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(Hypertension. 2008;51:239.)
© 2008 American Heart Association, Inc.

Original Articles

Low Concentrations of Sphingosylphosphorylcholine Enhance Pulmonary Artery Vasoreactivity

The Role of Protein Kinase C and Ca²⁺ Entry

Vladimir A. Snetkov; Gavin D. Thomas; Bonnie Teague; Richard M. Leach; Yasin Shaifta; Greg A. Knock; Philip I. Aaronson; Jeremy P.T. Ward

From the King’s College London School of Medicine, Division of Asthma, Allergy and Lung Biology, London, United Kingdom.

Correspondence to Jeremy P.T. Ward, Department of Physiology, Henriette Raphael House, King’s College London, Guy’s Hospital Campus, London SE1 1UL, United Kingdom. E-mail Jeremy.ward{at}kcl.ac.uk

	Abstract

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Sphingosylphosphorylcholine (SPC) is a powerful vasoconstrictor, but in vitro its EC₅₀ is 100-fold more than plasma concentrations. We examined whether subcontractile concentrations of SPC (1 µmol/L) modulated vasoreactivity of rat intrapulmonary arteries using myography and measurement of intracellular [Ca²⁺]. SPC (1 µmol/L) had no effect on force or intracellular [Ca²⁺] on its own, but dramatically potentiated constrictions induced by 25 mmol/L [K⁺], such that at 40 minutes, force and intracellular [Ca²⁺] (Fura PE3 340/380 ratio) were increased by 429±96% and 134±26%, respectively. The potentiation was stereospecific, apparent at concentrations >100 nmol/L of SPC, and independent of the endothelium, 2-aminoethoxydiphenylborane–sensitive Ca²⁺ entry, and Rho kinase. It was abolished by the phospholipase C inhibitor U73122, the broad spectrum protein kinase C (PKC) inhibitor Ro31-8220, and the PKC inhibitor rottlerin, but not by Gö6976, which is ineffective against PKC. The potentiation could be attributed to enhancement of Ca²⁺ entry. SPC also potentiated the responses to prostaglandin F₂ and U436619, which activate a 2-aminoethoxydiphenylborane sensitive nonselective cation channel in intrapulmonary arteries. In this case, potentiation was partially inhibited by diltiazem but abolished by 2-aminoethoxydiphenylborane, Ro31-8220, and rottlerin. SPC (1 µmol/L) caused translocation of PKC to the perinuclear region and cytoskeleton of cultured intrapulmonary artery smooth muscle cells. We present the novel finding that low, subcontractile concentrations of SPC potentiate Ca²⁺ entry in intrapulmonary arteries through both voltage-dependent and independent pathways via a receptor-dependent mechanism involving PKC. This has implications for the physiological role of SPC, especially in cardiovascular disease, where SPC is reported to be elevated.

Key Words: pulmonary artery • lysosphingolipids • sphingosylphosphorylcholine • L-type Ca²⁺ channels • protein kinase C

	Introduction

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The lysosphingolipid sphingosylphosphorylcholine (SPC) is an important cardiovascular mediator derived from sphingomyelin and carried by plasma lipoproteins.^1,2 It has been associated with multifarious processes, including proliferation, angiogenesis, inflammation, and vasoconstriction,^1–7 and elevated SPC concentrations may contribute to cardiovascular disease,² including hypercholesterolemia and atherosclerosis,^3,8 cerebral and coronary vasospasm,^6,7 and hypertension.⁹ In systemic arteries, SPC-induced vasoconstriction is reported to be mediated via pertussis toxin–sensitive G_i proteins, inositol triphosphate–induced Ca²⁺ release, Rho kinase (RhoK)–mediated Ca²⁺ sensitization, and Ca²⁺ entry via voltage-dependent L-type channels,^1,2,10 although in some arteries Ca²⁺ sensitization may be the major mechanism.^6,7 In contrast, we reported recently that, in rat small intrapulmonary arteries (IPAs), the SPC-induced elevation of intracellular [Ca²⁺] is primarily because of activation of a voltage-independent Ca²⁺ entry pathway that is insensitive to pertussis toxin and Ca²⁺ release from stores.⁴

