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Mediation of serotonin hyperalgesia by the cAMP second messenger system
0306-4522/92 $5.00+ 0.00 Pergamon Press Lid © 1992 IBRO
Neuroscience Vol. 48, No. 2, pp. 479-483, 1992
Printed in Great Britain
MEDIATION OF SEROTONIN HYPERALGESIA BY THE cAMP SECOND MESSENGER SYSTEM Y. O. TAIWO,* P. H. HELLER and J. D. LEVINEt Departments of Medicine, Anatomy, and Oral and Maxillofacial Surgery, and Division of Neurosciences, University of California at San Francisco, CA 94143-0452A, U.S.A. Al~traet--In this study we have evaluated the second messenger system that might couple 5-HTt^ receptor activation to produce peripheral hyperalgesia. The intradermal injection of the serotonin (5-hydroxytryptamine; 5-HT) receptor agonist for the 1A receptor subset (5-HTIA), ( _+.)-2-dipropylamino8-hydroxy-l,2,3,4-tetrahydronaphthaline hydrobromide (8-OH DPAT) produces a dose-dependent hyperalgesia which was attenuated by a cAMP kinase inhibitor (the R-isomer of cyclic adenosine-Y-5'monophosphate), but prolonged by the inhibition of endogenous phosphodiesterase by rolipram, supporting a role for the cAMP second messenger system. The 5-HT~A receptor agonist, 8-OH-DPAT, and the adenyl cyclase activator, forskolin administered together, produced an additive hyperalgesia, suggesting that the 5-HT,A receptor in peripheral terminals of the primary afferent neurons is positively coupled to the cAMP second messenger system in producing hyperalgesia. The inability of pertussis toxin to inhibit 8-OH DPAT-induced hyperalgesia further supports this hypothesis. The coupling of the 5-HT~A receptor to the cAMP second messenger system appears to be through guanine regulatory proteins since guanosine 5'-O-(3-thiotriphosphate) and cholera toxin both markedly enhanced 8-OH DPAT hyperalgesia. In further support of the role of guanine nucleotide regulatory proteins, guanosine 5'-O-(2-thiodiphosphate), as well as activators of inhibitory guanine regulatory proteins (the mu-opioid agonist, [D-Ala2,N-Me-Phe4,GlyS-ol]-enkephalin, and the adenosine A~ agonist, N%yclopentyladenosine, significantly attenuated 8-OH DPAT hyperalgesia.
Serotonin is known to play a role in nociception by regulating the transmission of nociceptive information at various levels of the peripheral and the central nervous system, msIt is found in inflammatory lesions and has been reported to cause peripheral hyperalgesia3'l°.13.14'23'3°--a lowering of nociceptive threshold. In a companion study 31 we provided evidence to suggest that serotonin hyperalgesia is mediated by a direct action on the primary afferent, at the serotonin (5-hydroxytryptamine; 5-HT)IA type receptor (5-HTIA).3~ Previous studies by us have demonstrated that other hyperalgesic agents, which act directly on primary afferent nociceptors, such as prostaglandin E2 and 12, 8R,15S-dihydroxyicosatetraenoic acid and adenosine, all mediate hyperalgesia through the cAMP second messenger system. 32-34 *Present address: The Procter and Gamble Co., Cincinnati, OH 45239-8707, U.S.A. ~'To whom correspondence should be addressed at: Division of Rhenmatology and Clinical Immunology, S-1334/Box 0452A, University of California at San Francisco, San Francisco, CA 94143-0452A, U.S.A. Abbreviations: CPA, N%yclopentyladenosine; DAMGO, [D-Ala2,N-Me-Phe4,GlyS-ol]-enkephalin; DPDPE, IDPenS]-enkephalin; GDP-beta-S, guanosine 5'-O-(2-thiodiphosphate); G-proteins, guanine nucleotide regulatory proteins; GTP-gamma-S, guanosine 5'-O-(3-thiotriphosphate); 5-HT, 5-hydroxytryptamine or serotonin; 8-OH-DPAT, ( + )-2-dipropylamino-8-hydroxy-l,2,3,4tetrahydronaphthaline hydrobromide; Rp-cAMPs, Risomer of cyclic adenosine-Y5'-monophosphate; U50,488H, trans-3,4-dichloro-N-methyl-N[2-( l-pyrolidinyl)cyclohexyl] benzencacteamide. 479
