Brain Research, 528 (1990) 55-61 E!sevier

55

BRES 15859

5-Hydroxytryptamine releases adenosine and cyclic AMP from primary afferent nerve terminals in the spinal cord in vivo Marva I. Sweeney, Thomas D. White and Jana Sawynok Department of Pharmacology, Dalhousie University, Halifax, N.S. (Canada) (Accepted 20 March 1990) Key word~: Serotonin; Noradrenaline; Adenosine release; Cyclic AMP release; Spinal cord; Antinociception

5-Hydroxytryptamine (5-HT) releases a purine nucleotide, which is subsequently converted to adenosine, from primary afferent nerve terminals in vitro. This release may mediate spinal antinociception by 5-HT. In the present study, we have investigated whether release also occurs from the spinal cord in vivo using an intrathecal perfusion system in rats. Adenosine was quantitated using high performance liquid chromatography (HPLC) with fluorescence detection. Following perfusion of the spinal cord with 50 and 500/~M 5-HT, a 35-50% increase in the release of endogenous adenosine was observed. This release was completely blocked by 50 #M methysergide, and by intrathecal injection with 100/~g capsaicin 5-8 days prior to release experiments. Intrathecal perfusion with 50 and 500/~M 5-HT also released a nucleotide which eluted from the HPLC column at a retention time identical to that of cyclic AMP standards, and was reduced following incubation with cyclic AMP phosphodiesterase. This release of cyclicAMP also was eliminated following intrathecal pretreatment with capsaicin. In contrast to 5-HT, noradrenaline (NA, 500/~M and 5 mM) did not release adenosine or cyclic AMP from the intact spinal cord. These data demonstrate that release of nucleotide, probably cyclic AMP, and subsequent metabolism to adenosine, can be induced by 5-HT but not NA in vivo. This strengthens the hypothesis that release of adenosine from the spinal cord may mediate antinociception by intrathecal 5-HT but not NA.

INTRODUCTION There is increasing evidence to suggest that adenosine release is involved in antinociception produced by a variety of spinally active analgesics 19. The most supportive data are available for morphine, whereby antinociception produced by intrathecal (i.t.) morphine is blocked by spinal administration of the methylxanthine adenosine receptor antagonists theophylline and 8phenyltheophylline. In addition, morphine releases adenosine from primary afferent nerve terminals in the spinal cord in vitro and in vivo (reviewed in ref. 19). I.t. administration of 5-hydroxytryptamine (5-HT) produces elevations in nociceptive thresholds 2°'21"28, and recently such antinociception has been shown to be attenuated by i.t. theophylline 4. In addition, 5-HT in vitro increases the release of a nucleotide from dorsal spinal cord synaptosomes which is degraded extracellularly to adenosine 24. This purine release is Ca2+-depen dent, receptor-mediated, and originates in capsaicinsensitive small diameter primary afferent nerve termina|s 24. Together, these behavioural and biochemical observations have led to the hypothesis that a component of antinociception produced by i.t. 5-HT may involve the

release of a nucleotide which is degraded extracellularly to adenosine within the spinal cord 4'19'24. In contrast to morphine and 5-HT, i.t. noradrenaline (NA) produces spinal antinociception which is resistent to i.t. injection of theophylline or 8-phenyltheophylline 4' 23. However, exposure of spinal cord synaptosomes to NA also enhances the release of a nucleotide which is converted to adenosine following release2.3. This latter observation raises the question of whether NA-induced release of a nucleotide and its subsequent metabolism to adenosine occurs from the spinal cord in vivo. In the present study, we have examined the ability of both 5-HT and N A to release adenosine into peffusates of the spinal subarachnoid space of rats. Subsequently, the possible neuronal origin of adenosine released by 5-HT in vivo was determined. This was achieved by examining release of purines following i.t. pretreatment of adult rats with the neurotoxin capsaicin 3. Three to 7 days following i.t. injection, capsaicin induces a selective degeneration of small diameter primary afferent nerve terminals in superficial laminae of the dorsal spinal cord 16 and produces approximately 50% depletion of substance P within the same area 7,1°'13'27. Preliminary portions of this work have been published in abstract form 25.

