title: Neuronal and endothelial sites of acetylcholine synthesis and release associated with microvessels in rat cerebral cortex: ultrastructural and neurochemical studies.
abstract: We sought to establish what proportion of the cholinergic innervation of the cerebral cortex (CX) is associated with intraparenchymal blood vessels by using immunocytochemical and neurochemical techniques, and whether [3H]acetylcholine ([3H]ACh) is synthesized and released by elements associated with cortical microvessels (MV). MVs and, for comparison, tissue homogenates were prepared using sucrose gradient/differential ultracentrifugation methods. Efficacy of the separation technique was indicated by the activity of gamma-glutamyltranspeptidase (up to 29.2-fold enrichment), an endothelial cell marker enzyme, in the MV fraction and microscopy. The size of isolated microvessels ranged from 5 to 40 micron (o.d.) with 67.7% of the vessels less than 10 micron and 32.2% between 11 and 40 micron (690 vessels measured from 4 animals). By electron microscopy immunoreactive choline acetyltransferase (ChAT), the biosynthetic enzyme for ACh, was localized to: (a) axons and axon terminals opposed to the basal laminae of capillaries and small arterioles, and (b) capillary endothelial cells. ChAT-labeled elements associated with MVs were most prominent in layers I, III and V of the CX consistent with the local pattern of cholinergic innervation. The absolute amount of ACh synthesized (pmol Ach/100 mg wet wt.) by elements associated with cortical MVs was relatively small (2.3% total cortical homogenate activity). Inhibition of MV ChAT activity to 5% of control by the specific ChAT inhibitor, 4-naphthylvinylpyridine, and HPLC analysis of the product, indicated that authentic ACh was measured. Other tissues similarly synthesized small amounts of ACh relative to the CX, caudate nucleus (CN, 2.4%), cerebellum (CRB, 1.4%) and liver (LIV, 3.9%). Consistent with the known extent of the cholinergic innervation of the tissues examined, the rank order of ChAT associated for both MVs and homogenate were: CN greater than CX much greater than CRB greater than LIV. However, based on the specific activities of ChAT, cortical MVs have the remarkable capacity to synthesize ACh at rates 95% greater than cortical (S1 fraction) homogenate (59.0 +/- 3.5 nmol/mg protein/40 min; n = 7), which is enriched in nerve terminals. Except for LV (+11%), other tissues also had remarkably high ChAT activity in MV (% above corresponding homogenate; P less than 0.05, n = 5): CN (+269) and CRB (+313). Release of [3H]ACh from MVs and, for comparison, nerve terminals were graded to K+ depolarization stimulus (5-55 mM), maximal with 55 mM K+ and Ca2+ dependent.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Neurology, Cornell University Medical College, New York, NY 10021.
authors.forename: S P
authors.initials: SP
authors.lastname: Arnerić
authors.forename: M A
authors.initials: MA
authors.lastname: Honig
authors.forename: T A
authors.initials: TA
authors.lastname: Milner
authors.forename: S
authors.initials: S
authors.lastname: Greco
authors.forename: C
authors.initials: C
authors.lastname: Iadecola
authors.forename: D J
authors.initials: DJ
authors.lastname: Reis
mesh_terms: D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D002540:Cerebral Cortex / Q000098:blood supply* / Q000378:metabolism / Q000648:ultrastructure; D002560:Cerebrovascular Circulation; D002795:Choline O-Acetyltransferase / Q000032:analysis; D002799:Cholinergic Fibers / Q000378:metabolism* / Q000648:ultrastructure; D004727:Endothelium / Q000378:metabolism / Q000648:ultrastructure; D007150:Immunohistochemistry; D008297:Male; D008833:Microcirculation / Q000378:metabolism; D008854:Microscopy, Electron; D009411:Nerve Endings / Q000378:metabolism / Q000648:ultrastructure; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D002795:Choline O-Acetyltransferase; D000109:Acetylcholine