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Calcitonin gene-related peptide increases in the dorsal root ganglia of adjuvant arthritic rat
Peptides,Vol. 10, pp. 447~-52. ©Pergamon Press plc, 1989. Printed in the U.S.A.
0196-9781/89 $3.00 + .00
Calcitonin Gene-Related Peptide Increases in the Dorsal Root Ganglia of Adjuvant Arthritic Rat YASUSHI KURAISHI, TOYOMICHI NANAYAMA, HIROSHI OHNO, NOBUTAKA FUJII,* AKIRA OTAKA,* HARUAKI YAJIMA* AND MASAMICHI SATOH t
Department of Pharmacology and *Department of Pharmaceutical Manufacturing Chemistry Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606, Japan R e c e i v e d 18 April 1988
KURAISHI, Y., T. NANAYAMA, H. OHNO, N. FUJII, A. OTAKA, H. YAJIMA AND M. SATOH. Calcitoningene-related peptide increasesin the dorsal rootganglia of adjuvantarthriticrat. PEPTIDES 10(2) 447-452, 1989.--We examined the effect of adjuvant arthritis on the content of immunoreactive calcitonin gene-related peptide (iCGRP) in the dorsal root ganglia at LA-L6 levels and the spinal cord at a lumbar level in rats, Arthritis was induced by inoculating adjuvant into both hind-paws twice at a 10 day interval. In the arthritic rats 15 days after the first inoculation (day 15), the content of iCGRP was significantly increased in the dorsal root ganglia, with no change in the dorsal and ventral horns. The content in the dorsal root ganglia was still high on day 26 and had decreased by day 40. An intrathecal injection of colchicine (0.2 mg, 18 hr before killing) enhanced the increase of iCGRP in the dorsal root ganglia and decreased it in the dorsal horn of arthritic rats, although in noninoculated rats such treatment produced no significant changes in the content of iCGRP in both regions. The arthritis-induced increase in the content of iCGRP in the dorsal root ganglia was significantly reduced after treatment with the antiinflammatory analgesic, diclofenac sodium, in a dose of 3 mg/kg/day, PO for 10 days. Swelling and hyperalgesia in the hind-paw were depressed after such treatment. These results suggest that adjuvant arthritis with long-lasting inflammation with pain facilitates the turnover, especially biosynthesis, of CGRP in primary afferent neurons. Calcitonin gene-related peptide (CGRP)
Arthritis
Dorsal root ganglion
Spinal cord
Colchicine
Diclofenac
nous CGRP, especially that in the primary afferents. Recently we have found that intrathecal injection of an antiserum against CGRP improves hyperalgesia shown in adjuvant arthritic rats (16). If such a CGRP-associated hyperalgesia would be due to this neuropeptide present in the primary afferents, adjuvant arthritis would alter the turnover of this neuropeptide. We report here that adjuvant arthritis increases the content of iCGRP in the DRG, a finding which may reflect the enhancement of the biosynthesis of this neuropeptide in primary afferent neurons.
CALCITONIN gene-related peptide (CGRP) is composed of 37 amino acid residues and derived from the calcitonin gene predominantly in neural tissues (1,23). With regard to the primary sensory neurons, immunoreactive CGRP (iCGRP) is contained in a moderate or large population of the dorsal root ganglion (DRG) cells and in some colocalized with other neuropeptides such as substance P and somatostatin (8, 12, 19, 27, 34); the latter two peptides are implicated in nociceptive transmission (15, 21, 35, 36). The nerve terminals and varicosities containing iCGRP are densely distributed in the superficial layers of the dorsal horn (5, 8, 26 34). Thus, this peptide is thought to be related to sensory transmission in the spinal dorsal horn. In fact, an intrathecal injection of CGRP lowers the nociceptive threshold for acute mechanical stimulation (21) and enhances the effects of substance P and somatostatin on the nociceptive flexion reflex (35,36) in rats. In addition, CGRP promotes the release of immunoreactive substance P evoked by capsaicin, an algesic compound, from slices of the rat dorsal horn (21). These findings suggest the participation of CGRP in the facilitation of transmission of nociceptive information in the dorsal horn. However, it remains unknown whether such influences would be exerted by endoge-
METHOD
Induction of Adjuvant Arthritis Arthritis was produced by intradermally inoculating 0,04 ml of a suspension of heat-killed Mycobacterium butyricum (Difco Laboratories, Detroit, MI) in paraffin oil (12 mg/ml) into both hind-paws of male Sprague-Dawley rats at 7-8 weeks of age. This inoculation was given twice at 10-day intervals. Rats were kept on a 12-hr light/dark cycle (8:00 a.m./8:00 p.m.) at 23°C with free access to food and water. An average of the volume of both
1Requests for reprints should be addressed to M. Satoh, Ph.D.
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KURAISHI ET AL.
