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Am J Physiol Heart Circ Physiol 291: H231-H238, 2006. First published February 17, 2006; doi:10.1152/ajpheart.01281.2005
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Brimonidine evokes heterogeneous vasomotor response of retinal arterioles: diminished nitric oxide-mediated vasodilation when size goes small

Robert H. Rosa, Jr.,1 Travis W. Hein,1 Zhaoxu Yuan,1 Wenjuan Xu,1 Melissa I. Pechal,1 Ryan L. Geraets,1 Joseph M. Newman,1 and Lih Kuo1,2

1Departments of Ophthamology and Surgery, Scott and White Eye Institute; and 2Department of Systems Biology and Translational Medicine, Texas A&M University System Health Science Center, Temple, Texas

Submitted 6 December 2005 ; accepted in final form 10 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Brimonidine, an {alpha}2-adrenergic receptor (AR) agonist, has been employed in the treatment of glaucoma due to its beneficial effects on intraocular pressure reduction and neuroprotection. In addition, some studies have implicated that brimonidine might influence ocular blood flow; however, its effect on the retinal microcirculation has not been documented. Herein, we examined the vasomotor action of brimonidine on different branching orders of retinal arterioles in vitro and determined the contribution of the {alpha}2-AR subtype and the role of endothelium-derived nitric oxide (NO) in this vasomotor response. First- and second-order retinal arterioles of pigs were isolated, cannulated, and pressurized for functional studies. Videomicroscopic techniques were employed to record diameter changes in response to brimonidine. RT-PCR was performed for detection of {alpha}-AR and endothelial NO synthase (eNOS) mRNA in retinal arterioles. All first-order arterioles (82 ± 2 µm ID) dilated dose dependently to brimonidine (0.1 nM to 10 µM) with 10% dilation at the highest concentration. Second-order arterioles (50 ± 1 µm ID) responded heterogeneously with either dilation or constriction. The incidence and magnitude of vasoconstriction were increased with increasing brimonidine concentration. Administration of the NO synthase inhibitor NG-nitro-L-arginine methyl ester abolished the brimonidine-induced vasodilation in first- and second-order arterioles. Regardless of vessel size, vasomotor responses (i.e., vasodilation and vasoconstriction) of retinal arterioles were sensitive to the {alpha}2-AR antagonist rauwolscine. Consistent with the functional data, {alpha}2A-AR and eNOS mRNAs were detected in retinal arterioles. Collectively, our data demonstrate that brimonidine at clinical doses evokes a consistent NO-dependent vasodilation in first-order retinal arterioles but a heterogeneous response in second-order arterioles. These vasomotor responses are mediated by the activation of {alpha}2-AR. It appears that brimonidine, depending on the concentration and vessel size, may alter local retinal blood flow.

retinal blood flow; retinal microcirculation

BRIMONIDINE (Alphagan), an {alpha}2-adrenergic receptor (AR) agonist, has been employed in the clinical setting of glaucoma because of its therapeutic potential of reducing intraocular pressure (IOP) by aqueous suppression and enhanced uveoscleral outflow (8, 21). In addition to the IOP-lowering effects, several recent investigations in animal models suggest that brimonidine may exert a neuroprotective effect on retinal ganglion cells by preventing cell death secondary to ischemic injury (14, 18, 30). A potential mechanism of neuroprotection implicated in the literature involves the {alpha}2-AR agonist effect on the retinal and optic nerve head vasculature (25, 31, 36, 38, 49). Topical brimonidine administration has reportedly produced acute (12 h) and sustained (up to 6 mo) increases in pulsatile ocular blood flow in patients with primary open angle glaucoma (49) and normal tension glaucoma (36). Moreover, studies (25, 38) employing the Heidelberg retinal flowmeter also demonstrated an acute increase (90 min to 2 wk) in retinal blood flow after topical administration of brimonidine. Because brimonidine also reduces IOP, the increased retinal or ocular blood flow may be a consequence of IOP reduction and/or its association with alteration of ocular rigidity during pulsatile ocular blood flow measurements. In contrast to the above findings, other investigations utilizing either color Doppler imaging (24, 29, 41) or the Heidelberg retinal flowmeter (11, 40) have disclosed no significant acute and/or chronic hemodynamic effects of brimonidine on the retinal, choroidal, and/or optic nerve head circulation in humans.

