Pei Lai

Francesco Michelangeli
email: f.michelangeli@bham.ac.uk

Changes in Expression and Activity of the Secretory Pathway Ca 2+ ATPase 1 (SPCA1) in A7r5 Vascular Smooth Muscle Cells, Cultured at Different Glucose Concentrations

Keywords: Ca 2+ -ATPase, calcium homeostasis, diabetes, Golgi apparatus, vascular smooth muscle cells, SPCA1, SERCA NG, normal glucose (5.55mM) culture media, HG, high glucose (25mM) culture media, VSMCs, vascular smooth muscle cells, SERCA, sarcoplasmic-endoplasmic reticulum Ca 2+ ATPase, SPCA, secretory pathway Ca 2+ ATPase, AVP, Arginine vasopressin

Changes in expression and activity of the secretory pathway Ca2+ ATPase 1 (SPCA1) in A7r5 vascular

smooth muscle cells, cultured at different glucose concentrations.

Introduction

Cardiovascular diseases, including those associated with diabetes mellitus, have long been associated with the dis-regulation of Ca 2+ homeostasis in vascular cells [START_REF] Levy | Abnormal cell calcium homeostasis in type 2 diabetes mellitus -A new look on old disease[END_REF]. Comparisons between healthy and diabetic subjects in the past have shown distribution and use of Ca 2+ ions differ between the two conditions. For example, diabetes has been associated with higher than normal basal [Ca 2+ ] i in rat-derived cardiac myocytes [START_REF] Smogorzewski | High glucose concentration causes a rise in [Ca 2+ ](i) of cardiac myocytes[END_REF] and vascular smooth muscle cells (VSMCs) [START_REF] Barbagallo | Glucoseinduced alterations of cytosolic-free calcium in cultured rat tail artery vascular smooth-muscle cells[END_REF], demonstrated from their culture in high glucose media. Altered hormone-induced Ca 2+ signalling has also been noted in human VSMCs from diabetic patients [START_REF] Fleischhacker | Human diabetes is associated with hyperreactivity of vascular smooth muscle cells due to altered subcellular Ca 2+ distribution[END_REF].

Abnormal expression and / or activity of specific proteins that participate in Ca 2+ signalling and homeostasis have been linked to diabetes. These include Ca 2+ dependent enzymes, such as increased activity of both calpain in rat endothelial cells [START_REF] Stalker | The calcium-dependent protease calpain causes endothelial dysfunction in type 2 diabetes[END_REF] and Ca 2+ /calmodulin-dependent protein kinase II in rat whole carotid arteries [START_REF] Yousif | Role of Ca 2+ /calmodulin-dependent protein kinase II in development of vascular dysfunction in diabetic rats with hypertension[END_REF]. In addition, Ca 2+ transporters have shown to be influenced by the diabetic state in vascular cells. Examples of these include the inhibition of store-operated Ca 2+ channels in rat microvascular smooth muscle cells [START_REF] Curtis | Diabetes-induced activation of protein kinase C inhibits store-operated Ca 2+ uptake in rat retinal microvascular smooth muscle[END_REF] and increased activity of T-type Ca 2+ channels in rat cardiomyocytes [START_REF] Li | T-type Ca 2+ channels are involved in high glucose-induced rat neonatal cardiomyocyte proliferation[END_REF]. The sarcoplasmic endoplasmic reticulum Ca 2+ -ATPase (SERCA) has also been shown to differ between healthy and diabetic subjects, both in the form of decreased expression levels in heart from streptozotocininduced diabetic rats [START_REF] Teshima | Diminished expression of sarcoplasmic reticulum Ca 2+ -ATPase and ryanodine sensitive Ca 2+ channel mRNA in streptozotocin-induced diabetic rat heart[END_REF] and increased activity in rat primary VSMCs cultured in high glucose media [START_REF] Tong | High glucose oxidizes SERCA cysteine-674 and prevents inhibition by nitric oxide of smooth muscle cell migration[END_REF].

The secretory pathway Ca 2+ -ATPase 1 (SPCA1 or ATP2C1), a Golgi apparatuslocalised Ca 2+ ion pump which is relatively highly expressed in vascular smooth muscle cells [11], has not been investigated in relation to diabetes-related cardiovascular pathology. It is unlikely, however, that the other isoform, SPCA2, plays a substantial role in cardiovascular pathology since it is not significantly expressed in vascular tissue [START_REF] Vanoevelen | The secretory pathway Ca 2+ /Mn 2+ -ATPase 2 is a Golgi-localized pump with high affinity for Ca 2+ ions[END_REF]. However, previous studies that have linked SPCA1 expression to diabetes in pancreatic cells [START_REF] Mitchell | Role for plasma membrane-related Ca 2+ -ATPase-1 (ATP2C1) in pancreatic beta-cell Ca 2+ homeostasis revealed by RNA silencing[END_REF] and, furthermore, alterations in the Golgi apparatus has also been observed in VSMCs treated with kinase inhibitors to inhibit proliferation [START_REF] Thyberg | Tyrphostin A9 and wortmannin perturb the Golgi complex and block proliferation of vascular smooth muscle cells[END_REF]. SPCA1 is important for establishing a store of Ca 2+ ions in the Golgi apparatus, which is required by Ca 2+ -dependent enzymes essential for posttranslational modification of proteins in transit through the secretory pathway [15].

In this initial study, we utilize one of the most commonly employed vascular smooth muscle cell lines (A7r5 cells) to determine whether culturing them in either normal glucose (NG)-or high glucose (HG)-supplemented media, to mimic the 'normal' and 'hyperglycaemic' glucose homeostatic states, specifically alters the expression and activity of SPCA1. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Materials & Methods

A7r5 Cell Culture & Treatment A7r5 (rat embryonic thoracic aortic smooth muscle) cells (a gift from Prof. C.W. Taylor; University of Cambridge, UK) were cultured in DMEM (Lonza), which was supplemented with 10% FBS 4mM L-glutamine, 1% non-essential amino acids and 1% 1:1 penicillin-streptomycin (PAA). Growth was maintained under conditions of 37 o C and 5% CO 2 . Culture media contained 25mM D-glucose when cells were maintained for growth or for obtaining cells in the 'hyperglycemic' high glucose (HG) cultured state. Sub-culturing of cells was done every three to four days by trypsinisation using 0.05% trypsin-EDTA solution (Invitrogen). Cells in the normal glucose (NG) cultured state were initially obtained by seeding a new flask of subcultured cells from HG media into NG (5.55mM D-glucose) media. Both NG and HG cells were sub-cultured for at least a further two passages in their respective media before being used for experimentation.