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hind-paws served as an index of the severity of infiammantion. Rats with more than a 3 cm 3 average volume of the hind-paws on day 15 (15 days after the first inoculation) were considered to be arthritic; the volume of the hind-paw was 1.73 ± 0.18 cm 3 (mean ± SD, n = 38) before the inoculation. The nociceptive threshold of hind-paw was measured by the Randall-Selitto test (22) with a loading rate of 48 g/sec. Since in preliminary experiments, intradermal injections of saline alone into the hind-paws did not affect the content of iCGRP in the DRG, we used nontreated age-matched rats as control in the present experiments.
Extraction of CGRP Rats were decapitated and frozen in dry ice-ethanol, and the DRG of both sides at the I.A-L6 levels and the lumbar enlargement (1 cm long) were rapidly removed. The dorsal and ventral horns were excised, as described (14). The tissues were boiled in 1 ml of 2 M acetic acid for 5 min, sonicated and centrifuged at 27,000 × g for 15 min at 4°C. The supernatant was subjected to radioimmunoassay. Recovery of the added synthetic CGRP was 69.5 --_2.8% (n = 7), and the results were corrected for losses.
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Radioimmunoassay was conducted using anti-CGRP antiserum produced by immunizing rabbits against human CGRP(6-37) conjugated to bovine serum albumin using N-succinimidyl-p(2-nitrovinyl)-benzoate (6). [Tyr°]-rat et-CGRP (Peninsula Laboratories, Belmont, CA) labeled with Na~25I (NEN Products, Boston, MA) and rat c(-CGRP (Peptide Institute, Minoh, Japan) were used as tracer and standard, respectively. A buffer for antiserum and tracer was made up of 0.05 M phosphate buffer (pH 6.0), 0.15 M NaC1, 0.1% bovine serum albumin, 0.1% gelatin and 0.1% NAN3, and that for a sample 0.05 M phosphate buffer (pH 7.5), 0.1% bovine serum albumin, 0.1% Triton X-100 and 0.1% NaN 3. "Bound" and "free" fractions were separated with 20% polyethylene glycol 6,000, as described (14). Cross-reactivity of rat 13-CGRP was similar to rat a-CGRP and those of substance P, somatostatin-14 and somatostatin-28 were all less than 0.05%. The dilution curve of the extraction from the lumbar enlargement of rats was parallel to the standard curve. In addition, separation of the lumbar extraction by either gel chromatography or highperformance liquid chromatography showed a single peak of iCGRP, although the chromatography did not separate the authentic a- and f3-CGRP. These results show that iCGRP of the rat can be determined using the present radioimmunoassay and that iCGRP mainly corresponds to CGRP.
Drug Administration Diclofenac sodium (a gift from Ciba-Geigy Japan, Takarazuka, Japan) was given daily in a peroral dose of 3 mg/kg/day as a 0.5% carboxymethylcellulose suspension, about 1:00 p.m. from day 16 to day 25. The nociceptive threshold and the volume of the hind-paw were measured 1 hr before (day 16) or after (days 19, 22 and 25) the administration. Two hundred p.g of colchicine (Nacalai Tesque, Kyoto, Japan) was intrathecally administered in a volume of 10 p.1 through a lumbar puncture (24) on day 14, and 18 hr later the rats were killed.
Data Processing Results are expressed as mean ± SEM. Statistical comparisons was made using Student's t-test (two-tailed), simultaneous multiple comparisons by the Bonferroni method (33) or two-way
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FIG. 1. Effects of adjuvant arthritis on (a) the nociceptive threshold and (b) the volume of the hind-paw. Arthritis groups (O) were inoculated with adjuvant on the days indicated by arrows and control groups (©) were given no treatment. Values are means and SEM of 6 experiments. *p<0.01 as compared to the control on the same day (Student's t-test).
analysis of variance (ANOVA); p<0.05 was considered significant. RESULTS
Effects of Arthritis on the Content of iCGRP A remarkable swell and a decrease in the nociceptive threshold (hyperalgesia) of the hind-paw were observed from day 15 to day 40 (Fig. 1). The content of iCGRP in the DRG at the lumbar levels was increased from day 15 to day 26 and tended toward a recovery on day 40; the increase compared to the noninoculated control was 81% (p<0.05, Student's t-test), 100% (p<0.05) and 53% (0.05
Effect of Colchicine on the Content of iCGRP In this series of experiments we examined the effects on the content of iCGRP of colchicine, a compound which blocks axonal flow. An intrathecal injection of colchicine significantly facilitated an increase in the content of iCGRP in the DRG, as revealed by ANOVA, F(1,20)=4.515, p<0.05; percent increases of iCGRP by colchicine were 8.3 and 61.5 (p<0.005, simultaneous multiple comparisons) in noninoculated and arthritic groups, respectively (Fig. 3). On the contrary, iCGRP in the dorsal horn was decreased by colchicine, F(1,20)=8.488, p<0.01, and this decrease was
CGRP AND PAIN
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FIG. 2. Influence of adjuvant arthrits on the content of immunoreactive calcitonin gene-related peptide (iCGRP) in the dorsal root ganglia at the L4-L6 levels. Arthritis groups (0) were inoculated with adjuvant on the days indicated by arrows and control groups ((3) were given no treatment. Mean and SEM are represented and the values in parentheses indicate the number of rats used. *p
more remarkable in the arthritic rats (39%, p < 0 . 0 1 , simultaneous multiple comparisons) than in noninoculated ones (21%, not significant) (Fig. 4).
