Br. J. Pharmacol. (1990), 100, 656-660
(D Macmillan Press Ltd, 1990
Bradykinin-induced depolarization of primary afferent nerve terminals in the neonatal rat spinal cord in vitro 'P.M. Dunn & H.P. Rang The Sandoz Institute for Medical Research, 5 Gower Place, London WC1E 6BN 1 Application of bradykinin (BK) to the spinal cord of the neonatal rat evoked depolarizations which could be recorded via either the dorsal or ventral roots. However, responses recorded via the ventral root were abolished by removal of extracellular Ca2+ or the addition of Cd2", while responses recorded via the dorsal root were unaffected. 2 The response recorded via the ventral root was inhibited by the substance P antagonist spantide, while responses recorded via the dorsal root were unaffected. 3 Depolarizations recorded via the dorsal root were concentration-dependent with an EC50 of 30nM. These responses were not antagonized by the BK1 selective antagonist Leu8des-Arg9BK, but were antagonized by D-Arg0Hyp3Thi5"8D-Phe7BK with a pA2 of 6.8 + 0.6, which is similar to the values determined for other BK2-mediated responses. 4 Application of phorbol dibutyrate (PDBu) to the spinal cord also evoked a depolarization with respect to the dorsal root. This response to PDBu was enhanced by removal of extracellular Ca2 while the response to BK was unaffected. 5 The potent protein kinase inhibitor staurosporine reduced the response to PDBu, but did not affect the response to BK. 6 These results suggest that BK by acting on BK2 receptors can depolarize the central terminals of primary afferent nerve fibres. This response to BK does not appear to be mediated via the activation of protein kinase C. The depolarization to BK recorded via the ventral root of the spinal cord is indirect and may be secondary to the action of BK on the primary afferent terminals.
Introduction In the periphery, the nonapeptide bradykinin (BK) is formed from kininogen by the enzyme kallikrein in response to tissue injury. However, there are several pieces of evidence which suggest a role for BK in the central nervous system. Bradykinin and BK-like immunoreactivity have been detected in the brain (Correa et al., 1979; Perry & Snyder, 1984) and BK binding sites have been demonstrated in the spinal cord (Steranka et al., 1988). Intracerebroventricular and intrathecal injection of BK produces selective autonomic and behavioural effects and antinociception (Ribeiro & Rocha E Silva, 1973; Laneuville & Couture, 1987). However, central administration of BK receptor antagonists alone appear to have no effect on nociception (Laneuville & Couture, 1987; Dray et al., 1988a), indicating that BK does not play an important role in the central transmission of nociceptive information. The precise mechanism of the antinociceptive action of intrathecal BK is unknown, but the effect is blocked by a2-adrenoceptor antagonists, and it has been suggested that BK is acting on the terminals of bulbo-spinal noradrenergic neurones (Laneuville et al., 1989). Application of BK to the spinal cord of the neonatal rat evokes a depolarization of the ventral root (Dray et ql., 1988a). However, since BK binding sites are located mainly on primary afferent nerve fibres and in the dorsal horn of the spinal cord (Steranka et al., 1988), the response recorded via the ventral root is probably secondary to an action in the dorsal horn, possibly on the terminals of the primary afferent nerve fibres. In this study, we have examined further the actions of BK on the neonatal rat spinal cord in vitro. By recording responses via both the ventral and dorsal roots, we have attempted to determine the site of action of BK. In many cells, BK activates the enzyme phospholipase C (PLC) (Yano et al., 1985; Derian & Moskowitz, 1986; Thayer et al., 1988), giving rise to the formation of inositol trisphosphate (1P3), and diacylglycerol (DAG) which is the ' Author for correspondence at present address: Dept of Pharmacology, University College, Gower Street, London WC1E 6BT.
endogenous activator of protein kinase C (PKC) (see Nishizuka, 1988). In the periphery, BK stimulates the peripheral terminals of nociceptive sensory neurones (Erdos, 1979; Martin et al., 1987; Dray et al., 1988b). This activation of sensory neurones probably results from a depolarization of the nerve terminal due to an increase in sodium permeability of the membrane, which may be mediated via the activation of PKC by DAG (Burgess et al., 1989). We have therefore compared the action of BK in the spinal cord with its action on sensory neurones in the periphery, by looking at the ionic basis of the BK response and the possible involvement of PKC in this response. Some of these data have already been presented to the British Pharmacological Society (Dunn & Rang, 1989).
