Immortalization of cat iris sphincter smooth muscle cells by SV40 virus: Growth, morphological, biochemical and pharmacological characteristics

Exp. Eye Res. (1995) 61 : 535-545

Immortalization of Cat Iris Sphincter Smooth Muscle Cells by SV40 Virus" Growth, Morphological, Biochemical and Pharmacological Characteristics ANETTE O C K L I N D a , S A R D A R Y . K . Y O U S U F Z A I b,SIKHA GHOSH c, M I G U E L COCA-PRADOS c , J O H A N STJERNSCHANTZaI'ANO A T A A . A B D E L - L A T I F b*

Glaucoma Research Laboratories, Pharmacia Ophthalmics, Uppsala, Sweden, bDepartment of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, U.S.A., CDepartment of Ophthalmology and Visual Science, Yale University, New Haven, CT, U.S.A. (Received Columbia 19 January 1995 and accepted in revised form 5 May 1995) The purpose of this study was to establish immortalized cell cultures of cat iris sphincter smooth muscle cells for a model investigating ocular receptors and their signal transduction pathways. Cultured cat iris sphincter muscle cells were immortalized by viral transformation with SV40 virus and the morphological and immunocytochemical properties of the normal and immortalized cells were investigated. The transformed cell clone, SV-CISM-2, was further characterized biochemically and pharmacologically. The normal muscle cells showed characteristics of smooth muscle cells, as judged by their growth and the presence of smooth muscle ~-actin and desmin. After seven passages the normal cells ceased to proliferate. In contrast, the immortalized cells retained their proliferative ability for more than 220 population doublings over 55 passages. The transformation phenotype in these cells was confirmed by their expression of the large T-antigen, the incorporation of viral DNA into cellular DNA, growth in agarose and in low-serum medium, and complete loss of contact inhibition. The immortalized cells expressed smooth muscle ~-actin, desmin and MLC protein. Biochemical and pharmacological studies on the SV-CISMcells revealed the presence of several functional receptors including muscarinic cholinergic, fl-adrenergic, peptidergic (substance P and endothelin), Platelet-activating factor, and prostaglandin (PG). Muscarinic stimulation of these cells resulted in: (a) a dose-dependent increase in the release of arachidonic acid (AA) and (PGs) and enhancement in the production of inositol trisphosphate (IP~); and (b) a substantial increase in MLC phosphorylation (118 %), an indicator of smooth muscle contractility. The stimulatory effects of carbachol on these responses were completely blocked by atropine, a muscarinic receptor antagonist. This study constitutes the first successful immortalization of iris sphincter smooth muscle cells. The SV-CISM-2 cells can serve as an important model system for investigations on the biochemical and pharmacological properties of receptors and their signal transduction pathways in smooth muscle. The advantage of these cells over normal iris sphincter cells is that they can be propagated over many generations without alterations in their morphological, biochemical and physiological characteristics. © 1995 Academic Press Limited Key words: iris sphincter smooth muscle cells; cat; SV40 virus; immortalized cells; tissue culture; morphology; receptors; phosphoinositide hydrolysis; arachidonic acid release; myosin light chain phosphorylation.

1. Introduction The iris sphincter has long served as a model for studies on receptor pharmacology. Its physiology and innervation are well established (Davson, 1990). In addition to cholinergic muscarinic receptors, the iris sphincter is also enriched in fl-adrenergic, prostaglandin (PG), substance P, endothelin and plateletactivating factor receptors (for review, Abdel-Latif, 1995). Activation of these receptors in the sphincter muscle results in the stimulation of phospholipases (C,A~,D) and generation of the second messengers inositol trisphosphate (IP3) and 1,2-diacylglycerol (DAG), arachidonic acid (AA) release and PG synthesis, cAMP formation, and to muscle contractionrelaxation, depending on the receptor subtype and * For correspondence. 0014-4835/95/110535 + 11 $12.00/0

species being investigated (Abdel-Latif, 1989, 1991, 1995). In the past the unavailability of isolated smooth muscle cells from the iris sphincter has hindered detailed studies on the molecular mechanisms underlying receptor function and signal transduction pathways in this tissue. Tissue culture is a powerful research tool to investigate the properties of smooth muscle at the cellular level (Charnley-Campbell, Campbell and Ross, 19 79) and cultured cells provide a more homogeneous cell population. However, m a n y cultured cells usually stop proliferating after a few passages. This problem is overcome by immortalization of the cells. In the past decade several important findings which were reported on receptors and signal transduction pathways were derived from work carried out on isolated a n d / o r immortalized cells (Taylor, 1993). While cultured cells from ocular tissues have been © 1995 Academic Press Limited

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widely used in a number of studies (Polansky et al., 1979; Coca-Prados and Wax, 1986; Tamm et al., 1991 ; Woldemussie, Feldman and Chen, 1993; Araki et al., 1993 ; Andley et al., 1994) attempts in the past to culture and immortalize smooth muscle cells from the iris sphincter have been unsuccessful. This could be due to the fact that this unique muscle develops from the neuroectoderm (Davson, 1990), in contrast to other smooth muscle cells in the body which are derived from the mesoderm. Thus the aim of the present study was (a) To establish an immortalized smooth muscle cell line from the iris sphincter, and (b) to characterize this cell line morphologically, biochemically and pharmacologically. The availability of such a cell line would greatly enhance our understanding of receptor pharmacology and signal transduction pathways in this unique smooth muscle. In the present study we attempted to immortalize cat iris sphincter smooth muscle cells employing SV40 virus. Our results show that immortalized cat iris sphincter muscle (SV-CISM-2) cells can be maintained in culture for 55 passages over 220 doublings without changes in growth behavior. These cells still synthesize c~-actin and desmin as monitored by immunohistochemistry, and myosin light chain (MLC) protein as monitored by immunoblotting showing that immortalization has not altered their normal differentiating function. The SV-CISM-2 cells retained functional receptors as monitored by the generation of second messengers and MLC phosphorylation.