Effect of Diclofenac on the Content of iCGRP and Inflammation To determine whether arthritic inflammation with pain would be responsible for the increase in CGRP contents in the DRG, rats were given diclofenac sodium, a nonsteroidal antiinflammatory agent which exerts a potent analgesic action on rheumatoid arthritis (4, 25, 28). Although the degree of swelling and hyperalgesia of the hind-paw was constant in the arthritic group from day 16 to day 25, diclofenac partially but significantly improved the swelling, [F(1,30)= 25.927, p<0.0001, compared in arthritis without diclofenac] and hyperalgesia, F(1,30) = 14.404, p <0.001 (Fig. 5). Such treatment with diclofenac significantly, F(1,20) = 4.463, p < 0 . 0 5 , inhibited the arthritis-induced increase in the content of iCGRP in the DRG (Fig. 6). In the nonarthritic rats, diclofenac did not affect the content of iCGRP (Fig. 6), the volume of the paw and the nociceptive threshold (data not shown).
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FIG. 3. Effects of an intrathecal injection of colchicine (0.2 mg, 18 hr before killing) on the content of immunoreactive calcitonin gene-related peptide (iCGRP) in the dorsal root ganglia at the I.A-L6 levels in adjuvant arthritic and noninoculated rats. Values represent mean---SEM from 6 experiments. Significant effects were produced by arthritis, F(1,20)= 26.352, p<0.01, colchicine, F(1,20)= 6.277, p<0.05, and their interaction, F(1,20) = 4.515, p<0.05. *P<0.005 vs. arthritis without colchicine and p<0.0001 vs. colchicine alone (simultaneous multiple comparisons).
DISCUSSION We found a remarkable increase in the content of iCGRP in the DRG during adjuvant arthritis in rats. This increase may be due to changes in the content in the somata of primary afferent neurons rather than in terminals and vaficosities because the increase was
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TABLE 1 EFFECTS OF ADJUVANTARTHRITIS ON THE CONTENT OF IMMUNOREACTIVECGRP IN THE DORSALROOT GANGLIA, DORSAL HORN AND VENTRALHORN OF THE RAT ICGRP (pmol/mg tissue)
Control
Arthritis
0.095 - 0.013 0.82 4- 0.10 0.043 --- 0.012 7
0.172 - 0.030* 1.01 - 0.11 0.042 --- 0.006 6
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+ control
Dorsal root ganglia Dorsal horn Ventral horn n
The rats were killed on 15 days after the first inoculation and the immunoreactive CGRP measured. *p
-
+ arthritis
FIG. 4. Effects of an intrathecal injection of colchicine (0.2 mg, 18 hr before killing) on the content of immunoreactive calcitonin gene-related peptide (iCGRP) in the dorsal horn of the lumbar enlargement in adjuvant arthritic and noninoculated rats.Values represent mean_SEM from 6 experiments. A significant decrease was produced by colchicine, F(1,20) = 8.488, p
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FIG. 5. Effects of diclofenac sodium on the nociceptive threshold (a) and on the volume (b) of the hind-paw. The diclofenac plus arthritis group (O) was given diclofenac sodium (3 mg/kg/day, PO) on the days indicated by arrow heads (16-25 days after the first inoculation). The arthritis group (O) was given only the linoculation and the control (n) no treatment. Each group was 6 rats and values were mean- SEM. Diclofenac significantly improved the hyperalgesia, F(1,30)= 14.404, p<0.001, and the swelling, F(1,30) = 25.927, p<0.0001, during the period from 19 to 25 days after the first inoculation. *p<0.05 when compared with the group of arthritis without diclofenac on the same days (Student's t-test).
