Functional and structural reversibility of H-7 effects on the conventional aqueous outflow pathway in monkeys

To determine the mechanism of H-7-induced outflow resistance decrease, the reversibility of H-7 effects on outflow pathway was studied physiologically and morphologically in live monkey eyes. Total outflow facility was measured by two-level constant pressure perfusion before (baseline measurement) and after (post-drug measurement) anterior chamber (AC) exchange with 300microM H-7 or vehicle in opposite eyes of eight monkeys. H-7 was then removed by AC exchange with drug-free vehicle in both eyes, followed by a 2.5hr waiting period, after which outflow facility was measured again with (Group 2; n=4) or without (Group 1; n=4) another preceding drug-free AC exchange. For morphological study, five monkeys were initially perfused similarly to Group 1 in physiology, but the facility measurement beginning 2.5hr after drug removal was either omitted or replaced by gold solution infusion. Following baseline measurement, two of the five monkeys received H-7 or vehicle in opposite eyes, while three monkeys received H-7 in both eyes 2.5hr apart, contributing one H-7-treated 'recovery' eye and one H-7-treated 'acute' eye. After perfusion, both eyes of all five monkeys were studied by light and electron microscopy. Outflow facility during post-drug measurement in the H-7-treated eye was significantly increased by two-fold. However, the facility increase was reduced when measured beginning 2.5hr after drug removal, with the reduction being greater in Group 1. 'Recovered' outflow facility after drug removal gradually increased again under continuous AC infusion with drug-free vehicle. Morphologically, major changes in and around Schlemm's canal (SC) in the H-7-treated 'acute' eye included protrusion of the entire inner wall (IW) into SC, relaxation of the IW cells and reorganization of the IW cytoskeleton. The changes in IW cells and juxtacanalicular region of the H-7-treated 'recovery' eye were non-uniform, with areas resembling the vehicle-treated eye ('contracted areas') and areas resembling the H-7-treated 'acute' eye ('relaxed areas'). The average junction-to-junction distances in the IW cells of the H-7-treated 'recovery' eye were intermediate between the vehicle-treated eye and the H-7-treated 'acute' eye. In conclusion, H-7's effect on outflow facility seems reversible, but AC exchange or continuous infusion with drug-free vehicle can re-elevate the 'recovered' outflow facility. Major morphological changes in the TM immediately after H-7 include IW protrusion, cellular relaxation and cytoskeleton reorganization. The decrease in 'relaxed areas' in the TM, in conjunction with the reversed outflow facility, 2.5hr after drug removal suggests that cellular relaxation in the TM is the structural basis for H-7-induced increase in outflow facility.     

Relationships between increased aqueous outflow facility during washout with the changes in hydrodynamic pattern and morphology in bovine aqueous outflow pathways

Previous studies suggest that the structural correlate for the increased outflow facility (C) during washout in the bovine eye is separation between the inner wall (IW) and underlying juxtacanalicular connective tissue (JCT). However, how these structural changes affect hydrodynamic patterns of outflow during washout has not been studied. We hypothesize that an increase in the outflow facility during washout is associated with an increase in the effective filtration area (EFA) of aqueous outflow, which is regulated by a loss of the connectivity between the IW and JCT. To test this hypothesis, the relationship between C and the hydrodynamic patterns of outflow as well as the morphological changes in JCT and IW during the washout were investigated. Ten bovine eyes were perfused at 15 mmHg with Dulbecco's PBS + 5.5 mM glucose (DPBS) for 30 min to establish stable baseline C. After measuring baseline C, five eyes (short-duration group) were perfused with 0.5 mL DPBS containing 0.002% microspheres (0.5 μm) to trace the hydrodynamic pattern of outflow. Five other eyes (long-duration group) were perfused for 3 h to elicit a significant washout effect followed by subsequent perfusion of the same volume (0.5 mL) of microspheres to map out the outflow pattern after washout. All eyes were then perfusion-fixed. Anterior segments were sectioned and prepared for confocal and light microscopy. Total length (TL) and filtration length (FL) of the IW were measured in ≥15 images/eye to calculate percent effective filtration length (PEFL = FL/TL) while TL and length exhibiting JCT/IW separation (SL) were measured in ≥13 images/eye to calculate percent separation length (PSL = SL/TL). In long-duration eyes, C increased 170.5 ± 21.3% (mean ± SEM, 1.55 ± 0.24 vs 4.13 ± 0.55 μl/min/mmHg, p = 0.001) above baseline. Pre-fixation C (4.13 ± 0.55 μl/min/mmHg) in long-duration was 1.6-fold greater than that (2.14 ± 0.61 μl/min/mmHg; p = 0.042) in short-duration. A more uniform tracer labeling was observed in the JCT/IW of long-duration eyes compared to short-duration. PEFL was 2.3-fold larger (52.82 ± 6.06 vs. 22.2 ± 6.0%; p = 0.007) and PSL was 2.6-fold larger (54.2 ± 6.0 vs. 20.5 ± 1.3%; p = 0.004) in long-duration eyes compared to short-duration. Data from all eyes revealed a positive correlation between PEFL and PSL (p = 0.02). Both PEFL and PSL demonstrated significant positive correlations with the relative increase in C due to washout (p ≤ 0.05). An additional experiment was performed in which unequal volumes of tracer (0.5 and 1.0 mL) were perfused in paired eyes for both short- and long-duration (N = 2 for each condition) to examine the affect on PEFL. No significant change in PEFL was found in eyes perfused with 0.5 and 1.0 mL within the same group. These data support our hypothesis that separations between the IW and JCT result in an increase in the EFA that then influences C. Altogether, these data suggest that outflow hydrodynamics and the tissue structure work together to regulate outflow resistance.     