2. Materials and Methods

Primary Cultures Iris smooth muscle cells were isolated from 4-6month-old cats. The cats were treated according to the recommendations of the NIH Guidelines for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 8 5 - 2 3 , 1985). The animals were killed using an overdose of pentobarbital and the iris sphincter muscle was dissected out from freshly removed eyes. Each eye was opened by a circumferential incision at the ora serrata. After placing the anterior segment in a Petri dish, the cornea was removed by cutting along the limbus. The stroma anterior to the muscle was thoroughly removed with fine forceps and scissors under a dissection microscope. The muscle was then everted and the iris pigment epithelium attached was carefully scraped off with a surgical sponge. The sphincter muscle fibers, running circumferentially, could then easily be distinguished from the radial fibers of the dilator muscle. The sphincter muscle was dissected out, further cleaned and cut into 1-2 mm a pieces. The explants were placed in Dulbecco's modified eagle medium (DMEM) containing 2 mg ml -~ collagenase, type 1A, 10% fetal calf serum (FCS) and 50 #g ml -~ gentamicin and then incubated for 2 hr at 37°C with occasional gentle

A. O C K L I N D ET AL.

agitation. The major parts of the explants were then dispersed into single cells or groups of 2-5 cells. The cell suspension was centrifuged at 200 g for 7 min and resuspended in equal parts of DMEM and Ham's F-12 supplemented with 10% FCS and 5 0 / z g m l ~ gentamicin ( + / S M medium). To selectively remove contaminating fibroblasts, which are reported to be more adhesive than smooth muscle cells in primary culture (Polinger, 1970) the cell suspension was incubated in a 25 cm 2 tissue culture flask containing + / S M medium for 30 min. The cells that were still in suspension at this time were transferred to another flask and cultured at 37°C in 5 % CO~, humidified air. After 3 days, one third of the culture medium was replaced with fresh medium and after 1 week the cells were confluent. The smooth muscle cells were subcultured at a split ratio of 1 : 4 using 0.05% trypsin and 0"02% EDTA. For routine culture 10 ng ml -~ h u m a n recombinant PDGF (isotype BB) was added.

Immortalization of the Smooth Muscle Ceils A primary culture of the cat iris sphincter muscle cells at subconfluency was infected with wild-type SV40 at multiplicities of infection of about 25 plaqueforming units per cell. Immortalization was carried out according to standard procedures (Coca-Prados and Wax, 1986). The culture medium was changed to DMEM supplemented with 10% FCS and 50 #g m1-1 gentamicin and the cells were inspected for the formation of transformed-like foci for 4 - 8 weeks. Individual foci were selected and maintained in DMEM supplemented with 10% FCS and 5 0 # g m l 1 gentamicin at 37°C in 5 % CO2, humidified air. One clone, designated SV-CISM-2, was isolated from one focus and studied here in detail.

Immunocytochemical Demonstration of Viral Large TAntigen SV-CISM-2 cells were grown on glass coverslips, fixed in methanol-acetone (1:2) for 20 min at - 2 0 ° C and stained by indirect immunofluorescence. Fixed cells were incubated with hamster antiserum to the large T-antigen of SV40 (1:10) for 1 hr at 37°C. After three washes in PBS, ceils were incubated with rhodamine-conjugated goat anti-hamster IgG (1:100). The coverslips were mounted and photographed under epifluorescent light. Cells incubated with preimmune hamster antiserum served as controls.

Southern Blot Analysis Southern blot analysis was performed to test the presence of integrated viral DNA in SV-CISM-2 cells. For this purpose, high molecular weight cellular DNA

IRIS SPHINC'TER S M O O T H MUSCLE CELL LINE

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was prepared and digested with two different restriction enzymes: Eco RI and Bill II. The first restriction enzyme cleaves once within the SV40 genome. The second restriction enzyme cleaves cellular sequences adjacent to the integrated viral DNA. SV40 DNA labeled with 32p was used as a probe to hybridize a Southern blot containing cellular DNA, digested with ~co RI or Bgl II, as described previously (Coca-Prado s and Wax, 1986).

gentamicin (culture medium) and subcultured every 4 days at a split ratio of 1 : 4. Aliquots of early passages were stored below - 135°C for further studies. For all studies examining the effects of carbachol (CCh) and other agonists on AA metabolism, second messenger formation and MLC phosphorylation, SV-CISM-2 cells were plated in culture medium at a density of 2 x 106 cells per 25 cm 2 flask and used when confluent. Cells from passages 2 0 - 3 5 were employed.

