Muscarinic (M) Receptors in Coronary Circulation

Kathryn G. Lamping; Jürgen Wess; Yinghong Cui; Daniel W. Nuno; Frank M. Faraci

From the Department of Internal Medicine and Pharmacology (K.G.L., D.W.N., F.M.F.), University of Iowa, Roy J. and Lucille A. Carver School of Medicine and the Veterans Administration Medical Center (K.G.L., D.W.N.), Iowa City, IA; and the Laboratory of Bioorganic Chemistry (J.W., Y.C.), National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD.

ABSTRACT

Objective— Determining the role of specific muscarinic (M) receptor subtypes mediating responses to acetylcholine (ACh) has been limited by the specificity of pharmacological agents. Deletion of the gene for M5 receptors abolished response to ACh in cerebral blood vessels but did not affect dilation of coronary arteries. The goal of this study was to determine the M receptors mediating responses to ACh in coronary circulation using mice deficient in M2 or M3 receptors (M2–/–, M3–/–, respectively).

Methods and Results— Coronary arteries from respective wild-type, M2–/–, or M3–/– mice were isolated, cannulated, and pressurized. Diameter was measured with video microscopy. After preconstriction with U46619, ACh produced dose-dependent dilation of coronary arteries that was similar in wild-type and M2–/– mice. In contrast, dilation of coronary arteries from M3–/– mice to ACh was reduced by 80% compared with wild type. The residual response to ACh was atropine insensitive. Relaxation of coronary arteries to other stimuli was similar in M2–/– and M3–/– mice. Similar results were obtained in aorta rings.

Conclusion— These findings provide the first direct evidence that relaxation to ACh in coronary circulation is mediated predominantly by activation of M3 receptors.

This study examined the M receptor subtype (M2 versus M3 receptors) involved in the response of coronary circulation to ACh using mice deficient in the genes for M2 and M3 receptors. M3 receptor activation and not M2 receptors primarily mediates responses to ACh in the coronary circulation.

Key Words: acetylcholine ? muscarinic receptors ? genetically altered mice

Introduction

Acetylcholine (ACh) is an important mediator of neurogenic vasodilation in coronary circulation1,2 as well as a major investigative tool for studies of endothelial function in experimental animals and in patients.3–5 Endothelial dysfunction, defined as impaired vasodilation to ACh, has been observed in many studies of cardiovascular diseases and may be associated with risk factors for coronary artery disease.3–5 In some cases, vascular responses to ACh are selectively altered,3–5 whereas responses to other endothelium-dependent dilators are minimally affected. To understand these mechanisms, including changes that occur with vascular dysfunction, it is important to define muscarinic (M) signaling at the molecular level. Although M receptor antagonists prevent ACh-induced relaxation,6,7 identification of specific M receptor subtypes mediating vascular relaxation to ACh has long been hampered by the lack of M antagonists with high selectivity for a single receptor subtype.8 In addition, overlapping expression patterns of different M receptors and identification of multiple M receptors within a given tissue contribute to the difficulty in linking specific M receptors with a specific physiological or pathophysiological response.9

Five major classes of M receptors, denoted M1 through M5, have been identified using pharmacological and molecular approaches.8 Depending on the specific receptor subtype, a wide variety of phenotypes has been observed in M receptor–deficient mice.10 These phenotypes have been reviewed recently in detail.10 Depending on the vascular bed and animal species, vascular relaxation or contraction to ACh has been suggested to be mediated by activation of different M receptors on the basis of pharmacological data.6,11–21 In cerebral circulation, we demonstrated that relaxation in response to ACh is mediated by activation of M5 receptors using mice deficient in M5 receptors.22 However, M5 receptor activation did not mediate responses to ACh in coronary circulation.22 Previous studies using pharmacological approaches suggest that M2 and M3 receptor activation may mediate responses to ACh in coronary circulation.6,20 Genes for both receptors are expressed in vascular tissue.23 However, the relative importance of M2 and M3 receptors in coronary circulation is not clear. Conclusions regarding the physiological role of the individual M receptors are hampered by the limited specificity of the pharmacological agents tested because even "selective" M2 and M3 antagonists show high affinity for M4 and M1 receptors, respectively.9 Moreover, the pharmacological properties of the M3 receptor are very similar to those of the M5 subtype, raising the possibility that responses thought previously to be mediated by M3 receptors may in fact involve the activation of M5 receptors.24 Thus, the goal of this study was to determine the role of M2 and M3 receptors in mediating ACh-dependent vasodilation in coronary circulation using mice deficient in the expression of either receptor subtype.25,26 For comparison, we conducted analogous studies with mouse aorta, by far the most commonly used blood vessel for studies of vascular biology in mice.27

