JOURNAL OF CELLULAR PHYSIOLOGY 143:26-38 (1990)

Contraction of Vascular Smooth Muscle in Cell Culture T H O M A S R. MURRAY,* BRYAN E. MARSHALL,

AND

EDWARD

I.

MACARAK

Center for Research in Anesthesia, School of Medicine (T.R.M., B.E.M.) and Department of Histology and Embryology, School of Dental Medicine (E./.M.), University of Pennsylvania, Philadelphia, Pennsylvania 19 104; Connective Tissue Research Institute, University City Science Center, Phi/dde/phia,Pennsy/vania 7 9 I04 (€.).M.) The use of cultured vascular smooth muscle cells for the study of events related to excitation and contraction of smooth muscle has been limited by the inability to reliably induce contractile responses after subculturing of t h e cells. This limitation has been overcome by t h e cell culture preparation described herein. We demonstrate that appropriate responses to both smooth muscle agonists and vasodilators were preserved in cells that were serially subcultured. Fetal bovine pulmonary artery and aortic cell cultures were established following enzymatic dispersion of the medial portion of freshly harvested vessels. At various times after isolation, cells were transferred to microscope coverslips coated with a polymerized silicone preparation (polydimethylsiloxane).Tension forces generated by t h e cells were manifested as wrinkles and distortions of this flexible growth surface. Visual evidence of cell contraction in the form of increased wrinkling was documented for cells exposed to angiotensin II, carbachol, and KCI. Decreases in cell tension occurred following treatment with isoproterenol, and those relaxing effects were overcome by subsequent treatment with the agonist carbachol. The contractile responses did not diminish with prolonged maintenance in culture or repeated subculturing. Phosphorylation of the light chains on the contractile protein myosin was also measured as a biochemical index of agonist-induced contraction. Cells depolarized with KCI or exposed to carbachol showed increased myosin phosphorylation when analyzed by 2-dimensional gel electrophoresis. The responses remained intact through 7 passages and 9 weeks in culture. These results show that cultured vascular smooth muscle cells do not necessarily undergo a phenotypic modulation with loss of contractility under prolonged maintenance in culture. Cell cultures from isolated muscle cells have become valuable for the study of some basic physiological and biochemical properties of muscle (Lieberman, 1987). These preparations can offer a variety of advantages. The cell cultures contain only a single cell type, the extracellular environment is easily controlled, and all cells have unrestricted access to superfusate solutions, i.e., diffusion distances present in multilayered muscle preparations are eliminated. Further, in the case of vascular smooth muscle (VSM), the endothelial cells, normally closely associated with the medial smooth muscle layer, can be isolated from the muscle preparation and maintained separately or in a mixed culture. This allows for controlled experimentation with VSM in the presence and absence of endothelial influences. The discovery of several endothelial-derived vasoactive substances underscores the importance of controlling for endothelial influences on VSM when studying muscle physiology (Furchgott, 1983; Vanhoutte, 1987). A significant obstacle to the use of VSM cells in culture for work involving cell contraction has been the difficulty in getting cultured cells to contract reliably in the presence of vasoactive stimuli when subcultured or maintained in culture for long periods (Chamley et 0 1990 WILEY-LISS, INC

al., 1977; Mauger et al., 1975). This has been related to the changes in morphology and organelle composition that are sometimes seen when VSM cells are placed in cell culture. It has been suggested (Chamley-Campbell et al., 1979) that these cells undergo a “phenotypic modulation” a s they replicate in culture and convert to a cell type that is characterized by proliferation, synthesis, and secretion of extracellular proteins (Sjolund et al., 1986) rather than contraction. If measures are taken to growth arrest the cultures before they are allowed to proliferate extensively, these changes are sometimes reversed (Chamley-Campbell et al., 1981). Nevertheless, despite the apparent return to a morphology characteristic of the “contractile” phenotype, VSM cell contraction could not be demonstrated once the cells were passaged in culture (Chamley et al., 1977). Several recent studies have caused the concept of “contractile” versus “synthetic” phenotype to be ques-

Received September 14, 1989; accepted November 29, 1989

*To whom reprint requestsicorrespondence should be addressed.

CONTRACTION OF VSM CELLS IN CELL CULTURE

tioned as being overly simplistic in describing the state of differentiation of VSM cells in culture (Owens et a]., 1986; Rovner et al., 1986). Cells that had been maintained in culture or subcultured for long periods (and therefore presumably in the “synthetic” phenotype) continued to show some contractile properties and responses (Gunther et al., 1982; Colucci et al., 1984; Martin and Gordon, 1983; Owens et al., 1986). This evidence from several independent laboratories, taken collectively, raises considerable doubt about the idea of a global, coordinated conversion from one phenotype to another in all vascular smooth muscle cells. Efforts were therefore directed at developing a VSM culture preparation that would reliably contract to agonists in long-term culture and after subcultivation. The present work describes cultures of smooth muscle cells that can be made to contract in culture to a variety of vasoactive stimuli. Both morphologic (wrinkling of the culture surface) and biochemical (phosphorylation of myosin light chains) changes were observed and constitute our evidence for contraction of fetal bovine VSM cells in vitro. We demonstrate that appropriate responses to several vasoactive agents (KC1, angiotensin 11, carbachol, and isoproterenol) were maintained in cultured cells for up to 9 weeks and even with repeated subculture. This indicates that the capacity for VSM cells to contract in culture is not necessarily lost after extensive cellular replication as had been previously reported (Mauger et al., 1975; Chamley et al., 1977). Further, the demonstration that the contractile responses were maintained in these cultures indicates their potential usefulness as a convenient model system for studies examining various aspects of excitation-contraction coupling in VSM.

