Cholinergic Responses of Ophthalmic Arteries in M₃ and M₅ Muscarinic Acetylcholine Receptor Knockout Mice

Abstract

purpose. To determine the functional role of M₃ and M₅ muscarinic acetylcholine receptor subtypes in ophthalmic arteries using gene-targeted mice.

methods. Muscarinic receptor gene expression was quantified in murine ophthalmic arteries using real-time PCR. To test the functional relevance of M₃ and M₅ receptors, ophthalmic arteries from mice deficient in either subtype (M3R^−/−, M5R^−/−, respectively) and wild-type controls were isolated, cannulated with micropipettes, and pressurized. Changes in luminal vessel diameter in response to muscarinic and nonmuscarinic receptor agonists were measured by video microscopy.

results. With the use of real-time PCR, all five muscarinic receptor subtypes were detected in ophthalmic arteries. However, mRNA levels of M₁, M₃, and M₅ receptors were higher than those of M₂ and M₄ receptors. In functional studies, after preconstriction with phenylephrine, acetylcholine and carbachol produced concentration-dependent dilations of ophthalmic arteries that were similar in M5R^−/− and wild-type mice. Strikingly, cholinergic dilation of ophthalmic arteries was almost completely abolished in M3R^−/− mice. Deletion of either M₃ or M₅ receptor did not affect responses to nonmuscarinic vasodilators such as bradykinin or nitroprusside.

conclusions. These findings provide the first evidence that M₃ receptors are critically involved in cholinergic regulation of diameter in murine ophthalmic arteries.

Disturbed ocular and retrobulbar hemodynamics have been observed in a variety of eye diseases, including age-related macular degeneration, ^{1 2 3} diabetic retinopathy, ^{4 5 6} nonarteritic anterior ischemic optic neuropathy, ^{7 8} and glaucoma. ^{9 10} Endothelial dysfunction, defined as impaired endothelium-dependent vasodilation to specific stimuli, ¹¹ has been implicated in the pathophysiology of these diseases. ^{12 13 14 15} Acetylcholine is a powerful dilator of most vascular beds and a major investigative and diagnostic tool for the assessment of endothelial function. ^{16 17 18 19} Its activity is mediated by endothelial muscarinic receptors triggering the release of vasorelaxation agents, such as nitric oxide (NO). ^{20 21}  

Systemically or topically applied cholinergic agents, including muscarinic receptor agonists, were shown to increase ocular blood flow in experimental animals ^{22 23} and humans, ²⁴ suggesting that acetylcholine is involved in the regulation of ocular perfusion by the activation of muscarinic receptors. Hence, it is important to define muscarinic receptor signaling at the molecular level in ocular arteries to understand the pathophysiological changes in the eye that occur with endothelial dysfunction. 

Five muscarinic receptor subtypes, denoted M₁ through M₅, have been identified. ²⁵ They are generally grouped according to their functional coupling, either to the mobilization of intracellular calcium through the activation of phospholipase Cβ (M₁, M₃, M₅) or to the inhibition of adenylyl cyclase (M₂, M₄). ²⁶ Remarkably, the expression pattern of muscarinic receptor subtypes and their role in mediating vascular responses differ substantially between individual vascular beds. ^{27 28 29 30} Thus, they may represent an attractive therapeutic target for the selective treatment of local ischemic disorders. 

To date, muscarinic acetylcholine receptor expression has not been determined in ocular arteries. Therefore, we used real-time PCR to quantify mRNA expression of individual muscarinic receptor subtypes in isolated murine ophthalmic arteries. 

Previous studies making use of pharmacologic approaches and electrical stimulation of parasympathetic nerve pathways suggest the involvement of M₃ receptors in mediating choroidal vasodilation in pigeons, ³¹ and of M₃ and M₅ receptors in chronically sympathectomized rats. ³² However, conclusions regarding the physiological role of individual muscarinic receptor subtypes are hampered by the limited specificity of the pharmacologic agents tested. ^{25 33} For example, the pharmacologic properties of the M₃ receptor are similar to those of the M₅ subtype, ³⁴ raising the possibility that responses previously thought to be mediated by M₃ receptors may involve the activation of M₅ receptors. To circumvent these difficulties, we used mice deficient in the expression of M₃ and M₅ receptors to determine the role of either subtype in mediating cholinergic vasodilation in ophthalmic arteries. 

