Functional Role of α₁-Adrenoceptor Subtypes in Murine Ophthalmic Arteries

Abstract

Purpose.: To identify the α₁-adrenoceptor (α₁-AR) subtypes mediating vascular adrenergic responses in murine ophthalmic arteries.

Methods.: Expression of mRNA was quantified for individual α₁-AR subtypes in murine ophthalmic arteries using real-time PCR. To assess the functional relevance of α₁-ARs for mediating vascular responses, ophthalmic arteries from mice deficient in one of the three α₁-AR subtypes (α_1A-AR^−/−, α_1B-AR^−/−, and α_1D-AR^−/−, respectively) and wild-type controls were isolated, cannulated with micropipettes, and pressurized. Changes in luminal artery diameter in response to the α₁-AR agonist phenylephrine, the sympathetic transmitter noradrenaline, and to the nonadrenergic vasoconstrictor arginine vasopressin (AVP) were measured by video microscopy.

Results.: Using real-time PCR, mRNA for all three α₁-AR subtypes was detected in ophthalmic arteries from wild-type mice. In functional studies, phenylephrine and noradrenaline produced dose-dependent constriction of ophthalmic arteries that was similar in wild-type, α_1B-AR^−/−, and α_1D-AR^−/− mice. Strikingly, responses to phenylephrine and noradrenaline were almost completely abolished in α_1A-AR^−/− mice. In contrast, the nonadrenergic agonist AVP produced dose-dependent vasoconstrictor responses that did not differ between any of the mouse genotypes tested.

Conclusions.: These findings provide evidence that the α_1A-AR subtype mediates adrenergic vasoconstriction in murine ophthalmic arteries.

Disturbances in ocular perfusion have been implicated in the pathophysiology of various eye diseases, including diabetic retinopathy, nonarteritic anterior ischemic optic neuropathy, and glaucoma. ^{1 –6} The α₁-adrenoceptor (α₁-AR) family plays a critical role in regulating ocular vascular tone and blood flow by mediating vasoconstrictor responses of catecholamines in the ocular circulation. ^{7 –11}  

Pharmacologic studies and molecular cloning techniques have revealed the existence of three α₁-AR subtypes: α_1A, α_1B, and α_1D. ^{12 –16} All three receptor subtypes are expressed in blood vessels and can mediate vasoconstriction via G_q/11 protein–mediated increases of inositol phosphates and intracellular calcium in vascular smooth muscle cells. ¹⁷ However, the expression pattern of individual α₁-AR subtypes and their role in mediating vascular responses to catecholamines differs considerably between individual vascular beds. ^18,19 Based on these findings, selective pharmacologic activation or blockade of individual α₁-AR subtypes may provide a useful tool to selectively modulate perfusion of various organs, including the eye. Thus, we designed this study to identify the α₁-AR subtypes that mediate adrenergic vascular responses in ophthalmic arteries. Since the expression pattern of α₁-ARs is unknown in ocular vessels, we used real-time (RT) PCR to quantify mRNA expression of individual α₁-AR subtypes in murine ophthalmic arteries. Due to the lack of highly selective agonists and antagonists for individual α₁-AR subtypes, we used gene-targeted mice deficient in one of the three α₁-ARs (α_1A-AR^−/−, α_1B-AR^−/−, α_1D-AR^−/−, respectively) to determine the role of each receptor subtype in mediating adrenergic vasoconstriction in ophthalmic arteries. 

Materials and Methods

Animals

All studies were performed in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the local government. The generation of α_1A-AR^−/−, α_1B-AR^−/−, and α_1D-AR^−/− mice has been described previously. ^{20 –22} Each genotype has been backcrossed with C57BL/6Slc mice for more than eight times and maintained on a C57BL/6Slc background. For experiments, male mice at the age of 7 to 9 months were used. Mice were fed with standard mouse chow and allowed free access to tap water. 

