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Am J Physiol Gastrointest Liver Physiol 294: G554-G566, 2008. First published December 13, 2007; doi:10.1152/ajpgi.00500.2007
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NEUROREGULATION AND MOTILITY

Dual purinergic synaptic transmission in the human enteric nervous system

J. E. Wunderlich,1 B. J. Needleman,3 Z. Chen,1 J. G. Yu,1 Y. Wang,2 I. Grants,1 D. J. Mikami,3 W. S. Melvin,3 H. J. Cooke,2 and F. L. Christofi1

Departments of 1Anesthesiology, 2Neuroscience, and 3Surgery, Division of GI Surgery, College of Medicine and Public Health, The Ohio State University, Columbus, Ohio

Submitted 30 October 2007 ; accepted in final form 13 December 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Based on findings in rodents, we sought to test the hypothesis that purinergic modulation of synaptic transmission occurs in the human intestine. Time series analysis of intraneuronal free Ca2+ levels in submucosal plexus (SMP) from Roux-en-Y specimens was done using Zeiss LSM laser-scanning confocal fluo-4 AM Ca2+ imaging. A 3-s fiber tract stimulation (FTS) was used to elicit a synaptic Ca2+ response. Short-circuit current (Isc = chloride secretion) was recorded in mucosa-SMP in flux chambers. A distension reflex or electrical field stimulation was used to study Isc responses. Ca2+ imaging was done in 1,222 neurons responding to high-K+ depolarization from 61 surgical cases. FTS evoked synaptic Ca2+ responses in 62% of recorded neurons. FTS caused frequency-dependent Ca2+ responses (0.1–100 Hz). FTS Ca2+ responses were inhibited by {Omega}-conotoxin (70%), hexamethonium (50%), TTX, high Mg2+/low Ca2+ (≤100%), or capsaicin (25%). A P2Y1 receptor (P2Y1R) antagonist, MRS-2179 or PLC inhibitor U-73122, blocked FTS responses (75–90%). P2Y1R-immunoreactivity occurred in 39% of vasoactive intestinal peptide-positive neurons. The selective adenosine A3 receptor (AdoA3R) agonist 2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (2-Cl-IBMECA) caused concentration- and frequency-dependent inhibition of FTS Ca2+ responses (IC50 = 8.5 x 10–8 M). The AdoA3R antagonist MRS-1220 augmented such Ca2+ responses; 2-Cl-IBMECA competed with MRS-1220. Knockdown of AdoA1R with 8-cyclopentyl-3-N-(3-{[3-(4-fluorosulphonyl)benzoyl]-oxy}-propyl)-1-N-propyl-xanthine did not prevent 2-Cl-IBMECA effects. MRS-1220 caused 31% augmentation of TTX-sensitive distension Isc responses. The SMP from Roux-en-Y patients is a suitable model to study synaptic transmission in human enteric nervous system (huENS). The P2Y1/G{alpha}q/PLC/inositol 1,3,5-trisphosphate/Ca2+ signaling pathway, N-type Ca2+ channels, nicotinic receptors, and extrinsic nerves contribute to neurotransmission in huENS. Inhibitory AdoA3R inhibit nucleotide or cholinergic transmission in the huENS.

purinergic transmission; calcium signaling; adenosine A3 receptors; P2Y1 receptors; submucous nerve plexus

ADENOSINE (ADO) RECEPTORS may offer a novel therapeutic target in gut inflammation, inflammatory bowel disease (IBD) (24), or other diseases (33, 34, 43, 54). Ado is a metabolite of ATP that acts at AdoA1 receptor (AdoA1R), AdoA2aR, AdoA2bR, or AdoA3R on neural and nonneural layers of the gut to influence synaptic transmission, gut reflexes, and function (14, 17, 18). ATP itself or other nucleotides, like ADP, AMP, UTP, or UTP, act at additional receptors belonging to ionotropic channel P2X1-P2X7 or metabotropic G-protein-coupled P2Y receptors P2Y1, P2Y4, P2Y6, P2Y11-P2Y14 with specific actions in gut neurophysiology (16, 18).

In general, Ado modulates neural, immune, or sensory signals and may provide cytoprotection or neuroprotection in both the enteric nervous system (ENS) and central nervous system (14, 37, 48, 49). In the ENS, Ado suppresses synaptic transmission (10), efferent function of extrinsic capsaicin-sensitive sensory nerves (64), mucosal reflexes (19), neuroeffector transmission (11), and morphine withdrawal diarrhea (38).

Increasing the concentration of endogenous Ado (eAdo) is one mechanism by which several drugs may reduce gut inflammation (1, 2, 6, 28). An Ado kinase inhibitor or an AdoA3R agonist, N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA), may be beneficial in murine models of colitis (42, 56), but the mechanisms involved remain poorly understood. The sites of action of these drugs and, in particular, whether AdoA3Rs are involved remain unclear. AdoA3Rs have a wide range of physiological and disease-related effects (18, 21, 34, 41) with promise for treatment of a variety of heart disease, uveitis, colorectal cancer, and inflammation (23, 29, 43, 51). IB-MECA is in Phase I and II clinical trials for a chronic inflammatory disease, rheumatoid arthritis, and is apparently without toxicity (www.canfite.com/develop.html).

It is increasingly more evident that abnormalities in the ENS, or "little brain of the gut," are involved in IBD or functional bowel diseases (irritable bowel syndrome) that underlie symptoms of constipation, diarrhea, dysmotility, malabsorption and transport, and painful sensations (59). Purinergic signaling pathways play an important role in sensory signaling in enterochromaffin cells (EC) and secretomotor reflexes in the intestinal tract; purinergic signaling operates at all levels of the EC-neural-secretomotor axis in gut reflexes (16, 18). Abnormalities in purinergic signaling, such as those that occur in experimental IBD (30), are expected to influence gut neural reflexes and contribute to symptomology in disease states.

Our recent study (30) assessed the protective effect of an AdoA3R agonist, IB-MECA, on gene dysregulation and injury in a rat chronic model of 2,4,6-trinitrobenzene sulfonic acid-induced colitis. Oral IB-MECA prevented abnormal gene expression in 92% of these genes, histopathology, gut injury, and weight loss. IB-MECA or Ado could suppress elevated free radicals in ex vivo inflamed gut. Oral IB-MECA blocked the colitis-induced upregulation of benzodiazepine, P2X1 receptor (P2X1R), P2X4R, P2X7R, P2Y2R, and P2Y6R, A2aR/A2bR, but not A1R or A3R genes, or downregulated P2X2R, P2Y1R, and P2Y4R (30).

Neural hypoxia elevates interstitial Ado in networks of enteric ganglia or CA1 hippocampal neurons that suppress transmitter release (26, 47) via AdoA1R and possibly AdoA3R, based on indirect pharmacological analysis. Such activation of low-affinity AdoA3R could protect the ENS by limiting excitability.

