Low Concentrations of Sphingosylphosphorylcholine Enhance Pulmonary Artery Vasoreactivity
The Role of Protein Kinase Cδ and Ca2+ Entry
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 PKCδ inhibitor rottlerin, but not by Gö6976, which is ineffective against PKCδ. The potentiation could be attributed to enhancement of Ca2+ entry. SPC also potentiated the responses to prostaglandin F2α 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 PKCδ 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 PKCδ. This has implications for the physiological role of SPC, especially in cardiovascular disease, where SPC is reported to be elevated.
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 Gi 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 inflammation1,2,6,7; 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)δ–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
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 α-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 (F340/380).
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 BKCa 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.
α-Toxin Permeabilization of IPA
Isometric force was recorded in α-toxin–permeabilized arteries, as described previously.4 IPAs were mounted as above, but incubated at 26°C and permeabilized with 60 μg/mL of α-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.
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-PKCδ 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-PKCδ 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; F340/380) were normalized to the response to a 2-minute exposure to KPSS4,17 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).
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 (F340/380) 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).
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 KV 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 PKCδ; 3 μmol/L), and rottlerin (PKCδ; 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 PKCδ, but not endothelium-derived mediators, store release or 2-APB-sensitive Ca2+ entry, RhoK-mediated Ca2+ sensitization, or other PKC isozymes.
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 [F340/380]: 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 α-toxin–permeabilized IPAs constricted to ≈30% of the maximum response with a pCa of 6.7, obviating any contribution from PKC-mediated Ca2+ sensitization.
Effect of 1 μmol/L of SPC on Agonist-Induced IPA Vasoconstriction
SPC (1 μmol/L) caused a significant leftward shift of the PGF2α concentration-response relationship (Figure 5A). PGF2α-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 PGF2α but only reduced and did not abolish the SPC-induced leftward shift of the PGF2α concentration-response relationship (Figure 5B). Consistent with this, and using the sequential challenge protocol shown in Figure 1 but with PGF2α, SPC (1 μmol/L) in the presence of diltiazem potentiated 3 μmol/L of PGF2α-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+] [F340/380]: 212±57%; n=3; Figure 4D), indicating that SPC also potentiates voltage-independent Ca2+ entry.
Repeated challenges with PGF2α showed less consistency than for K+, so to characterize in more detail the SPC-mediated potentiation of the response to PGF2α, we applied SPC during a stable, established PGF2α-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.
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; PGF2α was then applied to induce a constriction of ≈25% KPSS (3 to 10 μmol/L of PGF2α). 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 5⇑B, 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 PKCδ Translocation in Cultured PASMCs
The data obtained in the presence of the 3 PKC inhibitors strongly suggested a role for PKCδ; we, therefore, examined the effect of 1 μmol/L of SPC on PKCδ subcellular localization in PASMCs cultured from IPAs. In untreated cells, PKCδ 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 PKCδ to the perinuclear region, cytoskeleton, and possibly membrane.
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 KV 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 KV currents in IPA smooth muscle cells. This suggests that neither KV 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 α-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 (α, βI, βII, and γ), novel (δ, ε, η, θ, and μ), and atypical (ι and λ) PKC isozymes (IC50 <1 μmol/L), whereas Gö6976 has selectivity for conventional isozymes (IC50 <1 μmol/L) but is relatively ineffective against PKCδ (IC50 ≥30 μmol/L). Rottlerin has a 10-fold greater potency for PKCδ (IC50 ≈1 to 6 μmol/L) than for PKC α, β, and γ and ≈50-fold greater potency for ε, θ, and ζ.19,23 Ro31-8220 and rottlerin abolished the SPC-induced potentiation of vasoreactivity, whereas Gö6976 had no effect, suggesting that PKCδ is a critical component of the signal transduction pathway. Accordingly, 1 μmol/L of SPC induced PKCδ translocation in PASMCs from the nucleus to the perinuclear region, cytoskeleton, and possibly the membrane (Figure 7); activation of PKCδ has been associated previously with a similar subcellular translocation in vascular smooth muscle and cardiomyocytes.24,25
The potentiation of PGF2α-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 PKCδ 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 PKCδ and PLC in pancreatic islet endothelial cells,26 and PKCδ 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 PKCδ by diacylglycerol and PKCδ-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 PKCδ-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.
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.
Sources of Funding
This work was supported by the Wellcome Trust (grants 078075 and 068160) and British Heart Foundation (FS/06/003/20402).
The first 2 authors contributed equally to this article.
- Received November 9, 2007.
- Revision received November 26, 2007.
- Accepted November 29, 2007.
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