Although a precise explanation for the above apparent discrepancies is not currently available, factors involving differences in patient demography and drug administration (frequency and duration), experimental/instrumental variations, and uncertainties in the concentration and distribution of the drug in retinal tissue should be considered. Furthermore, a heterogeneous vascular response to {alpha}2-AR agonists may exist in the ocular circulation. Nonetheless, it remains unclear whether brimonidine exhibits any vasomotor action on retinal microvessels. Herein, using isolated vessel approaches, we investigated the direct effect of brimonidine on retinal microvascular diameter and determined the contribution of the {alpha}2-AR subtype and the endothelium-derived vasodilator nitric oxide (NO) to the observed vasomotor reaction. We also examined the gene expression of {alpha}1- and {alpha}2-ARs and endothelial NO synthase (eNOS) in retinal arterioles.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal preparation. All animal procedures were performed in accordance with the Association for Research in Vision and Ophthalmology 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, 27 male and 5 female (8–12 wk old, 10–15 kg) purchased from Barfield Farms (Rogers, TX) were sedated with a cocktail of Telazol (4.4 mg/kg im) and xylazine (2.2 mg/kg im), anesthetized with pentobarbital sodium (30 mg/kg iv), intubated, and ventilated with room air. Heparin (1,000 U/kg) was administered into the marginal ear vein to prevent clotting. The eyes were enucleated and immediately placed into a moist chamber on ice.

Isolation and cannulation of retinal arterioles. The anterior segment and vitreous body were removed carefully under a dissecting microscope. The posterior segment or eyecup was placed in a cooled dissection chamber (6°C) containing a physiological salt solution (PSS; in mM: 145.0 NaCl, 4.7 KCl, 2.0 CaCl2, 1.17 MgSO4, 1.2 NaH2PO4, 5.0 glucose, 2.0 pyruvate, 0.02 EDTA, and 3.0 MOPS) with 1% albumin (USB, Cleveland, OH). First-order and second-order retinal arterioles (~120 and 80 µm in maximal internal diameter, respectively, and 0.6–1.0 mm in length) were carefully dissected out by using a pair of Dumont microdissection forceps (Fine Science Tools, Foster City, CA) with the aid of a stereomicroscope (model SZX12; Olympus, Melville, NY). After careful removal of any remaining neural/connective tissue within 1 h after enucleation, an arteriole was then transferred for cannulation to a Lucite vessel chamber containing PSS-albumin solution equilibrated with room air at ambient temperature. One end of the arteriole was cannulated by using a glass micropipette (tip outer diameter of 30–40 µm) filled with PSS-albumin solution, 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 with a second micropipette and also secured with suture. After cannulation, the vessel and pipettes were transferred to the stage of an inverted microscope (Olympus CKX41) coupled to a video camera (Sony DXC-190, Labtek, Campbell, CA) and video micrometer (Cardiovascular Research Institute, Texas A&M University System Health Science Center, College Station, TX) for continuous measurement of the internal diameter throughout the experiment. The cannulating pipettes were connected to independent pressure reservoirs. By adjusting the height of the reservoirs, the vessel was pressurized to 55 cmH2O intraluminal pressure without flow. This level of pressure was used based on pressure ranges that have been documented in retinal arterioles in vivo (20) and in the isolated, perfused retinal microcirculation (27). Preparations with side branches and leaks were excluded from further investigation.

Experimental protocols. Cannulated retinal arterioles were bathed in PSS-albumin at 36–37°C to allow the development of stable basal tone. We have consistently observed that vessels constrict to 60–70% of maximal diameter within a 60-min equilibration period and maintain a stable resting diameter without vasomotion. This basal tone does not change significantly and can be maintained (<3% change from initial level) >8 h under control conditions. After the development of stable basal tone at the end of the 60-min equilibration period, the dose-dependent responses to brimonidine [5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline, 0.1 nM to 10 µM] or sodium nitroprusside (10 nM to 0.1 mM) were constructed in different vessels by cumulative administration of agonist to the vessel bath. First- and second-order arterioles were exposed to each concentration of agonist for 5 min until a stable diameter was established. After the control responses were completed, the {alpha}2-AR antagonist rauwolscine (5 µM) (7, 42) was administered to the vessel bath for 30 min, and vasomotor reactivity elicited by brimonidine or sodium nitroprusside was then reexamined in the same vessel. In another set of vessels, the vasomotor response to brimonidine was studied two times with a 30-min equilibration period in between to examine whether the agonist exhibited tachyphylaxis and to confirm the reproducibility. The vasomotor effect of {alpha}1-AR agonist phenylephrine on basal tone was also examined in some vessels.

