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Vol. 284, Issue 1, 370-377, 1998
The Johns Hopkins Asthma and Allergy Center (B.J.C., B.J.U.), Baltimore, Maryland and Justus-Liebig University, Institute for Anatomy and Cell Biology (A.F.), Giessen, Germany
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Abstract |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Previous studies indicated that antidromic stimulation of capsaicin-sensitive vagal afferent fibers activated, via peripheral release of tachykinins, nonadrenergic, noncholinergic parasympathetic ganglion neurons that mediate relaxations of guinea pig trachealis. On the basis of the effects of selective agonists and inhibition with a nonselective receptor antagonist (SR 48968), we speculated that tachykinin-mediated activation of neurokinin3 (NK3) receptors might be involved. Using the recently developed NK3-selective receptor antagonist SR 142801, we further assessed the role of NK3 receptors in these relaxant responses. Relaxations of the guinea pig trachea elicited by antidromic stimulation of capsaicin-sensitive vagal afferent nerves were markedly inhibited by 0.3 µM SR 142801 and were abolished by a combination of SR 142801 and either of the NK1-selective receptor antagonists SR 140333 and CP 99994 (0.3 µM each). The NK3 receptor antagonist had similar effects on the relaxant responses elicited by capsaicin and substance P, but it had no effect on relaxations of the trachealis elicited by electrical field stimulation of the postganglionic nerves that innervate the trachealis or by stimulation of the preganglionic parasympathetic vagal nerves that innervate the trachea. These results and the observation that the ganglion neurons that mediate these responses are densely innervated by substance P-containing nerve fibers lead us conclude that stimulation of capsaicin-sensitive visceral afferent fibers activates, upon peripheral release of tachykinins, nonadrenergic, noncholinergic inhibitory neurons innervating guinea pig trachealis via activation of both NK3 and NK1 receptors.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Studies
carried out in the heart (Thorén, 1979
), airways (Coleridge et
al., 1989), GI tract (Szurzewski and King, 1989), gallbladder (Mawe, 1995) and bladder (de Groat et al., 1993; de Groat
and Booth, 1993) indicate that capsaicin-sensitive afferent fibers regulate the activity of autonomic nerves that innervate the viscera. In many instances, activation of the capsaicin-sensitive afferent fibers elicits changes in end organ function only when the
preganglionic nerves that innervate the relevant autonomic ganglia are
intact and functional (Thorén, 1979; Szurzewski and King, 1989;
Coleridge et al., 1989; de Groat et al., 1993).
These observations indicate that capsaicin-sensitive afferent nerves
elicit changes in autonomic nerve activity via centrally
mediated autonomic reflexes.
It is equally apparent, however, that another site of action of these
afferent nerves may be in the periphery, within autonomic ganglia
(Kreulen and Peters, 1986; Myers and Undem, 1993; de Groat and Booth,
1993; Myers et al., 1996). Anatomical studies have demonstrated the presence of axon collaterals from these visceral afferent fibers in autonomic ganglia (Szurzewski and King, 1989; de
Groat and Booth, 1993; Myers et al., 1996). Furthermore,
studies carried out in isolated ganglia have demonstrated effects of
these afferent nerves on the electrophysiological properties of the autonomic ganglion neurons after selective stimulation of their sensory/afferent nerve terminals (Szurzewski and King, 1989; de Groat
and Booth, 1993; Myers et al., 1996). In addition to
centrally mediated reflexes, then, capsaicin-sensitive afferent nerves
may modulate autonomic nerve activity by modulating neuronal
excitability through peripheral reflexes.
Tachykinins are transmitters that are frequently associated with the
nerve terminals of visceral afferent nerves (Canning, 1997). Acting on
NK1, NK2 and NK3 receptors, which
are selectively (but not specifically) activated by substance P, NKA
and NKB, respectively, the tachykinins are thought to mediate many of
the central and peripheral actions of afferent nerves (Canning, 1997).