PCR

mRNA was extracted from cells and converted to cDNA copies via reverse transcription polymerase chain reaction (RT-PCR), using a Superscript III CellsDirect™ cDNA synthesis kit (Invitrogen). Cells were harvested by trysinisation, washed with cell culture PBS, and re-suspended in 0.1% diethylpyrocarbonate (DEPC)-treated PBS. 2x10 4 cells were used to prepare each cDNA sample. BIOTAQ™ Taq DNA polymerase and dNTP mix were purchased from BIOLINE. Primers were all made by Alta Bioscience (University of Birmingham, UK). Primer sequences and their specific PCR conditions were obtained from the following associated references: for SPCA1 [START_REF] Missiaen | Ca 2+ uptake and release properties of a thapsigargin-insensitive nonmitochondrial Ca 2+ store in A7r5 and 16HBE14ocells[END_REF], SERCA [START_REF] Varadi | Isoforms of endoplasmic reticulum Ca 2+ -ATPase are differentially expressed in normal and diabetic islets of Langerhans[END_REF], glyceraldehyde 3phosphate dehydrogenase (GAPDH) [START_REF] Di Napoli | Trimetazidine improves post-ischemic recovery by preserving endothelial nitric oxide synthase expression in isolated working rat hearts[END_REF], β-actin [START_REF] Varadi | Isoforms of endoplasmic reticulum Ca 2+ -ATPase are differentially expressed in normal and diabetic islets of Langerhans[END_REF] and ribosomal protein L19 (RPL19) [START_REF] Al-Bader | Housekeeping gene expression during fetal brain development in the rat -validation by semi-quantitative RT-PCR[END_REF]. The SPCA1 primers only recognised mRNA corresponding to the SPCA1 isoform (all splice variants). The SERCA primers recognised mRNA corresponding to all SERCA isoforms. Each 50µl PCR reaction contained 2.5 units of BIOTAQ™, 2mM dNTP mix and 1µl cDNA sample. SPCA1 and SERCA PCRs required 35 cycles. GAPDH, β-actin and RPL19 required 28 cycles. All PCR products were separated by agarose gel electrophoresis, stained with SYBR® Safe DNA gel stain (Invitrogen) and visualised using a UV transluminator. PCR product band sizes were checked using a 100bp DNA ladder (NEB).

Images of stained gels were taken using a UVP ImageStore 5000 system. Analysis was undertaken using ImageJ (version 1.41e; National Institutes of Health USA). Mean pixel intensity values were measured for the product bands, each of which were corrected for background and made relative to those for RPL19 (a commonly used internal control for PCR reactions [START_REF] Al-Bader | Housekeeping gene expression during fetal brain development in the rat -validation by semi-quantitative RT-PCR[END_REF]) from the same sample to correct for differences in total cDNA quantity. The expression of RPL19, a housekeeping protein, was unlikely to be affected by different glucose culture conditions in A7r5 cells, when compared to other control markers, such as GAPDH (a glycolytic enzyme) and β-actin (a contractility marker [20]). A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

SDS-PAGE & Western Blotting

Cell lysate samples were prepared using a proportion of the same cells used for cDNA preparation. For each sample, 1-1.25x10 6 cells were incubated on ice for two hours in lysis buffer (50mM Tris-HCl (pH 7.4), 150mM NaCl, 1mM EDTA, 1% Trition-X100, 1% sodium deoxycholate, 0.1% SDS and 1mM PMSF). The cell suspension was then homogenised by passing it through a 21-guage needle via a syringe, centrifuged at 2400g for 5 minutes, and the supernatant was collected.

Proteins in all cell lysate samples were separated by SDS-PAGE, transferred onto nitrocellulose sheets by Western blotting, and probed with antibodies for SPCA1, SERCA and α-actin. The methodology was as described previously in [11] with the following modifications: 20µg of protein was loaded of each sample for SDS-PAGE; anti-SPCA1 (custom antibody from BioCarta GmbH, [11]), anti-SERCA (YIF4) (a gift from Dr. J.M. East, Southampton University) and anti-α-actin (clone 1A4; Sigma) antibodies were diluted in TTBS at ratios of 1:75, 1:800 and 1:800, respectively, and incubated with the blots for 90 minutes; following incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000 dilution), Immobilon™ Western chemiluminescent HRP substrate (Millipore) was used to visualise antibody-bound protein bands; HRP-substrate-treated blots were viewed and images captured using a BIO-RAD Fluor-S Max MultiImager. Images were analysed with ImageJ software to determine pixel intensity values for each product band. These were corrected for background and values for the HG samples were made relative to their counterpart NG samples.

Microsomal Membrane Preparation

The method used was as previously described [11] with the following modifications: cells were harvested by trypsinisation, washed with PBS and centrifuged at 900g for 10 minutes at 4 o C; the pellet was homogenised with both Polytron and Potter homogenisers after re-suspension in membrane preparation buffer; the homogenate was centrifuged at 10,000g for 15 minutes at 4 o C, the pellet was re-homogenised and centrifuged again, and the two supernatants were combined and centrifuged at 100,000g for 50 minutes at 4 o C. The final pellet, which contained the microsomal membranes, was re-suspended in fresh buffer, aliquoted and snap frozen in liquid nitrogen at -80 o C.

Ca 2+ -dependent ATPase activity.

Ca 2+ dependent ATPase activity was measured using the phosphate liberation method, as previously described [21], with minor modifications. The assay buffer included sodium azide (2mM) and vanadate (2µM) to inhibit mitochondrial Ca 2+ uptake and PMCA activity, respectively, the reaction period was 90 minutes and each reaction contained 20µg of microsomal membrane proteins. In order to distinguish between Ca 2+ ATPase activity from SERCA and SPCA, experiments were repeated in the presence and absence of 1µM thapsigargin (Sigma) as this would completely inhibit SERCA and have minimal effects on SPCA activity [21,[START_REF] Dode | Dissection of the functional differences between human secretory pathway Ca 2+ /Mn 2+ -ATPase (SPCA) 1 and 2 isoenzymes by steadystate and transient kinetic analyses[END_REF].

Fluo-3-based intracellular Ca 2+ imaging

This method was as described previously [START_REF] Ogunbayo | Tetrabromobisphenol A (TBBPA), induces cell death in TM4 Sertoli cells by modulating Ca 2+ transport proteins and causing dysregulation of Ca 2+ homeostasis[END_REF], but with minor modifications. The cells were grown at a density of 1x10 4 cells per coverslip and loaded with Fluo-3 AM (Sigma) at a final concentration of 6µM. After 45 minutes incubation, the cells were incubated in Hank's buffered saline solution, containing sulfinpyrazone (0.08µM), for a further 10 minutes. Fluo-3-loaded cells were then viewed using an inverted epifluorescence microscope (Nikon TS-100F) with filters specifically designed to monitor Fluo-3 fluorescence and a 20x Plan objective. The video images were recorded using StellaCam Astrovid 3 Camera connected to the computer using a Hauppage USB TV live video capture device and software (set at 1 frame /sec). Following initiation of the recordings, the cells were treated with 1µM thapsigargin for 5 minutes prior to the addition of 10nM arginine-vasopressin (AVP) (Bachem Biosciences, Germany). The latter treatment was recorded for a further 5 minutes.

Changes in Fluo-3 fluorescence intensity induced by AVP were measured using ImageJ software, and corrected for both background and photo-bleaching as previously described [START_REF] Ogunbayo | Tetrabromobisphenol A (TBBPA), induces cell death in TM4 Sertoli cells by modulating Ca 2+ transport proteins and causing dysregulation of Ca 2+ homeostasis[END_REF]. Values for each cell were made relative to their basal fluorescence intensity value at the time point of AVP application. These relative values were used to produce Ca 2+ transient plots, from which the kinetics of the rise and decay phases could be calculated.

Immunohistochemistry & Lectin Staining

The method used was as described previously [11] with the following modifications: cells were seeded at a density of 1.5-3x10 4 cells per coverslip; prior to antibody treatment, they were fixed with 2% v/v formaldehyde; anti-SPCA1 and anti-SERCA working dilutions were 1:30 and 1:100, respectively; tetramethylrhodamine (TMR)conjugated wheat germ agglutinin (WGA) (Invitrogen) (for Golgi apparatus labelling) was used at 10µg/ml per coverslip. The labelled cells were viewed using fluorescence microscopy, with appropriate filters. Digital photographs of the cells were collected, using the same imaging system described above, except that the images were recorded at a 640 x 480 pixel resolution.