27

0.5 g/L bactopeptone (Difco, Detroit), BME amino acids (Hazelton), BME vitamins (Flow Laboratories, McLean, VA), and 15 mM HEPES (Research Organics, Cleveland). Culture medium contained 5.8 mM KCl under standard conditions, and KC1 levels mentioned in experimental treatments are in addition to this amount. Cells were maintained in a humidified atmosphere of 5% CO, in air at 37°C and passaged ( 1 : l O ratio) at weekly intervals. Absence of endothelial contamination in the VSM cell cultures was confirmed by negative staining with antibody directed against the endothelial-derived von Willebrand protein (Jaffe et al., 1973).

Assessing cellular contraction The responsiveness of individual cultured PA VSM cells to vasoactive substances was evaluated using a very sensitive procedure as described by Harris et al. (1980). A thin layer of silicone fluid, polydimethyl siloxane (30,000 centipoise, Hopkins & Williams, Essex, England), was spread on a microscope coverslip, which was then inverted, and the silicone fluid layer was briefly exposed (1-3 seconds) to a low flame from a Bunsen burner. With this short heating, crosslinking of the outermost portion of the linear polymeric chains occurred and resulted in a very thin rubber “skin,” under which remained a non-cross-linked fluid, which served a s a lubricant. Traction forces on the silicone rubber surface (such as those caused by contracting cells) generated wrinkles in the polymerized layer, which were readily apparent under phase-contrast microscopy. Cultures were prepared by placing coverslips, coated as described above, on the bottom of standard plastic cell culture dishes and seeding the dishes with VSM cells (5-20 x lo3 cellsicm’). Cells were allowed to MATERIALS AND METHODS attach for a minimum of 16 hours before challenge with agonists. Responses to vasoactive stimuli were docuIsolation and culture of VSM cells mented with a Nikon Diaphot inverted phase-contrast Pulmonary arteries (PA) and aortas were obtained microscope equipped with a Nikon 35 mm camera. from second- or third-trimester fetal calves shortly af- Cells were photographed before and a t various times ter cows were sacrificed at a local abattoir (Moyer after the addition of vasoactive agents to the culture Packing Co., Souderton, PA). Vessels were transported medium bathing the cells. The microscope stage and to the laboratory in chilled (4°C) medium 199 (M199) cultures were maintained a t 37°C with a continuous (Hazelton, Lenexa, Kansas). Pulmonary arteries and flow of heated air. aortas were separated from surrounding tissue and cut Two-dimensional gel electrophoresis into longitudinal strips under aseptic conditions. The medial portion of the vessel wall, which contains only Cultures were seeded at a density of 5-10 x lo3 VSM cells (Pease and Paule, 19601, was isolated by cells/cm2 in standard 100 mm plastic culture dishes dissection. A suspension of dispersed single VSM cells (Corning, Corning, NY) and maintained for at least 1 was produced by first mincing the muscle strips into 1 week post-confIuence. Three hours prior to use, culture mm pieces followed by a n enzymatic digestion of the medium was changed from M199 containing 10% FBS extracellular matrix (Van Dijk and Laird, 1984). The to 4.5 ml of serum-free medium. Vasoactive agents digestion solution consisted of collagenase (Cooper Bio- were dissolved in M199 and added as 0.5 ml aliquots. chemical, Freehold, NJ, 170 U/ml), soybean trypsin in- Incubations were halted at the indicated times by sihibitor (Sigma Chemical Co., St. Louis, 0.05 mg/ml), multaneously adding ice-cold perchloric acid with and DNase I (Sigma, 100 Ulml) all dissolved in tissue EGTA (final concentrations 1 N and 1 mM, respecculture medium M199. Digestion and culture medium tively) and freezing the dishes in a n ethanol/dry ice also contained Fungizone (2.5 pglml) and gentamicin bath. Dishes were thawed and rinsed successively with (10 pg/ml) (GIBCO, Grand Island, NY). Digestions ice-cold solutions of 1 N perchloric acid with 1 mM were carried out for 16 hours at 37°C on a gyratory EGTA and then 0.5 N perchloric acid. Cells were harshaker a t 200 rpm. Isolated cells were washed and re- vested with a plastic scraper and collected by centrifususpended in M199 containing 10% fetal bovine serum gation at 1,OOOg for 5 min. The pellets were resus(FBS) and seeded into culture vessels. Cells were fed pended in 0.25 ml of buffer (1%sodium dodecyl sulfate, twice weekly with M199 supplemented with 10% FBS 20 mM dithiothreitol, and 10%glycerol) and disrupted (Sigma), 0.29 g/L L-glutamine (Sigma), 3 g/L glucose, by sonication.