Thus agents that cause hyperalgesia indirectly, such as bradykinin and leukotriene B4, cause release of one of the direct-acting agents which ultimately employ cAMP as a second messenger. Since serotonin appears to be a direct-acting hyperalgesic agent, there is a likelihood that cAMP is a second messenger. Indeed, serotonin has been demonstrated to enhance brain adenyl cyclase activity ll'12 by an action at a 5-HT1^ receptor. 2°,27-29The inhibition of forskolin-stimulated adenyl cyclase activity in cell-free preparations from guinea-pig and rat hippocampus by 5-HT agonists is also mediated by action at the 5-HT~A receptor. 7-9 Therefore, it appears that the 5-HTtA receptor can be positively or negatively linked to adenyl cyclase, perhaps as has been previously proposed through linkage to differing guanine nucleotide regulatory proteins (G-proteins) in the tissue or species of interest. 6'36'37 In this study we evaluate the contribution of the cAMP second messenger system to hyperalgesia induced by the activation of 5-HT~A receptors. EXPERIMENTAL PROCEDURES
The experiments were performed on 250-300-g male Sprague-Dawley rats (Bantin and Kingman, Fremont, CA). The nociceptive flexion reflex was quantified with a Ugo Basile Analgesymeter (Stoelting, Chicago), which applies linearly increasing mechanical force to the dorsum of the rat's hindpaw. During the week prior to the experiments, rats were trained, each day, in the paw-withdrawal reflex at 5-rain intervals for a period of 2 h. This adaptation procedure produces a stable baseline threshold
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measurement. 35On the day of the experiments, the rats were again exposed to the test stimulus at 5-min intervals for 2 h. The baseline threshold was defined as the mean of the last six determinations. The mean threshold of the rats used in this study was 134.5 __+1.6 g (n = 128). The baselines of the various treatment groups did not differ significantly from that of the control group. The selective 5-HT~Aagonist ( 4- )2-dipropylamino-8-hydroxy- 1,2,3,4-tetrahydronaphthaline hydrobromide (8-OH-DPAT) I,jS'zl was then injected in a 2.5-#1 volume on to the dorsum of the paw at the point at which the nociceptive threshold was determined. Three threshold measurements were made, 10, 15 and 20 min after injection, and the mean of these three values was defined as the nociceptive threshold. Ten-fold higher doses of 8-OH-DPAT were then sequentially administered every 25 rain and the effect on nociceptive thresholds similarly determined. This multiple injection procedure is not expected to, by itself, produce hyperatgesia since multiple injections of saline in this paradigm, in trained rats, have been observed to produce no lowering of nociceptive thresholds. A contribution of any tachyphylaxis to sequential 8-OH-DPAT injection should be similar in the two groups. The involvement of second messenger systems was evaluated by co-injecting, with 8-OH-DPAT, the cAMP protein kinase and protein kinase C inhibitors [R-isomer of cyclic adenosine-Y5'monophosphate; (RpcAMPS) z4,24 and PKCI ( 1 9 - 3 1 ) ] , 16't7'25 the phosphodiesterase inhibitor (Rolipram), 22'26 the G-protein analogs (GTPgammaS and GDPbetaS), the G-protein activator (cholera toxin) and ligands that are able to activate cAMP coupled inhibitory receptors, the mu-opioid [D-Ala2,N-Me-Phe4,GlyS-d]-en kephalin (DAMGO) and the adenosine A~-agonist N6-cyclopentylodenosine (CPA). The effect of 5-HTIA receptor activation on forskolin-induced increase in c A M P 7-9 w a s evaluated by co-injecting 8-OH-DPAT with forskolin. 8-OH-DPAT was obtained from Research Biochemicals Inc. (RBI; Natick, MA). Forskolin, CPA, guanosine 5'-0(3-thiotriphosphate) (GTP-gamma-S), guanosine 5'-0-(2thiodiphosphate) (GDP-beta-S), cholera toxin, pertussis toxin [D-Pen2'5]-enkephalin (DPDPE) and DAMGO were obtained from Sigma (St Louis, MO). PKCI(19-31), Auspep (Australia); RpcAMPS from BIOLOG (F.R.G.), Rolipram and trans-3,4-dichloro-N-methyl-N[2-(1-pyroolidinyl)cyclohexyl] henzeneacetamide (U50,488H) were generous gifts from Berlex (Ceder Knoll, N J) and Upjohn Co. (Kalamazoo, MI), respectively.
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Fig. 1. Dose-dependent effects of intradermally injected 8-OH-DPAT on paw-withdrawal nociceptive thresholds alone ( 0 ; n = 20), or when co-injected with 1 #g PKCI (19-31) (O; n = 6 ) or with l n g (A; n - 6 ) , 100ng (C); n = 6 ) or l # g (I-q; n = 6 ) of RpcAMPS. In this and subsequent figures, responses are graphed as percentage change from baseline threshold after intradermal injection of the various agents. Each point represents the mean 4- S.E.M. in a group of rats.
