Departments of 1 Pharmacology and 2 Anesthesiology, The Ohio State University, Columbus, Ohio 43210
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The role of adenosine A1 receptors (A1R) in reflex-evoked short-circuit current (Isc) indicative of chloride secretion was studied in the guinea pig colon. The A1R antagonist 8-cyclopentyltheophylline (CPT) enhanced reflex-evoked Isc. Adenosine deaminase and the nucleoside transport inhibitor S-(4-nitrobenzyl)-6-thioinosine enhanced and reduced reflex-induced Isc, respectively. The A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA) inhibited reflex-evoked Isc at nanomolar concentrations, and its action was antagonized by CPT. In the presence of either N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide to block the 5-hydroxytryptamine (5-HT)-mediated pathway or piroxicam to block the prostaglandin-mediated pathway, CCPA reduced the residual reflex-evoked Isc. CCPA reduced the response to a 5-HT pulse without affecting the tetrodotoxin-insensitive Isc responses to carbachol or forskolin. Immunoreactivity for A1R was detected in the membrane (10% of neurons) and cytoplasm (90% of neurons) of neural protein gene product 9.5-immunoreactive (or S-100-negative) submucosal neurons, in glia, and in the muscularis mucosa. A1R immunoreactivity in a majority of neurons remained elevated in the cytoplasm despite preincubation with adenosine deaminase or CPT. A1R immunoreactivity colocalized in synaptophysin-immunoreactive presynaptic varicose nerve terminals. The results indicate that endogenous adenosine binding to high-affinity A1R on submucosal neurons acts as a physiological brake to suppress reflex-evoked Isc indicative of chloride secretion.
5-hydroxytryptamine; prostaglandin; submucous plexus; chloride secretion
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ADENOSINE IS A UBIQUITOUS molecule that has diverse actions in cardiopulmonary, renal, and gastrointestinal systems. Adenosine, a key metabolite of ATP, is distributed throughout the intracellular and extracellular compartments. Its concentration is dependent on processes related to production, release, reuptake, and metabolism (23). Extracellular levels of adenosine can rise as a consequence of increased intracellular hydrolysis of adenine nucleotides and its subsequent transport to the extracellular space or by increased ATP release into the extracellular compartment and its subsequent hydrolysis. Adenosine exerts its actions by binding to cell surface receptors belonging to the P1 purinoceptor family of receptors which includes A1, A2A, A2B, and A3 receptors cloned from human or animal sources (25). Highly specific affinity-purified anti-A1, A2A, A2B, and A3 receptor antibodies to peptide sequences of each receptor have been produced for immunohistochemistry (13, 30, 44). Because of substantial interspecies sequence homology among these receptors, the antibodies cross-react with receptors from several different species.
Adenosine A1 receptors (A1R) have been identified on isolated myenteric varicosities by ligand binding techniques (6, 7). Biochemical studies indicate that A1R activation leads to inhibition of acetylcholine and tachykinin release from presynaptic varicose nerve terminals in enteric ganglia (32). Furthermore, activation of this receptor on myenteric neurons leads to suppression of cholinergic and tachykinergic transmission to longitudinal muscle (6, 9). Electrophysiological studies provide evidence for both pre- and postsynaptic A1R inhibition of slow synaptic transmission [slow excitatory postsynaptic potentials (EPSP)] (10, 11) and presynaptic inhibition of fast synaptic transmission (fast EPSP) (11, 12). In AH/type 2 neurons, the main postsynaptic action of adenosine is A1R-mediated suppression of neuronal excitability, associated with a decrease in cell input resistance and a sustained membrane hyperpolarization lasting seconds to minutes (5, 12). In addition, adenosinergic suppression of the predominant slow excitatory synaptic inputs to myenteric neurons often reveals a robust slow inhibitory postsynaptic potential in AH/type 2 neurons (11). In contrast to findings in AH/type 2 neurons, A1R are not present in a significant proportion of cell somas of S/type 1 neurons in enteric ganglia, and, in these neurons, adenosine elevates excitability and causes a slow EPSP-like effect by activating another receptor subtype (2). In submucosal neurons of the guinea pig small intestine, adenosine also acts at presynaptic A1 sites to inhibit voltage-activated calcium currents and release of acetylcholine, a transmitter involved in nicotinic fast excitatory transmission (1, 2).
A1R belong to the family of G protein-coupled receptors. Thus far, two types of G protein-coupled receptors, namely, opioid and tachykinin receptors, have been shown to undergo receptor internalization into enteric neurons after being activated by agonists (24, 35, 40, 42). Within 30-60 min, these receptors are recycled to the membrane of enteric neurons. Because endogenous adenosine is continuously released and provides an ongoing inhibitory tone on neuronal excitability, neurotransmitter release, and synaptic transmission in the myenteric plexus (11, 32), it seems likely that a significant proportion of A1R should be internalized at all times, if indeed A1R undergo internalization and recycling.
Because activation of A1R on
enteric neurons in some regions of the bowel is linked to inhibition of
both neurotransmitter release and neuroeffector transmission, we
investigated the role of neural
A1R in the submucous plexus in the
regulation of 5-HT- and prostaglandin-mediated chloride secretory
reflexes in the colon. Chloride secretion by colonic epithelial cells
is modulated by submucosal secretomotor neurons that release the
neurotransmitters vasoactive intestinal polypeptide (VIP) and
acetylcholine (Fig. 1) (15, 38, 39). VIP
and acetylcholine bind to VIP and muscarinic M3 receptors on epithelial crypt
cells to elevate intracellular cAMP and intracellular
Ca2+ levels leading to chloride
secretion. Synaptic input to these secretomotor neurons can be
initiated by mechanical stimulation or mucosal stroking that releases
5-hydroxytryptamine (5-HT) from enterochromaffin cells and
prostaglandins from an unknown cell type (26, 39). In one pathway (Fig.
1B), 5-HT activates
5-HT1P receptors on submucosal
primary afferents containing substance P, acetylcholine, and glutamate
(15, 26, 29). The presence of NK1
receptors on cholinergic secretomotor neurons in the ileum suggests
that these neurons receive synaptic input directly from primary
afferents via release of substance P (33). Activation of primary
afferents also triggers VIP secretomotor neurons, but it is uncertain
whether this occurs directly or indirectly via interneurons (38, 39).
