Involvement of Src Family Protein Tyrosine Kinases in Ca2+ Sensitization of Coronary Artery Contraction Mediated by a Sphingosylphosphorylcholine-Rho-Kinase Pathway
We recently reported that sphingosylphosphorylcholine (SPC) is a novel messenger for Rho-kinase–mediated Ca2+ sensitization of vascular smooth muscle (VSM) contraction. Subcellular localization and kinase activity of Src family protein kinases (SrcPTKs), except for c-Src, is controlled by a reversible S-palmitoylation, an event inhibited by eicosapentaenoic acid (EPA). We examined the possible involvement of SrcPTKs in SPC-induced Ca2+ sensitization and effects of EPA. We used porcine coronary VSM and rat aortic VSM cells (VSMCs) in primary culture. An SrcPTKs inhibitor, PP1, and EPA inhibited SPC-induced contraction, concentration-dependently, without affecting [Ca2+]i levels and the Ca2+-dependent contraction induced by high K+ depolarization. A digitized immunocytochemical analysis in VSMCs revealed that SPC induced translocation of Fyn, but not of c-Src, from the cytosol to the cell membrane, an event abolished by EPA. Translocation of Rho-kinase from the cytosol to the cell membrane by SPC was also inhibited by EPA and PP1. The SPC-induced activation of SrcPTKs was blocked by EPA and PP1, but not by Y27632, an Rho-kinase inhibitor. Rho-kinase–dependent phosphorylation of myosin phosphatase induced by SPC was inhibited by EPA, PP1, and Y27632. Translocation and activation of SrcPTKs, including Fyn, play an important role in Ca2+ sensitization of VSM contractions mediated by a SPC-Rho-kinase pathway.
- Ca2+ sensitization
- Src family protein tyrosine kinase
- eicosapentaenoic acid
Sphingosylphosphorylcholine (SPC) generated by N-deacylation of sphingomyelin, one of the most abundant lipids in cell membrane, is a phospholipid mediator in blood plasma and has a physiological role in regulation of the heart.1 LDL and other lipoproteins function as carriers for SPC thereby contributing to the development of coronary artery disease.2 We recently reported that SPC is a novel messenger for Ca2+ sensitization of cerebral vascular smooth muscle (VSM) contraction, which was completely abolished by Rho-kinase inhibitor or a dominant-negative Rho-kinase.3,4⇓ It was proposed that the SPC-induced Ca2+ sensitization might play an important role in the pathogenesis of vasospasm.5 The potentiation of Ca2+ sensitivity of the contractile apparatus in VSM is associated with vasospasm that plays an important role in the pathogenesis of ischemic heart diseases and cerebral vascular diseases.6–9⇓⇓⇓ Evidence for the involvement of a Rho/Rho-kinase pathway and its inhibition of myosin phosphatase in the Ca2+ sensitization of VSM contraction has been reported.10–12⇓⇓ Rho-kinase inhibits myosin phosphatase by phosphorylation at Thr695 of myosin phosphatase target subunit 1 (MYPT1).13
Src family protein tyrosine kinases (SrcPTKs) are involved in the Gα12-Rho/Rho-kinase pathway in HEK293 cells.14 Cellular redistribution of PKC, Rho-kinase, and other kinases plays an important role in Ca2+ sensitization.12 The N-terminal sequence of SrcPTKs was found to associate with the inner leaflet of cell membranes and dual fatty acylation with myristate at Gly2 and palmitate at Cys3 (and Cys5 or Cys6) regulates the membrane binding and subcellular distribution of SrcPTKs.15,16⇓ As S-palmitoylation is a reversible modification, it may play an important role in the biological activity, including membrane association and kinase activity.17 It was reported that polyunsaturated fatty acids (PUFAs), particularly the n-3 series, eicosapentaenoic acid (EPA), inhibit T-cell signal transduction by displacing Fyn, a member of SrcPTKs, from rafts.18 Other investigators reported that Fyn plays an important role in angiotensin II–induced tyrosine phosphorylation and nuclear translocation of STAT1 in VSM cells (VSMCs).19,20⇓
We asked if SrcPTKs may be involved in Rho-kinase–mediated Ca2+ sensitization of VSM contraction induced by SPC and if so, to investigate effects of EPA on the translocation and kinase activation of SrcPTKs.
Materials and Methods
SPC was purchased from Biomol. EPA, docosahexaenoic acid (DHA), and 2-bromopalmitate were from Sigma. 4-Amino-5-(methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) was from Alexis. 4-Amino-7-phenylpyrazolo[3,4-d]pyrimidine (PP3) was from Calbiochem. Y27632 was generous gift from Welfide, Osaka, Japan.