To elucidate the role of the endothelium-derived vasodilator NO in the retinal arteriolar reactivity to brimonidine, the vascular responses were assessed in another series of studies before and after the incubation of first- and second-order retinal arterioles with the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 10 µM). To confirm the efficacy of L-NAME, vasodilation to the NO-mediated agonist bradykinin (22) was examined. At the end of each isolated vessel study, a complete dose-dependent vasodilation to sodium nitroprusside was examined to ensure that the smooth muscle vasodilatory function was intact. The vessels were excluded from data analysis if the responsiveness to sodium nitroprusside was compromised. All drugs were administered extraluminally, and each antagonist (i.e., rauwolscine and L-NAME) was incubated for at least 30 min.

Chemicals. Drugs were obtained from Sigma-Aldrich (St. Louis, MO). Bradykinin, sodium nitroprusside, and L-NAME were dissolved in PSS. Rauwolscine and brimonidine were dissolved in water and DMSO, respectively, as stock solutions (10 mM), and subsequent concentrations were diluted in PSS. The final concentration of DMSO in the vessel bath was 0.1%. Vehicle control studies indicated that the final concentration of solvent had no effect on arteriolar function.

RNA isolation and reverse transcription polymerase chain reaction. Because low AR message level was detected in our preliminary studies, first- (6 to 8 per sample) and second-order retinal arterioles (12 to 16 per sample) were dissected under 6°C PSS and pooled together from both eyes for each RT-PCR experiment. These vessels were homogenized in 1 ml TRIzol reagent (Invitrogen, Carlsbad, CA), and total RNA was isolated according to the manufacturer’s instructions. Because the {alpha}2A-AR subtype is the predominant {alpha}2-AR in porcine and human retina (9, 50), we determined whether retinal arterioles express this AR subtype. Porcine neural retina tissue was used as a positive control for {alpha}2A-AR mRNA. Sets of primers specific for the {alpha}1-AR (gene accession no. U03864, sense: 5'-CAA AGT CTC CAG CCT GTC GCA CAA-3', antisense: 5'-ATC GGT CTC CCG TAG GTT GCT GTA-3'), {alpha}2A-AR (gene accession no. NM000681, sense: 5'-GGT TCC CCT TCT TCT TCA CCT ACA-3', antisense: 5'-TCG TGG TTG AAG ATG GTG TAG ATG-3'), eNOS (gene accession no. M93718, sense: 5'-GTG TTT GGA CGC GTC CTC ACC-3', antisense: CTC CTG CAG GGA AAA GCT CTG-3'), and GAPDH (gene accession no. U48832, sense: 5'-CCA CCC ACG GCA AGT TCC ACG GCA-3', antisense: 5'-GGT GGT GCA GGA GGC ATT GCT GAC-3') genes were engineered (Sigma-Genosys, The Woodlands, TX), and RT-PCR was constructed. With the use of 0.1 and 1 µg/µl of total RNA for eNOS/GAPDH and {alpha}-AR samples, respectively, RT (Thermoscript reverse transcriptase, Invitrogen) and PCR (Expand High Fidelity PCR enzyme, Roche, Indianapolis, IN) reactions were conducted as delineated previously (51). To determine whether the PCR reaction was amplifying genomic DNA, a first-strand cDNA synthesis reaction was performed with and without RT. The PCR reaction was optimized and run for 40 cycles for {alpha}1-AR, {alpha}2A-AR, and eNOS genes and 35 cycles for GAPDH genes. The PCR-amplified products were electrophoresed on a 1.8% agarose gel and visualized with ethidium bromide staining. Images of ethidium bromide-stained products were acquired with the Gel Doc 1000 system (Bio-Rad, Hercules, CA) and quantified by using volume integration (Multi-Analyst software/Macintosh, Bio-Rad). The level of expression of {alpha}2A-AR transcripts was normalized to that of GAPDH transcripts.

