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Originally published In Press as doi:10.1167/iovs.08-3129 on January 17, 2009
 (Investigative Ophthalmology and Visual Science. 2009;50:3329-3336.)
© 2009 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.08-3129
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Functional and Molecular Characterization of the Endothelin System in Retinal Arterioles

Travis W. Hein,1,2 Yi Ren,3 Zhaoxu Yuan,1,2 Wenjuan Xu,1,2 Sonal Somvanshi,3 Taiji Nagaoka,4 Akitoshi Yoshida,4 and Lih Kuo1,2,3

1From the Departments of Ophthalmology and 2Surgery, Scott and White Eye Institute, Temple, Texas; the 3Department of Systems Biology and Translational Medicine, College of Medicine, Texas A&M Health Science Center, Temple, Texas; and the 4Department of Ophthalmology, Asahikawa Medical College, Asahikawa, Japan.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Activation of the endothelin (ET) system has been implicated in the pathogenesis of retinal ischemic disease. Although ET-1, the predominant endogenous isoform of ET, has been shown to cause constriction of retinal vessels, the expression and functional significance of its synthesis and the involved specific ET receptors in retinal arterioles remain unknown. The authors examined the roles of ETA and ETB receptors and of endothelin-converting enzyme (ECE)-1 in ET-1–induced vasomotor responses of single retinal arterioles.

METHODS. To exclude systemic confounding effects, porcine retinal arterioles were isolated for vasoreactivity and molecular studies.

RESULTS. Isolated and pressurized retinal arterioles developed basal tone and constricted in a manner dependent on concentration to ET-1. ET-1 precursor big ET-1 elicited time-dependent vasoconstriction over 20 minutes, which was blocked by the ECE-1 inhibitor phosphoramidon. ETA receptor antagonist BQ123 inhibited most (approximately 90%) of vasoconstrictions to ET-1 and big ET-1. ETB receptor agonist sarafotoxin also elicited concentration-dependent constriction of retinal arterioles but with significantly less potency than ET-1. ETB receptor antagonist BQ788 abolished vasoconstriction to sarafotoxin but only slightly reduced responses to ET-1 and big ET-1. Protein and mRNA expressions of ETA, ETB, and ECE-1 were detected in retinal arterioles. Immunohistochemistry revealed ETA and ETB receptors predominantly in smooth muscle and ECE-1 predominantly in endothelium and smooth muscle.

CONCLUSIONS. ET-1 elicits constriction of retinal arterioles predominantly through the activation of smooth muscle ETA receptors. Endogenous production of ET-1 from vascular ECE-1 is sufficient to evoke ETA receptor–dependent constriction in retinal arterioles.

Endothelin (ET) was originally discovered as a potent vasoconstrictor peptide produced by and released from vascular endothelial cells.1 However, accumulating evidence also shows that ET can be synthesized from vascular smooth muscle cells in nonocular vasculature2 3 4 and from nonvascular cells such as neuronal and glial cells in the retina.5 Three isoforms of ET—ET-1, ET-2, and ET-3—have been identified, with ET-1 the predominant biologically active peptide.6 Cells initially produce the ET-1 precursor, preproendothelin-1, which is subsequently processed to yield a biologically inactive intermediate, big ET-1.6 This precursor peptide is proteolytically cleaved by endothelin-converting enzyme (ECE)-1 to generate ET-1.6 7 8 The functional effects of ET-1 are mediated by activation of two receptors, ETA and ETB. Activation of both receptors on the vascular smooth muscle leads to sustained vasoconstriction,1 whereas the activation of endothelial ETB receptors promotes vasodilation.9 10

