From the Neuronal Gene Expression Unit, Pain and
Neurosensory Mechanisms Branch, NIDCR, the
Laboratory of
Cellular Carcinogenesis and Tumor Promotion, NCI, the ** Neuronal
Excitability Section, Epilepsy Branch, NINDS, and the
Laboratory of Developmental Neurobiology,
NICHD, National Institutes of Health, Bethesda, Maryland 20892
Received for publication, September 13, 2000, and in revised form, December 11, 2000
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ABSTRACT |
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The real time dynamics of vanilloid-induced
cytotoxicity and the specific deletion of nociceptive neurons
expressing the wild-type vanilloid receptor (VR1) were investigated.
VR1 was C-terminally tagged with either the 27-kDa enhanced green
fluorescent protein (eGFP) or a 12-amino acid Capsaicin (CAP),1 a well
characterized membrane-permeable vanilloid agonist, has potent
stimulatory actions on nociceptive neurons and causes loss of
unmyelinated "C"-type sensory afferents when administered to
newborn animals (1, 2). Similarly, resiniferatoxin (RTX), an
ultrapotent capsaicin analogue, can deplete
[3H]RTX-binding sites from the brain stem, the sensory
ganglia (dorsal root and trigeminal), and the spinal cord with a
mechanism that has not been fully elucidated (3, 4). Both systemic and epidural administration of RTX to adult rats produce analgesia to
subsequent noxious thermal stimulation (5). Depending on concentration,
duration of exposure, and route of administration, vanilloid ligands
may either desensitize the nociceptive primary afferent nerve ending or
completely delete the neuron itself (6-8). Previous studies
investigating the processes of desensitization and cell loss mainly
examined whole animals or primary cultures of dorsal root ganglia,
where nociceptive neuronal cell bodies are located. The cloning of the
first isotype of the vanilloid receptor (VR1) provides a means to
investigate more fully the molecular and cell biological mechanisms of
pain signal transduction and the processes underlying susceptibility of
VR1-expressing cells to impairment upon exposure to vanilloid agonists
(9). The VR1 has been characterized as a Ca2+ ionophore (9,
10); thus, it also provides a very specific molecule with which to
investigate the role of ligand-activated transmembrane calcium fluxes
in cellular toxicity.
The VR1 exhibits homology to Ca2+
store-dependent TRP channels. In contrast to TRPs, which
regulate intracellular calcium stores, VR1 confers two pivotal sensory
functions. VR1 transduces chemical (vanilloids and pH) and physical
(heat) stimuli at the molecular level, generating action potentials in
nociceptive nerve endings, and ultimately leading to sensations of
heat, thermal pain, and inflammatory pain (10, 11). Loss of moderately
noxious heat and vanilloid-stimulated sensory functions has been
verified in animals lacking VR1 (11, 12). VR1 mRNA is expressed
with remarkable cell and tissue specificity in the DRG.
Immunocytochemistry demonstrates VR1 throughout the peripheral endings,
cell body, and presynaptic terminals of small size dorsal root ganglion
(DRG) neurons (10, 13). These sites coincide well with regional
localization of [3H]RTX binding (3, 4). The ability of
CAP and RTX to displace [3H]RTX binding in the spinal
cord and DRG as well as similarities in [3H]RTX binding
studies with recombinantly expressed VR1 suggest that both vanilloids
act on the same receptors (14).
Previous studies with vanilloids have shown that vanilloid
administration can lead to cell type-specific membrane damage. Long
term exposure to vanilloids causes profound ultrastructural changes in
DRG neurons (15, 16). VR1-specific antibodies have been used in
intracellular localization by light and electron microscopy, but the
functional relationship of these morphological observations to the
observed cell damage has not been explored (10, 13). Immunostaining of
fixed cells often limits detailed visual observation of rapid
intracellular processes. In the present paper, we used fluorescence
confocal microscopy and real time imaging of enhanced green fluorescent
protein (eGFP)-tagged VR1 (VR1eGFP) to examine simultaneously both the
morphological and functional molecular processes underlying the
immediate effects of vanilloid exposure on the perikarya of cells and
neurons with high temporal and spatial resolution. Biochemical
functions in the plasma membrane (membrane potential changes, calcium
uptake, and [3H]RTX binding) similar to wild-type VR1
were readily observed with the chimeric VR1eGFP. In addition, another
active pool of VR1 was located at the endoplasmic reticulum (ER), which
also reacted within seconds to vanilloid treatment.
Exposure of VR1-expressing cells to vanilloids produced a rapidly
evolving cytotoxicity. This commenced with a rise in
[Ca2+]i, which quickly surpassed the
Ca2+ tolerance or sequestration capacity of the
mitochondria. Subsequently, nuclear envelope shrinkage and blebbing
occurred, followed by cell death. The effect of elevated
[Ca2+]i on vital organelles suggests that
targeted administration of vanilloid agents to the cell body can
rapidly compromise and then eliminate (within hours) VR1-expressing
nociceptive neurons. In fact, immunoblot analysis of protein extracts
from primary DRG cultures showed that treatment with any of several
vanilloid agonists eliminated cells expressing the VR1.
