Direct cutaneous hyperalgesia induced by adenosine

Neuroscience

Vol. 38, No. Printed in Great Britain

0306-4522/90$3.00+ 0.00

3, pp. 757-762, 1990

DIRECT

Pergamon Press plc 0 1990 IBRO

CUTANEOUS

HYPERALGESIA ADENOSINE

INDUCED

BY

Y. 0. TAIWO and J. D. LEVINE* Departments of Medicine, Anatomy and Oral Surgery and Division of Neurosciences, University of California at San Francisco, CA 94143, U.S.A. Abstract-The intradermal injection of adenosine produces a dose-dependent decrease in mechanical nociceptive threshold in the hindpaw of the rat that is not attenuated by elimination of indirect pathways for the production of hyperalgesia. Adenosine-induced hyperalgesia is mimicked by the AZ-agonists, S-(N-ethyl)-carboxamido-adenosine and 2-phenylaminoadenosine but not by the A,-agonist, N6-cyclopentyladenosine and antagonized by the adenosine A,-receptor antagonist, PD 081360-0002 but not by the A,-antagonist, 1,3-dipropyl-8-(2-amino-4-chlorphenyl)xanthine. The latency to onset of adenosine and 2-phenylaminoadenosine hyperalgesia is similar to that produced by prostaglandin E,, a directly acting hyperalgesic agent but shorter than that produced by leukotriene B,, which acts indirectly. 2-Phenylaminoadenosine hyperalgesia is prolonged by rolipram, a phosphodiesterase inhibitor. Both 2-phenylaminoadenosine and prostaglandin EZ hyperalgesia are antagonized by the A,-agonist N6-cyclopentyladenosine and the mu-agonist, [D-Ala*, NMe-Phe4, Gly-ollenkephalin. However, I-acetyl-2-(8chloro-lO,l l-dihydrodibenz[b,f]oxazepine-lO-carbonyl)hydrazine, a prostaglandin-receptor antagonist, inhibits _ nrostaelandin E,. (Taiwo and Levine, Brain Res. 458, 402406, 1988) but not 2-phenylamino_ . adenosine hyperalgesia and PD 081360-0002, the adenosine &eptor antagonist, inhibits 2-phenylaminoadenosine but not prostaglandin E, hyperalgesia. These data suggest that adenosine is a directly acting agent that produces hyperalgesia by an action at the AZ-receptor and that this hyperalgesia is mediated by the CAMP second messenger.

observed in the setting of inflammation is a reflection of a decrease in threshold in primary afferent nociceptors.‘s2*6 Some hyperalgesic inflammatory mediators, such as bradykinin, exert their effects indirectly by acting on other cells to produce mediators that, in turn, act directly on primary afferents.28.~~32*5’ So far, all known directly acting hyperalgesic agents-prostaglandin E, @GE,), prostaglandin I, (PGI,) and 8(R), 15(S)-dihydroxyicosa-(5E, 9, 11, 13Z)-tetraenoic acid (8(R), 15(S)diHETEFare eicosanoid products of the cyclooxygenase or lipoxygenase pathway of arachidonic acid metabolism.‘~19~5’~s3 The hyperalgesia induced by all of these directly acting agents is thought to be mediated by the CAMP second messenger system.‘8.49 Adenosine, which is generated in large amounts in hypoxic and ischemic tissue, such as occurs in inflammatory lesions, i’s*’has been shown to activate

unmyelinated afferents7~2s~36.39to produce pain in humans3s4’ and to elicit nociceptive behaviors in animals.’ Since adenosine increases CAMP, by an

The hyperalgesia

*To whom correspondence should be addressed at: Division of Rhematology and Clinical Immunology, U-426/ Box 0724, University of California at San Francisco, San Francisco, CA 94143-0724, U.S.A.

