November 2006
Volume 47, Issue 11
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Glaucoma  |   November 2006
Prostaglandin F2α, but Not Latanoprost, Increases the Ca2+ Sensitivity of the Pig Iris Sphincter Muscle
Author Affiliations
  • Yuhei Hasegawa
    From the Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, the
    Department of Ophthalmology, Graduate School of Medical Sciences, and the
  • Junji Nishimura
    From the Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, the
  • Naohisa Niiro
    From the Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, the
  • Katsuya Hirano
    From the Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, the
  • Tatsuro Ishibashi
    Department of Ophthalmology, Graduate School of Medical Sciences, and the
  • Hideo Kanaide
    From the Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, the
    Kyushu University COE Program on Lifestyle-Related Diseases, Kyushu University, Fukuoka, Japan.
Investigative Ophthalmology & Visual Science November 2006, Vol.47, 4865-4871. doi:10.1167/iovs.05-1518
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      Yuhei Hasegawa, Junji Nishimura, Naohisa Niiro, Katsuya Hirano, Tatsuro Ishibashi, Hideo Kanaide; Prostaglandin F2α, but Not Latanoprost, Increases the Ca2+ Sensitivity of the Pig Iris Sphincter Muscle. Invest. Ophthalmol. Vis. Sci. 2006;47(11):4865-4871. doi: 10.1167/iovs.05-1518.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To determine the mechanisms underlying prostaglandin (PG) F2α-, carbachol (CCh)-, or latanoprost (a PGF2α analogue)-induced contraction of the pig iris sphincter muscle.

methods. Effects of these agents on myofilament Ca2+ sensitivity were evaluated and compared with the use of receptor-coupled permeabilized preparations by α-toxin. The effects of PGF2α and CCh on the phosphorylation of myosin light chain (MLC) were also analyzed.

results. In the intact strips, all three of these agents induced contractions. In permeabilized strips, PGF2α and CCh, but not latanoprost, caused an additional tension development at a fixed intracellular Ca2+ concentration ([Ca2+]i) and also shifted the [Ca2+]i-tension curve to the left, thus indicating that PGF2α and CCh, but not latanoprost, induced increases in Ca2+ sensitivity (Ca2+ sensitization). This Ca2+ sensitization could have been inhibited by Y27632, a rho kinase inhibitor, but not by GF109203X, a protein kinase C (PKC) inhibitor or by PD98059, a mitogen-activated protein (MAP) kinase inhibitor. PGF2α increased the level of MLC phosphorylation at a constant [Ca2+]i.

conclusions. PGF2α, but not latanoprost, induced Ca2+ sensitization of the pig iris sphincter muscle in an MLC phosphorylation–dependent manner through the rho–rho kinase pathway. The effect of latanoprost on the Ca2+ sensitization mechanism was different from that of PGF2α and was thought to play a beneficial role in glaucoma treatment.