Prostaglandins also appear to activate submucous neurons independent of
the 5-HT-activated pathway (Fig. 1A) (16, 17, 20). VIP secretomotor
neurons in the ileum and cholinergic neurons in the colon are reported
to be targets of prostaglandins (16, 17, 20). Understanding how these reflex circuits regulate chloride secretion is important because chloride secretion provides an essential driving force for sodium movement as well as fluid accumulation necessary for lubrication or for
flushing the intestinal contents during host defense against microbial
invasion.
|
The physiological role of adenosine in the modulation of these reflexes was explored with treatments that either limit or increase the availability of endogenous adenosine at A1R or with exogenous application of agonists or antagonists. These treatments were also used to test whether activation of A1R by endogenous adenosine undergoes internalization and recycling to the membrane like other G protein-coupled receptors. Laser confocal microscopy and immunofluorescent colabeling for A1R and neuronal protein gene product 9.5 (PGP 9.5), S-100 (glial), or synaptophysin (presynaptic varicosities which surround the cell soma) were used to identify the distribution of A1R that may be involved in the reflexes. The results indicate that endogenous adenosine binding to A1R on submucous neurons acts as a physiological brake to suppress short-circuit current (Isc) indicative of chloride secretion through the 5-HT- and prostaglandin-activated neural reflex pathways in the colon. The kinetics of internalization/recycling of A1R appear to behave differently from other G protein-coupled receptors in enteric ganglia.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissues for reflex studies. Male albino Hartley guinea pigs (Harlan Sprague-Dawley, Indianapolis, IN) weighing 250-600 g were allowed food and water ad libitum. Animals were stunned and exsanguinated, a method approved by The Ohio State University Institutional Laboratory Animal Care and Use Committee. A 10- to 15-cm segment of the colon 5 cm proximal to the anus was removed, flushed with cold Krebs-Ringer solution, and cut along the mesenteric border. The longitudinal and circular muscle layers with the myenteric plexus were removed by blunt dissection to give sheets of submucosa-mucosa containing intact submucosal ganglia. All solutions were gassed with 95% O2-5% CO2 mixture and buffered at pH 7.2-7.4.
Experimental design. Conventional or modified flux chambers were used. The flux chambers were equipped with Krebs-Ringer-agar bridges connected to calomel half cells for measurement of transmural potential difference (PD) and aluminum foil electrodes for passing Isc from a voltage-clamp apparatus. Solution resistance between the PD-sensing bridges was compensated. The current necessary to change the transepithelial PD by 8 mV was used to monitor tissue conductance, a measure of tissue viability, and was calculated from Ohm's law.
To determine whether drugs had effects on basal transport, or whether they affected calcium- or cAMP-mediated secretion, conventional flux chambers were used. Drugs were added to the mucosal or serosal compartment (10 ml), and Isc, a measure of active ion transport, was monitored by a voltage-clamp apparatus (VCC600, Physiologic Instruments, Houston, TX). For studies of the effects of drugs on neural reflex-evoked secretion, muscle-stripped colonic segments were mounted in modified Ussing flux chambers with the mucosal side oriented upward (38). Both mucosal and serosal compartments (1.5 ml) were continuously perfused at a rate of 1.6 ml/min with Krebs-Ringer solution warmed to 37°C by a heat exchanger. This flow rate allowed a rapid washout of drugs from the mucosal compartment. For experiments designed to evoke neural reflexes, a 2-mm-wide brush attached to a micromanipulator was lowered to the mucosal surface. Stroking occurred with a forward or backward motion for 1 s. After two to three strokes at 5-min intervals, drugs were perfused either in the serosal or mucosal bath, and, 30-60 min later, a second stroke was applied to assess the effects of the drugs.Mucosal 5-HT pulse. A pulse of 5-HT onto the mucosal surface was previously shown to activate 5-HT1P receptors on submucosal primary afferent neurons and to activate the reflex without the complication of using a physical stimulus such as stroking that could release mediators from other sources (16, 39). A 15-µl pulse of 100 µM 5-HT into the mucosal bath (1.5 ml) was applied from a pipette held in a micromanipulator. The pipette tip was positioned at a fixed distance (2-3 mm) from the epithelial surface. An initial 5-HT pulse was given followed 30-60 min later by a second pulse.
Immunofluorescent labeling of A1R and imaging with laser scan confocal miscroscopy. Guinea pigs were stunned and exsanguinated as previously described (11). Segments of colon were removed, placed in an ice-cold Krebs solution with 2 µM nicardipine, and bubbled with a mixture of 95% O2-5% CO2. The tissue was opened and pinned flat with the luminal side up on the Sylgard base of a culture dish. Fine microdissection was performed to remove mucosa, circular muscle, myenteric plexus and longitudinal muscle layers, leaving the submucous layer intact. In the initial experiments, the tissue was fixed for 5 h at 4°C with either a modified Zamboni's fixative (2% paraformaldehyde plus 0.2% picric acid) or 0.5% paraformaldehyde and then processed for immunofluorescent labeling. Because 0.5% paraformaldehyde was not suitable for colabeling studies with anti-PGP 9.5 or anti-S-100 protein antibodies, the modified Zamboni's fixative was used in most experiments.
Colabeling experiments were conducted using antibodies against PGP 9.5 that label neurons, synaptophysin that labels varicose nerve terminals, or S-100 that labels enteric glial cells. After fixation, the tissue was washed sequentially with dimethyl sulfoxide and PBS, treated with goat serum (1:10 dilution) for 30 min at room temperature, and then incubated with primary antibodies overnight at 4°C. When PGP 9.5 was used to label neurons, the tissue was also exposed to 0.1% Triton X-100 while being incubated with goat serum. The dilution of the antibodies was 1:25-100 for A1R and 1:50-100 for other proteins. After removal of the primary antibody and washes with PBS, the tissues were simultaneously incubated with anti-rabbit Texas red-conjugated secondary antibody (1:100 dilution) and anti-mouse FITC-conjugated secondary antibody for 3 h at room temperature, washed with PBS, and mounted on slides.A1R internalization. The role of internalization and recycling to the membrane was investigated, as has been shown to occur for other G protein-coupled receptors in the gut (35, 40, 41). Two protocols were used to assess the dynamics of A1R in submucous ganglia. 1) To reduce the fluidity of the cell membrane and minimize any internalization of receptors, immediately after the colon was removed from the animal, a segment was microdissected in perfused ice-cold Krebs buffer supplemented with 2 µM nicardipine and fixed for staining. 2) Immediately after the tissues were removed from the animals, the colon segments were slit open and placed in a warm (37°C) Krebs supplemented with 2 µM nicardipine, 1 µM tetrodotoxin, and either 10 µM 8-cyclopentyltheophylline (CPT) to block the action of endogenous adenosine at A1R or 5 U/ml adenosine deaminase for 2 h to inactivate adenosine. Denaturization of the enzyme was prevented by minimizing bubbling. Under these conditions at 37°C, any internalized A1R may recycle back to the membrane and remain there since adenosine can no longer activate A1R. Tetrodotoxin blocks any neural contribution to released endogenous adenosine. After the 2-h incubation, tissues were immediately immersed in ice-cold Krebs supplemented with the same agents. Tissues were then stretched and fixed with an appropriate fixative for designated time before being dissected.
