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Institute for Anatomy and Cell Biology, Justus-Liebig-University, Giessen, Germany (N.S., S.S., S.W., A.G., W.K., R.V.H.); and Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive Kidney Diseases, Department of Health and Human Services, Bethesda, Maryland (M.Y., J.W.)
Received April 4, 2003; accepted September 5, 2003
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Abstract |
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; Fryer and Jacoby, 1998; Barnes, 2001). An increase in pulmonary cholinergic nerve activity is associated with chronic obstructive pulmonary disease (COPD) and asthma (Zaagsma et al., 1997; Fryer and Jacoby, 1998; Barnes, 2001), and asthmatic patients are hypersensitive to the bronchoconstricting actions of muscarinic agonists (Jacoby and Fryer, 2001). Moreover, muscarinic antagonists are highly useful drugs in treating COPD and certain forms of asthma (Watson and Eglen, 1999; Jacoby and Fryer, 2001). Taken together, these findings underscore the high clinical relevance of pulmonary MRs.
Molecular cloning studies have led to the identification of five molecularly distinct MR subtypes (Caulfield and Birdsall, 1998). The M1, M3, and M5 receptor subtypes couple preferentially to the Gq/11 protein, whereas the M2 and M4 receptors are preferentially connected with Gi (Caulfield, 1993; Felder, 1995). Stimulation of these receptors leads to a vast array of intracellular events, including the hydrolysis of phosphatidylinositol, an increase of [Ca2+]i, the activation of mitogen-activated protein kinases, and the inhibition of cAMP synthesis (Felder, 1995).
The M1, M2, and M3 receptor subtypes have been detected in murine, porcine, and human airways (Fryer and el-Fakahany, 1990; Mak et al., 1992; Garssen et al., 1993; Hislop et al., 1998). Previous work suggests that the muscarinic constriction of tracheal smooth muscle and the main bronchi is mainly mediated by the M3 receptor subtype (Roffel et al., 1990; Stengel et al., 2000, 2002). On the other hand, the contribution of individual MR subtypes to the constriction of smaller intrapulmonary airways has not been analyzed to date. However, such knowledge is of particular pathophysiological relevance because these smaller airways are primarily responsible for airway resistance (Wohlsen et al., 2001; Martin, 2002). To address this issue, we used the model of precision-cut lung slices (PCLS). This preparation allows for the study of small peripheral murine airways that are comparable in structure to the terminal bronchioles in human lung (Martin et al., 1996; Wohlsen et al., 2001). Specifically, we used videomicroscopy and digital imaging to study muscarine-mediated responses of intraparenchymal murine airways. Because multiple MRs are expressed in the lung (Zaagsma et al., 1997; Fryer and Jacoby 1998; Barnes, 2001) and it is difficult to distinguish between the different MR subtypes by classic pharmacological tools (Caulfield and Birdsall, 1998), elucidating the physiological and pathophysiological roles of the individual MR subtypes in pulmonary function represents a considerable challenge. To overcome these difficulties, we obtained PCLS preparations from recently developed MR mutant mice. To asses the functional roles of the M1, M2, and M3 receptor subtypes in peripheral airway responses, we analyzed airways of different sizes in mice deficient in either the M1 (mAChR1-/- mice), M2 (mAChR2-/- mice), or the M3 subtype (mAChR3-/- mice) (Gomeza et al., 1999; Miyakawa et al., 2001; Yamada et al., 2001; Fisahn et al., 2002). In addition, we also generated and studied mice lacking both the M2 and M3 subtypes (mAChR2/3-/- double-knockout mice).
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Materials and Methods |
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; Miyakawa et al., 2001; Yamada et al., 2001; Fisahn et al., 2002). The mAChR1-/- and mAChR3-/- mice and the corresponding wild-type mice (mAChR+/+ mice) had the following genetic background: 129SvEv (50%) x CF1 (50%). The mAChR2-/- mice and the corresponding wild-type mice (mAChR2+/+ mice) had a slightly different genetic background (Gomeza et al., 1999): 129J1 (50%) x CF1 (50%).
