Evokes Retinal Arteriolar Dilation: Dual Pathways Via NOS Activation and Phosphodiesterase Inhibition

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Sildenaﬁl (Viagra) Evokes Retinal Arteriolar Dilation: Dual Pathways via NOS Activation and Phosphodiesterase Inhibition

Zhaoxu Yuan,1 Travis W. Hein,1 Robert H. Rosa, Jr,1 and Lih Kuo1,2

PURPOSE. Sildenafil (Viagra; Pfizer, New York, NY), a selective (Invest Ophthalmol Vis Sci. 2008;49:720–725) DOI:10.1167/ phosphodiesterase type-5 (PDE5) inhibitor, is widely used to iovs.07-1208 treat impotence by improving penile blood flow via elevation of cGMP. However, its effect on ocular circulation is contro- ildenafil (Viagra; Pfizer, New York, NY) is the first commer- versial and whether retinal arterioles are responsive to this Scially available selective inhibitor of phosphodiesterase-5 drug remains unclear. In this study, the direct reaction of (PDE5) to be widely used for the treatment of erectile dysfunc- retinal arterioles to sildenafil was examined and the signaling tion. The inhibition of PDE5 can enhance the relaxation effect pathway underlying this vasomotor activity was probed. of nitric oxide (NO) on the smooth muscle lining of both the arteries and the sinusoidal spaces of the corpora cavernosa by METHODS. Retinal arterioles from porcine eyes were isolated, reducing cyclic guanosine monophosphate (cGMP) degrada- cannulated, and pressurized without flow. Diameter changes in tion and thus increase arterial blood flow into penile sinusoids response to sildenafil were recorded using videomicroscopic for erection. However, visual disturbances (e.g., changes in techniques. color and light perception, alterations in retinal electrophysi- RESULTS. Retinal arterioles (67 Ϯ 2 ␮m) dilated dose depen- ology, conjunctival hyperemia, and blurred vision)1–3 and non- dently to sildenafil (1 ng/mL to 1 ␮g/mL). This dilation was arteritic anterior ischemic optic neuropathy4,5 have been re- inhibited by the nitric oxide (NO) synthase inhibitor NG-nitro- ported in association with the use of sildenafil. Although it has L-arginine methyl ester (L-NAME), the guanylyl cyclase inhibitor been suggested that some of these symptoms may be related to 1H- [1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), the extra- the additional inhibition of PDE6 by sildenafil in photoreceptor cellular signal-regulated kinase (ERK) pathway inhibitor cells,1,3 it remains unclear whether alteration of the ocular PD98059, the nonselective potassium channel blocker tetra- circulation, directly or indirectly through local and/or systemic ethylammonium (TEA), and the selective adenosine triphos- blood pressure changes, by sildenafil is involved.4 In the past 5 years, several investigators have studied the phate (ATP)-sensitive potassium (KATP) channel blocker glibenclamide. The vasodilation elicited by the NO donor S-nitroso- effect of sildenafil on the ocular circulation in humans. An N-acetylpenicillamine (SNAP) was inhibited by ODQ and TEA increase in pulsatile ocular–choroidal blood flow and retinal but was insensitive to PD98059. In the presence of L-NAME, the microcirculatory flow velocity by sildenafil has been reported ␮ in healthy human subjects.6,7 However, the ineffectiveness of addition of SNAP (1 M) produced modest vasodilation and the 8 8,9 inhibited sildenafil response was subsequently restored. The sildenafil on optic nerve rim and choroidal circulation was also observed in healthy human subjects. In a small cohort of restored dilation was insensitive to PD98059 but was blocked 10 11 by TEA. healthy persons or patients with erectile dysfunction, sildenafil had no effect on blood velocity in the central retinal CONCLUSIONS. Activation of NO synthase, through ERK signal- artery but caused a significant increase in mean velocity in the ing, leading to NO production and subsequent guanylyl cyclase ophthalmic artery.10,11 In addition to these inconsistent find- activation and KATP channel opening is the major vasodilatory ings, direct measurements of retinal vessel diameter in healthy pathway for sildenafil in retinal arterioles. Moreover, the ele- human subjects also yield conflicting results. Dilation of the vated cGMP, from endogenous or exogenous NO, plays a superior temporal artery12 and vein12,13 in the human retina in permissive role for sildenafil to exert vasodilation through response to sildenafil was reported by some investigators, but inhibition of the PDE5 pathway independent of ERK signaling. others found the absence of vasomotor action of sildenafil.14 Although these apparent discrepancies may be a result of different methods, techniques, and/or sensitivities in measur-

