 |
INTRODUCTION |
Acetylcholine (ACh)
is a potent algogen that produces burning pain when applied to the
human suction blister base or applied iontophoretically to human skin
(Magerl et al. 1990
; Steen and Reeh 1993
;
Vogelsang et al. 1995
). Although widespread excitatory effects have been reported to explain this algesic action, these effects were frequently postulated to be secondary to vasodilation (Akoev 1981
; Diamond 1959
). Possible
sources of ACh in the close vicinity of primary afferent nerve
terminals have been identified, but at present it is not known if ACh
at all appears in the inflammatory environment to excite nociceptors.
In the cornea, epithelial cells contain a high concentration of ACh and
ACh excited corneal nerve endings (Pesin and Candia
1982
; Tanelian 1991
). In the skin,
keratinocytes synthesize, store, and secrete the substance
(Grando et al. 1993
) and, cutaneous unmyelinated
nociceptors respond to application of ACh both via nicotinic and
muscarinic receptors in an in vitro model (Steen and Reeh
1993
). While the excitatory effect of nicotinic receptor
activation is easily explained by the opening of cation channels and
consecutive depolarization of the neuron, nociceptor activation via
muscarinic ACh receptors is somewhat surprising. Even more so since in
contrast to the excitatory action in the periphery, the intrathecal
application of muscarinic agonists lead to a prolonged analgesia, which
indicates the presence of inhibitory muscarinic effects in the CNS
(Abram and Winne 1995
; Bleazard and Morris
1993
). These opposing effects may be explained by the
differential expression of receptor subtypes. Pharmacologically, muscarinic receptors can be subdivided into four subtypes, whereas five
different muscarinic receptors genes (M1-M5) have been described (Caulfield and Birdsall 1998
; Felder
1995
). The M1-, M3-, and M5-receptor subtypes are coupled to
the inositoltriphosphate/diacyl glycerol (IP3/DAG) pathway resulting in
release of Ca2+ from internal stores and
activation of protein kinase C (PKC) while M2- and M4-receptor subtype
activation results in the inhibition of adenylate cyclase activity
(Felder 1995
). Functional studies in cultured DRG
neurons demonstrated the coupling of muscarinic receptor activation to
the inhibition of voltage-activated calcium channels (Formenti
and Sansone 1991
; Wanke et al. 1994
) and
to the calcium dependent stimulation of nitric oxide synthase (NOS) and
consecutive cyclic GMP (cGMP) production (Bauer et al.
1994
). None of the previous studies has attempted to identify
and localize the muscarinic receptor subtypes that contribute to these
functional changes in DRG neurons. Since both suggest an involvement of
calcium ions, it was the aim of the present study to investigate the
effects of muscarine on the intracellular calcium concentration
([Ca2+]i) in DRG neurons.
For this purpose, the calcium indicator dye Fura-2 was
nondisruptively loaded into the neurons, and calcium concentration was
monitored microfluorimetrically (Tsien 1981
). The
muscarinic antagonists atropine, gallamine, and 4-DAMP were used to
pharmacologically identify the receptor subtypes involved. For
detection of the mRNA, we used reverse transcription-polymerase chain
reaction (RT-PCR) and single-cell RT-PCR. Immunohistochemical and
histochemical techniques allowed the further classification of the
neurons. Double labeling of M2R/M3R with I-B4 identified putative
nociceptive neurons.
 |
METHODS |
Cell culture
Details of dissociation procedures have been published elsewhere
(Zeilhofer et al. 1996
). Briefly, lumbar DRG
(L1-L5) were harvested
from adult Wistar rats of either sex (100-160 g) that had been killed
by respiration of 100% CO2. After dissection, ganglia were transferred into Dulbecco's modified eagle medium (DMEM,
Gibco, Karlsruhe, Germany) supplemented with 25 mg/500 ml gentamycin
(Sigma, Deisenhofen, Germany). The connective tissue was removed and
ganglia were treated with collagenase (0.28 U/ml in DMEM, 75 min,
Boehringer Mannheim, Germany) and trypsin (25,000 U/ml in DMEM, 12 min,
Sigma). After dissociation, the cell suspension was centrifuged at 2000 g and finally resuspended in supplemented culture medium.
