The depolarising action of capsaicin on rat isolated sciatic nerve

Life Sciences, Vol. 35, pp. 1561-1568 Printed in the U.S.A.

Pergamon Press

THE DEPOLARISING ACTION OY CAPSAICIN ON RAT ISOLATED SCIATIC NERVE A.G. Hayes, A.S. Hawcock and R.G. Hill* Department ot Pharmacology, Glaxo Group Research Ltd., Greenford, Mddlesex. (Received in final form July 24, 1984) Summary The effect of capsaicin on the Isolated sciatic nerve of rat was studied by extracellular recording of membrane polarisation. Capsaicrn depolarised the sciatic nerve, but desensrtizatlon occurred rapidly upon repeated admrnistratron. Several other neuroactive substances, including substance P, were inactive. The depolarisation was reduced in nerves depleted of unmyelinated fibres by neonatal capsaicin treatment, suggesting that it occurs mainly in C-fibres. This depolarising action of capsarcin could explain the irritant and acute antinociceptive properties of capsaicin. Capsaicin is the pungent algesic substance found in red peppers. When applied topically or systemically, it produces initially an intense irritation, which has been attributed to the release of Substance P (SP) from smalldlameter primary afferent fibres (6,231. This is followed by a later phase in which the InJected animal shows lnsensitivlty to noxious stimuli. If capsaicin is given neonatally, it causes a degeneration of unmyelinated primary afferent fibres (14, 19) and a permanent marked reduction in the levels of various peptides contained in these fibres (5, 13). The only known behavioural changes accompanying these anatomical changes are rncreases in Repeated dosrng of adult rats with nociceptive thresholds (9, 12, 19). capsaicin causes a similar increase in nociceptive thresholds and loss of peptides from primary sensory neurons (5, 11). but these effects, although are not permanent, long-lasting, and are not accompanied by any fibre degeneratron. Small single doses of capsaicin given to adult rats also cause raised nociceptive thresholds lasting for a few hours (10). but this eftect is not accompanied by changes in peptide levels (17). The efkects of systemically administered capsaicrn on nociceptive thresholds and peptide levels can be mimicked by local application to a peripheral nerve (1, 15). It is thus of interest to determine the effects of capsaicin on the membrane properties of primary afferent fibres. In the present study, the effects of capsaicin on the isolated sciatic nerve of the rat were investigated, using the technique of extracellular membrane polarisatlon recording. methods Rats (AH random-bred Hooded, male, 250-3009) were anaesthetised with chloral hydrate (300mg/kg 1.p.)and a piece of sciatic nerve, approximately 1.5cm long

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~-

* Present address: Parke-Davis Research Unit, Hills Road, CambrIdge, CH2 2QB. Reprint requests to A.G. Hayes at the above address. 0024-3205184 $3.00 + .OO Copyright (c) 1984 Pergamon Press Ltd.

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and O.5-0.7mm I" diameter, was dlssected from lust above the knee on each side. 'The pieces of nerve were desheathed under a mlcroscope and mounted I" 3compartment perspex baths. 'i'henerve was threaded through 2 greased slots such that the 2 cut ends lay 1" the outer compartments, while the remainder lay 1" the centre compartment. The tissues were contlnously supertused with hrebsHenselelt solution at 27OC. Drugs were applied at known concentration vld the perfusion stream into the centre compartment only. The D.C. potential between the centre and one outer compartment was recorded using Hg/AgCl electrodes connected to the tissue "la agar/fllter paper brldyes, and dlsplayed on a potentrometrlc chart recorder. The Krebs-Henselelt ,"zf,' 2:ipf;;2cn.?5,

solution used contained (mrl) rJa+ 143, Y+ 5.9, Mg2+ 1.2, H2P04-1.2 and D-glucose 11. 'The tollow~ny drugs were 8-methyl-N-vanlllyl-b-nonenamlde, bradyklnln, hlstamlne dlhydrochlorlde, muscarlne chloride, 5-hydroxytryptamlne creatlnlne sulphate, tetrodotoxin, (Sigma Chemicals Ltd.) and substance P (Peninsula Laboratories Ltd.). 'l'hecapsalcl" analogues used were synthesized in the Chemistry Research Departments, Glaxo Group Research LImIted, Ware and Greenford. I'helr structures are shown below.-