Materials and Methods

M2–/– and M3–/– Mice

The generation of M2–/– and M3–/– mice has been described previously.25,26 The M3–/– mice and the corresponding wild-type mice had the following genetic background: 129SvEv (50%)xCFI (50%). The M2–/– mice and the corresponding wild-type mice had a slightly different genetic background:25 129J1 (50%)xCF1 (50%). For all experiments, adult male M2–/– and M3–/– mice and wild-type mice with the same respective genetic backgrounds (14 to 23 weeks of age) were used. Animal procedures were in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee at the University of Iowa and the Veterans Affairs Medical Center.

Measurements of Vascular Reactivity

Responses of coronary arteries and aorta were measured using methods published previously.22,28–30 Mice were heparinized (400 U/kg IP) and anesthetized with pentobarbital (100 mg/kg IP). The heart and thoracic aorta were removed rapidly and placed in ice-cold Krebs buffer. The anterior descending or circumflex arteries were isolated from the left ventricle under a dissecting microscope, cannulated and sutured onto micropipettes filled with Krebs buffer in an organ bath, and pressurized to 40 mm Hg. The vessel was imaged using a microscope and video camera, and lumen diameter measured from the video image with an electronic dimension analyzer. Arteries were allowed to equilibrate for 60 minutes before study. Isolated arteries were preconstricted submaximally with thromboxane mimetic U46619 (0.05 to 0.1 μg/mL) to 50% to 60% of maximal KCl responses. After development of stable constrictions, cumulative dose-response curves to ACh, sodium nitroprusside, or papavarine were obtained. Responses to ACh and sodium nitroprusside in arteries from wild-type and M3–/– mice were also compared before and after atropine (30 μmol/L), a nonselective M receptor blocker.

Responses of mouse aorta were examined as described previously.30,31 Aorta were cut into rings (3 to 4 mm in length), mounted on wires, and connected to a force transducer in an organ bath. Tension was increased over 60 minutes to 0.75g. Vessels were allowed to equilibrate 60 minutes before study. Rings were precontracted with U46619 (3 to 5 nmol/L) to maintain a stable precontraction of 50% to 60% of maximal KCl response before dose-response curves to ACh, calcium ionophore A23187, or papavarine were measured. Responses to ACh and sodium nitroprusside in aorta from wild-type and M3–/– mice were also compared before and after atropine (30 μmol/L). Relaxation responses were expressed as percentage decrease in tension from preconstriction values.

RT-PCR Analysis

Total RNA was extracted from aorta and brain using the total RNA isolation kit from Invitrogen. Extracted RNA samples were treated with 4 U of RNase-free DNase (Ambion) at 37°C for 1 hour to remove residual genomic DNA. The RNA was then reversed transcribed with an oligo-dT16 primer and murine leukemia virus reverse transcriptase using the GeneAmp RNA PCR kit, as described by the manufacturer (Applied Biosystems). The RT step was omitted in control samples to test for the presence of contaminating genomic DNA. The reverse-transcribed products were screened for the presence of M1 through M5 cDNA by PCR using the GeneAmp RNA PCR kit (Applied Biosystems) and an Eppendorf Mastercycler thermal cycler (40 cycles of 94°C at 1 minute, 56°C at 2 minutes, and 72°C at 3 minutes). PCRs were performed in a final volume of 50 μL containing 10 μL of the RT reaction product (corresponding to 0.2 μg RNA), 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 2 mmol/L MgCl2, 1 mmol/L of each 2'-deoxynucleoside 5'-triphosphate, 100 ng each of the sense and the corresponding antisense primers, and 1.25 U of AmpliTaq DNA polymerase. The identity of the PCR products was confirmed by restriction analysis (data not shown). The RT-PCR products were separated on 1.5% agarose gels containing ethidium bromide and photographed under UV illumination.

