`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 AND TIMOTHY S. 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 may be involved 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 I se, 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 'Sc with an EC50 (half-maximal effective concentration) value of 7.5 nm.
`The maximum (mean + s.E.M.) increase in /se (µA/cm2) for NKA was 111± 10.
`5. Tetrodotoxin (0.5 pm) and bumetanide (10 pm), but not atropine (1.0 pm),
`hexamethonium (100 pm) or pyrilamine (10µM), significantly inhibited NKA-
`induced increases in Is,.
`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 ISe 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 fl-preprotachykinin A and y-preprotachykinin A
`(Maggio, 1988). It shares a common C-terminus and 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.
`t 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
`
`MYLAN EXHIBIT - 1040
`Mylan Pharmaceuticals, Inc. v. Bausch Health Ireland, Ltd. - IPR2022-00722
`
`
`
`342
`
`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
`enzyme release by neutrophils (Shanahan & Anton, 1988).
`Recently, NKA was shown under short-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 NaCI). 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 cm2) and bathed
`with buffered physiological saline solution composed of (mm): NaCl, 120.2 ; KCI, 5.9; CaC12, 2.5;
`MgC12, 1.2; NaH2PO4, 1.2; NaHCO,, 25; glucose, 11.1 (pH 7.4, 37 °C), gassed with 95% O2 and
`5 % CO,.
`
`Electrical measurements
`Electrical potential difference (PD) was monitored at the beginning of each experiment with a
`pair of saturated KC1—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 (4c) was applied through silver—silver
`chloride electrodes to the tissue to clamp open-circuit PD (potential difference) at zero, and
`compensation was made for fluid resistance. Initial tissue conductance (Gt.) was calculated from the
`values of 'Sc and PD according to Ohm's law. The reciprocal of Gt (mS/cm2) times 1000 equals the
`tissue resistance (R, R cm2).
`
`Concentration—response curves
`After mounting, the tissues were allowed to equilibrate for at least 30-60 min; effects on
`presumed electrogenic chloride secretion were monitored as changes in i s,. The concentration
`required to increase Ise by 50% (EC„) was calculated from the curves by non-linear least-squares
`regression.
`
`
`
`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 poor tissue
`viability, was responsible for the lack of response. Antagonist controls were conducted on a
`separate tissue from the same animals.
`
`A
`
`B
`
`0.05
`
`0.01
`
`1.0 µM-NKA
`
`0.1
`
`5 min
`
`E OO
`
`
`
`Fig. 1. Representative effect of NKA (UM) on I„ 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 /se 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 /„. 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 t test was used
`for testing statistical significance.
`
`RESULTS
`
`Effect of NKA on 'Sc
`NKA evoked a rapid but transient increase in /se upon serosal addition (Fig. 1).
`The responses were concentration dependent over a range of 0.5-100 nivi (Fig. 2); the
`EC50 value was determined to be 7.5 nM. The maximum response to NKA was
`111 + 10 itA/cm2 (n = 26 tissues from thirteen animals).
`
`
`
`344
`
`X. - Y. TIEN AND OTHERS
`
`9
`
`7
`8
`-log [NKA] (M)
`
`I
`
`6
`
`140-
`
`120
`
`100-
`
`80-
`
`60-
`
`40
`
`20
`
`0
`
`Increase in 'Sc (µA/cm2)
`
`Fig. 2. Concentration-response curve (serosal addition) for NKA-induced increases I se
`across rat colonic mucosa. Results are expressed as the change in I se (itA/cm2) above
`baseline vs. log concentration of NKA. Symbols and bars represent the means + S.E.M. of
`determinations for at least twelve animals. The maximal increase in Ise was achieved at
`10-7-10-6 M.
`
`*
`
`TTX
`(0.5µM)
`
`Hexamethonium
`(100µM)
`
`Atropine
`(1 µM)
`
`150
`
`100
`
`50
`
`Percentage of maximal NKA response
`
`Fig. 3. Effects of tetrodotoxin (TTX), atropine and hexamethonium against NKA
`(50 nM). Inhibitors were added 10 min before NKA. Results are expressed as
`means + S.E.M. of three to eight animals. *P < 0.05.
`
`Effects of inhibitors
`To substantiate that the NKA-evoked increase in 'Sc was due to active chloride ion
`secretion, tissues were incubated with bumetanide (10 µM, 30 min) before adding
`NKA to the tissue bath. Responses to a maximally effective concentration (50 nM)
`
`
`
`NEUROKININ A INCREASES SHORT-CIRCUIT CURRENT
`
`345
`
`of NEA were significantly (P < 0.001) reduced by serosal addition of bumetanide to
`14+4% of control. Unidirectional flux measurements with 36C1- could not be
`conducted due to the transient nature of the response.