Materials and Methods

Animals

All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the federal animal rights committee. The generation of M3R^−/− and M5R^−/− mice has been described previously. ^{29 35} Briefly, the M₃ or the M₅ receptor gene was inactivated with the use of mouse embryonic stem cells derived from 129SvEv mice. The resultant chimeric mice were then mated with CF-1 mice to generate M3R^−/−, M5R^−/−, and wild-type mice with the following genetic contribution: 129SvEv (50%) × CF-1 (50%). In all experiments, male mice at the age of 3 to 5 months were used. 

Real-Time PCR Analysis in Isolated Ophthalmic Arteries

Muscarinic receptor gene expression was quantified in isolated ophthalmic arteries of wild-type mice using real-time PCR. After mice were killed by CO₂ inhalation, ophthalmic arteries were carefully isolated with the use of fine-point tweezers under a dissecting microscope, added to a 1.5-mL tube, and immediately snap frozen. To increase RNA yield, arteries were pooled from five mice. Subsequently, vessels were homogenized in lysis buffer with a homogenizing device (FastPrep; 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 an RT-PCR kit (Superscript; Invitrogen, Karlsruhe, Germany) and random hexamers. Quantitative PCR analysis was performed (GeneAmp StepOne Plus; Applied Biosystems, Darmstadt, Germany). 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 25 μL with 0.4 pmol/μL of each primer and 2× ready-to-use master mix (SYBR Green Master Mix; Bioline); 2 μL cDNA corresponding to 10 ng RNA was used as template. Published sequences for mouse M₁ (NM_007698), M₂ (NM_203491), M₃ (NM_033269), M₄ (NM_007699), and M₅ (NM_205783) were used to design primers for PCR amplification. Primer sequences were M₁ sense 5′-TGA CAG GCA ACC TGC TGG TGC T-3′ and antisense 5′-AAT CAT CAG AGC TGC CCT GCG G-3′; M₂ sense 5′-CGG ACC ACA AAA ATG GCA GGC AT-3′and antisense 5′-CCA TCA CCA CCA GGC ATG TTG TTG T-3′; M₃ sense 5′-CCT CTT GAA GTG CTG CGT TCT GAC C-3′ and antisense 5′-TGC CAG GAA GCC AGT CAA GAA TGC-3′; M₄ sense 5′-TGT GGT GAG CAA TGC CTC TGT CAT G-3′ and antisense 5′-GGC TTC ATC AGA GGG CTC TTG AGG A-3′; M₅ sense 5′-ACC ACT GAC ATA CCG AGC CAA GCG-3′ and antisense 5′-TTC CCG TTG TTG AGG TGC TTC TAC G-3′; and β-actin sense 5′-CAC CCG CGA GCA CAG CTT CTT T-3′ and antisense 5′-AAT ACA GCC CGG GGA GCA TC-3′. Expression levels of M₁, M₂, M₃, M₄, and M₅ mRNA were normalized to β-actin using the ΔCt method. 

