1 Center for Gastroenterological Research and 2 Department of Physiology, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We studied the effect of excitatory
neurotransmitters (105 M) on the
intracellular Ca2+ concentration
([Ca2+]i) of cultured myenteric
neurons. ACh evoked a response in 48.6% of the neurons. This response
consisted of a fast and a slow component, respectively mediated by
nicotinic and muscarinic receptors, as revealed by specific agonists
and antagonists. Substance P evoked a
[Ca2+]i rise in 68.2% of the
neurons, which was highly dependent on Ca2+ release from
intracellular stores, since after thapsigargin (5 µM) pretreatment
only 8% responded. The responses to serotonin, present in 90.7%, were
completely blocked by ondansetron (10
5
M), a 5-HT3 receptor antagonist. Specific agonists of other
serotonin receptors were not able to induce a
[Ca2+]i rise. Removing
extracellular Ca2+ abolished all serotonin and fast ACh
responses, whereas substance P and slow ACh responses were more
persistent. We conclude that ACh-induced signaling involves both
nicotinic and muscarinic receptors responsible for a fast and a more
delayed component, respectively. Substance P-induced signaling requires
functional intracellular Ca2+ stores, and the
5-HT3 receptor mediates the serotonin-induced Ca2+ signaling in cultured myenteric neurons.
enteric nervous system; calcium channel; enteric neuron
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE MYENTERIC PLEXUS NEURONS control various functions of the gastrointestinal tract. Intracellular Ca2+ concentration ([Ca2+]i) can be used to monitor activation of these neurons (32). In culture, myenteric neurons form new networks that can be used as a model for the enteric nervous system. Focal electrical stimulation of the neuronal processes induces a transient [Ca2+]i rise in a subset of the cultured neurons. These [Ca2+]i changes require neuronal conduction and excitatory synaptic transmission (40). The constitutive elements of synaptic communication between neurons are the fast and slow excitatory postsynaptic potentials (EPSPs). In whole-mount myenteric plexus preparations, it has been established that the majority of fast EPSPs are due to the activation of nicotinic ACh receptors, allowing cations to flow through the receptor channel (38). However, fast synaptic events are not restricted to nicotinic receptors. The activation of the serotonin 5-HT3 receptor and the purinergic P2x receptor, as well as ligand-gated cation channels, induce similar brief depolarizations (3, 5, 8, 24, 34, 35).
At present, there is still debate on which neurotransmitters control slow EPSPs. The main candidates are substance P (SP) and serotonin (5-HT) (6, 17, 25, 42, 43). The activation of the neurokinin (NK)1 receptor by SP induces a slow depolarization quite similar to the electrically evoked slow EPSPs (10, 16). Although NK1 seems to be predominant in the enteric nervous system, there is also evidence that NK3 receptors are involved in neurotransmission (15, 31). The activation of the atypical 5-HT1p receptor induces a similar slow depolarization in several types of myenteric neurons (1, 7, 12, 23, 24, 35). Besides SP and 5-HT, it is also suggested that ACh mediates slow EPSPs by activating muscarinic receptors (26, 28).
