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Direct pharmacological comparison of the muscarinic receptors mediating relaxation and contraction in the rabbit thoracic aorta
General Pharmacology 32 (1999) 445–452
Direct pharmacological comparison of the muscarinic receptors mediating relaxation and contraction in the rabbit thoracic aorta Barry D. Sawyer, Frank P. Bymaster, David O. Calligaro, Julie Falcone, Charles H. Mitch, John S. Ward, Celia Whitesitt, Harlan E. Shannon * Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, USA Manuscript received April 28, 1998; accepted manuscript July 7, 1998
Abstract The purpose of the present studies was to directly compare the pharmacology of the muscarinic cholinergic receptors coupled to carbachol-induced relaxation and contraction of the intact and the endothelium-denuded rabbit thoracic aorta, respectively. The order of potencies of agonists for producing relaxation in the intact aorta was similar to that for producing contraction in the denuded aorta. In both preparations, the partial agonists pilocarpine, McN-A-343, and RS86 functioned as antagonists, indicating a lack of receptor reserve in both preparations. Further, the pA2 values for antagonists in both tissues were virtually identical and were consistent with the pharmacology of M3 receptors. 1999 Elsevier Science Inc. All rights reserved. Keywords: Muscarinic M3 receptors; Muscarinic M2 receptors; Thoracic aorta (rabbit); Muscarinic receptor partial agonists; Carbachol; Pilocarpine; RS86
Cholinergic agonists produce relaxation of various endothelium-intact vascular smooth muscle preparations in vitro including the rabbit aorta (Angus et al., 1983; Furchgott and Zawadski, 1980). The nonselective muscarinic antagonist atropine blocks cholinergic agonistinduced relaxation of the rabbit aorta, thus indicating that relaxation is mediated through muscarinic receptors. The affinities of selective muscarinic antagonists at the receptor mediating muscarinic agonist-induced relaxation support the interpretation that M3 receptors mediate relaxation in the rabbit aorta (Choo et al., 1986; Eglen and Whiting, 1985a; Eglen et al., 1994). Stimulation of muscarinic receptors located on endothelial cells produces relaxation of the rabbit aorta through synthesis of nitric oxide (Furchgott and Zawadski, 1980). Accordingly, removal of endothelium or exposure to nitric oxide synthase inhibitors eliminates muscarinic receptor-mediated relaxation (Furchgott and Zawadski, 1980). In the absence of endothelium, muscarinic agonists can produce contraction of vascular preparations by activation of receptors located directly on the smooth muscle (Eglen and Whiting, 1990; Furchgott and Cherry, 1984). The muscarinic receptor subtype * Corresponding author. Tel.: (317) 276-0749; Fax: (317) 2765546; E-mail: [email protected].
mediating contraction appears to be species and tissue dependent (Eglen and Whiting, 1990). In the rabbit aorta, the identity of the subtype of muscarinic receptor mediating contraction is controversial. The rank order of affinities of the muscarinic antagonists atropine and pirenzepine (Choo et al., 1986) obtained in vitro from static muscle bath experiments indicates that M3 receptors mediate muscarinic agonistinduced contraction in the rabbit aorta. However, more recent studies, with the use of superfusion techniques, provide some evidence that muscarinic agonist-induced contraction may be mediated by M2 receptors (Jaiswal et al., 1991). Most recently, Watson and Eglen (1994), using a broad range of muscarinic antagonists to inhibit (1)cis-dioxolane-induced contraction in static muscle bath preparations, concluded that muscarinic M3 receptors mediate contraction of the endothelium-denuded rabbit aorta. Data reported by different workers from experiments in the endothelium-denuded aorta cannot be directly compared, owing to the use of different preparations (partially contracted vs. noncontracted aorta), different test systems (superfusion vs. static baths), different agonists and antagonists, different experimental design, and different statistical analyses of data (Choo et al., 1986; Jaiswal et al., 1991; Watson and Eglen, 1994).
0306-3623/99/$–see front matter 1999 Elsevier Science Inc. All rights reserved. PII: S0306-3623(98)00215-8
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Therefore, the apparent discrepancies between results from different laboratories may be due to the use of different methodologies. The purpose of the present study was to directly compare the effects of muscarinic agonists and antagonists on the endothelium-intact and endotheliumdenuded rabbit thoracic aorta by using the same test system, drugs, experimental design, and statistical analysis. First, we characterized the subtype(s) of muscarinic receptor(s) that mediate muscarinic agonistinduced responses in the endothelium-intact and the endothelium-denuded rabbit aorta by using six muscarinic agonists and four muscarinic antagonists. In experiments with agonists, McN-A-343, pilocarpine, and RS86 demonstrated no agonist activity in either the endothelium-intact or the endothelium-denuded aorta However, at micromolar concentrations, all thee of these low-efficacy agonists decreased responses to a maximally effective concentration of carbachol in the denuded aorta. Consequently, we evaluated pilocarpine, McN-A-343, and RS86 as antagonists of carbachol in both the endothelium-intact and the endotheliumdenuded aorta. Finally, to better understand the lack of agonist activity of pilocarpine, McN-A-343, and RS86, we compared relative muscarinic receptor densities in the rabbit aorta, the guinea pig urinary bladder, and the guinea pig ileum longitudinal muscle by measuring [3H]N-met-scopolamine binding under saturating conditions.
1. Materials and methods Male New Zealand White rabbits (Hazelton Research Products, Denver, PA) were sacrificed by IV sodium pentobarbital overdose. Thoracic aortas were dissected free of surrounding tissue, cut into segments 3–5 mm in length, and placed in modified Krebs’ solution of the following composition (in millimolar): NaCl, 134; KCl, 3.4; CaCl2, 2.8; KH2PO4, 1.3; NaHCO3, 16; MgSO4, 0.6; and glucose, 7.7. The pH of the Krebs’ solution was maintained at 7.4 during all experiments by constant bubbling with 95% O2/5% CO2. Intact segments were used for relaxation experiments. For contraction experiments, segments were denuded of endothelium by placing them on a pair of fine-point forceps, endothelium side in, and rotating them ten times under light finger pressure. In all experiments, segments were threaded on triangular wire tissue hangers, placed in 10-ml organ baths, and attached to Grass FT.03 force transducers. Organ baths were maintained at 378C. A force of approximately 4 g was applied to each segment two or three times over a 30-min period. Then, segments were equilibrated for at least 60 min before the addition of compounds. Muscarinic agonist-induced relaxation was investigated in the endothelium-intact aorta precontracted with 100 nM norepinephrine. Responses to norepineph-
rine were allowed to reach equilibrium. Then, cumulative concentration–response curves for acetylcholine, carbachol, oxotremorine, McN-A-343, pilocarpine, and RS86 were constructed in half-log increments by using a dosing interval of 2–5 min. Buffer was added to control tissues on the same time schedule. Responses were expressed as a percentage of the response to 100 nM norepinephrine. Muscarinic agonist-induced contraction was investigated in the endothelium-denuded aorta. After an initial test with 100 mM carbachol, the carbachol was washed out and tissues were allowed to equilibrate for at least 20 min. Cumulative concentration-response curves for acetylcholine, carbachol, oxotremorine, pilocarpine, McN-A-343, and RS86 were constructed in half-log increments by using a dosing interval of 2–5 min. In experiments in which no contractile response to agonists occurred (i.e., pilocarpine, McN-A-343, and RS86), 100 mM carbachol was added a second time at the end of the protocol. Responses in each tissue were expressed as a percentage of the initial response to 100 mM carbachol. In antagonism experiments, buffer (control tissues) or various concentrations of the nonselective muscarinic antagonist atropine or the relatively selective cholinergic antagonist pirenzepine (M1), AF DX-116 (M2), or 4-DAMP (M1/M3) were added to either the intact aorta prior to contraction with 100 nM norepinephrine or the endothelium-denuded aorta after the initial test and washout of 100 mM carbachol. Tissues were allowed to equilibrate for 30 min. Cumulative concentration–response curves for carbachol were then constructed by using half-log increments and a dosing interval of 2–5 min. Responses of each tissue were expressed as a percentage of the response to 100 nM norepinephrine for the intact aorta or 100 mM carbachol for the endothelium-denuded aorta. Only one concentration–response curve was determined per tissue. In addition, the muscarinic agonists McN-A-343, pilocarpine, and RS86 were evaluated as antagonists of carbachol by using the same protocol. 1.1. Data acquisition and analysis Changes in isometric tension were recorded and analyzed with an M5000 Signal Processing Center with the use of XYZ Realtime software (Modular Instruments, Inc., Malvern, PA) and a Compaq Deskpro 386 computer (Compaq Computer Corporation, Houston, TX). Mean concentration–response curves for agonists were analyzed by using a four-parameter logistic model (De Lean et al., 1978; Shannon et al., 1993). IC50 values (endothelium-intact tissues) or EC50 values (endothelium-denuded tissues) were defined as the molar concentration of agonist required to produce a half-maximal effect. Apparent dissociation constants of antagonists were estimated by generalizing the approach of Waud and