Growth in Semisolid Medium

Prelabeling of SV-CISM-2 Ceils with [3H]AA and Assay for the Release of Labeled AA and PGs by CCh

The anchorage-independent growth ability of the cells w a s investigated by culturing immortalized (passage 4 0 ) a n d normal cells (passage 4) in + / S M medium containing 0"33% agarose (Freshney, 1987) for 10 days.

Growth in Low Serum Medium Requirement of serum for growth was investigated by culturing both immortalized (passage 40) and normal cells (passage 4) in + / S M medium containing 0.5% and 2% FCS for 10 days.

Phase Contrast Microscopy Cells from primary cultures, subcultures and SVCISM-2 cells were examined periodically under phase contrast illumination and photographs were taken.

Immunocytochemistry of the Cytoskeleton Confirmation of the identity of the smooth muscle cells was performed by immunostaining with antibodies to cytoskeletal proteins. The staining patterns for the normal (passage 2-4) and SV-CISM-2 cells (passage 33-40) were compared. For this purpose, cells were cultured in glass chamber slides and starved for 1-2 days in low serum + / S M medium, fixed in icecold methanol or acetone for 5 min at - 20°C and air dried. Non-specific binding was blocked by 20% goat serum (Dakopatts AB). Monolayers were then stained with mouse monoclonal antibodies to h u m a n smooth muscle a-actin (1:200), desmin (prediluted) and cytokeratin (1 : 50) for 90 min. After washing in PBS, the cells were further incubated with FITC-labelled goat anti-mouse IgG (1:130) for 30 min. Non-specific binding was tested with preimmune mouse IgG (Dakopatts) or PBS. Acetone-fixed cryostat sections of the cat iris sphincter with attached iris pigment epithelium served as positive controls and cultured cat scleral fibroblasts (Harrison, Mondino and Mayer, 1990) served as negative controls.

Maintenance of SV-CISM-2 Cells in Culture Cells were routinely cultured in + / S M medium or DMEM supplemented with 10% FCS and 50 #g m1-1

To assay for muscarinic stimulation of phosphollpase A 2 SV-CISM-2 cells (2 mg protein per flask) were incubated in culture medium containing 3H-AA (2-5 #Ci per flask) for 24 hr at 37°C in a COz incubator, washed three times with non-radioactive DMEM, then incubated in 5 ml medium for 10 min in absence and presence of various concentrations of CCh. At the end of incubation the medium was analyzed for the release of radioactive AA and PGs by means of thin-layer chromatography (TLC) as described previously (Yousufzai and Abdel-Latif, 1985).

Prelabeling of SV-CISM-2 Cells with myo[aH]Inositol and Assay for Inositol Phosphates Production by CCh and the Effects of Isoproterenol To assay for the effects of muscarinic and adrenergic stimulation of phospholipase C SV-CISM-2 cells (2 mg protein per flask) were incubated in 5 ml inositoldeficient DMEM that contained aH-inositol (20 #Ci per flask) for 24 hr. The labeled cells were washed three times with non-radioactive medium then incubated in 5 ml medium that contained LiC1 (10 mM) for 5 min. Isoproterenol a n d / o r 3-isobutyl-l-methylxanthine (IBMX) were then added for 5 min, followed by addition of CCh for 10 min. The experiment was terminated by siphoning of the medium followed by addition of cold methanol. The 3H-inositol phosphates, which were recovered in the aqueous layer, were analyzed by means of anion-exchange chromatography as described previously (Howe et al., 1986).

Assay for Endogenous Release of PGs by CCh in SVCISM-2 Cells Incubation conditions and extraction of PGs from the medium were the same as described above except that no 3H-AA was included in the incubation medium. Release of endogenous PGF2~ and PGE2 from the cells into the medium (5 ml) by CCh were determined by radioimmunoassay (RIA) as previously described (Yousufzai and Abdel-Latif, 1984). The amount of PGs in each sample was determined by interpolation from the standard curve. Rate of PG release is presented as the amount of PG 10 min -1 (mg protein) -1.

538

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kb

--

23.1

--

9.4

--

6.6

--

4.4

--

2.3

FIG. 2. Southern blot analysis of SV40 DNA in SV-CISM2 cells. High molecular weight DNA from SV-CISM-2 cells digested with Eco RI (lane 1) or Bgl H (lane 2) separated by electrophoresis in 0'7% agarose gel, transferred to nitrocellulose filter and probed with a2P-labeled SV40 DNA. Arrow at left marks the position of linear SV40 DNA (5.2 kb) and arrow at right a 14-kb fragment of cellular DNA with integrated SV40 DNA. At right is shown the size (in kb) of several fragments of DNA digested with Hind Ill.

FIG. 1. (A) Immunofluorescence micrograph of SV-CISM2 cells showing T-antigen-specific staining. (B) Phase contrast micrograph of the same field shown in (A). x 1000.

Identification of MLC Protein by Western Immunoblotting SV-CISM-2 cells and rabbit iris sphincter muscle were homogenized in ice-cold 5 % trichloroacetic acid. The acid-insoluble material was solubilized in 2 0 0 / t l of SDS sample buffer by boiling for 30 rain in sealed tubes. Samples containing 2 0 / t g of protein were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) by the method of Laemmli (1970) using 12 % polyacrylamide gels. Following electrophoresis, the protein bands were electrophoretically transferred to nitrocellulose membranes. The non-specific sites on the m e m b r a n e were blocked by incubating it for 1 hr at room temperature in buffer containing 20 mM Tris-HC1, (pH 7"5), 0"5 M NaC1 and 3% BSA. The membranes were then blotted with monoclonal antibody anti-myosin (light chains 20 K). The MLC was detected with the use of alkaline phosphataseconjugated goat anti-mouse IgG and the chromogenic substrate 5-bromo-4-chloro-3-indoyl phosphate and Nitro Blue Tetrazolium.