Statistical Analysis

Standard errors of the means (S.E.M) were calculated for data from PCRs, Western blots, Ca 2+ -ATPase assays and kinetics measurements from Fluo-3 fluorescence Ca 2+ imaging experiments. Two-tailed equal variance Student's T-test and analysis of variance (Minitab) were used to test for statistical significance (P). A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Results

Figure 1 shows the PCR products, which relates to mRNA expression levels, for SPCA1 and SERCA, GAPDH and β-actin. The data were obtained from triplicate paired culture experiments, where the PCR products of the transcripts in HG cultured cells were compared to their corresponding paired NG cultured cells and then expressed as a percentage. The data showed that the relative expression of SPCA1 and SERCA, in HG versus NG cultured cells, were found have increased by 60 ± 13% and 19 ± 5%, respectively. Furthermore, these changes in expression were statistically significant between the two conditions (i.e. P <0.01 for SPCA1 and P <0.05 for SERCA). In contrast, the mRNA expression levels for GAPDH and β-actin were not substantially affected (i.e. an increase of 7 ± 4% and decrease of 4 ± 3%, for GAPDH and β-actin, respectively). Figure 1 also shows representative blots of the PCR products for the different mRNA transcripts isolated from cells grown in high (HG) and normal (NG) glucose culture media.

Figure 2 shows Western blots representing the protein levels of SPCA1, SERCA and α-actin in NG and HG cultured A7r5 cells. The mean percentage differences in protein levels were determined using total cell lysate samples from three paired cultures and expressed as a percentage of the HG cells compared to NG cells. The results confirmed the PCR study by showing that HG cultured cells had an increased abundance of SPCA1 (by 15.5 ± 5%), SERCA (by 9 ± 4%) and α-actin (by 2 ± 4%), when compared to NG cells. The increase in SPCA1 protein levels between HG and NG cultured cells was found to be statistically significant (P <0.05).

To determine whether increased SPCA1 expression resulted in increased SPCAspecific Ca 2+ -ATPase activity, microsomal membranes were isolated from HG and NG cultured cells, and measured using the phosphate liberation assay (Table 1). 1µM thapsigargin was used to distinguish the contribution made by SERCA and SPCA. The assay buffer also contained both 2mM azide and 1µM vanadate to inhibit mitochondrial Ca 2+ uptake / activity and plasma membrane Ca 2+ -ATPase activity, respectively. Under these experimental conditions, the Ca 2+ -dependent ATPase activities were deemed to be predominantly due to either a combination of SERCA and SPCA (in the absence of thapsigargin) or SPCA alone (in the presence of thapsigargin) [20]. Due to the inherent variability in Ca 2+ -ATPase activities from different membrane preparations, experiments were done using samples of microsomal membranes isolated from three replicate pairs of HG and NG cell cultures. Table 1 shows that the Ca 2+ -ATPase activities from the three membrane preparations, isolated from NG and HG cultured cells, were similar in the absence of thapsigargin. (The mean activities were 3.9 ± 0.5 and 3.2 ± 0.4 nmol/min/mg protein for NG and HG cultured cells, respectively). On the other hand, in the presence of 1µM thapsigargin, the Ca 2+ -ATPase activities did show significant differences between the membranes isolated from NG and HG cultured cells, with higher activities consistently obtained from HG cultured cell membranes. Mean activities for the three membrane preparations were 0.75 ± 0.27 nmol/min/mg protein for NG cells and 1.86 ± 0.55 nmol/min/mg protein for HG cells. Statistical analysis showed that the activity differences between the two culture conditions were significant (P < 0.05). A comparison of total Ca 2+ -ATPase activity (from SERCA and SPCA) in the membranes, with the activity for SPCA alone, showed that SPCA accounted for between 20-60% of the Ca 2+ -ATPase activity in A7r5 cultured cells. This is not dissimilar to our earlier findings in aortic VSMC membranes, where we showed that about 50% of total ATP-dependent Ca 2+ uptake into these membranes was due to SPCA [11]. Therefore, these findings again highlight that SPCA plays a significant role in Ca 2+ uptake into Ca 2+ stores within VSMCs.

To assess whether increased expression of SPCA1 can alter the profile of agoinstinduced cytosolic [Ca 2+ ] transients, NG and HG cultured cells, loaded with Fluo-3, were treated with 10nM AVP. These cells were first pre-treated with 1µM thapsigargin in order to minimise the effects of SERCA on their response to AVP. Figure 3 shows the averaged traces (with each trace comprising of data from ten individual cells) of Fluo-3 fluorescence change in both NG (figure 3A) and HG (figure 3B) cells. Although the peak values in AVP-induced Ca 2+ transients were similar in cells from the two glucose culture conditions, their kinetic profiles were notably different. The Ca 2+ transients observed for NG cultured cells where much slower than those for HG cells, for both the rise and the decay phases of the transients. However if the cells were not initially pre-treated with thapsigargin, the kinetics of the Ca 2+ transients for both NG and HG cells were very similar to each other and more rapid (insets), consistent with observations from previous studies [24]. The mean peak heights between cells not treated and those pre-treated with thapsigargin, for both NG and HG culture conditions were each reduced by about 60%. This shows that the contribution made by SERCA-containing Ca 2+ stores to the AVP mediated Ca 2+ responses in these cells is not overwhelmingly dominant since SPCA-containing Ca 2+ stores also contributes significantly.

The kinetic measurements taken from the Ca 2+ transient traces representing postthapsigargin AVP responses showed a quantitative difference between NG and HG cells (Table 2). The mean time taken to increase from basal to peak [Ca 2+ ] i levels of the AVP response was approximately 3-fold slower in NG cultured cells compared to HG cultured cells. The half-life for the decay in [Ca 2+ ] i from the peak back towards basal levels was also about 2-fold slower in NG cells. Statistical analysis of these kinetic measurements for both HG and NG cells, showed that these differences were significant (i.e. P<0.05, for both the rise and decay phases when comparing NG and HG cultured cells).

Immuno-localisation of SPCA1, together with WGA-based lectin affinity staining, in NG and HG cultured cells showed SPCA1 and the Golgi apparatus share a very similar distribution pattern, based on its characteristic perinuclear staining (figure 4).

[WGA binding is commonly used as a marker for the Golgi apparatus because it binds to glycoproteins highly abundant in this organelle, [START_REF] Wright | Structural comparison of the 2 distinct sugar bindingsites in wheat-germ-agglutinin isolectin-ii[END_REF]]. Comparison of the staining patterns for SPCA1 showed no notable differences between NG and HG cultured cells. Thus localisation of SPCA1 was largely unaffected by the glucose culturing conditions in A7r5 cells. Immuno-staining for SERCA showed a more uniform distribution throughout the cell, that was distinct from those observed for both SPCA1 and WGA, and which we have previously reported elsewhere for primary VSMCs [11].

Discussion

Data obtained by Wootton et al.,[11] showed rat aortic tissue and primary human aortic VSMCs express relatively high levels of SPCA1 protein compared to many other tissues and cell types. From the Ca 2+ -dependent ATPase measurements we show that between 20-60% of the Ca 2+ -ATPase activity, associated with intracellular membranes of A7r5 cells, is derived from SPCA. Again this is similar with our previous findings, which show that in aortic VSMC membranes about 50% of the ATP-dependent Ca 2+ uptake is via SPCA [11]. This is also corroborated by the fact that substantial increases in AVP-induced intracellular [Ca 2+ ] are observed in cells previously treated with thapsigargin, which suggests that these VSMCs must contain a sizeable SERCA-independent mobilizable Ca 2+ store. Therefore taken together, these findings highlight the substantial role SPCA plays in Ca 2+ store re-filling within VSMCs.

