Vasoactive Responses of U46619, PGF_2α, Latanoprost, and Travoprost in Isolated Porcine Ciliary Arteries

Abstract

purpose. To compare the vasoactive properties of the prostanoids U46619 (thromboxane A₂ analogue), prostaglandin F_2α (PGF_2α), latanoprost free acid, and travoprost free acid in isolated porcine ciliary arteries.

methods. In a myograph system (isometric force measurement), quiescent vessels were exposed (cumulatively) to U46619, PGF_2α, latanoprost, or travoprost (0.1 nM–0.1 mM). Experiments were also conducted in the presence of SQ 29548 (TP-receptor antagonist; 3–10 μM) or AL-8810 (FP-receptor antagonist; 3–30 μM). Contractions were expressed as the percentage of 100 mM potassium chloride-induced contractions.

results. In quiescent vessels, contractions (concentration–response curves) induced by (0.1 mM) PGF_2α (87.9% ± 3.5%), U46619 (66.7% ± 4.1%), and latanoprost (62.9% ± 3.6%) were more pronounced (P ≤ 0.001) than those induced by travoprost (23.0% ± 4.4%). Concentration–response curves for PGF_2α, latanoprost, and travoprost were preceded by a smaller contraction peak (0.1 μM) that was higher (P ≤ 0.05) for travoprost (24.4% ± 2.8%) than for PGF_2α (12.9% ± 4.6%), but not different (P = 0.58) from latanoprost (22.0% ± 3.0%). The 50% maximal contraction (PD₅₀: negative log M concentration) of U46619 (−8.05 ± 0.13) was lower (P ≤ 0.001) than those of latanoprost (−5.65 ± 0.10), PGF_2α (−5.49 ± 0.14), and travoprost (−5.12 ± 0.52). Contractions were inhibited (P ≤ 0.05–0.001) either by SQ 29548 or AL-8810.

conclusions. In isolated porcine ciliary arteries, all prostanoids tested induced contractions. Among them, travoprost appeared to be the least potent and U46619 the most efficient.

The thromboxane A₂ analogue U46619 and prostaglandin F_2α (PGF_2α) belong to the prostanoid family. ¹ U46619 is regarded as a thromboxane A-receptor (TP) agonist and PGF_2α as a prostaglandin F-receptor (FP) agonist. ¹ U46619 and PGF_2α are known to be strong vasoconstrictors, particularly in the ocular circulation. ^{2 3}  

Latanoprost and travoprost are potent ocular hypotensive drugs that share some chemical structural similarities with PGF_2α. ⁴ In the present study, we compared the vasoactive responses of U46619, PGF_2α, latanoprost, and travoprost in isolated porcine ciliary arteries and additionally assessed how these responses can be modulated by SQ 29548 (TP-receptor antagonist) or AL-8810 (FP-receptor antagonist). 

Material and Methods

Vessels Preparation

Porcine eyes were obtained from an abattoir immediately after death. In cold modified Krebs-Ringer solution (NaCl 118 mM, KCl 4.7 mM, CaCl₂ 2.5 mM, K₂PO₄ 1.2 mM, MgSO₄ 1.2 mM, NaHCO₃ 25 mM, glucose 11.1 mM, and EDTA 0.026 mM), ciliary arteries were dissected and cut into 2-mm segments. ⁵ In an organ chamber, two 45-μm tungsten wires were passed through the vessel’s lumen and attached to a force transducer for isometric force measurements (Myo-Interface; JP Trading, Aarhus, Denmark). Vessels were left in place for 30 minutes in modified Krebs-Ringer solution (95% O₂, 5% CO₂, 37°C) before being stretched in a stepwise manner and exposed between stretch steps to 100 mM potassium chloride (KCl) until the optimum passive tension was reached (i.e., <10% variation of KCl-induced contractions between two different stretch steps). ⁶ To assess the functional integrity of the endothelium, vessels were precontracted with 0.1 μM U46619 and then exposed to bradykinin. The endothelial function was considered to be preserved if bradykinin (100 nM–1 μM) evoked at least 80% relaxation. ⁷ Vessels were then washed out with modified Krebs-Ringer solution before any of the following experimental protocols were conducted. 

