Europeat

9

Jurmal

of Pharmacology,

221 ( 199.2)

17~ 177

175

1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00

EJP 21130

Short communication

The effect of nedocromil sodium on the isolated rabbit vagus nerve Dale M. Jackson, Christopher

E. ?ollard and Susan M. Roberts

Received 5 August 1992. accepted

Nedocromil

sodium

dcpolarizcd

the isolated

rabbit

vagus nerve.

I I August 1992

The

depolarization

was blocked

by DIDS

(4,4’-diiso-

thiocyanostilbcnc-2,2’-disulphonic dcpolarizntion chloride

acid1 and did not occur when the nerve was bathed in solutions low in chloride. After the nerve was refractory !o nedocromil sodium for an hour. It is suggested that nedocromil sodium can affect a

chanr,:!. Vagus ncrvc; Ncdocromil

sodium; Ci-

channels; DIDS

1. Introduction Nedocromil sodium has been shown to stimu!ak: bronchial C-fibre endings in the dog lung, and it haps been suggested that it is this stimulation that acccunts for some of its beneficial action in man (Jackson et al., 1989). In view of this action nedocromil sodium has been applied to isolated vagus nerves taken from rabbits since the majority of the fibrcs in this nerve are type C-fibres.

2. Materials and methods Cervical vagi were dissected from young, adult New Zealand white rabbits (1.5-3 kg) killed by the i.v. injection of sodium pentobarbitone (200 mg/ml). The nerve trunks were dcsheathed and maintained in Krebs solution bubbled with 95% 0,-50/c CO, and chilled to about 5°C. The dcsheathed nerve was mounted in a 3-chambered perspex bath (Marsh et al., 1987). The chambers, separated by 1 mm wide partitions sealed with silicone grease, were filled with Krebs solution. The centre chamber was continuously superfused with Krebs solution (5 ml mine.‘) previously bubbled with 95% O,-5% CO, and at ambient temperature (1820°C). Changes in the DC potential recorded across the first partition with Ag/AgCl electrodes were assumed

(4,4’-diisothiocyanostilbcnc-2,2’-disulphonic

acid)

to represent changes in membrane potential. These were amplified and recorded on a potentiometric chart recorder (Sekonic SS 250F). Twenty minutes after setting up, an ED5(, dose of 5-HT (3 X lo-” M) was then given to assess the viability of the nerve. If the response to this dose of 5-HT was less than 0.6 mV the nerve was discarded. Agonists were added to the supcrfusing solution and left in contact with the nerve until a maximal response had been achieved. The superfusion solution was then ret:rrned to normal Krebs. The viability of the nerve was checked throughout the experiment by giving the standard dose of 5-HT (3 X lo-” M). Composition of the Krebs solution: NaCl 117.6 mM, KCI 5.36 mM, MgSO, * 7H,O 1.18 mM, CaClz 2.55 mM, NaH,PO, 0.89 mM, NaHCO, 25.0 mM, glucose 1I.10 mM. Tris replaced NaCl to obtain Na+-free Krebs, sodium isothionate replaced NaCl to obtain low chloride Krebs, and CaCI, was omitted and I mM EGTA added to obtain Caz+-free Krebs. Krebs deficient in either Na+, Ca*+ or Cl- was allowed to superfuse for at least an hour before agonist responses were studied. Drugs: pentobarbitone sodium (Animalcare); ncdocromil sodium (Fisons); r-arninobutyric acid (GABA) (Sigma); 4,4’-diisothiocyanostilbenc-2,2’-disulphonic acid (DIDS) (Aldrich).

3. Results Correspondence to: D.M. Jxtckson, Dcpartmcnt of Phwmacology. Fisons Research and Development Laboratories. Bakewell Road. Louyhborough. Leicestcrshirr LEI I ORII. UK.

Compared with 5-HT, nodocromil sodium caused a slow depolarization of the ncrvc (fig. 1A) which was not maintained during continuous perfusion of the

(

A /

G

S-HT3xld*M

I

0.5mV

1

-’ Nadocramil

6.6i

6

A---eaadium

ZixdM

T

The cffcct of removing Na’. CiI”, and Cl- on the rcsponsc of the vagus ncrvc to ncdocromil sodium wcrc studied by using Krcbs solution doficicnt in the appropriate ion. The vagal ncrvc pr~p~ir~ti~n TCspondcd normally lo ncdocmmil sodium in sodium fret irnd calcium free solutions, hut low chloride solutions prevcntcd responses to ncdocmmil sodium and to GABA ifig. I B). itfthough thz r:crvcs still responded to 5-HT. The chloride channel blocking drug DIDS (fig. 10 and IO ’ h! NPPB (S-nitrr~-‘-(3-phcnylprctpylamino)hcnzu;itc) (data not shown) iilstl inhihitcd the rcsponscs to ncdocromil sodium.