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Parker (1971) to allow maximum responses to vary (Shannon et al., 1993). Mean concentration–response curves for carbachol alone or in the presence of from one to three concentrations of antagonists were simultaneously fitted by a four-parameter logistic model (De Lean et al., 1978). IC50 or EC50 values, steepness, and dose ratios (ratios of IC50 or EC50 values) were estimated. Dose ratios were used to calculate apparent dissociation constants for antagonists in a manner analogous to determining pA2 and KB by the method of Arunlakshana and Schild (1959), as implemented by Waud and Parker (1971). 1.2. [3H]N-met-scopolamine binding Rabbit aorta, guinea pig bladder, and guinea pig ileum longitudinal muscle/myenteric plexus were dissected and placed in cold 50 mM NaPO4 buffer containing 2 mM MgCl2. Tissues were homogenized in 70 volumes of buffer for 30 s at setting 7 on a polytron. In addition, aorta and ileum homogenates were filtered through a double layer of cheesecloth. The suspensions were then centrifuged twice at 17,000g for 10 min. Tissue pellets were resuspended at 40 mg/ml tissue wet weight, and 1 ml was incubated in quadruplicate with 4 nM [3H]N-met-scopolamine (84 Ci/mmol, New England Nuclear, Boston, MA) for 2 h at 258C. To terminate binding, the homogenates were filtered with vacuum through glass-fiber filters (Whatman, GF/C) that had been soaked in 0.1% polyethylenimine for 2 h. The filters were washed three times with 2 ml of cold buffer and placed in scintillation vials containing 10 ml of scintillation fluid (Ready Protein1, Beckman, Fullerton, CA). Radioactivity on the filters was determined by liquid scintillation spectrometry. Nonspecific binding was determined in the presence and absence of 1 mM atropine. Protein was determined by the method of Bradford (1976). 1.3. Drugs The drugs used in this study were obtained from the following sources: Carbachol (Aldrich Chemical Co., Milwaukee, WI); acetylcholine chloride, atropine sulfate, oxotremorine sesquifumarate, and pilocarpine hydrochloride (Sigma Chemical Co., St. Louis, MO); 4-DAMP (4-diphenylacetoxy-N-methyl-piperidine methiodide) and McN-A-343 [(4-hydroxy-2-butynyl)trimethylammonium chloride] (Research Biochemicals, Inc., Natick, MA); AF DX-116 (11-2[[2-[(diethylamino)methyl]1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4] benzodiazepin-6-one) and (6)-RS86 (2-ethyl-8-methyl2,8-diazospiro-[4,5]-decane-1,3-dione) were provided by Lilly Research Laboratories. 2. Results 2.1. Agonist-induced relaxation Norepinephrine produced concentration-dependent contractions of the intact rabbit thoracic aorta with
Fig. 1. Muscarinic agonist-induced relaxation and contraction in the rabbit thoracic aorta. (A) Cumulative concentration–response curves for buffer (s), acetylcholine (d), carbachol (h), oxotremorine (j), pilocarpine (r), McN-A-343 (n), and RS86 (m) in intact rabbit thoracic aorta. (B) Cumulative concentration–response curves for acetylcholine (d), carbachol (h), oxotremorine (j), pilocarpine (r), McN-A-343 (n), and RS86 (m) in the endothelium-denuded rabbit thoracic aorta. Each point represents the mean of six tissues. Vertical lines represent 61 SEM and are absent when less than the size of the point. Drug concentrations are molar. Data are expressed as a percentage of the response to 100 nM norepinephrine in intact tissues or 100 mM carbachol in endothelium-denuded tissues.
an EC50 of 160 6 14 nM (n 5 11; data not shown). A concentration of 100 nM was thus chosen as the concentration to precontract intact tissue in relaxation experiments. At equilibrium, the response to 100 nM norepinephrine was equal to 3.52 6 0.11 grams (n 5 49). Tissues treated with buffer, to control for any fade in the response to norepinephrine, relaxed by only 6 6 3% for the duration of experiments. Acetylcholine, oxotremorine, and carbachol produced concentration-dependent relaxation of the intact aorta that had been precontracted with 100 nM norepinephrine (Fig. 1A). The maximum relaxation observed in acetylcholine-, oxotremorine-, or carbachol-treated tissues was equal to 89 6 4, 86 6 7, or 75 6 6% of their responses to 100 nM norepinephrine, respectively. IC50 values for acetylcholine, oxotremorine, and carbachol were 96 6 4, 272 6 14, and 505 6 27 nM, respectively. The muscarinic partial agonists pilocarpine, McN-A343, and RS86 did not produce relaxation of the intact aorta at concentrations as high as 100 mM (Fig. 1A). 2.2. Agonist-induced contraction Carbachol contracted the endothelium-denuded rabbit thoracic aorta in a concentration-dependent manner (Fig. 1B). The response to 100 mM carbachol was equal to 3.64 6 0.24 g (n 5 36). Acetylcholine, oxotremorine, and carbachol produced concentration-dependent contraction of the endothelium-denuded rabbit thoracic aorta. Maximum responses to acetylcholine and carbachol occurred at 100 mM and were equal to 101 6 3 and 103 6 4% of the initial response to 100 mM carbachol.
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EC50 values for acetylcholine and carbachol were 2.7 6 0.1 and 4.6 6 0.1 mM, respectively. Unlike those of acetylcholine and carbachol, the maximum response to oxotremorine (100 mM) was only 34 6 10% of the initial response to 100 mM carbachol (Fig. 1B). The EC50 value for oxotremorine was 8.6 6 1.7 mM. Similar to their lack of agonist activity in the intact aorta, the muscarinic partial agonists pilocarpine, McNA-343, and RS86 did not contract the endotheliumdenuded aorta at concentrations as high as 100 mM (Fig. 1B). However, in the presence of 100 mM pilocarpine, McN-A-343, or RS86, the response to 100 mM carbachol, added to the bath at the end of the assay, was decreased by 85 6 3, 14 6 3, and 44 6 10%, respectively, compared with the response to 100 mM carbachol added to the bath at the beginning of the experiment. 2.3. Antagonism of carbachol-induced responses by the muscarinic antagonists atropine, pirenzepine, AF DX-116, and 4-DAMP The nonselective muscarinic antagonist atropine antagonized carbachol-induced relaxation of the precon-
tracted intact rabbit aorta as well as carbachol-induced contraction of the endothelium-denuded aorta (data not shown). Pretreatment of the intact aorta with 10 nM atropine produced a shift to the right of the carbachol concentration–response curve. The apparent 2log KB for atropine in the intact preparation was 9.3. Pretreatment of the endothelium-denuded aorta with 10 nM atropine also produced a shift to the right of the carbachol concentration–response curve (data not shown). The apparent 2log KB for atropine in the denuded aorta was 9.4. In another series of experiments, the relatively selective M1 receptor antagonist pirenzepine, the somewhat selective M2 receptor antagonist AF DX-116, and the relatively selective M1/M3 receptor antagonist 4-DAMP all antagonized carbachol-induced relaxation of intact rabbit aorta (Fig. 2A–C). Pretreatment of the intact aorta with pirenzepine, AF DX-116, or 4-DAMP produced concentration-dependent shifts to the right of the carbachol concentration–response curve. The pA2 values for pirenzepine, AF DX-116, and 4-DAMP in the intact rabbit aorta are presented in Table 1. Carbachol-induced contraction of the endothelium-
Fig. 2. Antagonism of carbachol-induced responses by pirenzepine, AF DX-116, and 4-DAMP in the (A–C) intact and the (D–F) endotheliumdenuded rabbit thoracic aorta. Each point represents the mean of five to six tissues. Vertical lines represent 61 SEM and are absent when less than the size of the point. Drug concentrations are molar. Data are expressed as a percentage of the response to 100 nM norepinephrine in intact tissues or 100 mM carbachol in endothelium-denuded tissues. (All panels) Carbachol alone (s); (A and D) carbachol plus pirenzepine, 0.3 mM (d), 1.0 mM (j), 3.0 mM (m); (B and E) carbachol plus AF DX-116, 10 mM (d), 30 mM (j), 100 mM (m); (C and F) carbachol plus 4-DAMP, 0.01 mM (d), 0.03 mM (j), 0.10 mM (m).