Methods Preparation One day old Sprague-Dawley rat pups were killed by decapitation and the entire vertebral column removed rapidly, and placed in a dish containing Locke solution. A dorsal laminectomy was performed and the dura overlying the cord was removed. The cord with attached dorsal and ventral roots was then removed. The cord was hemisected sagitally and cut transversely, to produce a section of lumbar cord extending to one root either side of the root selected for recording (usually
Recording The section of spinal cord was placed in a 2 chamber bath, with the spinal cord in one compartment and a single dorsal root projecting into the second compartment. The d.c. potential between the two compartments was recorded via silversilver chloride electrodes connected to a differential amplifier (WPI; DAM50) and displayed on a chart recorder (Gould BS272). The compartment containing the cord (volume -1 ml) was perfused by gravity with Locke solution, at room temperature (22 to 25°C), at a constant rate of
BRADYKININ DEPOLARIZATION OF NERVE TERMINALS a
4~~~. BK 10
-i en C 0 Q. en
[BK] (nM) a) en
[BK1 (nM) Figure 1 Responses to bradykinin (BK) recorded via the dorsal root of the neonatal rat spinal cord. (a) Depolarizations evoked by 1 min application of 10, 30 and l0OnM BK. (b) Log concentration-response curve for the depolarization to BK; combined data from 11 experiments with 3-5 concentrations applied in each experiment. Points represent the mean and vertical lines s.e.mean of 4-11 observations normalized with respect to the response evoked by 30nM BK. The EC50 computed by an iterative curve fitting routine was 30 nM.
2 ml per min. Drugs were dissolved in the perfusate at the required final concentration. In some experiments, the same technique was used to record responses from a single ventral root instead of a dorsal root.
The BK concentration-response curve was determined by recording responses to 3-5 concentrations applied in a pseudo-random order, in each experiment. The responses were
(B3824] (>M) Figure 3 Effect of the antagonist D-Arg0Hyp3Thi5'8D-Phe7BK (B3824) on the response to bradykinin (BK). (a) Log concentrationresponse curves to BK recorded in a single experiment, before (0), during (0) and after (El) perfusion with solution containing 3pM B3824. (b) A Schild plot for the antagonism produced by B3824. The data were fitted well by a straight line (r = 0.93, n = 8) with a slope of 0.97 which was not significantly different from unity (P > 0.05 by one sample Student's t test). The pA2 value determined from this line was 6.8 + 0.6.
then normalized with respect to the response produced by 30 nm BK (a concentration used in every experiment). The normalized data were fitted with an unconstrained mass action curve by an iterative curve fitting routine (RS/1, BBN Software Products Corp.)
Analysis of results a
Means were compared by Student's t test (paired or unpaired as stated).
Drugs and solutions 200
BK 30 nM
des Arg9 BK 3 FM
Leu8 des Arg I BK 10 FIM
0 BK 30 nM Figure 2 Action of selective BK1 receptor agonists and antagonists on the neonatal rat spinal cord. (a) Comparison of the effect of bradykinin (BK, 30nM) with the selective BK1 agonist des-Arg9BK (3pM). (b) Responses to 30nM BK recorded in the absence and presence of the selective BK1 antagonist Leu8des-Arg9BK. The antagonist at concentrations up to 10pUM failed to alter responses to BK.
The composition of the normal Locke solution was (mM): NaCl 154, KCI 5.6, CaCl2 2, HEPES 10 and D-glucose 5; buffered to pH 7.4 with NaOH. Sodium-free solution contained (mM): N-methyl glucamine 154, KCI 0.7, CaCl2 2, HEPES 10 and D-glucose 5; buffered to pH 7.4 with 4.9 mm KOH. Drugs used were bradykinin (Sandoz, Basle), DArg0Hyp3Thi5'8D-Phe7BK (B3824), des-Arg9BK, Leu8desArg9BK (Bachem, AG), staurosporine (Fluka), tetrodotoxin (TTX), spantide ([D-Arg'D-Trp7 9Leu" ]-substance P), 4/)phorbol-12,13-dibutyrate (PDBu), capsaicin (Sigma). Stock solutions (10mM) of staurosporine, PDBu and capsaicin were made up in dimethylsulphoxide. All other drugs were dissolved in Locke solution.
