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Cardiovascular Research 1998 40(3):564-572; doi:10.1016/S0008-6363(98)00193-X
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Identification of phospholipase C β isoforms and their location in cultured vascular smooth muscle cells of pig, human and rat

Lynda Blayney*, Peter Gapper and Colin Rix

Department of Cardiology, University of Wales College of Medicine, Heath Park, Cardiff, CF4 4XN, UK

* Corresponding author. Tel.: +44-1222-744256; fax: +44-1222-743500.

Received 15 January 1998; accepted 28 April 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: Four phospholipase C (PLC) β isoforms have been described in pig aortic vascular smooth muscle. The aim was to determine if all four PLC β isoforms are commonly expressed in vascular smooth muscle cells (VSMC) of three species, i.e. pig, human and rat, and if the individual isoforms had distinctive intracellular distributions. Methods: Vascular smooth muscle cell cultures were derived from explants of porcine and rat aorta and a human renal artery cell line. PLC β isoform content was resolved using Western blotting. Intracellular location was determined by immunocytochemistry and confocal microscopy. Results: All three species expressed PLCs β1, β2, β3 and β4. In all species, PLC β1 demonstrated foci of concentration throughout the cytoplasm; PLC β2 demonstrated a punctate pattern that was principally at the cell periphery or was in the Golgi, depending upon the antibody used; PLC β3 was also cytoplasmic but showed a different pattern from PLC β1 and PLC β4 was cytoplasmic, except in pig quiescent cells, where it was associated with filamentous structures at the intersection with the plasma membrane. Conclusions: VSMCs of three different species express all four PLC β isoforms. Each isoform has a unique and consistent signature of distribution that is generally common to all species.

KEYWORDS Rat; Pig; Human; Vascular smooth muscle cell cultures; Phospholipase C; Intracellular localisation; Confocal microscopy


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
There are three families of phosphatidyl-D-inositol, 4,5-bisphosphate (PIP2)-directed phospholipase C (PLC) isoforms, which are classified according to their cDNA sequences [1]. These enzymes break down PIP2 into the second messengers inositol trisphosphate (IP3) and diacylglycerol (DAG). The PLC {gamma} isoforms are activated by tyrosine kinase type receptors, PLC β isoforms (β1, β2, β3 and β4) are activated by seven membrane-spanning receptors via G-proteins and the mechanism of activation of PLC {delta} is unknown. Studies with porcine smooth muscle showed that four PLC β isoforms, PLC {gamma}1 and PLC {delta}1 were present in soluble fractions prepared from medial tissue [2]. The role of so many PLC β isoforms in one homogeneous cell type is unclear from their biochemistry. Studies with purified proteins have shown that all four PLC β isoforms are activated via G-proteins of the G{alpha}q family and also (with the exception of PLC β4) by G-protein β{gamma} subunits [3]. More specificity of signalling is observed in isolated membrane fractions [4], or in permeabilised whole cells where specific receptor pathways are associated with activation of specific PLC β isoforms [5–7]. This difference may reflect the contribution of compartmentalisation to the specificity of PLC signalling pathways in intact cells, enabling some protein–protein interactions but barring others [8]. In previous studies, distinct cellular locations have been observed for PLC {gamma}1 and PLC β1 isoforms. This varied with cell type and with growth conditions. PLC {gamma}1 has been associated with the cytoskeleton [9, 10]and has also been observed in the nucleus of proliferating cells [11]. PLC β1 has been shown to be located primarily in the nucleus in some cell types [9, 11, 12], but not in others [13], and to vary with the state of differentiation of cells [12, 14]. PLCs β1 and β2 have also been found in the nuclear fraction [15].

The purpose of this study was to compare the PLC β isoforms and their location in vascular smooth muscle cells (VSMCs) from different species. This was to determine whether compartmentalisation, i.e. distinct intracellular distributions, may contribute to different physiological roles. The PLC isoforms present were determined using lauryl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) and Western blotting; their intracellular location was determined by immunocytochemistry and confocal laser scanning microscopy. To indicate the architecture of the cells, antibodies to proteins associated with specific intracellular locations were used as controls. These included {alpha}-actin for the smooth muscle cytoskeleton [16], p58 for the Golgi [17], G-protein β subunit for the plasma membrane [18]and caveolin-1 for caveoli [19].