A conceptual problem regarding any physiological role for SPC is that the EC₅₀ for most of its effects in vitro is in the range of 7 to 18 µmol/L,^1,2,4–6 whereas plasma concentrations may be as low as 50 nmol/L, although in serum this rises to 130 nmol/L, suggesting release from activated platelets.¹¹ It has been argued that lysophospholipids act in a paracrine or autocrine fashion, with higher local concentrations than in plasma, especially at sites of thrombus formation, atherosclerosis, and inflammation^1,2,6,7; tissue concentrations of 10 µmol/L of SPC are reported for certain types of inflammation.¹² Studies on SPC are hindered by a lack of specific antagonists, and its receptors remain unidentified. SPC is a low-affinity ligand for sphingosine-1-phosphate receptors, but this cannot account for the majority of its actions. Although GPR4, OGR1, and G2A have been proposed as SPC receptors, recent evidence suggests that this family responds to protons and not SPC.^13,14

We considered whether concentrations of SPC insufficient to exert direct vasoactive effects might potentiate vasoconstriction induced by other means, as demonstrated for some other agonists in IPAs,¹⁵ and examined the effects of subcontractile concentrations of SPC (1 µmol/L) on depolarization- and agonist-induced vasoconstriction of rat IPAs. We report the novel and potentially important finding that these low concentrations of SPC substantially potentiate IPA vasoreactivity via a protein kinase C (PKC)–dependent enhancement of both voltage-dependent and independent Ca²⁺ entry and that this mechanism differs from that underlying vasoconstriction induced by higher concentrations of SPC.

	Materials and Methods

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Male Wistar rats (200 to 300 g) were killed by cervical dislocation; the investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996). Small IPAs (third to fourth branch; 150 to 450 µm ID) were mounted in a myograph (Danish MyoTechnology) containing physiological salt solution (PSS) gassed with 95% air/5% CO₂ (pH 7.4) at 37°C, as described previously.¹⁶ Endothelial denudation was achieved by rubbing the lumen and confirmed by loss of relaxation to acetylcholine. Pulmonary artery smooth muscle cells (PASMCs) were dispersed from similar-sized IPAs using collagenase (type XI; 2 mg/mL) and papain (1 mg/mL)¹⁶ and used immediately for electrophysiology or cultured in DMEM containing 10% FCS. PASMCs from passages 3 to 4 were grown on 13-mm coverslips and growth arrested in serum-free medium for 24 hours before use; each cell line was verified as smooth muscle by immunostaining for smooth muscle -actin, calponin, and desmin (Santa Cruz Biotechnology).

Estimation of Intracellular [Ca²⁺]
IPAs were incubated for 1 hour at 37°C in PSS with 4 µmol/L of Fura PE-3/AM followed by washing with PSS. The myograph was mounted on an inverted microscope and microfluorometer (Cairn Ltd). Force was recorded simultaneously with the ratio of emission intensities at >510 nm from excitation wavelengths of 340 and 380 nm (F_340/380).

Electrophysiology
Freshly dispersed PASMCs were studied at 20°C using a whole-cell patch clamp (Axopatch-200c, Axon Instruments Inc). The bath was continuously perfused with HEPES-buffered PSS containing 2 mmol/L of tetraethylammonium to block the BK_Ca current. The pipette solution contained (in mmol/L): KCl, 140; MgCl₂, 2; EGTA, 5; HEPES, 10; MgATP, 2.0; and Li₂GTP, 0.2, with the pH adjusted to 7.2 with KOH. Current-voltage relationships were obtained using a voltage ramp protocol, with a holding potential of –60 mV and a 0.5-second ramp from –90 to +100mV every 5 seconds.

-Toxin Permeabilization of IPA
Isometric force was recorded in -toxin–permeabilized arteries, as described previously.⁴ IPAs were mounted as above, but incubated at 26°C and permeabilized with 60 µg/mL of -toxin at pCa 6.5. IPAs were relaxed with solution containing 10 mmol/L of EGTA before submaximal vasoconstriction was induced by increasing [Ca²⁺] to 200 nmol/L (pCa 6.7) by adjusting the K₂ EGTA:Ca EGTA ratio.

PKC Translocation
Cultured PASMCs were exposed to 1 µmol/L of SPC for 10 minutes before fixation with 4% paraformaldehyde and 4% PEG6000 and permeabilization with 0.1% Triton. Cells were stained with anti-PKC antibody (Santa Cruz Biotechnology) overnight at 4C, followed by Alexa 488–labeled secondary antibody (goat anti-rabbit immunoglobulin, Invitrogen) for 2 hours at room temperature. Coverslips were viewed using a CARVII confocal imager (BD Biosciences) and Zeiss Axovert microscope. Specificity of anti-PKC was confirmed by Western blot; staining was negative with secondary antibody alone.