The sizes of the expected RT-PCR products were M1 (497 bp), M2 (480 bp), M3 (498 bp), M4 (474 bp), and M5 (485 bp). Subtype-specific primers were designed on the basis of the mouse M1 through M5 receptor sequences (GenBank data accession numbers: M1 J04192; M2 AF264049; M3 AF264050; M4 X63473; and M5 AF264051). The following primers were used: M1: 5'-TCTGCTCATCAGCTTTGACCG-3' (forward); 5'-CATCCTCTTCCTCTTCTTCTTTCC-3' (reverse); M2: 5'-TGTCAGCAATGCCTCCGTTATG-3' (forward); 5'-GCCTTGCCATTCTGGATCTTG-3' (reverse); M3: 5'-GGTGTGATGATTGGTCTGGCTTG-3' (forward); 5'-GGAAGCAGAGTTTTCCAGGGAG-3' (reverse); M4: 5'-TCAAGAGCCCTCTGATGAAGCC-3' (forward); 5'-AGATTGTCCGAGTCACTTTGCG-3' (reverse); M5: 5'-GCTGACCTCCAAGGTTCCGATTC-3' (forward); and 5'-CCGTCAGCTTTTACCACCAATC-3' (reverse).

Statistical Analysis

Data are presented as mean±SEM. Constrictions are presented as percentage of change in diameter from baseline diameter. Responses of vessels from the same mouse were averaged, and n represents the number of mice per group. Comparisons were made using a 1-way ANOVA with repeated measures followed by Student/Newman–Keuls test to detect individual differences. P<0.05 was defined as significant.

Results

Responses of Coronary Arteries from M2–/– and M3–/– Mice

To determine whether M2 receptors are involved in responses to ACh, we compared responses of coronary arteries from wild-type mice and M2–/– mice. ACh produced relaxation of coronary arteries from M2–/– mice that was not different from relaxation obtained in wild-type mice (Figure 1, left). For example, maximal dilation of arteries from wild-type and M2–/– mice to 10 μmol/L ACh was 36±9% (n=7) and 36±6% (n=6), respectively. Nitroprusside also produced dose-dependent dilation of coronary arteries not different in wild-type and M2–/– mice (data not shown). Thus, activation of M2 receptors is not involved in mediating dilator responses to ACh in coronary circulation of the mouse.

Figure 1. Responses of coronary arteries from wild-type and M2–/– mice (left) and M3–/– mice (right) to ACh. Deletion of the gene for M2 receptors had no significant effect on dilation of coronary arteries in response to ACh (wild type n=7; M2–/– n=6). Dilation in response to ACh was decreased in coronary arteries from M3–/– mice (n=17) compared with wild type (n=18; *P<0.05 vs wild type).

To determine the role of M3 receptors in mediating responses to ACh in coronary circulation, we compared responses of coronary arteries from wild-type mice and M3–/– mice to ACh. In wild-type mice, ACh produced dose-dependent vasodilation that was maximal at 10 μmol/L (31±5%; n=18; Figure 1, right). Dilation of coronary arteries from M3–/– mice to ACh was reduced by 80% compared with responses in wild-type mice (percentage of change diameter of 6±3% at 10 μmol/L, not statistically different from 0; n=17; Figure 1, right). Thus, activation of the M3 receptor is the primary mechanism that mediates dilation to ACh in coronary arteries.

To study whether deletion of the gene for M3 receptors affects responses to non-M vasodilators, we measured responses of coronary arteries from wild-type and M3–/– mice to nitroprusside and papavarine. Nitroprusside and papavarine produced dose-dependent dilation of coronary arteries from wild-type mice. Maximal dilation to papavarine (percentage of change diameter at 200 μmol/L 67±2% n=13; 72±6% n=10) was not different in wild-type and M3–/– mice, respectively. Similar results were obtained with nitroprusside (data not shown). Thus, deletion of the M3 receptor gene selectively decreased responses to ACh.

Responses of Aorta from M2–/– and M3–/– Mice

To determine whether activation of M2 receptors is involved in relaxation of aorta to ACh, we compared responses from wild-type and M2–/– mice. Relaxation of aorta in response to ACh (Figure 2, left), A23187 (Figure 3, left), and papavarine (maximal response at 10 μmol/L papavarine: wild type 103±3%, n=4; M2–/– 106±2%, n=4) was not different in wild-type and M2–/– mice. Thus, as observed in coronary circulation, activation of M2 receptors is not involved in mediating relaxation of aorta in response to ACh.

Figure 2. Response of aorta from wild-type and M2–/– mice (left) and M3–/– mice (right) to ACh. Deletion of the gene for M2 receptors had no effect on relaxation of aorta in response to ACh (wild type n=4; M2 receptor–/– n=4). Relaxation in response to ACh was decreased in M3–/– mice (n=11) compared with wild type (n=12; *P<0.05 vs wild type).

