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(Investigative Ophthalmology and Visual Science. 2002;43:1127-1134.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Alterations in Arachidonic Acid Release and Phospholipase C-ß1 Expression in Glaucomatous Human Ciliary Muscle Cells

Shahid Husain, Ismail Kaddour-Djebbar and Ata A. Abdel-Latif

From the Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Prostaglandin (PG) F2{alpha} and other Ca2+-mobilizing agonists, such as carbachol (CCh) and endothelin (ET)-1, induce an increase in uveoscleral outflow, in part through receptor-mediated mechanisms in the ciliary muscle. Because changes in uveoscleral outflow across the ciliary muscle could cause elevation of intraocular pressure (IOP) in patients with glaucoma, the present study was conducted to investigate the possibility that basal and agonist-induced second-messenger formation may be altered in glaucomatous human ciliary muscle (g-HCM) cells compared with normal (n)-HCM cells.

METHODS. Normal and glaucomatous HCM cells were cultured from donor eyes, the cells were identified based on their positive immunostaining with smooth muscle–specific anti-{alpha}-actin (SM), anti-SM-myosin, and anti-desmin antibodies. Activation of phospholipase A2 (PLA2) was measured by the release of [3H] arachidonic acid (AA) into the medium, accumulation of PGE2 was measured by radioimmunoassay, [3H]myo-inositol phosphate production was measured by ion-exchange chromatography, and phospholipase C (PLC)-ß1 expression was determined by immunoblot analysis with polyclonal antibodies specific for PLC-ß1.

RESULTS. Homogenous primary cell cultures from normal and glaucomatous human ciliary muscle were established and characterized. The data obtained can be summarized as follows: Both n- and g-HCM cells exhibited similar morphologic characteristics and immunoreactivities. The effects of the agonists on AA release in both n- and g-HCM cells were in the following order: PGF2{alpha} > ET-1 > CCh; their effects on PGE2 release were in the following order: PGF2{alpha} > CCh > ET-1; and their effects on inositol phosphate production were in the following order: CCh > ET-1 > PGF2{alpha}. Both the basal- and stimulated release of AA were significantly higher in the g-HCM cells than in the n-HCM cells (for PGF2{alpha}, 60% vs. 151%). The basal release of PGE2 in g-HCM cells was two- to fivefold higher than that of n-HCM cells, and there are alterations in the effects of the agonists on PGE2 release. Agonist-induced inositol phosphate production in g-HCM cells was considerably lower than that of n-HCM cells (CCh, 58% vs. 421%), and the amount of PLC-ß1 expressed in g-HCM cells, compared with that in n-HCM cells, was markedly reduced (by 44%).

CONCLUSIONS. These data are the first to show that basal and agonist-induced AA release and inositol phosphate production as well as expression of PLC-ß1 are altered in g-HCM cells compared with that of n-HCM cells. The molecular mechanisms underlying these alterations in g-HCM cells could include changes in sensitivity and number of receptors, overexpression of PLA2 and the cyclooxygenases, and underexpression of PLC-ß1. Alterations in these signaling pathways in g-HCM cells could contribute to changes in the uveoscleral outflow pathway, which may lead to an increase in IOP in patients with glaucoma. Comparative studies on the signaling pathways in g- and n-HCM cells can provide important information about the regulation of uveoscleral outflow and the pathologic course of glaucoma.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Glaucoma is a group of diseases that have in common a characteristic optic cup neuropathy with associated visual field loss for which elevated intraocular pressure (IOP) is considered one of the major risk factors.1 2 Although the cellular mechanisms for the regulation of IOP in normal and glaucomatous eyes remain unknown, the current pharmacologic therapy for primary open-angle glaucoma (POAG), the most common form of glaucoma, relies almost exclusively on drugs that lower IOP.3 In fact, at this time, there is no direct treatment for the optic neuropathy of glaucoma with proven effectiveness.4 IOP is dependent on aqueous humor formation and outflow. The latter consists of trabecular outflow and uveoscleral outflow. Recent research suggests that uveoscleral outflow may be a more important route of aqueous outflow than previously thought, possibly accounting for up to 50% in normal eyes of young people.2 It is increased by cycloplegic and adrenergic agents and prostaglandins (PGs) and is decreased by agents that cause contraction of the pupil of the eye (miotics). The main resistance in the aqueous outflow is constituted by the ciliary muscle.5 Physiological studies in animals and humans indicate that the IOP reduction induced by PGF2{alpha} or its analogues, including latanoprost and PGF2{alpha}-isopropylester, reflects increased uveoscleral outflow without significant changes in conventional outflow or aqueous production.5 6 7 8 9 PGF2{alpha} increases uveoscleral outflow through the iris root and ciliary body, either by decreasing the extracellular matrix that surrounds the muscle bundles,10 or by relaxing the ciliary musculature.5 These observations demonstrate that the ciliary muscle plays an important role in the regulation of aqueous outflow in the mammalian eye.

