Functional acetylcholine muscarinic receptor subtypes in human brain microcirculation: Identification and cellular localization

Acetylcholine is an important regulator of local cerebral blood flow. There is, however, limited information available on the possible sites of action of this neurotransmitter on brain intraparenchymal microvessels. In this s tudy, a combination of molecular and functional approaches was used to iden tify which of the five muscarinic acetylcholine receptors (mAChR) are prese nt in human brain microvessels and their intimately associated astroglial c ells. Microvessel and capillary fractions isolated from human cerebral cort ex were found by reverse transcriptase-polymerase chain reaction to express m2, m3, and, occasionally, mi and m5 receptor subtypes. To localize these receptors to a specific cellular compartment of the Vessel wall, cultures o f human brain microvascular endothelial and smooth muscle cells were used, together with cultured human brain astrocytes. Endothelial cells invariably expressed m2 and m5 receptors, and occasionally the mi receptor; smooth mu scle cells exhibited messages for all except the m4 mAChR subtypes, whereas messages for all five muscarinic receptors were identified in astrocytes. In all three cell types studied, acetylcholine induced a pirenzepine-sensit ive increase (62% to 176%, P < 0.05 to 0.01) in inositol trisphosphate, sug gesting functional coupling of mi, m3, or m5 mAChR to a phospholipase C sig naling cascade. Similarly, coupling of m2 or m4 mAChR to adenylate cyclase inhibition in endothelial cells and astrocytes, but not in smooth muscle ce lls, was demonstrated by the ability of carbachol to significantly reduce ( 44% to 50%, P < 0.05 to 0.01) the forskolin-stimulated increase in cAMP lev els. This effect was reversed by the mAChR antagonist AF-DX 384. The result s indicate that microvessels are able to respond to neurally released acety lcholine and that mAChR, distributed in different vascular and astroglial c ompartments, could regulate cortical perfusion and, possibly, blood-brain b arrier permeability, functions that could become jeopardized in neurodegene rative disorders such as Alzheimer's disease.