B.D. Sawyer et al./General Pharmacology 32 (1999) 445–452 Table 1 pA2 values (and 95% confidence intervals) of muscarinic receptor antagonists and partial agonists at receptors mediating carbacholinduced responses in the intact and the endothelium-denuded rabbit thoracic aorta pA2 (95% confidence interval) Drug 4-DAMP AF DX-116 Pirenzepine Pilocarpine McN-A-343 RS86
Intact aorta (relaxation)
Denuded aorta (contraction)
9.2 (9.1–9.3) 5.9 (5.8–6.0) 7.1 (7.0–7.2) 6.1 (6.0–6.3) 5.0 (4.9–5.1) 5.6 (5.4–5.7)
9.2 (9.1–9.4) 6.1 (6.0–6.3) 7.1 (7.0–7.2) 5.6 (5.5–5.7) 5.0 (4.9–5.1) 5.6 (5.4–5.7)
denuded rabbit aorta also was antagonized by pirenzepine, AF DX-116, and 4-DAMP (Fig. 2D–F). Pretreatment of the endothelium-denuded aorta with pirenzepine, AF DX-116, or 4-DAMP produced concentration-dependent shifts to the right of the carbachol concentration–response curve. The pA2 values for pirenzepine, AF DX-116, and 4-DAMP in the endothelium-denuded aorta are presented in Table 1. 2.4. Antagonism of carbachol-induced responses in the rabbit aorta by pilocarpine, McN-A-343, and RS86 The muscarinic low-efficacy agonists pilocarpine, McN-A-343, and RS86 antagonized carbachol-induced relaxation of the intact rabbit aorta (Fig. 3A–C). Pretreatment of the intact aorta with pilocarpine, McN-A343, or RS86 produced concentration-dependent shifts to the right of the carbachol concentration–response curve. The pA2 values for pilocarpine, McN-A-343, and RS86 in the intact rabbit aorta are presented in Table 1. Pilocarpine, McN-A-343, and RS86 also antagonized carbachol-induced contraction of the endotheliumdenuded rabbit aorta. Pretreatment of the endothelium-denuded aorta with pilocarpine, McN-A-343, or RS86 produced shifts to the right of the carbachol concentration–response curve (Fig. 3D–F). The pA2 values for pilocarpine, McN-A-343, and RS86 in endotheliumdenuded aorta are presented in Table 1. 2.5. Density of muscarinic receptors in the rabbit thoracic aorta, the guinea pig urinary bladder, and the guinea pig ileum myenteric plexus-longitudinal muscle as measured by [3H]N-met-scopolamine binding The density of muscarinic receptors in the rabbit thoracic aorta was low compared with the guinea pig urinary bladder or the guinea pig ileum myenteric plexuslongitudinal muscle. Under saturating conditions, the
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concentration of muscarinic receptors in the rabbit aorta as measured by [3H]N-met-scopolamine binding was 51 fmol/mg protein compared with 362 fmol/mg protein in the guinea pig urinary bladder and 1494 fmol/ mg protein in the guinea pig myenteric plexus-longitudinal muscle.
3. Discussion There is general agreement that the M3 subtype of muscarinic receptor mediates vasorelaxation in the rabbit thoracic aorta. However, controversy exists concerning the identity of the subtype of muscarinic receptor that mediates vasoconstriction in the rabbit aorta. For example, Jaiswal et al. (1991) concluded that M2 receptors mediate contraction, whereas Watson and Eglen (1994) concluded that M3 receptors mediate contraction. Results from previous studies were generated by using different preparations, test systems, pharmacologic agents, experimental designs, and statistical analyses, which might account for the apparent differences observed. The present study sought to clarify the pharmacology of the receptor subtypes in the rabbit aorta by directly comparing the effects of six muscarinic agonists and four muscarinic antagonists on the intact and the endothelium-denuded rabbit aorta by using comparable methodologies in intact and denuded preparations. In the present studies, there were differences between intact and denuded aorta preparations in the absolute potencies of acetylcholine, carbachol, and oxotremorine, in the orders of potencies of these agonists, and in the efficacy of oxotremorine. The three agonists were from 9 to 30 times as potent in relaxing the precontracted intact aorta as in contracting the denuded aorta. Higher potencies of muscarinic agonists in intact compared with denuded aorta preparations were observed previously (Furchgott and Zawadski, 1980; Jaiswal et al., 1991; Jellife, 1962; Watson and Eglen, 1994). Although such differences in potencies could be due to different muscarinic receptor subtypes mediating relaxation and contraction, a more parsimonious explanation is that the size of the receptor population mediating relaxation is greater than that mediating contraction. The density of muscarinic receptors in the rabbit thoracic aorta is relatively low compared with other smooth muscles used for cholinergic smooth muscle assays. In the present study, the density of muscarinic receptors, as measured by [3H]N-met-scopolamine binding, in the intact rabbit aorta (51 fmol/mg protein) was approximately 7 and 30 times lower than in the guinea pig urinary bladder (362 fmol/mg protein) and the guinea pig ileum myenteric plexus-longitudinal muscle (1494 fmol/mg protein), respectively. Our results comparing all three tissues are similar to previously published values for these tissues (Nilvebrant and Sparf, 1983; Sim and Manjeet, 1989; Yamamura and Snyder,
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Fig. 3. Antagonism of carbachol-induced responses by the muscarinic partial agonists pilocarpine, McN-A-343, and RS86 in the (A–C) intact and the (D–F) endothelium-denuded rabbit thoracic aorta. Each point represents the mean of five to six tissues. Vertical lines represent 61 SEM and are absent when less than the size of the point. Drug concentrations are molar. Data are expressed as a percentage of the response to 100 nM norepinephrine in intact tissues or 100 mM carbachol in endothelium-denuded tissues. (All panels) Carbachol alone (s); (A) carbachol plus pilocarpine, 0.3 mM (d), 1.0 mM (j), 3.0 mM (m); (D) carbachol plus pilocarpine, 10 mM (d), 30 mM (j), 100 mM (m); (B and E) carbachol plus McN-A-343, 30 mM (d), 100 mM (j), 300 mM (m); (C and F) carbachol plus RS86, 30 mM (d), 100 mM (j), 300 mM (m).
