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(Investigative Ophthalmology and Visual Science. 2006;47:938-945.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.05-0527
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Endothelin Antagonism: Effects of FP Receptor Agonists Prostaglandin F2{alpha} and Fluprostenol on Trabecular Meshwork Contractility

Hagen Thieme,1,2 Christin Schimmat,3 Galina Münzer,3 Marianne Boxberger,3 Michael Fromm,3 Norbert Pfeiffer,1 and Rita Rosenthal2,3

1From the Augenklinik und Augenpoliklinik, Johannes Gutenberg-Universität Mainz, Mainz, Germany; and 2Augenklinik und Hochschulambulanz and 3Institut für Klinische Physiologie, Charité–Universitätsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. This study analyzes additional mechanisms behind the ocular hypotensive effect of prostaglandin F (PGF) receptor (FP receptor) agonists PGF2{alpha} and fluprostenol (fluprostenol-isopropyl ester [travoprost]), which reduce intraocular pressure (IOP) in patients with glaucoma probably by enhancing uveoscleral flow. The trabecular meshwork (TM) is actively involved in IOP regulation through contractile mechanisms. Contractility of TM is induced by endothelin (ET)-1, a possible pathogenic factor in glaucoma. The involvement of FP receptor agonists in the ET-1 effects on TM function was studied.

METHODS. The effects of FP receptor agonists on contractility of bovine TM (BTM) were investigated using a force-length transducer. The effects of PGF2{alpha} on intracellular Ca2+ ([Ca2+]i) mobilization in cultured cells were measured using fura-2AM. The expression of the FP receptor protein was examined using Western blot analysis.

RESULTS. The ET-1–induced (10–8 M) contraction in isolated BTM was inhibited by PGF2{alpha} (10–6 M) and fluprostenol (10–6 M). This effect was blocked by FP receptor antagonists. Carbachol-induced contraction or baseline tension was not affected by PGF2{alpha} or fluprostenol. In cultured TM cells, ET-1 caused a transient increase in [Ca2+]i that was reduced by PGF2{alpha}. No reduction occurred in the presence of the FP receptor antagonist Al-8810. Western blot analysis revealed the expression of the FP receptor in native and cultured TM.

CONCLUSIONS. FP receptor agonists operate by direct interaction with ET-1–induced contractility of TM. This effect is mediated by the FP receptor. Thus, FP receptor agonists may decrease IOP by enhancing aqueous humor outflow through the TM by inhibiting ET-1–dependent mechanisms.

The effects of prostaglandin F2{alpha} (PGF2{alpha}) and FP receptor agonists on intraocular pressure (IOP) have been investigated extensively in the past few years. It has been shown that prostaglandins reduce IOP in patients with glaucoma or ocular hypertension by enhancing aqueous humor outflow through the uveoscleral pathway.1 2 3 However, the mechanisms of action of these agents on IOP remain partially unclear. Evidence shows increased secretion of matrix metalloproteinases (MMPs) by the ciliary muscle after treatment with PGF2{alpha} and PGF2{alpha}-related compounds.4 5 The activation of MMP in the extracellular space results in a reduction of extracellular matrix (ECM) components such as collagen and laminin.6 7 The decrease of ECM within the interbundle spaces of the ciliary muscle reduces hydraulic resistance to aqueous humor outflow and thereby probably contributes to the PGF2{alpha}-mediated increase of uveoscleral outflow. Increased immunoreactivity for MMPs was detected in the sclera and iris root of monkey eyes as well as in the ciliary muscle after PGF2{alpha} treatment.8 In organ cultures of human sclera, increased scleral permeability was found after exposure to PGF2{alpha}-related compounds, which was accompanied by increased expression of MMPs.9

Other authors10 11 have described the involvement of the myosin light chain (MLC) kinase signaling pathway in the IOP-lowering effect of PGF2{alpha} and its analogues, and they have examined the effect of PGF2{alpha} and latanoprost on phosphoinositide turnover, MLC phosphorylation, and contraction in cat and bovine iris sphincter. In these tissues, PGF2{alpha} and latanoprost increased inositol phosphate production, MLC phosphorylation, and contraction. In addition, they suggest that changes in the contraction–relaxation of smooth muscles of the anterior segment could facilitate aqueous humor outflow and thus contribute to the IOP-lowering effects of FP-class prostaglandins.