28

MURRAY ET AL

The extent of myosin light chain (MLC) phosphorylation was determined using a modification of the 2dimensional gel electrophoresis procedure described by OFarrell (1975). Aliquots of the sonicated samples were subjected to isoelectric focusing (3.5 mm x 130 mm gels) under reducing conditions in the first dimension. Ampholytes in the pH range of 4.0 to 6.5 were used. This was followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis in 12% gels in the second dimension. These slab gels were then stained with Coomassie Blue. Evaluation of the MLC bands with an LKB laser densitometer allowed quantitation of the proportion of the total amount of light chain that was phosphorylated.

Statistical analysis MLC phosphorylation is expressed as the percent of the total MLC that is contained in the bands corresponding to phosphorylated forms of the protein. Values are means * S.D. (n), where n is the number of observations. Data are compared using Student’s t test.

RESULTS Wrinkling of the silicone rubber “skin” PA VSM cells attached and spread on thin sheets of polymerized polydimethyl siloxane (silicone rubber). Traction forces exerted by these cells on the growth surface were manifested as distortions and wrinkles in the silicone rubber and were seen in unstimulated cultures under standard growth conditions (Fig. 1A). The cells in Figure 1A were photographed immediately prior to the addition of angiotensin 11. Upon exposure to angiotensin I1 (320 nM), a marked increase in wrinkling of the silicon substrate underneath the muscle cells was seen, indicating a contractile response (Fig. 1B). The wrinkling appeared quickly and progressively increased during the 2 minute exposure to the drug. Appropriate responses to other agents that affect muscle tone via different mechanisms were also noted. Potassium chloride (20 mM), which causes contraction of VSM through effects on voltage-operated ion channels, likewise brought about increases in tension during a 3 minute exposure (Fig. 2). Similarly, the response to isoproterenol, a f%-agonistthat causes vasodilation in vivo, is preserved in these cells in culture. A decrease in tension is evidenced by the reduction of surface distortion following treatment of another culture with 110 pM isoproterenol (Fig. 3). Attempts to reverse the responses to the various vasoactive agents tested by removal of the drugs were not readily feasible owing to the highly hydrophobic and delicate nature of the polymerized polydimethyl siloxane growth surface. Complete removal of the culture medium and exposure of the surface to air during efforts to wash out previously administered drugs caused marked distortion and damage to the polymerized layer. However, vasodilators and vasoconstrictors could be given sequentially to the same cell preparation to show that a drug from one class could functionally overcome the antagonistic effects of a previously administered drug. Resting tension in an unstimulated culture (Fig. 4A) was reduced by a 3 minute exposure to 75 pM isoproterenol (Fig. 4B). The relaxation caused by the isoproterenol was reversed in this culture within

112 minute after the addition of the muscarinic agent carbachol (70 pM). As shown in Figure 4C, there was an increased amount of substrate wrinkling, even compared to the initial resting level. Raising the carbachol concentration 2 minutes later to 130 pM brought about increased contraction of these VSM cells during the next minute (Fig. 4D). Contractile responses are maintained during prolonged culture and subculture The results using the silicone rubber surface to assess changes in cell tension suggested that this was a very sensitive method for detecting contractile responses in isolated cultured VSM cells. This method was therefore used to determine if cells that had been maintained in culture for several weeks or cells that had been repeatedly subcultured could still react appropriately to vasoactive stimuli. The first of these issues was addressed by maintaining PA VSM cells in culture for 7 weeks prior to testing. Cells were grown in standard plastic tissue culture flasks for the first 10 days in primary culture. The cultures were then subcultured onto glass microscope coverslips coated with polymerized polydimethyl siloxane and maintained for an additional 32 days. Contractile responses to carbachol(60 pM) were preserved during this 7 week period and appeared identical with those noted in primary cultures (Fig. 5). Similarly, cells passaged four times a t 7 to 10 day intervals over the same 42 day period also demonstrated contraction when exposed to carbachol (Fig. 6). Comparable long-term studies were carried out with aortic VSM cells isolated from the same fetal calves as the PA cells. These cells had the same morphologic appearance and exhibited observable traction forces in unstimulated cultures similar to the PA VSM cells (Fig. 7A). Exposure of these 6 week old cultures (which had been subcultured on day 10) to 60 pM carbachol raised tone in the cells (Fig. 7B). Other cultures of aortic VSM that were established 42 days earlier and passaged four times showed decreases in tone when treated with 110 pM isoproterenol (Fig. 8). Cell replication Inspection of VSM cells seeded on polydimethyl siloxane under phase contrast microscopy revealed the morphology of the cells to be comparable to cells seeded on standard plastic culture dishes. Cells attached and spread within hours of being seeded on either surface. At low cell densities, VSM cells typically appeared elongated with foot-like processes extending from many of them. However, over the next several days after seeding, it became strikingly apparent that there were many more cells in areas of the culture surface devoid of the silicone rubber coating compared with those coated with it. This was especially true for the cultures used in studies involving long-term maintenance that were kept for a period of weeks. A growth curve was therefore determined to compare plating efficiency and replication rates on microscope coverslips with and without polydimethyl siloxane coating. Cells were seeded onto round coverslips (25 mm diameter) a t 25,000 cells each and placed in 35 mm diameter plastic culture dishes. After allowing cells to attach overnight to the coverslips the dishes were flooded with growth