Figure 3. Response of aorta from wild-type and M2–/– mice (wild type n=4; M2–/– n=4, left) and M3–/– mice (wild type n=7; M3–/– n=5; right) to calcium ionophore A23187. Deletion of the gene for M2 or M3 receptors had no effect on relaxation of aorta in response to A23187, which releases NO via non-M receptor mechanism.

To determine whether the role of M3 receptors in mediating responses to ACh was specific for coronary circulation, we also compared responses of aorta from wild-type and M3–/– mice. ACh produced marked relaxation of aorta from wild-type mice (n=12; Figure 2, right). Relaxation to ACh at 10 μmol/L was reduced by 60% in aorta from M3–/– mice (n=11; Figure 2, right). In contrast, relaxation to papavarine (maximal response at 10 μmol/L: wild type 103±2%, n=7; M3–/– 105±1%, n=6) and the calcium ionophore A23187 (Figure 3, right) were not different in aorta from wild-type and M3–/– mice. Thus, vascular responses to activation of a non-M receptor mechanism, including another endothelium-dependent agonist, were normal in M3–/– mice.

Effects of Atropine on Responses to ACh

To confirm that the responses to ACh were because of activation of M receptors, we compared responses before and after atropine (30 μmol/L), a nonselective M receptor blocker. In coronary circulation (n=6) and aorta (n=5) of wild-type mice, atropine reduced the dilation to ACh by 60% to 70% in both vessels (Figure 4) but had no effect on the dilation to ACh in M3–/– mice (coronary arteries n=7; aorta n=5). Atropine had no effect on responses to nitroprusside in either coronary arteries or aorta (data not shown). Thus, the responses to ACh in the aorta and coronary circulation appear to be mediated primarily by activation of M receptors.

Figure 4. Responses of coronary artery (left) and aorta (right) from wild-type and M3–/– mice to ACh before and after addition of atropine (30 μmol/L). Atropine decreased responses in both coronary arteries (n=6) and aorta (n=5) from wild-type mice (*P<0.05 vs without atropine) but had no significant effect on vessels from M3–/– mice (coronary arteries n=7; aorta n=5).

RT-PCR Analysis of M Receptor Expression in Mouse Aorta

A recent RT-PCR study showed that coronary arteries not only express M3 but also M2 receptors.23 To examine which M receptor subtypes are expressed in the mouse vascular tissue, we conducted analogous studies using mouse aorta total RNA. Because all 5 M receptors are known to be expressed in the brain, mouse brain total RNA served as a positive control. cDNA was prepared from total RNA samples and subjected to PCR amplification by using primer pairs specific to the individual mouse M receptor genes, as described in Materials and Methods. The identity of all amplified PCR products was confirmed by restriction enzyme analysis (data not shown). As expected, all 5 M receptors were found to be expressed in the brain (Figure 5). In contrast, only M2 and M3 receptor cDNA could be detected consistently in samples from mouse aorta (Figure 5). Whereas the M2 signal was always very strong, the M3 signal was usually weaker. Occasionally, very faint bands were also observed with the M1-, M4-, or M5-specific primers. However, these signals were not always reproducible, probably because of the very low expression levels of these receptors in the mouse aorta.

Figure 5. RT-PCR analysis of M1 through M5 receptor expression in mouse aorta and total brain. A representative 1.5% agarose gel (stained with ethidium bromide) is shown. Primers specific for the individual mouse M receptors were used to amplify cDNA prepared from mouse aorta and brain total RNA (see Materials and Methods). As expected, all 5 M receptor subtypes were found to be expressed in the brain (positive control). In the aorta, only M2 and M3 receptor mRNA could be detected. Control samples that had not been treated with reverse transcriptase did not result in any detectable RT-PCR products, confirming the absence of contaminating genomic DNA. Four separate experiments gave similar results. M indicates marker DNA.

Discussion

The major goal of the present study was to use gene-targeted mice to identify the M receptor subtype that mediates responses of coronary circulation to ACh. Deletion of the M3 receptor in the mouse reduced coronary vasodilation to ACh by 80%. The response to ACh was primarily mediated by activation of M receptors because blockade of M receptor activation with atropine decreased responses to levels similar to deletion of the gene expressing M3 receptors. Relaxation of the aorta in response to ACh was also reduced to a large extent in M3–/– mice. In contrast, deletion of M2 receptors had no effect on responses to ACh in either coronary circulation or the aorta. Deletion of either M2 or M3 receptor did not affect responses to non-M vasodilators, such as papavarine and nitroprusside, or the endothelium-dependent agonist A23187. These data provide the first direct evidence regarding the importance (or lack thereof) of activation of M2 and M3 receptors in mediating vasodilation to ACh.