specifically seen in the DRG but not in the dorsal horn. In the former region, iCGRP is present in a large population of the cells and the distribution of the iCGRP-containing terminals and varicosities is relatively sparse (7, 12, 27). On the contrary, the latter has dense terminals and varicosities (27,34). Thus, the increase might not result from a suppression in the release of iCGRP from the nerve terminals. Therefore, two factors can be considered to influence the content of iCGRP; one is the biosynthesis of CGRP in the somata and another is the axonal transport of CGRP from the somata to the nerve terminals. The increase of iCGRP in the DRG in the presence of adjuvant arthritis and its augmentation by intrathecal colchicine (inhibition of axonal transport) suggest that the biosynthesis of iCGRP was facilitated in the arthritic rats. Measurement of the content of mRNA encoding CGRP in the DRG provides pertinent evidence for changes in the biosynthesis of this peptide, and such experiments are ongoing in our laboratory. As opposite changes in the content of iCGRP in the DRG and the dorsal horn were greater after colchicine treatment in the arthritic rats than in noninoculated ones, an augmented axonal transport in the arthritic rats was also suggested. The adjuvant arthritis is an autoimmune disease in the joint (31,32) and systemic immunologic changes may also occur in the inoculated animals. In the present experiments, the arthritisinduced increase in the content of iCGRP was seen in the DRG but not in the ventral horn, in which CGRP is present in the motoneurons (8,26), suggesting that CGRP-containing motoneurons may not be affected by systemic immunologic changes. The arthritis-induced increase in the content of iCGRP in the DRG was
FIG. 6. The effect of diclofenac sodium on the arthritis-induced increase in the content of immunoreactive calcitonin gene-related peptide (iCGRP) in the dorsal root ganglia at the L4-L6 levels in the rats. Values represent mean ___SEM from 6 experiments. Either diclofenac sodium (3 mg/kg/day, PO) or vehicle was administered for 10 days, which correspond to 16-25 days after the first inoculation in arthritic group, and the control groups were without inoculation. The arthritis-induced increase was significantly, F(1,20) =4.463, p<0.05, inhibited by diclofenac. *p<0.01 vs. untreated control and arthritis plus diclofenac (simultaneous multiple comparisons). suppressed by diclofenac, which also inhibited the swelling and hyperalgesia of the hind-paw. At low concentrations, diclofenac selectively inhibits cyclooxygenase but not 5-1ipoxygenase (13), and thus selectively inhibits biosynthesis of prostaglandins which are implicated as endogenous inhibitors of humoral and cellular immunity. In addition, nonsteroidal antiinflammatory agents do not suppress the immune responses (9, 17, 30). These findings, taken together, suggest that the increase in the content of iCGRP in the DRG did not result from systemic immunologic changes. Although CGRP is mainly present in the small- to mediumsized DRG neurons, this peptide also occurs in the large DRG neurons (12), which may be related to sensations other than a nociceptive one (11). Adjuvant arthritic rats show a decrease in locomotor activity (3), which, one may consider, would be responsible for the increase in the content of iCGRP in the DRG. However, such an increase was probably due to the augmented biosynthesis of this peptide, a phenomenon suggesting that the activity of iCGRP-containing primary neurons is greater in adjuvant arthritis. On the other hand, the decreased locomotor activity probably reduces the incidence of mechanical stimuli in the hind-paws and then diminishes the activity of mechanical nociceptive afferents (18). Thus, it is unlikely that the increase in the content of iCGRP in the DRG was due to a decrease in locomotor activity. The present results suggest the increased synthesis and axonal transport of CGRP in primary sensory neurons of adjuvant arthritic rats. Recently we found that capsaicin-evoked release of iCGRP from slices of the lumbar cord was greater in adjuvant arthritic rats than in nonarthritic ones (unpublished observation), suggesting the increase in the content of iCGRP in the nerve terminals of the capsaicin-sensitive, probably nociceptive (37), primary afferents. Many CGRP-positive fibers in the spinal dorsal horn are lost after treatment with capsaicin (5), whereas CGRP-positive fibers in the dorsal column nuclei are mainly derived from the medium- to large-sized DRG neurons and insensitive to capsaicin (29). Taken together, these findings suggest that some populations of the CGRP-containing primary afferents are related to nociception.
CGRP AND PAIN
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Considering a finding that the antinociceptive effect of intrathecally injected anti-CGRP antiserum was greater in adjuvant arthritic rats than in nonarthritic ones (16), the increased turnover of CGRP in the primary sensory neurons may be related to the hyperalgesia of the adjuvant arthritic rat. Furthermore, no increase in the content of iCGRP in the dorsal horn of arthritic rats may be attributed to an increase in the release of iCGRP from the nerve terminals there. In the DRG, immunoreactive substance P is localized in subpopulations of small or intermediate-sized neurons (2,10), most of which also contain CGRP (5, 27, 34). In the adjuvant arthritic rats, the spontaneous release of immunoreactive substance P from the spinal dorsal horn was enhanced (20). As CGRP potentiates the capsaicin-induced release of immunoreactive sub-
stance P from the primary afferent terminals (21), the increased content of iCGRP in the primary afferents of arthritic rats may contribute to the enhanced release of immunoreactive substance P in those rats. In conclusion, the present results provide evidence for the enhanced turnover of iCGRP in the primary afferent neurons of adjuvant arthritic rats and suggest the possibility that at least some populations of the CGRP-containing primary afferents are activated by inflammatory, including noxious, stimulation. ACKNOWLEDGEMENTS We thank M. Ohara for comments. Part of this study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture (Japan) and the Ministry of Public Welfare (Japan).