Capsaicin-sensitive vagal afferent fibers that innervate the airways
contain NKA and substance P (Kummer et al., 1992). In several species, including humans, tachykinin-containing nerve endings
innervate many structures within the airway wall, including airway
parasympathetic ganglia (Kummer et al., 1992; Fischer and Hoffman, 1996; Dey et al., 1996; Myers et al.,
1996). Because activation of these afferent nerves can modulate
parasympathetic nerve activity both in vivo (Martling
et al., 1984) and in vitro (Myers and Undem,
1991, 1993; Watson et al., 1993; Myers et al., 1996) and because their effects are mimicked by exogenously
administered tachykinins and blocked by tachykinin receptor
antagonists, it seems likely that airway parasympathetic nerve activity
is modulated by peripherally released tachykinins.
In a previous study of the parasympathetic relaxant innervation of
guinea pig airways, we presented evidence that collaterals from
capsaicin-sensitive vagal afferent fibers can activate the NANC
parasympathetic ganglion neurons that innervate the trachealis, thus
eliciting, via noncholinergic synaptic neurotransmission, NANC relaxations of the trachealis (Canning and Undem, 1994). On the
basis of the effects of receptor-selective agonists and antagonists
available at the time of those studies, we suggested that these
responses of the NANC parasympathetic ganglion neurons were mediated by
tachykinins acting on both NK3 and NK1
receptors. Taking advantage of the newly developed NK1 and
NK3 receptor-selective antagonists SR 140333 (Edmonds-Alt
et al., 1993) and SR 142801 (Emonds-Alt et al.,
1995), respectively, we undertook the present study to examine this
hypothesis further.
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Materials and Methods |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Functional studies.
Guinea pigs were asphyxiated in a vessel
filled with 100% CO2 and exsanguinated. The trachea,
esophagus and associated nerves were removed in toto and
pinned dorsal side down to the bottom of a water-jacketed dissecting
dish continuously overfilled (20 ml/min) with warmed (37°C),
oxygenated Krebs' buffer of the following composition (mM): NaCl
(118), KCl (5.4), Mg2SO4 (1.2),
NaH2PO4 (1), CaCl2 (1.9),
NaHCO3 (25) and dextrose (11.1). The trachea, esophagus and
associated nerves were dissected free from extraneous tissues and
prepared for measurement of isometric tension of the trachealis as
previously described (Canning and Undem, 1993a). Two adjacent cartilage
rings (rings 6 and 7 caudal to the larynx) were cut open opposite the
trachealis. One end of the opened rings was sutured to a pin in the
bottom of the dissecting dish, and the other end was sutured to a force
transducer (Grass FT03; Grass Instruments, Cambridge, MA) connected to
a Grass polygraph (Model 7D) for measurement of isometric tension.
Optimal base-line tone (1.5 g) was set and was continually adjusted
during the ensuing 90-min equilibration period.
). These distinct vagal pathways
were stimulated as previously described (Canning and Undem, 1993a,
1993b, 1994). The vagus nerves were cut caudal to the nodose ganglia,
and suction electrodes were placed on the cut ends of the vagus nerves
rostral (stimulating only those fibers that project axons to the
superior laryngeal nerves) and caudal (stimulating only those fibers
that project axons to the recurrent laryngeal nerves) to the nodose
ganglia. The suction electrodes were connected to a Grass stimulator
(Model S44) that delivered square pulses of optimum stimulus
intensities (150 V, pulse duration 1 msec). The nerves were stimulated
bilaterally for 10 sec at frequencies between 4 and 24 Hz.
Relaxations were elicited after addition of atropine and contraction of
the trachealis with 5 µM histamine. The magnitude of the vagally
mediated relaxations were expressed as a percentage of the maximum
relaxant response elicited by 0.1 mM papaverine administered at the end
of each experiment.
The effects of SR 142801, SR 140333 and CP 99994 on vagally mediated
relaxations of the trachealis were assessed in nonpaired experiments
(this was necessary because of the long equilibration period required
for SR 142801; see Emonds-Alt et al., 1995). Preparations were pretreated with vehicle, SR 142801 or both SR 142801 and either SR
140333 or CP 99994 (0.3 µM each) at the outset of each experiment,
and the relaxations elicited in tissues treated with vehicle were
compared with those elicited in tissues treated with the antagonists.