1974). Although we were unable to dissect endothelium from muscle to determine the size of the receptor populations in each tissue separately, pervious studies suggest that either the number of receptors or receptor– effector coupling in muscle is less than in endothelial tissue (Furchgott and Zawadski, 1980; Jaiswal et al., 1991). It is well known that, as the size of the receptor population decreases or the efficiency of receptor–effector coupling decreases, the efficacy and potency of agonists decrease (Kenakin, 1993; Tallarida and Jacob, 1979). The lower relative efficacy of oxotremorine in the denuded compared with the intact aorta in the present studies is consistent with differences in receptor population size or with differences in receptor–effector coupling in the denuded compared with the intact aorta. Moreover, the present findings are consistent with the partial agonist activity of oxotremorine in other tissues (Ringdahl, 1987). Similarly, differences in receptor population size or receptor–effector coupling likely also account for the differences in the orders of potencies of the three agonists for producing relaxation of the intact aorta (acetylcholine . oxotremorine . carbachol) compared with that for producing contraction of the denuded aorta (acetylcholine . carbachol . oxotremo-
rine). Thus, differences in absolute potency, order of potency, and efficacy for muscarinic agonists between the intact and the denuded aorta are most likely due to differences in receptor density or receptor–effector coupling efficiency rather than to differences in receptor subtype. As noted by Watson and Eglen (1994), determination of antagonist affinity constants in rabbit aorta preparations is complicated by the trend toward an increased maximum response to carbachol in the presence of some concentrations of antagonists. Unequal maximum values violate assumptions for calculating pA2 values by the method of Arunlakshana and Schild (1959). We therefore chose to calculate pA2 values by generalizing the approach of Waud and Parker (1971), a method that allows maximum responses to vary (Shannon et al., 1993) and permitted us to calculate the more robust pA2 statistic for all antagonists rather than 2log KB values when maximum responses vary, as was done by Watson and Eglen (1994). In the present studies, the pA2 values for the relatively selective M1 receptor antagonist pirenzepine, the somewhat selective M2 receptor antagonist AF DX-116, and the M1/M3 receptor antagonist 4-DAMP in antagonizing carbachol-
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induced relaxation in the intact rabbit aorta were virtually identical with their pA2 values for antagonizing carbachol-induced contraction in the endotheliumdenuded rabbit aorta. The pA2 values of pirenzepine and 4-DAMP for antagonism of carbachol-induced relaxation and contraction in the present studies are in good agreement with pA2 and 2log KB values obtained by Eglen and co-workers from experiments conducted in static organ baths (Eglen and Whiting, 1985a; Watson and Eglen, 1994). However, the pA2 values that we obtained for AF DX-116 in both intact and denuded are at least an order of magnitude lower than the pA2 value reported by Jaiswal et al. (1991) for AF DX-116 in the denuded aorta; Jaiswal et al. (1991) did not report a pA2 value of AF-DX-116 for relaxation of the rabbit aorta. Rather, they investigated inhibition of the contractile response to a single concentration of acetylcholine by various concentrations of AF DX-116 in aortas that had been moderately contracted with 10 nM norepinephrine. The reasons for the apparent discrepancies between the results of Jaiswal et al. (1991) and the present as well as other (Watson and Eglen, 1994) reports are most likely methodological differences. Further, the affinities of these antagonists are consistent with the interpretation that the muscarinic receptor subtype in both the intact and the denuded aorta is of the M3 subtype. Thus, most of the results with muscarinic antagonists support the conclusions of Watson and Eglen (1994), but not those of Jaiswal et al. (1991), that M3 muscarinic receptors mediate both relaxation and contraction in the rabbit thoracic aorta. We found that pilocarpine and McN-A-343 neither contracted nor relaxed the rabbit aorta, confirming and extending previous results (Eglen and Whiting 1985b; Furchgott and Cherry, 1984; Jaiswal et al., 1991). To our knowledge, this is the first report that the muscarinic partial agonist RS86 also lacks efficacy in the rabbit aorta. Consistent with the present findings, pilocarpine, McN-A-343, and RS86 are low-efficacy agonists in tissues that have considerably larger muscarinic receptor populations [(Furchgott and Bursztyn, 1967) Sawyer and Shannon, unpublished observations]. Tissues with a low receptor density, such as the rabbit aorta, however, offer a unique opportunity to classify receptor subtypes by using low-efficacy agonists such as pilocarpine, McNA-343, and RS86 to antagonize responses to full agonists. pA2 values of McN-A-343 and RS86 for antagonism of carbachol-induced relaxation in the intact aorta were virtually identical with their pA2 values for antagonism of carbachol-induced contraction the in endothelium-denuded aorta. In contrast, the pA2 value of pilocarpine for antagonism of carbachol-induced relaxation in the intact rabbit aorta (6.1; 95% confidence interval, 6.0–6.3) was significantly different from the pA2 value for antagonism of carbachol-induced contraction in the endothelium-denuded aorta (5.6; 95% confidence inter-
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val, 5.5–5.7). The present pA2 values are consistent with KB values previously reported for pilocarpine (Furchgott and Cherry, 1984; Hawcock and Roberts, 1987). The reason for the apparent difference in affinity of pilocarpine for the muscarinic receptors mediating carbachol-induced relaxation and contraction is not readily apparent, although Furchgott and Cherry (1984) suggested that the apparent difference may be the result of an action of pilocarpine at an alternative site to the agonist-binding sight. 4. Summary In the endothelium-intact aorta that had been precontracted with 100 nM norepinephrine, the order of potencies for producing relaxation by muscarinic agonists was acetylcholine . oxotremorine . carbachol. In addition, the partial muscarinic agonists pilocarpine, McN-A-343, and RS86 did not produce relaxation. Similarly, in the endothelium-denuded aorta, acetylcholine and carbachol produced contractions and were approximately equipotent, whereas oxotremorine was a partial agonist and pilocarpine, McN-A-343, and RS86 failed to produce contractions. The pA2 values of the muscarinic antagonists 4-DAMP, AF DX-116, and pirenzepine were virtually identical in the two preparations and were consistent with the pharmacology of M3 receptors. In addition, pilocarpine, McN-A-343, and RS86 functioned as competitive antagonists of carbachol in both the intact and the denuded aorta, consistent with the relatively small size of the muscarinic receptor population in this tissue determined by [3H]N-met-scopolamine binding under saturating conditions. The pA2 values for McN-A-343 and RS86 were identical in the two preparations, but pilocarpine was approximately 30-fold as potent in the denuded as in the intact preparation. We conclude that muscarinic receptors on endothelium and smooth muscle of the rabbit thoracic aorta are very similar, if not identical, and both are of the M3 subtype. References Angus, J.A., Campbell, G.R., Cocks, T.M., Manderson, J.A., 1983. Vasodilation of acetylcholine is endothelium dependent: a study by sonomicrometry in canine femoral artery in vivo. J Physiol (Lond) 344, 209–222. Arunlkshana, O., Schild, O., 1959. Some quantitative uses of drug antagonists. Br J Pharmac Chemother 14, 58–98. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72, 248–254. Choo, L.K., Malta, E., Mitchelson, F., 1986. The affinity of some selective muscarinic receptor antagonists for the muscarinic receptor mediating endothelial-dependent relaxation of the rabbit and rat thoracic aorta. J Pharm Pharmac 38, 843–845. De Lean, A., Munson, P.J., Rodbard D., 1978. Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay and physiological dose-response relationships. Am J Physiol 235, E97–E102.
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Eglen, R.M., Whiting, R., 1985a. Determination of the muscarinic receptor subtype mediating vasodilation. Br J Pharmac 84, 3–5. Eglen, R.M., Whiting, R., 1985b. Comparison of the activities of muscarinic agonists on rabbit aorta and guinea pig ileum. Br J Pharmac 83, 386P. Eglen, R.M., Whiting, R., 1990. Heterogeneity of vascular muscarinic receptors. J Auton Pharmac 19, 233–245. Eglen, R.M., Reddy, H., Watson, N., Challis, R.A.J., 1994. Muscarinic acetylcholine receptor subtypes in smooth muscle. Trends Pharmac Sci 15, 114–119. Furchgott, R.F., Bursztyn, P., 1967. Comparison of dissociation constants and of relative potencies of selected agonists acting on parasympathetic receptors. Ann N Y Acad Sci 144, 882–899. Furchgott, R.F., Cherry, P.D., 1984. The muscarinic receptor of the vascular endothelium that subserves vasodilation. Trends Pharmac Sci 5 (suppl.), 45–48. Furchgott, R.F., Zawadski, J.V., 1980. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288, 373–376. Hawcock, A.B., Roberts, F.F., 1987. Comparison of the activities of muscarinic agonists on rabbit thoracic aorta preparations. Br J Pharmac 92, 722P. Jaiswal, N., Lambrecht, G., Mutschler, E., Tacke, R., Malik K.U., 1991. Pharmacological characterization of the vascular muscarinic receptors mediating relaxation and contraction in rabbit aorta. J Pharmac Exp Ther 258, 842–850.
