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) 215-225

215

Elsevier BRE 10201

Action of Intrathecal Capsaicin and its Structural Analogues on the Content and Release of Spinal Substance P: Selectivity of Action and Relationship to Analgesia KHEM JHAMANDAS 1, T. L. YAKSH3. G. HARTY 3, J. SZOLCSANYI2 and V. L. W. GO 3

1Department of Pharmacology, Queen's University, Kingston, Ontario (Canada), 2Departmentof Pharmacology, Universityof Peds, Pe~s (Hungary) and 3Department of Neurosurgical Research, Mayo Clinic, Rochester, MI55905 (U.S.A.) (Accepted December 27th, 1983)

Key words: capsaicin-- homovaniUic acid analogues - - spinal substance P

Intrathecal injections of capsaicin (CAP) and 4 other homovaniUic acid (HMV) derivatives related to the structure of CAP were carded out. Capsaicin, 1-nonenoylvanillylamide (NVA), HMV-dodecylamide (DCA) (but not HMV-cyclohexylamide (CHA) or HMV-hexadecylamide (HDC)) reduced the spinal content of substance P (SP), as measured by radioimmunoassay (RIA), and increased the tail-flick latency. Similar injection of kainic acid and piperine reduced levels of SP but failed to affect the tail-flick latency. None of the agents used affected spinal levels of cholecystokinin (CCK) or vasoactive intestinal peptide (VIP) as measured by RIA. In experiments using in vivo superfusion of the rat spinal cord, CAP, DCA and NVA were found to stimulate release of SP. Capsaicin had no effect on the levels of CCK or VIP immunoreactivity in the spinal superfusate. A tachyphylaxis to the effect of CAP and DCA on spinal SP release was demonstrated. Pretreatment with either agent blocked the releasing effect of the second. Pretreatment with an inactive analogue (HDC) had no effect on the subsequent activity of CAP. Kainic acid and piperine did not induce release of SP from the spinal cord. The relative selectivity of spinally administered capsaicinoids with regard to their effects on the content and release of peptides known to be contained in primary afferents and the presence of a similar structure-activity relationship for depletion and release of SP, desensitization and antinociception suggest the presence of a specific receptor site associated with a specific population of primary afferents through which pain information may pass. Whether SP is an 'afferent pain transmitter' is not clear, but at the least, it appears to serve as a marker for a population of afferents acted upon by spinally administered capsaicinoids.

INTRODUCTION Early studies with extracts of p e p p e r containing capsaicin ( C A P ) , a derivative of vanillylamide, demonstrated that direct application to the skin o r tongue would p r o d u c e a burning sensation and sharp taste, respectively19. Subsequent investigations r e v e a l e d that either topical or systemically a d m i n i s t e r e d capsaicin would result in the powerful activation of nociceptive afferents, pain b e h a v i o r in m a n and animals and a p r o m i n e n t desensitization22,26,sL Intrathecal injection of exceedingly low doses of C A P also causes a long lasting desensitization of the rat's response to t h e r m a l and chemical algogenic stimuli42A4,64, and G a m s e et al. 14 and H a y e s and Zyers 17 suggest that desensitization can be achieved by a focal action of the drug at both central and p e r i p h e r a l sites.

Systemically or intrathecally a d m i n i s t e r e d C A P causes a selective and p r o f o u n d d e p l e t i o n of SP in the dorsal horn of the rat spinal c0rdl,7.13,32, 63. In acute experiments C A P has been found to p r o v o k e a calciu m - d e p e n d e n t release of SP from the spinal cord in vitro and in viv02,5,11,12,56,64. These observations, in conjunction with the hypothesis that SP m a y serve as an afferent pain transmitter18, 31 a p p e a r e d to offer an explanation for the algogenic and desensitizing effects of C A P . M o r e recent observations on C A P have, however, suggested that its effects are not limited to an action on SP-containing neurons. Thus, C A P reduces the content of SP, somatostatin and the activity of glutamate decarboxylase 44 and causes a significant reduction in the levels of spinal horn cholecystokinin (CCK) as d e t e r m i n e d by immunohistochemistry, but not by r a d i o i m m u n o a s s a y (RIA)27,38,40.

Correspondence: T. L. Yaksh, Mayo Clinic, Rochester, MI 55905, U.S.A.