4. Discussion

Ncdocromil sodium ciiuscd a dose rclatcd SIOWdcp~)l~iriziiti~~nof the rabbit vagus IWVC. The d~p~~l~lriz~tion could not he maintained and once it had occurred it could not bc rcpcatcd fi;r ;:n hour.. During the refractory period to ncdocromil sodium the nerve re10-d spondcd n~~rrn~lllyto GABA itnd 5-HT. The dcpolarConcentration of NedPcromit Sodium ization caused by ncdocromil sodium was significantly 5; reduced by lowering tbc chloride concentration in tbc pa-fusing solution but it was Iitrgcly unaffcctcd by r~rn~~vin~Nit ’ itnd Cit’ ‘. Similar results wcrc found by Brown and Marsh (1978) when investigating the action of GABA on the isolated rat vagus. The depolarization to n~d~~~r~~rnilsodium was blocked by the chloride channel blocking drug DIDS. The results suggest that ncdocromil sodium dcpolarizcs the rabbit viigus ncrvc by opening it chloride channel. and after this opening it is possible that the chenncl is then blocked. The channel affcctcd is not the GABA-itctiv;lted chloride channel sincc GABA still products ii depolarization during the refractory pcriud to n~d~~r~~rnilsodium. Many mechanisms hiWc been offcrcd to cxplrrin the actions of ncdocromil sodium, but the possibility that it can block a chloride channel is attractive, since it could Explain how the drug affects mast cells and sensory ncffcs. compound. The return of the DC potential to basciinc Expcrimcnts using simultnncous mcasurcmcnt of took approximately IO min and fur about I h the ncrv~ mcmbranc current and intracellular calcium ([Gail) in ~2’: rcfrrtctory to n~d~~r~~rnilsodium. During this pcrat pcritara,::tl mast cells have shown that three ionic riud normal responses cc~~ldhc obtained to S-HT and mechanisms arc activated following rcccpfor stimulaGABA. By lcaving an hour bctwccn responses to nction which enhance secretion by maintaining clcvittcd docromil sodium. 3 dose response curve’ could bc oh[CrtJ First, a voltage dcpcndcnt cation channel through taincd. In order to compare relative maxima the dose which divaicnt ions can pass. Secondly, a llyp~rp~l~rrcsponsc Iinc to GABA is included on the sitmc graph ization driven calcium current activated by both cxtcr(fig. IBI. lntcrcstingly the rclatcd compound sodium nitI stimuli and by intrilccllularly applied IP,. Thirdly, a ~r:rmcgly~at~ did not cvtrkc dopolarising rcsponscs cvcti x”- chloride current, activated following rcccptor stimulaat a c~~n~~ntr~ti~~n of I mM. However IO J M sodium tion and also induced by i!it~rn~~llyapplied cAMP or by cromoglycato did inhibit the dcpolarisations caused by high [Cai]a This chloride current will clamp the mast nedocromil sodium. cc!! mcmhranc potential to ncgativc values thus provid-

177

ing the driving force for calcium influx through the first and second conductances (Penner et al., 1988). The ability of nedocromil sodium to stabilize mast cells (Eady et al., 19855)may result from its ability to block this chloride channel and thercforc limit calcium entry. Indeed, there is direct evidence that sodium cromoglycats, a compound similar to nedocromil sodium. can block chloride channels in mast cells (Romanin et al., 1991). In experiments not reported here, sodium cromoglycate, although not producing a depolarisation itself. did block the depolarisation produced by ncdocromil sodium. This observation would also support the idea that sodium cromoglycate can block a chloride channel. Inhalation of solutions with a low chloride concentration cause coughing in man and this effect is blocked by prior treatment with nedocromil sodium (Eschenbather et al., 1984). Furthermore in puppies low chloride solutions instilled into the larynx cause a prolonged reflex apnoca by stimulation of afferents in the superior laryngeal nerve (Boggs and Barllett 1982). There is evidence, therefore, from experiments in animals and man, that low chloride solutions can stimulate airway sensory nerves. This suggests that diffusion of chloride ions from sensory nerves results in their depolarization and the generation of action potentials. Chloride ions are normally abundant in tracheal fluid into which they are actively secreted by the tracheal epithelium. The dilution of this fluid by inhalation of nebulised distilled water may result in the diffusion of chloride ions from the sensory nerves and this may cause cough. Nedocromil sodium may block fog in-

duced cough by blocking chloride channels on sensory nerves in the lung. The results presented in this paper offer a mode of

action for nedocromil sodium which could explain its actions in man. Single cell and patch clamp experiments will be required before these theories can be confirmed.

References Boggs. O.F. and D. Ba<.

1982. Chemical

apnoeic reflex in puppies, Exercise Physiol. 53. 455. Brown, D.A.

and S. Marsh,

malian peripheral

Respir.

Environ.

1978. Axonal GABA-receptors

in mam-

nerve trunks, Brain Res. 156, 187.

Eady, R.P.. B. Greenwood.

D.M.

Jackson, T.S.C.

19%. The effect of nedocromil choconstriction

specificity of a laryngeal

Appl. Physiol.

J.

Orr and E. Wells,

sodium on antigen-induced

in the Ascaris-sensitive

bron-

monkey, Br. J. Pharmacol.

85, 323. Eschenbacher,

W. L.. I1.A.

Boushey and D. Sheppard,

1984, Alter-

ation in osmolarity of inhaled aeros-11s cause bronchoconstriction aad cough. but absence of a permeant Am. Rev. Respir. Jackson, D.M.,

A.A.

anion causes cough alone,

Dis. IZY, 211. Norris and R.P. fidy,

1YXY. Nedocromil

acd sensory nerves in the dog lung, Pulm. Pharmacol. Marsh, S.J.. D.A.

Stansfield,

sodium

2, 179.

R. Brown, R. Davey and D. McCarthy,

19x7, The mechanism of the action of capsaicin on sensory C-type neurons and their axons in vitro, Neuroscience Penner,

R.P.,

G.

Matthews

and E.

Neher,

23, 275.

1988,

Regulation

of

calcium influx by second messengers in ml mast cells, Nature 334, 40’). Romanin,

C..

M.

Immunologically

Reinsprecht.

I. Pecht

and

H.

Schindler,

iYY1.

activated chloride channels involved in degranu-

lation of rat mucosal mast cells, EMBO

J. 10, 3603.