After plating on cover slips coated with poly-L-lysine (200 µg/ml, Sigma) cultures were kept in serum free TNB 100 medium (Biochrom, Berlin) supplemented with penicillin/streptomycin (each 20,000 IU/100 ml), 2 mM L-glutamine (both from Gibco) and
100 ng/ml nerve growth factor (Alomone Labs, Jerusalem, Israel) at 37°C in a humid atmosphere containing 5% CO2.
Calcium measurement in isolated DRG neurons
Recordings of the intracellular free calcium concentration were
performed in isolated neurons between 20 and 36 h in primary culture. External solution consisted of (in mM) 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose,
and 10 HEPES (Merck, Darmstadt, Germany) at pH 7.3 adjusted with NaOH.
For calcium measurements, neurons were loaded nondisruptively with 3 µM Fura-2 for 30 min (Molecular Probes, Leiden, Netherlands).
Background-corrected fluorescent images were taken with a slow scan CCD
camera system with fast monochromator (PTI, Monmouth Junction, New
Jersey) coupled to an Axiovert microscope with ×40 fluotar
oil-immersion objective (Zeiss, Jena, Germany). Fura-2 was excited at
340 and 380 nm wavelengths (
) and fluorescence was collected at
> 420 nm at a frequency of 1 Hz with equal exposure time at
each wavelength (200 ms). [Ca2+]i was calculated as
previously published (Zeilhofer et al. 1996
), and
calibration constants obtained in vitro were
Rmin = 0.44, Rmax = 8.0, and
Keff = 1.2 µM. For application of
chemicals, a fast 10-channel system with common outlet was used.
Solenoids were controlled manually from a switch board (Dittert
et al. 1998
). To determine the source of
[Ca2+]i increase, five
cells were exposed to muscarine in calcium-free extracellular solution
consisting of 145 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 EGTA (all from Sigma), and 10 HEPES
(Merck) at pH 7.3 adjusted with NaOH. Chemicals (atropine, muscarine,
4-DAMP, gallamine) were purchased from Biotrend (Köln,
Germany). Intraindividual comparisons were performed using the Wilcoxon
matched pairs test and differences were considered significant at
P < 0.05.
RT-PCR
For RT-PCR, lumbar DRG and pieces of large intestine that served
as positive control of five Wistar rats were quick-frozen in RNazol
(WAK-Chemie, Bad-Homburg, Germany) and homogenized after thawing using
a turrax. The total RNA was isolated using the RNazol reagent technique
according to the recommended protocol. Contaminating DNA was removed
using DNase (1 U/µg total RNA, Gibco-BRL, Life Technologies GmbH,
Karlsruhe, Germany) in the presence of 20 mM Tris-HCl (pH 8.4), 2 mM
MgCl2, and 50 mM KCl for 15 min at 25°C. Equal
amounts of RNA were reverse transcribed in the presence of (in mM) 3 MgCl2, 75 KCl, 50 Tris-HCl, pH 8.3, 10 dithiothreitol, and 0.5 dNTPs (Gibco-BRL) and 25 µg oligo(dT) (MWG
Biotech, Ebersberg, Germany), with 200 U of Superscript RNase
H
reverse transcriptase (Gibco-BRL) for 50 min
at 42°C. For the PCR reaction 4 µl buffer II, 4 µl
MgCl2, 1 µl dNTP (10 mM each), 0.4 µl (2 U)
AmpliTaq Gold polymerase (all reagents from Perkin Elmer), and 1 µl
of each primer (20 µM, M2R, Genbank Accession No. J03025 forward
primer 5'AGCCCGCAAAATCGTGAA3' position 1534; reverse primer
5'GACATTGTATGGCGCCCAC3', position 1666, product 132 bp, M3R Genbank
Accession No. M16407, forward primer 5'ACCAACTCCTCGGCAGACAA3', position
1364; reverse primer 5'GCGACATCCTCTTCCGCTT3', position 1485, product
121 bp, MWG Biotech) were mixed. Cycling conditions for PCR were 10 min
at 95°C, 35 cycles of 45 s at 94°C, 60 s at 58°C, and
45 s at 73°C followed by 7 min at 73°C. Control reactions for
RT-PCR included the absence of the RT reaction before PCR and the
absence of template that was replaced by water. Control reactions
showed no amplification products.
Single-cell RT-PCR
Neurons were identified after establishing the whole cell
configuration by sodium currents elicited with voltage steps (150 ms,
E = +20 mV) from a holding potential of
80 mV.