216 Though the mechanisms of CAP action are not known, it appears certain that the algogenic, neurotoxic and desensitizing actions are subject to specific drug-structure requirementsS.28.s:,54,55. Similar structure-activity profiles were described for the depleting effects of intrathecai capsaicinoids on immunoreactive SP and CCK in spinal cord 40 and their apparent potency in releasing spinal SP in vitroS. In the present study, the action of intrathecally administered CAP and 4 structurally related analogues was examined. In each animal we assessed by RIA, the spinal content of SP, VIP and CCK, and the latency of the thermally evoked tail-flick. The effects of these CAP-like agents have been compared with two other agents, kainic acid and piperine, a neurotoxin and a pungent agent, respectively, that are not structurally related to CAP. In a separate series of experiments the effects of CAP and these other agents have been tested on the in vivo release of SP from the spinal cord in the rat.

placed in a stereotaxic frame and respired artificially. A polyethylene inflow catheter (PE-10) was inserted about 10 cm to the caudal lumbar cord and a short stainless-steel outflow cannula was placed in the cisternal opening. Artificial cerebrospinal fluid (CSF) 6~ containing bacitracin (30pg/ml) and bovine serum albumin (120 ktg/ml) was infused through the inflow catheter at the rate of 100/A/rain using a two-channel peristaltic pump (Scientific Industries). The outflow of CSF was collected directly by the second peristaltic channel into a polypropylene tube containing (1.2 ml acetic acid and kept on ice. Sequential 20-min samples of perfusate were collected, frozen immediately and lyophilized. The lyophilized samples were reconstituted (0.5 ml volume) in the assay buffer and amount of SP was estimated by RIA (see below). The drugs to be tested were added to the superfusate entering the inflow catheter. Blank assay volumes were measured with perfusate and examined with perfusate plus releasing agent.

METHODS

Assay

Preparation All experiments were performed on male Sprague-Dawley rats (300-500 g). In animals maintained under halothane anesthesia, a polyethylene catheter (8 cm long, PE-10) was inserted through a cut in the cisterna magna, to the rostral edge of the lumbar enlargement 61. Following a period of recovery (7 days), the animals received injections of CAP, or one of the other agents, in 10-pl volumes. CAP and other agents were dissolved in the vehicle 50% dimethylsulfoxide and saline. Control injections were made using the vehicle (10/ll) only. Seven days following drug injection the nociceptive thresholds of all animals were measured using the tail-flick reflex test. In the absence of a response, the tail was removed after 6 s. After this the animals were sacrificed by decapitation and the lumbar region of the spinal cord rapidly removed, cleaned of meninges, dorsal and ventral roots, and frozen. In a separate series of experiments the effects of intrathecal CAP and related agents were tested on the release in vivo of SP from the superfused rat spinal cord using the technique described previously65. Briefly, a rat was anesthetized with chloraiose-urethane, a tracheal tube was inserted, and the animal

The peptides were extracted from cord tissues by boiling in 0.1 N HCI for 5 min. The extracts were lyophilized and the content of 3 peptides. SP, CCK-8 and VIP, was measured by RIA. Antiserum for SP (No. R4892) identifies the C-terminal of the undecapeptide and cross-reacts significantly with the hexapeptide. Assay sensitivity was 3 fmol/tube. Antisera for CCK (No. 3440) recognized the C-terminal sequence of the octapeptide and cross-reacted equally well with the tetrapeptide. Assay sensitivity was 2 fmol/tube. Antisera for VIP (courtesy of Professor V. Mutt, GIH Research Laboratory, Karolinska Institute) recognized the C O O H terminal. Assay sensitivity was 1 fmol/tube. The RIAs were carried out with the double antibody technique using [125I]TyrSSP; and I-VIPl_28 were the respective ligands. As the CCK antiserum reacts equally well to gastrin17, [12q]gastrin17 was used as the ligand. Further details of the extraction procedures and the SP, VIP and CCK assays used for tissue and spinal superfusate have been previously given41,62. Characterization of the immunoreactivity was carfled out with serial dilution curves and column chromatography. Displacement curves carried out with dilutions of the tissue extract or the spinal superfusate were observed to be essentially parallel with

217 eak). The structures of these agents have originally been shown elsewhere 53.

those generated by authentic CCK-8, VIPl_28 and SPI-ll with their respective antisera. Concentrated perfusion samples were placed in a Sephadex G-50 column (1 x 100 cm) and eluted with 0.01 phosphate buffer with 0.5% bovine serum albumin (BSA) at pH 7.6. Void volumes and salt peaks were calibrated with Blue Dextran 2000 (Pharmacia) and Na125I (New England Nuclear), respectively. VIP and SP immunoreactivity co-migrated in a single peak with authentic VIPl_28 and SPI_ll. CCK immunoreactivity appeared to co-migrate as a single peak with CCK-8. Subsequent studies carried out on a Sephadex G-10 column confirmed that the CCK immunoreactivity was a single peak corresponding to the octapeptide. In the absence of a chemical identification of the immunoreactive products, our designation of SP, VIP or CCK only implies the immunoreactive properties of the unknown material.