The physiological role of AdoA3Rs in the brain or ENS has been questioned (13, 21, 50), because the AdoA3R is a low-affinity receptor that requires high/micromolar concentrations of eAdo for activation: high levels of eAdo occur at sites of inflammation, infection, and metabolic stress (44, 48, 57, 58). However, in vitro studies demonstrated that ongoing release of eAdo differentially affects excitatory and inhibitory transmission to S or AH neurons in the gut. These effects at high affinity A1 or non-A1/putative A2 or A3 receptors (13) serve to complement the ability of Ado to shut down excitatory neural activity in gut microcircuits through its dual pre- and postsynaptic actions (9, 11, 19, 26).

The AdoA3R shows a species-specific distribution (40), pharmacology, function, and diversity of structure (40, 46). Differential expression of AdoA3R mRNAs occurs in the mammalian intestines (13, 27, 40), and AdoA3Rs, as well as AdoA2aR, AdoA2bR, and AdoA1Rs, are discretely expressed in human myenteric and submucosal neurons (13). Our laboratory's initial studies also identified an inhibitory limb of the neural motor reflex that may be activated by putative AdoA3Rs in rat distal colon (3). In a guinea pig model of neurogenic diarrhea involving dimaprit/H2 activation of gut reflexes, neural AdoA3Rs are involved in inhibitory modulation of a stereotype cyclical pattern of coordinated motility and secretion (5). In addition, non-A1/A2 inhibitory receptors that may represent putative AdoA3Rs gate excitability of enteric AH neurons (14). Our findings in normal rodent gut, as well as reports by others (25), suggest a physiological role of low micromolar levels of eAdo at AdoA3Rs in intact neural circuits. A functional role for IB-MECA and AdoA3Rs is also indicated in synaptic plasticity that represents the likely substrates for learning and memory (21) that may operate in AH/intrinsic primary afferent neurons (IPANs) (18, 32, 65).

AdoA3Rs are, in general, inhibitory receptors in the ENS (F. L. Christofi, unpublished observations; Ref. 16). In contrast, the P2Y1R is emerging as a significant target for nucleotides in excitatory modulation of EC cell secretion of 5-HT to trigger gut reflexes (8), excitatory purinergic signaling in mucosal stroking reflexes and synaptic transmission in submucous ganglia (12, 20), and slow synaptic transmission in submucous plexus (SMP) neurons (67). P2Y1Rs are coupled to G{alpha}q/phospholipase C (PLC)/inositol 1,4,5-trisphosphate (IP3)/Ca2+ signaling in enteric neurons or EC cells (F. L. Christofi, unpublished observations; Refs. 8, 12, 16, 20, 67).

Purinergic receptor mechanisms involved in the modulation of neuronal excitability or synaptic transmission in the integrated neural circuits of the human ENS (huENS) remain unknown. AdoA1R, AdoA2aR, AdoA2bR, and AdoA3R gene transcripts and proteins are differentially expressed in neural and nonneural layers of the human and rodent intestine, suggesting a role in secretomotor reflexes (13, 18). We tested the hypothesis that purines modulate synaptic transmission in the huENS by acting at inhibitory AdoA3R and excitatory P2Y1Rs. It was necessary to first establish an optical recording technique suitable for monitoring synaptic transmission in the ENS. Fluo 4-acetylmethyl ester (AM) laser confocal Ca2+ imaging was used to record synaptic responses in postsynaptic neurons generated by focal electrical fiber tract stimulation (FTS) of internodal strands, connecting two ganglia in the SMP. Some of the data were presented in preliminary form in two abstracts at the American Gastroenterology Association Meetings (52, 69).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Collection of human jejunum specimens. Specimens of human jejunum were collected from obese patients who underwent laparoscopic gastric bypass surgery. The study was approved by the biomedical science institutional review board of The Ohio State University. The human jejunum specimen was immediately given to the investigator by the surgeon within seconds after removal from the patient in the operating room and placed in 4°C oxygenated Krebs solution (in mM: 120 NaCl, 6.0 KCl, 1.2 MgCl2, 1.35 NaH2PO4, 14.4 NaHCO3, 2.5 CaCl2, 12.7 glucose). After approval of a pathologist, the specimen was used to carry out biological studies in gut neural layers. The time elapsed from tissue removal from the patient to the laboratory bench was 15 min maximum. The tissue was transferred to a large volume of Krebs (2 liters) at room temperature for 1 h initially, to remove anesthetic medications.

Microdissection and laser scanning confocal microscope fluo-4 AM Ca2+ imaging in human SMP after electrical FTS. The jejunum was opened along the mesenterial border, and nonviable tissue that was damaged by surgical instruments or cauterization was removed after careful macroscopic inspection by an experienced physician scientist. The whole thickness tissue was placed and perfused in oxygenated Krebs at room temperature, pinned and stretched to Sylgard, and carefully microdissected to remove mucosa, circular muscle, and myenteric plexus-longitudinal muscle layers. The dissection took ~1 h. The remaining SMP with intact network of ganglia in multiple layers was cut into ~1-cm SMP pieces for loading with Ca2+ indicator for separate experiments. We could routinely obtain 8–12 separate pieces of SMP from one jejunal specimen. Each piece of SMP was loaded with 30 µM fluo-4 AM (Molecular Probes, Eugene, OR) for 2 h and then incubated an additional 1 hr in oxygenated Krebs solution at room temperature to cleave the AM from fluo-4 AM. The total time elapse from the tissue removal from the patient to placement of the tissue preparation on the stage of the confocal microscope was ~5 h.

Time series analysis of intracellular free calcium at 2-s intervals was done in fluo-4 AM loaded submucous neurons that were monitored by a Zeiss laser scanning confocal microscope (LSM) 410 laser-scanning confocal imaging system through a x40 oil immersion objective (numerical aperture 1.3, working distance = 170 µm). LSM Ca2+ imaging was done in single submucous ganglia of the SMP at 2-s intervals (12, 20). A schematic diagram of the preparation is illustrated in Fig. 1A.


Figure 1
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  		Fig. 1. Optical recordings of synaptic Ca2+ signals in the human enteric nervous system with the use of a Zeiss LSM/REN 410 imaging system. A: submucosal plexus (SMP) from the jejunum of Roux-en-Y specimens was loaded with 30 µM fluo-4 AM Ca2+. A 3-s fiber tract electrical stimulation (FTS) was applied by a 25-µm tip titanium electrode placed 0.3 mm away from the recording ganglion to elicit a synaptic Ca2+ response in neurons of the recorded ganglion of the SMP. B: neuronal responses were captured by time series Ca2+ analysis in response to FTS at different frequencies. Representative images of peak Ca2+ responses occurring in neurons from one ganglion are shown for 0.1–100 Hz. C: FTS caused a frequency-dependent synaptic Ca2+ response peaking at 10-25 Hz and declining at 50–100 Hz. Values are means ± SE for 10 ganglia; n = 52 neurons. P < 0.05.