CONTRACTION OF VSM CELLS IN CELL CULTURE

Fig. 1. Contractile response of PA VSM cells to angiotensin 11. A primary culture seeded onto polymerized polydimethyl siloxane is shown before (A) and 2 minutes after (B) exposure to 320 nM angiotensin 11. Basal tension in the cell, as indicated by the wrinkling of

29

the substrate under the unstimulated cells, is markedly increased by this treatment. Changes were documented by phase-contrast microscopy a t 37°C. Magnification = x 112.

30

MURRAY ET AL

Fig. 2. Effect of increased KCL concentration on VSM tone. PA VSM cells are shown before (A) and 3 minutes after (B) raising the KCl in the medium by 20 mM. Increased wrinkling of the substrate is noted under the cluster of cells (arrowheads) as well a s in the areas adjacent to them. Magnification = X 112.

Fig. 3. Decreases in VSM tone due to isoproterenol. Cultures of PA VSM frequently demonstrated some basal tone (arrowheads) under standard growth conditions (A). Addition of isoproterenol (110 JLM final concentration) resulted in a progressive diminution of wrinkling over the following 9 minutes (B).Magnification = x 112.

medium. Cell number per coverslip was determined at the indicated times after plating (Fig. 9). Plating e f f ciency was comparable for coverslips with and without

the silicone rubber coating (78%). However, cell replication occurred more quickly and continued to a higher cell density on the uncoated coverslips.

CONTRACTION OF VSM CELLS IN CELL CULTURE

31

Fig. 4. Carbachol reverses the relaxing effects of isoproterenol. Basal tone (A) in cultures of PA VSM cells was progressively reduced over 3 minutes by the addition of isoproterenol (75 p M final concentration) (arrowheads) (B). Addition of carbachol (70 FM) 1 minute later overcame the relaxing effects of isoproterenol and raised tone

above the basal level within 1/2 minute (C). The carbachol concentration was then raised further to 130 kM, and marked increases in contraction were noted over the next 2/3 minute (D).Magnification

Myosin light chain phosphorylation

proteins. Cultures harvested without prior stimulation showed a majority of the myosin to be in a n unphosphorylated form (Fig. 10A). This form of the protein migrated to a point of the gel that corresponded to a n isoelectric point with a PI = 5.1. Myosin light chains from resting cultures typically contained 20-30’3 of the total myosin in the phosphorylated species. Brief exposure (2 minutes) to 100 mM KC1 prior to harvesting caused a n increase in the percent of

Phosphorylation of the 20,000 dalton light chains of the contractile protein myosin by a calcium- and calmodulin-dependent kinase was employed here a s a biochemical marker for contraction of isolated PA VSM cells in culture. Homogenates of PA VSM cells subjected to 2-dimensional electrophoresis showed myosin light chains to be resolved from other cellular

=

~ 3 3 .

MURRAY

32

ET AL.

Fig, 5. Contractile response to carbachol in 7 week old cultures. Confluent primary cultures were passaged once 10 days after VSM cells were isolated from fetal bovine PA and seeded onto the polymerized silicone surface. Subcultured cells were maintained for an additional 32 days before testing. Resting tone was apparent in these 42

day old cultures prior to stimulation (A). Progressive increases in cell tension were noted (arrowheads) over the 8 minutes following addition of carbachol(60 pM) to the culture medium (B).Magnification = X 39.

myosin that was phosphorylated and a corresponding decrease in the unphosphorylated bands (Fig. 10B). Identification of the bands on the 2-dimensional gels that were the myosin light chains was based on three lines of evidence. First, the proteins co-migrated with purified turkey gizzard myosin light chains. Second, the bands reacted with smooth muscle-specific myosin light chain antibody. And, last, spots corresponding to positions believed to be phosphor lated forms of the myosin showed incorporation of [3 PI into those spots when cells were incubated with the label before and during stimulation. Phosphorylation patterns for MLC during a 2 minute exposure of cultures to 100 mM KC1 showed a concentration-dependent increase in the percentage of the light chains in the phosphorylated form (Fig. 11). Levels of the phosphorylated myosin species in maximally stimulated cultures (100 mM KC1) reached amounts of 140% of those in parallel unstimulated cultures. Likewise, phosphorylation in cultures treated with 100 FM carbachol for 5 minutes increased to 143 5.0% (n =

5 ) of levels seen in untreated controls (P < .01). Following introduction of 100 mM KC1 to the culture medium, phosphorylation increased to maximal levels within 1minute (Fig. 12). This level of phosphorylation was maintained for at least 10 minutes without significant decline. The effects of prolonged culture periods and repeated passage of cells on phosphorylation levels in resting and stimulated cultures were addressed next. Cultures were passaged at weekly intervals for 8 weeks with basal and KC1-stimulated (100 mM for 2-10 minutes) percentages of phosphorylated myosin light chains determined for each group. Levels of phosphorylated myosin in untreated cells did not vary systematically with time in culture and ranged from 14 to 30% (Fig. 13). KC1 induced statistically significant (P 5 .04) increases in phosphorylation for cells from all passages and a t all times tested without any apparent decrease in response with extended time in cult,ure. There were no statistically significant differences in the magnitude of the response among the various treated groups.