Labeling was then viewed with the Zeiss LSM 410 laser scanning confocal imaging system (Carl Zeiss). The argon-krypton laser was used to excite tissues at 488 nm (FITC) and 568 nm (Texas red), respectively. The fluorescence emission was first separated by a 560-nm dichromic mirror. The FITC fluorescence was further selected by a 515- to 540-nm band-pass filter and that of Texas red was selected by a long-pass filter of 590 nm. Under such conditions, the crossover fluorescence between the two channels was negligible (<2%). Specimens were viewed through a ×40 oil immersion fluor-objective (1.3 numerical aperture). The pinhole was set at 30, which gave rise to a section thickness of ~1.0 µm. The averaged image of two to four consecutive scans was saved as a 512 × 512 RGB tif image for later analysis. For simultaneous immunofluorescent colabeling of A1R and S-100 protein or other proteins, the images were acquired as 512 × 512 overlay RGB tif images displayed as single or dual fluorescence images. The final color of the RGB dual images depends on the extent of colocalization of the two antigens (labeled by green or red fluorochromes) in the same cells, i.e., yellow denotes strong colocalization. These images provide qualitative information about colocalization. The colocalization of A1R immunoreactivity with other proteins was further analyzed by colocalization software on the Zeiss LSM 410 computer. This software computes a scatter diagram from the two images that are acquired in overlay mode and stored separately under image 1 (red) and image 2 (green) in the video memory. The scatter diagram treats all pixels that are in the same position in both images as pairs and displays each pixel pair as x, y coordinates corresponding to the respective fluorescence intensities for images 1 and 2. The threshold fluorescent intensity value of each individual image (red and green) was first determined by a separate measure function of the software. Colocalization was defined as follows. True colocalization occurred when paired pixels had fluorescence intensities that were at least 40 units higher than the intensity of each negative control image for each fluorochrome (i.e., secondary antibody conjugated to FITC or Texas red without primary antibody). Colocalized pixels typically had intensities >110 units. An area was then outlined on the scatter diagram that included all pixels whose intensity values were >110 (which represent colocalization). The regions that demonstrated positive labeling in both images according to our exclusion criteria (i.e., for both molecules) were assigned a blue color, and this was superimposed on the overlay RGB source image. A combined RGB color scale with blue mask overlay was constructed and included with each figure image displayed in this manuscript. In appropriate figures, images are displayed as single channel (red or green), dual channel (red and green), and transformed colocalization image of RGB overlay image together with blue mask distribution of colocalization sites in the tissue.Antisera. Anti-A1R affinity-purified rabbit polyclonal antibody (Alpha Diagnostic International, San Antonio, TX) to a 14-amino acid sequence corresponding to the third extracellular domain of the rat A1R was used. The peptide used for making A1R antibodies shows 100% amino acid homology with canine, rabbit, and human A1R sequences and 85% homology with the bovine sequence. The antibody to A1R cross-reacts with human, pig, lamb, and rat. The glial-specific mouse monoclonal anti-S-100 antibody, clone 15E2E2 (Biogenex, San Ramon, CA), was used as well as the mouse monoclonal antibody for neuron-specific PGP 9.5 (clone 13C4) against human brain PGP 9.5 (Biogenesis, Sandown, NH) which reacts with human, rat, and guinea pig. The mouse monoclonal anti-synaptophysin antibody (clone SVP-38) against the rat retinal synaptosomal-derived antigen (Sigma Immunochemicals, St. Louis, MO), which reacts with human, guinea pig, and pig synaptophysin, was used to stain neurons.
Bound antibodies were visualized by incubating tissues in Texas red (A1 receptor)-labeled or FITC-labeled (S-100 or PGP 9.5 or synaptophysin) secondary antibodies to rabbit IgG. In parallel control experiments, tissues were incubated with normal rabbit antiserum instead of the primary antibodies. The specificity of the labeling to A1R was tested in experiments in which anti-A1R antibody was first preabsorbed with a peptide corresponding to the antigenic site on the A1R (Alpha Diagnostic International, San Antonio, TX). Double label immunohistochemistry was used to identify neurons (absence of S-100 staining or positive PGP 9.5 staining), glial cells (positive S-100 staining), or varicosities (positive synaptophysin) that expressed the A1 receptor.Statistics. Means ± SE are reported. The n values refer to the number of tissues, which approximates the number of animals unless otherwise stated. Student's t-test was used to determine statistical significance at P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Basal and reflex-evoked Isc. Basal Isc in 64 control groups averaged 4 ± 3 µA/cm2 and was not statistically different from zero. The Isc was previously shown to be due to the algebraic sum of small net fluxes of sodium, chloride, and a residual ion (27). During stroking, Isc increased over baseline levels by 82 ± 5 µA/cm2. The stroking-induced change in Isc was shown previously to be due to stimulation of electrogenic chloride secretion (38).
Role for endogenous adenosine in reflex-evoked
Isc.
To determine whether endogenous adenosine modulates
Isc evoked by
mucosal stroking, which elicits a neural reflex, the selective A1R antagonist CPT was added to
the serosal bath. CPT (0.5 µM) had no effect on baseline
Isc compared with
vehicle controls (Table 1). However, CPT
caused a concentration-dependent enhancement of the neurally evoked
reflex response due to stroking (Fig.
2A). To
rule out the possibility that any adenosine displaced from the
A1R by CPT could affect excitatory
A2A receptors on neurons to
increase Isc and
thereby also to contribute to the CPT response, 8-(3-chlorostyrl)caffeine, a specific
A2A receptor antagonist, was
added. CPT (0.5 µM) enhanced stroking-evoked
Isc to 153 ± 20% (n = 12;
P < 0.05) of control response
despite the presence of 1 µM 8-(3-chlorostyrl)caffeine; the
enhancement is comparable to that obtained with CPT alone. The
A2A receptor antagonist alone had
little effect on baseline
Isc (Table 1).
|
|
Effects of adenosine A1R analogs on
reflex-evoked Isc.