Solutions and Drugs
PSS contained (in mmol/L): NaCl, 118; NaHCO₃, 24; KCl, 4; CaCl₂, 1.8; MgSO₄, 1; NaH₂PO₄, 0.434; and glucose, 5.56. In experiments where [K⁺] was altered, osmolarity was maintained by equimolar substitution of K⁺ for Na⁺. HEPES-buffered PSS contained (in mmol/L): NaCl, 130; KCl, 4: CaCl₂, 2; MgCl₂, 2; glucose, 5; and HEPES, 10, buffered to pH 7.4 with NaOH. Chemicals were obtained from Calbiochem, except for 2-aminoethoxydiphenylborane (2-APB; Tocris Cookson Ltd), and D-erythro-SPC and L-threo-SPC (Avanti Lipids Inc). Unless otherwise stated, diastereomeric SPC was used in the experiments. Lysosphingolipids were dissolved in 2:1 chloroform:methanol for storage at –80°C. Before use, solvent was evaporated and the residue dissolved in PSS.

Calculations and Statistical Analysis
Changes in force and intracellular [Ca²⁺] ([Ca²⁺]_i; F_340/380) were normalized to the response to a 2-minute exposure to KPSS^4,17 and expressed as the percentage of KPSS. Values for EC₅₀ (IC₅₀) and maximum response were derived from fitting to the Hill equation (Sigmaplot, Systat Software Inc). Results are shown as mean±SEM and compared using paired or unpaired Student’s t test or ANOVA with a Holm-Sidak posthoc as appropriate (SigmaStat, Systat Software Inc).

	Results

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As we reported previously for diastereomeric SPC,⁴ D-erythro- SPC induced vasoconstriction of IPA only at concentrations >1 µmol/L, with a similar Hill coefficient (2.4±0.9), maximum vasoconstriction (88±6% KPSS), and EC₅₀ (7.7±0.2 µmol/L; n=3). L-threo-SPC only elicited constriction at 30 µmol/L (11±4% KPSS; n=3).

Subcontractile Concentrations of SPC and Depolarization-Induced IPA Vasoconstriction
IPAs were challenged with sequential 5-minute applications of PSS containing 21 to 25 mmol/L of [K⁺] to give a rise in tension of 15% of that induced by KPSS. SPC (1 µmol/L) was added to the bath, and the procedure was repeated at 20-minute intervals in the continued presence of SPC. Alone, 1 µmol/L of SPC had no effect on tension and caused only a very small, transient (<3-minute) elevation in [Ca²⁺]_i (1.9±0.7% KPSS; n=13). However, it subsequently caused a dramatic potentiation of the response to depolarization that gradually increased to a plateau at 40 to 60 minutes (Figure 1), such that at 20 minutes the force was increased to 294±47% and [Ca²⁺]_i (F_340/380) to 198±14% (n=7; P<0.01), and at 40 minutes they were increased to 529±96% and 234±26%, respectively (n=6; P<0.001). In the absence of SPC, the response to repeated depolarization was unchanged over the entire experimental period (95±10% of control at 80 minutes; n=4). The SPC-induced potentiation of force was apparent at concentrations >100 nmol/L and was concentration dependent and stereospecific (Figure 2A).

View larger version (15K): [in this window] [in a new window]   Figure 1. A typical experimental trace of simultaneous force and [Ca2+]i (expressed as F340/380) recordings in a small IPA, illustrating the sequential challenge protocol with PSS containing 25 mmol/L of [K+]. Note that 1 µmol/L of SPC had no effect on force or [Ca2+]i on its own but caused a substantial, slowly developing potentiation of the constriction elicited by 25 mmol/L of [K+]. The response to depolarization with KPSS is shown for comparison.