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CONTRACTION OF VSM CELLS IN CELL CULTURE

Fig. 6. Effect of repeated subculture on contractile response to carbachol. Cells isolated from the same harvest as those in Figure 5 were passaged four times at 7-10 day intervals over the same 42 day culture period. Cells were grown in standard plastic tissue culture flasks for 39 days during which time cultures became confluent four times prior to being subcultured. On day 39, cultures were split for the last time and seeded onto coverslips coated with polydimethyl siloxane. Cells were photographed before (A)and 2 minutes after (B) treatment with carbachol (30 FM). Note increased wrinkling in carbacholtreated cultures (arrowhead). Magnification = x 165.

33

Fig. 7. Contractile responses in 7 week old aortic VSM cell cultures. Aortic VSM cells were isolated from the same fetal calves as those used for studies shown in Figures 5 and 6. These cells have a morphology that is similar to the PA cells. Primary isolates of aortic VSM cells were grown to confluence in plastic culture flasks prior to being subcultured once onto the silicone rubber on day 10. Cells were then maintained an additional 32 days under the same conditions as those used for the PA cells in Figure 5. Unstimulated aortic cells showed some basal tone (A). This baseline level of tension (arrowheads) was increased by the addition of carbachol (60 pM) as shown in a photograph taken 1 minute later (B). Magnification = X 83.

DISCUSSION Prior work has suggested that VSM cells lose the ability to contract when subcultured (Chamley et al., 1977; Mauger et al., 19751, and this has been a limiting factor in their usefulness for studying certain aspects of excitation-contraction coupling. The present report suggests that this is not true for all VSM cells in culture. A fetal bovine VSM cell preparation is described that demonstrates the capacity to respond appropriately to several vasoactive stimuli and that retains this property during prolonged cell culture and repeated passage. Increases in traction forces exerted by the

cells on the elastic growth surface (polydimethyl siloxane) were observed for angiotensin 11, carbachol, and KCI. These agents function via distinctly different mechanisms of action, indicating that multiple mechanisms for stimulating contraction remained intact in these cultured cells. Also, P-adrenergic receptor-mediated responses can be elicited as shown by the decreased wrinkling noted when isoproterenol was added to cultures (Figs. 3,8). The effects of the smooth muscle relaxant isoproterenol on cell tone were reversed by addition of the agonist carbachol (Fig. 4). A capacity to

34

MURRAY ET AL

Fig. 8. Preservation of VSM relaxation response in long-term aortic culture. Cultures of aortic VSM cells were established and maintained similar to those described in Figure 6. Forty-two day old cultures that had been passaged four times showed varying amounts of basal tone. A culture is depicted before (A) and 5 minutes after (B)

addition of isoproterenol (110 pM) to the culture medium. Note the decrease in wrinkling after addition of isoproterenol. Responsiveness to the vasodilator isoproterenol was diminished neither by the length of time in culture nor the number of subcultivations. Magnification = x 83.

demonstrate contractile responses in this sensitive assay was not diminished by prolonged time in culture or repeated passage of either PA or aortic VSM cells. It is apparent from observing cultures that not all cells contract when exposed to agonists. The proportion of cells responding varied from culture to culture but did not change systematically with time in culture or passage number. This observation is similar to one by Gunther et al. (1982) where contractile responses to angiotensin 11 were seen in only about 30% of cells in primary culture. The reason for the lack of response in other cells of the same culture is not clear and remains a point for further investigation. The use of crosslinked polydimethyl siloxane to as-

sess changes in cell tension is a very sensitive method capable of detecting small changes in sheer forces exerted by cells. Indeed, the method was first described in conjunction with time-lapse photography as a means to observe the weak traction forces generated by fibroblasts as they slowly migrate across a culture surface (Harris et al., 1980; Harris, 1984). However, it is a qualitative type of analysis that does not readily lend itself to measurement of the amount of force generated by the stimulated cells. Conversion of myosin light chains from an unphosphorylated to a phosphorylated form correlates well with initial force development for several smooth muscle preparations (for review, see Silver, 1986). Therefore, phosphorylation of myosin