Real-Time PCR Analysis

Expression of α₁-AR mRNA was quantified in isolated ophthalmic arteries from wild-type mice (C57BL/6Slc) using RT-PCR. After mice had been killed by CO₂ inhalation, the eyes were immediately removed together with the retrobulbar tissue and placed in ice-cold PBS (Invitrogen, Karlsruhe, Germany). Then, ophthalmic arteries were isolated by the use of fine-point tweezers under a dissecting microscope, transferred into a 1.5-mL tube, and immediately snap frozen. To increase the RNA yield, arteries were pooled from three mice. Subsequently, vessels were homogenized in lysis buffer using a homogenizing device (Schwingmühle MM 300; Retsch GmbH, Haan, Germany; Lysing Matrix D MP, MP Biomedicals, Illkirch, France). After homogenization, total RNA was extracted with a kit (Absolutely RNA Nanoprep; Stratagene, La Jolla, CA) according to the manufacturer's protocol. After complete DNA digestion, the RNA was reverse transcribed with the use of a reverse transcription kit and random hexamers (High Capacity cDNA Reverse Transcription Kit; Applied Biosystems, Darmstadt, Germany). Quantitative PCR analysis was performed (GeneAmp StepOne Plus; Applied Biosystems). Nucleic acid stain (SYBR Green; Bioline, Luckenwalde, Germany) was used for the fluorescent detection of DNA generated during PCR. The PCR reaction was performed in a total volume of 12.5 μL with 0.4 pmol/μL of each primer and ready-to-use 2× reaction mix (ImmoMix; Bioline); 2 μL cDNA corresponding to 10 ng RNA was used as a template. Published sequences for mouse α_1A-AR (NM_013461), α_1B-AR (NM_007416), and α_1D-AR (NM_013460) were used to design primers for PCR amplification. Primer sequences were α_1A-AR sense 5′-TGC GAG GAC TGA AGG TCC GCT-3′ and antisense 5′-CAG GGA CGC TGG GCG AAT GG-3′; α_1B-AR sense 5′-TCC AGG GAA AAG AAA GCA GCC AA-3′ and antisense 5′-GGG TAG ATG ATG GGG TTG AGG CA-3′; α_1D-AR sense 5′-TAA GGC TGC TCA AGT TTT CCC GC-3′ and antisense 5′-TGA GCG GGT TCA CAC AGC TAT TGA-3′; ß-actin sense 5′-CAC CCG CGA GCA CAG CTT CTT T-3′ and antisense 5′-AAT ACA GCC CGG GGA GCA TC-3′. The expression levels of α_1A-AR, α_1B-AR, and α_1D-AR mRNA were normalized to ß-actin using the ΔCt method. Parallelism of standard curves was confirmed. 

Measurements of Vascular Reactivity

Mice were killed by CO₂ inhalation and the eyes were rapidly removed, together with the retrobulbar tissue, and placed in ice-cold Krebs buffer with the following ionic composition (in mM): 118.3 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 25 NaHCO₃, and 11 glucose (Carl Roth GmbH, Karlsruhe, Germany). Then, ophthalmic arteries were isolated under a dissecting microscope, placed in an organ chamber filled with cold Krebs solution, and cannulated and sutured onto micropipettes, as described previously. ²³ Vessels were pressurized via the micropipettes to 50 mm Hg under no-flow conditions using two reservoirs filled with Krebs solution and imaged using a video camera mounted on an inverted microscope; video sequences were captured to a personal computer for offline analysis. The organ chamber was continuously circulated with oxygenated and carbonated Krebs buffer at 37°C and pH 7.4. Arteries were allowed to equilibrate for 30 to 40 minutes before study. During this time, ophthalmic arteries of all groups developed spontaneous myogenic tone by constricting to 86% to 81% of initial diameter recorded immediately after pressurization. Reduction in luminal artery diameter during the equilibration period was similar in all four mouse genotypes. Viability of vessels was assessed as satisfactory when at least 50% constriction from stabilized resting diameter in response to high KCl solution (100 mM) was achieved. Then, cumulative concentration–response curves were obtained to phenylephrine (1 nM to 300 μM), a non-subtype–selective α₁-AR agonist; to noradrenaline (1 nM to 300 μM), a major neurotransmitter of sympathetic nerves; and to arginine vasopressin (AVP, 1 pM to 300 nM), a nonadrenergic receptor agonist, which induces vasoconstricton via V _1a-receptor–mediated increases of inositol phosphates and intracellular calcium in vascular smooth muscle. Responses to noradrenaline (10 μM) were also compared before and after addition of prazosin (100 nM), a competitive non-subtype–selective α₁-AR antagonist. 