The effects of SR 142801 on capsaicin-induced, esophagus-dependent
relaxations of the trachealis were also assessed. Tissues were prepared
as described above (after removal of the extrinsic nerves; see Canning
and Undem, 1994) and suspended in water-jacketed organ baths filled
with warmed (37°C), oxygenated Krebs' buffer. One end of the
preparation was sutured to the bottom of the organ bath, and the other
end was sutured to an isometric force transducer connected to a Grass
polygraph. Base-line tone was set at 1.5 g, and the buffer bathing
the tissues was changed at 15-min intervals during the 90-min
equilibration. After equilibration, the tissues were contracted with 5 µM histamine. When the contraction to histamine had stabilized, the
NK3-selective agonist senktide analog (0.3 µM) was added
to assess the integrity of the neural projections from the esophagus to
the trachea (senktide analog elicits relaxations of the trachealis only
when the adjacent esophagus, and consequently the NANC relaxant nerve
pathways to the trachealis, are intact; Canning and Undem, 1994).
Preparations that did not relax to senktide analog (<5%) were not
studied further.
When the response to senktide analog reached maximum, the tissues were
washed repeatedly for 45 min and then treated with vehicle (DMSO), TTX
(1 µM) or 0.3 µM SR 142801 for 2 hr. At the end of the drug
equilibration period, the trachealis was recontracted with 5 µM
histamine, and cumulative concentration-response curves to capsaicin
(0.01 µM-10 µM) were constructed. Each successive dose of
capsaicin was added when the response to the previous dose had peaked.
The magnitude of these relaxant responses was expressed as a percentage
of the contraction elicited by histamine.
The effects of tachykinin receptor antagonists on senktide analog- and
substance P-induced relaxations of the trachealis were also assessed.
Experiments were carried out as described above, except that rather
than adding capsaicin after antagonist administration and recontraction
of the trachealis with histamine, we added 0.3 µM senktide analog or
1 µM substance P. The responses elicited were expressed as a
percentage of the relaxant response elicited by senktide analog at the
outset of the experiment.
Previous studies indicated that NK2 receptors are not
involved in the relaxations elicited by stimulation of the
capsaicin-sensitive nerves or by exogenously administered tachykinins
(Canning and Undem, 1994). Accordingly, to eliminate any possible
functional antagonism mediated by activation of the NK2
receptors on the smooth muscle (Ellis and Undem, 1994), unless
otherwise stated we carried out all of our functional experiments in
the presence of the NK2 receptor antagonist SR 48968 (0.1 µM).
To test for selectivity of the receptor antagonists, we assessed their
effects on contractions elicited by capsaicin (predominantly an
NK2 receptor-mediated response; Ellis and Undem, 1994), the NK2-selective agonist
Ala8NKA4-10 and/or the
NK1-selective agonist ASMSP. Antagonists were added at
least 2 hr before construction of the concentration-response curves. If
significant shifts in the curves were generated, a pKb for the
antagonists was calculated as previously described (Renzetti et
al., 1992).
To test for effects of the antagonists on NANC relaxations independent
of tachykinin receptor antagonism (i.e., pre- or
postjunctional effects), EFS-induced NANC relaxations were elicited in
the absence and presence of the antagonists. Tracheal strips were
suspended in an organ bath as described above. After a 2-hr
pretreatment with the tachykinin receptor antagonists or vehicle, the
trachealis was contracted with 5 µM histamine. When the contraction
to histamine had stabilized, EFS was delivered through bipolar platinum
ring electrodes placed on either side of the tracheal strips. The
electrodes were connected to a Grass stimulator (Model S44) connected
in series to a Med-Lab Stimusplitter (Med-Lab Instruments, Fort
Collins, CO) that amplified and measured the stimulus intensity.
Tissues were stimulated with optimum stimulus intensities (12 V, 1 msec, 200-400 mA) at frequencies of 0.1 to 32 Hz. Frequency-response curves were constructed in a cumulative manner, the frequency being
increased when the response to the ongoing stimulus had stabilized. The
magnitude of relaxations elicited by EFS were expressed as a percentage
of the maximum relaxation elicited by 0.1 mM papaverine (added at the
end of each experiment).
