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Calcitonin gene-related peptide (8–37) does not antagonize calcitonin gene-related peptide in rat spinal cord
ELSEVIER
Neuroscience Letters 204 (1996) 185-188
H[UROSClHCI lETTERS
Calcitonin gene-related peptide (8-37) does not antagonize calcitonin gene-related peptide in rat spinal cord Xiao-Jun Xu, Zsuzsanna Wiesenfeld-Hallin* Department t¢f Medical Laboratory Sciences and Technology, Section of Clinical Neurophysiology, Karolinska Institute, Huddinge University Hospital, S-141 86 Huddinge, Sweden Received 11 December 1995; revised version received 5 January 1996; accepted 5 January 1996
Abstract
We have examined the effects of intrathecal (i.t.) human calcitonin gene-related peptide (hCGRP) and its C-terminal fragment hCGRP(8-37), a proposed CGRP antagonist, on the flexor reflex in decerebrate, spinalized, unanesthetized rats. I.t. hCGRP at 26 pmol caused a moderate facilitation of the reflex which was not antagonized by hCGRP(8-37) at doses ranging from 26 pmol to 5.2 nmol. Furthermore, hCGRP(8-37) by itself facilitated the reflex, with no signs of inhibition. It is concluded that the spinal CGRP receptor mediating the spinal facilitatory effect of hCGRP is not antagonized by hCGRP(8-37). Thus, it is unlikely that hCGRP(8-37) can be useful as a spinal analgesic.
Keywords: Antagonist; Antinociception; Calcitonin gene-related peptide (CGRP); CGRP(8-37); Flexor reflex; Intrathecal; Pain; Spinal cord
Calcitonin gene-related peptide (CGRP) contains 37 amino acids and is widely distributed in the nervous system [7]. CGRP-Iike immunoreactivity (LI) has been observed in many dorsal root ganglion cells of various sizes and in fiber networks of the superficial laminae of the dorsal horn [ 14,15]. These observations, together with the finding that CGRP binding sites can be visualized in the dorsal spinal cord [17], have led to the suggestion that CGRP may have a role in nociceptive transmission and modulation. Indeed, noxious peripheral stimulation evoked release of CGRP-LI in the dorsal horn of the spinal cord [101 and intrathecal (i.t.) administration of CGRP elicited hyperalgesia in some, but not all, behavioral studies [3,11,18]. CGRP also potentiated the spinal excitatory effect of substance P (SP), further supporting a pronociceptive role for this peptide [ 15,16]. In 1989, Chiba et al. showed that a C-terminal fragment of human CGRP, hCGRP(8-37), bound with high affinity to CGRP receptors in rat liver plasma membranes and acted as a competitive antagonist of CGRP [2]. Since then, numerous studies have shown that CGRP(8-37) antagonized effects mediated by exogenous and endoge* Corresponding author. Tel.: +46 8 7461975; fax: +46 8 7748856.
nous CGRP in a number of preparations [4-6,8]. However, behavioral studies on the effect of i.t. CGRP(8-37) on nociception have generated conflicting results. While Yu et al. recently reported a potent antinociceptive effect of hCGRP(8-37) in rats on the hot plate and paw pressure tests [ 18], Saxen et al. failed to observe any effect of this fragment on the tail flick test in mice [13]. Furthermore, Saxen et al. also reported that i.t. hCGRP(8-37) did not block the spinal effects of CGRP, but rather acted like an agonist [13]. CGRP(8-37) also failed to block the effect of CGRP in numerous test systems, particularly in the central nervous system [4,12]. The present studies were carried out to examine the effects of i.t. hCGRP(8-37) in a physiological preparation, on the flexor reflex in decerebrate, spinalized, unanesthetized rats. We wanted to establish (1) whether the spinal facilitatory effect of hCGRP is blocked by hCGRP(8-37), and (2) whether i.t. hCGRP(8-37) induced a spinally mediated antinociception. The experiments were performed on female SpragueDawley rats weighing 200-250 g (B&K Universal, Stockholm, Sweden). They were anesthetized with methohexital (Brietal, Lilly, Indianapolis, USA; 70 mg/kg, i.p.), ventilated and decerebrated by aspiration of the forebrain