Correspondence: M. Sweeney. Present address: Department of Pharmacology, St. George's Hospital Medical School, Cranmer Terrace, Tooting, London SWl7 ORE, U.K. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

56 MATERIALS AND METHODS An i.t. perfusion system was used as described previously 22. Briefly, male Sprague-Dawley rats (Canadian Hybrid Farms, Nova Scotia) weighing 500-600 g were anesthetized with urethane (1.7 g/kg) and mounted in a stereotaxic apparatus. A 10 cm inflow cathether (PE-10 tubing) was inserted into the i.t. space through the open cisterna while an outflow catheter (PE-50 tubing) was placed below the cerebrospinal fluid (CSF) surface in the open cisterna. Both catheters were attached to a Minipuls 2 peristaltic pump (Gilson), and the spinal cord perfused for 15-20 min at 67/~l/min with artificial CSF (containing (in mM) NaC1 118, NaH2PO 4 2.5, NaHCO 3 21, KC1 2.6, CaCI 2 1.3 and MgC12 0.9 and gassed with 95% 02/5% CO 2 to maintain a pH of 7.4). Subsequently, 5 serial 15 min samples of the perfusate were collected on ice in the following sequence: two basal collections, two collections in the presence of the drug to be tested, and one post-drug collection. The drugs used were 5-HT (creatinine sulfate, Sigma) and NA (bitartrate, Sigma), dissolved in ascorbic acid (0.1% w/v in artificial CSF, Sigma) and methysergide (hydrogen maleate, Sandoz), dissolved either in CSF or the ascorbic acid vehicle. When used, methysergide was present in the perfusing medium for the duration of the 75 min period. In experiments with cyclic AMP phosphodiesterase (Sigma), the enzyme was dissolved in distilled water and 10 /~1 (5 × 10 -4 units) added to aliquots of the perfusates or standards containing cyclic AMP and adenosine. One unit of cyclic AMP phosphodiesterase will convert 1/~mol cyclic AMP/min to 5"-AMP at 30 °C and pH 7.5. Samples (containing phosphodiesterase or controls) were subsequently incubated for 5 min at 37 °C to permit phosphodiesterase-induced metabolism of cyclic AMP to 5"-AMP. Aliquots (500 ~1) of all samples were deproteinated and assayed for adenosine or cyclic AMP content using high performance liquid chromatography (HPLC) with fluorescence detection (sensitivity 2 pmol) 23. Heights and areas of peaks produced by both adenosine

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AMP T i m e (rain) Fig. 5. Effect of i.t. pretreatment with 100/~g capsaicin on release of adenosine (A) and cyclic AMP (B) from the spinal cord in vivo evoked by 500/~M 5-HT. Rats were injected i.t. with vehicle (60% dimethylsulfoxide in saline) or 100/~g capsaicin 5-8 days prior to release experiments. The second basal adenosine (ADO) release values were 165.3 ± 22.1 and 323.9 ± 34.4 pmol/15 min in vehicle- and capsaicin-pretreated rats, respectively (P < 0.01, unpaired t-test, n = 6). The second basal cyclic AMP release values were 51.3 ± 8.2 and 144.1 + 74.2 pmol/15 min, respectively (P < 0.01, unpaired t-test, n = 3-4). 5-HT 500/aM was present in the perfusate where indicated by the bars (A and B). Values represent means ± S.E.M. of release over the 15-min perfusion period directly preceding the point (A and B) or the sum of percentage change in release from basal release values during the two perfusion periods in the presence of 5-HT (C). *P < 0.05, **P < 0.01, unpaired t-test compared to vehicle pretreatment. capsaicin 3 5 - 8 days prior to release experiments. I.t. pretreatment with vehicle had no significant effect on nociceptive latencies in the tail-flick test (P > 0.1, Table II). In these animals, 5 0 0 / t M 5-HT released adenosine from the spinal cord in vivo (Fig. 5A) with a profile which was similar to that seen before (Fig. 1A). I.t. pretreatment with capsaicin produced significant elevations in

TABLE II Effect of i.t. pretreatment with 100 l~g capsaicin or vehicle on nociceptive latencies in the tail-flick test

Values are means + S.E.M. of number of adenosine (A) or cyclic AMP (B) release experiments expressed in parentheses. Release data from these animals are displayed in Fig. 5. Capsaicin was dissolved in 60% dimethylsulfoxide in saline. Day refers to the number of days following pretreatment that tail-flick nociceptive latencies were recorded. Intrathecal pretreatment

