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POTASSIUM CONCENTRATION IS REDUCED IN CULTURED RABBIT TRACHEAL SMOOTH MUSCLE CELLS AFTER WITHDRAWAL OF SERUM
Cell Biology International 2001, Vol. 25, No. 7, 691–695 doi:10.1006/cbir.2000.0711, available online at http://www.idealibrary.com on
SHORT COMMUNICATION
POTASSIUM CONCENTRATION IS REDUCED IN CULTURED RABBIT TRACHEAL SMOOTH MUSCLE CELLS AFTER WITHDRAWAL OF SERUM ALICE WARLEY* EM Unit, Department of Ophthalmology, The Rayne Institute, St Thomas’ Hospital, London SE1 7EH Received 13 November 2000; accepted 17 November 2000
Comparison of elemental concentrations in growth-arrested airway smooth muscle cells with those in their proliferating counterpart showed that potassium (K + ) was significantly reduced, whereas concentrations of other elements sodium (Na + ), magnesium (Mg2+ ), phosphorus (P), and chlorine (Cl ) remained unchanged. Reduced K + concentration was associated with a change in the cells from a spindle shape to a flattened form. 2001 Academic Press K: smooth muscle; cell culture; potassium; sodium; X-ray microanalysis.
INTRODUCTION The suggestion that hyperplasia of airway smooth muscle cells may contribute to the pathogenesis of asthma (Hirst, 1996) has led to the use of cultured airway smooth muscle to investigate factors which affect the proliferation of these cells. Numerous studies have shown increases in the fluxes of ions across the membranes of various different types of cell after stimulation into proliferation (Moolenhaar et al., 1986; Smith and Cameron, 1999), but the occurrence of such changes in airway smooth muscle (ASM) has not previously been documented. MATERIALS AND METHODS Cell cultures Smooth muscle cells were isolated from the trachealis of adult male New Zealand White rabbits (2–3 kg) and cultured as previously described (Warley et al., 1994). For X-ray microanalysis cells from passage 6 to 8 were seeded onto Thermanox coverslips bearing 100 mesh gold grids which had *To whom correspondence should be addressed: EM Unit, Dept of Ophthalmology, GKT Medical and Dental Schools, The Rayne Institute, St Thomas’ Hospital, London SE1, 7EH. Email: [email protected]. Fax: 020 7401 9206. 1065–6995/01/070691+05 $35.00/0
been coated with Pioloform, sterilised under UV and coated with laminin before use. Growth arrest and stimulation with 10% FCS After subculture, cells were allowed to settle overnight in the presence of growth medium containing 10% foetal calf serum (FCS). The cells were growth-arrested by replacing the medium with Dulbecco’s modified Eagle’s medium (DMEM) containing insulin, ascorbate and transferrin in place of the FCS. The cells remained in this medium for 72 h before some were stimulated by exposure to DMEM containing FCS, the remainder being maintained in the growth-arrest medium as controls. In both experimental and control cultures, the medium was changed every 24 h. Samples for X-ray microanalysis were taken after 72 h of growth arrest (zero time) and every 24 h. X-ray microanalysis and electron microscopy At the end of the experimental period, grids bearing the cells were removed from culture and the overlying medium was removed by dipping the cells rapidly into ice-cold distilled water. The cells were cryofixed by plunging into liquid nitrogen, freezedried overnight and coated with carbon before analysis. X-ray microanalysis was performed using 2001 Academic Press
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Cell Biology International, Vol. 25, No. 7, 2001
700
700 A
B K+
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500 [] mmoles/kg
500 [] mmoles/kg
P
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300
400
300 Cl
200
200 Na
+
100
0
−
100
24 48 72 Hours after addition of FCS
96
0
24 48 72 Hours after addition of FCS
96
Fig. 1. Concentrations of (A) sodium and potassium, and (B) phosphorus and chlorine in growth-arrested cultured rabbit airway smooth muscle cells (open symbols) and in cells stimulated to divide by the presence of foetal calf serum (closed symbols). Cells were plated at a density of 104 cells/well, allowed to settle overnight in the presence of DMEM containing 10% foetal calf serum, and then growth-arrested by FCS deprivation for 72 h. Cells were then either maintained in a growth-arrested state or stimulated into division by replacement of the growth-arrest medium with DMEM containing 10% FCS. Samples for X-ray micro-analysis were taken every 24 h and treated as described in the methods. Data represent the meanSE of cells from 9 animals, 12 to 15 cells were analysed from each animal.
a Zeiss EM 10 electron microscope. Areas of cell cytoplasm, approximately 66 m, were analysed for 100 s live time at 80 kV accelerating voltage and 1 nA beam current in STEM mode at ambient temperature. Spectra were processed using the FLS fitting routine, and quantification was achieved by use of the continuum-normalisation procedure, with reference to standards composed of gelatin containing known amounts of inorganic salts. Statistical comparisons were made by analysis of variance followed by Tukey’s t-test (for full details see Warley et al., 1994).