Cytoplasm of these neurons was aspirated under visual control into
a patch pipette (3-9 M
) filled with 6 µl sterile filtered
recording solution [(in mM) 151 KCl, 10 NaCl, 0.5-3 EGTA, and 10 HEPES, pH 7.2] (Scholz et al. 1998b
). The pipette
contents were subsequently ejected into a RNase-free 0.2 ml tube
containing 10 µl lysis buffer (Kummer et al. 1998
).
Sterile gloves were worn and the capillaries used were
fire-polished directly before the experiment on a microforge. RT of one
aliquot (7 µl) was performed after heating the tubes (10 min,
70°C) and cooling on ice for 5 min. The other aliquot was diluted in
10 µl sterile water and served as the control without RT. The
RT-mix contained 1 µl dNTP-mix (10 mM each), 0.8 µl random hexamer
(50 µM), 0.5 µl RNase inhibitor (10 U), 0.8 µl murine leukemia
virus reverse transcriptase, EC 2.7.7.49 (all reagents Perkin Elmer), and H2O added to the total RNA to
a final volume of 17.5 µl. The steps for the RT were 10 min
20°C, 60 min 43°C, and 5 min 99°C. Subsequent time release PCR
(Perkin Elmer) was performed using three 5 µl aliquots of the RT
mix at 2 min 45 s at 95°C, 60 cycles of 45 s at 94°C,
60 s at 58°C and 45 s at 73°C followed by 7 min at
73°C. Three 5 µl aliquots without RT were added to the PCR mix.
Aliquots were run on a 2% agarose gel buffered with Tris-acetate
EDTA containing ethidium bromide. As a control for the presence of
cDNA, we used primers corresponding to the housekeeping gene,
phosphobilinogen deaminase (PBGD) (Fink et al. 1999
) and
for glycerin aldehyde 3-phosphat dehydrogenase (GAPDH)
(Haberberger et al. 1999
). The PCR products were tested for their identity by cycle sequencing (AbiPrism Cycler, MWG
Biotech). Control reactions for single-cell RT-PCR included the absence of the RT reaction before PCR and the absence of template that was
replaced by water. Control reactions showed no amplification products.
Immunohistochemistry
Five young adult Wistar rats of either sex were deeply
anesthetized and perfused transcardially with polyvinylpyrrolidone- and
procainamide HCl-containing rinsing solution (Forssmann et al.
1977
) followed by 300 ml Zamboni's fixative (Zamboni
and de Martino 1967
). For the embedding in polyethylenglycol
(PEG), animals were perfused and tissues removed as previously
described (Haberberger et al. 1999
). Ganglia were
dehydrated (4 × 10 min in 80% EtOH, 2 × 15 min in 100%
EtOH, 3 × 10 min in dimethylsulphoxide and 2 × 10 min in
80% EtOH) and incubated at 55°C in PEG (mw 1,000) followed by
embedding in PEG (mw 1,450, Anderson et al. 1997
). The PEG blocks were
sectioned at 6 µm with a tetrander (Jung, Heidelberg, Germany).
Histochemistry and immunohistochemistry were performed at free-floating
sections. Sections were incubated overnight at room temperature with
polyclonal rabbit antisera raised against amino acid residues 457-466
of the human M2R or against the amino acid residues 580-589 of the
human M3R or biotinylated I-B4-lectin (Table
1) Secondary reagents were
fluoresceinisothiocyanate (FITC)-conjugated anti-rabbit IgG from goat
and streptavidin conjugated to Cy-3 (Table 1). Control sections,
exposed to M2R or M3R antiserum that had been preabsorbed with the
corresponding synthetic antigen (20-100 µg antigen/ml diluted
antiserum; antigen from Biotrend), showed no immunolabeling. For each
muscarinic receptor subtype, two sections (at least 50 µm apart) from
one ganglion were used for image analysis (ScionImage, Scion, Las
Vegas, NV). Only cells with clear visible nuclei were used.
Dissociated sensory neurons after 20-36 h in primary culture were
fixed using Zamboni's fixative. The unspecific binding sites were
blocked with PBS, containing 10% normal swine serum, 0.1% bovine
serum albumin, and Tween-20 for 1 h. Subsequent incubation was
performed as previously described. Sections and cells were examined
under epifluorescence (BX60 microscope, Olympus, Hamburg, Germany)
using appropriate filter combinations for Cy-3 (excitation filter
525-560 nm, barrier filter 570-650 nm) and FITC (excitation filter
460-490 nm, barrier filter 515-550 nm).
 |
RESULTS |
Exposure of dissociated neurons (20-35 µm diam) to muscarine
for 30 s (in 1 µM concentration) increased
[Ca2+ ]i (Fig.