HO /C"3 CH30

CH2NHCO(CH2)4

CH=CH

Capsalcl"

CH \CH

3

HO

CH30

CH2NHCO(CH2)7CH3

AH 23491X

HO

CH30

CH2NHCO(CH2j4CH3

AH

23602X

CH30

CH30

kor superfusion I" 10% ethanol solutlo".

CH2CONH(CH2)

llCH3

onto sclatlc nerves, capsalcl" to make up a lmd solution, and

cc1

19382

and Its analogues were dissolved this was then diluted with Krebs

A few rats were treated on day 2 of life with capsacl" 50mg/kg s.c.. kor ln]ectlon, the capsalcl" was ground with gum acacia, made up to volume with saline and dispersed using a" ultra-sonicator. Controls were ln]ected with an appropriate volume of vehicle. The rats were allowed to grow to maturity and their sclatlc nerves were removed when the rats achieved a body weight ot 2503oog.

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I

10%

0.2mV

I

Capsalcin

3x 14%

Isolated

Sclatlc

Nerve

1563

I

Capsaicin

10%

Capsaicin

L -c----=-----

10 min

B

on Rat

Capsalcln

Vehicle

10%

Substance P

loo-

80-

Molar

FIG.

concenlratlon

1

A representative trace showing the depolarlslng effect of capsalcln on the A) rat Isolated sciatic nerve. The vehicle in which the capsalcln was dissolved was wlthout effect on membrane polarlsatlon. Also substance P, lpM, was Concentration-response curve showing the depolarlslng without eftect. to Each point represents the mean value calculated from actlon of capsalcln. four different preparations and standard error bars are shown.

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Results Capsalcln (300nl'l to 10~11) produced a dose-related depolarlsatlon of the Isolated sclatlc nerve, whereas the vehicle alone was wlthout effect (h'lg. 1). Tachyphylaxls rapldly developed unless an Inter-dose interval of greater than and even at this interval It was apparent with higher one hour was used, concentrations of drug (big. 2~). However, the depolarlslng action of a single dose of capsaicln did not appear to fade during a sustained application of the In capsalcln desensltlzed tissues, the dose-response curve to drug (Ug 2B). potassium depolarisatlon was unchanged. A variety ot other substances were also applied to sclatlc nerves, but of these, only IncreasIng the potassium Ion concentration to 10.6mM consistently caused depolarlsatlon. Substance P, bradyklnln, hlstamlne, muscarlne and 5hydroxytryptamlne, In concentrations up to lOOPPI, were all without dlscernable effect. In four experiments, Krebs solution contalnlng lOpPI tetrodotoxln was superfused over the nerve and It was consistently found that the depolarlsatlon produced by capsalcln perslsted In the presence of tetrodotoxln. Three

capsalcln

analogues,

AH 23491X.

AH 23602Xand

CC1

-I

A

/

/

19382,

were

examined

0 5mV

10mm

-

l’b 10 &A K+

10% Capsalcm

lO?Y 10 5rnM Capsalcm K+

1

0.2

mV

10 mm

1 Om5Y Capsaicm

FIG.

2

A representative trace showing tachyphylaxls resulting from appllcatlon of A) 55mln later, dose, admInIstered a single dose of capsalcln, lOPPi. A subsequent the depolarlslng response resultlnq had a conslderably reduced eftect, whereas from IncreasIng the potassium ion concentration was unchanged. A representative trace showlnq that the depolarlslng response to capsa1cln B) did not fade when the drug was left In contact with the tissue for 45 ml". A slmllar result was obtalned =n four dltterent preparations.

