Brain Research 903 (2001) 231–236www.elsevier.com/ locate /bres

Short communication

Distribution of various calcium channel a subunits in murine DRG1

neurons and antinociceptive effect of v-conotoxin SVIB in micea , b c b*Manabu Murakami , Takashi Suzuki , Osamu Nakagawasai , Hiroshi Murakami ,

a c c aShinobu Murakami , Akihisa Esashi , Ryoo Taniguchi , Teruyuki Yanagisawa ,c d b cKoichi Tan-No , Ichiro Miyoshi , Hironobu Sasano , Takeshi Tadano

aDepartment of Molecular Pharmacology, Tohoku University School of Medicine, Seiryoumachi, Aoba-ku, Sendai 980-8575, JapanbDepartment of Pathology, Tohoku University School of Medicine, Sendai, JapancDepartment of Pharmacology, Tohoku Pharmaceutical University, Sendai, Japan

dInstitute for Animal Experimentation, Tohoku University School of Medicine, Sendai, Japan

Accepted 20 March 2001

Abstract

Immunohistological study revealed the differential localization of subtypes of voltage-dependent calcium channels in the dorsal rootganglion neurons. Intrathecal injection of v-conotoxin SVIB, an analogue of v-conotoxin GVIA, which acts on N-type voltage-dependentcalcium channels, significantly shortened the licking time in the late phase of a formalin test. 2001 Elsevier Science B.V. All rightsreserved.

Theme: Excitable membranes and synaptic transmission

Topic: Calcium channel physiology, pharmacology, and modulation

Keywords: Calcium channel; v-Conotoxin SVIB; Nociception

Voltage-dependent calcium channels play pivotal roles neurons are known to contain the cell bodies of thein the control of calcium-linked cellular functions includ- primary nociceptive afferent fibers and it has been electro-ing neurotransmitter release. There are at least five differ- physiologically demonstrated that there are also fiveent voltage-activated calcium channels, L-, N-, P/Q-, R- different voltage-activated calcium channels, L-, N-, P/Q-,and T-types, in the central nervous system [9]. Of these R- and T-types, in the DRG [2,5,9,23]. However, little ischannels, N and P/Q-type channels are localized at the known about the channel distribution in the DRG althoughpresynaptic terminals of neurons and mediate calcium- the significance of the N-type channels in the nociceptiveinflux, which is necessary for the release of neurotrans- pathway has been confirmed by the effect of an antagonist,mitters [1,3,5,6,10,16,25]. In a previous study, we have v-conotoxin GVIA, which is relatively specific but alsoshown that a and a subunits are highly expressed in attenuates other calcium channel conductances [20].1B 1A

murine dorsal-root ganglion (DRG) by in situ hybridiza- To analyze the importance of the voltage-dependenttion, suggesting that N- and P-type channels have im- calcium channels in the nociceptive pathway, we deter-portant roles in the signal transduction in nociceptive mined the distribution of the a , a , a , a , and a1B 1A 1E 1C 1D

afferent fibers [17]. Blockade of N-type channels at subunits, which are thought to form pore-forming subunitspresynaptic terminals by v-conotoxin GVIA has been of N-, P/Q-, R- and L-type calcium channels, respectively,shown to inhibit glutaminergic transmission between the in the DRG. Furthermore, we examined the antinociceptiveneurons of the DRG and the spinal cord [7]. The DRG action of v-conotoxin SVIB, which is known to act on

N-type channels, and compared its analgesic effect withother agents. For this purpose, we intrathecally delivered*Corresponding author. Tel.: 181-22-717-8063; fax: 181-22-717-agents and examined the behavioral responses to nocicep-8065.

E-mail address: mmura@mail.cc.tohoku.ac.jp (M. Murakami). tive stimulation by formalin.