We aimed to demonstrate the existence of these candidate neurotransmitters in the myenteric neuron cultures and to confirm that they mimic the [Ca2+]i changes evoked by electrical stimulation of interconnecting fibers in cultured myenteric networks (40). Furthermore, we wanted to explore in more detail the underlying mechanism of Ca2+ signaling in myenteric neurons.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Myenteric Neuron Cultures
Primary cultures of myenteric neurons were prepared from adult guinea pig small intestine according to previously described methods (11, 14). In brief, the longitudinal muscle and the adherent myenteric plexus (LMMP) were dissected from the circular muscle and the mucosa; the LMMP was digested in an enzymatic solution containing protease (1 mg/ml) and collagenase (1.25 mg/ml). After a 30-min incubation (37°C), the suspension was placed on ice and spun at 1,600 rpm. Ganglia were picked up and plated in Lab-tek culture dishes (GIBCO, Merelbeke, Belgium), in which they adhered to the cover-glass bottom. After a few days, neurons started growing in networklike structures reminiscent of the ganglionated plexus. The culture medium was changed every two days. Medium 199 enriched with 10% fetal bovine serum and 50 ng/ml 7s nerve growth factor (7sNGF) was used. Antibiotics (streptomycin, 100 µg/ml; penicillin, 100 U/ml; amphotericin B, 1 µg/ml; gentamicin, 50 µg/ml) were added to the medium, glucose concentration was elevated to 30 mM, and the Ca2+ concentration was adjusted to 2.5 mM by adding CaCl2. The culture chambers were kept in an incubator at 37°C and continuously gassed with 95% O2-5% CO2. Arabinose-C-furanoside (10 µM) was added to prevent the proliferation of dividing cells (glial cells, fibroblasts).Experimental Medium
Experiments were performed in a modified Krebs solution containing (in mM) 150 NaCl, 6 KCl, 1.5 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES (pH 7.38). The Ca2+-free solution contained 2.5 mM MgCl2 and 2 mM EDTA. The high-K+ medium contained 75 mM K+. Osmolarity was adjusted by lowering the Na+ concentration to 81 mM. The pH of the modified Krebs solution was checked after solving the agonists and adjusted when necessary.[Ca2+]i Measurements
The neurons were loaded for 30-45 min at room temperature in a modified Krebs solution containing 5 µM indo 1-AM and 2.5 µM Pluronic F-127 (25% wt/wt). The AM is cleaved by esterases in the cytoplasm, and the free indo 1 molecule is trapped in the cell. On binding to Ca2+, the emission peak of the indo 1 molecule is shifted from 480 nm to 405 nm. The ratio of these two signals is directly proportional to the free [Ca2+]i. Experiments were performed at 22°C on a Bio-Rad MRC 1024 confocal microscope equipped with an Ar+ ion laser. Sample frequency depended on the number of "regions of interest" selected and ranged between 1.25 and 1.75 Hz. All time experiments were monitored using Laser-sharp software. The results were expressed as a ratio of emitted fluorescence. The amplitude of the signals and the peak duration, defined as the width at half-height, were analyzed and compared with a Student's t-test.Immunocytochemistry
Cultured cells were fixed in a freshly prepared phosphate buffered (pH 8.0) 2%/0.2% paraformaldehyde/picric acid solution. The cells were permeabilized (2 h), and nonspecific binding sites were blocked by treating the cells for 1 h in a 0.1 M PBS solution containing 0.5% (wt/vol) Triton X-100 and 4% (vol/vol) goat serum. Primary antisera were diluted in this blocking solution, and preparations were exposed for ~20 h at room temperature. To prevent bacterial growth, NaN3 was added to the incubation solution in a 0.3% (wt/vol) concentration. After incubation in the primary antibody solution, dishes were rinsed 3 × 5 min in a 0.1 M PBS solution and incubated in the appropriate fluorescence-labeled secondary antibodies (2 h, 0.1 M PBS, 4% goat serum). The fluorophores were visualized under a Nikon microscope equipped with a fluorescence unit. For Cy3, filtercube Y-2EC (EX BP540-580, DM RK595, EM BA 600-660) was used. Filtercube UV-2EC (EX BP340-380, DM NDM400, EM BA 435-485) and B-2A (EX BP470-490, DM 505, EM BA 510/20) were used for the visualization of aminomethylcoumarin and FITC, respectively.Chemicals
Medium 199 and antibiotics were from GIBCO; ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Differential Properties of Neurons and Nonneuronal Cells in Culture
Morphology and immunocytochemistry. The myenteric neuron cultures were not completely void of other cell types such as glial cells and fibroblasts. The latter two cell types had flat cell bodies and stuck closely to the glass. On the contrary, neuronal cell bodies were rather thick and could easily be distinguished under phase-contrast optics. Subsequent immunocytochemical processing for NSE (data not shown) confirmed the morphological identification.
Voltage-operated Ca2+
channels.
All cells were briefly depolarized with 75 mM K+, which
opened the voltage-operated Ca2+ channels (VOCC) and led to
a Ca2+ influx. A one-to-one correlation was observed
between the cells with a high-K+-induced
[Ca2+]i response and the neurons as
identified morphologically and by NSE staining. Nonneuronal cells
seemed to lack VOCCs, and therefore the high-K+-induced
[Ca2+]i response (Fig.