View larger version (17K): [in this window] [in a new window]   Figure 2. A, Concentration dependence of SPC-induced potentiation at 40 minutes of constriction elicited by 25 mmol/L of [K+], showing stereospecificity. Diastereomeric SPC (open bars); D-erythro-SPC (black bars); L-threo-SPC (gray bars). Bars are mean±SEM; n=4 to 5. *P<0.05, **P<0.01 vs control (100%); ##P<0.01 D-erythro-SPC vs L-threo-SPC. B, Cumulative concentration-response relationship for K+ (equimolar substitution for Na+) in IPA (filled circles; EC50: 36±2 mmol/L; maximum: 109±3% KPSS; n=9), and after preincubation with 1 µmol/L of SPC (open circles; EC50: 31±1 mmol/L, P<0.001; maximum: 120±4% KPSS, P<0.001; n=9). Symbols are mean±SEM.

We examined the effects of SPC on the concentration-response relationship to [K⁺] (Figure 2B). Preincubation with 1 µmol/L of SPC for 40 minutes shifted this relationship to the left (EC₅₀: control: 36±2 mmol/L of [K⁺]; SPC: 31±1 mmol/L of [K⁺]; n=9; P<0.001). The maximum response was increased (control: 109±3% of KPSS; SPC: 120±4% of KPSS; P<0.001), indicating enhancement of voltage-gated Ca²⁺ entry and/or increased Ca²⁺ sensitivity rather than depolarization. SPC (1 µmol/L) had no effect on K_V current amplitude in myocytes from IPA, with an unchanged 0 current potential (control: –24±1mV; SPC: –22±1mV; n=5). Direct examination of voltage-activated Ca²⁺ current proved impossible, because this current is small and highly variable in IPA and ran down over the period required (>30 minutes).

Mechanism of SPC-Induced Potentiation of Vasoconstriction
Using the sequential K⁺ challenge protocol shown in Figure 1, we examined the effects of endothelial denudation, 2-APB (75 µmol/L), cyclopiazonic acid (Sarco-endoplasmic reticulum Ca²⁺ ATPase inhibitor; 10 µmol/L), Y27632 (RhoK inhibitor; 3 µmol/L), U73122 (phospholipase C [PLC] inhibitor; 10 µmol/L), and 3 PKC inhibitors, Ro31-8220 (broad spectrum; 3 µmol/L), Gö6976 (conventional and some novel isozymes, but not PKC; 3 µmol/L), and rottlerin (PKC; 1 µmol/L). IPAs were preincubated with inhibitor for 20 minutes and then challenged 4 times at 20-minute intervals with PSS containing 24 mmol/L of [K⁺], with SPC (1 µmol/L) applied during the final 2 challenges. Tension developed during these last 2 challenges was compared with the paired controls, ie, after preincubation with inhibitor but before the addition of SPC.

SPC-induced potentiation of tension was unaffected by removal of the endothelium, 2-APB, cyclopiazonic acid, Y27632, or Gö6976 but was substantially reduced or abolished by U73122, Ro31-8220, and rottlerin (Figure 3). This suggests that the mechanism of potentiation involves PLC and PKC, but not endothelium-derived mediators, store release or 2-APB-sensitive Ca²⁺ entry, RhoK-mediated Ca²⁺ sensitization, or other PKC isozymes.

View larger version (22K): [in this window] [in a new window]   Figure 3. Potentiation of depolarization (25 mmol/L of [K+])-induced vasoconstriction of IPA by 1 µmol/L of SPC measured at 40 minutes and after removal of the endothelium or preincubation with inhibitors. Data were compared with their respective paired controls (100%), ie, after preincubation with inhibitor but before the addition of SPC. Bars are mean±SEM; n=4 to 31. *P<0.05, **P<0.01 vs control; P<0.05, P<0.01 vs SPC.

Quantification of changes in [Ca²⁺]_i in the presence of PKC inhibitors was complicated by their fluorescence, but in 3 experiments we were able to show that Ro31-8220 abolished both the SPC-induced potentiation of tension and the rise in [Ca²⁺]_i (tension: 99±15% control; [Ca²⁺]_i [F_340/380]: 104±20% control; Figure 4A and 4B). PdBu (100 nmol/L), an activator of conventional and novel PKC isozymes, also potentiated the depolarization-induced rise in [Ca²⁺]_i (147±11% control; n=3; P<0.05; Figure 4C). SPC (1 µmol/L) had no detectable effect on tension (n=4) in -toxin–permeabilized IPAs constricted to 30% of the maximum response with a pCa of 6.7, obviating any contribution from PKC-mediated Ca²⁺ sensitization.