To generate animals deficient in both the M2 and M3 subtypes, homozygous mAChR2-/- mice (Gomeza et al., 1999) were crossed with homozygous mAChR3-/- mice (Yamada et al., 2001; Zhang et al., 2002). The resulting F1 compound heterozygotes were then intercrossed to generate F2 mice. mAChR2/3-/- mice were obtained at the expected Mendelian ratio and were subsequently interbred to generate the animals used for the experiments described here. F2 wild-type mice were interbred to obtain control mice (mAChR2/3+/+) with an equivalent genetic background [129/J1 (25%) x 129SvEv (25%) x CF1 (50%)]. All experiments were carried out with male mice that were 6 to 12 weeks old.
RT-PCR. For RT-PCR studies, lung slices from MR-deficient and wild-type mice and brains from wild-type mice (mAChR+/+ mice; positive control) were transferred into lysis buffer (QIAGEN GmbH, Hilden, Germany) and homogenized using a mixer mill with a frequency of 300 Hz (QIAGEN). Total RNA was isolated using spin columns according to the protocol recommended by the manufacturer (RNeasy Kit, QIAGEN). Contaminating DNA was removed using DNase (1 U/µg total RNA; Invitrogen, Carlsbad, CA) in the presence of 20 mM Tris-HCl, pH 8.4, 2 mM MgCl2, and 50 mM KCl for 15 min at 25°C. Equal amounts of RNA were reverse-transcribed in the presence of 3 mM MgCl2, 75 mM KCl, 50 mM Tris-HCl, pH 8.3, 10 mM dithiothreitol, 0.5 mM dNTPs (Invitrogen), and 25 µg oligo(dT) (MWG Biotech, Ebersberg, Germany), with 200 U of Superscript RNase H- reverse transcriptase (Invitrogen) for 50 min at 42°C. For the PCR, 2 mM MgCl2 (10 mM), 0.25 mM dNTP (10 mM), 0.5 U/25 µl AmpliTaq Gold DNA Polymerase (all reagents from PerkinElmer Life Sciences, Boston, MA), and 20 µM of each primer (Table 1; MWG Biotech) were mixed (buffer 2). Cycling conditions for PCR were 10 min at 95°C, 40 cycles of 30 s at 94°C, 20 s at 62°C, and 30 s at 73°C, followed by 7 min at 73°C.
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Quantitative RT-PCR. Real-time quantitative PCR (iCycler; Bio-Rad, Munich, Germany) was used to quantify levels of mAChR1, -2, and -3 mRNAs in PCLS of MR subtype-deficient lungs. Preparation of cDNA was done as described for RT-PCR. Gene-specific Taq-Man PCR primers and probes specific for mouse mAChR1, -2, and -3 and GAPDH (Table 1) were designed using Primer Express software (Applied Biosystems, Foster City, CA). All PCRs were prepared in triplicate from four to eight animals using a ready-to-use kit according to the manufacturer's protocol (QuantiTect, QIAGEN). Primers specific for GAPDH were used for standardization. The data were normalized by subtracting the threshold cycle levels between the mAChRs and GAPDH.