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60
90
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Fig. 2. The hyperalgesia induced by the intradermal injection of l #g 8-OH-DPAT alone ( 0 ; n = 8) or when co-injected with 1 # g of the phosphodiestvrase inhibitor rolipram (©; n = 8) at 10, 30, 60, 90 and 120rain after injection. GTP-gamma-S and GDP-heta-S were dissolved in distilled water and were injected subsequent to an injection of distilled water causing type-osmotic shock which renders the cells transiently permeable to the agents. 5 Stock solutions (4 mg/ml) of rolipram and forskolin were made in t: l ethanol : saline. Further dilutions of the stocks were made in saline. All other substances were dissolved in saline. Statistical analyses of the dose-response effects o f 8-OHDPAT and forskolin were performed using a repeated measures analysis of variance (ANOVA). The effect of the second messenger modulators on 8-OH-DPAT hyperalgesia, as well as that of 8-OH-DPAT on forskolin hypexalgesia, were assessed using a two-factor, repeated measures ANOVA. The effect of rolipram on the duration of 8-OHDPAT hyperalgesia was determined using a two-factor repeated measures ANOVA with two within subject factors (i.e. effect and time (30-180rain)). Differences were considered statistically significantly at the P < 0.05 level. RESULTS The intradermal injection of the 5-HTt^ agonist, 8 - O H - D P A T , produced a dose-dependent hyperalgesia [F(3,60) = 49.67; P < 0.001); Fig. 1]. This hyperalgesia was not attenuated by the protein kinase C inhibitor, P K C I (19-31) [ F ( I , 2 4 ) = 0 . 3 3 ; P >0.05; Fig. IA]. In contrast, the c A M P kinase inhibitor R p c A M P S (which has been shown to attenuate the cAMP-dependent hyperalgesia produced by PGE2 and by 8-bromo c A M P ) within the dose range 1 n g - l g g attenuated 8 - O H - D P A T hyperalgesia in a dose-dependent fashion [F(3,34) = 6.19; P < 0.005]. The phosphodiesterase inhibitor rolipram, at a dose (1 #g) which, by itself, did not affect nociceptive threshold ( - 1 . 1 3 + 1.84; t ( 2 3 ) = 1.16; P > 0.05)significantly prolonged the duration o f 8 - O H - D P A T - i n duced hyperalgesia as demonstrated by a significant main effect of time [F(5,70) = 4.99; P < 0.01] and a significant group x time interaction [F(5,70) = 3.64; P < 0.001; Fig. 2]. To evaluate whether 5-HT~^ activation affects forskolin-induced hyperalgosia, different doses of 8O H - D P A T were co-injected with forskolin. The intradermal injection of forskolin by itself produced a dose-dependent hypvralgesia [F(3,36)=39.77; P < 0.001; Fig. 3]. The co-injection of 8 - O H - D P A T , in a dose-dependent fashion did not inhibit forskolin
cAMP mediates serotonin hyperalgesia 0 40- • • • ~; 30" ,
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tive thresholds of forskolin alone (C); n = 12) or when co-injected with 2.5 #1 (4) of saline (0; n = 6) or with l n g ( m ; n = 6 ) , 10ng ( & ; n = 6 ) or l/~g (V; n = 6 ) of 8-OH-DPAT. hyperalgesia, rather, it enhanced forskolin hyperalgesia [F(3,26)=5.08; P <0.01; Fig. 3]. The 8-OHDPAT vehicle, saline, did not significantly enhance forskolin hyperalgesia [F(1,16)= 1.395; P < 0 . 0 5 ; Fig. 3]. To evaluate the involvement of G-proteins in 5HT~^ induced hyperalgesia modulators of G-protein activity were co-injected with 8-OH-DPAT. GTPgamma-S, GDP-beta-S, and cholera toxin, administered alone, within the dose range I ng-1 #g, did not significantly affect nociceptive threshold [F(4,28) = 3.2, F(4,28) = 0.69, and F(4,28) = 5.32, respectively; all P > 0.05). Similarly, intradermal injection of distilled water to produce hypo-osmotic shock also did not significandy affect nociceptive thresholds (+3.2 +4.9%; P >0.05; n = 8). When co-injected with 8-OHDPAT, however, GTP-gamma-S and cholera toxin significantly enhanced 8-OH-DPAT hyperalgesia [F(1,26)= 11.44, P <0.005 and F(1,26)= 15.08, P < 0.001, respectively; Fig. 4). In contrast, GDPbeta-S significantly decreased 8-OH-DPAT hyperal• 8-OH-DPAT O +1 I~gOholora Toxin 40-1[] +1 p,go'T'P-,zr-S | A +1 p.ggDP-,~-S
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481
gesia [F(1,26)=8.95; P < 0 . 0 1 ; Fig. 4]. The intradermal injection of pertussis toxin (1 pg), which by itself did not sit, nificantly affect nociceptive threshold [t(5) -- 0.86; P > 0.05], did not significantly alter 8-OH-DPAT hyperalgesia [F(1,24)=3.78; P > 0.05]. To further evaluate the role of G-proteins, the effects of other agents known to activate inhibitory G-proteins, were also evaluated. The intradermal injection of the mu-opiate D A M G O and of the adenosine AI agonist CPA attenuated hyperalgesia induced by 8-OH-DPAT [F(1,24)= 5.67, P < 0.05 and F(1,24) = 10.15, P < 0.005, respectively; Fig. 5]. The intradermal injection of either the delta opioid agonist DPDPE or the kappa-opioid agonist U50,488H did not significantly affect 8-OH-DPAT hyperalgesia [F(1,24)= 0.001, P > 0.05 and F(1,24) --0.85, P > 0.05, respectively; Fig. 5].
