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The inhibition of cyclo-oxygenase (COX) enzymes and the blockade of Ca2+ channels play an important role in the regulation of smooth muscle relaxation. This study was designed to investigate the relaxant effects of celecoxib, DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone), and indomethacin, cyclo-oxygenase (COX-1 and −2) inhibitors, in the absence or presence of a nifedipine, l-type Ca2+ channel blocker, on bovine ciliary muscle.
Ciliary muscle strips (n = 12) were mounted in organ baths and tested for changes in isometric tension in response to celecoxib, DFU, and indomethacin. The relaxant effects of celecoxib, DFU, and indomethacin on carbachol-induced contractions in the presence or absence of nifedipine were investigated.
Celecoxib (10−7–10−4 M), DFU (10−7–10−4 M), indomethacin (10−7–10−4 M), and nifedipine (10−7–10−4 M) inhibited the carbachol-induced contractions in a concentration-dependent manner. The Emax value of indomethacin was significantly higher than the Emax values of celecoxib and DFU in ciliary muscle (P < 0.05), with no significant change in pD2 values (P > 0.05). The relaxation responses by celecoxib, DFU, and indomethacin were significantly increased in the presence of nifedipine (10−6 M). There were no significant differences between pEC50 and values of celecoxib, DFU, and indomethacin in the absence of nifedipine (10−6 M) (P > 0.05), but Emax values were significantly increased (P < 0.05).
These results suggest that the celecoxib, DFU, and indomethacin cause relaxation in ciliary muscle precontracted with carbachol. Blockade of calcium channels with nifedipine in ciliary muscle may increase the relaxant effect of celecoxib, DFU, and indomethacin. The topical or systemic use of celecoxib, DFU, and indomethacin with nifedipine can cause blurred near vision due to ciliary muscle relaxation, and in ocular pain conditions caused by ciliary spasm, the pain can be decreased more easily by combined use of these drugs.
The ciliary muscle is a smooth muscle that affects position of the lens during accommodation and enables the changes in lens shape for light focusing,1 and also, it has been believed that the ciliary muscle plays an important role in regulating aqueous-humor dynamics in the eye. Contraction of the ciliary muscle inhibits the spaces between the fiber bundles and by loosening the trabecular meshwork, respectively. Conversely, relaxation of the ciliary muscle facilitates uveoscleral outflow while it inhibits conventional outflow.2 There are many humoral and neural factors responsible for the relaxation and contraction of the ciliary muscle.3
Prostaglandins (PGs) are autacoids, that is, hormones that are synthesized, released, and act locally and, especially, prostaglandin E2 (PGE2) and prostaglandin F2a (PGF2a) have well-characterized physiologic and pathophysiologic roles in the eye.4–8 They elicit receptor-mediated responses, including vasodilatation, alteration of intraocular pressure (IOP), and the constriction of iris sphincter and ciliary muscles.9,10 A lot of efforts have been directed at identifying the type and distribution of prostaglandin-specific binding sites in ocular tissues.11–14 The principal therapeutic effects of nonsteroidal anti-inflammatory drugs (NSAIDs) derive from their ability to inhibit prostaglandin production. The first enzyme in the prostaglandin synthetic pathway is prostaglandin G/H synthase, also known as cyclo-oxygenase (COX). This enzyme converts arachidonic acid (AA) to the unstable intermediates, PGG2 and PGH2, and leads to the production of thromboxane A2 (TXA2) and a variety of prostaglandins. There are two forms of COX: cyclo-oxygenase-1 (COX-1) and cyclo-oxygenase-2 (COX-2). Most NSAIDs inhibit both COX-1 and COX-2 with little selectivity, while celecoxib and DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)-phenyl-2(5H)-furanone) exhibit selectivity for COX-2, but indomethacin is a nonselective COX inhibitor in vitro.15
The calcium messenger system has a central role in mediating muscle contraction. There are two branches in this system: calmodulin leads to transient effect, whereas protein kinase C leads to a more sustained cellular response. The rise in calcium concentration from intracellular or extracellular sources, or both, leads to the activation of calmodulin-dependent myosin light-chain kinase. The active kinase catalyzes phosphorylation of a myosin light chain, which permits myosin to interact with actin, causing smooth muscle contraction. However, after this increase, the calcium concentration falls back to its basal level, and the content of phosphorylated myosin declines slowly to its resting state, even though the muscle remains contracted.16 During this sustained phase, contraction depends on the efflux of calcium ion, indicating that the calcium ion still has a messenger function, despite that its concentration is no longer elevated.17
In some biologic systems, PGF2α consistently increases smooth muscle cell contractility. In rat aorta smooth muscle cells, PGF2α, at concentrations from 1.4 × 10−8 to 1.4 × 10−5 mol/L, causes a dose-dependent increase of cytosolic free-calcium concentration.18 In hen uterine smooth muscle cells, there is a dose-dependent increase of calcium efflux, with PGF2α concentrations ranging between 10−10 and 10−6 mol/l.19 Both of these effects, an increase in cytosolic free-calcium concentration and increase in calcium efflux, are known to be associated with smooth muscle contraction.16 Hence, these data suggest that PGF2α causes a dose-dependent contraction of smooth muscle cells, not relaxation, as proposed in the ciliary muscle. In the ciliary muscle. the tonic contraction requires a sustained influx of Ca+2 through the cell membrane. But little has been known about the route(s) of Ca+2 influxes in this tissue that lacks voltage-gated Ca+2 channels.20
Nifedipine is a dihydropyridine, which preferentially blocks Ca2+ channels in vascular smooth muscle, and it is used generally in the management of hypertension. While these patients use nifedipine systemically, at the same time, they can also use COX inhibitors topically or systemically with several indications. Therefore, in this study, we aimed to investigate the relaxant effects of COX inhibitors in bovine ciliary muscle and to show changes in this effect in the presence or absence of nifedipine.