RESULTS AND DISCUSSION Element content in the growth cycle There were no significant differences in the concentrations of Na + , Cl or Mg2+ between growth arrested and stimulated cells throughout the growth period (Fig. 1A and B; data for Mg2+ not shown). These findings are in agreement with the idea that in proliferating cells increases in inward flux of Na + are balanced by outward flux leading to no overall increase in intracellular Na + concentration (Moolenhaar et al., 1986).
After addition of FCS, the concentration of K + increased slight and remained steady throughout the period of proliferation, whereas the concentration of K + gradually declined in growth arrested cells (Fig. 1A). After 48 h, the difference in [K + ] between the growth arrested and serum stimulated cells was significant (P<0.001, 48 h; and P<0.05 at 72 and 96 h). There was no change in [P] in the stimulated cells throughout the growth cycle, but the concentration of this element showed a gradual decrease in the non-stimulated cells, being significantly different (P<0.05) at 72 h after stimulation (Fig. 1B). In addition to differences in K + concentration, cells subject to serum withdrawal showed changes in overall morphology. Cells from cultures taken at zero time (i.e. after growth arrest for 72 h) showed a mixture of spindle and flattened shapes under low power STEM (Fig. 2A). After addition of FCS, the cells became narrow and spindle-shaped and appeared to grow in parallel with one another, and after 48 h the increase in cell number is apparent (Fig. 2C). At higher magnification, the spindleshape of the rapidly growing cells and their alignment with the tapered cytoplasmic end of one cell adjacent to the broad nuclear region of the next cell is more clearly seen (Fig. 2E). As the period of
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Fig. 2. A, B, C. Lower power STEM images of rabbit airway smooth muscle cells from an identical source. (A) Cells growth arrested in serum-depleted medium for 72 h. Inclusions resembling apoptotic bodies, or apoptotic nuclei are sometimes seen (arrow). (B, C) Cells 48 h later in the presence of (B) growth-arrest medium or (C) after stimulation with 10% FCS. (D) Higher-power image of cells from B, the cells are flattened, and stress fibres can be seen running through the cytoplasm. Whereas cells which have been stimulated by the addition of FCS appear spindle-shaped, narrower and are more dense. Marker=5 m.
Cell Biology International, Vol. 25, No. 7, 2001
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40 Frequency
Frequency
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30
30
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20
10
10
0
100
300 500 700 900 + [K ] Unstimulated cells
0
100
300 500 700 900 + [K ] Unstimulated cells
Fig. 3. Frequency histogram showing the distribution of [K + ] in rabbit airway smooth muscle cells after serum withdrawal (A) and after 48 h stimulation (B).