DISCUSSION
The data from this study demonstrate that the hyperalgesia produced by serotonin, at the 5-HT1A receptor, 31is mediated through coupling to the cAMP second messenger system via guanine nucleotide stimulatory proteins. The observation that RpcAMPS but not PKCI (19-31) inhibited 8-OH-DPAT hyperalgesia suggests that the cAMP second messenger system and not protein kinase C is involved in the hyperalgesia produced by 8-OH-DPAT. Further support for cAMP involvement is the observation that rolipram inhibition of endogenous phosphodiesterase, the enzyme which breaks down cAMP, significantly prolongs the duration of 8-OH-DPAT hyperalgesia. The rolipram effect on serotonin hyperalgesia is similar to its effect on other direct-acting hyperalgesic agents. 33 A similar prolongation of duration of hyperalgesia by other direct-acting agents is seen with isobutylmethylxanthine, 34 which also inhibits phosphodiesterase.
T~* 40- • 8-OH-DPAT O +1 PODPDPE A +11~gU50488H ~ 30" V +lpgDAMGO 22
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Fig. 4. Dose-dependent effects on paw-withdrawal nocicep-
five thresholds of intradermally injected 8-OH-DPAT alone ( 0 ; n = 20) or when co-injected with 1/t 8 cholera toxin (C); n = 8), 1/~8 pertussin toxin (<~; n = 6) or when hypoosmotically shocked and then co-injected with 1 P8 GTP-gammaS (l-1; n -- 8) or 1/~g GDP-beta-S (A; n = 8).
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1000
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Fig. 5. Dose-dependent effects on paw-withdrawal nocicepfive thresholds of intradermafiy injected 8-OH-DPAT alone (0; n = 20) or when co-injected with 1/~g DAMGO (V; n = 6), I pg CPA ([7; n = 6), 1/tg U-50,488H (A; n = 6) or I pg DPDPE (O; n -- 6).
482
Y.O. TAIWOet al.
We also observed that 8-OH-DPAT and forskolin prostaglandin E2 and adenosine hyperalgesia ~ ' (a direct adenyl cyclase activator) were at least addi- also had no effect on 8-OH-DPAT hyperalgesia. tive in producing hyperalgesia. The increase in intraIn summary, the present study demonstrates that cellular cAMP-mediated events after 5-HT1A receptor the activation of 5-HT1A receptors on primary afferactivation we observed is in line with reports of ent terminals (see companion paper 3j) is positively elevations in intracellular cAMP levels by 5-HT~A in linked to the cAMP second messenger through a studies using the guinea-pig hippocampus 27 29 and rat stimulatory, cholera toxin-sensitive, guanine regulathippocampal membranes2° but contrasts with the ory protein (G~). Our conclusion that these changes studies of DeVivo and Maayani 7 9 which suggests a are occurring specifically in the primary afferent is negative coupling of 5-HTIA activation to adenyl based on our observations3~ that elimination of all cyclase. It may well be, as proposed previously,6,36'37 components that are currently known to indirectly that the 5-HTjA receptor can be either negatively or contribute to hyperalgesia did not significantly affect positively coupled to adenyl cyclase depending on serotonin hyperalgesia. An obvious limitation of this tissue and species, and determined by the specific technique is that there may be unidentified mechanG-protein to which the receptor couples. 36The obser- isms of indirect hyperalgesia. vation that the irreversible analogs, GTP-gamma-S and GDP-beta-S, respectively, enhanced and attenuCONCLUSIONS ated the hyperalgesia produced by intradermally injected 8-OH-DPAT is compatible with the suggestion We make the tentative conclusion that serotonin that the production of peripheral hyperalgesia by the hyperalgesia appears to be mediated by the cAMP activation of the 5-HTjA receptor appears to be second messenger system in the primary afferent coupled to a G-protein. That cholera toxin, which nociceptor. The attenuation of 5-HT hyperalgesia by selectively activates stimulatory proteins, significantly activators of inhibitory G-proteins (e.g. mu-opioids enhanced the hyperalgesia induced by 8-OH-DPAT, and adenosine A~ agonists) further reinforces the while pertussis toxin--which prevents the activation proposal of these agents as novel strategies for the of inhibitory G-proteins--failed to prevent 8-OHrelief of inflammatory pain. Various inflammatory DPAT hyperalgesia suggests that the G-protein incells contain significant quantities of serotonin. volved is the excitatory (Gs) G-protein. Stimulation Should these be released in significant quantities in of G-inhibitory proteins by adenosine Aragonists inflammatory lesions, its direct hyperalgesic effect and the mu-opioid D A M G O significantly modified may explain the lack of response in certain inflammathe hyperalgesia produced by 8-OH-DPAT in a tory states to nonsteroidal anti-inflammatory drugs similar way to that by which these agents modify or to corticosteroids. the hyperalgesia of other directly-acting hyperalgesic agents such as prostaglandin E 2 and adenosine Acknowledgements--This research was supported by NIH (A2-agonist).w'33 The delta (DPDPE) and kappa grant NS21647, AM32634 and DE08973. JDL is a Rita (U50,488H) opiates, which have no effect on Allen Foundation fellow.
