Capsaicin inhibits intestinal Cl- secretion and promotes Na+ absorption by blocking TRPV4 channels in healthy and colitic mice
2022; Elsevier BV; Volume: 298; Issue: 5 Linguagem: Inglês
10.1016/j.jbc.2022.101847
ISSN1083-351X
AutoresHanxing Wan, Xiong Ying Chen, Fenglian Zhang, Jun Chen, Fenglan Chu, Zachary M. Sellers, Feng Xu, Hui Dong,
Tópico(s)Gastrointestinal motility and disorders
ResumoAlthough capsaicin has been studied extensively as an activator of the transient receptor potential vanilloid cation channel subtype 1 (TRPV1) channels in sensory neurons, little is known about its TRPV1-independent actions in gastrointestinal health and disease. Here, we aimed to investigate the pharmacological actions of capsaicin as a food additive and medication on intestinal ion transporters in mouse models of ulcerative colitis (UC). The short-circuit current (Isc) of the intestine from WT, TRPV1-, and TRPV4-KO mice were measured in Ussing chambers, and Ca2+ imaging was performed on small intestinal epithelial cells. We also performed Western blots, immunohistochemistry, and immunofluorescence on intestinal epithelial cells and on intestinal tissues following UC induction with dextran sodium sulfate. We found that capsaicin did not affect basal intestinal Isc but significantly inhibited carbachol- and caffeine-induced intestinal Isc in WT mice. Capsaicin similarly inhibited the intestinal Isc in TRPV1 KO mice, but this inhibition was absent in TRPV4 KO mice. We also determined that Ca2+ influx via TRPV4 was required for cholinergic signaling–mediated intestinal anion secretion, which was inhibited by capsaicin. Moreover, the glucose-induced jejunal Isc via Na+/glucose cotransporter was suppressed by TRPV4 activation, which could be relieved by capsaicin. Capsaicin also stimulated ouabain- and amiloride-sensitive colonic Isc. Finally, we found that dietary capsaicin ameliorated the UC phenotype, suppressed hyperaction of TRPV4 channels, and rescued the reduced ouabain- and amiloride-sensitive Isc. We therefore conclude that capsaicin inhibits intestinal Cl- secretion and promotes Na+ absorption predominantly by blocking TRPV4 channels to exert its beneficial anti-colitic action. Although capsaicin has been studied extensively as an activator of the transient receptor potential vanilloid cation channel subtype 1 (TRPV1) channels in sensory neurons, little is known about its TRPV1-independent actions in gastrointestinal health and disease. Here, we aimed to investigate the pharmacological actions of capsaicin as a food additive and medication on intestinal ion transporters in mouse models of ulcerative colitis (UC). The short-circuit current (Isc) of the intestine from WT, TRPV1-, and TRPV4-KO mice were measured in Ussing chambers, and Ca2+ imaging was performed on small intestinal epithelial cells. We also performed Western blots, immunohistochemistry, and immunofluorescence on intestinal epithelial cells and on intestinal tissues following UC induction with dextran sodium sulfate. We found that capsaicin did not affect basal intestinal Isc but significantly inhibited carbachol- and caffeine-induced intestinal Isc in WT mice. Capsaicin similarly inhibited the intestinal Isc in TRPV1 KO mice, but this inhibition was absent in TRPV4 KO mice. We also determined that Ca2+ influx via TRPV4 was required for cholinergic signaling–mediated intestinal anion secretion, which was inhibited by capsaicin. Moreover, the glucose-induced jejunal Isc via Na+/glucose cotransporter was suppressed by TRPV4 activation, which could be relieved by capsaicin. Capsaicin also stimulated ouabain- and amiloride-sensitive colonic