Videomorphometry. Mice were killed by cervical dislocation, and precision-cut lung slices were prepared using a slight modification of the protocol described by Martin et al. (1996). Briefly, the lungs were perfused via the right ventricle with Krebs-Ringer buffer containing heparin (1000 IU), penicillin/streptomycin (1%), and sodium nitroprusside (0.075 µM). The airways were filled via the cannulated trachea with agarose (low-melting-point agarose, 1.6% in Krebs-Ringer buffer; Sigma Chemie, Deisenhofen, Germany). Subsequently, the lungs and heart were removed en bloc and placed in ice-cold Krebs-Ringer buffer. The cranial lobe of the right lung was cut into 200- to 250-µm thick slices using a vibratome (VT1000S; Leica, Wetzlar, Germany). For the removal of the agarose, the slices were incubated in minimal essential medium at 37°C under normoxic conditions for 2 to 4 h. Experiments were performed under normoxic conditions in a lung-slice superfusion chamber (Hugo Sachs Elektronik-Harvard Apparatus GmbH, March-Hugstetten, Germany) mounted on an inverted microscope (Leica) equipped with a camera (Stemmer Imaging, Puchheim, Germany). The MR agonist muscarine was purchased from Sigma Chemie. Viable airways of 100 to 400 µm in diameter were examined and incubated in the slide chamber for 5 min in minimal essential medium until the first image was acquired. The area of the airway lumen at the beginning of the experiment was defined as 100%, and bronchoconstriction or dilation was expressed as a percentage decrease or increase of this area. The studies were performed using slices after 4 to 8 h in culture. For the analysis, airways were subsequently analyzed using the Optimas 6.5 Image Analysis software program (Media Cybernetics, Silver Spring, MD).
Statistical Analysis. Data are presented as means ± S.E.M. of 4 to 18 slices obtained from three to six animals. Because it is principally impossible to test for a normal (Gaussian) distribution at such numbers, nonparametric statistical tests were used (SPSS software; SPSS Inc., Chicago, IL). Matched pairs were evaluated with use of the Wilcoxon rank sum test. In the case of more than two non-matched groups, Mann-Whitney U test for comparison between two groups was conducted only when statistically significant differences were reached by the global Kruskal-Wallis test that was performed first. Differences were considered statistically significant when p < 0.05.
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Results |
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Videomorphometry of Wild-Type Mice. In lung slices from wild-type mice (mAChR+/+, mAChR2+/+, and mAChR2/+/+ mice), the cumulative administration of muscarine (10-8-10-4 M) resulted in concentration-dependent bronchoconstrictor responses (decreases in luminal airway area) (Figs. 2, 3, 4). The basal airway diameters were the following: mAChR+/+, 215 ± 31 µm; mAChR2+/+, 216 ± 13 µm; and mAChR2/3+/+, 205 ± 24 µm. The muscarine responses consisted of three distinct phases (Figs. 2B and 5). First, a robust bronchoconstriction developed within the first minute after muscarine administration. This initial bronchoconstriction was followed by a transient relaxation response. Finally, this activity was followed by a sustained bronchoconstriction. Approximately 10 min after administration, the highest muscarine concentration used (10-4 M) led to a 
50 to 60% reduction of the luminal area (luminal area of 48 ± 7% in mAChR+/+ mice, 43 ± 3% in mAChR2+/+ mice, and 40 ± 9% in mAChR2/3+/+ mice) (Figs. 2D, 3D, and 4D).
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A single application of 10-4 M muscarine to smaller airways with a diameter of approximately 160 µm (mAChR+/+, 155 ± 11 µm; mAChR2+/+, 167 ± 7 µm) caused a pronounced bronchoconstrictor response (remaining luminal area: 16 ± 6%) (Fig. 5). The secondary transient relaxation was more pronounced in mAChR+/+ than in mAChR2+/+ mice (Fig. 5).
mAChR1-/- Mice. In mAChR1-/- mice (airway diameter, 277 ± 21 µm), cumulative administration of muscarine (10-8-10-4 M) induced concentration-dependent decreases in airway area, as observed with wild-type mice (Fig. 2B). However, compared with bronchi from wild-type animals, muscarine caused significantly stronger bronchoconstriction responses in mAChR1-/- bronchi at concentrations of 10-8 M (luminal area of 69 ± 7% in mAChR1-/- mice versus 88 ± 6% in mAChR+/+ mice, p = 0.036, Mann-Whitney U test) (Fig. 2, C and D) and 10-6 M (luminal area of 52 ± 8% in mAChR1-/- mice versus 62 ± 13% in mAChR+/+ mice, p = 0.021, Mann-Whitney U test) (Fig. 2, C and D). At the highest concentration of muscarine used (10-4 M), however, the response of mAChR1-/- bronchi was not significantly different from that of mAChR+/+ preparations (luminal area of 39 ± 9% versus 46 ± 11%, respectively; p = 0.145, Mann-Whitney U test) (Fig. 2, C and D). Strikingly, the transient bronchorelaxation response after the initial bronchoconstriction in wild-type preparations was absent in smaller bronchi from mAChR1-/- mice (airway diameter, 139 ± 12 µm) (Figs. 2B and 5).
