Pflügers Archiv - European Journal of Physiology

, Volume 467, Issue 12, pp 2485–2493 | Cite as

Analgesic effect of a broad-spectrum dihydropyridine inhibitor of voltage-gated calcium channels

  • Vinicius M. Gadotti
  • Chris Bladen
  • Fang Xiong Zhang
  • Lina Chen
  • Miyase Gözde Gündüz
  • Rahime Şimşek
  • Cihat Şafak
  • Gerald W. ZamponiEmail author
Ion channels, receptors and transporters


Voltage-activated calcium channels are important facilitators of nociceptive transmission in the primary afferent pathway. Consequently, molecules that block these channels are of potential use as pain therapeutics. Our group has recently reported on the identification of a novel class of dihydropyridines (DHPs) that included compounds with preferential selectivity for T-type over L-type channels. Among those compounds, M4 was found to be an equipotent inhibitor of both Cav1.2 L- and Cav3.2 T-type calcium channels. Here, we have further characterized the effects of this compound on other types of calcium channels and examined its analgesic effect when delivered either spinally (i.t.) or systemically (i.p.) to mice. Both delivery routes resulted in antinociception in a model of acute pain. Furthermore, M4 was able to reverse mechanical hyperalgesia produced by nerve injury when delivered intrathecally. M4 retained partial activity when delivered to Cav3.2 null mice, indicating that this compound acts on multiple targets. Additional whole-cell patch clamp experiments in transfected tsA-201 cells revealed that M4 also effectively blocks Cav3.3 (T-type) and Cav2.2 (N-type) currents. Altogether, our data indicate that broad-spectrum inhibition of multiple calcium channel subtypes can lead to potent analgesia in rodents.


T-type calcium channel N-type calcium channel Neuropathic pain Dihydropyridine analogues Electrophysiology 



This work was supported by operating grants to GWZ from the Canadian Institutes of Health Research and by the Vi Riddell Child Pain program of the Alberta Children’s Hospital Research Institute. GWZ is a Canada Research Chair. VG held a Canadian Diabetes Association fellowship. CB held a T. Chen Fong studentship and an Alberta Innovates-Health Solutions (AI-HS) studentship award. FXZ holds an AI-HS fellowship.


  1. 1.
    Altier C, Zamponi GW (2004) Targeting Ca2+ channels to treat pain: T-type versus N-type. Trends Pharmacol Sci 25:465–470. doi: 10.1016/ CrossRefPubMedGoogle Scholar
  2. 2.
    Atanassoff PG, Hartmannsgruber MW, Thrasher J, Wermeling D, Longton W, Gaeta R, Singh T, Mayo M, McGuire D, Luther RR (2000) Ziconotide, a new N-type calcium channel blocker, administered intrathecally for acute postoperative pain. Reg Anesth Pain Med 25:274–278PubMedGoogle Scholar
  3. 3.
    Basbaum AI (1999) Distinct neurochemical features of acute and persistent pain. Proc Natl Acad Sci U S A 96:7739–7743PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Bennett GJ, Xie YK (1988) A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 33:87–107CrossRefPubMedGoogle Scholar
  5. 5.
    Bladen C, Gadotti VM, Gunduz MG, Berger ND, Simsek R, Safak C, Zamponi GW (2015) 1,4-Dihydropyridine derivatives with T-type calcium channel blocking activity attenuate inflammatory and neuropathic pain. Pflugers Arch 467:1237–1247. doi: 10.1007/s00424-014-1566-3 CrossRefPubMedGoogle Scholar
  6. 6.
    Bladen C, Gunduz MG, Simsek R, Safak C, Zamponi GW (2014) Synthesis and evaluation of 1,4-dihydropyridine derivatives with calcium channel blocking activity. Pflugers Arch 466:1355–1363. doi: 10.1007/s00424-013-1376-z CrossRefPubMedGoogle Scholar
  7. 7.