Because many of the drugs that are currently used to lower IOP through the uveoscleral outflow pathway, such as latanoprost and muscarinic cholinergic agonists, act through specific receptors on the ciliary muscle, several researchers have investigated the effects of these drugs on the biochemical events that lead from receptor activation to cellular response in this tissue. In general, these studies were conducted either on intact ciliary muscle or on cultured human ciliary muscle (HCM) cells. Among the studies reported on cultured HCM cells are the following: (1) PGF2{alpha}-induced c-Fos,11 increased matrix metalloproteinase release,12 and increased intracellular Ca2+ ([Ca2+]i) mobilization in a concentration-dependent manner13 ; (2) cholinergic muscarinic agonists, such as carbachol (Cch), activates phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) and generate the two second messengers, inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG), increased [Ca2+]i through an M3-like muscarinic receptor subtype,14 and downregulated M3 mRNA expression and decreased [3H] 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) binding15 ; (3) endothelin depolarizes membrane voltage and increases [Ca2+]i,16 stimulates PLC activity and increases [Ca2+]i,17 and stimulates the release of arachidonic acid (AA) and PGs18 ; (4) histamine activates PLC and increased [Ca2+]i19 and induces contraction.20 These observations demonstrate the stimulatory effects of Ca2+-mobilizing agonists on AA release and PG synthesis, inositol phosphate production, [Ca2+]i mobilization, and contraction in normal (n)-HCM cells. Although the stimulatory effects of Ca2+-mobilizing agonists on these responses has been investigated in n-HCM cells, there is little known about agonist-induced second-messenger formation in glaucomatous (g)- HCM cells. To fill this gap, in the present study we established primary cultures of ciliary muscle cells from normal human donors and human donors with glaucoma and investigated the effects of the Ca2+-mobilizing agonists PGF2{alpha}, CCh, and endothelin (ET)-1 on AA release, PGE2 synthesis, inositol phosphate production, and expression of the PLC-ß1 isoform.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Myo-[3H]inositol (specific activity, 22.3 Ci/mmol), [3H]AA (specific activity, 184.6 Ci/mmol) and PGE2 [125I]radioimmunoassay (RIA) kits were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). ET-1 was obtained from Peptide International (Louisville, KY) and PGF2{alpha} from Cayman (Ann Arbor, MI). CCh, monoclonal {alpha}-actin smooth muscle (SM) antibodies (Clone no. 1A4), and bovine serum albumin (fraction V) were obtained from Sigma Chemical Co. (St. Louis, MO), and anti-SM myosin, anti-desmin, and anti-PLC-ß1 antibodies were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Fluorescein-conjugated secondary antibodies were obtained from Southern Biotechnology Associates, Inc. (Birmingham, AL). Fetal bovine serum (FBS) was obtained from HyClone (Logan, UT) and all other tissue culture materials were obtained from Cellgro (Herndon, VA). All other chemicals were of reagent grade.

Cell Culture
Use of human tissue in this study conformed to the tenets of the Declaration of Helsinki. Ciliary smooth muscle (SM) cultures were established from normal and glaucomatous human eyes (donors age range, 50–78 years). The human eyes were obtained from the National Disease Research Interchange (Philadelphia, PA) and from the Glaucoma Research Foundation (San Francisco, CA) within 24 hours after death. Seven pairs of eyes were obtained from individuals with a documented history of POAG (age range, 55–80 years). Enucleation was completed within 2 to 6 hours after death, and eyes were preserved in moist chambers at 4°C. Ciliary muscles were dissected with the aid of a dissecting microscope under sterile conditions, and cultures were prepared as described by others.21 22 23 Briefly, ciliary muscles were dissected, further cleaned, and cut into 1- to 2-mm pieces. The explants were placed in DMEM containing 2 mg/mL collagenase type IA, 10% FBS, and 50 µg/mL gentamicin and incubated for 1 to 2 hours at 37°C, with occasional shaking. The major parts of the explants were dispersed into single cells or group of cells, centrifuged at 200g for 10 minutes and resuspended in DMEM 199 supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B in 5% CO2 in humidified air. The cells were then subcultured at a split ratio of 1:4 using 0.05% trypsin and 0.02% EDTA. To selectively remove contaminating fibroblasts that are more adhesive than SM cells in primary culture,24 the cell suspensions were incubated in a 25-cm2 tissue culture flask for 1 hour at 37°C. The cells that remained in suspension were transferred to another flask (marked type "A" SM cells) and cultured at 37°C in 5% CO2 in humidified air. Complete medium was then added to the original flask that contained mostly fibroblasts (marked type "B" fibroblast-like cells) and cultured at 37°C in 5% CO2 in humidified air. After 3 days, one third of the culture medium was replaced with fresh medium and the morphology of the cells was routinely monitored under the phase contrast microscope. In general, type "A" cells were different from type "B" cells in morphology. HCM cells obtained from each pair of normal and glaucomatous eyes were grown and maintained separately. Cells of passages 3 to 9 were used in the present study.

Immunocytochemistry
HCM cells were identified from nonmuscle cells by the presence of SM-specific {alpha}-actin, using a procedure described by others.21 22 23 For the staining of cultures, cells were seeded in tissue culture chamber mounted slides (Laboratory-Tek II; Nunc, Inc., Naperville, IL). Culture medium was removed by rinsing three times with PBS, and cells were fixed with ice-cold methanol for 15 minutes. Cells were washed and incubated with monoclonal anti-{alpha}-SM actin (1:50), anti-SM myosin (1:50), or anti-desmin (1:20) antibodies diluted in PBS with 1% goat serum for 2 hours at room temperature. The cells were washed three times with PBS and incubated with fluorescein-conjugated secondary antibodies (1:100) for 1 hour. The cells were then washed with PBS, and tissue chambers were removed from glass slides and mounted (Gel-Mount; Biomedia, Foster City, CA). Control experiments were performed using serum albumin instead of the primary antibodies. Stained cells were viewed under a fluorescence microscope (Axiophot; Carl Zeiss, Oberkochen, Germany).