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No 85.23, revised 1985).

2.1 Cell culture
VSMC cultures were established from the media of rat and porcine aorta using an explant method [20]. For immunocytochemistry, the VSMCs were grown on coverslips in six-well plates and for cell lysates in T75 flasks. Growth was stimulated by including 10% (v/v) foetal calf serum (FCS)+10% new born calf serum (NBCS) in the culture medium and cells were used for immunocytochemistry when they were judged to be 40–60% confluent. HRA cells were from an established cell line [21]and were at passage 14 or 15 when used for immunocytochemistry and at passage 15–18 for extraction for Western blots. To quiesce cultures, they were depleted of FCS and NBCS for 72 h. This was sufficient to sustain cell viability in rat and pig cultures, but human renal artery required a more complex quiescing medium [21]. The cells were either used for immunocytochemistry or FCS and NBCS were added again and the cells were cultured for a further 72 h. Microvascular endothelial cell cultures were a gift [22].

2.2 Determination of actively growing cells
To establish whether the smooth muscle cells cultured with or without FCS+NBCS were proliferating or quiescent, 2 µCi of [6-3H] thymidine/ml were added to the culture medium [23]. The cells were cultured on slides and tritiated thymidine was added for the final 48 h of each culture condition. The slides were then treated with 4% (v/v) paraformaldehyde, followed by photographic emulsion and exposed for seven days at 4°C. They were developed, counterstained with haematoxylin and viewed under the light microscope. The number of black nuclei (those taking up thymidine) and the total number of nuclei were recorded. Proliferation was assessed from the% of black to total nuclei.

2.3 SDS–PAGE and Western blotting
Cell cultures were lysed by adding 1 ml of boiling sodium dodecyl sulphate (SDS) buffer [10 mM Tris, pH 7.5, plus 1% (w/v) SDS] containing ‘Complete’, a protease inhibitor cocktail (Calbiochem) and calpain I (15 µg/ml) and II (7 µg/ml) inhibitors (Calbiochem) to a T75 tissue culture flask. The contents were scraped into an eppendorf microcentrifuge tube and then passed through a 23 gauge needle 10x to break up strings of DNA. The sample was centrifuged at 10 000xg in a bench centrifuge for 5 min and concentrated 40-fold by centrifugal ultrafiltration, using Amicon centricon 10 tubes. An aliquot was used for a Bradford-based protein determination [24]and the remainder was stored at –70°C until use. It was adjusted to an appropriate protein concentration (See legends to figures), and taken up with SDS Laemmli buffer and loaded onto 10% polyacrylamide gels for electrophoresis (PAGE). Thereafter, the proteins were transferred to nitrocellulose membranes and Western blotting was performed to determine the PLC isoforms [2]. For PLC β1, β2 and β3, incubations were for 1 h at 37°C. For PLC β4, the Western blotting protocol was different and, to detect this isoform, the incubations with primary and secondary antibody were overnight at 4°C, and washes (also at 4°C) were extended to 30 min.

2.4 Immunocytochemistry
VSMCs on coverslips were permeabilised and fixed using methanol at –20°C for 5 min [25]. The buffer used to make up all solutions and to wash between additions was phosphate-buffered saline (PBS), pH 7.4, prepared from tablets (Sigma). The incubations with antibody were for 1 h at 37°C. The coverslips were washed and incubated with 20% FCS in PBS, followed by primary antibody (details given below) in PBS with 0.1% bovine serum albumin (Sigma) and 0.01% azide (PBS–BSA–azide). After washing, the secondary antibody was added and this was diluted (x1000 of the original supplied) in PBS–BSA–azide as before; these were anti-rabbit cy3 conjugate (Amersham) or antimouse fluorescein isothiocyanate (FITC) conjugate (TCS). The control slide for background fluorescence was incubated with rabbit IgG (Sigma) at 10 µg/ml instead of primary antibody. The coverslips were mounted on glass slides using FluorSaveTM Reagent (Calbiochem) containing 2.5% (w/v) 1,4-diazabicycl-O[2.2.2]octane.

