Characterization of endogenous noradrenaline release from intact and epithelium‐denuded rat isolated trachea

Abstract

Overflow of endogenous noradrenaline (NA) from the in vitro incubated rat trachea evoked by two periods of electrical field stimulation (S1, S2, at 3 or 15 Hz) or by high potassium (60 mm) was determined by high performance liquid chromatography (h.p.l.c.) with electrochemical detection. In the presence of the neuronal uptake inhibitor desipramine, the α2‐adrenoceptor antagonist, yohimbine, enhanced the overflow of NA evoked by stimulation at 3 Hz by about 100% suggesting the presence of presynaptic inhibitory autoreceptors on the sympathetic nerves innervating the trachea. When desipramine and yohimbine were present throughout the experiments, the overflow of NA evoked by the second period of electrical stimulation (S2) was significantly smaller than that evoked by the first (S1). This decline of overflow was prevented when the NA precursor, tyrosine, was additionally present throughout the experiments. After removal of the epithelium, the tissue content of NA was reduced by about 30%, suggesting that part of the NA may be present and released within the epithelium. However, the overflow of NA evoked by stimulation at 3 Hz or 15 Hz was reduced by 70–80%, indicating that the epithelium may additionally exert a permissive role on the release of NA within the airways, possibly by suppressing inhibitory factors. Stimulation by high potassium (60 mm for 10 min) caused a large overflow of NA (about 45% of the tissue NA), both from epithelium‐free and epithelium‐denuded tracheae. Thus the ‘endogenous inhibition’ of NA release after removal of the epithelium is surmountable when a high potassium stimulus is applied. An enhanced muscarinic inhibition of NA release in epithelium‐denuded tracheae may not be responsible for the reduction in NA overflow, since the muscarinic receptor antagonist, scopolamine, did not significantly affect the overflow of NA evoked by 3 or 15 Hz, either in the presence or the absence of the epithelium. The muscarinic receptor agonist, oxotremorine, inhibited the overflow of NA evoked by stimulation at 3 Hz almost completely, an effect antagonized by scopolamine, demonstrating specific, inhibitory muscarinic receptors. Since damage to the epithelium is known to occur in obstructive bronchial disease, the present experiments suggest that an impaired sympathetic (broncho‐relaxant) neurotransmission could be involved in the pathogenesis of bronchoconstriction.

Mucosa‐dependent muscarinic liberation of prostaglandins from rat isolated trachea

“…before washing period) by gently rubbing the luminal surface of the trachea with a pipe cleaner. Removal of the mucosa by this procedure has previously been confirmed by light microscopy after staining with haematoxylin and eosin (Racke et al, 1991;1992).…”

“…for at least 1 week before use. As described in detail previously (Racke et al, 1991), the animals were killed by stunning followed by exsanguination and the whole tracheae were dissected, opened by a cut along the ventral side, fixed in a glass organ bath and finally incubated in 1.7 ml Krebs-HEPES solution of the following composition (mM): NaCl 118.5, KCl 5.7, CaCl2 1.25, MgCl2 1.2, sodium EDTA 0.03, (+)-ascorbic acid 0.06, HEPES 20.0 and phosphate buffer 1.0 (both adjusted to pH 7.4) and D-glucose 11.1. The incubation medium was kept at 370C and continuously gassed with 100% 02.…”

Endogenous noradrenaline release from guinea‐pig isolated trachea is inhibited by activation of M2 receptors

“…As described in detail for rats (Racke et al, 1991), the isolated tracheae were fixed between two platinum wire field electrodes and incubated in 1.7 ml Krebs-HEPES solution (composition, mM): NaCl 118.5, KCI 5.7, CaCI2 1.2, MgCl2 1.2, sodium EDTA 0.03, (+ )-ascorbic acid 0.06, HEPES 20.0 (pH 7.4) and D-glucose 11.1. The medium also contained desipramine (1 gM), tyrosine (1O gM) and yohimbine (1I M) (see Racke et al, 1991). The bath fluid (37°C and gassed with 100% 02) was collected every 10 min.…”

“…Antagonism was quantified by calculating apparent pA2 values of EC" values according to equation (4) of Furchgott (1972). EC50 values (concentrations causing 50% inhibition of NA overflow) were calculated with a computer programme (see Racke et al, 1991). Mean values of n observations are given ± s.e.mean.…”

“…Biphasic pulses of 1 ms, 250 mA, 3 Hz (Grass S9 stimulator) were applied 3 times for 1 min with 1 min intervals. NA was determined by high performance liquid chromatography with electrochemical detection and the evoked overflow of NA was calculated as described previously (Racke et al, 1991). Antagonism was quantified by calculating apparent pA2 values of EC" values according to equation (4) of Furchgott (1972).…”