serum withdrawal increased, the cells appeared to flatten and take on a triangular shape. Few spindleshaped cells were seen (Fig. 2B). At higher magnification, stress fibres were seen running through the cytoplasm at the edge of the flattened cells (Fig. 2B). These changes in morphology are unlikely to result from the well-documented modulation of phenotype which occurs when smooth muscle cells are placed in primary culture, because the cells used here were carried through serial passage and are unlikely to be able to revert fully to the contractile state. The flattened form of ASM described here resembles that of cultured vascular smooth muscle cells treated to inhibit proliferation (Weissberg et al., 1993) and may be indicative of the nonproliferating state in vitro. The changes in K + concentration cannot be ascribed to the change in thickness of the proliferating cells since absorption of X-ray would lead to a decrease, rather than an increase in concentration. Specimen thickness would also be expected to affect the concentration of other elements. The association of higher concentrations of K + with cells in the proliferative state agrees with results from studies of tissues in vivo (Grundin et al., 1985; Smith and Cameron, 1999). However, the concentration of potassium found in the actively proliferating ASM is similar to values recorded in VSM in vivo from various different sources (see Warley et al., 1994). Since both the spindle shape and the higher potassium concentrations are seen in smooth muscle in vivo, which is contractile and non-proliferating, and in actively
proliferating muscle in vitro, it is unlikely that these are characteristic of proliferation per se. The results presented here show that for ASM in culture loss of K + from the growth arrested cells accounts in part for the differences between the proliferating and non-proliferating cells. This is further borne out by examination of K + concentrations in individual cells (Fig. 3). At 48 h after stimulation, when the cells are in the log phase of growth, [K + ] shows a homogeneous distribution with the mean, median and modal concentrations identical (mean 555 mmoles/kg, median and mode 556 mmoles/kg), whereas in the cells subject to serum withdrawal there is much wider distribution of K + concentrations with the mean value, 491.9 mmoles/kg dry weight higher than the median (485 mmoles/kg) and modal (436 mmoles/kg) values. The finding of decreased [K + ] without a concomitant increase in [Na + ] fits a pattern that is beginning to be recognised as being characteristic of cells entering apoptosis. Low concentrations of K + are known to be associated with the final stages of apoptosis (Warley and Morris, 1988; FernandezSegura et al., 1999), but there is evidence that loss of intracellular potassium begins earlier (Skepper et al., 1999), before morphological changes become apparent, and that this loss enhances the apoptotic process (Bortner et al., 1997). Serum provides factors necessary to support growth in vitro, and also plays an important role in the survival of cells in culture (Collins et al., 1994). Withdrawal of serum is known to promote apoptosis in a number
Cell Biology International, Vol. 25, No. 7, 2001
of different cell types including vascular smooth muscle (Bennett et al., 1995). The results presented here show a lowering of intracellular potassium in cultured ASM cells deprived of serum, the small number of cells (2–3%) exhibiting the lowest concentrations of potassium agree with reported rates of apoptosis in VSM after serum withdrawal (Bennet et al., 1995). The occasional finding of inclusions resembling either apoptotic nuclei or engulfed apoptotic bodies in cells after serum withdrawal (Fig. 2A) further adds support to this idea. ACKNOWLEDGEMENTS The author would like to thank Drs S. J. Hirst and J. P. T. Ward for their help with this work. This work was supported by the Wellcome Trust and a Sharpey-Shafer Award from the Special Trustees of St Thomas’ Hospital. REFERENCES B MR, E GI, S SM, 1995. Apoptosis of human vascular smooth muscle cells derived from normal vessels and coronary atherosclerotic plaques. J Clin Invest 95: 2266–2274. B CD, H FM J, C JA, 1997. A primary role for K + and Na + efflux in the activation of apoptosis. J Biol Chem 272: 32436–32442. C MKL, P GR, R-T G, N MA, L-R A, 1994. Growth factors as survival factors: regulation of apoptosis. Bioessays 16: 133–138.
695
F-S E, C FJ, C MA, W A, C A, 1999. Changes in elemental content during apoptotic cell death studies by electron probe X-ray microanalysis. Exp Cell Res 252: 454–462. G TG, R GM, F B, L M, W Y, 1985. X-ray microanalysis of psoriatic skin. J Invest Dermatol 85: 378–380. H SJ, 1996. Airway smooth muscle cell culture: application to studies of airway wall remodelling and phenotype plasticity in asthma. Eu Resp J 9: 808–820. M WH, D LHK, D SW, 1986. Ionic signalling by growth factor receptors. J Exp Biol 124: 359–373. S JN, K I, G MR, H L, H SJ, W A, M MJ, C NRB, 1999. Changes in elemental concentrations are associated with early stages of apoptosis in human monocyte-macrophages exposed to oxidised low-density lipoprotein: an X-ray microanalytical study. J Pathol 188: 100–106. S NKR, C IL, 1999. Ionic regulation of proliferation in normal and cancer cells. In: Ingram P, Shelburne J, Roggli V, LeFurgey A, eds. Biomedical Applications of Microprobe Analysis. San Diego, Academic Press. 445– 459. W A, C KPB, C HB, T CHC, W JPT, H SJ, 1994. Preparation of cultured airway smooth muscle for study of intracellular element concentrations by X-ray microanalysis: comparison of whole cells with cryosections. J Microsc 175: 143–153. W A, M IW, 1988. Concentrations of elements in dying thymocytes from the thymus gland of diabetic rats. Scanning Microscopy 2: 2227–2232. W PL, G DJ, S CM, M JC, 1993. Approaches to the development of selective inhibitors of vascular smooth muscle cell proliferation. Cardiovascular Res 27: 1191–1198.