    Bourinet E, Alloui A, Monteil A, Barrere C, Couette B, Poirot O, Pages A, McRory J, Snutch TP, Eschalier A, Nargeot J (2005) Silencing of the Cav3.2 T-type calcium channel gene in sensory neurons demonstrates its major role in nociception. EMBO J 24:315–324. doi: 10.1038/sj.emboj.7600515 PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Bourinet E, Soong TW, Stea A, Snutch TP (1996) Determinants of the G protein-dependent opioid modulation of neuronal calcium channels. Proc Natl Acad Sci U S A 93:1486–1491PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Cao XH, Byun HS, Chen SR, Pan HL (2011) Diabetic neuropathy enhances voltage-activated Ca2+ channel activity and its control by M4 muscarinic receptors in primary sensory neurons. J Neurochem 119:594–603. doi: 10.1111/j.1471-4159.2011.07456.x PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Cheng JK, Lin CS, Chen CC, Yang JR, Chiou LC (2007) Effects of intrathecal injection of T-type calcium channel blockers in the rat formalin test. Behav Pharmacol 18:1–8. doi: 10.1097/FBP.0b013e3280141375 CrossRefPubMedGoogle Scholar
  11. 11.
    Choi S, Na HS, Kim J, Lee J, Lee S, Kim D, Park J, Chen CC, Campbell KP, Shin HS (2007) Attenuated pain responses in mice lacking Ca(V)3.2 T-type channels. Genes Brain Behav 6:425–431. doi: 10.1111/j.1601-183X.2006.00268.x CrossRefPubMedGoogle Scholar
  12. 12.
    Dobremez E, Bouali-Benazzouz R, Fossat P, Monteils L, Dulluc J, Nagy F, Landry M (2005) Distribution and regulation of L-type calcium channels in deep dorsal horn neurons after sciatic nerve injury in rats. Eur J Neurosci 21:3321–3333. doi: 10.1111/j.1460-9568.2005.04177.x CrossRefPubMedGoogle Scholar
  13. 13.
    Dubreuil AS, Boukhaddaoui H, Desmadryl G, Martinez-Salgado C, Moshourab R, Lewin GR, Carroll P, Valmier J, Scamps F (2004) Role of T-type calcium current in identified D-hair mechanoreceptor neurons studied in vitro. J Neurosci 24:8480–8484. doi: 10.1523/JNEUROSCI.1598-04.2004 CrossRefPubMedGoogle Scholar
  14. 14.
    Evans AR, Nicol GD, Vasko MR (1996) Differential regulation of evoked peptide release by voltage-sensitive calcium channels in rat sensory neurons. Brain Res 712:265–273CrossRefPubMedGoogle Scholar
  15. 15.
    Favereaux A, Thoumine O, Bouali-Benazzouz R, Roques V, Papon MA, Salam SA, Drutel G, Leger C, Calas A, Nagy F, Landry M (2011) Bidirectional integrative regulation of Cav1.2 calcium channel by microRNA miR-103: role in pain. EMBO J 30:3830–3841. doi: 10.1038/emboj.2011.249 PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Flatters SJ, Bennett GJ (2004) Ethosuximide reverses paclitaxel- and vincristine-induced painful peripheral neuropathy. Pain 109:150–161. doi: 10.1016/j.pain.2004.01.029 CrossRefPubMedGoogle Scholar
  17. 17.
    Fossat P, Dobremez E, Bouali-Benazzouz R, Favereaux A, Bertrand SS, Kilk K, Leger C, Cazalets JR, Langel U, Landry M, Nagy F (2010) Knockdown of L calcium channel subtypes: differential effects in neuropathic pain. J Neurosci 30:1073–1085. doi: 10.1523/JNEUROSCI.3145-09.2010 CrossRefPubMedGoogle Scholar
  18. 18.
    Francois A, Schuetter N, Laffray S, Sanguesa J, Pizzoccaro A, Dubel S, Mantilleri A, Nargeot J, Noel J, Wood JN, Moqrich A, Pongs O, Bourinet E (2015). The low-threshold calcium channel Cav3.2 determines low-threshold mechanoreceptor function. Cell Rep  10.1016/j.celrep.2014.12.042.
  19. 19.
