Cellular pathways mediating tachykinin-evoked secretomotor responses in guinea pig ileum

W. MacNaughton1, B. Moore2, and S. Vanner2

1 Gastrointestinal Research Group, University of Calgary, Calgary, Alberta T2N 4N1; and 2 Gastrointestinal Diseases Research Unit, Queen's University, Kingston, Ontario, Canada K7L 5G2

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

This study characterized tachykinin-evoked secretomotor responses in in vitro submucosal and mucosal-submucosal preparations of the guinea pig ileum using combined intracellular and Ussing chamber recording techniques. Superfusion of endogenous tachykinins substance P (SP), neurokinin A (NKA), and neurokinin B depolarized single submucosal neurons and evoked increased short-circuit current (Isc) responses in Ussing chamber preparations. The NK1-receptor agonist [Sar9,Met(O2)11]SP [50% effective concentration (EC50) = 2 nM] depolarized all submucosal neurons examined. The NK3-receptor agonist senktide (EC50 = 20 nM) depolarized ~50% of neurons examined, whereas the NK2-receptor agonist [Ala5,beta -Ala8]NKA-(4---10) had no effect on membrane potential. [Sar9,Met(O2)11]SP and senktide evoked similar increases in Isc that were tetrodotoxin sensitive (91 and 100%, respectively) and were selectively blocked by the NK1 antagonist CP-99,994 and the NK3 antagonist SR-142801, respectively. Capsaicin-evoked increases in Isc were significantly inhibited (54%, P < 0.05) by CP-99,994 but not by SR-142801. Neither antagonist inhibited slow excitatory postsynaptic potentials. These findings suggest that tachykinin-evoked secretion in guinea pig ileum is mediated by NK1 and NK3 receptors on submucosal secretomotor neurons and that capsaicin-sensitive nerves release tachykinin(s) that activate the NK1 receptors.

substance P; ion transport; submucosal neurons; neurokinins; secretion

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

TACHYKININS ARE POTENT secretagogues in the gastrointestinal tract (4, 10, 11, 13, 18, 19). In the small intestine, several studies have shown that tachykinins stimulate chloride secretion from mucosal enterocytes, which results in the passive movement of water into the intestinal lumen (10, 13, 18, 19). This tachykinin-evoked secretion appears to result almost entirely from activation of neurokinin receptors located on submucosal secretomotor neurons (13, 19). These neurons in turn innervate mucosal enterocytes through the release of both cholinergic and noncholinergic neurotransmitters (4, 19). These studies have established that tachykinins are important modulators of this neurosecretory pathway, but the nature of the tachykinins and receptors involved remains unclear.

The tachykinins substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) are a complex family of endogenous peptides that share a common COOH-terminal amino acid sequence (11, 17, 20). These tachykinins are active at three cloned and functionally characterized tachykinin receptors, termed NK1, NK2, and NK3 (20). SP is most potent at NK1, NKA at NK2, and NKB at NK3 receptors. Within the enteric nervous system both NK1 and NK3 receptors have been identified (1, 11, 15, 19, 21, 26). These receptor subtypes are not uniformly distributed, however, and appear to be confined to specific organs and subsets of neurons within enteric ganglia (1, 15, 19, 21). In the submucosal plexus, the study of tachykinin-evoked secretion has focused predominantly on the actions of SP (4, 10, 13, 19, 23), but a systematic study of the tachykinin family and their receptors has not been conducted. It has been suggested that the NK1 receptor mediates intestinal ion transport because the actions of SP were partially blocked by the NK1-receptor antagonist CP-96,345 (19). Interpretation of these findings, however, has since proven problematic because micromolar concentrations of this antagonist are now recognized to result in nonselective inhibition, including blockade of NK3 receptors (11, 25). Furthermore, in other studies, activation of submucosal secretomotor neurons by SP was not inhibited by the SP analog [D-Arg1,D-Pro2,D-Trp7,9,Leu1]SP (10). However, peptide analog antagonists have also been proven to lack specificity and potency (10, 11, 20, 28, 31). Consequently, although SP has been shown to evoke important secretomotor responses in the small intestine, further study is needed to clarify the role of other endogenous tachykinins and the receptor subtype(s) that mediate tachykinin-evoked secretion.

The recent development of more selective tachykinin agonists and antagonists has enabled a more detailed characterization of tachykinin-evoked responses (20, 31). These pharmacological tools also provide an opportunity to examine cellular sources of endogenous tachykinins. Within the intestine, neurons are the principal source of tachykinins (11). Immunohistochemical studies employing antibodies directed against SP or a related tachykinin have demonstrated the presence of immunoreactivity in both intrinsic enteric and extrinsic sensory nerves (5, 7). SP-immunoreactive enteric neurons have been localized within the myenteric plexus with axonal projections to submucosal neurons in the submucosal plexus (5). A role for SP as a putative neurotransmitter has been further supported by electrophysiological studies in the submucosal plexus that have shown that electrical stimulation of axonal projections in fiber tracts, which include projections from the myenteric plexus, evokes a slow excitatory postsynaptic potential (EPSP), and the exogenous application of SP can mimic the slow EPSP (23, 24). SP immunoreactivity is also found in nerve terminals of extrinsic sensory nerves projecting to the submucosal plexus (7). Recent studies have shown that these fibers are capsaicin sensitive (8, 27) and can be selectively activated by this neurochemical probe. Capsaicin-evoked stimulation of these nerves results in local release of neurotransmitter(s) from nerve terminals within the submucosal plexus, which in turn activates submucosal secretomotor neurons (27). These fibers have been shown to release SP or a related tachykinin in some neuronal circuits, but in others calcitonin gene-related peptide (CGRP) appears to be the principal neurotransmitter (8). Together, these data imply that both intrinsic and extrinsic nerves may activate this submucosal secretomotor pathway, but direct evidence remains to be shown.