1A) could be used as a
physiological means to distinguish between the cells.
|
Capacitative Ca2+ entry. Thapsigargin (Tg) prevents the refilling of the intracellular Ca2+ stores by irreversibly blocking their ATP-dependent Ca2+ pump. The acute administration of 5 µM Tg resulted in a [Ca2+]i rise in 96% of the cultured myenteric neurons (n = 48), indicating that the stores were Tg sensitive. In 60% of the neurons, the responses had a typical oscillatory character (Fig. 1B). Tg-pretreated (5 µM, 1 h) neurons were then used to screen for the presence of capacitative Ca2+ entry, which allows the Ca2+ entry in cells with depleted Ca2+ stores. Tg-pretreated cells with capacitative Ca2+ entry display characteristic [Ca2+]i signaling. When the extracellular Ca2+ concentration ([Ca2+]e) is changed from 1.5 mM to 0 mM, a decrease in [Ca2+]i can be observed. When 1.5 mM Ca2+ is restored, the [Ca2+]i suddenly increases (37). In all nonneuronal Tg-treated cells (n = 25), the [Ca2+]i dropped below the baseline when 0 mM [Ca2+]e was administered and a [Ca2+]i overshoot could be observed after readjusting the [Ca2+]e to 1.5 mM (Fig. 1A). Myenteric neurons did not display this typical signaling. A small [Ca2+]i rise was only observed in 20% of the neurons. Because of the low amplitude and the lack of a preceding [Ca2+]i drop, these responses were judged not characteristic of capacitative Ca2+ entry. In the majority of the myenteric neurons (80%), no [Ca2+]i changes were observed (n = 109) (Fig. 1A).
Presence of the Candidate Neurotransmitters
Fixed culture cells were processed for ChAT, SP, and 5-HT immunoreactivity. The majority of the neurons expressed ChAT, the main enzyme in the synthesis of ACh, and could therefore be considered cholinergic (Fig. 2). Immunoreactivity for SP was readily observed in the majority of fibers in the network. Although 5-HT occurrence was more limited, some fibers were clearly 5-HT positive.
|
Neurotransmitters and [Ca2+]i Signaling
ACh-induced
[Ca2+]i
signaling.
ACh induced a [Ca2+]i rise in a
subset of the myenteric neurons in the network. The amplitude of the
[Ca2+]i rise, as well as the number
of responding cells, increased dose dependently. Application of
107 M ACh (n = 50) caused a
detectable rise in 2.0% of the neurons, whereas 93.0% of the cells
displayed a [Ca2+]i rise with
10
4 M ACh (n = 86) (Fig.
3A). The blockade of neuronal
conduction by 10
6 M TTX did not alter
the number of responding cells (n = 20). Selective nicotinic
activation with 1,1-dimethyl-4-phenyl-piperazine (DMPP) evoked a
[Ca2+]i rise in 28% (n = 68) and 93.5% (n = 154) of the neurons for 10
6 M and
10
5 M, respectively. Repeated
application of ACh and DMPP revealed no significant reduction in the
number of responding cells. The shape of the
[Ca2+]i transient induced by DMPP
and ACh clearly differed. Almost all responses to DMPP reached the
maximum within 1-2 s (98.8%, n = 79), whereas ACh also
evoked slow responses. This slow response subgroup, which was absent
for DMPP, accounted for 9.8% (n = 81).
|
|
|
Substance P-induced
[Ca2+]i
signaling.
SP induced a [Ca2+]i rise in a
subset of the cultured myenteric neurons. Low concentrations
(1010 and
10
9 M) did not evoke a detectable
[Ca2+]i rise (n = 43 and
63). The number of neurons responding to higher concentrations
increased logarithmically (12.9% for
10
8 M, 17.6% for
10
7 M, 54.1% for
10
6 M, and 68.2% for
10
5 M, n = 85), reaching 85% of
the cells for 10
4 M (n = 20)
(Fig. 6A). The relative
amplitude compared with the K+-induced response also
increased (5.4 ± 1.3%, 7.6 ± 1.8%, 14.1 ± 3.0%, and 18.9 ± 2.9% for 10
8 M,
10
7 M,
10
6 M,
10
5 M, respectively, n = 7). TTX
(10
6 M) did not alter the number of
responses to SP (n = 39). In the absence of extracellular
Ca2+, SP still induced responses in a number of cells;
however, the number of responses to SP was significantly reduced (16.6 vs. 62.6%, P = 0.01). The amplitude of the remaining responses
was reduced to 47.3 ± 12%. The L-type Ca2+-channel
blocker nifedipine (10
6 M) did not alter
either the number of responses or the amplitude of the response (58.3%
and 93.6 ± 12%, respectively). Similarly, blockade of N-type
Ca2+ channels by
-conotoxin (5 × 10
7 M) did not significantly affect the
number and the amplitude of the SP-induced
[Ca2+]i rises (45.5% and 77 ± 16%, respectively).
|
5-HT-induced
[Ca2+]i
signaling.
