had no effect on resting force. Removal of the endothelium had the same effect as L-NAME (Max: 110 ± 7% KPSS; EC50: 10.5 ± 0.8 µM; n = 4).

3.2 Comparison with S1P and lysophosphatidic acid
The S1P concentration–response relationship was flatter than for SPC (Fig. 2B), and although the threshold was lower (<0.1 µM), vasoconstriction at 30 µM S1P was significantly smaller (p<0.001; n = 5). LPA did not elicit vasoconstriction in IPA up to 100 µM (n = 4; Fig. 2B).

3.3 Signaling pathways underlying SPC-mediated vasoconstriction of IPA
PTX did not affect SPC-mediated vasoconstriction of IPA (Fig. 3A); an identical methodology has been used successfully to ablate Gi-mediated responses to C-natiuretic peptide in small mesenteric arteries by ourselves (unpublished observations), and C-natiuretic peptide, adenosine and angiotensin II in small mesenteric arteries and renal afferent arterioles by others [22,23]. Pre-incubation with the PLC inhibitor U73122 [GenBank] (3 µM) had no effect on 30 µM SPC-induced vasoconstriction (control: 61.1 ± 6.7% KPSS; U73122 [GenBank] : 72.1 ± 5.7% KPSS; n = 5; NS). Additionally, the elevation in [Ca2+]i induced by SPC lacked the fast transient component characteristic of IP3-mediated Ca2+ release, as seen for example for 100 nM prostaglandin (PGF2{alpha}; Fig. 3B); U73122 [GenBank] did abolish the response to PGF2{alpha} (n>10; data not shown). In the absence of extracellular Ca2+ SPC failed to elicit any rise in [Ca2+]i apart from a very small transient, although the response to PGF2{alpha} retained a classical large transient (Fig. 3B). These data strongly suggest that SPC does not induce PLC/IP3-mediated Ca2+ release in IPA.

3.4 SPC-induced Ca2+ sensitisation in IPA
The Rho kinase inhibitor Y27632 caused a concentration-dependent relaxation of SPC-induced vasoconstriction (Fig. 4A); there was no difference in maximum relaxation or IC50 whether IPA were preconstricted with 10 or 30 µM SPC (10 µM SPC: 72 ± 4%, 1.3 ± 0.1 µM, n = 6; 30 µM SPC: 65 ± 9%, 1.0 ± 0.2 µM, n = 9; NS). Y27632 (10 µM) also abolished the associated increase in MYPT1 phosphorylation (p<0.05; Fig. 5B). Y27632 alone reduced control phosphorylation (p<0.05), implying a basal level of Rho kinase activity.


Figure 4
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  		Fig. 4 Panel A shows the relaxing effect of the Rho kinase inhibitor Y27632 on vasoconstriction induced by 10 µM (filled circles) and 30 µM (open circles) SPC. Symbols represent mean ± S.E.M. of 6–9 experiments. Panel B shows MYPT1 phosphorylation in the presence of SPC (30 µM), SPC with the Rho kinase inhibitor Y27632 (10 µM), and Y27632 alone. Bars represent mean ± S.E.M. of 6 experiments. *=p<0.05 compared to control; #=p<0.05 compared to SPC alone.

 

Figure 5
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  		Fig. 5 Panel A shows the Ca2+–force relationship for {alpha}-toxin permeabilized IPA (n = 3). The EC50 was 240 nM. Panel B shows a trace, typical of 15 others, of the effects of SPC on permeabilized IPA in the absence of GTP. Y27632 was added in 4 experiments.

 
SPC-mediated Ca2+ sensitisation was investigated directly using {alpha}-toxin permeabilized IPA. The plot of force against [Ca2+] shown in Fig. 5A demonstrates that adequate permeabilization was achieved, with an EC50 of 240 nM [Ca2+]. Maximum force at pCa 4.5 was 13.6 ± 2.3 mN. Subsequent experiments were performed at pCa ~6.8 to provide an initial basal force of 21.5 ± 3.1% of maximum; this was stable for >45 min. It has been suggested that SPC-induced Ca2+ sensitisation is GTP-independent [13,15], so experiments were performed with and without 2 µM GTP. In the absence of GTP, SPC (50 µM) caused an increase in force over 20–25 min to ~200% of initial force (n = 16); this was abolished and reduced below initial force by Y27632 (n = 4; Fig. 5B). The more selective Rho kinase inhibitor H1152 was similarly effective (n = 3; Fig. 6A). The presence or absence of exogenous GTP made no difference to the increase in force elicited by SPC or the degree of suppression induced by Y27632 (n = 5; Fig. 6A).