The aim of this study was to first systematically characterize the actions of endogenous tachykinins that evoke intestinal ion secretion and to establish the neurokinin receptor subtypes that mediate these actions. Subsequent studies examined potential sources of endogenous tachykinin(s) that activate the defined neurokinin receptors in this secretomotor reflex, using selective neurokinin antagonists. A combined approach of intracellular recording and Ussing chamber studies was used to enable cellular properties to be studied in a functionally defined secretomotor pathway.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Experiments were conducted on in vitro submucosal and mucosal-submucosal preparations from the guinea pig ileum using intracellular and Ussing chamber recording techniques. Approval of the Animal Care Committees of Queen's University and the University of Ottawa was obtained.

Intracellular Recording

Guinea pigs (125-175 g) were rendered immediately unconscious by a blow to the head and were killed by decapitation. In vitro submucosal preparations were dissected from the ileum, as previously described (22, 26). Preparations were continuously perfused in a 0.5-ml organ bath with a physiological saline solution containing (in mM) 126 NaCl, 1.2 NaH2PO4, 1.2 MgCl2, 2.5 CaCl2, 5 KCl, 25 NaHCO3, and 11 glucose gassed with 95% O2-5% CO2 and maintained at 35-36°C. All drugs were added to the bath by superfusion.

Recordings were obtained from submucosal neurons with intracellular electrodes filled with 2 M KCl (60-120 MOmega ), as previously described (22). Membrane potential was measured with an Axoclamp 2A amplifier (Axon Instruments, Foster City, CA). Fiber tracts were stimulated with small bipolar tungsten electrodes (20 Hz, pulse duration 0.7 ms, train duration 400-500 ms).

Ussing Chamber Recordings

Guinea pigs (200-250 g) were killed by lethal injection of pentobarbital sodium, and in vitro mucosal-submucosal preparations were dissected from the ileum as previously described (27). Preparations were mounted in standard Ussing chambers and bathed with a physiological saline solution containing (in mM) 115 NaCl, 2 KH2PO4, 2.4 MgCl2, 1.3 CaCl2, 8 KCl, and 25 NaHCO3. The mucosal buffer contained 10 mM mannitol and the serosal buffer contained 10 mM glucose. Solutions were gassed with 95% O2-5% CO2 and maintained at 37°C. All drugs were added to the serosal side of the tissue after a 20-min equilibration period.

Tissue responses were recorded by clamping the potential difference across the tissue to 0 mV by applying a short-circuit current (Isc) with a voltage-clamp apparatus (DCV-1000, World Precision Instruments, Sarasota, FL). Isc was recorded as an indicator of net active electrolyte transport across the tissue. Electrical field stimulation (EFS; 100 V, pulse duration 500 µs, 25 Hz, 3 s) was delivered with a dual-impedance stimulator (Harvard Instruments). Baseline Isc was reestablished between different applications of EFS or drugs.

Drugs

The following drugs were used: SP, NKA, NKB, [Sar9,Met(O2)11]SP, [Ala5,beta -Ala8]NKA-(4---10), and senktide (from Bachem); CP-99,994 and CP-96,345 (gifts from Pfizer); SR-142801 (a gift from Sanofi Recherche); tetrodotoxin (TTX), capsaicin, 5-hydroxytryptamine (5-HT), atropine, and histamine (from Sigma); thioperamide and LY-53857 (Research Biochemicals); BRL-43694 (a gift from SmithKline Beecham); and SDZ-205-557 (a gift from Sandoz). Capsaicin was dissolved in Tween-80, alcohol, and saline (10:10:80, vol/vol/vol). SR-142801 was dissolved in a 100% ethanol 10 mM stock solution. [Ala5,beta -Ala8]NKA-(4---10) was dissolved in 10% acetic acid stock solution, and NKA was dissolved in 2.5% acetic acid in Ussing chamber studies. All other peptides were dissolved in distilled water. Vehicle effects were negligible and any minimal effects on Isc were subtracted from agonist-evoked changes in Isc in Ussing chamber studies.

Statistics

Data are expressed as means ± SE. Comparisons between matched pairs of tissues in Ussing chamber studies were made using Student's t-test for paired data and repeated-measures analysis of variance where appropriate. An associated P value of <0.05 was considered to be significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Tachykinin-Evoked Depolarizations of Submucosal Neurons

Intracellular recordings were obtained from submucosal neurons (n = 75) that had resting membrane potentials ranging from -48 to -65 mV. Fiber tract stimulation elicited slow excitatory postsynaptic potentials (EPSPs; >5 mV) in all neurons. Stable impalements were typically obtained for >1 h, enabling multiple agonists to be examined on a single neuron. Agonists were applied in a noncumulative fashion, and 10-min washouts were allowed between applications to prevent desensitization.

