Department of Physiology and Pharmacology, State University of New York Downstate Medical Center, Brooklyn, New York 11203
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ABSTRACT |
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The enteric nervous system
(ENS) contains functional ionotropic and group I metabotropic glutamate
(mGlu) receptors. In this study, we determined whether enteric neurons
express group II mGlu receptors and the effects of mGlu receptor
activation on voltage-gated Ca2+ currents in these cells.
(2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC), a group
II mGlu receptor agonist, reversibly suppressed the Ba2+
current in myenteric neurons isolated from the guinea pig ileum. Significant inhibition was also produced by L-glutamate and
the group II mGlu receptor agonists,
(2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) and
(2S,1'S,2'S)-2-(2-carboxycyclopropyl)glycine (L-CCG-I), with a rank order potency of 2R,4R-APDC > DCG-IV > L-glutamate > L-CCG-I, and was reduced by
the group II mGlu receptor antagonist LY-341495. Pretreatment of
neurons with pertussis toxin (PTX) reduced the action of mGlu receptor
agonists, suggesting participation of Gi/Go
proteins. Finally, -conotoxin GVIA blocked current suppression by
DCG-IV, suggesting modulation of N-type calcium channels. mGlu2/3 receptor immunoreactivity was displayed by neurons in culture and in
the submucosal and myenteric plexus of the ileum. A subset of these
cells displayed a glutamatergic phenotype as shown by the expression of
vesicular glutamate transporter 2. These results provide the first
evidence for functional group II mGlu receptors in the ENS and show
that these receptors are PTX sensitive and negatively coupled to N-type
calcium channels. Inhibition of N-type calcium channels produced by
activation of group II mGlu receptors may modulate enteric neurotransmission.
enteric neurons; N-type calcium channels; pertussis toxin; vesicular glutamate transporter 2
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INTRODUCTION |
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SEVERAL SUBTYPES OF GLUTAMATE receptors have been identified in the central nervous system (CNS). These include the ionotropic glutamate receptors, which mediate excitatory synaptic transmission through ligand-gated ion channels, and the metabotropic glutamate (mGlu) receptors, which are coupled to G proteins and act on various intracellular second messenger systems (1, 9, 25). mGlu receptors are located pre- and postsynaptically, and glutamate acting via mGlu receptors can exert not only an excitatory but also an inhibitory action. Eight mGlu receptors have been cloned and are classified into three groups. Group I mGlu receptors (mGlu1 and mGlu5) activate phospholipase C through coupling to the Gq class of G proteins. Group II (mGlu2 and -3) and group III (mGlu4, -6, -7, and -8) mGlu receptors inhibit cAMP formation through coupling to the Gi/Go class of G proteins, reduce Ca2+ currents, and presynaptically inhibit the release of glutamate or other neurotransmitters (for review, see Ref. 5).
Neurons that contain glutamate and express the vesicular glutamate transporter 2 (VGLUT2) are also present in the enteric nervous system (ENS) (15, 32), which consists of neuronal microcircuits within the bowel wall. Enteric neurons release glutamate in a Ca2+-dependent manner (27, 34), and activation of both ionotropic and group I mGlu receptors have been associated with the excitation of enteric neurons (14, 21, 22). In addition, glutamate receptor ligands affect intestinal motility (4, 10, 12, 22) and secretion (28, 33); therefore, glutamate receptors may play a role in the modulation of enteric reflexes (15).
In this study, we used the patch-clamp recording method to determine whether enteric neurons express functional group II mGlu receptors. We investigated the effects of group II mGlu receptor agonists on Ca2+ currents in acutely isolated myenteric neurons of the guinea pig ileum. mGlu2/3 receptor immunoreactivity was localized in the ENS by confocal microscopy. Our results provide the first evidence for functional group II mGlu receptors in the gut and show that these receptors are pertussis toxin (PTX) sensitive and negatively coupled to N-type Ca2+ channels.