Adjacent to the smooth muscle of the iris, the trabecular meshwork (TM)—with its smooth muscle-like properties—is located in the anterior segment of the eye. It is now widely accepted that the TM contributes actively to the regulation of conventional outflow and thus to IOP.12 Contraction of TM decreases outflow, whereas relaxation increases this parameter. Contraction of TM is induced by muscarinic agonists and by endothelin (ET)-1. Accumulating evidence indicates a role for ET-1 in the pathogenesis of glaucoma.13 14 15 16 17 The antiglaucoma drug unoprostone, a docosanoid with affinity to the FP receptor, appears to decrease IOP by an anti-endothelin effect on TM contractility.15 It has been shown that unoprostone increases the facility of outflow through the TM.18

Until now, nothing has been known about the effects of other FP receptor agonists on TM contractility or the involvement of this tissue in the IOP-lowering effect of these agents. This study was performed to investigate the effect of PGF2{alpha} and fluprostenol (fluprostenol–isopropylester [travoprost]) on TM contractility.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Contractility Measurements
TM strips were carefully dissected from freshly enucleated bovine eyes according to methods described previously.19 Isolated strips (2- to 4-mm long) rested under control conditions (Ringer’s solution) for at least 1 hour before various agents were applied. Only strips showing a stable basic contractile tone were used for experiments. Direct isometric tension measurements of single TM strips were performed using a force-length transducer. Tissue strip contractions induced by ET-1 were expressed relative to the response obtained with a maximal effective carbachol concentration (10–6 M), which was tested in each strip as a control and set to 100% force obtainable. Determination of ET-1 activity in the presence of inhibitory substances was accomplished after preincubation with these substances and an ET-1 peak in the presence of inhibitors. The ionic concentrations (in 10–3 M) of Ringer’s solution were: 151 Na+, 5 K+, 1.7 Ca2+, 0.9 Mg2+, 131 Cl–, 0.9 SO42–, 1 H2PO4–, 28 HCO3–, and 5 glucose. All solutions were kept at 37°C, and a stable pH (7.4) achieved by gassing with 5% CO2 in air.

Cell Cultures
BTM strips were used for cell cultures as described previously.20 BTM cell cultures were incubated in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, and 100 µg/mL streptomycin. Human TM (HTM) strips were excised from eyes previously enucleated for posterior malignant melanoma without history of glaucoma. HTM cell cultures were established and characterized as previously described21 22 and were incubated in DMEM supplemented with 20% FCS, 100 µg/mL kanamycin, and 50 µg/mL gentamicin. Cell cultures were maintained at 37°C and 5% CO2 in air. The medium was changed twice a week. Confluent cultures were passaged using the trypsin/EGTA method and split in a ratio 1:2. Only well-characterized cells from early passages (2–4) were used. Tenets of the Declaration of Helsinki were followed, informed consent was obtained, and institutional human experimentation committee approval was granted for the studies.

Measurement of [Ca2+]i
Measurements of [Ca2+]i were performed using the Ca2+-sensitive dye fura-2AM based on methods described by Grynkiewicz et al.23 Cells in culture flasks were trypsinated and thereafter cultured on coverslips for at least 1 week. Before each experiment, semiconfluent cells were incubated in control solution (HEPES-Ringer) with 10 µM fura-2AM for 30 minutes at room temperature. The dye was loaded by diffusion and intracellular cleavage of fura-2AM to fura-2. Then the coverslip was placed into the perfusion chamber on the stage of an inverted microscope. Cells were perfused with control solution for 30 minutes to wash out extracellular dye before the measurement was started. The excitation light was generated by a xenon lamp (XPO 75 W/2; Osram, Munich, Germany) filtered by two rotating filters (6/s) at 340 and 380 nm. Relative fluorescence of fura-2 after excitation was registered at 510 nm by a photomultiplier (928 SF; Hamamatsu, Hamamatsu, Japan) with consequent signal detection with an EPC-9 patch-clamp amplifier. For data storage and processing, TIDA for Windows was used. Changes in the 340/380-nm fluorescence ratio represent relative changes in [Ca2+]i. Absolute [Ca2+]i was calculated using the equation and dissociation constant of Grynkiewicz.23 The ionic concentrations (in 10–3 M) of HEPES-Ringer solution were: 151 Na+, 5 K+, 1.7 Ca2+, 0.9 Mg2+, 156.7 Cl–, 0.9 SO42–, 1 H2PO4–, 10 HEPES, and 5 glucose.