RT-PCR Cloning and Epitope Tagging--
To obtain VR1-specific
mRNA, 100 DRGs were rapidly removed from 12 adult Harlan
Sprague-Dawley rats. Total RNA was isolated with the TRI REAGENT
(Molecular Research Center Inc., Cincinnati, OH). A fragment,
comprising the sequence between the XbaI and AflIII sites of rat VR1, was amplified first by the Access
RT-PCR system (Promega) and then cloned into the BlueScript vector
(Stratagene). The missing 5'-sequence was added likewise with the
SacI and XbaI sites. At the 5' ends of the N- and
C-terminal fragments, the SacI and AflIII sites
(underlined) were incorporated with forward primers
AGATCTCGAGCTCAAATGGAACAACGGGCTAGCTTAGACTC and
CTGTATTCCACATGTCTGGAGCTGTTCAAGTTC, respectively. As reverse
primers ACTGAGTCCCGGGCGCTGATGTCTGCAGGCT and
CACACAGTCGACTTTCTCCCCTGGGACCATGGAATCCTT were used, in which the XbaI and SalI sites were incorporated,
respectively. The SacI-AflIII and the
RT-PCR- generated AflIII-SalI fragments were
triple-ligated into a SacI and SalI cut pEGFP-N3
vector (CLONTECH). The immediate early promoter of
the cytomegalovirus in the pEGFP-N3 vector was employed to produce the
full-length VR1 with the eGFP tag. Rat VR1 with the short, 12- amino
acid Electrophysiology--
For patch clamp studies,
VR1eGFP-expressing COS7 and HEK293 cells were voltage-clamped in Krebs
buffer containing (in mM) NaCl (124), KCl (4.9),
KH2PO4 (1.2), MgSO4 (2.4),
CaCl2 (2.5), NaHCO3 (25.6), and glucose (10),
using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA).
Recordings were carried out with patch electrodes (2-10 M Determination of Ca2+ Uptake--
Cells were
transfected at 80% confluence in 75-cm2 T-flasks with 20 µg of VR1eGFP or VR1 [3H]RTX Binding--
48 h after transfection in
75-cm2 T-flasks, cells were detached from the plastic
surface by serum-free DMEM containing 1 mM EDTA and then
washed and resuspended in 10 mM HEPES (pH 7.4) buffer, containing (in mM) KCl (5), NaCl (5.8), MgCl2
(2), CaCl2 (0.75), glucose (12), and sucrose (137). Intact
cells were incubated (105/well) in a filtration plate with
200 pM [3H]RTX for 60 min at 37 °C and
then processed as described earlier (5). Data were analyzed by computer
fit to the Hill equation as noted previously (18, 19).
Fluorescent Confocal Microscopy--
COS7, NIH 3T3, and HEK293
cells were seeded on 25-mm coverslips and transfected with 1 µg each
of the plasmid constructs, cultured for 24 h post-transfection at
35 °C, then mounted in a 1-ml chamber and examined with a MRC-1024
Bio-Rad confocal microscope. To study the two- and three-dimensional
distribution of fluorescent chimeric proteins, each x-y
plane was scanned over 1 s and at 0.2-µm increments in
the z axis mode. To label different subcellular compartments
of live cells fluorescently, the ER marker eGFP-KDEL (CLONTECH) was transiently transfected in COS7 and
NIH 3T3 cells. To label mitochondria, MitoTracker (Molecular Probes)
dye was incubated for 30 min at a 250 nM concentration; the
cells were then washed with Hanks' balanced salt solution supplemented
with 1 mM CaCl2 and 0.8 mM
MgCl2, buffered with 15 mM HEPES (pH 7.4) (HBSSH).
[Ca2+]i Microfluorometry--
For
determination of [Ca2+]i, cells were cultured in
glass bottom dishes (MatTek Corp., Ashland, MA) and then transfected with 2 µg of VR1eGFP plasmid. After 24 h in culture, cells were loaded with Fura 2/AM (Molecular Probes, Eugene, OR) for 30 min at
37 °C. Single cells expressing the VR1eGFP construct were identified by eGFP fluorescence and were selected based on visual inspection of
fluorescence intensity. In all experiments, cells exhibiting intermediate fluorescence in comparison to other cells in the same dish
were picked for analysis. To determine the
[Ca2+]i, the excitation ratio of Fura-2 at 340 and 380 nm was recorded photometrically in Krebs' buffer at a 10-Hz
sampling rate and integrated over 0.5 s, as described previously
(20). [Ca2+]i was calculated using the ratio
based equation (21).
Ratiometric Imaging of [Ca2+]i Employing
Confocal Microscopy--
Cells cultured on
poly-D-lysine-coated coverslips were pre-loaded with 5 µM Indo-1 AM dye. After incubation for 30 min at 34 °C, the cells were washed three times in HBSSH to remove excess dye and examined under the confocal microscope. To record in "zero" extracellular Ca2+ cells were washed four times (5 min
each) in HBSSH containing no CaCl2 and 1 mM
EGTA and imaged in the same medium. Groups of cells expressing VR1eGFP
or small size neurons were selected under the microscope. To quantitate
the fluorescence ratio, perikarya of the cells were marked with the
graphic tools of the LaserSharp software in the field of a × 40 objective of the Bio-Rad confocal system. Ratiometric imaging was
performed at 10-s intervals with an UV laser, and the ratio of
fluorescence intensity emitted at 405 and 485 was calculated.