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MATERIALS AND METHODS |
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Culture of myenteric neurons. Myenteric ganglia were isolated from guinea pig small intestine as previously described (16, 35). Briefly, male guinea pigs (1 wk old) were anesthetized by CO2 inhalation and exsanguinated. The Animal Care and Use Committee of the State University of New York Downstate Medical Center has approved this procedure. The small intestine was removed, cleaned, and placed in iced Krebs solution of the following composition (mM): 121.3 NaCl, 5.95 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.34 NaH2PO4, 14.3 NaHCO3, and 12.7 glucose. The bowel was mounted on a pipette placed in the intestinal lumen. The longitudinal muscle and adherent myenteric plexus (LMMP) were then rapidly stripped from the gut with a fine cotton swab. Resulting LMMP preparations were minced, and tissue suspension was incubated in an enzyme solution containing collagenase type 1A (40 mg; Sigma) in 10 ml Krebs solution for 20-25 min at 37°C in a shaker bath. The digested tissue was gently triturated with a fire-polished Pasteur pipette and centrifuged at medium speed (1,000 g) for 7-10 min. The final pellet was resuspended in 3.0 ml of DMEM/F-12K nutrient mixture (GIBCO, Grand Island, NY) containing 10% fetal bovine serum, gentamycin (10 µg/ml), penicillin (100 U/ml), and streptomycin (50 µg/ml) (all additives from Sigma). Individual ganglia were plated in 35-mm plastic petri dishes (Corning Glass, Corning, NY). Cultures were incubated at 37°C in an atmosphere of 5% CO2 and used for patch-clamp studies the next day.
Whole cell patch-clamp recording.
Neurons were viewed with an inverted microscope (Axiovert S100; Zeiss)
by using phase-contrast optics. Whole cell patch-clamp recordings were
performed with pipettes of 2-6 M in resistance, containing (in
mM) 117 tetraethylammonium chloride (TEACl), 14 phosphocreatine, 4.5 MgCl2, 10 HEPES, 10 EGTA, 4.0 ATP, and 0.3 GTP, adjusted to
pH 7.2 with tetraethylammonium hydroxide (TEAOH); the
osmolarity was 310 mosM. To record Ba2+ currents through
voltage-gated Ca2+ channels, the neurons were superfused
with a solution containing (in mM) 25 BaCl2, 145 TEACl, 0.1 EGTA, 10 HEPES, and 10 D-glucose; pH was adjusted to 7.4 (with TEAOH) and osmolarity to 340 mosM (with sucrose). Under these
conditions, sodium and potassium currents were suppressed with
tetrodotoxin (TTX, 0.001 µM) and TEACl, respectively.
Data analysis.
Averaged values in the text and figures are expressed as means ± SE. Peak amplitudes of Ca2+ currents recorded before and
after the administration of drugs were compared statistically by ANOVA
or paired Student's t-test. Agonist concentration-response
curves obtained from individual neurons were fit with the following
logistic function: y =
[YminYmax/1+(x/EC50)n] + Ymax, where Ymin and Ymax are the
minimum and maximum responses, respectively, and n is the
slope factor.
Immunocytochemistry.
Cultured neurons were fixed (overnight at 4°C) with 4% formaldehyde
in 0.1 M sodium phosphate buffer (pH 7.4) and washed 3 times with PBS.
After fixation, preparations were exposed to PBS containing 1.0%
Triton X-100 and 4.0% horse serum for 30 min to permeabilize the
tissue and reduce background staining. Immunoreactivity was then
demonstrated by incubating the cultures with polyclonal antibodies
raised in rabbit against a COOH-terminal peptide of mGlu2 (it
recognizes both mGlu2 and mGlu3 diluted 1:500; Chemicon International,
Temecula, CA) (2, 20, 24), antibodies raised against the
bacterial fusion protein containing the cytoplasmic COOH terminus of
rat VGLUT2 (rabbit polyclonal, diluted 1:2,000) (32), or
antibodies raised against the 1B-subunit of
-conotoxin GVIA-sensitive N-type Ca2+ channels
(17). Bound antibodies were located by incubating preparations with secondary antibodies to rabbit IgG coupled to FITC
(diluted 1:300; Jackson ImmunoResearch) or Cy3 (diluted 1:300; Jackson
ImmunoResearch). Preparations were then washed in PBS and coverslipped
with Vectashield (Vector, Burlingame, CA).
Confocal microscopy. Confocal images were obtained by using a Radiance 2000 confocal microscope (Bio-Rad, Hercules, CA) attached to a Zeiss Axioskop 2 microscope. Usually, 5-10 optical sections were taken at 1.0-µm intervals. Images of 1,024 × 1,024 pixels were obtained and processed by using Adobe Photoshop 6.0 (Adobe Systems, Mountain View, CA) and printed by using a Tektronix Phaser 440 printer.
Drugs.
BaCl2, TTX, CdCl2, TEA, -conotoxin GVIA, and
nifedipine were obtained from Sigma.
(2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC),
(2S,2'R,3'R)-2- (2',3'-dicarboxycyclopropyl)glycine (DCG-IV),
(2S,1'S,2'S)-2-(2-carboxycyclopropyl)glycine (L-CCG-I), (S)-3,5-dihydroxyphenylglycine (S-DHPG),
L-glutamate, LY-341495, and 1-aminoindan-1,5-dicarboxylic
acid (AIDA) were obtained from Tocris Cookson (St. Louis, MO).