The next series of experiments investigated the effects of exogenous
application of a highly selective and potent
A1R agonist on baseline
Isc and on
neurally evoked responses. Baseline
Isc was not
altered by 0.1 µM
2-chloro-N6-cyclopentyladenosine
(CCPA) when neurons were blocked with 0.2 µM tetrodotoxin (Table 1).
In the absence of neural blockade, the ability of CCPA to reduce
Isc was highly
correlated (r = 0.84; slope 1.0) with
the degree of ongoing neural activity in the basal state (Fig.
3). When ongoing neural activity, defined
by the reduction in
Isc in response
to 0.2 µM tetrodotoxin, was low, CCPA's effect on
Isc was small; on
the other hand, when the tetrodotoxin-induced reduction in
Isc was large, so
was the effect of CCPA.
|
|
Determination of sites of action of A1R
analogs on reflex-evoked Isc.
Another series of experiments was done to determine whether CCPA
inhibited reflex-evoked
Isc by acting on
epithelial cells. Two secretagogues, carbachol and forskolin, known to
increase Isc and
cause chloride secretion by calcium- and cAMP-mediated pathways,
respectively, were added to the serosal bath (4, 28). Because both
carbachol and forskolin activate epithelial cells directly as well as
indirectly via submucosal nerves, 0.2 µM tetrodotoxin was added to
block the neurally mediated
Isc response, and
these responses were compared with responses in the absence of
tetrodotoxin. In the absence of tetrodotoxin, 10 µM carbachol evoked
a large increase in
Isc that was
significantly reduced by 0.1 µM CCPA (Fig.
5A,
left). In the presence of
tetrodotoxin, carbachol produced a smaller increase in
Isc, and this was
unaffected by CCPA (Fig. 5A,
right). The results are consistent
with an effect of CCPA on the neural pathway to the epithelium and not
directly on the epithelial cells.
|
|
|
Immunofluorescent distribution of A1R immunoreactivity in submucous ganglia. The distribution of A1R in submucosal ganglia of the guinea pig colon was further characterized in immunofluorescent colabeling studies with anti-A1R antibodies. Data analysis was from 7 guinea pigs and 35 separate microdissected tissues. Five additional animals were used in initial studies to optimize the staining procedures.
Colocalization of A1R and PGP 9.5 immunoreactivity.
A1R immunoreactivity was prominent
in many but not all PGP 9.5 immunoreactive cells as shown in Fig.
8, B and
C. Similar results were obtained in
tissues fixed with modified Zamboni's fixative (n = 3 tissues) or 0.5%
paraformaldehyde (n = 3), although the staining for PGP 9.5 was stronger with the former fixation method. A
mild 5-h fixation method was used to prevent destruction of the
extracellular antigenic site for
A1R. Preliminary studies (data not
shown; n = 5 animals) showed that
overnight fixations of tissues for
A1R greatly increased background
and labeled tissues indiscriminately. Overnight fixation for PGP 9.5 produced the strongest specific staining, and this finding explains the
somewhat weaker immunofluorescence staining seen for PGP 9.5 in
colabeling studies for A1R (Fig.
8C). A 5-h incubation was adopted
for all subsequent dual labeling studies involving
A1R. Single immunofluorescence labeling studies for A1R revealed
a similar distribution profile of
A1R immunoreactivity in large
cells ranging in neuronal size from 20 to 50 µm (data not shown).
|
Colocalization of A1R and S-100
immunoreactivity.
The distribution of A1R in
S-100-immunoreactive glial cells is shown in Fig.
9. Nine tissues were colabeled with S-100
and A1R, six tissues were fixed in
modified Zamboni's fixative, and three were fixed in 0.5%
paraformaldehyde. No significant differences in immunoreactive
distribution/intensity were evident between the two fixation methods.
Colocalization analysis revealed that A1R immunoreactivity is
colocalized in glial cells (Fig. 9, C and D).
A1R immunoreactivity was unevenly
distributed in glia, with some ganglia having more immunoreactive glia
than others. All
A1R-immunoreactive cells lacking
S-100 immunoreactivity have larger cell diameters and represent
submucosal neurons (Fig. 9B). Not
all neurons had A1R
immunoreactivity.
|
Cellular distribution of A1R
immunoreactivity.
Thin optical sectioning of tissues (i.e., 1 µm thick) with the laser
confocal scanning microscope and viewing of the specimens with a high
numerical aperture (1.3 numerical aperture) ×40 or ×100
objective revealed that A1R
immunoreactivity was present in the cell soma of the neurons and
distributed both in the cell membranes and cytoplasm of the neurons.
Exclusive membrane localization of the
A1R immunoreactivity was evident
in ~10% of the neurons and occurred as a thin ring of fluorescence
around the membrane (Fig.
10A).
The size of the nonimmunoreactive area inside the ring of fluorescence
is clearly bigger than the nucleus (i.e., twice the size of nucleus),
indicating that the cytoplasm in addition to the nucleus is lacking
A1R immunoreactivity. The
edge/outline of the cell membrane was further identified by colabeling
of the neurons for synaptophysin (Fig. 10,
B and
C). In the majority (90%) of the
neurons, A1R immunoreactivity was
also present in the cytosol of the submucosal neurons (Figs. 8,
B-D,
11D, and
12, A
and B). A1R immunoreactivity codistributed
with a significant proportion of synaptophysin immunoreactive
varicosities (Fig. 8D). Similar data
were obtained in six different tissues from three animals.
|
|
|
Selectivity of the anti-A1R antibody for adenosine A1R. In tissues in which the primary antibody was preabsorbed with a peptide (at 2-5 times the anti-A1R antibody concentration) corresponding to the immunogenic peptide recognized by the A1R antibody, all A1R immunoreactivity was abolished (Figs. 10D and 12, C and D), whereas immunoreactivity for either S-100 or synaptophysin was not affected. Similar results were obtained in six tissues from two different animals.
A1R immunoreactivity in other submucosal cell types. Prominent A1R immunoreactivity was present in the muscularis mucosae (Fig. 11D). A1R immunoreactivity was absent in the mucosal crypt cells of the colon (Fig. 11, A-C). This was the case in both whole-mount tissues and frozen sections. Less than 5% of frozen sections showed some nonselective labeling that is sometimes seen with frozen sections, especially at the edge of tissues; no such immunoreactivity was present in whole-mount tissues (n = 4 tissues from 3 animals).