DRG Culture--
DRG neuron-enriched cultures were prepared from
embryonic rats (E16). Briefly, embryos were removed from the uterus and
placed in Petri dishes containing Lebowitz medium (Life Technologies, Inc.). The cords were dissected, and the DRGs were stripped off with
the meninges. Cells were digested in 0.125% trypsin at 37 °C for 20 min. For plating, dissociated cells were changed into minimal essential
medium containing 5% horse serum and 50 ng/ml nerve growth factor
(NGF). Cells were seeded on 25-mm glass coverslips or on multiwell
plates. Surfaces were coated with poly-D-lysine and
laminin. DRG cultures were maintained in DMEM containing 20 mM HEPES, 7.5% fetal bovine serum, 7.5% horse serum, 5 mg/ml uridine supplemented with 2 mg/ml FUDR to inhibit cell division
and 50 ng/ml NGF to promote neuronal survival and differentiation.
Cultures were selected in this medium for 1 week, at which point well
differentiated neurons and nondividing cells dominated the population.
Primary DRG cultures in this stage were used in confocal microscopy.
Western Blotting--
Total protein extracts were prepared in
denaturing SDS buffer and analyzed for immunoreactivity by Western
blotting similar to that described by us previously (17). VR1-specific
antibody was raised in rabbits employing the N-terminal
MEQRASLDSEESESPPQE peptide of rat VR1 conjugated to keyhole limpet
hemocyanin as immunogen. Immune sera were affinity purified against the
peptide used for immobilization. The specific antibody fraction eluted from the affinity column was diluted 1000-fold for further
characterization in Western blot and immunocytochemistry experiments.
This antibody fraction recognizes the native rat VR1, as illustrated in
Fig. 11, and the chimeric VR1eGFP and VR1 VR1-epitope. Upon
exposure to resiniferatoxin, VR1eGFP- or VR1
-expressing cells
exhibited pharmacological responses similar to those of cells
expressing the untagged VR1. Within seconds of vanilloid exposure, the
intracellular free calcium ([Ca2+]i) was
elevated in cells expressing VR1. A functional pool of VR1 also was
localized to the endoplasmic reticulum that, in the absence of
extracellular calcium, also was capable of releasing calcium upon
agonist treatment. Confocal imaging disclosed that resiniferatoxin
treatment induced vesiculation of the mitochondria and the endoplasmic
reticulum (~1 min), nuclear membrane disruption (5-10 min), and cell
lysis (1-2 h). Nociceptive primary sensory neurons endogenously
express VR1, and resiniferatoxin treatment induced a sudden increase in
[Ca2+]i and mitochondrial disruption which was
cell-selective, as glia and non-VR1-expressing neurons were unaffected.
Early hallmarks of cytotoxicity were followed by specific deletion of VR1-expressing cells. These data demonstrate that vanilloids disrupt vital organelles within the cell body and, if administered to sensory
ganglia, may be employed to rapidly and selectively delete nociceptive neurons.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tag (KGFSYFGEDLMP) was constructed in a vector, p
MTH,
driven by the metallothionein promoter (17). Briefly, SalI
and MluI restriction endonuclease sites were incorporated into the VR1 PCR fragment amplified using the forward
AGTAGTGTCGACGAACAACGGGCTAGCTTAGACTCA and reverse
TTGTTGACGCGTTTTCTCCCCTGGGACCATGGAATC primers, respectively. After cutting the PCR fragment with these enzymes the
size-separated cDNA insert was ligated in p
MTH at the compatible
XhoI and MluI sites (17). The chimeric constructs
were verified by sequencing and transiently transfected in COS7,
HEK293, and NIH 3T3 cells employing the protocol provided for the
LipofectAMINE reagent (Life Technologies, Inc.). The basal activity of
the p
MTH promoter was used in NIH 3T3 cells to produce VR1
, yet
prevent toxicity from long term, high level expression.
) filled
with 10 mM HEPES buffer (pH 7.4) containing (in
mM) CsCl (120), tetraethylammonium chloride (20),
CaCl2 (1), MgCl2 (2), EGTA (10), ATP (4), and GTP (0.5).
plasmids. After 48 h, 5 × 104 cells were detached from the plastic surface by
serum-free DMEM containing 1 mM EDTA and then washed two
times and resuspended in medium without EDTA. Cell suspensions were
incubated in serum-free DMEM containing 1 µCi/ml
45Ca2+ and ligands as indicated for 15 min at
35 °C in 96-well filtration plates (MultiScreen-DV, Millipore,
Marlborough, MA). Ca2+ uptake was terminated on ice, and
samples were processed and analyzed as described (18).
(data not shown).
Stripping of nitrocellulose blotting filters (Bio-Rad) was carried out
in 200 ml of 50 mM Tris-HCl buffer (pH 7.5) containing 2%
SDS and 0.1
-mercaptoethanol at 65 °C for 1 h. Stripped
blots were reanalyzed for tagged fusion proteins either with the GFP
(CLONTECH) or the
PKC-specific antibodies (Life
Technologies, Inc.), prepared in rabbits. To assess equal loading,
filters were re-probed with a rat cytochrome c-specific
monoclonal antibody (6H2.B4) as suggested by the manufacturer
(Transduction Laboratories). Antibodies for Western blotting were used
at a 1:1000 dilution. To visualize the interaction with the primary
antibodies, enhanced chemiluminescence technology was employed
according to the protocol provided by the manufacturer (New England Biolabs).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and VR1eGFP plasmid constructs were expressed in COS7,
HEK293, and NIH 3T3 cells by transient transfection. Western blot analysis with polyclonal eGFP and
-tag-specific antibodies
demonstrated that the VR1eGFP and VR1
chimeric proteins expressed in
HEK293 and NIH 3T3 cells were of the appropriate sizes, 120 and 93 kDa, respectively (Fig. 1, 1st and
3rd lanes). No tag-related immunoreactivity was found in the
nontransfected host cells (Fig. 1, 2nd and 4th lanes). Repeated Western analyses with GFP- and
-tag-specific antibodies showed no proteolytic cleavage of the VR1
chimeric proteins (data not shown). These transiently transfected
plasmid constructs expressed proteins that exhibited identical
molecular weights after the tag-specific antibodies were removed and
the blots re-probed with the N-terminal specific VR1 antibody (data not
shown).