It has also been well-established that vascular tissues prone to developing atherosclerotic plaques (as observed in diabetes mellitus [26]) have a tendency to change their VSMCs from a contractile to a secretory phenotype [27]. This is characterised by their increased ability to produce secreted proteins, such as collagen and proteoglycans found in the extra-cellular matrix [START_REF] Mazzone | Cardiovascular disease risk in type 2 diabetes mellitus: insights from mechanistic studies[END_REF]. Since SPCA1 plays an important role in both post-translational modification of proteins in the Golgi apparatus and transit of proteins through the secretory pathway, the aims of this study was to investigate whether SPCA1 expression and / or activity was different between normal and high glucose cultured A7r5 cells, a commonly used vascular smooth muscle cell line.

Measurements of mRNA and protein levels in this study showed A7r5 cells grown in HG culture media expressed measurably more SPCA1 than cells grown in NG culture media, which suggests glucose availability can influence the expression of this protein. There was a notable difference, however, in the extent of change between SPCA1 mRNA and protein expression levels, whereby a 60% increase in mRNA resulted in only about 16% increase in protein. A similar discrepancy was observed by Reinhardt et al., [START_REF] Reinhardt | Ca 2+ -ATPase protein expression in mammary tissue[END_REF], when they compared SPCA expression levels between rat mammary and brain tissue, and attributed their finding to a difference in protein turnover. A similar explanation may also, at least in part, explain our observation in A7r5 cells. Transcriptional control of SPCA1 may also play a role, since Sp1 and YY1, which are the transcription factors of the SPCA1 gene [30], possess increased activity in cells grown in high, compared to normal, glucose media [START_REF] Han | Reduced O glycosylation of Sp1 is associated with increased proteasome susceptibility[END_REF][START_REF] Hiromura | YY1 is regulated by O-linked N-acetylglucosaminylation (O-GlcNAcylation)[END_REF]. One possibility could be that a combination of enhanced transcription and near-maximal translation of the SPCA1 gene in hyperglycaemic A7r5 cells may result in an increase in its mRNA, but not necessarily increase in its protein levels to the same extent.

Consistent with data from PCR-based mRNA measurement and immunochemical detection of SPCA1 protein, thapsigargin-insensitive Ca 2+ -dependent ATPase activity in microsomal membranes (representative of SPCA1 activity) was also found to be higher from HG, compared to NG, cultured cells. However, the mean activity for the HG cultured cells was about 2-fold higher than that for the NG cultured cells, which would also appear to be disproportionate to the increase in SPCA1 protein levels observed. Such differences may be explained by the differences in Ca 2+ transport activities between different SPCA1 splice variants [START_REF] Dode | Functional comparison between secretory pathway Ca 2+ /Mn 2+ -ATPase (SPCA) 1 and sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) 1 isoforms by steady-state and transient kinetic analyses[END_REF]. In our study we measured all SPCA activity and the expression of all SPCA1 slice-variants. However, one could envisage that an increased expression of SPCA1 splice variants that have higher levels of activity, could contribute to the higher than normal SPCA activity observed in HG cultured cells. However, this possibility needs to be further explored.

In order to assess whether higher than normal expression of SPCA1 within HG cultured A7r5 cells can alter agonist-induced cytosolic [Ca 2+ ] transients, the effects of AVP on cytosolic [Ca 2+ ] profiles were investigated. AVP (working through the phosphoinositide pathway) initiates Ca 2+ mobilisation from internal Ca 2+ stores and stimulates Ca 2+ entry from the extra-cellular medium. Cytosolic Ca 2+ removal (by uptake into intra-cellular stores and extrusion out of the cell) then follows, resulting in a typical Ca 2+ transient profile [24]. In our study, using thapsigargin pre-treated A7r5 cells, we showed that although the peak heights of SERCA-independent AVP-induced Ca 2+ transients were not significantly different between NG and HG cultured cells, these transients were different with regards to both their rise and decay phase kinetics. The differences in the rise and decay phase kinetics could be attributed to differences in the abundance of thapsigargin-insensitive IP 3 -releaseable Ca 2+ -stores (i.e. Golgi Ca 2+ stores). Thus if HG cultured cells had more of these Ca 2+ stores than NG cultured cells, this could result in faster rates of Ca 2+ release, as well as faster rates of Ca 2+ re-uptake (due to their higher levels of SPCA). This becomes even more apparent once SERCA-containing Ca 2+ stores have been emptied by thapsigargin treatment.

Our immuno-staining studies confirm that SPCA1 remains located to the distinct regions within the cell consistent with being the Golgi apparatus, rather than more widely distributed throughout the ER like SERCA. Furthermore, increased SPCA expression, as observed in HG cultured cells, does not appear to affect its subcellular location (figure 4).

In summary, this study has shown that culturing A7r5 cells (a commonly used VSMC model) in high glucose media (which mimics the hyperglycaemic state associated with severe diabetes mellitus) can lead to: (1) higher SPCA1 expression levels (both at the mRNA and protein level); (2) increased thapsigargin-insensitive Ca 2+ -ATPase activity, which is comparable to the activity observed for SERCA; and (3) faster rates of both the rise and decay phases of AVP-induced Ca 2+ transients in thapsigargin-pretreated cells. These findings suggest that glucose availability can substantially affect the Ca 2+ handling properties within the Golgi apparatus, and therefore affect the secretory pathway and post-translational protein modification processes, as well as Ca 2+ -mediated hormonal responses, in A7r5 VSMCs. If these changes in SPCA1 expression and activity also occur in primary VSMCs, this might potentially contribute towards cardiovascular diseases often associated with diabetes. Therefore further studies are required, using primary VSMCs cultured in different glucose media, as well as studying cells isolated from normal and severely diabetic animals, in order to corroborate this hypothesis. For each sample used, mean relative band intensities (where values from HG cells were made relative to those from NG cells, after correcting for background) for both SPCA1 and SERCA were calculated from blots derived from cells of three replicate culture experiments. These were then used to calculate a final mean relative band intensity value for each protein of interest and percentage changes in these proteins for HG cells relative to NG cells were calculated. Error bars show the S.E.M of the differences. Statistically significant P-values (where P <0.05) are indicated by *. The images under each data bar show the associated typical protein band intensities observed for each of the target proteins, whereby HG and NG represents the proteins derived from cells grown under hyperglycaemic and normal glucose culture conditions, respectively. The molecular mass for each protein was, from left to right, ~100kDa for both SPCA1 and SERCA and 42kDa for α-actin, respectively. A c c e p t e d M a n u s c r i p t

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Bioscience

  Reports Immediate Publication. Published on 16 Jun 2009 as manuscript BSR20090058 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090058

Bioscience

  Reports Immediate Publication. Published on 16 Jun 2009 as manuscript BSR20090058 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090058A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  Bioscience Reports Immediate Publication. Published on 16 Jun 2009 as manuscript BSR20090058 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090058

Bioscience

  Reports Immediate Publication. Published on 16 Jun 2009 as manuscript BSR20090058 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090058A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Bioscience

  Reports Immediate Publication. Published on 16 Jun 2009 as manuscript BSR20090058 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090058A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Bioscience

  Reports Immediate Publication. Published on 16 Jun 2009 as manuscript BSR20090058 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090058A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

BioscienceLegendsFigure 1 .

