Journal of Physiology (1991), 437, pp. 341-350
`With 6 figures
`Printed in Great Britain
`
`341
`
`NEUROKININ A INCREASES SHORT-CIRCUIT CURRENT ACROSS
`
`RAT COLONIC MUCOSA: A ROLE FOR VASOACTIVE
`
`INTESTINAL POLYPEPTIDE
`
`By XIAO-YING TIEN*, LANE J. WALLACE, JAMES F. KACHUR,
`SUZANNE WON-KIM anp TIMOTHYS. GAGINELLAT
`From the Division of Pharmacology, College of Pharmacy, The Ohio State University,
`Columbus, OH 43210 and Searle Research and Development, Skokie, IL 60077, USA
`
`(Received 4 October 1990)
`
`SUMMARY
`
`1. Neurokinin A (NKA)is a mammalian tachykinin distributed principally in the
`nervous system, including the myenteric innervation of the gut.
`2. NKA maybeinvolved in neurogenic inflammation and as a modulatory factor
`in the diarrhoea associated with mucosal
`inflammation of inflammatory bowel
`disease (ulcerative colitis).
`3. We evaluated the effect of NKA on the short-circuit current J,,, assumed to
`reflect electrogenic chloride secretion, across muscle-stripped rat colonic mucosa
`mounted in Ussing chambers.
`4. Serosal addition of NKA produced a concentration-dependent (0-1-100 nm)
`increase in J,, with an EC,, (half-maximal effective concentration) value of 7:5 nM.
`The maximum (mean+s.£.M.) increase in J,, (wA/cm?) for NKA was 111+10.
`5. Tetrodotoxin (0°5 wm) and bumetanide (10 4m), but not atropine (1:0 “M),
`hexamethonium (100m) or pyrilamine (10 um), significantly inhibited NKA-
`induced increasesin J,,.
`6. The response to NKA was attenuated by 45 min pre-treatment with antisera
`raised against vasoactive intestinal polypeptide (VIP). Moreover, prior desen-
`sitization to VIP attenuated the effect of NKA.
`7. These studies suggest that NKA increases J,, in rat colon, in part, through a
`non-cholinergic neural mechanism involving VIP.
`
`INTRODUCTION
`
`Neurokinin A (NKA, substance k, neuromedin L) is a mammalian tachykinin
`(Kangawa, Minamino, Fukuda & Matsuo, 1983; Kimura, Okada, Sugita, Kanagawa
`& Manekata, 1983), derived from £-preprotachykinin A and y-preprotachykinin A
`(Maggio, 1988). It shares a common C-terminusand co-exists with other tachykinins
`
`* Present address: Department of Physiology/Biophysiology, College of Medicine, Case Western
`Reserve University, 2119 Abington Road, Cleveland, OH 44106, USA.
`+ To whom reprint requests should be sent at the Department of Gastrointestinal Disease
`Research, Searle Research and Development, 4901 Searle Parkway, Skokie, IL 60077, USA.
`MS 8841
`
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`342
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`X.-Y. TIEN AND OTHERS
`
`in the same subpopulation of neurons, and therefore may also act as a
`neurotransmitter
`(Minamino, Kangawa, Fukuda & Matsuo, 1984; Dalsgaard,
`Haegerstrand, Theodorsson-Norheim, Brodin & Hokfelt, 1985; Dockray, 1987).
`Neurogenic inflammation, characterized by vasodilatation, plasma extravasation
`and smooth muscle contraction initiated by release of proinflammatory mediators
`from sensory unmyelinated afferent nerve endings, may be a contributing factor in
`the pathophysiology of inflammatory bowel disease (Mayer, Raybould & Koelbel,
`1988). Neurokinin A is a candidate mediator in neurogenic inflammation. It is
`localized in sensory neurons (Hua, Theodorsson-Norheim, Brodin, Lundberg &
`Hokfelt, 1985), increases vascular permeability and is a vasodilator (Foreman, 1987).
`Receptor binding sites for neurokinin A are expressed by cells mediating
`inflammatory and immune responses (Mantyh, Mantyh, Gates, Virna & Maggio,
`1988). Tachykinins may affect
`inflammation by increasing mast cell secretion,
`promoting monocyte chemotaxis and stimulating phagocytosis and lysosomal
`enzymerelease by neutrophils (Shanahan & Anton, 1988).
`Recently, NKA was shown undershort-circuit conditions to cause net electrolyte
`secretion in the canine tracheal epithelium (Rangachari, McWade & Donoff, 1987;
`Tamaoki, Ueki, Widdicombe & Nadel, 1988) and the guinea-pig jejunum (Mathison
`& Davidson, 1989). Since the colonic mucosal secretory response to NKA has not
`been reported, and secretory diarrhoea is associated with ulcerative colitis, we
`investigated the effects and mechanism of action of NKA on rat colonic mucosa in
`vitro.
