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Sphingosine-1-phosphate activation of TRPC5 in vascular smooth muscle cells

Sphingosine-1-phosphate activation of TRPC5 in vascular smooth muscle cells Volume 1 † Number 2 † June 2008 10.1093/biohorizons/hzn013 ......................................................................................................................................................................................................................................... Research article Sphingosine-1-phosphate activation of TRPC5 in vascular smooth muscle cells Samantha Fahy University of Leeds, Leeds, UK. Supervisor: Prof. David Beech, Institute of Membrane and Systems Biology, University of Leeds, Leeds, UK. ........................................................................................................................................................................................................................................ Calcium signalling is a complex and diverse system utilized in many cellular processes and in the transmission of cellular information. A number of transient receptor potential (TRP) proteins have been identified in humans and other mammals; these proteins are impli- cated as having a role in calcium signalling. TRPC5 is a member of this protein family which combines with TRPC1 to form non-selective cation channels in human saphenous vein cells, a type of smooth muscle cell. The exact function of TRPC5 remains elusive, however, it can be activated by sphingosine-1-phosphate (S1P), an endogenous signalling phospholipid involved in SMC migration. 2þ The aim of these experiments was to investigate the effects of S1P on the intracellular calcium concentration, [Ca ] , in HSV cells, utilizing dominant-negative (DN)-TRPC5 transfected cells to establish the role played by TRPC5 in this response. A secondary aim was to establish the effect of SMC migration on the above response parameters to S1P using the HSV scratch assay, where a 2 mm line of cells was scraped away from the surface of a glass cover slip and the remaining cells incubated for 24 h. Concurrent with the literature, S1P evoked a significant response in HSV cells (n¼23; P¼0.001). The baseline was significantly lower in the DN-TRPC5 cells compared with the control cells (P,0.001), and the maximum response in the DN-TRPC5 transfected HSV cells reached only 60% of the maximum response in control cells. This suggested that TRPC5 was involved with maintaining basal [Ca2þ] levels and indicated the proportion of the response for which TRPC5 was responsible. The response to S1P was significantly larger in migrated (n¼7) compared with static (n¼11) HSV cells (P¼,0.001) and this response was delayed by 2.3 min; the baseline was also higher in the latter group. This suggested a functional change in the cell following migration that may have been attributable to TRPC5, for example, channel up-regulation. In conclusion, TRPC5-like channels are responsible for a proportion of the S1P response and are implicated in SMC migration. This highlights potential pharmacological targets for the treatment of atherosclerosis, neointimal hyperplasia and coronary heart disease. Key words: TRPC5, S1P, saphenous, Fura-2, calcium. ........................................................................................................................................................................................................................................ TRP proteins are divided into six families; it is the fifth Introduction member of the canonical TRP proteins (TRPC5) that will be the main focus here. Sossey-Alaoui et al. cloned and Calcium signalling is a complex and diverse system, part of characterized human TRPC5 from the encoding gene many cellular processes and the transmission of cellular located on the X chromosome (Xq23). They identified the information. The main channels permeable to these ions in protein as TRPC5; a novel member of the TRP protein mammalian cells include stretch-activated channels (SACs) family. and voltage-, receptor- and store- operated channels TRPC5 proteins assemble as homomeric or heteromeric (VOCs, ROCs and SOCs, respectively). However, relatively tetratamers around a central pore, permeable to cations. little is known about the mechanisms that underlie and TRPC5-like channels can be activated in a number of ways unify this process. A number of transient receptor potential including via receptors. For example, a G-protein coupled (TRP) proteins have been identified in humans and other receptor, such as the endogenous muscarinic M2/3 acetyl- mammals; these proteins are implicated as having a role choline receptor, is activated by an agonist causing in calcium signalling. ......................................................................................................................................................................................................................................... 2008 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 92 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... dissociation of the Gbg subunit from the Ga subunit. This a number of vascular diseases including those putatively subunit activates phospholipase (PL) Cb which causes the linked to TRPC1 channel upregulation: atherosclerosis, hydrolysis of the membrane-bound phosphatidylinositol neointimal hyperplasia and coronary heart disease. bisphosphate (PIP ) into the lipophylic diacylglycerol While S1P application may cause cellular responses such (DAG) and the hydrophilic inositol triphosphate (IP ). The as changes in calcium concentration and cell migration, first of these components remains within the membrane, these responses may not solely be caused by activation of where it is involved in the activation and translocation of a TRPC5-like channels. Xu et al. utilized a dominant- number of proteins including protein kinase (PK)C. The negative (DN) transfection to impair the function of second component travels through the cytoplasm to IP TRPC5. This allowed for a comparison to be made 2þ receptors (IP Rs) located on intracellular Ca stores; the between the wild-type (or mock-transfected) proteins and endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) the DN-transfected proteins; where the apparent loss of cel- in muscle cells. Activation of these receptors leads to lular response was representative of the contribution to the 2þ release of these Ca stores which putatively leads to the cellular effect made by TRPC5-like channels. The opening of SOCs. Zeng et al. described TRPC5 channels TRPC5-DN transfect is a triple alanine mutation of the con- can be activated by a multiplicity of signals in human served sequence of residues leucine, phenylalanine and embryonic kidney (HEK)293 cells; TRPC5 may be activated tryptophan (LFW) located in the channel pore which as an ROC, as an SOC, by external ionic activation and by results in impaired channel function. 2þ intracellular Ca . The literature proposes a number of different methods 2þ The exact function of TRPC5 is yet to be defined; evidence that are used in investigating TRPC5’s involvement in Ca 2þ suggests a number of possibilities. For example, the location signalling. Changes in intracellular Ca concentration, 2þ of the gene encoding this protein is associated with certain [Ca ] , can be measured using conventional fluorescence mental retardation disorders which may suggest a further videomicroscopy and high-throughput, automated fluoro- role. This group also suggested its importance in the basic metric imaging. Conventional imaging, as it will be referred 2þ developmental process and polymorphisms of the encoding to here, utilizes videomicroscopy and Ca sensitive dyes to gene might hinder this development, leading to the afore- measure and visualize these changes. A xenon light source in mentioned disorders. Alternatively, Greka et al. conjectured a monochromator is used to select the appropriate wave- that TRPC5 homomer channels have a role in hippocampal lengths for the fluorescent dye used. Temporally controlled growth cone morphology and motility. ratiometric imaging exploits the intrinsic properties of duel This involvement in cell motility has also been