    Fujii S, Kameyama K, Hosono M, Hayashi Y, Kitamura K (1997) Effect of cilnidipine, a novel dihydropyridine Ca++−channel antagonist, on N-type Ca++ channel in rat dorsal root ganglion neurons. J Pharmacol Exp Ther 280:1184–1191PubMedGoogle Scholar
  20. 20.
    Furukawa T, Miura R, Honda M, Kamiya N, Mori Y, Takeshita S, Isshiki T, Nukada T (2004) Identification of R(−)-isomer of efonidipine as a selective blocker of T-type Ca2+ channels. Br J Pharmacol 143:1050–1057. doi: 10.1038/sj.bjp.0705944 PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Gadotti VM, Caballero AG, Berger ND, Gladding CM, Chen L, Pfeifer TA, Zamponi GW (2015) Small organic molecule disruptors of Cav3.2–USP5 interactions reverse inflammatory and neuropathic pain. Mol Pain 11:12. doi: 10.1186/s12990-015-0011-8 PubMedCentralCrossRefPubMedGoogle Scholar
  22. 22.
    Gadotti VM, You H, Petrov RR, Berger ND, Diaz P, Zamponi GW (2013) Analgesic effect of a mixed T-type channel inhibitor/CB2 receptor agonist. Mol Pain 9:32. doi: 10.1186/1744-8069-9-32 PubMedCentralCrossRefPubMedGoogle Scholar
  23. 23.
    Gadotti VM, Zamponi GW (2011) Cellular prion protein protects from inflammatory and neuropathic pain. Mol Pain 7:59. doi: 10.1186/1744-8069-7-59 PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Garcia-Caballero A, Gadotti VM, Stemkowski P, Weiss N, Souza IA, Hodgkinson V, Bladen C, Chen L, Hamid J, Pizzoccaro A, Deage M, Francois A, Bourinet E, Zamponi GW (2014) The deubiquitinating enzyme USP5 modulates neuropathic and inflammatory pain by enhancing Cav3.2 channel activity. Neuron 83:1144–1158. doi: 10.1016/j.neuron.2014.07.036 CrossRefPubMedGoogle Scholar
  25. 25.
    Gover TD, Kao JP, Weinreich D (2003) Calcium signaling in single peripheral sensory nerve terminals. J Neurosci 23:4793–4797PubMedGoogle Scholar
  26. 26.
    Hatakeyama S, Wakamori M, Ino M, Miyamoto N, Takahashi E, Yoshinaga T, Sawada K, Imoto K, Tanaka I, Yoshizawa T, Nishizawa Y, Mori Y, Niidome T, Shoji S (2001) Differential nociceptive responses in mice lacking the alpha(1B) subunit of N-type Ca(2+) channels. Neuroreport 12:2423–2427CrossRefPubMedGoogle Scholar
  27. 27.
    Hunskaar S, Hole K (1987) The formalin test in mice: dissociation between inflammatory and non-inflammatory pain. Pain 30:103–114CrossRefPubMedGoogle Scholar
  28. 28.
    Hylden JL, Wilcox GL (1980) Intrathecal morphine in mice: a new technique. Eur J Pharmacol 67:313–316CrossRefPubMedGoogle Scholar
  29. 29.
    Jacus MO, Uebele VN, Renger JJ, Todorovic SM (2012) Presynaptic Cav3.2 channels regulate excitatory neurotransmission in nociceptive dorsal horn neurons. J Neurosci 32:9374–9382. doi: 10.1523/JNEUROSCI.0068-12.2012 PubMedCentralCrossRefPubMedGoogle Scholar
  30. 30.
    Jagodic MM, Pathirathna S, Joksovic PM, Lee W, Nelson MT, Naik AK, Su P, Jevtovic-Todorovic V, Todorovic SM (2008) Upregulation of the T-type calcium current in small rat sensory neurons after chronic constrictive injury of the sciatic nerve. J Neurophysiol 99:3151–3156. doi: 10.1152/jn.01031.2007 PubMedCentralCrossRefPubMedGoogle Scholar
  31. 31.
    Kaster MP, Gadotti VM, Calixto JB, Santos AR, Rodrigues AL (2012) Depressive-like behavior induced by tumor necrosis factor-alpha in mice. Neuropharmacology 62:419–426. doi: 10.1016/j.neuropharm.2011.08.018 CrossRefPubMedGoogle Scholar
  32. 32.