The application of 106 M 5-HT induced a
[Ca2+]i rise in 25.6% of the
neurons (n = 122), and 90.7% responded when
10
5 M 5-HT was applied (n = 388). The relative amplitude of the response also increased dose
dependently. The application of 5-HT induced a
[Ca2+]i rise, with a relative
amplitude of 9.1 ± 1.0% (10
6 M,
n = 9) and of 45.3 ± 3.6%
(10
5 M, n = 43) compared with
the K+-induced response. No 5-HT-induced responses could be
observed when Ca2+ was removed from the extracellular
medium (n = 14). TTX did not alter the number of responses to
5-HT (n = 27). Nifedipine (10
6
M) and
-conotoxin (5 × 10
7 M) did
not alter the number of responses but significantly reduced the
amplitude of the 5-HT-induced responses to 47.2 ± 5% (P < 0.01) and 87 ± 4.4% (P = 0.04), respectively. The response
to 5-HT was also tested in Tg-pretreated (5 µM, 1 h) neurons. The number of responses to 5-HT was not significantly altered for 10
6 M (28.57% vs. 25.41%)
(n = 28) and for 10
5 M
(81.48% vs. 90.7%).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The results of this study add further to the understanding of how neurotransmitters induce a [Ca2+]i rise in myenteric neurons. Excitatory neurotransmitters (ACh, SP, and 5-HT) are endogenously present in the extensively branching networks of cultured myenteric neurons. The exogenous application of these neurotransmitters induced a [Ca2+]i rise via different mechanisms, mimicking the transient [Ca2+]i changes observed by electrically-induced synaptic transmission (40).
The basics of [Ca2+]i signaling in myenteric neurons are incompletely known. Previously, we demonstrated that several types of Ca2+ channels contribute to the high-K+-induced Ca2+ entry in cultured myenteric neurons (40). With respect to intracellular Ca2+ stores, inositol trisphosphate (IP3) as well as ryanodine-sensitive stores have been described (20). At present, the exact role of IP3 and RyR in myenteric neurons is poorly understood. A third possible Ca2+ source would be the entry of extracellular Ca2+ through capacitative Ca2+ entry. As far as intestinal cells are concerned, capacitative Ca2+ entry has been described for myocytes (39, 44) and enteric glial cells (29, 45). In line with other neuronal cells, we found no evidence for capacitative Ca2+ entry in myenteric neurons.
ACh, SP, and 5-HT induced a [Ca2+]i rise in a dose-dependently increasing number of cells. Kimball and Mulholland (19) reported a similar dose-dependent increase. This effect might be explained by the fact that, especially for the low concentrations, small responses might be lost in the noise of the signal, resulting in an underestimation of the number of responses. Another possible reason might be that secondary neurotransmitters were released, inducing a [Ca2+]i rise in other neurons. If present, this should be an action potential-independent mechanism, since TTX had no effect on the number of responses. Finally, agonists applied in high concentration may activate neurons with a limited receptor density.
Although Simeone et al. (33) reported that the M1 receptor
controlled the ACh-induced [Ca2+]i
signaling in myenteric neurons, our data show that both nicotinic and
muscarinic receptors are involved. Selective activation of the
nicotinic receptor, a ligand-gated cation channel, induced an increase
in [Ca2+]i. It has been shown that
DMPP activates a Ca2+ current through the receptor channel
itself (38). Since in 0 mM [Ca2+]e
no responses to DMPP persisted, we may conclude that the fast phase of
the response was due to direct Ca2+ influx through the
receptor channel. The muscarinic receptor apparently induced a
prolonged [Ca2+]i elevation, since
atropine significantly reduced the duration of the responses. Moreover,
when atropine was administered together with ACh, the
[Ca2+]i instantly dropped to its
basal concentration, mimicking the nicotinic response. Only few
responses persisted in the presence of C6, and these were all slow
responses. The fact that ACh in the presence of atropine mimicked the
shape of the DMPP response provides evidence for the hypothesized role
of both receptors (Fig. 9). In line with
data of Simeone et al. (33), we also found that it was indeed the
muscarinic M1 receptor that was involved in the muscarinic
[Ca2+]i signaling in myenteric
neurons, whereas the M2 receptor did not contribute.