Effects of SP, NKA, and NKB on submucosal neurons. Superfusion of SP [1-100 nM; 50% effective concentration (EC50) = 6 nM] depolarized all neurons (Fig. 1; n = 4) and evoked action potential discharge, as previously described (16, 22, 24). NKA (30-300 nM; EC50 = 80 nM) and NKB (30-300 nM; EC50 = 90 nM) superfusion also depolarized submucosal neurons and activated action potential discharge (Fig. 1; n = 4).


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Fig. 1.   Endogenous tachykinins substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) depolarize submucosal neurons. A: representative intracellular recordings from single submucosal neurons comparing responses to superfusion of SP, NKA, and NKB. Resting membrane potential (Vm) of each cell was (left to right) -63, -63, and -59 mV, respectively. B: summary of concentration-response for SP, NKA, and NKB. Values are means ± SE for >= 4 cells.

Effects of selective tachykinin agonists on submucosal neurons. All neurons examined (n = 35) were depolarized by superfusion of the NK1 agonist [Sar9,Met(O2)11]SP (Fig. 2). Noncumulative applications (n = 4) evoked concentration-dependent depolarizations (1-50 nM; EC50 = 2 nM). Depolarizations were associated with an increase in membrane resistance (mean = 50%, n = 7) and typically evoked action potential discharge (Fig. 2) with bursts of fast EPSPs. The NK3 agonist senktide (3-100 nM; EC50 = 20 nM) depolarized 25 of 55 cells examined (Fig. 2). Senktide (30 nM) increased membrane resistance (mean = 47%, n = 3). Maximal effective concentrations (EC100) (30-100 nM) evoked mean maximal depolarization of 8 mV with a range of 5-16 mV. The effects of both senktide and the NK1 agonist [Sar9,Met(O2)11]SP were examined in 21 cells. In 11 cells, the NK1 agonist (30 nM) depolarized the neuron (mean = 12 mV), but senktide (30-100 nM) had no effect on membrane potential. The NK2 agonist [Ala5,beta -Ala8]NKA-(4---10) (100-300 nM; n = 4) had no effect on membrane potential. The effects of both senktide (100 nM) and [Sar9,Met(O2)11]SP (30 nM) were also examined in the presence and absence of TTX to determine whether the depolarizations resulted solely from activation of receptors on the postsynaptic membrane. TTX (1 µM) blocked all synaptic potentials evoked by electrical stimulation of fiber tracts (n = 8). Compared with controls, TTX had no effect on depolarizations evoked by [Sar9,Met(O2)11]SP (13.7 ± 2.6 vs. 15.0 ± 2.9 mV, respectively, n = 3) or senktide (5.7 ± 0.5 vs. 5.3 ± 0.3 mV, respectively, n = 3).


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Fig. 2.   NK1- and NK3-receptor agonists depolarize submucosal neurons. A: representative traces of intracellular recordings from single submucosal neurons comparing responses to superfusion of NK1 agonist [Sar9,Met(O2)11]SP, NK2 agonist [Ala5,beta -Ala8]NKA-(4---10), and NK3 agonist senktide. The NK1 agonist (30 nM) evokes a large depolarization and discharge of action potentials (action potentials are truncated in recording). The NK3 agonist (200 nM) depolarizes the neuron and evokes a few action potentials. The NK2 agonist has no effect on membrane potential. Resting Vm of each cell was (left to right) -63, -55, and -60 mV, respectively. B: summary of concentration-response of NK receptor agonists. Values are means ± SE of >= 4 recordings.

Effects of NK1 and NK3 antagonists on tachykinin-evoked depolarizations. CP-99,994 (30 nM) completely inhibited maximal depolarizations evoked by SP (30 nM) and caused a parallel shift to the right in the concentration-response curve (Fig. 3A). Maximal depolarizations evoked by [Sar9,Met(O2)11]SP (30 nM) were also completely blocked by CP-99,994 (30 nM; n = 3) (Fig. 3B), but depolarizations evoked by senktide (30 nM; n = 3; mean depolarization 8 vs. 7 mV, respectively) were unaffected. Senktide-evoked depolarizations (30-100 nM) were completely blocked by the selective NK3 antagonist SR-142801 (100 nM; n = 3) (Fig. 3B). SP-evoked depolarizations (10 nM) were unaffected by the NK3 antagonist (100 nM; mean depolarization 10 vs. 9 mV, respectively; n = 3).


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Fig. 3.   Effect of NK1-receptor antagonist CP-99,994 and NK3-receptor antagonist SR-142801 on agonist-evoked depolarizations of submucosal neurons. A: CP-99,994 (30 nM) causes parallel shift to right in SP dose-response curve. Values are means ± SE of >= 3 cells. B: representative recordings from single submucosal neurons show that CP-99,994 (30 nM) and SR-142801 (100 nM) block NK1- and NK3-agonist-evoked responses, respectively. In top 3 traces (resting Vm, -52 mV), NK1 agonist [Sar9,Met(O2)11]SP is superfused for duration of black bar. Depolarization evoked by NK1 agonist (top left) is completely blocked when agonist is reapplied in presence of CP-99,994 (top middle). After 10-min washout, reapplication of agonist evokes response similar to control (top right). In bottom 3 traces (resting Vm, -56 mV), NK3 agonist senktide is superfused for duration of black bar. Depolarization evoked by senktide (bottom left) is completely blocked when agonist is reapplied with SR-142801 (bottom middle). After prolonged 45-min washout, reapplication of senktide evokes only a minimal response (bottom right).