In previous studies from our laboratory, we tested the role of M5 receptors in mediating dilation to ACh in cerebral and coronary circulation. Cerebral blood vessels from M5–/– mice failed to dilate in response to ACh.22 In contrast, responses to ACh in coronary circulation from M5–/– mice were intact. These data provided the first physiological evidence of a role for M5 receptors in the blood vessels but suggest that this receptor subtype is not functionally important in coronary arteries.

Pharmacological studies and studies of mRNA expression have suggested diverse expression of M receptor subtypes in vascular tissue6,11–21,23. In coronary circulation, studies with subtype-preferring M antagonists suggested a role for M3 receptors in mediating endothelium-dependent relaxation.6,18,20 However, the proper interpretation of such classical pharmacological studies is complicated by the fact that the subtype selectivity of the M antagonists used in these studies is relatively small.8 Moreover, the pharmacological properties of the M3 receptor are very similar to those of the M5 subtype, raising the possibility that responses thought previously to be mediated by M3 receptors may in fact involve the activation of M5 receptors.24 Finally, it is generally very difficult to predict the simultaneous involvement of 2 or more M receptor subtypes in a specific functional response by using antagonists of limited subtype selectivity.

The recent development of gene-targeted mice lacking specific M receptor subtypes allows a more definitive approach to defining the physiological roles of M2 and M3 receptor subtypes.10,32 Recent studies have examined the role of these receptors in function of nonvascular smooth muscle.10,33–35 Using a similar approach, the present study clearly indicates that M3 receptors mediate the majority of the response to ACh in coronary circulation and in aorta. The findings in this study and our previous study,22 indicating that vascular responses to ACh are mediated by M5 receptors in brain and predominantly by M3 receptors in heart, highlight the importance of studies designed to define M signaling at the molecular level in different vascular beds. Identification of tissue-specific mechanisms that mediate vascular responses may be beneficial in designing organ-specific pharmacological approaches for investigative study or to treat vascular disease.

Although a major component of the vasodilator response to ACh was mediated by M3 receptors, a minor component was M3 receptor independent. Consistent with a previous RT-PCR study using RNA isolated from human coronary arteries,23 we found that mouse aorta does not only express M3 but also M2 M receptors. However, as observed recently with M5–/– mice,22 ACh-induced vasodilation responses in aorta and in coronary circulation were not significantly affected by the absence of M2 receptors, suggesting that the residual vascular responses remaining in the M3–/– mice are probably not mediated by M5 or M2 receptors. However, given the apparent abundance of M2 receptor mRNA in vascular tissue,36 we cannot rule out the possibility that a potential involvement of M2 receptors in this response may have remained undetected in the M2–/– mice because of the presence of the predominant M3 receptor pathway. One possibility is that M2 receptors are expressed by vascular muscle rather than endothelial cells and play a role in modulating signal pathways in smooth muscle. However, in coronary circulation, our results indicate that an atropine-insensitive mechanism mediates a very small (statistically insignificant) portion of the ACh response in wild-type and M3–/– mice. Although this small residual response may be mediated by nicotinic receptors, we feel this is unlikely because previous studies did not find evidence for nicotinic receptor activation in responses of normal arteries to ACh.37

Studies in knockout models for isoforms of nitric-oxide synthase (NOS), including our own,28,38,39 have demonstrated compensatory upregulation of other pathways in the absence of endothelial NOS. An analogous compensation does not appear to occur after deletion of a single M receptor.10 Because the majority of the response to ACh was absent in coronary arteries of M3–/– mice, any compensatory expression of other M receptors must be very modest and does not result in preservation of a functional response.

In conclusion, the results of the present study provide direct evidence that ACh-induced vasodilation in aorta and coronary circulation is mediated predominantly by M3 receptors. These findings highlight the usefulness of using M receptor mutant mice to study the complex mechanisms regulating responses in different vascular beds.

Acknowledgments

Supported by grants from the National Institutes of Health (HL39050, HL62984, and NS-24621), and a grant-in-aid from the American Heart Association. K.G.L. and F.M.F. are established investigators of the American Heart Association.

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