Statistics In the perfusion experiments, data were expressed as percent of control (e.g. the level of SP measured in the sample collected immediately prior to the addition of neurotoxin and the neurotoxin sample). A simple one-way analysis of variance was carried out. The error variance was then used to construct a Newman-Keuls test for multiple comparisons 60. For tissue levels, similar comparisons were made between peptide levels in control and in neurotoxin-treated animals. For simple comparison between pre- and post-treatment, a t-test for paired samples was performed. The injection of capsaicinoids resulted in an agitation response. The magnitude of the response was ranked as: 0 (no change in behavior); 1 (squeaking during injection, with occasional efforts to scratch hind flank); 2 (intense squeaking, biting/scratching). The ranking was made in the absence of any knowledge of the drugs' characteristics. The scores were summed, the means taken and the drugs ranked accordingly. A Zz contingency analysis was carried out to determine whether the observed frequency of assigned scores varied from the expected 16.

Drugs Drugs used in this study were: capsaicin, kainic acid (Sigma, St. Louis, MO); capsaicin analogues 1-nonenoylvaniUylamide (NVA), HMV-dodecylamide (DCA), HMV-cyclohexylamide (CHA) and HMV-hexadecylamide (HDC); and piperine (capsaicin analogues were synthesized and provided through the courtesy of Drs. P. Hegyes and S. Fold1 -Nonanoyl-

Control 200

Capsaicin

HMV-Dode-

Vanillamide

cylamide

HMV-Cyclo-

hexylamide

HMV-Hexadecylamide 15

1 150 co t--

Q.

u} > ¢} _J

0

100

0 O. ff~

r" ® <

Ii!iii: 50

•

2.4tO. 1 I (n=16) I

6.0=0.4 t (n=7) j

5.51'0.4 I (n=9) I

~

4.14"0.5 I(n=lO) I

2.1 ~'0.2 I(n=11) I

2.4~04 I (n=9) I

Fig. 1. Effect of an intrathecal injection capsaicin and 4 structurally related analogues on the content of substance P (SP), cholecystokinin (CCK) and vasoactiveintestinal peptide (VIP) in the rat lumbar spinal cord, and on the tail-flicklatency. The tail-flicklatencies given below each set of histograms were measured 7 days after a single injection (75 #g) of capsaicin or its derivatives, n = the number of animals used. Note the difference between the vertical scale on the left and right sides.

218 RESULTS

Effect of intrathecal agents on spinal peptide content and tail-flick latencies Fig. 1 illustrates the effect of capsaicin or one of its 4 analogues on the content of SP, VIP and CCK, and the tail-flick latency, 7 days after a single intrathecal injection (75 pg). Following CAP treatment, levels of SP in the lumbar cord after 7 days were reduced from a control value of 166 + 13.7 ng/g to 90.5 + 5.8 ng/g tissue weight (P < 0.05), representing a 45% decrease in peptide content. The tail-flick latency in these CAP-treated animals was increased to 6.0 s (P < 0.05), the time limit set for the stimulus cut-off in this test. The 4 analogues of CAP, NVA, DCA, C H A and HDC, affected SP levels and tail-flick latency to different degrees. After treatment with N V A the levels of SP were reduced to 75 + 7.5 ng/g (P < 0.05) and the tail-flick latency significantly increased to 5.5 s (P < 0.05). Agent D C A produced a small, but statistically significant reduction (P < 0.05) in the levels of spinal SP (128 + 10.1 ng/g) and raised tail-flick latency to 4.1 s (P < 0.05). In contrast to the action of these 3 agents, the two analogues C H A and H D C exerted minimal effects either on SP or the tail-flick latency. Neither CAP nor any of the other 4 H M V derivatives tested had a significant action on the spinal levels of VIP or CCK (P > 0.10). Fig. 2 shows the effects of intrathecal kainic acid and piperine treatment on the spinal content of the 3 peptides and on the tail-flick latency. Intrathecal injection of kainic acid and piperine reduced levels of

SP to 91 _+ 5.2 and 80 + 2.4 ng/g, respectively. Despite a clear reduction in the spinal levels of SP, the tail-flick latency was not significantly affected by either agent. Neither agent influenced the spinal levels of VIP or CCK. Higher doses of intrathecal kainate could not be tested since these produced strong body contractures resulting in asphyxia and death.