mAChR2-/- Mice. In mAChR2-/- mice (airway diameter, 242 ± 8 µm), cumulative application of muscarine (10-8-10-4 M) led to concentration-dependent bronchoconstriction, as was observed with the corresponding wild-type mouse strain (mAChR2+/+ mice) (Fig. 3B). Compared with bronchi from wild-type animals, mAChR2-/- bronchi showed similar rapid bronchoconstriction responses (Fig. 3C) but a subsequent relaxation in the presence of 10-4 M muscarine (luminal area 10 min after muscarine, 69 ± 8% in mAChR2-/- mice versus 55 ± 5% in mAChR2+/+ mice, p = 0.023, Mann-Whitney U test) (Fig. 3D). Similarly, the sustained bronchoconstriction of smaller airways, measuring 168 ± 7 µm in diameter, after a single application of 10-4 M muscarine was greatly reduced in mAChR2-/- mice (luminal area 15 min after muscarine, 57 ± 7% in mAChR2-/- mice versus 33 ± 8% in mAChR2+/+ mice, p = 0.049, Mann-Whitney U test) (Fig. 5). In contrast, the initial rapid bronchoconstriction response did no differ between mAChR2+/+ and mAChR2-/- mice (Fig. 5).
mAChR3-/- Mice. In mAChR3-/- mice (airway diameter, 212 ± 14 µm), cumulative muscarine application (10-8-10-4 M) led to concentration-dependent bronchoconstriction responses, as was observed with mAChR+/+ mice (Fig. 2B). The application of a low concentration of muscarine (10-8 M) led to a bronchoconstriction response that did not differ significantly from that obtained with mAChR+/+ mice (Fig. 2, B-D). However, higher muscarine concentrations (10-6 M and 10-4 M) resulted in significantly reduced bronchoconstriction responses in mAChR3-/- mice (10-6 M muscarine: luminal area of 64 ± 5% in mAChR3-/- mice versus 44 ± 8% in mAChR+/+ mice, p = 0.029, Mann-Whitney U test; 10-4 M muscarine: luminal area of 60 ± 4% in mAChR3-/- mice versus 46 ± 11% in mAChR+/+ mice, p = 0.018, Mann-Whitney U test) (Fig. 2, B and C).
Compared with preparations from wild-type mice, single administration of 10-4 M muscarine to smaller airways (168 ± 11 µm in diameter) from mAChR3-/- mice caused a significantly reduced rapid bronchoconstriction response (by approximately 60%; luminal area of 68 ± 5 µm in mAChR3-/- mice versus 16 ± 6 µm in mAChR+/+ mice, p = 0.04, Mann-Whitney U test) (Fig. 5). The transient relaxation response observed with preparations from mAChR3-/- mice (39 ± 10% relaxation after initial constriction) was not different from that seen with mAChR+/+ mice (35 ± 10%) (Fig. 5).
mAChR2/3-/- Mice. We also generated (see Materials and Methods) and studied mAChR2/3-/- double-knockout mice. Strikingly, the bronchoconstriction response to the cumulative application of muscarine (10-8-10-4 M) was completely abolished in PCLS preparations from mAChR2/3-/- mice (airway diameter, 192 ± 23 µm) (Fig. 4, A-D). The corresponding wild-type control mice (mAChR2/3+/+) gave the same spectrum of responses as found with the mAChR+/+ mice (Figs. 2 and 4).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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; Fryer and Jacoby, 1998; Barnes, 2001). Moreover, increased cholinergic activity is known to be associated with pulmonary diseases such as COPD and certain forms of asthma (Zaagsma et al., 1997; Fryer and Jacoby, 1998; Barnes, 2001). Airway resistance is determined largely by the diameter of smaller intrapulmonary airways (Martin 2002; Escolar et al., 2003). A better understanding of which MR subtypes contribute to the constriction of these intrapulmonary airways is therefore of considerable clinical relevance. To address this question, we used the PCLS model, which has been shown to maintain the integrity of all components of the peripheral lung, including viable peripheral airways (Martin et al., 1996; Wohlsen et al., 2001).