    Kim C, Jun K, Lee T, Kim SS, McEnery MW, Chin H, Kim HL, Park JM, Kim DK, Jung SJ, Kim J, Shin HS (2001) Altered nociceptive response in mice deficient in the alpha(1B) subunit of the voltage-dependent calcium channel. Mol Cell Neurosci 18:235–245. doi: 10.1006/mcne.2001.1013 CrossRefPubMedGoogle Scholar
  33. 33.
    Kumar PP, Stotz SC, Paramashivappa R, Beedle AM, Zamponi GW, Rao AS (2002) Synthesis and evaluation of a new class of nifedipine analogs with T-type calcium channel blocking activity. Mol Pharmacol 61:649–658CrossRefPubMedGoogle Scholar
  34. 34.
    Malmberg AB, Basbaum AI (1998) Partial sciatic nerve injury in the mouse as a model of neuropathic pain: behavioral and neuroanatomical correlates. Pain 76:215–222CrossRefPubMedGoogle Scholar
  35. 35.
    Marger F, Gelot A, Alloui A, Matricon J, Ferrer JF, Barrere C, Pizzoccaro A, Muller E, Nargeot J, Snutch TP, Eschalier A, Bourinet E, Ardid D (2011) T-type calcium channels contribute to colonic hypersensitivity in a rat model of irritable bowel syndrome. Proc Natl Acad Sci U S A 108:11268–11273. doi: 10.1073/pnas.1100869108 PubMedCentralCrossRefPubMedGoogle Scholar
  36. 36.
    Minami M, Katayama T, Satoh M (2006) Brain cytokines and chemokines: roles in ischemic injury and pain. J Pharmacol Sci 100:461–470CrossRefPubMedGoogle Scholar
  37. 37.
    Munro G, Erichsen HK, Mirza NR (2007) Pharmacological comparison of anticonvulsant drugs in animal models of persistent pain and anxiety. Neuropharmacology 53:609–618. doi: 10.1016/j.neuropharm.2007.07.002 CrossRefPubMedGoogle Scholar
  38. 38.
    Pertovaara A (2006) Noradrenergic pain modulation. Prog Neurobiol 80:53–83. doi: 10.1016/j.pneurobio.2006.08.001 CrossRefPubMedGoogle Scholar
  39. 39.
    Saegusa H, Kurihara T, Zong S, Kazuno A, Matsuda Y, Nonaka T, Han W, Toriyama H, Tanabe T (2001) Suppression of inflammatory and neuropathic pain symptoms in mice lacking the N-type Ca2+ channel. EMBO J 20:2349–2356. doi: 10.1093/emboj/20.10.2349 PubMedCentralCrossRefPubMedGoogle Scholar
  40. 40.
    Salter MW, Zamponi GW (2014) Calcium-permeable ion channels in pain signaling. Physiol Rev 94:81–140. doi: 10.1152/physrev.00023.2013 CrossRefPubMedGoogle Scholar
  41. 41.
    Smith MT, Cabot PJ, Ross FB, Robertson AD, Lewis RJ (2002) The novel N-type calcium channel blocker, AM336, produces potent dose-dependent antinociception after intrathecal dosing in rats and inhibits substance P release in rat spinal cord slices. Pain 96:119–127CrossRefPubMedGoogle Scholar
  42. 42.
    Staats PS, Yearwood T, Charapata SG, Presley RW, Wallace MS, Byas-Smith M, Fisher R, Bryce DA, Mangieri EA, Luther RR, Mayo M, McGuire D, Ellis D (2004) Intrathecal ziconotide in the treatment of refractory pain in patients with cancer or AIDS: a randomized controlled trial. JAMA 291:63–70. doi: 10.1001/jama.291.1.63 CrossRefPubMedGoogle Scholar
  43. 43.
    Sukiasyan N, Hultborn H, Zhang M (2009) Distribution of calcium channel Ca(V)1.3 immunoreactivity in the rat spinal cord and brain stem. Neuroscience 159:217–235. doi: 10.1016/j.neuroscience.2008.12.011 CrossRefPubMedGoogle Scholar
  44. 44.
