COLOCALIZATION OF VASOACTIVE-INTESTINAL-PEPTIDE AND NITRIC-OXIDE SYNTHASE IN NEURONS OF THE FERRET TRACHEA

Neurally-mediated relaxation of smooth muscle in human,26 guinea-pig,7 cat,14 and pig18 airways is largely attributed to a nonadrenergic, noncholinergic mechanism. While the specific transmitter(s) of this relaxant system have not been conclusively identified, vasoactive intestinal peptide and nitric oxide have emerged as likely mediators in airway smooth muscle. Both vasoactive intestinal peptide and nitric oxide relax guinea-pig3,22,28 pig18,19 and human2,24 smooth muscle. Vasoactive,intestinal peptide is present in nerve fibers associated with airway smooth muscle in humans and several animal species.12,13,33 In guinea-pigs, vasoactive intestinal peptide is released during electrical field stimulation of airway strips and the release correlates with the nonadrenergic relaxation.23 This relaxation is markedly reduced after incubation of tracheal tissue with a specific VIP antibody23 and by immunization to vasoactive intestinal peptide.16 Similarly, nonadrenergic relaxations induced by electrical field stimulation are reduced in human,2,15 pig,19 guinea-pig22 and bovine32 airways by nitric oxide synthesis inhibitors. Vasoactive intestinal peptide is present in nerve cell bodies of airway ganglia,12,13 suggesting that these nerves in airway smooth muscle originate from intrinsic neurons.10 It is stored in dense-core vesicles of nerve terminals near airway smooth muscle,21 suggesting that preformed vasoactive intestinal peptide is released by fusion of the vesicles with the cell membrane of the nerve terminal. Nitric oxide is probably generated by a novel mechanism involving de novo synthesis at the nerve terminal during neural activation by the action of the enzyme nitric oxide synthase.29,30 However, since nitric oxide is also produced by both endothelial cells17,25 and macrophages,31 it is important to show that neurons in the airways are capable of producing nitric oxide in order to establish it as a neural mediator. Therefore, we have examined the localization of nitric oxide synthase and its possible colocalization with vasoactive intestinal peptide in the anatomically well characterized plexus of ferret trachea.1 We found that nitric oxide synthase and vasoactive intestinal peptide are present in a subpopulation of neurons within the plexus, with nerve cell bodies located in specific ganglia and in nerve fibers associated with tracheal smooth muscle and the walls of blood vessels. These findings prove that nitric oxide synthase and vasoactive intestinal peptide are present in the same neurons and that the nerve fibers are processes of motor neurons with nerve cell bodies located in intramural ganglia of the tracheal plexus.