1). Application of
10
6 M muscarine was
followed by an increase of [Ca2+
]i by 334 ± 85 (SE) nM in 76% neurons
(13/17 cells). The [Ca2+
]i increase showed no significant tachyphylaxis,
but there was a tendency to be less pronounced after the second and the
third stimulation with the agonist in the concentration of
10
6 M (Fig. 1). The
effect of muscarine was antagonized by the muscarinic receptor
antagonist atropine (10
6
M, P < 0.01) and by the M3R-antagonist 4-DAMP (1 µM,
P < 0.01), whereas the allosteric M2-receptor
antagonist gallamine (10
6
M) showed no significant effect on the increase in
[Ca2+ ]i (Figs.
2-4).
The [Ca2+ ]i increased by
153 ± 24 nM after muscarine application and 106 ± 35 nM after
application of muscarine together with gallamine (Fig. 3). No rise in
[Ca2+ ]i was observed
when the neurons were exposed to muscarine in the absence of
extracellular calcium (n = 5), which suggests calcium influx as a possible source of the increase.

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Fig. 1.
Muscarine induced rises in [Ca2+]i in 80% of
small- to medium-sized dissociated dorsal root ganglion (DRG) neurons.
B: repeated application of 10 6
M muscarine did not induce significant tachyphylaxis of the response in
the neuron depicted in A. C: the average
responses of 7 neurons.
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Fig. 2.
Muscarine-induced increases in [Ca2+]i were
completely inhibited by the nonsubtype selective muscarinic receptor
antagonist atropine. B: the neuron depicted
A responded to 10 6 M muscarine
with a rise in [Ca2+ ]i which was blocked by
10 6 M atropine. The average effects are
depicted in C; *, the significant reduction by atropine
(P < 0.01).
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Fig. 3.
Muscarine-induced rises in [Ca2+]i were not
inhibited by the allosteric M2 inhibitor gallamine. B:
specimen of a single DRG neuron (see arrow in A) and
average results from 9 neurons (C).
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Fig. 4.
Muscarine-induced calcium responses were significantly blocked by the
M3 receptor antagonist 4-DAMP. B: specimen of a single
DRG neuron that is depicted in A. C: the
average inhibition by 4-DAMP; *, the significant block
(P < 0.01).
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The presence of M2R and M3R protein was demonstrated by means of
immunohistochemistry. Sensory neurons in lumbar DRG showed immunoreactivity (IR) for both receptor subtypes (Fig.
5). M2R- and M3R-IR neurons were
distributed throughout the ganglion. The intensity of immunoreactivity
for both receptor subtypes varied over a wide range. Intense
intracellular M2R-IR occurred in somata and axonal processes of small-
to medium-sized neurons (20-35 µm diam). M2R-IR was present in 41%
(153/373 cells) of lumbar DRG neurons. The vast majority of the cells
(97%, 149/153 cells) had diameters between 20 and 30 µm. Most of
these M2R-IR neuronal profiles (83.6%, 128/153 cells) showed also I-B4
labeling, but I-B4-positive cells without M2R-IR were also present
(Figs. 5 and 6). The labeling with I-B4
varied from intense to weak or absent. Both, intensely and weakly
labeled cells expressed M2R-IR. Intense M3R-IR was mainly present in
somata of small- to medium-sized neurons, whereas axonal processes
showed no or only faint staining. The M3R-IR was present in about 90%
(350/385 cells) of the perikarya including I-B4-positive neurons
(33.2%, 128/390 cells) of all neuronal profiles (Figs. 5 and 6).
Smooth muscle cells of intraganglionic blood vessels expressed intense
M3R-IR but no M2R-IR (Fig. 5).

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Fig. 5.
Localization of M2R- and M3R-immunoreactivity (IR) and I-B4 binding
sites in DRG in situ. A and B: sensory
neurons displaying intense M2R-IR are also I-B4-positive (arrows).
Occassionally M2R-IR was present without IB-4 labeling (arrowhead).
Some I-B4 labeled neurons are devoid of M2R-IR (double arrowhead). Note
that M2R-IR is also present in neuronal processes. C and
D: M3R-IR is present in I-B4-labeled neurons (arrows).