Effects of intrathecal agents on spinal SP release Fig. 3 shows the effect of CAP on the in vivo release of SP from the intact spinal cord of the chloraloseurethane anesthetized rat. Perfusion of the spinal cord with CSF containing CAP (2 × 10-4 M) produced vasodilation (as evidenced by pink ears), mydriasis and an immediate increase in the release of SP. Following deletion of CAP from the perfusate, the levels of SP fell to predrug baseline. This effect of spinal CAP on spinal SP release was dose dependent (see Fig. 4). Thus, over the range of 2-200pM, levels of SP were increased from 10 to 120% over baseline. In subsequent release experiments, we compared the effect of CAP and its pungent analogues NVA, DCA, CHA, H D C and piperine at doses of 200/~M. As shown in Fig. 4, the CAP analogues DCA, N V A and C H A evoked statistically significant increases in the collected levels of SP in the spinal superfusates a release from the spinal cord. Multiple comparisons carried out with the Newman-Keuls test indicated CAP = D C A = NVA = C H A > HDC = PIP = KAI = 0; (P < 0.05). As in the content experiments, the effects of kainate were tested at a dose lower than that used in tests with other agents. A higher dose of kainate could not be tested successfully because of 350

200

.~

Piperine

Kainic

Acid

300 u~ 2 5 0

150

¢o

S

o [ ~13~O,~(n=lO),l

I 2 1 t 0 , 1 (n=g)

0 0

Jo.

i

Fig. 2. Effect of an intrathecal injection of kainic acid (0.075 #g) and piperine (75 #g) on the content of SP, CCK and VIP of the rat spinal cord, and on the tail-flick latency. The latter was measured 7 days after a single injection of these agents. Results of control experiments are illustrated in Fig. 1. n = number of animals used. Note the difference between left and right vertical scales.

I&

_~ 2 0 0 oJ t'r O,. 150 '~ .E

~

100

I

so 0

| I

I

I

,

Capsaicin

i 0

I

I

i 40

i

i 80

CaDsalcm i i

120

160

T i m e (rain)

Fig. 3. The stimulatory effect of capsaicin on the release of SPlike immunoreactivity from the supcrfused spinal cord of chloralosc-u/ethane anesthetized rat. Capsaicin (g~O~M) was applied to the spinal cord during periochi r e p r e s e ~ e d b y h 0 r b zontal bars. Each value represents the mean + S,E.M., n = 5. Triangle, P < 0.05.

219 group. As can be seen, there was a reduction by the preceding administration of either agent to the SP releasing actions of the second agent, whether the first agent were N V A or CAP. This suggests that both agents were acting to deplete similar stores of spinal SP. A n inactive agent, H D C , neither released nor desensitized.

200 ~8O

"~ 16o ,20 8 ~ c &

100

~. ~

40

80

20 o

r.L.I 2

200

20 Clip

200

200

200

200

200

OCA

NVA

CHA

HDC

Pip

0.1

0.5 Kill

Fig. 4. Comparative effects of capsaicin (CAP), HMV-dodecylamide (DCA), 1-nonenoylvanillylamide (NVA), HMV-cyclohexylamide (CHA), HMV-hexadecylamide (HDC), piperine (PIP) and kainic acid (KAI) on the release of SP-like immunoreactivity from the superfused rat spinal cord in vivo. Doses below each histogram are given in/~M. The release values have been standardized by expressing release in the presence of the drug as a percentage of release measured in the sample immediately preceding the drug application. Each value shown represents the mean + S.E.M., n = 4-6 experiments. Triangle, P < 0.05.

marked tachycardia and hypertension which induced bleeding from the spinal cord.

