Sensitization of recombinant vanilloid receptor-1 by various neurotrophic factors

doi:10.1016/j.lfs.2003.11.023

Life Sciences

Volume 75, Issue 2, 28 May 2004, Pages 153-163

https://doi.org/10.1016/j.lfs.2003.11.023 Get rights and content

Abstract

The vanilloid receptor (VR1) is a central integrator molecule of nociceptive stimuli. In this study, we have measured the effects of various neurotrophins (nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and -4) on recombinant rat VR1-mediated intracellular calcium rise in response to capsaicin in VR1/C6 cells. Our results clearly show that all neurotrophins sensitize the VR1 to capsaicin. Furthermore, using K252a, an inhibitor of tyrosine kinases, we present that actions of neurotrophins are mediated by the trk (A, B, C) receptors expressed in these cells. These data argue for the putative roles of neurotrophins in inducing inflammatory (thermal) hyperalgesia via VR1.

Introduction

The vanilloid receptor (VR1) is a ligand-gated cation channel localized mainly in a subset of sensory (nociceptive) neurons Caterina et al., 1997, Caterina and Julius, 2001. The receptor can be activated by vanilloids such as capsaicin and its ultrapotent analog, resiniferatoxin Szallasi and Blumberg, 1999, Caterina and Julius, 2001. Besides such exogenous factors, endogenous stimuli such as low-threshold heat, acidosis, bradykinin, eicosanoids, and other arachidonic acid derivatives Caterina and Julius, 2001, Di Marzo et al., 2002 may also act on VR1. The effect of these “endovanilloids” may represent either a direct activation of the receptor or indirect sensitization of VR1 subsequent to modulation of various intracellular signaling pathways (Di Marzo et al., 2002). The process of sensitization, by decreasing the heat-threshold of VR1, therefore, may have a key role in initiating inflammatory (thermal) hyperalgesia (Caterina et al., 2000).

The neurotrophins (e.g., nerve growth factor, NGF; brain-derived growth factor, BDNF; neurotrophin-3, -4, NT3, NT4) are trophic factors critical in embryonic development, differentiation, survival and regeneration Snider, 1994, Lewin and Barde, 1996 of various nerve cell types, including VR1-expressing sensory neurons (Kirstein and Farinas, 2002). Moreover, the in vivo and in vitro functional properties of such nociceptive neurons strongly depend on the presence of neurotrophins, particularly NGF Winter et al., 1988, Crowley et al., 1994, Winston et al., 2001.

Recently, NGF was suggested to exert an acute effect on nociceptive sensory neurons in addition to its trophic effect (Shu and Mendell, 1999a). It was postulated that NGF, either indirectly (via the degranulation of mast cells) Mazurek et al., 1986, Shu and Mendell, 1999a or directly (via binding to its tyrosine kinase receptors which are expressed in such neurons) (McMahon et al., 1994) was able to sensitize the VR1-mediated cellular responses, e.g., increase the sensitivity of VR1 to capsaicin Shu and Mendell, 1999b, Shu et al., 1999, Chuang et al., 2001. Levels of NGF are increased in inflamed tissues (Weskamp and Otten, 1987), and this effect may contribute to the role of neurotrophin(s) in inflammatory hyperalgesia Shu and Mendell, 1999a, Chuang et al., 2001, Di Marzo et al., 2002.

Since only a few papers have explored the direct action of neurotrophins on VR1-expressing (native or recombinant) cells, in this study we sought to describe the effect of various neurotrophins on VR1-mediated calcium responses to capsaicin in a heterologous expression system stably expressing the rat VR1.

Section snippets

Establishment of VR1/C6 cells stably expressing the rat VR1

A previously constructed metallothionein promoter-based pεMTH vector (2–4 μg cDNA) (Oláh et al., 1994) encoding the cDNA of the rat VR1 was used Oláh et al., 2001, Lázár et al., 2003 to transfect C6 rat glioma cells growing in 6-well tissue culture dishes (VR1/C6 cells) by the LipofectAMINE (Invitrogen, Paisley, UK) transfection reagent, using the protocol suggested by the manufacturer. Cells were then selected in Dulbecco's Modified Eagle's Medium (DMEM) (Sigma, St. Louis, MO, USA)

Results and discussion

First, we examined neurotrophin receptor expression in the rVR1/C6 glioma cells. As was revealed by Western blotting (Fig. 1B), all of the high-affinity neurotrophin receptors, namely the trkA, B, C, as well as the low-affinity receptor p75 are expressed in the cells. Immunocytochemistry experiments corroborated these observations (Fig. 1C). These findings confirm previous data (using mainly RT-PCR) that all members of the neurotrophin receptor family are expressed in C6 cells Hutton et al.,

Conclusions

In summary we can conclude that various neurotrophins are all capable of sensitizing the recombinant VR1-mediated intracellular calcium response to the action of capsaicin. Furthermore, we have also shown that the sensitization process may occur via either trk receptors (i.e., A, B, or C) suggesting a common intracellular mechanisms related to the activation of the trk receptor. Since all trk receptors may alter the activity of the phospholipase Cγ (PLCγ) Kaplan and Miller, 1997, Huang and

Acknowledgements

The authors are indebted to Ms. Ibolya Varga for helpful technical assistance. This work was supported by Hungarian research grants: OTKA F035036, NKFP 00088/2001, OMFB 00200/2002. Tamás Bı́ró is a recipient of the György Békésy Postdoctoral Scholarship of the Hungarian Ministry of Education.

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Sensitization of recombinant vanilloid receptor-1 by various neurotrophic factors

Abstract

Introduction

Section snippets

Establishment of VR1/C6 cells stably expressing the rat VR1

Results and discussion

Conclusions

Acknowledgements

Neuron

Neuroscience Letters

Neuron

Brain Research and Molecular Brain Research

Cell

Current Opinion in Neurobiology

Current Opinion in Cell Biology

Neuroscience Letters

FEBS Letters

Neuron

Analytical Biochemistry

Journal of Biological Chemistry

Neuroscience Letters

Pain

Cell

Pain

Neuron

Distinct sub-populations in HaCaT cells as revealed by the characteristics of intracellular calcium release induced by phosphoinositide-coupled agonists

Archives of Dermatological Research

A simple immunfluorescence technique for simultaneous visualization of mast cells and nerve fibers reveals selectivity and hair cycle — dependent changes in mast cell — nerve fiber contacts in murine skin

Archives in Dermatological Research

Brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4 act as “epitheliotrophins” in murine skin

Laboratory Investigation