Results Application of BK (3 to 1000 nM) to the spinal cord for 1 min evoked depolarizations which could be recorded via the dorsal root (Figure la). These responses reached a peak in
P.M. DUNN & H.P. RANG
approximately 2 min, and then declined during the following 5 to 10min. Longer applications of BK did not produce significantly larger responses and to prevent problems of desensitization, BK applications were limited to 1 min and responses were recorded at 40 min intervals. This effect of BK was concentration-dependent, with an EC50 of 30 nm (Figure lb).
* BK 100 nM
0 Ca2+, 4 Mg2+
We used some of the selective BK receptor agonists and antagonists to investigate the type of receptor mediating the depolarization recorded via the dorsal root. The selective BK1 agonist des Arg9 BK at concentrations up to 3 ,UM failed to evoke any depolarization (Figure 2a), and the BK1 antagonist Leu8des Arg9BK at a concentration of 10pM failed to antagonize the response to BK (Figure 2b). However, the response to BK was antagonized by the antagonist D-Arg0Hyp3Thi5'8DPhe7BK (B3824). This compound produced a parallel displacement of the BK log concentration-response curve (Figure 3a). The Schild plot for the antagonism by B3824 was linear with a slope of 0.97, which was not significantly different from unity (Figure 3b) and yielded a pA2 value of 6.8 + 0.6.
2.0 T a) (n c
r 1.0 N a)
Ionic mechanism We examined the dependence of the dorsal root response to BK on extracellular sodium. When extracellular sodium was replaced by N-methylglucamine, there was a reversible reduction in the amplitude of the response to BK (Figure 4a). Application of TTX to the preparation produced a small hyperpolarization and abolished any spontaneous activity, but the response to BK was unaffected (Figure 4b). Removal of extracellular Ca2 + produced no change in the response to BK (see below and Figure 5a).
E o z
BK + Stau
PDBu + Stau
Figure 5 Role of second messengers in the response to bradykinin (BK). (a) Responses to 100 nM BK and 1 FM phorbol dibutyrate (PDBu) recorded from the same preparation, in control (2mm Ca2") solution and in Omm Ca2', 4mM Mg2+ solution. While the response to BK was essentially unaltered in Ca2 '-free solution, the response to PDBu was greatly enhanced. (b) Comparison of the effect of the protein kinase C inhibitor staurosporine (Stau, 200nM) on responses to BK (30nM) and PDBu (1,1M). Responses were normalized with respect to an initial response to 30nM BK. Each column represents the mean and bars s.e.mean from 4 experiments. *Significantly different from control, P < 0.05 by unpaired Student's t test.
501 0 %O
Control 0 Na Wash
TTX 2 b
[100 BK 100 nM
Figure 4 Dependence of the response to bradykinin (BK) on Na+ ions. (a) The response amplitude before (control), during (ONa) and after (Wash) perfusion with solution in which Na+ was replaced by an equimolar amount of N-methylglucamine. Columns represent mean and bars s.e.mean from 4 experiments. (b) Responses to BK recorded before and during perfusion with a solution containing 2pM tetrodotoxin (TTX). TTX produced a small hyperpolarization and abolished the spontaneous activity, but did not affect the depolarization evoked by BK. *Significantly different from control, P < 0.05 by paired Student's t test.