Statistical Analysis

Data are presented as mean ± SE and n represents the number of mice per group. Vascular responses are presented as percentage of change in diameter from stabilized resting diameter. Comparisons of concentration–response curves were made using the Brunner test for nonparametric analysis of longitudinal data. ²⁴ The Bonferroni adjustment was used to correct for multiple comparisons. To compare vascular responses to noradrenaline before and after prazosin treatment, the Wilcoxon signed-rank test was used. Comparisons of α₁-AR mRNA expression levels were made using the Kruskal–Wallis test. The level of significance was set at 0.05. 

Results

α₁-Adrenoceptor mRNA Expression in Ophthalmic Arteries

Expression of α₁-AR mRNA was determined in ophthalmic arteries (five pooled samples) from wild-type mice (n = 15) by the use of quantitative RT-PCR. Remarkably, mRNA of all three α₁-AR subtypes was found to be expressed at high levels, although there was no difference between mRNA expression levels of individual receptor subtypes (Fig. 1). 

Figure 1.

 

Relative mRNA expression of individual α₁-AR subtypes (α_1A, α_1B, and α_1D) normalized to β-actin transcripts in ophthalmic arteries from wild-type mice. Values are averages of five independent experiments and are expressed as mean ± SE.

Responses of Ophthalmic Arteries

Baseline luminal diameters of ophthalmic artery segments (after development of stable myogenic tone) were (in μm) 118 ± 13, 130 ± 9, 125 ± 8, and 132 ± 10 in α_1A-AR^−/−, α_1B-AR^−/−, α_1D-AR^−/−, and wild-type mice and did not differ between individual mouse genotypes (P > 0.05, one-way ANOVA, n = 8–10 per genotype). To identify the α₁-AR subtypes that mediate adrenergic vasoconstriction of ophthalmic arteries, we compared vascular responses from α_1A-AR^−/−, α_1B-AR^−/−, α_1D-AR^−/−, and wild-type mice to the non-subtype–selective α₁-AR agonist phenylephrine. Phenylephrine elicited concentration-dependent vasoconstriction in arteries from wild-type, α_1B-AR^−/−, and α_1D-AR^−/− mice that was similar in the three groups (Fig. 2). Maximal reduction in luminal diameter in response to phenylephrine was 41% ± 6%, 43% ± 8%, and 38% ± 4% in wild-type (n = 10), α_1B-AR^−/− (n = 8), and α_1D-AR^−/− (n = 10) mice, respectively. The pD2 values (mean negative log of the vasoconstrictor concentration producing 50% of maximal response) were 5.79 ± 0.16, 5.75 ± 0.19, and 5.68 ± 0.19 in wild-type, α_1B-AR^−/−, and α_1D-AR^−/− mice, respectively. In contrast, phenylephrine-induced vasconstriction was almost completely abolished in ophthalmic arteries from α_1A-AR^−/− mice (n = 8), differing significantly from responses of all other genotypes. Maximal reduction in luminal diameter to phenylephrine was only 6% ± 5% in this group (Fig. 2). 

Figure 2.

 

Responses of ophthalmic arteries from wild-type, α_1A-AR^−/−, α_1B-AR^−/−, and α_1D-AR^−/− mice to the α₁-AR agonist phenylephrine. Vasoconstriction to phenylephrine was almost completely abolished in ophthalmic arteries from α_1A-AR^−/− mice. In contrast, deletion of α_1B-AR and α_1D-AR genes had no significant effect on vascular reactivity. Values are expressed as mean ± SE (#P < 0.01, α_1A-AR^−/− mice versus all other groups, n = 8 to 10 per concentration and genotype).

Moreover, we examined responses to the sympathetic transmitter noradrenaline that, apart from α₁-ARs, can also activate α₂-ARs and β-ARs. Noradrenaline also produced concentration-dependent vasoconstriction in ophthalmic arteries from wild-type, α_1B-AR^−/−, and α_1D-AR^−/− mice that did not differ between the three genotypes. Maximal constriction to noradrenaline was 42% ± 7%, 52% ± 7%, and 44% ± 5% in wild-type (n = 10), α_1B-AR^−/− (n = 8), and α_1D-AR^−/− (n = 10) mice, respectively. The pD2 values were 5.97 ± 0.20, 5.75 ± 0.17, and 5.91 ± 0.17 in wild-type, α_1B-AR^−/−, and α_1D-AR^−/− mice, respectively. In α_1A-AR^−/− mice (n = 8), however, maximal reduction in luminal artery diameter in response to noradrenaline was negligible and differed significantly from responses of all other genotypes. Maximal reduction in luminal diameter was only 1% ± 3% in this group (Fig. 3A). After incubation with the competitive non-subtype–selective α₁-AR antagonist prazosin (100 nM), responses to noradrenaline (10 μM) were negligible in all groups of mice (Fig. 3B). 