`TTX (0.5 ym) inhibited by 93% the J. response to 50 nM-NKA (Fig. 3). Atropine
`(1 µm, 10 min), which completely inhibited the response to 0.4 mM-bethanechol, had
`
`400 -
`
`300-
`
`200 -
`
`100-
`
`0
`
`.c
`4.)
`
`U
`
`Hist
`
`Hist
`pyrilamine
`
`NKA NKA
`pyrilamine
`
`Fig. 4. Effects of the H, receptor antagonist pyrilamine (10µm) 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.
`
`no significant effect on responses elicited by NKA; the ganglionic blocker,
`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
`determine if it would block the NKA-induced response. Pyrilamine (10 µm, 15 min)
`significantly blocked the histamine (1 mm) but not the NKA (50 nM) response
`(Fig. 4).
`The effect of VIP antisera on the 'Sc 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
`
`
`
`346
`
`X. - Y. TIEN AND OTHERS
`
`desensitization. The desensitized tissues were responsive to subsequent application of
`bethanechol (0.4 mm) and aminophylline (20 mm, data not shown). We also
`investigated whether desensitization to VIP could block the effect of NKA. After the
`response to 0.5 pm-VIP had returned to baseline, the response to a second addition
`
`Control
`+ VIP Ab (1 : 200)
`
`7
`8
`-log [NKA] (M)
`
`6
`
`Control
`+ VIP Ab (1 : 200)
`
`A
`
`120
`
`E
`
`80
`
`G)
`
`40
`
`200
`
`150
`
`E
`
`100
`
`a)
`
`50
`
`9
`
`7
`8
`-log [VIP] (M)
`
`Fig. 5. Effect of pre-treatment of rat colon with VIP antisera (VIP Ab) (1:200) on NKA-
`induced (A) and VIP-induced (B) increases in I
`. Symbols and bars are means+ S.E.M.,
`n = 4 animals. *P < 0.05.
`
`
`
`NEUROKININ A INCREASES SHORT-CIRCUIT CURRENT
`
`347
`
`of 0.5 ,um-VIP was reduced by 50% or greater. The subsequent addition of NKA
`after desensitization to VIP was also attenuated (Fig. 6).
`
`<.7".
`E
`ic
`
`J
`c _
`a) ca
`co
`a)
`U
`C
`
`100
`
`80—
`
`60-
`
`40-
`
`20-
`
`0
`
`----.O---
`--IF—
`
`Control
`Desensitized to 0.5 µM-VIP *
`
`*
`
`*
`
`9
`
`8
`-log [NKA] (M)
`
`7
`
`6
`
`Fig. 6. Effect of desensitization to VIP on the response to NKA-induced increases in 'Sc.
`Symbols and bars are means+ S.E.M., n = 4 animals. *P < 0.05.
`
`DISCUSSION
`The present study demonstrates that NKA stimulates 'Sc 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 secretagogue in this tissue. Because
`the transient nature of the Is, response elicited by NKA precluded measurement of
`ion fluxes, we examined the ionic basis of action of NKA with bumetanide, a chloride
`transport inhibitor. Bumetanide inhibited the NKA-induced increase in I se,
`suggesting that electrogenic chloride secretion is a major contributor to the is,
`response. NKA was ineffective 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 is, (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 is, 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 due to the release of acetylcholine.
`At a concentration sufficient to completely block the increase in I se due to
`bethanechol, atropine failed to prevent the is, response to NKA. This suggests that
`acetylcholine is not likely to be responsible for the TTX-sensitive component of NKA
`action.
`
`
`
`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 Hi 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 NKA in the rat colon. Other investigators (Kachur,
`Miller, Field & Rivier, 1982; Keast et al. 1985) also reported that H, blockers failed
`to inhibit increases in 'Sc elicited by another tachykinin, substance P.
`Our studies implicate VIP in the response to NKA. VIP-like immunoreactivity is
`present in submucous neurons (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 serosal fluid from segments of 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 IS, is similar to NKA in our study and VIP antiserum blocked the effect
`of NKA. VIP is 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 NKA receptors (Maggio, 1988), we do not know if VIP release from submucous
`neurons is physiologically regulated by NKA, substance P or both peptides.
`The argument for neuronal release of VIP by NKA is 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.
`Our studies do not rule out the possibility that other mediators are involved in the
`NKA response. Pre-treatment with indomethacin also attenuates the NKA response
`in our preparation (data not shown). This is consistent with previous observations
`that NKA releases prostacyclin from cultured endothelial cells (Marceau, Tremblay,
`Couture & Regoli, 1989). The role of prostanoids in mediating the effects of
`tachykinins certainly deserves future study.
`
`We are 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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