Tachykinin-Evoked Secretomotor Responses

The effects of tachykinins on Isc recorded from submucosal-mucosal preparations in Ussing chambers were examined to extend the findings of the intracellular recording studies to a functionally defined set of neurons. Previous studies (19) have shown that SP-evoked increases in Isc result almost entirely from activation of submucosal secretomotor neurons. These secretomotor neurons consist of two populations, cholinergic and noncholinergic neurons.

SP-, NKA-, and NKB-evoked changes in Isc. SP (1-100 nM; n = 5) evoked concentration-dependent changes in Isc (Fig. 4), as previously reported (10, 18, 19). NKA (1-100 nM; n = 5) and NKB (1-100 nM; n = 5) also caused a concentration-dependent increase in Isc. Repeat application of SP and NKA up to 100 nM did not desensitize, but reapplication of NKB (10-100 nM) resulted in a significant attenuation of the response.


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Fig. 4.   Endogenous tachykinins SP, NKA, and NKB evoke a concentration-dependent increase in short-circuit current (Isc). Values are means ± SE of 5 preparations. Inset: representative traces of changes in Isc in response to application of SP, NKA, and NKB in separate preparations. Vertical bar, 50 µA/cm2; horizontal bar, 5 min.

Changes in Isc evoked by selective activation of tachykinin receptors. The NK1 agonist [Sar9,Met(O2)11]SP (n = 5) and the NK3 agonist senktide (0.1-100 nM; n = 5) evoked concentration-dependent increases in Isc (Fig. 5). The NK2 agonist [Ala5,beta -Ala8]NKA-(4---10) (10-100 nM) did not significantly alter Isc compared with baseline (Fig. 5). The NK1 antagonist CP-99,994 (50 nM) completely blocked maximal Isc responses evoked by [Sar9,Met(O2)11]SP (30 nM; n = 4) but had no effect on Isc responses evoked by senktide (l nM, mean Isc = 88 ± 31 vs. 54 ± 11 µA/cm2, respectively) compared with control responses (Fig. 5B). Maximal senktide-evoked changes in Isc (100 nM) were completely blocked by the NK3 antagonist SR-142801 (100 nM), but this antagonist had no effect on submaximal responses evoked by [Sar9,Met(O2)11]SP compared with controls (10 nM; 114 ± 29 vs. 84 ± 24 µA/cm2, respectively, n = 5) (Fig. 5B). EFS-evoked changes in Isc were not altered by CP-99,994 (50 nM; n = 5) or SR-142801 (100 nM; n = 6).


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Fig. 5.   Tachykinin-evoked increases in Isc are mediated by NK1 and NK3 receptors. A: NK1 agonist [Sar9,Met(O2)11]SP and NK3 agonist senktide evoke a concentration-dependent increase in Isc. NK2 agonist [Ala5,beta -Ala8]NKA-(4---10) has virtually no effect. Values are means ± SE of 5 preparations. B: comparison of NK agonist and electrical field stimulation (EFS)-evoked increases in Isc in presence (filled bars) and absence (open bars) of NK1 antagonist CP-99,994 (CP; 100 nM) and NK3 antagonist SR-142801 (SR; 100 nM). CP-99,994 (100 nM) completely blocks increases in Isc evoked by [Sar9,Met(O2)11]SP (30 nM) but has no effect on responses evoked by EFS. Similarly, SR-142801 (100 nM) completely blocks senktide (100 nM)-evoked increases in Isc but has no effect on EFS-evoked responses. Bars represent means ± SE of 5 preparations. ** P < 0.01.

Effect of TTX on tachykinin-evoked changes in Isc. The effects of TTX on tachykinin-evoked increases in Isc were examined to determine whether these increases resulted from activation of receptors on submucosal secretomotor neurons (TTX sensitive) and/or the enterocyte (TTX insensitive). Pretreatment with TTX (l µM) inhibited mean NKA-evoked Isc by 91% (100 nM; n = 8) and [Sar9,Met(O2)11]SP-evoked Isc by 91% (100 nM; n = 7) and completely blocked Isc responses evoked by SP (100 nM; n = 3), senktide (100 nM; n = 5), and NKB (100 nM; n = 5) (Fig. 6). EFS-evoked changes in Isc were completely blocked by TTX (l µM), but responses to carbachol (500 nM) alone were not significantly different from those obtained in the presence of TTX (P = 0.093).


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Fig. 6.   NK1- and NK3-receptor-mediated increases in Isc result predominantly from activation of receptors located on submucosal neurons. Left to right: SP, NKA, NKB, NK1 agonist, NK3 agonist, carbachol, and EFS responses were compared in presence and absence (control) of tetrodotoxin (TTX). TTX (1 µM) blocks neurally evoked secretion (EFS) but has no effect on carbachol responses mediated by muscarinic receptors directly on the enterocyte. Bars represent means ± SE of 5 experiments. ** P < 0.01.