Explants were prepared from at least three fresh batches of aorta and all slides were prepared from cells at passages two and three. For each of the primary antibodies, 12 slides (six duplicates) were examined for each species under each growth condition. Two ‘lots’ of each of the PLC primary antibodies (see below) were purchased.

2.5 Primary antibodies
Antibodies to the PLC isoforms, G-protein β subunits and caveolin-1 were all raised in rabbit and purchased from Santa Cruz. The antibodies to p58 and {alpha}-actin were raised in mouse and both were purchased from Sigma. Catalogue numbers for all antibodies are as listed and the dilution used (of the original) is given in parentheses, PLCβ1, sc-205 (x20); PLCβ2, sc-206 (x100); PLCβ3, sc-403 (x20); PLCβ4, sc-404 (x20); Gβ, sc378 (x100); caveolin-1, sc894 (x20); p58, G-2404 (x20) and {alpha}-actin, A2547 (x100).

2.6 Confocal microscopy and image capture
The experiments were performed using a Leica TCS4D confocal laser scanning microscope, which was based on a Leitz DM upright microscope controlled by a VME Bus os9 operated computer and image processing software. It used an argon/krypton laser, which enabled the cy3 conjugate to be visualized with an excitation wavelength of 568 nm and the FITC conjugate with an excitation wavelength of 488 nm using the relevant beam splitters and barrier filters to allow detection through a confocal pinhole set to 60 µm. This set-up allowed a depth of focus of 1 µm with the x40 and of 0.5 µm with the x100 oil immersion objectives. Serial optical sections were taken through the XY or YZ axis of the cells, and the images, in a 512x512 pixel format, were stored on disc. The brightest mid-cell Z-section was selected for XY comparison. The field was 200 µm2, which sampled three–four cell lengths and gave an image containing a representative number of cells. Transverse YZ sections were used to distinguish the interface of the cell with the coverslip. The use of antibodies to proteins of established intracellular distribution (p58, Golgi; G-protein β subunit, plasma membrane; caveolin-1, caveoli and {alpha}-actin, cytoskeleton) enabled the architecture of the cell to be defined. The images were archived as TIFF files. They were transferred to Adobe Photoshop software (Adobe Systems Inc, CA, USA), where they were converted to grayscale and contrast enhanced by 50%. The images were printed at 300 dots per inch on a dye sublimation printer.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Cell proliferation
The number of nuclei taking up [3H]thymidine compared to the total number of nuclei (expressed as a percentage) was used as a measure of the number of proliferating cells in the cultures. The removal of growth factors by the omission of FCS+NBCS significantly decreased proliferation in pig and rat VSMC cultures, but HRA VSMC cultures were unaffected (Fig. 1). The readdition of FCS+NBC to porcine and rat 72 h quiesced VSMC cultures resulted in the return to the same percentage of black nuclei associated with proliferating cells, thus demonstrating that the quiescence did not affect cell viability. Proliferation of HRA VSMCs was not diminished by the removal of FCS+NBCS and the readdition of medium after the ‘quiescent’ time period apparently resulted in loss of tritiated thymidine uptake. However, microscopic examination of these cultures (not shown) indicated that they attained confluence during this period and ceased to proliferate for this reason. Thus, it was not possible to obtain HRA VSMC cultures that were comparable to porcine and rat quiescent VSMC cultures.


Figure 1
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  		Fig. 1 Cell proliferation. This was determined as the uptake of [6-3H]thymidine, as detailed in Section 2, and was expressed as the percentage of black nuclei to total nuclei. For each experiment, three different explants of rat and pig were used and, for HRA, nine equivalent slides were set up from the same cell line culture (three for each experiment) to give n=3. For each explant, three slides were set up together: the first was processed after three days of culture with FCS+NBCS. The slides were then washed and the culture medium was changed to a quiescing medium for three days, when the second slide was processed. The culture medium was then changed to one containing FCS+NBCS and the final slide was processed after a further three days of culture. Three fields per processed slide were counted for black and total nuclei using the x40 objective and the results were averaged. n=3±SEM, *=p<0.05 using Student's paired t-test.