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Neuroscience Vol. 48, No. 2, pp. 479-483, 1992
Printed in Great Britain
MEDIATION OF SEROTONIN HYPERALGESIA BY THE cAMP SECOND MESSENGER SYSTEM Y. O. TAIWO,* P. H. HELLER and J. D. LEVINEt Departments of Medicine, Anatomy, and Oral and Maxillofacial Surgery, and Division of Neurosciences, University of California at San Francisco, CA 94143-0452A, U.S.A. Al~traet--In this study we have evaluated the second messenger system that might couple 5-HTt^ receptor activation to produce peripheral hyperalgesia. The intradermal injection of the serotonin (5-hydroxytryptamine; 5-HT) receptor agonist for the 1A receptor subset (5-HTIA), ( _+.)-2-dipropylamino8-hydroxy-l,2,3,4-tetrahydronaphthaline hydrobromide (8-OH DPAT) produces a dose-dependent hyperalgesia which was attenuated by a cAMP kinase inhibitor (the R-isomer of cyclic adenosine-Y-5'monophosphate), but prolonged by the inhibition of endogenous phosphodiesterase by rolipram, supporting a role for the cAMP second messenger system. The 5-HT~A receptor agonist, 8-OH-DPAT, and the adenyl cyclase activator, forskolin administered together, produced an additive hyperalgesia, suggesting that the 5-HT,A receptor in peripheral terminals of the primary afferent neurons is positively coupled to the cAMP second messenger system in producing hyperalgesia. The inability of pertussis toxin to inhibit 8-OH DPAT-induced hyperalgesia further supports this hypothesis. The coupling of the 5-HT~A receptor to the cAMP second messenger system appears to be through guanine regulatory proteins since guanosine 5'-O-(3-thiotriphosphate) and cholera toxin both markedly enhanced 8-OH DPAT hyperalgesia. In further support of the role of guanine nucleotide regulatory proteins, guanosine 5'-O-(2-thiodiphosphate), as well as activators of inhibitory guanine regulatory proteins (the mu-opioid agonist, [D-Ala2,N-Me-Phe4,GlyS-ol]-enkephalin, and the adenosine A~ agonist, N%yclopentyladenosine, significantly attenuated 8-OH DPAT hyperalgesia.
Serotonin is known to play a role in nociception by regulating the transmission of nociceptive information at various levels of the peripheral and the central nervous system, msIt is found in inflammatory lesions and has been reported to cause peripheral hyperalgesia3'l°.13.14'23'3°--a lowering of nociceptive threshold. In a companion study 31 we provided evidence to suggest that serotonin hyperalgesia is mediated by a direct action on the primary afferent, at the serotonin (5-hydroxytryptamine; 5-HT)IA type receptor (5-HTIA).3~ Previous studies by us have demonstrated that other hyperalgesic agents, which act directly on primary afferent nociceptors, such as prostaglandin E2 and 12, 8R,15S-dihydroxyicosatetraenoic acid and adenosine, all mediate hyperalgesia through the cAMP second messenger system. 32-34 *Present address: The Procter and Gamble Co., Cincinnati, OH 45239-8707, U.S.A. ~'To whom correspondence should be addressed at: Division of Rhenmatology and Clinical Immunology, S-1334/Box 0452A, University of California at San Francisco, San Francisco, CA 94143-0452A, U.S.A. Abbreviations: CPA, N%yclopentyladenosine; DAMGO, [D-Ala2,N-Me-Phe4,GlyS-ol]-enkephalin; DPDPE, IDPenS]-enkephalin; GDP-beta-S, guanosine 5'-O-(2-thiodiphosphate); G-proteins, guanine nucleotide regulatory proteins; GTP-gamma-S, guanosine 5'-O-(3-thiotriphosphate); 5-HT, 5-hydroxytryptamine or serotonin; 8-OH-DPAT, ( + )-2-dipropylamino-8-hydroxy-l,2,3,4tetrahydronaphthaline hydrobromide; Rp-cAMPs, Risomer of cyclic adenosine-Y5'-monophosphate; U50,488H, trans-3,4-dichloro-N-methyl-N[2-( l-pyrolidinyl)cyclohexyl] benzencacteamide. 479
Thus agents that cause hyperalgesia indirectly, such as bradykinin and leukotriene B4, cause release of one of the direct-acting agents which ultimately employ cAMP as a second messenger. Since serotonin appears to be a direct-acting hyperalgesic agent, there is a likelihood that cAMP is a second messenger. Indeed, serotonin has been demonstrated to enhance brain adenyl cyclase activity ll'12 by an action at a 5-HT1^ receptor. 2°,27-29The inhibition of forskolin-stimulated adenyl cyclase activity in cell-free preparations from guinea-pig and rat hippocampus by 5-HT agonists is also mediated by action at the 5-HT~A receptor. 7-9 Therefore, it appears that the 5-HTtA receptor can be positively or negatively linked to adenyl cyclase, perhaps as has been previously proposed through linkage to differing guanine nucleotide regulatory proteins (G-proteins) in the tissue or species of interest. 6'36'37 In this study we evaluate the contribution of the cAMP second messenger system to hyperalgesia induced by the activation of 5-HT~A receptors. EXPERIMENTAL PROCEDURES