Influence of endogenous adenosine on A1R internalization. The percentage of submucous neurons displaying cytosolic A1R immunoreactivity remained at near 90% after treatment of tissues with either 10 µM CPT (n = 4 tissues, 2 animals) or 5 U/ml adenosine deaminase (n = 6 tissues, 3 animals) for 1-2 h (Fig. 12, A and B) These treatments are sufficient to maximally block the interaction of endogenous adenosine with A1R.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Endogenous adenosine, through its action on A1R, appears to have little effect on baseline Isc but inhibits reflex-evoked Isc that was previously shown to be due to electrogenic chloride secretion (7, 15, 38, 39). This conclusion comes from the observations that the selective A1R antagonist enhanced the reflex-evoked change in Isc independent of the blockade of A2A receptors. Furthermore, lowering endogenous adenosine by addition of adenosine deaminase or increasing its concentration by inhibiting transporters that regulate transmembrane movements of nucleosides appropriately enhanced or inhibited reflex-evoked changes in Isc, respectively.
The finding that the A1R antagonist had little effect on baseline Isc suggests that endogenous adenosine plays a small role, if any, in modulating basal electrogenic transport rates. We cannot rule out effects of adenosine A1R activation on electroneutral transport that is not reflected by Isc. It is only after stroking that the antagonist enhances reflex-evoked Isc. It is unclear whether stroking itself releases adenosine and its purine precursors. Although the source of endogenous adenosine is unknown, others have suggested that ATP and its major metabolites including adenosine are released from enteric neurons by electrical stimulation (31). Because adenosine is found in many different cells, we cannot rule out the possibility this nucleoside is released from nonneural cells as well (23).
The inhibitory effect of adenosine A1R activation on reflex-evoked Isc was verified by using a specific A1R agonist, CCPA. The effective concentration to achieve a half-maximal response was in the nanomolar range, consistent with an action of this agonist at A1R (25). Additional evidence for specificity of the agonist at A1R is provided by the ability of the selective A1 antagonist CPT to attenuate its inhibitory effect.
Functional investigation of the location of A1R suggests that they are unlikely to be found in abundance on epithelial cells, because CCPA was ineffective in altering carbachol- or forskolin-evoked Isc when the neurons were blocked with tetrodotoxin. Furthermore, A1R immunoreactivity was not detected in crypt cells that secrete chloride. Other studies suggest that excitatory A2B receptors are present on T84 colonic epithelial cells and their activation stimulates chloride secretion (43). Because both carbachol and forskolin are known to activate neural pathways to the epithelium as well as to trigger secretion by acting directly on epithelial cells, CCPA must be acting somewhere within the neural reflex circuit that innervates the epithelium (4, 28). Possible sites include enterochromaffin cells that release 5-HT and prostaglandin-producing cells or the neural pathways that they activate (15, 16, 38, 39). It is unknown whether enterochromaffin cells in the guinea pig colon express A1R, although A2 receptors have been reported for porcine intestine (36).
A pulse of 5-HT was used to bypass the enterochromaffin cells by directly activating intrinsic primary afferents that are synaptically coupled to secretomotor neurons (33, 39). Previous studies have clearly demonstrated that the mucosal pulse activates 5-HT1P and not other 5-HT receptors (16, 38). Therefore, maximal blockade of this receptor is thought to block the 5-HT-activated limb of the reflex (16, 26). The inhibition of the 5-HT pulse by CCPA is consistent with an effect on neural circuits in the 5-HT-mediated pathway. Further support for the concept that adenosine's effect on the reflex is to modulate neuronal activity comes from the observation that during neural blockade CCPA had no effect on baseline Isc nor on carbachol- or forskolin-stimulated secretion. However, when there was spontaneous neural activity, the CCPA-evoked reduction in Isc correlated with the degree of ongoing activity. Furthermore, CCPA was able to reduce the neurally mediated carbachol- and forskolin-stimulated secretory response (i.e., the response in the absence of neural blockade).
A1R immunoreactivity is evident in a significant population of submucous neurons that are immunoreactive for PGP 9.5 or lack immunoreactivity to the glial protein S-100. This provides further support for our conclusion that neural A1 purinoceptors on submucosal neurons inhibit the 5-HT- and prostaglandin-mediated limbs of the secretory reflex. Blockade of A1R immunoreactivity by preabsorption with the blocking peptide indicates that the anti-A1R antibody is binding specifically to A1R in submucosal neurons. A1R immunoreactivity was localized to both membranes and cytosolic regions of the ganglion cells, and only a minority of neurons show exclusively a membrane localization of the A1R immunoreactivity, seen as a thin ring around the cell soma that is well marked by a dense network of synaptophysin-immunoreactive varicosities.
A1R antibody binding to sites on intracellular enzymes involved in intermediary metabolism found in all cells that have affinity for adenosine cannot explain the A1R immunoreactivity in the cytoplasm, because not all cells/types had cytoplasmic staining for A1R. A1R were absent in many glia, numerous submucosal neurons and their varicosities, all crypt cells in this study, as well as many myenteric neurons in the human and guinea pig small intestine (Christofi, unpublished observations). Unlike NK1 receptors, A1R are expressed in glial cells in submucous ganglia and are usually located in close apposition to the edges of submucosal neurons, making it exceedingly more difficult to reveal membrane staining for A1R; this is even more so in the smaller neurons even with laser confocal imaging of thin optical sections. Although the ubiquitous distribution of A1R in both neurons and glia make it difficult to resolve nerve terminal sites, the strong colocalization of A1R and synaptophysin immunoreactivities in submucous ganglia is consistent with the presence of A1R on presynaptic varicose nerve terminals.
Internalization/recycling has been studied in enteric neurons only for two members of the G protein-coupled receptor family, namely, opioid and tachykinin receptors (24, 35, 40-42). After being activated by substance P, NK1 receptors are rapidly internalized via clathrin-coated endosomes and within 30-60 min recycle back to the membrane (40, 42). Previous electrophysiological studies suggested that A1R are expressed on the surface of the cell somas of enteric neurons (1, 2, 12). Consistent with these findings was the observation of a thin ring of A1R immunoreactivity around some neurons. Our results also suggest that the A1R also exists in the internalized form in our in vitro microdissected submucous plexus preparations. Our finding that endogenous adenosine provides an ongoing tonic suppression of neural reflexes indicates that the endogenous accumulation of adenosine is sufficient to activate A1 sites on submucous neurons, which could lead to subsequent receptor internalization and the observed cytosolic immunoreactivity.
