Confocal analysis of the whole-mount rat mesenteric branch arteries (MBA) revealed nucleated structures with axonal processes which immunostained for calcitonin gene-related peptide (CGRP). Immunocytochemistry ruled out the possibility that these were immune elements (macrophages and mast or dendritic cells) in close proximity with nerve fibers. To test our hypothesis that beta-CGRP is expressed in the rat MBA, we performed RT-PCR using total RNA isolated from the mesenteric artery arcade and intron spanning primers designed to amplify 188 bp of the beta-CGRP and 333 bp of alpha-CGRP cDNA. The PCR yielded an amplicon of the predicted size which was cloned into the pCR 3.1 vector. DNA sequence analysis of the insert showed 100% homology with the beta-CGRP cDNA, indicating that mRNA encoding beta-CGRP is expressed in the vessel. To learn whether neuronal cell bodies are located in the adventitia of MBA, we performed a limited collagenase digestion of isolated segments and plated the resulting cells in Ham's F12 medium with 10% horse serum on polyornithine-coated cover glasses. The medium was replaced after 48 h with Ham's F12 nutrient mixture containing N2 supplement. This resulted in a mixed population of fibroblasts, a small number of smooth muscle cells and a subset of cells that sprouted axons and immunostained positively for neuronal cell adhesion molecule and CGRP antigens. Fibroblasts and smooth muscle cells did not label with these antibodies. These data demonstrate, for the first time, that a population of adventitial neuronal somata (termed ANNIES), possibly of sensory nerve origin, is located in small mesenteric arteries.
Copyright 2006 S. Karger AG, Basel.