Incubation and Measurement of the Effect of CCh and Atropine on 32pi Incorporation into MLC of SV-CISM-2 Cells To assay for the effects of CCh and atropine on MLC phosphorylation SV-CISM-2 cells (2 mg protein per flask) were incubated in 5 ml DMEM that contained 32Pi (75/,Ci ml 1) for 24 hr. The labeled cells were washed three times with non-radioactive medium then incubated in 5 ml of the same medium in the absence and presence of CCh (25/~,M) for 2 min. In experiments where atropine (2'5/~M) was used, the cells were incubated for 5 min with the drug before the addition of the agonist. The reaction was stopped by immersion in a methanol-solid-CO 2 slurry at - 8 0 ° C . MLC phosphorylation was analysed by SDS-PAGE and autoradiography as described previously (Howe et al., 1986).

Determination of Proteins Protein content was determined by the method of Lowry et al. (1951) with bovine serum albumin as a standard.

Materials Materials used were obtained from the following sources: all cell culture reagents and trypsin-

IRIS SPHIN(~TER SMOOTH MUSCLE CELL LINE

539

FIG. 3. Phase-contrast micrographs of cat iris sphincter smooth muscle cells. (A) Confluent primary cultures of normal cells. (B) Subconfluent tertiary cultures of non-transformed cells. (C) Subconfluent SV-CISM-2 cells at passage 33. (D) Confluent SVCISM-2 cells at passage 33. Typical appearance of transformed cells. × 1OO. ethylenediamine tetra-acetic acid (EDTA) (Gibco/Life Technologies Ltd, Paisley, U.K. or Grand Island, NY, U.S.A.); collagenase, platelet-derived growth factor (PDGF) (isotype BB), mouse monoclonal antibodies against h u m a n smooth muscle a-actin and antimyosin (light chains 2OK), FITC-labeled goat anti-mouse IgG, CCh, agarose, norepinephrine, substance P, atropine, platelet-activating factor (PAF), L-isoproterenol-HC1 and IBMX (Sigma Chemical Company, St. Louis, MO, U.S.A.); glass chamber slides (Labtek, Naperville, IL, U.S.A.) and cell culture plastic from Nunc, Nunclon Delta, Roskilde, Denmark. Mouse monoclonal antibodies to h u m a n desmin, ~-actin and cytokeratin were obtained from Immunotech, Marseille, France and preimmune sera from Dakopatts AB, Stockholm, Sweden. The monoclonal anti 56 kDa keratin antibody was produced against keratin from epidermal stratum corneum and reacts with keratin polypeptides no. 1, 2, 5, 6, 7, 8, 11, 14, 16, 17 and 18. Antiserum to large T-antigen was a gift from Dr Robert Carrol, (Department of Pathology, New York University) and wild-type simian virus 40 (SV40) was a gift from

Dr P. K. Ghosh (Yale University, CT, U.S.A.). Restriction enzymes were purchased from United States Biochemical, Cleveland, OH, U.S.A. endothelin-1 (human and porcine) from Peptide International, Louisville, KY, U.S.A. : PGs from Cayman Chemical Corporation, Ann Arbor, MI. U.S.A.: 3H-PGE2- and 3H-PGF2~ RIA kits from Advanced Magnetics, Cambridge, MA, U.S.A.; myo-['~H]-inositol (18 Ci mmol ~) and ~H-AA (1OO Ci mmo1-1) from Dupont, NEN Products, Boston, MA, U.S.A. and prestained SDS-PAGE standards (low range) from Bio-Rad Laboratories, Hercules, CA, U.S.A.

3. Results

Immortalization of Cat Iris Sphincter Smooth Muscle Cells We have used SV40 virus to induce cell proliferation of a primary culture of cat iris sphincter smooth muscle cells. After their transformation with SV40 virus, we isolated and characterized a clone, SV-CISM-

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Fro. 4. Immunofluorescence micrographs of normal smooth muscle cells at passage 7 intensively labeled with antibodies to smooth muscle a-actin (A) and to desmin (B); and micrographs of SV-CISM-2 cells at passage 34 labelled with antibodies to a-actin (C) and to desmin (D); x 200. The negative controls exhibited no staining (data not shown).

2. The successful incorporation of viral DNA into the host cell genome was confirmed by the immunocytochemical demonstration of viral large T-antigen. More than 9 9 % of the cells were transformed, as demonstrated by nuclear staining (Fig. 1), while no staining was seen in non-transformed cells or w h e n preimmnne serum was used. SV40 DNA labeled with 32p was used as a probe to hybridize a Southern blot containing cellular DNA digested with Eco RI or Bgl II. When cellular DNA was digested with Eco RI, the probe hybridized to two major bands of about 5-2 kb and 2-8 kb, respectively (Fig. 2). The 5-2 kb band corresponds to the size of the full length SV40 DNA. The smaller band corresponds to the electrophoretic mobility of supercoil SV40 DNA. However, a fragment of about 14 kb was detected when Bgl II was used, indicating that indeed the viral SV40 DNA was integrated into a single site in the cellular DNA of SV-CISM-2 cells. The SV-CISM-2 cells characterized and described in the present work have been grown over a period of several months.