1 ing ocular blood velocity/flow and vascular diameter, those From the Departments of Ophthalmology and Surgery, Scott & observations may also have been confounded by the activation White Eye Institute, Temple, Texas; and the 2Department of Systems Biology and Translational Medicine, College of Medicine, Texas A&M of neural, humoral, local, and/or autoregulatory flow regulation Health Science Center, Temple, Texas. mechanisms secondary to sildenafil administration. Therefore, Supported by the Scott & White Research Foundation (LK, TWH), it remains unclear whether sildenafil exhibits direct vasomotor the Ophthalmic Vascular Research Program, the Kruse Family Endow- action in the ocular circulation. To investigate, we used in vitro ment Fund (LK), K08EY016143 (RHR), and the Retina Research Foun- isolated vessel techniques to determine directly whether sil- dation (TWH). denafil exerts vasodilatory action in retinal arterioles and to Submitted for publication September 15, 2007; revised October elucidate the underlying signaling pathways responsible for 27, 2007; accepted December 18, 2007. this vasomotor activity. Disclosure: Z. Yuan, None; T.W. Hein, None; R.H. Rosa, Jr, None; L. Kuo, None The publication costs of this article were defrayed in part by page METHODS charge payment. This article must therefore be marked “advertise- ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. Animal Preparation Corresponding author: Zhaoxu Yuan, Departments of Ophthal- mology and Surgery, Scott & White Eye Institute, College of Medicine, All animal procedures were performed in accordance with the ARVO Texas A&M Health Science Center, 702 Southwest H. K. Dodgen Loop, Statement for the Use of Animals in Ophthalmic and Vision Research Temple, TX 76504; [email protected]. and were approved by the Scott & White Institutional Animal Care and

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Use Committee. Pigs (8–12 weeks old of either sex; 7–10 kg) pur- Chemicals chased from Barfield Farms (Rogers, TX) were sedated with tiletamine- Sildenafil citrate was obtained from Pfizer. Other drugs were obtained zolazepam (4.4 mg/kg, IM; Telazol, Fort Dodge Animal Health, Fort from Sigma-Aldrich (St. Louis, MO) except where specifically stated Dodge, IA) and xylazine (2.2 mg/kg, IM), anesthetized with pentobar- otherwise. L-NAME, TEA, and sodium nitroprusside were dissolved in bital sodium (30 mg/kg, IV), intubated, and ventilated with room air. PSS, and SNAP was dissolved in water. Sildenafil, ODQ, and PD98059 Heparin (1000 U/kg) was administered into the marginal ear vein to were dissolved in ethanol and subsequently diluted in PSS. Gliben- prevent clotting, and the eyes were enucleated and immediately placed clamide was dissolved in dimethyl sulfoxide (DMSO). Vehicle control in a moist chamber on ice. studies were performed in parallel with drug administration, and the highest concentration of organic solvents (0.1% ethanol and 0.03% Isolation and Cannulation of Microvessels DMSO) had no effect on the basal tone or arteriolar function, as The preparation of isolated retinal arterioles has been described in our reported in our previous studies.15–17 previous studies.15,16 In brief, the anterior segment and vitreous body from the porcine eye were removed carefully under a dissection Data Analysis microscope. The eye cup was placed in a cooled dissection chamber The changes in inner diameters of arterioles in response to agonists in (8°C) containing a physiological salt solution (PSS; in mM: NaCl 145.0, the absence and presence of various inhibitors were recorded through- KCl 4.7, CaCl2 2.0, MgSO4 1.17, NaH2PO4 1.2, glucose 5.0, pyruvate out the experiments. At the end of each experiment, the vessel was 2.0, EDTA 0.02, and MOPS 3.0) with 1% albumin (USB, Cleveland, OH). relaxed with 0.1 mM sodium nitroprusside in calcium-free PSS contain- Second-order retinal arterioles (40–50 ␮m in internal diameter in situ ing EDTA (1 mM) to obtain its maximal diameter. The dilations of and 0.6–1.0 mm in length without branches) in the temporal region of retinal arterioles to agonists were normalized to their maximal vasodi- the retina were