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII: S 0 3 0 4 - 3 9 4 0 ( 9 6 ) 1 2 3 5 1 - 8
186
X.-J. Xu, Z. Wiesenfeld-Hallin / Neuroscience Letters 204 (1996) 185-188
150-
A
±
e-
.o ,od
I
catheter (PE 10) was implanted caudal to the transection with its tip on the lumbar spinal cord (L4-5). The flexor reflex was elicited by supramaximal electric shocks applied to the sural nerve innervation area in the left foot (0.5 ms, 10mA, 1/min) which activated A- and Cafferents. A stable reflex baseline (defined as less than 15% variability) was established for at least 20-30 min before administration of drugs. The flexor reflex was recorded as EMG activity with stainless steel needle electrodes inserted into the ipsilateral posterior biceps femoris/semitendinosus muscles. The number of action potentials exceeding the level of spontaneous EMG activity was integrated over 2 s and recorded on a chart recorder (Gould 2400 S). During the experiments the heart rate and rectal temperature of the rat were monitored. hCGRP(8-37) (Peninsula Laboratories Europe and Bachem) and hCGRP (Peninsula Laboratories Europe) were dissolved in 0.9% saline and injected i.t. in 10/~1 followed by 10/~1 saline to flush the catheter. In experiments where the antagonistic effect of hCGRP(8-37) on i.t. hCGRP-induced reflex facilitation was examined, the various doses of hCGRP(8-37) were injected in ascending order 5-30 min prior to the administration of CGRP. I.t. hCGRP at 26 pmoi elicited a moderate and brief facilitation of the flexor reflex (maximal facilitation 80 _+ 7% over baseline level lasting 6-+ 2 min; Figs. 1 and 2). Pretreatment with hCGRP(8-37) at doses ranging from 26 pmol to 2.6 nmol did not antagonize the facilitatory effect of hCGRP (Figs. 1 and 2). In fact, the reflex facilitatory effect of 26 pmol hCGRP was significantly prolonged after pretreatment with 2.6 nmol hCGRP(8-37) (Figs. 1 and 2). Further increasing the dose of hCGRP(837) to 5.2 nmol still did not reduce the reflex facilitatory
I
100"
,m
o
50"
0 (6) 30-
26 (5)
260
2600
(6)
(5)
26 (5)
260 (6)
2600 (5)
B
A
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E
20-
i
t-
.2 10Cl
0 (8)
Dose of hCGRP(8-37) (pmol) Fig. 1. Peak increase in reflex magnitude (A) and duration of reflex facilitation (B) induced by 26 pmol i.t. hCGRP by itself or after pretreatment with various doses of hCGRP(8-37). The doses of hCGRP(837) and the number of experiments for each dose are indicated under the columns. The data are expressed as mean _+ SEM. ANOVA indicated that there was no overall significant difference among the groups with respect to the magnitude or duration of facilitation (F3,20 = 1.2 and 2.7, respectively, P > 0.05). The Fisher PLSD test indicated a significant difference between the group that received only hCGRP (dose 0) and that which received 2.6 nmol hCGRP(8-37), *P < 0.05.
and midbrain. The spinal cord was exposed by iaminectomy at mid-thoracic level and sectioned at Th8-9. An i.t.
hCGRP 26 pmol 250
hCGRP 26 pmol hCGRP (8-37) | 2600 pmol ~
hCGRP (8-37) 5200 pmol
200 "0 C
150
E x o ~=
100
50
0
I
0
i
20
I
40
I
~
I
i
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i
100
120
140
160
180
200
i
220
Time (rain) Fig. 2. Illustration of the effects of i.t. hCGRP and hCGRP(8-37) on the flexor reflex in a single experiment. Baseline reflex magnitude is defined as 100%. Note that 2600 pmol hCGRP (8-37) did not block, but rather enhanced, the reflex facilitatory effect of 26 pmol i.t. CGRP. Moreover, further addition of 5.2 nmol CGRP(8-37) did not produce any reflex inhibition, but facilitation.