CPA, N6cyclopentyladenosine; CV1808, 2phenylaminoadenosine; DAMGO lo-Ala*, NMe-Phe4,

Abbreviations:

Gly-ollenkephalin; 8(R), 15(S)-di HETE, 8(R), lS(S)-dihydroxyicosa-(5E. 9. 11. 132)-tetraenoic acid: HTOZ. PD 081360~OtJO2;NECA, 5’-(N-ethyl)-carboxamidoi adenosine; 6-OHDA, 6-hydroxydopamine; PACPX, 1,3-dipropyl-8-(2-amino-4-chlorphenyl)xanthine; PGE,, prostaglandin E,,PGI,, prostaglandin I,; SC19220, lacetyl-2-(8-chloro-10, 1I-dihydrodibenz[b, floxazepine10carbonyl)hydrazine.

action on the adenosine A2-receptor,‘o~‘2*42~47 we investigated whether adenosine would also produce hyperalgesia. EXPERIMENTAL PROCEDURES A Basile Analgesymeter@ (Stoelting, Chicago, IL) was used to quantify the paw-withdrawal threshold reflex in 250300g male SpragucDawley rats (Bantin and Kingman, Fremont, CA). This device generates a mechanical force that increases linearly with time. The force is applied, by a dome-shaped plunger, to the dorsum of the rat’s hindpaw. The nociceptive threshold is defined as the force, in grams, at which the rat withdraws its paw. During the week prior to gathering the reported data, rats were trained in the paw-withdrawal test, at S-min intervals, for a period of 2 h each day. This procedure increases the sensitivity of this test.” On the day of the experiment, the rats were exposed to the test stimulus at 5-min intervals for 2 h prior to intradermal injection of the test agent. The baseline threshold is defined as the mean of the last six determinations just prior to intradermal injection of the test agent. The test agent was then injected intradermally, in a volume of 2.5~1, in each hindpaw. Three withdrawal thresholds were then measured at 5-min intervals. The mean of these three values is defined as the paw-withdrawal threshold in the presence of the test agent. The effect of the test agent is calculated as the percentage change from baseline paw-withdrawal threshold. Further doses of test agents, each one order of magnitude greater than the previous, were administered, at 25-min intervals, and the effect on pawwithdrawal threshold similarly defined in relationship to baseline paw-withdrawal threshold. To evaluate the onset latency of hyperalgesia produced by a test agent, the average

757

758

Y.0.TAIWOand J. D. LEVINE

of eight to 10 paw-withdrawal thresholds measured at I-min intervals was defined as baseline threshold. The test agent was then injected intradermally, in a volume of 2.5 ~1 and the paw-withdrawal threshold measured at I-min intervals over the next 20 min. To assess the effect of phosphodiesterase inhibitionwhich has previously been demonstrated to prolong the duration of hyperalgesia induced by directly acting hyperalgesic agentss-n the duration of 2-phenylaminoadenosine (CVl808) hyperalgesia, 1 pg of rolipram was co-injected with I pg CVl808 and nociceptive thresholds were measured at 5-min intervals for 120 min. Mean responses at 10,30,60, 90 and 120 min were then compared with similar responses in a group of rats that received just 1 pg of CVl808. The test agents used in this study were: adenosine; N6-cyclopentyladenosine (CPA), an adenosine A,-agonist’.*“.59(Sigma, St Louis, MO); CV1808, an adenosine A,-agonist5~3s~54~w (R esearch Biochemical Inc., Natick, MA); 5’-(N-ethyl)-carboxamido-adenosine (NECA) (Boehringer Mannheim, F.R.G.; 1,3-dipropyl-8-(2-amino-4-chlorphenyl)xanthine (PACPX), an adenosine A,-antagonists,” (Research Biochemicals Inc., Natick, MA); PD 081360-0002 (HTQZ), an adenosine A,-antagonist4 (Warner-Lambert Pharmaceuticals, Ann Arbor, MI); PGE,, a directly acting hyperalgesic agent’~51~s3 (Sigma, St Louis, MO); I-acetyl-2(8-chloro- 10,ll -dihydrodibenz[b,f]oxazepine- lO-carbonyl)hydrazine (SCl9220), an E-type prostaglandin-receptor antagonist’[email protected]’ (courtesy, Dr Donna Hammond, G. D. Searle, Skokie, IL), rolipram, a phosphodiesterase inhibitor’3.37.45(a generous gift of Berlex Laboratories, Cedar Knolls, NJ) and [D-Ala2, NMe-Phe4, Gly-ollenkephalin (DAMGO), a mu-opiate receptor agonist23.26 (Peninsula Laboratories, Belmont, CA). PACPX and HTQZ were dissolved in dimethylsulfoxide, stock solution (4 mg/ml) of CV 1808, rolipram and PGE, were made in 1:1, 1: 9 and I : 9. ethanol: saline solutions, respectively, with further dilutions in saline. All other test agents were dissolved in saline. To eliminate sympathetic postganglionic neuron terminals in the skin, 6-hydroxydopamine (6-OHDA) in a vehicle of chilled ascorbic saline (1 .O%), was injected intraperitoneally over a seven-day period ending 24 h before the experiment. The rats received a 50mg/kg dose on days 1 and 2, and lOOmg/kg on days 3, 4 and 7. This protocol destroys sympathetic efferents, producing in various tissues, an 80-95% depletion of peripheral adrenergic transmittersS5 and abolishing the hyperalgesic effects of bradykinin and norepinephrine.j2 In addition, these rats also received hydroxyurea in doses of 200 mg/kg on day 5 and 100 mg/kg on days 6 and 7, to eliminate polymorphonuclear leukocytes that have been implicated in the effects of leukotriene B,, an indirectly acting hyperalgesic agent.29 To eliminate products of the cyclooxygenase pathway of arachidonic acid metabolism, rats received indomethacin, in a vehicle of 2% sodium bicarbonate titrated to pH 7.2 with monosodium phosphate, at a dose of 4 mg/kg, i.p. This eliminates the cyclooxygenase pathway of arachidonic acid metabolism35,57 and abolishes the hyperalgesic effects of bradykinin and norepinephrine.32 Dose-response curves were statistically compared using a two-factor repeated measures analysis of variance.