View larger version (19K): [in this window] [in a new window]   Figure 4. Representative experimental traces of simultaneous force and [Ca2+]i (expressed as F340/380) recordings, showing the control response superimposed on the response in the same IPA after 40 minutes of incubation with 1 µmol/L of SPC (A, B, and D) or 100 nmol/L of PdBu (C). In A through C, constriction was elicited by PSS containing 23 mmol/L of [K+] and in D by the TP agonist U-46619 (20 nmol/L) in the presence of diltiazem (10 µmol/L).

Effect of 1 µmol/L of SPC on Agonist-Induced IPA Vasoconstriction
SPC (1 µmol/L) caused a significant leftward shift of the PGF₂ concentration-response relationship (Figure 5A). PGF₂-induced vasoconstriction of IPAs is mediated via TP receptors and associated with Ca²⁺ entry via a 2-APB–sensitive voltage-independent pathway and diltiazem-sensitive L-type channels.¹⁷ As expected, diltiazem (10 µmol/L) partially suppressed the response to PGF₂ but only reduced and did not abolish the SPC-induced leftward shift of the PGF₂ concentration-response relationship (Figure 5B). Consistent with this, and using the sequential challenge protocol shown in Figure 1 but with PGF₂, SPC (1 µmol/L) in the presence of diltiazem potentiated 3 µmol/L of PGF₂-induced constriction to 341±125% (n=3) at 40 minutes and U436619 (20 nmol/L)-induced tension and elevation in [Ca²⁺]_i (tension: 563±159%; [Ca²⁺] [F_340/380]: 212±57%; n=3; Figure 4D), indicating that SPC also potentiates voltage-independent Ca²⁺ entry.

View larger version (17K): [in this window] [in a new window]   Figure 5. Cumulative concentration-response relationships for PGF2 in IPAs. The left panel shows the effect of preincubation with 1 µmol/L of SPC (Control, filled circles; SPC, open circles); the right panel shows the response in the presence of 10 µmol/L of diltiazem. In both cases. control and experimental curves were significantly different (–diltiazem: P<0.01, n=4; +diltiazem: P<0.05, n=5; 2-way ANOVA); symbols are mean±SEM; *P<0.01 (Holm-Sidak posthoc).

Repeated challenges with PGF₂ showed less consistency than for K⁺, so to characterize in more detail the SPC-mediated potentiation of the response to PGF₂, we applied SPC during a stable, established PGF₂-induced constriction equivalent to 25% KPSS, with potentiation measured at 10 minutes (Figure 6A). Because of the slowly developing nature of the SPC-induced potentiation, its magnitude using this shortened protocol was smaller than for the repeated challenge protocol but was still highly significant (140±6.2% control; n=12; P<0.001). For comparison, SPC-induced potentiation of K⁺-induced constriction was 165±26% (n=3) at 10 minutes using this protocol.

View larger version (15K): [in this window] [in a new window]   Figure 6. A, Representative trace illustrating the second, shorter protocol where SPC is added on top of an established stable vasoconstriction induced by PGF2. SPC was added once developed force reached a plateau and caused a further increase in force; in the absence of SPC, tension was stable for 10 minutes, as illustrated by the dashed line. B, Increase in PGF2-induced force caused by 1 µmol/L of SPC measured after 10 minutes using the protocol shown in A and after preincubation with inhibitors; 100% represents tension in the presence of inhibitors but before the addition of SPC. Bars are mean±SEM; n=4 to 12. *P<0.05, **P<0.01 vs control; P<0.05, P<0.01 vs SPC.

Using the above protocol we investigated the effects of diltiazem (10 µmol/L), 2-APB (75 µmol/L), Y27632 (3 µmol/L), Ro31-8220 (3 µmol/L), rottlerin (1 µmol/L), and cyclopiazonic acid (10 µmol/L) on the potentiation induced by 1 mol/L of SPC. IPAs were preincubated with inhibitors for 20 minutes; PGF₂ was then applied to induce a constriction of 25% KPSS (3 to 10 µmol/L of PGF₂). Figure 6B shows the effects of these inhibitors on SPC-induced potentiation. Consistent with the data shown in Figure 3 for depolarization-induced constriction, the potentiation was unaffected by Y27632 but substantially reduced or abolished by Ro31-8220 and rottlerin, and, consistent with Figures 4D and 5B, diltiazem significantly reduced the potentiation by 50%, whereas 2-APB abolished it. After constriction with U-46619 (20 nmol/L) in the presence of diltiazem, SPC (1 µmol/L) caused a further rise in both tension (148±10% control; n=6; P<0.01) and [Ca²⁺]_i (184±16%; P<0.01), again confirming that SPC potentiates voltage-independent Ca²⁺ entry.