 
Ca2+ imaging was carried out using an Ar-Kr laser to excite the cell at 488 nm, and fluorescence emissions were passed through a FT510 dichroic mirror and collected through a photomultiplier tube equipped with a BP 505–550 filter, positioned in front of the pinhole and light path. Therefore, fluorescence emission is restricted to between 505 and 550 nm. Imaging was done using time series analysis LSM software. Scanned sections had a thickness of 17.5 µm. During the experiment, the tissue was perfused with oxygenated Krebs solution at 36 ± 0.5°C with a "solution in-line heater" (Warner Instruments, Hamden, CT) at a perfusion rate of 20 ml/min. An electrode, which was guided by a computerized Eppendorf Micromanipulator 5171, was placed on a fiber tract (or internodal strand connecting 2 ganglia) 0.3 mm away from the recorded ganglion (Fig. 1). The number of neurons that responded to FTS could be altered by switching the electrode to another fiber tract, indicative of differences in synaptic inputs from different sites.

After an equilibration period, a 3-s fiber tract electrical stimulus (0.1, 0.5, 1, 5, 10, 25, 50, 75, and 100 Hz) was applied by a 25-µm titanium tip (voltage 50 V, duration 0.4 ms). The effects of superfused agonists and antagonists (competitive and noncompetitive) on FTS-evoked Ca2+ transients were assessed on FTS before and 20–30 min after treatment. At the end of each experiment, the recorded ganglion was exposed to 75 mM high-potassium (K+) solution. Neurons were included in the Ca2+ analysis when they responded to FTS before drug treatment and to high-K+ solution at the end of the experiments.

Short-circuit current (chloride secretion) recordings. Short-circuit current (Isc = chloride secretion) was recorded in mucosa-submucosa preparations in Ussing flux chambers under voltage clamp. For these experiments, longitudinal muscle with attached myenteric plexus and circular muscle layers were removed from the jejunum specimen. The remaining mucosa-submucosa tissue (M-SMP) was used as a flat sheet, which was pinned down on the inside of an Ussing chamber with a surface area of 1.767 cm2. Voltage clamp allowed us to record Isc, which is a measurement of chloride secretion. M-SMP were set up in flux chambers, according to published reports in rodent tissue (39).

Distension reflexes, electrical field stimulation, and Isc responses. Distension, caused by the removal of 150 µl fluid for 30 s from the submucosal compartment, evoked a reflex Isc response. Electrical field stimulation (EFS) with 10 or 20 Hz for 30 s (voltage 15 V, duration 0.5 ms) evoked an Isc in the M-SMP. These frequencies were shown previously to evoke optimal EFS responses that can be used in pharmacological studies. Based on our frequency-response curves to FTS in the Ca2+ studies, a peak response occurred at 10–20 Hz (see below). Agonists and antagonists drugs were added to the submucosal side after an initial Isc response was evoked by mechanical or electrical stimulation. Synaptic transmission was confirmed by blocking nerve conduction with tetrodotoxin in the end of each experiment.

Colabeling for P2Y1 and vasoactive intestinal peptide in SMP. Colabeling studies were done for P2Y1R and vasoactive intestinal peptide (VIP) immunoreactivity (ir) in human submucous neurons with polyclonal antibodies. The submucosa was first incubated with P2Y1R (Alamo, Jerusalem, Israel) and VIP primary antibodies (Peninsula, San Carlos, CA) overnight. FITC and Texas Red-conjugated secondary antibodies were used to show P2Y1R or VIP-positive neurons. Dual-labeling techniques were applied as previously described (13).

Statistics. Mean values ± SE were reported. Statistical significance was evaluated by paired or unpaired Student's t-test, depending on the experimental design. The EC50/IC50 values are obtained from sigmoid cumulative concentration-response curves fitted by a nonlinear curve-fitting program (GraphPad Prism 3). For multiple comparisons between different frequencies of FTS, ANOVA followed by post hoc (Dunnett's and Newman-Keuls) tests was used. Some of the pharmacology (i.e., Cl-IBMECA concentration-response curve) was also tested with ANOVA. Statistical analysis was performed using StatView 54.51 (Abacus Concepts, Calabasas, CA). Differences are considered statistically significant for P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tissue was obtained from postsurgical specimens in 61 patients undergoing Roux-en-Y gastric bypass surgery (age, 22–65 yr old; body mass index, 35–68 kg/m2). Laser confocal Ca2+ imaging was done in 143 separate experiments in 143 ganglia in different tissues. The n values include all neurons that respond to FTS to the first electrical stimulus without treatment. Responses to agonists or antagonists included all neurons that had an initial FTS response, whether they had small or large effects. This included neurons with a very small response (i.e., even if it was 10%) vs. neurons that had a large response (i.e., 60%).

Each ganglion displayed three to four fiber tracts under the microscope. By stimulation of one fiber tract, a certain number of neurons within the visualized ganglion responded with a synaptic Ca2+ response. Our experimental protocol was restricted to stimulation of a single-fiber tract per recorded ganglion. The ganglia in the human SMP (as is the case in rat SMP) are oriented in all directions; a fiber tract was randomly chosen for stimulation, with no preconception of orientation or direction. Seven hundred and sixty-three neurons (62.44%) out of 1,222 neurons that all responded to high-K+ depolarization gave a synaptic Ca2+ response to FTS stimulation. If FTS gave a response in two or more neurons in a ganglion, this was used for full analysis. FTS evoked responses in 2–20 neurons/ganglion.

High-K+ depolarization was used at the end of the experiment to confirm the number of neurons in the recorded ganglion that are "viable neurons"; this number was usually greater than that evoked by FTS. The total number of neurons in a ganglion in relation to the number of responsive neurons to high-K+ depolarization was not determined in our study. It is likely that only 60% of viable neurons respond to FTS, because we only stimulated one internodal strand. In fact, when we switch the FTS electrode to a different fiber tract, some different neurons are often activated, suggesting that differential recruitment of neurons occurs by stimulating different fiber tracts. The question of recruitment of neurons, convergence and divergence of synaptic responses, deserves further consideration, but was not analyzed further here. To date, Dr. Jan Tack's group has analyzed such responses in guinea pig myenteric neurons using Ca2+ imaging (62). EFS or distension experiments were done in 19 M-SMP from 8 postsurgical specimens.

FTS and synaptic Ca2+ responses. A frequency-dependent Ca2+ response curve was obtained in submucous neurons in response to FTS. Examples of time series images that depict neural Ca2+ responses at different frequencies are shown in Fig. 1B. The Ca2+ response increased in a frequency-dependent manner from 0.1 to 100 Hz, peaking at 10–25 Hz and gradually declining by 30–40% by 100 Hz (Fig. 1C).

