ABSTRACT
Uptake of choline by the high-affinity choline transporter CHT1 is the rate-limiting step in neuronal acetylcholine (ACh) synthesis. Here, we investigated by RT-PCR, in-situ hybridisation, immunohistochemistry, and Western blotting whether CHT1 is also expressed in cholinergic epithelia. CHT1-mRNA and -protein were detected in keratinocytes of human skin, rat skin and tongue, the human keratinocyte cell line HaCaT, and the ciliated cells of the rat tracheal epithelium. Immunohistochemically, CHT1 was predominantly localized to the epithelial cell membranes, in case of ciliated tracheal cells it was restricted to the apical membrane. This is the first study to demonstrate the expression of CHT1 in non-neuronal cells. The close apposition of CHT1 to reported sites of localization of choline acetyltransferase in these cells is strongly in favour of ACh synthesis being fuelled by choline uptake via CHT1 in these epithelia.
Subject(s)
Epithelial Cells/metabolism , Membrane Transport Proteins/biosynthesis , Animals , Cholinergic Fibers/metabolism , Cilia/metabolism , Female , Humans , Male , RNA, Messenger/biosynthesis , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Trachea/cytology , Trachea/drug effects , Trachea/metabolismABSTRACT
The rate limiting step in neuronal acetylcholine (ACh) synthesis is the uptake of choline by the high-affinity choline transporter (CHT1). Here, we investigated the distribution of CHT1 in the rat trachea. CHT1-mRNA was detected by reverse transcriptase-polymerase chain reaction in trachea without epithelium, abraded tracheal mucosa, and in epithelial cells obtained by laser-assisted cell-picking. Accordingly, CHT1-mRNA could also be detected in tracheal epithelial cells by in situ hybridization. Recently obtained polyclonal rabbit and guinea-pig antisera against a synthetic peptide corresponding to amino acid residues 29-40 of the rat CHT1 sequence localized CHT1 protein in combination with antisera against the vesicular acetylcholine transporter in cholinergic fibers innervating tracheal glands and the tracheal muscle. In case of the tracheal epithelium, CHT1 was restricted to the apical membrane of the ciliated cells, as demonstrated by confocal laser scanning and electron microscopy using an affinity-purified CHT1 antiserum. The close apposition of CHT1 to reported sites of localization of choline acetyltransferase in these cells is strongly in favor of ACh synthesis being fueled by choline uptake via CHT1 after release and breakdown of ACh at the luminal surface. Accordingly, cholinergic regulation of tracheal epithelial function is governed by local release and recycling of ACh by ciliated cells.
