PHARMACOLOGICAL CHARACTERIZATION OF MUSCARINIC ACETYLCHOLINE BINDING-SITES IN HUMAN AND BOVINE CEREBRAL MICROVESSELS

The aim of this study was to investigate the presence of muscarinic receptors in human brain microvessels (MVs) and capillaries (CAPs) and, further, to pharmacologically characterize these receptors as well as those in bovine cerebral microvascular beds. For this purpose, the binding of [H-3]N-methyl scopolamine ([H-3]NMS) was assessed in isolated human and bovine cerebral MVs and CAPs and competition studies were performed against [H-3]NMS binding with several well characterized muscarinic antagonists. The antagonist cerebrovascular affinity constants (pK(D)) were determined with the computer-fitting software LIGAND and then compared by correlation analyses to their reported affinities (pK(i)) at the five cloned muscarinic receptors. The specific binding of [H-3]NMS to human and bovine MVs and CAPs was saturable, of high affinity and competitively inhibited by muscarinic antagonists. Heterogeneous populations of muscarinic binding sites were found in the microvascular tissues from both species. In human cortical MVs, the pharmacological binding profile obtained from various muscarinic receptor antagonists was best correlated to that of the cloned mi (r = 0.95; p < 0.001) and less so m5 (r = 0.77; p = 0.025) receptor subtypes while in bovine MVs, the presence of the mi subtype was strongly suggested. Cerebrovascular affinities obtained for selected muscarinic antagonists in single preparations of human and bovine CAPs were suggestive of the presence of M(1)/ml and M(3)/m3 receptor subtypes, and possibly the m5 subtype in bovine CAPs. The detection of M(1)/ml, M(3)/m3 and possibly m5 muscarinic receptor subtypes in brain microcirculation is consistent with reports where these receptors have been shown to mediate vasoconstriction, vasodilatation, and activation of nitric oxide synthase, respectively. The physiological role of the putative m5 receptor in the cerebrovascular bed is, as yet, unclear. Altogether, these results indicate the presence of specific muscarinic binding sites in human and bovine brain microcirculation and are supportive of a role for these muscarinic receptors in the neurogenic regulation of cortical cerebral blood flow and possibly blood-brain-barrier functions by acetylcholine.