Figure 6
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  		Fig. 6 Panel A shows data derived from experiments similar to that in Fig. 5B. Bars represent mean ± S.E.M. of 3–15 experiments. #=p<0.01, ##=p<0.001 compared with force at pCa 6.8 (100%); **=p<0.01 compared with SPC. In panel B time matched experiments for 1 mM GDPβS alone (filled circles) and GDPβS plus SPC (50 µM; open circles) are shown on the left; the difference between these means is replotted on the right (open squares) against time-matched experiments with SPC alone (filled squares). Symbols represent mean ± S.E.M. of 5 experiments.

 
The lack of requirement for exogenous GTP could be due to retention of sufficient GTP following permeabilization to support G-proteins. PGF2{alpha} and U46619 [GenBank] also induce Ca2+ sensitisation in this preparation without exogenous GTP (own unpublished observations), although both are known to act via GPCR. We therefore examined the effect of GDPβS, an irreversible inhibitor of G-proteins. GDPβS (1 mM) itself caused a slow variable vasoconstriction, as previously reported in intact arteries [26]. To allow for this, we compared time-matched experiments in the presence of GDPβS, with and without addition of SPC. SPC caused a ~20% increase in force over that induced by GDPβS alone (Fig. 6B), though this did not reach significance (repeated measures ANOVA, p>0.3, n = 5). The difference between the GDPβS and GDPβS plus SPC curves, (i.e. the additional force elicited by SPC) was also replotted against time-matched experiments with SPC alone, suggesting that at least 70% of SPC-induced Ca2+ sensitisation is G-protein dependent (Fig. 6B).

3.5 Mechanisms of SPC-induced Ca2+ entry
The suppression of both SPC-induced vasoconstriction and elevation of [Ca2+]i by removal of extracellular Ca2+ implies a requirement for Ca2+ entry. However, the L-type Ca2+ channel blocker diltiazem (10 µM) was relatively ineffective against vasoconstriction induced by 30 µM SPC (n = 7; Fig. 7), whereas La3+ caused a more profound concentration-dependent relaxation (69 ± 7%; IC50 1.9 ± 0.2 µM; n = 5). In the presence of 10 µM La3+, SPC-induced vasoconstriction was similar to that obtained in the absence of extracellular Ca2+ (Fig. 7). The putative CCE blocker 2-APB (75 µM) depressed SPC-induced vasoconstriction to a similar extent (Fig. 7), and residual force was abolished by Y27632 (Fig. 7). Consistent with this, 2-APB abolished the rise in [Ca2+]i induced by 30 µM SPC, whilst only reducing force by ~70% (Fig. 8A).


Figure 7
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  		Fig. 7 Effect of Ca2+ entry antagonists on SPC-induced vasoconstriction. The open bar shows the effect of zero extracellular Ca2+ for comparison. Bars represent mean ± S.E.M. of 4–7 experiments. *=p<0.05, **=p<0.001 (compared to SPC alone); #=p<0.01 compared to SPC plus 2-APB.

 

Figure 8
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  		Fig. 8 Panel A shows the effect of 2-APB on SPC (30 µM)-induced vasoconstriction and elevation of [Ca2+]i in an IPA; trace typical of 3 others. Panel B shows the effect of thapsigargin and subsequent addition of SPC (30 µM) on [Ca2+]i; trace typical of 5 others.

 
We have previously shown that 2-APB and La3+ at the concentrations used here are effective blockers of CCE in IPA [e.g. 21]. Nevertheless, our results suggest that SPC does not cause significant Ca2+ release in IPA, and would therefore be unlikely to activate CCE via this mechanism (see Fig. 3B). However a slow, non-IP3-mediated Ca2+ release or inhibition of SERCA could potentially empty stores and activate CCE without necessarily being detected as an elevation of [Ca2+]i. To examine this hypothesis we therefore examined the effect of SPC after activation of CCE following emptying of intracellular stores with 1 µM thapsigargin, in the presence of diltiazem to prevent Ca2+ entry via L-type channels. Application of thapsigargin elicited a large, stable increase in [Ca2+]i of 45 ± 8% KPSS–F340/380 (n = 6; Fig. 8B). Subsequent application of SPC (30 µM) caused a further elevation in [Ca2+]i that was not significantly different from that in the absence of thapsigargin (SPC: 34 ± 3%, n = 7; SPC and thapsigargin: 38 ± 9% KPSS–F340/380). This suggests that SPC does not activate CCE via release from a thapsigargin-sensitive store.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
5. Conclusions
 Acknowledgments
 References
 
