Essential role of mitochondrial permeability transition in vanilloid receptor 1-dependent cell death of sensory neurons

doi:10.1016/S1044-7431(03)00121-0

Molecular and Cellular Neuroscience

Volume 24, Issue 1, September 2003, Pages 57-68

https://doi.org/10.1016/S1044-7431(03)00121-0 Get rights and content

Abstract

Capsaicin causes pain by activating VR1, a cloned capsaicin receptor, in sensory neurons. After the initial excitatory responses, capsaicin produces prolonged analgesia, presumably because of the neurotoxic effect that leads to the death of sensory neurons. However, the mechanism underlying capsaicin-induced cell death of sensory neurons is not known. Here we report that capsaicin induces cell death in VR1-expressing sensory neurons and VR1-transfected human embryonic kidney cells. Cell death of sensory neurons induced by capsaicin is accompanied by DNA fragmentation, TUNEL staining, and shrinkage of the nucleus in a caspase-dependent manner, indicating the apoptotic nature of the cell death. Mitochondrial permeability transition is likely to be a major component of capsaicin-induced cell death because bonkrekic acid and cyclosporin A, inhibitors of mitochondrial permeability transition, block this cell death. These results imply that capsaicin induces mitochondrial dysfunction in VR1-expressing cells, leading to apoptotic cell death, which is a well-known neurotoxic effect of capsaicin.

Introduction

Capsaicin (CAP), the pungent ingredient in hot peppers, has been widely used for the study of pain or the study of properties of a subpopulation of primary afferent nerves (Wood, 1993). CAP causes immediate and severe pain by exciting sensory neurons. The excitation of sensory neurons by CAP is mediated by the activation of CAP receptors, which are nonselective and ligand-gated channels present in small sensory neurons Oh et al 1996, Caterina et al 1997. Oddly enough, after initial excitation, sensory neurons do not respond to CAP on application of repeated or high doses of CAP Cholewinski et al 1993, Szallasi and Blumberg 1999. Thus, CAP and its analogs produce a prolonged analgesic effect that draws clinical attention as potentially useful analgesics (Holzer, 1991). The unresponsiveness of sensory neurons to CAP or other noxious stimuli involves two processes (Holzer, 1991). The immediate effect is induced by the desensitization of the CAP receptor to further CAP, which is believed to require dephosphorylation of the CAP receptors (Docherty et al., 1996). However, the persistent unresponsiveness of sensory nerves after high doses of CAP is believed to be associated with morphological, neurochemical, and histochemical changes in sensory neurons (Holzer, 1991). High-dose application of CAP degenerates a large fraction of unmyelinated axons and the cell bodies of sensory neurons, which leads to deficits in nociception and other physiological reflexes McDougal et al 1985, Arvidsson and Ygge 1986, Hylden et al 1992. Because of its neurotoxic effects, CAP is often called a neurotoxin and is used as a chemical tool for dissecting small afferent fibers from mixed nerves Holzer 1991, Szallasi and Blumberg 1999.

Although the neurotoxic effects of CAP on sensory neurons are well documented, the detailed signals that cause its neurotoxic effects are not known. After CAP application, sensory neurons were found to exhibit a large increase in cytosolic Ca²⁺ via influx through VR1, a cloned CAP receptor Wood et al 1988, Bleakman et al 1990, Caterina et al 1997, Savidge et al 2001. Consistent with this idea, VR1 is known to have a high permeability to Ca²⁺ (Caterina et al., 1997), and in addition to the increase in cytosolic Ca²⁺, CAP application also induces the accumulation of Ca²⁺ in the mitochondria of dorsal root ganglion neurons (Dedov and Roufogalis, 2000). It is now known that mitochondral Ca²⁺ overload initiates apoptotic cascades and results in cytotoxicity. The apoptotic cascades include an increase in reactive oxygen species (ROS) or mitochondrial dysfunction, such as mitochondrial permeability transition (MPT) induced by opening permeability transition pores Green and Reed 1999, Kruman and Mattson 1999, Duchen 2000.

Even though CAP-induced apoptotic cell death in cancer cells has been well established (Surh, 2002), CAP-induced apoptosis in sensory neurons is elusive. Because neurotoxins, such as glutamate, cause cell death accompanied by elevation in intracellular and mitochondrial Ca²⁺ (Duchen, 2000), it is likely that the excessive Ca²⁺ overload in the mitochondria induced by CAP disrupts mitochondrial function in sensory neurons (Szoke et al., 2002). Therefore, we hypothesized that CAP induces cell death of sensory neurons via alterations in mitochondrial function.