Although the amplitude of the response was slightly reduced, the number
of responses to ACh was not altered in the presence of an L- or N-type
Ca2+-channel blocker. This suggests that VOCCs mainly
contribute to the amplitude and maintenance of the elevated
[Ca2+]i rather than to the
initiation of the response. The activation of VOCCs was probably due to
membrane depolarization induced by ACh. Involvement of N-type
Ca2+ channels was also suggested by Takahashi et al. (36).
|
We showed that SP induced a dose-dependent [Ca2+]i rise in myenteric neurons. Sarosi et al. (30) reported that this is mediated through an NK1 receptor. L-type and N-type Ca2+-channel blockade did not alter either the number of responses to SP or the amplitude of the signal. These data contrast with the findings of Sarosi et al. (30), who reported that [Ca2+]i signaling induced by SP was mainly due to influx through an L-type Ca2+ channel, whereas the intracellular stores were less important. However, the inhibition by Tg pretreatment suggests that the SP-induced [Ca2+]i rise did rely on Ca2+ release from intracellular Ca2+ stores. In addition, since the inactivation of RyR had no effect, we indirectly demonstrated that SP, also in myenteric neurons, activates an IP3-dependent pathway. Similar to other cell types (18), a PTX-insensitive G protein is involved in the signaling cascade induced by SP.
The intracellular Ca2+ stores do not play a crucial role in
the control of the 5-HT-induced signaling. The 5-HT3
receptor mediates short-lived depolarizations in the myenteric neurons
of several regions in the gut. We showed that this 5-HT receptor was
essential for the 5-HT-induced
[Ca2+]i signaling. Specific
blockade abolished the [Ca2+]i
response in almost all cells. Therefore, we can conclude that at least
the onset of the [Ca2+]i rise was
mediated by this receptor. Since the 5-HT3 receptor is a
ligand-gated cation channel (5), we may expect that a direct influx
through the receptor channel is responsible for the initiation of the
response. Activation of VOCCs might lead to an additional influx of
extracellular Ca2+, which might explain the reduced
amplitude in the presence of nifedipine and -conotoxin.
We observed no [Ca2+]i changes in response to either 5-HT1a or 5-HT1p receptor activation. The absence of a 5-HT1a response is in line with the general hyperpolarizing action of this receptor. Surprisingly, the activation of the 5-HT1p receptor, which induces a depolarization in a subset of myenteric neurons, did not induce a [Ca2+]i rise in the cultured neurons. This might be due to the fact that the 5-HT1p-induced depolarization was too small to activate VOCCs or, alternatively, that the 5-HT1p receptor was not expressed in culture. We observed that 5-HT1-2 blockade with methiothepin suppressed the [Ca2+]i rise induced by 5-HT. Although 5-HT2a-c receptors are present in the gastrointestinal tract, these receptors seem to be located on smooth muscle cells (13, 21). The only evidence for neuronal 5-HT2-like receptors was found in the guinea pig colon (2). However, electrophysiological studies failed to demonstrate the presence of a 5-HT2-like receptor on guinea pig myenteric neurons. Alternatively, Crespi et al. (4) also described a nonspecific effect of methiothepin on an intracellular target common to the 5-HT and dopamine release mechanisms. Although in snail neurons, methiothepin inhibits the cAMP-dependent L-type Ca2+ channel phosphorylation (22), its mode of action in myenteric neurons remains to be further elucidated. 5-HT4 receptor antagonism did not influence the response to 5-HT. The 5-HT4 receptor is positively coupled to adenylate cyclase. Direct activation of the adenylate cyclase with forskolin depolarizes the membrane of AH/type II neurons both in the myenteric plexus in situ (27) and in culture (11). However, forskolin did not induce a [Ca2+]i rise in the cultured cells. This may imply that no [Ca2+]i rise is involved in the cAMP pathway, or, alternatively, that this pathway was absent in the cultures because of a phenotypic shift.