Experimental Protocols

In a first set of experiments, quiescent ciliary arteries were exposed cumulatively, to increasing concentrations (0.1 nM–0.1 mM) of U46619, PGF_2α, latanoprost, or travoprost. In a second set of experiments, quiescent vessels were first incubated for 30 minutes with different concentrations (3–10 μM) of SQ 29548 (TP-receptor antagonist) and then exposed to increasing concentrations (0.1 nM–0.1 mM) of U46619, PGF_2α, latanoprost, or travoprost. In a third set of experiments, quiescent vessels were first incubated for 30 minutes with different concentrations (3–30 μM) of AL-8810 (FP-receptor antagonist) and then exposed to increasing concentrations (0.1 nM–0.1 mM) of U46619, PGF_2α, latanoprost, or travoprost. Timed control experiments were conducted in parallel with only the vehicle solution. 

Drugs and Statistical Analysis

U46619, PGF_2α, latanoprost free acid, and travoprost free acid were obtained from Cayman Chemicals (Ann Arbor, MI) and dissolved in pure ethanol (maximal final concentration in the organ chamber, ≤1%). Bradykinin and SQ 29548 were purchased from Sigma-Aldrich (Buchs, Switzerland). AL-8810 was provided by Alcon Laboratories (Fort Worth, TX). Concentrations are expressed as final molar concentrations in the organ chambers. Contractions are given as percent of 100 mM KCl-induced maximal contraction. Results are provided as mean ± SEM with n representing the number of vessels studied (one vessel per eye). The concentration causing 50% of the maximal contraction is expressed as a negative log M concentration (PD₅₀). Statistical comparisons were conducted with a one-way ANOVA multiple comparison followed by the Bonferroni test, with a two-tailed P ≤ 0.05 considered to be statistically significant. 

Results

Effect of Prostanoids in Quiescent Vessels

In quiescent vessels (Fig. 1) , all prostanoids induced contractions (concentration–response curve). At a 0.1 mM concentration, U46619, PGF_2α, latanoprost free acid, and travoprost free acid evoked contractions that were significantly different from those in timed control experiments. Contractions induced by PGF_2α (87.9% ± 3.5%), U46619 (66.7% ± 4.1%), and latanoprost (62.9% ± 3.6%) were significantly (P ≤ 0.001) more pronounced than those induced by travoprost (23.0% ± 4.4%). The PD₅₀ of U46619 (−8.05 ± 0.13) was significantly (P ≤ 0.001) lower than that of latanoprost (−5.65 ± 0.10), PGF_2α (−5.49 ± 0.14), and travoprost (−5.12 ± 0.52). It has to be noted that the concentration–response curves evoked by PGF_2α, latanoprost free acid, and travoprost free acid were preceded by a small peak contraction that occurred at a concentration of 0.1 μM and that was significantly different from that in the timed control. This first peak contraction was significantly (P ≤ 0.05) different for travoprost than for PGF_2α but not significantly (P = 0.58) different for latanoprost. 

Effect of SQ 29548 on Prostanoid-Induced Contractions

In the presence of different concentrations of SQ 29548 (TP-receptor antagonist), the concentration-response curve of U46619 (TP-receptor agonist) was significantly (P ≤ 0.001) shifted to the right, without any decrease in the maximal contraction observed, indicating that SQ 29548 is likely to act as a competitive inhibitor for U46619 (Fig. 2) . In the presence of 10 μM SQ 29548, the concentration–response curve evoked by PGF_2α also tended to be shifted to the right, whereas those evoked by latanoprost and travoprost approached zero. 