Measurements of [3H]AA Release
n-HCM and g-HCM cells (passages 3–9) were grown to confluence in 12-well plates and incubated with 0.75 µCi/mL (300 nM) [3H]AA for 24 hours at 37°C in DMEM containing 0.1 mg/mL bovine serum albumin (BSA). After labeling, the cells were washed three times with nonradioactive DMEM to remove unincorporated AA and then incubated in the absence or presence of PGF2{alpha} (1 µM), ET-1 (0.1 µM), or CCh (10 µM) in 1 mL serum-free DMEM for the indicated time interval. At the end of incubation, the medium was collected, centrifuged, and the radioactivity determined as previously described.25

Assay of Release of Endogenous PGE2
n-HCM and g-HCM cells were grown to confluence in 12-well plates and starved for 24 hours in serum-free medium. The cells were incubated in the absence or presence of PGF2{alpha} (1 µM), CCh (10 µM), or ET-1 (0.1 µM) for 5, 10, and 5 minutes, respectively. After incubation, PGE2 was assayed in the medium by RIA, as described previously.26 The amount of PGE2 in each sample was determined by interpolation from the standard curve. The rate of PGE2 release is presented as the amount of PGE2 released per milligram protein per 15 minutes. The amount of PGE2 released is a function of protein in the cells.

Radiolabeling of n- and g-HCM Cells with Myo-[3H]Inositol and Analysis of Inositol Phosphates
To assay for the effects of agonists on PLC activity, n- and g-HCM cells were grown in 12-well plates. The confluent cells were incubated in inositol-deficient DMEM that contained [3H]inositol (5 µCi, 290 nM/well) for 24 hours. The labeled cells were washed three times with nonradioactive medium and preincubated in medium that contained 10 mM LiCl for 10 minutes. LiCl is an inhibitor of inositol phosphatases, and thus it acts to protect the inositol phosphates from hydrolysis by these enzymes.27 At this time, PGF2{alpha} (1 µM), CCh (10 µM), or ET-1 (0.1 µM) was added as indicated and incubation continued for 5, 10, and 5 minutes, respectively. The reactions were terminated by aspirating the medium and adding 1 mL ice-cold 10% (wt/vol) trichloroacetic acid (TCA) to the cells. The cells were scraped off the wells and centrifuged at 1000g for 10 minutes, and the inositol phosphates were analyzed as described previously.28 Briefly, the supernatant containing inositol phosphates was extracted four times with water-saturated diethyl ether and neutralized with 0.1 M NaOH. The inositol phosphates were analyzed by anion-exchange chromatography (with Dowex AG 1-X8 resin, formate form, 200–400 mesh; Bio-Rad, Hercules, CA). The pellet was solubilized in 0.5 M NaOH and proteins determined according to the method of Lowry et al.29

Western Blot Analysis
Cells were lysed in 50 mM Tris-HCl buffer (pH 7.5) containing 1% Triton X-100, 0.5% deoxycholate, 10 mM EDTA, 50 mM NaF, 2 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM sodium pyrophosphate, 10 µg/mL leupeptin, and 50 µg/mL aprotinin for 20 minutes on ice. Cell lysates were centrifuged at 2500g for 10 minutes and the supernatant used for immunodetection of PLC-ß1 isoform. Equal amounts of proteins were then resolved on 10% SDS/PAGE. Proteins were then transferred to nitrocellulose membranes and probed with antibodies specific for PLC-ß1, followed by an incubation with secondary antibodies (horseradish peroxidase–conjugated goat anti-rabbit IgG at 1:3000) for 1 hour at 20°C, as described previously.30 For chemiluminescence detection, the membranes were treated with enhanced chemiluminescence detection reagent (ECL; Amersham Pharmacia Biotech) for 1 minute and subsequently exposed to ECL hyperfilm for 1 to 2 minutes.

Experimental Design
n-HCM and g-HCM cells were grown under identical conditions. For comparative studies, we have used similar passages of n- and g-HCM cells. For PGE2 analysis, cells were starved in serum-free medium to minimize the nonspecific activation of enzymes and signaling pathway components. For analysis of [3H]AA release and [3H]inositol phosphate production, cells were prelabeled with [3H]AA or [3H]inositol for 24 hours in serum-free medium. After treatment with the agonists PGE2 release into the medium was analyzed by RIA, [3H]AA release into the medium was analyzed by counting in the scintillation counter, and [3H]inositol phosphates were extracted from the cells and analyzed by ion-exchange chromatography and counted in the scintillation counter.