Western Blot Analysis
Cultured cells were washed with ice-cold PBS, scraped from the culture dish in ice-cold lysis buffer containing (in 10–3 M) 20 Tris, 5 MgCl2, 1 EDTA, and 0.3 EGTA supplemented with protease inhibitors. Small BTM strips were homogenized in lysis buffer using a homogenizer (Polytron; Kinematik, Littau, Switzerland). Homogenate was obtained by three freeze-thaw cycles and subsequent passage through a 26G1/2 needle. The membrane fraction was separated by two centrifugation steps. Samples were first centrifuged for 5 minutes at 500g, and then the supernatant was centrifuged for 30 minutes at 43,000g (4°C). The pellet containing the membrane fraction was resuspended in lysis buffer. Protein content was determined using a protein assay reagent (BCA; Pierce, Rockford, IL) and was quantified with a plate reader (Tecan Group Ltd., Zurich, Switzerland). Lysate with 20 µg total protein was loaded on an 8.5% SDS polyacrylamide gel. Membrane lysates and molecular weight markers (Fermentas International Inc., Burlington, ON, Canada) were separated by electrophoresis in Mini Protean electrophoresis cells (Bio-Rad Life Science Group, Hercules, CA). After blotting of proteins to nitrocellulose filter screens (NEN Life Science Products, Boston, MA) for 1 hour at 100 V (4°C), blot membranes were blocked with 5% nonfat milk in PBS-Tween for 2 hours at room temperature and overnight with 5% BSA in PBS-Tween at 4°C. Membranes were then incubated in polyclonal antibody raised against the FP receptor. After incubation with peroxidase-conjugated secondary antibody and use of detection reagent (Lumi light Western blotting substrate; Roche, Nutley, NJ) specific signals were visualized by means of luminescence imaging (LAS-1000; Fujifilm, Sendai, Japan). Specific staining was confirmed using the corresponding blocking peptide combined with the polyclonal antibody in accordance with the manufacturer’s instructions.

Chemicals and Solution
The following reagents were used for the experiments: endothelin-1 (Alexis Deutschland GmbH, Grünberg, Germany), PGF2{alpha}, PGF2{alpha} dimethylamine, PGF2{alpha} dimethylamide, fluprostenol, and FP receptor polyclonal antibody (Cayman Chemicals, Ann Arbor, MI). All other chemicals were purchased from Merck (Darmstadt, Germany), Sigma (Deisenhofen, Germany), and Serva (Heidelberg, Germany).

Calculations and Statistical Analysis
Data are presented as mean ± SEM and were analyzed for significance using Student’s t-test. For multiple testing Bonferroni correction was applied. Significance levels: n.s., not significantly different; *P < 0.05; **P < 0.01; ***P < 0.001. Number (n) refers to the number of experiments. Western blot experiments were performed at least three times on individual cell cultures or on native tissues; results of one representative experiment are shown.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Effects of PGF2{alpha} and Fluprostenol on Carbachol- and ET-1–Induced Contractility
As has been shown,15 24 25 26 27 the muscarinic agonist carbachol (10–6 M) led to a contraction in BTM strips that was set to 100% force (Fig. 1) . ET-1 caused contractions from baseline level in a dose-dependent manner (10–9 M ET-1, 22.9% ± 5.9%; 10–8 M ET-1, 61.5% ± 8.4%; both versus carbachol). To test the effect of PGF2{alpha} and fluprostenol on the ET-1–induced contraction, tissue was preincubated for 20 minutes with FP receptor agonists before ET-1 was applied in the presence of the agonists (Fig. 1) . ET-1–induced contractions were partially blocked by 10–6 M PGF2{alpha} (10–9 M ET-1, 4.1% ± 2.1%; 10–8 M ET-1, 31.0% ± 10.6%; both versus carbachol; Fig. 1A ) and 10–6 M fluprostenol to 25.0% ± 6.5% (Fig. 2A) . PGF2{alpha} and fluprostenol had no influence on baseline tension or carbachol-induced contraction (114% ± 11.4%; Fig. 1B ; 127.1% ± 18.6%; Fig. 2B ). A summary of these effects is shown in Figure 3 .