In summary, we studied in detail the Ca2+ signaling induced by three major excitatory neurotransmitters on cultured myenteric neurons of the small intestine. For each of the three neurotransmitters, we have investigated the mechanism by which the [Ca2+]i rise is induced. Both muscarinic and nicotinic receptors were involved in the control of the ACh-induced [Ca2+]i rise. Activation of nicotinic receptors, which are ligand-gated channels, was accompanied by Ca2+ influx generating a fast rise in [Ca2+]i. Activation of M1 receptors induces a prolonged [Ca2+]i rise. Release of Ca2+ from intracellular stores was essential in the SP-induced signaling, whereas 5-HT-induced Ca2+ signaling was activated by the 5-HT3 receptor, also a ligand-gated channel. It is clear that these data obtained from cultured neurons should be interpreted with caution, especially when extrapolating to in vivo situations. Studies on neurons in situ will be needed to assess the extent to which [Ca2+]i signaling was affected by the culturing procedure.
![]() |
ACKNOWLEDGEMENTS |
---|
The antibody for ChAT was kindly provided by Dr. Michael Schemann, Hannover, Germany.
![]() |
FOOTNOTES |
---|
Parts of this study were already published in abstract form in Neurogastroenterol Motil (10: 106 and 107, 1998) and Gastroenterology (114: A1187 and A1187, 1998).
The "Principles of Laboratory Animal Care" were followed as well as the specific national laws of the Ministerie van Landbouw, Belgium.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. Tack, Center for Gastroenterological Research, Catholic Univ. Leuven, B-3000 Leuven, Belgium (E-mail: jan.tack{at}med.kuleuven.ac.be).
Received 27 September 1999; accepted in final form 6 January 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Branchek, TA,
Mawe GM,
and
Gershon MD.
Characterization and localization of a peripheral neural 5-hydroxytryptamine receptor subtype (5-HT1P) with a selective agonist, 3H-5-hydroxyindalpine.
J Neurosci
8:
2582-2595,
1988[Abstract].
2.
Briejer, MR,
Akkermans LM,
Lefebvre RA,
and
Schuurkes JA.
Novel 5-HT2-like receptor mediates neurogenic relaxation of the guinea-pig proximal colon.
Eur J Pharmacol
279:
123-133,
1995[ISI][Medline].
3.
Christofi, FL,
Guan Z,
Wood JD,
Baidan LV,
and
Stokes BT.
Purinergic Ca2+ signaling in myenteric neurons via P2 purinoceptors.
Am J Physiol Gastrointest Liver Physiol
272:
G463-G473,
1997
4.
Crespi, D,
Mennini T,
and
Gobbi M.
Carrier-dependent and Ca2+-dependent 5-HT and dopamine release induced by (+)-amphetamine, 3,4-methylendioxymethamphetamine, p-chloroamphetamine and (+)-fenfluramine.
Br J Pharmacol
121:
1735-1743,
1997[Abstract].
5.
Derkach, V,
Surprenant A,
and
North RA.
5-HT3 receptors are membrane ion channels.
Nature
339:
706-709,
1989[ISI][Medline].
6.
Erde, SM,
Sherman D,
and
Gershon MD.
Morphology and serotonergic innervation of physiologically identified cells of the guinea pig's myenteric plexus.
J Neurosci
5:
617-633,
1985[Abstract].
7.
Frieling, T,
Cooke HJ,
and
Wood JD.
Serotonin receptors on submucous neurons in guinea pig colon.
Am J Physiol Gastrointest Liver Physiol
261:
G1017-G1023,
1991
8.
Galligan, JJ,
and
Bertrand PP.
ATP mediates fast synaptic potentials in enteric neurons.
J Neurosci
14:
7563-7571,
1994[Abstract].
9.
Gershon, MD.
Serotonin: its role and receptors in enteric neurotransmission.
Adv Exp Med Biol
294:
221-230,
1991[Medline].
10.
Hanani, M,
and
Burnstock G.
The actions of substance P and serotonin on myenteric neurons in tissue culture.
Brain Res
358:
276-281,
1985[ISI][Medline].
11.
Hanani, M,
Xia Y,
and
Wood JD.
Myenteric ganglia from the adult guinea-pig small intestine in tissue culture.
Neurogastroenterol Motil
6:
103-118,
1994[ISI].