Isc. Finally, we found that dietary capsaicin ameliorated the UC phenotype, suppressed hyperaction of TRPV4 channels, and rescued the reduced ouabain- and amiloride-sensitive Isc. We therefore conclude that capsaicin inhibits intestinal Cl- secretion and promotes Na+ absorption predominantly by blocking TRPV4 channels to exert its beneficial anti-colitic action. Inflammatory bowel disease (IBD) is a group of chronic inflammatory intestinal disorders, including Crohn's disease (CD) and ulcerative colitis (UC). CD can affect any part of the digestive tract characterized by transmural inflammation, but UC is limited to the colon. Ulcerations and bloody diarrhea are the characteristic symptoms in UC (1Chang J.T. Pathophysiology of inflammatory bowel diseases.N. Engl. J. Med. 2020; 383: 2652-2664Crossref PubMed Scopus (130) Google Scholar). IBD is thought to be triggered by environmental factors in genetically susceptible individuals (2de Mattos B.R. Garcia M.P. Nogueira J.B. Paiatto L.N. Albuquerque C.G. Souza C.L. Fernandes L.G. Tamashiro W.M. Simioni P.U. Inflammatory bowel disease: An overview of immune mechanisms and biological treatments.Mediators Inflamm. 2015; 2015: 493012Crossref PubMed Scopus (116) Google Scholar). Hallmarks of the disease include immune cell infiltration and activation in the mucosa leading to inflammation and ulceration of the intestinal wall (3Keita Å V. Lindqvist C.M. Öst Å. Magana C.D.L. Schoultz I. Halfvarson J. Gut barrier dysfunction-a primary defect in twins with Crohn's disease predominantly caused by genetic predisposition.J. Crohn's colitis. 2018; 12: 1200-1209Crossref PubMed Google Scholar, 4Michielan A. D'Incà R. Intestinal permeability in inflammatory bowel disease: Pathogenesis, clinical evaluation, and therapy of leaky gut.Mediators Inflamm. 2015; 2015: 628157Crossref PubMed Scopus (305) Google Scholar). So far, there are limited clinical therapies for IBD, but currently available therapies (such as anti-TNF-α biologics) cannot cure IBD due to the lack of in-depth understanding of its pathogenesis (5Baumgart D.C. Le Berre C. Newer biologic and small-molecule therapies for inflammatory bowel disease.N. Engl. J. Med. 2021; 385: 1302-1315Crossref PubMed Scopus (15) Google Scholar). Therefore, it is urgent to elucidate the detailed pathogenesis and develop new cures for IBD patients. Chili pepper has a long history of flavoring, preserving food, as well as medication worldwide (6Kraft K.H. Brown C.H. Nabhan G.P. Luedeling E. Luna Ruiz Jde J. Coppens d'Eeckenbrugge G. Hijmans R.J. Gepts P. Multiple lines of evidence for the origin of domesticated chili pepper, Capsicum annuum, in Mexico.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 6165-6170Crossref PubMed Scopus (143) Google Scholar). As an active compound from chili pepper, capsaicin has numerous beneficial roles outside of human gastrointestinal (GI) tract. Capsaicin has been used for the prevention/treatment of pain, hypertension, and inflammation (7Richards B.L. Whittle S.L. Buchbinder R. Neuromodulators for pain management in rheumatoid arthritis.Cochrane database Syst. Rev. 2012; 1: Cd008921PubMed Google Scholar, 8Singh U. Bernstein J.A. Intranasal capsaicin in management of nonallergic (vasomotor) rhinitis.Prog. Drug Res. Fortschritte der Arzneimittelforschung. Progres des recherches pharmaceutiques. 2014; 68: 147-170Google Scholar). Even more interestingly as reported recently, dietary capsaicin could significantly enhance hematopoietic stem cells mobilization to improve their yield for stem cell-based therapeutic agents (9Gao X. Zhang D. Xu C. Li H. Caron K.M. Frenette P.S. Nociceptive nerves regulate haematopoietic stem cell mobilization.Nature. 