`Part of this work has been presented in abstract form (Tien, Wallace & Gaginella,
`1988).
`
`METHODS
`
`Tissue preparation
`Male Sprague-Dawley rats (250-400 g) were killed by cervical dislocation, and the distal colon
`was excised. The colon was opened and rinsed with ice-cold saline solution (0°9% w/v NaCl). The
`circular and longitudinal muscle layers were stripped off with a pair of fine forceps. Pieces of
`muscle-stripped mucosa were mounted as flat sheets in Ussing chambers (0-64 cm?) and bathed
`with buffered physiological saline solution composed of (mm): NaCl, 120-2; KCl, 5:9; CaCl,, 2:5;
`MgCl,, 1:2; NaH,PO,, 1:2; NaHCO,, 25; glucose, 11:1 (pH 7:4, 37 °C), gassed with 95% O, and
`5% CO,.
`
`Electrical measurements
`
`Electrical potential difference (PD) was monitored at the beginning of each experiment with a
`pair of saturated KCl-agar (3%) bridges placed on each side of the tissue. The circuit was
`completed with calomel half-cell electrodes connected to a DVC 1000 voltage clamp (WPI
`Instruments, New Haven, CT, USA). Short-circuit current (/,,) was applied through silver—silver
`chloride electrodes to the tissue to clamp open-circuit PD (potential difference) at zero, and
`compensation was madefor fluid resistance. Initial tissue conductance (G,) was calculated from the
`values of I,, and PD according to Ohm’s law. The reciprocal of G, (mS/cm?) times 1000 equals the
`tissue resistance (R, Q cm?).
`
`Concentration—response curves
`After mounting, the tissues were allowed to equilibrate for at least 30-60 min; effects on
`presumedelectrogenic chloride secretion were monitored as changes in J,,. The concentration
`required to increase I,, by 50% (EC,,) was calculated from the curves by non-linear least-squares
`regression,
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`NEUROKININ A INCREASES SHORT-CIRCUIT CURRENT
`
`343
`
`Effects of antagonists
`Antagonists were added to the serosal side of the tissue for various times (see individual drugs
`below) prior to agonist addition. If blockade was observed, bethanechol (0-4 mM) or aminophylline
`(20 mm) was added after the agonist response to ensure that antagonism, rather than poortissue
`viability, was responsible for the lack of response. Antagonist controls were conducted on a
`“ separate tissue from the same animals.
`
`A
`
`B
`
`0.05
`
`Y
`
`0-01
`'
`
`5
`<
`OooO
`“ye
`
`5 min
`
`TOHM-NKA
`01
`tat
`
`Fig. 1. Representative effect of NKA (um) on J/,, after a single concentration (A) and
`following cumulative addition (B).
`
`VIP antisera studies
`
`VIP antisera was used to assess a role for VIP in the response to NKA. The VIP antiserum was
`used at a final dilution of 1: 200. Each tissue preparation treated with VIP antiserum was compared
`with a control tissue from the same animal. Basal /,, values and conductance were comparable in
`VIP-antisera treated and control tissues.
`
`Materials
`
`NKA, VIP, TTX, hexamethonium and pyrilamine were obtained from Sigma (St Louis, MO,
`USA), and bumetanide was obtained from Hoffmann-La Roche Inc., Nutley, NJ, USA. Agents not
`soluble in distilled water or saline were dissolved in dimethyl sulphoxide (DMSO). Equivalent
`amounts of DMSO (without drug) added to the serosal side did not affect J,,. VIP antisera (Ras
`71651-N, rabbit) was purchased from Penninsula Laboratories, San Carlos, CA, USA.
`
`Statistical analysis
`All values are expressed as the means+s.E.M. The Student’s paired or unpaired ¢ test was used
`for testing statistical significance.
`
`RESULTS
`
`Effect of NKA on I,,
`NKAevokeda rapid but transient increase in J,, upon serosal addition (Fig. 1).
`The responses were concentration dependent over a range of 0:5-100 nm (Fig. 2); the
`EC,, value was determined to be 75nm. The maximum response to NKA was
`111+10 wA/cm? (n = 26 tissues from thirteen animals).
`
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`344
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`X.-Y. TIEN AND OTHERS
`
`140
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`IncreaseinIke(uA/cm2)
`
`9
`
`8
`
`7
`
`6
`
`~log [NKA] (mM)
`
`Fig. 2. Concentration—-response curve (serosal addition) for NKA-induced increases [,,
`across rat colonic mucosa. Results are expressed as the change in J,, (uA/cm?) above
`baseline vs. log concentration of NKA. Symbols and bars represent the means+s.E.M. of
`determinations for at least twelve animals. The maximalincrease in J,, was achieved at
`10-7-10-° M.