carefully dissected with Vannas spring scissors and a latory capacity and expressed as a percentage of maximal dilation, pair of Dumont microdissection forceps (Fine Science Tools, Foster based on the following formula: (response diameter Ϫ baseline diam- City, CA) with the aid of a stereomicroscope (model SZX12; Olympus, eter)/(maximal diameter Ϫ baseline diameter) ϫ 100 ϭ % maximal Melville, NY). An arteriole was then transferred for cannulation to a dilation. Data are reported as the mean Ϯ SEM, and n represents the Lucite vessel chamber containing PSS-albumin solution. Both ends of number of vessels studied. Statistical comparisons were performed by the arteriole were cannulated with glass micropipettes (tip outer di- two-way ANOVA followed by the Bonferroni multiple-range test. The ameter of 30–40 ␮m) filled with PSS-albumin solution, and the outside changes in resting tone by pharmacologic antagonists were analyzed of the arteriole was securely tied to the pipettes with 11-0 nylon by paired Student’s t-test. P Ͻ 0.05 was considered significant. Taking sutures (Alcon, Fort Worth, TX). After cannulation, the vessel and into account the observed variability in responses (SD) when applying pipettes were transferred to the stage of an inverted microscope ANOVA, the sample size for each protocol in the present study is (model CKX41; Olympus) coupled to a video camera (Sony DXC-190; sufficient to detect a 5% change in dilation with the power of at least Labtek, Campbell, CA) and video micrometer (Cardiovascular Research 0.80 when ␣ ϭ 0.05. Institute, Texas A&M Health Science Center, College Station, TX) for continuous measurement of the internal diameter. The arterioles were RESULTS pressurized to 55 cmH2O without flow by two independent reser- 15,17 voirs based on pressure ranges that have been documented in Dilation of Retinal Arterioles to Sildenafil retinal arterioles in vivo18 and in the isolated, perfused retinal microcirculation.19 Preparations with leaks were excluded from further In this study, all vessels developed a similar level of basal tone study. (constricted to 69% Ϯ 1% of their maximal diameter) at 36 to 37°C bath temperature with 55 cmH2O transluminal pressure. Experimental Protocols The average resting and maximal diameters of the vessels were 67 Ϯ 2 and 95 Ϯ 2 ␮m, respectively. Sildenafil produced Cannulated arterioles were bathed in PSS-albumin at 36 to 37°C, to dose-dependent (1 ng/mL to 1 ␮g/mL) dilation of retinal arte- allow development of basal tone. After a stable basal tone was achieved rioles, and the dilation was completely developed within 2 to (ϳ30–40 minutes), the concentration-dependent response to sildenafil 3 minutes at each concentration. The highest concentration of (1 ng/mL to 1 ␮g/mL) was established. In some vessels, the response sildenafil (1 ␮g/mL) elicited 31% Ϯ 2% of maximum dilation was re-examined after 30 to 60 minutes, to confirm the reproducibility. (Fig. 1). To avoid the confounding effects from the high con- To determine the possible signaling pathways involved in the retinal centration of solvent (i.e., ethanol), the concentration of sil- arteriolar response to sildenafil, the following series of experiments denafil higher than 1 ␮g/mL was not examined. The sildenafil- were performed. The role of nitric oxide synthase (NOS) in mediating elicited dilation was reproducible and did not deteriorate with the vascular response was assessed in the presence of a known effec- repetitive applications (Fig. 1). tive concentration of the specific inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 10 ␮M).15 The involvement of soluble guanylyl cyclase, Role of NOS and Guanylyl Cyclase in Vasodilation extracellular signal-regulated kinase (ERK), and potassium channel to Sildenafil activation was assessed by the specific inhibitors 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 0.1 ␮M),20 PD98059 (0.3 ␮M),21 Incubation of retinal arterioles with the NOS inhibitor L-NAME for 30 minutes did not significantly alter basal tone (control: and the nonselective potassium channel inhibitor tetraethylammonium Ϯ Ϯ ϭ (TEA, 20 mM)22 or the selective adenosine triphosphate (ATP)-sensi- 67% 5% vs. L-NAME: 67% 5%, P 0.08) but abolished the ␮ 15,16 vasodilatory response to sildenafil (Fig. 1). In a separate series tive potassium (KATP) channel inhibitor glibenclamide (5 M), respectively. To determine whether NO-induced vasodilation