Jelliffe, R.W., 1962. Dilator and constrictor effects of acetylcholine on isolated rabbit aortic chains. J Pharmac Exp Ther 135, 349–353. Kenakin, T., 1993. Pharmacologic analysis of drug-receptor interaction. 2d ed. Raven Press, New York. Nilvebrant, L., Sparf, B., 1983. Muscarinic receptor binding in the guinea pig urinary bladder. Acta Pharmac Toxicol 52, 30–38. Ringdahl, B., 1987. Selectivity of partial agonists related to oxotremorine based on differences in muscarinic receptor reserve between the guinea pig ileum and urinary bladder. Mol Pharmac 31, 351–356. Shannon, H.E., Sawyer, B.D., Bemis, K.G., Bymaster, F.P., Health, I., Mitch, C.H., Ward, J.S., 1993. Muscarinic M1 receptor agonist actions of muscarinic receptor agonists in rabbi vas deferens. Eur J Pharmac 232, 47–57. Sim, M.K., Manjeet, S., 1989. Endothelial muscarinic receptors in the rabbit aorta. Eur J Pharmac 163, 399–404. Tallarida, R.J., Jacob, L.S., 1979. Kinetics of drug-receptor interaction: interpreting dose–response data. In: The dose–response relation in pharmacology. Springer-Verlag, New York, pp. 49–84. Watson, N., Eglen, R.M., 1994. Muscarinic M3 receptors mediate contractions in rabbit endothelium-denuded aorta in vitro. J Auton Pharmac 14, 283–293. Waud, D.R., Parker, R.B., 1971. Pharmacological estimation of drugreceptor dissociation constants. Statistical evaluation: II. Competitive antagonists. J Pharmac Exp Ther 177, 13–24. Yamamura, H.I., Snyder, S.H., 1974. Muscarinic cholinergic receptor binding in the longitudinal muscle of the guinea pig ileum with [3H]quinuclidinyl benzilate. Mol Pharmacol 10, 861–867.
Direct pharmacological comparison of the muscarinic receptors mediating relaxation and contraction in the rabbit thoracic aorta Barry D. Sawyer, Frank P. Bymaster, David O. Calligaro, Julie Falcone, Charles H. Mitch, John S. Ward, Celia Whitesitt, Harlan E. Shannon * Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, USA Manuscript received April 28, 1998; accepted manuscript July 7, 1998
Abstract The purpose of the present studies was to directly compare the pharmacology of the muscarinic cholinergic receptors coupled to carbachol-induced relaxation and contraction of the intact and the endothelium-denuded rabbit thoracic aorta, respectively. The order of potencies of agonists for producing relaxation in the intact aorta was similar to that for producing contraction in the denuded aorta. In both preparations, the partial agonists pilocarpine, McN-A-343, and RS86 functioned as antagonists, indicating a lack of receptor reserve in both preparations. Further, the pA2 values for antagonists in both tissues were virtually identical and were consistent with the pharmacology of M3 receptors. 1999 Elsevier Science Inc. All rights reserved. Keywords: Muscarinic M3 receptors; Muscarinic M2 receptors; Thoracic aorta (rabbit); Muscarinic receptor partial agonists; Carbachol; Pilocarpine; RS86
Cholinergic agonists produce relaxation of various endothelium-intact vascular smooth muscle preparations in vitro including the rabbit aorta (Angus et al., 1983; Furchgott and Zawadski, 1980). The nonselective muscarinic antagonist atropine blocks cholinergic agonistinduced relaxation of the rabbit aorta, thus indicating that relaxation is mediated through muscarinic receptors. The affinities of selective muscarinic antagonists at the receptor mediating muscarinic agonist-induced relaxation support the interpretation that M3 receptors mediate relaxation in the rabbit aorta (Choo et al., 1986; Eglen and Whiting, 1985a; Eglen et al., 1994). Stimulation of muscarinic receptors located on endothelial cells produces relaxation of the rabbit aorta through synthesis of nitric oxide (Furchgott and Zawadski, 1980). Accordingly, removal of endothelium or exposure to nitric oxide synthase inhibitors eliminates muscarinic receptor-mediated relaxation (Furchgott and Zawadski, 1980). In the absence of endothelium, muscarinic agonists can produce contraction of vascular preparations by activation of receptors located directly on the smooth muscle (Eglen and Whiting, 1990; Furchgott and Cherry, 1984). The muscarinic receptor subtype * Corresponding author. Tel.: (317) 276-0749; Fax: (317) 2765546; E-mail: [email protected].
mediating contraction appears to be species and tissue dependent (Eglen and Whiting, 1990). In the rabbit aorta, the identity of the subtype of muscarinic receptor mediating contraction is controversial. The rank order of affinities of the muscarinic antagonists atropine and pirenzepine (Choo et al., 1986) obtained in vitro from static muscle bath experiments indicates that M3 receptors mediate muscarinic agonistinduced contraction in the rabbit aorta. However, more recent studies, with the use of superfusion techniques, provide some evidence that muscarinic agonist-induced contraction may be mediated by M2 receptors (Jaiswal et al., 1991). Most recently, Watson and Eglen (1994), using a broad range of muscarinic antagonists to inhibit (1)cis-dioxolane-induced contraction in static muscle bath preparations, concluded that muscarinic M3 receptors mediate contraction of the endothelium-denuded rabbit aorta. Data reported by different workers from experiments in the endothelium-denuded aorta cannot be directly compared, owing to the use of different preparations (partially contracted vs. noncontracted aorta), different test systems (superfusion vs. static baths), different agonists and antagonists, different experimental design, and different statistical analyses of data (Choo et al., 1986; Jaiswal et al., 1991; Watson and Eglen, 1994).
0306-3623/99/$–see front matter 1999 Elsevier Science Inc. All rights reserved. PII: S0306-3623(98)00215-8
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Therefore, the apparent discrepancies between results from different laboratories may be due to the use of different methodologies. The purpose of the present study was to directly compare the effects of muscarinic agonists and antagonists on the endothelium-intact and endotheliumdenuded rabbit thoracic aorta by using the same test system, drugs, experimental design, and statistical analysis. First, we characterized the subtype(s) of muscarinic receptor(s) that mediate muscarinic agonistinduced responses in the endothelium-intact and the endothelium-denuded rabbit aorta by using six muscarinic agonists and four muscarinic antagonists. In experiments with agonists, McN-A-343, pilocarpine, and RS86 demonstrated no agonist activity in either the endothelium-intact or the endothelium-denuded aorta However, at micromolar concentrations, all thee of these low-efficacy agonists decreased responses to a maximally effective concentration of carbachol in the denuded aorta. Consequently, we evaluated pilocarpine, McN-A-343, and RS86 as antagonists of carbachol in both the endothelium-intact and the endotheliumdenuded aorta. Finally, to better understand the lack of agonist activity of pilocarpine, McN-A-343, and RS86, we compared relative muscarinic receptor densities in the rabbit aorta, the guinea pig urinary bladder, and the guinea pig ileum longitudinal muscle by measuring [3H]N-met-scopolamine binding under saturating conditions.