The experiments were performed on 250-300-g male Sprague-Dawley rats (Bantin and Kingman, Fremont, CA). The nociceptive flexion reflex was quantified with a Ugo Basile Analgesymeter (Stoelting, Chicago), which applies linearly increasing mechanical force to the dorsum of the rat's hindpaw. During the week prior to the experiments, rats were trained, each day, in the paw-withdrawal reflex at 5-rain intervals for a period of 2 h. This adaptation procedure produces a stable baseline threshold
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measurement. 35On the day of the experiments, the rats were again exposed to the test stimulus at 5-min intervals for 2 h. The baseline threshold was defined as the mean of the last six determinations. The mean threshold of the rats used in this study was 134.5 __+1.6 g (n = 128). The baselines of the various treatment groups did not differ significantly from that of the control group. The selective 5-HT~Aagonist ( 4- )2-dipropylamino-8-hydroxy- 1,2,3,4-tetrahydronaphthaline hydrobromide (8-OH-DPAT) I,jS'zl was then injected in a 2.5-#1 volume on to the dorsum of the paw at the point at which the nociceptive threshold was determined. Three threshold measurements were made, 10, 15 and 20 min after injection, and the mean of these three values was defined as the nociceptive threshold. Ten-fold higher doses of 8-OH-DPAT were then sequentially administered every 25 rain and the effect on nociceptive thresholds similarly determined. This multiple injection procedure is not expected to, by itself, produce hyperatgesia since multiple injections of saline in this paradigm, in trained rats, have been observed to produce no lowering of nociceptive thresholds. A contribution of any tachyphylaxis to sequential 8-OH-DPAT injection should be similar in the two groups. The involvement of second messenger systems was evaluated by co-injecting, with 8-OH-DPAT, the cAMP protein kinase and protein kinase C inhibitors [R-isomer of cyclic adenosine-Y5'monophosphate; (RpcAMPS) z4,24 and PKCI ( 1 9 - 3 1 ) ] , 16't7'25 the phosphodiesterase inhibitor (Rolipram), 22'26 the G-protein analogs (GTPgammaS and GDPbetaS), the G-protein activator (cholera toxin) and ligands that are able to activate cAMP coupled inhibitory receptors, the mu-opioid [D-Ala2,N-Me-Phe4,GlyS-d]-en kephalin (DAMGO) and the adenosine A~-agonist N6-cyclopentylodenosine (CPA). The effect of 5-HTIA receptor activation on forskolin-induced increase in c A M P 7-9 w a s evaluated by co-injecting 8-OH-DPAT with forskolin. 8-OH-DPAT was obtained from Research Biochemicals Inc. (RBI; Natick, MA). Forskolin, CPA, guanosine 5'-0(3-thiotriphosphate) (GTP-gamma-S), guanosine 5'-0-(2thiodiphosphate) (GDP-beta-S), cholera toxin, pertussis toxin [D-Pen2'5]-enkephalin (DPDPE) and DAMGO were obtained from Sigma (St Louis, MO). PKCI(19-31), Auspep (Australia); RpcAMPS from BIOLOG (F.R.G.), Rolipram and trans-3,4-dichloro-N-methyl-N[2-(1-pyroolidinyl)cyclohexyl] henzeneacetamide (U50,488H) were generous gifts from Berlex (Ceder Knoll, N J) and Upjohn Co. (Kalamazoo, MI), respectively.
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100
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Fig. 1. Dose-dependent effects of intradermally injected 8-OH-DPAT on paw-withdrawal nociceptive thresholds alone ( 0 ; n = 20), or when co-injected with 1 #g PKCI (19-31) (O; n = 6 ) or with l n g (A; n - 6 ) , 100ng (C); n = 6 ) or l # g (I-q; n = 6 ) of RpcAMPS. In this and subsequent figures, responses are graphed as percentage change from baseline threshold after intradermal injection of the various agents. Each point represents the mean 4- S.E.M. in a group of rats.
al.