Several in vitro (bovine, porcine, human)11 12 13 and in vivo (rabbit, rat, cat, human)14 15 16 17 18 19 studies have demonstrated potent ET-1–mediated vasoconstriction in the normal retinal circulation. Enhanced activation of the ET system has been implicated in the reduction of retinal vessel diameter during isometric exercise20 and of retinal blood flow under conditions of retinal hyperoxia,21 22 23 ischemia,24 and diabetes.19 25 Furthermore, several ocular diseases are associated with ET system activation; elevated levels of ET-1 have been observed in the plasma of patients with retinal occlusions of the ischemic type26 27 and with progressive open-angle glaucoma28 and in the vitreous humor of patients with diabetic retinopathy.29 30 Although autoradiography and immunoreactivity of ET receptors and ECE-1 have been suggested in the retinal tissue of animals and humans,31 32 33 the cellular localization/expression and the functional significance of specific ET receptors and ECE-1 in small retinal arterioles, the site for flow regulation, have not been established. To address these issues directly without the confounding influences of metabolic, hemodynamic, humoral, and glial/neuronal factors associated with in vivo preparations, porcine retinal arterioles were isolated and pressurized without flow for in vitro functional and molecular studies. We examined the vasomotor action of ET-1 and big ET-1 and elucidated the relative functional role and molecular distribution of ETA/ETB receptors and ECE-1 in single retinal arterioles.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animal Preparation
All animal procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Scott and White Institutional Animal Care and Use Committee. Pigs of either sex (age range, 8–12 weeks; weight range, 7–10 kg) purchased from Barfield Farms (Rogers, TX) were sedated with telazol (4.4 mg/kg, intramuscularly) and xylazine (2.2 mg/kg, intramuscularly), anesthetized with sodium pentobarbital (30 mg/kg, intravenously), intubated, and ventilated with room air. Heparin (1000 U/kg) was administered into the marginal ear vein to prevent clotting. Eyes were enucleated and immediately placed in a moist chamber on ice.

Isolation and Cannulation of Microvessels
Techniques for identifying and isolating retinal microvessels were described previously.34 35 36 After enucleation, the eyecup was placed in a cooled dissection chamber (approximately 8°C) containing a physiological salt solution (PSS; 145.0 mM NaCl, 4.7 mM KCl, 2.0 mM CaCl2, 1.17 mM MgSO4, 1.2 mM NaH2PO4, 5.0 mM glucose, 2.0 mM pyruvate, 0.02 mM EDTA, and 3.0 mM MOPS) with 0.1% albumin (USB, Cleveland, OH). Single second-order retinal arterioles (ranges: 40–60 µm internal diameter in situ, 0.6–1.0 mm length) were carefully dissected with the aid of a stereomicroscope (model SZX12; Olympus, Melville, NY). After the removal of any remaining neural/connective tissues, the arteriole was transferred to a polymethylmethacrylate vessel chamber containing PSS-albumin solution equilibrated with room air at ambient temperature. One end of the arteriole was cannulated using a glass micropipette filled with PSS containing 0.1% albumin, and the outside of the arteriole was securely tied to the pipette with 11-0 ophthalmic suture (Alcon, Fort Worth, TX). The other end of the vessel was cannulated and secured with suture. After cannulation, the vessel and pipettes were transferred to the stage of an inverted microscope (model CKX41; Olympus) coupled to a video camera (DXC-190; Sony, Tokyo, Japan), video micrometer (Cardiovascular Research Institute, Texas A&M Health Science Center, College Station, TX), and data acquisition system (PowerLab; ADInstruments, Colorado Springs, CO) for recording of internal diameter.34 The vessels were pressurized to 55 cm H2O (40 mm Hg) intraluminal pressure without flow by two independent pressure reservoirs. This level of pressure was used based on pressure ranges documented in retinal arterioles in vivo37 and in the isolated, perfused retinal microcirculation,38 and was consistent with estimated ocular perfusion pressure in humans, as reported previously.39

Experimental Protocols
Cannulated arterioles were bathed in PSS-albumin at 36°C to 37°C to allow development (approximately 60 minutes) of basal tone. In one series of studies, the vasomotor response to cumulative administration of ET-1 (0.1 pM to 10 nM), selective ETB receptor agonist sarafotoxin S6c (0.1 pM to 10 nM; Tocris Cookson, Ellisville, MO),40 or ET-1 precursor big ET-1 (10 nM, 50 nM, 100 nM) was evaluated. Concentrations of ET-1 used in the present study were within the clinical and experimental range reported for vitreous fluid (picomolar range)30 and the predicted level for local vasculature (nanomolar range).41 Retinal arterioles were exposed to each concentration of ET receptor agonists for 5 minutes or to big ET-1 for 20 minutes until a stable diameter was established. Because the vasoconstrictor actions of ET-1, sarafotoxin, and big ET-1 were maintained after washout, only one dose-response curve was constructed in each vessel for these drugs.