 
3.2 Immunoblotting for PLC β isoforms
SDS–PAGE and Western blotting were used to compare the PLC isoforms expressed in VSMCs of different species and under different growth conditions (Fig. 2). PLC β1, PLC β2, PLC β3 and PLC β4 were present in VSMCs of pig, human and rat. Each species was recognised by the respective antibodies and the bands had the same molecular mass for each PLC isoform. Identical lanes were run using pooled lysates from HRA cells and the lanes were blotted individually for PLCs β1, β2 and β3. The arrows indicating the molecular mass markers and the relationship between the three isoforms are those taken from this blot. There were no interspecies differences in the molecular masses of the individual isoforms run on the same gel; the bands always lined up at the same point. PLC β4 showed a different distribution in proliferating and quiescent porcine VSMCs viewed by confocal microscopy (see below). For this reason, lysates from quiescent and proliferating porcine VSMC cultures were compared for PLC β4 content and there was no apparent difference in the quantity of the PLC β4 expressed (Fig. 2). The PLC β4 isoform was difficult to detect by SDS–PAGE and Western blotting. It was necessary to load 200 µg of protein from lysed cells onto each lane of the gel. The molecular mass of PLC β4 appeared to be greater than that expected and this ran with the 205 kDa marker, however, calculation of Rf values using the whole marker range suggested that its molecular mass was in the region of 140–160 kDa and that the top marker was consistently below the linear value extrapolated (data not shown). Antibodies to the Gβ subunit and caveolin-1 recognised bands at about 40 and 27 kDa, respectively, confirming specificity and cross-reaction in each species.


Figure 2
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  		Fig. 2 SDS–PAGE and Western blotting for PCL β isoforms. All blots were performed at least twice and, for rat and pig, these duplicate cell lysates were from cultures derived from different explants. For PLC β1, β2 and β3, 100 µg of protein were loaded onto each lane of the gel and 200 µg were used for PLC β4. The blotting conditions for PLC β4 were also different (see Section 2). The HRA lanes for PLC β1, β2 and β3 were from the same blot, together with the arrows which represent the position of Biorad prestained molecular mass markers (kDa). For each PLC isoform, the bands were detected in the same place irrespective of species.

 
3.3 Localisation of PLC β isoforms observed by confocal microscopy
The arrangement for viewing confocal sections is outlined in Fig. 3. Immunocytochemistry for all the PLC β isoforms confirmed the findings of Western blotting, i.e. that all isoforms were observed in all species.


Figure 3
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  		Fig. 3 Diagramatic representation of confocal ‘cell sections’. (a) This represents the relationship of the coverslip, on which the cells were cultured, to the slide, on which it was mounted, and the direction of the laser beam and mechanical advance of the microscope to generate longitudinal XY sections. (b) This demonstrates an XY section as viewed from the ‘top’ looking down through the coverslip. (c) This demonstrates a transverse YZ section as if viewed from the side of the coverslip and slide.

 
Fig. 4 shows XY sections of cells from proliferating cultures, comparing all species for the localisation of PLC β1, β2 and β3. Quiescent cultures gave the same result (data not shown). PLC β1 showed a fairly random distribution of foci of concentration throughout the cytoplasm, except in HRA cells where its signal was confined to the cell periphery: there was, however, less fluorescence in these cells. PLC β2 demonstrated two locations that were dependent upon the ‘lot’ of antibody purchased. Each antibody was raised against the same peptide and these gave different results for immunocytochemistry. The first ‘lot’ of antibody demonstrated exclusive localisation to the Golgi, which was consistent in all species and coincident with p58 localisation in dual-labelled slides (Fig. 4). The second ‘lot’ of antibody showed, in XY sections, punctate localisation that was associated with the cell periphery in all species, which appeared to be associated with the intersection of filamentous structures with the plasma membrane. PLC β3 demonstrated a diffuse fluorescent signal in the cytoplasm, which was most concentrated in the perinuclear region.