Effect of NK1 and NK3 Antagonists on Intrinsic and Extrinsic Nerve-Evoked Responses

Capsaicin-sensitive extrinsic sensory nerves. Our previous studies have demonstrated that capsaicin-sensitive nerves evoke a biphasic change in Isc in submucosal-mucosal preparations (27). In these studies, 200 nM capsaicin was shown to selectively activate extrinsic sensory nerves. These nerves release neurotransmitter(s) from nerve terminals within the submucosal plexus, which in turn activates submucosal secretomotor neurons. In the present study, the possibility that these neurotransmitters might include one or more tachykinins acting at either NK1 and/or NK3 receptors was examined. Capsaicin (200 nM) evoked typical biphasic increases in Isc (mean phase 1 response 41 ± 6 µA/cm2, mean phase 2 response 82 ± 18 µA/cm2; n = 10), as previously described (27). The larger phase 2 responses were inhibited 53% (P < 0.05; n = 5) by the nonpeptide NK1 antagonist CP-99,994 (50 nM) compared with controls (Fig. 7). Phase 1 responses were unaffected. The nonpeptide NK1 antagonist CP-96,345 (200 nM) also significantly reduced (71%) the second phase of the response (107 ± 20 vs. 31 ± 10 µA/cm2, control vs. CP-96,345 treated, respectively, P < 0.01; n = 5) but had no effect on the first phase. Responses to EFS (84 ± 18 vs. 72 ± 12 µA/cm2, control vs. drug, respectively) and carbachol (1 µM) (130 ± 31 vs. 178 ± 29 µA/cm2, control vs. drug, respectively) were not affected by either NK1 antagonist. In contrast, the NK3 antagonist SR-142801 (100 nM), which completely blocked Isc responses evoked by the NK3 agonist senktide (see above), had no effect on capsaicin-evoked changes in Isc (mean phase 1 Isc = 59 ± 10 vs. 42 ± 6 µA/cm2, P = 0.116, control vs. antagonist, respectively; mean phase 2 Isc = 106 ± 15 vs. 119 ± 26 µA/cm2, control vs. antagonist, respectively; n = 6) (Fig. 7A).


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Fig. 7.   Evidence for endogenous release of tachykinins from extrinsic capsaicin-sensitive nerves but not from intrinsic enteric nerves. A: representative trace of biphasic changes in Isc evoked by capsaicin in Ussing chamber studies. As previously described, capsaicin evokes a biphasic increase in Isc (27) characterized by a brief first phase and a larger, more prolonged second phase. B: capsaicin-evoked increases in Isc were compared in presence and absence (control) of NK1, NK3, and calcitonin gene-related peptide (CGRP) antagonists. NK1 antagonist CP-99,994 (100 nM) inhibits the second phase by >50% (* P < 0.05). NK3 antagonist SR-142801 (100 nM) and CGRP antagonist CGRP-(8---37) (2 µM) have no effect on either phase of capsaicin-evoked response. Bars represent means ± SE of 5 experiments. C: comparison of effect of NK1 (top traces) and NK3 antagonists (bottom traces) on slow excitatory postsynaptic potentials (EPSPs) evoked by fiber tract stimulation of axons of intrinsic enteric neurons. Neither antagonist has any effect on amplitude or time course of slow EPSP compared with control. Resting Vm values were -55 (top traces) and -52 mV (bottom traces).

Previous studies suggest that, in some tissues, SP or a related tachykinin degranulates mast cells through a receptor-independent mechanism (11). Mast cell mediators histamine and 5-HT evoke a secretory response through activation of submucosal secretomotor neurons (2, 30). Consequently, we examined the possibility that the capsaicin-evoked increase in Isc may in part be mediated by tachykinin-evoked mast cell degranulation. Histamine (1 µM-1 mM; EC100 sime  100 µM, n = 6) and 5-HT (0.1-100 µM; EC100 sime  100 µM; n = 6) evoked a dose-dependent increase in Isc, as previously described (4, 9). Antagonist concentrations were derived from our studies of the H1 antagonist pyrilamine (50 nM), which completely blocked histamine (10 µM)-evoked changes in Isc (n = 5), and from previous studies of 5-HT secretomotor responses (9). In the present study, antagonists to H1 (pyrilamine, 50 nM; n = 8), H2 (cimetidine, 10 µM; n = 7), and H3 (thioperamide, 300 nM; n = 7) receptors and to 5-HT2 (LY-53857, 10 µM; n = 7), 5-HT3 [BRL-43694 (granisetron), 10 µM; n = 6], and 5-HT4 (SDZ-205-557, 1 µM; n = 6) receptors had no effect on the Isc response evoked by capsaicin (200 nM) (data not shown). Our previous studies have shown that capsaicin (200 nM) selectively activates capsaicin-sensitive nerves because in preparations where capsaicin-sensitive nerves were surgically denervated, capsaicin had no effect on Isc (27).

CGRP is a putative neurotransmitter released from capsaicin-sensitive nerves in some neural circuits (8, 26). In the present study, superfusion of human CGRP-II (200 nM-2 µM) had no effect on membrane potential in intracellular recording studies (mean change in membrane potential compared with control responses 0.5 ± 0.4 mV; n = 7), and CGRP-II (100 nM) did not alter Isc (mean change in Isc compared with control responses 0.0 ± 0 µA/cm2; n = 3) in Ussing chamber studies. Pretreatment with supramaximal concentrations (26) of the CGRP antagonist CGRP-(8---37) (2 µM) did not affect responses to capsaicin (200 nM), EFS, or carbachol (1 µM) (n = 5 for each group).