肌动蛋白药物对小梁细胞的作用及对房水引流的影响

正常房水引流阻力主要来自于小梁、近管结缔组织和Schlemm管内壁细胞。细胞骨架肌动蛋白在调节细胞形态的稳定性、细胞与细胞、细胞与细胞基质间的附着连接方面起着重要作用。肌动蛋白药物能够引起小梁细胞细胞骨架的改变。蛋白激酶抑制剂、肌动蛋白降解剂等几类肌动蛋白药物均可作用于小梁细胞细胞骨架，影响房水引流，降低眼压．说明肌动蛋白药物可能成为一种有用的抗青光眼治疗药物。就肌动蛋白药物降眼压作用及其作用机制进行综述。     

Effects of the Rho kinase inhibitor Y-27632 and the phosphatase inhibitor calyculin A on outflow facility in monkeys

Previous studies have shown that the inhibition of Rho kinase is involved in the regulation of outflow facility in the live rabbit eye and the enucleated porcine eye. However, it is unknown whether the Rho kinase inhibition will do the same in non-human primates. To determine if the Rho kinase inhibitor Y-27632 will reduce outflow resistance in the live monkey eye, if Y-27632 and the phosphatase inhibitor calyculin A (Caly-A which antagonises Y-27632-induced MLC dephosphorylation) will affect outflow facility differently, and if the latter will inhibit effect of the former on facility, we studied effects of Y-27632 and Caly-A on outflow facility in living monkeys separately and concurrently. Total outflow facility was measured by 2-level constant pressure perfusion of the anterior chamber (AC) before and after exchange with different doses of Y-27632 (1, 10 and 100 microM) or Caly-A (10, 50 and 100 nM), or vehicles, followed by continuous AC infusion of corresponding drug/vehicle solution, in opposite eyes of cynomolgus or rhesus monkeys. The effect of 100 microM Y-27632 or 100 nM Caly-A vs vehicle and the effect of 100 microM Y-27632+100 nM Caly-A vs 100 microM Y-27632 alone on outflow facility were also determined in monkeys pre-treated topically with 10 microl of 1% atropine in both eyes 1 hr before perfusion. Both Y-27632 and Caly-A dose-dependently increased outflow facility by up to 2-3 fold in monkeys, adjusted for baseline and contralateral control eye washout. Pre-treatment with 1% topical atropine partially inhibited the effect of 100 nM Caly-A, but not 100 microM Y-27632, on outflow facility. 100 nM Caly-A gradually and partially inhibited the Y-27632-induced facility increase. In conclusion, Y-27632 increases outflow facility in monkeys presumably by inhibiting cellular contractility in the TM. Caly-A increases outflow facility by complicated mechanisms perhaps including drug-induced ciliary muscle contraction and cytoskeletal reorganisation in TM cells. The partial inhibitory effect of Caly-A on the Y-27632-induced increase in outflow facility may reflect the former partially inhibiting the latter's relaxation of cells in the TM.     