Figure 4
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  		Fig. 4 Immunocytochemistry for PLCs β1, β2 and β3 showing comparative XY sections of proliferating VSMCs of pig, human and rat cultures. Slides were prepared as described in Section 2. The field was 200 µm2 and the bar represents 25 µm. The results of two different ‘lots’ of PLC β2 antibody are shown. Lot A consistently localised to the Golgi and the insert in the pig image is for a dual-labelled PLC β2/p58 section. p58, a Golgi marker (a mouse polyclonal antibody), was detected with an anti-mouse FITC conjugate secondary antibody and PLC β2 was detected with an anti-rabbit cy3 conjugate.

 
In proliferating cells of all species, PLC β4, in XY sections, was evenly distributed throughout the cytoplasm (Fig. 5). The intensity of the fluorescent signal suggested that binding was present, since this was above the background levels detected with rabbit IgG incubation instead of primary antibody (Fig. 5). In quiescent cultures of pig VSMCs (but not in quiescent rat VSMCs), PLC β4 was associated with filamentous structures, particularly at the periphery of the cells. This effect could be abolished by the inclusion of the β4 peptide antigen in the primary antibody incubation and was, therefore, a specific binding effect. In YZ sections, PLC β4 was localised to the cell periphery, but only at the plasma membrane face that was adjacent to the coverslip (Fig. 5).


Figure 5
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  		Fig. 5 Immunocytochemistry for PLC β4 showing comparative XY and YZ sections for proliferating and quiescent VSMCs of pig, human and rat cultures. Slides were prepared as described in Section 2. The X,Y and Z bars represent 10 µm. PLC β4 demonstrated no particular localisation in proliferating cells from any species. In (quiescent) pig cultures, PLC β4 was localised to particular regions of the cell periphery. This was a specific effect of the PLC β4 antibody because inclusion of the antigen PLC β4 peptide (+peptide) in the primary antibody incubation abolished the fluorescent signal. YZ sections were used to distinguish the interface of the coverslip with the cultured cell. The XY and YZ fields for pig (quiescent) cultures are shown as a corresponding pair with a composite of YZ sections lined up along the X axis. Rabbit IgG was used instead of primary antibody for the control slides, to show background fluorescence due to the Cy3-conjugated secondary antibody.

 
3.4 Localisation of ‘marker’ proteins for known intracellular locations
The G-protein β subunit antibody was chosen because it had a broad spectrum recognition of four, Gβ1–4, isoforms. In XY sections of all species, it gave a clear pattern of high concentration at the cell periphery (Fig. 6). In YZ sections of porcine and rat cells, this peripheral staining gave a good definition of the perimeter of the cell (Fig. 6). Caveolin-1 was used as a marker for caveoli. This gave a distinctive pattern of distribution in all three species, which was most similar in rat and pig XY sections (Fig. 6). In YZ sections of both porcine and rat cells, this signal was confined to the plasma membrane interface with the coverslip (Fig. 6). For comparison, as a methodological control, some cultured rat microvascular endothelial cells were examined and these showed caveolin-1 associated with the entire plasma membrane, Fig. 6. The actin {alpha} isoform is known to be specific for smooth muscle cells and immunocytochemistry with this isoform is generally used to confirm VSMC identity. In this study, the antibody detected cytoskeletal filaments in all species (Fig. 6).


