Alterations in Arachidonic Acid Release and Phospholipase C-β₁ Expression in Glaucomatous Human Ciliary Muscle Cells

Abstract

purpose. Prostaglandin (PG) F_2α 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-α-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)-β₁ expression was determined by immunoblot analysis with polyclonal antibodies specific for PLC-β₁.

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: PGF_2α > ET-1 > CCh; their effects on PGE₂ release were in the following order: PGF_2α > CCh > ET-1; and their effects on inositol phosphate production were in the following order: CCh > ET-1 > PGF_2α. Both the basal- and stimulated release of AA were significantly higher in the g-HCM cells than in the n-HCM cells (for PGF_2α, 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-β₁ 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-β₁ 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-β₁. 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.

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 PGF_2α or its analogues, including latanoprost and PGF_2α-isopropylester, reflects increased uveoscleral outflow without significant changes in conventional outflow or aqueous production. ^{5 6 7 8 9} PGF_2α 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) PGF_2α-induced c-Fos, ¹¹ increased matrix metalloproteinase release, ¹² and increased intracellular Ca²⁺ ([Ca²⁺]_i) mobilization in a concentration-dependent manner ¹³ ; (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) binding ¹⁵ ; (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 PGs ¹⁸ ; (4) histamine activates PLC and increased [Ca²⁺]_i ¹⁹ 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 PGF_2α, CCh, and endothelin (ET)-1 on AA release, PGE₂ synthesis, inositol phosphate production, and expression of the PLC-β₁ isoform. 

Materials and Methods

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 PGF_2α from Cayman (Ann Arbor, MI). CCh, monoclonal α-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-β₁ 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 α-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-α-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 PGF_2α (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 PGF_2α (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, PGF_2α (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-β₁ 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-β₁, 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. 

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

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) . 

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 α-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 α-SM-actin antibodies in both n- and g-HCM cells. 

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 PGF_2α, 100 nM ET-1, and 10 μM CCh to be the optimal concentrations. As shown in Table 1 , PGF_2α 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, PGF_2α 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 PGF_2α, CCh, and ET-1 increased PGE₂ release by 207%, 99%, and 59%, respectively (Table 2) . However, in g-HCM cells, PGF_2α 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 PGF_2α, CCh, and ET-1 on inositol phosphate production were determined in n-HCM cells, PGF_2α, 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, PGF_2α 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-β₁ 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-β₁ in n- and g-HCM cells by using polyclonal antibodies specific for this isoform. Antibodies directed against PLC-β₁ revealed two immunoreactive bands of approximate molecular masses of 100 and 150 kDa, indicating the presence of PLC-β₁ 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-β₁ isoforms. The expression level of PLC-β₁ was also determined by densitometry. Arbitrary units for PLC-β₁ isoforms in n-HCM cells were 1494 ± 122, whereas in g-HCM cells they were 831 ± 75 (Fig. 4B) . The expression of PLC-β₁ 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 β-actin. Both n- and g-HCM samples contained comparable amounts of proteins (data not shown). These data show that the amount of PLC-β₁ expressed in g-HCM cells, compared with n-HCM cells, was markedly reduced, and that the differences observed in PLC-β₁ expression in n-HCM cells and g-HCM cells were not due to variations in the amounts of proteins loaded in the gel. 

Discussion

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-α-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 PGF_2α, CCh, and ET-1 on AA release, PGE₂ synthesis, inositol phosphate production, and PLC-β₁ 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-β₁ 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: PGF_2α > ET-1 > CCh, their effects on PGE₂ release were in the following order: PGF_2α > CCh > ET-1, and their effects on inositol phosphate production were in the following order CCh > ET-1 > PGF_2α. 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 SM ^{25 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-β₁ expressed in the g-HCM cells was markedly reduced (Fig. 4) . The decrease in PLC-β₁ protein expression in g-HCM cells from the seven glaucoma donors ranged between 16% and 50% compared with the n-HCM cells. PLC-β₁ 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 α subunits of the G_q 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 metabolism ^{35 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-β₁ 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-β₁, 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. 

 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.

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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, ×200.

Figure 2.

 

α-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, ×200.

Figure 3.

 

α-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, ×200.

Table 1.