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Fig. 1.
a, schematic representation of
C-terminally tagged VR1. b, Western blot analysis of the
chimeric proteins. To generate VR1eGFP and VR1 , the stop codon of
the wild-type VR1 was deleted and the C-terminal domain extended with
the cDNA coding for the enhanced green fluorescent protein or the
-tag as described under "Experimental Procedures." The
intracellular ankyrin repeats, the enhanced green fluorescent protein-,
and
-epitope-tagged chimera proteins are abbreviated as A, VR1eGFP,
and VR1
, respectively. b, VR1eGFP and VR1
plasmids
were transfected in HEK293 and NIH 3T3 cells, respectively. After
48 h 35 µg of total protein extract, prepared in SDS sample
buffer, was analyzed for the eGFP (lanes 1 and 2)
and
-tag immunoreactivities (lanes 3 and 4) by
Western blotting, employing eGFP and
-tag specific antibodies,
respectively. Enhanced chemiluminescence was employed to visualize the
interaction with the primary antibodies, as described under
"Experimental Procedures." The molecular weight was calculated from
the mobility of the immunoreactive protein band compared with protein
standards.
To study the electrophysiological properties of C-terminally
eGFP-tagged VR1, plasmid constructs producing VR1eGFP and eGFP as a
control were transiently expressed in HEK293 cells. Green fluorescent
cells of medium fluorescence intensity were voltage-clamped, and the
holding potential was adjusted to 60 mV. Capsaicin (10 µM) induced a large inward current (Fig.
2a). Similar currents were
also evoked by administration of 125 pM RTX to the cells; however, the currents rapidly desensitized to repeated applications (n = 6) (data not shown). Capsaicin was noted to be
less effective at inducing desensitization than RTX; therefore, CAP was
used in experiments that required repeated application of vanilloid ligands (e.g. Fig. 2). As expected for a functional
recombinant, the VR1eGFP-mediated current was attenuated by
coincubation of an antagonist, 10 µM capsazepine (CPZ).
The current versus voltage relationship demonstrated that
the VR1eGFP-mediated current was not particularly voltage-sensitive.
The reversal potential was near 0 mV, suggesting mixed cation
selectivity for the channel (data not shown). Cells transfected with
eGFP did not demonstrate currents when exposed to either CAP or RTX
(Fig. 2b and data not shown). Likewise, nontransfected
HEK293 cells did not demonstrate RTX-evoked currents. Overall, the
electrophysiological properties of the eGFP-tagged VR1 were very
similar to those described for nontagged VR1 (9).
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In accordance with the electrophysiological data, exposure to RTX
induced Ca2+ uptake in VR1eGFP-expressing HEK293 and COS7
cells. This ligand-induced Ca2+ influx (Fig.
3a) further confirms the
presence of VR1eGFP at the plasma membrane. RTX induced
45Ca2+ uptake with an ED50 = 100 ± 50 pM (n = 3) whereas that for
CAP was 0.5 ± 0.15 µM (data not shown). Similar
results were obtained for VR1 tagged with the 12-amino acid -epitope
in place of the eGFP tag. This indicates that a C-terminal tag,
per se, does not significantly change Ca2+
uptake parameters. In addition, RTX-induced
45Ca2+ uptake was completely blocked by 10 µM CPZ in VR1eGFP- (Fig. 3a) and
VR1
expressing cells (not shown).
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The curves in Fig. 3b demonstrate the quantitative
characteristics of [3H]RTX binding to eGFP-tagged and
-tagged VR1 expressed in COS7 cells. Both tagged recombinants
exhibited a high affinity, dose-dependent interaction
(Kd ~ 150 ± 10 pM,
n = 6) and cooperativity among the receptors (Hill
coefficient = 1.5-2). [3H]RTX binding was almost
completely inhibited by coincubation of 10 µM CPZ. No
significant [3H]RTX binding was detected in cells
transfected with the plasmid expressing only eGFP (Fig.
3b).
Confocal fluorescence microscopy was employed to analyze the
intracellular distribution of VR1eGFP. Optical sections taken at the
plane of cell attachment to the glass surface show VR1eGFP fluorescence
in the plasma membrane, where microvilli were labeled (Fig.
4a, VR1 accumulation also is
present at the focal points in this plane, not seen with fluorescent
markers of ER). Optical sections taken through the middle of the cell
nucleus, disclosed VR1eGFP in intracellular structures consistent with
the ER (Fig. 4, b versus d). To
confirm ER localization, cells were transfected with an eGFP that was
C-terminally tagged with the ER retention signal (i.e. the
KDEL motif, Fig. 4b). Visualization of the transiently expressed eGFP-KDEL chimera protein in COS7 cells verified that VR1eGFP
indeed stained the same ER compartment within the cytoplasm and around
the nucleus (Fig. 4, b versus d).