The source of adenosine is unknown, although it might be released from nerves or other surrounding cells under normal, damaged (microdissection), or ischemic situations in which metabolic demand exceeds metabolic availability. However, in experiments in which the receptor was blocked by the antagonist CPT or adenosine was degraded by the enzyme adenosine deaminase, recycling was not detected despite assay conditions that were shown previously to favor recycling of tachykinin receptors to the cell somal membranes of enteric neurons (40). Therefore, A1R on submucous neurons behave differently from other G protein-coupled receptors shown previously to undergo receptor internalization/recycling in enteric neurons. A1R are also different in that they represent the smallest of the G protein-coupled receptors cloned to date (i.e., 326 amino acids; Refs. 14, 30, 37). In contrast to NK1 receptor activation by substance P, the inhibitory response to CCPA is reproducible without any appreciable desensitization following a prolonged occupancy of the A1R by CCPA and a subsequent 30-min washout period. Therefore, sufficient numbers of functional high-affinity A1R must be present in the membrane during this period of time, since their activation leads to suppression of secretory reflexes. One possibility is that the amount of A1R recycled compared with the amount remaining in the cytosol may be too small to be detectable. The work of Ciruela et al. (14) in a vas deferens smooth muscle cell line, DDT1MF-2, and of Ruiz et al. (37) in rat brain sheds some light on why blockade of the interaction of endogenous adenosine with A1R did not result in detectable recycling to the membrane of a majority of submucous neurons. They report slow kinetics for agonist-induced internalization/recycling of the A1R. Chronic treatment with agonist resulted in a time-dependent translocation of A1R to intracellular vesicles that was evident at 5-12 h and maximal at 12-48 h (14). In the case of DDT1MF-2, 30% of the receptors were internalized. The slow kinetics of the A1R may be related to the lack of serine/threonine residues in the carboxy-terminal cytoplasmic tail, a finding that makes it unique in the family of G protein-coupled receptors (14).
Several neural sites of action of adenosinergic A1R inhibition of secretory reflexes are possible. Experiments designed to evaluate which limb of the reflex is modulated by A1R activation provide evidence that both pathways are affected. This conclusion is evidenced by CCPA's inhibition of the residual reflex-evoked Isc in tissues treated with piroxicam, which blocks the prostaglandin-mediated limb, or with HTP, which antagonizes 5-HT1P receptors, a gateway to the 5-HT-mediated limb (16, 26). Concentrations used were those shown to be maximally effective in previous studies, and therefore, appropriate blockade should have been achieved (16, 38).
The mechanism of inhibition when neural A1R are stimulated has not been investigated in submucosal neurons in the guinea pig colon. Current information is derived from electrophysiological studies on myenteric and submucosal neurons of the ileum (1, 2, 8, 11). Functional studies did not allow us to exclude the possibility that A1 receptor activation on enterochromaffin cells contributes to the overall inhibitory response. The apparent lack of A1R immunoreactivity in crypt glands would argue against the presence of A1R on enterochromaffin cells. However, if receptor density is low on these sparsely distributed cells, A1R may not have been readily detected. Another possibility is that the A1R agonist CCPA was also acting at other inhibitory P1 purinoceptors on enterochromaffin cells that are not recognized by the anti-A1R antibody. Indeed, recent observations in a carcinoid tumor cell line indicate the presence of transcripts of other P1 purinoceptors on these cells (Cooke, unpublished observations).
That neural A1R play a significant role in attenuating reflex-evoked Isc is clear from pulse experiments in which the enterochromaffin cell was bypassed by a pulse of 5-HT. Another potential target of adenosine at A1R is submucosal primary afferent neurons believed to be AH/type 2 neurons with Dogiel type II multipolar morphology (22). Suppression of the somal excitability of the primary afferent neuron would block or attenuate the neural reflex. Indeed, in both the myenteric and submucous plexuses, A1R activation suppresses the excitability of most AH/type 2 neurons (12; Christofi, unpublished observations).
It is also possible that adenosine is acting presynaptically at nerve terminals to inhibit release of transmitters for slow excitatory synaptic transmission (i.e., slow EPSP) as has been reported for myenteric neurons (8, 10). Inhibitory A1R on the primary afferent ending would prevent the relay of sensory information to the postsynaptic cell body when 5-HT activates 5-HT1P receptors to initiate neural reflex activity (22). Adenosine would in essence short-circuit the reflex at the neural activation site.
The submucosal primary afferent neurons, which contain substance P, acetylcholine, and glutamate as putative neurotransmitters, are likely to synapse directly with cholinergic secretomotor neurons and either directly or via an interneuron to VIP secretomotor neurons (Figs. 1 and 10) (15, 26, 29). Exogenous application of each of these putative neurotransmitters evokes slow EPSP-like responses in submucosal neurons. Adenosine has been shown to suppress all slow EPSP in both S/type 1 myenteric neurons and AH/type 2 neurons by acting at pre- and postsynaptic sites in AH/type 2 and only presynaptic sites in S/type 1 neurons (8). Secretomotor neurons are S/type 1 neurons and therefore would not be expected to have A1R on cell somas. Nevertheless, we cannot rule out the possibility that transmitter release at neuroepithelial junctions could be inhibited by A1R on secretomotor nerve terminals.
The sites of action of CCPA in the submucous plexus to suppress the
prostaglandin-mediated neural reflex are unknown.
PGE2 is reported to activate
directly VIP secretomotor neurons in the guinea pig ileum (17). The
chloride secretory effects of
PGE2, PGF2, and
PGI2 in the guinea pig colon are
mediated in part by activation of submucosal neurons and release of
transmitters that cause postsynaptic depolarization of the membrane
potential associated with an enhanced spike discharge. Although part of the excitatory response is mediated by the activation of nicotinic cholinergic circuits that drive responsive neurons synaptically, nicotinic receptors are not involved in the stroking reflex (38). Therefore, attenuation of nicotinic cholinergic transmission as a
mechanism for A1R-mediated
inhibition of prostaglandin effects can be excluded from consideration
(19-21). Neuronal effects of PGE2,
PGD2, and
PGI2 are, in part, mediated by
muscarinic receptors (18). Adenosine could suppress the
prostaglandin-mediated neural secretory reflex by acting at pre- or
postsynaptic A1R on submucosal neurons that display prostaglandin-mediated excitatory responses.
The role of glial A1R in submucous ganglia is unclear, but these receptors are unlikely to contribute to the inhibition of the secretory reflex responses studied here. There was strong labeling of the muscularis mucosae for A1R immunoreactivity, but the relevance of this receptors, if any, in secretory reflexes is unknown.