Force Recording of VSM
All procedures were approved by the Institutional Animal Care and Use Committee and were conducted in conformity with institutional guidelines. Porcine coronary arteries (20 to 30 mm from the origin of the proximal portion of left anterior descending arteries) were obtained from a local abattoir.21 Tissue specimens were placed in ice-cold physiological salt solution (PSS; in mmol/L: 123 NaCl, 4.7 KCl, 15.5 NaHCO3, 1.2 KH2PO4, 1.2 MgCl2, 1.25 CaCl2, and 11.5 D-glucose) and transported to our laboratory. The arteries were cut into strips (1×4 mm) without endothelium and adventitia.3 These strips were mounted vertically at the organ bath filled with PSS, gassed with 5% CO2/95% O2, and maintained at 37°C. A force-transducer (TB-612T, Nihon Koden) was used to measure isometric force.3 Effects of some drugs on the force were examined at the maximum and steady state of the precontraction induced by 30 μmol/L SPC or 40 mmol/L K+ PSS.
Simultaneous Measurement of [Ca2+]i and Force of VSM In Situ
Changes in [Ca2+]i and force were simultaneously measured using porcine coronary arterial strips, as described.3 Briefly, the medial strips of porcine coronary arteries were loaded with 12.5 μmol/L fura-2/AM (Dojindo). Changes in [Ca2+]i were continuously monitored with fluorescence intensities (510 nm) at alternating 340 nm (F340) and 380 nm (F380) excitation and their ratio (F340/F380), using a spectrofluorometer (CAM-230, Japan Spectroscopic) equipped with a randomized optical fiber system.
Western Blot Analysis
To detect Fyn, c-Src, and ROKα, porcine coronary arterial and rat aortic media were freeze-fractured and taken up in sample buffer. Protein extract was separated by 10% SDS-PAGE and subjected to immunoblotting with anti-Fyn, anti-c-Src (Santa Cruz), or anti-ROKα, (Transduction Laboratories) antibodies, and the signals were visualized using enhanced chemiluminescence (Amersham). With only secondary antibodies (without primary antibodies), signals indicating the existence of each protein were not detectable.
To detect phosphorylation of SrcPTKs and MYPT1, porcine coronary arterial media were freeze-fractured and taken up in ice-cold gentle lysis buffer containing (in mmol/L) 25 Tris, pH 7.5, 10% glycerol, 1% Nonidet P-40, 140 NaCl, 4 benzamidine, 1 4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride, 2 Na3VO4, and 10 mg/mL aprotinin. Lysates were equalized for protein concentrations using BCA Protein Assay kits (Pierce), separated by 10% SDS-PAGE, and subjected to immunoblotting with anti-phospho-Src(Y416) antibody (Upstate Biothechnology)22 or pM133T695 (a generous gift from Dr Masaaki Ito).13 The signals were visualized as above and analyzed by scanning the films using a flatbed office scanner and evaluating intensity and the program NIH Image 1.62.
Primary Culture of VSMCs
VSMCs in primary culture were obtained from the aortic media of male Wistar rats weighing 200 to 250 g, as described.23–25⇓⇓ Briefly, the aortic media were dispersed into single cells by incubation with collagenase type I (1 mg/mL) and elastase (10 U/mL) and seeded on a gelatin-coated low-fluorescence cell desk (Sumitomo Bakelite) in DMEM containing 10% heat-inactivated fetal bovine serum and antibiotics. All cells were synchronized in the G0 phase by culturing in serum-free medium for 24 hours before starting each experiment and were not contaminated with fibroblasts or endothelial cells.23–25⇓⇓
Primary-cultured rat VSMCs were serum-starved in DMEM for 24 hours before drug stimulation. After stimulation, cells were immunostained with primary antibodies (anti-Fyn, anti–c-Src, or anti-ROKα), as described.4,26⇓ Laser confocal microscope (LSM 510, Axiovert 100 mol/L; Carl Zeiss) equipped with an objective lens (Plan-Neofluar 40×/0.75 Ph2; Carl Zeiss) and appropriate filter combination (HFT488 and LP505; Carl Zeiss) was used to observe the staining. For each isolated cell, the ratios of signals on cell membrane and cytosol (without nucleus and perinuclear region),12,27⇓ on the 8 lines per cell, were measured by using the NIH image version 1.62 image analysis system. The mean of the 8 ratios (M/C ratio) was used to rule out intracellular bias and M/C ratio was compared with cancel the intercellular bias. Using this method, we previously demonstrated and digitized the subcellular localization of Rho-kinase in VSMCs.4 The surface plot is a bird’s-eye view and a method that shows the distribution of density more clearly. However, in some cases a bird’s-eye view would give us the apparent and slight rotation of the cells, without affecting the cell shape per se.