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Calcitonin Gene-Related Peptide Increases in the Dorsal Root Ganglia of Adjuvant Arthritic Rat YASUSHI KURAISHI, TOYOMICHI NANAYAMA, HIROSHI OHNO, NOBUTAKA FUJII,* AKIRA OTAKA,* HARUAKI YAJIMA* AND MASAMICHI SATOH t
Department of Pharmacology and *Department of Pharmaceutical Manufacturing Chemistry Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606, Japan R e c e i v e d 18 April 1988
KURAISHI, Y., T. NANAYAMA, H. OHNO, N. FUJII, A. OTAKA, H. YAJIMA AND M. SATOH. Calcitoningene-related peptide increasesin the dorsal rootganglia of adjuvantarthriticrat. PEPTIDES 10(2) 447-452, 1989.--We examined the effect of adjuvant arthritis on the content of immunoreactive calcitonin gene-related peptide (iCGRP) in the dorsal root ganglia at LA-L6 levels and the spinal cord at a lumbar level in rats, Arthritis was induced by inoculating adjuvant into both hind-paws twice at a 10 day interval. In the arthritic rats 15 days after the first inoculation (day 15), the content of iCGRP was significantly increased in the dorsal root ganglia, with no change in the dorsal and ventral horns. The content in the dorsal root ganglia was still high on day 26 and had decreased by day 40. An intrathecal injection of colchicine (0.2 mg, 18 hr before killing) enhanced the increase of iCGRP in the dorsal root ganglia and decreased it in the dorsal horn of arthritic rats, although in noninoculated rats such treatment produced no significant changes in the content of iCGRP in both regions. The arthritis-induced increase in the content of iCGRP in the dorsal root ganglia was significantly reduced after treatment with the antiinflammatory analgesic, diclofenac sodium, in a dose of 3 mg/kg/day, PO for 10 days. Swelling and hyperalgesia in the hind-paw were depressed after such treatment. These results suggest that adjuvant arthritis with long-lasting inflammation with pain facilitates the turnover, especially biosynthesis, of CGRP in primary afferent neurons. Calcitonin gene-related peptide (CGRP)
Arthritis
Dorsal root ganglion
Spinal cord
Colchicine
Diclofenac
nous CGRP, especially that in the primary afferents. Recently we have found that intrathecal injection of an antiserum against CGRP improves hyperalgesia shown in adjuvant arthritic rats (16). If such a CGRP-associated hyperalgesia would be due to this neuropeptide present in the primary afferents, adjuvant arthritis would alter the turnover of this neuropeptide. We report here that adjuvant arthritis increases the content of iCGRP in the DRG, a finding which may reflect the enhancement of the biosynthesis of this neuropeptide in primary afferent neurons.
CALCITONIN gene-related peptide (CGRP) is composed of 37 amino acid residues and derived from the calcitonin gene predominantly in neural tissues (1,23). With regard to the primary sensory neurons, immunoreactive CGRP (iCGRP) is contained in a moderate or large population of the dorsal root ganglion (DRG) cells and in some colocalized with other neuropeptides such as substance P and somatostatin (8, 12, 19, 27, 34); the latter two peptides are implicated in nociceptive transmission (15, 21, 35, 36). The nerve terminals and varicosities containing iCGRP are densely distributed in the superficial layers of the dorsal horn (5, 8, 26 34). Thus, this peptide is thought to be related to sensory transmission in the spinal dorsal horn. In fact, an intrathecal injection of CGRP lowers the nociceptive threshold for acute mechanical stimulation (21) and enhances the effects of substance P and somatostatin on the nociceptive flexion reflex (35,36) in rats. In addition, CGRP promotes the release of immunoreactive substance P evoked by capsaicin, an algesic compound, from slices of the rat dorsal horn (21). These findings suggest the participation of CGRP in the facilitation of transmission of nociceptive information in the dorsal horn. However, it remains unknown whether such influences would be exerted by endoge-
METHOD
Induction of Adjuvant Arthritis Arthritis was produced by intradermally inoculating 0,04 ml of a suspension of heat-killed Mycobacterium butyricum (Difco Laboratories, Detroit, MI) in paraffin oil (12 mg/ml) into both hind-paws of male Sprague-Dawley rats at 7-8 weeks of age. This inoculation was given twice at 10-day intervals. Rats were kept on a 12-hr light/dark cycle (8:00 a.m./8:00 p.m.) at 23°C with free access to food and water. An average of the volume of both
1Requests for reprints should be addressed to M. Satoh, Ph.D.
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hind-paws served as an index of the severity of infiammantion. Rats with more than a 3 cm 3 average volume of the hind-paws on day 15 (15 days after the first inoculation) were considered to be arthritic; the volume of the hind-paw was 1.73 ± 0.18 cm 3 (mean ± SD, n = 38) before the inoculation. The nociceptive threshold of hind-paw was measured by the Randall-Selitto test (22) with a loading rate of 48 g/sec. Since in preliminary experiments, intradermal injections of saline alone into the hind-paws did not affect the content of iCGRP in the DRG, we used nontreated age-matched rats as control in the present experiments.