Prostaglandins (PGs) are derived from arachidonic acid and display a wide range of biologic functions. 1 2 3 4 Although there are enormous species differences regarding their physiological effects, in human ocular tissue, PGF2α, PGE2, 5 and thromboxane B2 5 6 have been shown to be involved in the pathogenesis of the neurogenic inflammation of the eye, which is characterized by noncholinergic long-lasting miosis, anterior uveal hyperemia, and increased intraocular pressure (IOP) because of a breakdown of the blood–aqueous barrier. 7 A mechanistically similar phenomenon called surgically induced miosis sometimes occurs during surgical procedures of the eye, in spite of the use of the sympathomimetic and anticholinergic agents. 8 The trauma of ocular surgery may stimulate prostaglandin synthesis to induce papillary constriction, which thus makes the operation more difficult and increases the risk for surgical trauma. Although nonsteroidal anti-inflammatory drugs have been used to block the production of prostaglandins by inhibiting cyclo-oxygenase in addition to making use of sympathomimetic and anticholinergic agents to prevent surgically induced miosis, the mechanisms underlying such PGF2α–induced contraction of the iris sphincter muscle are still poorly understood. 
Although PGF2α at high doses had been thought to increase IOP, a PGF2α analogue, latanoprost (13,14-dihydro-17-phenyl-18,19,20-trinor- PGF2α-isopropyl ester), has been widely used for the treatment of glaucoma as an ocular hypotensive agent. Thus, it has been suggested that latanoprost might increase the uveoscleral outflow of aqueous humor. However, the mechanism for this is still not well understood. Although Poyer et al. 9 and Yousufzai et al. 10 have suggested the PGF2α-mediated increase in aqueous humor outflow is accompanied by relaxation of the ciliary muscle, Yoshitomi et al. 11 reported that neither PGF2α nor latanoprost had a relaxant effect on this muscle. Weinreb et al., 12 however, reported that these agents could induce a reduction of the extracellular matrix within the ciliary muscle by increasing the release of matrix metalloproteinases. In addition, PGF2α is well known to induce miosis in pigs, bovines, and rabbits, but this effect is not as apparent as in humans. 13 14 Even if PGF2α-induced miosis is not a major mechanism for its antiglaucoma effect, it is still important to understand the effect of PGF2α and its analogue, latanoprost, on the contractility of the iris sphincter muscle. Therefore, we considered that it would be of great interest to know the mechanism for the PGF2α- and latanoprost-induced contraction of the iris sphincter muscle. 
It is generally accepted that smooth muscle contraction is primarily regulated by an increase in the intracellular Ca2+ concentration ([Ca2+]i) and by subsequent phosphorylation of 20 kDa myosin light chain (MLC) by Ca2+-calmodulin–dependent MLC kinase (MLCK). 15 It is also known that Ca2+ sensitivity of the contractile apparatus changes during stimulation by various agonists, indicating that secondary regulatory pathways are likely to play a role in the regulation of smooth muscle contraction. 16 17 18 19 However, the role of Ca2+ sensitivity in the contraction of the iris smooth muscle has not yet been elucidated. 
In the present study, we investigated the mechanisms underlying PGF2α-, latanoprost-, and carbachol (CCh)-induced contraction of the pig iris sphincter muscle. In particular, we investigated whether these agents affect Ca2+ sensitivity on the contractile apparatus in the α-toxin–permeabilized pig iris sphincter muscle strips. We also investigated MLC phosphorylation in the α-toxin–permeabilized pig iris sphincter muscle during activation by PGF2α at a fixed [Ca2+]i. We obtained evidence that latanoprost has a different effect on the Ca2+ sensitivity from PGF2α and would thus play a beneficial role in glaucoma treatment. 
Materials and Methods
Tissue Preparation
All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Porcine eyes were enucleated at a local slaughterhouse after the animals had been killed. After the cornea was removed, iris sphincter muscle preparations were cut, under a binocular microscope, into strips measuring approximately 1.0 mm in width and 2.5 mm length. The strips were placed in normal physiological saline solution (PSS) consisting of the following: NaCl, 123 mM; KCl, 4.7 mM; CaCl2, 1.25 mM; MgCl2, 1.2 mM; H2PO4, 1.2 mM; NaHCO3, 15.5 mM; d-glucose, 11.5 mM, 95% O2, and 5% CO2
Tension Measurement
The strips were mounted between two tungsten wires, one fixed and the other attached to a force transducer (UL2; Minebea Co., Japan). Tension experiments were performed at room temperature. The developed tension was expressed as a percentage, assigning the 118 mM K+-induced contraction to be 100%. 
Permeabilization with α-Toxin
Iris sphincter muscle strips were permeabilized by α-toxin according to the methods described by Nishimura et al. 16 19 Tension measurements of permeabilized tissues were taken at room temperature. The developed tension was expressed as a percentage, and values in the relaxing solution ([Ca2+]i < 10 nM) and in the activating solution ([Ca2+]i = 10 μM; maximal tension) were assigned as 0% and 100%, respectively. 