Data and Statistical Analysis
In general, three pairs of healthy donor eyes were used to prepare the n-HCM cells, and seven pairs of glaucomatous donor eyes were used to prepare the g-HCM cells. For determinations of AA, PGE2, and inositol phosphates, g-HCM cells from each donor were used. Parallel experiments were run using the n-HCM cells. All data obtained from the n-HCM cells for AA, PGE2, and inositol phosphates were pooled and presented in Tables 1 2 and 3 , respectively, whereas data for each glaucoma donor eye are presented separately.


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  		Table 1. Effects of PGF2{alpha}, CCh, and ET-1 on [3H]AA Release in n- and g-HCM Cells

 

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  		Table 2. Effects of PGF2{alpha}, CCh, and ET-1 on the Release of Endogenous PGE2 in n- and g-HCM cells

 

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  		Table 3. Effects of PGF2{alpha}, CCh, and ET-1 on [3H]Inositol Phosphate Production in n- and g-HCM Cells

 
Data are presented as a percentage of the respective control (in absence of agonist) ± SEM. Statistical analysis was performed on the absolute values using paired Student’s t-tests. Statistical analysis of absolute values was also determined between n- and g-HCM cells in the absence and presence of agonists using unpaired Student’s t-test. P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell Morphology
Ciliary muscle cells in culture were identified by their pattern of growth, morphology and immunocytochemical staining characteristics. After collagenase treatment, a suspension of ciliary muscle cells was transferred to tissue culture flasks, and cells started to divide in 5 to 7 days, especially in areas where the cells settled in clumps. Initially, small cell clones of bipolar ribbon or spindle-shaped cells were formed. When cells grow to higher densities, they form longitudinal bands of parallel cells and become thinner. This growth pattern was observed in all primary cell cultures of glaucomatous (Fig. 1A) and normal (Fig. 1B) eyes, regardless of the age of the donor. When the cells became confluent, they grew in a hill-and-valley pattern, as seen under phase-contrast microscope (Figs. 1A 1B) .



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  		Figure 1. Phase image of g-HCM cells (A) and n-HCM cells (B). Cells have typical hill-and-valley arrangement of growth, as described by other investigators.21 22 23 32 Magnification, x200.

 
Immunocytochemistry
To demonstrate that the cultured HCM cells are not contaminated with fibroblasts we used immunologic staining with SM-specific antibodies. HCM cells stained positively against SM-specific {alpha}-actin in both n-HCM cells (Fig. 2B) and g-HCM cells (Fig. 3B) . The staining revealed typical straight, noninterrupted, cablelike fibers running parallel to each other along the long axis of the cells. When the cells were confluent, the cytoplasm of the cells was filled with densely arranged parallel fibers, staining strongly for {alpha}-SM-actin antibodies in both n- and g-HCM cells.



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  		Figure 2. {alpha}-SM actin (B), SM-myosin (D) and desmin (F) immunofluorescence of n-HCM cells. The negative control cultures (A, C, E) showed no positive staining. Magnification, x200.

 


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  		Figure 3. {alpha}-SM actin (B), SM-myosin (D) and desmin (F) immunofluorescence of g-HCM cells. The negative control cultures (A, C, E) showed no positive staining. Magnification, x200.

 
To further ascertain the purity of the HCM cells, we used the SM-specific antibody SM-myosin and the intermediate filament protein desmin. SM-myosin (heavy chain) antibody reacts specifically with SM cells, but not with myosin isoforms of fibroblasts or myofibroblasts.31 32 n-HCM and g-HCM cells, respectively, showed positive staining with SM-myosin antibodies (Figs. 2D 3D) . Furthermore, both n- and g-HCM cells, respectively, showed positive staining with anti-desmin antibodies (Figs. 2F 3F) . Negative control cultures in all cases showed no positive staining (Figs. 2A 2C 2E 3A 3C 3E) . These data demonstrate that these were pure SM cells.

Effects of the Agonists on [3H]AA Release in n- and g-HCM Cells
The purpose of this experiment was to compare the effects of the agonists on AA release in n- and g-HCM cells. The cells were labeled with [3H]AA and then treated with the agonists. In preliminary studies, we found 1 µM PGF2{alpha}, 100 nM ET-1, and 10 µM CCh to be the optimal concentrations. As shown in Table 1 , PGF2{alpha} increased AA release in n-HCM cells by 60%, and in g-HCM cells by up to 151% (the range among the seven donors was 85%–151%); CCh increased it by 25% in n-HCM cells, and by up to 73% in g-HCM cells (range, 30%–73%); and ET-1 increased it by 29% in n-HCM cells, and by up to 63% in g-HCM cells (range, 37%– 63%). In general, the basal release of AA in g-HCM cells was 42% (average of seven donors) higher than that of n-HCM cells. Of the three agonists, PGF2{alpha} was most potent in inducing AA release in both n- and g-HCM cells. The biochemical basis for the variations in basal and agonist-stimulated AA remains to be established. It can be concluded from these results that both the basal- and stimulated release of AA is significantly higher in the g- than in the n-HCM cells.