12.
Hillsley, K,
and
Mawe GM.
5-HT is present in nerves of guinea pig sphincter of Oddi and depolarizes sphincter of Oddi neurons.
Am J Physiol Gastrointest Liver Physiol
275:
G1018-G1027,
1998
13.
Hoyer, D,
and
Martin GR.
Classification and nomenclature of 5-HT receptors: a comment on current issues.
Behav Brain Res
73:
263-268,
1996[ISI][Medline].
14.
Jessen, KR,
Saffrey MJ,
and
Burnstock G.
The enteric nervous system in tissue culture. I. Cell types and their interactions in explants of the myenteric and submucous plexuses from guinea pig, rabbit and rat.
Brain Res
262:
17-35,
1983[ISI][Medline].
15.
Johnson, PJ,
Bornstein JC,
and
Burcher E.
Roles of neuronal NK1 and NK3 receptors in synaptic transmission during motility reflexes in the guinea-pig ileum.
Br J Pharmacol
124:
1375-1384,
1998[Abstract].
16.
Johnson, SM,
Katayama Y,
Morita K,
and
North RA.
Mediators of slow synaptic potentials in the myenteric plexus of the guinea-pig ileum.
J Physiol (Lond)
320:
175-186,
1981[ISI][Medline].
17.
Katayama, Y,
and
North RA.
Does substance P mediate slow synaptic excitation within the myenteric plexus?
Nature
274:
387-388,
1978[ISI][Medline].
18.
Khawaja, AM,
and
Rogers DF.
Tachykinins: receptor to effector.
Int J Biochem Cell Biol
28:
721-738,
1996[ISI][Medline].
19.
Kimball, BC,
and
Mulholland MW.
Neuroligands evoke calcium signaling in cultured myenteric neurons.
Surgery
118:
162-170,
1995[ISI][Medline].
20.
Kimball, BC,
Yule DI,
and
Mulholland MW.
Caffeine- and ryanodine-sensitive Ca2+ stores in cultured guinea pig myenteric neurons.
Am J Physiol Gastrointest Liver Physiol
270:
G594-G603,
1996
21.
Kuemmerle, JF,
Murthy KS,
Grider JR,
Martin DC,
and
Makhlouf GM.
Coexpression of 5-HT2A and 5-HT4 receptors coupled to distinct signaling pathways in human intestinal muscle cells.
Gastroenterology
109:
1791-1800,
1995[ISI][Medline].
22.
Lukyanetz, EA,
and
Kostyuk PG.
Two distinct receptors operate the cAMP cascade to up-regulate L-type Ca channels.
Pflügers Arch
432:
174-181,
1996[ISI][Medline].
23.
Mawe, GM,
Branchek TA,
and
Gershon MD.
Blockade of 5-HT-mediated enteric slow EPSPs by BRL 24924: gastrokinetic effects.
Am J Physiol Gastrointest Liver Physiol
257:
G386-G396,
1989
24.
Michel, K,
Sann H,
Schaaf C,
and
Schemann M.
Subpopulations of gastric myenteric neurons are differentially activated via distinct serotonin receptors: projection, neurochemical coding, and functional implications.
J Neurosci
17:
8009-8017,
1997
25.
Morita, K,
North RA,
and
Katayama Y.
Evidence that substance P is a neurotransmitter in the myenteric plexus.
Nature
287:
151-152,
1980[ISI][Medline].
26.
Morita, K,
North RA,
and
Tokimasa T.
Muscarinic agonists inactivate potassium conductance of guinea-pig myenteric neurones.
J Physiol (Lond)
333:
125-139,
1982[Abstract].
27.
Nemeth, PR,
Palmer JM,
Wood JD,
and
Zafirov DH.
Effects of forskolin on electrical behaviour of myenteric neurones in guinea-pig small intestine.
J Physiol (Lond)
376:
439-450,
1986[Abstract].
28.
North, RA,
Slack BE,
and
Surprenant A.
Muscarinic M1 and M2 receptors mediate depolarization and presynaptic inhibition in guinea-pig enteric nervous system.
J Physiol (Lond)
368:
435-452,
1985[Abstract].
29.
Sarosi, GA,
Barnhart DC,
Turner DJ,
and
Mulholland MW.
Capacitative Ca2+ entry in enteric glia induced by thapsigargin and extracellular ATP.