Figure 6
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  		Fig. 6 Immunocytochemistry for ‘markers’ of cell architecture, i.e. {alpha}-actin, G-protein β subunit and caveolin-1, showing comparative XY sections of proliferating VSMCs of pig, human and rat cultures and of rat microvascular endothelial cells (MVEs). Slides were prepared as described in Section 2. The bar represents 10 µm. G-protein β subunit was used to show the plasma membrane, caveolin-1 the caveolae and {alpha}-actin the cytoskeleton. The XY and YZ fields are shown as a corresponding pair, with a composite of YZ sections lined up along the X axis. YZ sections were used to distinguish the interface of the coverslip with the cultured cell. G-protein β subunits demonstrated an even distribution at all plasma membrane interfaces in both pig and rat VSMCs. In contrast, caveolin-1 was confined to the plasma membrane at the coverslip interface. Rat microvascular endothelial cells (MVEs), where caveolin-1 has been detected at the plasma membrane, were used as a preparative control for the VSMCs and, in these cells, caveolin-1 was distributed at all of the plasma membrane interfaces. {alpha}-Actin, an actin isoform characteristic of smooth muscle cells, was used to identify VSMC phenotype and was present in cultured cells of all three species, demonstrating a pattern that was typical of cytoskeletal fibres. {alpha}-Actin (a mouse polyclonal) was detected with an anti-mouse FITC conjugate secondary antibody, and an anti-rabbit cy3 conjugate was used as the secondary antibody for the other primary antibodies.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In a previous study, we identified the four PLC β isoforms β1, β2, β3 and β4 in medial tissue from porcine aorta [2]. This number of isoforms had been described in bovine brain [26, 27], but these could have originated from the heterogeneous cell types constituting this tissue. In this study, we have confirmed that multiple expression of PLC β isoforms is a general rule in vascular smooth muscle by comparison of the isoforms present in different species using SDS–PAGE and Western blotting. The expression of different isoforms may reflect different physiological roles, including the specificity of the receptor to effector pathways, accomplished either by specific protein–protein interactions or by physical compartmentalisation. In support of the latter possibility, immunocytochemistry and confocal laser scanning microscopy have demonstrated that the PLC β1 β2 β3 and β4 isoforms have unique intracellular distributions, which are consistent irrespective of species.

The antibodies to the PLC β isoforms used in this study cross-react with rat and human isoforms (suppliers information) and with porcine PLC β isoforms [2]. SDS–PAGE and Western blotting showed that the individual antibodies cross-reacted with a protein of the appropriate molecular mass for each PLC β isoform in each species. Immunocytochemistry demonstrated that these same antibodies also recognised the folded proteins and the use of two ‘lots’ of antibody for each isoform gave the same pattern of intracellular localisation. The exception was PLC β2 and there was a notable difference in the distribution pattern between the two ‘lots’ of antibody. One gave a distribution that fitted the expectations for a PLC isoform, namely a distinctive location at the cell periphery. The other ‘lot’ of PLC β2 antibody, however, associated with the Golgi, which was confirmed by dual labelling with the Golgi marker protein p58 [17], a distribution pattern that was the same in all three species and in both proliferating and quiescent cells. The explanation offered by the supplier was that one antibody had an exclusive preference for the immature protein. The ‘lot’ of PLC β2 recognising the Golgi is no longer available, so non-reducing gels cannot be run for verification. The SDS–PAGE and Western blotting experiments were performed with the ‘lot’ of antibody that gave the non-Golgi distribution. This discrepancy indicates the importance of caution in using antibodies to localise proteins within cells and necessitates the routine use of at least two ‘lots’ of antibody for confirmation. This would also apply to the use of antibodies to determine the distribution of proteins in fractionation studies. The use of antibodies to proteins of established intracellular distribution enabled the architecture of the cell to be defined. The G protein β({gamma}) subunit proteins are integral to the plasma membrane and is associated with the cytoplasmic face. It gave a clear picture of the plasma membrane, marking the periphery of the cell in both XY and YZ sections, which was observed in both porcine and rat cultures, indicating that the methanol treatment used to permeabilise and fix the cultured cells did not disrupt the plasma membrane structure but allowed antibodies to diffuse into the cell.

In previous studies, PLC β isoforms have been found in the plasma membrane, the cytoplasm or the nucleus. Stimulation via receptor/G-protein pathways to produce IP3 and DAG from plasma membrane PIP2 [28]is the convention for PLC β isoform activation. A fractionation study demonstrated increased PLC β2 in the plasma membrane after stimulation of porcine trachaelis smooth muscle with carbachol [29], supporting the hypothesis of translocation from the cytoplasm. However, a number of studies have reported PLCs β1 [11, 13, 15, 30, 31], β2 [15]and β3 [15]in the nucleus, and the distribution between nucleus and cytoplasm may depend upon the differentiated state of the cell [12, 14, 15]. In our study, PLC β1 was distributed in foci throughout the cell, but was never observed in the nucleus. We observed PLC β2 at the plasma membrane, apparently at the point of intersection with filamentous structures. PLC β3, like PLC β1, appeared to be distributed within the cytoplasm, although the pattern was different.