Furthermore, the same ER colocalization result was obtained when
VR1eGFP-expressing cells were costained with the ER-tracker vital dye
(Molecular Probes) (not shown). Fig. 4 illustrates localization of VR1
in COS7 cells but a similar ER localization was seen in HEK293 and NIH
3T3 cells, indicating that it is not a cell type-specific anomaly. ER
localization was observed over a range of transfection efficiencies,
and the distribution between the plasma membrane and ER was similar in
cells expressing VR1eGFP at different levels (not shown). The
proportionality between plasma membrane and ER also was maintained in a
cell line stably expressing relatively lower levels of VR1 from the
noninduced MTH promoter. These data, as well as results from
immunocytochemical staining of fixed DRG neurons, which show a high
density of staining throughout the neuronal cytoplasm (10), suggest the
distribution is not simply due to overexpression. Dual wavelength
imaging studies demonstrated that the intracellular compartment
containing VR1eGFP (green) was distinct from the
filamentous mitochondria (red), which were labeled by the
red MitoTracker dye and are generally much thicker than the ER (Fig.
4f).
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Previously it was noted that addition of ionomycin to cells induces intracellular membrane fragmentation due to permeabilization of the plasma membrane to cations (22). This was verified in baseline control studies using the eGFP-KDEL-expressing plasmid, which showed that ionomycin treatment induced membrane fragmentation of the ER (Fig. 4, b versus c). This fragmentation was identical to what occurred with VR1eGFP-expressing cells upon exposure to 1 nM RTX. In these cells a 20-s exposure induced fragmentation of the ER (Fig. 4, d and e) and, simultaneously, a rounding up of the filamentous mitochondria (Fig. 4, f versus g). Testing of a second vanilloid ligand, CAP (1 µM), also demonstrated membrane fragmentation with similar dynamics as noted with RTX (not shown). With the appropriate sets of fluorescence filters, we observed no mixing between the VR1eGFP vesicles (green) and the mitochondrial membranes (red) (data not shown). In cells expressing only eGFP, the structure of the mitochondria and ER did not change upon exposure to vanilloids, indicating the dependence for these effects on the presence of the VR1 receptor. Thus, within the first few seconds after vanilloid exposure, coincident and structurally similar intracellular organelle remodeling occurs for both the ER and the mitochondria.
The effect of RTX on cytosolic Ca2+ was studied by
microfluorometry in transfected cells loaded with the
Ca2+-monitoring dye, Fura-2 AM. The resting
[Ca2+]i was similar (~50 nM) in
COS7 cells transfected with either VR1eGFP or eGFP plasmids. Addition
of 1 nM RTX induced a rapid (within 10 s) elevation of
[Ca2+]i in VR1eGFP-expressing cells which peaked
at 500 nM at ~1 min (n = 3) and,
consistent with the concurrent ER and mitochondrial damage (Fig. 4,
e and g), did not return to resting levels (Fig. 5a). In the absence of
external Ca2+, the RTX-induced increase in
[Ca2+]i was temporally delayed from 10 s to
~1 min (see Figs. 7 and 8). No increase in
[Ca2+]i was observed in cells expressing only
eGFP (n = 3) (not shown).
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The preceding experiments focused on events occurring within the first
few seconds following exposure to VR1 agonists. To extend the time of
cellular observation, serial 1-s confocal microscopy scans were
performed at 1-min intervals on live VR1eGFP-expressing cells. In these
experiments the early events occurred as described (e.g.
transmembrane Ca2+ flux, intracellular remodeling, within
30 s in 13 out of 15 cells). Within 3 min after RTX
administration, the nuclear membrane was outlined with VR1eGFP
fluorescence, and ER membrane vesicles were observed around the nucleus
(Fig. 5c). Then, progressively growing blebs were noted in
the nuclear membrane (2 out of 10 at 5 min and 9 out of 10 cells at 10 min). In the cell shown, the membrane degradation concluded with
bursting of the plasma membrane at 43 min (Fig. 5c). Other
cells displayed similar nuclear changes and cell disruption within 1-2
h (3 out of 5 within 1 h and 5 out of 5 monitored for 2 h in
one experiment). Lower doses of RTX (0.1 nM) evoked
slower nuclear membrane fragmentation but eventually resulted in cell
lysis (4 out of 4 cells within 3 h). The effects were not due to
the repetitive scanning, since single scans of transfected cells
performed at 30 min after RTX administration also revealed (8 out of 8)
identical effects of RTX on intracellular membrane structures.
Fig. 6 summarizes quantitatively the
fluorescence scanning confocal microscopy data collected in repeated
experiments. ER fragmentation was noted in 16 cells within 6 s of
adding 1 nM RTX; vesiculation was completed within 1 min in
each cell monitored (scans were done every 6 s, n = 30). ER vesiculation directly corresponded to the time course
determined for RTX-induced [Ca2+]i accumulation
(Fig. 5a). The first nuclear bleb appeared 2 min after RTX
treatment (2 out of 30) and then each observed cell (n = 30) showed at least 1 bleb by 10 min. Cell lysis started as early as
10 min after RTX addition (1 out of 30 cells) and was complete within
2 h (30 out of 30 cells).
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To explore potential activity of the ER-localized VR1, COS7 cells
expressing VR1eGFP were treated with 1 nM RTX in the
absence of extracellular Ca2+. VR1eGFP-expressing cells,
pre-washed extensively in 1 mM EGTA, showed vesiculation of
the ER and disruption of mitochondria similar to that determined in the
presence of 1 mM extracellular Ca2+ (Figs. 4
and 5). However, the time required for these changes was much longer,
on the order of minutes rather than seconds. The first obvious signs of
ER fragmentation and round up of mitochondria were noted at ~3 min
after 1 nM RTX addition (5 out of 5). Nontransfected cells
(n = 2) in the same microscopic field showed an intact
mitochondrial structure as determined by MitoTracker fluorescence,
indicating that the vanilloid effects on the intracellular membranes
were specific to those cells expressing VR1eGFP (Fig.