Immunoblot
Strips from the pig iris sphincter muscle and coronary artery were subjected to immunoblot analysis, as previously described. 20 Regarding the tissue samples, freshly dissected iris sphincter muscle and coronary artery strips were rapidly frozen and then were shattered by hammering. Ten micrograms total protein was separated with SDS-PAGE and then was transferred to the polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). Membranes were incubated with anti–CPI-17 polyclonal antibody 21 or anti–MYPT1 monoclonal antibody. 22 Antigen detection was performed with a chemiluminescent substrate (SuperSignal West Pico [Pierce, Rockford, IL] and ChemicDoc XRS-J [Bio-Rad]). 
Measurement of MLC Phosphorylation
After incubation in 180 nM Ca2+ solution for 5 minutes (0 second), iris sphincter muscle strips were exposed to 10 μM PGF2α (10, 30, 60, 120 seconds) or to 10 μM CCh (10, 20, 30, 60 seconds). At the indicated times, the phosphorylation of MLC was determined using urea–glycerol gel electrophoresis. 23 24  
Drugs and Solutions
The composition of the normal physiological saline solution (PSS) and the permeabilized preparations have been described. PSS (118 mM K+) was made by an equimolar substitution of KCl for NaCl. The following were obtained from commercial manufacturers: PGF2α, carbachol, α-toxin, U46619, phorbol 12,13 dibutyrate (PDBu), and thapsigargin (Sigma Chemical, St. Louis, MO); latanoprost and PD98059 (2′-amino-3′-methoxyflavone; Cayman Chemical, Ann Arbor, MI); guanosine 5′-triphosphate (GTP), Y27632 ((R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecalboxamide dihydrochloride, monohydrate), and GF109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (Calbiochem, La Jolla, CA). 
Statistical Analysis
All data were expressed as the mean ± SEM along with the number of observations (n). One strip obtained from one animal was used for each experiment; therefore, the number of experiments (n value) also indicated the number of animals. Student t test was used to determine any statistical differences between the two mean values. P < 0.05 was considered significant. The four-parameter logistic model was used to fit the sigmoidal curve to the concentration response of each drug. 24 All data were collected using a computerized data acquisition system (MacLab [Analogue Digital Instruments, Australia]; Macintosh [Apple Computer, Cupertino, CA]). 
Results
Effect of PGF2α, CCh, and Latanoprost on the Tension Development of the Intact Iris Sphincter Muscle in Normal PSS
The application of 10 μM PGF2α induced a sustained increase in tension in normal PSS (32.6% ± 6.2%; n = 8; Fig. 1A ). On the other hand, 10 μM latanoprost induced only a small contraction (9.60% ± 3.6%; n = 8; Fig. 1B ). The addition of 10 μM CCh induced the development of tension significantly greater than that induced by PGF2α or latanoprost (257% ± 22%; n = 8; P < 0.01). Figure 1Dsummarizes the data obtained by the cumulative application of various concentrations of PGF2α, latanoprost, and CCh (10 pM∼30 μM). EC50 values were 33.7 ± 2.36 μM for PGF2α (n = 6), 53.1 ± 2.3 μM for latanoprost (n = 6), and 429 ± 78 nM for CCh (n = 6). The tension induced by the cumulative application of each drug tended to be greater than that obtained by the single application of the same concentration. 
Effect of PGF2α, CCh, and Latanoprost on the Tension Development of the α-Toxin–Permeabilized Iris Sphincter Muscle
Figures 2A 2B and 2Cshow the representative recordings of the effect of 10 μM PGF2α, 10 μM latanoprost, and 1 μM CCh, respectively, on the tension development induced by 500 nM Ca2+ and 10 μM GTP in the α-toxin–permeabilized pig iris sphincter muscle. The application of PGF2α or CCh during the steady state contraction evoked by the mixture of 500 nM Ca2+ and 10 μM GTP induced an additional tension development at a constant [Ca2+]i. In contrast, the application of latanoprost did not induce any tension development (–2.88 ± 2.1%; n = 4) at the concentration of 100 μM, which induced a comparable contraction to PGF2α in the intact strips (Fig. 2C) . In Figures 2A and 2B , the additional tension development was composed of two phases, the initial transient phase and the sustained phase. To rule out the possibility that the intracellular Ca2+ release from the sarcoplasmic reticulum (SR) might have contributed to this biphasic nature, we treated the strips with 10 μM thapsigargin, an inhibitor of SR ATPase (Ca2+ pump), for 10 minutes before and during the protocol. As shown in Figure 2D , thapsigargin had no effect on the tension development induced by PGF2α at a constant [Ca2+]i, thus indicating that the intracellular Ca2+ release may not be responsible for the biphasic tension development induced by PGF2α. Thapsigargin had no effect in either case of CCh stimulation (data not shown). Both PGF2α and CCh induced significant increases in tension at a fixed [Ca2+]i during the initial transient and the sustained phases. However, the CCh-induced initial transient phase (77.0% ± 5.7%; n = 8) was significantly (P < 0.05) greater than the PGF2α-induced phase (39.8% ± 2.9%; n = 14), whereas no significant difference was found in the sustained phase (25.3% ± 2.8%; n = 14 [PGF2α] and 24.8% ± 4.5%; n = 8 [CCh]). Thapsigargin had no significant effect on the tension development induced by PGF2α or CCh in either phase (PGF2α: peak, 36.2% ± 8.4%; sustained, 22.9% ± 3.5% [n = 4]; CCh: peak, 71.6% ± 8.3%; sustained, 20.8% ± 4.1% [n = 4]). In addition, PGE2, U46619 (a thromboxane A2 analogue), and thromboxane B2 also induced further tension development at a constant [Ca2+]i (traces not shown). 
