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P2X3 receptors mediate visceral hypersensitivity during acute chemically-induced colitis and in the post-inflammatory phase via different mechanisms of sensitization.

Deiteren A, van der Linden L, de Wit A, Ceuleers H, Buckinx R, Timmermans JP, Moreels TG, Pelckmans PA, De Man JG, De Winter BY - PLoS ONE (2015)

Bottom Line: Hypersenstivity was accompanied by an increased colonic release of ATP.A-317491 did not modify visceral sensitivity in controls.These findings indicate that P2X3 receptors are not involved in sensory signaling under physiological conditions whereas they modulate visceral hypersensitivity during acute TNBS-colitis and even more so in the post-inflammatory phase, albeit via different mechanisms of sensitization, validating P2X3 receptors as potential new targets in the treatment of abdominal pain syndromes.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Medicine and Pediatrics, Division of Gastroenterology, University of Antwerp, Antwerp, Belgium.

ABSTRACT

Objectives: Experiments using P2X3 knock-out mice or more general P2X receptor antagonists suggest that P2X3 receptors contribute to visceral hypersensitivity. We aimed to investigate the effect of the selective P2X3 antagonist A-317491 on visceral sensitivity under physiological conditions, during acute colitis and in the post-inflammatory phase of colitis.

Methods: Trinitrobenzene sulphonic-acid colitis was monitored by colonoscopy: on day 3 to confirm the presence of colitis and then every 4 days, starting from day 10, to monitor convalescence and determine the exact timepoint of endoscopic healing in each rat. Visceral sensitivity was assessed by quantifying visceromotor responses to colorectal distension in controls, rats with acute colitis and post-colitis rats. A-317491 was administered 30 min prior to visceral sensitivity testing. Expression of P2X3 receptors (RT-PCR and immunohistochemistry) and the intracellular signalling molecules cdk5, csk and CASK (RT-PCR) were quantified in colonic tissue and dorsal root ganglia. ATP release in response to colorectal distension was measured by luminiscence.

Results: Rats with acute TNBS-colitis displayed significant visceral hypersensitivity that was dose-dependently, but not fully, reversed by A-317491. Hypersenstivity was accompanied by an increased colonic release of ATP. Post-colitis rats also displayed visceral hypersensitivity that was dose-dependently reduced and fully normalized by A-317491 without increased release of ATP. A-317491 did not modify visceral sensitivity in controls. P2X3 mRNA and protein expression in the colon and dorsal root ganglia were similar in control, acute colitis and post-colitis groups, while colonic mRNA expression of cdk5, csk and CASK was increased in the post-colitis group only.

Conclusions: These findings indicate that P2X3 receptors are not involved in sensory signaling under physiological conditions whereas they modulate visceral hypersensitivity during acute TNBS-colitis and even more so in the post-inflammatory phase, albeit via different mechanisms of sensitization, validating P2X3 receptors as potential new targets in the treatment of abdominal pain syndromes.