suggested wavelength ratiometric dyes. for TRPC5 heteromer (TRPC5/TRPC1) channels located Fura-2 acetoxymethyl ester (AM) is a dual-wavelength 5 2þ 7 in vascular SMCs. Sphingosine-1-phosphate (S1P), an ratiometric Ca indicator excited by UV light. Unlike endogenous signalling phospholipid, has been demonstrated Fura-2 sodium and potassium salts, the AM can passively to have a role in the migration of vascular SMCs. Xu diffuse across cell membranes. The esters are then cleaved et al. identified S1P as a novel activator of heteromeric by intracellular esterases and the membrane-impermeant TRPC1/TRPC5 and homomeric TRPC5 channels. S1P con- Fura-2 remains in the interior of the cell. Fura-2 is a divalent centrations 1, 3 and 10 mM evoked graded responses in metal ion chelator that has a high affinity for and binds rever- 2þ HEK293 cells within a 5-min activation period; the median sibly to Ca at physiologically relevant concentrations; 3 mM was selected for further experiments. As has been pre- 100 nm. To a lesser extent, Fura-2 may also bind to 2þ viously mentioned, this group conjectured a mechanism for other bivalent cations such as Mg leading to quenching vascular SMC motility, controlled by TRPC5 that was of the fluorescent signal. Complexation of Fura-2 with 2þ evoked by S1P. Ca causes a conformational change in Fura-2 that is Xu et al. used human saphenous vein (HSV) cells, a type detected through the ratio of the fluorescence intensities at of SMC obtained from the saphenous vein in the leg in prep- the excitation wavelengths 340 and 380 nm; this indicates 2þ aration for coronary artery bi-pass surgery. These cells can the [Ca ] . These experiments are carried out under dark be cultured to produce a high-throughput and cost-effective conditions so as to prevent bleaching of the fluorescent in vitro simulation of physiological conditions. The so-called dye. Frequent illumination of the dye may also lead to ‘scratch assay’ was used by this group to study SMC bleaching; furthermore, bleaching may give false negative 2þ migration; where a line of cells is scraped away from the results as it may appear that the [Ca ] is decreasing when plated monolayer of cultured HSV cells. Over a 24-h it is actually the fluorescent signal that is decreasing. period, the surrounding cells migrated into this scratch; There are several advantages of using such ratiometric these cells were then identified using fluorescence video- dyes, such as Fura-2, over single-excitation dyes, such as microscopy and their responses to S1P compared with Flou 3. For example, the intensity of the fluorescence of 2þ static cells from the same monolayer. It follows that single excitation dyes is dependent upon the [Ca ] while TRPC5 may be key to the understanding and treatment of the ratio of the spectral shift produced by ratiometric dyes ......................................................................................................................................................................................................................................... 93 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... 2þ is proportional to [Ca ]. Therefore, the exact concentration combination with a monochromator (Till Photonics, of the dye does not need to be known as it will not affect the Germany), which selected the excitation wavelength pro- experimental outcome. vided by a xenon arc lamp. Cells were continuously perfused The principle aim of my experiments was to investigate the with SBS at a rate of 2ml/min; following the establishment effects of S1P on TRPC5 and TRPC5-like channels in HSV of a stable baseline with SBS, the perfusion was changed to cells. The use of DN-TRPC5 transfected SMC cells would 3 mM S1P (Sigma, UK), applied at the same rate for the be used identify the components of the S1P response for below specified period of time. Cells in the scratch assay which TRPC5 is responsible in the cells. A secondary aim were identified as migrated or static depending on their of this experiment was to establish the effect, if any, of spatial positioning to the visible scratch area. Emissions SMC migration on the above response parameters to S1P. were collected by a 510 nm filter and an image was taken at each of the excitation wavelengths via a CCD camera (Orca ER; Hamamatsu, Japan) at 10-s intervals. Imaging Methods and materials was controlled using Openlab 4 software (Image Processing & Vision Company Ltd, UK). Cell culture As approved by the Leeds teaching hospital research ethics High-throughput fluorometric imaging committee, HSV tissue samples were collected anonymously At a confluency of 60%, cells were seeded into clear 96-well from patients at Leeds General Infirmary. These patients plates. Following a 24-h incubation period, cells were loaded were undergoing open heart surgery and had given their with 2 mM Fura-2 AM in SBS for 1 h at 378C before being informed consent. Tissue samples were transported to the manually washed three times with SBS. SBS was replaced in laboratory in Hanks’ solution (see ‘Materials’) and the each well in varying volumes according to the protocol 2þ median layer of the tissue was extracted by dissection on being followed. Changes in [Ca ] in response to application the day of removal from the host. These cells were then cul- of various substances were read in a 96-well plate FlexStation tured in 75 cm flasks in Dulbecco’s modified Eagle’s II (Molecular Devices, USA). Fura-2 was excited at medium (DMEM)-F12 media (Invitrogen, UK) sup- 340/380 nm every 10 s for a total duration of 300 s. plemented with 10% foetal bovine serum and 1% penicil- lin/streptomycin (Sigma, UK), and maintained at 378Cina Materials 5% CO2 incubator. Cells were passaged 1:2 every 3–4 days. Hanks’ solution contained (in mmol/L): 137 NaCl, 5.4 KCl, Where indicated, cells were transfected with DN-TRPC5 0.01 CaCl2, 0.34 NaH2PO4, 0.44 K2HPO4, 8 D-glucose, 5 plasmid using the Basic Nucleofector Kit for primary HEPES. SBS contained (in mmol/L): 130 NaCl, 5 KCl, 8 smooth muscle cells and Amaxa Transfection System D-glucose, 10 HEPES, 1.2 MgCl2 and 1.5 CaCl2, pH (Amaxa Biosystems, Germany). Mock-transfected HSV titrated to 7.4 and osmolarity corrected to 290 mOsm cells were used as a positive control. The transfection was using D-mannitol where necessary. validated previously and found to be 80–90% effective using the green fluorescent protein. Methods of analysis Passage of HSV cells was carried out by washing with Recordings from conventional fluorescence videomicroscopy 2 ml DPBS (Invitrogen, UK) prior to exposure to 2ml were analysed off-line using Openlab 4. The means of five 1% Trypsin/EDTA (Invitrogen, UK) for 3–7 mins depend- regions of interest from each cell were taken, as specified 2þ ing upon the intrinsic properties of the cells. below. [Ca ] is expressed as the fluorescence intensity ratio of the cells at both excitation wavelengths (340 and Conventional fluorescence videomicroscopy 380 nm). For high-throughput fluorometric imaging, real- At a confluence of 50%, HSV cells were split onto 10 mm time data from the Flexstation were collected by SoftMax glass cover-slips in 24-well plates and sub-cultured for 24 h Pro (Molecular Devices, USA). prior to imaging. For cells in the scratch assay, a linear All data were analysed using Origin Pro 7.5 (Origin Lab scrape of 2 mm was made with a pipette tip through the Corporation, USA). Conventional fluorescence microscopy monolayer of cells on the cover-slip before being incubated data were expressed as mean+ SEM for n cells (in paren- for a further 24 h prior to imaging. Following transfection, theses); n is number of cells analysed per experiment, parenth- cells in the DN paradigm were seeded onto cover-slips 48 h eses are the number of cover-slips. High-throughput prior to imaging. fluorometric imaging data were expressed as mean+ SEM Prepared cells were loaded with 3 mM Fura-2 AM (Sigma, for n cells, where n is the number of wells from which that UK) in standard bath solution (SBS; see below) for 1 h at data set was collated. The statistical significance, if any, was 378C then washed with SBS for a minimum of 30 min at investigated using a one-way ANOVA test for three data sets room temperature. Loaded cells were viewed using an and two-sample paired or independent Student’s t-test for inverted microscope (Axiovert 25 CF I; Zeiss, Germany) in two data sets, P, 0.05 being regarded as significant. ......................................................................................................................................................................................................................................... 