is medi- of studies, treating the vessels with the soluble guanylyl cyclase inhibitor ODQ did not alter the resting tone (control: 70% Ϯ ated by the activation of ERK and guanylyl cyclase and the opening of Ϯ ϭ potassium channels, the vascular response to the NO donor S-nitroso- 2% vs. ODQ: 69% 2%, P 0.14). However, ODQ abolished N-acetylpenicillamine (SNAP, 0.1 ␮M to 0.1 mM)23 was examined in the vasodilatory response to sildenafil in a manner similar to the presence of PD98059, ODQ, and TEA, respectively. In a separate L-NAME (Fig. 1). series of experiments, the vascular response to sildenafil was examined Role of ERK Signaling and Potassium Channels in in the presence of L-NAME after adding SNAP (1 ␮M). The effects of PD98059 and TEA on sildenafil-induced vasodilation were subse- the Vasodilatory Response to Sildenafil quently examined. All drugs were administered extraluminally, and The ERK kinase inhibitor PD98059 abolished the vasodilatory each antagonist was incubated with the vessels for at least 30 minutes. response to sildenafil, except at the highest concentration (i.e.,

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FIGURE 1. Role of NOS and guanylyl cyclase in isolated retinal arte- FIGURE 2. Role of ERK signaling and potassium channels in isolated riolar dilation to sildenafil. Dose-dependent sildenafil-induced vasodila- retinal arteriolar dilation to sildenafil. Dose-dependent sildenafil-in- tion was initially examined (n ϭ 20) and then repeated in some vessels duced vasodilation (Control, n ϭ 22) was inhibited by the ERK kinase after a 30-minute washout period (n ϭ 8) or after incubation with the inhibitor PD98059 (0.3 ␮M, 30 minutes, n ϭ 11), the nonselective NOS inhibitor L-NAME (10 ␮M, 30 minutes, n ϭ 6) or the soluble potassium channel inhibitor TEA (20 mM, 30 minutes, n ϭ 5), or the ␮ ϭ ␮ ϭ guanylyl cyclase inhibitor ODQ (0.1 M, 30 minutes, n 6). There selective KATP channel inhibitor glibenclamide (5 M, 30 minutes, n was no significant difference between the initial and repeat responses, 6). *P Ͻ 0.05 versus Control. but the vasodilation was abolished by L-NAME or ODQ. *P Ͻ 0.05 versus Control. exerts a vasodilatory effect on retinal arterioles. In the present ϳ6% remained; Fig. 2). Incubation with either the nonselective study, we used a videomicroscopic technique for diameter potassium channel blocker TEA or the selective KATP channel measurement and provide the first direct evidence that sil- blocker glibenclamide significantly inhibited the sildenafil-in- denafil produces dose-dependent dilation of retinal arterioles at duced vasodilation (Fig. 2). PD98059, TEA, and glibenclamide concentrations between 1 ng/mL and 1 ␮g/mL. It has been did not change the basal tone (control: 67% Ϯ 1% vs. PD98059: 65% Ϯ 2%, P ϭ 0.30; control: 70% Ϯ 4% vs. TEA: 70% Ϯ 4%, P ϭ 0.61; control: 68% Ϯ 4% vs. glibenclamide: 68% Ϯ 4%, P ϭ 0.90). Role of ERK Signaling and Potassium Channels in the Vasodilatory Response to NO The NO donor SNAP dilated the retinal arterioles in a concentration-dependent manner, and this dilation was not affected by PD98059. However, both TEA and ODQ inhibited retinal arteriolar dilation in response to SNAP in a similar manner (Fig. 3). Role of the PDE5 Pathway To determine the role of PDE5 in sildenafil-induced vasodilation, the following series of experiments were performed in the presence of L-NAME to eliminate the NOS-dependent pathway. As indicated in Figure 1, the vasodilation elicited by sildenafil was abolished by L-NAME. In the presence of L-NAME (30 minutes), the addition of SNAP (1 ␮M) produced a mod- erate dilation (26% Ϯ 6%) of retinal arterioles within 1 minute. Subsequent administration of sildenafil produced concentration-dependent dilation of the vessels in a manner comparable to that observed in control conditions (i.e., without L-NAME; Fig. 4). This restored vasodilation was insensitive to PD98059 but was abolished by TEA (Fig. 4). FIGURE 3. The role of ERK signaling and potassium channels in NO- DISCUSSION mediated vasodilation. Dose-dependent vasodilation in response to the NO donor SNAP (Control, n ϭ 19) was not affected by PD98059 (0.3 ␮M, The reports of the clinical effects of sildenafil on ocular circu- 30 minutes, n ϭ 8) but was inhibited by both TEA (20 mM, 30 minutes, lation are contradictory, and it is unclear whether this drug n ϭ 5) and ODQ (0.1 ␮M, 30 minutes, n ϭ 6). *P Ͻ 0.05 versus Control.