Effect of PGF2α and CCh on the Relationship between [Ca2+]i and Tension in the α-Toxin–Permeabilized Preparation
Figure 3shows a representative plot of 12 independent experiments of 10 μM PGF2α or 10 μM CCh on the tension development induced by the cumulative application of the increasing function of [Ca2+]i. The Ca2+–tension relationship in the presence of 10 μM PGF2α or 10 μM CCh shifted to the left. Under control conditions, EC50 was 2.66 ± 0.04 μM, which was significantly (P < 0.05) greater than that in the presence of 10 μM PGF2α (1.70 ± 0.09 μM) or 10 μM CCh (1.03 ± 0.04 μM) by the paired t test (n = 12). 
Effects of the Selected Kinase Inhibitors on the Increase in Ca2+ Sensitivity Induced by PGF2α or CCh in the α-Toxin–Permeabilized Preparation
As shown in Figures 4A and 4B , 1 μM Y27632 inhibited 10 μM PGF2α– and 1 μM CCh–induced tension by 22.5% ± 6.0% (n = 4) and 22.7% ± 4.9% (n = 4), respectively. However, as shown in Figures 4C 4D 4E 4F , neither GF109203X (10 μM), a protein kinase C (PKC) inhibitor, nor PD98059 (10 μM), a mitogen-activated protein (MAP) kinase inhibitor, had an effect (PGF2α-sustained phase, 16.4% ± 4.1%; GF109203X, 17.6% ± 5.9% [n = 4]; PD98059, 16.2% ± 5.1% [n = 4]; CCh-sustained phase, 14.9% ± 6.0%; GF109203X, 14.2% ± 2.5% [n = 4]; PD98059, 12.6% ± 2.4% [n = 4]). We next examined the effect of Y27632 on the tension induced by 500 nM [Ca2+]i with 10 μM GTP. We chose a concentration of [Ca2+]i, which produces a tension comparable to that induced by 300 nM Ca2+ plus 10 μM PGF2α. As shown in Figures 5A 5B 5C 5D , 1 μM Y27632 significantly inhibited PGF2α- and Ca2+-induced contractions by 23.3% ± 4.1% for PGF2α and 36.2% ± 8.7% for 500 nM [Ca2+]i, respectively (P < 0.01). 
Lack of CPI-17 Expression in the Pig Iris Sphincter Muscle
Given that a protein kinase C (PKC) inhibitor had no effect on the PGF2α- induced increase in the Ca2+ sensitivity of the pig iris sphincter muscle, we next examined the effect of an activator of PKC (PDBu). PDBu did not induce any further tension development in the intact and permeabilized pig iris sphincter muscle (n = 4; Fig. 6A ). We thus investigated the expression of PKC-potentiated protein phosphatase-1 inhibitory protein (CPI-17) and an isoform of a large subunit of MLC phosphatase (MYPT1). As shown in Figure 6B , CPI-17 expression was under detectable levels in the pig iris sphincter muscle (n = 4), whereas MYPT-1 expression was observed to be similar to that of the coronary smooth muscle (114% ± 5.6% of coronary artery; n = 4). 
Effect of PGF2α and CCh on the Level of MLC Phosphorylation in the α-Toxin–Permeabilized Preparation
To determine whether the PGF2α- or CCh- induced increase in the Ca2+ sensitivity of pig iris sphincter muscle was accompanied by an increase in MLC phosphorylation, a protocol similar to that shown in Figure 2was used and the level of MLC phosphorylation was measured at 180 nM [Ca2+]i. As shown in Figure 7 , the tension development induced by PGF2α and CCh at steady state ([Ca2+]i = 180 nM) was accompanied by an increase in MLC phosphorylation. 
Discussion
In the present study, the mechanisms underlying PGF2α-induced contraction of the pig iris sphincter muscle were investigated, particularly in comparison with the contraction induced by latanoprost or CCh, a major constrictor agent of the iris sphincter muscle. The major findings are as follows: (1) In the intact strips, latanoprost was the least potent of the three agents for inducing contraction. (2) In the permeabilized preparation, PGF2α and CCh, but not latanoprost, caused Ca2+ sensitization. (3) The PGF2α-induced enhancement of contraction at a fixed [Ca2+]i was blocked only by a rho kinase inhibitor (Y27632) but not by a PKC inhibitor or a MAP kinase inhibitor. (4) Y27632 also inhibited Ca2+-induced contraction. (5) Although MYPT1 was expressed, CPI-17 was not expressed in this tissue. (6) The additional tension development induced at a fixed [Ca2+]i by PGF2α or CCh was accompanied by an increase in MLC phosphorylation. These results indicated that PGF2α and CCh induced Ca2+ sensitization of the pig iris sphincter muscle in an MLC phosphorylation–dependent manner through the rho–rho kinase pathway, whereas latanoprost—at the concentration that induced a contraction comparable to that of PGF2α in the intact strips—had no effect on Ca2+ sensitivity. This is the first report describing the PGF2α- or CCh-induced increase in Ca2+ sensitivity of the myofilament and its underlying mechanism in the α-toxin–permeabilized pig iris sphincter muscle. 