Section snippets

CAP-induced Ca²⁺ influx

To characterize VR1-dependent cell death, human embryonic kidney (HEK) 293T cells were transfected with VR1 (Hwang et al., 2000). Western blot with polyclonal antibody raised against the N terminus of rat VR1 revealed doublet bands with an apparent molecular weight of ∼95 kDa in cells transfected with pcDNA3.1-VR1, but not in the mock-transfected cells (Fig. 1A). The VR1-transfected cells exhibited VR1 immunofluorescence localized in the plasma membrane and in the cytoplasm (Fig. 1B) (Olah et

Discussion

The primary action of CAP is to excite sensory neurons via the opening of the ligand-gated channel, VR1. However, a prolonged or high-dose application of CAP produces loss of a subpopulation of small sensory neurons, which leads to long-lasting analgesia. Morphological and biochemical changes occurring after prolonged CAP application account for the loss of sensory neurons Palermo et al 1981, Hammond and Ruda 1991, Kwan et al 1999. Due to the neurotoxic action on small sensory nerves, CAP is

Cell culture and transfection

HEK 293T cells were cultured in minimum essential medium supplemented with nonessential amino acids, 10% fetal bovine serum, sodium bicarbonate (2.4 g/liter), 15 mM HEPES, and 1 mM sodium pyruvate (pH 7.0). Cells were routinely maintained at 37°C in a humid atmosphere with 5% CO₂ and fed with culture medium every 2–3 days. For transfection, cells were harvested after a brief trypsin digestion and seeded onto 24-well culture plates (Nunc, Rochester, NY, USA) or 8-well chambered coverglasses

Acknowledgements

This work was supported by Creative Research Initiatives of the Ministry of Science and Technology of Korea and in part by a BK21 program.

References (58)

J. Arvidsson et al.
A quantitative study of the effects of neonatal capsaicin treatment and of subsequent peripheral nerve transection in the adult rat
Brain Res.
(1986)
M.R. Castillo et al.
Ca²⁺-dependent mechanisms of cell injury in cultured cortical neurons
Neuroscience
(1998)
A. Cholewinski et al.
The role of calcium in capsaicin-induced desensitization in rat cultured dorsal root ganglion neurons
Neuroscience
(1993)
L. Genestier et al.
Caspase-dependent ceramide production in Fas- and HLA class I-mediated peripheral T cell apoptosis
J. Biol. Chem.
(1998)
M.G. Grutter
Caspaseskey players in programmed cell death
Curr. Opin. Struct. Biol.
(2000)
B.J. Gwag et al.
Calcium ionophores can induce either apoptosis or necrosis in cultured cortical neurons
Neuroscience
(1999)
D. Jiang et al.
Zn²⁺ induces permeability transition pore opening and release of pro-apoptotic peptide from neuronal mitochondria
J. Biol. Chem.
(2001)
J. Jung et al.
Agonist recognition sites in the cytosolic tails of vanilloid receptor 1
J. Biol. Chem.
(2002)
J.Y. Koh et al.
Staurosporine-induced neuronal apoptosis
Exp. Neurol.
(1995)
J.J. Lemasters et al.
The mitochondrial permeability transition in cell deatha common mechanism in necrosis, apoptosis and autophagy
Biochim. Biophys. Acta
(1998)

M. Maccarrone et al.

Anandamide induces apoptosis in human cells via vanilloid receptors

J. Biol. Chem.

(2000)

D.B. McDougal et al.

Effect of capsaicin upon fluoride sensitive acid phosphatases in selected ganglia and spinal cord and upon neuronal size and number in dorsal root ganglion

Brain Res.

(1985)

P. Nicotera et al.

The role of calcium in apoptosis

Cell Calcium

(1998)

P. Nicotera et al.

Nuclear calcium transport and the role of calcium in apoptosis

Cell Calcium

(1994)

Z. Olah et al.

Ligand-induced dynamic membrane changes and cell deletion conferred by vanilloid receptor 1

J. Biol. Chem.

(2001)

N.N. Palermo et al.

Selective neurotoxic action of capsaicin on glomerular C-type terminals in rat substantia gelatinosa

Brain Res.

(1981)

J.R. Savidge et al.