Relative roles of ETA and ETB receptors in the retinal arteriolar response to ET agonists were evaluated after incubation of the vessels with respective antagonists BQ123 (1 µM)42 43 and BQ788 (0.1 µM).43 44 In another set of vessels, the specificity of the ET receptor antagonists was assessed by examining their ineffectiveness on vasoconstriction to thrombin (0.1 U/mL). To differentiate the vasomotor effect of ET-1 from big ET-1 and to determine the functional importance of endogenously synthesized ET-1 for vasoconstriction, another series of experiments was performed in the presence of ECE-1 inhibitor phosphoramidon (50 µM), which can prevent the conversion of big ET-1 to ET-1.4 45 Given that phosphoramidon can also inhibit neutral endopeptidase (NEP), some vessels were examined in the presence of the selective NEP inhibitor thiorphan (10 µM)46 for comparison. All vessels were pretreated with antagonists or inhibitors extraluminally for at least 30 minutes.

Chemicals
Drugs were obtained from Sigma-Aldrich (St. Louis, MO) except where specifically stated otherwise. ET-1, big ET-1, sarafotoxin, BQ788, phosphoramidon, and thrombin were dissolved in water, whereas BQ123 and thiorphan were dissolved in ethanol. Subsequent concentrations of these drugs were diluted in PSS. The final concentration of ethanol in the vessel bath was less than 0.1%. Vehicle control studies indicated that this final concentration of solvent had no effect on the arteriolar function.

RNA Isolation and Reverse Transcription–Polymerase Chain Reaction Analysis
Total RNA was isolated from retinal arterioles (12–16 vessels/sample pooled from both eyes) and neural retina tissue, as described previously.36 Sets of primers specific for ETA receptor (GenBank accession no. NM214229; sense, 1102–1125 bp; antisense, 1321–1344 bp), ETB receptor (GenBank accession no. L06623; sense, 1200–1223 bp; antisense, 1521–1544 bp), ECE-1 (GenBank accession no. NM001397; sense, 2041–2064 bp; antisense, 2290–2313 bp), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; GenBank accession no. U48832; sense, 106–129 bp; antisense, 393–416 bp) genes were engineered (Sigma-Genosys, The Woodlands, TX). With the use of 0.1 and 0.3 µg total RNA for GAPDH and ET receptor/ECE-1 samples, respectively, RT-PCR was conducted as described previously.36 The PCR reaction was optimized and run for 35 cycles for all genes. The level of expression of ET receptor or ECE-1 transcripts was normalized to that of GAPDH transcripts. For some samples, the relative purity of vessels and neural retina was verified by detection of endothelial nitric oxide synthase (eNOS) and {alpha}1-adrenergic receptor ({alpha}1-AR) transcripts, respectively, as shown in our previous study.36

Western Blot Analysis
Retinal arterioles (12–16 vessels/sample) and neural retina tissue were isolated and sonicated in lysis buffer. Porcine lung tissue was used as positive control for the detection of ET receptors and ECE-1. The protein content of each lysate was determined with the BCA protein assay kit (Pierce, Rockford, IL). Equal amounts of protein (10 µg) were separated by Tris-glycine SDS-PAGE (4%–15% Tris-HCl Ready Gels; Bio-Rad, Hercules, CA), transferred onto a nitrocellulose membrane, and incubated with rabbit anti–ETA or anti–ETB receptor polyclonal antibody (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) or with rat anti–ECE-1 monoclonal antibody (1:250; R&D Systems, Minneapolis, MN). Membranes were stripped and reprobed with rabbit anti–{alpha}-smooth muscle actin antibody (1:1000; Sigma-Aldrich) to verify the relative purity of samples or with rabbit anti–p38 antibody (1:1000; Santa Cruz Biotechnology), which has been shown to be highly expressed in retinal homogenates.47 After incubation with appropriate secondary antibody, the membranes were washed and developed by enhanced chemiluminescence (Pierce). Densitometric analyses of immunoblots were performed by ImageJ software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html). Results for total ETA and ETB in vessel samples were normalized to total {alpha}-smooth muscle actin.