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Related in: MedlinePlus

Scheme of the two experimental set-ups.In set-up 1 (acute colitis), rats were instilled with TNBS (colitis) or saline (control) and further experiments were conducted 3 days later, during the acute phase of colitis. In set-up 2 (post-colitis), rats were instilled with TNBS or saline and the extent of colitis and the healing process were monitored individually by repeated colonoscopy: first on day 3 to confirm the presence of colitis and thereafter, starting from day 10, every 4 days until complete mucosal healing (score = 0) occurred. Further experiments were conducted 3 days after colonoscopically proven mucosal healing. A-317491 (10–25 mg/kg) or vehicle (saline), denoted by the injection needle, was administered 30 min prior to the start of the VMR protocol. Evaluation of the inflammatory status entailed colonoscopy, macroscopic and microscopic assessment of the colonic tissue in addition to a myeloperoxidase activity (MPO) assay. ATP, adenosine 5’-triphosphate; DRG, dorsal root ganglion; IHC, immunohistochemistry; RT-PCR, reverse transcription polymerase chain reaction; TNBS, trinitrobenzene sulphonic acid; VMR, visceromotor response.
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pone.0123810.g001: Scheme of the two experimental set-ups.In set-up 1 (acute colitis), rats were instilled with TNBS (colitis) or saline (control) and further experiments were conducted 3 days later, during the acute phase of colitis. In set-up 2 (post-colitis), rats were instilled with TNBS or saline and the extent of colitis and the healing process were monitored individually by repeated colonoscopy: first on day 3 to confirm the presence of colitis and thereafter, starting from day 10, every 4 days until complete mucosal healing (score = 0) occurred. Further experiments were conducted 3 days after colonoscopically proven mucosal healing. A-317491 (10–25 mg/kg) or vehicle (saline), denoted by the injection needle, was administered 30 min prior to the start of the VMR protocol. Evaluation of the inflammatory status entailed colonoscopy, macroscopic and microscopic assessment of the colonic tissue in addition to a myeloperoxidase activity (MPO) assay. ATP, adenosine 5’-triphosphate; DRG, dorsal root ganglion; IHC, immunohistochemistry; RT-PCR, reverse transcription polymerase chain reaction; TNBS, trinitrobenzene sulphonic acid; VMR, visceromotor response.

Mentions: Experiments were performed either during acute colitis or in the post-colitis phase (Fig 1). In set-up 1, the role of P2X3 receptors in sensory signaling was investigated during acute colitis. Rats were randomized to receive a saline (control) or TNBS enema and all experiments were conducted 3 days later, during acute inflammation. In set-up 2, the contribution of P2X3 to visceral hypersensitivity was studied in the post-inflammatory phase of colitis. Rats were randomized to receive a saline (control) or TNBS (colitis) enema and the extent of inflammation was verified colonoscopically on day 3. From day 10 onwards, convalescence was monitored individually by repeated colonoscopy which was performed every 4 days. If at any time point colonoscopy still showed signs of mucosal inflammation, the animal was allowed to recover further and colonoscopy was repeated 4 days later. If colonoscopy showed complete mucosal healing, all experiments were conducted 3 days later.


P2X3 receptors mediate visceral hypersensitivity during acute chemically-induced colitis and in the post-inflammatory phase via different mechanisms of sensitization.

Deiteren A, van der Linden L, de Wit A, Ceuleers H, Buckinx R, Timmermans JP, Moreels TG, Pelckmans PA, De Man JG, De Winter BY - PLoS ONE (2015)

Scheme of the two experimental set-ups.In set-up 1 (acute colitis), rats were instilled with TNBS (colitis) or saline (control) and further experiments were conducted 3 days later, during the acute phase of colitis. In set-up 2 (post-colitis), rats were instilled with TNBS or saline and the extent of colitis and the healing process were monitored individually by repeated colonoscopy: first on day 3 to confirm the presence of colitis and thereafter, starting from day 10, every 4 days until complete mucosal healing (score = 0) occurred. Further experiments were conducted 3 days after colonoscopically proven mucosal healing. A-317491 (10–25 mg/kg) or vehicle (saline), denoted by the injection needle, was administered 30 min prior to the start of the VMR protocol. Evaluation of the inflammatory status entailed colonoscopy, macroscopic and microscopic assessment of the colonic tissue in addition to a myeloperoxidase activity (MPO) assay. ATP, adenosine 5’-triphosphate; DRG, dorsal root ganglion; IHC, immunohistochemistry; RT-PCR, reverse transcription polymerase chain reaction; TNBS, trinitrobenzene sulphonic acid; VMR, visceromotor response.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401691&req=5

