European Journal of Pharmacology, 184 (1990) 333-337
Elsevier EJP 20678
Sensory nerve depletion potentiates inhibitory non-adrenerg~c, non-cholinergic nerves in guinea-pig airways C. D a v i d S t r e t t o n , M a r i a G. Belvisi a n d P e t e r J. B a r n e s Department of Thoracic Medicine, National Heart and Lung Institute, Dooehouse Street, London SW3 6L Y, U.K.
Received 19 June 1990, accepted 26 June 1990
The effect of sensory neuropeptide depletion by systemic capsaicin pretreatment was studied both on inhibitory non-adrenergic, non-eholinergic responses elicited to electrical field stimulation and on bronchodilator responses to vasoactive intestinal peptide (the putative transmitter of these nerves), in guinea-pig trachea. Capsaicin pretreatment enhanced the relaxation responses induced by both electrical field stimulation and vasoactive intestinal peptide, whereas these reponses were not significantly increased in vehicle-pretreated or in untreated animals. The bronchodilatot response to isoprenaline was unaffected by capsaicin pretreatment. Sensory neuropeptide depletion therefore augments both inhibitory non-adrenergic, non-cholinergic responses and responses to exogenous vasoactive intestinal peptide, suggesting an effect on vasoactive intestinal peptide receptors. Capsaicin; Non-adrenergic, non-chohnergic (NANC) responses (inhibitory); Airways (guinea-pig)
1. Introduction Neurogenic responses to electrical field stimulation (EFS) in the guinea-pig trachea are biphasic with an initial rapid, atropine-sensitive, cholinerglc contractile component which is followed by a prolonged relaxation. The relaxation response is only slightly reduced by propranolol and is therefore believed to be predominantly non-adrenergic, non-cholinerglc ( N A N C ) (Coburn and Tomita, 1973). The neurotransmitter of these inhibitory N A N C (i-NANC) nerves, while not certain, m a y be vasoactive intestinal peptide (VIP) or a related peptide (Ellis and Farmer, 1989). Systemic capsaicin pretreatment of guinea-pigs leads to the selective depletion of sensory neuropeptides such as substance P and neurokinin A
Correspondence to: P.J. Barnes, Department of Thoracic Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, U.K.
from C-fibre primary afferent nerves with the subsequent degeneration of these fibres (Jansco et al., 1977), while cholinergic neurones are reported to be unaffected (Martling et al., 1984). In order to study the interaction between nerves in the guinea-pig trachea we have examined the effect of capsaicin pretreatment on i - N A N C responses and the responses to exogenous VIP.
2. Materials and methods 2.1. Capsaicin p r e t r e a t m e n t
Three groups of male Dunkin-Hartley guineapigs (270-350 g) from the same batch were subjected to either pretreatment with capsaicin (50 mg kg-1), using the single systemic injection protocol developed by Lundberg et al. (Lundberg et al., 1983), pretreatment with the vehicle for capsaicin (10% Tween 80 and 10% ethanol in saline) (vehicle control animals) or no pretreat-
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334 ment (normal control animals). All animals were used one week later. Completeness of sensory denervation was tested by lack of contractile response to capsaicin (10 -5 M) at the end of the experiment.
X 1 0 - 6 M) were constructed. Only one concentration-response relationship was constructed in a single tissue. Relaxation responses were expressed as a percentage of the m a x i m u m relaxation response induced in each tissue to 10 -4 M papaverine.
2.2. Tissue preparation 2.5. Analysis of results Animals were killed by cervical dislocation and the trachea rapidly removed and placed in KrebsHenseleit solution of the following composition (mM): NaC1 118, KC1 5.9, MgSO 4 1.2, CaC12 2.5, N a H 2 P O 4 1.2, N a H C O 3 25.5 and glucose 5.05. Indomethacin (10 -5 M) and atropine and propranolol (each 10 -6 M) were present in all experiments. The solution was gassed with a 95% 02-5% CO 2 mixture, p H 7.4. The trachea was opened longitudinally by cutting through the cartilage and the upper two sections of trachea, containing three to four cartilaginous strips were suspended between parallel platinum field electrodes in 10 ml organ baths for the measurement of isometric changes in tension. The tissues were allowed to equilibrate for 1 h, with frequent washing, under a resting tension of 1 g.
2.3. Frequency-response relationships
Relaxation responses were expressed as absolute changes in tension, and then converted to a percentage of the maximal relaxation for each tissue. Comparison of results between different treatment groups was analysed using Student's t-test for unpaired data. Statistical analysis was performed using absolute values. Probability values < 0.05 were considered significant.
3.1. Inhibitory N A N C responses In animals pretreated with capsaicin, the maximal relaxation response to EFS at 20 Hz was 51.6 + 5.0% compared with 26.4 +__2.9% in vehicle (Tween)-treated animals (P < 0.001, n = 10) (fig. la, table 1). There was no significant difference between relaxation responses in tissues from vehicle-treated and untreated animals. All i - N A N C responses were blocked by tetrodotoxin (10-6 M), indicating that the relaxation responses induced by EFS were due to nerve stimulation.
Tissues were exposed to biphasic square wave pulses of supramaximal voltage at source of 40 V and duration of 0.5 ms, and frequency-response relationships were constructed using the frequency range of 1-50 Hz, each stimulation being applied for 30 s. The magnitude of the stimulus frequencies were randomised. Inhibitory responses to EFS are prolonged and succesive frequencies were applied once the tissues had returned to their basal levels of tension. Only one complete range of frequencies was tested on each preparation. The inhibitory responses obtained to EFS were expressed as absolute changes in tension and then converted to a percentage of the maximum relaxation response induced in the tissues by papaverine (10 -4 M).