Figure 1
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  		FIGURE 1. Effect of PGF2{alpha} on ET-1– and carbachol-induced contraction in BTM strips. Original recordings of isometric force. (A) After a carbachol (10–6 M)–induced peak, a second contraction was provoked either by application of ET-1 (10–8 M) (black curve) or application of ET-1 (10–8 M) after preincubation of the tissue for 20 minutes with PGF2{alpha} (10–6 M) (gray curve). The ET-1–induced contraction in the presence of PGF2{alpha} was strongly reduced. (B) After a first carbachol peak, a second contraction was provoked by application of carbachol (black curve) or application of carbachol after preincubation for 20 minutes with PGF2{alpha} (gray curve). PGF2{alpha} had no inhibitory effect on the carbachol-induced contraction.

 

Figure 2
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  		FIGURE 2. Effect of fluprostenol on ET-1– and carbachol-induced contraction in BTM strips. Original recordings of isometric force. Similar experiments as described for Figure 1 . ET-1–induced contraction was inhibited in the presence of fluprostenol (10–6 M) (A), which had no inhibitory effect on the carbachol-induced contraction (B).

 

Figure 3
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  		FIGURE 3. Effect of PGF2{alpha} and fluprostenol on carbachol- and ET-1–induced contraction in BTM. Summary of data obtained with all BTM strips under the influence of carbachol and ET-1 in the presence and absence of PGF2{alpha} and fluprostenol. Number of experiments (n) is given in brackets within the bars. n.s., not significantly different; *P < 0.05; ***P < 0.001.

 
Effects of PGF2{alpha} and Fluprostenol in Combination with FP Receptor Antagonists on ET-1–Induced Contractility
To test the involvement of the FP receptor in the inhibiting action of PGF2{alpha} on contractility, PGF2{alpha} was applied in the presence of the FP receptor antagonists PGF2{alpha} dimethylamide (Fig. 4A) or PGF2{alpha} dimethylamine. Both substances had no effect on baseline tension of BTM strips. In the presence PGF2{alpha} dimethylamide (10–6 M), the 10–8 M ET-1–induced contraction of BTM was not altered by PGF2{alpha} (10–6 M; 55.3% ± 4.1%). The same effect was observed in the presence of PGF2{alpha} dimethylamine (10–6 M; 61.7% ± 11.8%; n = 7). In addition, the inhibiting effect of fluprostenol on ET-1–induced contraction was abrogated in the presence of Al-8810 (10–6 M; 50.3% ± 6.1%; Fig. 4B ). A summary of these data is shown in Figure 5 .


Figure 4
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  		FIGURE 4. Effect of FP receptor blockade on PGF2{alpha} or fluprostenol action on ET-1–induced contraction in BTM. Original recordings of isometric force. After a carbachol (10–6 M)–induced peak, the tissue was preincubated for 20 minutes with the FP receptor blocker PGF2{alpha} dimethylamide (10–6 M) (A) or Al-8810 (10–6 M) (B) before PGF2{alpha} (10–6 M) and ET-1 (10–8 M) (A) or fluprostenol (10–6 M) and ET-1 (10–8 M) (B) were applied. Under these conditions, PGF2{alpha} and fluprostenol failed to inhibit the ET-1–induced contraction.

 

Figure 5
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  		FIGURE 5. Effects of PGF2{alpha} and fluprostenol in combination with FP receptor antagonists on ET-1–induced contraction in BTM. Summary of data obtained with all BTM strips under the influence of FP receptor blockers, PGF2{alpha} or fluprostenol, and ET-1. Number of experiments (n) is given in brackets within the bars. n.s., not significantly different.