Effects of Agonists on the Release of Endogenous PGE2 in n- and g-HCM Cells
To confirm the finding that agonist-induced AA release is higher in g- than in n-HCM cells, we investigated the effects of the agonists on the release of endogenous PGE2, a major metabolite of AA in ocular tissues. The release of PGE2 was determined by RIA. The basal release of PGE2 in g-HCM cells was two- to fivefold higher than that of n-HCM cells (Table 2 , see legend for description). In contrast, the magnitude of the stimulatory effects of the agonists on PGE2 release in n- and g-HCM cells varied with the agonist. Thus, in n-HCM cells PGF2{alpha}, CCh, and ET-1 increased PGE2 release by 207%, 99%, and 59%, respectively (Table 2) . However, in g-HCM cells, PGF2{alpha} reduced significantly PGE2 release compared with that in n-HCM cells, CCh had comparable effects on PGE2 release in both n- and g-HCM cells, and the stimulatory effects of ET-1 on PGE2 release were considerably higher in the g- than in the n-HCM cells. These data clearly show that under basal conditions the release of PGE2 in g-HCM cells is considerably higher than that in n-HCM cells. In addition, these results indicate that there are alterations in the effects of the agonists on PGE2 release in n- and g-HCM cells and that these effects are agonist specific.

Effects of Agonists on Inositol Phosphate Production in n- and g-HCM Cells
Another second-messenger system that we investigated in the n- and g-HCM cells is the phosphoinositide-signaling pathway. HCM cells were labeled with [3H]inositol, and the effects of PGF2{alpha}, CCh, and ET-1 on inositol phosphate production were determined in n-HCM cells, PGF2{alpha}, CCh, and ET-1 increased inositol phosphate production by 33%, 421%, and 153%, respectively (Table 3) . In contrast, in g-HCM cells the effects of these agonists were significantly suppressed. Thus, PGF2{alpha} increased inositol phosphate production by only up to 19% (range, 2%–19%); CCh increased it by up to 217% (range, 58%–217%); and ET-1 increased it by up to 127% (range, 36%–127%). The levels of basal inositol phosphate production varied considerably, both in n-HCM cells (7179–17,220 dpm/mg protein) and in g-HCM cells (8136–15,003 dpm/mg protein; Table 3 ). These data show that agonist-induced inositol phosphate production in g-HCM cells is considerably lower than that of n-HCM cells.

Immunochemical Identification and Densitometric Analysis of PLC-ß1 in n- and g-HCM Cells
The finding that agonist-induced inositol phosphate production is suppressed in the g-HCM cells could suggest a reduction in the expression of PLC-isoforms. To answer this possibility, we examined the presence of PLC-ß1 in n- and g-HCM cells by using polyclonal antibodies specific for this isoform. Antibodies directed against PLC-ß1 revealed two immunoreactive bands of approximate molecular masses of 100 and 150 kDa, indicating the presence of PLC-ß1 in these cells (Fig. 4A) . The specificity of each band was confirmed by deletion of the immunoreactive band on incubation with appropriate blocking peptides. To further confirm the specificity of these bands, we used various combinations of antibodies and blocking peptides. Both bands completely disappeared, even when 1:10 ratios of antibodies and blocking peptides were used, suggesting that both bands correspond to PLC-ß1 isoforms. The expression level of PLC-ß1 was also determined by densitometry. Arbitrary units for PLC-ß1 isoforms in n-HCM cells were 1494 ± 122, whereas in g-HCM cells they were 831 ± 75 (Fig. 4B) . The expression of PLC-ß1 was detected by loading equal amounts of proteins from n- and g-HCM cell lysates in gels under identical experimental conditions. Furthermore, to rule out the possibility of variation in the loaded amounts of proteins, we used anti-actin-antibodies as a protein marker. The nitrocellulose membrane was either reprobed with anti-actin antibodies, or another membrane containing the same amount of loaded proteins was proved. This antibody recognizes all isoforms of actin including ß-actin. Both n- and g-HCM samples contained comparable amounts of proteins (data not shown). These data show that the amount of PLC-ß1 expressed in g-HCM cells, compared with n-HCM cells, was markedly reduced, and that the differences observed in PLC-ß1 expression in n-HCM cells and g-HCM cells were not due to variations in the amounts of proteins loaded in the gel.



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  		Figure 4. Immunochemical identification (A) and quantitation (B) of PLC-ß1 in n- and g-HCM cells. Equal amounts of proteins (10 µg) of cell lysates obtained from n- and g-HCM cells were analyzed by 10% SDS-PAGE and immunoblotted with specific anti-PLC-ß1 polyclonal antibodies. In the presence of the inhibitory peptide, both bands were abolished. The intensity of each band was quantitated by densitometry and expressed as arbitrary units. Quantitative data shown for PLC-ß1 represent the sum of two bands of PLC-ß1. Results are from one experiment (donor 1) that is a representative of 6 to 8 separate experiments. Comparable decreases in PLC-ß1 expression were observed in g-HCM cells from other donors.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we established homogeneous primary cell cultures derived from n- and g-HCM. The n- and g-HCM cells used exhibited morphologic and immunoreactivity consistent with observations made by other investigators in cultured human ciliary muscle cells.21 22 23 Thus, similar growth patterns were observed in all primary cell cultures of normal and glaucomatous eyes, regardless of the age of the donor (Fig. 1) , and a similar pattern of positive staining was observed in n- and g-HCM cells immunostained with anti-{alpha}-SM actin, anti-SM-myosin, and anti-desmin antibodies (Figs. 2 3) . The data presented on the characterization of the HCM cells clearly show that these cells were SM cells (Figs. 2 3) . Consequently, we used these cells to study the effects of the Ca2+-mobilizing agonists PGF2{alpha}, CCh, and ET-1 on AA release, PGE2 synthesis, inositol phosphate production, and PLC-ß1 expression.