1. Materials and methods Male New Zealand White rabbits (Hazelton Research Products, Denver, PA) were sacrificed by IV sodium pentobarbital overdose. Thoracic aortas were dissected free of surrounding tissue, cut into segments 3–5 mm in length, and placed in modified Krebs’ solution of the following composition (in millimolar): NaCl, 134; KCl, 3.4; CaCl2, 2.8; KH2PO4, 1.3; NaHCO3, 16; MgSO4, 0.6; and glucose, 7.7. The pH of the Krebs’ solution was maintained at 7.4 during all experiments by constant bubbling with 95% O2/5% CO2. Intact segments were used for relaxation experiments. For contraction experiments, segments were denuded of endothelium by placing them on a pair of fine-point forceps, endothelium side in, and rotating them ten times under light finger pressure. In all experiments, segments were threaded on triangular wire tissue hangers, placed in 10-ml organ baths, and attached to Grass FT.03 force transducers. Organ baths were maintained at 378C. A force of approximately 4 g was applied to each segment two or three times over a 30-min period. Then, segments were equilibrated for at least 60 min before the addition of compounds. Muscarinic agonist-induced relaxation was investigated in the endothelium-intact aorta precontracted with 100 nM norepinephrine. Responses to norepineph-
rine were allowed to reach equilibrium. Then, cumulative concentration–response curves for acetylcholine, carbachol, oxotremorine, McN-A-343, pilocarpine, and RS86 were constructed in half-log increments by using a dosing interval of 2–5 min. Buffer was added to control tissues on the same time schedule. Responses were expressed as a percentage of the response to 100 nM norepinephrine. Muscarinic agonist-induced contraction was investigated in the endothelium-denuded aorta. After an initial test with 100 mM carbachol, the carbachol was washed out and tissues were allowed to equilibrate for at least 20 min. Cumulative concentration-response curves for acetylcholine, carbachol, oxotremorine, pilocarpine, McN-A-343, and RS86 were constructed in half-log increments by using a dosing interval of 2–5 min. In experiments in which no contractile response to agonists occurred (i.e., pilocarpine, McN-A-343, and RS86), 100 mM carbachol was added a second time at the end of the protocol. Responses in each tissue were expressed as a percentage of the initial response to 100 mM carbachol. In antagonism experiments, buffer (control tissues) or various concentrations of the nonselective muscarinic antagonist atropine or the relatively selective cholinergic antagonist pirenzepine (M1), AF DX-116 (M2), or 4-DAMP (M1/M3) were added to either the intact aorta prior to contraction with 100 nM norepinephrine or the endothelium-denuded aorta after the initial test and washout of 100 mM carbachol. Tissues were allowed to equilibrate for 30 min. Cumulative concentration–response curves for carbachol were then constructed by using half-log increments and a dosing interval of 2–5 min. Responses of each tissue were expressed as a percentage of the response to 100 nM norepinephrine for the intact aorta or 100 mM carbachol for the endothelium-denuded aorta. Only one concentration–response curve was determined per tissue. In addition, the muscarinic agonists McN-A-343, pilocarpine, and RS86 were evaluated as antagonists of carbachol by using the same protocol. 1.1. Data acquisition and analysis Changes in isometric tension were recorded and analyzed with an M5000 Signal Processing Center with the use of XYZ Realtime software (Modular Instruments, Inc., Malvern, PA) and a Compaq Deskpro 386 computer (Compaq Computer Corporation, Houston, TX). Mean concentration–response curves for agonists were analyzed by using a four-parameter logistic model (De Lean et al., 1978; Shannon et al., 1993). IC50 values (endothelium-intact tissues) or EC50 values (endothelium-denuded tissues) were defined as the molar concentration of agonist required to produce a half-maximal effect. Apparent dissociation constants of antagonists were estimated by generalizing the approach of Waud and
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Parker (1971) to allow maximum responses to vary (Shannon et al., 1993). Mean concentration–response curves for carbachol alone or in the presence of from one to three concentrations of antagonists were simultaneously fitted by a four-parameter logistic model (De Lean et al., 1978). IC50 or EC50 values, steepness, and dose ratios (ratios of IC50 or EC50 values) were estimated. Dose ratios were used to calculate apparent dissociation constants for antagonists in a manner analogous to determining pA2 and KB by the method of Arunlakshana and Schild (1959), as implemented by Waud and Parker (1971). 1.2. [3H]N-met-scopolamine binding Rabbit aorta, guinea pig bladder, and guinea pig ileum longitudinal muscle/myenteric plexus were dissected and placed in cold 50 mM NaPO4 buffer containing 2 mM MgCl2. Tissues were homogenized in 70 volumes of buffer for 30 s at setting 7 on a polytron. In addition, aorta and ileum homogenates were filtered through a double layer of cheesecloth. The suspensions were then centrifuged twice at 17,000g for 10 min. Tissue pellets were resuspended at 40 mg/ml tissue wet weight, and 1 ml was incubated in quadruplicate with 4 nM [3H]N-met-scopolamine (84 Ci/mmol, New England Nuclear, Boston, MA) for 2 h at 258C. To terminate binding, the homogenates were filtered with vacuum through glass-fiber filters (Whatman, GF/C) that had been soaked in 0.1% polyethylenimine for 2 h. The filters were washed three times with 2 ml of cold buffer and placed in scintillation vials containing 10 ml of scintillation fluid (Ready Protein1, Beckman, Fullerton, CA). Radioactivity on the filters was determined by liquid scintillation spectrometry. Nonspecific binding was determined in the presence and absence of 1 mM atropine. Protein was determined by the method of Bradford (1976). 1.3. Drugs The drugs used in this study were obtained from the following sources: Carbachol (Aldrich Chemical Co., Milwaukee, WI); acetylcholine chloride, atropine sulfate, oxotremorine sesquifumarate, and pilocarpine hydrochloride (Sigma Chemical Co., St. Louis, MO); 4-DAMP (4-diphenylacetoxy-N-methyl-piperidine methiodide) and McN-A-343 [(4-hydroxy-2-butynyl)trimethylammonium chloride] (Research Biochemicals, Inc., Natick, MA); AF DX-116 (11-2[[2-[(diethylamino)methyl]1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4] benzodiazepin-6-one) and (6)-RS86 (2-ethyl-8-methyl2,8-diazospiro-[4,5]-decane-1,3-dione) were provided by Lilly Research Laboratories. 2. Results 2.1. Agonist-induced relaxation Norepinephrine produced concentration-dependent contractions of the intact rabbit thoracic aorta with
Fig. 1. Muscarinic agonist-induced relaxation and contraction in the rabbit thoracic aorta. (A) Cumulative concentration–response curves for buffer (s), acetylcholine (d), carbachol (h), oxotremorine (j), pilocarpine (r), McN-A-343 (n), and RS86 (m) in intact rabbit thoracic aorta. (B) Cumulative concentration–response curves for acetylcholine (d), carbachol (h), oxotremorine (j), pilocarpine (r), McN-A-343 (n), and RS86 (m) in the endothelium-denuded rabbit thoracic aorta. Each point represents the mean of six tissues. Vertical lines represent 61 SEM and are absent when less than the size of the point. Drug concentrations are molar. Data are expressed as a percentage of the response to 100 nM norepinephrine in intact tissues or 100 mM carbachol in endothelium-denuded tissues.
an EC50 of 160 6 14 nM (n 5 11; data not shown). A concentration of 100 nM was thus chosen as the concentration to precontract intact tissue in relaxation experiments. At equilibrium, the response to 100 nM norepinephrine was equal to 3.52 6 0.11 grams (n 5 49). Tissues treated with buffer, to control for any fade in the response to norepinephrine, relaxed by only 6 6 3% for the duration of experiments. Acetylcholine, oxotremorine, and carbachol produced concentration-dependent relaxation of the intact aorta that had been precontracted with 100 nM norepinephrine (Fig. 1A). The maximum relaxation observed in acetylcholine-, oxotremorine-, or carbachol-treated tissues was equal to 89 6 4, 86 6 7, or 75 6 6% of their responses to 100 nM norepinephrine, respectively. IC50 values for acetylcholine, oxotremorine, and carbachol were 96 6 4, 272 6 14, and 505 6 27 nM, respectively. The muscarinic partial agonists pilocarpine, McN-A343, and RS86 did not produce relaxation of the intact aorta at concentrations as high as 100 mM (Fig. 1A). 2.2. Agonist-induced contraction Carbachol contracted the endothelium-denuded rabbit thoracic aorta in a concentration-dependent manner (Fig. 1B). The response to 100 mM carbachol was equal to 3.64 6 0.24 g (n 5 36). Acetylcholine, oxotremorine, and carbachol produced concentration-dependent contraction of the endothelium-denuded rabbit thoracic aorta. Maximum responses to acetylcholine and carbachol occurred at 100 mM and were equal to 101 6 3 and 103 6 4% of the initial response to 100 mM carbachol.