7). If the extracellular Ca2+
concentration was adjusted back to 1 mM in the presence of
1 nM RTX, it triggered an abrupt ER fragmentation (within
1-3 s in 5 out of 5 cells) demonstrating the pivotal function of
Ca2+ in this process (data not shown). These data suggest
that the ER-localized VR1 mobilizes calcium from the ER but that
[Ca2+]i accumulation occurred more slowly or that
the amounts were such that the [Ca2+]i
accumulation was buffered and more time was required to reach toxic
cytoplasmic concentrations.
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The quantitative aspects of calcium release from internal stores are
presented in Fig. 8, which examines both
DRG neurons, and transiently transfected COS7 cells. In recording
medium containing zero extracellular Ca2+, ratio
imaging of VR1eGFP-positive COS7 cells (exhibiting medium or low green
fluorescence intensity, Fig. 8a) or small size DRG cells
(Fig. 8b) demonstrated dramatic increases in the 405/485 ratio after administration of RTX. Control nontransfected COS7 cells
did not show any change in the 405/485 ratio when exposed to RTX. As
noted above, the increase in [Ca2+]i in the zero
extracellular Ca2+ conditions was temporally delayed,
requiring ~1 min. By comparison, cells exposed to RTX in the presence
of 1 mM extracellular Ca2+ exhibit an increase
within 10 s. The ~45-50-s delay also occurred in small size DRG
neurons (Fig. 8b). The RTX-induced ratio increase in cells
expressing VR1eGFP, and small size DRG neurons indicates Ca2+ release from internal stores and
[Ca2+]i accumulation in the cytosol. These
experiments suggest that the intracellular VR1 is functional and that
RTX, a lipid-soluble agonist of VR1, can indeed act as an agonist at
these intracellular sites.
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To verify that the rapid intracellular membrane changes observed in
heterologous cells expressing VR1 also occurred in small-size DRG
neurons, experiments were carried out on primary cultures from DRG
(Fig. 9). Cultures were preincubated with
the MitoTracker dye and then exposed to 1 nM RTX. Small
size neurons were selected and monitored by confocal microscopy
employing 1-s scans every 20 s before and after treatment with
vanilloids. Neurons in this size range were previously determined to be
VR1-positive with an antibody raised against the N-terminal 18 amino
acids of VR1 (data not shown). The MitoTracker dye revealed normal,
slightly elongated mitochondria in untreated small size DRG neurons
(Fig. 9a). Treatment with RTX induced the mitochondria to
fragment within 20 s (Fig. 9b), similar to the kinetics
observed in COS7 cells ectopically expressing VR1eGFP (Fig. 4,
d versus e). RTX at 1 nM
concentration was without effect on mitochondria localized in the glial
cell population (Fig. 9c) or in nearby large neurons within
the field of view (data not shown).
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The above experiments demonstrated that vanilloids rapidly target the
ER and mitochondria at the cell body in small size DRG neurons
expressing VR1, producing effects within seconds. To address the
effects on DRG neurons of longer exposure to RTX, DRG cultures were
subjected to dual wavelength fluorescent imaging. Cultures were loaded
with Indo-1 AM and then incubated in HBSSH containing 1 mM
CaCl2. Propidium iodide (PI) was added to the imaging
chamber just before the cultures were examined with confocal
microscopy. In repeated experiments, specific neurons in the field of
view demonstrated instant increases in [Ca2+]i
(within seconds) upon administration of 25 nM RTX. Neurons
exhibiting the RTX-induced increase in [Ca2+]i
started to accumulate PI in the nucleus ~40 min after addition of
RTX; a time that coincides with loss of plasma membrane integrity in
VR1eGFP-expressing COS7 cells exposed to RTX (Figs. 5 and 6). In the
same microscopic field there were neurons that neither responded to RTX
with an elevation in [Ca2+]i nor accumulated PI
even after 2 h of exposure (Fig. 10). This result is consistent with the
idea that these neurons do not express VR1 and reinforces the cellular
and molecular specificity of vanilloid actions on DRG neurons. Repeated
time course observations with DRG cultures indicated similar dynamics
for DRG neuronal death as described for transiently (see Fig. 6) or
stably transfected NIH 3T3 cells (not shown) expressing recombinant
VR1.
|
Elevated [Ca2+]i can induce cytotoxicity within
minutes to hours in VR1-expressing cells or specific DRG neurons, as demonstrated in previous experiments. This suggests that vanilloid application to the perikarya may be an effective means for specific deletion of nociceptive neurons. To study this phenomenon, 1-week-old DRG cultures from rats were treated with 50 µM olvanil
(OLV, a long chain fatty acid modified synthetic vanilloid) or 25 nM RTX for 48 h. From these cultures, total protein
extracts were prepared in denaturing SDS sample buffer (Fig.
11). Western blot of VR1 protein and
densitometry of films after enhanced chemiluminescence visualization
revealed that olvanil treatment almost completely eliminated (99%
decrease) neurons expressing VR1 from the culture. RTX, employed at a
concentration 2,000-fold less than OLV, eliminated ~80% of the
neurons expressing VR1. Other cell types present in the DRG culture
were not affected after 48 h of treatment with OLV or RTX, as
assessed by re-probing the same Western blots with an antibody
recognizing the common tissue protein cytochrome c (Fig.