Data analysis. At the end of each functional experiment, the vessel was relaxed with 0.1 mM sodium nitroprusside in ethylenediaminetetraacetic acid (1 mM)-calcium-free PSS to obtain its maximal diameter at 55 cmH2O intraluminal pressure. For the analysis of brimonidine-induced responses, diameter changes were normalized to the resting basal diameter and expressed as a percent change in resting diameter. Data are reported as means ± SE, and the n value represents the number of vessels (one per pig) studied. Statistical comparisons of data were performed by Student’s t-test or by analysis of variance followed by the Bonferroni multiple-range test, as appropriate. Regression analysis and Pearson correlation were performed to assess the relationship between resting diameter and percent change in resting diameter at various concentrations of brimonidine. A value of P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Vasomotor response to brimonidine. First-order (n = 18) and second-order (n = 16) retinal arterioles developed a comparable level of basal tone (first-order: constricted to 67 ± 1% of their maximal diameter vs. second-order: constricted to 63 ± 2% of their maximal diameter; P = 0.09) at a bath temperature of 36–37°C and 55 cmH2O intraluminal pressure. The average resting diameters of the first- and second-order arterioles were 82 ± 2 µm and 50 ± 1 µm, respectively. Figure 1 shows the relationship between resting diameter and individual vascular response to various concentrations of brimonidine. The slope and the correlation coefficient (r) for the diameter-response relationship were increased with increasing brimonidine concentrations, indicating a size-dependent vasomotor response. The plot of individual responses revealed that a majority of first-order arterioles (66–96 µm) dilated to brimonidine at all concentrations. At downstream second-order arterioles (42–60 µm), lower concentrations of brimonidine (≤10–8 M) dilated ~30% of the vessels. However, these vessels constricted consistently to higher concentrations of brimonidine (≥10–7 M). Brimonidine elicited a reproducible dose-dependent dilation and constriction of first-order (Fig. 2A) and second-order (Fig. 2B) arterioles, respectively. To determine the contribution of {alpha}2-AR in these vascular responses, some vessels were treated with the {alpha}2-AR antagonist rauwolscine and the dose-dependent responses to brimonidine were reexamined. Rauwolscine did not significantly alter basal tone of first-order (control: 65 ± 2% vs. rauwolscine: 67 ± 4%) or second-order (control: 66 ± 3% vs. rauwolscine: 63 ± 4%) arterioles but blocked the respective dilation (Fig. 2A) and constriction (Fig. 2B) of these vessels to brimonidine. In contrast to the varied response to brimonidine, dose-dependent vasodilation to the endothelium-independent agonist sodium nitroprusside was not significantly different between first- and second-order retinal arterioles (Fig. 3A). Furthermore, rauwolscine did not affect basal tone (first-order arterioles, control: 66 ± 3% vs. rauwolscine: 68 ± 2%; second-order arterioles, control: 60 ± 2% vs. rauwolscine: 60 ± 2%) or sodium nitroprusside-induced dilation (Fig. 3B), indicating that vasodilatory function of this group of vessels was not compromised by the {alpha}2-AR antagonist.


Figure 1
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  		Fig. 1. Scatterplots showing relationship between resting diameter and percent change in resting diameter at various concentrations of brimonidine. Vasomotor responses are plotted for individual first-order (n = 18) and second-order (n = 16) retinal arterioles. Solid line, regression analysis; vertical dashed line, separation between first-order (>60 µm ID; closed circles) and second-order (≤60 µm ID; open circles) retinal arterioles; horizontal dashed line, separation between vasodilation (positive change in resting diameter) and vasoconstriction (negative change in resting diameter); r, correlation coefficient.

 

Figure 2
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  		Fig. 2. Vasomotor response of retinal arterioles to brimonidine. A: dose-dependent dilation of first-order retinal arterioles (81 ± 4 µm resting diameter) in response to brimonidine was examined (control) and then repeated after a 30-min washout period in the absence (repeat) or presence of {alpha}2-adrenergic receptor (AR) antagonist rauwolscine (5 µM). B: dose-dependent constriction of second-order arterioles (50 ± 2 µm resting diameter) in response to brimonidine was examined (control) and then repeated after a 30-min washout period in the absence (repeat) or presence of {alpha}2-AR antagonist rauwolscine (5 µM). *P < 0.05 vs. control.