94 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... Results Figure 1A is an example image of fura-2 AM loaded HSV cells as viewed on the conventional fluorometric imaging rig. Application of S1P to such HSV cells caused a significant 2þ change in [Ca ] (P, 0.001; Fig. 1B). Following this transi- ent response in the HSV cells, the fluorescence ratio declined towards the baseline. As has been previously mentioned, HSV cells contain a number of receptors on which S1P could be acting and the TRPC5-DN transfection allows for information to be gained about the response mediated by TRPC5 and TRPC5-like channels. Figure 2A shows the response to S1P in DN- and mock-transfected HSV cells. The baseline 2þ [Ca ] was significantly lower in the DN- compared to the mock-transfected cells (P, 0.001). S1P application to the DN-transfected cells caused the transient response, followed 2þ by a return to the baseline [Ca ] . A transient response was 2þ also observed in the mock-transfected cells but the [Ca ] at Figure 2. Effect of TRPC5-DN transfection on the response to S1P and the endpoint was significantly higher than the baseline (P, methanol in HSV cells. (A) Responses to 3 mM S1P in mock-transfected 0.05). While the amplitude of the responses to S1P was HSV cells (closed squares (B); n ¼ 24) and in DN-transfected HSV cells found to be significant (P, 0.001) for both cell types, the (open squares (A); n ¼ 24). (B) Responses to methanol in mock-transfected maximum response in the DN-transfected cells reached HSV cells (closed squares (B); n ¼ 24) and in DN-transfected HSV cells (open squares (A); n ¼ 24). only 60% of the maximum response in the mock-transfected cells; this difference was significant (P, 0.001). Figure 2B shows the response to methanol in DN- com- a role in such migration. The purpose of the HSV scratch 2þ pared to mock-transfected cells. A peak, small in comparison assay was to establish the differences, if any, in the Ca to those for S1P (Fig. 2A), was observed in the mock- response to S1P in static cells compared with cells that had transfected cells in response to methanol; this was significant migrated into a 2 mm linear scratch. As a general point (P ¼ 0.05). A non-significant peak was also observed in the of observation, cells located within the scratch were more DN-transfected cells following methanol addition. easily identifiable by a brighter fluorescence compared with To allude once more to the ongoing theme; S1P initiates other cells (Fig. 3A). This was not considered to be diagnos- SMC migration and TRPC5 has been implicated as having tic and cells were initially deemed to be either ‘static’ or ‘migrated’ by opinion. The initial analysis of results from these two groups resulted in high errors and confusing data (data not shown). Peak responses for each cell were binned according to their values to produce two distinct popu- lations. These populations define the meaning of ‘static’ and ‘migrated’ cells for the purposes of this discussion. There were many note-worthy differences between the static and migrated cells, as illustrated in Fig. 3B. The base- 2þ line [Ca ] was significantly higher in the static compared with the migrated cells (P, 0.001) and the response in the static cells occurred almost immediately after the application of S1P at t ¼ 1.5 min and peaks at t ¼ 2.8 min. There was a 2þ change in the [Ca ] in the migrated cells around this point which was found to be significant compared with the base- line (P ¼ 0.001), but was small in comparison to the maximum response. The main peak in the migrated cells 2þ occurred at t ¼ 5 min; this change in the [Ca ] was found Figure 1. Establishing a response to S1P in HSV cells using conventional to be significant compared with the baseline (P, 0.001). videomicroscopy. (A) Example image showing HSV cells loaded with Following the transient response in the static cells, the Fura-2 AM indicator dye. (B) Response to 3 mM S1P in HSV cells n¼23(3) 2þ [Ca ] did not return to baseline; this sustained component where n is the number of cells, parentheses indicate the number of of the response was significantly higher than the cover slips. ......................................................................................................................................................................................................................................... 95 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... of activation by agonists such as S1P, suggesting a spon- taneous activity of these channels. Secondly, the difference between the amplitudes of the responses to S1P in the DN- compared with the mock-transfected cells is also relevant to this discussion. The maximum response reached in the DN-transfected cells reached only 60% of the maximum response reached in the mock-transfected cells. It seems 2þ intuitive that the discrepancy of 40% was due to Ca current mediated by normally functioning TRPC5-like chan- nels. It follows that the response in the DN-transfected cells was mediated by the normally functioning TRPC5-like chan- nels, i.e. those not affected by the transfection and by other endogenous non-selective cation channels activated by S1P. There is a paucity of data in the literature to support this latter conjecture. The HSV scratch assay is an in vitro simulation of SMC migration that was utilized here to further characterize the Figure 3. Effect of cell migration on the response to S1P in HSV cells response of SMC cells to S1P. A marked difference was observed where a 2 mm line of cells had been scraped from the surface of a glass coverslip and the remainder incubated for 24 h. (A) Annotated in the intensity of the fluorescence of the cells located in or example image showing HSV scratch assay with cells loaded with Fura-2 near the scratch (Fig. 3A). The relatively small differences in AM indicator dye demonstrating scratch (white perforated line) and puta- 2þ the basal [Ca ] (Fig. 3B) are unlikely to account for this tive migrated cells with increased fluorescence compared with other cells higher intensity of fluorescence. It is likely that changes in the (white arrows). (B) Responses to S1P in HSV cells in the scratch assay; cel- cell-shape due to migration affected the fluorescent signal lular responses were binned according to the maximum peak response per cell into these two distinct populations; ‘static’ (closed squares (B); intensity as is seen during blood vessel contraction. n ¼ 11(4)) and ‘migrated’ (open squares (A); n ¼ 7(4)). There are several possible factors that may have given rise to the aforementioned difficulties in identifying whether baseline (P, 0.05) and was followed by another small, these cells were migrated or static. It is important to bear non-significant peak at t ¼ 7.5 min. Following the transient in mind that this identification was by opinion and therefore 2þ response in the migrated cells, it was unclear if the [Ca ] subjective. Following the initial scratch, there was a residual returned to the baseline. It can, however, be said that the layer of loose cells in the surrounding area. These loose cells 2þ [Ca ] in these cells does fall below the sustained phase of were washed away 24 h later when the cells were loaded with the static cells. Fura-2 AM, however, in the meantime, it is feasible that these multi-layered cells may have acted together in a tissue- like manner. This may have affected the properties of cells Discussion contacting the glass-cover slip. The above results were sufficient to satisfy the initial aims of Although this design was the same as that used by Xu the work and, as is fully discussed below, highlighted some et al., this idea highlights a limitation in the experimental intriguing properties of TRPC5-like channels. Consistent design and an area for improvement with this type of with