The results of our studies demonstrated for the first time abnormalities in AA metabolism and the phosphoinositide signaling pathway in HCM cells isolated from glaucomatous eyes. This conclusion is based on the following observations: Both basal and stimulated release of AA were significantly higher in the g- than in the n-HCM cells (Table 1) . The basal release of PGE2 in g-HCM cells was considerably higher than that of n-HCM cells, and this was accompanied by significant alterations in the effects of the agonists on the release of the prostaglandin (Table 2) . Agonist-induced inositol phosphate production in g-HCM cells was considerably lower than that in n-HCM cells (Table 3) . The amount of PLC-ß1 expressed in g-HCM cells, compared with that in n-HCM cells, was markedly reduced (Fig. 4) . In general, the effects of the agonists on AA release in both the n- and g-HCM cells were in the following order: PGF2{alpha} > ET-1 > CCh, their effects on PGE2 release were in the following order: PGF2{alpha} > CCh > ET-1, and their effects on inositol phosphate production were in the following order CCh > ET-1 > PGF2{alpha}. The higher agonist-induced AA release in g-HCM cells could be due to an increase in sensitivity or number of receptors or to an upregulation of PLA2, the enzyme responsible for AA release in SM25 30 in the glaucomatous eyes. The marked suppression of both basal and agonist-induced inositol phosphate production in the g-HCM cells (Table 3) could be explained by the observation that the amount of PLC-ß1 expressed in the g-HCM cells was markedly reduced (Fig. 4) . The decrease in PLC-ß1 protein expression in g-HCM cells from the seven glaucoma donors ranged between 16% and 50% compared with the n-HCM cells. PLC-ß1 is localized in the plasma membrane, and several lines of evidence have demonstrated, both in vitro and in vivo, that the guanosine triphosphate (GTP)-bound {alpha} subunits of the Gq family activate this enzyme.33 34

The data presented in this report demonstrate for the first time significant alterations in agonist-induced second-messenger formation in g-HCM cells. In the past, physiological, pharmacologic, and biochemical differences have been observed between cells of normal and glaucomatous (POAG) eyes. Thus, glaucomatous trabecular meshwork (TM) cells differ from normal TM cells in glycosaminoglycan synthesis and secretion, resting volume, and cortisol metabolism35 36 37 38 39 and show an increased expression of TIGR/myocilin.40 More recently, Putney et al.41 investigated Na-K-Cl in normal and glaucomatous human TM cells. They found that Na-K-Cl cotransport activity of glaucomatous TM cells was reduced by 32% ± 2% compared with that in normal TM cells, whereas Western blot analyses showed that cotransporter protein expression in glaucomatous TM cells was reduced by 64% ± 14%, compared with expression in normal TM cells. Also, exposure of normal TM cells to 10 µM norepinephrine or 50 µM 8-bromo-cAMP was found to diminish Na-K-Cl cotransport activity, whereas these agents were without effect on glaucomatous TM cell cotransport.

In summary, the results of this study provide the first evidence for alterations in the basal and stimulated release of AA and PGE2, in the stimulated release of inositol phosphates, and in the protein expression of PLC-ß1 in g-HCM cells compared with that in n-HCM cells. Some of the molecular mechanisms underlying these alterations in the g-HCM cells may include underexpression of PLC-ß1, overexpression of PLA2 and the cyclooxygenases, and alterations in the sensitivity and number of receptors. These findings add further support to the observations of other investigators mentioned earlier who reported on alterations of various metabolic pathways in cultures of human trabecular meshwork cells from glaucomatous tissue. Alterations in signaling pathways in g-HCM cells could contribute to changes in the uveoscleral outflow pathway that may lead to an increase in IOP in patients with glaucoma. Comparative studies on the signaling pathways in g-HCM and n-HCM cells can provide important information about the regulation of uveoscleral outflow and the pathologic course of glaucoma. Furthermore, knowledge of changes in the phosphoinositide signaling system in glaucomatous eyes could provide the basis for a better understanding of the molecular mechanisms underlying the alterations in uveoscleral outflow in patients with glaucoma.


   Acknowledgements
 
The authors thank Eric Miller for technical assistance, and GuiLin Zhan for advice and suggestions on culturing human ciliary muscle cells.


   Footnotes
 
Supported by National Eye Institute Grants RO1-EY04171 and RO1-EY04387.