pone.0123810.g001: Scheme of the two experimental set-ups.In set-up 1 (acute colitis), rats were instilled with TNBS (colitis) or saline (control) and further experiments were conducted 3 days later, during the acute phase of colitis. In set-up 2 (post-colitis), rats were instilled with TNBS or saline and the extent of colitis and the healing process were monitored individually by repeated colonoscopy: first on day 3 to confirm the presence of colitis and thereafter, starting from day 10, every 4 days until complete mucosal healing (score = 0) occurred. Further experiments were conducted 3 days after colonoscopically proven mucosal healing. A-317491 (10–25 mg/kg) or vehicle (saline), denoted by the injection needle, was administered 30 min prior to the start of the VMR protocol. Evaluation of the inflammatory status entailed colonoscopy, macroscopic and microscopic assessment of the colonic tissue in addition to a myeloperoxidase activity (MPO) assay. ATP, adenosine 5’-triphosphate; DRG, dorsal root ganglion; IHC, immunohistochemistry; RT-PCR, reverse transcription polymerase chain reaction; TNBS, trinitrobenzene sulphonic acid; VMR, visceromotor response.
Mentions: Experiments were performed either during acute colitis or in the post-colitis phase (Fig 1). In set-up 1, the role of P2X3 receptors in sensory signaling was investigated during acute colitis. Rats were randomized to receive a saline (control) or TNBS enema and all experiments were conducted 3 days later, during acute inflammation. In set-up 2, the contribution of P2X3 to visceral hypersensitivity was studied in the post-inflammatory phase of colitis. Rats were randomized to receive a saline (control) or TNBS (colitis) enema and the extent of inflammation was verified colonoscopically on day 3. From day 10 onwards, convalescence was monitored individually by repeated colonoscopy which was performed every 4 days. If at any time point colonoscopy still showed signs of mucosal inflammation, the animal was allowed to recover further and colonoscopy was repeated 4 days later. If colonoscopy showed complete mucosal healing, all experiments were conducted 3 days later.

Bottom Line: Hypersenstivity was accompanied by an increased colonic release of ATP.A-317491 did not modify visceral sensitivity in controls.These findings indicate that P2X3 receptors are not involved in sensory signaling under physiological conditions whereas they modulate visceral hypersensitivity during acute TNBS-colitis and even more so in the post-inflammatory phase, albeit via different mechanisms of sensitization, validating P2X3 receptors as potential new targets in the treatment of abdominal pain syndromes.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Experimental Medicine and Pediatrics, Division of Gastroenterology, University of Antwerp, Antwerp, Belgium.

ABSTRACT

Objectives: Experiments using P2X3 knock-out mice or more general P2X receptor antagonists suggest that P2X3 receptors contribute to visceral hypersensitivity. We aimed to investigate the effect of the selective P2X3 antagonist A-317491 on visceral sensitivity under physiological conditions, during acute colitis and in the post-inflammatory phase of colitis.

Methods: Trinitrobenzene sulphonic-acid colitis was monitored by colonoscopy: on day 3 to confirm the presence of colitis and then every 4 days, starting from day 10, to monitor convalescence and determine the exact timepoint of endoscopic healing in each rat. Visceral sensitivity was assessed by quantifying visceromotor responses to colorectal distension in controls, rats with acute colitis and post-colitis rats. A-317491 was administered 30 min prior to visceral sensitivity testing. Expression of P2X3 receptors (RT-PCR and immunohistochemistry) and the intracellular signalling molecules cdk5, csk and CASK (RT-PCR) were quantified in colonic tissue and dorsal root ganglia. ATP release in response to colorectal distension was measured by luminiscence.

Results: Rats with acute TNBS-colitis displayed significant visceral hypersensitivity that was dose-dependently, but not fully, reversed by A-317491. Hypersenstivity was accompanied by an increased colonic release of ATP. Post-colitis rats also displayed visceral hypersensitivity that was dose-dependently reduced and fully normalized by A-317491 without increased release of ATP. A-317491 did not modify visceral sensitivity in controls. P2X3 mRNA and protein expression in the colon and dorsal root ganglia were similar in control, acute colitis and post-colitis groups, while colonic mRNA expression of cdk5, csk and CASK was increased in the post-colitis group only.

Conclusions: These findings indicate that P2X3 receptors are not involved in sensory signaling under physiological conditions whereas they modulate visceral hypersensitivity during acute TNBS-colitis and even more so in the post-inflammatory phase, albeit via different mechanisms of sensitization, validating P2X3 receptors as potential new targets in the treatment of abdominal pain syndromes.

Show MeSH
Related in: MedlinePlus