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tail-flick latencies (Table IIA). In rats pretreated with capsaicin, release of adenosine from the intact spinal cord induced by 500 # M 5-HT was inhibited by 8 0 - 9 0 % (Fig. 5A,C). Basal release of adenosine was not reduced following i.t. pretreatment with capsaicin but in fact was significantly increased (P < 0.01, unpaired t-test, Fig. 5). In a separate experiment, i.t. pretreatment with capsaicin also completely attenuated the release of cyclic A M P induced by 500 # M 5-HT (Fig. 5B,C) and produced modest, though statistically significant, increases in nociceptive thresholds in the tail-flick test (Table IIB). Vehicle administration had no significant affect on pain thresholds (P > 0.5; Table liB). In a m a n n e r similar to adenosine release, the basal release of cyclic A M P also was increased by i.t. pretreatment with capsaicin (Fig. 5). Previously, i.t. pretreatment with capsaicin had no effect on basal release of adenosine from the spinal cord in vivo 26, and the reason for the increase seen here is unclear. Nonetheless, the increase in basal release and reduction of 5-HT-induced release of cyclic A M P and adenosine suggests that purines released under basal conditions originate from a different pool to that stimulated by 5-HT. DISCUSSION The present study demonstrates that 5-HT can increase the extracellular content of endogenous adenosine

60 within the spinal cord in vivo via activation of a methysergide-sensitive receptor. This adenosine likely originates from small diameter primary afferent nerve terminals since i.t. pretreatment with capsaicin eliminated the accumulation of adenosine. Previously, i.t. pretreatment with capsaicin was shown to produce specific degeneration of the spinal terminals of C-fibers located in the substantia gelatinosa with no evidence of damage to glial cells3'16. In addition, i.t. capsaicin generally produces a plateau 40-60% depletion of immunoreactive substance P in the dorsal spinal cord 7' 10,13,27, consistent with the observation that primary afferent neurons contain about half of the substance P in the dorsal horn of the spinal cord TM. These observations using the intact spinal cord confirm data obtained in vitro 24 and indicate that nucleotide released by 5-HT from the spinal cord also can be metabolized extracellulady to adenosine in vivo. These biochemical results support the behavioural observation that antinociception produced by i.t. 5-HT is inhibited by i.t. theophylline, presumably via blockade of spinal adenosine receptors 4. Together, these data strengthen the hypothesis that adenosine release from the spinal cord may partially mediate antinociception by i.t. 5-HT 4'19'24. In the present study, the adenosine-releasing effect of 5-HT in vivo is similar to that of morphine with regard to neuronal origin 26. However, morphine releases adenosine per se ~3 while 5-HT releases nucleotide in vitro 24 and in vivo (Figs. 2-5). 5-HT does not release ATP in vitro (unpublished observations). The retention time for the nucleotide peak obtained by perfusion with 5-HT is identical to that of cyclic AMP standards and the height of this peak was reduced by at least 50% following incubation in the presence of cyclic AMP phosphodiesterase (Fig. 3). This indicates that the nucleotide released is probably cyclic AMP. There is some evidence to suggest that 5-HT stimulates the production of cyclic AMP. A 5-HT receptor-mediated increase in the activity of adenylate cyclase has been demonstrated previously in whole brain synaptosomal membranes 15 as well as in the cortex 1, hypothalamus 5, striatum 6 and hippocampus 2"12. 5-HT also stimulates the basal production of cyclic AMP in homogenates 5 and explanted cultures of spinal cord 11. However, many investigators have reported that 5-HT also has inhibitory actions on adenylate cyclase activity in various brain regions (reviewed in ref. 17) as well as on forskolin-stimulated adenylate cyclase activity in the spinal cord 11. It seems possible that release of cyclic AMP induced by i.t. 5-HT acts as the primary source for extracellular adenosine. This hypothesis is based on the observation that 5-HT-evoked accumulation of adenosine from spinal cord synaptosomes is eliminated when ecto-5"-nucleo-