Extraction of CGRP Rats were decapitated and frozen in dry ice-ethanol, and the DRG of both sides at the I.A-L6 levels and the lumbar enlargement (1 cm long) were rapidly removed. The dorsal and ventral horns were excised, as described (14). The tissues were boiled in 1 ml of 2 M acetic acid for 5 min, sonicated and centrifuged at 27,000 × g for 15 min at 4°C. The supernatant was subjected to radioimmunoassay. Recovery of the added synthetic CGRP was 69.5 --_2.8% (n = 7), and the results were corrected for losses.
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Radioimmunoassay was conducted using anti-CGRP antiserum produced by immunizing rabbits against human CGRP(6-37) conjugated to bovine serum albumin using N-succinimidyl-p(2-nitrovinyl)-benzoate (6). [Tyr°]-rat et-CGRP (Peninsula Laboratories, Belmont, CA) labeled with Na~25I (NEN Products, Boston, MA) and rat c(-CGRP (Peptide Institute, Minoh, Japan) were used as tracer and standard, respectively. A buffer for antiserum and tracer was made up of 0.05 M phosphate buffer (pH 6.0), 0.15 M NaC1, 0.1% bovine serum albumin, 0.1% gelatin and 0.1% NAN3, and that for a sample 0.05 M phosphate buffer (pH 7.5), 0.1% bovine serum albumin, 0.1% Triton X-100 and 0.1% NaN 3. "Bound" and "free" fractions were separated with 20% polyethylene glycol 6,000, as described (14). Cross-reactivity of rat 13-CGRP was similar to rat a-CGRP and those of substance P, somatostatin-14 and somatostatin-28 were all less than 0.05%. The dilution curve of the extraction from the lumbar enlargement of rats was parallel to the standard curve. In addition, separation of the lumbar extraction by either gel chromatography or highperformance liquid chromatography showed a single peak of iCGRP, although the chromatography did not separate the authentic a- and f3-CGRP. These results show that iCGRP of the rat can be determined using the present radioimmunoassay and that iCGRP mainly corresponds to CGRP.
Drug Administration Diclofenac sodium (a gift from Ciba-Geigy Japan, Takarazuka, Japan) was given daily in a peroral dose of 3 mg/kg/day as a 0.5% carboxymethylcellulose suspension, about 1:00 p.m. from day 16 to day 25. The nociceptive threshold and the volume of the hind-paw were measured 1 hr before (day 16) or after (days 19, 22 and 25) the administration. Two hundred p.g of colchicine (Nacalai Tesque, Kyoto, Japan) was intrathecally administered in a volume of 10 p.1 through a lumbar puncture (24) on day 14, and 18 hr later the rats were killed.
Data Processing Results are expressed as mean ± SEM. Statistical comparisons was made using Student's t-test (two-tailed), simultaneous multiple comparisons by the Bonferroni method (33) or two-way
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FIG. 1. Effects of adjuvant arthritis on (a) the nociceptive threshold and (b) the volume of the hind-paw. Arthritis groups (O) were inoculated with adjuvant on the days indicated by arrows and control groups (©) were given no treatment. Values are means and SEM of 6 experiments. *p<0.01 as compared to the control on the same day (Student's t-test).
analysis of variance (ANOVA); p<0.05 was considered significant. RESULTS
Effects of Arthritis on the Content of iCGRP A remarkable swell and a decrease in the nociceptive threshold (hyperalgesia) of the hind-paw were observed from day 15 to day 40 (Fig. 1). The content of iCGRP in the DRG at the lumbar levels was increased from day 15 to day 26 and tended toward a recovery on day 40; the increase compared to the noninoculated control was 81% (p<0.05, Student's t-test), 100% (p<0.05) and 53% (0.05
Effect of Colchicine on the Content of iCGRP In this series of experiments we examined the effects on the content of iCGRP of colchicine, a compound which blocks axonal flow. An intrathecal injection of colchicine significantly facilitated an increase in the content of iCGRP in the DRG, as revealed by ANOVA, F(1,20)=4.515, p<0.05; percent increases of iCGRP by colchicine were 8.3 and 61.5 (p<0.005, simultaneous multiple comparisons) in noninoculated and arthritic groups, respectively (Fig. 3). On the contrary, iCGRP in the dorsal horn was decreased by colchicine, F(1,20)=8.488, p<0.01, and this decrease was
CGRP AND PAIN
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FIG. 2. Influence of adjuvant arthrits on the content of immunoreactive calcitonin gene-related peptide (iCGRP) in the dorsal root ganglia at the L4-L6 levels. Arthritis groups (0) were inoculated with adjuvant on the days indicated by arrows and control groups ((3) were given no treatment. Mean and SEM are represented and the values in parentheses indicate the number of rats used. *p
more remarkable in the arthritic rats (39%, p < 0 . 0 1 , simultaneous multiple comparisons) than in noninoculated ones (21%, not significant) (Fig. 4).