the published data, S1P did elicit a significant response work. Currently, there is no equipment available to enable in these cells producing the characteristic transient response less subjective, controlled measurements of calcium 5 3 that has been previously observed. Unlike in the literature, responses in migrated cells. For example, while calcium it is unclear from Fig. 1B whether or not the S1P response changes can be automatically measured using an equipment had a sustained component. However, this phase was observed such as the FlexStation, and SMC migration can be measured in other experiments on this cell type (data not shown). using Matrigel (BD Biosciences, UK), which is highly valid to While these data show that S1P evoked a response in the the in vivo situation, there is no system that combines the 2þ form of a change in [Ca ] , it could not be unequivocally two. Arguably, the human errors experienced with this established from these results if this response was caused assay would have been reduced with further practise. by S1P activating TRPC5. The DN-transfection was used The results from the HSV scratch assay revealed a number to examine the effects of S1P in the absence of functional of differences between static and migrated SMCs. The higher TRPC5-like channels. The implications of the results from baseline observed in the static cells suggested a difference in 2þ this novel work were 2-fold. First, the lower baseline Ca permeabilities between the two cell types in a manner observed in the DN- compared with the mock-transfected that is independent of channel activation. This finding may cells suggested that TRPC5-like channels contribute to suggest down-regulation of a non-selective cation channel 2þ basal Ca levels. This was in a manner that is independent in the cell membrane that does not necessarily contain ......................................................................................................................................................................................................................................... 96 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... TRPC5. Following on from work by Kumar et al., which pharmacologically prevent SMC migration in vascular suggested that TRPC1 is up-regulated in SMC migration, it disease and other associated conditions. It remains is feasible that TRPC5 up-regulation is associated with this unknown whether there is an association between the X phenomenon also. This is backed up by Xu et al. who chromosome loci associated with certain mental retardation found co-assembly of TRPC1 and TRPC5 sub-units in disorders and the region encoding TRPC5. Understandably, SMCs and is supported by the results showing that S1P acti- a link between these regions of the gene highlights some con- vates TRPC5 (Fig. 2A). This latter point is relevant due to siderations if TRPC5 is to be targeted pharmacologically. the aforementioned tendency of this phospholipid to induce Presently, TRPC5 knowledge can only assist in understand- SMC migration. This could have been explored further by ing SMC migration-related disorders. incubating the SMCs in S1P with the aim of causing SMC The experimental data provide strong evidence to suggest migration. The effect of this induced migration on the ampli- that S1P activates TRPC5-like channels in HSV cells. tude of the response to S1P could then be compared with Additionally, that migrated SMC react differently to S1P responses in cells incubated in SBS. compared with static cells. This provides a rationale for In agreement with the aforementioned literature, the differ- declaring the aims of the investigation as satisfied and con- ences in the amplitudes of the responses between the static and cludes this report. migrated cells may also have been due to differences in channel expression in the migrated cells. Alternatively, migration of Acknowledgements SMCs may have increased the affinity of the GPCR for S1P to the effect of causing an increased response. Xu et al. I would like to thank all members of the Beech Lab for their suggested that external S1P activation is via activation of a help and support throughout this project, with special thanks G-protein coupled receptor linked to PLC but that TRPC5 to David Beech, Eman Al-Shawaf and Jing Li. may act as an ionotropic receptor in response to intracellular S1P. Intracellular S1P may have been contributing to this 2þ Funding response, causing a difference in the [Ca ]. The delayed response observed in the migrated cells has This project was funded by in part by the Faculty of not been previously demonstrated and indicates a further Biological Sciences, University of Leeds and also by the functional change following migration. As the amplitude of Wellcome Trust. the response remains the same, it is unlikely that a down- regulation of the GPCR for S1P has occurred but the delay References could be due to a decrease in sensitivity to S1P. Indeed these receptors may not be affected at all, but coupling of 1. Clapham DE (2003) TRP channels as cellular sensors. Nature 426: 517–524. these receptors to TRCP5-like channels has in some way 2. Sossey-Alaoui K, Lyon JA, Jones L et al. (1999) Molecular cloning and charac- been altered. A DN-TRPC5 scratch assay may have assisted terization of TRPC5 (HTRP5), the human homologue of a mouse brain 2þ receptor-activated capacitative Ca entry channel. Genomics 60: 330–340. in finding out the reasons underlying this delay. 3. Zeng F, Xu S-Z, Jackson PK et al. (2004) Human TRPC5 channel activated by a Besides the aforementioned technical limitations of the multiplicity of signals in a single cell. J Phys 590: 739–750. equipment and techniques used for these experiments, it is 4. Greka A, Navarro B, Oancea E et al. (2003) TRPC5 is a regulator of hippo- also important to note that calcium indicator dye experiments campal neurite length and growth cone morphology. Nat Neuro 6: 837–845. are not direct measures of ion-channel function. This is 5. Xu S-Z, Muraki K, Zeng FA et al. (2006) Sphingosine-1-phosphate-activated 2þ because they only reflect the level of free Ca , regardless of 2þ Ca channel controlling vascular smooth muscle cell motility. Circ Res 98: the source. Additionally, attention should be drawn to the 1381–1389. notion that TRPC5 channels are non-selective cation chan- 6. Kumar B, Dreja K, Shah SS et al. (2006) Upregulated TRPC1 channel in vascu- 2þ nels, so the use of Ca indicators may not reflect complete lar injury in vivo and its role in Human neointimal hyperplasia. Circ Res 98: 557–563. channel function. These experiments serve only as approxi- 2þ 7. Grynkiewicz G, Peonie M, Tsein RY. (1985) A new generation of Ca indi- mations to demonstrate principals based on manipulation of cators with greatly improved fluorescence properties. J Biol Chem 260: TRPC5 using S1P. To directly test TRPC5 channel function, 3440–3450. patch-clamping could have been carried out. 8. Haugland RP (1995) The Handbook 10th Edition. Carlsbad, CA: Invitrogen The clinical use of this information about the involvement Corporation. of TRPC5 is presently minimal. Limited information about 9. Himpens B, Lydrup ML, Hellstrand P et al. (1990) Free cytosolic calcium the functional role of TRPC5-like channels means that selec- during spontaneous contractions in smooth muscle of the guinea-pig meso- tive targeting of this protein cannot yet be used to tubarium. Euro J Phys 417: 404–409. ........................................................................................................................................................................................................................................ Submitted on 17 January 2008; accepted on 11 February 2008; advance access publication 23 April 2008 ......................................................................................................................................................................................................................................... http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

Sphingosine-1-phosphate activation of TRPC5 in vascular smooth muscle cells

Bioscience Horizons , Volume 1 (2) – Jun 23, 2008

