Molecular Vision 2015; 21:699-705
Received 12 July 2014 | Accepted 10 June 2015 | Published 12 June 2015
Ichiro Tanano, Taiji Nagaoka, Shinji Ono, Tsuneaki Omae, Shinichi Otani, Akitoshi Yoshida
Department of Ophthalmology, Asahikawa Medical University, Asahikawa, Japan
Correspondence to: Taiji Nagaoka, Department of Ophthalmology, Asahikawa Medical University, Midorigaoka Higashi 2-1-1-1, Asahikawa, 078-8510, Japan; Phone: +81-166-68-2543; FAX: +81-166-68-2549; email: email@example.com
Purpose: To clarify the vasodilatory mechanism of unoprostone isopropyl (UI), we examined its effects on the retinal microvascular diameter to determine the dependence on the endothelium and/or smooth muscle to reveal the signaling mechanisms involved in this vasomotor activity.
Methods: Porcine retinal arterioles were isolated, cannulated, and pressurized without flow in vitro. Video microscopic techniques recorded the diametric responses to UI.
Results: The retinal arterioles dilated in response to UI in a dose-dependent (100 pM-10 µM) manner. The nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) inhibited UI-induced vasodilation. The large-conductance Ca2+-activated K channel (BKCa channel) blocker iberiotoxin also inhibited UI-induced vasodilation. The residual vasodilation after L-NAME was eliminated with co-administration of iberiotoxin.
Conclusions: UI elicits dilation of the retinal arterioles mediated by NO release and BKCa channel activation.
Unoprostone isopropyl (UI), a prostaglandin F2α-related compound, has been introduced recently to treat glaucoma . This drug is thought to reduce intraocular pressure by increasing conventional or uveoscleral outflow . Recently, UI was also reported to affect the ocular blood flow [3-6] and retinal microcirculation in vivo  and in vitro . These reports supported the idea that topical UI may increase ocular blood flow. Because we recently reported that retinal blood flow (RBF) is impaired in patients with type 2 diabetes mellitus with mild and no diabetic retinopathy (DR) , topical UI may be useful as a novel treatment of DR by improving retinal circulation in patients with diabetes. However, the underlying mechanisms of the effect of UI on retinal circulation remain unclear. In the current study, we examined the effect of UI on retinal microvascular diameter using an isolated vessel technique to exclude the confounding effects of metabolic, hemodynamic, humoral, and glial/neuronal factors associated with in vivo experiments.
The Animal Care Committee of Asahikawa Medical University approved all animal procedures, which were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. The eyes were enucleated immediately from pigs of either sex (age, 16–24 weeks; weight, 10–15 kg) after they were killed in a local slaughterhouse; the eyes were transported to the laboratory in a moist chamber on ice.
The techniques for identifying and isolating retinal microvessels have been described previously [9-13]. Briefly, the isolated retinal arterioles (90–130 µm in situ) were cannulated with a pair of glass micropipettes and pressurized to 55 cmH2O (~40 mmHg) intraluminal pressure without flow using two independent pressure reservoir systems . The vasomotor activity of the isolated vessels was continuously recorded throughout the experiments via videomicroscopic techniques .
Cannulated, pressurized arterioles were bathed in physiologic salt solution (PSS) with albumin (0.1%) at 36 to 37 °C to allow development of stable basal tone; after about 30 to 40 min, concentration-dependent vasodilation developed in response to UI (100 pM-10 µM). After measurement of the control dose response of UI without drugs, the vessels were washed with PSS to allow redevelopment of the basal tone. In some studies, the vasodilation elicited by UI was re-examined after 30 min to conﬁrm the reproducibility of the response (n=6).
In the first series of studies, we examined the role of the endothelium in UI-induced dilation by comparing the responses before and after removal of the endothelium via intraluminal perfusion of the nonionic detergent 3-[(3-cholamidopropyl) dimethylammonio]-1 propane sulfonate (CHAPS; 0.4%) as described previously . We also assessed the role of endothelium-derived relaxing factors (EDRF), i.e., prostaglandins (PG), nitric oxide (NO), and cytochrome P450 metabolites, in mediating the vascular response in the presence of known effective concentrations of the speciﬁc enzyme inhibitors indomethacin (10 µM) [10,15], NG-nitro-L-arginine methyl ester (L-NAME; 10 µM) [9,10], and sulfaphenazole (10 µM) , respectively. To study the role of the PGI2 receptor (IP) on UI-induced dilation, we assessed the arterioles pre-incubated with the IP antagonist CAY10441 (0.1 μM) . To study the involvement of the K channels, we examined these pathways by treating the vessels with K channel inhibitors, i.e., the nonselective K channel blocker tetraethylammonium (TEA; 10 mM)  and the large-conductance Ca2+-activated K channel (BKCa channel) blocker iberiotoxin (0.1 µM) [12,19-21].