It is now generally accepted that although smooth muscle contraction is primarily regulated by [Ca2+]i, 15 the modulation of Ca2+ sensitivity also plays an important role. 15 16 17 18 The mechanism for such an increased Ca2+ sensitivity (Ca2+ sensitization) is still unclear. However, this mechanism can be classified into two distinct mechanisms. One is increased Ca2+ sensitivity with increased MLC phosphorylation (MLC phosphorylation–dependent Ca2+ sensitization), 18 and the other is without increased MLC phosphorylation (MLC phosphorylation–independent Ca2+ sensitization). 25 26 27 The present study clearly demonstrated that the PGF2α- or CCh-induced contraction of the pig iris sphincter muscle involves the MLC phosphorylation-dependent Ca2+ sensitization because PGF2α and CCh induced additional tension development with increased MLC phosphorylation at a constant [Ca2+]i. In addition, the tension levels induced by CCh were greater than those induced by PGF2α in the intact strips, but, in the permeabilized preparation, the sustained levels of PGF2α-induced contraction were similar to those of the CCh-induced contraction. This observation indicated that PGF2α-induced contraction is more dependent on the Ca2+ sensitization than on the increase in [Ca2+]i compared with CCh-induced contraction. In addition, we showed that thromboxane B2 increased Ca2+ sensitivity in the iris sphincter muscle, which has not yet been previously described. We consider that it may thus be one of the possible causes of uncontrollable miosis, including surgically induced miosis. 
The signal transduction pathway for MLC phosphorylation–dependent Ca2+ sensitization has recently been elucidated. The small guanosine triphosphatase rho is implicated in this type of Ca2+ sensitization of smooth muscle contraction. The GTP-bound active form of rhoA activates a downstream kinase, rho kinase, which phosphorylates the myosin-binding subunit (MBS) of myosin phosphatase to inhibit its activity. 28 Another possible pathway for MLC phosphorylation–dependent Ca2+ sensitization involves PKC and CPI-17. This novel protein has been reported to inhibit MLC phosphatase when phosphorylated by PKC. 29 In addition, MAP kinase has been implicated in iris sphincter muscle contraction. 30 31 Based on these considerations, we examined the effect of rho kinase inhibitor (Y27632), PKC inhibitor (GF109203X), and MAP kinase inhibitor (PD98059). We showed that the PGF2α-induced Ca2+ sensitization of the contractile apparatus in the α-toxin–permeabilized iris sphincter muscle, which is likely to depend on MLC phosphorylation, was inhibited by Y27632, but not by GF109203X or PD98059. These results indicated that the pathway of the PGF2α- or CCh-induced increase in Ca2+ sensitivity involves the rho–rho kinase system but not the PKC-CPI-17 pathway. The facts that PDBu had no effect on tension development and that CPI-17 did not exist in the pig iris sphincter muscle supported this hypothesis. In addition, Y27632 also depressed the Ca2+-induced contraction of the pig iris sphincter muscle, thus indicating that the rho–rho kinase pathway may be involved in Ca2+-calmodulin–dependent contraction. In close agreement with this result, Sakurada et al. 32 recently reported that membrane depolarization by 60 mM KCl and noradrenalin stimulation induced a similarly sustained contraction in the rabbit aorta, whereas both stimuli induced similar time-dependent, sustained increases in the amount of an active GTP-bound form of rhoA. 
Concerning the regulation of IOP, the rho kinase inhibitor has been reported to reduce IOP through an effect on the actin cytoskeleton by interfering with the actomyosin system. 33 34 Similarly, MLCK inhibitors have been reported to increase the outflow facility so that they might reduce IOP through the inhibition of MLC phosphorylation. 35 36 37 It can thus be speculated that these agents, which are coupled with the rho–rho kinase or the Ca2+-MLCK pathway, or both, may increase IOP. Latanoprost, a PGF2α analogue, has been widely used for the treatment of glaucoma, whereas PGF2α is thought to increase IOP. This discrepancy can thus be clearly explained by the present results, which show the rho–rho kinase pathway to be involved in the PGF2α-mediated pathway but not in the latanoprost-mediated pathway. In other words, PGF2α might have a bidirectional effect on IOP, increasing IOP by increasing the effect on MLC phosphorylation and decreasing IOP by increasing the uveoscleral outflow of aqueous humor. In the case of latanoprost, the former action was weaker than PGF2α and therefore was considered to play a beneficial role in the treatment of glaucoma. In the present study, we chose esterified latanoprost, which has already been clinically used. However, it should be noted that free-acid latanoprost might have a greater effect because it is reported to be 100 times more potent than the esterified latanoprost in a rabbit uterus preparation. 38 39  
In summary, PGF2α, but not latanoprost, was found to induce an increase in the Ca2+ sensitivity of the contractile apparatus in an MLC phosphorylation–dependent manner through the activation of the rho–rho kinase signaling pathway. The lack of any effect on Ca2+ sensitivity by latanoprost is thus considered to be beneficial in the treatment of glaucoma. 
 