`
`150
`
`100
`
`00
`
`0
`
`*
`
`
`
`oa
`¢c
`6Qano
`
`2 S
`
`2 @ £x@ E 6@
`
`DOo
`
`< 8o
`
`a
`
`TTX
`(0-5 uM)
`
`Hexamethonium
`(100 um)
`
`Atropine
`(1 uM)
`
`Fig. 3. Effects of tetrodotoxin (TTX), atropine and hexamethonium against NKA
`(50 nm).
`Inhibitors were added 10min before NKA. Results are expressed as
`means+s.z.M. of three to eight animals. *P < 0-05.
`
`Effects of inhibitors
`To substantiate that the NKA-evokedincreasein J,, was due to active chloride ion
`secretion, tissues were incubated with bumetanide (10 wm, 30 min) before adding
`NKAto the tissue bath. Responses to a maximally effective concentration (50 nm)
`
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`NEUROKININ A INCREASES SHORT-CIRCUIT CURRENT
`
`345
`
`of NKA weresignificantly (P < 0-001) reduced by serosal addition of bumetanide to
`14+4% of control. Unidirectional flux measurements with **Cl” could not be
`conducted due to the transient nature of the response.
`TTX (0-5 um) inhibited by 93% the /,, response to 50 nM-NKA(Fig. 3). Atropine
`‘(1 wm, 10 min), which completely inhibited the response to 0-4 mm-bethanechol, had
`
`(uA/em2)
`
`NKA
`
`NKA~
`pyrilamine
`
`IncreaseinIs,
`
`Hist
`
`Hist
`pyrilamine
`
`Fig. 4. Effects of the H, receptor antagonist pyrilamine (10 uM) against 1 mm-histamine
`and NKA (50 nm). The antagonist was added 10 min prior to agonists. All drugs were
`added to the serosal side. Data are expressed as means+s.£E.M. for determinations from
`at least six animals. *P < 0-001.
`
`the ganglionic blocker,
`no significant effect on responses elicited by NKA;
`hexamethonium, also failed to inhibit the response to NKA (Fig. 1).
`Because other neurokinins are reported to release histamine from mast cells
`(Erjavec, Lembeck, Florjanc-Irman, Skofttsch, Donnerer, Saria & Holzer, 1981;
`Foreman, 1987),
`the histamine receptor H, antagonist, pyrilamine, was used to
`determineif it would block the NKA-induced response. Pyrilamine (10 “Mm, 15 min)
`significantly blocked the histamine (1 mm) but not
`the NKA (50 nm) response
`(Fig. 4).
`The effect of VIP antisera on the J,, response to NKA was examined. A dilution
`of 1:200 attenuated both the NKA response (Fig. 5A) and the response to VIP
`(Fig. 5B). However, pre-treatment with non-specific rabbit serum (1:200) or pre-
`neutralized VIP antiserum did not affect the response to NKA.
`
`Desensitization studies
`
`After stimulation of the tissues with NKA, subsequent additions of the same
`concentration of the peptide failed to produce a response, indicating homologous
`
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`346
`
`X.-Y. TIEN AND OTHERS
`
`desensitization. The desensitized tissues were responsive to subsequent application of
`bethanechol
`(04mm) and aminophylline (20mm, data not shown). We also
`investigated whether desensitization to VIP could block the effect of NKA. After the
`response to 0-5 um-VIP had returned to baseline, the response to a second addition
`
`A
`
`120
`
`s+----- Control
`—m— + VIP Ab (1: 200)
`
`<
`
`x8&o
`
`Oa
`@&@
`
`S£
`
`
`
`9
`
`8
`
`7
`
`6
`
`-log [NKA] (mM)
`
`B
`
`200
`
`150
`
`s------ Control
`——m— + VIP Ab (1: 200)
`
`
`
`—
`
`<3& 3o 2o
`
`O£
`
`Fig. 5. Effect of pre-treatmentof rat colon with VIP antisera (VIP Ab) (1:200) on NKA-
`induced (A) and VIP-induced (B) increases in J,,. Symbols and bars are means+S8.E.M.,
`n = 4 animals. *P < 0:05.
`
`-log [VIP] (mM)
`
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`NEUROKININ A INCREASES SHORT-CIRCUIT CURRENT
`
`347
`
`of 0-5 wm-VIP was reduced by 50% or greater. The subsequent addition of NKA
`after desensitization to VIP was also attenuated (Fig. 6).
`
`100
`
`se-Q--- Control
`—ws— Desensitized to0-5 uM-VIP
`
`(uA/em?)
`Increasein[gc
`
`9
`
`8
`
`7
`
`6
`
`Fig. 6. Effect of desensitization to VIP on the response to NKA-inducedincreasesin J,,.
`Symbols and bars are means+s.E.M., n = 4 animals. *P < 0:05.
