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. 2011 Sep 27;52(10):7479-84.
doi: 10.1167/iovs.11-7370.

Identification of the muscarinic acetylcholine receptor subtype mediating cholinergic vasodilation in murine retinal arterioles

Adrian Gericke  1 , Jan J Sniatecki, Evgeny Goloborodko, Andreas Steege, Olga Zavaritskaya, Jan M Vetter, Franz H Grus, Andreas Patzak, Jürgen Wess, Norbert Pfeiffer
Affiliations

Identification of the muscarinic acetylcholine receptor subtype mediating cholinergic vasodilation in murine retinal arterioles

Adrian Gericke et al. Invest Ophthalmol Vis Sci. .

Abstract

Purpose: To identify the muscarinic acetylcholine receptor subtype that mediates cholinergic vasodilation in murine retinal arterioles.

Methods: Muscarinic receptor gene expression was determined in murine retinal arterioles using real-time PCR. To assess the functional relevance of muscarinic receptors for mediating vascular responses, retinal vascular preparations from muscarinic receptor-deficient mice were studied in vitro. Changes in luminal arteriole diameter in response to muscarinic and nonmuscarinic vasoactive substances were measured by video microscopy.

Results: Only mRNA for the M(3) receptor was detected in retinal arterioles. Thus, M(3) receptor-deficient mice (M3R(-/-)) and respective wild-type controls were used for functional studies. Acetylcholine concentration-dependently dilated retinal arterioles from wild-type mice. In contrast, vasodilation to acetylcholine was almost completely abolished in retinal arterioles from M3R(-/-) mice, whereas responses to the nitric oxide (NO) donor nitroprusside were retained. Carbachol, an acetylcholinesterase-resistant analog of acetylcholine, also evoked dilation in retinal arterioles from wild-type, but not from M3R(-/-), mice. Vasodilation responses from wild-type mice to acetylcholine were negligible after incubation with the non-subtype-selective muscarinic receptor blocker atropine or the NO synthase inhibitor N(ω)-nitro-L-arginine methyl ester, and were even reversed to contraction after endothelial damage with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.

Conclusions: These findings provide evidence that endothelial M(3) receptors mediate cholinergic vasodilation in murine retinal arterioles via activation of NO synthase.

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Figures

Figure 1.
Figure 1.
(A) Sketch of the experimental setup for videomicroscopic measurements of retinal arteriole responses. An isolated retina with the optic nerve and the ophthalmic artery left attached was mounted onto a transparent plastic platform and fixed with a steel ring. To pressurize vessels, the ophthalmic artery was cannulated with a micropipette that was connected to a reservoir filled with Krebs solution. (B) Photograph from above showing the experimental setup. At the 6 o'clock position, a micropipette is visible that was used to pressurize retinal arterioles via the ophthalmic artery. (C) First-order retinal arteriole with a red blood cell inside.
Figure 2.
Figure 2.
Relative mRNA expression of individual muscarinic receptor subtypes (M1–M5) normalized to β-actin transcripts in whole brain (positive control) (A) and retinal arterioles (B) from wild-type mice. As expected, mRNA of all five muscarinic receptor subtypes was detected in the brain. However, mRNA of the M3 receptor subtype only was found to be expressed in retinal arterioles. The values are averages of triplicate measurements and expressed as mean ± SE.
Figure 3.
Figure 3.
Responses of retinal arterioles from wild-type and M3R−/− mice to acetylcholine and nitroprusside. (A) Vasodilation to acetylcholine was virtually abolished in retinal arterioles from M3R−/− mice. (B) In contrast, responses of retinal arterioles to the NO donor nitroprusside did not differ between wild-type and M3R−/− mice. Values are expressed as mean ± SE (n = 6 per concentration and genotype; **P < 0.01, M3R−/− vs. wild-type mice).
Figure 4.
Figure 4.
Responses of retinal arterioles from wild-type and M3R−/− mice to carbachol. Vasodilation in response to carbachol (10−5 M) was almost completely abolished in retinal arterioles from M3R−/− mice. Values are expressed as mean ± SE (n = 6 per genotype; **P < 0.01, M3R−/− vs. wild-type mice).
Figure 5.
Figure 5.
Responses of retinal arterioles from wild-type mice to acetylcholine (10−5 M) before and after incubation with the non–subtype-selective muscarinic receptor blocker atropine (10−5 M) (A) and the NO synthase inhibitor L-NAME (10−4 M) (B). Remarkably, vasodilation responses were negligible after incubation with either of the two blockers. Values are expressed as mean ± SE (n = 5 per group; *P < 0.05, treated versus nontreated).
Figure 6.
Figure 6.
Responses of retinal arterioles from wild-type mice to acetylcholine (10−5 M) (A) and to the NO donor nitroprusside (10−5 M) (B) that had been perfused either with Krebs buffer (vehicle) only or with Krebs buffer containing 0.3% of CHAPS before. Remarkably, acetylcholine produced vasoconstriction after perfusion with CHAPS, whereas vasodilation responses to nitroprusside were retained. Values are expressed as mean ± SE (n = 5 per group; **P < 0.01, CHAPS-treated versus vehicle-treated).
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