Effect of 1 µmol/L of SPC on PKC Translocation in Cultured PASMCs
The data obtained in the presence of the 3 PKC inhibitors strongly suggested a role for PKC; we, therefore, examined the effect of 1 µmol/L of SPC on PKC subcellular localization in PASMCs cultured from IPAs. In untreated cells, PKC staining was strongest within the nucleus, with diffuse staining throughout the cytoplasm (Figure 7). In SPC-treated cells, staining in the nucleus was reduced, with translocation of PKC to the perinuclear region, cytoskeleton, and possibly membrane.

View larger version (129K): [in this window] [in a new window]   Figure 7. SPC induced translocation of PKC immunoreactivity in PASMCs from the nucleus to the perinuclear regions (eg, black arrows) and cytoskeleton, with some evidence for translocation to the membrane (eg, white arrows). A and B, control; C and D, treatment with 1 µmol/L of SPC for 10 minutes. B and D are at higher magnification (oil immersion objective) to show detail. Figures typical for 6 to 10 coverslips from each of 6 cell lines derived from different animals; >90% cells demonstrated translocation.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our key finding is that concentrations of SPC insufficient to cause vasoconstriction or elevation of [Ca²⁺]_i in IPA can nevertheless substantially enhance vasoconstriction elicited by other means. Such concentrations (300 to 1000 nmol/L) are >10-fold smaller than the EC₅₀ reported for SPC-induced vasoconstriction, per se, or, indeed, most other actions of SPC (12 µmol/L)^1,2,4–6 and are consequently much closer to those reported for plasma.¹¹ Notably, SPC was stereospecific for the effects elicited by both high and low concentrations, implying that both are receptor mediated (Figure 2A).

The signaling pathways underlying potentiation of IPA vasoreactivity by 1 µmol/L of SPC differ from those underlying vasoconstriction to higher concentrations. We have shown that the latter is mediated by increased RhoK-mediated Ca²⁺ sensitivity and activation of the 2-APB–sensitive, voltage-independent Ca²⁺ entry pathway but is independent of Ca²⁺ release from stores and PLC.⁴ In contrast, the potentiation of depolarization-induced constriction by 1 µmol/L of SPC was unaffected by 2-APB or inhibition of RhoK but was strongly suppressed by inhibition of PLC and PKC (Figure 3).

The related sphingolipid sphingosine-1-phosphate inhibits K_V channels and depolarizes cerebral arteries via a PKC-dependent mechanism.¹⁸ However, although the [K⁺]/force relationship was shifted to the left by 1 µmol/L of SPC, the maximum response was also enhanced (Figure 2B), and there was no effect on K_V currents in IPA smooth muscle cells. This suggests that neither K_V channel inhibition nor depolarization can account for the potentiation of vasoreactivity. Both RhoK and PKC modulate Ca²⁺ sensitivity in the pulmonary artery,¹⁹ but because inhibition of RhoK was without effect, and 1 µmol/L SPC did not alter Ca²⁺ sensitivity of -toxin–permeabilized IPAs, this suggests that the potentiation is mediated entirely via the observed enhancement of the elevation of [Ca²⁺]_i (Figures 1 and 4).

PKC enhances Ca²⁺ entry via L-type channels in smooth muscle²⁰ by increasing channel availability and prolonging open time,^21,22 and there is some evidence for differential effects of conventional and novel PKC isozymes.²⁰ Ro31-8220 has equivalent potencies against conventional (, β_I, β_II, and ), novel (, , , , and µ), and atypical ( and ) PKC isozymes (IC₅₀ <1 µmol/L), whereas Gö6976 has selectivity for conventional isozymes (IC₅₀ <1 µmol/L) but is relatively ineffective against PKC (IC₅₀ 30 µmol/L). Rottlerin has a 10-fold greater potency for PKC (IC₅₀ 1 to 6 µmol/L) than for PKC , β, and and 50-fold greater potency for , , and .^19,23 Ro31-8220 and rottlerin abolished the SPC-induced potentiation of vasoreactivity, whereas Gö6976 had no effect, suggesting that PKC is a critical component of the signal transduction pathway. Accordingly, 1 µmol/L of SPC induced PKC translocation in PASMCs from the nucleus to the perinuclear region, cytoskeleton, and possibly the membrane (Figure 7); activation of PKC has been associated previously with a similar subcellular translocation in vascular smooth muscle and cardiomyocytes.^24,25

