Neuropharmacology of purinergic receptors in human submucous plexus: Involvement of P2X₁, P2X₂, P2X₃ channels, P2Y and A₃ metabotropic receptors in neurotransmission.
BACKGROUND
The role of purinergic signaling in human ENS is not well understood. We sought to further characterize the neuropharmacology of purinergic receptors in human ENS and test the hypothesis that endogenous purines are critical regulators of neurotransmission.


METHODS
LSCM-Fluo-4/(Ca(2+))-imaging of postsynaptic Ca(2+) transients (PSCaTs) was used as a reporter of synaptic transmission evoked by fiber tract electrical stimulation in human SMP surgical preparations. Pharmacological analysis of purinergic signaling was done in 1,556 neurons (identified by HuC/D-immunoreactivity) in 235 ganglia from 107 patients; P2XR-immunoreactivity was evaluated in 19 patients. Real-time MSORT (Di-8-ANEPPS) imaging tested effects of adenosine on fast excitatory synaptic potentials (fEPSPs).


RESULTS
Synaptic transmission is sensitive to pharmacological manipulations that alter accumulation of extracellular purines: Apyrase blocks PSCaTs in a majority of neurons. An ecto-NTPDase-inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP or adenosine deaminase augments PSCaTs. Blockade of reuptake/deamination of eADO inhibits PSCaTs. Adenosine inhibits fEPSPs and PSCaTs (IC50 = 25 µM), sensitive to MRS1220-antagonism (A3AR). A P2Y agonist ADPβS inhibits PSCaTs (IC50 = 111 nM) in neurons without stimulatory ADPbS responses (EC50 = 960 nM). ATP or a P2X1,2,2/3 (α,β-MeATP) agonist evokes fast, slow, biphasic Ca(2+) transients or Ca(2+) oscillations (ATP,EC50 = 400 mM). PSCaTs are sensitive to P2X1 antagonist NF279. Low (20 nM) or high (5 µM) concentrations of P2X antagonist TNP-ATP block PSCaTs in different neurons; proportions of neurons with P2XR-immunoreactivity follow the order P2X2 > P2X1 >> P2X3; P2X1 + P2X2 and P2X3 + P2X2 are co-localized. RT-PCR identified mRNA-transcripts for P2X1-7, P2Y1,2,12-14R.


CONCLUSIONS
Purines are critical regulators of neurotransmission in human ENS. Purinergic signaling involves P2X1, P2X2, P2X3 channels, P2X1 + P2X2 co-localization and inhibitory P2Y or A3 receptors. These are potential novel therapeutic targets for neurogastroenterology.
Department of Anesthesiology, The Wexner Medical Center at The Ohio State University, Columbus, OH, USA.
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Liñán-Rico
Department of Anesthesiology, The Wexner Medical Center at The Ohio State University, Columbus, OH, USA.
J E
JE
Wunderlich
Department of Anesthesiology, The Wexner Medical Center at The Ohio State University, Columbus, OH, USA.
J T
JT
Enneking
Department of Anesthesiology, The Wexner Medical Center at The Ohio State University, Columbus, OH, USA.
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Tso
Department of Anesthesiology, The Wexner Medical Center at The Ohio State University, Columbus, OH, USA.
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Grants
Division of Pediatric Gastroenterology, The Research Institute at Nationwide Children's Hospital, Columbus, OH, USA.
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KC
Williams
Department of Anesthesiology, The Wexner Medical Center at The Ohio State University, Columbus, OH, USA.
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Otey
Human Biology, Technische Universität München, Freising, Germany.
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K
Michel
Human Biology, Technische Universität München, Freising, Germany.
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M
Schemann
Department of Surgery, The Wexner Medical Center at The Ohio State University, Columbus, OH, USA.
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B
Needleman
Department of Surgery, The Wexner Medical Center at The Ohio State University, Columbus, OH, USA.
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A
Harzman
Department of Anesthesiology, The Wexner Medical Center at The Ohio State University, Columbus, OH, USA. Electronic address: Fedias.Christofi@osumc.edu.
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Christofi
D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D003082:Colectomy; D004558:Electric Stimulation; D019706:Excitatory Postsynaptic Potentials / Q000502:physiology; D006801:Humans; D007150:Immunohistochemistry; D009474:Neurons / Q000187:drug effects* / Q000502:physiology*; D058905:Purinergic Agents / Q000494:pharmacology; D011983:Receptors, Purinergic / Q000378:metabolism*; D013368:Submucous Plexus / Q000187:drug effects* / Q000502:physiology*; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology; D056969:Voltage-Sensitive Dye Imaging
D058905:Purinergic Agents; D011983:Receptors, Purinergic; D000255:Adenosine Triphosphate; D002118:Calcium
A(3) inhibitory receptors; ATP; Endogenous adenosine; Human enteric nervous system; Inhibitory P2Y receptors; P2X channels; Purinergic synaptic transmission; Submucous plexus