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for their ablllty to depolarlse the isolated sciatic nerve. AH 23491X produced (mean capsalcln depolarlsation = slmllar depolarlsatlons to capsalcln at lpM cross60'12 V; mean AH 23491 depolarlsatlon = 63 t 19 V, n=3) and showed were without desensltlsatlon to capsalcln (Ylg. 3). The two latter compounds effect on membrane polarisation (n=4). Capsalcln treated

obtalned

depolarisatlons of sclatlc nerves drssected from rats that had been neonatally with capsalcln were slgnlflcantly smaller than those from control rats (Pig. 4).

Previous studies have shown that capsalcln depolarlses the cell bodies of sensory neurones ln the dorsal root ganglion (24) and also their central terminals III the dorsal horn of the splnal cord (2, 25). The present study extends these findlngs to show that capsaicln also depolarlses fibres along the length of the perlpheralnerve. This depolarlsation 1s not mediated via SP, bradyklnln, muscarlne, hlstamlne or serotonin receptors. The type of flbre within the sciatic nerve which 1s depolarlsed by capsaicln cannot be determined with any precision from our experiments. In rats treated neonatally with capsalcln, 90% of the C-flbres are destroyed (18). In nerves from rats treated thus, the capsaicln depolarisatlon was substantially reduced suggesting that a large part of the depolarlsation is mediated via C-flbres. 'The remalnlng depolarisation may be medlated either via any undamaged C-fibres or via myelinated flbres. Other studies have also shed some light on the types ot tlbres attected by capsalcln. Szolcsanyi (21), using multifibre recordings tram cat saphenous nerve III vlvo, found that capsaicin excited the slowlyconducting C2 flbres, whilst not affecting Aa, AB, A& or C, flbres.

A1;;23491x 1o-6 M k

FIG.

After

10-6M desensltlslng

•i

3

Cross-desensltlsatlon between capsalcln and AH 23491X an the Isolated sclatlc On the left-hand side of the figure are control responses, on the nerve. right-hand side of the figure 1s shown the lack of depolarlslng effect of capsalcln and AH 23491X when admlnlstered concurrently with a desensltlslng dose of capsalcin, 3U11.

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Kecordlngs from single sensory nerve tlbres in the rat (16) and cat (4) have respond to capsalcln appllcatlon by an shown that only polymodal noclceptors floth of these studies also observed the rapid tachyphylaxls evoked discharge. which occurs with a subsequent appllcatlon of capsalcln and which was particularly noticeable ln our own experiments. The mechanism

100

underlyIng

the capsalcln

depolarlsatlon

1s unlikely

to involve

an

1

90 80

1

70

30 20 10 0l-

M concentration FIG.4 to capsalcln of sclatlc nerves taken from control Mean depolarlslng responses rats (open columns) and rats treated neonatally with capsalcln 50mg/kg S.C. *represents values slgnlflcantly dltferent from control at (black columns). p < 0.05.

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Increase rn axonal sodium conductance, as we were able to demonstrate a clear depolarlsatron rn the presence of tetrodotoxrn. It LS therefore of rnterest to note that rn voltage clamped sclatlc nerve frbres from the frog, the predomrnant eftect of capsarcrn 1s to block one of the rast components of potassium conductance (3).