tidase is inhibited, implying that release is exclusively in the form of a purine nucleotide 24. In addition, the time course for the release of cyclic AMP (Fig. 4A) closely parallels that for the appearance of adenosine (Fig. 1A). In view of the rapid conversion of released nucleotide to adenosine by spinal cord synaptosomes 23'24'26, it is surprising that cyclic AMP released in vivo w a s not completely degraded to adenosine: One possible explanation is that a large amount of nucleotide released saturates ecto-enzymes responsible for extracellular conversion to adenosine. The results shown in Fig. 5 support this possibility where all of the cyclic AMP presumably released during the initial 15-min perfusion with 5-HT may have been metabolized to adenosine. However, as more cyclic AMP is released, less degradation occurs as reflected by an increase in the content of extracellular cyclic AMP and a decrease in the accumulation of extracellular adenosine. This theory of saturation of ecto-enzymes may also explain why release of cyclic AMP induced by 5-HT seems to be dose-dependent (Fig. 4B), while that of adenosine does not (Fig. 1B). It is possible that the greater amount of nucleotide released by 500 ktM 5-HT cannot be completely metabolized to nucleoside because of saturation of the ecto-enzymes responsible for their interconversion. Although the mechanism by which 5-HT releases purines, from primary afferent nerves is not clear, it may involve depolarization of these terminals. A slow depolarization of type C dorsal root ganglia 9 and their spinal terminals 8'1s by 5-HT has been reported. Previously, depolarization of capsaicin-sensitive neurons by addition of K + or capsaicin has been shown to release adenosine and phosphorylated nucleotide(s) from spinal cord synaptosomes 26. It is likely that 5-HT-induced release of purine in vivo is a direct consequence of receptor activation since the 5-HT receptor antagonist methysergide completely blocked release stimulated by 5-HT. In contrast to 5-HT, NA did not enhance the release of cyclic AMP or adenosine from the spinal cord in vivo even at a dose as high as 5 mM. Although NA releases adenosine from spinal cord synaptosomes, this adenosine is derived from released nucleotide 23. It is possible that NA released a nucleotide from the spinal cord in vivo which was not detected due to sensitivity of our HPLC system or the deproteination method employed. Nucleotide released may not have been dephosphorylated to adenosine since the enzymes required for this conversion may not be accessible to the site of release. NA and 5-HT release nucleotides from different sources. NA-evoked purine release in vitro originates from neurons located in both dorsal and ventral halves of the spinal cord which are resistant to capsaicin pretreatment 26 while 5-HT acts at small diameter primary afferent neurons in the dorsal

61 spinal cord to release purines 24 (also Fig. 5). This lack of d e t e c t a b l e changes in purine release by i.t. N A strengthens the hypothesis that adenosine release is not directly involved in antinociception p r o d u c e d by i.t. N A 4'19'23. T h e possible identity and role of purine(s) released by N A from an in vitro p r e p a r a t i o n of the spinal cord is not clear at present. In contrast, it a p p e a r s that direct application of 5-HT to the spinal cord can induce the release of cyclic A M P from a p o p u l a t i o n of small REFERENCES 1 Ahn, H.S. and Makman, M.H., Stimulation of adenylate cyclase activity in monkey anterior limbic cortex by serotonin, Brain Research, 153 (1978) 636-640. 2 Barbaccia, M.L., Brunello, N., Chuang, D.M. and Costa, E., Serotonin elicited amplification of adenylase cyclase activity in hippocampal membranes from adult rat, J. Neurochem., 40 (1982) 1671-1679. 3 Buck, S.H. and Burks, T.E, The neuropharmacology of capsaicin: review of some recent observations, Pharmacol. Rev., 38 (1986) 179-226. 4 DeLander, G.E. and Hopkins, C.J., Interdependence of spinal adenosinergic, serotonergic and noradrenergic systems mediating antinociception, Neuropharmacology, 26 (1987) 1791-1794. 5 Enjalbert, A., Bourgoin, S., Hamon, M., Adrien, J. and Bockaert, J., Post synaptic serotonin-sensitive adenylate cyclase in the CNS I, Mol. Pharmacol., 14 (1978) 2-10. 6 Fillion, G., Rouselle, J.C., Beaudoin, D., Pradelles, P., Goiny, M., Dray, F. and Jacobs, J., Serotonin sensitive adenylate cyclase in horse brain synaptosomal membranes, Life Sci., 24 (1979) 1813-1822. 7 Gamse, R., Capsaicin and nociception in the rat and mouse: possible role of substance P, Naunyn Schrniederberg's Arch. Pharmacol., 320 (1982) 205-216. 8 Holz, IV, G.G. and Anderson, E.G., The actions of serotonin on frog primary afferent terminals and cell bodies, Comp. Biochem. Physiol., 77C (1984) 13-21. 9 Holz, IV, G.G., Shefner, S.A. and Anderson, E.G., Serotonin depolarizes type A and C primary afferents: an intracellular study in bullfrog dorsal root ganglion, Brain Research, 327 (1985) 71-79. 10 Jhamandas, K., Yaksh, T.L., Harty, G., Szolcsanyi, J. and Go, V.L.W., Action of intrathecal capsaicin and its structural analogues on the content and release of spinal substance P: selectivity of action and relationship to analgesia, Brain Research, 306 (1984) 215-226. 11 Makman, M.H., Dvorkin, B., and Crain, S.M., Modulation of adenylate cyclase activity of mouse spinal cord-ganglion explants by opioids, serotonin and pertussis toxin, Brain Research, 445 (1988) 303-313. 12 Markstein, R., Hoyer, D. and Engel, G., 5-HT1A-receptors mediate stimulation of adenylate cyclase in rat hippocampus, Naunyn Schmiederberg's Arch. Pharmacol., 333 (1986) 335-341. 13 Nagy, J.I., Emson, P.C. and Iversen, L.L., A re-evaluation of the neurochemical and antinociceptive effects of intrathecal capsaicin in the rat, Brain Research, 211 (1981) 497-502.