Intrinsic enteric neurons. The effect of the NK1 and NK3 antagonists on nerve-evoked slow EPSPs was examined to determine if the release of tachykinins acting at these receptors mediated this synaptic potential. Previous studies have shown that slow EPSPs in the submucosal plexus are resistant to cholinergic blockade (22, 23). These studies were confined to cells in which the nerve-evoked slow EPSP was reproducible in amplitude and duration in control experiments. Slow EPSPs were not inhibited (Fig. 7B) by CP-99,994 (100 nM, n = 7; control mean amplitude 11 ± 2 mV and mean half-duration 6 ± 0.06 s vs. CP-99,994 mean amplitude 12 ± 2 mV and mean half-duration 7 ± 0.4 s) or SR-142801 (100 nM, n = 6; control mean amplitude 11 ± 2 mV and mean half-duration 7 ± 0.6 s vs. SR-142801 mean amplitude 11 ± 2 mV and mean half-duration 7 ± 0.9 s).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

This study characterized tachykinin responses in a defined neural secretory pathway in the submucosal plexus. This pathway consists of cholinergic and noncholinergic secretomotor neurons that innervate mucosal enterocytes in the small intestine (4). Activation of this pathway stimulates chloride secretion from intestinal enterocytes, which results in the concomitant passive movement of water into the lumen. In the present study, the results of intracellular recording from single submucosal neurons demonstrate that both NK1 and NK3 receptors are found on submucosal neurons. Ussing chamber studies provided direct evidence that these receptors are located on submucosal secretomotor neurons, which in turn stimulate mucosal enterocytes. Capsaicin-sensitive nerves release endogenous tachykinins, which activate NK1 receptors in this pathway as well as other as yet unidentified neurotransmitter(s).

Systematic studies were conducted to identify NK1- and NK3-receptor subtypes and to localize these receptors to submucosal secretomotor neurons. In these studies, the NK1 agonist [Sar9,Met(O2)11]SP and the NK3 agonist senktide activated single submucosal neurons during intracellular recording. Senktide, however, did not depolarize all neurons examined, which may suggest that NK3 receptors are not homogeneously distributed among submucosal neurons and/or that the receptor density varies between individual neurons. Nonetheless, both NK1 and NK3 agonists evoked large secretory responses in Ussing chamber studies, demonstrating that both receptors mediate functional responses. The selectivity of these agonists was demonstrated in both intracellular and Ussing chamber studies in which NK1 and NK3 agonist responses were selectively blocked by the nonpeptide antagonists CP-99,994 and SR-142801, respectively. There was no evidence, however, that NK2-receptor-mediated secretory responses exist in this neural pathway. The location of the receptors within this neural pathway was further studied in Ussing chamber studies to determine whether the tachykinin-evoked response resulted solely from activation of tachykinin receptors on the submucosal neurons or whether there may in addition be receptors located directly on the enterocyte. Responses to the selective agonists were studied in the presence and absence of TTX, which blocks action potential conduction in axons of the submucosal secretomotor neurons innervating the enterocyte. In these studies, TTX completely blocked NK3-mediated responses and 91% of the NK1-mediated response. These studies suggest, therefore, that tachykinin-evoked secretory responses are almost exclusively mediated by secretomotor neurons. They do suggest, however, that NK1 receptors are also found directly on the enterocyte. These findings are consistent with previous studies, which had shown that a small component of SP-evoked secretion is mediated by actions directly on the enterocyte (4, 10, 18). Previous studies had suggested that these neurokinin receptors may differ from those on the secretomotor neuron (10), but the findings of the present study suggest that both effector cells are activated by tachykinins acting at NK1 receptors.

There is considerable indirect evidence to suggest that both intrinsic enteric neurons and extrinsic capsaicin-sensitive nerves release tachykinins, which could activate the NK1 and NK3 receptors described in this study (4, 16, 21, 24). The extrinsic capsaicin-sensitive nerves are known to release SP or a related tachykinin in some tissues, but in other neural circuits CGRP appears to be the predominant neurotransmitter (8). In the submucosal plexus, capsaicin selectively activates extrinsic nerve fibers, which innervate both cholinergic and noncholinergic submucosal secretomotor neurons (27). In the present study, capsaicin-evoked changes in Isc were significantly blocked by NK1 antagonists but not by CGRP antagonists. These data demonstrate that capsaicin-sensitive nerves release tachykinin(s), which activates this neural secretory pathway, and that CGRP appears to play little role. The possibility that intrinsic neurons also released endogenous tachykinins was examined by studying the effects of the tachykinin antagonist CP-99,994 on the slow EPSP. Previous studies (22, 23) have shown that exogenous application of SP can mimic the time course and conductance changes characteristic of the slow EPSP. Furthermore, immunohistochemical studies have demonstrated that SP-immunoreactive fibers project from the myenteric plexus to the submucosal plexus (5) and that after selective lesioning studies (3), which destroyed fibers containing other putative neurotransmitters such as 5-HT, somatostatin, and vasoactive intestinal peptide, but not SP, the electrically evoked slow EPSP appeared unchanged. Despite these suggestive data, the present study failed to find evidence that the slow EPSP was mediated by the release of tachykinins acting at either the NK1 or NK3 receptor. These data may imply that tachykinins are not the predominant mediator of the slow EPSP but do not preclude that tachykinins mediate some EPSPs in the submucosal plexus. Indeed, it has been suggested (23, 24) that it is unlikely that the slow EPSP results from the release of neurotransmitter from one or a few discrete fibers, but rather results from the release of neurotransmitter from multiple fibers containing a number of different neuropeptides. Therefore, the removal of one peptide input might not be expected to markedly alter the amplitude or time course of the nerve-evoked slow EPSP (23, 24).