In airways smooth muscle obtained from vehicle-treated animals, the concentration-response relationships to exogenous VIP were not significantly different from those obtained in normal, untreated tissues. VIP responses from capsaicin-pretreated animals were significantly a u g m e n t e d however, with a mean ECs0 value of 31.5 nM compared with 47.3 nM in control animals (P < 0.001, n = 8) (fig. lb).
2. 4. Concentration-response studies
3.3. lsoprenaline responses
Concentration-response relationships to VIP 3 X 1 0 - 7 M) and isoprenaline ( 1 0 - 1 1 -- 3
( 1 0 - 9 --
3.2. VIP responses
C o n c e n t r a t i o n - r e s p o n s e relationships constructed to exogenous isoprenaline ( 1 0 - 1 1 - 3 x
Relaxation (~ maximum) 100-
Relaxation (~ maximum) 90=
F r e q u e n c y (Hz)
Fig. 1. (a) Frequency-response relationships for inhibitory N A N C responses in tracheal strips from capsaicin-treated (A), vehicle (Tween)-treated (zx) and untreated (13) guinea-pigs. Relaxation is expressed as the % maximal response to papaverine (10 -4 M). All points represent the means±S.E.M, of 10 animals. Significance of difference from vehicle-treated animals: * P < 0.05, * * P < 0.01 and * * * P < 0.001, as compared with eapsaicin-treated and untreated values. Statistical analysis was performed using absolute values. (b) Concentration-response relationships for relaxation induced by vasoactive intestinal peptide (VIP) in tracheal strips from eapsaicin-treated (A), vehicle-treated (zx) and untreated (13) animals. All points represent the m e a n s ± S.E.M. of eight animals. Significance of difference from vehicle-treated animals: * P < 0.05, * * P < 0.01 and * * * P < 0.001, as compared with capsalcintreated and untreated values. Statistical analysis was performed using absolute data.
TABLE 1 Frequency-response relationships to electrical field stimulation expressed as mg tension in capsaicin-treated, vehicle-treated and untreated guinea-pig trachea. Data are expressed as the mean relaxations (rag tension)± S.E. of 10 observations. Relaxations (mg)
Max. relax, to papaverine (10 -4 M)
1 Hz a
523 + 69 b
698 + 81 c
973 + 56 d
1281 ± 63 d
1522 + 78 a
1902 + 233 ¢
409 + 73
421 + 113
577 ± 111
681 ± 131
1136 + 98
2271 ± 321
398 ± 25
506 ± 49
717 + 68
813 ± 148
905 ± 83
2011 ± 214
a Frequency (Hz). b p < 0.05, c p < 0.01 and a p < 0.001, as compared with vehicle-treated animals.
336 10 -6 M) were not significantly different in tissues obtained from all three different animal groups, such that there was no significant difference in the ECs0 values (9.5 nM in capsaicin animals compared with 9.7 nM in vehicle-treated controls and 10.2 nM in untreated animals, n = 8).
We have demonstrated that the i - N A N C response elicited by EFS in the guinea-pig trachea is significantly enhanced after depletion of neuropeptides from sensory nerves, suggesting that these neuropeptides may modulate the activity of iN A N C nerves. Furthermore, the relaxation response induced by exogenous VIP was also enhanced when compared with responses in control animals. Since VIP is a possible neurotransmitter of i-NANC nerves in guinea-pig, this indicates that the mechanism of i-NANC facilitation may involve enhanced bronchodilator responsiveness in airway smooth muscle. Relaxation responses to isoprenaline were not, however, significantly different in capsaicin-pretreated tissues as compared with either vehicle or normal control tissues, suggesting that the facilitation of i-NANC responses may be due to a selective effect on post-junctional VIP receptors. The reason for the more pronounced augmentation of the i - N A N C responses elicited to EFS relative to the enhanced responsiveness to exogenously applied VIP in tissues from capsaicin-pretreated animals is unclear but may be explained in part by the fact that capsaicin pretreatment abolishes the excitatory N A N C component elicited to EFS, which is due to the release of sensory neuropeptides. The removal of this functional antagonism may contribute to the exaggerated iN A N C response, and would not be evident with exogenous VIP. Sensory nerve degeneration might remove a source of neutral endopeptidase which has been shown to degrade SP (Skidgel et al., 1984) and VIP (Rhoden and Barnes, 1990). Exogenous SP addition has not, however, been shown to be affected in tissues from capsaicinised animals (Martling et al., 1984; Stretton et al., 1989), suggesting that responses to VIP may also be unaf-
fected if VIP and SP share the same degradative enzymes. The mechanism by which selective depletion of neuropeptides from sensory nerves leads to specific increase in VIP responsiveness of smooth muscle is not certain but may involve either VIP receptors or their coupling to adenylyl cyclase. This study emphasises the importance of interactions between airway nerves and raises the possibility that abnormal function of a nerve type may influence other neural pathways. Neuropeptides in sensory nerves may be involved in inflammatory conditions of the airways such as asthma (Barnes, 1986) and i - N A N C nerves are the only bronchodilator mechanism in human airways (Palmer et al., 1986). Neurogenic inflammation might therefore lead to impaired function of i - N A N C nerves in asthma and thus contribute to exaggerated bronchoconstrictor responses.
This work was supported by the Medical Research Council and Fisons pie.
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1984, Hydrolysis of substance P and neurotensin by converting enzyme and neutral endopeptidase, Peptides 5, 769. Stretton, C.D., M.G. Belvisi and P.J. Barnes, 1989, The effect of sensory nerve depletion on cholinergic neurotransmission in guinea-pig airways, Br. J. Pharmacol. 98, 782P.