Reproducibility of the Ca2+ response at different frequencies. FTS-Ca2+ responses were evoked repeatedly for 10 consecutive trials at 5-min intervals over a 45-min period at 5, 10, 25, and 35 Hz to assess reproducibility of the response (Fig. 2). At frequencies of 5, 10, and 35 Hz, ANOVA analysis revealed that the response declined significantly after repeated FTS trials. When stimuli of 25 Hz were applied to fiber tracks, no significant difference was seen during 10 repeated identical stimuli. This frequency was chosen for most pharmacological studies. At 35 Hz, repeated FTS caused a complete inhibition of the elicited Ca2+ response (Fig. 2D).


Figure 2
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  		Fig. 2. Reproducibility of synaptic Ca2+ responses in submucous neurons. FTS was used to elicit a Ca2+ response 10 consecutive times at 5-min intervals at 5, 10, 25, and 35 Hz. A: a frequency of 5 Hz was applied to fiber tracts of different ganglia. ANOVA analysis of neuronal Ca2+ responses revealed a significant difference between single stimuli (n = 6; P < 0.05). The Ca2+ response decreased gradually after 5 stimuli. B: a frequency of 10 Hz also showed a significant decrease of the Ca2+ response (ANOVA, n = 13; P < 0.05). The Ca2+ responses decreased gradually after the fourth stimulus. C: when stimuli of 25 Hz were applied to fiber tracks, no significant difference was seen during 10 repeated identical stimuli (ANOVA, n = 6; P = 0.25). D: FTS at 35 Hz was the least reproducible after the fourth consecutive stimulus, and responses could no longer occur from the seventh to the tenth stimulus (ANOVA, n = 6; P < 0.01). {Delta}, Change.

 
P2Y1R/G{alpha}q/PLC signaling mechanism. To assess the role of the PLC signaling pathway in the FTS Ca2+ response, we sought to investigate the effects of inhibition of PLC using U-73122 or a nucleotide P2Y1R-selective antagonist to suppress FTS Ca2+ responses, because P2Y1Rs are known to be coupled to a G{alpha}q/PLC/Ca2+ signaling in submucous neurons of the rat and guinea pig intestine and in human ECs (F. L. Christofi, unpublished observations; Refs. 12, 16, 17, 20, 68).

The selective P2Y1R antagonist 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS-2179; 20 µM) or the PLC inhibitor U-73122 (2 µM) could block 75–90% of the peak FTS Ca2+ response (Fig. 3, A–C). Examples of FTS Ca2+ responses in submucous neurons that were blocked by MRS-2179 are shown in Fig. 3A (time series of selected pseudocolor images displaying fluo-4 AM-Ca2+ fluorescence responses) and Fig. 3B (displaying the Ca2+ transients before and after MRS-2179).


Figure 3
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  		Fig. 3. Role of the P2Y1/G{alpha}q/PLC/inositol 1,4,5-trisphosphate (IP3) signaling pathway in synaptic transmission. A: time-lapse images of FTS-evoked synaptic Ca2+ responses before (1), during control stimuli (2, 3), and after 25-min perfusion of the P2Y1 receptor (P2Y1R) antagonist 20 µM MRS-2179 (4, 5). FTS was at 25 Hz for 3 s. In the end of the experiment, the ganglion was exposed to a 75 mM K+ Krebs solution (6). Arrows mark 2 neurons that responded to FTS. B: Ca2+ transients for the 2 neurons depicted by white and red arrows in A. Both neurons showed Ca2+ responses to FTS before treatment with 20 µM MRS-2179. The Ca2+ response in both neurons marked with arrows in A were abolished by MRS-2179 (red and black curves). Neurons that were sensitive to MRS-2179 and other neurons in the same ganglion responded to high K+ at the end of the experiment. Overall, maximum responses to MRS-2179 occurred at the third or fourth FTS, and those responses were used in statistical analysis. C: neuronal Ca2+ responses to FTS ({Delta}peak Ca2+ response over baseline = 66.1 ± 4.0, n = 20) were 70–95% reduced after treatment with the PLC inhibitor U-73122 (2 µM, {Delta}peak Ca2+ response over baseline = 18.85 ± 5.8, n = 11; ***P < 0.0001), as well as with the selective P2Y1R antagonist MRS-2179 (20 µM, {Delta}peak Ca2+ response over baseline = 4.15 ± 1.3, n = 12; ***P < 0.0001). D: colocalization of P2Y1R-immunoreactivity (ir) (secondary antibody conjugated to FITC) in vasoactive intestinal peptide (VIP)-positive neurons (secondary antibody conjugated to Texas Red) of the SMP. Yellow neurons represent strong colocalization of P2Y1Rs and VIP. Curved arrows show red VIP-positive neuron. Arrowheads indicate green P2Y1R-positive cell. Yellow neurons show colocalization of P2Y1Rs and VIP. E: subsets of neurons with P2Y1R-ir. There are 29.6% P2Y1R-ir cells, 30.9% VIP-ir only cells, and 39.5% neurons that are P2Y1R+/VIP+ (n = 89 neurons).

 
Colocalization of P2Y1R-ir in VIP-positive neurons. Colabeling experiments revealed that 39% of VIP-ir neurons in the human SMP displayed strong ir for P2Y1R (Fig. 3, D and E). An additional 30% of neurons were P2Y1R+/VIP–, and 31% of neurons were VIP+/P2Y1R–. An exhaustive immunochemical study of P2Y1R distribution and chemical coding of submucous neurons was not attempted, as this was not the primary focus of our study.

Functional viability of human submucous neurons. Membrane depolarization and functional viability of human submucous neurons was assessed by K+ depolarization of the neurons. A high-K+ (75 mM) modified Krebs solution was used to depolarize human submucous neurons in intact M-SMP that served to identify them as neurons and their viability and ability to respond to stimulation. High K+ causes membrane depolarization and activation via Ca2+ influx through voltage-operated Ca2+ channels (VOCC). All neurons used in the FTS Ca2+ analysis (n = 722) displayed a robust high-K+ Ca2+ response (Fig. 3A, image 6; Fig. 3B, transient 6).

Synaptic Ca2+ mechanisms. The FTS-evoked Ca2+ response in submucous neurons of the human jejunum was sensitive to blockade of nicotinic synaptic transmission by hexamethonium, blockade of Ca2+-dependent synaptic transmission by high Mg2+/low Ca2+, or blockade of N-VOCC using {Omega}-conotoxin MVIIA (Fig. 4). In control experiments, repeated FTS Ca2+ responses at 0, 5, 25, and 30 min caused only a very modest reduction in peak Ca2+ response (<10%, Fig. 4A). Hexamethonium caused a 50% reduction in the FTS Ca2+ response (Fig. 4B), and {Omega}-conotoxin blocked 70–80% of the response. High-Mg2+/low-Ca2+ perfusion caused an 80% suppression of the response that was reversible with washout (Fig. 4D).