Effect of AL-8810 on Prostanoid-Induced Contractions

In the presence of increasing concentrations of AL-8810 (FP-receptor antagonist), the concentration-response curves of PGF_2α (FP-receptor agonist), but also of U46619 and latanoprost tended to be shifted to the right in a concentration-dependent manner (Fig. 3) . In the presence of AL-8810, the concentration–response curve of travoprost tended to be abolished. 

Discussion

The results of the present study indicate that U46619 (TP-receptor agonist), PGF_2α (FP-receptor agonist), latanoprost free acid, and travoprost free acid have vasoconstrictive properties in isolated porcine ciliary arteries. They also show that SQ 29548 (TP-receptor antagonist) and AL-8810 (FP-receptor antagonist) had an inhibitory effect on the contractions evoked by these drugs. 

Among the four vasoconstrictors tested, U46619 (thromboxane A₂ agonist) appeared to be the most efficient vasoconstrictor, with a PD₅₀ that was more than twofold lower than the PD₅₀ of PGF_2α. In addition, U46619 was able to produce highly potent contractions that were almost equivalent to those observed with PGF_2α, which was, among the four prostanoids tested, the one that induced the most potent contractions. It also has to be noted that the vasoconstrictive response of U46619 differed from the three other prostanoids in the sense that the contractions evoked by PGF_2α, latanoprost free acid, and travoprost free acid were preceded by a small peak contraction. This peak contraction was observed at a concentration as low as 0.1 μM for the three drugs. The peak-contraction induced by latanoprost and travoprost at a concentration of 0.1 μM did not differ significantly. In contrast, at higher concentrations (0.1 mM), the contractions evoked by PGF_2α and latanoprost free acid were significantly higher than those induced by travoprost. The present study further demonstrates that when the vasoconstrictive effects of latanoprost and travoprost are compared, it clearly appears that latanoprost is a stronger vasoconstrictor than travoprost in isolated porcine ciliary arteries, at least at high concentrations that might not necessarily be clinically relevant. 

The results of the present study are in agreement with different publications indicating that U46619 and PGF_2α are strong vasoconstrictors in vitro. ^{2 3} Such studies have shown that high concentrations of PGF_2α can for example cause vasoconstriction in rabbit posterior ciliary arteries, bovine retinal arteries, and monkey ophthalmic arteries. ^{8 9 10} The present study is also in agreement with reports indicating that latanoprost can act as a vasoconstrictor. ^{2 11} Indeed, Astin ² reported similar contractions in bovine ciliary artery preparations with latanoprost at concentrations above 1 μM. These contractions were inhibited by the TP-receptor antagonist GR 32191B. 

It has to be mentioned that, in humans, about 2 hours after a single topical ocular instillation of latanoprost eye drop solution, the maximal concentration of latanoprost measured in the aqueous humor was ∼0.1 μM. At this concentration, as shown in the present study, this drug was able to induce significant contractions in isolated porcine ciliary arteries (first peak contraction). ¹² However, the use of radioactively labeled microspheres to assess ocular blood flow showed no major circulatory changes in the posterior pole of monkeys, feline, and rabbit eyes after a single topical administration of 6 to 10 μg latanoprost (corresponding to four times the topical dose applied in humans). ¹³ Nevertheless, this observation does not exclude that this type of drug could lead, in some predisposed patients, to a vasoconstriction. Indeed, it has been reported that latanoprost could exacerbate angina pectoris. ¹⁴  

Because PGF_2α, which is considered to be a potent FP-receptor agonist, ¹ shares some close chemical structural similarities with latanoprost and travoprost, one would expect that the contractions induced by these drugs would be specifically inhibited by an FP-receptor antagonist such as AL-8810 and not by a TP-receptor antagonist such as SQ 29548. Similarly, U46619, considered to be a potent TP-receptor agonist, ¹ should be inhibited by SQ 29548 (TP-receptor antagonist) and not by AL-8810 (FP-receptor antagonist). The present study illustrated that this is definitely not the case regarding the vasoconstrictive response of PGF_2α and U46619 in isolated porcine ciliary arteries. Indeed, both AL-8810 (FP-receptor antagonist) and SQ 29548 (TP-receptor antagonist) were able to inhibit the contraction induced by the FP-receptor agonist PGF_2α as well as by the TP-receptor agonist U46619. 