Tachyphylaxis As shown in Fig. 3, a second application of C A P (5 x 10--4 M) failed to evoke a second release of SP. As shown in Table I, similar results were also observed with N V A (5 x 10-~ M). To determine whether the actions of these two compounds were subject to a cross-tachyphylaxis, a subsequent series of experiments were carried out in which C A P or N V A (5 x 10 -4 M each) was administered into the spinal superfusate followed two samples later by the administration of the second agent. Table I presents the mean results of 3 animals in each

Effects o f intrathecal capsaicin on C C K and V I P release In separate experiments, we examined the effects of intrathecal C A P on V I P and C C K release from spinal cord. At concentrations of up to 200/~M C A P , no changes in the resting levels of either V I P or C C K were observed. In view of the absence of activity, further studies on the effects of the other analogues were not pursued. General behavioral observations Injection of C A P in animals produced characteristic behavioral signs: an initial contracture, followed by vocalization, biting and scratching. These latter signs lasted for about 7-10 min after which the animals reassumed the normal posture and behavior, and were overtly indistinguishable from control or untreated animals. The magnitude (mean of the total ranks for each group) of the post-injection behavioral syndrome evoked by similar doses of the pungent agents was: C A P (3.0); N V A (1.7); D C A (1.4); C H A (1.2); H D C (1.1) PIP (0.8). Z2 analysis indicated that the distribution of expected frequencies of rating (0, 1 or 2) was associated with the intrathecal drug used (x 2 = 14.79, df = 10, P < 0.10). Kainic acid evoked a powerful behavioral reaction which in-

TABLE I

Tachyphylaxis and cross-tachyphylaxis at capsaicin (CAP), 1-nonenoylvanillylamide (NVA) and HMV-hexadecylamide (HDC) administered spinally on the spinal release of substance P Following 2 control samples, the first drug (5 x 10-4 M) was added to the spinal superfusate only during the third sample, the second drug (5 x 10-4 M) was added only during the fifth sample.

Order of drug presentation

CAP CAP DCA DCA HDC *P < 0.05.

CAP DCA DCA CAP CAP

n (number of animals)

Baseline levels of SP in spinal superfusate (nmol; mean +_S.E.)

Evoked release of spinal SP (% of predrug baseline) Fi~tdrug

Second drug

4 3 3 3 3

98 +- 13 102 + 18 71 + 14 53 + 12 87 + 15

258 + 28' 202 + 19" 168 + 23* 143 + 31" 107 + 15

106 + 12 113 + 16 118 + 12 128 --+13 176 --+26*

220 cluded marked body contractures and scratching. At the time of sacrifice (7 days) all animals appeared behaviorally normal with the exception of those animals which were analgesic secondary to the CAP and NVA treatments. These animals reliably showed urine-stained abdomens. At autopsy these animals all displayed markedly distended bladders. DISCUSSION The addition of capsaicinoid agents during in vivo spinal superfusion experiments results in a dose-dependent increase in the levels of SP but not CCK or VIP immunoreactivity in the collected effluent. The release of spinal SP by CAP and its active analogues shows a complete and rapidly developing tachyphylaxis which is observed during multiple presentations of the drug at short intervalsS,11,56. We believe the SP whose release is evoked by CAP derives from primary afferent terminals. This hypothesis is supported by 4 observations. First, SP-like immunoreactivity is in high concentrations in the dorsal gray, particularly in the substantia gelatinosa. Though some of the immunoactivity is co-contained in spinopetal 5-HT fibers2t, 50 much of the immunoreactivity, particularly in the dorsal gray, disappears after rhizotomy20. Second, CAP is known to have a powerful depolarizing effect on dorsal roots and ganglials,sg, 66. In single unit studies CAP evokes a prolonged opening of the Ca channel of spinal ganglion cells and significant depolarization15,s9. For type B cells, the measured depolarization does not appear to recover during the period of the experiment59. Such prolonged effects could account for the powerful releasing action and subsequent acute tachyphylaxis observed with CAP. Third, the release evoked by intrathecal CAP is not significantly diminished by pretreatment with intrathecal 5,6-dihydroxytryptamine (see ref. 31) or kainic acid (Yaksh, Harry and Go, unpublished observations), manipulations which produce depletionS0,63,64 of SP stores co-contained in the terminals of the spinopetal serotonergic system3, 6 and intrinsic neurons, respectively2. Finally, kainic acid produces a powerful depolarizing effect on the spinal cord which is mediated by glutamate receptors post-synaptic to the primary afferents 67. In spite of the evident signs of spinal excita-