Figure 4
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  		Fig. 4. FTS-evoked Ca2+ signaling mechanisms in human SMP. A: FTS-evoked Ca2+ responses in regular Krebs solution are only modestly reduced by repetition at 25-min intervals (25 Hz, 3 s) (n = 38, P < 0.01). B: sensitivity of the FTS-induced Ca2+ response to the nicotinic ganglionic blocker hexamethonium (n = 15, P < 0.0001). C: sensitivity of the FTS-induced Ca2+ response to the N-type Ca2+ channel blocker {omega}-conotoxin MVIIA (n = 26, P < 0.0001). D: time-dependent and reversible inhibition of the FTS-induced Ca2+ response by high-Mg2+/low-Ca2+ solution. The effect was reversible when the high-Mg2+/low-Ca2+ solutions was exchanged for regular Krebs solution in the end of the experiment (n = 14, P < 0.001, P < 0.0001).

 
Blockade of the extrinsic and intrinsic pathways with capsaicin and TTX. Capsaicin (10 µM) inhibited the FTS-evoked Ca2+ response by ~25% in submucous neurons of the intact SMP (Fig. 5A). Blockade of nerve conduction with TTX could abolish FTS-evoked Ca2+ responses (Fig. 5B). The distension-evoked Isc response was reduced 29% by 10 µM capsaicin alone. Capsaicin, together with 1 µM TTX, reduced the Isc response by 64% (Fig. 5C).


Figure 5
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  		Fig. 5. Involvement of extrinsic and intrinsic neural pathways in synaptic and distension-evoked secretory responses. A: capsaicin caused a modest reduction in the FTS-evoked Ca2+ response (n = 26, P < 0.001). B: sensitivity of the Ca2+ response to TTX (n = 43, P < 0.0001). C: the distension-evoked short-circuit current (Isc) response was reduced 29.3 ± 7.1% by 10 µM capsaicin alone. 10 µM capsaicin plus 1 µM TTX reduced the Isc response by 63.7 ± 7.0% [N = 5 patients, 7 mucosa-SMP (M-SMP) tissues; P < 0.05].

 
MRS-1220 augments synaptic and distension-evoked responses in human intestine. The human AdoA3R-selective antagonist MRS-1220 alone at 1 or 10 µM concentrations could augment the FTS-Ca2+ response (Fig. 6A). The rodent-selective AdoA3R antagonist MRS-1191 had no effect on synaptic Ca2+ responses; the synaptic Ca2+ response was not augmented by 1 µM MRS-1191 (38.2 ± 2.6 to 41.7 ± 4.1 pixels, P = 0.06; 8 neurons) or 10 µM MRS-1191 (44.9 ± 3.1 to 49.7 ± 2.4 pixels, P = 0.081; 43 neurons). MRS-1220 also enhanced the distension-evoked Isc response by 31%, and enhancement was blocked and slightly further reduced by 1 µM TTX (Fig. 7B).


Figure 6
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  		Fig. 6. The human adenosine A3 receptor (AdoA3R) antagonist MRS-1220 enhances synaptic transmission and distension-evoked neural secretion. A: MRS-1220 (1 µM, 10 µM) caused a 50–70% augmentation of the synaptic Ca2+ response (n = 62, P < 0.001). B: MRS-1220 enhanced the distension-evoked Isc response by 30.7 ± 1.5%, and enhancement was blocked by 1 µM TTX (N = 3 patients; 3 M-SMP tissues; P < 0.01).

 

Figure 7
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  		Fig. 7. 2-Chloro-N6-(3-iodobenzyl)adenosine-5'-N- methylcarboxamide (2-Cl-IBMECA) or endogenous adenosine act at AdoA3Rs to suppress synaptic Ca2+ responses in human submucous neurons. A: the selective AdoA3R agonist 2-Cl-IBMECA caused a frequency-dependent suppression of synaptic Ca2+ responses. It was more effective between 0.1 and 25 Hz. B: 2-Cl-IBMECA caused a concentration-dependent inhibition of synaptic Ca2+ responses at 25 Hz (ANOVA, P < 0.01). The IC50 = 8.5 x 10–8 M. C and D: the AdoA3R antagonist MRS-1220 augmented the FTS frequency-dependent Ca2+ responses. E: 2-Cl-IBMECA competes with AdoA3Rs to block the augmentation caused by MRS-1220. F: knockdown of AdoA1R with the irreversible antagonist 8-cyclopentyl-3-N-(3-{[3-(4-fluorosulphonyl)benzoyl]-oxy}-propyl)-1-N-propyl-xanthine (FSCPX) did not augment the frequency-dependent Ca2+ response. G: the AdoA3R agonist 2-Cl-IBMECA caused a robust inhibition of the synaptic Ca2+ response after knockdown of AdoA1R with FSCPX. Three hundred and twenty neurons were separately analyzed in these experiments, 6–10 neurons were included at each data point, and ganglia from 3–6 different Roux-en-Y patients were used to generate each curve. Data represent means ± SE. No symbol, P > 0.05; *P < 0.01; **P < 0.001; ***P < 0.0001.

 
Inhibition of synaptic Ca2+ responses by activation of AdoA3Rs. The selective AdoA3R agonist 2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyl-carboxamide (2-Cl-IBMECA) (5 µM) could abolish the FTS-Ca2+ response at frequencies ranging from 0.5 to 25 Hz. At higher frequencies, 2-Cl-IBMECA was less effective, but significantly inhibited the FTS-Ca2+ response (Fig. 7A).

2-Cl-IBMECA caused a concentration-dependent inhibition of the 25-Hz FTS-evoked Ca2+ response. At a concentration of 5 µM 2-Cl-IBMECA, the Ca2+ response was nearly abolished. The apparent IC50 for 2-Cl-IBMECA inhibition of the FTS-evoked Ca2+ response is ~8.5 x 10–8 M (Fig. 7B).

The human AdoA3R selective antagonist MRS-1220 augmented the frequency-Ca2+ response at 1–35 Hz in human submucous neurons in response to FTS. The effect of MRS-1220 was greatest at 25 and 35 Hz. In contrast, in control experiments, vehicle instead of MRS-1220 did not augment the FTS-Ca2+ response (Fig. 7, C and D). In the presence of 5 µM 2-Cl-IBMECA, MRS-1220 could no longer augment the FTS-Ca2+ response (Fig. 7E).