SHORT COMMUNICATION
POTASSIUM CONCENTRATION IS REDUCED IN CULTURED RABBIT TRACHEAL SMOOTH MUSCLE CELLS AFTER WITHDRAWAL OF SERUM ALICE WARLEY* EM Unit, Department of Ophthalmology, The Rayne Institute, St Thomas’ Hospital, London SE1 7EH Received 13 November 2000; accepted 17 November 2000
Comparison of elemental concentrations in growth-arrested airway smooth muscle cells with those in their proliferating counterpart showed that potassium (K + ) was significantly reduced, whereas concentrations of other elements sodium (Na + ), magnesium (Mg2+ ), phosphorus (P), and chlorine (Cl ) remained unchanged. Reduced K + concentration was associated with a change in the cells from a spindle shape to a flattened form. 2001 Academic Press K: smooth muscle; cell culture; potassium; sodium; X-ray microanalysis.
INTRODUCTION The suggestion that hyperplasia of airway smooth muscle cells may contribute to the pathogenesis of asthma (Hirst, 1996) has led to the use of cultured airway smooth muscle to investigate factors which affect the proliferation of these cells. Numerous studies have shown increases in the fluxes of ions across the membranes of various different types of cell after stimulation into proliferation (Moolenhaar et al., 1986; Smith and Cameron, 1999), but the occurrence of such changes in airway smooth muscle (ASM) has not previously been documented. MATERIALS AND METHODS Cell cultures Smooth muscle cells were isolated from the trachealis of adult male New Zealand White rabbits (2–3 kg) and cultured as previously described (Warley et al., 1994). For X-ray microanalysis cells from passage 6 to 8 were seeded onto Thermanox coverslips bearing 100 mesh gold grids which had *To whom correspondence should be addressed: EM Unit, Dept of Ophthalmology, GKT Medical and Dental Schools, The Rayne Institute, St Thomas’ Hospital, London SE1, 7EH. Email: [email protected]. Fax: 020 7401 9206. 1065–6995/01/070691+05 $35.00/0
been coated with Pioloform, sterilised under UV and coated with laminin before use. Growth arrest and stimulation with 10% FCS After subculture, cells were allowed to settle overnight in the presence of growth medium containing 10% foetal calf serum (FCS). The cells were growth-arrested by replacing the medium with Dulbecco’s modified Eagle’s medium (DMEM) containing insulin, ascorbate and transferrin in place of the FCS. The cells remained in this medium for 72 h before some were stimulated by exposure to DMEM containing FCS, the remainder being maintained in the growth-arrest medium as controls. In both experimental and control cultures, the medium was changed every 24 h. Samples for X-ray microanalysis were taken after 72 h of growth arrest (zero time) and every 24 h. X-ray microanalysis and electron microscopy At the end of the experimental period, grids bearing the cells were removed from culture and the overlying medium was removed by dipping the cells rapidly into ice-cold distilled water. The cells were cryofixed by plunging into liquid nitrogen, freezedried overnight and coated with carbon before analysis. X-ray microanalysis was performed using 2001 Academic Press
692
Cell Biology International, Vol. 25, No. 7, 2001
700
700 A
B K+
600
500 [] mmoles/kg
500 [] mmoles/kg
P
600
400
300
400
300 Cl
200
200 Na
+
100
0
−
100
24 48 72 Hours after addition of FCS
96
0
24 48 72 Hours after addition of FCS
96
Fig. 1. Concentrations of (A) sodium and potassium, and (B) phosphorus and chlorine in growth-arrested cultured rabbit airway smooth muscle cells (open symbols) and in cells stimulated to divide by the presence of foetal calf serum (closed symbols). Cells were plated at a density of 104 cells/well, allowed to settle overnight in the presence of DMEM containing 10% foetal calf serum, and then growth-arrested by FCS deprivation for 72 h. Cells were then either maintained in a growth-arrested state or stimulated into division by replacement of the growth-arrest medium with DMEM containing 10% FCS. Samples for X-ray micro-analysis were taken every 24 h and treated as described in the methods. Data represent the meanSE of cells from 9 animals, 12 to 15 cells were analysed from each animal.
a Zeiss EM 10 electron microscope. Areas of cell cytoplasm, approximately 66 m, were analysed for 100 s live time at 80 kV accelerating voltage and 1 nA beam current in STEM mode at ambient temperature. Spectra were processed using the FLS fitting routine, and quantification was achieved by use of the continuum-normalisation procedure, with reference to standards composed of gelatin containing known amounts of inorganic salts. Statistical comparisons were made by analysis of variance followed by Tukey’s t-test (for full details see Warley et al., 1994).