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EC50 values for acetylcholine and carbachol were 2.7 6 0.1 and 4.6 6 0.1 mM, respectively. Unlike those of acetylcholine and carbachol, the maximum response to oxotremorine (100 mM) was only 34 6 10% of the initial response to 100 mM carbachol (Fig. 1B). The EC50 value for oxotremorine was 8.6 6 1.7 mM. Similar to their lack of agonist activity in the intact aorta, the muscarinic partial agonists pilocarpine, McNA-343, and RS86 did not contract the endotheliumdenuded aorta at concentrations as high as 100 mM (Fig. 1B). However, in the presence of 100 mM pilocarpine, McN-A-343, or RS86, the response to 100 mM carbachol, added to the bath at the end of the assay, was decreased by 85 6 3, 14 6 3, and 44 6 10%, respectively, compared with the response to 100 mM carbachol added to the bath at the beginning of the experiment. 2.3. Antagonism of carbachol-induced responses by the muscarinic antagonists atropine, pirenzepine, AF DX-116, and 4-DAMP The nonselective muscarinic antagonist atropine antagonized carbachol-induced relaxation of the precon-
tracted intact rabbit aorta as well as carbachol-induced contraction of the endothelium-denuded aorta (data not shown). Pretreatment of the intact aorta with 10 nM atropine produced a shift to the right of the carbachol concentration–response curve. The apparent 2log KB for atropine in the intact preparation was 9.3. Pretreatment of the endothelium-denuded aorta with 10 nM atropine also produced a shift to the right of the carbachol concentration–response curve (data not shown). The apparent 2log KB for atropine in the denuded aorta was 9.4. In another series of experiments, the relatively selective M1 receptor antagonist pirenzepine, the somewhat selective M2 receptor antagonist AF DX-116, and the relatively selective M1/M3 receptor antagonist 4-DAMP all antagonized carbachol-induced relaxation of intact rabbit aorta (Fig. 2A–C). Pretreatment of the intact aorta with pirenzepine, AF DX-116, or 4-DAMP produced concentration-dependent shifts to the right of the carbachol concentration–response curve. The pA2 values for pirenzepine, AF DX-116, and 4-DAMP in the intact rabbit aorta are presented in Table 1. Carbachol-induced contraction of the endothelium-
Fig. 2. Antagonism of carbachol-induced responses by pirenzepine, AF DX-116, and 4-DAMP in the (A–C) intact and the (D–F) endotheliumdenuded rabbit thoracic aorta. Each point represents the mean of five to six tissues. Vertical lines represent 61 SEM and are absent when less than the size of the point. Drug concentrations are molar. Data are expressed as a percentage of the response to 100 nM norepinephrine in intact tissues or 100 mM carbachol in endothelium-denuded tissues. (All panels) Carbachol alone (s); (A and D) carbachol plus pirenzepine, 0.3 mM (d), 1.0 mM (j), 3.0 mM (m); (B and E) carbachol plus AF DX-116, 10 mM (d), 30 mM (j), 100 mM (m); (C and F) carbachol plus 4-DAMP, 0.01 mM (d), 0.03 mM (j), 0.10 mM (m).
B.D. Sawyer et al./General Pharmacology 32 (1999) 445–452 Table 1 pA2 values (and 95% confidence intervals) of muscarinic receptor antagonists and partial agonists at receptors mediating carbacholinduced responses in the intact and the endothelium-denuded rabbit thoracic aorta pA2 (95% confidence interval) Drug 4-DAMP AF DX-116 Pirenzepine Pilocarpine McN-A-343 RS86
Intact aorta (relaxation)
Denuded aorta (contraction)
9.2 (9.1–9.3) 5.9 (5.8–6.0) 7.1 (7.0–7.2) 6.1 (6.0–6.3) 5.0 (4.9–5.1) 5.6 (5.4–5.7)
9.2 (9.1–9.4) 6.1 (6.0–6.3) 7.1 (7.0–7.2) 5.6 (5.5–5.7) 5.0 (4.9–5.1) 5.6 (5.4–5.7)
denuded rabbit aorta also was antagonized by pirenzepine, AF DX-116, and 4-DAMP (Fig. 2D–F). Pretreatment of the endothelium-denuded aorta with pirenzepine, AF DX-116, or 4-DAMP produced concentration-dependent shifts to the right of the carbachol concentration–response curve. The pA2 values for pirenzepine, AF DX-116, and 4-DAMP in the endothelium-denuded aorta are presented in Table 1. 2.4. Antagonism of carbachol-induced responses in the rabbit aorta by pilocarpine, McN-A-343, and RS86 The muscarinic low-efficacy agonists pilocarpine, McN-A-343, and RS86 antagonized carbachol-induced relaxation of the intact rabbit aorta (Fig. 3A–C). Pretreatment of the intact aorta with pilocarpine, McN-A343, or RS86 produced concentration-dependent shifts to the right of the carbachol concentration–response curve. The pA2 values for pilocarpine, McN-A-343, and RS86 in the intact rabbit aorta are presented in Table 1. Pilocarpine, McN-A-343, and RS86 also antagonized carbachol-induced contraction of the endotheliumdenuded rabbit aorta. Pretreatment of the endothelium-denuded aorta with pilocarpine, McN-A-343, or RS86 produced shifts to the right of the carbachol concentration–response curve (Fig. 3D–F). The pA2 values for pilocarpine, McN-A-343, and RS86 in endotheliumdenuded aorta are presented in Table 1. 2.5. Density of muscarinic receptors in the rabbit thoracic aorta, the guinea pig urinary bladder, and the guinea pig ileum myenteric plexus-longitudinal muscle as measured by [3H]N-met-scopolamine binding The density of muscarinic receptors in the rabbit thoracic aorta was low compared with the guinea pig urinary bladder or the guinea pig ileum myenteric plexuslongitudinal muscle. Under saturating conditions, the
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concentration of muscarinic receptors in the rabbit aorta as measured by [3H]N-met-scopolamine binding was 51 fmol/mg protein compared with 362 fmol/mg protein in the guinea pig urinary bladder and 1494 fmol/ mg protein in the guinea pig myenteric plexus-longitudinal muscle.
3. Discussion There is general agreement that the M3 subtype of muscarinic receptor mediates vasorelaxation in the rabbit thoracic aorta. However, controversy exists concerning the identity of the subtype of muscarinic receptor that mediates vasoconstriction in the rabbit aorta. For example, Jaiswal et al. (1991) concluded that M2 receptors mediate contraction, whereas Watson and Eglen (1994) concluded that M3 receptors mediate contraction. Results from previous studies were generated by using different preparations, test systems, pharmacologic agents, experimental designs, and statistical analyses, which might account for the apparent differences observed. The present study sought to clarify the pharmacology of the receptor subtypes in the rabbit aorta by directly comparing the effects of six muscarinic agonists and four muscarinic antagonists on the intact and the endothelium-denuded rabbit aorta by using comparable methodologies in intact and denuded preparations. In the present studies, there were differences between intact and denuded aorta preparations in the absolute potencies of acetylcholine, carbachol, and oxotremorine, in the orders of potencies of these agonists, and in the efficacy of oxotremorine. The three agonists were from 9 to 30 times as potent in relaxing the precontracted intact aorta as in contracting the denuded aorta. Higher potencies of muscarinic agonists in intact compared with denuded aorta preparations were observed previously (Furchgott and Zawadski, 1980; Jaiswal et al., 1991; Jellife, 1962; Watson and Eglen, 1994). Although such differences in potencies could be due to different muscarinic receptor subtypes mediating relaxation and contraction, a more parsimonious explanation is that the size of the receptor population mediating relaxation is greater than that mediating contraction. The density of muscarinic receptors in the rabbit thoracic aorta is relatively low compared with other smooth muscles used for cholinergic smooth muscle assays. In the present study, the density of muscarinic receptors, as measured by [3H]N-met-scopolamine binding, in the intact rabbit aorta (51 fmol/mg protein) was approximately 7 and 30 times lower than in the guinea pig urinary bladder (362 fmol/mg protein) and the guinea pig ileum myenteric plexus-longitudinal muscle (1494 fmol/mg protein), respectively. Our results comparing all three tissues are similar to previously published values for these tissues (Nilvebrant and Sparf, 1983; Sim and Manjeet, 1989; Yamamura and Snyder,
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Fig. 3. Antagonism of carbachol-induced responses by the muscarinic partial agonists pilocarpine, McN-A-343, and RS86 in the (A–C) intact and the (D–F) endothelium-denuded rabbit thoracic aorta. Each point represents the mean of five to six tissues. Vertical lines represent 61 SEM and are absent when less than the size of the point. Drug concentrations are molar. Data are expressed as a percentage of the response to 100 nM norepinephrine in intact tissues or 100 mM carbachol in endothelium-denuded tissues. (All panels) Carbachol alone (s); (A) carbachol plus pilocarpine, 0.3 mM (d), 1.0 mM (j), 3.0 mM (m); (D) carbachol plus pilocarpine, 10 mM (d), 30 mM (j), 100 mM (m); (B and E) carbachol plus McN-A-343, 30 mM (d), 100 mM (j), 300 mM (m); (C and F) carbachol plus RS86, 30 mM (d), 100 mM (j), 300 mM (m).