These results provide compelling evidence that endogenous adenosine suppresses secretory reflexes in the submucosal plexus of the guinea pig colon. The internalized A1R apparently behave differently from other G protein-coupled receptors in enteric neurons. Understanding the integrated response to elevated adenosine levels will have to take into account the balance between its role in intermediary metabolism and its extracellular actions at excitatory and inhibitory P1 (A1, A2A, A2B , A3) purinoceptors on submucosal neurons, as well as the contribution of adenosine from ATP release and breakdown to adenosine.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by National Institutes of Health Grants DK-37240 (to H. J. Cooke), DK-44179 (to F. L. Christofi), and National Center for Research Resources Grant 1S10-RR-11434-01 (to F. L. Christofi).
![]() |
FOOTNOTES |
---|
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: H. J. Cooke, Dept. of Pharmacology, The Ohio State University, 333 W. 10th Ave., Columbus, OH 43210.
Received 22 June 1998; accepted in final form 26 October 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Barajas-Lopez, C.,
A. L. Peres,
and
R. Espinosa-Luna.
Cellular mechanisms underlying adenosine actions on cholinergic transmission in enteric neurons.
Am. J. Physiol.
271 (Cell Physiol. 40):
C264-C275,
1996
2.
Barajas-Lopez, C.,
A. Surprenant,
and
R. A. North.
Adenosine A1 and A2 receptors mediate presynaptic inhibition and postsynaptic excitation in guinea pig submucosal neurons.
J. Pharmacol. Exp. Ther.
258:
490-495,
1991[Abstract].
3.
Broad, R. M.,
T. J. McDonald,
E. Brodin,
and
M. A. Cook.
Adenosine A1 receptors mediate inhibition of tachykinin release from perifused enteric nerve endings.
Am. J. Physiol.
262 (Gastrointest. Liver Physiol. 25):
G525-G531,
1992
4.
Carey, H. V.,
H. J. Cooke,
P. Nemeth,
and
J. D. Wood.
Nerve-mediated action of forskolin on guinea pig ileal mucosa.
Experientia
41:
1156-1158,
1985[Medline].
5.
Christofi, F. L.,
L. V. Baidan,
R. H. Fertel,
and
J. D. Wood.
Adenosine A2 receptor-mediated excitation of a subset of AH/type 2 neurons and elevation of cAMP levels in myenteric ganglia of guinea pig ileum.
Neurogastroenterol. Motil.
6:
67-78,
1994.
6.
Christofi, F. L.,
and
M. A. Cook.
The affinity of various purine nucleosides for adenosine receptors on purified myenteric varicosities compared with their efficacy as presynaptic inhibitors of acetylcholine release.
J. Pharmacol. Exp. Ther.
237:
305-311,
1986[Abstract].
7.
Christofi, F. L.,
and
M. A. Cook.
Possible heterogeneity of adenosine receptors on myenteric nerve endings.
J. Pharmacol. Exp. Ther.
243:
302-309,
1987[Abstract].
8.
Christofi, F. L.,
and
M. A. Cook.
Purinergic modulation of gastrointestinal function.
In: Purinergic Approaches in Experimental Therapeutics, edited by K. A. Jacobson,
and M. F. Jarvis. New York: Wiley-Liss, 1997, p. 261-282.
9.
Christofi, F. L.,
T. J. McDonald,
and
M. A. Cook.
Adenosine receptors are negatively coupled to release of tachykinin(s) from enteric nerve endings.
J. Pharmacol. Exp. Ther.
253:
290-295,
1990[Abstract].
10.
Christofi, F. L.,
and
J. D. Wood.
Effects of PACAP on morphologically identified myenteric neurons in guinea pig small bowel.
Am. J. Physiol.
264 (Gastrointest. Liver Physiol. 27):
G414-G421,
1993
11.
Christofi, F. L.,
and
J. D. Wood.
Presynaptic inhibition by adenosine A1 receptors on guinea pig small intestinal myenteric neurons.
Gastroenterology
104:
1420-1429,
1993[Medline].
12.
Christofi, F. L.,
and
J. D. Wood.
Electrophysiological subtypes of inhibitory P1 purinoceptors on myenteric neurones of guinea pig small bowel.
Br. J. Pharmacol.
113:
703-710,
1994[Abstract].
13.
Ciruela, F.,
V. Casado,
J. Mallol,
E. I. Canela,
C. Lluis,
and
R. Franco.
Immunological identification of A1 adenosine receptors in brain cortex.
J. Neurosci. Res.
42:
818-828,
1995[Medline].
14.
Ciruela, F.,
C. Saura,
E. I. Canela,
J. Mallol,
C. Lluis,
and
R. Franco.
Ligand-induced phosphorylation, clustering, and desensitization of A1 receptors.
Mol. Pharmacol.
52:
788-797,
1997
15.
Cooke, H. J.,
M. Sidhu,
P. Fox,
Y.-Z. Wang,
and
E. Zimmermann.
Substance P as a mediator of colonic secretory reflexes.
Am. J. Physiol.
273 (Gastrointest. Liver Physiol. 36):
G238-G245,
1997.
16.
Cooke, H. J.,
M. Sidhu,
and
Y.-Z. Wang.
5-HT activates neural reflexes regulating secretion in guinea pig colon.
Neurogastroenterol. Motil.
9:
181-186,
1997[Medline].
17.
Dekkers, J. A. J. M.,
A. B. A. Kroese,
C. M. Keenan,
W. K. MacNaughton,
and
K. A. Sharkey.
Prostaglandin E2 activation of VIP secretomotor neurons in the guinea pig ileum.
J. Auton. Nerv. Syst.
66:
131-137,
1997[Medline].
18.
Diener, M.,
R. J. Bridges,
S. F. Knobloch,
and
W. Rummel.
Neuronally mediated and direct effects of prostaglandins on ion transport in rat colon descendens.
Naunyn-Schmeidebergs Arch. Pharmacol.
337:
74-78,
1988[Medline].
19.
Frieling, T.,
C. Rupprecht,
G. Dobreva,
D. Haussinger,
and
M. Schemann.
Effects of prostaglandin F2 (PGF2
) and prostaglandin I2 (PGI2) on nerve-mediated secretion in guinea pig colon.
Eur. J. Physiol.
431:
212-220,
1995[Medline].
20.
Frieling, T.,
C. Rupprecht,
G. Dobreva,
and
M. Schemann.
Differential effects of inflammatory mediators on ion secretion in the guinea pig colon.
Comp. Biochem. Physiol. A Physiol.
118:
341-343,
1997[Medline].
21.
Frieling, T.,
C. Rupprecht,
A. B. A. Kroese,
and
M. Schemann.
Effects of the inflammatory mediator prostaglandin D2 on submucosal neurons and secretion in guinea pig colon.
Am. J. Physiol.
266 (Gastrointest. Liver Physiol. 29):
G132-G139,
1994
22.