RESULTS AND DISCUSSION Element content in the growth cycle There were no significant differences in the concentrations of Na + , Cl or Mg2+ between growth arrested and stimulated cells throughout the growth period (Fig. 1A and B; data for Mg2+ not shown). These findings are in agreement with the idea that in proliferating cells increases in inward flux of Na + are balanced by outward flux leading to no overall increase in intracellular Na + concentration (Moolenhaar et al., 1986).
After addition of FCS, the concentration of K + increased slight and remained steady throughout the period of proliferation, whereas the concentration of K + gradually declined in growth arrested cells (Fig. 1A). After 48 h, the difference in [K + ] between the growth arrested and serum stimulated cells was significant (P<0.001, 48 h; and P<0.05 at 72 and 96 h). There was no change in [P] in the stimulated cells throughout the growth cycle, but the concentration of this element showed a gradual decrease in the non-stimulated cells, being significantly different (P<0.05) at 72 h after stimulation (Fig. 1B). In addition to differences in K + concentration, cells subject to serum withdrawal showed changes in overall morphology. Cells from cultures taken at zero time (i.e. after growth arrest for 72 h) showed a mixture of spindle and flattened shapes under low power STEM (Fig. 2A). After addition of FCS, the cells became narrow and spindle-shaped and appeared to grow in parallel with one another, and after 48 h the increase in cell number is apparent (Fig. 2C). At higher magnification, the spindleshape of the rapidly growing cells and their alignment with the tapered cytoplasmic end of one cell adjacent to the broad nuclear region of the next cell is more clearly seen (Fig. 2E). As the period of
Cell Biology International, Vol. 25, No. 7, 2001
693
Fig. 2. A, B, C. Lower power STEM images of rabbit airway smooth muscle cells from an identical source. (A) Cells growth arrested in serum-depleted medium for 72 h. Inclusions resembling apoptotic bodies, or apoptotic nuclei are sometimes seen (arrow). (B, C) Cells 48 h later in the presence of (B) growth-arrest medium or (C) after stimulation with 10% FCS. (D) Higher-power image of cells from B, the cells are flattened, and stress fibres can be seen running through the cytoplasm. Whereas cells which have been stimulated by the addition of FCS appear spindle-shaped, narrower and are more dense. Marker=5 m.
Cell Biology International, Vol. 25, No. 7, 2001
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60
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40 Frequency
Frequency
694
30
30
20
20
10
10
0
100
300 500 700 900 + [K ] Unstimulated cells
0
100
300 500 700 900 + [K ] Unstimulated cells
Fig. 3. Frequency histogram showing the distribution of [K + ] in rabbit airway smooth muscle cells after serum withdrawal (A) and after 48 h stimulation (B).