Immunohistochemistry
To identify and localize vascular ET system components, retinal arterioles were embedded and frozen in OCT compound (Tissue-Tek; Sakura Finetek, Torrance, CA). Frozen sections (10-µm thick) were fixed in 4% paraformaldehyde and immunolabeled with specific primary antibodies (ETA and ETB [Sigma-Aldrich]; ECE-1 [Santa Cruz Biotechnology]) and either an anti–{alpha}-smooth muscle actin antibody (Sigma-Aldrich) or an anti–eNOS (Santa Cruz Biotechnology) antibody. Afterward, the slides were incubated with rhodamine red-labeled (Jackson Laboratory, Bar Harbor, ME) or FITC-labeled (Jackson Laboratory) secondary antibodies. Staining control tissues were exposed for the same duration to nonimmune serum (Jackson Laboratory) in place of primary antibody. Slides were observed for red (rhodamine red) and green (FITC) images, and were analyzed under a fluorescence microscope (Axiovert 200; Zeiss, Thornwood, NY). Merged images were created with ImageJ software (NIH).

Data Analysis
At the end of each functional experiment, the vessel was relaxed with 0.1 mM sodium nitroprusside in EDTA (1 mM)-calcium-free PSS to obtain its maximal diameter at 55 cm H2O intraluminal pressure.34 Diameter changes in response to the ET agonists were normalized to the resting diameter and expressed as percentage changes in diameter. The median effective concentration (EC50) value for the response to ET-1 was calculated (Prism software; GraphPad, San Diego, CA). Data are reported as mean ± SEM, and n represents the number of vessels (one per pig) studied. One-way analysis of variance (ANOVA) followed by Dunnett post hoc comparison was used to determine the significance of changes in baseline diameter by different concentrations of ET agonists. Student’s t-test or two-way ANOVA followed by Bonferroni multiple-range test was used to determine the significance of experimental interventions and gene/protein expression, as appropriate. P < 0.05 was considered significant.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Vasomotor Response to ET Receptor Activation
In this study, all vessels developed a similar level of basal tone (constricted to 61% ± 1% of maximal diameter) at 36°C to 37°C bath temperature with 55 cm H2O intraluminal pressure. Average resting and maximal diameters of the vessels were 55 ± 1 µm (range, 31–78 µm) and 90 ± 1 µm (range, 48–110 µm), respectively. Administration of ET-1 (1 nM) produced rapid (initiated within 1 minute) and sustained constriction of an isolated arteriole from the baseline diameter of 62 µm to 36 µm (Fig. 1A) . Further study showed that ET-1 produced concentration-dependent constriction of retinal arterioles with threshold response at 1 pM. The EC50 of ET-1 was 0.14 nM, and the highest concentration (10 nM) of ET-1 caused a 75% reduction in resting diameter (Fig. 1B) . The ETA receptor antagonist BQ123 abolished the vasoconstriction, but 15% vasoconstriction remained at the highest concentration of ET-1 (10 nM; Fig. 1B ). In vessels treated with BQ123, lower concentrations of ET-1 (1–100 pM) tended to cause vasodilation (5%–10% increase in resting diameter) but did not reach statistical significance. In another group of vessels, the ETB receptor antagonist BQ788 only attenuated the vasoconstriction to higher concentrations of ET-1 (i.e., 1 nM and 10 nM) by approximately 10% to 15% (Fig. 1B) . In the presence of BQ123 and BQ788, the vasomotor responses to ET-1 were abolished (Fig. 1B) . Inhibitory effects appeared to be specific for ET receptor activation because, in the presence of both antagonists, the vasoconstriction to thrombin (0.1 U/mL) was not altered (control, 46% ± 5%; BQ123 + BQ788, 44% ± 2%; n = 4; P = 0.74, unpaired t-test). We also found that administration of the selective ETB agonist sarafotoxin caused a concentration-dependent constriction of retinal arterioles with a threshold response at 10 pM, but the response was modest, and the highest concentration (10 nM) only caused approximately 15% reduction in resting diameter (Fig. 2) . The vasoconstrictor response was abolished by BQ788 (Fig. 2) but unaffected by BQ123 (Fig. 2) .


Figure 1
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  		FIGURE 1. Vasomotor response of isolated retinal arterioles to ET-1. (A) Representative tracing shows ET-1 (1 nM)-induced constriction of retinal arterioles initiated within 1 minute. (B) Dose-dependent change in resting diameter from baseline (B) in response to ET-1 was examined in the absence (control, n = 8) or presence of ETB receptor antagonist BQ788 (0.1 µM, n = 8), ETA receptor antagonist BQ123 (1 µM, n = 8), or both BQ123 and BQ788 (n = 5). *P < 0.05 versus baseline. {dagger}P < 0.05 versus control.