0-oControl .-•6OHM + hydroxyurea A-Alndomsthacin

1 ng

The intraderrnal injection of adenosine produced a dose-dependent decrease in nociceptive threshold (i.e. hyperalgesia) (Fig. 1) [main effect of dose, F(3, 51) = 37.60, P < O.OOl]. This hyperalgesia was not significantly attenuated after sympathectomy and neutroPhil-depletion or after indomethacin [main effects of treatment F(1,28) = 3.29 and F(1,21) = 0.16, P ZS0.05, P > 0.05, respectively] (Fig. 1).

100ng

1JLg

Fig. I. Dose-dependent effects of intradermally injected adenosine on paw-withdrawal nociceptive thresholds in control rats (0, n = 18) or in rats that had been pretreated with 6-OHDA and hydroxyurea (0, n = 12) or with indomethacin (A, n = 6). In this and subsequent figures, responses are graphed as percentage change from baseline threshold after intradermal injection of various agents. Each point represents the mean & S.E.M. o-ob-ledm

* -15

1 1w

long

100 ng

’ Irg

Fig. 2. Dose-dependent antagonism of adenosine-induced hyperalgesia (0, n = 18) by the A,-antagonist HTQZ [lo ng (0, n = 6) 100 ng (A, n = 6) and 1 pg (A, n = 6)].

1w RESULTS

long

10 “g

100 ng

1w

Fig. 3. Lack of effect on the dose-dependent hyperalgesia induced by adenosine (0, n = 18) of 1 /Ig of the A, adenosine antagonist PACPX (0. n = 4). To evaluate the relative contribution of A,- and A,-receptors to adenosine-induced hyperalgesia, ligands showing selectivity for these receptors were employed. Adenosine-induced hyperalgesia was antagonized in a dose-dependent fashion by the A,receptor antagonist HTQZ [main effect of treatment

159

Adenosine hyperalgesia in rat skin A--hmlol+rh,-

A-AwmB+loh,wm q--owlme+l~wm

2

I

15

a 3

0

B II

-15 1 1 ng

Fig. 4. Dose-dependent effects of adenosine (0, n = 18), CV1808(@, n = 14),NECA (A, n = 8) and CPA (A, n = 8) on paw-withdrawal threshold. o-ocv

o-m.%

10 ng

lpg

Fig. 7. Lack of effect on CVlIOB-induced hyperalgesia of the At-antagonist PACPX [l ng (0, n = 12), 10 ng (A, n = 12), 1OOng (A, n = 12) and 1 pg (0, n = 12)].

mm lmn

1Oong

w3gnl

+ -

A-APGEZ A-A LTB4 2_

wT

-101 1 ng

long

1OOng

lpg

Fig. 5. Lack of effect of indomethacin (0, n = 6) on the dosedependent effects of the AZ-agonist CV1808 on nociceptive paw-withdrawal thresholds (0, n = 141.