Pretreatments.
Unless otherwise stated, atropine (1 µM),
propranolol (1 µM) and indomethacin (3 µM) were added to the buffer
to limit the influence of cholinergic and adrenergic nerves and the
effects of neuromodulatory prostanoids, respectively (Canning and
Undem, 1993a, 1994). Also, unless otherwise stated, 0.1 µM SR 48968 was added to block tachykinin-mediated contractions of the trachealis via NK2 receptor activation.
Immunohistochemistry.
Tissue preparation for the
immunohistochemical analyses was carried out as previously described
(Kummer et al., 1992). Guinea pigs were sacrificed by
inhalation of CO2 and exsanguinated. The esophagus (the
location of the cell bodies of the neurons that mediate NANC
relaxations of the trachealis; Canning and Undem, 1993b; Canning
et al., 1996) was removed in toto, fixed
overnight in 4% paraformaldehyde in 0.1% phosphate buffer, washed in
0.1% PBS and stored until freezing in 0.1% phosphate buffer
containing 18% sucrose for cryoprotection. The esophagus was then
frozen rostral end up in quickmount on filter paper, and serial
transverse sections (12 µm) were made on a cryostat and placed on
chromalum-coated slides.
; Fischer et al., 1993).
Sections were incubated for 1 hr in blocking solution consisting of 1% bovine serum albumin, 10% normal swine serum and Tween 20 (to enhance
tissue penetration of the antisera) in PBS. After several washes in
PBS, the slides were coated with buffer containing monoclonal mouse
antibodies to substance P (1:800; kindly provided by Dr. J.-Y. Couraud,
Gif-sur-Yvette, France) and polyclonal rabbit antibodies to either the
neuronal isoform of NO synthase (1:1000; kindly provided by B. Mayer,
Graz, Austria) or VIP (1:1000; Immuno Nuclear, Stillwater, MN) for 16 hr. The tissues were then washed twice and incubated for 1 hr with the
secondary antisera to the anti-substance P (biotinylated sheep
anti-mouse; Amersham, Buchler, Braunschweig FRG) followed by a 1-hr
incubation in the secondary antisera to VIP and NOS (FITC-labeled goat
anti-rabbit; Organon Teknika, Eppelheim, FRG) and strepavidin Texas Red
(Amersham). The slides were then washed and coverslipped in
carbonate-buffered glycerol (pH 8.6). FITC- and Texas Red-labeled nerve
fibers and nerve cell bodies were visualized and photographed as
previously described (Kummer et al., 1992).
Presentation of data and statistical analysis.
Results are
presented as a mean ± (S.E.M.) for n experiments,
where n is the number of animals studied. Relaxations
elicited by capsaicin are expressed as a percentage of the contraction elicited by histamine. Contractions elicited by capsaicin under conditions that normally favor relaxations (trachea isolated with the
adjacent esophagus intact, precontracted with histamine) were recorded
as no response or 0% of the histamine-induced contraction. Relaxations
elicited by vagus nerve stimulation or EFS are expressed as a
percentage of the maximum relaxation elicited by 0.1 mM papaverine. Relaxations elicited by senktide analog or substance P are expressed as
a percentage of the pretreatment (control) response to senktide analog.
Contractions elicited by Ala8NKA4-10 and
ASMSP are expressed as a percentage of the maximum contraction elicited
by the agonists. EC50 values for ASMSP and Ala8NKA4-10 were determined by visual
inspection of the dose-response curves. For the antagonists studies,
pKb values were calculated as previously described (Renzetti et
al., 1992).
Drugs.
Atropine sulfate, capsaicin, indomethacin,
DL-propranolol, papaverine hydrochloride, histamine
diphosphate and (TTX) were bought from Sigma (St. Louis, MO). CP 99994 was a generous gift from Merck Frosst (Quebec, Canada). SR 142801 and
SR 142806 were gifts from SmithKline Beecham (King of Prussia, PA).
Substance P, Ala8NKA4-10, ASMSP, senktide
analog, SR 140333 and SR 48968 were gifts from Zeneca, Inc.