 

Figure 3
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  		Fig. 3. Effects of vessel size and {alpha}2-AR blockade on retinal arteriolar dilation to sodium nitroprusside. A: sodium nitroprusside caused dose-dependent dilation of isolated first-order (78 ± 4 µm resting diameter) and second-order (49 ± 2 µm resting diameter) retinal arterioles. B: dose-dependent dilation of first-order (n = 5) and second-order (n = 6) retinal arterioles in response to sodium nitroprusside was examined before (control) and after incubation with rauwolscine (5 µM).

 
Role of endothelium-derived NO in retinal arteriolar dilation to brimonidine. The relative contribution of endothelium-derived NO to brimonidine-induced vasodilation of first-order retinal arterioles was examined in the absence and presence of L-NAME, a NO synthase inhibitor. L-NAME had a tendency to increase basal tone (control: 67 ± 2% vs. L-NAME: 64 ± 3%; P = 0.13) but not in a significant manner. The retinal arteriolar dilation to an endothelium-dependent, NO-mediated agonist bradykinin (10 nM) was significantly inhibited by L-NAME (control: 80 ± 5% of maximal dilation vs. L-NAME: 26 ± 5% of maximal dilation; n = 4). Similar to rauwolscine, L-NAME completely blocked the vasodilatory response to brimonidine in first-order arterioles (Fig. 4). The dilation of retinal arterioles to the endothelium-independent NO donor sodium nitroprusside was not altered by L-NAME (data not shown), which is consistent with our previous findings (22).


Figure 4
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  		Fig. 4. Role of endothelium-derived nitric oxide (NO) in retinal arteriolar dilation to brimonidine. Dose-dependent dilation of isolated first-order retinal arterioles (76 ± 5 µm resting diameter) in response to brimonidine was examined before (control) and after incubation with NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (10 µM). *P < 0.05 vs. control.

 
{alpha}-AR and eNOS mRNA expression in retinal arterioles. To support the results from functional studies above, we performed RT-PCR to detect {alpha}-AR and eNOS mRNAs in isolated retinal arterioles and neural retina tissue. Figure 5 shows that the {alpha}2A-AR and eNOS transcripts, but not the {alpha}1-AR transcript, were detected in retinal arterioles. In contrast, both {alpha}2A-AR and {alpha}1-AR transcripts were detected in the neural retina tissue devoid of retinal vessels. The expression of {alpha}2A-AR mRNA, after normalization with GAPDH, was consistently slightly higher in retinal arterioles than in neural retina tissue (Fig. 5). It appears that the retinal arteriole sample was relatively homogeneous because {alpha}1-AR was observed only in the neural retina tissue.


Figure 5
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  		Fig. 5. RT-PCR analysis of {alpha}-AR and endothelial NOS (eNOS) mRNA expression in porcine retinal arterioles. Top: total RNA from isolated retinal arterioles and neural retina tissue was reversed transcribed by using gene-specific primers for {alpha}2A-AR (136 bp), {alpha}1-AR (178 bp), eNOS (342 bp), and GAPDH (311 bp) mRNAs. After the PCR reaction, gene products were electrophoresed on a 1.8% agarose gel and visualized with ethidium bromide staining. Hae III-restricted {phi}X174-DNA was used as a size marker. Data are representative of 3 independent experiments. Bottom: {alpha}2A-AR transcripts for retinal arterioles and neural retina tissue were normalized with corresponding GAPDH transcripts. *P < 0.05 vs. neural retina tissue.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, with an established videomicroscopic technique (22), we examined the direct action of brimonidine on isolated porcine retinal arterioles. Brimonidine caused dose-dependent dilation of all first-order retinal arterioles and mild dilation of some second-order retinal arterioles at the low concentrations (≤10 nM) (Fig. 1). It should be noted that this vasodilatory response was evident at therapeutic concentrations of brimonidine (i.e., vitreous concentrations ~10–100 nM after topical administration) (1, 26). At these presumed therapeutic doses, brimonidine produced up to an 8% increase in resting diameter of first-order retinal arterioles (Figs. 2A and 4) and up to a 10% decrease in diameter in second-order arterioles (Fig. 2B). This magnitude of vasodilation or vasoconstriction would have a significant impact on local retinal blood flow, because Poiseuille’s law indicates that blood flow, at a constant driving pressure, is directly proportional to the fourth power of the vessel radius.