When 1 JUM PDBu was applied to the spinal cord, a small slow depolarization was recorded via the dorsal root (Figure 5a). As found by Rang & Ritchie (1988), this response was greatly enhanced by removal of Ca2+ from the bathing solution (Figure 5a), with the response in Ca2"-free solution being 210 + 50% (mean + s.e.mean, n = 5) of the response recorded in normal (2 mm Ca2+) solution. In contrast, the response to BK was unaffected in Ca2 +-free solution (Figure 5a). We examined the effect of the PKC inhibitor staurosporine on responses to BK and PDBu. In these experiments an initial normalizing response to 30nm BK was recorded, followed by a response to either PDBu alone, BK in the presence of staurosporine, or PDBu in the presence of staurosporine. Equilibration of the preparation with staurosporine (200nM) for 20min produced a large reduction in the response to PDBu, but had no effect on the response to BK (Figure Sb).
Site of action When the same recording technique was applied to the isolated vagus nerve (n = 2), dorsal roots (n = 2), or dorsal root ganglia (n = 2), we were unable to detect any response to BK, although depolarizations to y-aminobutyric acid (GABA) and capsaicin were observed. In agreement with the observations of Dray et al. (1988a), when BK was applied to the spinal cord
BRADYKININ DEPOLARIZATION OF NERVE TERMINALS Cd2+ 10 p.M
substance P antagonist, spantide, on the responses recorded via the dorsal and ventral roots. The response to 30nm BK recorded via the ventral root was reversibly reduced by 5 UM spantide, while responses recorded via the dorsal root were unaffected (Figure 6b). To see whether the depolarization recorded via the dorsal root was due to an action of BK on the central projections of nociceptive (capsaicin-sensitive) neurones, we examined the effect of capsaicin on the response to BK. Capsaicin 1OPM produced a large depolarization and when BK was applied during the capsaicin response the effect of BK was greatly reduced (Figure 7), indicating that there was at least a considerable overlap in the populations of afferent fibres responding to BK and capsaicin.
BK 30 nM
Figure 6 Comparison of the effects of Cd2+ (a) and the substance P antagonist spantide (Span) (b) on responses to bradykinin (BK) recorded via the dorsal and ventral roots from 4 preparations. Cd2+ reversibly inhibited both the spontaneous activity and the response to BK recorded via the ventral root, while the response to BK recorded via the dorsal root was unaffected. Similarly, the response to BK recorded via the ventral root was abolished by spantide, while the response recorded via the dorsal root was unaffected.
depolarizations could also be recorded via the ventral root (Figure 6). However, when synaptic transmission was blocked by the removal of calcium from the bathing solution, or by addition of cadmium (10-1OOpM), responses recorded via the ventral root were abolished, while responses recorded via the dorsal root were unaltered (Figure 6a), suggesting that the response recorded via the dorsal root was direct, while the response recorded via the ventral root was secondary to the release of neurotransmitter within the spinal cord. To see whether substance P might have a transmitter role in the ventral root response to BK, we examined the effect of the
200 p.V BK 30 nM
Figure 7 Effect of capsaicin on the response to bradykinin (BK) recorded via the dorsal root. Responses to bradykinin (BK) were recorded before, during and after perfusion with a solution containing 10pM capsaicin (Caps). Capsaicin evoked a depolarization of approximately 4000#V (not shown). Application of BK during the slowly declining capsaicin depolarization evoked a considerably smaller response.