(Wilmington, DE). Atropine (10 mM), propranolol (10 mM), papaverine (50 mM), substance P (1 mM), senktide analog (1 mM), TTX (1 mM) and
histamine (20 mM) were dissolved in water. ASMSP (1 mM),
Ala8NKA4-10 (1 mM), CP 99994 (1 mM), SR
142801 (1 mM), SR 142806 (1 mM) and SR 48968 (10 mM) were dissolved in
DMSO. Indomethacin (30 mM) and capsaicin were dissolved in ethanol.
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Results |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Immunohistochemistry.
Neuronal cell bodies possessing
immunoreactivity to VIP and NOS were found throughout the myenteric
plexus of the guinea pig esophagus (fig.
1). This plexus is located between the
outer longitudinal and inner circular striated muscle layers of the
esophagus and is the probable location of the neurons that mediate NANC
relaxations of the trachealis (Canning and Undem, 1993b; Canning
et al., 1996). Double-labeling studies revealed that neurons
within the myenteric plexus expressing VIP and NOS-IR were enveloped by
substance P-containing nerve fibers (fig. 1). Substance
P-immunoreactivity was not seen in any perikarya within the esophageal
myenteric plexus.
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Effect of tachykinin receptor antagonists on vagally mediated relaxations. Stimulation (24 Hz, 10 sec) of the rostral (capsaicin-sensitive) and caudal (parasympathetic) vagal pathways that innervate the guinea pig trachea elicited relaxations of the precontracted (5 µM histamine) trachealis that averaged 18 ± 8% and 35 ± 4%, respectively, of the maximum relaxation elicited by 0.1 mM papaverine (n = 8). The NK3-selective antagonist SR 142801 (0.3 µM) markedly inhibited relaxations of the guinea pig trachea elicited by stimulation of the capsaicin-sensitive vagal pathways. Addition of both SR 142801 and either of the NK1-selective antagonists SR 140333 (0.3 µM) and CP 99994 (0.3 µM) virtually abolished relaxations elicited by stimulation of the rostral (capsaicin-sensitive) vagal pathways (fig. 2A). The combination of SR 140333 and SR 142801 also abolished capsaicin-induced (0.1-10 µM) relaxations in this preparation (n = 4).
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Effect of tachykinin receptor antagonists on capsaicin-induced
relaxations.
In results consistent with our previous studies
(Canning and Undem, 1994), capsaicin elicited concentration-dependent
relaxations of the guinea pig trachea isolated with the adjacent
esophagus intact that were abolished by 1 µM TTX but were unaffected
by NK2 receptor antagonism with 0.1 µM SR 48968 (fig.
3A). The NK3-selective receptor antagonist SR 142801 (0.3 µM) markedly inhibited
capsaicin-induced relaxations of the trachealis (fig. 3B). Indeed, 4 of
11 preparations failed to relax to capsaicin in the presence of SR
142801 (and SR 48968). The less active isomer of SR 142801, SR 142806 (0.3 µM), was ineffective at inhibiting capsaicin-induced relaxations of the trachealis (fig. 3C).
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Effects of tachykinin receptor antagonists on tachykinin-mediated responses. Substance P (1 µM) elicited esophagus-dependent, TTX-sensitive relaxations of the precontracted guinea pig trachea (fig. 4). The NK1-selective receptor antagonists CP 99994 and SR 140333 and the NK3-selective receptor antagonist SR 142801 (each at 0.3 µM) inhibited relaxations of the trachealis elicited by substance P. Addition of both SR 140333 and SR 142801 abolished substance P-induced relaxations (fig. 4).