X.-J. Xu, Z. Wiesenfeld-Hallin / Neuroscience Letters 204 (1996) 185-188
150O *=
100-
'*-
50
15-
,• Z,=
"9
A
i'l
26
260
2600
5200
(6)
(8)
(7)
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26
260
2600
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6
(6)
(8)
(7)
5200
(6)
Dose of hCGRP(8-37) (pmol) Fig. 3. Summary of the magnitude (A) and duration (B) of reflex facilitation induced by various doses of i.t. hCGRP(8-37). The doses of hCGRP(8-37) and the number of experiments for each dose are indicated under the columns. The data are expressed as mean _ SEM.
effect of hCGRP (maximal facilitation 103 _+7% lasting 5 _+2 min, n = 3). I.t. hCGRP(8-37) alone facilitated the flexor reflex at all doses (Figs. 2 and 3). No significant reflex depression was observed after any dose of hCGRP(8-37) as illustrated in Fig. 2. CGRP(8-37) binds with high affinity to CGRP receptors with K i in the sub-nanomolar range [2,12]. This antagonist blocked the effect of CGRP in various assays at doses which are usually lO--100-fold greater than CGRP [2,4--6,8,12]. In the present study we observed that even at doses which are 100-200-fold greater than hCGRP, hCGRP(8-37) failed to block the moderate facilitation of the flexor reflex induced by i.t. CGRP. It appears that failure of antagonism by hCGRP(8-37) reflects lack of effect of this drug upon the spinal receptor mediating the reflex facilitatory effect of hCGRP. Previous studies have suggested the possible existence of subtypes of CGRP receptors based on differential sensitivity to the antagonistic effect of CGRP(8-37) [4,12]. Our data indicate that the CGRP receptor in rat spinal cord mediating the hyperalgesic effect of CGRP is probably the CGRP2 receptor subtype, which is less sensitive to C-terminal CGRP fragments [12]. Saxen et al. also failed to observe an antagonism of the spinal effects of CGRP by CGRP(8-37) in behavioral studies [ 13]. hCGRP(8-37) by itself caused dose-dependent facilitation of the flexor reflex, albeit with lower potency compared to hCGRP. Although in most preparations
187
CGRP(8-37) appears to be devoid of agonistic action [12], there are reports indicating that C-terminal CGRP fragments may produce an agonistic effect at high doses in the spinal cord and stomach [5,13]. It is thus possible that the reflex facilitatory effect of hCGRP(8-37) observed here reflects an agonism. Furthermore, C-terminal CGRP fragments have been suggested to be agonists at cholecystokinin [9] and tachykinin [1] receptors, which may also account for the reflex facilitatory effect of hCGRP(8-37). I.t. hCGRP(8-37) up to 5.2 nmol failed to produce any measurable depression of the flexor reflex, which further supports the notion that this CGRP fragment is probably not an antagonist of the CGRP receptor mediating the pronociceptive effect of CGRP. In a behavioral study, Yu et al. have reported that i.t. administration of 5 or 10 nmol hCGRP(8-37) produced potent and prolonged antinociception in tests involving both mechanical and thermal stimulation [18]. It is puzzling that such behavioral antinociception is not reflected in the flexor reflex preparation as one can expect a close correlation between the behavioral and electrophysiological studies following i.t. drug administration. One possibility is that in the behavioral experiments i.t. hCGRP(8-37) may be a nociceptive stimulus which in turn activates descending inhibitory control, resulting in antinociception. Such a mechanism does not operate in our physiological preparation, as the rats are spinalized. In conclusion, the present study showed that i.t. administration of hCGRP(8-37), a proposed CGRP receptor antagonist, did not block the facilitatory effect of i.t. hCGRP on the flexor reflex in rats. Thus, the spinal CGRP receptor mediating the pronociceptive effect of CGRP is probably of the CGRP 2 subtype, which is less sensitive to the antagonistic effect of hCGRP(8-37). Furthermore, as hCGRP(8-37) did not depress the flexor reflex, it is unlikely that hCGRP(8-37) has a spinal analgesic function. Finally, tools other than hCGRP(8-37) are needed to define the role of CGRP and its receptors in mediating spinal nociception. This study was supported by the Swedish Medical Research Council (project no. 07913), the Bank of Sweden Tercentenary Foundation, Astra Pain Control AB, the Swedish Society of Medicine, Lars Hiertas Memory Foundation and research funds of the Karolinska Institute. [1] Andersson, S.E. and Almeg,~rd, B., CGRP(8-37) and CGRP(3237) contract the iris sphincter in the rabbit eye: antagonism by spantide and GR82334, Regul. Peptides, 49 (1993) 73-80 (1993). [2] Chiba, T., Yamaguchi, A., Yamatani, T., Naksmura, A., Morishita, T., Inui, T., Fukase, M., Noda, T. and Fujita, T., Calcitonin gene-related peptide antagonist human CGRP(8-37), Am. J. Physiol., 256 (1989) E331-E335. [3] Cridland, R.A. and Henry, J.L., Effects of intrathecal administration of neuropeptides on a spinal nociceptive reflex in the rat: VIP, galanin, CGRP, TRH, somatostatin and angiotensin I1, Neuropeptides, 11 (1989) 23-32.