Differences were analyzed by analysis of variance followed by Fisher’s protected least significance test for multiple samples comparisons. A value of P<0.05 was considered to be statistically significant.
Effect of SrcPTK Inhibitor on SPC-Induced Ca2+ Sensitization
We investigated the involvement of SrcPTKs in the SPC-induced Ca2+ sensitization of VSM contraction, using a specific inhibitor of SrcPTKs, PP1 and its inactive analogue, PP3 (Figures 1A through 1C). PP1 (20 μmol/L), but not PP3 (20 μmol/L), abolished the SPC-induced contraction, without affecting the accompanying slight elevation of [Ca2+]i (Figures 1B through 1D and 2⇓A), whereas PP1 had no apparent effect on [Ca2+]i elevation and contraction induced by 40 mmol/L K+ depolarization, a typical Ca2+-dependent contraction (Figure 1E). PP1 inhibited the SPC-induced contraction in a concentration-dependent manner (Figure 2B). PP1 (20 μmol/L) inhibited the VSM contraction induced by the vasoconstrictor, U46619 (100 nmol/L), which is known to activate Rho-kinase (data not shown).
Effect of Acylation Inhibitor on SPC-Induced Contraction
Protein acylation is an important mechanism for translocation and activation of SrcPTKs.17 The palmitate derivative, 2-bromopalmitate, inhibited Fyn fatty acylation in general and S-palmitoylation, with some degree of specificity.28 Therefore, we investigated the effect of 2-bromopalmitate on the SPC-induced contraction. 2-Bromopalmitate (100 μmol/L) abolished the SPC-induced contraction (Figures 3A and 4⇓A).
Effect of PUFAs for SPC-Induced Ca2+ Sensitization
It was reported that EPA displaced SrcPTKs from the cell membrane as an inhibitor of S-palmitoylation of SrcPTKs.18,28⇓ Compared with 2-bromopalmitate, EPA does not affect N-myristoylation but does specifically inhibit S-palmitoylation.28 EPA (60 μmol/L) more rapidly abolished the SPC-induced contraction than DHA (60 μmol/L), without affecting the accompanying slight elevation of [Ca2+]i (Figures 3B through 3D and 4⇑A), whereas EPA had no effect on the [Ca2+]i elevation and contraction induced by 40 mmol/L K+ depolarization (Figure 3E). EPA inhibited the SPC-induced contraction in a concentration-dependence manner (Figure 4B). In addition, the SPC-induced contraction was not affected by another PUFA, arachidonic acid (60 μmol/L), and EPA (60 μmol/L) also inhibited VSM contraction induced by the vasoconstrictor, U46619 (100 nmol/L) (data not shown). A blocker of fatty acid translocator, 4,4′-diisothiocyanostilbene-2,2′-disulfonate blocked the relaxing effect of EPA (data not shown), thereby supporting the intracellular action of EPA. The trans isomer and the ethylester form of EPA (supplied by Drs M. Soma and T. Yano, Mochida Pharmaceutical Co, Ltd, Gotemba, Japan) had no significant effect on the SPC-induced contraction, indicating the structural stereo-specificity of the EPA effect (data not shown).
Translocation of SrcPTK and Rho-Kinase
Although the expression of most SrcPTKs is restricted to hematopoietic cells, among them only Fyn and c-Src are widely expressed.29–31⇓⇓ We confirmed the expression of Fyn, c-Src, and ROKα proteins in porcine and rat VSM in a Western blot analysis (Figure 5). Subsequently, translocation of Fyn and c-Src in primary-cultured rat VSMCs was examined using a digitized immunocytochemical analysis of confocal images (Figure 6). VSMCs were used because the abundant extracellular matrix in vascular tissue prohibited a good confocal image of each VSMC. SPC (30 μmol/L, 5 minutes) increased the M/C ratio of Fyn, but not of c-Src, indicating that SPC induces the translocation of Fyn, but not of c-Src, from the cytosol to the cell membrane in VSM (Figure 6). Preincubation with EPA (60 μmol/L, 15 minutes) abolished the elevation of M/C ratio of Fyn by SPC. In contrast, EPA had no apparent effect on the subcellular localization of c-Src and was not affected by SPC (Figures 6B and 6C). As shown in Figure 7, SPC (30 μmol/L) induced translocation of Rho-kinase from the cytosol to the cell membrane and this event was abolished by PP1 (20 μmol/L) and EPA (60 μmol/L), but not by PP3 (20 μmol/L).