serum withdrawal increased, the cells appeared to flatten and take on a triangular shape. Few spindleshaped cells were seen (Fig. 2B). At higher magnification, stress fibres were seen running through the cytoplasm at the edge of the flattened cells (Fig. 2B). These changes in morphology are unlikely to result from the well-documented modulation of phenotype which occurs when smooth muscle cells are placed in primary culture, because the cells used here were carried through serial passage and are unlikely to be able to revert fully to the contractile state. The flattened form of ASM described here resembles that of cultured vascular smooth muscle cells treated to inhibit proliferation (Weissberg et al., 1993) and may be indicative of the nonproliferating state in vitro. The changes in K + concentration cannot be ascribed to the change in thickness of the proliferating cells since absorption of X-ray would lead to a decrease, rather than an increase in concentration. Specimen thickness would also be expected to affect the concentration of other elements. The association of higher concentrations of K + with cells in the proliferative state agrees with results from studies of tissues in vivo (Grundin et al., 1985; Smith and Cameron, 1999). However, the concentration of potassium found in the actively proliferating ASM is similar to values recorded in VSM in vivo from various different sources (see Warley et al., 1994). Since both the spindle shape and the higher potassium concentrations are seen in smooth muscle in vivo, which is contractile and non-proliferating, and in actively
proliferating muscle in vitro, it is unlikely that these are characteristic of proliferation per se. The results presented here show that for ASM in culture loss of K + from the growth arrested cells accounts in part for the differences between the proliferating and non-proliferating cells. This is further borne out by examination of K + concentrations in individual cells (Fig. 3). At 48 h after stimulation, when the cells are in the log phase of growth, [K + ] shows a homogeneous distribution with the mean, median and modal concentrations identical (mean 555 mmoles/kg, median and mode 556 mmoles/kg), whereas in the cells subject to serum withdrawal there is much wider distribution of K + concentrations with the mean value, 491.9 mmoles/kg dry weight higher than the median (485 mmoles/kg) and modal (436 mmoles/kg) values. The finding of decreased [K + ] without a concomitant increase in [Na + ] fits a pattern that is beginning to be recognised as being characteristic of cells entering apoptosis. Low concentrations of K + are known to be associated with the final stages of apoptosis (Warley and Morris, 1988; FernandezSegura et al., 1999), but there is evidence that loss of intracellular potassium begins earlier (Skepper et al., 1999), before morphological changes become apparent, and that this loss enhances the apoptotic process (Bortner et al., 1997). Serum provides factors necessary to support growth in vitro, and also plays an important role in the survival of cells in culture (Collins et al., 1994). Withdrawal of serum is known to promote apoptosis in a number
Cell Biology International, Vol. 25, No. 7, 2001
of different cell types including vascular smooth muscle (Bennett et al., 1995). The results presented here show a lowering of intracellular potassium in cultured ASM cells deprived of serum, the small number of cells (2–3%) exhibiting the lowest concentrations of potassium agree with reported rates of apoptosis in VSM after serum withdrawal (Bennet et al., 1995). The occasional finding of inclusions resembling either apoptotic nuclei or engulfed apoptotic bodies in cells after serum withdrawal (Fig. 2A) further adds support to this idea. ACKNOWLEDGEMENTS The author would like to thank Drs S. J. Hirst and J. P. T. Ward for their help with this work. This work was supported by the Wellcome Trust and a Sharpey-Shafer Award from the Special Trustees of St Thomas’ Hospital. REFERENCES B MR, E GI, S SM, 1995. Apoptosis of human vascular smooth muscle cells derived from normal vessels and coronary atherosclerotic plaques. J Clin Invest 95: 2266–2274. B CD, H FM J, C JA, 1997. A primary role for K + and Na + efflux in the activation of apoptosis. J Biol Chem 272: 32436–32442. C MKL, P GR, R-T G, N MA, L-R A, 1994. Growth factors as survival factors: regulation of apoptosis. Bioessays 16: 133–138.
695
F-S E, C FJ, C MA, W A, C A, 1999. Changes in elemental content during apoptotic cell death studies by electron probe X-ray microanalysis. Exp Cell Res 252: 454–462. G TG, R GM, F B, L M, W Y, 1985. X-ray microanalysis of psoriatic skin. J Invest Dermatol 85: 378–380. H SJ, 1996. Airway smooth muscle cell culture: application to studies of airway wall remodelling and phenotype plasticity in asthma. Eu Resp J 9: 808–820. M WH, D LHK, D SW, 1986. Ionic signalling by growth factor receptors. J Exp Biol 124: 359–373. S JN, K I, G MR, H L, H SJ, W A, M MJ, C NRB, 1999. Changes in elemental concentrations are associated with early stages of apoptosis in human monocyte-macrophages exposed to oxidised low-density lipoprotein: an X-ray microanalytical study. J Pathol 188: 100–106. S NKR, C IL, 1999. Ionic regulation of proliferation in normal and cancer cells. In: Ingram P, Shelburne J, Roggli V, LeFurgey A, eds. Biomedical Applications of Microprobe Analysis. San Diego, Academic Press. 445– 459. W A, C KPB, C HB, T CHC, W JPT, H SJ, 1994. Preparation of cultured airway smooth muscle for study of intracellular element concentrations by X-ray microanalysis: comparison of whole cells with cryosections. J Microsc 175: 143–153. W A, M IW, 1988. Concentrations of elements in dying thymocytes from the thymus gland of diabetic rats. Scanning Microscopy 2: 2227–2232. W PL, G DJ, S CM, M JC, 1993. Approaches to the development of selective inhibitors of vascular smooth muscle cell proliferation. Cardiovascular Res 27: 1191–1198.