Am J Physiol Gastrointest Liver Physiol
275:
G550-G555,
1998
30.
Sarosi, GA, Jr,
Kimball BC,
Barnhart DC,
Zhang W,
and
Mulholland MW.
Tachykinin neuropeptide-evoked intracellular calcium transients in cultured guinea-pig myenteric neurons.
Peptides
19:
75-84,
1998[ISI][Medline].
31.
Schemann, M,
and
Kayser H.
Effects of tachykinins on myenteric neurones of the guinea-pig gastric corpus: involvement of NK-3 receptors.
Pflügers Arch
419:
566-571,
1991[ISI][Medline].
32.
Shuttleworth, CW,
and
Smith TK.
Action potential-dependent calcium transients in myenteric S neurons of the guinea-pig ileum.
Neuroscience
92:
751-762,
1999[ISI][Medline].
33.
Simeone, DM,
Kimball BC,
and
Mulholland MW.
Acetylcholine-induced calcium signaling associated with muscarinic receptor activation in cultured myenteric neurons.
J Am Coll Surg
182:
473-481,
1996[ISI][Medline].
34.
Surprenant, A,
and
Crist J.
Electrophysiological characterization of functionally distinct 5-hydroxytryptamine receptors on guinea-pig submucous plexus.
Neuroscience
24:
283-295,
1988[ISI][Medline].
35.
Tack, JF,
Janssens J,
Vantrappen G,
and
Wood JD.
Actions of 5-hydroxytryptamine on myenteric neurons in guinea pig gastric antrum.
Am J Physiol Gastrointest Liver Physiol
263:
G838-G846,
1992
36.
Takahashi, T,
Tsunoda Y,
Lu Y,
Wiley J,
and
Owyang C.
Nicotinic receptor-evoked release of acetylcholine and somatostatin in the myenteric plexus is coupled to calcium influx via N-type calcium channels.
J Pharmacol Exp Ther
263:
1-5,
1992[Abstract].
37.
Takemura, H,
and
Putney JW, Jr.
Capacitative calcium entry in parotid acinar cells.
Biochem J
258:
409-412,
1989[ISI][Medline].
38.
Trouslard, J,
Mirsky R,
Jessen KR,
Burnstock G,
and
Brown DA.
Intracellular calcium changes associated with cholinergic nicotinic receptor activation in cultured myenteric plexus neurones.
Brain Res
624:
103-108,
1993[ISI][Medline].
39.
Van Assche, G,
Depoortere I,
Missiaen L,
Janssens J,
and
Peeters TL.
Agonist-induce Ca2+ signalling in rabbit antrum and colon circular smooth muscle muscle cultures (Abstract).
Neurogastroenterol Motil
10:
106,
1998.
40.
Vanden Berghe, P,
Tack J,
Coulie B,
Andrioli A,
Bellon E,
and
Janssens J.
Synaptic transmission induces transient Ca2+ concentration changes in cultured myenteric neurons.
Neurogastroenterol Motil.
12:
117-124,
2000[ISI][Medline].
41.
Vanden Berghe, P,
Tack J,
Coulie B,
and
Janssens J.
Sumatriptan is an agonist at 5-HT1p receptors on myenteric neurons in the guinea-pig gastric antrum (Abstract).
Neurogastroenterol Motil
7:
291,
1995.
42.
Willard, AL.
Substance P mediates synaptic transmission between rat myenteric neurones in cell culture.
J Physiol (Lond)
426:
453-471,
1990[Abstract].
43.
Wood, JD,
and
Mayer CJ.
Slow synaptic excitation mediated by serotonin in Auerbach's plexus.
Nature
276:
836-837,
1978[ISI][Medline].
44.
Young, SH,
Ennes HS,
McRoberts JA,
Chaban VV,
Dea SK,
and
Mayer EA.
Calcium waves in colonic myocytes produced by mechanical and receptor-mediated stimulation.
Am J Physiol Gastrointest Liver Physiol
276:
G1204-G1212,
1999
45.
Zhang, W,
Sarosi GA, Jr,
Barnhart DC,
and
Mulholland MW.
Endothelin-stimulated capacitative calcium entry in enteric glial cells: synergistic effects of protein kinase C activity and nitric oxide.
J Neurochem
71:
205-212,
1998[ISI][Medline].