`
`-log [NKA] (mM)
`
`DISCUSSION
`
`The present study demonstrates that NKA stimulatesJ,, in rat colonic mucosa, in
`part through a non-cholinergic mechanism. VIP is a candidate mediator for the
`neural-associated component of NKA action as a secretagoguein this tissue. Because
`the transient nature of the J,, response elicited by NKA precluded measurement of
`ion fluxes, we examinedtheionic basis of action of NKA with bumetanide,a chloride
`transport
`inhibitor. Bumetanide inhibited the NKA-induced increase in [,,,
`suggesting that electrogenic chloride secretion is a major contributor to the J,,
`response. NKA wasineffective when added to the mucosal solution, suggesting that
`its receptors are located on the basolateral side of the cell membrane. Furthermore,
`pre-incubation of the tissues with TTX completely blocked the NKA-induced
`increases in J,, (about 95%). This suggests that the majority of the NKA-induced
`secretion is due to stimulation of neurotransmitter release from enteric neurons.
`Others have noted that NKA may act via a neural mechanism to stimulate J,, in
`rabbit ileum (Mandel & Eichold, 1988).
`The muscarinic receptor antagonist atropine was used to determine whether the
`TTX-sensitive portion of the NKA response was dueto the release of acetylcholine.
`At a concentration sufficient
`to completely block the increase in J,, due to
`bethanechol, atropine failed to prevent the J,, response to NKA. This suggests that
`acetylcholineis not likely to be responsible for the TTX-sensitive component of NKA
`action.
`
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`348
`
`X.-Y. TIEN AND OTHERS
`
`Tachykinins can release histamine from peritoneal mast cells (Johnson & Erdos,
`1973; Erjavec et al. 1981; Keast, Furness & Costa, 1985), and histamine stimulates
`intestinal electrolyte secretion through activation of H, receptors from different
`species (Fromm & Halpern, 1979; Linaker, McCay, Higgs & Turnberg, 1981; Cooks,
`Nemeth & Wood, 1984; McCabe & Smith, 1984; Hardcastle & Hardcastle, 1988;
`Kachur, Allbee & Gaginella, 1988). However, based on the fact that pyrilamine (an
`H,receptor antagonist) did not prevent the NKA response, histamine is not likely
`to be responsible for the effects of NKAin the rat colon. Other investigators (Kachur,
`Miller, Field & Rivier, 1982; Keast et al. 1985) also reported that H, blockers failed
`to inhibit increases in J,, elicited by another tachykinin, substance P.
`Our studies implicate VIP in the response to NKA. VIP-like immunoreactivity is
`present in submucousneurons(Ferri, Adriah, Ghatei, O'Shaughnessy, Probert, Lee,
`Buchan, Polak & Bloom, 1983) and VIP is well known to stimulate intestinal
`epithelial chloride secretion both in vivo and in vitro. Electrical field stimulation has
`been demonstrated to release VIP into the serosalfluid from segmentsof rabbit ileum
`mounted in Ussing chambers (Gaginella, O’Dorisio & Hubel, 1981) and NKA may
`evoke the neural release of VIP from the cat jejunum in response to noxious stimuli
`(Brunsson, Fahrenkrug, Jodal, Sjoqvist, Theodorsson & Lundgren, 1990). The effect
`of VIP on J,, is similar to NKA in our study and VIP antiserum blocked theeffect
`of NKA. VIPis also suggested to be involved in neurokinin inhibition of cholinergic
`myenteric neurons in the canine antrum (Mayer, Koelbel, Snape, Vandeventer &
`Leduc, 1990). Since NKA, depending on concentration, may affect both substance P
`and NKAreceptors (Maggio, 1988), we do not knowif VIP release from submucous
`neurons is physiologically regulated by NKA, substance P or both peptides.
`The argumentfor neuronal release of VIP by NKAis strengthened by results from
`the VIP-NKA cross-desensitization studies, which demonstrated that prior
`desensitization of epithelial VIP receptors by VIP decreased the sensitivity of the
`tissue to NKA.
`Ourstudies do not rule out the possibility that other mediators are involved in the
`NKAresponse. Pre-treatment with indomethacin also attenuates the NKA response
`in our preparation (data not shown). This is consistent with previous observations
`that NKAreleases prostacyclin from cultured endothelial cells (Marceau, Tremblay,
`Couture & Regoli, 1989). The role of prostanoids in mediating the effects of
`tachykinins certainly deserves future study.
`
`Weare grateful to Mary Lasquety for expert secretarial assistance and to Dr Jack Grider for
`helpful advice on the use of the VIP antibody.
`
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`NEUROKININ A INCREASES SHORT-CIRCUIT CURRENT—349
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`SHANAHAN, F. & Anton, P. (1988). Neuroendocrine modulation of the immune system. Possible
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