11).
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DISCUSSION |
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The present paper investigates the early cellular dynamics of
vanilloid-triggered events through in vivo expression of
fluorescent- and epitope-tagged recombinants of rat VR1. Extension of
the C terminus with the 27-kDa eGFP did not compromise the
electrophysiological function of VR1 upon expression in heterologous
cell systems and tagging with either eGFP or a short 12-amino acid
-epitope did not affect vanilloid-induced ionophore function as
assessed by 45Ca2+ uptake. The ultrapotent
vanilloid, RTX, binds with nanomolar affinity to both VR1eGFP and
VR1
. The binding and 45Ca2+ uptake
parameters are similar to those reported either for the nonchimeric VR1
or for rat DRG primary cultures (3, 4, 14, 23-26). The
electrophysiology of VR1eGFP-expressing cells showed that vanilloids
rapidly induced an inward current flow that was blocked by CPZ, as
described for wild-type VR1 (9). Vanilloid-induced 45Ca2+ uptake and [3H]RTX binding
were also inhibited by CPZ, further supporting the specific interaction
of vanilloids with the tagged VR1 variants. VR1eGFP and VR1
mimic
the wild-type receptor and are thus useful reporters with which to
study the dynamics of VR1 activation by vanilloids, other modulators,
and physical stimuli.
In addition to localization at the plasma membrane, VR1eGFP was demonstrated in the ER by colocalization with the fluorescent ER-tracker dye in live COS7, HEK293, and NIH 3T3 cells, as determined using high resolution confocal microscopy. One of the benefits of this localization and fluorescent tagging was the elucidation of the abrupt ER fragmentation due to agonist treatment. Addition of RTX to VR1-expressing cells induced ~10-fold accumulation of [Ca2+]i. This coincided with the rapid transformation of the normal lattice-like ER membrane structure into rounded vesicles, the earliest hallmark of Ca2+ cytotoxicity observed in living cells so far (Fig. 6). Calcium-induced ER vesiculation occurred throughout the cytoplasm (i.e. the z-stack of confocal acquisition), similar to that described previously for ionomycin (22). This type of membrane rearrangement (Figs. 4, 5, and 7) is clearly distinct from the agonist-mediated endocytosis, which can occur with nonion channel transmembrane receptors. In addition to the ER, the live cell imaging experiments revealed other membrane compartments that reacted to the vanilloid-induced elevated [Ca2+]i. These include the following: (i) shedding of the plasma membrane; (ii) intracellular fragmentation of mitochondria; and later (iii) blebbing of the nuclear envelope; (iv) accumulation of propidium iodide in the cell nucleus; and (v) rupture of the plasma membrane (Figs. 4, 5, 9, and 10).
The intracellular location of VR1 was separable from the mitochondrial compartment as assessed concurrently with the red MitoTracker dye (Figs. 4 and 7). Although spatially distinct, both the ER and mitochondria fragmented in seconds upon the large increase in [Ca2+]i through VR1 (Figs. 4, 5, 9, and 10). The staining of the mitochondrial compartment with the MitoTracker vital dye provided a surrogate end point to extend our findings in transiently transfected cells to DRG neurons in primary culture where specific small sized neurons showed the same immediate early changes in mitochondrial structure when exposed to vanilloids (Figs. 4, 7 versus 9). However, the perikarya of the responding neurons are spheroid rather than flat and extended, and a much smaller volume is available for confocal microscopy due to the high nucleus/cytosol ratio. Thus, in contrast to the flattened COS7 cells, geometry and size make nociceptive neurons less favorable subjects for live cell imaging of elongated organelles such as the mitochondria and the ER. As expected for the restricted cellular expression of VR1 in the DRG (9), RTX did not affect the mitochondria of adjacent large neurons or glia, imaged at the same time (Fig. 9).
The potential role of ER-localized VR1 is not yet clear, and we continue to investigate it. Our studies in calcium-free media show that a similar vanilloid-induced, VR1-specific remodeling of ER can occur with Ca2+ mobilized from intracellular sources, although with a much slower time course. Orientation or folding of VR1 in the ER may be factors, but the lag period in heterologous systems and DRG neurons (Figs. 7 and 8) also suggests buffering mechanisms are present (i.e. mitochondrial Ca2+ uptake (27)) or that multiple steps are required for activation of ER-localized ion channels (22, 28). The delay is not due to lack of membrane penetration of vanilloids, which are highly lipid-soluble. In fact, the water-soluble CAP analogue DA-5018 can only bind to VR1 when applied intracellularly, implying that vanilloids first must traverse the plasma membrane for activation of VR1 Ca2+ signaling (29). Other investigations (30-32) have shown that removal of extracellular calcium diminishes but does not eliminate physiological effects in a variety of systems including vanilloid-evoked inward currents with repeated administration of vanilloids in cultured DRG neurons. Furthermore, it was observed that capsaicin stimulated release of substance P from C-fibers in the absence of extracellular Ca2+ (33). Here we positively identified a delayed but RTX-induced Ca2+ release from intracellular stores in zero extracellular Ca2+, implying an important role of ER-localized VR1 (Fig. 8). These observations, together with previous electrophysiological data (30, 31), indicate that, in addition to the plasma membrane exposed VR1, a novel, functional, intracellular (i.e. ER) pool exists in DRG neurons. The results are consistent with the interpretation that, in the ER, VR1 gates Ca2+ stores which respond to vanilloids by releasing Ca2+ to the cytosol (Figs. 7-10).