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Volume 1 † Number 2 † June 2008 10.1093/biohorizons/hzn013 ......................................................................................................................................................................................................................................... Research article Sphingosine-1-phosphate activation of TRPC5 in vascular smooth muscle cells Samantha Fahy University of Leeds, Leeds, UK. Supervisor: Prof. David Beech, Institute of Membrane and Systems Biology, University of Leeds, Leeds, UK. ........................................................................................................................................................................................................................................ Calcium signalling is a complex and diverse system utilized in many cellular processes and in the transmission of cellular information. A number of transient receptor potential (TRP) proteins have been identified in humans and other mammals; these proteins are impli- cated as having a role in calcium signalling. TRPC5 is a member of this protein family which combines with TRPC1 to form non-selective cation channels in human saphenous vein cells, a type of smooth muscle cell. The exact function of TRPC5 remains elusive, however, it can be activated by sphingosine-1-phosphate (S1P), an endogenous signalling phospholipid involved in SMC migration. 2þ The aim of these experiments was to investigate the effects of S1P on the intracellular calcium concentration, [Ca ] , in HSV cells, utilizing dominant-negative (DN)-TRPC5 transfected cells to establish the role played by TRPC5 in this response. A secondary aim was to establish the effect of SMC migration on the above response parameters to S1P using the HSV scratch assay, where a 2 mm line of cells was scraped away from the surface of a glass cover slip and the remaining cells incubated for 24 h. Concurrent with the literature, S1P evoked a significant response in HSV cells (n¼23; P¼0.001). The baseline was significantly lower in the DN-TRPC5 cells compared with the control cells (P,0.001), and the maximum response in the DN-TRPC5 transfected HSV cells reached only 60% of the maximum response in control cells. This suggested that TRPC5 was involved with maintaining basal [Ca2þ] levels and indicated the proportion of the response for which TRPC5 was responsible. The response to S1P was significantly larger in migrated (n¼7) compared with static (n¼11) HSV cells (P¼,0.001) and this response was delayed by 2.3 min; the baseline was also higher in the latter group. This suggested a functional change in the cell following migration that may have been attributable to TRPC5, for example, channel up-regulation. In conclusion, TRPC5-like channels are responsible for a proportion of the S1P response and are implicated in SMC migration. This highlights potential pharmacological targets for the treatment of atherosclerosis, neointimal hyperplasia and coronary heart disease. Key words: TRPC5, S1P, saphenous, Fura-2, calcium. ........................................................................................................................................................................................................................................ TRP proteins are divided into six families; it is the fifth Introduction member of the canonical TRP proteins (TRPC5) that will be the main focus here. Sossey-Alaoui et al. cloned and Calcium signalling is a complex and diverse system, part of characterized human TRPC5 from the encoding gene many cellular processes and the transmission of cellular located on the X chromosome (Xq23). They identified the information. The main channels permeable to these ions in protein as TRPC5; a novel member of the TRP protein mammalian cells include stretch-activated channels (SACs) family. and voltage-, receptor- and store- operated channels TRPC5 proteins assemble as homomeric or heteromeric (VOCs, ROCs and SOCs, respectively). However, relatively tetratamers around a central pore, permeable to cations. little is known about the mechanisms that underlie and TRPC5-like channels can be activated in a number of ways unify this process. A number of transient receptor potential including via receptors. For example, a G-protein coupled (TRP) proteins have been identified in humans and other receptor, such as the endogenous muscarinic M2/3 acetyl- mammals; these proteins are implicated as having a role choline receptor, is activated by an agonist causing in calcium signalling. ......................................................................................................................................................................................................................................... 2008 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 92 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... dissociation of the Gbg subunit from the Ga subunit. This a number of vascular diseases including those putatively subunit activates phospholipase (PL) Cb which causes the linked to TRPC1 channel upregulation: atherosclerosis, hydrolysis of the membrane-bound phosphatidylinositol neointimal hyperplasia and coronary heart disease. bisphosphate (PIP ) into the lipophylic diacylglycerol While S1P application may cause cellular responses such (DAG) and the hydrophilic inositol triphosphate (IP ). The as changes in calcium concentration and cell migration, first of these components remains within the membrane, these responses may not solely be caused by activation of where it is involved in the activation and translocation of a TRPC5-like channels. Xu et al. utilized a dominant- number of proteins including protein kinase (PK)C. The negative (DN) transfection to impair the function of second component travels through the cytoplasm to IP TRPC5. This allowed for a comparison to be made 2þ receptors (IP Rs) located on intracellular Ca stores; the between the wild-type (or mock-transfected) proteins and endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) the DN-transfected proteins; where the apparent loss of cel- in muscle cells. Activation of these receptors leads to lular response was representative of the contribution to the 2þ release of these Ca stores which putatively leads to the cellular effect made by TRPC5-like channels. The opening of SOCs. Zeng et al. described TRPC5 channels TRPC5-DN transfect is a triple alanine mutation of the con- can be activated by a multiplicity of signals in human served sequence of residues leucine, phenylalanine and embryonic kidney (HEK)293 cells; TRPC5 may be activated tryptophan (LFW) located in the channel pore which as an ROC, as an SOC, by external ionic activation and by results in impaired channel function. 2þ intracellular Ca . The literature proposes a number of different methods 2þ The exact function of TRPC5 is yet to be defined; evidence that are used in investigating TRPC5’s involvement in Ca 2þ suggests a number of possibilities. For example, the location signalling. Changes in intracellular Ca concentration, 2þ of the gene encoding this protein is associated with certain [Ca ] , can be measured using conventional fluorescence mental retardation disorders which may suggest a further videomicroscopy and high-throughput, automated fluoro- role. This group also suggested its importance in the basic metric imaging. Conventional imaging, as it will be referred 2þ developmental process and polymorphisms of the encoding to here, utilizes videomicroscopy and Ca sensitive dyes to gene might hinder this development, leading to the afore- measure and visualize these changes. A xenon light source in mentioned disorders. Alternatively, Greka et al. conjectured a monochromator is used to select the appropriate wave- that TRPC5 homomer channels have a role in hippocampal lengths for the fluorescent dye used. Temporally controlled growth cone morphology and motility. ratiometric imaging exploits the intrinsic properties of duel This involvement in cell motility has also been suggested wavelength ratiometric dyes. for TRPC5 heteromer (TRPC5/TRPC1) channels located Fura-2 acetoxymethyl ester (AM) is a dual-wavelength 5 2þ 7 in vascular SMCs. Sphingosine-1-phosphate (S1P), an ratiometric