 

Effects of PGF_2α, CCh, and ET-1 on [³H]AA Release in n- and g-HCM Cells

Additions	n-HCM Cells	g-HCM Cells
				Donor 1	Donor 2	Donor 3	Donor 4	Donor 5	Donor 6	Donor 7
None	100	100	100	100	100	100	100	100
PGF2α (1 μM)	160 ± 11*	210 ± 12* , †	185 ± 10*	195 ± 7* , †	199 ± 11* , †	251 ± 9* , †	238 ± 24* , †	233 ± 18* , †
CCh (10 μM)	125 ± 4*	148 ± 6* , †	130 ± 6*	153 ± 5* , †	146 ± 15* , †	151 ± 12* , †	151 ± 9* , †	173 ± 19* , †
ET-1 (100 nM)	129 ± 4*	151 ± 8* , †	143 ± 5* , †	163 ± 9* , †	145 ± 8* , †	137 ± 4*	161 ± 15* , †	146 ± 8* , †

 Data are presented as percentage of [³H]AA released in relation to control levels (in absence of the agonist) ± SEM. For each donor three to five experiments were performed in triplicate. Parallel experiments were run using n-HCM cells. Basal values of [³H]AA (dpm/mg protein) for the n-HCM cells were: 1815 ± 46 and for the g-HCM cells were as follows: donor 1, 3031 ± 337; donor 2, 3127 ± 847; donor 3, 2386 ± 252; donor 4, 1699 ± 72; donor 5, 2266 ± 78; donor 6, 2742 ± 242; donor 7, 2844 ± 198 (average, 2585 ± 190). Statistical analysis was performed on the absolute values.

*  Significantly different from basal values using paired Student’s t-test (P < 0.005).

†  Significantly different from n-HCM cells using unpaired Student’s t-test (P < 0.05).

Table 2.

 

Effects of PGF_2α, CCh, and ET-1 on the Release of Endogenous PGE₂ in n- and g-HCM cells

Additions	n-HCM Cells	g-HCM Cells
				Donor 1	Donor 2	Donor 3	Donor 4	Donor 5	Donor 6	Donor 7
None	100	100	100	100	100	100	100	100
PGF2α (1 μM)	307 ± 41*	216 ± 15* , †	145 ± 12* , †	199 ± 13* , †	266 ± 36* , †	245 ± 18* , †	232 ± 25* , †	229 ± 17* , †
CCh (10 μM)	199 ± 18*	183 ± 8*	207 ± 18*	203 ± 16*	283 ± 90* , †	206 ± 22*	193 ± 31*	182 ± 23*
ET-1 (100 nM)	159 ± 15*	168 ± 10*	148 ± 9*	243 ± 70* , †	391 ± 37* , †	187 ± 36* , †	316 ± 46* , †	186 ± 18* , †

 Data presentation and number of experiments are as in Table I . Mean ± SEM basal values of PGE₂ release (pg/mg protein · 15 min) for the n-HCM cells were 687 ± 33 and for g-HCM cells were as follows: donor 1, 1893 ± 283; donor 2, 1740 ± 103; donor 3, 1752 ± 277; donor 4, 3332 ± 172; donor 5, 1487 ± 150; donor 6, 1293 ± 182; donor 7, 1383 ± 189 (average, 1840 ± 261). Statistical analysis was performed on the absolute values.

*  Significantly different from basal values using paired Student’s t-test (P < 0.005).

†  Significantly different from n-HCM cells using unpaired Student’s t-test (P < 0.05).

Table 3.

 

Effects of PGF_2α, CCh, and ET-1 on [³H]Inositol Phosphate Production in n- and g-HCM Cells

Additions	n-HCM Cells	g-HCM Cells
				Donor 1	Donor 2	Donor 3	Donor 4	Donor 5	Donor 6	Donor 7
None	100	100	100	100	100	100	100	100
PGF2α (1 μM)	133 ± 11*	102 ± 5, †	114 ± 6	116 ± 7	106 ± 5, †	103 ± 7, †	113 ± 6	119 ± 8
CCh (10 μM)	521 ± 63*	158 ± 12* , †	308 ± 50* , †	317 ± 60* , †	217 ± 55* , †	207 ± 15* , †	248 ± 28* , †	160 ± 15* , †
ET-1 (100 nM)	253 ± 12*	162 ± 23* , †	210 ± 33* , †	178 ± 32* , †	136 ± 22* , †	209 ± 8* , †	227 ± 15*	173 ± 8* , †

 Data presentation and number of experiments are as in Table I . Mean ± SEM basal values of [³H]inositol phosphates (dpm/mg protein) in the n-HCM cells ranged from 7179 to 17220 (mean, 12040 ± 626), and in the g-HCM were as follows: donor 1, 11049 ± 1760; donor 2, 15003 ± 2194; donor 3, 14409 ± 1679; donor 4, 11062 ± 765; donor 5, 8136 ± 1796; donor 6, 14500 ± 1108; donor 7, 13368 ± 798. Statistical analysis was performed on the absolute values.

*  Significantly different from basal values using paired Student’s t-test (P < 0.005)

†  Significantly different from n-HCM cells using unpaired Student’s t-test (P < 0.05)

Figure 4.

 

Immunochemical identification (A) and quantitation (B) of PLC-β₁ 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-β₁ 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-β₁ represent the sum of two bands of PLC-β₁. Results are from one experiment (donor 1) that is a representative of 6 to 8 separate experiments. Comparable decreases in PLC-β₁ expression were observed in g-HCM cells from other donors.

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