Effects of chemical inhibition of N-WASP, a critical regulator of actin polymerization on aqueous humor outflow through the conventional pathway

The integrity of actin cytoskeletal organization in aqueous humor outflow pathway is thought to play a critical role in modulation of aqueous humor outflow through the trabecular meshwork. Our understanding of the regulation of actin cytoskeletal dynamics in outflow pathway, however, is very limited. To explore the potential importance of Neural Wiskott-Aldrich syndrome protein (N-WASP), a critical regulator of actin polymerization/nucleation in aqueous humor outflow pathway, the effects of Wiskostatin, a selective pharmacological inhibitor of N-WASP, on aqueous humor outflow facility were evaluated using enucleated porcine eyes and a constant pressure perfusion system. Further, drug induced effects on actin cytoskeletal organization, cell adhesions, myosin II phosphorylation, matrix metalloproteinase (MMP) activity, and cytoskeletal protein profile in porcine trabecular meshwork (TM) cells were determined by immunofluorescence, zymography, and mass spectrometry. Aqueous humor outflow facility was increased significantly and progressively in the Wiskostatin perfused porcine eyes. The Wiskostatin perfused eyes appear to exhibit increased giant vacuoles in the inner wall of aqueous plexi and deformation of aqueous plexi. The Wiskostatin treated TM cells demonstrated extensive vacuoles in their cytosol, and both actin stress fibers and focal adhesions were decreased in a reversible manner. The drug-treated TM cells also revealed decreased myosin II and actin in the cytoskeletal enriched triton insoluble fraction but did not affect myosin II phosphorylation or MMP-2 activity. These data demonstrate that the chemical inhibition of N-WASP increases aqueous humor outflow facility in association with decreased actomyosin interaction and cell adhesive interactions revealing the importance of N-WASP in homeostasis of aqueous humor outflow.     

The changing paradigm of outflow resistance generation: Towards synergistic models of the JCT and inner wall endothelium

Aqueous humor outflow resistance is the primary determinant of intraocular pressure (IOP), and increased outflow resistance is the basis for elevated IOP associated with glaucoma. Experimental evidence suggests that the bulk of outflow resistance is generated in the vicinity of the inner wall endothelium of Schlemm's canal, its basement membrane and the juxtacanalicular connective tissue (JCT). However, attempts to sort out the contribution of each of these tissues to total outflow resistance have not been successful. Conventional understanding of outflow resistance assumes that the resistance of each tissue strata (i.e., the inner wall endothelium, its basement membrane and JCT) in the outflow pathway adds in series to contribute to total outflow resistance generation. However, this perspective leads to a paradox where the apparent resistances of all tissues in the outflow pathway are much lower than the measured total resistance. To resolve this paradox, we explore synergistic models of outflow resistance generation where hydrodynamic interactions between different tissue strata lead to a total resistance that is greater than the sum of the individual tissue resistances. We closely examine the “funneling” hypothesis that has emerged as a leading synergistic model, and we review the basis of funneling, mechanical and biological requirements for funneling and evidence in support of this hypothesis. We also propose refinements to the funneling model and describe how funneling may relate to segmental variability of aqueous humor outflow patterns observed within the trabecular meshwork. Pressure gradients across the JCT and inner wall endothelium will generate mechanical loads that influence the morphology of these tissues. Because tissue morphology may in turn affect outflow resistance, there exists the potential for a two-way coupling or a “fluid-solid interaction” between outflow hydrodynamics and the mechanical behavior of the inner wall and JCT. Furthermore, the adhesions and tethers between the inner wall and JCT must be physically capable of supporting such loads. We examine the structure and mechanical strength of these adhesions, and provide evidence that these adhesions and tethers are unable to support the full load imposed by the bulk of outflow resistance generation unless a substantial fraction of outflow resistance is generated within the JCT, consistent with the funneling model. This indicates that these attachments between the inner wall and JCT have considerable physiological importance for outflow resistance regulation, by maintaining the proximity between these two tissues to facilitate funneling. Further study is greatly needed to better characterize these important interactions.     