It is possible that AdoA1Rs contribute to the inhibitory effect of the AdoA3R agonist 2-Cl-IBMECA, and, therefore, we carried out additional experiments in the presence of the irreversible AdoA1R antagonist 8-cyclopentyl-3-N-(3-{[3-(4-fluorosulphonyl)benzoyl]-oxy}-propyl)-1-N-propyl-xanthine (FSCPX) to knock down AdoA1R. FSCPX alone did not augment the FTS-Ca2+ response in submucous neurons (Fig. 7F). At a concentration of 1 µM FSCPX, which is sufficient to block all high-affinity AdoA1Rs (60), the AdoA3R agonist 2-Cl-IBMECA could still suppress the FTS-evoked Ca2+ response at frequencies ranging from 1 to 35 Hz (Fig. 7G).

EFS does not reveal an AdoA3R. EFS at 10 or 20 Hz was used to elicit a TTX-sensitive Cl– secretory response in M-SMP. Knockdown of AdoA1Rs with FSCPX caused a modest but significant augmentation of the 20-Hz EFS-induced Isc response in M-SMP; no effect was observed at 10 Hz. After knockdown, 2-Cl-IBMECA had no effect on the 10- or 20-Hz EFS-induced Isc response in M-SMP (Fig. 8).


Figure 8
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  		Fig. 8. Electrical field stimulation (EFS) reveals AdoA1R but not AdoA3Rs. A and B: EFS (10 or 20 Hz, 15, 0.5 ms, 30 s) increased the Isc response in each of two M-SMP preparations from a Roux-en-Y specimen. Knockdown of AdoA1Rs with FSCPX (0.8 µM) augments the Isc response, but the AdoA3R agonist 2-Cl-IBMECA (2.5 µM) does not inhibit the TTX-sensitive/neural-evoked Isc response. C and D: pooled data reveal that EFS responses are TTX sensitive and, therefore, are mediated by activation of submucous neurons. FSCPX caused a significant augmentation of the Isc response, but 2-Cl-IBMECA was without effect in the presence of FSCPX knockdown of AdoA1R (N = 4 patients; 6 M-SMP tissues at 10 Hz and 6 M-SMP tissues at 20 Hz EFS).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To our knowledge, this is the first study to provide proof that purinergic modulation of synaptic transmission occurs in the human SMP, which may be important in mucosal reflexes and distension reflexes (F. L. Christofi, unpublished observations; Ref. 16). Our study demonstrated that dual modulation of synaptic Ca2+ signaling occurs via activation of inhibitory AdoA3R or excitatory P2Y1Rs. The P2Y1/G{alpha}q/PLC/IP3/Ca2+ signaling is a major pathway in synaptic transmission of the huENS.

We developed a suitable approach to study synaptic transmission in the huENS. LSM Ca2+ imaging can be used to monitor synaptic Ca2+ responses evoked by electrical FTS of interganglionic connectives in the human SMP. Neurons in the intact ganglia are easily visualized by their fluo 4 fluorescence and are viable, responding with a robust Ca2+ transient to high-K+ depolarization and activation of VOCC or to FTS. Focal electrical stimulation of a neighboring interganglionic connective leads to a synaptic Ca2+ response in 64% of viable neurons in recorded ganglia; this implies that a majority of neurons within each ganglion receive significant synaptic inputs. The FTS response recorded in jejunum ganglia obtained from 61 different Roux-en-Y surgery cases involves neuronal conduction and synaptic transmission because it is sensitive to TTX, high-Mg2+/low-Ca2+ solutions, {omega}-conotoxin, and hexamethonium and displays inhibition to high-frequency stimulation. Previous work using calcium indicators revealed activity-dependent changes in intracellular calcium levels in response to electrical stimulation in both AH and S electrophysiological types of enteric neurons (4, 31, 35, 36, 55, 62). The FTS frequencies used in the present study are known to generate multiple action potentials associated with a transient elevation in Ca2+ levels. A high frequency or train stimulus (this study) would elicit a slow excitatory postsynaptic potential (EPSP) response with superimposed action potentials; the hexamethonium response observed would further imply that multiple fast nicotinic EPSPs contribute to the overall EPSP response. These fast EPSP responses are more easily recorded and distinguished by multisite optical recording techniques with a voltage-sensitive dye (53). Synaptic Ca2+ responses in the huENS are repeatable, reproducible, and quantifiable.

Synaptic transmission in human submucous ganglia. Our study provides proof for nerve conduction (TTX sensitivity), neurotransmission (high Mg2+/low Ca2+, and {omega}-conotoxin sensitivity), the types of neurotransmitters involved in neurotransmission (nucleotides, ACh), as well as the signaling pathways and receptors involved in synaptic transmission in the intact neural circuits of human submucous ganglia. High-Mg2+/low-Ca2+ solutions remove Ca2+ involved in neurotransmission, or they would be acting to induce Ca2+-induced Ca2+ release from intracellular ryanodine-sensitive or IP3-sensitive stores. N-type Ca2+ channels that are sensitive to {omega}-conotoxin are involved in the synaptic Ca2+ response in human submucous neurons. At presynaptic sites, N-type Ca2+ channels may play an important role in transmitter release (36, 63, 66). Therefore, the Ca2+ response is synaptically driven, since it is sensitive to {omega}-conotoxin. In guinea pig myenteric plexus, synaptic Ca2+ transients were sensitive to the same manipulations of TTX, {omega}-conotoxin, and high Mg2+/low Ca2+ (4). N-type Ca2+ channels could also be operating in the cell somas or enteric neurons. Action-potential-dependent Ca2+ transients in the cell somas of enteric S neurons were shown to be blocked by ~70% by {omega}-conotoxin (55), and it has been suggested that {omega}-conotoxin-resistant Ca2+ responses may represent antidromic activation of neurons, such as could occur with FTS.

Species differences in synaptic Ca2+ signals between human and rat SMP. In an earlier preliminary study, we characterized synaptic Ca2+ signaling pathways to electrical FTS in a rat preparation of SMP that is similar to human, at least anatomically, with several layers of submucous ganglia as in human (7). It is noteworthy that synaptic Ca2+ signaling mechanisms in the rat SMP differ significantly than those in the human SMP, stressing the importance of carrying out a study in human SMP, albeit a much more complex preparation. In rat SMP, unlike the human SMP, the synaptic Ca2+ response is mediated primarily by a cAMP-dependent mechanism and is blocked by a protein kinase A inhibitor. In human SMP (this study), the P2Y1/G{alpha}q/PLC/IP3/Ca2+ signaling pathway predominates. In rat SMP, MRS-2179 could only block synaptic Ca2+ responses in 23% of neurons. Also different, nicotinic cholinergic transmission plays a minor role in synaptic Ca2+ responses in rat SMP. Such differences deserve further study in human SMP.