d i a m e t e r p r i m a r y afferent neurons. R e l e a s e d cyclic A M P m a y then be d e g r a d e d to a d e n o s i n e in the synaptic cleft via an action of e c t o p h o s p h o d i e s t e r a s e s and 5"-nucleotidases. A d e n o s i n e subsequently binds to methylxanthinesensitive receptors to m o d u l a t e nociceptive processing.

Acknowledgements. This work was supported by the Medical Research Council of Canada in a grant to J.S. and T.D.W. 14 Nagy, J.I., Iversen, L.L., Goedert, M., Chapman, D. and Hunt, S.P., Dose-dependent effects of capsaicin on primary sensory neurons in the neonatal rat, J. Neurosci., 3 (1983) 399-406. 15 Pagel, J., Christian, S.T., Quayle, E.S. and Monti, J.A., A serotonin sensitive adenylate cyclase in mature rat brain synaptosomal membranes, Life Sci., 19 (1976) 819-824. 16 Palermo, N.N., Brown, H.K. and Smith, D.L., Selective neurotoxic action of capsaicin on glomerular C-type terminals in rat substantia gelatinosa, Brain Research, 208 (1981) 506-510. 17 Peroutka, S.J., Serotonin receptors. In H. Meltzer (Ed.), Psychopharmacology: The Third Generation of Progress, Raven, New York, 1987, pp. 303-311. 18 Phillis, J.W. and Kirkpatrick, J.R., Action of biogenic amines on isolated toad spinal cord, Gen. PharmacoL, 10 (1979) 115-119. 19 Sawynok, J. and Sweeney, M.I., Role of purines in nociception, Neuroscience, 32 (1989) 557-569. 20 Schmauss, C., Hammond, D.L., Ochi, J.W. and Yaksh, T.L., Pharmacological antagonism of the antinociceptive effects of serotonin in the rat spinal cord, Eur. J. Pharmacol., 90 (1983) 349-357. 21 Solomon, R.E. and Gebhart, G.F., Mechanisms of effects of intrathecal serotonin on nociception and blood pressure in rats, J. Pharmacol. Exp. Ther., 245 (1988) 905-912. 22 Sweeney, M.I., White, T.D., Jhamandas, K.H. and Sawynok, J., Morphine releases endogenous adenosine from the spinal cord in vivo, Eur. J. Pharmacol., 141 (1987) 169-170. 23 Sweeney, M.I., White, T.D. and Sawynok, J., Involvement of adenosine in the spinal antinociceptive effects of morphine and noradrenaline, J. Pharmacol. Exp. Ther., 243 (1987) 657-665. 24 Sweeney, M.I., White, T.D. and Sawynok, J., 5-Hydroxytryptamine releases adenosine from primary afferent nerve terminals in the spinal cord, Brain Research, 462 (1988) 346-349. 25 Sweeney, M.I., White, T.D. and Sawynok, J., Serotonin releases adenosine from primary afferent nerve terminals in the spinal cord: possible involvement in spinal antinociception, Soc. Neurosci. Abstr., 14 (1988) 852. 26 Sweeney, M.I., White, T.D. and Sawynok, J., Morphine, capsaicin and K + release purines from capsaicin-sensitive nerve terminals in the spinal cord, J. Pharmacol. Exp. Ther., 248 (1989) 447-454. 27 Yaksh, T.L., Farb, D.H., Leeman, S.E. and Jessell, T.M., Intrathecal capsaicin depletes substance P in the rat spinal cord and produces prolonged thermal analgesia, Science, 206 (1979) 481-483. 28 Yaksh, T.L. and Wilson, P.R., Spinal serotonin terminal system mediates antinociception, J. Pharmacol. Exp. Ther., 208 (1979) 446-453.