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[4] Dennis, T., Foumier, A., Cadieux, A., Pomerleau, F., Jolicoeur, F., St. Pierre, S. and Quirion, R., hCGRP(8-37), a calcitonin gene-related peptide antagonist revealing calcitonin gene-related peptide receptor heterogeneity in brain and periphery, J. Pharmacol. Exp. Ther., 254 (1990) 123-128. [5] Evangelista, S., Tramontana, M. and Maggi, C.A., Pharmacological evidence for the involvement of multiple calcitonin generelated peptide (CGRP) receptors in the antisecretory and antiulcer effect of CGRP in rat stomach, Life Sci., 50 (1991) PLI3PLI8. [6] Hughes, S.R. and Brain, S.D., A calcitonin gene-related peptide (CGRP) antagonist CGRP(8-37) inhibits microvascular responses induced by CGRP and capsaicin in the skin, Br. J. Pharmacol., 104 (1991) 738-742. [7] lshida-Yamamoto, A. and Tohyama, M., Calcitonin gene-related peptide in the nervous tissue, Prog. Neurobiol., 33 (1989) 335386. [8] Maggi, C.A., Chiba, T. and Giuliani, S., Human a-calcitonin gene-related peptide-(8-37) as an antagonist of exogenous and endogenous calcitonin gene-related peptide, Eur. J. Pharmacol., 192 (1991) 85-88. [9] Maton, P.N., Pradhan, T. and Moore, S., C-terminal peptides of calcitonin gene-related peptide act as agonists at the cholecystokinin receptor, Peptides, 11 (1990) 1163-1167. [10] Morton, C.R. and Hutchison, W.D., Release of sensory neuropeptides in the spinal cord: studies with calcitonin gene-related peptide and galanin, Neurosciencc, 31 (1989) 807-815. [I 1] Oku, R., Satoh, M., Fujii, N., Otaka, A., Yajima, H. and Takagi, H., Calcitonin gene-related peptide promotes mechanical no-
[12]
[13]
[14]
[15]
[16]
[17]
[18]
ciception by potentiating release of substance P from the spinal dorsal horn in rats, Brain Res., 403 (1987) 350-354. Quirion, R., Von Rossum, D., Dumont, Y., St-Pierre, S. and Fournier, A., Characterization of CGRP I and CGRP 2 receptor subtypes, Ann. N. Y. Acad. Sci., 657 (1992) 88-105. Saxen, M.A., Smith, F.L., Dunlow, L.D., Dombrowski, D.S. and Welch, S.P., The hypothermic and antinociceptive effects of intrathecal injection of CGRP(8-37) in mice, Life Sci., 55 (1994) 1665-1674. Skofitsch, G. and Jacobowitz, D.M., Calcitonin gene-related peptide coexists with substance P in capsaicin-sensitive neurons and sensory ganglia of the rat, Peptides, 6 (1985) 747-754. Wiesenfeld-Hallin, Z., HiSkfelt, T., Lundberg, J.M., Forssmann, W.G., Reinecke, M., Tschopp. F.A. and Fischer, J.A., lmmunoreactive calcitonin gene-related peptide and substance P co-exist in sensory neurons to the spinal cord and interact in spinal behavioral responses of the rat, Neurosci. Lett., 52 (1994) 199-204. Woolf, C.J. and Wiesenfeld-Hallin, Z., Substance P and calcitonin gene-related peptide synergistically modulate the gain of the nociceptive flexor withdrawal reflex in the rat, Neurosci. Lett., 66 (1986) 226-230. Yashpal, K., Kar, S., Dennis, T., Quirion, R., Quantitative autoradiographic distribution of calcitonin gene-related peptide (hCGRPa) binding sites in the rat and monkey spinal cord, J. Comp. Neurol., 322 (1992) 224-232. Yu, L.C., Hansson, P. and Lundeberg, T., The calcitonin generelated peptide antagonist CGRP8_37 increases the latency to withdrawal responses in rats, Brain Res., 653 (1994) 223-230.