Ca indicator excited by UV light. Unlike endogenous signalling phospholipid, has been demonstrated Fura-2 sodium and potassium salts, the AM can passively to have a role in the migration of vascular SMCs. Xu diffuse across cell membranes. The esters are then cleaved et al. identified S1P as a novel activator of heteromeric by intracellular esterases and the membrane-impermeant TRPC1/TRPC5 and homomeric TRPC5 channels. S1P con- Fura-2 remains in the interior of the cell. Fura-2 is a divalent centrations 1, 3 and 10 mM evoked graded responses in metal ion chelator that has a high affinity for and binds rever- 2þ HEK293 cells within a 5-min activation period; the median sibly to Ca at physiologically relevant concentrations; 3 mM was selected for further experiments. As has been pre- 100 nm. To a lesser extent, Fura-2 may also bind to 2þ viously mentioned, this group conjectured a mechanism for other bivalent cations such as Mg leading to quenching vascular SMC motility, controlled by TRPC5 that was of the fluorescent signal. Complexation of Fura-2 with 2þ evoked by S1P. Ca causes a conformational change in Fura-2 that is Xu et al. used human saphenous vein (HSV) cells, a type detected through the ratio of the fluorescence intensities at of SMC obtained from the saphenous vein in the leg in prep- the excitation wavelengths 340 and 380 nm; this indicates 2þ aration for coronary artery bi-pass surgery. These cells can the [Ca ] . These experiments are carried out under dark be cultured to produce a high-throughput and cost-effective conditions so as to prevent bleaching of the fluorescent in vitro simulation of physiological conditions. The so-called dye. Frequent illumination of the dye may also lead to ‘scratch assay’ was used by this group to study SMC bleaching; furthermore, bleaching may give false negative 2þ migration; where a line of cells is scraped away from the results as it may appear that the [Ca ] is decreasing when plated monolayer of cultured HSV cells. Over a 24-h it is actually the fluorescent signal that is decreasing. period, the surrounding cells migrated into this scratch; There are several advantages of using such ratiometric these cells were then identified using fluorescence video- dyes, such as Fura-2, over single-excitation dyes, such as microscopy and their responses to S1P compared with Flou 3. For example, the intensity of the fluorescence of 2þ static cells from the same monolayer. It follows that single excitation dyes is dependent upon the [Ca ] while TRPC5 may be key to the understanding and treatment of the ratio of the spectral shift produced by ratiometric dyes ......................................................................................................................................................................................................................................... 93 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... 2þ is proportional to [Ca ]. Therefore, the exact concentration combination with a monochromator (Till Photonics, of the dye does not need to be known as it will not affect the Germany), which selected the excitation wavelength pro- experimental outcome. vided by a xenon arc lamp. Cells were continuously perfused The principle aim of my experiments was to investigate the with SBS at a rate of 2ml/min; following the establishment effects of S1P on TRPC5 and TRPC5-like channels in HSV of a stable baseline with SBS, the perfusion was changed to cells. The use of DN-TRPC5 transfected SMC cells would 3 mM S1P (Sigma, UK), applied at the same rate for the be used identify the components of the S1P response for below specified period of time. Cells in the scratch assay which TRPC5 is responsible in the cells. A secondary aim were identified as migrated or static depending on their of this experiment was to establish the effect, if any, of spatial positioning to the visible scratch area. Emissions SMC migration on the above response parameters to S1P. were collected by a 510 nm filter and an image was taken at each of the excitation wavelengths via a CCD camera (Orca ER; Hamamatsu, Japan) at 10-s intervals. Imaging Methods and materials was controlled using Openlab 4 software (Image Processing & Vision Company Ltd, UK). Cell culture As approved by the Leeds teaching hospital research ethics High-throughput fluorometric imaging committee, HSV tissue samples were collected anonymously At a confluency of 60%, cells were seeded into clear 96-well from patients at Leeds General Infirmary. These patients plates. Following a 24-h incubation period, cells were loaded were undergoing open heart surgery and had given their with 2 mM Fura-2 AM in SBS for 1 h at 378C before being informed consent. Tissue samples were transported to the manually washed three times with SBS. SBS was replaced in laboratory in Hanks’ solution (see ‘Materials’) and the each well in varying volumes according to the protocol 2þ median layer of the tissue was extracted by dissection on being followed. Changes in [Ca ] in response to application the day of removal from the host. These cells were then cul- of various substances were read in a 96-well plate FlexStation tured in 75 cm flasks in Dulbecco’s modified Eagle’s II (Molecular Devices, USA). Fura-2 was excited at medium (DMEM)-F12 media (Invitrogen, UK) sup- 340/380 nm every 10 s for a total duration of 300 s. plemented with 10% foetal bovine serum and 1% penicil- lin/streptomycin (Sigma, UK), and maintained at 378Cina Materials 5% CO2 incubator. Cells were passaged 1:2 every 3–4 days. Hanks’ solution contained (in mmol/L): 137 NaCl, 5.4 KCl, Where indicated, cells were transfected with DN-TRPC5 0.01 CaCl2, 0.34 NaH2PO4, 0.44 K2HPO4, 8 D-glucose, 5 plasmid using the Basic Nucleofector Kit for primary HEPES. SBS contained (in mmol/L): 130 NaCl, 5 KCl, 8 smooth muscle cells and Amaxa Transfection System D-glucose, 10 HEPES, 1.2 MgCl2 and 1.5 CaCl2, pH (Amaxa Biosystems, Germany). Mock-transfected HSV titrated to 7.4 and osmolarity corrected to 290 mOsm cells were used as a positive control. The transfection was using D-mannitol where necessary. validated previously and found to be 80–90% effective using the green fluorescent protein. Methods of analysis Passage of HSV cells was carried out by washing with Recordings from conventional fluorescence videomicroscopy 2 ml DPBS (Invitrogen, UK) prior to exposure to 2ml were analysed off-line using Openlab 4. The means of five 1% Trypsin/EDTA (Invitrogen, UK) for 3–7 mins depend- regions of interest from each cell were taken, as specified 2þ ing upon the intrinsic properties of the cells. below. [Ca ] is expressed as the fluorescence intensity ratio of the cells at both excitation wavelengths (340 and Conventional fluorescence videomicroscopy 380 nm). For high-throughput fluorometric imaging, real- At a confluence of 50%, HSV cells were split onto 10 mm time data from the Flexstation were collected by SoftMax glass cover-slips in 24-well plates and sub-cultured for 24 h Pro (Molecular Devices, USA). prior to imaging. For cells in the scratch assay, a linear All data were analysed using Origin Pro 7.5 (Origin Lab scrape of 2 mm was made with a pipette tip through the Corporation, USA). Conventional fluorescence microscopy monolayer of cells on the cover-slip before being incubated data were expressed as mean+ SEM for n cells (in paren- for a further 24 h prior to imaging. Following transfection, theses); n is number of cells analysed per experiment, parenth- cells in the DN paradigm were seeded onto cover-slips 48 h eses are the number of cover-slips. High-throughput prior to imaging. fluorometric imaging data were expressed as mean+ SEM Prepared cells were loaded with 3 mM Fura-2 AM (Sigma, for n cells, where n is the number of wells from which that UK) in standard bath solution (SBS; see below) for 1 h at data set was collated. The statistical significance, if any, was 378C then washed with SBS for a minimum of 30 min at investigated using a one-way ANOVA test for three data sets room temperature. Loaded cells were viewed using an and two-sample paired or independent Student’s t-test for inverted microscope (Axiovert 25 CF I; Zeiss, Germany) in two data sets, P, 0.05 being regarded as significant. ......................................................................................................................................................................................................................................... 