We initially demonstrated with the use of an RT-PCR strategy that the mAChR1, mAChR2, and mAChR3 subtypes are expressed in mouse peripheral airways. In agreement with this finding, these receptor subtypes are present in various other pulmonary preparations from different species, including humans (Zaagsma et al., 1997; Fryer and Jacoby, 1998; Barnes, 2001). The lack of ligands endowed with a high degree of MR subtype selectivity (except for some recently discovered snake toxins) represents a considerable obstacle in identifying the physiological roles of the individual MR subtypes (Caulfield and Birdsall, 1998). To examine the importance of individual MR subtypes in airway function in a more direct fashion, we therefore took advantage of the recent availability of mutant mouse strains deficient in specific mAChR subtypes. Specifically, we studied muscarine-mediated responses of mouse peripheral airways from mAChR1-/-, mAChR2-/-, and mAChR3-/- mice (Gomeza et al., 1999; Miyakawa et al., 2001; Yamada et al., 2001; Fisahn et al., 2002) and from newly generated mAChR2/3-/- mice. This analysis allowed us to determine the relative contributions made by the M1, M2, and M3 receptor subtypes to the constriction of peripheral airways. Quantitative RT-PCR studies showed that the disruption of individual mAChR genes did not lead to compensatory changes in the expression levels of the remaining MR subtypes.
Previous pharmacological studies indicated that the M3 receptor subtype is responsible for the receptor-mediated constriction of mouse and human trachea and human larger bronchi (Roffel et al., 1990; Stengel et al., 2000). In the present study, we observed a markedly reduced cholinergic constriction in mAChR3-/- bronchi of the peripheral lung similar to that observed in the trachea of the same mouse strain (Stengel et al., 2002). In the trachea, mAChR3 deletion reduced the constriction to the ACh-receptor agonist carbamylcholine by 50% (Stengel et al., 2002), whereas the reduction amounted to up to 60% in peripheral bronchi of different sizes (this study). These findings clearly indicate that muscarinic airway constriction involves both M3 and non-M3 MR subtypes. Despite the general reduction in muscarine-induced bronchoconstriction in mAChR3-/- mice, a triphasic response, characterized by an initial constriction, a transient relaxation, and a sustained constriction, still persisted in this mutant mouse strain.
Previous studies have shown that airway smooth-muscle cells express both the M2 and M3 receptor subtypes (Zaagsma et al., 1997; Fryer and Jacoby 1998; Barnes, 2001). To test the hypothesis that the M2 receptor subtype contributes to cholinergic constriction of peripheral airways, we studied muscarine-mediated airway responses in mAChR2-/- mice and mAChR2/3-/- mice. Strikingly, muscarine-mediated bronchoconstriction was completely abolished in mAChR2/3-/- mice. This observation clearly indicates that MR-mediated constriction of mouse peripheral airways involves the activation of both the M2 and M3 receptor subtypes and that no additional MR subtypes are involved. Using independently generated mAChR3-/- and mAChR2/3-/- mice, Matsui et al. (2002) recently showed that activation of both the M2 receptor and M3 receptor subtypes also fully accounts for muscarinic agonist-mediated contractile responses of ileal and bladder smooth-muscle preparations. This concerted action of the M2 and M3 receptor subtypes in regulating smooth muscle tone therefore seems to represent a rather general phenomenon.