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Ala8NKA4-10)
receptors and by examining their effects on capsaicin-induced contractions of the trachealis (a predominantly NK2
receptor-mediated response; Ellis and Undem, 1994). SR 142801 (0.3 µM) was without effect on Ala8NKA4-10-
and ASMSP-mediated contractions of the guinea pig trachea [pA2 < 6.5 at both NK2 (n = 2) and NK1
receptors (n = 3)] and was without effect on
contractions of the guinea pig trachea elicited by capsaicin (1 µM
capsaicin elicited contractions of the guinea pig trachea that averaged
71 ± 6% and 62 ± 6% in the absence and presence of 0.3 µM SR 142801, respectively; n = 4, P > .1). By
contrast, SR 140333 and CP 99994 (each studied at 0.3 µM) caused
marked leftward shifts in the concentration-response curve for ASMSP
(estimated pKb values of 8.8 ± 0.4 and 9.1 ± 0.1, respectively; n = 3) but were without marked effect on
capsaicin-induced contractions of the trachealis (1 µM capsaicin
elicited contractions that averaged 72 ± 5%, 71 ± 4% and
45 ± 8% in the presence of vehicle, 0.3 µM SR 140333 and 0.3 µM CP 99994, respectively; n = 3).
The effects of the tachykinin receptor antagonists on relaxations of
the guinea pig trachea elicited by the NK3-selective agonist senktide analog are illustrated in figure
5. Senktide analog (0.3 µM) elicited
esophagus-dependent, TTX-sensitive relaxations of the trachealis that
were virtually abolished by 0.3 µM SR 142801 but were not affected by
0.3 µM SR 142806, SR 140333 or 0.3 µM CP 99994. In results
consistent with previous studies (Canning and Undem, 1994), senktide
analog-induced relaxations were also unaffected by a selective
concentration of the NK2 receptor antagonist SR 48968. Senktide analog elicited relaxations that averaged 38 ± 4% and
36 ± 3% of 5 µM histamine-induced contractions in the absence
(n = 31) and presence (n = 61) of 0.1 µM SR 48968, respectively (P > .1).
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Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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In the present study, we provide anatomical and pharmacological evidence for tachykinin-mediated activation of neurons that mediate NANC relaxations of guinea pig trachealis. More specifically, we demonstrate the presence of a dense network of tachykinin-containing nerve fibers innervating the neurons that are thought to mediate these relaxations and show that either electrical or chemical stimulation of these nerve endings elicits relaxations of the guinea pig trachealis that are inhibited by tachykinin receptor antagonists. Finally, using antagonists selective for each of the identified tachykinin receptor subtypes, we present evidence that relaxations of the guinea pig trachealis elicited by peripheral activation of the capsaicin-sensitive vagal afferent nerves involve activation of both NK1 and NK3 receptors.
Interactions between capsaicin-sensitive afferents and NANC
relaxant nerves.
NANC relaxations of the guinea pig trachea are
thought to be mediated by VIP and/or NO coreleased from postganglionic
parasympathetic nerves (Tucker et al., 1990; Li and Rand,
1991; Canning and Undem, 1993a, Canning et al., 1996). Using
an isolated, innervated guinea pig tracheal preparation, we had
previously described two vagal pathways that mediate NANC relaxations
of the guinea pig trachea: a parasympathetic pathway with preganglionic
fibers carried by the recurrent laryngeal nerves and a
capsaicin-sensitive pathway made up of afferent fibers carried by the
superior laryngeal nerves (Canning and Undem, 1993a). Surprisingly,
both of the vagal pathways that mediate relaxations of the guinea pig
trachea were disrupted when the trachea was separated from the adjacent
esophagus. On the basis of the results of experiments in which
ganglionic blockers were administered selectively to the trachea and
subsequent experiments with organotypic cultures, pharmacological
analyses and immunohistochemistry, we concluded that NANC relaxations
of the guinea pig trachea are mediated by nerve fibers emanating from
parasympathetic ganglia that are intrinsic to the adjacent esophagus
(Canning and Undem, 1993b; Canning et al., 1996).
; Ellis
and Undem, 1994). On the basis of our studies of the parasympathetic
pathways that mediate relaxations of the guinea pig trachea, we
reasoned that the capsaicin-sensitive nerve fibers may act to elicit
relaxations through intermediate neurons (e.g.,
parasympathetic ganglion neurons) that are intrinsic to the esophagus.
Subsequent studies demonstrating that both capsaicin and exogenously
administered tachykinins can elicit relaxations of the trachealis that
are TTX-sensitive and esophagus-dependent provided compelling support
for this hypothesis (Canning and Undem, 1994).