    Sun J, Triggle DJ (1995) Calcium channel antagonists: cardiovascular selectivity of action. J Pharmacol Exp Ther 274:419–426PubMedGoogle Scholar
  45. 45.
    Tjolsen A, Berge OG, Hunskaar S, Rosland JH, Hole K (1992) The formalin test: an evaluation of the method. Pain 51:5–17CrossRefPubMedGoogle Scholar
  46. 46.
    Uneyama H, Takahara A, Dohmoto H, Yoshimoto R, Inoue K, Akaike N (1997) Blockade of N-type Ca2+ current by cilnidipine (FRC-8653) in acutely dissociated rat sympathetic neurones. Br J Pharmacol 122:37–42. doi: 10.1038/sj.bjp.0701342 PubMedCentralCrossRefPubMedGoogle Scholar
  47. 47.
    Wang R, Lewin GR (2011) The Cav3.2 T-type calcium channel regulates temporal coding in mouse mechanoreceptors. J Physiol 589:2229–2243. doi: 10.1113/jphysiol.2010.203463 PubMedCentralCrossRefPubMedGoogle Scholar
  48. 48.
    Wappl E, Mitterdorfer J, Glossmann H, Striessnig J (2001) Mechanism of dihydropyridine interaction with critical binding residues of L-type Ca2+ channel alpha 1 subunits. J Biol Chem 276:12730–12735. doi: 10.1074/jbc.M010164200 CrossRefPubMedGoogle Scholar
  49. 49.
    Waxman SG, Zamponi GW (2014) Regulating excitability of peripheral afferents: emerging ion channel targets. Nat Neurosci 17:153–163. doi: 10.1038/nn.3602 CrossRefPubMedGoogle Scholar
  50. 50.
    Wen XJ, Li ZJ, Chen ZX, Fang ZY, Yang CX, Li H, Zeng YM (2006) Intrathecal administration of Cav3.2 and Cav3.3 antisense oligonucleotide reverses tactile allodynia and thermal hyperalgesia in rats following chronic compression of dorsal root of ganglion. Acta Pharmacol Sin 27:1547–1552. doi: 10.1111/j.1745-7254.2006.00461.x CrossRefPubMedGoogle Scholar
  51. 51.
    Wen XJ, Xu SY, Chen ZX, Yang CX, Liang H, Li H (2010) The roles of T-type calcium channel in the development of neuropathic pain following chronic compression of rat dorsal root ganglia. Pharmacology 85:295–300. doi: 10.1159/000276981 CrossRefPubMedGoogle Scholar
  52. 52.
    Yue J, Liu L, Liu Z, Shu B, Zhang Y (2013) Upregulation of T-type Ca2+ channels in primary sensory neurons in spinal nerve injury. Spine (Phila Pa 1976) 38:463–470. doi: 10.1097/BRS.0b013e318272fbf8 CrossRefGoogle Scholar
  53. 53.
    Zamponi GW, Stotz SC, Staples RJ, Andro TM, Nelson JK, Hulubei V, Blumenfeld A, Natale NR (2003) Unique structure-activity relationship for 4-isoxazolyl-1,4-dihydropyridines. J Med Chem 46:87–96. doi: 10.1021/jm020354w CrossRefPubMedGoogle Scholar
  54. 54.
    Zimmermann M (2001) Pathobiology of neuropathic pain. Eur J Pharmacol 429:23–37CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Vinicius M. Gadotti
    • 1
  • Chris Bladen
    • 1
  • Fang Xiong Zhang
    • 1
  • Lina Chen
    • 1
  • Miyase Gözde Gündüz
    • 2
  • Rahime Şimşek
    • 2
  • Cihat Şafak
    • 2
  • Gerald W. Zamponi
    • 1
    Email author
  1. 1.Department of Physiology and Pharmacology, Cumming School of Medicine, Hotchkiss Brain Institute and Alberta Children’s Hospital Research InstituteUniversity of CalgaryCalgaryCanada
  2. 2.Department of Pharmaceutical Chemistry, Faculty of PharmacyHacettepe UniversityAnkaraTurkey

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