94 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... Results Figure 1A is an example image of fura-2 AM loaded HSV cells as viewed on the conventional fluorometric imaging rig. Application of S1P to such HSV cells caused a significant 2þ change in [Ca ] (P, 0.001; Fig. 1B). Following this transi- ent response in the HSV cells, the fluorescence ratio declined towards the baseline. As has been previously mentioned, HSV cells contain a number of receptors on which S1P could be acting and the TRPC5-DN transfection allows for information to be gained about the response mediated by TRPC5 and TRPC5-like channels. Figure 2A shows the response to S1P in DN- and mock-transfected HSV cells. The baseline 2þ [Ca ] was significantly lower in the DN- compared to the mock-transfected cells (P, 0.001). S1P application to the DN-transfected cells caused the transient response, followed 2þ by a return to the baseline [Ca ] . A transient response was 2þ also observed in the mock-transfected cells but the [Ca ] at Figure 2. Effect of TRPC5-DN transfection on the response to S1P and the endpoint was significantly higher than the baseline (P, methanol in HSV cells. (A) Responses to 3 mM S1P in mock-transfected 0.05). While the amplitude of the responses to S1P was HSV cells (closed squares (B); n ¼ 24) and in DN-transfected HSV cells found to be significant (P, 0.001) for both cell types, the (open squares (A); n ¼ 24). (B) Responses to methanol in mock-transfected maximum response in the DN-transfected cells reached HSV cells (closed squares (B); n ¼ 24) and in DN-transfected HSV cells (open squares (A); n ¼ 24). only 60% of the maximum response in the mock-transfected cells; this difference was significant (P, 0.001). Figure 2B shows the response to methanol in DN- com- a role in such migration. The purpose of the HSV scratch 2þ pared to mock-transfected cells. A peak, small in comparison assay was to establish the differences, if any, in the Ca to those for S1P (Fig. 2A), was observed in the mock- response to S1P in static cells compared with cells that had transfected cells in response to methanol; this was significant migrated into a 2 mm linear scratch. As a general point (P ¼ 0.05). A non-significant peak was also observed in the of observation, cells located within the scratch were more DN-transfected cells following methanol addition. easily identifiable by a brighter fluorescence compared with To allude once more to the ongoing theme; S1P initiates other cells (Fig. 3A). This was not considered to be diagnos- SMC migration and TRPC5 has been implicated as having tic and cells were initially deemed to be either ‘static’ or ‘migrated’ by opinion. The initial analysis of results from these two groups resulted in high errors and confusing data (data not shown). Peak responses for each cell were binned according to their values to produce two distinct popu- lations. These populations define the meaning of ‘static’ and ‘migrated’ cells for the purposes of this discussion. There were many note-worthy differences between the static and migrated cells, as illustrated in Fig. 3B. The base- 2þ line [Ca ] was significantly higher in the static compared with the migrated cells (P, 0.001) and the response in the static cells occurred almost immediately after the application of S1P at t ¼ 1.5 min and peaks at t ¼ 2.8 min. There was a 2þ change in the [Ca ] in the migrated cells around this point which was found to be significant compared with the base- line (P ¼ 0.001), but was small in comparison to the maximum response. The main peak in the migrated cells 2þ occurred at t ¼ 5 min; this change in the [Ca ] was found Figure 1. Establishing a response to S1P in HSV cells using conventional to be significant compared with the baseline (P, 0.001). videomicroscopy. (A) Example image showing HSV cells loaded with Following the transient response in the static cells, the Fura-2 AM indicator dye. (B) Response to 3 mM S1P in HSV cells n¼23(3) 2þ [Ca ] did not return to baseline; this sustained component where n is the number of cells, parentheses indicate the number of of the response was significantly higher than the cover slips. ......................................................................................................................................................................................................................................... 95 Research article Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... of activation by agonists such as S1P, suggesting a spon- taneous activity of these channels. Secondly, the difference between the amplitudes of the responses to S1P in the DN- compared with the mock-transfected cells is also relevant to this discussion. The maximum response reached in the DN-transfected cells reached only 60% of the maximum response reached in the mock-transfected cells. It seems 2þ intuitive that the discrepancy of 40% was due to Ca current mediated by normally functioning TRPC5-like chan- nels. It follows that the response in the DN-transfected cells was mediated by the normally functioning TRPC5-like chan- nels, i.e. those not affected by the transfection and by other endogenous non-selective cation channels activated by S1P. There is a paucity of data in the literature to support this latter conjecture. The HSV scratch assay is an in vitro simulation of SMC migration that was utilized here to further characterize the Figure 3. Effect of cell migration on the response to S1P in HSV cells response of SMC cells to S1P. A marked difference was observed where a 2 mm line of cells had been scraped from the surface of a glass coverslip and the remainder incubated for 24 h. (A) Annotated in the intensity of the fluorescence of the cells located in or example image showing HSV scratch assay with cells loaded with Fura-2 near the scratch (Fig. 3A). The relatively small differences in AM indicator dye demonstrating scratch (white perforated line) and puta- 2þ the basal [Ca ] (Fig. 3B) are unlikely to account for this tive migrated cells with increased fluorescence compared with other cells higher intensity of fluorescence. It is likely that changes in the (white arrows). (B) Responses to S1P in HSV cells in the scratch assay; cel- cell-shape due to migration affected the fluorescent signal lular responses were binned according to the maximum peak response per cell into these two distinct populations; ‘static’ (closed squares (B); intensity as is seen during blood vessel contraction. n ¼ 11(4)) and ‘migrated’ (open squares (A); n ¼ 7(4)). There are several possible factors that may have given rise to the aforementioned difficulties in identifying whether baseline (P, 0.05) and was followed by another small, these cells were migrated or static. It is important to bear non-significant peak at t ¼ 7.5 min. Following the transient in mind that this identification was by opinion and therefore 2þ response in the migrated cells, it was unclear if the [Ca ] subjective. Following the initial scratch, there was a residual returned to the baseline. It can, however, be said that the layer of loose cells in the surrounding area. These loose cells 2þ [Ca ] in these cells does fall below the sustained phase of were washed away 24 h later when the cells were loaded with the static cells. Fura-2 AM, however, in the meantime, it is feasible that these multi-layered cells may have acted together in a tissue- like manner. This may have affected the properties of cells Discussion contacting the glass-cover slip. The above results were sufficient to satisfy the initial aims of Although this design was the same as that used by Xu the work and, as is fully discussed below, highlighted some et al., this idea highlights a limitation in the experimental intriguing properties of TRPC5-like channels. Consistent design and an area for improvement with this type of with the published data, S1P did elicit a significant response work. Currently, there is no equipment available to enable in these cells producing the characteristic transient response less