*

-15

1

Minutes

Fig. 8. Paw-withdrawal at 1 min intervals after adenosine (0, n = 12) PGE, (A, n = 18) and

Oi

1 "9

10 "'1

1OOng

lag

Fig. 6. Dose-dependent antagonism of CV1808 induced hyperalgesia (0, n = 14) by the AZ-antagonist HTQZ [l ng (O,n=6), lOng(A,n=6), lOOng(A,n=6)and ipg (CL n = 6)].

F(3, 32)=4.96, P c 0.01; P < 0.05 at 10 ng, P < 0.01 at 100 ng and P < 0.01 at 1 pg, by Dunnett’spost-hoc comparison] (Fig. 2) but not by the A,-receptor antagonist PACPX [main effect of treatment F( 1,20) = 1.68, P > 0.051 (Fig. 3). In addition, the adenosine AZ-agonists NECA and CV1808 produced dosedependent decreases in nociceptive threshold [significant main effects of dose F(3,21)= 14.48, P < 0.001 and F(3, 39 = 17.72, P < 0.001, respectively] (Fig. 4)

thresholds measured before and the intradermal injection of 1 pg 1 pg CV1808 (0, n = 12) 108 ng 1OOngleukotriene B, (A, n = 12).

”

0

30

60

90

I

120

150

nm* (mhuta)

Fig. 9. The hyperalgesia induced by the intradermal injection of 1 pg CV1808 alone (0, n = 6) or when co-injected with 1 pg of the phosphodiesterase inhibitor rolipram (0, n = 6) at 10, 30, 60, 90 and 120min after injection.

of peak magnitude similar to adenosine hyperalgesia while the adenosine Al-agonist CPA, up to a dose of 1 pg, was without significant effect [main effect of dose F(3,21) = 0.81, P > 0.051. The hyperalgesic effect of CV1808, like that of adenosine, was not attenuated in indomethacin pretreated rats [main effects of treatment F(1, 18)=0.31, P > 0.051 (Fig. 5). Like adenosine-induced hyperalgesia, CVl808-induced

Y. 0. TAIWO

and J. D.

LEVINE 0-_Ocvr*a l -.mNm A-ACWlc*+ A-AWad P

w

1 ng

10

“g

100 ng

1 fJg

Fig. 10. (A) The dose-dependent antagonism of CV1808hyperalgesia (0, n = 14) by CPA [l ng (0, n = 6), long (A, n = 6) and 100 ng (A, n = 6)].

30

+

rceg ouwo

llsIwro0 + rQ@ruum

T

-10 1 1 “9

10 “g

100ng

l/q

Fig. 11. The dose-dependent inhibition of CV 1808-induced hypemlgesia (0, n = 14) by the mu-opioid agonist DAMGO [lo0 ng (0, n = 6). 1 pg (A, n = 6) and 1Opg (A, n = 6)].

P > 0.05 by Dunnett’s]. The antagonism of PGE2induced hyperalgesia by the A,-agonist CPA was, however, blocked by co-injection of the A,-antagonist PACPX [main effect of treatment F(l, 18) = 0.29, P z=-0.05 by Dunnett’s test] (Fig. 10B). DISCUSSION