Sodium nitroprusside (SNP; 0.1 µM-100 µM) was used to probe endothelium-independent NO-mediated vasodilation. The vascular response to SNP was examined in the presence of various interventions (Table 1).
All drugs were administered extraluminally unless otherwise stated. Each pharmacologic inhibitor was incubated with the vessels for at least 30 min.
All chemicals were obtained from Sigma-Aldrich (St. Louis, MO), except UI and CAY10441, which were provided by R-Teck Ueno, Ltd. (Tokyo, Japan) and Cayman Chemical (Ann Arbor, MI), respectively. L-NAME, TEA, iberiotoxin, and SNP were dissolved in PSS. Indomethacin and sulfaphenazole were dissolved in ethanol. UI was dissolved in dimethyl sulfoxide (DMSO). Subsequent dilutions of these drugs were prepared in PSS. The final concentration of ethanol or DMSO in the vessel bath was 0.1% . Vehicle control studies indicated that this final concentration of solvent had no effect on arteriolar function.
At the end of each experiment, the vessels were relaxed in EDTA (1 mM) calcium-free PSS to obtain the maximal diameter at 55 cmH2O intraluminal pressure [9,11]. The diametric changes in response to UI and SNP were normalized to the resting diameters and expressed as the percent changes in diameter compared to the maximal dilation [9,11]. The data are expressed as the mean ± standard error of the mean, and n represents the number of vessels studied. Statistical comparisons of the changes in resting tone caused by antagonists were performed using the non-parametric Wilcoxon signed-rank test. Two-way ANOVA, followed by the Bonferroni multiple-range test, was used to determine the signiﬁcance of the difference between control and experimental interventions. p<0.05 was considered signiﬁcant.
The basal tone in all vessels (n=38) ranged from 53% to 78% (average, ~59% ± 4%) of their maximal diameters (Table 1). The average resting and maximal vessel diameters were 57±6 and 94±5 µm, respectively. There were no significant changes in the resting tone after any interventions. UI induced consistent concentration-dependent dilation of the retinal arterioles. The highest concentration (10 µM) elicited about 30% of the maximal dilation (Figure 1). Further study showed that UI-induced dilation was reproducible and did not deteriorate after repeated applications (Figure 1).
In the denuded vessels (n=4), the UI-induced dilation decreased partly and the response to the highest UI concentration decreased significantly (p<0.01) from 30% to 10% (Figure 2). The NOS inhibitor L-NAME significantly (p<0.001; Figure 2) reduced UI-induced vasodilation, which was comparable to that produced by denudation (L-NAME versus denudation, p>0.05). In contrast, the inhibition of cytochrome P450 epoxygenase and prostaglandins by sulfaphenazole and indomethacin, respectively, did not affect the vasodilatory responses to UI. Blockage of the IP receptor by CAY10441 did not affect the vasodilatory response to UI (Figure 2).
TEA significantly (p<0.01) inhibited UI-induced vasodilation of the retinal arterioles (Figure 3). In addition, iberiotoxin attenuated UI-induced dilation of the retinal arterioles in a manner similar to that of TEA. The residual vasodilation in the presence of iberiotoxin was almost eliminated with subsequent L-NAME treatment (Figure 4).
Various interventions (denudation, co-incubated with L-NAME, TEA, iberiotoxin, iberiotoxin +L-NAME ) did not affect the SNP-induced dilation of the retinal arterioles (88.3 ±2.5 versus 88.8 ±2.2, 86.8 ±3.3, 90.2 ±4.2, 88.7 ±2.8, 88.7 ±2.8 µm; Table 1).
Based on two-way analysis of variance, the responses to SNP were unaffected by any perturbations compared with the control.