tion produced by intrathecal kainate, no SP release was evoked by this glutamate analogue in the present experiments. Capsaicinoid applied into the spinal intrathecal space in doses which are not systemically active results in a significant depletion in the spinal levels of SP but not VIP or CCK activity as measured by RIA. Histochemical studies on the effect of intrathecally administered CAP and its analogues indicate that depletion of immunoreactivity occurs in the region of the dorsal gray known to contain the terminals of primary afferents27,4°. Studies on the neurotoxic effects of systemically administered CAP indicate that the obvious degeneration produced by neonatal treatment is limited largely to unmyelinated fiber populations/8,19,46. These observations, showing a depletion of SP by CAP, are consistent with the observation that SP immunoreactivity is found in type B ganglion cells 11 which give rise to small diameter primary afferents. The longterm changes in SP content as measured by RIA and immunohistochemistry probably result from changes in dorsal horn terminal morphology and afferent fiber loss which results from CAP treatment, particularly in neonates 22,23,34. The mechanism of these changes is not known, but may be related to irreversible depolarization of the small sensory afferents59 Kainic acid also evokes a powerful depolarization by a glutamate receptor mediated event which leads subsequently to degeneration of the nerve cell48. lntrathecal kainate results in a significant reduction in spinal levels of peptides thought to be contained in interneurons, e.g. neurotensin, enkephalin and SP 1.24, 49, which presumably possess glutamate receptors. We think it is significant therefore that. m general, the releasing potencies of capsaicinoids, and presumably their ability to depolarize primary afferents, parallel their ability to produce terminal degeneration (see Table I). The possibility that the two classes of agents (capsaicinoids and glutamate analogues) exert their relatively selective neurotoxic effects by a similar mechanism, through separate receptors associated with different neuronal populations, represents a unifying hypothesis for these observations. Alternately, as CAP can inhibit afferent axon transpot0 4, and degeneration could result from the loss of a transported neurotrophic factor, perhaps SP 58. It is important to stress that these effects of intrathecal CAP appear relatively selective for certain

221 neuronal populations. Systemically administered CAP does not alter brain levels of SP nor the SP immunoreactivity co-contained within spinopetal serotonergic terminals (see above). Intrathecal or systemic administration of doses of CAP in excess of those required to significantly diminish afferent stores of SP have no effect on the assayed spinal levels of 5-HT, NE (as measured by high-performance liquid chromatography), neurotensin, Met-enkephalin, VIP or CCK (as measured by RIA) (refs. 13, 40, 51, 62 and unpublished observations), the spinal stores of the latter two being contained in part in primary afferents35,37,62. In contrast, CAP treatment has been shown to diminish spinal levels of SP (as measured by RIA and histochemistry, see above), soma-. tostatin (as measured by RIA and histochemistry13,40), and CCK (as measured by immunohistochemistry, see below). This relative selectivity suggests that CAP and its analogues are exerting their actions on selected neuronal populations. The distinction between the results after CAP treatment obtained with RIA and immunohistochemistry for CCK has been shown reliably in rat and cat, in several laboratories27,38-40,47,62. This difference between RIA and histochemistry has also been reported in rat after rhizotomy where denervation had no effect on CCK levels measured by RIA but did reduce the intensity of the immunohistochemical measure 39. w e do not know the reason for this difference between RIA and immunohistochemistry vis-a-vis CCK levels. Based on the characterization of our antisera, the similarity of the column migration profile of CCK immunoreactivity extracted from rat spinal cord samples of CAPand vehicle-treated rats and the parallelism in the ligand displacement curves in the CCK RIA obtained with extracted spinal cord samples, we conclude that the assay is measuring the presence of carboxy terminus CCK fragments (ref. 62 and Yaksh and Go, unpublished observations). Recently, it has been reported with immunohistochemistry that CCK immunoreactivity may be cocontained with SP in dorsal root ganglion ceUs8. Disappearance of the CCK immunohistochemical reaction product after CAP treatment is at least consistent with the finding that CAP has a neurotoxic effect on SP-containing afferents. Behavioral effects of intrathecal CAP are mediated by an effect on central processes of small primary af-