 

Figure 2
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  		FIGURE 2. Vasomotor response of isolated retinal arterioles to sarafotoxin. Dose-dependent change in resting diameter from baseline (B) in response to ETB receptor agonist sarafotoxin was examined in the absence (control, n = 8) or presence of ETB receptor antagonist BQ788 (0.1 µM, n = 6) or ETA receptor antagonist BQ123 (1 µM, n = 5). *P < 0.05 versus baseline. {dagger}P < 0.05 versus control.

 
Vasoconstriction of Retinal Arterioles to Big ET-1
To determine whether retinal arterioles exhibit functional ECE-1 activity, vasomotor response to the ECE-1 substrate big ET-1 was examined. Figure 3A shows a representative tracing of the vasoconstrictor response of an isolated retinal arteriole to big ET-1. Big ET-1 (100 nM) elicited relatively slow vasoconstriction (initiated within 5 minutes) from the baseline diameter of 50 µm and stabilized at 25 µm after 15 to 20 minutes (Fig. 3A) . Figure 3B shows concentration-dependent constriction of retinal arterioles to big ET-1. Vasoconstrictor responses to 50 nM and 100 nM big ET-1 were significantly attenuated by the ECE-1 inhibitor phosphoramidon and by BQ123. By contrast, BQ788 reduced vasoconstriction to 100 nM big ET-1 by approximately 10% without affecting the response to 50 nM big ET-1 (Fig. 3B) . Because the selective NEP inhibitor thiorphan did not alter vasoconstriction to big ET-1 (100 nM; control, 56% ± 4%, n = 9; thiorphan, 48% ± 5%, n = 5; P = 0.32, unpaired t-test) and phosphoramidon did not alter vasoconstriction to ET-1 (10 nM; control, 75% ± 4%, n = 8; phosphoramidon, 65% ± 8%, n = 5; P = 0.24, unpaired t-test), these results suggest that phosphoramidon selectively blocked the action of ECE-1.


Figure 3
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  		FIGURE 3. Vasomotor response of isolated retinal arterioles to big ET-1. (A) Representative tracing shows big ET-1 (100 nM)-induced constriction of retinal arterioles initiated within 5 minutes. (B) Dose-dependent change in resting diameter from baseline (B) in response to big ET-1 was examined in the absence (control, n = 9) or presence of ECE-1 inhibitor phosphoramidon (50 µM, n = 5), ETB receptor antagonist BQ788 (0.1 µM, n = 5), or ETA receptor antagonist BQ123 (1 µM, n = 5). *P < 0.05 versus baseline. {dagger}P < 0.05 versus control.

 
ET Receptor and ECE-1 mRNA Expression in Retinal Arterioles
Figure 4 shows that ETA, ETB, and ECE-1 mRNA were detected in retinal arterioles and in the neural retina tissue devoid of retinal vessels. Samples were relatively homogeneous because eNOS transcripts were detected only in the retinal arterioles, whereas {alpha}1-AR was observed only in the neural retina tissue (Fig. 4A) . The expression of ETB receptor mRNA in retinal arterioles and neural retina, after normalization with GAPDH, was consistently higher than was the corresponding ETA receptor expression (Fig. 4B) . Normalization of ECE-1 and both ET receptors showed greater expression of all three transcripts in retinal arterioles than in neural retina tissue (Fig. 4B) .


Figure 4
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  		FIGURE 4. RT/PCR analyses of ET receptors and ECE-1 mRNA expression in porcine retinal arterioles. (A, upper) Equal amounts of total RNA isolated from normal retinal arterioles and neural retina tissue were reversed transcribed using gene-specific primers for ETA receptor (243 bp), ETB receptor (345 bp), and ECE-1 (273 bp) mRNA. After 35 cycles of PCR, gene products were electrophoresed on a 1.8% agarose gel and visualized with ethidium bromide staining. HaeIII-restricted {phi}X174-DNA was used as a size marker. Data are representative of three independent experiments. Lower: RT/PCR gene products for eNOS (342 bp) or {alpha}1-AR receptor (178 bp) mRNA from retinal arterioles and neural retina tissue. (B) ETA/B and ECE-1 transcripts for neural retina tissue and retinal arterioles were normalized with corresponding GAPDH transcripts. *P < 0.05 versus neural retina tissue.