1974). Although we were unable to dissect endothelium from muscle to determine the size of the receptor populations in each tissue separately, pervious studies suggest that either the number of receptors or receptor– effector coupling in muscle is less than in endothelial tissue (Furchgott and Zawadski, 1980; Jaiswal et al., 1991). It is well known that, as the size of the receptor population decreases or the efficiency of receptor–effector coupling decreases, the efficacy and potency of agonists decrease (Kenakin, 1993; Tallarida and Jacob, 1979). The lower relative efficacy of oxotremorine in the denuded compared with the intact aorta in the present studies is consistent with differences in receptor population size or with differences in receptor–effector coupling in the denuded compared with the intact aorta. Moreover, the present findings are consistent with the partial agonist activity of oxotremorine in other tissues (Ringdahl, 1987). Similarly, differences in receptor population size or receptor–effector coupling likely also account for the differences in the orders of potencies of the three agonists for producing relaxation of the intact aorta (acetylcholine . oxotremorine . carbachol) compared with that for producing contraction of the denuded aorta (acetylcholine . carbachol . oxotremo-
rine). Thus, differences in absolute potency, order of potency, and efficacy for muscarinic agonists between the intact and the denuded aorta are most likely due to differences in receptor density or receptor–effector coupling efficiency rather than to differences in receptor subtype. As noted by Watson and Eglen (1994), determination of antagonist affinity constants in rabbit aorta preparations is complicated by the trend toward an increased maximum response to carbachol in the presence of some concentrations of antagonists. Unequal maximum values violate assumptions for calculating pA2 values by the method of Arunlakshana and Schild (1959). We therefore chose to calculate pA2 values by generalizing the approach of Waud and Parker (1971), a method that allows maximum responses to vary (Shannon et al., 1993) and permitted us to calculate the more robust pA2 statistic for all antagonists rather than 2log KB values when maximum responses vary, as was done by Watson and Eglen (1994). In the present studies, the pA2 values for the relatively selective M1 receptor antagonist pirenzepine, the somewhat selective M2 receptor antagonist AF DX-116, and the M1/M3 receptor antagonist 4-DAMP in antagonizing carbachol-
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induced relaxation in the intact rabbit aorta were virtually identical with their pA2 values for antagonizing carbachol-induced contraction in the endotheliumdenuded rabbit aorta. The pA2 values of pirenzepine and 4-DAMP for antagonism of carbachol-induced relaxation and contraction in the present studies are in good agreement with pA2 and 2log KB values obtained by Eglen and co-workers from experiments conducted in static organ baths (Eglen and Whiting, 1985a; Watson and Eglen, 1994). However, the pA2 values that we obtained for AF DX-116 in both intact and denuded are at least an order of magnitude lower than the pA2 value reported by Jaiswal et al. (1991) for AF DX-116 in the denuded aorta; Jaiswal et al. (1991) did not report a pA2 value of AF-DX-116 for relaxation of the rabbit aorta. Rather, they investigated inhibition of the contractile response to a single concentration of acetylcholine by various concentrations of AF DX-116 in aortas that had been moderately contracted with 10 nM norepinephrine. The reasons for the apparent discrepancies between the results of Jaiswal et al. (1991) and the present as well as other (Watson and Eglen, 1994) reports are most likely methodological differences. Further, the affinities of these antagonists are consistent with the interpretation that the muscarinic receptor subtype in both the intact and the denuded aorta is of the M3 subtype. Thus, most of the results with muscarinic antagonists support the conclusions of Watson and Eglen (1994), but not those of Jaiswal et al. (1991), that M3 muscarinic receptors mediate both relaxation and contraction in the rabbit thoracic aorta. We found that pilocarpine and McN-A-343 neither contracted nor relaxed the rabbit aorta, confirming and extending previous results (Eglen and Whiting 1985b; Furchgott and Cherry, 1984; Jaiswal et al., 1991). To our knowledge, this is the first report that the muscarinic partial agonist RS86 also lacks efficacy in the rabbit aorta. Consistent with the present findings, pilocarpine, McN-A-343, and RS86 are low-efficacy agonists in tissues that have considerably larger muscarinic receptor populations [(Furchgott and Bursztyn, 1967) Sawyer and Shannon, unpublished observations]. Tissues with a low receptor density, such as the rabbit aorta, however, offer a unique opportunity to classify receptor subtypes by using low-efficacy agonists such as pilocarpine, McNA-343, and RS86 to antagonize responses to full agonists. pA2 values of McN-A-343 and RS86 for antagonism of carbachol-induced relaxation in the intact aorta were virtually identical with their pA2 values for antagonism of carbachol-induced contraction the in endothelium-denuded aorta. In contrast, the pA2 value of pilocarpine for antagonism of carbachol-induced relaxation in the intact rabbit aorta (6.1; 95% confidence interval, 6.0–6.3) was significantly different from the pA2 value for antagonism of carbachol-induced contraction in the endothelium-denuded aorta (5.6; 95% confidence inter-
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val, 5.5–5.7). The present pA2 values are consistent with KB values previously reported for pilocarpine (Furchgott and Cherry, 1984; Hawcock and Roberts, 1987). The reason for the apparent difference in affinity of pilocarpine for the muscarinic receptors mediating carbachol-induced relaxation and contraction is not readily apparent, although Furchgott and Cherry (1984) suggested that the apparent difference may be the result of an action of pilocarpine at an alternative site to the agonist-binding sight. 4. Summary In the endothelium-intact aorta that had been precontracted with 100 nM norepinephrine, the order of potencies for producing relaxation by muscarinic agonists was acetylcholine . oxotremorine . carbachol. In addition, the partial muscarinic agonists pilocarpine, McN-A-343, and RS86 did not produce relaxation. Similarly, in the endothelium-denuded aorta, acetylcholine and carbachol produced contractions and were approximately equipotent, whereas oxotremorine was a partial agonist and pilocarpine, McN-A-343, and RS86 failed to produce contractions. The pA2 values of the muscarinic antagonists 4-DAMP, AF DX-116, and pirenzepine were virtually identical in the two preparations and were consistent with the pharmacology of M3 receptors. In addition, pilocarpine, McN-A-343, and RS86 functioned as competitive antagonists of carbachol in both the intact and the denuded aorta, consistent with the relatively small size of the muscarinic receptor population in this tissue determined by [3H]N-met-scopolamine binding under saturating conditions. The pA2 values for McN-A-343 and RS86 were identical in the two preparations, but pilocarpine was approximately 30-fold as potent in the denuded as in the intact preparation. We conclude that muscarinic receptors on endothelium and smooth muscle of the rabbit thoracic aorta are very similar, if not identical, and both are of the M3 subtype. References Angus, J.A., Campbell, G.R., Cocks, T.M., Manderson, J.A., 1983. Vasodilation of acetylcholine is endothelium dependent: a study by sonomicrometry in canine femoral artery in vivo. J Physiol (Lond) 344, 209–222. Arunlkshana, O., Schild, O., 1959. Some quantitative uses of drug antagonists. Br J Pharmac Chemother 14, 58–98. Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72, 248–254. Choo, L.K., Malta, E., Mitchelson, F., 1986. The affinity of some selective muscarinic receptor antagonists for the muscarinic receptor mediating endothelial-dependent relaxation of the rabbit and rat thoracic aorta. J Pharm Pharmac 38, 843–845. De Lean, A., Munson, P.J., Rodbard D., 1978. Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay and physiological dose-response relationships. Am J Physiol 235, E97–E102.
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