It 1s possrble to hypothesize on the sequence of events whrch occur wlthrn prrmary sensory neurones following capsarcln admrnlstratron. Presumably, the depolarlsatlon lnltrally generates actron potentrals wlthln polymodal nocrceptors and these travel orthodromrcally to the dorsal horn and result in Thus could produce the release of substance P and possibly other peptldes. lnitral intense rrritation. The short duration of the rrrltatlon 1s unlikely to be because the depolarlsatron 1s not marntalned, as our experiments suggested that rt drd not fade rn the presence of the drug. It could, however, be a result of depolarlsatlon block of the nerve frbres and the subsequent acute antlnocrceptlve effect could depend II-I a slmrlar mechanism. In fact, Petsche, blelscher, Lembeck and Handwerker (20) have shown that capsarcrn blocks rmpulse traffic In those C-flbres respondrng to noxrous heat and mechanical stlmulr. Our data on the capsalcrn analogues also ravour the effect 1s a result of lnterpretatron that the acute antlnociceptrve depolarrsatlon block of parn flbres. Thus, AH 23491X, which produced an equrvalent depolarlsatron to capsarcln on the sciatic nerve and crossdesensltlzed to It, 1s approximately equlpotent with capsalcln In producrng antinoclceptlon (8). AH 23602X 1s very much less active than capsalcln as an antrnoclceptlve agent and CC1 19382 1s rnactrve, which correlates well with therr lnabllrty to depolarlse peripheral nerve frbres rn the present experiments. The way rn whrch this rnltlaldepolarlslng effect may lead to the prolonged rnsensrtlvrty to pain produced by capsalcrn 1s still unknown, although recent evrdence suggests that this may result from a depletron of termrnal transmitter stores consequent on a block of axoplasmlc transport (7).

References 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

A. AINSWOR'I'H,P. HALL, P.D. WALL, G. ALLT, M.L. MACKENZIE, S. GIBSON and J.M. POLAK, Pain 11 379-388 (1981). B. AUL'I and R.H. EVANS, J.Physrol. 306 22-23P (1980). J.M. DUBOIS, Brran Res. -245 372-375 (1982). and A.G. RANAGE, Neuropharmac. 20 191-198 (1981). R.W. POS'l'ER R. GAMSE., s.E. LEEMAN, P. HOLZER and Y. LErltlECK,Naunyn-Schmiedeberg's Arch. Pharmac. -317 140-148 (19811. R.GAMbb, A. MOLNAR and P. LElIBECK, Life Scl. 25 629-636 (1979). R. GAMSE, u. PETbCHE, F. LEmBhCK and G. JANEO, Brain Res. 239 447-462 (19821. A.G. HAYES, A. OXFORD, M. REYNOLDS, A.H. bHINGLER, M. SKINGLE, C. SMITH and M.B. TYERS, Life Scl. 34 1241-1248 (1984). A.G. HAYES, J.W. SCADDING, M. SKINGLE and M.& TYERS, J. Pharm. Pharmac. 33 183-185 (1981). XG. HAYES, M. SKINGLE and M.B. TYERS, Neuropharmac. 20 505-511 (1981). A.G. HAYES and M.B. 'TYERS,Brarn Res . 189 561-564 (19801. p. HOLZER, I. JURNA, R. GAi4SE and F. LEMBECK, Eur.J.Pharmac. -58 51 l-514 (1979).

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Pharmac. 313 91-94 11980). P. KENINS, Neuroscl. Lett. 29 83-88 (1982). F. LbMBECK and J. UONNhRER, Taunyn-Schmiedeberg's Arch. Pharmac. 316 240243 (1981). J.I. NAGY, S.P. HUN'I. L.L. IVtRSEN and P.C. hllSON, Weurosclence 5 19231934 (1981). S.R. VINCENT, W.A. STAIUES, H.C. kIBIGER, T.D. REISINE and J.I. NAGY, Brain Res. -186 435-444 (1980). H.I. YHMAMURA, U. PETSCHE, E. FLEISCHER, F. LEMBECKand H.O. HANUWERKER, Brain Res. 265 233-240 (1983). J. SZOLCSANYI, J. Physiol. (Paris) 73 251-259 (1977). J. SZOLCSANYI and A. JANCSO-GABOR, zznelm. Forsch. 26 33-37 (1976). E. THERIAULT, M. OTSUKH and 1'.J&SELL, Brain Res.17?209-213 (1979). J.T. WILLIAllS and W. ZIEGLGANSBERGER, Braln Res. -253 125-131 (1982). M. YANAGISAWA, 5. NAKANO and M. OTSUKA, niomed. Res. -1 Supplement BB-90 (19SO).