2021; 589: 591-596Crossref PubMed Scopus (40) Google Scholar). Although dietary capsaicin is absorbed with a great efficiency in GI tract, its exact pharmacological actions have not been well studied in this organ. After capsaicin is passively absorbed in the upper GI tract (10Kawada T. Suzuki T. Takahashi M. Iwai K. Gastrointestinal absorption and metabolism of capsaicin and dihydrocapsaicin in rats.Toxicol. Appl. Pharmacol. 1984; 72: 449-456Crossref PubMed Scopus (106) Google Scholar), it stimulates gut mucosal afferent nerves and blood flow rates (11Leung F.W. Capsaicin-sensitive intestinal mucosal afferent mechanism and body fat distribution.Life Sci. 2008; 83: 1-5Crossref PubMed Scopus (56) Google Scholar) and also ameliorates abnormal glucose homeostasis (12Wang P. Yan Z. Zhong J. Chen J. Ni Y. Li L. Ma L. Zhao Z. Liu D. Zhu Z. Transient receptor potential vanilloid 1 activation enhances gut glucagon-like peptide-1 secretion and improves glucose homeostasis.Diabetes. 2012; 61: 2155-2165Crossref PubMed Scopus (95) Google Scholar). Numerous studies have revealed that capsaicin has both transient receptor potential vanilloid 1 (TRPV1)-dependent and -independent actions in mammals. The former includes such as capsaicin triggering a painful and burning sensation throughout human GI tract (13Domotor A. Peidl Z. Vincze A. Hunyady B. Szolcsanyi J. Kereskay L. Szekeres G. Mozsik G. Immunohistochemical distribution of vanilloid receptor, calcitonin-gene related peptide and substance P in gastrointestinal mucosa of patients with different gastrointestinal disorders.Inflammopharmacology. 2005; 13: 161-177Crossref PubMed Scopus (59) Google Scholar, 14Holzer P. Transient receptor potential (TRP) channels as drug targets for diseases of the digestive system.Pharmacol. Ther. 2011; 131: 142-170Crossref PubMed Scopus (167) Google Scholar) and increasing glucagon-like peptide-1 levels in the plasma and the ileum (12Wang P. Yan Z. Zhong J. Chen J. Ni Y. Li L. Ma L. Zhao Z. Liu D. Zhu Z. Transient receptor potential vanilloid 1 activation enhances gut glucagon-like peptide-1 secretion and improves glucose homeostasis.Diabetes. 2012; 61: 2155-2165Crossref PubMed Scopus (95) Google Scholar), but the latter includes such as capsaicin inhibiting intestinal epithelial anion secretion and inducing apoptosis of gastric cancer cells (15Bouyer P.G. Tang X. Weber C.R. Shen L. Turner J.R. Matthews J.B. Capsaicin induces NKCC1 internalization and inhibits chloride secretion in colonic epithelial cells independently of TRPV1.Am. J. Physiol. Gastrointest. Liver Physiol. 2013; 304: G142-G156Crossref PubMed Scopus (15) Google Scholar, 16Braga Ferreira L.G. Faria J.V. Dos Santos J.P.S. Faria R.X. Capsaicin: TRPV1-independent mechanisms and novel therapeutic possibilities.Eur. J. Pharmacol. 2020; 887: 173356Crossref PubMed Scopus (17) Google Scholar). While the TRPV1-dependent action of capsaicin has been studied extensively on sensory neurons mostly, little is known about its TRPV1-independent actions on epithelial cells in GI health and disease. Moreover, growing evidence suggests that TRPV4 channels, another important member of TRPV family, play a critical role in the pathogenesis of UC (17D'Aldebert E. Cenac N. Rousset P. Martin L. Rolland C. Chapman K. Selves J. Alric L. Vinel J.P. Vergnolle N. Transient receptor potential vanilloid 4 activated inflammatory signals by intestinal epithelial cells and colitis in mice.Gastroenterology. 