Comparison of intracellular calcium signals evoked by heat and capsaicin in cultured rat dorsal root ganglion neurons and in a cell line expressing the rat vanilloid receptor, VR1

Neuroscience

(2001)

R.C. Scaduto et al.

Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives

Biophys. J.

(1999)

X.M. Sun et al.

Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis

J. Biol. Chem.

(1999)

S.A. Susin et al.

Mitochondria as regulators of apoptosisdoubt no more

Biochim. Biophys. Acta.

(1998)

E. Szoke et al.

Neonatal capsaicin treatment results in prolonged mitochondrial damage and delayed cell death of B cells in the rat trigeminal ganglia

Neuroscience

(2002)

P. Bernardi

Mitochondrial transport of cationschannels, exchangers, and permeability transition

Physiol. Rev.

(1999)

V.P. Bindokas et al.

Superoxide production in rat hippocampal neuronsselective imaging with hydroethidine

J. Neurosci.

(1996)

D. Bleakman et al.

The effect of capsaicin on voltage-gated calcium currents and calcium signals in cultured dorsal root ganglion cells

Br. J. Pharmacol.

(1990)

M.J. Caterina et al.

The capsaicin receptora heat-activated ion channel in the pain pathway

Nature

(1997)

V.N. Dedov et al.

Mitochondrial calcium accumulation following activation of vanilloid (VR1) receptors by capsaicin in dorsal root ganglion neurons

Neuroscience

(2000)

R.J. Docherty et al.

Inhibition of calcineurin inhibits the desensitization of capsaicin-evoked currents in cultured dorsal root ganglion neurons from adult rats

Pflugers Arch.

(1996)

M.R. Duchen

Mitochondria and calciumfrom cell signaling to cell death

J. Physiol.

(2000)

J.A. Dykens

Isolated cerebral and cerebellar mitochondria produce free radicals when exposed to elevated Ca²⁺ and Na⁺implications for neurodegeneration

J. Neurochem.

(1994)

Cited by (64)