In the present study, we have demonstrated that the cell bodies of
neurons with VIP-IR and/or NOS-IR phenotypes in the myenteric plexus of
the esophagus are enveloped by substance P-containing nerve fibers.
Indeed, a striking observation is the density of substance P-IR nerve
fibers innervating all the neurons of the myenteric plexus
of the esophagus (B. J. Canning, A. Fischer and B. J. Undem,
unpublished observations). This suggests an important role for
tachykinins in regulating neurons localized to the esophagus and
provides an anatomical basis for an interaction between neurons that
mediate NANC relaxations of the trachealis and capsaicin-sensitive visceral afferent fibers that innervate the ganglia in which they are
contained.
Role of tachykinins in mediating relaxations of guinea pig tracheal
smooth muscle.
Previous studies indicated that relaxations of the
guinea pig trachea elicited by capsaicin or antidromic stimulation of
the capsaicin-sensitive vagal afferent nerves are mediated by
tachykinins (Canning and Undem, 1994). At the time of our previous
study, however, potent and selective NK3 receptor
antagonists to carry out the necessary pharmacological analyses were
not available. Nevertheless, on the basis of the effects of
NK3-selective agonists and the inhibitory effects of a
nonselective NK3 receptor antagonist (SR 48968), we
proposed that the relaxations elicited by stimulation of the
capsaicin-sensitive nerves were mediated at least in part by
NK3 receptors.
). The involvement of NK1 receptors in this
response is further supported by the observation in the present study
that the NK1 receptor-selective antagonists SR 140333 and
CP 99994 inhibited both vagally mediated and substance P-induced
relaxations of the trachealis.
The present study also provides further evidence for NK2
receptor-independent mechanisms of relaxations of the guinea pig trachealis elicited by stimulation of capsaicin-sensitive vagal afferent nerves: capsaicin, substance P and senktide analog elicit TTX-sensitive relaxations of the trachealis in the presence of the
NK2-selective antagonist SR 48968.
Having found that adding either CP 99994 or a concentration of SR 48968 that blocks NK3 receptors (in addition to NK2
receptors) could in many instances abolish relaxations elicited by
stimulation of the capsaicin-sensitive nerves, we speculated that
relaxations mediated by the endogenously released tachykinins might
require activation of both receptor subtypes on the
noncholinergic parasympathetic ganglion neurons (Canning and Undem,
1994). The results of the present study are, however, inconsistent with
the hypothesis that the relaxations are mediated by such a synergistic
interaction between NK1 and NK3 receptors.
Blocking NK2 receptors leaves the NK3 receptor
antagonist SR 142801, when administered alone, unable to abolish
completely the relaxations elicited by stimulation of the
capsaicin-sensitive nerves or by substance P. This may explain in part
the observation that the effects of only NK1 or only
NK3 receptor blockade were not consistent, causing
inhibition that varied from no effect whatsoever to complete
abolishment of the responses to capsaicin, substance P or nerve
stimulation. It seems likely that factors such as the local
concentrations of the tachykinins (each with its own relative potency
at NK1 and NK3 receptors), their access to both
receptor subtypes and the relative level of expression of these
subtypes within the ganglia of any given preparation all contribute to
the observed variability and the relative inhibitory effects of the
antagonists. In any event, it is clear that tachykinins can elicit
activation of the parasympathetic ganglion neurons by activating either
NK1 or NK3 receptors.
Contribution of other cells and other mechanisms to
capsaicin-mediated relaxations.
Although the results of the
present study argue strongly for the involvement of tachykinin
receptors on the NANC inhibitory neurons in the relaxations elicited by
stimulation of the capsaicin-sensitive nerves, the possibility that
other cells and mechanisms are involved cannot be discounted. Thus
stimulation of the capsaicin-sensitive nerves may induce relaxations by
activating, via tachykinins or other transmitters found in
the capsaicin-sensitive nerve endings, non-neural cells associated with
the airways that might subsequently release a substance that relaxes
the guinea pig trachealis (Figini et al., 1996).
Alternatively, capsaicin may also act directly on the smooth muscle
(Ellis et al., 1996) or induce the release of a relaxant
neurotransmitter that acts directly on the smooth muscle (Bhogal
et al., 1994; Pinto et al., 1996).