Bovine eyes (n = 12) were freshly enucleated after the slaughter of the animal and kept in Krebs–Henseleit solution. The experimental protocol applied has been approved by the Animals Research Ethics Committee of our medical school. Eyes were used within 2 h after enucleation. After removal of the cornea, the ciliary muscle was rapidly excised and transferred to the laboratory in previously aerated (95% O2 and 5% CO2) Krebs' bicarbonate solution (composition in mmol/L: NaCl 118; KCl 4.7; MgCl2 1.2; CaCl2 2.5; NaHCO3 25; KH2PO4 1.2; glucose 11; pH 7.4).
The ciliary muscles were dissected into ciliary muscle strips (approximately 8 × 2 × 2 mm) placed in 10-mL tissue baths, and filled with preaerated Krebs' bicarbonate solution at 37°C. The upper end of the preparation was tied to an isometric transducer (Grass FT 03; Grass Medical Instuments, Quincy, MA) and preloaded with 1 g. The strips in tissue baths were allowed to equilibrate for at least 1 h. The Krebs–Henseleit physiologic salt solution in the tissue baths was changed every 15 min during the equilibration period. After equilibration, concentration-dependent contraction responses of carbachol (10−9–10−5 M) were obtained. Submaximal contraction was obtained with 10−6 M of carbachol in both ciliary muscle strips (Fig. 1). Following contraction by carbachol (10−6 M), the relaxation responses to COX-1-inhibitor indomethacin (10−7–10−4 M) and COX-2 inhibitors celecoxib (10−7–10−4 M) or DFU (10−7–10−4 M) in the presence or absence of l-type Ca2+ channel blocker nifedipine (10−6 M), were obtained in a cumulative manner in ciliary muscle strips. Nifedipine (10−6 M) was added to the organ bath 20 min before the precontraction with carbachol to allow it to take over all of the receptors (10−6 M). After the addition of each drug concentration, we waited until there was a plateau response before adding the next one. Concentration-dependent relaxation responses of celecoxib, DFU, and indomethacin in the presence or absence of nifedipine were obtained in different organ baths. The effect of each drug was examined in 12 muscle strips.
Chemicals used in the current experiments were celecoxib (Celebrex; Pfizer, New York, NY), DFU from Merck Sharp and Dohme (Rahway, NJ), indomethacin from ICN Pharmaceuticals (Costa Mesa), and nifedipine and carbachol from Sigma (St. Louis, MO). All chemicals were dissolved in distilled water, except for DFU, which was dissolved in dimethylsulfoxide. Drugs added to the organ bath never exceeded 1% of the total volume. All drugs were freshly prepared on the day of the experiments.
Carbachol-induced (10−6 M) contractions were considered as the reference response. Relaxation responses were expressed as the percentage of the carbachol-induced contractions. The effects of cumulative concentrations celecoxib, DFU, and indomethacin on carbachol-induced contractions in the presence or absence of nifedipine were measured, and values for −log10 EC50 (pD2) and mean maximal inhibition (Emax) were compared. Maximal inhibitor effects were calculated for each concentration-response curve. The EC50 value of each drug represents 50% of the maximal inhibitor effect. EC50 values were calculated by linear regression of the probit of response versus log10 molar concentration for each of the celecoxib, DFU, and indomethacin. Data were presented as the means ± standard error of the mean and analyzed by repeated measures of analysis of variance with the Dunnett's test. A P-value of <0.05 was considered significant.
Maximal contraction of carbachol was obtained at the 10−5 M concentration. Nifedipine (10−7–10−4 M, n = 12)-induced concentration-dependent relaxations on the isolated bovine ciliary muscle strips precontracted by submaximal carbachol concentration (10−6 M; Fig. 2). The relaxation responses in ciliary muscle by nifedipine reached statistical significance beginning from the concentrations of 10−5 M (P < 0.05; Fig. 2).