Neuroscience Letters 204 (1996) 185-188
H[UROSClHCI lETTERS
Calcitonin gene-related peptide (8-37) does not antagonize calcitonin gene-related peptide in rat spinal cord Xiao-Jun Xu, Zsuzsanna Wiesenfeld-Hallin* Department t¢f Medical Laboratory Sciences and Technology, Section of Clinical Neurophysiology, Karolinska Institute, Huddinge University Hospital, S-141 86 Huddinge, Sweden Received 11 December 1995; revised version received 5 January 1996; accepted 5 January 1996
Abstract
We have examined the effects of intrathecal (i.t.) human calcitonin gene-related peptide (hCGRP) and its C-terminal fragment hCGRP(8-37), a proposed CGRP antagonist, on the flexor reflex in decerebrate, spinalized, unanesthetized rats. I.t. hCGRP at 26 pmol caused a moderate facilitation of the reflex which was not antagonized by hCGRP(8-37) at doses ranging from 26 pmol to 5.2 nmol. Furthermore, hCGRP(8-37) by itself facilitated the reflex, with no signs of inhibition. It is concluded that the spinal CGRP receptor mediating the spinal facilitatory effect of hCGRP is not antagonized by hCGRP(8-37). Thus, it is unlikely that hCGRP(8-37) can be useful as a spinal analgesic.
Keywords: Antagonist; Antinociception; Calcitonin gene-related peptide (CGRP); CGRP(8-37); Flexor reflex; Intrathecal; Pain; Spinal cord
Calcitonin gene-related peptide (CGRP) contains 37 amino acids and is widely distributed in the nervous system [7]. CGRP-Iike immunoreactivity (LI) has been observed in many dorsal root ganglion cells of various sizes and in fiber networks of the superficial laminae of the dorsal horn [ 14,15]. These observations, together with the finding that CGRP binding sites can be visualized in the dorsal spinal cord [17], have led to the suggestion that CGRP may have a role in nociceptive transmission and modulation. Indeed, noxious peripheral stimulation evoked release of CGRP-LI in the dorsal horn of the spinal cord [101 and intrathecal (i.t.) administration of CGRP elicited hyperalgesia in some, but not all, behavioral studies [3,11,18]. CGRP also potentiated the spinal excitatory effect of substance P (SP), further supporting a pronociceptive role for this peptide [ 15,16]. In 1989, Chiba et al. showed that a C-terminal fragment of human CGRP, hCGRP(8-37), bound with high affinity to CGRP receptors in rat liver plasma membranes and acted as a competitive antagonist of CGRP [2]. Since then, numerous studies have shown that CGRP(8-37) antagonized effects mediated by exogenous and endoge* Corresponding author. Tel.: +46 8 7461975; fax: +46 8 7748856.
nous CGRP in a number of preparations [4-6,8]. However, behavioral studies on the effect of i.t. CGRP(8-37) on nociception have generated conflicting results. While Yu et al. recently reported a potent antinociceptive effect of hCGRP(8-37) in rats on the hot plate and paw pressure tests [ 18], Saxen et al. failed to observe any effect of this fragment on the tail flick test in mice [13]. Furthermore, Saxen et al. also reported that i.t. hCGRP(8-37) did not block the spinal effects of CGRP, but rather acted like an agonist [13]. CGRP(8-37) also failed to block the effect of CGRP in numerous test systems, particularly in the central nervous system [4,12]. The present studies were carried out to examine the effects of i.t. hCGRP(8-37) in a physiological preparation, on the flexor reflex in decerebrate, spinalized, unanesthetized rats. We wanted to establish (1) whether the spinal facilitatory effect of hCGRP is blocked by hCGRP(8-37), and (2) whether i.t. hCGRP(8-37) induced a spinally mediated antinociception. The experiments were performed on female SpragueDawley rats weighing 200-250 g (B&K Universal, Stockholm, Sweden). They were anesthetized with methohexital (Brietal, Lilly, Indianapolis, USA; 70 mg/kg, i.p.), ventilated and decerebrated by aspiration of the forebrain
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PII: S 0 3 0 4 - 3 9 4 0 ( 9 6 ) 1 2 3 5 1 - 8