Studies of Growth and Morphological Characteristics of Cat Iris Sphincter Muscle Cells in Primary Culture and of SV-CISM-2 Cells Under phase-contrast illumination individual cells in primary culture were ribbon- or spindle-shaped with phase-dense cytoplasm, central oval nuclei and long narrow cytoplasmic processes. After about 1 week a confluent monolayer had formed in which the cells were closely attached and arranged in slightly curved arrays [Fig. 3 (A)]. After subculture at a split ratio of 1:4, they reached confluency within 5 days. Approximately 3 - 4 weeks post-confluency the culture became organized into irregular bands of parallel monolayered cells, interspersed with multilayered hillocks. In subsequent subcultures [Fig. 3 (B)], cell morphology and growth pattern remained the same but growth was successively retarded, the population densities were lower and at about passage 7 the cells ceased to divide, despite the presence of PDGF. The morphology and growth behavior of SV-CISM-

IRIS SPHINCI'ER S M O O T H MUSCLE CELL LINE

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240

8 25

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220 -

200

i

180 o

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140 -

180

-~ 160

PGD 2 AA

~

120 o

100

200

140

120 0

7

6 5 -Log carbachol (M)

4

FIG. 5. Dose-response to CCh for release of aH-labeled AA and PGs in SV-CISM-2 cells. Incubations with the muscarinic agonist were carried out for 10 rain. Typical control values (in absence of agonist) for radioactive AA ( , ) , PGD2 (IN), PGE2 ( • ) and PGF2~ (C)) (dpm per flask) were 3474 _+302, 29_+ 5, 39 4- 5 and 51_+4, respectively. ECs0 s (FM): AA, 1"3; PGD2, 1.5; PGE2, 2-5; PGFz~, 3-5. The effects of CCh on the release of radioactive AA and PGs are expressed as percentages of their respective controls. Each point represents _+SEM from 2 or 3 experiments each run in duplicate. 2 cells were typical for immortalized cells (Pastan, 1979). Prior to confluency, the cells exhibited a bipolar but less elongated morphology than their parent cells [Fig. 3(C)], they were smaller, more refractive and had an irregular growth pattern. The nuclei had usually more t h a n four nucleoli, which indicates a high metabolic activity. Numerous round cells, characteristic for dividing cells, were seen. At confiuency (Fig. 3 (D)] they became thinner and longer and had a more ordered growth pattern. In postconfluent cultures the cells were even smaller, multilayered and not contact inhibited. SV-CISM-2, but not the normal cells, were capable of colony formation in a semi-solid medium such as agarose and exhibited a more t h a n doubled growth rate after the transformation. In contrast to the normal cells, SV-CISM-2 cells still proliferated after 10 days in + / S M medium containing 0.5 % FCS. Immunocytochemically, the smooth muscle cells in primary culture stained brightly for smooth muscle eactin [Fig. 4 (A)]. The stained microfilament bundles were oriented longitudinally in parallel. The cells were weakly positive for desmin [Fig. 4 (B)], as compared to cells stained with preimmune antibodies and PBS (data not shown). At 3 weeks post-confluency the desmin content had increased. None of the cells expressed cytokeratin, the intermediate filament typical for epithelial cells. Both smooth muscle a-actin and desmin were present in the intact cat iris sphincter while 5 6 kDa cytokeratin was found in the iris pigment epithelium (data not shown). In the scleral fibroblast cultures, used as negative control, about 5 % of the cell

i00

11

7

6 5 -Log carbaehol (M)

4

FIG. 6. Dose-response to CCh for release of endogenous PGF2~ in SV-CISM-2 cells. The average basal release of PGF2~ after 10 min of incubation was 259_+ 18 ng per flask (ECs0 = 4.5/ZM). Atropine (1/~M) was added 5 re.in before the addition of CCh (10/zM). Each point represents mean _+SEM from two experiments each run in duplicate.

population stained for smooth muscle a-actin and desmin (data not shown). These cells probably represent smooth muscle cells from scleral vessels. The remaining cells were unstained. Similarly, SV-CISM-2 cells stained for smooth muscle a-actin in a similar pattern as the normal cells but the staining was weaker [Fig. 4(C)]. Desmin (Fig. 4) was faintly expressed and was mostly perinuclear while normal cells also stained in the peripheral cytoplasm. Specificity of the immunoreactions was assured by comparison with the fibroblasts which were used as negative controls, and by staining with the preimmune antibodies.

Studies of Biochemical and Pharmacological Properties of SV-CISM-2 Cells Dose-response to CGh for release of all-labeled AA and PGs. The effects of different concentrations of CCh on AA release and PGs by cultured cells prelabeled with aH-AA are shown in Fig. 5. The muscarinic agonist increased AA release and the PGs in a dose-dependent manner. The ECs0 values obtained for AA release and PGD 2, PGE 2 and PGF2~ were 1.3, 1.5, 2.5 and 3"5/ZM, respectively. At 10/ZM CCh the increases in the release of AA, PGD2, PGE2 and PGF2~ were 26, 50, 66 and 150%, respectively.