subjective, controlled measurements of calcium 5 3 that has been previously observed. Unlike in the literature, responses in migrated cells. For example, while calcium it is unclear from Fig. 1B whether or not the S1P response changes can be automatically measured using an equipment had a sustained component. However, this phase was observed such as the FlexStation, and SMC migration can be measured in other experiments on this cell type (data not shown). using Matrigel (BD Biosciences, UK), which is highly valid to While these data show that S1P evoked a response in the the in vivo situation, there is no system that combines the 2þ form of a change in [Ca ] , it could not be unequivocally two. Arguably, the human errors experienced with this established from these results if this response was caused assay would have been reduced with further practise. by S1P activating TRPC5. The DN-transfection was used The results from the HSV scratch assay revealed a number to examine the effects of S1P in the absence of functional of differences between static and migrated SMCs. The higher TRPC5-like channels. The implications of the results from baseline observed in the static cells suggested a difference in 2þ this novel work were 2-fold. First, the lower baseline Ca permeabilities between the two cell types in a manner observed in the DN- compared with the mock-transfected that is independent of channel activation. This finding may cells suggested that TRPC5-like channels contribute to suggest down-regulation of a non-selective cation channel 2þ basal Ca levels. This was in a manner that is independent in the cell membrane that does not necessarily contain ......................................................................................................................................................................................................................................... 96 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Research article ......................................................................................................................................................................................................................................... TRPC5. Following on from work by Kumar et al., which pharmacologically prevent SMC migration in vascular suggested that TRPC1 is up-regulated in SMC migration, it disease and other associated conditions. It remains is feasible that TRPC5 up-regulation is associated with this unknown whether there is an association between the X phenomenon also. This is backed up by Xu et al. who chromosome loci associated with certain mental retardation found co-assembly of TRPC1 and TRPC5 sub-units in disorders and the region encoding TRPC5. Understandably, SMCs and is supported by the results showing that S1P acti- a link between these regions of the gene highlights some con- vates TRPC5 (Fig. 2A). This latter point is relevant due to siderations if TRPC5 is to be targeted pharmacologically. the aforementioned tendency of this phospholipid to induce Presently, TRPC5 knowledge can only assist in understand- SMC migration. This could have been explored further by ing SMC migration-related disorders. incubating the SMCs in S1P with the aim of causing SMC The experimental data provide strong evidence to suggest migration. The effect of this induced migration on the ampli- that S1P activates TRPC5-like channels in HSV cells. tude of the response to S1P could then be compared with Additionally, that migrated SMC react differently to S1P responses in cells incubated in SBS. compared with static cells. This provides a rationale for In agreement with the aforementioned literature, the differ- declaring the aims of the investigation as satisfied and con- ences in the amplitudes of the responses between the static and cludes this report. migrated cells may also have been due to differences in channel expression in the migrated cells. Alternatively, migration of Acknowledgements SMCs may have increased the affinity of the GPCR for S1P to the effect of causing an increased response. Xu et al. I would like to thank all members of the Beech Lab for their suggested that external S1P activation is via activation of a help and support throughout this project, with special thanks G-protein coupled receptor linked to PLC but that TRPC5 to David Beech, Eman Al-Shawaf and Jing Li. may act as an ionotropic receptor in response to intracellular S1P. Intracellular S1P may have been contributing to this 2þ Funding response, causing a difference in the [Ca ]. The delayed response observed in the migrated cells has This project was funded by in part by the Faculty of not been previously demonstrated and indicates a further Biological Sciences, University of Leeds and also by the functional change following migration. As the amplitude of Wellcome Trust. the response remains the same, it is unlikely that a down- regulation of the GPCR for S1P has occurred but the delay References could be due to a decrease in sensitivity to S1P. Indeed these receptors may not be affected at all, but coupling of 1. Clapham DE (2003) TRP channels as cellular sensors. Nature 426: 517–524. these receptors to TRCP5-like channels has in some way 2. Sossey-Alaoui K, Lyon JA, Jones L et al. (1999) Molecular cloning and charac- been altered. A DN-TRPC5 scratch assay may have assisted terization of TRPC5 (HTRP5), the human homologue of a mouse brain 2þ receptor-activated capacitative Ca entry channel. Genomics 60: 330–340. in finding out the reasons underlying this delay. 3. Zeng F, Xu S-Z, Jackson PK et al. (2004) Human TRPC5 channel activated by a Besides the aforementioned technical limitations of the multiplicity of signals in a single cell. J Phys 590: 739–750. equipment and techniques used for these experiments, it is 4. Greka A, Navarro B, Oancea E et al. (2003) TRPC5 is a regulator of hippo- also important to note that calcium indicator dye experiments campal neurite length and growth cone morphology. Nat Neuro 6: 837–845. are not direct measures of ion-channel function. This is 5. Xu S-Z, Muraki K, Zeng FA et al. (2006) Sphingosine-1-phosphate-activated 2þ because they only reflect the level of free Ca , regardless of 2þ Ca channel controlling vascular smooth muscle cell motility. Circ Res 98: the source. Additionally, attention should be drawn to the 1381–1389. notion that TRPC5 channels are non-selective cation chan- 6. Kumar B, Dreja K, Shah SS et al. (2006) Upregulated TRPC1 channel in vascu- 2þ nels, so the use of Ca indicators may not reflect complete lar injury in vivo and its role in Human neointimal hyperplasia. Circ Res 98: 557–563. channel function. These experiments serve only as approxi- 2þ 7. Grynkiewicz G, Peonie M, Tsein RY. (1985) A new generation of Ca indi- mations to demonstrate principals based on manipulation of cators with greatly improved fluorescence properties. J Biol Chem 260: TRPC5 using S1P. To directly test TRPC5 channel function, 3440–3450. patch-clamping could have been carried out. 8. Haugland RP (1995) The Handbook 10th Edition. Carlsbad, CA: Invitrogen The clinical use of this information about the involvement Corporation. of TRPC5 is presently minimal. Limited information about 9. Himpens B, Lydrup ML, Hellstrand P et al. (1990) Free cytosolic calcium the functional role of TRPC5-like channels means that selec- during spontaneous contractions in smooth muscle of the guinea-pig meso- tive targeting of this protein cannot yet be used to tubarium. Euro J Phys 417: 404–409. ........................................................................................................................................................................................................................................ Submitted on 17 January 2008; accepted on 11 February 2008; advance access publication 23 April 2008 .........................................................................................................................................................................................................................................

Journal

Bioscience HorizonsOxford University Press

Published: Jun 23, 2008

Keywords: Key words TRPC5 S1P saphenous Fura-2 calcium

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