CONTRACTION OF VSM CELLS IN CELL CULTURE

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Fig. 9. Effect of growth surface on replication rate. Microscope coverslips (25 mm) with ( 0 )or without ( 0 )a coating of polymerized polydimethyl siloxane were attached to the bottom of 35 mm culture dishes. One milliliter of a cell suspension of PA VSM cells containing 25,000celldm1 was placed on each coverslip. This was enough to cover the entire coverslip without spilling over onto the adjacent culture dish surface. Cells were allowed to attach overnight before 2 ml of additional media was added. Cells were harvested at the indicated times, and cell number per coverslip was determined. Points represent the mean ? S.D. for triplicate cultures except the day 10 point for the control curve, which is the mean of duplicate dishes.

light chains was employed here as a biochemical marker for cell contraction. This permitted a more quantitative assessment of drug responses as well as a means to confirm that the changes in wrinkling on the silicone rubber surface did represent cell contraction. The present study demonstrates that myosin light chain phosphorylation can be stimulated in cultures of PA VSM cells by agents that stimulate cell contraction. The percentage of the total myosin that is phosphorylated increases as the amount of KCl in the bathing medium is raised from 20 to 100 mM. Maximal levels of about 50% myosin phosphorylation with the KC1-induced contraction seen here in culture are comparable to the 50% maximum seen with strips from swine carotid arteries (Driska et al., 1981) and with rabbit PA muscle strips (Himpens et al., 1988). There is no diminution in the capacity for our cells to phosphorylate myosin in response t o agonists over long-term culture or subculture (Fig. 131, and agonist-induced increases to carbachol are preserved. These studies complement our results with the silicone rubber technique to demonstrate for the first time that cultured VSM cells can be made to contract despite repeated passage in culture. The time course of phosphorylation, in response to the 100 mM KCl, showed an initial rapid rise to maximal percentage phosphorylation with little or no decrease in phosphorylation levels during longer stimulation periods. This pattern differs from that sometimes seen with VSM strips where typically there is a rapid transient rise during the first 1-2 minutes followed by a drop to intermediate or low levels of phosphorylation without a concomitant decrease in stress generated by the muscle (Aksoy et al., 1983; Silver, 1986;Himpens et al., 1988).The decreases in phosphorylation in muscle strips over time have been shown to vary with several factors including the tissue source,

35

the nature of the stimulus, changes in [Ca+’ I, and temperature and may also reflect a role for other regulatory mechanisms (Hai et al., 1988). Unstimulated cultures typically had 1 5 3 0 %of the total myosin in the phosphorylated form, consistent with the presence of basal tone in the cells. Evidence for some tension in resting cultures is also apparent with cells grown on the polymerized silicone rubber. Most cultures contained regions of cells where minor wrinkling of the surface was evident prior to stimulation (see Figs. 1-8). Anderson et al. (1981) also saw significant amounts of basal phosphorylation in their primary cultures of mesenteric VSM cells, and it could be markedly decreased below basal levels with dibutyryl cyclic AMP or chlorpromazine in their hands. The higher basal levels of phosphorylation in cultured cells seen here and by Anderson et al. (1981) compared with levels seen in arterial strips (Aksoy et al., 1983) is likely related to the presence of considerably more basal tone in cells attached to the culture surface. Unlike muscle strips, which can be stretched or shortened to vary tone prior to testing, the length of the cultured cells cannot readily be manipulated. However, it is possible that differences in methodology for harvesting the cultured cells could also be responsible for some of the higher basal phosphorylation observed here (Driska et al., 1981; Aksoy et al., 1983). We did not observe any decreases in the responsiveness to KC1, carbachol, and isoproterenol of cells that have been serially passaged or maintained in culture for up to 9 weeks. This is at odds with the concept of “dedifferentiation” or phenotypic modulation to an irreversible “synthetic” state as had been postulated to occur with extensive replication in culture (Chamley et al., 1977; Chamley-Campbell et al., 1979. A revised hypothesis (Schwartz et al., 1986) suggested that the “contractile” and “synthetic” phenotypes were only two points on a “continuous spectrum of smooth muscle phenotypic expression,” and this seems far more plausible. The data presented here, as well as the work of several other independent laboratories (Gunther et al., 1982; Martin and Gordon, 1983; Larson et al., 1984; Colucci et al., 1984, Owens et al., 1986; Balmforth et al., 1988) are consistent with this revised view and collectively argue that repeatedly subcultured VSM cells may continue to express several characteristics associated with contractile responsiveness. The issue of what controls expression of the contractile apparatus in cultured VSM cells has been further clarified by the work from Owens’ laboratory (Owens et al., 1986; Rovner et al., 1986). Based upon analyses of fractional synthesis and content of smooth muscle-specifica-actin and myosin, these investigators postulated that expression of contractile functions is coupled to cell replication, with it being expressed to a much greater extent in non-dividing cells. All of our findings are consistent with this since our work was conducted using postconfluent cultures (myosin phosphorylation studies) or cells whose growth rates were significantly retarded by culturing on the polydimethyl siloxane (Fig. 9). Our results demonstrating contraction of repeatedly subcultured VSM cells contrasts with earlier reports where VSM cells were said to no longer contract following serial passage (Mauger et al., 1975; Chamley et al., 1977). There are several possible explanations for

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MURRAY ET AL.