ferents. Intrathecally administered CAP results in an initial muscle spasm which is followed by vigorous biting and scratching directed at the flanks and hind paws. Subsequently, there is a dimunition of the reflex and organized behavioral response to otherwise algogenic chemical and thermal stimuli. Application of capsaicinoid agents onto the peripheral nerve terminal, e.g. the cornea52,54,55, results in nocisponsive evoked behavior directed at the eye (wiping of the eye). Several lines of evidence suggest that these effects are mediated by an action of small primary afferent fibers. Firstly, small myelinated and unmyelinated fibers are thought to mediate the transmission of noxious thermal and chemical stimuli57. The fact that innervation of the cornea is limited to free nerve endings 36 makes it likely that the desensitization is mediated by that class of fibers. Secondly, a variety of physiological experiments have demonstrated that CAP applied topically will excite small diameter primary afferents when applied peripherally or centrally10,33,34,59. Thirdly, systematic studies on the effect of peripherally administered CAP has clearly shown that the neurotoxic effects appear limited to unmyelinated afferent populations 46. We stress that these observations do not mean that the effects of CAP are only in spinal afferent systems which transmit nociceptive information. The urine-stained belly noted in this paper corresponds to urinary overflow. In recent studies we noted that the intrathecal CAP in unanesthetized rats prepared with chronic indwelling bladder catheters would block the volume-evoked micturition reflex (Brent and Yaksh, in preparation). This magnitude of the micturition blockade in these animals correlated absolutely with the degree of failure of the thermally evoked nociceptive responses. It is important to note that Hoyes and Barber 23 have reported that systemic CAP would produce extensive degeneration of axons in the ureteric plexi. Many of the axons appeared to have terminals with dense cored vesicles. The afferent system mediating the bladder reflex has long been a point of contention; but Milne and DeGroat 43 have argued for the importance of A6/C fiber caliber afferents. The apparent effects of spinal CAP on small but not large caliber systems supports this possibility vis-a-vis the afferent link of the volume-evoked micturition reflex.

222 in producing desensitization. Secondly, the characteristics of the spinal receptors acted upon by capsaicinoid agents to alter the nociceptive response were indistinguishable from the spinal receptor which produced changes in urinary function. As we do not commonly consider the volume-evoked micturition reflex to be mediated by pain afferents, such observations suggest that CAPsensitive afferents are not uniquely related to pain transmission. Thirdly, with regard to the biochemical endpoints, there is a gratifyingly close correspondence between the in vitro and in vivo ability of these agents to evoke the release of SP. In addition, the more potent the drug is as a releaser, the more potent is its ability to produce nerve degeneration and subsequent SP depletion. A n important exception is pipefine. Though it had little effect on SP release, it serves to significantly reduce dorsal horn SP 4°. The fact that piperine does not produce acute desensitization (ref. 41 and present experiments), indicates that it may act by a different mechanism than the capsaicinoids. Whether piperine blocks axon transport or produces long lasting depolarization is not known. Fourthly, examination of the structure-activity relationship across the anatomical, biochemical and be-

The structure--activity profile o f the capsaicinoids suggests that these agents are p r o d u c i n g their behavioral and biochemical effects at a m e m b r a n e site with c o m m o n characteristics. If the effects of a class of

agents on several biological systems are mediated by the same type of receptor, a minimum criterium is that agents thought to act through that receptor show a comparable structure-activity relationship and similar patterns of cross-desensitization. Table II summarizes several of the behavioral and biochemical end-points measured in in vivo and in vitro after administration of the several capsaicinoid analogues used in this and other studies. Although presently there are relatively few analogues systematically examined so far, several points appear clear. Firstly, the relative ordering of activity for spinal vs peripherally administered drugs do not significantly differ in their ability to evoke the respective nocisponsive behaviors (agitation, corneal wiping) or to produce subsequent desensitization. This suggests that the characteristics of the sites acted upon by the central vs peripheral action of these drugs cannot be distinguished. With the apparent exception of C H A after corneal application, agents which were strong producers of pain behavior after either intrathecal or corneal administration were also respectively active TABLE 11

Summary showing ranking of the potency of capsaicin analogues and piperine with regard to afferent degeneration, spinal SP depletion, spinal SP release in vivo and in vitro, and the pain-evoking and desensitizing action

MPP = minimum pain producing concentration (ug/ml) as defined by wiping response evoked by instillation onto cornea; MDC = minimum desensitizin_gconcentration (ug/ml) as measured with wiping response 5<55. Ag = ranked magnitude of agitation response induced by intrathecal injection of fixed dose of capsaicinoid; TF = ranked magnitude of tail-flick inhibition observed 7 days after intrathecal injection of capsaicinoid. "Jr = no statistical difference between two adjacent rankings. (0) = no detectable effect in the doses used. Behavioral end-point

Biochemical end-point Nerve degeneration *

Spinal SP depletion ** RIA

Capsaicin (CAP) 1-Nonenoyivanillylamide (NVA) HMV-dodecylamide (DCA) HMV-octylester (HVO) HMV-~yclohexylamide (CHA)