Measurements of Vascular Reactivity

Mice were killed by CO₂ inhalation, and the eyes were rapidly removed, together with the retrobulbar tissue, and were placed in ice-cold Krebs buffer with the following ionic composition: 118.3 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, 11 mM glucose (Carl Roth GmbH, Karlsruhe, Germany). Then ophthalmic arteries from wild-type, M3R^−/−, and M5R^−/− mice were isolated under a dissecting microscope, placed in an organ chamber filled with cold Krebs solution, cannulated onto glass micropipettes, and secured with 10–0 nylon monofilament suture, as described previously for arteries of other vascular beds. ²⁹ Vessels were pressurized through the micropipettes to 50 mm Hg under no-flow conditions using two reservoirs filled with Krebs solution and were imaged using a video camera mounted on an inverted microscope (DM IL; Leica, Wetzlar, Germany). Video sequences were captured to a personal computer for analysis using imaging software (Khoros; Khoral Research, Inc., Albuquerque, NM). 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 60 minutes before the experiments were started. Viability of vessels was assessed as satisfactory when at least 50% constriction from resting diameter in response to membrane depolarization with KCl (100 mM) was achieved. Then contractile responses to the α₁-adrenergic receptor agonist phenylephrine (10⁻⁹–10⁻⁴ M; Sigma-Aldrich, Munich, Germany) were tested. Responses to KCl and phenylephrine did not differ between the three groups of mice (data not shown). Subsequently, arteries were preconstricted with phenylephrine to 50% to 60% of the initial vessel diameter, and cumulative concentration-response curves to acetylcholine (10⁻⁹–10⁻³ M), carbachol (10⁻⁹–10⁻³ M), bradykinin (10⁻¹¹–10⁻⁵ M), and nitroprusside (10⁻¹⁰–10⁻⁴ M) were obtained (all drugs from Sigma-Aldrich, Munich, Germany). Responses to acetylcholine were also compared before and after the addition of atropine (3 × 10⁻⁵ M; Sigma-Aldrich), a nonselective muscarinic receptor antagonist. 

Statistical Analysis

Data are presented as mean ± SE. Vascular responses are presented as percentage of change in diameter from the preconstricted diameter. When multiple vessels from a single mouse were studied, responses were averaged so that n represented the number of mice per group. Comparisons between concentration-response curves were made using ANOVA for repeated measures, and individual differences were detected by the Bonferroni test. For comparisons of vascular responses to acetylcholine before and after atropine treatment, the Wilcoxon signed-rank test was used. P < 0.05 was defined as significant. 

Results

Muscarinic Receptor mRNA Expression in Ophthalmic Arteries

Expression of muscarinic receptor mRNA was determined in ophthalmic arteries from wild-type mice using real-time PCR. All five muscarinic receptor subtypes were expressed in ophthalmic arteries. However, mRNA levels of M₁, M₃, and M₅ receptors, which couple to phospholipase Cβ and intracellular calcium mobilization, were higher than those of M₂ and M₄ receptors, which couple to the inhibition of adenylyl cyclase (Fig. 1) . 

Responses of Ophthalmic Arteries to Acetylcholine and Carbachol

Baseline luminal diameters of ophthalmic arteries (before preconstriction) were 109 ± 8 μm, 111 ± 8 μm, and 107 ± 10 μm in M3R^−/−, M5R^−/−, and wild-type mice and did not differ between individual groups (P > 0.05, ANOVA). 

To examine whether M₃ or M₅ receptors were involved in cholinergic responses of ophthalmic arteries, we compared vascular responses from M3R^−/−, M5R^−/−, and wild-type mice to acetylcholine (10⁻⁹–10⁻³ M) and carbachol (10⁻⁹–10⁻³ M). Acetylcholine elicited dose-dependent dilation of ophthalmic arteries from M5R^−/− mice that was not different from vasodilation obtained in wild-type mice (Fig. 2A) . Maximal dilation to 10⁻⁴ M acetylcholine was 44% ± 7% (n = 11) and 56% ± 9% (n = 11) in M5R^−/− and wild-type mice, respectively. 

Strikingly, acetylcholine-induced vasodilation was almost completely abolished in M3R^−/− mice. Dilation to 10⁻⁴ M acetylcholine was only 4% ± 2% (n = 11) in this group and was not statistically different from baseline values (Fig. 2A) . 

To test whether cholinergic responses of ophthalmic arteries were mediated by muscarinic receptors, we examined responses to acetylcholine after the addition of atropine (3 × 10⁻⁵ M), a nonselective muscarinic receptor blocker. After atropine treatment, responses to acetylcholine were virtually abolished in all groups of mice (Fig. 2B) , indicative of the involvement of muscarinic receptors. 