Effect of Diclofenac on the Content of iCGRP and Inflammation To determine whether arthritic inflammation with pain would be responsible for the increase in CGRP contents in the DRG, rats were given diclofenac sodium, a nonsteroidal antiinflammatory agent which exerts a potent analgesic action on rheumatoid arthritis (4, 25, 28). Although the degree of swelling and hyperalgesia of the hind-paw was constant in the arthritic group from day 16 to day 25, diclofenac partially but significantly improved the swelling, [F(1,30)= 25.927, p<0.0001, compared in arthritis without diclofenac] and hyperalgesia, F(1,30) = 14.404, p <0.001 (Fig. 5). Such treatment with diclofenac significantly, F(1,20) = 4.463, p < 0 . 0 5 , inhibited the arthritis-induced increase in the content of iCGRP in the DRG (Fig. 6). In the nonarthritic rats, diclofenac did not affect the content of iCGRP (Fig. 6), the volume of the paw and the nociceptive threshold (data not shown).
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FIG. 3. Effects of an intrathecal injection of colchicine (0.2 mg, 18 hr before killing) on the content of immunoreactive calcitonin gene-related peptide (iCGRP) in the dorsal root ganglia at the I.A-L6 levels in adjuvant arthritic and noninoculated rats. Values represent mean---SEM from 6 experiments. Significant effects were produced by arthritis, F(1,20)= 26.352, p<0.01, colchicine, F(1,20)= 6.277, p<0.05, and their interaction, F(1,20) = 4.515, p<0.05. *P<0.005 vs. arthritis without colchicine and p<0.0001 vs. colchicine alone (simultaneous multiple comparisons).
DISCUSSION We found a remarkable increase in the content of iCGRP in the DRG during adjuvant arthritis in rats. This increase may be due to changes in the content in the somata of primary afferent neurons rather than in terminals and vaficosities because the increase was
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TABLE 1 EFFECTS OF ADJUVANTARTHRITIS ON THE CONTENT OF IMMUNOREACTIVECGRP IN THE DORSALROOT GANGLIA, DORSAL HORN AND VENTRALHORN OF THE RAT ICGRP (pmol/mg tissue)
Control
Arthritis
0.095 - 0.013 0.82 4- 0.10 0.043 --- 0.012 7
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Dorsal root ganglia Dorsal horn Ventral horn n
The rats were killed on 15 days after the first inoculation and the immunoreactive CGRP measured. *p
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FIG. 4. Effects of an intrathecal injection of colchicine (0.2 mg, 18 hr before killing) on the content of immunoreactive calcitonin gene-related peptide (iCGRP) in the dorsal horn of the lumbar enlargement in adjuvant arthritic and noninoculated rats.Values represent mean_SEM from 6 experiments. A significant decrease was produced by colchicine, F(1,20) = 8.488, p
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FIG. 5. Effects of diclofenac sodium on the nociceptive threshold (a) and on the volume (b) of the hind-paw. The diclofenac plus arthritis group (O) was given diclofenac sodium (3 mg/kg/day, PO) on the days indicated by arrow heads (16-25 days after the first inoculation). The arthritis group (O) was given only the linoculation and the control (n) no treatment. Each group was 6 rats and values were mean- SEM. Diclofenac significantly improved the hyperalgesia, F(1,30)= 14.404, p<0.001, and the swelling, F(1,30) = 25.927, p<0.0001, during the period from 19 to 25 days after the first inoculation. *p<0.05 when compared with the group of arthritis without diclofenac on the same days (Student's t-test).
specifically seen in the DRG but not in the dorsal horn. In the former region, iCGRP is present in a large population of the cells and the distribution of the iCGRP-containing terminals and varicosities is relatively sparse (7, 12, 27). On the contrary, the latter has dense terminals and varicosities (27,34). Thus, the increase might not result from a suppression in the release of iCGRP from the nerve terminals. Therefore, two factors can be considered to influence the content of iCGRP; one is the biosynthesis of CGRP in the somata and another is the axonal transport of CGRP from the somata to the nerve terminals. The increase of iCGRP in the DRG in the presence of adjuvant arthritis and its augmentation by intrathecal colchicine (inhibition of axonal transport) suggest that the biosynthesis of iCGRP was facilitated in the arthritic rats. Measurement of the content of mRNA encoding CGRP in the DRG provides pertinent evidence for changes in the biosynthesis of this peptide, and such experiments are ongoing in our laboratory. As opposite changes in the content of iCGRP in the DRG and the dorsal horn were greater after colchicine treatment in the arthritic rats than in noninoculated ones, an augmented axonal transport in the arthritic rats was also suggested. The adjuvant arthritis is an autoimmune disease in the joint (31,32) and systemic immunologic changes may also occur in the inoculated animals. In the present experiments, the arthritisinduced increase in the content of iCGRP was seen in the DRG but not in the ventral horn, in which CGRP is present in the motoneurons (8,26), suggesting that CGRP-containing motoneurons may not be affected by systemic immunologic changes. The arthritis-induced increase in the content of iCGRP in the DRG was