 
Lack of Effect of PGF2{alpha} on ET-1–Induced Contraction
As mentioned, PGF2{alpha} has no relaxing effect on baseline tension in TM. Furthermore, no relaxing effect of PGF2{alpha} on ET-1–induced contraction was observed. When PGF2{alpha} was applied after the tissue was treated with ET-1 and contraction was initiated, no reduction of the ET-1 effect occurred (Fig. 6) . In this case, contraction provoked by ET-1 was 69.1% ± 8.9% (n = 4) and thus not significantly different from ET-1–induced contraction without PGF2.


Figure 6
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  		FIGURE 6. Application of PGF2{alpha} after the onset of ET-1–induced contraction in BTM strips. Original recording of isometric force. After a carbachol (10–6 M)–induced peak, ET-1 (10–8 M) was applied. In the ascending phase of the ET-1–induced contraction, PGF2{alpha} (10–6 M) was added. Under these conditions, the prostaglandin had no inhibitory effect.

 
Effects of PGF2{alpha} and ET-1 on [Ca2+]i
In cultured BTM cells, the baseline [Ca2+]i was 89.4 ± 14.0 nM (n = 12). Application of ET-1 (10–8 M) resulted in an increase of [Ca2+]i to 203.8% ± 23.3% of the baseline level (Figs. 7A 7C) . PGF2{alpha} (10–6 M) reduced the ET-1–induced increase to 146.9% ± 8.9% (Figs. 7B 7C) . Baseline [Ca2+]i in HTM cells was 105.5 ± 12.3 nM (n = 24) and is within the range published by our and other groups.22 28 ET-1 (5 x 10–8 M) caused an increase of [Ca2+]i to 221.7% ± 19.3% from the baseline level (Fig. 8A) . In the presence of PGF2{alpha} (10–5 M), the ET-1–induced enhancement of [Ca2+]i was 139.0% ± 15.8% of the baseline level (Fig. 8B) . In the presence of Al-8810 (10–6 M), PGF2{alpha} (10–5 M) had no inhibiting effect, and the ET-1–induced [Ca2+]i peak reached 249.7% ± 49.3% of the baseline level (Fig. 8C) . A summary of these data is shown in Figure 8D . Application of PGF2{alpha} (10–5–10–6 M) or Al-8810 (10–6 M) had no effect on baseline [Ca2+]i in cultured BTM and HTM cells.


Figure 7
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  		FIGURE 7. Effect of ET-1 and PGF2{alpha} on [Ca2+]i in cultured BTM cells. Measurements of [Ca2+]i were performed using the Ca2+-sensitive dye fura-2AM. For investigation of PGF2{alpha} on the ET-1 effect, cells were preincubated for 5 minutes with the prostaglandin. (A) ET-1 (10–8 M)–induced increase in [Ca2+]. (B) Reduction of the ET-1–induced increase in [Ca2+]i in the presence of PGF2{alpha} (10–6 M). (C) Summary of data obtained with cultured BTM cells. Number of experiments (n) is given in brackets within the bars. *P < 0.05; **P < 0.01.

 

Figure 8
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  		FIGURE 8. Effect of ET-1, PGF2{alpha}, and All-8810 with PGF2{alpha} on [Ca2+]i in cultured HTM cells. Measurements of [Ca2+]i were performed using the Ca2+-sensitive dye fura-2AM. Cells were preincubated for 5 minutes with PGF2{alpha} (10–5 M) or AL-8810 (10–6 M) before the substances were applied in combination with others. (A) ET-1 (5 x 10–8 M)–induced increase in [Ca2+]i. (B) Reduction of the ET-1–induced increase in [Ca2+]i in the presence of PGF2{alpha}. (C) No inhibiting effect of PGF2{alpha} in the presence of Al-8810. (D) Summary of data obtained with cultured HTM cells. Number of experiments (n) is given in brackets within the bars. n.s., not significantly different; *P < 0.05; ***P < 0.001.

 
FP Receptor Detection by Western Blot Analysis
Western blot analysis was used to detect the FP receptor on the protein level. Membrane lysates of native BTM strips and cultured BTM and HTM cells were investigated. In all preparations, the FP receptor was identified at 64 kDa (Fig. 9) . Corresponding blocking peptide confirmed the specificity of the detected bands.