Different conditions could lead to such observations. For example, although some agents may activate one receptor preferentially, they may exhibit some modest affinity for other prostaglandin-receptor subtypes. In the case of the FP-receptor antagonist AL-8810 it has been shown that it can produce a slight inhibition of TP-receptor-mediated phosphoinositide turnover in human nonpigmented ciliary epithelial cells at a concentration of 100 μM. ¹⁵ Another condition that may affect the vasoactive response of these ocular blood vessels is the possible existence of a heterogeneous population of TP receptors. ¹⁶ For example, it is known that the human TP receptor isoforms TP_α and TP_β show different sensitivities to inhibition by endogenous and exogenous mediators. ¹⁷ In any event, one has to keep in mind that, since the specificity of the TP- and FP-receptor antagonists tested can always be questioned, no definite conclusion can be drawn regarding the possible stimulation of different receptors by the prostanoids tested in the present study. Nevertheless, it clearly appears that SQ 29548 and AL-8810 could strongly inhibit the contractions induced by all prostanoids studied in the present trial. 

In conclusion, in isolated porcine ciliary arteries and at the concentrations tested, the maximal contractions evoked by U46619, PGF_2α, and latanoprost free acid were much higher than those evoked by travoprost free acid, and the contractions evoked by any of the four drugs were inhibited either by the TP-receptor antagonist SQ 29548 or the FP-receptor antagonist AL-8810. 

 Supported in part by Alcon Laboratories, Fort Worth, Texas.

 Submitted for publication June 16, 2005; revised July 15, 2005; accepted November 21, 2005.

 Disclosure: I. Vysniauskiene, Alcon Laboratories (F); R. Allemann, Alcon Laboratories (F); J. Flammer, Alcon Laboratories (F); I.O. Haefliger, Alcon Laboratories (F)

 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: Ivan O. Haefliger, Laboratory of Ocular Pharmacology and Physiology, University Eye Clinic, Mittlere Strasse 91, PO Box, CH-4012 Basel, Switzerland.

Figure 1.

 

Contracting effect of the prostanoids U46619, PGF_2α, latanoprost free acid (latanoprost), and travoprost free acid (travoprost) in quiescent isolated porcine ciliary arteries. Statistical comparisons were made versus timed control (control) and U46619 (§), PGF_2α (‡), latanoprost (†), or travoprost (*), with a single, double, or triple symbol corresponding to P ≤ 0.05, P ≤ 0.01, or P ≤ 0.001, respectively.

Figure 2.

 

Effect of the TP-receptor antagonist SQ 29548 on contractions induced by U46619, PGF_2α, latanoprost free acid (latanoprost), and travoprost free acid (travoprost) in quiescent isolated porcine ciliary arteries. Statistical comparisons were made between timed control (control) and the effect of 3 μM (*) and 10 μM (†) SQ 29548 on the contraction induced by the prostanoids, with a single, double, or triple symbol corresponding to P ≤ 0.05, P ≤ 0.01, or P ≤ 0.001, respectively.

Figure 3.

 

Effect of the FP-receptor antagonist AL-8810 on contractions induced by U46619, PGF_2α, latanoprost free acid (latanoprost), and travoprost free acid (travoprost) in quiescent isolated porcine ciliary arteries. Statistical comparisons were made between timed control (control) and the effect of 3 μM (*), 10 μM (†), 20 μM (‡), and 30 μM (§) AL-8810 on the contraction induced by the prostanoids, with a single, double, or triple symbol corresponding to P ≤ 0.05, P ≤ 0.01, or P ≤ 0.001, respectively.

 

The authors thank Julie Crider (Alcon Laboratories) for providing critical, fair, and constructive comments regarding the manuscript.