 
ET Receptor and ECE-1 Protein Expression and Localization in Retinal Arterioles
At the protein level, immunoblotting showed that ETA and ETB receptors were expressed in retinal arterioles and in neural retina tissue, with a markedly higher amount in the former (Fig. 5A) . Moreover, ETA expression was significantly higher than ETB expression in retinal arterioles (P = 0.03; Fig. 5B ). ECE-1 protein was detected in retinal arterioles (Fig. 5A) , but a greater amount (50 µg vs. 10 µg) was required to detect ECE-1 in neural retina than in the vessels (data not shown). Vascular and neural tissue samples were relatively homogeneous because {alpha}-smooth muscle actin protein was detected only in retinal arterioles (Fig. 5A) . Sufficient neural retina protein was present because p38 expression was consistently higher in neural retina tissue than in retinal arterioles (Fig. 5A) . For cellular localization of proteins, immunohistochemical data show that eNOS and {alpha}-smooth muscle actin were distinctly distributed in the endothelium and smooth muscle, respectively, as evident by the lack of overlap in the merged image (Fig. 6A) . ETA receptor protein appeared to be localized exclusively to the smooth muscle layer, as evident by the overlap with {alpha}-smooth muscle actin (yellow staining in merged image; Fig. 6B ) but not with eNOS (Fig. 6C) . Modest ETB receptor staining was observed in the outer aspect of the smooth muscle layer (Fig. 6D) , and only slight overlap with eNOS was detected. On the other hand, ECE-1 staining was detected in smooth muscle and endothelial layers, with the latter showing overlap with eNOS (yellow staining in merged image; Fig. 6E ).


Figure 5
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  		FIGURE 5. Western blot analyses of ET receptors and ECE-1 protein expression in porcine retinal arterioles. (A) Immunoblots show detection of ETA and ETB in retinal arterioles (RA) and in neural retina tissue (RT) but ECE-1 expression only in retinal arterioles. ET receptors and ECE-1 were also detected in the porcine lung tissue (positive control). {alpha}-Smooth muscle actin (SMA) was detected only in retinal arterioles; greater expression of p38 was present in neural retina than in retinal arterioles. (B) ET receptors for retinal arterioles were normalized with corresponding SMA. Data represent three independent experiments. *P < 0.05 versus ETA.

 

Figure 6
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  		FIGURE 6. Immunohistochemical detection of ET receptors and ECE-1 in isolated retinal arterioles. (A) In the presence of anti-eNOS (red) or anti–{alpha}-smooth muscle actin (SMA, green) antibodies, high levels of immunostaining were detected in endothelial (arrowhead) and smooth muscle (arrow) layers, respectively. The merged image shows the lack of overlap staining. (B) In the presence of anti–ETA receptor (green) or anti–SMA (red) antibodies, high levels of immunostaining were detected in the smooth muscle for both proteins, which was confirmed by overlap staining (yellow). (C) In the presence of anti–ETA receptor (green) or anti–eNOS (red) antibodies, selective immunostaining was detected in smooth muscle for ETA receptors and endothelium for eNOS, respectively. The merged image shows the lack of overlap staining. (D) In the presence of anti–ETB receptor (green) or anti–eNOS (red) antibodies, immunostaining was detected in smooth muscle and endothelium for ETB receptor and in endothelium for eNOS. The merged image shows slight overlap staining. (E) In the presence of anti–ECE-1 (green) or anti–eNOS (red) antibodies, immunostaining was detected in the endothelium and smooth muscle for ECE-1 and in the endothelium for eNOS, respectively. The merged image shows overlap staining (yellow) in the endothelial layer. White arrowheads: endothelial cells. Arrows: vascular smooth muscle cells. Data shown are representative of three separate experiments.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
ET-1 is a potent vasoactive peptide that has been implicated in modulating retinal microvascular tone. The putative regulatory role of ET-1 is based on observations in animal and human studies showing that intravitreal14 15 16 17 19 or intravenous18 administration of ET-1 constricts retinal arteries and reduces retinal blood flow, respectively, in vivo. In addition, direct evidence for the vasoconstrictor action of ET-1 has been described in isolated bovine retinal conduit arteries12 and large porcine arterioles (first-order feeding arterioles without tone).11 However, the sensitivity and threshold concentrations of ET-1 and the receptor subtypes responsible for its vasomotor action have not been documented. It is also unclear whether retinal arterioles are capable of synthesizing ET-1 to exert vasomotor activity. Our present results demonstrate that exposure of isolated small porcine retinal arterioles to ET-1 causes constriction, predominantly through smooth muscle ETA receptor activation, with sensitivity and threshold levels of 0.14 nM and 1 pM, respectively. We also provide the first direct evidence that retinal arterioles have ECE-1 and that its activation is sufficient to elicit ET-1–mediated, ETA-dependent constriction of retinal arterioles.