Figure 3.

 

Responses of ophthalmic arteries from wild-type, α_1A-AR^−/−, α_1B-AR^−/−, and α_1D-AR^−/− mice to the sympathetic transmitter noradrenaline. (A) Vasoconstriction to noradrenaline was almost completely abolished in ophthalmic arteries from α_1A-AR^−/− mice. In contrast, deletion of α_1B-AR and α_1D-AR genes had no significant effect on vascular reactivity. Values are expressed as mean ± SE (#P < 0.01, α_1A-AR^−/− mice versus all other groups, n = 8 to 10 per concentration and genotype). (B) Vasoconstrictor responses to noradrenaline (10⁻⁵ M) were virtually abolished after incubation with prazosin (10⁻⁷ M). Values are expressed as mean ± SE (*P < 0.05, prazosin-treated versus nontreated arteries, n = 6 per genotype).

The nonadrenergic vasoconstrictor AVP elicited concentration-dependent vasoconstricton in ophthalmic arteries from all four mouse genotypes, which did not differ between individual groups (Fig. 4). Maximal constriction to AVP was 48% ± 8%, 49% ± 8%, 52% ± 8%, and 45% ± 7% in wild-type (n = 10), α_1A-AR^−/− (n = 8), α_1B-AR^−/− (n = 8), and α_1D-AR^−/− (n = 10) mice, respectively. The pD2 values were 9.28 ± 0.17, 9.30 ± 0.20, 9.17 ± 0.25, and 9.06 ± 0.27 in wild-type, α_1A-AR^−/−, α_1B-AR^−/−, and α_1D-AR^−/− mice, respectively. 

Figure 4.

 

Responses of ophthalmic arteries from wild-type, α_1A-AR^−/−, α_1B-AR^−/−, and α_1D-AR^−/− mice to the nonadrenergic vasoconstrictor AVP. Deletion of α_1A-AR, α_1B-AR, or α_1D-AR genes did not affect responses to AVP. Values are expressed as mean ± SE (n = 8 to 10 per concentration and genotype).

Discussion

The purpose of the present study was to identify the α₁-AR subtypes that mediate adrenergic responses in ophthalmic arteries. Using RT-PCR, we found mRNA of all three α₁-AR subtypes to be expressed at similar levels in ophthalmic arteries of wild-type mice. Since highly selective agonists and antagonists are not available for all three subtypes, we used mice with targeted disruption of single α₁-AR subtype genes to assess the functional relevance of each receptor subtype. Strikingly, ophthalmic arteries from mice deficient in the α_1A-AR gene showed almost no reactivity to phenylephrine and noradrenaline. The α₁-AR antagonist prazosin almost completely abolished noradrenaline-induced responses, indicative of the predominant involvement of α₁-ARs in adrenergic vasoconstriction of ophthalmic arteries. Deletion of either receptor subtype did not affect vasoconstriction induced by AVP, suggesting that the lack of a single α₁-AR subtype does not affect the downstream signaling cascades that ultimately mediate vasoconstriction. 

Previous studies using electrical stimulation of sympathetic nerve pathways and intravenous application of α₁-AR antagonists demonstrated that α₁-ARs mediate neurogenic vasoconstriction in the anterior choroid of rats and in long posterior ciliary arteries of cats. ^8,10 Another study using transmural electrical stimulation in isolated vascular strips showed that α₁-ARs contributed to neurogenic vasoconstriction in dog short posterior ciliary and ophthalmic arteries. ⁹ The present study is the first to demonstrate that adrenergic vasoconstriction of murine ophthalmic arteries is mediated predominantly by the α_1A-AR subtype. 