Role of inhibitory AdoA3R and excitatory P2Y1Rs in synaptic transmission. All previous work on purinergic regulation in the gut and gut reflexes was done in rodents (16, 18). The physiological role of AdoA3Rs in the brain or ENS has been questioned (13, 21, 50) because the AdoA3R is a low-affinity receptor that requires high/micromolar concentrations of eAdo for activation; high levels of eAdo occur at sites of inflammation, infection, and metabolic stress (22, 44, 48, 57, 58). Earlier studies showed that ongoing release of eAdo in micromolar levels differentially affects excitatory and inhibitory transmission in the gut. These effects at high affinity A1 or non-A1/putative A2 or A3Rs (13) serve to complement the ability of Ado to shut down excitatory neural activity in gut microcircuits through its dual pre- and postsynaptic actions (9, 11, 19, 26).

In this study, activation of the AdoA3R inhibits synaptic transmission in the human SMP. Several lines of evidence support the hypothesis that AdoA3Rs are negatively coupled to synaptic transmission in the huENS. First of all, it is known that AdoA3R-ir is discretely localized in human submucous neurons (13). The IC50 concentration of the potent and selective AdoA3R agonist 2-Cl-IBMECA in suppressing the synaptic Ca2+ response was 8.5 x 10–8 M. To rule out the possibility that 2-Cl-IBMECA is acting at high-affinity AdoA1R (known to exist in submucous neurons) instead of AdoA3Rs, the irreversible AdoA1R antagonist FSCPX that forms a covalent bond with its receptor (60) was used to knock down AdoA1R. The ability of 2-Cl-IBMECA to suppress or abolish the synaptic Ca2+ response after knockdown and the antagonistic effect of the selective AdoA3R antagonist MRS-1220 strongly argue for an AdoA3R, not AdoA1R, in the 2-Cl-IBMECA effect. The potency of 2-Cl-IBMECA in human submucous neurons was 10-fold greater than that reported for guinea pig colonic enteric neurons (IC50 = 0.8 µM) in a model of secretory diarrhea, in which 2-Cl-IBMECA was able to suppress the dimaprit/H2-evoked cyclical coordinated pattern of motility and secretion (5). AdoA3R shows a species-specific distribution (40), pharmacology, and diversity of structure (41) that could explain the difference in potency of the AdoA3R agonist between human (this study) and guinea pig (5) submucous neurons. Other studies also identified an inhibitory limb of the neural motor reflex that may be activated by putative AdoA3Rs in rat distal colon, further arguing for a physiological role of AdoA3R in the ENS (3). In addition, non-A1/A2 inhibitory receptors that may represent putative AdoA3Rs gate excitability of enteric AH neurons (14). Our findings in normal rodent gut, as well as reports by others (25), suggest a physiological role of low micromolar levels of eAdo at AdoA3Rs in intact neural circuits. A functional role for IB-MECA and AdoA3Rs is also indicated in synaptic plasticity that represent the likely substrates for learning and memory (21) that may operate in AH/IPANs (18, 32, 65).

AdoA3Rs are negatively coupled to nucleotide and cholinergic synaptic transmission. 2-Cl-IBMECA was most effective at lower frequencies of FTS, and, in the range of 0.5-to 25-Hz frequencies, activation of AdoA3R could abolish synaptic transmission. At higher frequencies, it could only partially inhibit synaptic transmission, indicating an AdoA3R-resistant component to synaptic transmission. We did not do an exhaustive analysis of the putative transmitters released at each of the frequencies that could be inhibited by AdoA3Rs. However, at 25-Hz frequency, we can conclude that nucleotides (ATP or ADP) are a major contributor to the FTS synaptic Ca2+ response, since it could be blocked by a P2Y1R antagonist. Nicotinic cholinergic transmission is involved in ~50% of responsive neurons that were sensitive to hexamethonium blockade of synaptic Ca2+ responses. Since 2-Cl-IBMECA could abolish the 25-Hz FTS response, it would follow that activation of AdoA3Rs leads to suppression of purinergic and cholinergic transmission in the human SMP. At higher frequencies, 35–100 Hz, the identity of the transmitter(s) that is not sensitive to 2-Cl-IBMECA inhibition remains unknown. Also, we could not delineate whether effects of 2-Cl-IBMECA were at pre- and/or postsynaptic AdoA3Rs. Both sites are possible, since AdoA3R-ir has been identified on substance P varicose fibers and cell somas of SP or VIP neurons in rat and human intestine (F. L. Christofi, unpublished observations; Ref. 16). In rodent ENS, AdoA3R-ir is expressed in both cell somas and processes of neurons, revealing their simple uniaxonal morphology (Dogiel Type I) or their multipolar Dogiel Type II morphology (F. L. Christofi, unpublished observations; Ref. 16).

Our study provides strong pharmacological evidence for activation of AdoA3R in intact neural circuits of the human SMP by eAdo; the AdoA3R antagonist MRS-1220 alone that prefers the human AdoA3R (but not a rodent selective antagonist MRS-1191) was effective in revealing an inhibitory influence of eAdo at AdoA3R on synaptic transmission in human submucous ganglia. Collectively, this study in human submucous ganglia, our laboratory's preliminary data in normal rodent gut (8), as well as reports by others (25), suggest a physiological role of low-micromolar levels of eAdo at AdoA3Rs in intact neural circuits. A functional role for IB-MECA and AdoA3Rs is also suggested in synaptic plasticity that represents the likely substrates for learning and memory (29) that may operate in AH/IPANs (18, 32, 65). However, it is also very likely that these receptors are important in abnormal or disease states of the gut, given that low-affinity AdoA3Rs can be more fully activated by high levels of Ado that can be released during disease and inflammation (22, 44, 48, 57, 58). AdoA3Rs are subject to dysregulation in a profilin transgenic mouse with gut smooth muscle hypertrophy (F. L. Christofi and H. H. Hassanain, unpublished observations), a rabbit Crohn's/ileitis model (61), gut tissue placed in organotypic culture (F. L. Christofi, unpublished observations; Ref. 16), and 2,4,6-trinitrobenzene sulfonic acid colitis (30), and A3R overexpression could be useful as a genetic therapy (13). Nerve terminals, neutrophils, and endothelial cells are known to release high levels of eAdo at sites of inflammation, infection, and metabolic stress that can activate the low-affinity AdoA3R. Studies are underway to assess whether gut neural AdoA3Rs behave differently in IBD (ulcerative colitis and Crohn's disease).

Transgenic manipulation of A3Rs provides a unique opportunity to study A3R signaling and proof of physiological effect of A3R agonists in gut reflexes (or protective effect in experimental colitis). Our current focus is on the use of transgenic A3R knockout and profilin mouse models for the study of the physiological functions of the A3R, including secretomotor function and synaptic transmission in intact neural circuits of the SMP, as well as the consequences of A3R inhibition on animal models of inflammatory bowel disease (45).