0006-8993/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 01 )02427-1

232 M. Murakami et al. / Brain Research 903 (2001) 231 –236

Immunohistochemical analysis was performed using the v-conotoxin GVIA blocks the release of other neuro-streptavidin–biotin amplification method. The DRG con- transmitters, such as acetylcholine and calcitonin gene-tains cell bodies of the primary afferent sensory fibers, related peptide from peripheral nerve terminals [12,13,21].including myelinated A-d and unmyelinated C fibers. a and a subunits of dihydropyridine sensitive L-type1C 1D

Hence, the distribution of voltage-dependent calcium chan- calcium channels, and the a subunit, which is thought to1E

nels was investigated. Heterogeneous staining of a form the R-type calcium channel, are located predominant-1A

subunits was found throughout the DRG neurons, with ly on the cell body [26].different levels in the cell bodies of some neuronal cells We found that the distribution of the a subunits of1

(arrow), and with low intensities in some adjacent nerve calcium channels in murine DRG was for the most partfibers (asterisk) (Fig. 1A,F). The a subunits were similar to that observed in rat spinal cord. a and a1B 1A 1B

heterogeneously localized on the cell bodies in the proxim- subunits were unevenly expressed in the cell bodies ofal regions of the DRG neurons (Fig. 1B,G, arrow). Some some neurons and in some adjacent nerve fibers. We foundadjacent nerve fibers in the proximal portion were also relatively homogeneous expressions of a and a1C 1D

positively stained (Fig. 1G, asterisk). The distributions of subunits in the cell bodies in the murine DRG neurons.the a and a subunits were similar (Fig. 1C,D). Their Although we could not completely analyze the channel1C 1D

immunoreactivities were homogeneous throughout the distributions at the nerve terminals, our results showedDRG neurons. a subunits were heterogeneously local- fewer N- and P/Q-types in the cell bodies compared with1E

ized in the cell bodies of the DRG neurons. The staining L-type calcium channels. As the a subunit showed a1E

pattern was different from those of other subunits. The different distribution, it might have quite different roles insizes of the cells and the intensities were various and some nociceptive neurotransmission.nerve fibers were also positively stained (Fig. 1E). Intrathecal administration of v-conotoxin GVIA and

The formalin test was done by subcutaneously injecting v-agatoxin-IVA, but not L-type calcium channel antago-20 ml of a 2% formaldehyde solution after intrathecal nists, inhibits the excitability of dorsal horn neurons [1].injection (i.t.) at the L5 and L6 intervertebral space, as we v-Conotoxin GVIA, derived from the venom of Conuspreviously described [22]. The subjects were 4–5-week- geogrophus, is a potent blocker of neuronal calciumold mice maintained with an alternating 12-h light–dark channels (N-type) [8,15,18] and its analgesic action hascycle. Intrathecal v-conotoxin SVIB (8.0 nmol /kg) 10 min been well characterized [14]. It blocks neurotransmitterprior to subcutaneous formalin significantly reduced the release at the presynaptic terminals and its effect islicking time in the late phase. Intrathecal injection with generally irreversible [4,11,27]. In the present study, wev-conotoxin GVIA (0.5 nmol /kg) decreased the licking examined the effect of a novel N-type channel blocker,time in both the early and late phases. Nicardipine (300 v-conotoxin SVIB. v-Conotoxin SVIB from Conusnmol /kg), which acts preferentially on L-type channels, striatus is an analogue of v-conotoxin GVIA. It does notshowed no apparent effect on either phase, while cil- share the same binding site of v-conotoxin GVIA, and hasnidipine (300 nmol /kg), which acts on both L- and N-type different pharmacological characteristics, such as its re-channels, significantly reduced the licking time in the late versible inhibitory effect on evoked postsynaptic responsesphase (Fig. 2A). v-Conotoxin SVIB showed a dose-depen- of a frog neuromuscular preparation [19]. Although thedent antinociceptive effect in the late phase (Fig. 2B). blocking effect of this toxin on N-type channels is known,

In the present study we have elucidated for the first time its analgesic effects have not been examined.the distribution of calcium channel a subunits in the DRG The present study demonstrated that v-conotoxin SVIB1

neurons. As calcium influx through the voltage-dependent has a potent antinociceptive effect in mice. Considering thecalcium channels is a trigger of neurotransmission at the presence of the a (N-type) subunit in DRG and the1B

synapse, it would be quite informative to understand the localization of N-type channels at the presynaptic termi-distribution of each calcium channel a subunit in the nals, v-conotoxin SVIB exerts its analgesic effect by1