4.1 Characteristics of SPC-induced vasoconstriction in rat small IPA
SPC induced a slowly developing but powerful vasoconstriction in IPA with an EC50 of ~12 µM and maximum response at ~30 µM SPC similar to that elicited by KPSS (Fig. 1); the concentration response curve was steep (Hill coefficient >2). Similar results have been reported for other arteries, with EC50 s between 3–18 µM [10–12,15], a slow response [12,13,15], and steep concentration response relationship [10,11,15]. The increase in force was associated with a sustained elevation in [Ca2+]i, which at 30 µM SPC reached ~34% of the response to KPSS (Fig. 1). An increase in [Ca2+]i of ~25% KPSS was observed in one report on coronary artery [15], whilst in another and in cerebral artery the rise was small or absent [12,13], and in aortic vascular smooth muscle cells the elevation was transient or with a small plateau [11,14,27]. In IPA removal of extracellular Ca2+ substantially reduced vasoconstriction (Fig. 1), consistent with reports from rat mesenteric and renal arteries [11], but not cerebral or coronary arteries [12,13]. It also abolished the elevation in [Ca2+]i in IPA (Fig. 3B), implying a role for Ca2+ entry.

Although acting over a similar concentration range, S1P induced ~50% less force than SPC at 30 µM, and the concentration–response relationship was much flatter (Fig. 2). A similar comparison was reported for mesenteric and renal arteries [11]. The EC50 of SPC-induced vasoconstriction reported here and elsewhere (2–20 µM) are substantially greater than those reported for identified SPC or S1P receptors (2–65 nM; [7]). This is consistent with an unidentified, SPC-specific receptor, and suggests SPC is not acting via S1P receptors. Similarly, the lack of response to LPA obviates against LPA as an intracellular mediator, as previously proposed [28].

4.2 Role of nitric oxide and endothelium
Inhibition of NO synthase enhanced SPC-induced vasoconstriction (Fig. 2). As removal of the endothelium had the same effect, it is unlikely that endothelial-derived mediators other than NO play a significant role. This could be interpreted either as activation of NO synthesis by SPC, or suppression of basal NO release by L-NAME, though L-NAME did not affect basal force. Although Bischoff et al. [11] reported no effect of NO synthase inhibition in rat mesenteric and renal arteries, Mogami et al. [29] showed that SPC caused NO-dependent relaxation in bovine coronary artery, and S1P3 receptors have been implicated in activation of NO synthase in human aorta [30].

4.3 Signaling pathways and Ca2+ elevating mechanisms
Previous studies suggest that SPC elevates [Ca2+]i in vascular smooth muscle primarily via PTX-sensitive Gi proteins coupled to PLC and IP3-induced Ca2+ release (reviewed in Refs. [2,3,8]). However, although Bischoff et al. observed that PTX abolished the transient elevation in [Ca2+]i induced by SPC in aortic smooth muscle cells, the smaller sustained elevation was retained [11]. PLC inhibition is also reported to abrogate the response to SPC in mesenteric artery and intact aortic smooth muscle cells [10,11,31]. SPC may also directly induce Ca2+ release from thapsigargin-sensitive stores in permeabilized cells, though the physiological relevance of this is debatable (reviewed in Ref. [8]).

In direct contrast to the above, we found that neither PTX nor inhibition of PLC with U73122 [GenBank] had any effect on SPC-induced vasoconstriction of IPA. Moreover, the profile of the SPC-induced elevation of [Ca2+]i in IPA differed from that induced by agonists such as PGF2{alpha} which do cause IP3-mediated store release, lacking the large transient elevation in [Ca2+]i in the presence of external Ca2+, and showing no significant elevation in its absence (Fig. 3B). Also, thapsigargin in the presence of diltiazem did not alter the elevation in [Ca2+]i elicited by SPC (Fig. 8B). These data strongly suggest that in IPA SPC does not cause Ca2+ release from thapsigargin-sensitive stores, whether via PLC/IP3 or otherwise, and that Ca2+ entry via L-type channels is unimportant. The latter is consistent with the relatively minor effect of diltiazem on SPC-induced vasoconstriction (Fig. 7). We have previously shown that thapsigargin causes complete emptying of IPA intracellular stores, as subsequent application of either agonists (e.g. PGF2{alpha}) or caffeine caused no further release [21].