Submitted for publication September 14, 2001; accepted December 11, 2001.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Ata A. Abdel-Latif, Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912-2100; labdel{at}mail.mcg.edu


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Quigley, HA, Nickells, RW, Kerrigan, LA, Pease, ME, Thibault, DJ, Zack, DJ. (1995) Retinal ganglion cell death in experimental glaucoma and after axotomy occurs by apoptosis Invest Ophthalmol Vis Sci 36,774-786[Abstract/Free Full Text]
  2. Cantor, L Fechtner, RD Michael, AJet al eds. Basic and Clinical Science Course: Glaucoma 2000–2001;Section 10,5-24 The Foundation of the American Academy of Ophthalmology San Francisco.
  3. Mittag, TW, Danias, J, Poborenec, G, et al (2000) Retinal damage after 3 to 4 months of elevated intraocular pressure in a rat glaucoma model Invest Ophthalmol Vis Sci 41,3451-3459[Abstract/Free Full Text]
  4. Alward, WLM. (1998) Medical management of glaucoma N Engl J Med 339,1298-1307[Free Full Text]
  5. Crawford, K, Kaufman, PL. (1987) Pilocarpine antagonizes PGF2{alpha}-induced ocular hypotension: evidence for enhancement of uveoscleral outflow by PGF2{alpha} Arch Ophthalmol 105,1112-1116[Abstract/Free Full Text]
  6. Camras, C, Alm, A, Watson, P, Stjernschantz, J, . and the Latanoprost study group (1996) Latanoprost, a prostaglandin analog, for glaucoma therapy: efficiency and safety after one year treatment in 198 patients Ophthalmology 103,1916-1924[Medline][Order article via Infotrieve]
  7. Bito, LZ, Stjernschantz, J, Resul, B, Miranda, OC, Basu, S. (1993) The ocular effects of prostaglandins and the therapeutic potential of a new PGF2{alpha} analog, Ph XA41 (latanoprost), for glaucoma management J Lipid Mediat 6,535-543[Medline][Order article via Infotrieve]
  8. Gabelt, BT, Kaufman, PL. (1989) Prostaglandin F2{alpha} increases uveoscleral outflow in the cynomolgus monkey Exp Eye Res 49,389-402[Medline][Order article via Infotrieve]
  9. Nilsson, SFE, Samuelsson, M, Bill, A, Stjernschantz, J. (1989) Increased uveoscleral outflow as a possible mechanism of ocular hypotension caused by prostaglandin F2{alpha}-1-isopropyl ester in the cynomolgus monkey Exp Eye Res 48,707-716[Medline][Order article via Infotrieve]
  10. Lütjen-Drecoll, E, Tamm, E. (1998) Morphological study of the anterior segment of cynomolgus monkey eyes following treatment with prostaglandin F2{alpha} Exp Eye Res 47,761-769
  11. Lindsey, JD, To, HD, Weinreb, RN. (1994) Induction of c-Fos by prostaglandin F2{alpha} in human ciliary smooth muscle cells Invest Ophthalmol Vis Sci 35,242-250[Abstract/Free Full Text]
  12. Weinreb, RN, Kashiwagi, K, Kashiwagi, F, Tsukahara, S, Lindsey, JD. (1997) Prostaglandins increase matrix metalloproteinase release from human ciliary smooth muscle cells Invest Ophthalmol Vis Sci 38,2772-2780[Abstract/Free Full Text]
  13. Chen, W, Andom, T, Bhattacherjee, P, Paterson, C. (1997) Intracellular calcium mobilization following prostaglandin receptor activation in human ciliary muscle cells Curr Eye Res 16,847-853[Medline][Order article via Infotrieve]
  14. Matsumoto, S, Yorio, T, De Santis, L, Pang, I-H. (1994) Muscarinic effects on cellular functions in cultured ciliary muscle cells Invest Ophthalmol Vis Sci 35,3732-3738[Abstract/Free Full Text]
  15. Zhang, X, Schroeder, A, Erickson, KA. (1999) Effect of continuous administration of a cholinergic agonist on [3H]4-DAMP binding and m3 mRNA expression in cultured human ciliary muscle cells J Ocul Pharmacol Ther 15,153-163[Medline][Order article via Infotrieve]
  16. Korbmacher, C, Helbig, H, Haller, H, Erickson-Lamy, KA, Wiederholt, M. (1989) Endothelin depolarizes membrane voltage and increases intracellular calcium concentration in human ciliary muscle cells Biochem Biophys Res Commun 164,1031-1039[Medline][Order article via Infotrieve]
  17. Matsumoto, S, Yorio, T, Mangino, PE, Desantis, L, Pang, I-H. (1996) Endothelin-induced changes of second messengers in cultured human ciliary muscle cells Invest Ophthalmol Vis Sci 37,1058-1066[Abstract/Free Full Text]
  18. Yousufzai, SYK, Abdel-Latif, AA. (1997) Endothelin-1 stimulates the release of arachidonic acid and prostaglandins in cultured ciliary muscle cells: activation of phospholipase A2 Exp Eye Res 65,73-81[Medline][Order article via Infotrieve]
  19. Markwardt, KL, Magnino, PE, Pang, I-H. (1996) Effect of histamine on phosphoinositide turnover and intracellular calcium in human ciliary muscle cells Exp Eye Res 62,511-520[Medline][Order article via Infotrieve]
  20. Markwardt, KL, Magnino, PE, Pang, I-H. (1997) Histamine induced contraction of human ciliary muscle cells Exp Eye Res 64,713-717[Medline][Order article via Infotrieve]