To our knowledge, only one other study has evaluated the retinal vasomotor reactivity associated with brimonidine. Spada et al. (42) studied the effect of locally administered brimonidine on microvessel caliber with intravital microscopy in human retinal tissues grafted into the hamster cheek pouch. These investigators found that brimonidine (1 nM to 0.1 mM) caused dose-dependent constriction in the microvessels of the naive hamster cheek pouch but did not cause significant constriction or dilation of selected arteriolar segments (~12–25 µm in resting diameter) in human retinal xenografts. Only a transient (1 min) and modest vasoconstriction (~10%) was observed when a high concentration of brimonidine (0.1 mM) was administered. Although Spada et al. (42) did not investigate vasomotor response in larger arterioles, their results support our findings on the increased incidence of vasoconstriction to a high concentration of brimonidine in smaller retinal arterioles. It is worth mentioning that the growth of new vessels in the xenograft, confounding influences from hemodynamic factors, and the potential release of local vasomotor chemicals from hamster native tissue might play a role in modulating brimonidine-induced vasoreactivity in the above hamster cheek pouch preparation. In our study, we obviated these potential confounding influences by characterizing the vasomotor reactivity to brimonidine in a controlled environment using the isolated vessel preparation.

Interestingly, Hughes et al. (23) found size-dependent response of isolated resistance arteries to brimonidine in human splanchnic, peripheral, coronary, pulmonary, and uterine tissue. The magnitude of the contractile response was reported to be inversely related to vessel size (23). Our findings suggest that the retinal arteriolar network exhibits a unique size-dependent vasomotor response to brimonidine with homogeneous vasodilation in first-order arterioles and a heterogeneous vasomotor reaction in second-order arterioles with predominantly vasoconstriction at higher concentrations. In this regard, the effect of brimonidine on overall retinal blood flow seemingly depends on the local concentration and the reaction of large versus small arterioles to this drug. It appears that a general increase in retinal blood flow is expected at lower doses of brimonidine due to dilation of first- and second-order arterioles. However, this increased flow might be counterbalanced or even reversed by the increased vasoconstriction in second-order arterioles when local concentration of brimonidine is further elevated. This may explain the apparent inconsistent results of retinal arterial blood flow in response to brimonidine in vivo because the size of vessels studied was inconsistent and the concentration and distribution of brimonidine in retinal tissue were uncertain during flow/velocity measurement. Moreover, because retinal tissue exhibits autoregulation of blood flow (16), the initiation of compensatory vasoregulatory mechanisms secondary to flow alteration by brimonidine cannot be excluded. Nevertheless, our present study demonstrates that brimonidine, at a clinical concentration, is vasoactive in retinal microvessels.

Despite the apparent lack of autonomic innervation in the human retinal vasculature, {alpha}1- and {alpha}2-ARs (9, 33, 50) have been shown to localize to the retina, and topically applied brimonidine can reach the posterior segment of the eye at nanomolar concentrations sufficient to activate {alpha}2-AR (1, 26). However, the expression of {alpha}2-ARs in retinal arterioles has not been reported, and the recent observation on the insignificant retinal flow response to brimonidine has raised the question of whether retinal arterioles express {alpha}2-AR (41). In the present study, we demonstrated for the first time that {alpha}2A-AR mRNA is expressed in retinal arterioles (Fig. 5). This is consistent with the report that {alpha}2A-AR subtype is the predominant subtype in the porcine and human retinal tissue (9, 50), supporting our use of the porcine model to investigate the effect of brimonidine on the retinal vasculature.