To exclude the possibility that the different responses to acetylcholine were caused by differences in acetylcholinesterase activity in the vascular wall, we tested the responses of ophthalmic arteries to carbachol, another muscarinic receptor agonist, which, in contrast to acetylcholine, is resistant to degradation by acetylcholinesterase. Similar to acetylcholine, carbachol induced dose-dependent relaxation of ophthalmic arteries from M5R^−/− and wild-type mice that did not differ between these two groups (Fig. 3) . For example, maximal dilation to 10⁻⁴ M carbachol was 38% ± 8% (n = 9) and 44% ± 9% (n = 11) in M5R^−/− and wild-type mice, respectively. In contrast, vasodilation to carbachol was only 4% ± 2% (n = 11) in M3R^−/− mice and was not statistically different from baseline values (Fig. 3) . 

Responses of Ophthalmic Arteries to Bradykinin and Nitroprusside

To test whether deletion of the M₃ and M₅ receptor genes affected responses to nonmuscarinic vasodilators in ophthalmic arteries, we examined vascular responses in M3R^−/−, M5R^−/−, and wild-type mice to the endothelium-dependent vasodilator bradykinin (10⁻¹¹–10⁻⁵ M). Bradykinin elicited concentration-dependent dilatory responses in arteries from M3R^−/−, M5R^−/−, and wild-type mice that did not differ between the three groups (Fig. 4A) . Maximal dilation to 10⁻⁶ M bradykinin was 18% ± 3% (n = 11), 22% ± 5% (n = 9), and 21% ± 4% (n = 11) in M3R^−/−, M5R^−/−, and wild-type mice, respectively. 

In addition, the endothelium-independent NO donor nitroprusside (10⁻¹⁰–10⁻⁴ M) produced concentration-dependent dilation of ophthalmic arteries that did not differ between the three groups of mice (Fig. 4B) . For example, maximal vasodilation to 10⁻⁵ M nitroprusside was 48% ± 8% (n = 11), 45% ± 9% (n = 9), and 53% ± 8% (n = 11) in M3R^−/−, M5R^−/− and wild-type mice, respectively. 

Discussion

The major goal of the present study was to identify the muscarinic receptor subtypes that mediate responses of ophthalmic arteries to acetylcholine. With the use of real-time PCR, we found all five muscarinic receptor subtypes to be expressed in ophthalmic arteries of wild-type mice. However, M₁, M₃ and M₅ receptors were expressed at higher levels than M₂ and M₄ receptors. Given that previous studies pointed toward an involvement of M₃ and M₅ receptors in the cholinergic regulation of ocular perfusion, ^{31 32} we used mice deficient in M₃ or M₅ receptors to assess the functional relevance of either subtype in isolated ophthalmic arteries. Strikingly, deletion of the M₃ receptor almost completely abolished cholinergic vasodilation, whereas the absence of M₅ receptors had no effect on responses to acetylcholine. Responses to acetylcholine were primarily mediated by muscarinic receptors because blockade of muscarinic receptor activation with atropine abolished responses in wild-type and M5R^−/− mice. In M3R^−/− mice, the dilation of ophthalmic arteries was also negligible in response to the muscarinic receptor agonist carbachol, which is resistant to degradation by acetylcholinesterase. Thus, increased acetylcholinesterase activity in the vascular wall is not likely to account for the strongly reduced cholinergic dilation of ophthalmic arteries in M3R^−/− mice. Deletion of either M₃ or M₅ receptor did not affect responses to nonmuscarinic vasodilators, such as the NO donor nitroprusside or the endothelium-dependent agonist bradykinin, suggesting that the absence of these receptors does not interfere with the downstream signaling cascades that ultimately mediate vasorelaxation. Thus, these data provide the first direct evidence that M₃ receptors are critically involved in cholinergic vasodilation of ophthalmic arteries. 

