Quantification and Potential Functions of Endogenous Agonists of Transient Receptor Potential Channels in Patients With Irritable Bowel Syndrome.
OBJECTIVE
In mice, activation of the transient receptor potential cation channels (TRP) TRPV1, TRPV4, and TRPA1 causes visceral hypersensitivity. These receptors and their agonists might be involved in development of irritable bowel syndrome (IBS). We investigated whether polyunsaturated fatty acid (PUFA) metabolites, which activate TRPs, are present in colon tissues from patients with IBS and act as endogenous agonists to induce hypersensitivity.


METHODS
We analyzed colon biopsy samples from 40 patients with IBS (IBS biopsies) and 11 healthy individuals undergoing colorectal cancer screening (controls), collected during colonoscopy at the University of Bologna, Italy. Levels of the PUFA metabolites that activate TRPV1 (12-hydroperoxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid, 5-hydroxyeicosatetraenoic acid, and leukotriene B4), TRPV4 (5,6-epoxyeicosatrienoic acid [EET] and 8,9-EET), and TRPA1 (PGA1, 8-iso-prostaglandin A2, and 15-deoxy-Δ-prostaglandin J2) were measured in biopsies and their supernatants using liquid chromatography and tandem mass spectrometry; we also measured levels of the PUFA metabolites prostaglandin E2 (PGE2) and resolvins. C57Bl6 mice were given intrathecal injections of small interfering RNAs to reduce levels of TRPV4, or control small interfering RNAs, along with colonic injections of biopsy supernatants; visceral hypersensitivity was measured based on response to colorectal distension. Mouse sensory neurons were cultured and incubated with biopsy supernatants and lipids extracted from biopsies or colons of mice. Immunohistochemistry was used to detect TRPV4 in human dorsal root ganglia samples (from the National Disease Research Interchange).


RESULTS
Levels of the TRPV4 agonist 5,6-EET, but not levels of TRPV1 or TRPA1 agonists, were increased in IBS biopsies compared with controls; increases correlated with pain and bloating scores. Supernatants from IBS biopsies, but not from controls, induced visceral hypersensitivity in mice. Small interfering RNA knockdown of TRPV4 in mouse primary afferent neurons inhibited the hypersensitivity caused by supernatants from IBS biopsies. Levels of 5,6-EET and 15-HETE were increased in colons of mice with, but not without, visceral hypersensitivity. PUFA metabolites extracted from IBS biopsies or colons of mice with visceral hypersensitivity activated mouse sensory neurons in vitro, by activating TRPV4. Mouse sensory neurons exposed to supernatants from IBS biopsies produced 5,6-EET via a mechanism that involved the proteinase-activated receptor-2 and cytochrome epoxygenase. In human dorsal root ganglia, TPV4 was expressed by 35% of neurons.


CONCLUSIONS
Colon tissues from patients with IBS have increased levels of specific PUFA metabolites. These stimulate sensory neurons from mice and generate visceral hypersensitivity via activation of TRPV4.
Inserm, U1043, Toulouse, France; CNRS, U5282, Toulouse, France; Université de Toulouse, Université Paul Sabatier, Centre de Physiopathologie de Toulouse Purpan (CPTP), Toulouse, France.
Nicolas
N
Cenac
Inserm, U1043, Toulouse, France; CNRS, U5282, Toulouse, France; Université de Toulouse, Université Paul Sabatier, Centre de Physiopathologie de Toulouse Purpan (CPTP), Toulouse, France.
Tereza
T
Bautzova
Inserm, U1043, Toulouse, France; CNRS, U5282, Toulouse, France; Université de Toulouse, Université Paul Sabatier, Centre de Physiopathologie de Toulouse Purpan (CPTP), Toulouse, France; Inserm U1048, Toulouse, France; Lipidomic Core Facility, Metatoul Platform, Université de Toulouse, Université Paul Sabatier, Toulouse, France.
Pauline
P
Le Faouder
Monash Institute of Pharmaceutical Sciences, Parkville, Victoria, Australia.
Nicholas A
NA
Veldhuis
Monash Institute of Pharmaceutical Sciences, Parkville, Victoria, Australia; Department of Anatomy and Neuroscience, The University of Melbourne, Parkville, Victoria, Australia.
Daniel P
DP
Poole
Inserm, U1043, Toulouse, France; CNRS, U5282, Toulouse, France; Université de Toulouse, Université Paul Sabatier, Centre de Physiopathologie de Toulouse Purpan (CPTP), Toulouse, France.
Corinne
C
Rolland
Inserm, U1043, Toulouse, France; CNRS, U5282, Toulouse, France; Université de Toulouse, Université Paul Sabatier, Centre de Physiopathologie de Toulouse Purpan (CPTP), Toulouse, France.
Jessica
J
Bertrand
Division of Neurology, Department of Medicine, Duke University Medical Center, Durham, North Carolina.
Wolfgang
W
Liedtke
Ambiotis SAS, Toulouse, France.
Marc
M
Dubourdeau
Inserm U1048, Toulouse, France; Lipidomic Core Facility, Metatoul Platform, Université de Toulouse, Université Paul Sabatier, Toulouse, France.
Justine
J
Bertrand-Michel
Departments of Internal Medicine and Gastroenterology, University of Bologna, Bologna, Italy.
Lisa
L
Zecchi
Departments of Internal Medicine and Gastroenterology, University of Bologna, Bologna, Italy.
Vincenzo
V
Stanghellini
Monash Institute of Pharmaceutical Sciences, Parkville, Victoria, Australia; Department of Pharmacology, The University of Melbourne, Parkville, Victoria, Australia.
Nigel W
NW
Bunnett
Departments of Internal Medicine and Gastroenterology, University of Bologna, Bologna, Italy.
Giovanni
G
Barbara
Inserm, U1043, Toulouse, France; CNRS, U5282, Toulouse, France; Université de Toulouse, Université Paul Sabatier, Centre de Physiopathologie de Toulouse Purpan (CPTP), Toulouse, France; University of Calgary, Department of Pharmacology and Physiology, Calgary, Alberta, Canada. Electronic address: nathalie.vergnolle@inserm.fr.
Nathalie
N
Vergnolle
D000328:Adult; D000368:Aged; D000818:Animals; D001706:Biopsy; D015220:Calcium Channels / Q000378:metabolism*; D002853:Chromatography, Liquid; D003106:Colon / Q000166:cytology / Q000294:innervation / Q000378:metabolism*; D015232:Dinoprostone / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000378:metabolism*; D005260:Female; D005727:Ganglia, Spinal / Q000166:cytology / Q000378:metabolism; D006801:Humans; D043183:Irritable Bowel Syndrome / Q000378:metabolism*; D007558:Italy; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008875:Middle Aged; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D011984:Sensory Receptor Cells / Q000166:cytology / Q000378:metabolism; D015398:Signal Transduction; D000074025:TRPA1 Cation Channel; D050916:TRPV Cation Channels / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D050051:Transient Receptor Potential Channels / Q000378:metabolism*; D055815:Young Adult
D015220:Calcium Channels; D005231:Fatty Acids, Unsaturated; D009419:Nerve Tissue Proteins; D034741:RNA, Small Interfering; D000074025:TRPA1 Cation Channel; C473350:TRPA1 protein, human; D050916:TRPV Cation Channels; C494482:TRPV1 protein, human; C416986:TRPV4 protein, human; D050051:Transient Receptor Potential Channels; D015232:Dinoprostone
PAR2; Pain; Protease; Proteolytic Activity