The mechanism of increasing outflow facility by rho-kinase inhibition with Y-27632 in bovine eyes

Rho-kinase inhibitor Y-27632 (Y-27) affects actomyosin cytoskeletal networks and has been shown to significantly increase outflow facility (C) in enucleated porcine and rabbit eyes, as well as in vivo monkey eyes without obvious toxicity. The mechanisms underlying these responses remain largely unknown. In this study, we investigate how Y-27 affects aqueous humor C, the hydrodynamic patterns of outflow, and the morphology of the inner wall (IW) and juxtacanalicular connective tissue (JCT). 12 bovine eyes were perfused at 15 mmHg with Dulbecco's PBS containing 5.5 mM glucose (DPBS) to establish stable baseline C. The anterior chamber was exchanged and perfused with DPBS containing 50 microM Y-27 in 7 eyes, while 5 eyes received DPBS alone. Eyes were then perfused with DPBS containing fluorescent microspheres (0.5 microm; 0.002% v/v) at a fixed volume to deliver equivalent amounts of tracer to label the hydrodynamic filtration patterns. All eyes were perfusion-fixed with Karnovsky's fixative. Radial and frontal sections were prepared in all quadrants and confocal images were taken along the IW of the aqueous plexus (AP). The total length (TL) and filtration length (FL) of the IW were measured in > or =16 images/eye, and the average percent effective filtration length (PEFL=FL/TL) was calculated. Sections with AP were processed and examined by light and electron microscopy. The TL of the IW and length exhibiting JCT/IW separation (SL) were measured in > or =16 micrographs/eye, and the average percent separation length (PSL=SL/TL) was also calculated. After Y-27 treatment, C increased from 1.54+/-0.34 (+/-SEM) to 2.36+/-0.54 microL/min per mmHg (58.2+/-18.9%) while control eyes changed from 1.67+/-0.41 to 1.71+/-0.39 microl/min per mmHg (6.0+/-9.3%) and the percent changes between the Y-27-treated and control eyes were significant (p=0.03). Control eyes showed segmental distribution of tracer in the trabecular meshwork tending to cluster near collector channel ostia, whereas Y-27-treated eyes showed a more uniform pattern and extensive labeling along the IW. In Y-27-treated eyes, PEFL was 3-fold larger (57.6+/-3.7%) compared to control eyes (18.2+/-4.5%; p<0.001). Light microscopic examination revealed that, with Y-27, the IW and JCT were significantly distended compared to control eyes, with discernible separation between the IW and JCT. PSL was 2.8-fold larger in Y-27-treated eyes (59.3+/-3.6%) than in controls (20.8+/-2.0%; p<0.001). A significant positive correlation was found between PEFL and PSL (p=0.003) suggesting that as connectivity between the JCT and IW decreases the available area for aqueous humor drainage increases along the AP. Our study also demonstrates a significant positive correlation between C and the PSL (p=0.01), a finding identical to what we reported to occur during the "washout" effect in bovine eyes. Our data suggests the structural correlate to the increase in C and PEFL after Y-27-treatment is physical separation between the JCT and IW. The increase in C after Y-27-treatment may share a similar mechanism comparable with the washout effect that occurs in bovine eyes. Overall, these findings support our hypothesis that JCT/IW connectivity influences local outflow hydrodynamics that regulate C, and suggest that strategies targeting JCT/IW connectivity are promising anti-glaucoma therapies to reduce IOP.     

Similar hydrodynamic and morphological changes in the aqueous humor outflow pathway after washout and Y27632 treatment in monkey eyes