 1 Figure 1. Differences in mRNA content for SPCA1, SERCA, GAPDH and βactin in hyperglycaemic compared to normal glucose-cultured A7r5 cells. For each sample, a mean band intensity value (corrected for background and made relative to their matching RPL19 band intensity) was determined from mRNA extracted from cells derived from three replicate culture conditions. The PCR reactions were also repeated in triplicate and for each primer pair. A final mean relative band intensity value was calculated from three replicates, for each primer pair and for each glucose culture condition. Relative percentage changes were calculated of values obtained from HG relative to the NG cells (HG/NG). Error bars show S.E.M. Statistical significant P-values are indicated by * (for P values <0.05) and ** (for P values <0.01). The images under each data bar show the associated typical PCR product band intensities observed for each of the primer pairs, whereby HG represents hyperglycaemic and NG represent normal glucose culture conditions. The PCR product band sizes for each mRNA of interest were, from left to right, 224bp (SPCA1), 288bp (SERCA), 400bp (GAPDH) and 255bp (β-actin).

Figure 2 .

 2 Figure 2. Differences in protein content for SPCA1, SERCA and α-actin in hyperglycaemic compared to normal glucose-cultured A7r5 cells.For each sample used, mean relative band intensities (where values from HG cells were made relative to those from NG cells, after correcting for background) for both SPCA1 and SERCA were calculated from blots derived from cells of three replicate culture experiments. These were then used to calculate a final mean relative band intensity value for each protein of interest and percentage changes in these proteins for HG cells relative to NG cells were calculated. Error bars show the S.E.M of the differences. Statistically significant P-values (where P <0.05) are indicated by *. The images under each data bar show the associated typical protein band intensities observed for each of the target proteins, whereby HG and NG represents the proteins derived from cells grown under hyperglycaemic and normal glucose culture conditions, respectively. The molecular mass for each protein was, from left to right, ~100kDa for both SPCA1 and SERCA and 42kDa for α-actin, respectively.

Figure 3 .

 3 Figure 3. Ca 2+ transients induced by AVP following pre-treatment with thapsigargin in hyperglycaemic and normal glucose-cultured A7r5 cells.For both (A) NG and (B) HG glucose culture conditions, the main figures show data from four coverslips, whereby each trace represents averaged fluorescence values from ten cells, analysed for Fluo-3 fluorescence changes in response to 10nM AVP, after pre-treatment of cells with thapsigargin. Insets show the typical response profile (averaged from ten cells) for 10nM AVP after pre-treatment with DMSO (vehicle control for thapsigargin) in cells from the same glucose culture conditions. Arrows indicate when AVP is added.

Figure 4 .

 4 Figure 4. Immuno-localisation of SPCA1, Golgi apparatus and SERCA in A7r5 cells.This figures shows immuno-staining for SPCA1 using the anti-SPCA1 antibody (1:30 dilution) in (A) NG and (B) HG cultured cells. NG cultured cells were used to identify the location of the Golgi apparatus with TMR-WGA (10µg/ml) (C), and the Y1F4 antibody (1:100 dilution) for SERCA (D). Cells were viewed at 200x magnification under the epi-fluorescence microscope.

  Bioscience Reports Immediate Publication. Published on 16 Jun 2009 as manuscript BSR20090058

Table 1 . Ca 2+ -dependent ATPase activities of microsomal membranes from normal glucose and hyperglycaemic cultured A7r5 cells. Ca 2+ -Dependent ATPase Activity (nmol/min/mg protein) ± S.E.M

 1 Bioscience Reports Immediate Publication. Published on 16 Jun 2009 as manuscript BSR20090058 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090058 A c c e p t e d M a n u s c r i p t

	Membrane	No Thapsigargin	1µM Thapsigargin
	Preparation
		NG	HG	NG	HG
	1	3.2 ± 0.5	3.1 ± 0.7	0.62 ± 0.09 1.05 ± 0.17
	2	4.1 ± 1.6	2.7 ± 0.4	0.38 ± 0.07 2.89 ± 1.21
	3	4.4 ± 0.5	3.7 ± 0.2	1.26 ± 1.17 1.63 ± 0.48

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Table 2 . Kinetics measurements from AVP-mediated Ca 2+ responses in normal glucose and hyperglycaemic cultured A7r5 cells, following pre-treatment with thapsigargin Kinetics Measurements from Ca 2+ Transient Profiles of AVP Responses in Thapsigargin Pre-treated Cells NG HG Time taken to reach half- maximal response (s)

 2 Bioscience Reports Immediate Publication. Published on 16 Jun 2009 as manuscript BSR20090058 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090058

	48.3 ± 5.1	18.2 ± 3.2

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Acknowledgements

The BBSRC are thanked for a PhD studentship to PL. Prof. Colin Taylor is thanked for supplying the A7r5 cells. Dr Jeremy Pritchard is thanked for help with the statistical analysis.


Paul G Crichton

Nadeene Parker

Antonio J Vidal-Puig

Martin D Brand

NOT ALL MITOCHONDRIAL CARRIER PROTEINS SUPPORT PERMEABILITY TRANSITION PORE FORMATION: NO INVOLVEMENT OF UNCOUPLING PROTEIN 1

come

Not all mitochondrial carrier proteins support permeability transition pore formation: no involvement of uncoupling protein 1

2

SYNOPSIS

The mitochondrial permeability transition pore (mPTP) is a non-specific channel that forms in the mitochondrial inner membrane in response to several stimuli, including elevated levels of matrix calcium. The pore is proposed to be comprised of the adenine nucleotide translocase (ANT), voltage dependent anion channel and cyclophilin D. Knockout studies, however, have demonstrated that ANT is not essential for permeability transition, which has led to the proposal that other members of the mitochondrial carrier protein family may be able to play a similar function to ANT in pore formation. To investigate this possibility we have studied the permeability transition properties of brown adipose tissue mitochondria in which levels of the mitochondrial carrier protein, uncoupling protein 1 (UCP1), can exceed those of ANT. Using an improved spectroscopic assay we have quantified mPTP formation in de-energised mitochondria from wild type and Ucp1 knockout mice and assessed the dependence of pore formation on UCP1. When correctly normalised for differences in mitochondrial morphology, we find that calcium-induced mPTP activity is the same in both types of mitochondria, with similar sensitivity to GDP (~50% inhibited), although the portion sensitive to cyclosporin A is higher in mitochondria lacking UCP1 (~80% inhibited, compared to ~60% in mitochondria containing UCP1). We conclude that UCP1 is not a component of the cyclosporin A-sensitive mPTP in brown adipose tissue and that a role in mPTP formation is not a general characteristic of the mitochondrial carrier protein family but is more likely to be restricted to specific members including ANT. KEY WORDS: adenine nucleotide translocase, brown adipose tissue mitochondria, mitochondrial carrier protein, mitochondrial swelling, permeability transition pore, uncoupling protein 1

The abbreviations used are: mPTP, mitochondrial permeability transition pore; ANT, adenine nucleotide translocase; VDAC, voltage dependent anion channel; BAT, brown adipose tissue; UCP1, uncoupling protein 1; Ucp1KO, Ucp1 knockout; FCCP, carbonylcyanide-ptrifluoromethoxyphenylhydrazone; NTA, nitrilotriacetic acid.

INTRODUCTION

Mitochondria possess a Ca 2+ -inducible non-specific pore that allows the free movement of solutes less than ~1.5 kDa across the mitochondrial inner membrane. Known as the mitochondrial permeability transition pore (mPTP), its activity is not compatible with the conventional chemiosmotic processes of mitochondria and causes matrix swelling, the collapse of protonmotive force and ATP depletion, leading to compromised cell viability. The mPTP has a clear involvement in necrotic cell death induced by calcium overload and oxidative stress, and may play a physiological role in the release of signalling factors from the mitochondrial intermembrane space during apoptosis (see [START_REF] Tsujimoto | Mitochondrial membrane permeability transition and cell death[END_REF][START_REF] Halestrap | Biochemistry: A pore way to die[END_REF][START_REF] Halestrap | Calcium, mitochondria and reperfusion injury: a pore way to die[END_REF][START_REF] Rasola | The mitochondrial permeability transition pore and its involvement in cell death and in disease pathogenesis[END_REF] for reviews).