Furness, J. B.,
W. A. A. Kunze,
P. P. Bertrand,
N. Clerc,
and
J. C. Bornstein.
Intrinsic primary afferent neurons of the intestine.
Prog. Neurobiol.
54:
1-18,
1998[Medline].
23.
Geiger, J. D.,
F. E. Parkinson,
and
E. A. Kowaluk.
Regulators of endogenous adenosine levels as therapeutic agents.
In: Purinergic Approaches in Experimental Therapeutics, edited by K. A. Jacobson,
and M. F. Jarvis. New York: Wiley-Liss, 1997, p. 55-84.
24.
Grady, E. F.,
P. Baluk,
S. Bohm,
P. D. Gamp,
H. Wong,
D. G. Payan,
J. Ansel,
A. L. Portbury,
J. B. Furness,
D. M. McDonald,
and
N. W. Bunnett.
Characterization of antisera specific to NK1, NK2, and NK3 neurokinin receptors and their utilization to localize receptors in the rat gastrointestinal tract.
J. Neurosci.
16:
6975-6986,
1996
25.
Jacobson, K. A.,
and
A. M. Van Rhee.
Development of selective purinoceptor agonists and antagonists.
In: Purinergic Approaches in Experimental Therapeutics, edited by K. A. Jacobson,
and M. F. Jarvis. New York: Wiley-Liss, 1997, p. 101-128.
26.
Kirchgessner, A. L.,
H. Tamir,
and
M. D. Gershon.
Identification and stimulation by serotonin of intrinsic sensory neurons of the submucosal plexus of the guinea pig gut: activity induced expression of fos immunoreactivity.
J. Neurosci.
12:
235-248,
1992[Abstract].
27.
Kuwahara, A.,
S. Bowen,
J. Wang,
C. Condon,
and
H. J. Cooke.
Epithelial responses evoked by stimulation of submucosal neurons in guinea pig distal colon.
Am. J. Physiol.
252 (Gastrointest. Liver Physiol. 15):
G667-G674,
1987
28.
Kuwahara, A.,
X. Y. Tien,
L. Wallace,
and
H. J. Cooke.
Cholinergic receptors mediating secretion in guinea pig colon.
J. Pharmacol. Exp. Ther.
242:
600-606,
1987[Abstract].
29.
Liu, M.-T.,
J. D. Rothstein,
M. D. Gershon,
and
A. L. Kirchgessner.
Glutamatergic enteric neurons.
J. Neurosci.
17:
4764-4784,
1997
30.
Mahan, L. C.,
L. D. McVittie,
E. M. Smyk-Randall,
H. Nakata,
F. J. Monsma,
C. R. Gerfen,
and
D. R. Sibley.
Cloning and expression of an A1 adenosine receptor from rat brain.
Mol. Pharmacol.
40:
1-7,
1991[Abstract].
31.
McCConalogue, K.,
L. Todorov,
J. B. Furness,
and
D. P. Westfall.
Direct measurement of the release of ATP and its major metabolites from the nerve fibers of the guinea pig taenia coli.
Clin. Exp. Pharmacol. Physiol.
23:
807-812,
1996[Medline].
32.
Moneta, N. A.,
T. J. McDonald,
and
M. A. Cook.
Endogenous adenosine inhibits substance P release from periperfused networks of myenteric ganglia.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G38-G44,
1997
33.
Moore, B. A.,
S. Vanner,
N. W. Bunnett,
and
K. A. Sharkey.
Characterization of neurokinin-1 receptors in the submucosal plexus of guinea pig ileum.
Am. J. Physiol.
273 (Gastrointest. Liver Physiol. 36):
G670-G678,
1997
34.
Nicholls, J.,
V. R. Brownhill,
and
S. M. Hourani.
Characterization of P1-purinoceptors on rat isolated duodenum longitudinal muscle and muscularis mucosae.
Br. J. Pharmacol.
117:
170-174,
1996[Abstract].
35.
Portbury, A. L.,
J. B. Furness,
H. M. Young,
B. R. Southwell,
and
S. R. Vigna.
Localization of NK1 receptor immunoreactivity to neurons and interstitial cells of the guinea pig gastrointestinal tract.
J. Comp. Neurol.
367:
342-351,
1996[Medline].
36.
Racke, K.,
A. Reimann,
H. Schworer,
and
H. Kilbinger.
Regulation of 5-HT release from enterochromaffin cells.
Behav. Brain Res.
73:
83-87,
1996[Medline].
37.
Ruiz, A.,
J. M. Sanz,
G. Gonzalez-Calero,
M. Fernandez,
A. Andres,
A. Cubero,
and
M. Ros.
Desensitization and internalization of adenosine A1 receptors in rat brain by in vivo treatment with R-PIA: involvement of coated vesicles.
Biochim. Biophys. Acta
1310:
168-174,
1996[Medline].
38.
Sidhu, M.,
and
H. J. Cooke.
Role for 5-HT and ACh in submucosal reflexes mediating colonic secretion.
Am. J. Physiol.
269 (Gastrointest. Liver Physiol. 32):
G346-G351,
1995
39.
Sidhu, M.,
and
H. J. Cooke.
Role for vasoactive intestinal polypeptide neurons in colonic secretory reflexes in the guinea pig.
J. Auton. Nerv. Syst.
66:
105-110,
1997[Medline].
40.
Southwell, B. R.,
H. L. Woodman,
R. Murphy,
S. J. Royal,
and
J. B. Furness.
Characterization of substance P-induced endocytosis of NK1 receptors on enteric neurons.
Cell Tissue Res.
106:
563-571,
1996.
41.
Sternini, C.,
J. Minnis,
M. Spann,
M. Balestra,
M. Tonini,
and
N. C. Brecha.
Activation and internalization of the µ-opioid receptor (MOR) by endomorphin-1 and -2 (Abstract).
Gastroenterology
114:
A1162,
1998.
42.
Sternini, C.,
D. Su,
P. D. Gamp,
and
N. W. Bunnett.
Cellular sites of expression of the neurokinin-1 receptor in the rat gastrointestinal tract.
J. Comp. Neurol.
358:
531-540,
1995[Medline].
43.
Tally, K. J.,
B. J. Hrnjez,
J. A. Smith,
E. C. Mun,
and
J. B. Matthews.
Adenosine scavenging: a novel mechanism of chloride secretory control in intestinal epithelial cells.
Surgery
120:
248-254,
1996[Medline].
44.
Tucker, A. L.,
and
J. Linden.
Cloned receptors and cardiovascular responses to adenosine.
Cardiol. Res.
27:
62-67,
1993.