186
X.-J. Xu, Z. Wiesenfeld-Hallin / Neuroscience Letters 204 (1996) 185-188
150-
A
±
e-
.o ,od
I
catheter (PE 10) was implanted caudal to the transection with its tip on the lumbar spinal cord (L4-5). The flexor reflex was elicited by supramaximal electric shocks applied to the sural nerve innervation area in the left foot (0.5 ms, 10mA, 1/min) which activated A- and Cafferents. A stable reflex baseline (defined as less than 15% variability) was established for at least 20-30 min before administration of drugs. The flexor reflex was recorded as EMG activity with stainless steel needle electrodes inserted into the ipsilateral posterior biceps femoris/semitendinosus muscles. The number of action potentials exceeding the level of spontaneous EMG activity was integrated over 2 s and recorded on a chart recorder (Gould 2400 S). During the experiments the heart rate and rectal temperature of the rat were monitored. hCGRP(8-37) (Peninsula Laboratories Europe and Bachem) and hCGRP (Peninsula Laboratories Europe) were dissolved in 0.9% saline and injected i.t. in 10/~1 followed by 10/~1 saline to flush the catheter. In experiments where the antagonistic effect of hCGRP(8-37) on i.t. hCGRP-induced reflex facilitation was examined, the various doses of hCGRP(8-37) were injected in ascending order 5-30 min prior to the administration of CGRP. I.t. hCGRP at 26 pmoi elicited a moderate and brief facilitation of the flexor reflex (maximal facilitation 80 _+ 7% over baseline level lasting 6-+ 2 min; Figs. 1 and 2). Pretreatment with hCGRP(8-37) at doses ranging from 26 pmol to 2.6 nmol did not antagonize the facilitatory effect of hCGRP (Figs. 1 and 2). In fact, the reflex facilitatory effect of 26 pmol hCGRP was significantly prolonged after pretreatment with 2.6 nmol hCGRP(8-37) (Figs. 1 and 2). Further increasing the dose of hCGRP(837) to 5.2 nmol still did not reduce the reflex facilitatory
I
100"
,m
o
50"
0 (6) 30-
26 (5)
260
2600
(6)
(5)
26 (5)
260 (6)
2600 (5)
B
A
t" .m
E
20-
i
t-
.2 10Cl
0 (8)
Dose of hCGRP(8-37) (pmol) Fig. 1. Peak increase in reflex magnitude (A) and duration of reflex facilitation (B) induced by 26 pmol i.t. hCGRP by itself or after pretreatment with various doses of hCGRP(8-37). The doses of hCGRP(837) and the number of experiments for each dose are indicated under the columns. The data are expressed as mean _+ SEM. ANOVA indicated that there was no overall significant difference among the groups with respect to the magnitude or duration of facilitation (F3,20 = 1.2 and 2.7, respectively, P > 0.05). The Fisher PLSD test indicated a significant difference between the group that received only hCGRP (dose 0) and that which received 2.6 nmol hCGRP(8-37), *P < 0.05.
and midbrain. The spinal cord was exposed by iaminectomy at mid-thoracic level and sectioned at Th8-9. An i.t.
hCGRP 26 pmol 250
hCGRP 26 pmol hCGRP (8-37) | 2600 pmol ~
hCGRP (8-37) 5200 pmol
200 "0 C
150
E x o ~=
100
50
0
I
0
i
20
I
40
I
~
I
i
1
i
100
120
140
160
180
200
i
220
Time (rain) Fig. 2. Illustration of the effects of i.t. hCGRP and hCGRP(8-37) on the flexor reflex in a single experiment. Baseline reflex magnitude is defined as 100%. Note that 2600 pmol hCGRP (8-37) did not block, but rather enhanced, the reflex facilitatory effect of 26 pmol i.t. CGRP. Moreover, further addition of 5.2 nmol CGRP(8-37) did not produce any reflex inhibition, but facilitation.
X.-J. Xu, Z. Wiesenfeld-Hallin / Neuroscience Letters 204 (1996) 185-188
150O *=
100-
'*-
50
15-
,• Z,=
"9
A
i'l
26
260
2600
5200
(6)
(8)
(7)
(6)
26
260
2600
B
10-
6
(6)
(8)
(7)
5200
(6)
Dose of hCGRP(8-37) (pmol) Fig. 3. Summary of the magnitude (A) and duration (B) of reflex facilitation induced by various doses of i.t. hCGRP(8-37). The doses of hCGRP(8-37) and the number of experiments for each dose are indicated under the columns. The data are expressed as mean _ SEM.