Although previous in vivo studies have shown ET-1 causes constriction and decreases blood flow,15 16 18 the unequivocal determination of the direct role of ET-1 in vascular regulation in vivo is difficult because diameter changes and blood flow regulation under this situation are influenced by hemodynamic (i.e., myogenic and flow-induced responses) and local metabolic control mechanisms.48 49 Additional limitations of these in vivo studies are the inability to measure diameter changes in small retinal arterioles, which play a predominant role in retinal blood flow regulation, because of the limited resolution of the detection instrument15 16 18 and the inability to distinguish between vascular and neural/glial ET system components. In the present study, we used an isolated vessel approach to study the direct vasomotor influence of ET receptor and ECE-1 activation on small retinal arterioles and to identify the molecular distribution of the ET system in these microvessels.

To characterize the functional role of specific ET receptor subtypes, we examined the vascular response to ET-1, a nonselective ETA and ETB agonist, and to sarafotoxin, a selective ETB agonist. Both agonists caused concentration-dependent constriction; however, the maximal response to ET-1 was approximately fivefold greater. ET-1–induced vasoconstriction appeared to be mediated primarily by the ETA receptor subtype because selective ETA blocker BQ123 abolished the vasoconstriction except at the highest concentration of ET-1. Given that the ETB antagonist BQ788 completely inhibited sarafotoxin-induced vasoconstriction and only slightly attenuated constriction in response to the higher concentrations of ET-1 (1 and 10 nM), it appears that high concentrations of ET-1 also activate ETB receptors for vasoconstriction. The vasoconstriction in response to ET-1 in the range of 1 pM to 1 nM observed in the present study is considered pathophysiologically relevant because the level of ET-1 in the vitreous and in the vascular wall can reach picomolar30 and nanomolar41 ranges, respectively. The EC50 (0.14 nM) of ET-1 was comparable to that reported for isolated, pressurized resistance arteries in other tissues, such as coronary (0.57 nM)50 and intracerebral (4.8 pM)51 arterioles. Our results are also consistent with the estimated EC50 (0.50 nM) of ET-1 in the retinal circulation in vivo.15 Interestingly, a recent clinical study in healthy subjects has shown that intravenous administration of ET-1 did not alter the diameters of large retinal arteries but significantly reduced retinal blood flow,18 suggesting that ET-1 exerts its vasoconstrictor action primarily on downstream small retinal arterioles. Consistent with BQ123 administration blunting the ET-1–induced reduction in human retinal blood flow,18 our findings support the predominant role of ETA receptors in mediating ET-1–induced constriction of small retinal arterioles.

Previous results in other vascular beds indicate that ETB receptors are located mainly on endothelial cells9 52 and that activation of these receptors mediate vasodilation by the release of nitric oxide.9 10 52 In the present study, we only observed a tendency for some retinal arterioles to increase (5%–10%) resting diameter in response to ET-1 (1–100 pM) in the presence of ETA receptor blockade (Fig. 1) , suggesting a minor role of ETB receptor in vasomotor regulation of retinal arterioles. Moreover, in the presence of ETA receptor blocker, the activation of ETB receptors by sarafotoxin only elicited vasoconstriction (Fig. 2) . It appears that the component of endothelial ETB receptor-mediated vasodilation contributes little, if at all, to the vasomotor action of ET-1 in the porcine retinal circulation.