Earlier pharmacologic studies making use of subtype-selective agents and functional studies in gene-targeted mice lacking one or more α₁-AR subtypes revealed that the contribution of individual α₁-AR subtypes to adrenergic vasoconstrictor responses differs considerably depending on the vascular bed. Based on these studies, the α_1A-AR is significantly involved in adrenergic vasoconstriction of rat and mouse small mesenteric and tail arteries, but plays only a minor role in α₁-AR–mediated contraction of large vessels, such as aorta, iliac, and carotid arteries. ^{18,19,25 –27} In contrast, the α_1D-AR was shown to play a major vasoconstrictor role in large vessels, but was also suggested to participate in α₁-AR–mediated vasoconstriction of some small arteries, such as coronary and femoral small arteries. ^{19,27 –30} The α_1B-AR was shown to play only a minor role in adrenergic vasoconstriction. In vivo studies have demonstrated that blood pressure responses to phenylephrine and to noradrenaline are reduced in gene-targeted mice lacking the α_1B-AR. However, no differences in resting blood pressure between α_1B-AR knockout and respective wild-type mice were detected. ²¹ Moreover, several in vitro studies using α₁-AR subtype-selective antagonists and mice with targeted disruption of the α_1B-AR gene revealed only a minor contribution of the α_1B-AR subtype to vasoconstrictor responses in mouse aorta, carotid, mesenteric, and tail arteries. ^18,21,25  

Studies of mRNA and protein expression revealed diverse distribution of α₁-AR receptor subtypes among vascular beds. ^18,19,31 Some of these studies reported that the mRNA expression levels of individual α₁-AR subtypes were in fairly good agreement with their protein levels or their contribution to adrenergic vasoconstrictor responses. ^18,19,31,32 Other studies, however, demonstrated that the presence of mRNA or even protein for a particular α₁-AR subtype does not ensure its participation in vasoconstriction. ^33,34 This can be explained either by differences in the efficiency of individual α₁-AR subtypes to accumulate intracellular inositol phosphate and calcium or by a different extent of receptor subtype expression in intracellular compartments, preventing them from interaction with the hydrophilic natural ligands. ^{34 –36} Although we found mRNA of all three α₁-AR subtypes to be expressed at similar levels in ophthalmic arteries, adrenergic vasoconstriction was predominantly mediated by the α_1A-AR, raising a question about the physiologic role of α_1B- and α_1D-ARs. A growing body of evidence suggests that each receptor subtype can activate independent signaling pathways, resulting in different physiologic functions. ¹⁵ For example, the α_1D-AR was shown to mediate generation of reactive oxygen species and induction of apoptosis in human aortic smooth muscle cells. ^37,38 Moreover, the α_1B-AR was reported to mediate trophic effects of catecholamines in mouse arteries, whereas the α_1D-AR does not appear to be involved in this process. ^39,40 Thus, although our data indicate that α_1B- and α_1D-ARs do not significantly contribute to adrenergic vasoconstricton in murine ophthalmic arteries, both receptor subtypes may be involved in regulation of other physiologic or pathophysiologic actions. 

In conclusion, our data provide evidence that the α_1A-AR subtype mediates adrenergic vasoconstriction in murine ophthalmic arteries. From a clinical point of view, selective α_1A-AR antagonists may become therapeutically useful to increase ocular perfusion in certain pathologic conditions, such as diabetic retinopathy and glaucoma. ^1,2,5,6  

Footnotes

 Supported in part by a grant from the Gertraud Maria Rzehulka Foundation.

Footnotes

 Disclosure: A. Gericke, None; M.L. Kordasz, None; A. Steege, None; A. Sanbe, None; E. Goloborodko, None; J.M. Vetter, None; A. Patzak, None; N. Pfeiffer, None

The authors thank Paul C. Simpson (Cardiology Section, San Francisco Veterans Affairs Medical Center; Department of Medicine, Cardiology Division, University of California, San Francisco, CA), Susanna Cotecchia (Department of General and Environmental Physiology, University of Bari, Italy, and Department of Pharmacology and Toxicology, University of Lausanne, Switzerland), and Akito Tanoue (Department of Pharmacology, National Research Institute for Child Health and Development, Tokyo, Japan) for making α_1A-, α_1B-, and α_1D-AR knockout mice, respectively, available for this study; and Ulrike Neumann (Institute of Vegetative Physiology, Charité-Universitätsmedizin Berlin, Berlin, Germany) for expert technical assistance with real-time PCR experiments.