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role of soluble guanylyl cyclase/cGMP signaling in mediating NOS-dependent dilation in response to sildenafil in retinal arterioles (Fig. 5). Of interest, ERK kinase inhibitor PD98059 significantly at- tenuated sildenafil-induced vasodilation, indicating the involvement of ERK signaling in this vasomotor response. Although ERK activation leading to increased NOS activity and subsequent rapid NO release has been reported,26–28 it also has been

FIGURE 4. The role of the PDE5 pathway in sildenaﬁl-induced retinal arteriolar dilation. The dilation (Control, n ϭ 21) was not affected after SNAP (1 ␮M, 5 minutes) was added to the vessels that had been treated with L-NAME (10 ␮M) for 30 minutes (n ϭ 8). This vasodilatory response to sildenaﬁl was not altered by PD98059 (0.3 ␮M, 30 minutes, n ϭ 8) but was abolished by TEA (20 mM, 30 minutes, n ϭ 5). *P Ͻ 0.05 versus Control.

shown that the plasma sildenafil concentration can reach 0.5 to 0.7 ␮g/mL in human subjects within an hour after a single oral administration of 100 mg.24,25 Therefore, the concentrations of sildenafil used in the present study are clinically relevant. The threshold concentration for sildenafil to dilate retinal arterioles is at 10 ng/mL and the highest concentration (1 ␮g/mL) produces up to 30% maximal dilation. Although the magnitude of dilation might be modest, this vasomotor response is expected to have a significant impact on local retinal perfusion because blood flow, under constant driving pressure, is a function of the fourth power of the vessel radius. In contrast, it should be noted that sildenafil, at clinical doses, is not a potent vasodilator in retinal arterioles (Fig. 1). Therefore, its direct vasomotor actions are likely to be modulated and/or masked by various vasoregulatory feedback mechanisms in vivo, which may partly explain the inconsistent findings in hemodynamics and vascular responses to sildenafil in the human ocular circulation. Based on the results derived from the present study, we propose that a signaling pathway exists for retinal arteriolar dilation in response to sildenafil, as shown in Figure 5 and discussed later herein. We first examined the possible role of the endothelium-derived vasodilator NO in sildenafil-induced vasodilation. We found that blockade of NOS abolished the sildenafil-induced response, suggesting that NO released from FIGURE 5. The proposed signaling pathways involved in sildenafil- NOS contributes mostly to the dilation of retinal arterioles. Our induced retinal arteriolar dilation. Evidence from our in vitro studies previous studies have shown that porcine retinal arterioles supports that sildenafil acts on the endothelium to elicit ERK signaling express endothelial NOS,17 supporting the possible role of and subsequent activation of NOS for NO production. The endothe- endothelial NO signaling in response to sildenafil. Since acti- lium-released NO diffuses to the underlying smooth muscle and stim- vation of the soluble guanylyl cyclase/cGMP pathway is gener- ulates guanylyl cyclase to produce cGMP. The guanylyl cyclase/cGMP signaling triggers opening of KATP channels, which leads to smooth ally considered as a major vasodilatory mechanism for NO, it is muscle hyperpolarization and retinal arteriolar dilation. In addition, expected that the selective soluble guanylyl cyclase inhibitor sildenafil can elevate the cellular level of cGMP and induce dilation ODQ can inhibit sildenafil-induced dilation. Indeed, ODQ abol- through the inhibition of PDE5 in an ERK-independent manner. Inhi- ished the dilation of retinal arterioles to sildenafil in a manner bition of these signaling pathways by their respective inhibitors is comparable to that produced by L-NAME, indicating the pivotal indicated by the vertical lines in reference to the direction of the arrows.