Figure 1.
 
Effect of PGF2α, latanoprost, and CCh on the tension development of the intact pig iris sphincter muscle. (AC) Representative traces of the contraction induced by 10 μM PGF2α (A), latanoprost (B), and CCh (C). (D) Summary of the effect of the cumulative application of PGF2α (▪), latanoprost, (▴), and CCh (•) obtained from six to eight independent experiments. Vertical bars represent SEM. Contractions induced by each drug were plotted by assigning the 118 mM K+-induced contraction a value of 100%.
Figure 1.
 
Effect of PGF2α, latanoprost, and CCh on the tension development of the intact pig iris sphincter muscle. (AC) Representative traces of the contraction induced by 10 μM PGF2α (A), latanoprost (B), and CCh (C). (D) Summary of the effect of the cumulative application of PGF2α (▪), latanoprost, (▴), and CCh (•) obtained from six to eight independent experiments. Vertical bars represent SEM. Contractions induced by each drug were plotted by assigning the 118 mM K+-induced contraction a value of 100%.
Figure 2.
 
Effects of PGF2α (A), CCh (B), and latanoprost (C) on the Ca2+-induced contractions of the α-toxin–permeabilized iris sphincter muscle strips. (D) Effect of 10 μM thapsigargin, an inhibitor of SR ATPase (Ca2+ pump), on the PGF2α-induced contraction. The developed tension was expressed as a percentage, assigning the values in the relaxing solution ([Ca2+]i < 10 nM) and in the activating solution ([Ca2+]i = 10 μM; maximal tension) to be 0% and 100%, respectively. Ten micromolar GTP was added during all protocols.
Figure 2.
 
Effects of PGF2α (A), CCh (B), and latanoprost (C) on the Ca2+-induced contractions of the α-toxin–permeabilized iris sphincter muscle strips. (D) Effect of 10 μM thapsigargin, an inhibitor of SR ATPase (Ca2+ pump), on the PGF2α-induced contraction. The developed tension was expressed as a percentage, assigning the values in the relaxing solution ([Ca2+]i < 10 nM) and in the activating solution ([Ca2+]i = 10 μM; maximal tension) to be 0% and 100%, respectively. Ten micromolar GTP was added during all protocols.
Figure 3.
 
Effect of PGF2α and CCh on the Ca2+ sensitivity of the myofilament. A representative plot obtained from three strips of one pig. Ca2+ tension–relationship curves were constructed by the cumulative application of the increasing function of Ca2+ solution in the absence (control, ▴) or presence of 10 μM PGF2α (▪) or 10 μM CCh (•). Ten micromolar GTP was added during all protocols.
Figure 3.
 
Effect of PGF2α and CCh on the Ca2+ sensitivity of the myofilament. A representative plot obtained from three strips of one pig. Ca2+ tension–relationship curves were constructed by the cumulative application of the increasing function of Ca2+ solution in the absence (control, ▴) or presence of 10 μM PGF2α (▪) or 10 μM CCh (•). Ten micromolar GTP was added during all protocols.
Figure 4.
 
Effect of 1 μM Y27632 (A, B), a rho kinase inhibitor, 10 μM GF109203X (C, D), a PKC inhibitor, and 10 μM PD98059 (E, F), a MAP kinase inhibitor on the PGF2α- or CCh-induced contraction at a constant [Ca2+]i.
Figure 4.
 
Effect of 1 μM Y27632 (A, B), a rho kinase inhibitor, 10 μM GF109203X (C, D), a PKC inhibitor, and 10 μM PD98059 (E, F), a MAP kinase inhibitor on the PGF2α- or CCh-induced contraction at a constant [Ca2+]i.
Figure 5.
 
Effect of 1 μM Y27632 on the contractions induced by 500 nM Ca2+ alone (A) or 300 nM Ca2+ plus 10 μM PGF2α (B) in the presence of 10 μM GTP. (C, D) Summary of the results obtained from four independent experiments (C, 500 nM Ca2+ alone; D, 300 nM Ca2+ plus 10 μM PGF2α). Tension levels induced by 500 nM Ca2+ alone or 300 nM Ca2+ plus PGF2α, those following the application of 1 μM Y27632, and maximal tension levels induced by 10 μM Ca2+ were plotted. *P < 0.01.
Figure 5.
 
