Role of the M3 muscarinic acetylcholine receptor subtype in murine ophthalmic arteries after endothelial removal

Abstract

Purpose: We tested the hypothesis that the M3 muscarinic acetylcholine receptor subtype mediates cholinergic responses in murine ophthalmic arteries after endothelial removal.

Methods: Muscarinic receptor gene expression was determined in ophthalmic arteries with intact and with removed endothelium using real-time PCR. To examine the role of the M3 receptor in mediating vascular responses, ophthalmic arteries from M3 receptor-deficient mice (M3R(-/-)) and respective wild-type controls were studied in vitro. Functional studies were performed in nonpreconstricted arteries with either intact or removed endothelium using video microscopy.

Results: In endothelium-intact ophthalmic arteries, mRNA for all five muscarinic receptor subtypes was detected, but M3 receptor mRNA was most abundant. In endothelium-removed ophthalmic arteries, M1, M2, and M3 receptors displayed similar mRNA expression levels, which were higher than those for M4 and M5 receptors. In functional studies, acetylcholine evoked vasoconstriction in endothelium-removed arteries from wild-type mice that was virtually abolished after incubation with the muscarinic receptor blocker atropine, indicative of the involvement of muscarinic receptors. In concentration-response experiments, acetylcholine and carbachol concentration-dependently constricted endothelium-removed ophthalmic arteries from wild-type mice, but produced only negligible responses in arteries from M3R(-/-) mice. In contrast, acetylcholine concentration-dependently dilated ophthalmic arteries with intact endothelium from wild-type mice, but not from M3R(-/-) mice. Responses to the nitric oxide donor nitroprusside and to KCl did not differ between ophthalmic arteries from wild-type and M3R(-/-) mice, neither in endothelium-intact nor in endothelium-removed arteries.

Conclusions: These findings provide evidence that in murine ophthalmic arteries the muscarinic M3 receptor subtype mediates cholinergic endothelium-dependent vasodilation and endothelium-independent vasoconstriction.

Keywords: acetylcholine; mice; muscarinic receptors; ophthalmic arteries; vasoconstriction.

Figure 1

Relative mRNA expression of individual muscarinic receptor subtypes (M₁–M₅) normalized to β-actin transcripts in ophthalmic arteries from wild-type mice. (A) In arteries with endothelium, mRNA for all receptor subtypes was expressed, but M₃ receptor mRNA was most abundant (M₃ vs. M₄ and M₅, ***P < 0.001; M₃ vs. M₂, *P < 0.05; n = 10). (B) In arteries without endothelium, also muscarinic receptor mRNA for all five subtypes was detected; however, M₁, M₂, and M₄ receptor mRNA expression increased, whereas the expression levels of M₃ and M₅ receptor mRNA remained unchanged. Values are expressed as means ± SE (M₁ vs. M₅, #P < 0.01; M₂ vs. M₅, +P < 0.05; M₃ vs. M₅, ***P < 0.001; M₃ vs. M_4, **P < 0.01; n = 10).

Figure 2

Responses of ophthalmic arteries from wild-type mice with removed endothelium to acetylcholine (10⁻⁴ M) before and after incubation with the nonsubtype-selective muscarinic receptor blocker atropine (10⁻⁵ M). Remarkably, atropine virtually abolished acetylcholine-induced vasoconstriction. Values are expressed as mean ± SE (*P < 0.05, treated versus nontreated, n = 6).

Figure 3

Responses of ophthalmic arteries from wild-type and M3R^−/− mice to acetylcholine, nitroprusside, and KCl. Values are expressed as means ± SE (n = 6 per drug, concentration and genotype). (A) After endothelial removal, acetylcholine evoked concentration-dependent constriction responses in arteries from wild-type mice, but only negligible responses in arteries from M3R^−/− mice (***P < 0.001, M3R^−/− versus wild type). (B) In contrast, nitroprusside produced similar vasodilation responses in endothelium-denuded arteries from wild-type and M3R^−/− mice. (C) Constriction responses to KCl also were similar in ophthalmic arteries from wild-type and M3R^−/− mice. (D) In arteries with intact endothelium, acetylcholine induced pronounced vasodilation in wild-type mice, but almost no responses in M3R^−/− mice (***P < 0.001, M3R^−/− versus wild type). (E) Vasodilation to nitroprusside was retained in endothelium-intact arteries from M3R^−/− mice. (F) Vasoconstriction to KCl also was similar in endothelium-intact ophthalmic arteries from wild-type and M3R^−/− mice.

Figure 4

Responses of ophthalmic arteries from wild-type and M3R^−/− mice with damaged endothelium to carbachol. Vasoconstriction in response to carbachol was almost completely abolished in ophthalmic arteries from M3R^−/− mice. Values are expressed as mean ± SE (**P < 0.01, M3R^−/− versus wild-type, n = 5 per concentration and genotype).