Although the components of the mPTP are yet to be conclusively identified, pore formation is proposed to involve cyclophilin D, the adenine nucleotide translocase (ANT) and the voltage dependent anion carrier (VDAC) [START_REF] Tsujimoto | Mitochondrial membrane permeability transition and cell death[END_REF][START_REF] Halestrap | Biochemistry: A pore way to die[END_REF]. Cyclophilin D is a mitochondrial matrix located, peptidyl prolyl cis-trans isomerase that associates with the mitochondrial inner membrane during conditions that promote permeability transition [START_REF] Connern | Recruitment of mitochondrial cyclophilin to the mitochondrial inner membrane under conditions of oxidative stress that enhance the opening of a calcium-sensitive non-specific channel[END_REF][START_REF] Connern | Chaotropic agents and increased matrix volume enhance binding of mitochondrial cyclophilin to the inner mitochondrial membrane and sensitize the mitochondrial permeability transition to [Ca 2+[END_REF]. It is thought to induce a conformational change in a membrane protein to bring about pore formation [START_REF] Halestrap | Elucidating the molecular mechanism of the permeability transition pore and its role in reperfusion injury of the heart[END_REF]. The drug cyclosporin A inhibits cyclophilin D and is the classic inhibitor of the permeability transition [START_REF] Crompton | Inhibition by cyclosporin A of a Ca 2+dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress[END_REF][START_REF] Halestrap | Inhibition of Ca 2+ -induced large-amplitude swelling of liver and heart mitochondria by cyclosporin is probably caused by the inhibitor binding to mitochondrial-matrix peptidyl-prolyl cis-trans isomerase and preventing it interacting with the adenine nucleotide translocase[END_REF]. Mice lacking cyclophilin D do not undergo cyclosporin A-sensitive permeability transition but still form mPTP at higher matrix Ca 2+ concentrations [10-12], indicating that cyclophilin D is not an essential component but may act to sensitise the Ca 2+ trigger site of the pore.

ANT has long been implicated in mPTP (see [START_REF] Halestrap | Elucidating the molecular mechanism of the permeability transition pore and its role in reperfusion injury of the heart[END_REF]) and is thought to be the membrane target of cyclophilin D. Ligands of ANT that promote the 'c' (carboxyatractyloside) or 'm' conformation (bongkrekic acid) stimulate or inhibit mPTP, respectively. Matrix ADP binding to ANT also inhibits mPTP and may be key in the mechanism by which mPTP formation is inhibited by ∆ψ [START_REF] Halestrap | Elucidating the molecular mechanism of the permeability transition pore and its role in reperfusion injury of the heart[END_REF]13]. Importantly, a direct interaction between cyclophilin D and ANT has been demonstrated using cyclophilin D affinity columns and co-immunoprecipitation [START_REF] Halestrap | Calcium, mitochondria and reperfusion injury: a pore way to die[END_REF][START_REF] Crompton | Cyclophilin-D binds strongly to complexes of the voltage-dependent anion channel and the adenine nucleotide translocase to form the permeability transition pore[END_REF][START_REF] Woodfield | Direct demonstration of a specific interaction between cyclophilin-D and the adenine nucleotide translocase confirms their role in the mitochondrial permeability transition[END_REF]. Strong evidence that supports ANT as the primary component of mPTP is provided by reconstitution studies. In response to high Ca 2+ concentrations, phospholipid membranes containing purified ANT generate a non-specific pore that may represent a basic mPTP structure [16,[START_REF] Brustovetsky | A large Ca 2+ -dependent channel formed by recombinant ADP/ATP carrier from Neurospora crassa resembles the mitochondrial permeability transition pore[END_REF]. Reconstitution of ANT as part of a cyclophilin D-ANT-VDAC complex, recovered from a cyclophilin D affinity column, also results in Ca 2+ -inducible pore formation that, importantly, can be inhibited by cyclosporin A [START_REF] Crompton | Cyclophilin-D binds strongly to complexes of the voltage-dependent anion channel and the adenine nucleotide translocase to form the permeability transition pore[END_REF]. The exact influence of VDAC in mPTP formation is not clear though mitochondria from mice ablated of VDAC1 exhibit unchanged permeability transition properties suggesting this isoform, at least, plays only a minor role [START_REF] Krauskopf | Properties of the permeability transition in VDAC1 -/mitochondria[END_REF].

Mouse mitochondria lacking ANT still exhibit cyclosporin A-sensitive mPTP formation, indicating that ANT is not an essential component of mPTP [START_REF] Kokoszka | The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore[END_REF]. A possible explanation of this finding is that other proteins, similar to ANT, can perform the same structural role. ANT is typically the most abundant member of a large family of mitochondrial carrier proteins that share general structural features [START_REF] Kunji | The role and structure of mitochondrial carriers[END_REF]. Formation of cyclosporin A-sensitive mPTP may be a general feature of this family and the well-established influence of ANT merely a reflection of its abundance over other mitochondrial carriers. To address this possibility, we have studied the permeability transition properties of brown adipose tissue (BAT) mitochondria from mice kept at 21ºC, which are atypical in that they contain a mitochondrial carrier protein, uncoupling protein 1 (UCP1), at concentrations comparable to ANT [START_REF] Stuart | A mitochondrial uncoupling artifact can be caused by expression of uncoupling protein 1 in yeast[END_REF]. Using an improved spectroscopic assay of mitochondrial swelling, we have quantified Ca 2+ -induced mPTP formation in BAT mitochondria isolated from both wild type and Ucp1KO mice. When correctly normalised for differences in A c c e p t e d M a n u s c r i p t 4 the relationship between mitochondrial absorbance and matrix volume, we find that mPTP formation in Ucp1KO mitochondria is the same as in wild type mitochondria, but the fraction sensitive to cyclosporin A is increased. These results indicate UCP1 does not catalyze cyclosporin A-sensitive mPTP and ANT's role in mPTP is not a general characteristic of the mitochondrial carrier protein family.

EXPERIMENTAL

Animals

Male and female mice were housed at 21 ± 2 ºC, 57 ± 5% humidity, 12/12 h light/dark, with standard chow and water ad libitum, following UK Home Office Guidelines for the Care and Use of Laboratory Animals. Ucp1 -/-(termed Ucp1KO) [START_REF] Enerback | Mice lacking mitochondrial uncoupling protein are coldsensitive but not obese[END_REF] and wild type, sibling paired mice were used at age 7 -12 months. Ucp1 ablation was confirmed by Western analysis of the protein and PCR analysis of the genomic loci.

Isolation of mitochondria

BAT mitochondria were isolated as described in [23] but with the omission of EGTA in the final resuspension medium. Mitochondrial protein was estimated by the biuret method.