Fig. 10. Two-dimensional gels of PA VSM cell culture homogenates. Confluent VSM cultures were harvested as described under “Materials and Methods,” and homogenates were subjected to isoelectric focusing in 3.5 x 130 mm gels (pH 4.0-6.5). Regions of the gel corresponding to a pH range of 4.7 to 5.5 were excised and used as samples for electrophoresis in 12%polyacrylarnide gels. Myosin bands are indicated by arrows. Homogenates of cultures harvested without pre-

treatment (A) had most of the myosin in a band corresponding to the unphosphorylated form (U)with a smaller fraction to the left of it (more acidic) corresponding to phosphorylated myosin light chain (P). Cultures exposed to 100 mM KCl during the 2 minutes prior to harvesting 03)showed a n increased proportion of myosin in the phosphorylated band with a corresponding decrease in the unphosphorylated myosin.

the differences noted. First, the methods described to assess contractility in this work are more sensitive than those used earlier. The silicone rubber surface is easily wrinkled, and there is no need for any cell attachment sites to be disrupted as stress generated by the cells causes distortions in the surface. Conversely, agonist-induced contraction on standard culture dishes might require some cell attachment sites to be broken if the cells are to shorten in length. Second, myosin phosphorylation, as an index of contractile responses, is not dependent on the absolute amount of myosin present, while the extent of cell shortening is more likely to be. A third potential reason for our success in observing responses may lie in the tissue source itself. Fetal bovine pulmonary artery and aortic VSM cells

were employed here whereas others have primarily used adult tissue from other species. A forth feasible explanation concerns the stimuli used to elicit contraction. Martin and Gordon (1983) and Mauger et al. (1975) showed that contractile responses to some agents were lost under certain growth conditions, while responses to others were preserved, indicating that the activity of all vasoactive agents were not simultaneously lost. Finally, it is also possible that the polydimethyl siloxane provides an environment conducive to retaining the ability to contract in culture via its effects on cell replication. Further work will be required to determine the significance of these factors in maintaining contractility in cultured cells. In conclusion, both visual and biochemical evidence

CONTRACTION OF VSM CELLS IN CELL CULTURE 150

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Fig. 12. Time course of myosin light chain phosphorylation. Confluent cultures of PA VSM were harvested before or 1-10 minutes after the addition of KCl(100 mM) to the culture medium and subjected to 2-dimensional electrophoresis. Results are the mean ? S.D. for n = 3, and all values are significantly greater than controls ( P < ,011.

is presented for VSM contraction in cell culture and the response is not diminished in long-term culture or serially subcultured cells. Using a culture system free of neuronal, humoral, hemodynamic, and endothelial influences on vascular tone, appropriate responses were seen to both smooth muscle agonists and vasodilators. Cultured VSM cells, therefore, can offer some unique advantages for studying a variety of factors involved in smooth muscle excitation and contraction.

ACKNOWLEDGMENTS The authors thank Robert Rogers for his valuable technical assistance. Turkey gizzard myosin was the generous gift of Dr. Samuel K. Chacko, myosin light chain antibody was donated by Dr. Joe R. Haeberle, and polydimethyl siloxane was kindly given to us by Dr. Albert K. Harris. We are also grateful to Drs.

the mean f S.D. Levels of phosphorylation were significantly different from controls at P 4 .04 in all cases.

Thomas M. Butler, Robert S. Moreland, Albert K. Harris, Mike A. Clark, Stephen F. Gorfien, and Pamela S . Howard for their helpful discussions and advice, and to Dr. Phyllis M. Sampson for the use of her densitometer. A preliminary report of this work was presented a t the 1987 meeting of the American Society of Anesthesiologists, Atlanta, GA. This work was supported in part by grants from the Pennsylvania Thoracic Society (T.R.M.) and the National Institutes of Health 5T32GM07612 (T.R.M.), GM29629 (B.E.M.), and HL34005 (E.J.M.).

LITERATURE CITED Aksoy, M.O., Mras, S., Kamm, K.E., and Murphy, R.A. (1983) Ca' + , CAMP, and changes in myosin phosphorylation during contraction of smooth muscle. Am. J . Physiol., 245:C255-C270. Anderson, J.M., Gimbrone, M.A., and Alexander, R.W. (1981) Angiotensin I1 stimulates phosphorylation of the myosin light chain in cultured vascular smooth muscle cells. J . Biol. Chem., 256:46934696. Balmforth., A.J.., Lvall. " , F.. , Morton. J.I.. andBal1. S.G. (1988) Cultured mesenteric vascular smooth muscle cells exoress doDamine DAlreceptors. Eur. J Pharmacol , 155t305-308. Chamley, J.H., Campbell, G.R., McConnell, J.D., and Groschel-Stewart, U. (1977) Comparison of vascular smooth muscle cells from adult human, monkey, and rabbit in primary culture and in subculture. Cell Tissue Res., 177.503-522. Chamley-Campbell, J., Campbell, G.R., and Ross, R. (1979) The smooth muscle cell in culture. Physiol. Rev., 59:l-61. Chamley-Campbell, J.H., Campbell, G.R., and Ross, R. (1981) Phenotype-dependent response of cultured aortic smooth muscle to serum mitogens. J. Cell Biol., 89t379-383. Colucci, W.S., Black, T.A., Gimbrone, M.A., and Alexander, R.W. (1984) Regulation of alpha-adrenergic receptor coupled calcium flux in cultured vascular smooth muscle cells. Hypertension, 6: 119-124. Driska, S.P., Aksoy, M.O., and Murphy, R.A. (1981) Myosin light chain phosphorylation associated with contraction in arterial smooth muscle. Am. J. Physiol., 240tC222-C233. Furchgott, R.F. (1983) Role of endothelium in responses of vascular smooth muscle to drugs. Circ. Res., 53557-573. Gunther, S., Alexander, R.W., Atkinson, W.J., and Gimbrone, M.A.