Some M3R-IR cells do not bind I-B4 (double arrowhead).
E: smooth muscle cells of intraganglionic vessels
(asterisk) display M3R-IR. Bar = 20 µm.
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Fig. 6.
Size distribution of DRG neurons and M2+ (A) and M3R+
(B) neuron subpopulations. Total number of neurons are
given.
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Sensory neurons (20-35 µm diam) expressed M2R-IR and M3R-IR after
20-36 h in culture (Fig. 7). Double
labeling showed M2R-IR/M3R-IR and I-B4 binding sites in single sensory
neurons (Fig. 7). The M2R-IR was present intracellularly. Binding sites
for I-B4 were found in the plasma membrane with and without additional
staining of the Golgi apparatus (Fig. 7). Occasionally M2R-IR was
present in I-B4-negative neurons (Fig. 7). I-B4-positive neurons
without immunoreactivity for M2R were also present (Fig. 5). Analysis of the mRNA of M2R and M3R showed the presence of both muscarinic receptor types in the cDNA of rat lumbar DRG. No difference occurred between RT-PCR and time-release RT-PCR using total RNA of lumbar DRG
(Fig. 8). The expected amplicons with 132 bp (M2R) and 121 bp (M3R) in length were amplified and subsequent
sequencing (AbiPrism) showed identity of the product with the published
rat M2R and M3R sequences (Fig. 8) (Bonner et al. 1987
).
Control PCR without RT or template showed no products. Single-cell
RT-PCR analysis of eight lumbar DRG neurons of the rat showed the
presence of M2R and M3R mRNA in four single sensory neurons. Cytosol
was aspirated from typically small- to medium-sized neurons,
20-35 µm in diameter. For the amplification time-release RT-PCR was
used (Fig. 9). To exclude aspiration from
satellite cells, the neurons were identified by sodium currents
elicited with voltage steps in the whole cell configuration (Fig. 9).
Amplicons with the expected size for M2R (132 bp), M3R (121 bp), or
both receptor subtypes were present in single sensory neurons (Fig. 9).
Control PCR without template or RT showed no products.

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Fig. 7.
Double labeling for either M2R- and M3R-IR and I-B4 binding sites in
dissociated sensory neurons. Medium-sized M2R- and M3R-IR sensory
neurons are also labeled with I-B4 (arrows, double arrowheads). I-B4
stained the plasma membrane (arrow) or the plasma membrane and
intracellular structures (double arrowhead). The arrow indicates an
I-B4-positive cells without M3R-IR. M3R-IR neurons without I-B4
labeling are also present (asterisk). Bar = 20 µm.
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Fig. 8.
RT-PCR analysis for M2R- and M3R-mRNA expression in rat DRG tissue. No
difference occurred between RT-PCR (35 cycles) and time-release RT-PCR
(65 cycles) using total RNA of lumbar DRG. Amplicons were M2R (132 bp),
M3R (121 bp), and the house keeping genes glycerin aldehyde 3-phosphat
dehydrogenase (GAPDH, 299 bp) and phosphobilinogen deaminase (PBGD, 128 bp). Amplification products were absent in controls of time-release
RT-PCR without reverse transcriptase ( RT) or template ( template).
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Fig. 9.
Identification of neurons by whole cell patch-clamp recordings.
A: whole cell currents were evoked in DRG neurons by
successive depolarisation steps of 10 mV (200 ms, ranging from 70 to
+70 mV) from a holding potential of 80 mV. B: action
potentials were evoked by current pulses (3 ms) of increasing amplitude
(400, 500, and 600 pA). Pipette solution: high
Kin, bath solution: Control-HEPES, 24°C.
C: single-cell time-release RT-PCR analysis of
individual lumbar DRG neuron of the rat. Amplicons with the expected
size for M2R (132 bp), M3R (121 bp) were present in single sensory
neurons.
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|
 |
DISCUSSION |
This study demonstrates for the first time that muscarine induces
rise in [Ca2+]i in rat
sensory neurons. The muscarine-mediated increase was seen in about 80%
of small- to medium-sized neurons (20-35 µm diam). The response was
mediated by the M3R but not the M2R subtype and did not exhibit
significant tachyphylaxis. These functional data were corroborated by
the isolation of M3R but also M2R mRNA in single DRG neurons as well as
by positive M3R and M2R immunostaining of sensory neurons in vitro and
in situ.