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shown that NO can induce ERK activation29 via a cGMP- as shown in Figure 3. On the other hand, similar to the NOS dependent mechanism30–32 in various cell types, including the pathway, the vasodilation through PDE5 inhibition was abol- endothelium.33 Therefore, ERK signaling can be at the up- ished by TEA (Fig. 4), indicating that potassium channels are stream or downstream of NOS. To determine the location of the final effector for sildenafil-induced dilation by increased ERK in mediating sildenafil-induced vasodilation, we per- cellular cGMP through the pathways converging from NOS formed another series of experiments by examining whether activation and PDE5 inhibition (Fig. 5). the vasodilation elicited by NO (i.e., activation of cGMP path- One of the important findings and implications of the way) is through ERK signaling. We found that the dilation of present study is that sildenafil did not evoke the classic PDE5- retinal arterioles to NO donor SNAP was insensitive to inhibition pathway for retinal arteriolar dilation unless a high PD98059 (Fig. 3), suggesting that ERK signaling underlying level of cGMP was supplied from either the blood vessel per se sildenafil-induced vasodilation is at the upstream of NO (i.e., (via endothelial NOS activation) or the surrounding parenchy- NOS activation) (Fig. 5). mal tissues. It has been shown that retinal neuronal cells Elevation of smooth muscle cGMP has been shown to acti- express NOS37,38 and soluble guanylyl cyclase.38,39 Although vate potassium channels in some types of vasculatures, leading the direct effect of NO on retinal arterioles is vasodilation, the to vasorelaxation by the inhibition of transmembrane influx of increased parenchymal NO, and thus cGMP, has been shown calcium through voltage-gated calcium channels during mem- to promote vasoconstriction by inhibiting the release of vaso- brane hyperpolarization.34–36 We have shown in other studies dilator epoxyeicosatrienoic acids from glial cells during neuro- that potassium channels play an important role in the retinal nal activation (by flickering white light) in the intact retina.40 arteriolar dilation in response to adenosine, lactate, and SNAP In this regard, the balance between vascular and parenchymal stimulation via the cGMP-mediated pathway.15,16 In the NO appears to determine the final vascular tone in intact present study, the effect of potassium channel blockade on retinal tissues. It is conceivable that under cardiovascular risks sildenafil-induced dilation (Fig. 2) was found to be comparable such as hyperlipidemia, diabetes, and hypertension (i.e., con- to that produced by L-NAME and ODQ (Fig. 1). It is possible ditions in which endothelial NOS function is generally jeopar- that sildenafil induces a signaling pathway linking NO/cGMP to dized), retinal arteriolar dilation to sildenafil may be directly potassium channels. More specifically, we assumed that potas- compromised and the glial cell-mediated vasoconstriction may sium channel activation is at the downstream of NO/cGMP be promoted by increasing or prolonging the action of NO via signaling. If this were the case, we would expect to see the PDE5 inhibition in parenchymal tissues. If similar conditions blockage of retinal arteriolar dilation to NO donors (via cGMP exist in the microcirculation of the optic nerve head, the net signaling) by both ODQ and TEA. Indeed, ODQ and TEA vasoconstriction may contribute to the development of the inhibited the dilation of retinal arterioles in response to SNAP nonarteritic anterior ischemic optic neuropathy that has been in a similar fashion (Fig. 3). These results support the cardinal described in association with sildenafil administration.41 In role of potassium channels in NO/cGMP-mediated, sildenafil- addition to the possible inhibition of PDE6, this may explain, in induced vasodilation (Fig. 5). It appears that the specific acti- part, the transient visual disturbances associated with using