Fight fire with fire: Neurobiology of capsaicin-induced analgesia for chronic pain
2021, Pharmacology and Therapeutics
Capsaicin, the pungent ingredient in chili peppers, produces intense burning pain in humans. Capsaicin selectively activates the transient receptor potential vanilloid 1 (TRPV1), which is enriched in nociceptive primary afferents, and underpins the mechanism for capsaicin-induced burning pain. Paradoxically, capsaicin has long been used as an analgesic. The development of topical patches and injectable formulations containing capsaicin has led to application in clinical settings to treat chronic pain conditions, such as neuropathic pain and the potential to treat osteoarthritis. More detailed determination of the neurobiological mechanisms of capsaicin-induced analgesia should provide the logical rationale for capsaicin therapy and help to overcome the treatment's limitations, which include individual differences in treatment outcome and procedural discomfort. Low concentrations of capsaicin induce short-term defunctionalization of nociceptor terminals. This phenomenon is reversible within hours and, hence, likely does not account for the clinical benefit. By contrast, high concentrations of capsaicin lead to long-term defunctionalization mediated by the ablation of TRPV1-expressing afferent terminals, resulting in long-lasting analgesia persisting for several months. Recent studies have shown that capsaicin-induced Ca²⁺/calpain-mediated ablation of axonal terminals is necessary to produce long-lasting analgesia in a mouse model of neuropathic pain. In combination with calpain, axonal mitochondrial dysfunction and microtubule disorganization may also contribute to the longer-term effects of capsaicin. The analgesic effects subside over time in association with the regeneration of the ablated afferent terminals. Further determination of the neurobiological mechanisms of capsaicin-induced analgesia should lead to more efficacious non-opioidergic analgesic options with fewer adverse side effects.
Activation of the capsaicin-receptor TRPV1 by the acetaminophen metabolite N-arachidonoylaminophenol results in cytotoxicity
2018, Life Sciences
The anandamide reuptake inhibitor N-arachidonoylaminophenol (AM404) and the reactive substance N-acetyl-p-benzoquinone imine (NAPQI) are both metabolites of acetaminophen and may contribute to acetaminophen-induced analgesia by acting at TRPV1 expressed in the peripheral or central nervous system. While NAPQI slowly sensitizes and activates TRPV1 by interacting with distinct intracellular cysteine residues, detailed properties of AM404 as an agonist of TRPV1 have not yet been reported on. We explored the effects of AM404 on recombinant human TRPV1 and in rodent dorsal root ganglion (DRG) neurons.
HEK 293 cells expressing different isoforms of recombinant TRPV1 and rodent DRG neurons were employed for patch clamp and calcium imaging experiments. Cytotoxicity was assessed by propidium iodide and Annexin V staining on TRPV1-HEK 293 cells and with trypan blue staining on DRG neurons.
AM404 activates hTRPV1 at concentrations > 1 μM and in a concentration-dependent manner. AM404 also potentiates TRPV1-mediated currents evoked by heat and anandamide. Moreover, AM404-evoked currents are potentiated by NAPQI. While the partly capsaicin-insensitive rabbit (o) TRPV1 fails to respond to AM404, AM404-sensitivity is restored by insertion of the capsaicin binding-domain of rat TRPV1 into oTRPV1. In DRG neurons, AM404-evoked calcium influx as well as cell death is mediated by TRPV1.
AM404 gates TRPV1 by interacting with the vanilloid-binding site, and TRPV1 is the main receptor for AM404 in DRG neurons. While direct activation of TRPV1 requires high concentrations of AM404, it is possible that synergistic effects of AM404 with further TRPV1-agonists may occur at clinically relevant concentrations.
TRPs and Ca<sup>2+</sup> in cell death and survival
2018, Cell Calcium
Transient Receptor Potential (TRP) family mediate the influx of monovalent and/or divalent cations into cells in response to environmental stimuli. Pharmacological or genetic manipulations of TRP channels demonstrate that TRP channels influence cell death rates, prolonging or shortening of cell survival. Due to their diverse cellular localization, TRP channels mediated Ca²⁺ influx generates distinct intracellular Ca²⁺ signals that regulate downstream pathways converging to apoptosis or survival. In this review, we summarize the accumulated knowledge focused on how TRP channel regulate cell fate and may affect different pathologies including cardiovascular, neurological, metabolic or neoplastic disorders.
Differential cytotoxicity and intracellular calcium-signalling following activation of the calcium-permeable ion channels TRPV1 and TRPA1
2017, Cell Calcium
Several members of the transient receptor channel (TRP) family can mediate a calcium-dependent cytotoxicity. In sensory neurons, vanilloids like capsaicin induce neurotoxicity by activating TRPV1. The closely related ion channel TRPA1 is also activated by irritants, but it is unclear if and how TRPA1 mediates cell death. In the present study we explored cytotoxicity and intracellular calcium signalling resulting from activation of TRPV1 and TRPA1, either heterologously expressed in HEK 293 cells or in native mouse dorsal root ganglion (DRG) neurons. While activation of TRPV1 by the vanilloids capsaicin, resiniferatoxin and anandamide results in calcium-dependent cell death, activation by protons and the oxidant chloramine-T failed to reduce cell viability. The TRPA1-agonists acrolein, carvacrol and capsazepine all induced cytotoxicity, but this effect is independent of TRPA1. Activation of both TRPA1 and TRPV1 triggers a strong influx of external calcium, but also a strong calcium-release from intracellular stores most likely including the endoplasmic reticulum (ER). Activation of TRPV1, but not TRPA1 also results in a strong increase of mitochondrial calcium both in HEK 293 cells and mouse DRG neurons. Our data demonstrate that activation of TRPV1, but not TRPA1 mediates a calcium-dependent cell death. While both receptors mediate a release of calcium from intracellular stores, only activation of TRPV1 seems to mediate a robust and probably lethal increase in mitochondrial calcium.
Ca<sup>2+</sup> and calpain mediate capsaicin-induced ablation of axonal terminals expressing transient receptor potential vanilloid 1
2017, Journal of Biological Chemistry
Capsaicin is an ingredient in spicy peppers that produces burning pain by activating transient receptor potential vanilloid 1 (TRPV1), a Ca²⁺-permeable ion channel in nociceptors. Capsaicin has also been used as an analgesic, and its topical administration is approved for the treatment of certain pain conditions. The mechanisms underlying capsaicin-induced analgesia likely involve reversible ablation of nociceptor terminals. However, the mechanisms underlying these effects are not well understood. To visualize TRPV1-lineage axons, a genetically engineered mouse model was used in which a fluorophore is expressed under the TRPV1 promoter. Using a combination of these TRPV1-lineage reporter mice and primary afferent cultures, we monitored capsaicin-induced effects on afferent terminals in real time. We found that Ca²⁺ influx through TRPV1 is necessary for capsaicin-induced ablation of nociceptive terminals. Although capsaicin-induced mitochondrial Ca²⁺ uptake was TRPV1-dependent, dissipation of the mitochondrial membrane potential, inhibition of the mitochondrial transition permeability pore, and scavengers of reactive oxygen species did not attenuate capsaicin-induced ablation. In contrast, MDL28170, an inhibitor of the Ca²⁺-dependent protease calpain, diminished ablation. Furthermore, overexpression of calpastatin, an endogenous inhibitor of calpain, or knockdown of calpain 2 also decreased ablation. Quantitative assessment of TRPV1-lineage afferents in the epidermis of the hind paws of the reporter mice showed that EGTA and MDL28170 diminished capsaicin-induced ablation. Moreover, MDL28170 prevented capsaicin-induced thermal hypoalgesia. These results suggest that TRPV1/Ca²⁺/calpain-dependent signaling plays a dominant role in capsaicin-induced ablation of nociceptive terminals and further our understanding of the molecular mechanisms underlying the effects of capsaicin on nociceptors.
Crosstalk between calcium and reactive oxygen species signaling in cancer
2017, Cell Calcium
The interplay between Ca²⁺ and reactive oxygen species (ROS) signaling pathways is well established, with reciprocal regulation occurring at a number of subcellular locations. Many Ca²⁺ channels at the cell surface and intracellular organelles, including the endoplasmic reticulum and mitochondria are regulated by redox modifications. In turn, Ca²⁺ signaling can influence the cellular generation of ROS, from sources such as NADPH oxidases and mitochondria. This relationship has been explored in great depth during the process of apoptosis, where surges of Ca²⁺ and ROS are important mediators of cell death. More recently, coordinated and localized Ca²⁺ and ROS transients appear to play a major role in a vast variety of pro-survival signaling pathways that may be crucial for both physiological and pathophysiological functions. While much work is required to firmly establish this Ca²⁺-ROS relationship in cancer, existing evidence from other disease models suggests this crosstalk is likely of significant importance in tumorigenesis. In this review, we describe the regulation of Ca²⁺ channels and transporters by oxidants and discuss the potential consequences of the ROS-Ca²⁺ interplay in tumor cells.