Figure 9
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  		FIGURE 9. Expression of prostaglandin F receptor (FP receptor) in TM. Western blot analysis of membrane lysates incubated with anti-FP receptor antibody detected a specific signal at approximately 64 kDa (arrows) in native bovine tissue strips, cultured bovine cells, and human TM cells. Specific staining of the antibody was verified by use of the corresponding blocking peptide (BP).

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Treatment of glaucoma still focuses on lowering IOP, either pharmacologically or surgically. New glaucoma medications, such as the selective FP receptor agonists, are now widely accepted as antiglaucomatous drugs, but ongoing discussion continues concerning the IOP-lowering effects of these compounds. Some investigators suggest a modulation of conventional outflow facility,15 others see the primary target site in the enhancement of the uveoscleral outflow.29 30 This study describes an anti-endothelin effect of PGF2{alpha} and fluprostenol on TM contractility and supports the thesis of an influence on conventional outflow.

ET-1 is one of the most potent vasoactive peptides known, and it has been shown to play an important role in vascular homeostasis31 and in a variety of pathologic processes.32 Additionally, ET-1 seems to be involved in the pathogenesis of glaucoma.16 Aqueous humor ET-1 levels are elevated in eyes with primary open-angle glaucoma14 and exfoliation syndrome33 compared with those in healthy subjects. This is consistent with the observation of increased ET-1 concentrations in blood plasma of patients with normal-tension glaucoma.34 Furthermore, ET-1 has been shown to be important for the regulation of the conventional outflow in the anterior segment of bovine15 35 and primate eyes.36 37 ET-1 has been found to cause contraction of vascular smooth muscle and pericytes and seems to play a role in retinal and choroidal blood flow.17 38 The effects of ET-1 are mediated through two receptors, ET-AR and ET-BR,39 40 both expressed in TM. The ET-1–induced contraction was mainly mediated by the ET-AR,41 whereas the function of the ET-BR in TM has not been clear until now. An inhibition of ET-1–induced TM contractility by FP receptor agonists probably increases outflow facility and might decrease IOP. The PGF2{alpha}-induced enhancement of ocular and retinal blood flow, and with it neuroprotective action, could be the result of a similar effect of PGF2{alpha} on vascular smooth muscle in the eye.

It is important to note that PGF2{alpha} and fluprostenol were unable to influence baseline contractility or pathways that involve G-protein–linked muscarinic receptors, suggesting the involvement of different G-proteins, G-protein–coupled receptor kinases, or protein kinases in ET and muscarinic receptor activation and desensitization. Another possible cause for the missing effect of PGF2{alpha} on carbachol-induced contraction could be differences in the signaling pathways from activated ET and muscarinic receptors. In TM, muscarinic receptors of the m1-, m2-, and m3-subtypes are expressed and involved in contractility.25 An effect of PGF2{alpha} on TM contractility mediated by these receptors could be excluded from our study.

The mechanisms of the anti-endothelin action of FP receptor agonists are not yet clarified. Investigations with FP receptor blockers indicate that the effect is mediated by the FP receptor. In other smooth muscle, such as bovine iris sphincter, PGF2{alpha} stimulates phosphoinositide turnover, myosin light-chain phosphorylation, and contraction through the FP receptor with EC50 values of 9, 42, and 140 nM, respectively.11 In our study, a PGF2{alpha} concentration of 1 to 10 µM was used. In this concentration range, no effect on TM contractility or baseline [Ca2+]i could be observed. The Western blot data confirm the protein expression of the FP receptor in bovine and human TM. The molecular weight (64 kDa) is within the range published by others.11 Until now, the expression of the FP receptor in human TM has been shown by immunofluorescence microscopy and RT-PCR methods only.42 Possibly, the FP receptor density is too low in TM to induce contractility in comparison with other smooth muscle systems. Another explanation could be the expression of different FP receptor isoforms in TM. Until now, two isoforms have been identified, the FPA and the FPB receptors, which are for the most part identical except for their carboxyl termini.43 FPB is essentially a truncated version of FPA that lacks the 46 carboxyl-terminal amino acids, including four putative protein kinase C (PKC) phosphorylation sites.44 The carboxyl terminus of the FPA is a substrate for PKC, and PKC-dependent phosphorylation is responsible for differential regulation of second-messenger pathways by FP receptor isoforms. Thus, differences in the expression pattern of receptor isoforms may cause different cellular signaling after receptor activation. It should also be mentioned that most tissues contain a heterogeneous population of prostaglandin receptor subtypes mediating relaxation and contraction of smooth muscles and that most prostaglandin agonists display activity at different prostaglandin receptors.45 46 The cellular response depends on agonist potency at the different receptors. Possibly, in TM, PGF2{alpha} induces contraction and relaxation by way of different receptors, and each effect compensates for the other so that no effect on contractility could be observed.