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30
60
90
120
150
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Fig. 2. The hyperalgesia induced by the intradermal injection of l #g 8-OH-DPAT alone ( 0 ; n = 8) or when co-injected with 1 # g of the phosphodiestvrase inhibitor rolipram (©; n = 8) at 10, 30, 60, 90 and 120rain after injection. GTP-gamma-S and GDP-heta-S were dissolved in distilled water and were injected subsequent to an injection of distilled water causing type-osmotic shock which renders the cells transiently permeable to the agents. 5 Stock solutions (4 mg/ml) of rolipram and forskolin were made in t: l ethanol : saline. Further dilutions of the stocks were made in saline. All other substances were dissolved in saline. Statistical analyses of the dose-response effects o f 8-OHDPAT and forskolin were performed using a repeated measures analysis of variance (ANOVA). The effect of the second messenger modulators on 8-OH-DPAT hyperalgesia, as well as that of 8-OH-DPAT on forskolin hypexalgesia, were assessed using a two-factor, repeated measures ANOVA. The effect of rolipram on the duration of 8-OHDPAT hyperalgesia was determined using a two-factor repeated measures ANOVA with two within subject factors (i.e. effect and time (30-180rain)). Differences were considered statistically significantly at the P < 0.05 level. RESULTS The intradermal injection of the 5-HTt^ agonist, 8 - O H - D P A T , produced a dose-dependent hyperalgesia [F(3,60) = 49.67; P < 0.001); Fig. 1]. This hyperalgesia was not attenuated by the protein kinase C inhibitor, P K C I (19-31) [ F ( I , 2 4 ) = 0 . 3 3 ; P >0.05; Fig. IA]. In contrast, the c A M P kinase inhibitor R p c A M P S (which has been shown to attenuate the cAMP-dependent hyperalgesia produced by PGE2 and by 8-bromo c A M P ) within the dose range 1 n g - l g g attenuated 8 - O H - D P A T hyperalgesia in a dose-dependent fashion [F(3,34) = 6.19; P < 0.005]. The phosphodiesterase inhibitor rolipram, at a dose (1 #g) which, by itself, did not affect nociceptive threshold ( - 1 . 1 3 + 1.84; t ( 2 3 ) = 1.16; P > 0.05)significantly prolonged the duration o f 8 - O H - D P A T - i n duced hyperalgesia as demonstrated by a significant main effect of time [F(5,70) = 4.99; P < 0.01] and a significant group x time interaction [F(5,70) = 3.64; P < 0.001; Fig. 2]. To evaluate whether 5-HT~^ activation affects forskolin-induced hyperalgosia, different doses of 8O H - D P A T were co-injected with forskolin. The intradermal injection of forskolin by itself produced a dose-dependent hypvralgesia [F(3,36)=39.77; P < 0.001; Fig. 3]. The co-injection of 8 - O H - D P A T , in a dose-dependent fashion did not inhibit forskolin
cAMP mediates serotonin hyperalgesia 0 40- • • • ~; 30" ,
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tive thresholds of forskolin alone (C); n = 12) or when co-injected with 2.5 #1 (4) of saline (0; n = 6) or with l n g ( m ; n = 6 ) , 10ng ( & ; n = 6 ) or l/~g (V; n = 6 ) of 8-OH-DPAT. hyperalgesia, rather, it enhanced forskolin hyperalgesia [F(3,26)=5.08; P <0.01; Fig. 3]. The 8-OHDPAT vehicle, saline, did not significantly enhance forskolin hyperalgesia [F(1,16)= 1.395; P < 0 . 0 5 ; Fig. 3]. To evaluate the involvement of G-proteins in 5HT~^ induced hyperalgesia modulators of G-protein activity were co-injected with 8-OH-DPAT. GTPgamma-S, GDP-beta-S, and cholera toxin, administered alone, within the dose range I ng-1 #g, did not significantly affect nociceptive threshold [F(4,28) = 3.2, F(4,28) = 0.69, and F(4,28) = 5.32, respectively; all P > 0.05). Similarly, intradermal injection of distilled water to produce hypo-osmotic shock also did not significandy affect nociceptive thresholds (+3.2 +4.9%; P >0.05; n = 8). When co-injected with 8-OHDPAT, however, GTP-gamma-S and cholera toxin significantly enhanced 8-OH-DPAT hyperalgesia [F(1,26)= 11.44, P <0.005 and F(1,26)= 15.08, P < 0.001, respectively; Fig. 4). In contrast, GDPbeta-S significantly decreased 8-OH-DPAT hyperal• 8-OH-DPAT O +1 I~gOholora Toxin 40-1[] +1 p,go'T'P-,zr-S | A +1 p.ggDP-,~-S
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481
gesia [F(1,26)=8.95; P < 0 . 0 1 ; Fig. 4]. The intradermal injection of pertussis toxin (1 pg), which by itself did not sit, nificantly affect nociceptive threshold [t(5) -- 0.86; P > 0.05], did not significantly alter 8-OH-DPAT hyperalgesia [F(1,24)=3.78; P > 0.05]. To further evaluate the role of G-proteins, the effects of other agents known to activate inhibitory G-proteins, were also evaluated. The intradermal injection of the mu-opiate D A M G O and of the adenosine AI agonist CPA attenuated hyperalgesia induced by 8-OH-DPAT [F(1,24)= 5.67, P < 0.05 and F(1,24) = 10.15, P < 0.005, respectively; Fig. 5]. The intradermal injection of either the delta opioid agonist DPDPE or the kappa-opioid agonist U50,488H did not significantly affect 8-OH-DPAT hyperalgesia [F(1,24)= 0.001, P > 0.05 and F(1,24) --0.85, P > 0.05, respectively; Fig. 5].