vation of KATP channels is involved, since glibenclamide abol- sildenafil or other PDE5 inhibitors for the treatment of erectile ished the vasodilatory response to sildenafil (Fig. 2). These dysfunction,2 especially in patients with cardiovascular results are consistent with our previous findings showing that risks.42,43 NO/cGMP-induced dilation in the porcine retinal arterioles is In summary, the present study demonstrates that sildenafil, 16 mediated by the opening of KATP channels. at clinical doses, elicits a modest dilation of porcine retinal The complete inhibition of sildenafil-induced dilation by arterioles via activation of NOS through ERK signaling and the L-NAME is unanticipated, because sildenafil is a PDE5 inhibitor subsequent activation of guanylyl cyclase. The guanylyl cy- and is expected to act downstream of NO to elevate cGMP for clase/cGMP signaling triggers the opening of the KATP channels vasodilation (Fig. 5), unless the source of cGMP is totally for vasodilation. Moreover, the elevated cGMP, by endogenous eliminated and the resting level of cGMP and/or the activity of or exogenous NO, plays a permissive role in sildenafil-induced PDE5 is too low to be functional. Since the resting level of dilation through the inhibition of PDE5 in an ERK-independent cGMP is determined by the activity of both guanylyl cyclase manner. (for synthesis) and PDE5 (for degradation), the slight effect of ODQ and L-NAME on resting tone and the failure of sildenafil to References produce dilation in the presence of these antagonists indicates 1. Vobig MA, Klotz T, Staak M, Bartz-Schmidt KU, Engelmann U, that the NO derived from NOS is the sole source for cGMP and Walter P. Retinal side-effects of sildenafil. Lancet. 1999;353:375. that the resting level of NO/cGMP may have been too low to 2. Fraunfelder FW. Visual side effects associated with erectile dys- allow sildenafil to exert its vasodilatory influence via the inhi- function agents. Am J Ophthalmol. 2005;140:723–724. bition of PDE5. Alternatively, the PDE5 may be inactive in 3. Marmor MF, Kessler R. Sildenafil (Viagra) and ophthalmology. Surv retinal arterioles. Of interest, in the presence of L-NAME, the Ophthalmol. 1999;44:153–162. retinal arterioles dilated in response to sildenafil when exoge- 4. Hayreh SS. Erectile dysfunction drugs and non-arteritic anterior nous NO (i.e., SNAP) was provided (Fig. 4). This result sup- ischemic optic neuropathy: is there a cause and effect relation- ports the functional activity of PDE5 in modulating NO-medi- ship? J Neuroophthalmol. 2005;25:295–298. ated vasodilation. It appears that a sufficient provision of NO, 5. Pomeranz HD, Smith KH, Hart WM Jr, Egan RA. Sildenafil-associ- thus cGMP, to the vessel is essential in terms of allowing retinal ated nonarteritic anterior ischemic optic neuropathy. Ophthalmol- arterioles to respond to sildenafil through the PDE5-inhibition ogy. 2002;109:584–587. pathway. Therefore, the release of NO from activated NOS 6. Sponsel WE, Paris G, Sandoval SS, et al. Sildenafil and ocular perfusion. N Engl J Med. 2000;342:1680. plays not only a direct role in sildenafil-induced vasodilation, 7. Paris G, Sponsel WE, Sandoval SS, et al. Sildenafil increases ocular but also a permissive role in modulating this dilation via inhi- perfusion. Int Ophthalmol. 2001;23:355–358. bition of PDE5. In contrast to NOS activation, this PDE5 inhi- 8. Grunwald JE, Siu KK, Jacob SS, Dupont J. Effect of sildenafil citrate bition pathway was insensitive to PD98059 (Fig. 4), suggesting (Viagra) on the ocular circulation. Am J Ophthalmol. 2001;131: that the vasodilation mediated by PDE5 inhibition is indepen- 751–755. dent of ERK signaling. This finding is consistent with the idea 9. McCulley TJ, Luu JK, Marmor MF, Feuer WJ. Effects of sildenafil that the vasodilation elicited by the NO/cGMP pathway (i.e., citrate (Viagra) on choroidal congestion. Ophthalmologica. 2002; downstream from NOS) is not mediated by the ERK activation 216:455–458.

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