The possibility that tachykinin-evoked secretion mediated by capsaicin-sensitive nerves involved, at least in part, a mast cell-dependent pathway was also examined in this study. Nerve-mast cell interactions have been widely reported (2, 4). Capsaicin-sensitive nerves are thought to be one of the principal neural networks innervating the mast cell, leading to release of histamine and possibly other mediators such as 5-HT, prostaglandins, and bradykinin. In studies in which SP has been shown to degranulate mast cells, this action appears to involve a receptor-independent mechanism (30). Therefore, in the present study, evidence for a role of a mast cell-dependent pathway was sought using antagonists of the putative mast cell mediators, histamine and 5-HT. However, these antagonists had no effect on the capsaicin-evoked response, supporting the findings of previous studies, which show that exogenous application of SP does not appear to degranulate mast cells in this tissue (10, 19), and extending this observation to include other related tachykinins or putative neurotransmitters of the capsaicin-evoked response. Further studies are needed to determine whether this finding also applies to inflamed tissue because many studies in which nerve-mast cell connections are described (14, 29) involve allergic or inflammation models.

Activation of the NK1 and NK3 receptors described in this study could play a significant role in modulating the homeostatic regulation of water and electrolyte transport in the intestine. In addition to this physiological role, there is considerable evidence that tachykinins play multiple roles in the expression of the inflammatory response. These actions may reflect exaggerated actions of tachykinins because there is evidence that during inflammation the expression and release of tachykinins from capsaicin-sensitive nerves is increased and upregulation of tachykinin receptors may occur (6, 12). Not surprisingly, tachykinin antagonists have shown considerable potential in the treatment of inflammatory responses (31). Diarrhea is a prominent feature of intestinal inflammatory responses, and, consequently, the NK1 and NK3 receptors described in this study could provide targets for altering this pathophysiological response.

    ACKNOWLEDGEMENTS

This work was supported by the Medical Research Council of Canada (MRCC; S. Vanner) and the Crohn's and Colitis Foundation of Canada (W. MacNaughton). B. Moore is a recipient of the MRC-Canadian Association of Gastroenterology-Janssen Research Fellowship.

    FOOTNOTES

Address for reprint requests: S. Vanner, Division of Gastroenterology, 166 Brock St., Kingston, ON, Canada K7L 5G2.

Received 14 April 1997; accepted in final form 5 August 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Bertrand, P., and J. J. Galligan. Signal-transduction pathways causing slow synaptic excitation in guinea pig myenteric AH neurons. Am. J. Physiol. 269 (Gastrointest. Liver Physiol. 32): G710-G720, 1995[Abstract/Free Full Text].

2.   Bienenstock, J., G. MacQueen, P. Sestini, J. Marchall, R. Stead, and M. Perdue. Inflammatory cell mechanisms: mast cell/nerve interactions in vitro and in vivo. Am. Rev. Respir. Dis. 143: S55-S58, 1991[Medline].

3.   Bornstein, J., R. A. North, M. Costa, and J. B. Furness. Excitatory synaptic potentials due to activation of neurons with short projections in the myenteric plexus. Neuroscience 11: 723-731, 1984[Medline].

4.   Cooke, H. Neuroimmune signaling in regulation of intestinal ion transport. Am. J. Physiol. 266 (Gastrointest. Liver Physiol. 29): G167-G178, 1994[Abstract/Free Full Text].

5.   Costa, M., J. B. Furness, I. J. Llewellyn-Smith, and A. C. Cuello. Projections of substance P neurons within the guinea-pig small intestine. Neuroscience 6: 411-424, 1981[Medline].

6.   Donnerer, J., R. Schuliogio, and C. Stein. Increased content and storage of substance P and calcitonin gene related peptide in sensory nerves innervating inflamed tissue: evidence for a regulatory function of nerve growth factor in vivo. Neuroscience 49: 693-698, 1992[Medline].

7.   Gibbins, I., J. Furness, and M. Costa. Pathway-specific patterns of the co-existence of substance P, calcitonin gene related peptide, and cholecystokinin in neurons of the dorsal root ganglia of the guinea pig. Cell Tissue Res. 248: 417-437, 1987[Medline].

8.   Holzer, P. Capsaicin: cellular targets, mechanisms of actions and selectivity for thin sensory neurons. Pharmacol. Rev. 43: 143-201, 1991[Medline].

9.   Johnson, P., J. Bornstein, J. B. Furness, D. J. Woollard, and S. L. Orrman-Rossiter. Characterization of 5-hydroxytryptamine receptors mediating mucosal secretion of guinea-pig ileum. Br. J. Pharmacol. 111: 1240-1244, 1994[Abstract].