21DRG and the involvement of voltage-dependent calcium inhibiting Ca -influx, probably through N-type channels,channels in nociceptive neurotransmission. Previous ex- and suppressing neurotransmitter release.periments using site-directed anti-peptide antibodies dem- Although both are N-type channel antagonists, ouronstrated distinct distributions of the different calcium results show some differences in the pharmacologicalchannel a subunits in rat neurons [24–26]. In rat tissues, effects between v-conotoxin SVIB and v-conotoxin1

a subunits of the P/Q-type calcium channels and a GVIA. v-Conotoxin SVIB showed antinociceptive effects1A 1B

subunits of the N-type calcium channels are located only in the late phase with high dose (8 nmol /kg), whereaspredominantly along the apical dendrites with substantially v-conotoxin GVIA showed marked antinociceptive effectslower levels of somal expression [25,26]. Westenbroek et in both the early and late phases at a lower dose (0.5al. also reported that the nerve terminals, which are nmol /kg). While v-conotoxin GVIA obviously causedimmunoreactive for a subunits, contained substance P, shaker tremors at a dose of 2.0 nmol /kg, few shaker1B

an important neuropeptide in nociceptive neurotrans- tremors were observed with v-conotoxin SVIB at the samemission in rat spinal cord [26]. It is also known that dose, indicating its antinociceptive effects. These differ-

M. Murakami et al. / Brain Research 903 (2001) 231 –236 233

234 M. Murakami et al. / Brain Research 903 (2001) 231 –236

Fig. 2. (A) The effect of intrathecal v-conotoxin SVIB (8.0 nmol /kg), nicardipine (300 nmol /kg), cilnidipine (300 nmol /kg), and v-conotoxin GVIA (0.5nmol /kg) on the behavior induced by subcutaneous formalin injection. Nociceptive behavior in both early (0–10 min; upper panel) and late phases (10–30min; lower panel) is shown as the amount of time spent licking the injected paw. The data are expressed as the mean6S.E.M. Significant differences fromthe control were isolated using the Student’s t-test (*P,0.05). (B) The effects of various doses of v-conotoxin SVIB on the duration of licking aftersubcutaneous formalin injections. The data are expressed as the mean6S.E.M. Significant differences were determined with Dunnett’s test (*P,0.05 vs.control). All of the behavioral experiments were conducted under the ethical guidelines for the study of experimental pain in conscious animals andperformed with the approval of the ethics committee for animal experiments of Tohoku Pharmaceutical University.

Fig. 1. Distribution of a , a , a , a and a subunits of calcium channels in DRG (A–E). All scale bars are 50 mm. Arrows point to regions of1A 1B 1C 1D 1E

strong signals in small cells. Asterisks point to positively stained nerve fibers (F,G). After deparaffinization, slides were pre-treated with an autoclave fora , boiled in a microwave oven (a ), treated with pronase (a ), and incubated with trypsin (a ). Commercially available antibodies that specifically1A 1B 1C 1D

recognize a , a , a , a and a subunits (Alomone, Jerusalem, Israel) were used. The dilutions of primary antibodies used in our study were as1A 1B 1C 1D 1E

follows: 1 /100, 1 /100, 1 /200, 1 /300 and 1/150, for a , a , a , a and a , respectively. The antigen–antibody complex was visualized as1A 1B 1C 1D 1E

brown-colored precipitation with 3.39-diaminobenzidine (DAB) solution (1 mM DAB, 50 mM Tris–HCl buffer (pH 7.6), and 0.006% H O ) and2 2

counterstained with hematoxylin.

M. Murakami et al. / Brain Research 903 (2001) 231 –236 235

[3] E.M. Elliot, A.T. Malof, W.A. Catterall, Role of calcium channelences may be due to the irreversible effect of v-conotoxinsubtypes in calcium transients in hippocampal CA3 neurons, J.GVIA, as it irreversibly inhibits N-type channel functions,Neurosci. 15 (1995) 6433–6444.

while v-conotoxin SVIB’s effects are reversible. It may be [4] D. Feldman, B. Olivera, D. Yoshikami, Omega Conus geographicusdue to the differences in the pharmacological potency, as toxin: a peptide that blocks calcium channels, FEBS Lett. 214v-conotoxin GVIA seems to be more potent than other (1987) 295–300.