Activation of {alpha}2-AR can cause constriction of arterioles in the skeletal muscle microcirculation in humans (39) and in some animal models (10, 35). However, vasodilation in response to {alpha}2-AR activation in mesenteric (10), coronary (6), and cerebral (47) vessels was also reported. Because endothelial removal or inhibition of NO synthase can augment vasoconstriction induced by {alpha}2-AR agonists, it has been suggested that endothelial release of NO (via endothelial {alpha}2-AR activation) can modulate the contractile action of {alpha}2-AR in smooth muscle cells (2, 48). In fact, it has been shown in a number of vascular beds that activation of {alpha}2-AR by brimonidine is associated with NO-mediated vasodilation (5, 43, 47). Moreover, endothelial release of vasodilator prostaglandins or hyperpolarizing factors also has been reported in some vasculatures (46). However, the vasomotor signaling pathways involved in {alpha}2-AR activation by brimonidine in retinal arterioles have not been determined. In the present study, we found that rauwolscine, an {alpha}2-AR antagonist, abolished brimonidine-induced vasodilation in both first- (Fig. 2A) and second-order (n = 3; data not shown) retinal arterioles and also blocked vasoconstriction in small arterioles (Fig. 2B). These results indicate that {alpha}2-AR activation is responsible for the vasomotor action of brimonidine in retinal arterioles. In addition, the NO synthase inhibitor L-NAME completely blocked the vasodilatory response to brimonidine in both first- (Fig. 4) and second-order (n = 3; data not shown) retinal arterioles, suggesting that {alpha}2-AR activation stimulates NO release and is mechanistically involved in the dilation of retinal arterioles to brimonidine. It does not appear that this NO release is sufficient to compete with the brimonidine-elicited constrictor response because L-NAME did not enhance constriction of second-order arterioles to brimonidine (n = 3; data not shown). In contrast to in vivo findings in the retinal circulation (15), it is important to note that L-NAME (10 µM) did not significantly increase basal tone of isolated retinal arterioles. This may be due to the absence of luminal flow in our in vitro study because it has been shown that endothelial cells respond to increased flow (or shear stress) by releasing NO (28). In this regard, it is expected that the NO component would be more pronounced in vivo (i.e., with luminal flow) compared with that in vitro (i.e., without luminal flow) under resting conditions. Therefore, the effect of L-NAME on basal vascular tone would be less apparent as observed in our present and previous (22) in vitro studies. The possible role of endothelial NO signaling in response to agonist stimulation was supported by our molecular evidence that eNOS mRNA was detected in the retinal arterioles. Because brimonidine did not elicit or enhance vasoconstriction of retinal arterioles in the presence of rauwolscine, it is unlikely that brimonidine has other vasomotor activity through activation of {alpha}1-AR besides {alpha}2-AR. This contention is supported by the inability of the {alpha}1-AR agonist phenylephrine to alter resting diameter of first- or second-order retinal arterioles (n = 3; data not shown), as well as the identification of {alpha}2-AR, but not {alpha}1-AR, mRNA expression in isolated retinal arterioles.

The vasoconstrictor action of brimonidine in second-order retinal arterioles is expected to compromise local retinal blood flow. However, it is worth noting that arteriolar constriction to {alpha}2-AR activation is selectively inhibited by moderate tissue acidosis (12, 44), flow reduction (34), hypoxia (44), increased tissue metabolic rate (3), and by release of adenosine (13). The prevailing inhibition of adrenergic {alpha}2-mediated constriction in second-order arterioles under metabolic stress might help to alleviate the impact of brimonidine-induced constriction in small retinal arterioles and consequently facilitate flow distribution to the stressed tissues. On the other hand, because accumulating evidence suggests that endothelin-1 can either prevent {alpha}2-AR-mediated NO release and vasodilation (4, 32) or reduce smooth muscle sensitivity to NO (19), the enhanced endothelin-1 production during disease states, such as glaucoma (17, 37, 45), may compromise the vasodilatory effect of brimonidine. It is reasonable to speculate that the beneficial effect of brimonidine, in terms of vasodilation and flow enhancement, may be dependent on the local concentration of brimonidine, distribution and function of {alpha}2-AR, endothelial NO synthase activity, endothelin-1 level, and the local metabolic environment.

In conclusion, we found molecular and pharmacological evidence of {alpha}2-ARs in the retinal arterioles. The {alpha}2-AR agonist brimonidine, at clinically relevant doses, evoked a modest vasodilatory response in first-order, and some second-order, porcine retinal arterioles via activation of {alpha}2-ARs and subsequent production of the vasodilator NO. Vasoconstriction of most second-order arterioles in response to brimonidine was also observed. This apparent heterogeneity of vascular responses implicates a potential influence of topical brimonidine administration on local retinal blood flow.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by the Scott and White Research Foundation, Ophthalmic Vascular Research Program, and the Kruse Family Endowment Fund.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. H. Rosa, Jr., Scott and White Eye Institute, 2401 South 31st St., Temple, TX 76508 (e-mail address: rrosa{at}swmail.sw.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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