Our previous studies in bovine eyes demonstrated that the structural correlate to the increase in outflow facility after either Rho-kinase inhibitor Y-27632 (Y27) treatment or washout appeared to be separation between the juxtacanalicular tissue (JCT) and inner wall (IW) of the aqueous plexus, the bovine equivalent of Schlemm’s canal (SC). While these findings suggest that Y27 and washout may increase outflow facility through a similar mechanism, the anatomy of bovine outflow pathway differs considerably from both the human and monkey outflow pathway; however, only the human eye does not exhibit washout. In light of this, we compared the effects of Y27 and washout on outflow facility, hydrodynamic patterns of outflow, and the morphology of the IW and JCT in monkey eyes, given that their anatomy is closer to human eyes.Twelve freshly enucleated monkey eyes were used in this study. Eyes were perfused with Dulbecco’s PBS containing 5.5 mM glucose (GPBS) to establish a baseline facility at 15 mmHg. Four eyes were perfused for a short-duration (30 min) as a control, 4 eyes for a long-duration (180 min) to induce washout, and 4 eyes with GPBS+50 μM Y27 for 30 min. All eyes were then perfused with fluorescent microspheres (0.5 μm; 0.002%) to label the hydrodynamic patterns of outflow and then perfusion-fixed. Confocal images of frontal sections were taken along the IW of SC. The total length (TL) and the tracer-decorated length (FL) of the IW were measured to calculate the average percent effective filtration length (PEFL = FL/TL). Sections with SC were examined by light and electron microscopy. The TL of the IW and the length exhibiting separation (SL) in the JCT were measured to calculate the average percent separation length (PSL = SL/TL).Outflow facility increased 149.2% (p < 0.01) from baseline after washout during long-duration perfusion, and 114.9% (p = 0.004) after Y27 treatment, but did not change significantly after short-duration perfusion in control eyes (p = 0.46). Distribution of the tracer labeling appeared punctate along the IW of control eyes, while a more uniform pattern was observed after washout and Y27 treatment. PEFL in washout (83.4 ± 2.1%) and Y27 treated eyes (82.5 ± 1.6%) was 3.4-fold larger compared to controls (24.2 ± 4.2%, P < 0.001). The JCT appeared distended with loss of connections between JCT cells and between JCT cells and their extracelluar matrix in eyes with washout or after Y-27 treatment. PSL in the JCT was 2.3-fold larger in washout eyes (77.4 ± 3.3%) and 2.2-fold larger in Y27 treated eyes (75.2 ± 5.3%) versus controls (33.5 ± 5.3%, p = 0.001). Significant positive correlations were found between outflow facility and PEFL, facility and PSL and between PEFL and PSL.Our data demonstrated that similar hydrodynamic and morphological changes occurred in the aqueous humor outflow pathway of monkey eyes after induction of washout and Y27 treatment. Both Y27 and washout increase outflow facility by redistributing aqueous outflow through a larger area in the JCT. These hydrodynamic changes are likely driven by morphologic changes associated with a decrease in cell–cell and cell–matrix connections in the JCT.     

The effect of colchicine on the rate of formation of aqueous humour and the gross outflow facility in the albino rabbit

Colchicine, a naturally occurring plant alkaloid which prevents the polymerisation of cytoplasmic microtubules, lowers the intraocular pressure after topical administration or intravitreal injection. In this study wer have examined the effect of topically administered colchicine on the rate of formation of aqueous humour and the gross outflow facility in the albino rabbit eye. The disappearance of [¹⁴C]inulin from the anterior chamber was measured to calculate the rate of aqueous humour formation and in a separate group of animals the gross outflow facility determined using a constant pressure perfusion technique.Topically administered colchicine (10, 20 and 40 μg/eye) inhibited the rate of aqueous humour formation dose-dependently. The mean rate of formation in control eyes was 3·68 ± 0·09 μl/min (n = 16) decreasing to 1·8 μl/min (n = 9) in eyes treated with 40 μg colchicine. Furthermore, the gross outflow facility of colchicine treated (10 μg/eye) eyes (0·49 ± 0·03 μl/min/mmHg) was significantly greater (P < 0·05) than that of contralateral control eyes (0·39 ± 0·03 μl/min/mmHg).The pharmacological evidence available indicates that colchicine is acting via mechanisms of microtubule disruption to produce an ocular hypotensive response, suggesting that microtubules may be involved in the formation of aqueous humour and possibly the maintainance of cell shape and form in the outflow vessels of the anterior chamber.     

Morphological changes in glaucomatous eyes and the role of TGFbeta2 for the pathogenesis of the disease

This review summarizes the Ernst H. Bárány Prize Lecture given at the XVI. meeting 2004 of the International Society of Eye Research in Sydney, Australia. The article describes the author's early studies starting with the determination of the site of aqueous humour outflow resistance and its regulation through ciliary muscle contraction, which were performed in collaborations with Bárány. It continues with the qualitative and quantitative evaluation of the trabecular meshwork (TM) changes seen in different kinds of glaucoma diseases. A comparison of correlations between meshwork pathology, IOP, and axon loss in the optic nerve between eyes with pseudoexfoliation glaucoma (PEXG) and primary open angle glaucoma (POAG) indicates that in the secondary glaucoma (PEXG) optic neuropathy is mainly induced by an increase in IOP. In eyes with POAG, the correlations point towards a more complex pathogenesis of the disease. Common factors might be involved in both the TM and the optic nerve changes. In vitro studies performed in cell cultures of human TM cells and optic nerve astrocytes as well as organ culture studies of the anterior eye segment indicate that TGFbeta2 might be one of the factors involved in the development of POAG. The paper is primarily focused on studies performed by the author and complete reference to other previous or contemporary studies is therefore not given as the purpose is not to present a comprehensive review article.     