10.   Keast, J. R., J. B. Furness, and M. Costa. Different substance P receptors are found on mucosal epithelial cells and submucous neurons of the guinea-pig small intestine. Naunyn Schmiedebergs Arch. Pharmacol. 329: 382-387, 1985[Medline].

11.   Maggi, C. A., R. Patacchini, P. Rovero, and A. Giachetti. Tachykinin receptors and tachykinin receptor antagonists. J. Auton. Pharmacol. 13: 23-93, 1993[Medline].

12.   Mantyh, P., C. Mantyh, T. Gastes, S. Vigna, and J. Maggio. Receptor binding sites for substance P and substance K in the canine gastrointestinal tract and their possible role in inflammatory bowel disease. Neuroscience 25: 817-837, 1988[Medline].

13.   Mathison, R., and J. S. Davison. Regulation of epithelial transport in the jejunal mucosa of the guinea pig by neurokinins. Life Sci. 45: 1057-1064, 1989[Medline].

14.   Mathison, R., and J. S. Davison. Regulation of jejunal arterioles by capsaicin-sensitive nerves in Nippostrongylus brasiliensus-sensitized rats. J. Pharmacol. Exp. Ther. 273: 337-343, 1995[Abstract].

15.   Mawe, G. Tachykinins as mediators of slow EPSPs in guinea-pig gall-bladder ganglia: involvement of neurokinin-3 receptors. J. Physiol. (Lond.) 485: 513-524, 1995[Abstract].

16.   Mihara, S. Intracellular recordings from neurons of the submucous plexus. Prog. Neurobiol. 40: 529-572, 1993[Medline].

17.   Otsuka, M., and K. Yoshioka. Neurotransmitter functions of mammalian tachykinins. Physiol. Rev. 73: 230-308, 1993.

18.   Perdue, M. H., R. Galbraith, and J. S. Davison. Evidence for SP as a functional neurotransmitter in the guinea pig small intestinal mucosa. Regul. Pept. 18: 63-74, 1987[Medline].

19.   Reddix, R. A., and H. J. Cooke. Neurokinin 1 receptors mediate substance P-induced changes in ion transport in guinea-pig ileum. Regul. Pept. 39: 215-225, 1992[Medline].

20.   Regoli, D., A. Boudon, and J.-L. Fauchere. Receptors and antagonists for substance P and related peptides. Pharmacol. Rev. 46: 551-599, 1994[Medline].

21.   Schemann, M., and H. Kayser. Effects of tachykinins on myenteric neurons of the guinea-pig gastric corpus: involvement of NK-3 receptors. Pflügers Arch. 419: 566-571, 1991[Medline].

22.   Surprenant, A. Slow excitatory synaptic potentials recorded from neurones of guinea-pig submucous plexus. J. Physiol. (Lond.) 351: 343-361, 1984[Abstract].

23.   Surprenant, A. Synaptic transmission in neurones of the submucous plexus. In: Nerves and the Gastrointestinal Tract, edited by M. V. Singer, and H. Goebell. London: Kluwer Academic, 1989, p. 253-263.

24.   Surprenant, A. Transmitter mechanisms in the enteric nervous system: an electrophysiological vantage point. In: Trends in Autonomic Pharmacology, edited by S. Kalsner. London: Taylor & Francis, 1985, chapt. 6, p. 71-98.

25.   Tamura, K., K. Mutabagani, and J. D. Wood. Analysis of a nonpeptide antagonist for substance P on myenteric neurons of guinea pig small intestine. Eur. J. Pharmacol. 232: 235-239, 1993[Medline].

26.   Vanner, S. Corelease of neuropeptides from capsaicin-sensitive afferents dilates submucosal arterioles in guinea pig ileum. Am. J. Physiol. 267 (Gastrointest. Liver Physiol. 30): G650-G655, 1994[Abstract/Free Full Text].

27.   Vanner, S., and W. K. MacNaughton. Capsaicin-sensitive afferent nerves activate submucosal secretomotor neurons in guinea pig ileum. Am. J. Physiol. 269 (Gastrointest. Liver Physiol. 32): G203-G209, 1995[Abstract/Free Full Text].

28.   Vanner, S., and A. Surprenant. Cholinergic and noncholinergic submucosal neurons dilate arterioles in the guinea pig colon. Am. J. Physiol. 261 (Gastrointest. Liver Physiol. 24): G136-G144, 1991[Abstract/Free Full Text].

29.   Wallace, J., G. W. McKnight, and A. D. Befus. Capsaicin-induced hyperemia in the stomach: possible contribution of mast cells. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G209-G214, 1992[Abstract/Free Full Text].

30.   Wang, L., A. Stanisz, B. Wershil, S. Galli, and M. H. Perdue. Substance P induces ion secretion in mouse small intestine through effects on enteric nerves and mast cells. Am. J. Physiol. 269 (Gastrointest. Liver Physiol. 32): G85-G92, 1995[Abstract/Free Full Text].

31.   Watling, K., and J. Krause. The rising sun shines on substance P and related peptides. Trends Pharmacol. Sci. 14: 81-84, 1993[Medline].


AJP Gastroint Liver Physiol 273(5):G1127-G1134
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