Dose-response to CCh for release of endogenous PGF2~. CCh increased the release of PGF2~ in a dose-dependent m a n n e r (Fig. 6). The ECs0 value for CCh-stimulated release of PGF2~ was 4.5 FM. Addition of atropine (1/~M) to the cells caused complete inhibition of the CCh-stimulated release of PGF2~ in these cells.

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TABLE I

Effects of various agonists on the release of endottenous PGF2~ and PGE 2 in SV-CISM-2 Ceils PG Released [ng per flask (10 min) -1] Concentration

Agonist added

(,/tM)

None CCh

PGF2~

-1.0 10 I0 0'01 0.1 0'01 0.1 O'lnM 1"0 nM 0"1 1"0 0"1 1"0 0"1 1-0

Norepinephrine Substance P Endothelin-1 Platelet-Activating Factor PGF2~ PGE 2 PGD2

Percentage of control

326±20 509_+36 688_+37 304-+8 469_+40 756_+67 535-+35 688-+46 606_+53 658 _+44 --551_+40 737_+39 469_+30 492_+38

100 156 211 93 144 232 164 211 186 202 169 226 144 151

Percentage of control

PGE2 810-+73 1120-+76 1580-+96 1000-+40 1288_+114 1600_+143 1247-+94 1820+151 1061_+130 1640_+ 143 1360-+121 1440_+ 74 --1264_+73 1520_+110

100 138 195 123 159 198 154 225 131 202 168 178 156 188

Data represent means ±SEM from two experiments each run in duplicate. TABLE II 170

Effects of isoproterenol on CCh-induced inositol phosphates production in SV-CISM-2 cells

'~ 160

i

15o i

140

-~ 130

S.

aH-Inositol phosphates (dpm/flask) Additions None Carbachol (10#M)

120 110 100

IBMX(0'lmM)

L

0

7

6

5

4

-Log earbaehol (M) FIG. 7. Dose-response to CCh for inositol phosphates accumulation in SV-CISM-2 cells. The average basal release of inositol phosphates after 10 min of incubation was 45746_+ 3969 dpm per flask (EC~0= l'5/zM). Atropine (1/~M) was added 5 min before the addition of CCh (10/zM). Each point represents _+SEMfrom 2--3 experiments each run in duplicate.

Effects of various agonists on the endogenous release of PGF2~ and PGE 2. In addition to m u s c a r i n i c receptors, these cells c o n t a i n receptors for s u b s t a n c e P, endothelin, platelet-activating factor, PGF2~, PGE 2 a n d PGD 2 t h a t a r e coupled to the release of A A a n d PG synthesis (Table I). The SV-CISM-2 cells do n o t a p p e a r to c o n t a i n a l - a d r e n e r g i c receptors since 10/zM n o r e p i n e p h r i n e h a d little effect on t h e release of PGs. It is interesting to n o t e t h a t t h e b a s a l release of PGE 2 by these cells is 2.5 times as h i g h as t h a t of PGF2~ (Table I).

Isoproterenol (5/~M)+ IBMX (0.1 mM) Carbachol (IO/~M)+ IBMX (0"1 m u ) + Isoproterenol (5 #u)

|P1

IP2

IP3

1040_+70 1545_+108 (149%)* 963_+68 (93%) 823_+63 (79%) 1158_+114 (111%)

440_+28 730--+21 (166%) 413___17 (94%) 363-+39 (83%) 489-+40 (111%)

134_+4 179_+6 (134%) 122_+9 (91%) 109_+9 (81%) 141_+6 (105%)

* Percentage of control. The data are averages of two separate experiments, each run in triplicate.

Dose-response to CCh for inositol phosphates accumulation. I n c u b a t i o n of SV-CISM-2 cells prelabeled w i t h 3H-inositol in a n o n - r a d i o a c t i v e m e d i u m resulted in a c c u m u l a t i o n of inositol p h o s p h a t e s i n d i c a t i n g the b r e a k d o w n of inositol c o n t a i n i n g phospholipids by p h o s p h o l i p a s e C. A d d i t i o n of different c o n c e n t r a t i o n s of CCh i n c r e a s e d inositol p h o s p h a t e s p r o d u c t i o n in a d o s e - d e p e n d e n t m a n n e r (Fig. 7). The EC~0 v a l u e for CCh-induced a c c u m u l a t i o n of inositol p h o s p h a t e s w a s 1-5 #M. A d d i t i o n of a t r o p i n e (1/ZM) to t h e cells c a u s e d complete inhibition of the CCh-induced a c c u m u l a t i o n of inositol p h o s p h a t e s in these cells.

IRIS SPHINCTE'R S M O O T H M U S C L E CELL LINE Standard proteins

543

(A) 1

2

3

20 kDa - -

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(B) 12{ 139.9 -86.8 -I0(

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33.3 --

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6 Ceh Atropine FIG. 8. Imnmnoblot of myosin light chain. Rabbit iris sphincter muscle (lane 1) and SV-CISM-2 cells (lanes 2 and 3) homogenates were subjected to electrophoresis on the SDS-PAGE and then transferred to nitrocellulose membranes and probed with antibodies against MLC.