FIG. 6. The effect of diclofenac sodium on the arthritis-induced increase in the content of immunoreactive calcitonin gene-related peptide (iCGRP) in the dorsal root ganglia at the L4-L6 levels in the rats. Values represent mean ___SEM from 6 experiments. Either diclofenac sodium (3 mg/kg/day, PO) or vehicle was administered for 10 days, which correspond to 16-25 days after the first inoculation in arthritic group, and the control groups were without inoculation. The arthritis-induced increase was significantly, F(1,20) =4.463, p<0.05, inhibited by diclofenac. *p<0.01 vs. untreated control and arthritis plus diclofenac (simultaneous multiple comparisons). suppressed by diclofenac, which also inhibited the swelling and hyperalgesia of the hind-paw. At low concentrations, diclofenac selectively inhibits cyclooxygenase but not 5-1ipoxygenase (13), and thus selectively inhibits biosynthesis of prostaglandins which are implicated as endogenous inhibitors of humoral and cellular immunity. In addition, nonsteroidal antiinflammatory agents do not suppress the immune responses (9, 17, 30). These findings, taken together, suggest that the increase in the content of iCGRP in the DRG did not result from systemic immunologic changes. Although CGRP is mainly present in the small- to mediumsized DRG neurons, this peptide also occurs in the large DRG neurons (12), which may be related to sensations other than a nociceptive one (11). Adjuvant arthritic rats show a decrease in locomotor activity (3), which, one may consider, would be responsible for the increase in the content of iCGRP in the DRG. However, such an increase was probably due to the augmented biosynthesis of this peptide, a phenomenon suggesting that the activity of iCGRP-containing primary neurons is greater in adjuvant arthritis. On the other hand, the decreased locomotor activity probably reduces the incidence of mechanical stimuli in the hind-paws and then diminishes the activity of mechanical nociceptive afferents (18). Thus, it is unlikely that the increase in the content of iCGRP in the DRG was due to a decrease in locomotor activity. The present results suggest the increased synthesis and axonal transport of CGRP in primary sensory neurons of adjuvant arthritic rats. Recently we found that capsaicin-evoked release of iCGRP from slices of the lumbar cord was greater in adjuvant arthritic rats than in nonarthritic ones (unpublished observation), suggesting the increase in the content of iCGRP in the nerve terminals of the capsaicin-sensitive, probably nociceptive (37), primary afferents. Many CGRP-positive fibers in the spinal dorsal horn are lost after treatment with capsaicin (5), whereas CGRP-positive fibers in the dorsal column nuclei are mainly derived from the medium- to large-sized DRG neurons and insensitive to capsaicin (29). Taken together, these findings suggest that some populations of the CGRP-containing primary afferents are related to nociception.
CGRP AND PAIN
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Considering a finding that the antinociceptive effect of intrathecally injected anti-CGRP antiserum was greater in adjuvant arthritic rats than in nonarthritic ones (16), the increased turnover of CGRP in the primary sensory neurons may be related to the hyperalgesia of the adjuvant arthritic rat. Furthermore, no increase in the content of iCGRP in the dorsal horn of arthritic rats may be attributed to an increase in the release of iCGRP from the nerve terminals there. In the DRG, immunoreactive substance P is localized in subpopulations of small or intermediate-sized neurons (2,10), most of which also contain CGRP (5, 27, 34). In the adjuvant arthritic rats, the spontaneous release of immunoreactive substance P from the spinal dorsal horn was enhanced (20). As CGRP potentiates the capsaicin-induced release of immunoreactive sub-
stance P from the primary afferent terminals (21), the increased content of iCGRP in the primary afferents of arthritic rats may contribute to the enhanced release of immunoreactive substance P in those rats. In conclusion, the present results provide evidence for the enhanced turnover of iCGRP in the primary afferent neurons of adjuvant arthritic rats and suggest the possibility that at least some populations of the CGRP-containing primary afferents are activated by inflammatory, including noxious, stimulation. ACKNOWLEDGEMENTS We thank M. Ohara for comments. Part of this study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture (Japan) and the Ministry of Public Welfare (Japan).
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Cohen, I. R. Arthritis induced by a T-lymphocyte clone that responds to Mycobacterium tuberculosis and to cartilage proteoglycans. Proc. Natl. Acad. Sci. USA 82:5117-5120; 1985. 32. Van Eden, W.; Thole, J. E. R.; van der Zee, R.; Noordzij, A.; van Embden, J. D. A.; Hensen, E. J.; Cohen, I. R. Cloning of the mycobacterial epitope recognized by T lymphocytes in adjuvant arthritis. Nature 331:171-173; 1988. 33. Wallenstein, S.; Zucker, C. L.; Fleiss, J. L. Some statistical methods useful in circulation research. Circ. Res. 47:1-9; 1980. 34. Wiesenfeld-Hallin, Z.; H6kfelt, T.; Lundberg, J. M.; Forssmann, W. G.; Reinecke, M.; Tschopp, F. A.; Fischer, J. A. Immunoreactive calcitonin gene-related peptide and substance P coexist in sensory neurons to the spinal cord and interact in spinal behavioral responses
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