Our data show that, as time progresses, the vanilloid-induced elevation in cytosolic Ca2+ produces an evolving cytotoxicity. Within 10 min, progressive nuclear blebbing is seen which coincides with PI incorporation in the nucleus, followed by lysis of the plasma membrane and cell death in 1-2 h in both VR1-expressing cells and DRG neurons. These more long term events are shown as a series of single frames in Figs. 5 and 10; however, upon replay of the frames from transfected cells or DRG neurons as a video clip (not shown), the vanilloid-induced PI accumulation in the cell nucleus and cell lysis highlights the dramatic nature of the cell death. The selectivity is also apparent since adjacent nontransfected or non-VR1-expressing cells remain resistant to PI permeation (Fig. 10).
Double wavelength imaging of consecutive accumulation of [Ca2+]i and propidium iodide due to RTX treatment and Western blot analysis of VR1 in primary DRG cultures all support the selectivity of vanilloid-induced cell deletion. Western blot data from DRG cultures (Fig. 11), together with images of cells heterologously expressing VR1eGFP (Fig. 5) or of DRG neurons (Fig. 10), indicate that RTX treatment first compromises the function of cell organelles and then kills the cell. These data argue against specific VR1 proteolysis or desensitization of VR1 by limited endo- or exocytosis as mechanisms mediating VR1 protein elimination from the primary cultures.
Several mechanisms can govern the intracellular remodeling and cell death that occur upon exposure to vanilloids. The most likely is rapid Ca2+ toxicity following VR1 activation, which is consistent with the data presented here from VR1-expressing cells and DRG neurons (Figs. 4-11) and by others (9, 27). Although the primary steps of Ca2+ toxicity are similar to apoptosis, including nuclear blebbing and PI staining of the nucleus (34), chromatin fragmentation, apoptotic body formation, and caspase activation were not apparent within 24 h (data not shown). Previous studies characterized only the terminal phase of vanilloid-induced cellular changes in detail. The longer term dynamics visualized with VR1eGFP in the present study are consistent with those noted with end point observations in the DRG, including intracellular membrane fragmentation, mitochondrial swelling, and nuclear envelope segmentation (8, 34-36). By extension, these results are generalized to a variety of ligand-operated Ca2+ channels such as the excitatory amino acid N-methyl-D-aspartic acid receptor, which can be activated by potent toxic ligands (37, 38).
The present data suggest a new potential therapeutic use of vanilloids, which is the targeted removal of nociceptive primary afferent neurons. Among the wide variety of VR1 agonists, OLV may have several advantages for clinical application. It is reported not to cause painful activation of the nociceptive afferent ending and, therefore, may be less inflammatory than other vanilloids (39). Our data also show that OLV is nontoxic to cells devoid of VR1 up to 50 µM, yet this concentration almost completely and specifically removes VR1-expressing cells in DRG culture (Fig. 11). Clinical application of vanilloids could be made regionally selective by the use of image-guided intra-ganglionic or nerve root administration to trigger specifically increases in [Ca2+]i at the level of the neuronal perikarya in sensory ganglia involved in a pathological pain state. Preclinical data based on our experience with epidural RTX or capsaicin administered to adult rats (5, 40) suggest that loss of C fibers via this approach would be an effective means for pain control.
In summary, our data refine the early dynamics of Ca2+
cytotoxicity in VR1-expressing cells. Fluorescent imaging in live cells revealed that at least three vital organelles are immediately damaged
due to increased [Ca2+]i conferred by the
agonist-activated VR1. One is the ER, which reacts with abrupt
fragmentation. The others are the mitochondria and cell nucleus. These
organelles are present in the cell body of sensory neurons; therefore,
disruption of their function equally can contribute to desensitization
and elimination of VR1-expressing cells (5). Our primary culture
experiments suggest that precise targeting of vanilloids to the
perikarya in sensory ganglia can induce specific elimination of
VR1-positive cells by Ca2+ cytotoxicity, characterized here
in detail. A similar strategy may have clinical utility in select
conditions to remove nociceptive neurons causing intractable pain in humans.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
¶ To whom correspondence should be addressed: Bldg. 49, Rm. 1A19, NIH, 49 Convent Dr., MSC-4410, Bethesda, MD 20892-4410. Tel.: 301-496-2755; Fax: 301-402-0667; E-mail: zoltan.olah@nih.gov.
Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M008392200
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ABBREVIATIONS |
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The abbreviations used are:
CAP, capsaicin;
CPZ, capsazepine;
OLV, olvanil;
RTX, resiniferatoxin;
VR1, vanilloid
receptor;
TRP, Ca2+ store-dependent channel;
DRG, dorsal root ganglion;
eGFP, enhanced green fluorescent protein;
VR1eGFP, C-terminally eGFP-tagged vanilloid receptor;
VR1, C-terminally
-tagged vanilloid receptor;
ER, endoplasmic reticulum;
[Ca2+]i, intracellular free calcium;
NGF, neuronal growth factor;
FUDR, 5-fluoro-2'-deoxyuridine;
DMEM, Dulbecco's modified Eagle's medium;
RT-PCR, reverse
transcriptase-polymerase chain reaction;
PI, propidium iodide;
MTH, metallothionein.
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