[5] S. Fujii, K. Kameyama, M. Hosono, Y. Hayashi, K. Kitamura, Effectagents. It also may be due to the difference in the binding21of cilnidipine, a novel dihydropyridine Ca -channel antagonist, onsite, as the high affinity binding site for v-conotoxin SVIB 21N-type Ca channel in rat dorsal root ganglion neurons, J.is known to be distinct from that for v-conotoxin GVIA

Pharmacol. Exp. Ther. 280 (1997) 1184–1191.[19], but the exact amino acid domain sequences, which [6] K. Gohil, J.R. Bell, J. Ramachandran, G.P. Milijanich, Neuro-are responsible for the interaction with each toxin, have not anatomical distribution of receptors for a novel voltage-sensitive

calcium channel antagonist, SNX-230 (v-conopeptide MVIIC),yet been identified. It is also important to note that theBrain Res. 653 (1994) 258–266.exact electrophysiological profile of v-conotoxin SVIB on

[7] W. Gruner, L.R. Silva, v-Conotoxin sensitivity and presynapticthe calcium channels has not been completely well char-inhibition of glutamatergic sensory neurotransmission in vitro, J.

acterized by patch-clamp method. Neurosci. 14 (1994) 2800–2808.Although we found a number of neurons containing [8] D.R. Hillyard, V.D. Monje, I.M. Mintz, B.N. Bean, L. Nadasdi, J.

Ramachandran, G. Miljanich, A. Azimi-Zoonooz, J.M. McIntosh,L-type calcium channels (a and a ) in their soma, we1C 1DL.J. Cruz, J.S. Imperial, B.M. Olivera, A new Conus peptide ligandfound little analgesic effect with nicardipine, which is an

21for mammalian presynaptic Ca channels, Neuron 9 (1992) 67–77.L-type-specific calcium channel antagonist. This suggests 21[9] F. Hofmann, M. Biel,V. Flockerzi, Molecular basis for Ca channelthe minor contribution of L-type channels in nociceptive diversity, Annu. Rev. Neurosci. 17 (1994) 399–418.neurotransmission, whereas there are other stimuli, such as [10] L.M. Kerr, F. Filloux, B.M. Olivera, H. Jackson, J.K. Wamsley,

125Autoradiographic localization of calcium channels with [I ]v-thermal and visceral inflammatory ones, which were notconotoxin in rat brain, Eur. J. Pharmacol. 146 (1988) 181–183.examined in the present study and should be studied in the

[11] L. Kerr, D. Yoshikami, A venom peptide with a novel presynapticfuture.blocking action, Nature 308 (1984) 282–284.

Taken together, we have shown that there were different [12] P.M. Lundy, R. Frew, Evidence of v-conotoxin GVIA-sensitive21localizations of various calcium channel a subunits in the Ca channels in mammalian peripheral nerve terminals, Eur. J.1

DRG neurons. It is also proved that i.t. injection of Pharmacol. 156 (1988) 325–330.[13] C.A. Maggi, M. Tramontana, R. Cecconi, P. Santicioli, Neurochemi-v-conotoxin SVIB resulted in a significant reduction of the

cal evidence for the involvement of N-type calcium channels inresponse in the late phase of the formalin test. Consideringtransmitter secretion from peripheral endings of sensory nerves in

the different pharmacological effects of L- and N-type guinea pigs, Neurosci. Lett. 114 (1990) 203–206.channel blockers and the different distribution of a [14] A.B. Malmberg, T.L. Yaksh, Voltage-sensitive calcium channels in1

calcium channel subunits, we conclude that each channel spinal nociceptive processing: blockade of N-type and P-typechannels inhibits formalin-induced nociception, J. Neurosci. 14subtype has a distinct role in nociceptive neurotrans-(1994) 4882–4890.mission.