Effects of isoproterenol on CCh-induced inositol phosphates production. Isoproterenol, w h i c h activates adenylate cyclase and increases the intracellular concentration of cAMP, inhibits CCh stimulation of IP 3 production and contraction in the iris sphincter (Tachado, A k h t a r and Abdel-Latif. 1989). The data given in Table 2 show that this adrenergic agonist also inhibits CCh stimulation of inositol phosphates in the SV-CISM-2 cells, the inhibitions of IP r IP 2 and IP 3 being 38, 55 and 29%, respectively.

Immunoblot of MLC in SV-CISM-2 ceils.

Since MLC is an important constituent of the contractile proteins in smooth muscle, we have identified it in the cultured cells by anti-myosin (light chains 20 K) antibodies. W h e n the cell h o m o g e n a t e s from SV-CISM-2 cells and rabbit iris sphincter were subjected to SDS-PAGE. several protein bands of varying molecular mass were observed (data not shown). The separated proteins were transferred to nitrocellulose paper and probed with antibodies against MLC. As can be seen in Fig. 8, MLC antibody recognized a band in the 20 K region.

Effects of CCh and atropine on MLC phosphorylation in SV-CISM-2 cells. W h e n the SV-CISM-2 cells were incubated in a m e d i u m containing 32Pi and then

-

+ -

+ +

FI6.9. Typical autoradiogram, after protein separation by SDS-PAGE, showing the effects of CCh and atropine on MLC phosphorylation in SV-CISM-2 cells. (A) Autoradiogram of a"P-labeled MLC (20 K band) following incubation of the a2p_ labeled cells in absence and presence of carbachol (25/*M) and atropine (2"5 tiM) as indicated. (B) Effects of carbachol and atropine on a2p incorporation into MLC of the smooth muscle cells. Incubation conditions and analysis of MLC phosphorylation were the same as described under Methods. *Significantly different from the corresponding control, P < 0.01. stimulated with 25/IM CCh there was a 118 % increase in a2p labeling of MLC (Fig. 9). The stimulatory effect of CCh was blocked by atropine. 4. Discussion At present, no immortalized cell lines exist for iris sphincter s m o o t h muscle cells. The data presented here demonstrate that we have successfully immortalized cat iris sphincter smooth muscle cells using the SV40 virus. SV40 virus has been extensively employed for immortalization of cells (Coca-Prados and Wax. 1 9 8 6 : Iujvidin et al., 1990). The SV-CISM2 cells continued to grow by more t h a n 2 2 0 generations in vitro over 55 passages w i t h o u t a n y alterations in their m o r p h o l o g y or growth. We have characterized this cell line morphologically, biochemically and pharmacologically. The m o r p h o l o g y of cells cultured from n o r m a l cat iris sphincter muscle reported in this study resemble those of cells previously cultured from h u m a n iris sphincter muscle (Woldemussie, Feldmann and Chen, 1993). The

544

isolated cat iris sphincter muscle cells in primary culture exhibited characteristics of smooth muscle cells as revealed by morphology, growth behavior and immunocytochemistry (Chamley-Campbell et al., 1979: Gimbrone and Cotran, 1975). These cells stained intensely with antibodies against the microfilament smooth muscle protein ~-actin. Non-muscle cells m a y express some smooth muscle ~-actin, but in these cells ~-actin tends to be arranged irregularly within the cells, which is unlike the parallel arrangement seen in the present work. Also the a m o u n t of ~-actin is m u c h lower in non-muscle cells (Gown et al., 1985). The normal cells also stained positively for the muscle-specific intermediate filament desmin, although the staining was weak. It is known from other studies (Skalli et al., 1986; T a m m et al., 1991) that smooth muscle cells in culture gradually lose desmin and that expression of this protein can be partially restored in quiescent cultures. The same phenomenon has also been observed with smooth muscle ~-actin (Owens, 1986). These observations suggest that the isolated cat iris sphincter smooth muscle cells we employed for immortalization are free of non-muscle cells. SV-CISM-2 cells exhibited several morphological and immunocytochemical properties which are characteristic of immortalized cells. These include: extended life-span, complete loss of contact inhibition, reduced anchorage dependence for proliferation and low serum requirement (Freshney, 1987). Cell transformation resulted in a higher growth rate and a more rounded morphology compared to the normal cells. These are common features of transformed cells (Pastan and Willingham, 1978). The expression of smooth muscle a-actin and desmin was lower in the SV-CISM-2 cells than in the normal cells. This decrease is probably due to the rapid growth rate of these cells. Apart from the above observations, we could not detect qualitative differences between these cells and their normal counterparts. In addition to ~-actin and desmin, SV-CISM-2 cells can synthesize MLC (Fig. 8), another protein which distinguishes smooth muscle cells. The finding that CCh can enhance MLC phosphorylation by 118% demonstrates that these cells have the ability, to contract when exposed to muscarinic agonists (Fig. 9). This stimulation of MLC phosphorylation by CCh is considerably higher t h a n that reported previously for the intact iris sphincter (Howe et al., 1986). The biochemical and pharmacological properties of the SV-CISM-2 cells are comparable to those reported for the intact iris sphincter (for reviews, Abdel-Latif, 1989, 1991, 1995). Thus, muscarinic stimulation in these cultured cells results in an increase in: (a) release of 3H-labeled AA and PGs from cells prelabeled with 3H-AA, an indicator of phospholipase A 2 activation; (b) release of endogenous PGs, measured by means of RIA, an indicator of the cyclo-oxygenase pathway; (c) IP 3 production, an indicator of phospho-