Mitochondrial swelling

BAT mitochondria (0.15 mg) were stirred in 1.5 mL reaction medium (0.25 M mannitol, 1 mM nitrilotriacetic acid (tetraethyl ammonium salt), 20 mM MOPS, 10 mM Tris, pH 7.2 at 25°C) and the absorbance at 520 nm was followed using a Perkin Elmer lambda 18 spectrophotometer. To fully de-energise mitochondria and equilibrate Ca 2+ across membranes, a cocktail of 0.2 µM rotenone, 0.2 µM antimycin A and 2 µM A23187 was added after mitochondrial addition, followed by 1.3 µM FCCP. Swelling was induced by addition of calcium gluconate to give the free Ca 2+ concentration required under buffered conditions. Free calcium concentrations in the reaction medium were determined by following calcium green-5N (Molecular probes, Invitrogen) fluorescence calibrated to calcium standards in reaction medium lacking nitrilotriacetic acid. Alternatively, immediate swelling was achieved upon addition of mitochondria to reaction medium in which mannitol was replaced by 0.25 M erythritol. Swelling associated with erythritol uptake, specifically, was estimated by subtracting equivalent values of A -1 obtained in the presence of mannitol medium. In all cases, A -1 data were fit using exponentials to estimate initial rates (r 2 ≥ 0.99).

Phospholipid estimations

Mitochondrial lipids were extracted using chloroform and methanol, and the phospholipid phosphorus content estimated as described in [START_REF] Porter | Allometry of mitochondrial proton leak: Influence of membrane surface area and fatty acid composition[END_REF]. Values given (see main text) are averages (±S.E.) from five separate mitochondrial preparations.

RESULTS & DISCUSSION

Time-resolved mPTP formation in isolated mitochondria has typically been assessed by monitoring the collapse of ∆ψ or by following the absorbance changes associated with the matrix volume changes that accompany solute uptake. With either approach, the use of energised mitochondria complicates mechanistic interpretation as any affect on mPTP may be indirect, via ∆ψ, particularly if UCP1 is involved. Also, neither approach is ideal for a quantitative analysis of the time course of mPTP formation. Mitochondrial absorbance measurements, for instance, suffer from a non-linear relationship with matrix volume that can vary between mitochondria from different sources. To address this deficit in methodology, we developed an improved spectroscopic method to quantify cyclosporin A-sensitive mPTP in BAT mitochondria under deenergised conditions.

Mitochondria behave as simple osmometers in that their matrix volume is proportional to the inverse osmolality of the support medium [START_REF] Beavis | Swelling and contraction of the mitochondrial matrix. I. A structural interpretation of the relationship between light scattering and matrix volume[END_REF][START_REF] Diresta | K + -H + exchange and volume homeostasis in brown adipose tissue mitochondria[END_REF][27]. In accordance with previous reports [START_REF] Diresta | K + -H + exchange and volume homeostasis in brown adipose tissue mitochondria[END_REF], we find that the inverse of the apparent absorbance of BAT mitochondria, equilibrated in media of various osmotic strengths, also changed linearly with inverse osmolality of the medium (Figure 1, inset). When expressed as A -1 and in the range measured, therefore, absorbance measurements can be used quantitatively to report relative changes in mitochondrial matrix water content. Addition of 500 µM free Ca 2+ (as calcium gluconate, the anion of which is not transported by UCP1 [START_REF] Jezek | New substrates and competitive inhibitors of the Cltranslocating pathway of the uncoupling protein of brown adipose tissue mitochondria[END_REF]) to BAT mitochondria pre-equilibrated in 0.25 M mannitol medium induced rapid swelling associated with the uptake of mannitol (trace a, Figure 1A). This activity was inhibited by cyclosporin A (trace b), confirming that it is a consequence of mPTP formation. Intriguingly, inhibition occurred in the absence of phosphate in the incubation medium, in contrast to situation in liver mitochondria where mPTP inhibition by cyclosporin A is dependent upon the presence of this anion (see [START_REF] Basso | Phosphate is essential for inhibition of the mitochondrial permeability transition pore by cyclosporin A and by cyclophilin D ablation[END_REF]). Phosphate regulation of mPTP inhibition by cyclosporin A, therefore, may not occur in BAT mitochondria or, instead, may just be dependent upon the assay conditions used. Importantly in our assay conditions, mitochondria were kept fully de-energised (see 'Experimental' section) to avoid indirect affects of conventional UCP1 activity on mPTP. Initial rates of Ca 2+ -induced swelling were linearly dependent on the free Ca 2+ -concentration and were ~60 % sensitive to cyclosporin A (Figure 1B). This simple relationship suggests that our measurements do, indeed, report relative mPTP activities.

An initial assessment of mPTP formation in BAT mitochondria from Ucp1KO mice suggested that the rates of Ca 2+ -induced swelling were lower than in mitochondria from wild type mice (traces b and a, respectively, Figure 2A). Caution must be taken, however, as these differences could have been due to general differences in the light scattering properties of Ucp1KO and wild type mitochondria that were unrelated to mPTP. Indeed, a preliminary assessment of the dependence of A -1 on osmolality -1 for Ucp1KO mitochondria, indicated a linear, but shallower, relationship compared to wild type mitochondria (data not shown). This suggests that the two mitochondrial populations were morphologically different and potentially exhibited different increments in A -1 for a specific amount of solute imported during permeability measurements.

To correct for these differences, we measured swelling rates induced by erythritol. Biological membranes are permeable to this sugar, which enters mitochondria independently of mPTP and other transporters (see [START_REF] Garlid | Swelling and contraction of the mitochondrial matrix. II. Quantitative application of the light scattering technique to solute transport across the inner membrane[END_REF]). Rates of permeation per mg of mitochondrial protein will depend on membrane lipid composition and will be proportional to membrane surface area. Wild type and Ucp1KO mitochondria have the same phospholipid fatty acyl group composition [START_REF] Ocloo | Cold-induced alterations of phospholipid fatty acyl composition in brown adipose tissue mitochondria are independent of uncoupling protein-1[END_REF] and our estimates of mitochondrial lipid phosphorus (2.7 ± 0.2 µg/mg protein for wild type and 2.4 ± 0.4 µg/mg protein for Ucp1KO mitochondria), as a marker of total lipid, suggested that they do not have significantly different surface areas. Therefore, the initial rate of erythritol entry into both mitochondrial types should be the same, regardless of A -1 signal. Upon addition of A c c e p t e d M a n u s c r i p t 6 mitochondria to 0.25 M erythritol medium, it can be seen from Figure 2B that the spontaneous increase of A -1 associated with erythritol uptake in Ucp1KO mitochondria (trace b relative to d) was lower than in wild type mitochondria (traces a relative to c). This confirms that the relationship between A -1 per mole of solute uptake was different in the two mitochondrial populations, which potentially accounts for the apparent decrease in the mPTP-dependent rates observed in Ucp1KO mitochondria (cf. Figure 2A). By expressing Ca 2+ -induced rates as a function of erythritol-induced rates, mPTP activity in both types of mitochondria can be compared in the same units of solute influx.