Kinase Activities of SrcPTKs and Rho-Kinase
On activation, SrcPTKs autophosphorylate the site corresponding to Tyr416 of c-Src.22 Activated Rho-kinase phosphorylates Thr695 of MYPT1 and thereby inhibits the phosphatase activity.13 Therefore, we evaluated SrcPTKs and Rho-kinase activities in porcine coronary arterial media, using Western blotting and antibodies against SrcPTKs phosphorylated at the site corresponding to Tyr416 of c-Src (anti–phospho-SrcY416) and MYPT1 phosphorylated at the site of Thr695 (pM133T695), respectively (Figure 8). SPC (30 μmol/L) induced activations of SrcPTKs and Rho-kinase, which were inhibited by EPA (60 μmol/L) and PP1 (20 μmol/L), but not by PP3 (20 μmol/L). Thus, EPA inhibits both Fyn translocation and SrcPTKs activation induced by SPC. In contrast, a Rho-kinase inhibitor, Y27632 (10 μmol/L), inhibited the activation of Rho-kinase, but not of SrcPTKs, as induced by SPC (Figure 8).
The present study demonstrated that an SrcPTK inhibitor and EPA inhibited the Ca2+-independent contraction induced by SPC, and translocation and activation of SrcPTKs, including Fyn, and Rho-kinase. An Rho-kinase inhibitor did not affect the activation of SrcPTKs by SPC.
PP1, a pyrazolopyrimidine derivative is a potent and specific inhibitor of SrcPTKs that competes with ATP to prevent phosphorylation and is selective for SrcPTKs; its affinity for SrcPTKs is >50 times, >1000 times, and >20000 times higher than that for EGF-receptor kinase, JAK2, and ZAP-70, respectively.32 The concentration of PP1 (20 μmol/L) used in the present study was within the range of concentrations previously used for the inhibition of Fyn in cultured rat VSMCs stimulated by angiotensin II19,20⇓ and for the concentration-dependent inhibition of Fyn in vitro.22 In the present study, PP1, but not PP3, inhibited SPC-induced Ca2+-independent contractions, ROKα translocation, and Rho-kinase activation (Figures 1B, 1C, 2, 7, and 8⇑⇑⇑). Because PP1 had no effect on the [Ca2+]i levels and the high K+ depolarization–induced contraction and [Ca2+]i elevation (Figures 1E), PP1 does not affect any step of the signal transduction cascade for VSM contraction, namely the calmodulin-MLCK-myosin ATPase pathway. These results suggest that SrcPTKs are involved in the Ca2+-independent contraction of VSM mediated by the SPC-Rho-kinase pathway. We propose that SrcPTKs function as a mediator for the Rho-kinase–regulating Ca2+ sensitization. It was reported that angiotensin II rapidly increased VSMCs c-Src activity and induced a biphasic [Ca2+]i response, and that the initial [Ca2+]i transient was attenuated by the SrcPTK inhibitor, PP2, but not by PP3.33 In contrast, in the present study, PP1 had no effect on [Ca2+]i elevation. The reason for this discrepancy is unknown. The two experimental systems are essentially different with respect to the sample (cultured cell line or vascular tissue), the stimulator (angiotensin II or SPC), and application of the inhibitor (before or after incubation).
All of the inhibitors showed the complete inhibition of the SPC-induced contraction, albeit at different rates. In Figure 4A, the averaged values were calculated at the time point of 15 minutes after the application of inhibitors for demonstration of the rate difference. In addition, in Figure 4A, the control decline of the force was also plotted and used for the statistical analysis of the differences between the control and the treatments of the inhibitors, because the control SPC-induced contraction showed very slow decline of the force in the relatively long observation. However, the sustained levels of the SPC-induced contractions have maintained the higher levels for the longer period of time (at least two hours). Such a long contraction may be compatible with clinically observed vasospasm.