Cultured vascular smooth muscle cells were chosen for this study because these were most amenable to the preparation of slides for confocal microscopy. However, it is well established that there are differences in the normal tissue ‘contractile’ non-proliferative VSMC phenotype and that of proliferating VSMCs [32]. In our study, loss of phenotype with passage [16]was minimised by using pig and rat cultures at passages two and three, and the marker of VSMC phenotype, {alpha}-actin, was apparent even in HRA VSMCs at passages 14 and 15. To ameliorate the phenotypic difference between cultured proliferating cells and those in contractile tissue, FCS+NBCS stimulated cultures were compared to those following 72 h of quiescence. The localisation pattern of all the proteins examined in all three species were remarkably consistent and only PLC β4 in pig VSMCs showed any difference in location with growth condition. In proliferating cultures, it showed a uniform cytoplasmic distribution in all three species, but in quiescent cultures of pig VSMCs, it was observed to be associated with filamentous structures, particularly at the cell periphery. YZ sections demonstrated that this was confined to the plasma membrane at the coverslip interface. PLC β4 was detected in cell lysates from all species and there was no difference in the quantity of PLC β4 in Western blots of lysates of proliferating and quiescent pig VSMCs. Thus, it is likely that PLC β4 redistributed in response to growth conditions in pig VSMCs, although it is not clear why this did not also happen in quiescent cultures of rat VSMCs. It was not possible to obtain quiescent cultures of HRA VSMCs, which may produce endogenous growth factors, since the removal of FCS+NBCS did not diminish tritiated-thymidine uptake.

Differential localisation of proteins such as G-proteins, receptors and effectors (like PLC β isoforms) may indicate compartmentalisation and may underpin the specificity of signal transduction [8, 33–36]. Indeed, there is biochemical evidence to support the compartmentalisation of PLC isoforms. There is greater specificity of PLC activation by G-proteins and receptors in permeabilised cells [6, 7]and whole membranes [4], where structural integrity is largely preserved, than that observed with purified PLC β and G{alpha}q and β{gamma} isoforms [3]. Compartmentalisation may be achieved by the creation of signalling complexes or microenvironments such as caveoli, which are specialized invaginations of the plasma membrane, which have both G-proteins and receptors associated with them, although their specific role is unclear [19, 37]. Thus, caveoli might be candidates for PLC β isoform association. It is clearly shown that, in both pig and rat, the caveoli are associated with the plasma membrane at the coverslip interface. In cultured rat microvascular endothelial cells, however, the caveoli were at all of the plasma membrane faces, as expected [38], indicating that the distribution in VSMCs was unlikely to be a preparative artifact. This polarisation of both pig PLC β4 and caveolin-1 may not be typical of VSMCs within tissues where the VSMC's plasma membrane interface would be surrounded by extracellular matrix, but, in culture, the latter may be confined to the adherent coverslip interface. Future comparison of PLC β4 distribution using freshly isolated cells or examination of medial tissue would clarify this point. PLC β4 in YZ sections of quiescent cells showed a similar distribution to that of caveolin-1, but comparison of XY sections indicated that these were not in fact similar.

In conclusion, this study has shown that the multiple expression of PLC β isoforms (PLC β1, β2 β3 and β4) was common to porcine, rat and human VSMCs. Each of the PLC β isoforms had a unique intracellular distribution, which was similar in all species, perhaps suggesting universal and well conserved roles in signal transduction. The experience with the PLC β2 antibodies indicated the necessity to carefully characterise the antibodies used to give consistent results, however, immunocytochemistry and confocal laser scanning microscopy have the resolution to localise proteins to specific cellular domains, particularly if information was used from both XY and YZ sections.

Time for primary review 35 days.


   Acknowledgements
 
We would like to thank G Newman and B Jasani, Department of Pathology, T Griffith, Department of Diagnostic Radiology, A Shah, Department of Cardiology and A Newby, Bristol Heart Institute for helpful discussion. We would also like to thank J Assender, Department of Molecular and Medical Biosciences, Cardiff, for the gift of HRA cells, and A Shah for microvascular endothelial cell cultures.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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