The potentiation of PGF₂-induced vasoconstriction was inhibited by diltiazem by only 50%, and unlike that for depolarization-induced vasoconstriction was abolished by 2-APB (Figure 6B). This implies that SPC also potentiates voltage-independent Ca²⁺ entry, and this was found to be the case (see Figure 4D). This is also PKC dependent, because the potentiation was abolished by Ro31-8220 and rottlerin (Figure 6B). The complete block by 2-APB and partial block by diltiazem are consistent with a model whereby TP receptor agonists activate nonselective cation channel, resulting in both Ca²⁺ and Na⁺ entry, with the latter causing depolarization and additional Ca²⁺ entry via L-type channels (see Figure 5 and Reference ¹⁷).

Consistent with our results, other studies have shown that SPC activates PKC and PLC in pancreatic islet endothelial cells,²⁶ and PKC has been reported to mediate the hypoxia-induced enhancement of Ca²⁺ entry via L-type channels in carotid body glomus cells.²⁷ Moreover, SPC has also been reported to potentiate 2-APB–sensitive Ca²⁺ entry in thyroid FRO cells via a PKC- and PLC-dependent mechanism, independent of intracellular Ca²⁺ release or any increase in inositol phosphates.²⁸ SPC-induced vasoconstriction in systemic arteries is in part mediated by PLC/inositol triphosphate–induced Ca²⁺ release,^1,10 although this is not the case for IPA⁴; this reflects our previous finding that activation of PLC/inositol triphosphate–mediated Ca²⁺ release does not couple to vasoconstriction in IPA.¹⁷ Considering the above, we speculate that the potentiation of IPA vasoreactivity elicited by SPC is mediated via a G-protein coupled receptor linked to PLC, with subsequent activation of PKC by diacylglycerol and PKC-dependent phosphorylation of L-type channels and a 2-APB-sensitive nonselective cation channel.

In summary, we report the novel and potentially important finding that subcontractile concentrations (1 µmol/L) of SPC cause significant potentiation of IPA vasoreactivity via a PKC-mediated enhancement of Ca²⁺ entry via both L-type channels and a voltage-independent, 2-APB–sensitive nonselective cation channel. This pathway involves different mechanisms from those shown previously to underlie vasoconstriction induced by higher concentrations of SPC in IPA⁴ and other vascular beds,^1,2,10 possibly implying the existence of >1 G-protein coupled receptor for SPC.

Perspectives
Our results have considerable implications for the pathophysiological role of SPC. Although lysosphingolipids may act in a paracrine or autocrine fashion, with localized concentrations considerably higher than in plasma, especially at sites of thrombus formation, atherosclerosis, and inflammation,^1,2,6,7 the discrepancy between plasma SPC concentrations¹¹ and those required for most of its reported effects is still in the order of 100-fold. The phenomenon that we describe here, however, was apparent at 300 nmol/L and could conceivably be active in healthy tissues and certainly if SPC increases in cardiovascular disease, as suggested.^2,9 This could be of particular importance in the lung, because pulmonary vascular disease is commonly associated with inflammation and microemboli, factors that would be expected to increase SPC. However, the phenomenon does not seem to be limited to IPA, because in preliminary experiments we have found that it also occurs in a range of small systemic arteries. As SPC potentiates the response to TP agonists and is released from activated platelets,¹¹ an intriguing speculation is that this phenomenon could also contribute to the initial vasoconstrictor response associated with hemostasis and platelet-derived thromboxane A₂.

	Acknowledgments

 
Sources of Funding

This work was supported by the Wellcome Trust (grants 078075 and 068160) and British Heart Foundation (FS/06/003/20402).

Disclosures

None.

	Footnotes

 
The first 2 authors contributed equally to this article.

Received November 9, 2007; first decision November 26, 2007; accepted November 29, 2007.

	References

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