Physiological implications.
The interaction described here
between tachykinin-containing visceral afferent nerves and the nerves
that mediate NANC relaxations of the guinea pig trachealis is one of
several reported instances where autonomic nerve function is regulated
peripherally by visceral afferent fibers. Similar interactions have
been reported in parasympathetic ganglia that innervate the bladder and
gallbladder (Mawe, 1995; de Groat and Booth, 1993) and in the
parasympathetic, sympathetic and myenteric ganglia that innervate the
GI tract (Kreulen and Peters, 1986; Szurzewski and King, 1989). In the
airways, both direct (Myers and Undem, 1993; Myers et al.,
1996) and indirect evidence for such interactions has been reported for
the nerves that regulate airway smooth muscle tone (Martling et
al., 1984; Myers and Undem, 1991; Watson et al., 1993;
Canning and Undem, 1994). Interestingly, in the ferret trachea,
substance P-containing nerve fibers preferentially innervate ganglia
that contain VIP-IR and NOS-IR neurons, whereas the cholinergic neurons
found in the longitudinal nerve trunks of the ferret trachea are
sparsely innervated by substance P-containing nerve fibers (Dey
et al., 1996). In the human airway, VIP-IR and NOS-IR
neurons are also innervated by substance P-containing nerves (Fischer
and Hoffman, 1996). The prevalence of these interactions in autonomic
ganglia suggests that peripheral reflexes may be an important aspect of
autonomic regulation.
;
Mansfield et al., 1981). Finally, given the recent focus on
tachykinin receptor antagonists as potential therapeutic agents
(Canning, 1997), the data presented in this study provide further
evidence that NK3 receptor antagonists might be useful in
the treatment of disorders that involve alterations in autonomic nerve
activity.
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Acknowledgments |
|---|
The authors thank Ms. Sandra Reynolds and Mr. Martin Bodenbenner for expert technical assistance.
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Footnotes |
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Accepted for publication September 19, 1997.
Received for publication February 27, 1997.
1 This research was made possible by grants and a cooperative agreement between the National Institutes of Health (Bethesda, MD) and the German Health Ministry (Bonn, Germany).
Send reprint requests to: Brendan J. Canning, Ph.D., The Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224.
| |
Abbreviations |
|---|
NKA, neurokinin A; NKB, neurokinin B; NK1 (2 or 3), neurokinin1 (2 or 3); EFS, electrical field stimulation; NANC, nonadrenergic, noncholinergic; VIP, vasoactive intestinal polypeptide; NOS, nitric oxide synthase; IR, immunoreactive; TTX, tetrodotoxin; PBS, phosphate-buffered saline; CP 99994, (+), (2S, 3S)-3-(2-methoxybenzyl-amino)-2-phenyl-piperidine); SR 142801, (S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)propyl)-4-phenylpiperidin-4-yl)-N-methylacetamide) ; SR 142806, (R)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl) piperidin-3-yl)propyl)-4-phenylpiperidin-4-yl)-N-methylacetamide ; ASMSP, Ac-[Arg6, Sar9, Met (O2)11]-SP6-11; senktide analog, [Asp6, Asp7, MePhe8]-SP6-11); SR 140333, (S)1-[2-[3-(3,4-dichlorophenyl)-1-(3-isoproproxy phenylacetyl)piperidin-3-yl]ethyl]-4-phenyl-1-azoniabicyclo[2.2.2.]octane chloride; SR 48968 (S)-N-methyl-N[4-(4-acetylamino-4-phenyl piperidino)-2-(3, 4-dichlorophenyl)butyl]benzamide; DMSO, dimethyl sulfoxide; FITC, Fluorescein-isothiocyanate.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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, n = 8) and in the presence (
) of 1 µM tetrodotoxin (n = 5; panel A), the NK3 receptor antagonist SR 142801 (0.3 µM, n = 10; panel B) and the less active isomer of SR
142801, SR 142806 (0.3 µM, n = 3; panel C). An
asterisk (*) denotes a statistically significant decrease in the
magnitude of capsaicin-induced relaxations compared with vehicle
control (P < .05).