Bradykinin binding sites have been demonstrated in the dorsal horn of the spinal cord (Steranka et al., 1988), although the precise location of these sites is unknown. Bradykinin is known to have several effects within the spinal cord; it can activate dorsal horn neurones (Henry, 1976), cause depolarization of motor neurones (Dray et al., 1988a) and produce antinociception (Laneuville & Couture, 1987). We have shown that BK can also depolarize primary afferent fibres within the spinal cord, although we have been unable to record similar responses from isolated vagus nerve, dorsal roots or dorsal root ganglia. Since these preparations did respond to GABA and capsaicin, it suggests that, although BK binding sites are present on dorsal roots and dorsal root ganglia (Steranka et al., 1988), the electrophysiological response to BK is in some way localized to the terminal region of the primary afferent nerve fibres. Because the response recorded via the ventral root can be inhibited by the susbtance P antagonist spantide, the response recorded via the ventral root may be secondary to the release of substance P, presumably from the primary afferents. It is possible that the BK binding sites in the dorsal horn are exclusively on the primary afferent terminals and that other effects of BK within the spinal cord are all secondary to the depolarization of the primary afferent terminals. Although it has been suggested that BK can act on the terminals of bulbo-spinal noradrenergic neurones (Laneuville et al., 1989), the depolarization recorded via the dorsal root was unaffected by TTX and Ca2+ free solution, indicating that it was the result of a direct action of BK on the afferent terminals and not via the release of noradrenaline within the spinal cord. Capsaicin can selectively depolarize unmyelinated sensory neurones (Heyman & Rang, 1985; Marsh et al., 1987; Bevan & Forbes, 1988) and activates the same polymodal nociceptive neurones as BK (Martin et al., 1987). The response to BK recorded via the dorsal root was greatly reduced in the presence of capsaicin, confirming that BK was acting on capsaicin-sensitive fibres. Whether BK receptors within the spinal cord have any physiological role is at present unclear. Small amounts of BK have been detected in the spinal cord (Perry & Snyder, 1984) and neurones containing BK-like immunoreactivity have been observed elsewhere in the central nervous system (Correa et al., 1979). However, BK receptor antagonists failed to modify nociception in vivo (Laneuville & Couture, 1987), or the ventral root response to peripheral noxious stimuli in vitro (Dray et al., 1988a). Thus if BK does have a role in the spinal cord, it is not directly involved in the transmission of nociceptive information. Bradykinin receptors have been divided into BK1, BK2 and BK3 subtypes, based on the activity of a number of BK analogues (Regoli & Barabe, 1980; Farmer et al., 1989). We have shown that the BK receptors present on the primary afferent terminals are of the BK2 subtype since the BK1 agonist desArg9BK and the BK1 antagonist Leu8desArg9BK were inac-
P.M. DUNN & H.P. RANG
tive, while the response to BK was antagonized by D-Arg0Hyp3Thi5'8D-Phe7BK, which is inactive at BK3 sites. The pA2 value of 6.8 for this antagonist is similar to values of 6.0 and 6.9 found for BK2 receptors in isolated smooth muscle (Schacter et al., 1987; Steranka et al., 1988). These observations are consistent with experiments on cultured sensory neurones (Burgess et al., 1989) and with other effects of BK on the spinal cord (Laneuville & Couture, 1987; Dray et al., 1988a), which are also mediated via BK2 receptors. The depolarization of cultured sensory neurones by BK is due mainly to an increase in sodium conductance as a result of the activation of PKC by DAG, although other mechanisms may also be involved in some cells (Burgess et al., 1989). Activation of PKC by phorbol esters also causes a depolarization of vagal C fibres, due to an increase in sodium conductance (Rang & Ritchie, 1988). The depolarization of afferent terminals by BK is also due, in part, to an increase in sodium conductance, since the response was attenuated by the removal of extracellular sodium, although it was unaffected by TTX. However, other ions may also be involved in this response since it was not completely abolished in sodium-free solution. There are two results which indicate that this response to BK is not mediated via activation of PKC. Although PDBu,
an activator of PKC (Castagna et al., 1982), evoked a similar depolarization to BK, the response to PDBu was greatly enhanced by the removal of extracellular Ca2", while the response to BK was unaffected. This effect of Ca2+ on the response to PDBu is similar to that observed by Rang & Ritchie (1988) and, although the underlying mechanism is unclear, the differential effect of Ca2+ on the response to PDBu and BK suggests that these responses do not share a common mechanism. More importantly, while the response to PDBu was greatly reduced by staurosporine, a potent though not very selective inhibitor of PKC (Ruegg & Burgess, 1989), the response to BK was unaffected, providing further evidence that this response to BK is not mediated via PKC. In conclusion, we have shown that BK by acting on BK2 receptors can depolarize the terminals of primary afferent fibres within the spinal cord. This depolarization is due mainly to an increase in sodium conductance, but does not appear to be mediated via the activation of PKC. It is possible that other effects of BK on the spinal cord occur as a consequence of the depolarization of the afferent terminals and the subsequent release of neurotransmitters. We are grateful to Dr A. Dray for his comments and advice during the preparation of this manuscript.
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(Received January 25, 1990 Revised March 19, 1990 Accepted March 27, 1990)