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RESULTS |
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Group II mGlu receptor agonists inhibit the
Ba2+ current in myenteric neurons.
Recordings were made from 119 myenteric neurons in ganglia acutely
isolated from the guinea pig ileum. Ba2+ currents through
voltage-gated Ca2+ channels were evoked in all neurons by
voltage ramps from 70 mV to +60 mV in 150 ms duration (Fig.
1). We tested the effects of the group II
mGlu receptor agonist 2R,4R-APDC in 51 of these neurons. Application of
2R,4R-APDC (100 µM) inhibited the Ba2+ current in 15 of
51 (29.4%) cells tested by 49.7 ± 5.7% (Fig. 1). Inhibition of
the Ba2+ current was reversible on removal of the agonist.
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G protein and Ca2+-dependent effects
by group II mGlu receptors.
Group II mGlu receptors have been previously shown to be
Gi-coupled, negative modulators of L- and N-type
Ca2+ channels in electrophysiological studies using brain
slices, neuronal cultures, and heterologous expression systems
(7, 30). To investigate whether a G protein is involved in
the group II mGlu receptor-induced inhibition of the Ba2+
current in myenteric neurons, we tested whether the inhibitory effects
of DCG-IV could be blocked by PTX. Preincubation of neurons with PTX
(400 ng/ml) resulted in a significant (P < 0.05)
reduction in the inhibition of the peak Ba2+ current
produced by DCG-IV (100 µM; Fig.
4A).
Whereas the inhibition produced by DCG-IV was
45.2 ± 8.3% (n = 14), in untreated cells, toxin-treated neurons responded to DCG-IV with a mean inhibition of
20.7 ± 3.1% (n = 6). These results indicate that
PTX-sensitive inhibitory G proteins participate in the inhibitory
effects of DCG-IV on the Ba2+ current.
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mGlu2/3 receptor immunoreactivity is displayed by enteric neurons. The observation that group II mGlu receptor agonists reduce the Ba2+ current in enteric neurons suggests that these cells express functional group II mGlu receptors. Immunocytochemistry was therefore employed to investigate the distribution of group II mGlu receptors in the guinea pig ENS.
mGlu2/3 receptor immunoreactivity was displayed by a subset of neurons in isolated myenteric ganglia (Fig. 5A). Immunoreactivity was primarily associated with the cytoplasm of cells, although clusters of immunoreactivity were sometimes found on neurites. A similar distribution was found in submucosal (Fig. 5B) and myenteric (Fig. 5C) ganglia in whole-mount preparations of ileum. In addition, a subset of the neurons that expressed mGlu2/3 receptors contained calbindin (Figs. 5D), a marker of intrinsic primary afferent neurons. Isolated myenteric ganglia also contained
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DISCUSSION |
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In this study, we demonstrate for the first time the expression of functional group II mGlu receptors in the guinea pig ENS. We have shown 1) that the group II mGlu receptor agonists, 2R,4R-APDC and DCG-IV, but not the group I agonist S-DHPG, inhibit voltage-gated Ca2+ currents in myenteric neurons; 2) that this inhibition appears to be due to the inhibition of Ca2+ current through N-type Ca2+ channels; and 3) that the inhibitory pathway involves the participation of PTX-sensitive G proteins. In addition, immunohistochemistry revealed that enteric neurons display mGlu2/3 receptor immunoreactivity and are contacted by VGLUT2-immunoreactive nerve terminals. These findings indicate that glutamatergic nerve terminals innervate enteric neurons and that the amino acid could modulate the activity of the cells by activating group II mGlu receptors.
Approximately 30% of myenteric neurons in isolated ganglia responded to group II mGlu receptor agonists with an inhibition of the Ba2+ current. The agonists' potency was 2R,4R-APDC > DCG-IV >L-glutamate > L-CCG-I, and this order is fairly similar to the pharmacological profile of the group II mGlu receptors in freshly dissociated rat cerebellar neurons (19). 2R,4R-APDC was the most potent of the group II mGlu receptor agonists examined, with an EC50 of 2 µM, and there was little desensitization or rundown in the inhibitory effect of the agonist. The maximum degree of inhibition was ~50%. 2R,4R-APDC is the most selective agonist of mGlu2/3 receptors (26); therefore, the potency of the compound in inhibiting the Ba2+ current in myenteric neurons is consistent with the expression of mGlu2/3 receptor immunoreactivity in these cells. Lack of inhibition of S-DHPG on Ca2+ currents was expected, because in general, group I mGlu receptors activate phospholipase C and increase neuronal excitability (9, 21).