In the presence or absence of nifedipine (10−6 M), celecoxib (10−7–10−4 M; n = 12), DFU (10−7–10−4 M; n = 12), or indomethacin (10−7–10−4 M; n = 12)-induced concentration-dependent relaxations on the isolated bovine ciliary muscle strips precontracted by carbachol (10−6 M) (Fig. 2A and 2B). Nifedipine, at concentrations with no effect on carbachol-induced contractility, were added to the bath 20 min before the addition of celecoxib, DFU, and indomethacin. The relaxation responses in ciliary muscle by celecoxib, DFU, and indomethacin reached statistical significance beginning from the concentrations of 10−6 M (P < 0.05). The calculated pD2 (−log EC50) and Emax values for carbachol, celecoxib, DFU, and indomethacin are shown in Table 1. There were no significant differences between pD2 values of celecoxib, DFU, and indomethacin in ciliary muscle (P > 0.05). However, the Emax value of indomethacin was significantly more than celecoxib and DFU in ciliary muscle (P < 0.05).
Preincubation with nifedipine (10−6 M) of the ciliary muscle strips significantly increased the relaxation responses of celecoxib, DFU, and indomethacin (Fig. 3A and 3B; P < 0.05). Preincubation of the ciliary muscle strips with nifedipine (10−6 mol/L) did not significantly alter the pD2 values of celecoxib, DFU, and indomethacin (P > 0.05; Table 1).However, the Emax values for celecoxib, DFU, and indomethacin were significantly different in the absence (43.1±2.0%; 45.6±1.5%; 56.3±2.5%) and presence of nifedipine (57.6±1.8%; 60.8±1.5%; 71.3±2.5%) in ciliary muscle, respectively (P < 0.05). In addition, the Emax value of indomethacin in ciliary muscle was significantly more than celecoxib and DFU in the presence of nifedipine (Table 1; P < 0.05).
Although there are many humoral and neural factors responsible for the relaxation and contraction of the ciliary muscle, findings of this study demonstrate that the selective COX-1 inhibitor, indomethacin, and the selective COX-2 inhibitors, celecoxib and DFU, have relaxant effects on bovine ciliary muscle and the relaxant effect of these drugs significantly increase in the presence of nifedipine on bovine ciliary muscle, whereas nifedipine had no relaxation effect on the carbachol-induced contraction of bovine ciliary muscles in the same doses alone similarly with the results of Lepple-Wienhues and colleagues and Kageyama and Shirasawa.21,22 We found that the pD2 values of celecoxib, DFU, and indomethacin were similar in ciliary muscle strips in the absence or presence of nifedipine. pD2 values show enzyme affinity of agonist drugs. In the present study, similar pD2 values show that affinity to COX enzymes of celecoxib, DFU, and indomethacin did not change in the absence or presence of nifedipine in ciliary muscle. The Emax values of celecoxib, DFU, and indomethacin significantly increased in presence of nifedipine in ciliary muscle strips. Emax values show that efficacy of celecoxib, DFU, and indomethacin in ciliary muscle strips. These results show that the efficacy of celecoxib, DFU, and indomethacin is significantly higher in the presence of nifedipine, but the potency of these drugs is not significantly different. In addition, the efficacy of indomethacin was higher than celecoxib and DFU in the presence of ineffective doses of nifedipine in ciliary muscle strips. This interaction can be called as pharmacologic potentiation, which is a special form of synergism. In cases of potentiation, one of two agents exerts no effect upon exposure; but when exposure to both together occurs, the effect of the active agent is increased.23 This significance increase on the relaxant effects on bovine ciliary muscle, in the presence of nifedipine, may be due to the increased effect of these drugs on the COX enzymes or their effect on another mechanism. This effect cannot be to the direct effect of nifedipine, because we used it at ineffective doses. And the extra relaxant effect of indomethacin, compared with celecoxib and DFU, may be due to the inhibitor effect of this drug to both of the COX enzymes.
The topical or systemic use of celecoxib, DFU, and indomethacin with nifedipine can cause blurred near vision due to the ciliary muscle relaxation, especially in prepresbiopic or latent hyperopic patients. This can occur under the treatment doses, especially with celecoxib and DFU, because of the facilitative relaxant effect of nifedipine on ciliary muscle. In the ocular pain conditions caused by ciliary spasm, the pain can be decreased more easily by these combined use the drugs. This effect can also occur during the use of either COX inhibitors or nifedipine under the treatment doses. Thus, in the future, especially topical forms of these combinations (especially COX-2 inhibitors and nifedipine) can be used in the treatment of ocular pain due to ciliary spasm with minimal systemic side effects after controlled, clinical studies.
In summary, the inhibitory effect of each COX inhibitor significantly increased in the presence of nifedipine in ciliary muscle. This might be explained by the potentiation of COX enzymes. Further work is needed to determine the cellular mechanism(s) of increased relaxant effect in ciliary muscle.