effect of hCGRP (maximal facilitation 103 _+7% lasting 5 _+2 min, n = 3). I.t. hCGRP(8-37) alone facilitated the flexor reflex at all doses (Figs. 2 and 3). No significant reflex depression was observed after any dose of hCGRP(8-37) as illustrated in Fig. 2. CGRP(8-37) binds with high affinity to CGRP receptors with K i in the sub-nanomolar range [2,12]. This antagonist blocked the effect of CGRP in various assays at doses which are usually lO--100-fold greater than CGRP [2,4--6,8,12]. In the present study we observed that even at doses which are 100-200-fold greater than hCGRP, hCGRP(8-37) failed to block the moderate facilitation of the flexor reflex induced by i.t. CGRP. It appears that failure of antagonism by hCGRP(8-37) reflects lack of effect of this drug upon the spinal receptor mediating the reflex facilitatory effect of hCGRP. Previous studies have suggested the possible existence of subtypes of CGRP receptors based on differential sensitivity to the antagonistic effect of CGRP(8-37) [4,12]. Our data indicate that the CGRP receptor in rat spinal cord mediating the hyperalgesic effect of CGRP is probably the CGRP2 receptor subtype, which is less sensitive to C-terminal CGRP fragments [12]. Saxen et al. also failed to observe an antagonism of the spinal effects of CGRP by CGRP(8-37) in behavioral studies [ 13]. hCGRP(8-37) by itself caused dose-dependent facilitation of the flexor reflex, albeit with lower potency compared to hCGRP. Although in most preparations
187
CGRP(8-37) appears to be devoid of agonistic action [12], there are reports indicating that C-terminal CGRP fragments may produce an agonistic effect at high doses in the spinal cord and stomach [5,13]. It is thus possible that the reflex facilitatory effect of hCGRP(8-37) observed here reflects an agonism. Furthermore, C-terminal CGRP fragments have been suggested to be agonists at cholecystokinin [9] and tachykinin [1] receptors, which may also account for the reflex facilitatory effect of hCGRP(8-37). I.t. hCGRP(8-37) up to 5.2 nmol failed to produce any measurable depression of the flexor reflex, which further supports the notion that this CGRP fragment is probably not an antagonist of the CGRP receptor mediating the pronociceptive effect of CGRP. In a behavioral study, Yu et al. have reported that i.t. administration of 5 or 10 nmol hCGRP(8-37) produced potent and prolonged antinociception in tests involving both mechanical and thermal stimulation [18]. It is puzzling that such behavioral antinociception is not reflected in the flexor reflex preparation as one can expect a close correlation between the behavioral and electrophysiological studies following i.t. drug administration. One possibility is that in the behavioral experiments i.t. hCGRP(8-37) may be a nociceptive stimulus which in turn activates descending inhibitory control, resulting in antinociception. Such a mechanism does not operate in our physiological preparation, as the rats are spinalized. In conclusion, the present study showed that i.t. administration of hCGRP(8-37), a proposed CGRP receptor antagonist, did not block the facilitatory effect of i.t. hCGRP on the flexor reflex in rats. Thus, the spinal CGRP receptor mediating the pronociceptive effect of CGRP is probably of the CGRP 2 subtype, which is less sensitive to the antagonistic effect of hCGRP(8-37). Furthermore, as hCGRP(8-37) did not depress the flexor reflex, it is unlikely that hCGRP(8-37) has a spinal analgesic function. Finally, tools other than hCGRP(8-37) are needed to define the role of CGRP and its receptors in mediating spinal nociception. This study was supported by the Swedish Medical Research Council (project no. 07913), the Bank of Sweden Tercentenary Foundation, Astra Pain Control AB, the Swedish Society of Medicine, Lars Hiertas Memory Foundation and research funds of the Karolinska Institute. [1] Andersson, S.E. and Almeg,~rd, B., CGRP(8-37) and CGRP(3237) contract the iris sphincter in the rabbit eye: antagonism by spantide and GR82334, Regul. Peptides, 49 (1993) 73-80 (1993). [2] Chiba, T., Yamaguchi, A., Yamatani, T., Naksmura, A., Morishita, T., Inui, T., Fukase, M., Noda, T. and Fujita, T., Calcitonin gene-related peptide antagonist human CGRP(8-37), Am. J. Physiol., 256 (1989) E331-E335. [3] Cridland, R.A. and Henry, J.L., Effects of intrathecal administration of neuropeptides on a spinal nociceptive reflex in the rat: VIP, galanin, CGRP, TRH, somatostatin and angiotensin I1, Neuropeptides, 11 (1989) 23-32.
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