View all citing articles on Scopus

¹: Present address: Division of Biotechnology, Kangwon National University, Chuncheon, Kangwon-do 200-701, Korea.

View full text

Regular articleEssential role of mitochondrial permeability transition in vanilloid receptor 1-dependent cell death of sensory neurons

Abstract

Introduction

Section snippets

CAP-induced Ca2+ influx

Discussion

Cell culture and transfection

Acknowledgements

Brain Res.

Neuroscience

Neuroscience

J. Biol. Chem.

Curr. Opin. Struct. Biol.

Neuroscience

J. Biol. Chem.

J. Biol. Chem.

Exp. Neurol.

Biochim. Biophys. Acta

J. Biol. Chem.

Brain Res.

Cell Calcium

Cell Calcium

J. Biol. Chem.

Brain Res.

Neuroscience

Biophys. J.

J. Biol. Chem.

Biochim. Biophys. Acta.

Neuroscience

Mitochondrial transport of cationschannels, exchangers, and permeability transition

Physiol. Rev.

Superoxide production in rat hippocampal neuronsselective imaging with hydroethidine

J. Neurosci.

The effect of capsaicin on voltage-gated calcium currents and calcium signals in cultured dorsal root ganglion cells

Br. J. Pharmacol.

The capsaicin receptora heat-activated ion channel in the pain pathway

Nature

Mitochondrial calcium accumulation following activation of vanilloid (VR1) receptors by capsaicin in dorsal root ganglion neurons

Neuroscience

Inhibition of calcineurin inhibits the desensitization of capsaicin-evoked currents in cultured dorsal root ganglion neurons from adult rats

Pflugers Arch.

Mitochondria and calciumfrom cell signaling to cell death

J. Physiol.

Isolated cerebral and cerebellar mitochondria produce free radicals when exposed to elevated Ca2+ and Na+implications for neurodegeneration

J. Neurochem.

Regular article
Essential role of mitochondrial permeability transition in vanilloid receptor 1-dependent cell death of sensory neurons

CAP-induced Ca²⁺ influx

Isolated cerebral and cerebellar mitochondria produce free radicals when exposed to elevated Ca²⁺ and Na⁺implications for neurodegeneration