Inp

,onp

*mnp

1w3

Fig. 10. (B) The dose-dependent effects of PGE, on pawwithdrawal thresholds (0, n = 12) and its modification by 1 ng CPA (0, n = 6), 10 ng CPA (A, n = 6), 100ng CPA (A, n =6), lpg CPA (0, n=6) and lpg CPA+ lpg PACPX (m, n = 8). hyperalgesia was antagonized by the A,-antagonist HTQZ [main effect of treatment F(4,28)=2.68, P < 0.05 by Dunnett’s in presence of 1 pg of HTQZ] (Fig. 6) but not by the A,-antagonist PACPX [main effect of treatment F(4, 52) = 0.78, P > 0.051 (Fig. 7). The latency to onset of CV1808-induced hyperalgesia was similar to that produced by adenosine or the directly acting agent PGE, (Fig. 8). In contrast, the latency to onset of the hyperalgesia induced by the indirectly acting hyperalgesic agent leukotriene B4 was much longer. The phosphodiesterase inhibitor rolipram significantly prolonged the duration of CVl808-hyperalgesia [main effect of treatment at 120 min F (1, lo)= 19.38, P = O.OOl](Fig. 9). The hyperalgesia induced by CV1808 (Fig. 10A) and PGE, (Fig. lOB), were both antagonized in a dose-dependent fashion by the A,-agonist CPA. The mu-opiate agonist DAMGO, at doses that have been shown to inhibit PGE,-hyperalgesia3’ also inhibited CV 1808-hyperalgesia [main effect of treatment F(2,23) = 6.983, P < 0.011 (Fig. 11). The PGE,-receptor antagonist SC19220 had no effect on CV1808induced hyperalgesia [main effect of treatment F(2,23) =0.12, P > 0.051. In addition, the A,-antagonist HTQZ did not inhibit PGE,-induced hyperalgesia [main effect of treatment F(4,31) = 3.25,

In this study we have shown that adenosine, which activates unmyelinated afferents7*25,36’39 and produces pain 3S48 also lowers nociceptive thresholds in the rat to produce hyperalgesia. While it is not possible to completely exclude an indirect mechanism of action, the observation that adenosine acts in the absence of known indirect pathways for the production of hyperalgesia (i.e. after sympathectomy, indomethacin and neutrophil depletion), that adenosine-induced hyperalgesia has a latency to onset comparable with other directly acting hyperalgesic agents5’ and that adenosine analogues inhibit Ca2+-dependent currents in dorsal root ganglion cells in culture,‘4.33 are compatible with the suggestion that adenosine produces hyperalgesia by a direct action on the primary afferent. Since adenosine is known to modulate CAMP levels in cells, increasing and decreasing CAMP, by actions at A,- and A,-receptors, respectively,22,56we investigated the hyperalgesic effects of adenosine using agents that selectively act on these two receptors. We observed that the hyperalgesic effect of adenosine is mediated by an action at a site with characteristics of the A,-type adenosine receptor, as demonstrated by the selective hyperalgesic effect of A,-agonists and selective antagonism by A,-antagonists. The observation that the phosphodiesterase inhibitor rolipram prolongs the duration of AZ-agonist induced hyperalgesia is compatible with the hypothesis that this action on the primary afferent, like that of the other directly acting eicosanoids, is mediated by the CAMP second messenger system. Further support for the hypothesis that CAMP is the second messenger in the primary afferent nociceptor, mediating hyperalgesia, is provided by the observations that adenosine A,-receptor agonists, which decrease intracellular cAMP,~,‘~,~’but have no

Adenosine hyperalgesia in rat skin effect on mechanical nociceptive threshold on their own, inhibits adenosine As-agonist and PGE,-hyperalgesia. In addition, DAMGO, which is known to act at mu-opioid receptors to decrease cAMP,*‘,“,~ also inhibits PGE,-3’ and CVlSO8-h~eralgesia. Ahhough

these data are consistent with the suggestion that adenosine acts directly on the primary efferent to produce hyperalgesia, and that this action is mediated by the CAMP second messenger system, alternative explanations exist. Thus, in addition to modulating CAMP levels, opioids are known to have other effects inciuding regulation of neuro~ptide, as we11 as adenosine, release.43~” In addition, opioids have also been demonstrated to effect Ca2+ currents, an action mediated by G-proteins linked directly to a Ca2+ channel.” The interactions between these various effects may also influence the hyperalgesia produced by adenosine. Of substantial clinical significance is that the discovery of adenosine hyperalgesia can explain

761

why blockade of the cyclooxygenase pathway of arachidonic acid metabolism by anti-inflammatory agents, or even blockade of both the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism by corticosteroids, are not always effective at alleviating hyperalgesic pain associated with tissue injury and inflammation. CAMP is found in almost all cells of the body, and therefore its action is not’ a rational target against which to direct analgesic therapy. However, A,-agonists or adenosine A,antagonists may be feasible novel peripherally acting analgesic agents. Since adenosine analogs have been shown also to act in the central nervous system to produce analgesia43,” such adenosine analogs would, however, have to be restricted in location to peripheral sites of action.

work was supported grant NS 21647 and the Rita Allen Foundation.

Acknowledgements-This

by NIH

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