DISCUSSION
The data from this study demonstrate that the hyperalgesia produced by serotonin, at the 5-HT1A receptor, 31is mediated through coupling to the cAMP second messenger system via guanine nucleotide stimulatory proteins. The observation that RpcAMPS but not PKCI (19-31) inhibited 8-OH-DPAT hyperalgesia suggests that the cAMP second messenger system and not protein kinase C is involved in the hyperalgesia produced by 8-OH-DPAT. Further support for cAMP involvement is the observation that rolipram inhibition of endogenous phosphodiesterase, the enzyme which breaks down cAMP, significantly prolongs the duration of 8-OH-DPAT hyperalgesia. The rolipram effect on serotonin hyperalgesia is similar to its effect on other direct-acting hyperalgesic agents. 33 A similar prolongation of duration of hyperalgesia by other direct-acting agents is seen with isobutylmethylxanthine, 34 which also inhibits phosphodiesterase.
T~* 40- • 8-OH-DPAT O +1 PODPDPE A +11~gU50488H ~ 30" V +lpgDAMGO 22
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| ~0 1
10 100 Dose(no)
1000
Fig. 4. Dose-dependent effects on paw-withdrawal nocicep-
five thresholds of intradermally injected 8-OH-DPAT alone ( 0 ; n = 20) or when co-injected with 1/t 8 cholera toxin (C); n = 8), 1/~8 pertussin toxin (<~; n = 6) or when hypoosmotically shocked and then co-injected with 1 P8 GTP-gammaS (l-1; n -- 8) or 1/~g GDP-beta-S (A; n = 8).
Tr
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1
10
. ~
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1000
Dora (no)
Fig. 5. Dose-dependent effects on paw-withdrawal nocicepfive thresholds of intradermafiy injected 8-OH-DPAT alone (0; n = 20) or when co-injected with 1/~g DAMGO (V; n = 6), I pg CPA ([7; n = 6), 1/tg U-50,488H (A; n = 6) or I pg DPDPE (O; n -- 6).
482
Y.O. TAIWOet al.
We also observed that 8-OH-DPAT and forskolin prostaglandin E2 and adenosine hyperalgesia ~ ' (a direct adenyl cyclase activator) were at least addi- also had no effect on 8-OH-DPAT hyperalgesia. tive in producing hyperalgesia. The increase in intraIn summary, the present study demonstrates that cellular cAMP-mediated events after 5-HT1A receptor the activation of 5-HT1A receptors on primary afferactivation we observed is in line with reports of ent terminals (see companion paper 3j) is positively elevations in intracellular cAMP levels by 5-HT~A in linked to the cAMP second messenger through a studies using the guinea-pig hippocampus 27 29 and rat stimulatory, cholera toxin-sensitive, guanine regulathippocampal membranes2° but contrasts with the ory protein (G~). Our conclusion that these changes studies of DeVivo and Maayani 7 9 which suggests a are occurring specifically in the primary afferent is negative coupling of 5-HTIA activation to adenyl based on our observations3~ that elimination of all cyclase. It may well be, as proposed previously,6,36'37 components that are currently known to indirectly that the 5-HTjA receptor can be either negatively or contribute to hyperalgesia did not significantly affect positively coupled to adenyl cyclase depending on serotonin hyperalgesia. An obvious limitation of this tissue and species, and determined by the specific technique is that there may be unidentified mechanG-protein to which the receptor couples. 36The obser- isms of indirect hyperalgesia. vation that the irreversible analogs, GTP-gamma-S and GDP-beta-S, respectively, enhanced and attenuCONCLUSIONS ated the hyperalgesia produced by intradermally injected 8-OH-DPAT is compatible with the suggestion We make the tentative conclusion that serotonin that the production of peripheral hyperalgesia by the hyperalgesia appears to be mediated by the cAMP activation of the 5-HTjA receptor appears to be second messenger system in the primary afferent coupled to a G-protein. That cholera toxin, which nociceptor. The attenuation of 5-HT hyperalgesia by selectively activates stimulatory proteins, significantly activators of inhibitory G-proteins (e.g. mu-opioids enhanced the hyperalgesia induced by 8-OH-DPAT, and adenosine A~ agonists) further reinforces the while pertussis toxin--which prevents the activation proposal of these agents as novel strategies for the of inhibitory G-proteins--failed to prevent 8-OHrelief of inflammatory pain. Various inflammatory DPAT hyperalgesia suggests that the G-protein incells contain significant quantities of serotonin. volved is the excitatory (Gs) G-protein. Stimulation Should these be released in significant quantities in of G-inhibitory proteins by adenosine Aragonists inflammatory lesions, its direct hyperalgesic effect and the mu-opioid D A M G O significantly modified may explain the lack of response in certain inflammathe hyperalgesia produced by 8-OH-DPAT in a tory states to nonsteroidal anti-inflammatory drugs similar way to that by which these agents modify or to corticosteroids. the hyperalgesia of other directly-acting hyperalgesic agents such as prostaglandin E 2 and adenosine Acknowledgements--This research was supported by NIH (A2-agonist).w'33 The delta (DPDPE) and kappa grant NS21647, AM32634 and DE08973. JDL is a Rita (U50,488H) opiates, which have no effect on Allen Foundation fellow.
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