In a previous study using intravenous administration of subtype-selective muscarinic receptor antagonists and electrical stimulation of parasympathetic nerve pathways, it was suggested that endothelial M₃ receptors mediate choroidal vasodilation in pigeons. ³¹ In another study, in which a similar experimental approach has been used, M₃ and M₅ receptors were proposed to be involved in parasympathetic-mediated choroidal vasodilation in chronically sympathectomized rats. ³² However, the specificity of the pharmacologic agents tested has shown to be limited. ^{33 36} For example, the pharmacological properties of the M₃ receptor are similar to those of the M₅ subtype, ³⁴ raising the possibility that responses previously thought to be mediated by M₃ receptors may involve the activation of M₅ receptors. Moreover, even selective M₁ and M₂ antagonists display high affinity for M₃ and M₄ receptors, respectively. ^{25 33} Consequently, it is difficult to discern the role of each muscarinic receptor subtype by using pharmacologic agents of limited specificity when two or more subtypes are simultaneously involved in a specific functional response. The use of genetically engineered mice lacking specific muscarinic receptor subtypes allows a more definitive determination of the physiological roles of M₃ and M₅ receptors in ocular vessels. Recently, the function of muscarinic receptor subtypes has been examined in other arterial beds of gene-targeted mice. In these studies, the M₃ subtype was shown to mediate cholinergic vasodilation in femoral ³⁷ and coronary ³⁰ arteries and in the aorta, ^{30 37 38} whereas the M₅ subtype mediated responses to acetylcholine in cerebral arteries and arterioles. ²⁹ Remarkably, similar to our present findings in ophthalmic arteries, no functional role of M₅ receptors has been demonstrated for any extracerebral murine vascular bed tested thus far. ²⁹  

We found mRNA of all five muscarinic receptor subtypes to be expressed in ophthalmic arteries of wild-type mice. However, cholinergic responses were predominantly mediated by M₃ receptors, raising a question about the physiological role of the other coexpressed receptor subtypes. One possibility is that the other subtypes were expressed by vascular smooth muscle or by autonomic nerve terminals rather than by endothelial cells and that they play a role in regulating signaling pathways in vascular smooth muscle ²⁸ or in modulating transmitter release from autonomic nerves. ^{31 32}  

Based on previous in vivo studies in healthy animals and humans, systemic pharmacologic blockade of muscarinic receptors does not appear to significantly affect ocular blood flow under resting conditions or during isometric exercise. ^{31 39 40 41} In contrast, pharmacologic activation of muscarinic receptors was shown to increase pulsatile ocular blood flow in humans with ocular hypertension ²⁴ and long posterior ciliary artery blood flow in rabbits, ²² suggesting that muscarinic mechanisms are involved in ocular blood flow regulation. In support of this concept, pharmacologic blockade of muscarinic receptors was demonstrated to attenuate increases in choroidal blood flow induced by parasympathetic activation in pigeons. ³¹ Hence, it remains to be established under which conditions muscarinic acetylcholine receptors contribute to ocular blood flow regulation. 

Our findings in ophthalmic arteries do not necessarily reflect the situation in all ocular vessels because substantial anatomic and functional differences exist within the ocular vascular bed. ^{42 43} For example, retinal arteries as opposed to the ophthalmic artery are not innervated by autonomic nerve fibers, which may result in differences in autonomic input between the ophthalmic artery and retinal vessels. Thus, acetylcholine-mediated vasodilation in the ophthalmic artery induced by the activation of autonomic nerve fibers may transmit a higher blood pressure to the retinal vascular system, which in turn may respond with a myogenic constriction of arterioles to keep retinal blood flow constant. Furthermore, we cannot rule out the possibility that cholinergic responses of retinal arteries are mediated by another muscarinic receptor subtype than in ophthalmic arteries. 

