Exposure to ozone (O3) induces airway hyperresponsiveness mediated partly through the release of substance P (SP) from nerve terminals in the airway wall. Although substantial evidence suggests that SP is released by sensory nerves, SP is also present in neurons of airway ganglia. The purpose of this study was to investigate the role of intrinsic airway neurons in O3-enhanced airway responsiveness in ferret trachea. To remove the effects of sensory innervation, segments of ferret trachea were maintained in culture conditions for 24 h before in vitro exposure to 2 parts/million of O3 or air for 1 h. Sensory nerve depletion was confirmed by showing that capsaicin did not affect tracheal smooth muscle responsiveness to cholinergic agonist or contractility responses to electrical field stimulation (EFS). Contractions of isolated tracheal smooth muscle to EFS were significantly increased after in vitro O3 exposure, but the constrictor response to cholinergic agonist was not altered. Pretreatment with CP-99994, an antagonist of the neurokinin 1 receptor, attenuated the increased contraction to EFS after O3 exposure but had no effect in the air exposure group. The number of SP-positive neurons in longitudinal trunk ganglia, the extent of SP innervation to superficial muscular plexus nerve cell bodies, and SP nerve fiber density in tracheal smooth muscle all increased significantly after O3 exposure. The results show that release of SP from intrinsic airway neurons contributes to O3-enhanced tracheal smooth muscle responsiveness by facilitating acetylcholine release from cholinergic nerve terminals.

Dey. Interleukin-1␤-induced airway hyperresponsiveness enhances substance P in intrinsic neurons of ferret airway. Am J Physiol Lung Cell Mol Physiol 283: L909-L917, 2002. First published April 12, 2002 10.1152/ajplung.00363.2001.-Interleukin (IL)-1␤ causes airway inflammation, enhances airway smooth muscle responsiveness, and alters neurotransmitter expression in sensory, sympathetic, and myenteric neurons. This study examines the role of intrinsic airway neurons in airway hyperresponsiveness (AHR) induced by IL-1␤. Ferrets were instilled intratracheally with IL-1␤ (0.3 g/0.3 ml) or saline (0.3 ml) once daily for 5 days. Tracheal smooth muscle contractility in vitro and substance P (SP) expression in tracheal neurons were assessed. Tracheal smooth muscle reactivity to acetylcholine (ACh) and methacholine (MCh) and smooth muscle contractions to electric field stimulation (EFS) both increased after IL-1␤. The IL-1␤-induced AHR was maintained in tracheal segments cultured for 24 h, a procedure that depletes SP from sensory nerves while maintaining viability of intrinsic airway neurons. Pretreatment with CP-99994, an antagonist of neurokinin 1 receptor, attenuated the IL-1␤-induced hyperreactivity to ACh and MCh and to EFS in cultured tracheal segments. SP-containing neurons in longitudinal trunk, SP innervation of superficial muscular plexus neurons, and SP nerve fiber density in tracheal smooth muscle all increased after treatment with IL-1␤. These results show that IL-1␤-enhanced cholinergic airway smooth muscle contractile responses are mediated by the actions of SP released from intrinsic airway neurons. airway inflammation; airway smooth muscle contraction; muscarinic agonists; neurokinin receptor; airway innervation

The neurochemical profiles of neurons in ferret tracheal ganglia has been characterized, but their projections to smooth muscle and epithelium in ferret trachea has not been examined. The purpose of this study is to determine the location of cell bodies that project VIP-, SP-, and NPY-containing fibers to the ferret tracheal smooth muscle and epithelium. Segments of ferret trachea were cultured for 0, 1, 3, or 7 days, some in the presence of 3 microm capsaicin. VIP, SP, or NPY nerve fiber density was measured using morphometric procedures. A retrograde tracer, rhodamine-labeled microspheres, identified neurons projecting to the epithelium. The density of SP fibers in the epithelium was reduced after culture, but VIP innervation was not different. In tracheal smooth muscle, the density of VIP- and SP-IR fibers was not different during the culture period, but NPY fiber density was reduced at all culture times. Capsaicin treatment did not affect nerve fiber density in the tracheal smooth muscle but produced a significant reduction in the density of epithelial VIP- and SP-IR nerve fibers after 1 day. Rhodamine-labeled microspheres were identified in VIP-containing nerve cell bodies of the ferret tracheal plexus. VIP innervation to the airway epithelium in ferret originates both from cell bodies in airway ganglia and cell bodies in sensory ganglia. The pathway from airway ganglia suggest the existence of a local reflex mechanisms initiated by epithelial irritation.

Inhalation of irritants, such as toluene diisocyanate (TDI), stimulates substance P (SP) release from peripheral processes of sensory neurons innervating the airways. The purpose of this study was to determine if TDI inhalation affects intraneuronal levels of SP and preprotachykinin (PPT) messenger RNA (mRNA) in the sensory neurons of the trigeminal ganglion (TG) which innervate the nasal epithelium. The nasal cavity of Fisher-344 rats was instilled with rhodamine-labeled latex microspheres. Ten days later, the rats were exposed to 60 ppb of 2,4-2,6-TDI vapor for 2 h. The TG were removed 1, 12, 24, 48, 72, and 96 h after TDI treatment and prepared for SP immunocytochemistry and PPT in situ hybridization. SP nerve fiber density in nasal epithelium was significantly increased 12, 24, and 48 h after TDI exposure. The proportion of microsphere-labeled cell bodies expressing high levels of SP immunoreactivity was decreased at 24 h but was increased above controls at 48 and 72 h. The proportion of microsphere-labeled cell bodies expressing high levels of PPT mRNA was increased above control levels at 24 and 48 h. The percentage of leukocytes observed in nasal lavage fluid was significantly increased 12, 24, 48, and 72 h after inhalation. These studies indicate that SP production in TG neurons projecting to the nasal epithelium is transiently increased after TDI exposure, suggesting that TDI inhalation not only causes SP release but also increased intraneuronal neuropeptide levels. Increased neuronal SP levels may be involved in maintaining neurogenic inflammation or the development of airway hyperresponsiveness.