Our results suggest that SPC activates a Ca2+ entry pathway in rat IPA that does not involve activation of CCE by Ca2+ release from intracellular stores. However, this pathway is sensitive to La3+ and 2-APB (Figs. 7 and 8Go) at concentrations we have previously shown to block CCE in IPA [21]. Although 2-APB was originally reported as an IP3 receptor antagonist, it is now seen as an effective blocker of the channels underlying CCE, probably members of the Trp family [32,33]. The effect of 2-APB on SPC-induced vasoconstriction of IPA cannot be attributed to inhibition of IP3-mediated Ca2+ release as inhibition of PLC was ineffective, there was no significant rise in [Ca2+]i in the absence of extracellular Ca2+, and 10 µM La3+, which does not affect Ca2+ release, was equally effective.

It is notable that a similar activation of Ca2+ influx has been reported for other lysophospholipids, though not in vascular smooth muscle. S1P has been shown to directly activate channels underlying CCE in human neutrophils and HL60 cells, and has been proposed as a "Ca2+ influx factor" linking store release to CCE [16]. The same group also reported that LPA activates a pathway distinct from CCE in the same cells [17], but with similar characteristics to that described here. Although current evidence suggests that 2-APB is a specific blocker of CCE and does not affect receptor or diacylglycerol-gated Ca2+ influx pathways [32,33], the fact that SPC induced the same elevation of [Ca2+]i after depletion of intracellular stores with thapsigargin suggests that channels distinct from those underlying CCE are involved, possibly the same as those reported to be activated by LPA [17]. Guibert et al. [34] have also reported that serotonin activates a Ca2+ influx pathway in pulmonary artery that is distinct from CCE, and mediated via arachidonate-regulated channels. Further studies are required to see whether these are the same as those activated by SPC.

Many voltage-independent Ca2+ entry pathways, including CCE [21], involve non-selective cation channels permeable to Na+, activation of which would cause some depolarisation. This probably explains why diltiazem has a small but significant effect on the response to SPC.

4.4 SPC-induced Ca2+ sensitisation in IPA
A key role for Rho kinase-mediated Ca2+ sensitisation in lysophospholipid-induced vasoconstriction seems common to all arteries (see Ref. [3]), and our data for SPC in rat small IPA are consistent with this. However, IPA exhibit significant basal Rho kinase activity, unlike coronary arteries [15], so the effect of Rho kinase inhibition could potentially be due to loss of basal activity. We have previously shown in IPA that 3 µM Y27632 suppresses 80 mM K+-induced vasoconstriction by ~25% [35]. Even if it is assumed that this was entirely due to inhibition of basal activity (which not certain, [18]), then the inhibition by Y27632 of ~50% of SPC-induced vasoconstriction observed here (Fig. 4) implies that SPC is indeed further activating Rho kinase, consistent with other studies.

It has been suggested that SPC-induced Ca2+ sensitisation in coronary artery is GTP-independent [13,15], implying activation of Rho kinase via a G-protein-independent pathway. However, in permeabilized IPA the response to SPC was suppressed ~70% by GDPβS, strongly suggesting that activation of G-proteins plays a central role, though possibly in addition to a smaller GTP-independent component. It is conceivable that the latter could involve arachidonate, which activates Rho kinase directly [18], an interesting parallel to the discussion on SPC-induced Ca2+ influx and arachidonate-regulated channels.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
Acknowledgments
 References
 
We show for the first time evidence that SPC is a powerful vasoconstrictor in rat small IPA, and that SPC-induced vasoconstriction in these arteries is dependent on activation of a La3+ and 2-APB sensitive Ca2+ entry pathway, and independent of Ca2+ release from thapsigargin-sensitive stores. In contrast to many previous studies, SPC does not exert its effects in IPA via PTX-sensitive Gi-proteins, PLC or L-type Ca2+ channels. Although there is an acknowledged disparity between reported circulating concentrations of lysophospholipids (mid-high nanomole) and those required for vasoconstriction (low micromole) [2,3], it has been strongly argued that lysophospholipids are likely to act in a paracrine or autocrine fashion, with localised concentrations considerably higher than in plasma, especially at sites of thrombus formation, atheroscelerosis and inflammation [2,3,12,13]. It has therefore been suggested that SPC may contribute to the pathogenesis of vascular diseases [3,12,13]; in the light of this study it should be examined whether the same may be true for the pulmonary circulation.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Acknowledgments
References
 
This work was supported by the Wellcome Trust; GDT is a Wellcome Clinical Research Training Fellow (grants 062554, 068160).


   Notes
 
Time for primary review 16 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 Acknowledgments
 References
 

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