A. O C K L I N D

ETAL.

lipase C activation, and (d) MLC phosphorylation, an indicator of muscle contraction. Muscarinic stimulations of PG release, IP 3 production and MLC phosphorylation were completely blocked by atropine (Figs 5, 6 and 9), suggesting that these effects are mediated through muscarinic cholinergic receptors. The effects of CCh on second messenger formation is dose-dependent (Figs. 5, 7). In addition to muscarinic receptors, these cells contain other types of functional receptors including peptidergic (substance P and endothelin), fl-adrenergic (but not oc-adrenergic), platelet-activating factor and PGs (PGF2~, PGE 2 and PGD2) (Tables I and II). In conclusion, the data presented demonstrate that SV-CISM-2 cells share properties which are consistent with those observed in the intact iris sphincter and since they are m u c h easier to handle in vitro, they can serve as an important model in elucidating the biochemical and pharmacological characteristics of receptors and their physiological roles in smooth muscle function.

Acknowledgements We thank Dr Zhi Ye for assistance in culturing the cells for the biochemical and pharmacological studies. Part of this study was supported by N1H grants EY-04387 and R37-EY04171 (AAA), and EY-04873 and EY-08672 (MC-P).

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IRIS SPHINC'TER S M O O T H MUSCLE CELL LINE

Freshney, R. I. (1987). Cloning and Selection of Specific Cell Types. The Transformed Phenotype, In: Culture of Animal Cells. A Manual of Basic Technique, (Ed. Freshney, R.I.), Chapters 11 and 15. Alan R. Liss: New York, U.S.A. Gimbrone, M. A. and Cotran, R. S. (1975). Human vascular smooth muscle in culture. Growth and ultrastructure. Lab. Invest. 33, 16-27. Gown, A. M., Vogel, A. M., Gordon, D. and Lu, P. L. (1985). A smooth muscle-specific monoclonal antibody recognizes smooth muscle actin isozymes. ]. Cell Biol. 100, 807-13. Harrison, 8., Mondino, B.J, and Mayer, F. J. (1990). Scleral fibroblasts. Human leukocyte antigen expression and complement activation. Invest. Ophthalmol. Vis. Sci. 31, 2412-19. Howe, P. H., Akhtar, R. A., Naderi, S. and Abdel-Latif, A. A. (1986). Correlative studies on the effect of carbachol on myo-inositol trisphosphate accumulation, myosin light chain phosphorylation and contraction in sphincter smooth muscle of rabbit iris. I. Pharmacol. Exp. Ther. 239, 574-83. Iujvidin, S., Fuchs, O., Nudel, U. and Yaffe, D. (1990). SV40 immortalizes myogenic cells: DNA synthesis and mitosis in differentiating myotubes. Differentiation 43, 192-203. Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-5. Lowry, O. H., Rosenbrough, J. N., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 1 9 3 , 2 6 5 - 7 5 . Owens, G. K. (1986). Expression of smooth muscle-specific isoactin in cultured smooth muscle cells: Relationship between growth and cytodifferentiation. Cell. Biol. 102, 343-52. Pastan, I. and Willingham, M. (1978). Cellular trans-

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formation and the 'morphologic phenotype' of transformed cells. Nature 274, 645-50. Pastan, I. (1979). Cell transformation. Meth. Enzymol. 58, 368-70. Polansky, J. R., Weinreb, R. N., Baxter, J. D. and Alvarado, J. (1979). Human trabecular cells I. Establishment in tissue culture and growth characteristics. Invest. Ophthalmol. Vis. Sci. 18, 1043-9. Polinger, I. S. (1970). Separation of cell types in embryonic heart cultures. Exp. Cell. Res. 63, 78-82. Skalli, O., Bloom, W.S., Ropraz, P., Azzarone, B. and Gabbiani, G. (1986). Cytoskeletal remodeling of rat aortic smooth muscle cells in vitro situations. I. Submicrosc. Cytol. 18, 481-93. Tachado, S. D., Akhtar, R. A. and Abdel-Latif, A. A. (1989). Activation of beta-adrenergic receptors causes stimulation of cyclic AMP, inhibition ofinositol trisphosphate, and relaxation of bovine iris sphincter smooth muscle. Invest. Ophthalmol. Vis. Sci. 30, 2232-9. Tamm, E., Flfigel, C., Baur, A. and Lfitjen-Drecoll, E. (1991). Cell cultures of human ciliary muscle, growth, ultrastructural and immunocytochemical characteristics. Exp. Eye Res. 53, 375-87. Taylor, C.W. (1993). Intracellular Messengers. Pergamon Press: Oxford. Woldemussie, E., Feldmann. B.J. and Chen, J. (1993). Characterization of muscarinic receptors in cultured human iris sphincter muscle and ciliary muscle cells. Exp. Eye Res. 56, 385-92. Yousufzai, S. Y. K. and Abdel-Latif, A. A. (1984). The effects of ~zl-adrenergic and muscarinic cholinergic stimulation on prostaglandin release by rabbit iris. Prostaglandins 28, 399-415. Yousufzai, S. Y. K. and Abdel-Latif, A. A. (1985). Effects of platelet-activating factor on the release of arachidonic acid and prostaglandins by rabbit iris smooth muscle. Biochem. ]. 228, 697-706.