In rat lumbar sensory neurons, muscarine induced rise in
[Ca2+]i that was similar
to the responses shown in chick parasympathetic ciliary ganglion cells
where muscarine increased
[Ca2+]i between 186 and
476 nM (Sorimachi et al. 1995
). In five cells, muscarine
was applied in calcium-free solution, and under these conditions no
rise in [Ca2+]i was
observed. This may suggest calcium influx via activation of muscarinic
receptors as the source of the calcium increase. M2R have been
described in rat DRG neurons by means of immunohistochemistry and in
situ hybridization (Haberberger et al. 1999
; Tata
et al. 1999
). The M2R-antagonist gallamine, however, did not
inhibit the response and none of the second-messenger pathways
connected to M2R are reported to increase calcium levels but rather
contribute to a decrease.
The inhibition of the evoked rises in
[Ca2+]i by administration
of the muscarinic receptor-antagonist atropine and the M3R-antagonist 4-DAMP suggested the involvement of M3R, which is coupled to activation of phospholipase C and the production of IP3 and
DAG. IP3 acts on intracellular receptors followed
by release of calcium from internal stores. In the present study, the
rise in [Ca2+]i fully
depends on the presence of extracellular Ca2+.
Therefore this may be due to calcium influx from extracellular space.
We abstain from any speculation on the nature of this pathway, but the
M3-receptor subtype may be involved in this effect. Because of the
absence of real subtype selectivity of muscarinic antagonists, we
cannot exclude the involvement of M1R or M5R. In a variety of neurons
such changes in intracellular calcium concentration regulate important
signaling pathways like the activity of phospholipases, adenylate
cyclases, or guanylate cyclase (Felder 1995
). Similarly, calcium may exert a variety of effects in nociceptive neurons: first,
inflammatory mediators like bradykinin and ATP, and algogens like
capsaicin increase
[Ca2+]i in sensory
neurons (Kress and Guenther 1999
). Such rises in [Ca2+]i that were similar
to those observed in the present study, induced heat sensitization of
nociceptors (Guenther et al. 1999
). Therefore the
activation of muscarinic receptors may cause similar changes in the
sensitivity of sensory neurons. Second, Huang and Neher (1996)
showed a calcium-dependent release of substance P (SP) from the perikarya of dissociated DRG neurons of similar size as in the
present study. The increase in
[Ca2+]i evoked by
muscarine could, therefore also stimulate the exocytosis of
neuropeptides, suggesting a role of ACh in modulating either the axon
reflex response in the target tissue or transmission at the central
terminal in the dorsal horn. Accordingly ACh increased [Ca2+]i and greatly
facilitated the electrically evoked
[3H]D-aspartate efflux through
M3-receptors in cultured cerebellar granule cells of the rat
(Beani et al. 1997
). Third, ion channels could be
modulated by the elevated [Ca2+
]i. It was shown that Ca2+
release from intracellular stores in cultured neonatal rat DRG neurons
activated calcium-activated chloride conductances (Ayar and
Scott 1999
). Such Ca2+ release
might also activate calcium-activated potassium conductances like the
BKCa channel in DRG neurons, which influenced the
action potentials and the refractory period between action potentials (Scholz et al. 1998a
). Fourth, the rise in
[Ca2+ ]i may play a role
in signaling pathways like the activation of phospholipases or NOS
(Felder 1995
; Hu and Fakahany 1993
). Both, muscarinic receptors and NOS have been described in rat DRG by
autoradiography, immunohistochemistry (M2R, M3R), histochemistry (NOS),
and pharmacological methods (M2R, M3R, M4R, NOS) (Aimi et al.
1991
; Aley et al. 1998
; Haberberger et
al. 1999
; Wamsley et al. 1981
; this study). The
NOS product nitric oxide has a variety of modulatory effects on
nociception including production and facilitation of hyperalgesia
(Aley et al. 1998
).
The muscarine-induced increase in
[Ca2+]i observed in the
present material is apparently in contrast to previous studies on dissociated paratracheal neurons, pyramidal cells, and dissociated DRG
neurons describing an inhibition of high-voltage activated Ca2+-channels through activation of muscarinic
receptors (M2R or M4R) (Murai et al. 1998
;
Stewart et al. 1999
; Wanke et al. 1994
).
However, such an inactivation of voltage-activated calcium channels can be calcium dependent (for review, see Levitan 1999
) and,
therefore may be the consequence of stimulation of M3R and subsequent
increase in [Ca2+]i.