It has been shown that the inhibiting effect of PGF2{alpha} on ET-1–induced contractility occurred only if the prostaglandin was applied before ET-1 was added. Application of PGF2{alpha} after the onset of the ET-1 contraction was ineffective. Based on this observation, we conclude that the FP receptor must be activated before ET-1 binds to its receptor to induce attenuation of the ET-1 effect. Given that in TM the ET-AR is mainly responsible for contraction,41 involvement of this receptor can be supposed. In consequence of PGF2{alpha} preincubation, the ET-1–induced increase in [Ca2+]i is diminished and contractile force is reduced. We cannot elucidate from our data whether this effect occurs at the level of the ET receptor or at intracellular pathways leading to an enhancement of [Ca2+]i.

Our data show an inhibition of the ET-1–induced increase in [Ca2+]i by PGF2{alpha} in bovine and human TM cells. We did not detect any effect on baseline [Ca2+]i by PGF2{alpha}, which underscores the missing effect of this compound on baseline contractility. In TM, contractility is partially dependent and partially independent of intracellular Ca2+. The Ca2+-independent contraction uses PKC and rho-A/ROCK–mediated pathways based on pharmacomechanical coupling events.24 47 We cannot exclude the existence of additional effects of PGF2{alpha} and fluprostenol on Ca2+-independent contractility.

In contrast to our results, Sharif et al.48 described a phosphoinositide turnover and intracellular Ca2+ mobilization in HTM cells induced by FP class prostaglandin analogs, such as travoprost, latanoprost, bimatoprost, unoprostone isopropyl ester, and PGF2{alpha}. The missing effects of PGF2{alpha} on [Ca2+]i we observed are in good agreement with our contractility measurements. In addition, Krauss et al.49 show that PGF2{alpha} has no effect on TM contractility. PGF2{alpha} does not induce changes in [Ca2+]i or affect baseline tension. It may be that we could not detect very small changes in [Ca2+]i with our experimental equipment. Irrespective of this, a PGF2{alpha}-induced increase in [Ca2+]i is insufficient for triggering contractions in TM.

It is well known that in humans the main part of aqueous humor is drained through conventional outflow. Direct measurements in human eyes have suggested that less than 15% of aqueous humor is drained by the uveoscleral routes. However, indirect calculations indicate that rate to be approximately 35% in young adults and of 3% in elderly persons (older than 60 years; for review, see 50 ). This suggests that in patients with primary open-angle glaucoma, the uveoscleral outflow contributes to aqueous humor drainage to only a minor degree. Therefore, we assume that the anti-endothelin effect on TM contractility, which increases aqueous humor outflow through the conventional route, is also involved in the IOP-lowering effect of FP receptor agonists.

In summary, this study suggests an additional hypotensive effect of PGF2{alpha} and fluprostenol. This could be the result of an intervention in ET-1–dependent pathways in the TM, namely inhibition of the ET-1–induced increase in [Ca2+]i, causing a reduction of contractility of this tissue.


   Acknowledgements
 
The authors thank Alcon (Freiburg, Germany) for financial support in purchasing fluprostenol and Ingrid Lichtenstein for expert technical assistance.


   Footnotes
 
Supported by Deutsche Forschungsgemeinschaft DFG Th751/4-1.

Submitted for publication April 28, 2005; revised September 15 and November 4, 2005; accepted January 3, 2006.

Disclosure: H. Thieme, Alcon (F); C. Schimmat, None; G. Münzer, None; M. Boxberger, None; M. Fromm, None; N. Pfeiffer, None; R. Rosenthal, None

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: Rita Rosenthal, Augenklinik und Hochschulambulanz, Charité–Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany; rita.rosenthal{at}charite.de.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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