  21. Tamm, E, Flugel, C, Baur, A, Lütjen-Drecoll, E. (1991) Cell cultures of human ciliary muscle, growth, ultrastructural and immunocytochemical characteristics Exp Eye Res 53,375-387[Medline][Order article via Infotrieve]
  22. Woldemussie, E, Feldmann, BJ, Chen, J. (1993) Characterization of muscarinic receptors in cultured human iris sphincter and ciliary smooth muscle cells Exp Eye Res 56,385-392[Medline][Order article via Infotrieve]
  23. Zhan, G-L, Camras, CB, Opere, C, Tang, L, Ohia, SE. (1998) Effect of prostaglandins on cyclic AMP production in cultured human ciliary muscle cells J Ocul Pharmacol Ther 14,45-55[Medline][Order article via Infotrieve]
  24. Polinger, IS. (1970) Separation of cell types in embryonic heart cell cultures Exp Cell Res 63,78-82[Medline][Order article via Infotrieve]
  25. Husain, S, Abdel-Latif, AA. (1998) Role of PKC{alpha} in endothelin-1 stimulation of cytosolic phospholipase A2 and arachidonic acid release in cultured cat iris sphincter smooth muscle cells Biochim Biophys Acta 1392,127-144[Medline][Order article via Infotrieve]
  26. Yousufzai, SYK, Chen, A-L, Abdel-Latif, AA. (1988) Species differences in the effects of prostaglandins on inositol triphosphates accumulation, phosphatidic acid formation, myosin light chain phosphorylation and contraction in iris sphincter of the mammalian eye: interaction with the cAMP system J Pharmacol Exp Ther 247,1064-1072[Abstract/Free Full Text]
  27. Berridge, MJ, Downes, CP, Hanley, MR. (1982) Lithicene amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands Biochem J 206,587-595[Medline][Order article via Infotrieve]
  28. Ding, K-H, Husain, S, Akhtar, RA, Isales, CM, Abdel-Latif, AA. (1997) Inhibition of muscarinic-stimulated polyphosphoinositide hydrolysis and Ca2+ mobilization in cat iris sphincter smooth muscle cells by cAMP-elevating agents Cell Sign 9,411-421[Medline][Order article via Infotrieve]
  29. Lowry, OH, Rosenbrough, NJ, Randal, R. (1951) Protein measurement with the folin phenol reagent J Biol Chem 193,264-275
  30. Husain, S, Abdel-Latif, AA. (1999) Endothelin-1 activates p38 MAP kinase and cPLA2 in cat iris sphincter smooth muscle cells Biochem J 342,87-96
  31. Groschel-Stewart, U, Chamley, JH, McConnell, JD, Burnstock, G. (1975) Comparison of the reaction of cultured smooth and cardiac muscle cells and fibroblasts to specific antibodies to myosin Histochemie 43,215-224
  32. Chamley-Cambell, J, Campbell, GR, Ross, R. (1979) The smooth muscle cell in culture Physiol Rev 59,1-55[Free Full Text]
  33. Bernstein, G, Blank, JL, Smrcka, AV, et al (1992) Reconstitution of agonist-stimulated PIP2 hydrolysis using purified m1 muscarinic receptor, Gq/11 and phospholipase C-ß1 J Biol Chem 267,8081-8088[Abstract/Free Full Text]
  34. Wu, D, Lee, CH, Rhee, SG, Simon, MI. (1992) Activation of phospholipase C by the {alpha} subunits of the Gq and G11 proteins in transfected Cos-7 cells J Biol Chem 267,1811-1817[Abstract/Free Full Text]
  35. Stamer, WD, Roberts, BC, Epstein, DL, Allingham, RR. (2000) Isolation of primary open-angle glaucomatous trabecular meshwork cells from whole eye tissue Curr Eye Res 20,347-350[Medline][Order article via Infotrieve]
  36. Weinstein, BI, Gordon, GG, Southren, AL. (1983) Potentiation of glucocorticoid activity by 5 beta-dihydrocortisol: its role in glaucoma Science 222,172-173[Abstract/Free Full Text]
  37. Southren, AL, Gordon, GG, Munnangi, PR, et al (1983) Altered cortisol metabolism in cells cultured from trabecular meshwork specimens obtained from patients with primary open-angle glaucoma Invest Ophthalmol Vis Sci 24,1413-1417[Abstract/Free Full Text]
  38. Hernandez, MR, Wenk, EJ, Weinstein, BI, et al (1983) Glucocorticoid target cells in human outflow pathway: autopsy and surgical specimens Invest Ophthalmol Vis Sci 24,1612-1616[Abstract/Free Full Text]
  39. Polansky, JR, Alvarado, JA. (1994) Cellular mechanisms influencing the aqueous humor outflow pathway Principles and Practice of Ophthalmology ,226-251 WB Saunders Co Philadelphia, PA.
  40. Lütjen-Drecoll, E, May, CA, Polansky, JR, Johnson, DH, Bloemendal, H, Nguyen, TD. (1998) Localization of the stress proteins {alpha}ß-crystallin and trabecular meshwork inducible glucocorticoid response protein in normal and glaucomatous trabecular meshwork Invest Ophthalmol Vis Sci 39,517-525[Abstract/Free Full Text]
  41. Putney, LK, Brandt, JD, O’Donnell, ME. (1999) Na-K-Cl cotransport in normal and glaucomatous human trabecular meshwork cells Invest Ophthalmol Vis Sci 40,425-434[Abstract/Free Full Text]



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