DCG-IV and its congener L-CCG-I were also effective in inhibiting the Ba2+ current in myenteric neurons, with EC50 values of 3 and 10 µM, respectively. DCG-IV and L-CCG-I are widely used as mGlu2/3 receptor agonists, and the inhibitory effect of these compounds on the Ba2+ current in myenteric neurons is consistent with reports about their inhibition of Ca2+ currents in CNS neurons (19). DCG-IV has also been reported to act as an antagonist at group III mGlu receptors (3). L-CCG-I can activate all mGlu receptor subtypes in the micromolar range (26). The EC50 value for L-CCG-I obtained in the present study is within the micromolar range; therefore, the effect of L-CCG-I may not be mediated exclusively by its affinity for group II mGlu receptors.
A major role for group II mGlu receptors in the inhibition of Ca2+ currents in myenteric neurons is also supported by the experiments with the potent and competitive group II mGlu receptor antagonist LY- 341495 (18). LY-341495 significantly blocked the inhibition of the Ba2+ current produced by 2R,4R-APDC and L-glutamate, confirming an involvement of group II mGlu receptors. In addition to its effect at group II mGlu receptors, LY-341495 has previously been shown to block the activation of group I mGlu receptors (8). However, LY-341495 has a much lower potency at group I mGlu receptors, which makes it unlikely that this subtype of mGlu receptors mediates the effect seen in the present study.
Our observations add to accumulating evidence that group II mGlu
receptors modulate distinct Ca2+ channel subtypes. At least
three types of Ca2+ channels have been reported in guinea
pig myenteric neurons: N-, L-, and P-type (13, 17, 31).
Our study provides evidence that the N-type Ca2+ channels
are the main targets of the inhibitory effects of the group II mGlu
receptors, because after the channels were blocked with -conotoxin
GVIA, the receptors produced a much smaller percent inhibition of the
small, remaining Ca2+ current. In addition, application of
nifedipine, a well-known L-type Ca2+ channel blocker, did
not antagonize the DCG-IV-induced inhibition of the Ba2+
current. The small effect remaining in the presence of
-conotoxin GVIA most likely represents residual unblocked N-type current, because
we used a low concentration of toxin (2.5 nM).
Inhibition of N-type channels by group II mGlu receptor agonists occurs through a G protein mechanism of action, because incubation of neurons with PTX significantly reduced the response. The PTX-sensitive Go and Gi proteins mediate most of the inhibitory effects on Ca2+ channels. Group II mGlu receptors are coupled to Gi proteins in heterologous expression systems, and activation of these receptors reduces cAMP formation (25). Thus it is likely that inhibition of the Ba2+ current in myenteric neurons involves Gi, although this needs to be demonstrated in future studies.
Due to the recording methods, we were not able to
electrophysiologically determine which type of neurons express group II mGlu receptors. If intrinsic primary afferent neurons are among the
cells that express group II mGlu receptors, glutamate could modulate
enteric reflexes by directly modifying the excitability and/or
transmission of these cells. In support of this idea, we observed that
a subset of mGlu2/3 receptor-immunoreactive neurons contain calbindin.
Calbindin is a marker of intrinsic primary afferent neurons in the
guinea pig ileum, which are characterized by a slow
afterhyperpolarization (11). In addition,
calbindin-containing neurons display 1B-immunoreactivity
(17), confirming recent electrophysiological data that
most of the Ca2+ channels in guinea pig myenteric sensory
neurons belong to the N-type (29). Interestingly,
intrinsic primary afferent neurons resemble those of nodose ganglion
cells in which activation of group II mGlu receptors has been shown to
modulate sensory transmission (6).
In summary, this study has demonstrated for the first time the presence of functional group II mGlu receptors in the guinea pig ENS. Future studies aimed at characterizing the neurons that express group II mGlu receptors and the effects of specific agonists and antagonists on intestinal secretion and motility will provide insight into their role in gut function.
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ACKNOWLEDGEMENTS |
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This study was supported by National Institute of Neurological Disorders and Stroke Grant NS-35951.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. Kirchgessner, GlaxoSmithKline, NFSP, Third Ave., Harlow, Essex CM19 5AW United Kingdom (E-mail: Annette_2_Kirchgessner{at}gsk.com).
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.
August 28, 2002;10.1152/ajpgi.00216.2002
Received 7 June 2002; accepted in final form 20 August 2002.
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