A recent study (Matsui et al., 2003) showed that the relaxant effects of forskolin on muscarinic agonist-induced contractions were significantly greater in tracheal preparations from mAChR2-/- mice than in the corresponding preparations from wild-type mice, suggesting that a component of the muscarinic agonist-induced contraction response in tracheal smooth muscle involves an M2 receptor-mediated inhibition of the relaxant effect of agents that increase cAMP levels. However, our data indicate that the M2 receptor subtype is also directly involved in maintaining bronchoconstriction because bronchi from mAChR2-/- mice showed an attenuation of the sustained constriction response observed after muscarine administration. The underlying molecular pathways are still unclear but may include M2 receptor-mediated activation of Ca2+-dependent potassium channels (Kotlikoff et al., 1992). The differences in airway contractility and MR expression levels observed between the mAChR2+/+ mice and the other two wild-type strains may be related to the slightly different genetic background of the mAChR2+/+ mice (see Materials and Methods).
In addition to the M2 and M3 receptor subtypes, the M1 receptor is also expressed in lung tissue (Barnes 1993; Reinheimer et al., 2000). In the present study, bronchi from mAChR1-/- mice showed a significantly increased bronchoconstriction in response to muscarine (10-8 M) and lacked the transient relaxation after muscarine administration independent of the airway caliber. These data suggest the existence of an M1 receptor-dependent pathway counteracting cholinergic bronchoconstriction, possibly via the release of a relaxing agent. Both respiratory epithelia and sympathetic nerve terminals within bronchial smooth muscle are equipped with M1 receptors (Maclagan et al., 1989; Shapiro et al., 2001) and releasable bronchodilating agents (epithelium: nitric oxide and prostaglandin E2) (Spicuzza et al., 2002; Tilley et al., 2003). Studies with the M1 receptor-preferring antagonist pirenzepine have also suggested the existence of pulmonary (ganglionic) M1 receptors modulating airway diameter (Bloom et al., 1988; Maclagan et al., 1989). The relative contributions to M1 receptor-dependent relaxation made by epithelial cells and nerve terminals, both of which are present in the prepared PCLS, cannot be deduced from the present data.
Previous studies have shown that the M4 receptor subtype is the predominant MR expressed in rabbit lung tissue (Lazareno et al., 1990). Moreover, work by Kilbinger et al. (1995) suggested the existence of presynaptic M4 receptors mediating the autoinhibition of acetylcholine release in the guinea pig trachea. However, the results of this study, together with the observation that the inactivation of the mAChR4 gene in mice has no significant effect on carbachol-mediated contraction responses in tracheal smooth muscle (Stengel et al., 2000), suggest that M4 receptors do not play a significant role in muscarinic agonist-induced bronchoconstriction, at least not in the mouse.
In conclusion, the present study demonstrates that cholinergic constriction of murine peripheral airways is mediated by the concerted action of the M2 and M3 receptor subtypes. Our data also strongly suggest the existence of pulmonary M1 receptors, the activation of which counteracts this bronchoconstrictor activity. These findings should be of considerable relevance for the development of novel muscarinic drugs useful for the treatment of COPD and asthma.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: ACh, acetylcholine; COPD, chronic obstructive pulmonary disease; MR, muscarinic acetylcholine receptor (protein); mAChR+/+, wild-type control (gene); mAChR2/3+/+, wild-type control for double-knockout mice; mAChR1-/-, muscarinic acetylcholine receptor 1-deficient mice; mAChR2-/-, muscarinic acetylcholine receptor 2-deficient mice; mAChR3-/-, muscarinic acetylcholine receptor 3-deficient mice; mAChR2/3-/-, mice deficient in muscarinic acetylcholine receptors 2 and 3, PCLS, precision-cut lung slices; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcriptase; PCR, polymerase chain reaction.
Address correspondence to: Dr. R.V. Haberberger, Institut fur Anatomie and Zellbiologie, Autweg 123, D-35385 Gliessen, Germany. E-mail: rainer.v.haberberger{at}anatomie.med.uni-giessen.de
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), mAChR1-/- (
), and mAChR3-/- (
) mice. Measurements were taken 1 min (C) and 10 min (D) after application of 10-8,10-6, and 10-4 M muscarine. 
, p 
0.05; 