Effects of antiglaucoma drugs timolol and GLC756, a novel dopamine D2 agonist and D1 antagonist, on endotoxin-induced-uveitis in rats

Antiglaucoma drugs with anti-inflammatory properties may be of particular value for the long-term treatment of glaucoma since they may reduce the risk for treatment-related inflammatory processes in outer compartments of the eye. The purpose of this study was, to evaluate the effect of systemic and topical administration of GLC756, a novel mixed dopamine D(2) receptor agonist and D(1) receptor antagonist which lowered intraocular pressure in man, and timolol on endotoxin-induced-uveitis (EIU) in rats. For EIU, 8-week-old Lewis rats received an intravenous injection of 160 microg lipopolysaccharide (LPS) from Salmonella typhimurium. GLC756, timolol, or betamethasone, as positive control, were administered either topically (0.4, 0.5, and 0.1%, respectively, 16-times 20 microl eye drops during 48 hr) or systemically (1 mg kg(-1) subcutaneously for 5 days). Protein content, released TNF-alpha, the number of cells as well as cells expressing TNF-alpha were determined in aqueous humor 48 hr after LPS-injection and served as parameters for inflammation. LPS induced an increase of protein content, infiltrating cells and cells expressing TNF-alpha in the aqueous humor. Topical and systemic administration of GLC756 and betamethasone, almost completely suppressed the increase of protein content and betamethasone in addition also suppressed the number of cells in aqueous humor. In conclusion, the almost complete suppression of LPS-induced protein increase in aqueous humor by GLC756 suggests an additional anti-inflammatory potential of dopaminergic compounds in glaucoma treatment. Timolol did not show any effect on EIU in rats.     

Effects of prostaglandin F2alpha, latanoprost and carbachol on phosphoinositide turnover, MAP kinases, myosin light chain phosphorylation and contraction and functional existence and expression of FP receptors in bovine iris sphincter

A potential role for myosin light chain kinase (MLCK) in regulating intraocular pressure and outflow function has recently been reported in living monkey eye and rabbit eye. There is little information about the effects of the ocular hypotensive agents, prostaglandin F2alpha (PGF2alpha) and latanoprost on this signaling pathway in ocular tissues. The aim of this study was to determine the agonist activity of PGF2alpha, latanoprost and carbachol (CCh) on the MLCK pathway in isolated bovine iris sphincter and furthermore to investigate the existence of the FP receptor in this tissue. In the present studies on the MLCK pathway four signal transduction mechanism assays were employed, phosphoinositide (PI) turnover, p42/p44 MAP kinase phosphorylation and activation, MLC phosphorylation and contraction. In the studies on the existence of the FP receptor in the bovine iris sphincter, the pharmacology and expression of the FP receptor protein, using a polyclonal anti-FP-receptor antibody and Western blot analysis, were determined. The data obtained on the MLCK pathway showed that the three agonists stimulated the biochemical and pharmacological responses in a concentration and time-dependent manner and that the order of potency and efficacy is PGF2alpha>latanoprost>CCh. The EC50 values in the PI turnover, MAP kinase phosphorylation, MLC phosphorylation and contraction assays were for PGF2alpha: 9, 42, 200 and 140 nM, respectively, for latanoprost: 13, 59, 250 and 828 nM, respectively, and for CCh: 22, 200, 630 and 910 nM, respectively. Wortmannin, a selective inhibitor of MLCK, dose-dependently inhibited MLC phosphorylation and contraction induced by PGF2alpha, demonstrating a close relationship between activation of the MLCK pathway and contraction. The pharmacological studies showed that in the concentration range of 1 nM to 10 microM, the FP-receptor agonists caused concentration-response curves with the following order of potencies: 17-phenyl trinor PGF2alpha (bimatoprost acid)>PGF2alpha>cloprostenol>latanoprost>latanoprost acid>bimatoprost amide>fluprostenol. Immunoblot analysis of the FP receptor demonstrated expression of the prostaglandin FP receptor protein in this smooth muscle. These results clearly indicate that the MLCK signaling pathway is involved in the FP-receptor function of the bovine iris sphincter and furthermore demonstrate that functional FP receptors exist and are expressed in this tissue.