Figure 3 shows average rates of Ca 2+ -induced swelling of wild type and Ucp1KO mitochondria in the absence or presence of GDP or cyclosporin A. When correctly normalised to erythritol-induced rates, there was no effect of UCP1 ablation on Ca 2+ -induced solute flux in the presence or absence of the UCP1 inhibitor GDP, suggesting that UCP1 is not involved in mPTP formation (Figure 3A). Note that UCP1-dependent mPTP formation in these results was not masked by compensatory changes in ANT concentration, as both mitochondrial types contain similar amounts of ANT as assayed by Western blot (data not shown). GDP inhibited mPTP formation by ~50% (Figure 3A andB). This presumably occurs via an interaction with ANT, similar to the inhibitory affects of adenine nucleotides on mPTP [START_REF] Halestrap | Elucidating the molecular mechanism of the permeability transition pore and its role in reperfusion injury of the heart[END_REF], which is not without precedent as GDP has been shown to inhibit ANT-mediated proton conductance [32]. Intriguingly, the cyclosporin-sensitive fraction of mPTP activity was significantly higher in the absence of UCP1 (~80 inhibited by cyclosporin, compared to ~60% in the presence of UCP1, Figure 3A andB). This could reflect a stress response that increases mPTP components, or their sensitivity to Ca 2+ , indirectly related to the absence of UCP1 or it could reflect a minor role of UCP1 in cyclosporin A-insensitive mPTP formation. If either explanation were true, however, why a similar amplitude difference was not seen in the absence of cyclosporin A is not clear (Figure 3A). Alternatively, it may indicate changes in factors that influence cyclosporin efficacy. An increased concentration of endogenous phosphate associated with the loss of UCP1 could be one possibility, assuming that phosphate does indeed influence cyclosporin inhibition of mPTP in BAT mitchondria. Most importantly, these results show that that Ca 2+ -induced solute uptake activity, as well as the fraction of activity sensitive to cyclosporin A, was not lowered by UCP1 ablation, ruling out a structural role of UCP1 in the cyclosporin A-sensitive mPTP.

ANT is proposed to play a primary role in the formation of mPTP. However, Kokoszka et al. [START_REF] Kokoszka | The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore[END_REF] have demonstrated that mouse liver mitochondria in which ANT has been ablated, still undergo cyclosporin A-sensitive permeability transition, albeit requiring higher calcium loads to do so. One interpretation of this finding is that other less abundant mitochondrial carrier proteins contribute to mPTP formation in the absence of ANT, reflecting a general role of this protein family in mPTP formation (e.g. [START_REF] Halestrap | Mitochondrial permeability: dual role for the ADP/ATP translocator?[END_REF]). In the present study, we developed a simple quantitative assay to study mPTP formation in mitochondria from wild type and Ucp1KO mice. Our results demonstrate that UCP1 does not facilitate the cyclosporin A-sensitive mPTP, indicating that a role in mPTP formation is not a general characteristic of the mitochondrial carrier protein family. There is strong evidence that ANT is a functional component of the mPTP, rather than just influencing pore activity. In particular, several studies have shown that mPTP-like activity can be achieved following reconstitution of purified ANT into lipid bilayers [START_REF] Crompton | Cyclophilin-D binds strongly to complexes of the voltage-dependent anion channel and the adenine nucleotide translocase to form the permeability transition pore[END_REF]16,[START_REF] Brustovetsky | A large Ca 2+ -dependent channel formed by recombinant ADP/ATP carrier from Neurospora crassa resembles the mitochondrial permeability transition pore[END_REF]. In view of our conclusions, a likely explanation of the finding of Kokoszaka et al. [START_REF] Kokoszka | The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore[END_REF] is that, in addition to ANT, only specific members of the mitochondrial carrier family are involved in mPTP formation. This may be restricted to carriers that are more widespread across various tissues. A particularly strong candidate is the mitochondrial phosphate carrier. This carrier has recently A c c e p t e d M a n u s c r i p t been shown to bind cyclophilin D, similarly to ANT, in affinity column and coimmunoprecipitation studies and may, therefore, contribute to cyclosporin A-sensitive mPTP in the absence of ANT [START_REF] Leung | The mitochondrial phosphate carrier interacts with cyclophilin D and may play a key role in the permeability transition[END_REF][START_REF] Leung | Recent progress in elucidating the molecular mechanism of the mitochondrial permeability transition pore[END_REF].

In summary, we have developed a simple quantitative assay of mPTP formation in BAT mitochondria and demonstrated that UCP1 does not partake in cyclosporin A-sensitive mPTP formation. This finding is of significant importance as it eliminates a popular hypothesis that a role in cyclosporin A-sensitive mPTP formation is a general feature of the mitochondrial carrier protein family.

A c c e p t e d M a n u s c r i p t A. Calcium-induced rates of swelling, obtained in the absence (Ca) or presence of 1.1 µM cyclosporin A (Cy A) or 1 mM GDP, are shown normalised to erythritol-induced rates (V cal /V eryth ) for wild type (dark columns) and Ucp1KO mitochondria (light columns). Initial rates of mitochondrial swelling (A -1 /min) were measured as described for Figure 2 (see 'Experimental' section). B. Calcium-induced rates of swelling (V) in the absence or presence of Cy A or GDP expressed as a fraction of the rate obtained with calcium alone (V Ca ). All values are averages (±S.E.) from three independent experiments. Values significantly different between wild type and Ucp1KO mitochondria are indicated (p < 0.05 by paired Student's t-test).

Bioscience

  Reports Immediate Publication. Published on 21 Jul 2009 as manuscript BSR20090063 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090063

Bioscience

  Reports Immediate Publication. Published on 21 Jul 2009 as manuscript BSR20090063 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090063 A c c e p t e d M a n u s c r i p t

Bioscience

  Reports Immediate Publication. Published on 21 Jul 2009 as manuscript BSR20090063 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090063

Bioscience

  Reports Immediate Publication. Published on 21 Jul 2009 as manuscript BSR20090063 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20090063

Figure 1 .

 1 Figure 1. Calcium-induced cyclosporin A-sensitive swelling of de-energised BAT mitochondria. A. Inverse of A 520 (A -1 ) associated with mitochondrial swelling induced by addition of 500 µM free Ca 2+ in the absence (a) or presence of 1.1 µM cyclosporin A (b). No calcium control (c). Inset: The dependence of A -1 associated with mitochondria, on inverse osmolality (Osm -1 ) of the suspension medium. De-energised mitochondria were equilibrated for 1 min in reaction medium containing various concentrations of mannitol to give the required osmolality. Values are averages (±S.E.) from four independent experiments. B. The dependence of initial rates of calcium-induced swelling on the free Ca 2+ concentration added in the absence (closed) or presence (open) of 1.1 µM cyclosporin A. Typical traces and rates are shown and are representative of at least one repeated experiment.

Figure 2 .

 2 Figure 2. Calcium-and erythritol-induced swelling of BAT mitochondria from wild type and Ucp1KO mice. Inverse of A 520 (A -1 ) associated with the swelling of wild type (solid lines) and Ucp1KO mitochondria (dashed lines). A. Swelling induced by 500 µM free Ca 2+ (a and b). No calcium controls (c and d). B. Spontaneous swelling induced by addition of mitochondria to erythritol (a and b) or mannitol medium (c and d). Typical traces are shown and are representative of three repeated experiments.

Figure 3 .

 3 Figure 3. Calcium-induced mPTP formation in BAT mitochondria from wild type and Ucp1KO mice.A. Calcium-induced rates of swelling, obtained in the absence (Ca) or presence of 1.1 µM cyclosporin A (Cy A) or 1 mM GDP, are shown normalised to erythritol-induced rates (V cal /V eryth ) for wild type (dark columns) and Ucp1KO mitochondria (light columns). Initial rates of mitochondrial swelling (A -1 /min) were measured as described for Figure2(see 'Experimental' section). B. Calcium-induced rates of swelling (V) in the absence or presence of Cy A or GDP expressed as a fraction of the rate obtained with calcium alone (V Ca ). All values are averages (±S.E.) from three independent experiments. Values significantly different between wild type and Ucp1KO mitochondria are indicated (p < 0.05 by paired Student's t-test).

Bioscience

  FIGURES

Figure 2 Bioscience 12 Figure 3

 2123 Figure 2

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ACKNOWLEDGEMENTS

We thank Prof. Barbara Cannon for Ucp1KO mice and Julie Buckingham for technical assistance. This work was supported by the Medical Research Council and the Wellcome Trust (grants 065326/Z/01/Z and 066750/B/01/Z).