With the use of autoradiography and pharmacologic binding analysis, previous studies demonstrated that ETA receptors are present in human and rabbit retinal blood vessels and that ETB receptors are localized in the neural/glial cells of both species.31 On the other hand, ETA and ETB receptors have been detected in the neural retina tissue of rats.53 Unfortunately, the size and type (i.e., arteries or veins) of blood vessels studied in those previous reports are unclear, as is the molecular distribution of the ET receptors in the retinal microcirculation. In the present study, we showed the expression of ETA and ETB receptor mRNA and protein in retinal arterioles, which provided additional evidence for the functional roles of these two subtypes in ET-1–induced vasomotor responses. The mRNA and the protein expression of both ET receptor subtypes was also detected in neural retina tissue but was strikingly lower, especially at the protein level, than in retinal arterioles. Because ETB expression was higher than ETA expression at the mRNA but not the protein level in retinal arterioles, it appeared that functional ET receptors might have been regulated at the translational level. Interestingly, it has been reported that ETB receptor-mediated vasoconstriction can be enhanced after ischemic stress,54 possibly through a mechanism that selectively regulates ETB receptor expression.55 Although ETB-mediated retinal arteriolar constriction to ET-1 was moderate in the present study, its pathophysiological potential may be implicated during disease development.

Immunohistochemical analysis of single intact retinal arterioles indicated that the ETA receptors are selectively expressed in the smooth muscle layer, whereas the ETB receptors are located mainly at the outer smooth muscle cells. There was only punctate, overlap staining for ETB with eNOS, indicating sparse expression of ETB in the endothelium and supporting the lack of a significant endothelial ETB-mediated dilation in response to ET-1 or sarafotoxin. Collectively, our molecular data corroborate the functional data by showing distinct cellular distribution of ETA and ETB receptors in the porcine retinal arteriolar wall.

Previous studies have shown that ECE-1 protein expression and activity are present in human32 and bovine33 retinal tissue, respectively. The present results, to our knowledge, provide the first evidence of ECE-1 mRNA/protein expression and ECE-1 activity in the retinal microvasculature. Support for functional ECE-1 activity was evident by the ability of ET-1 precursor, big ET-1, to constrict isolated retinal arterioles. The initiation of the vasoconstrictor response to big ET-1 was slower (5 minutes) than that to ET-1 (1–2 minutes), which is consistent with the required conversion of big ET-1 to ET-1 by ECE-1 to exert vasomotor activity. This conclusion was supported by the ability of the ECE-1 inhibitor phosphoramidon to inhibit vasoconstriction to big ET-1 but not to ET-1. The prevention of retinal arteriolar constriction to big ET-1 by BQ123 and the weak inhibitory effect of BQ788 indicates that ETA receptor activation is primarily responsible for the observed vasomotor activity after ECE-1 activation. The functional role of vascular ECE-1 is further supported by our molecular findings showing ECE-1 mRNA and protein expression in isolated retinal arterioles. We also detected ECE-1 mRNA and protein in neural retina tissue; however, the expression levels were significantly less than those in the retinal vessels. Interestingly, immunohistochemical analysis showed ECE-1 staining in endothelial and smooth muscle layers. It is speculated that the widespread distribution of ECE-1 in the vascular wall may contribute to the high susceptibility of the vasculature to pathophysiological insults during retinal disease development/progression in association with ET-1 elevation.19 25 26 28 29 30

In summary, the present study demonstrates that a functional ET system is expressed in porcine retinal arterioles. Endogenous production of ET-1 from endothelial/smooth muscle ECE-1 is capable of evoking the constriction of retinal arterioles through smooth muscle ETA receptor activation. Because of the potential influence of ET-1 on local retinal blood flow, better understanding of the ET system in the retinal circulation could lead to new therapeutic modalities for retinal vascular diseases elicited by ET-1.


   Footnotes
 
Supported by Scott and White Research Foundation (TWH), Retina Research Foundation (TWH, LK); National Institutes of Health/National Eye Institute Grant R01EY018420 (TWH); the Scott and White Research Foundation Ophthalmic Vascular Research Program (LK); and the Kruse Family Endowment Fund (LK).

Submitted for publication November 8, 2008; revised December 16, 2008; accepted April 6, 2009.

Disclosure: T.W. Hein, None; Y. Ren, None; Z. Yuan, None; W. Xu, None; S. Somvanshi, None; T. Nagaoka, None; A. Yoshida, None; L. Kuo, None

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: Travis W. Hein, Departments of Ophthalmology and Surgery, Scott and White Eye Institute, 702 Southwest H. K. Dodgen Loop, Temple, TX 76504; thein{at}tamu.edu.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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