[15] A. McCleskey, A.P. Fox, L.J. Cruz, B.M. Olivera, R.W. Tsien, D.Yoshikami, v-Conotoxin: direct and persistent blockade of specifictypes of calcium channels in neurons but not in muscle, Proc. Natl.Acad. Sci. USA 84 (1987) 4327–4331.Acknowledgements

[16] G.P. Miljamich, J. Ramachandran, Antagonists of neuronal calciumchannels: structure, function, and therapeutic implications, Annu.We thank Miss Kumiko Hidaka, Mrs Fumihiro Yama-Rev. Pharmacol. Toxicol. 35 (1995) 707–734.

dera and Pei Xiang Yuan for technical assistance, and Drs [17] M. Murakami, O. Nakagawasai, S. Fujii, M. Hosono, S. Hozumi, A.Kensuke Kisara and Kazuo Nunoki, and Mr. Brent Bell for Esashi, R. Taniguchi, K. Kisara, Antinociceptive effect of cil-

nidipine, a novel N-type calcium channel antagonist, Brain Res. 868critically reading the manuscript. We also thank Dr(2000) 123–127.Hidetoshi Shimauchi, Fuji Rebio Co. and Boehringer

[18] B.M. Olivera, J.M. McIntosh, L.J. Cruz, F.A. Luque, W.R. Gray,Ingelheim Co. for their help. This research was sponsoredPurification and sequence of a presynaptic peptide toxin from Conus

by grants-in-aid from the Ministry of Education, Science geographus venom, Biochemistry 23 (1984) 5087–5090.and Culture, Japan. [19] C.A. Ramilo, G.C. Zafaralla, L. Nadasdi, L.G. Hammerland, D.

Yoshikami, W.R. Gray, K. Ramasharma, J. Ramachandran, G.Miljanich, B.M. Olivera, L.J. Cruz, Novel a- and v-conotoxins fromConus striatus Venom, Biochemistry 31 (1992) 9919–9926.

References [20] L. River, R. Galyean, W.R. Gray, A. Azimi-Zonooz, J.M. McIntosh,L.J. Cruz, B.M. Olivera, Neuronal calcium channel inhibitors, J.

[1] A. Diaz, A.H. Dickenson, Blockade of spinal N- and P-type, but not Biol. Chem. 262 (1987) 1194–1198.L-type, calcium channels inhibits the excitabitity of rat dorsal horn [21] G.R. Seabrook, D.J. Adams, Inhibition of neurally evoked transmit-neurones produced by subcutaneous formalin inflammation, Pain 69 ter release by calcium channel antagonists in rat parasympathetic(1997) 93–100. ganglia, Br. J. Pharmacol. 97 (1989) 1125–1136.

[2] S. Diochot, S. Richard, M. Baldy-Moulinier, J. Nargeot, J. Valmier, [22] T. Sakurada, K. Katsumata, H. Yogo, K. Tan-No, S. Sakurada, M.Dihydropyridines, phenylalkylamines and benzothiazepines block Ohba, K. Kisara, The neurokinin-1 receptor antagonist, sendide,

¨N-, P/Q- and R-type calcium currents, Pflugers Arch. Eur. J. exhibits antinociceptive activity in the formalin test, Pain 60 (1995)Physiol. 431 (1995) 10–19. 175–180.

236 M. Murakami et al. / Brain Research 903 (2001) 231 –236

[23] R.S. Scroggs, A.P. Fox, Calcium current variation between a cutely [26] R.E. Westenbroek, T. Sakurai, E.M. Elliot, J.W. Hell, T.V.B. Starr,isolated adult rat dorsal root ganglion neurons of different size, J. T.P. Snutch, W.A. Catterall, Immunochemical identification andPhysiol. (Lond.) 445 (1992) 639–658. subcellular distribution of the a subunits of brain calcium1A

[24] R.E. Westenbroek, M.K. Ahlijanian, W. A Catterall, Clustering of channels, J. Neurosci. 15 (1995) 6403–6418.21L-type Ca channels at the base of major dendrites in hippocampal [27] D. Yoshikami, Z. Bagobaldo, B. Olivera, The inhibitory effects of

21pyramidal neurons, Nature 347 (1990) 281–284. v-conotoxins on Ca channels and synapses, Ann. N. Y. Acad Sci.[25] R.E. Westenbroek, J.W. Hell, C. Warner, S.J. Dubel, T.P. Snutch, 560 (1989) 230–248.

W.A. Catterall, Biochemical properties and subcellular distributionof an N-type calcium channel-a -subunit, Neuron 9 (1992) 1099–1

1115.