The current study showed that UI induced concentration-dependent vasodilation of isolated porcine retinal arterioles (Figure 1). These results seem to be comparable to those in the ciliary arteries in rabbits [22,23] and mice . Yu et al. reported that, in the retinal arterioles, UI caused vasodilation only in endothelin-1 (ET-1)-pre-contracted arterioles but did not change the retinal vessel diameter in normal-tone arterioles without ET-1 pre-contraction . Although the authors did not provide a definitive explanation for this discrepancy, the differences between their finding in normal-tone vessels without ET-1 and ours in spontaneous tone vessels without no pre-constrictive drug might be due to differences in experimental protocols with or without flow to the vessel. Indeed, UI also was reported to increase ocular blood flow in vivo [4,5]. These previous in vivo findings seem to be comparable to ours.
It is unlikely that UI can reach the retinal artery after penetrating the cornea or sclera because UI is metabolized to M1 and M2 while penetrating the cornea . However, Watanabe et al. reported that the concentration of 3H-UF-021 (UI) increased in various ocular tissues after topical application . In their study, the concentration of 3H-UF-021 was 153 ng/g of wet tissue in the retina and choroid and 500 ng/g of wet tissue in the posterior sclera. Because the molecular weight of UI is 424.62 , the concentrations of UI (100 pM-10 µM) used in the present study seem to induce vasodilation in the retinal artery and increase RBF. Further clinical study is warranted to investigate whether topical administration of UI increases RBF in patients with retinal vascular disorders, i.e., DR and retinal vein occlusion.
Although previous studies have reported conflicting results that UI-induced vasodilation is endothelium-dependent  or endothelium-independent [22,23], we for the first time observed the inhibitory effects of NOS blockade by L-NAME on UI-induced vasodilation (Figure 2). We also confirmed that removal of the endothelium by CHAPS  decreased the unoprostone-induced vasodilation of the retinal arterioles, which is comparable to L-NAME (Figure 2). Taken together, our findings suggested that UI may cause vasodilation via NO production from the endothelium in the retinal arterioles.
In contrast to L-NAME, the vasodilatory response to UI was unaffected by inhibition of prostaglandin synthesis, i.e., the specific PGI2 receptor blocker CAY10441  and the cytochrome P450 metabolite by pretreatment with indomethacin and sulfaphenazole (Figure 2). These results indicated that UI-induced vasodilation of the retinal arterioles was mediated primarily via NO derived from the endothelium, independent of other endothelium-derived vasodilators, i.e., prostacycline and the endothelium-derived hyperpolarizing factor.
It is well known that the mechanism of action of UI involves activation of the BKCa channel . The current study is the first to suggest that the BKCa channel is involved in UI-induced vasodilation of the retinal arterioles. Although there are contradictions between our results and other reports [7,22,23], the discrepancies may be due to differences in the experimental protocols or arterial segments, e.g., with or without flow to the vessel, species, and retinal or ciliary arterioles. Further study is needed to clarify the detailed mechanisms.
Previous studies have reported the involvement of various K channels in the vasodilation of the retinal arterioles [9,10,12,18,19,28,29]. The current data showed that the BKCa channel blocker iberiotoxin inhibited UI-induced vasodilation (Figure 3) in the same manner as the nonselective K channel blocker TEA. These results suggested that the BKCa channel, which relaxes the smooth muscle via membrane hyperpolarization , may be involved primarily in UI-induced vasodilation of the retinal arterioles. Although the endothelial KCa channel might regulate endothelial NO synthase activity , the current finding that the combination of L-NAME and iberiotoxin almost eliminated dilation in response to UI suggested that the BKCa channel may be involved in the endothelium-independent (smooth muscle-dependent) vasodilation of the retinal arterioles in response to UI.
In conclusion, we showed for the first time that UI, a prostaglandin F2α-related compound, elicits potent dilation of the retinal arterioles. UI-induced vasodilation may have two components of endothelium-dependent and endothelium-independent pathways, probably via the NO production from the vascular endothelium and activation of the BKCa channel in smooth muscle. Because RBF is impaired in early-stage DR in patients with type 2 diabetes mellitus , UI-induced vasodilation may be a novel potential drug for topical treatment of DR.
Supported by a Grant-in-Aid for Scientific Research (B) 25293352 and Challenging Exploratory Research 25670724 from the Ministry of Education, Science, and Culture, Tokyo (TN).