In a variety of cardiovascular diseases, acetylcholine-induced vasodilation is impaired. The factors underlying these altered responses include changes of postreceptor mechanisms such as reduction of endothelial nitric oxide synthase expression and increase in protein kinase C activity during hyperglycemia, a mechanism that has been implicated in the pathogenesis of diabetic retinopathy. ^{44 45} However, in some pathologic conditions, vasodilation to acetylcholine is selectively impaired, whereas responses to other endothelium-dependent vasodilators are barely affected. ^{46 47} Thus, specific changes in muscarinic acetylcholine receptor function, for example, by receptor downregulation or uncoupling from intracellular signaling pathways, may also contribute to abnormal cholinergic vasodilation in pathologic conditions. 

Because of several technical limitations of in vivo measurements of ocular and retrobulbar blood flow ^{48 49 50} and the limited availability of human vascular tissue, studies in ocular vascular preparations from animal models remain important for understanding the mechanisms that account for ischemic disorders in the eye. The use of gene-targeted mice offers an attractive opportunity to define the mechanisms leading to disturbed ocular perfusion at the molecular level. Moreover, such studies may help to design specific pharmacologic approaches to treat abnormal ocular perfusion. 

In conclusion, data in the present study provide the first direct evidence that cholinergic vasodilation of ophthalmic arteries is mediated by M₃ receptors. From a clinical point of view, selective M₃ muscarinic receptor agonists may become therapeutically useful to increase ocular perfusion in some pathophysiological conditions such as age-related macular degeneration and glaucoma. 

 Supported by Grant 5/08 from the Ernst und Berta Grimmke Stiftung.

 Submitted for publication February 21, 2009; revised April 9, 2009; accepted June 25, 2009.

 Disclosure: A. Gericke, None; V.G.A. Mayer, None; A. Steege, None; A. Patzak, None; U. Neumann, None; F.H. Grus, None; S.C. Joachim, None; L. Choritz, None; J. Wess, None; N. Pfeiffer, 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.

 

Figure 1.

 

Relative mRNA expression of individual muscarinic receptor subtypes (M₁–M₅) normalized to β-actin transcripts in ophthalmic arteries pooled from five wild-type mice. Values are averages of three independent PCR measurements and are expressed as mean ± SE.

Figure 2.

 

Responses of ophthalmic arteries from wild-type, M3R^−/−, and M5R^−/− mice to acetylcholine. (A) Vasodilation to acetylcholine was almost completely abolished in ophthalmic arteries from M3R^−/− mice. In contrast, deletion of the M₅ receptor gene had no significant effect on relaxation of ophthalmic arteries in response to acetylcholine. Values are expressed as mean ± SE (n = 11 per concentration and genotype). *P < 0.01 (M3R^−/− vs. M5R^−/− and wild-type mice). Absence of error bar indicates that the SE was less than the size of the symbol. (B) Responses of ophthalmic arteries to acetylcholine (10⁻⁴ M) were virtually abolished after the addition of atropine (3 × 10⁻⁵ M). Values are expressed as mean ± SE (n = 8–10 per group). *P < 0.01.

Figure 3.

 

Responses of ophthalmic arteries from wild-type, M3R^−/−, and M5R^−/− mice to carbachol. Relaxation in response to carbachol was almost completely abolished in ophthalmic arteries from M3R^−/− mice, whereas deletion of the M₅ receptor gene had no significant effect on carbachol-induced vasodilation. Values are expressed as mean ± SE (n = 9–11 per concentration and genotype). *P < 0.01 (M3R^−/− vs. M5R^−/− and wild-type mice). Absence of error bar indicates that the SE was less than the size of the symbol.

Figure 4.

 

Responses of ophthalmic arteries from wild-type, M3R^−/−, and M5R^−/− mice to bradykinin (A) and nitroprusside (B). Deletion of either M₃ or M₅ receptor did not affect responses to nonmuscarinic vasodilators, such as the endothelium-dependent agonist bradykinin or the endothelium-independent NO donor nitroprusside. Values are expressed as mean ± SE (n = 9–11 per concentration and genotype).