1" 2 3 4 --

HMV-hexadecylamide(HDC) Piperine (PIP) Vehicle

(0)

1~t 2* 3* -(0)

In vitro spinal SP release ***

In vivo spinal SP release **

Corneal application

Spinal administration

MPP

MDC

Ag

TF

12 3*

1~t 3* 2*

2.5 3.5 48

140 210 53

1 2 3

1~t 2* 3

4

--

15

(0)

--

--

(0)

---

4 (0)

20 980

8920 5760

4 6

(0) (0)

(0)

--

(0)

166

(0)

5

(0)

(o)

(o)

(o)

(o)

(o)

(o)

(o)

* Ranking of potency; systemically administered into newborn rats ~. ** Ranking of potency; measured by radioimmunoassay. *** Ranking of potency; release of SP from adult rat spinal cordL

223 havioral measures indicates a striking consistency. Capsaicinoid agents which produced a significant release, depleted SP and produced small afferent degeneration would show a comparable potency in producing agitation and a subsequent desensitization. As noted, intrathecal kainic acid which produces powerful depolarization showed none of these biochemical or behavioral characteristics. Even pipefine, though it appeared to decrease SP content, neither released SP nor showed the behavioral effects of the active members of this series. Finally, as shown in the systematic behavioral studies of Szolcsanyi and Janc~o-Gabor 54,55, there is a clear cross-densensitization between active, but not inactive members. Though on a more limited scale, the present experiments substantiated the existence of a selective cross-desensitization as measured by spinal SP release for the spinal action of this class of agents. HDC, an analogue which does not deplete or release spinal SP, did not result in cross-desensitization in the dose employed. Such observations offer further support that the active members of the family o f capsaicinoid agents are indeed acting on a common membrane site, presumably on the afferent terminal. While our observations suggest a correlation between the ability of an agent to alter the disposition of afferent SP content and the pain response (however, see ref. 4), it does not p r o v e that SP has a role in afferent transmission. Our failure to see behavioral desensitization with piperine in spite of a significant SP depletion emphasizes this point (ref. 40 and present experirqents). Intrathecally administered SP indeed produces some signs of agitation 25, but none as profound as that produced by CAP. This suggests that other agents, perhaps more potent than SP or in conjunction with SP, may be released by CAP. It is important to note that CAP will produce depolarization of large afferents, though unlike that seen in C-fibers, large fiber depolarization is reversible 59. Indeed, Dodd et al.9 have shown that fluoride resistant acid phosphatase (FRAP), thought to be a marker for small afferents, is contained in afferent populations which do n o t contain SP. It is well known that REFERENCES 1 Barber, R. P., Vaughn, J. E., Slemmon, J. R., Salvaterra,

FRAP is also depleted by CAP 30. Thus other populations of as yet pharmacologically undefined afferent neurons are affected by this class of neurotoxins. At the least, however, such observations indicate that SP may serve as a marker for a specific population of afferents which possess a receptor identified by this series of capsaicinoid agents, the activation of which receptor produces a depolarization of pain-associated afferents, and the loss of which results in at least a partial obtundation of the afferent pathways through which high intensity thermal information gains access to the spinal cord. In summary, the presence of the structure-activity relationship mediating the releasing and depleting effects and the relative selectivity of the action of this class of agents with regard to the release and depletion of spinal amines and peptides suggest that the observed effects are mediated by a specific site (receptor) which is iargely associated with a discrete population of primary afferents. As with any receptor activated by an exogenous compound, an important question is whether the recognized site serves as the point of interaction with an endogenous substrate. The structure-activity relationship observed for the capsaicinoids suggests a specific recognition site at the central afferent terminals. The similarity of the capsaicinoids to the naturally occurring homovanillic acid raises the possibility that these materials may naturally interact with central afferent transmission. ACKNOWLEDGEMENTS We would like to thank Dr. P. Hegyes and Dr. S. Foldeak for their synthesizing and provision of generous samples of the several capsaicin analogues. We would also like to thank Ms. Jane Bailey, Ms. Sandy Michener and Ms. Ann Rockafellow for their expert and enthusiastic help in these experiments. The work was supported by funds from the Mayo Foundation, Grant NS 14629 to T.L.Y. and Grant AM07198 to V . L . W . G . K . J . was supported as a Killam Foundation Scholar.

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