Effect of 1 μM Y27632 on the contractions induced by 500 nM Ca2+ alone (A) or 300 nM Ca2+ plus 10 μM PGF2α (B) in the presence of 10 μM GTP. (C, D) Summary of the results obtained from four independent experiments (C, 500 nM Ca2+ alone; D, 300 nM Ca2+ plus 10 μM PGF2α). Tension levels induced by 500 nM Ca2+ alone or 300 nM Ca2+ plus PGF2α, those following the application of 1 μM Y27632, and maximal tension levels induced by 10 μM Ca2+ were plotted. *P < 0.01.
Figure 6.
 
Effect of 10 μM PDBu on the intact iris sphincter muscle and the contraction induced by 500 nM Ca2+ in the presence of 10 μM GTP in the α-toxin–permeabilized strip (A). Expression of CPI-17 (B) and MYPT1 (C) in the pig iris sphincter muscle (ISM) compared with the pig coronary artery (CA).
Figure 6.
 
Effect of 10 μM PDBu on the intact iris sphincter muscle and the contraction induced by 500 nM Ca2+ in the presence of 10 μM GTP in the α-toxin–permeabilized strip (A). Expression of CPI-17 (B) and MYPT1 (C) in the pig iris sphincter muscle (ISM) compared with the pig coronary artery (CA).
Figure 7.
 
Temporal changes in MLC phosphorylation levels induced by 10 μM PGF2α and 1 μM CCh in the α-toxin–permeabilized iris sphincter muscle strip. (A, B) Representative image of Western blotting for MLC phosphorylation and a representative trace that shows the specific protocol. (C, D) Quantitative analysis of temporal changes in the MLC phosphorylation levels obtained from four experiments. Relative MLC phosphorylation levels were shown as ratios of phosphorylated MLC/total MLC (pMLC/tMLC) at each time point.
Figure 7.
 
Temporal changes in MLC phosphorylation levels induced by 10 μM PGF2α and 1 μM CCh in the α-toxin–permeabilized iris sphincter muscle strip. (A, B) Representative image of Western blotting for MLC phosphorylation and a representative trace that shows the specific protocol. (C, D) Quantitative analysis of temporal changes in the MLC phosphorylation levels obtained from four experiments. Relative MLC phosphorylation levels were shown as ratios of phosphorylated MLC/total MLC (pMLC/tMLC) at each time point.
The authors thank Brian Quinn for linguistic comments and help with the manuscript. 
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Figure 1.
 
Effect of PGF2α, latanoprost, and CCh on the tension development of the intact pig iris sphincter muscle. (AC) Representative traces of the contraction induced by 10 μM PGF2α (A), latanoprost (B), and CCh (C). (D) Summary of the effect of the cumulative application of PGF2α (▪), latanoprost, (▴), and CCh (•) obtained from six to eight independent experiments. Vertical bars represent SEM. Contractions induced by each drug were plotted by assigning the 118 mM K+-induced contraction a value of 100%.
Figure 1.
 
Effect of PGF2α, latanoprost, and CCh on the tension development of the intact pig iris sphincter muscle. (AC) Representative traces of the contraction induced by 10 μM PGF2α (A), latanoprost (B), and CCh (C). (D) Summary of the effect of the cumulative application of PGF2α (▪), latanoprost, (▴), and CCh (•) obtained from six to eight independent experiments. Vertical bars represent SEM. Contractions induced by each drug were plotted by assigning the 118 mM K+-induced contraction a value of 100%.
Figure 2.
 
Effects of PGF2α (A), CCh (B), and latanoprost (C) on the Ca2+-induced contractions of the α-toxin–permeabilized iris sphincter muscle strips. (D) Effect of 10 μM thapsigargin, an inhibitor of SR ATPase (Ca2+ pump), on the PGF2α-induced contraction. The developed tension was expressed as a percentage, assigning the values in the relaxing solution ([Ca2+]i < 10 nM) and in the activating solution ([Ca2+]i = 10 μM; maximal tension) to be 0% and 100%, respectively. Ten micromolar GTP was added during all protocols.
Figure 2.
 
Effects of PGF2α (A), CCh (B), and latanoprost (C) on the Ca2+-induced contractions of the α-toxin–permeabilized iris sphincter muscle strips. (D) Effect of 10 μM thapsigargin, an inhibitor of SR ATPase (Ca2+ pump), on the PGF2α-induced contraction. The developed tension was expressed as a percentage, assigning the values in the relaxing solution ([Ca2+]i < 10 nM) and in the activating solution ([Ca2+]i = 10 μM; maximal tension) to be 0% and 100%, respectively. Ten micromolar GTP was added during all protocols.
Figure 3.
 