Although muscarinic receptor antagonists lack a true subtype
selectivity (Caulfield and Birdsall 1998
), the presence
of M3R is likely since the functional results presented here were
corroborated by the demonstration of M3 receptor mRNA and protein in
small- to medium-sized neurons by means of RT-PCR and
immunohistochemistry. The cell diameters of M3R-positive neurons from
rat lumbar DRG in the present study are very similar to those of
muscarinic receptor immunoreactive profiles in thoracic DRG of the rat
and in chick DRG (Bernardini et al. 1998
;
Haberberger et al. 1999
). In addition, their size
corresponds well to the size of cells investigated by microfluorometric
calcium measurement. Small- to medium-sized neurons represent the
perikarya of thinly or nonmyelinated afferent neurons (Snider
and McMahon 1998
).
Not only M3R-mRNA but also M2R-mRNA was found in some of these neurons,
and this corresponds well with results from in situ hybridization,
which showed M2R-mRNA in medium-small neurons in rat DRG (Tata
et al. 1999
). The finding of M2R- in addition to M3R-mRNA is in
agreement with present immunohistochemical detection of muscarinic M2R-
and M3R-protein in small- to medium-sized neurons in situ, in sections
of lumbar DRG, and, in dissociated neurons. M3R-IR was found in 90% of
lumbar neurons including I-B4-positive cells, which were M2R-IR too.
Therefore it can be concluded that at least the subpopulation of
I-B4-positive neurons contain M2R- and M3R-protein. The presence of
multiple muscarinic receptor-subtypes in single neurons has previously
been demonstrated in cultured pyramidal neurons from rat sensimotor
cortex where the activation of different muscarinic receptors in single
neurons resulted in an inhibition of N-, P- and L-type currents (see
the preceding text) (Stewart et al. 1999
).
Nociceptive neurons can be subdivided in two classes. One class depends
on the presence of nerve growth factor and expresses the neuropeptides
SP and CGRP (Snider and McMahon 1998
). The other class
is sensitive to glial cell derived neurotrophic factor and binds the
plant lectin I-B4 (Bennett et al. 1996
; Molliver
et al. 1995
). I-B4 is a marker of thinly myelinated and
unmyelinated C-fiber afferent neurons that project mainly to the skin
and, to a lesser extent to viscera, that terminate in the spinal cord (Bennett et al. 1996
; Molliver et al.
1995
; Petruska et al. 1997
; Plenderleith
and Snow 1993
). The two groups of neurons show partial overlap
since Wang and co-workers (1994)
demonstrated SP- and/or CGRP-IR in a subpopulation of I-B4-positive cells. SP-positive cells
were present in weakly labeled I-B4 neurons, whereas intensely I-B4-labeled cells showed no SP/CGRP-IR (Wang et al.
1994
). We observed that M2R- and M3R-IR were present in
intensely as well as weakly I-B4-stained cells. In addition to the
strongly immunoreactive M2R sensory neurons I-B4-negative cells were
observed with minor but visible immunoreactivity for M2R and M3R.
Therefore it cannot be excluded that M2R and M3R were, in addition to
their occurrence on I-B4-positive cells, also present on SP-containing
sensory neurons.
It was not possible to use both, M2R- and M3R-antisera together for the
detection of both receptor proteins in single neurons. However, about
84% of M2R-IR neurons were I-B4 positive, and all I-B4-positive
neurons showed M3R-IR. Therefore we can conclude that both receptor
subtypes were present in I-B4-positive neurons. I-B4-positive neurons
were small- to medium-sized. The mRNA for both receptors was detectable
in single neurons of this size, suggesting the presence of both
receptor proteins on individual sensory neurons. The presence and
localization of muscarinic M2- and M3R-mRNA and protein in rat lumbar
putative nociceptive DRG sensory neurons suggest the involvement of
different muscarinic receptor subtypes in sensory nociceptive signal
transduction and processing.
The authors thank A. Wirth-Huecking, I. Izydorczyk, and M. Bodenbenner for expert technical assistance.
This work was supported by the Deutsche Forschungsgemeinschaft (SFB
547, C2 and SFB 353, A10) and the Sanderstiftung (96.058.1).
Address for reprint requests: R. Haberberger, Institute
for Anatomy and Cell Biology, Aulweg 123, D-35385 Giessen, Germany (E-mail:
rainer.v.haberberger{at}anatomie.med.uni-giessen.de).
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