Although the expression of most SrcPTK members is restricted to hematopoietic cells, Fyn and c-Src are widely expressed in various cell types and have functional redundancy.29–31⇓⇓ Whether or not other SrcPTKs are present and active in VSM is unknown. Fyn is palmitoylated on its Cys3/6 residue, whereas c-Src does not include Cys3/6 and therefore cannot be palmitoylated. Although N-myristoylation is a chemically stable amide bond,34,35⇓ S-palmitoylation is a reversible modification and therefore plays a modulatory role in protein function, membrane association, protein-protein interaction, and signal transduction.18,28⇓ 2-Bromopalmitate is an inhibitor of Fyn fatty acylation with some specificity for S-palmitoylation.28 In the present study, 2-bromopalmitate inhibited the SPC-induced VSM contraction (Figures 3A and 4⇑A), which suggests that protein acylation may also be involved in SPC-induced Rho-kinase–mediated Ca2+ sensitization.
It was reported that PUFAs have various common physiological effects.36 Arachidonic acid, n-6 PUFA, activates isolated Rho-kinase and contracts permeabilized smooth muscle fibers.37 In the present study, EPA inhibited the SPC-Rho-kinase pathway and the SPC-induced VSM contraction. It was reported that EPA, n-3 PUFAs, is an inhibitor of Fyn S-palmitoylation.18,28⇓ A discrepancy between the effects of arachidonic acid and those of EPA was apparent and may be based on differences in molecular structures because of the position of unsaturated double bonds. It is possible that the PUFAs may alter the lipid architecture of the membrane, thereby nonspecifically inducing the translocation of SrcPTKs from rafts. However, we have recently shown that caveolin, another palmitoylated protein localized to the inner leaflet of the rafts, is not displaced from this fraction in response to PUFA treatment.26 Moreover, PUFAs do not translocate other raft proteins, such as CD48, CD59, and GM1.18 These data suggest that PUFA-induced translocation of Fyn from rafts is primarily due to specific inhibition of fatty acylation rather than to a nonspecific effect on raft structure. In the present study, because EPA had no effect on the [Ca2+]i levels and the high K+ depolarization–induced contraction and [Ca2+]i elevation (Figures 3E), EPA does not affect the calmodulin-MLCK-myosin ATPase pathway. In the present study, EPA inhibited the SPC-induced Ca2+ sensitization and membrane localization of Fyn and ROKα induced by SPC (Figures 3, 4, 6, and 7⇑⇑⇑). It was reported that EPA inhibited to a greater extent Fyn translocation than did DHA,18 and this was also observed in the present study (Figures 3B, 3C, and 4⇑A). Although the activation of SrcPTKs requires S-palmitoylation17 and may be linked to translocation out, we measured kinase activities of Fyn and Rho-kinase to confirm this notion (Figure 8). PP1 and EPA inhibited the activation of SrcPTKs and Rho-kinase, as induced by SPC. Y27632 does not affect the activation of SrcPTKs induced by SPC. PP1 and EPA inhibited SrcPTKs activation and thereby diminished the activation of a downstream effecter, Rho-kinase, as induced by SPC.
Negative staining of Fyn in the nucleus was frequently observed in the EPA treatment. The mechanism for this has not been examined in the present study. However, EPA is known to inhibit the activation of a ligand-activated transcription factor, peroxisome proliferator-activated receptor (PPAR),38 and PPAR is linked to Src family tyrosine kinases in vascular smooth muscle39 and cardiac muscle contractility.40 Taken together, the negative staining of Fyn in the nucleus may be related to muscle relaxation, which may be mediated by the inhibition of PPAR by EPA. This notion is currently under the investigation.
By way of summary, a SrcPTKs kinase-inhibitor, PP1, and a SrcPTKs translocation-inhibitor, EPA, inhibited Ca2+ sensitization of VSM contraction mediated by a SPC-Rho-kinase pathway and translocation and activation of SrcPTKs, including Fyn, whereas Rho-kinase inhibitor Y27632 has no effect. EPA and PP1 inhibited translocation and activation of the Rho-kinase induced by SPC. Therefore, SrcPTKs, including Fyn, may act as an upstream regulator of Rho-kinase in Ca2+-independent contractions. Finally, the present study may provide insight into possible molecular targets to prevent vasospastic diseases, as related to Ca2+ sensitization.
This work was supported in part by Grants in Aid for Scientific Research from the Ministry of Education, Science, Technology, Sports and Culture of Japan. We thank Drs M. Soma and T. Yano for helpful advice and for their supplying the trans isomer and the ethylester form of EPA, and R. Kimura for secretarial assistance. Y27632 was a generous gift from Welfide Corporation (Osaka, Japan).
Original received March 25, 2002; resubmission received August 19, 2002; revised resubmission received October 10, 2002; accepted October 10, 2002.
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