Effect of PGF2α and CCh on the Ca2+ sensitivity of the myofilament. A representative plot obtained from three strips of one pig. Ca2+ tension–relationship curves were constructed by the cumulative application of the increasing function of Ca2+ solution in the absence (control, ▴) or presence of 10 μM PGF2α (▪) or 10 μM CCh (•). Ten micromolar GTP was added during all protocols.
Figure 3.
 
Effect of PGF2α and CCh on the Ca2+ sensitivity of the myofilament. A representative plot obtained from three strips of one pig. Ca2+ tension–relationship curves were constructed by the cumulative application of the increasing function of Ca2+ solution in the absence (control, ▴) or presence of 10 μM PGF2α (▪) or 10 μM CCh (•). Ten micromolar GTP was added during all protocols.
Figure 4.
 
Effect of 1 μM Y27632 (A, B), a rho kinase inhibitor, 10 μM GF109203X (C, D), a PKC inhibitor, and 10 μM PD98059 (E, F), a MAP kinase inhibitor on the PGF2α- or CCh-induced contraction at a constant [Ca2+]i.
Figure 4.
 
Effect of 1 μM Y27632 (A, B), a rho kinase inhibitor, 10 μM GF109203X (C, D), a PKC inhibitor, and 10 μM PD98059 (E, F), a MAP kinase inhibitor on the PGF2α- or CCh-induced contraction at a constant [Ca2+]i.
Figure 5.
 
Effect of 1 μM Y27632 on the contractions induced by 500 nM Ca2+ alone (A) or 300 nM Ca2+ plus 10 μM PGF2α (B) in the presence of 10 μM GTP. (C, D) Summary of the results obtained from four independent experiments (C, 500 nM Ca2+ alone; D, 300 nM Ca2+ plus 10 μM PGF2α). Tension levels induced by 500 nM Ca2+ alone or 300 nM Ca2+ plus PGF2α, those following the application of 1 μM Y27632, and maximal tension levels induced by 10 μM Ca2+ were plotted. *P < 0.01.
Figure 5.
 
Effect of 1 μM Y27632 on the contractions induced by 500 nM Ca2+ alone (A) or 300 nM Ca2+ plus 10 μM PGF2α (B) in the presence of 10 μM GTP. (C, D) Summary of the results obtained from four independent experiments (C, 500 nM Ca2+ alone; D, 300 nM Ca2+ plus 10 μM PGF2α). Tension levels induced by 500 nM Ca2+ alone or 300 nM Ca2+ plus PGF2α, those following the application of 1 μM Y27632, and maximal tension levels induced by 10 μM Ca2+ were plotted. *P < 0.01.
Figure 6.
 
Effect of 10 μM PDBu on the intact iris sphincter muscle and the contraction induced by 500 nM Ca2+ in the presence of 10 μM GTP in the α-toxin–permeabilized strip (A). Expression of CPI-17 (B) and MYPT1 (C) in the pig iris sphincter muscle (ISM) compared with the pig coronary artery (CA).
Figure 6.
 
Effect of 10 μM PDBu on the intact iris sphincter muscle and the contraction induced by 500 nM Ca2+ in the presence of 10 μM GTP in the α-toxin–permeabilized strip (A). Expression of CPI-17 (B) and MYPT1 (C) in the pig iris sphincter muscle (ISM) compared with the pig coronary artery (CA).
Figure 7.
 
Temporal changes in MLC phosphorylation levels induced by 10 μM PGF2α and 1 μM CCh in the α-toxin–permeabilized iris sphincter muscle strip. (A, B) Representative image of Western blotting for MLC phosphorylation and a representative trace that shows the specific protocol. (C, D) Quantitative analysis of temporal changes in the MLC phosphorylation levels obtained from four experiments. Relative MLC phosphorylation levels were shown as ratios of phosphorylated MLC/total MLC (pMLC/tMLC) at each time point.
Figure 7.
 
Temporal changes in MLC phosphorylation levels induced by 10 μM PGF2α and 1 μM CCh in the α-toxin–permeabilized iris sphincter muscle strip. (A, B) Representative image of Western blotting for MLC phosphorylation and a representative trace that shows the specific protocol. (C, D) Quantitative analysis of temporal changes in the MLC phosphorylation levels obtained from four experiments. Relative MLC phosphorylation levels were shown as ratios of phosphorylated MLC/total MLC (pMLC/tMLC) at each time point.
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