38

MURRAY ET AL.

(1982) Functional angiotensin I1 receptors in cultured vascular smooth muscle cells. J. Cell Biol., 92t289-298. Hai. C.-M.. and Murohv. R.A. (19881 Cross-bridge ohosuhorvlation and regulation of iatch state in smooth muscc. Am. 3.Physiol., 2.54:CI)B-a 06. ~ _ . _.. ~ _

.

~

Harris, A.K. (1984) Tissue culture cells on deformable substrata: Biomechanical implications. J. Biomech. Eng., 106t19-24. Harris, A.K., Wild, P., and Stopic, D. (1980) Silicone rubber substrata; A new wrinkle in the study of cell locomotion. Science, 208:177179. Himpens, B., Matthijs, G., Somlyo, A.V., Butler, T.M., and Somlyo, A.P. (1988) Cytoplasmic free calicum, myosin light chain phosphorylation, and force in phasic and tonic smooth muscle. J. Gen. Physi d . , 92:713-729. Jaffe, E.A., Hoyer, L.W. and Nachman, R.L. (1973) Synthesis of antihemophilic factor antigen by cultures human endothelial cells. J. Clin. Invest., 52t2757-2763. Kamm, K.E., and Stull, J.T. (1985)The function of myosin and myosin light chain kinase phosphorylation in smooth muscle. Annu. Rev. Pharmacol. Toxicol., 2 5 5 9 3 4 2 0 . Larson, D.M., Fujiwara, K., Alexander, R.W., and Gimbrone, M.A. (1984)Myosin in cultured vascular smooth muscle cells: Immunofluorescence and immunochemical studies of alterations in antigenic expression. J. Cell Biol., Y9:1582-1589. Lieberman, M., Hauschka, S.D., Hall, Z.W., Eisenberg, B.R., Horn, R., Walsh, J.V., Tsien, R.W., Jones, A.W., Walker, J.L., Poenie, M., Fay, F., Fabiato, F., and Ashley, C.C. (1987) Isolated muscle cells as a physiological model. Am. J. Physiol., 253:C349-C363. Martin, W., and Gordon, J.L. (1983) Spontaneous and agonist-induced Rb efflux from rabbit aortic smooth muscle cells in culture: A comparison with fresh tissue. J. Cell. Physiol., 115r53-60.

Mauger, J.P., Worcel, M., Tassin, J., and Courtois, Y. (1975) Contractility of smooth muscle cells of rabbit aorta in tissue culture. Nature, 255t337-338. OFarrell, P.H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem., 250t4007-4021. Owens, G.K., Loeb, A,, Gordon, D., and Thompson, M.M. (1986) Expression of smooth muscle-specific-isoactin in cultured vascular smooth muscle cells: Relationship between growth and cytodifferention. J. Cell Biol., 102:343-352. Pease, D.C., and Paule, W.J. (1960) Electron microscopy of elastic arteries; the thoracic aorta of the rat. J. Ultrastruc. Res., 3r469483. Rovner, A.S., Murphy, R.A., and Owens, G.K. (1986) Expression of smooth muscle and nonmuscle myosin heavy chains in cultured vascular smooth muscle cells. J. Biol. Chem., 26t14740-14745. Schwartz, S.M.,Campbell, G.R., and Campbell, J.H. (1986) Replication of smooth muscle cells in vascular disease. Circ. Res., 58:427444. Silver, P.J. (1986) Pharmacological modulation of cardiac and vascular contractile protein function. J. Cardiolvasc. Pharmacol., S(Supp1. 9):S344 4 4 6 . Sjolund, M., Madsen, K., von der Mark, K., and Thyberg, J. (1986) Phenotype modulation in primary cultures of smooth-muscle cells from rat aorta. Synthesis of collagen and elastin. Differentiation, 32:173-180. Van Dijk, A.M., and Laird, J.D. (1984) Characterization of single isolated vascular smooth muscle cells from bovine coronary artery. Blood Vessels, 21 :267-278. Vanhoutte, P.M. (1987) Endothelium-dependent contractions in arteries and veins. Blood Vessels, 24r141-144.