The P2Y1/G{alpha}q/PLC/IP3/Ca2+ signaling pathway in human submucous ganglia. AdoA3Rs are, in general, inhibitory receptors in the ENS (F. L. Christofi, unpublished observations; Ref. 16). In contrast, the P2Y1R is emerging as a significant target for nucleotides in excitatory modulation of EC cell secretion of 5-HT to trigger gut reflexes (8), excitatory purinergic signaling in mucosal stroking reflexes and synaptic transmission in submucous ganglia of the rodent intestine (12, 20), and slow synaptic transmission in SMP neurons (67). P2Y1Rs are coupled to G{alpha}q/PLC/IP3/Ca2+ signaling in enteric neurons or EC cells (Refs. 8, 12, 16, 18, 20, 67).

In this study, we provide pharmacological evidence to support the hypothesis that P2Y1/G{alpha}q/PLC/Ca2+ signaling is a predominant pathway involved in synaptic transmission in intact neural circuits of the human submucous nerve plexus. A P2Y1-selective antagonist MRS-2179 prevents synaptic Ca2+ signaling in submucous ganglia, indicating that nucleotide release (ATP or ADP) during FTS activates postsynaptic excitatory P2Y1R on the postsynaptic membrane of neurons to elevate intracellular free Ca2+ levels. The selective PLC inhibitor is effective in blocking ≥80% of the synaptic Ca2+ response. Furthermore, recent evidence from detailed pharmacological and molecular signaling studies supports the hypothesis that modulation of 5-HT release from human ECs occurs via excitatory P2Y1R coupled to the G{alpha}q/PLC/IP3/Ca2+ signaling pathway, leading to 5-HT release (68, 70). In parallel to this mechanism, during FTS in human submucous ganglia, calcium influx in neurons during synaptic activation and action potential generation involve {omega}-conotoxin-sensitive N-type Ca2+ channels.

Our study provided pharmacological evidence for purinergic excitatory transmission via P2Y1Rs. P2Y1R-ir is expressed on 39% of submucous neurons with VIP, and another subpopulation of neurons expresses P2Y1R but not VIP-ir (30% of neurons); the identity of these neurons remains unknown. In our laboratory's previous study, it was shown that <10% of VIP neurons in the human SMP expressed AdoA3R-ir, whereas most substance P immunoreactive neurons expressed AdoA3R-ir (13). Therefore, it is likely that separate populations of submucous neurons exist with P2Y1R and AdoA3R, although colocalization studies would be necessary to provide direct proof. This was not technically feasible with the antisera available to us.

Mechanical or chemical activation of the mucosa can lead to an intestinal neural reflex and an increase in ion transport and fluid secretion. To date, only a few studies in rodents have investigated purinergic P2Y1R regulation of secretomotor function (12, 17, 19, 20). Mucosal distortion by brush stroking or distension can elicit the reflex. P2Y1-ir was identified in a majority of VIP, nitric oxide synthase, calretinin, neuropeptide Y, or somatostatin neurons, but not SP or calbindin submucous neurons (12). Mucosal touch/distension-evoked fluo 4/Ca2+ responses in submucous neurons were also inhibited by apyrase or blocked completely by MRS-2179; MRS-2179 only reduced Isc in stroking reflexes. It was concluded from those studies that several nucleotides may contribute to mechanically evoked secretomotor reflexes, including P2Y1Rs. It is likely that purinergic (P2Y1R) transmission in human gut is also involved in secretomotor reflexes, but this awaits proof.

Distension reflexes, EFS, and intrinsic/extrinsic nerves in the AdoA3R responses. In the M-SMP preparation, distension-evoked mucosal secretion (i.e., Isc/chloride secretion) involves both intrinsic (TTX-sensitive) and extrinsic (capsaicin-sensitive) nerve fibers. Distension-evoked secretion is augmented by the human selective AdoA3R antagonist MRS-1220, lending support to the concept that distension releases sufficient Ado or a related nucleotide precursor (i.e., AMP, ADP, ATP) that breaks down to Ado to activate a low-affinity AdoA3R. Extrinsic nerves with sensitivity to capsaicin are minor contributors to the synaptic Ca2+ response in submucous neurons; these are presumed to be nerve fibers that remain intact in the ganglia after acute microdissection of the M-SMP from human jejunum. The effect of MRS-1220 is blocked completely by TTX, indicating that the AdoA3R mechanism is restricted to intrinsic nerves in the human SMP.

Interestingly, when EFS stimulation was used to evoke a neural Isc/chloride secretory response, no AdoA3R could be revealed by using an AdoA3R-selective agonist 2-Cl-IBMECA. EFS activates all neurons in the neural circuits of the SMP indiscriminately and in both the ortho- and antidromic directions, making it more difficult to reveal an inhibitory AdoA3R component at 10- or 20-Hz stimulation frequency. EFS did reveal an AdoA1R inhibitory component at 20 Hz with the AdoA1R antagonist FSCPX, suggestive of sufficient eAdo release to activate this high-affinity receptor. In contrast, FTS could not reveal an AdoA1R component. However, FTS stimulation of an internodal strand activates a more discrete synaptic pathway and can reveal a prominent AdoA3R component to synaptic transmission. In the intact mucosa-submucosa, AdoA3Rs are located on both ECs (sites of 5-HT release to trigger gut mucosal reflexes) and submucous neurons. In human EC cells, eAdo is sufficient to provide ongoing inhibition of basal and mechanically evoked 5-HT release via AdoA3Rs (8). Another possibility to explain the lack of A1R functional responses in synaptic transmission is the potential downregulation or internalization of the receptor during the postsurgical period and processing of the tissue: from tissue removal in the operating room to Ca2+ imaging, it takes ~5 h, and, during this time, the A1R could undergo changes in expression. Furthermore, it is know that the A1 becomes internalized after activation, and it takes up to 24 h for trafficking back to the membrane. In fact, we found that, in human EC cells, we could reveal an A1 inhibition response only after protection of the A1R from activation and internalization by eAdo by blocking it with an A1 antagonist overnight (8).

Conclusions. Collectively, data in human EC cells and the intact neural circuits of the human SMP (this study) support the unified hypothesis that inhibitory AdoA3Rs and excitatory P2Y1Rs modulate mucosal reflexes by acting at receptors in the EC-neural secretomotor reflex arc (8, 16, 18). In this study, 2-Cl-IBMECA or eAdo can activate AdoA3Rs to suppress distension reflexes, as well as synaptic cholinergic and purinergic transmission in the human SMP. Nucleotides released during electrical stimulation of internodal strands release neurotransmitters that activate a major excitatory P2Y1/G{alpha}q/PLC/IP3/Ca2+ signaling pathway and N-type Ca2+ channels in the postsynaptic neurons; these mechanisms are important for communication between neurons in the "little brain" of the human gut.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. L. Christofi, Dept. of Anesthesiology, College of Medicine and Public Health, The Ohio State Univ., 226 Tzagournis Medical Research Facility, 420 West 12thAve., Columbus, OH 43210 (e-mail: christofi.1{at}osu.edu)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Purinergic receptors as future targets for treatment of functional GI disorders
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