2011; 140: 275-285Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 18Matsumoto K. Yamaba R. Inoue K. Utsumi D. Tsukahara T. Amagase K. Tominaga M. Kato S. Transient receptor potential vanilloid 4 channel regulates vascular endothelial permeability during colonic inflammation in dextran sulphate sodium-induced murine colitis.Br. J. Pharmacol. 2018; 175: 84-99Crossref PubMed Scopus (26) Google Scholar, 19Yamawaki H. Mihara H. Suzuki N. Nishizono H. Uchida K. Watanabe S. Tominaga M. Sugiyama T. Role of transient receptor potential vanilloid 4 activation in indomethacin-induced intestinal damage.Am. J. Physiol. Gastrointest. Liver Physiol. 2014; 307: G33-G40Crossref PubMed Scopus (25) Google Scholar); however, it is unknown if capsaicin acts on aberrant TRPV4 channels to affect UC outcome. In addition, although pathological roles of TRPV4 channels have attracted more attention in GI disease, their physiological roles in intestinal epithelial ion transports are still obscure. Since capsaicin has long been used as daily flavoring worldwide and is orally delivered to GI tract, it is therefore important to investigate its actions in GI health and disease. In particular, the effects of dietary capsaicin on IBD and the underlying mechanisms need to be verified since it is still elusive if capsaicin is a friend or foe to UC. Capsaicin was reported previously to alleviate (20Kihara N. de la Fuente S.G. Fujino K. Takahashi T. Pappas T.N. Mantyh C.R. Vanilloid receptor-1 containing primary sensory neurones mediate dextran sulphate sodium induced colitis in rats.Gut. 2003; 52: 713-719Crossref PubMed Scopus (169) Google Scholar), exacerbate (21Engel M.A. Khalil M. Mueller-Tribbensee S.M. Becker C. Neuhuber W.L. Neurath M.F. Reeh P.W. The proximodistal aggravation of colitis depends on substance P released from TRPV1-expressing sensory neurons.J. Gastroenterol. 2012; 47: 256-265Crossref PubMed Scopus (66) Google Scholar), or not affect the severity of experimental UC (22Lee J. Yamamoto T. Kuramoto H. Kadowaki M. TRPV1 expressing extrinsic primary sensory neurons play a protective role in mouse oxazolone-induced colitis.Auton. Neurosci. 2012; 166: 72-76Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). However, clinical epidemiological studies indicate that the incidence rate of IBD, including UC and CD, is much lower in the high chili consuming regions in China (such as Chengdu and Xian) than the light/no chili consuming regions (such as Guangzhou and Hong Kong) (23Ng W.K. Wong S.H. Ng S.C. Changing epidemiological trends of inflammatory bowel disease in Asia.Intest Res. 2016; 14: 111-119Crossref PubMed Scopus (188) Google Scholar, 24Ng S.C. Kaplan G.G. Tang W. Banerjee R. Adigopula B. Underwood F.E. Tanyingoh D. Wei S.C. Lin W.C. Lin H.H. Li J. Bell S. Niewiadomski O. Kamm M.A. Zeng Z. et al.Population density and risk of inflammatory bowel disease: A prospective population-based study in 13 countries or regions in asia-pacific.Am. J. Gastroenterol. 2019; 114: 107-115Crossref PubMed Scopus (93) Google Scholar), suggesting that dietary capsaicin might prevent/treat human IBD. Consistently, in a previous study, we demonstrated that capsaicin ameliorated experimental colitis through vasorelaxation of submucosal arteries to likely increase blood perfusion to the colonic mucosae (25Zhang L. Lu W. Lu C. Guo Y. Chen X. Chen J. Xu F. Wan H. Dong H. Beneficial effect of capsaicin via TRPV4/EDH signals on mesenteric arterioles of normal and colitis mice.J. Adv. Res. 2021; https://doi.org/10.1016/j.jare.2021.11.001Crossref Scopus (1) Google Scholar); however, it is largely unknown for the direct actions of capsaicin on intestinal epithelia in health and UC and the underlying molecular mechanisms. Therefore, this fellow-up study sought to further investigate these issues. Using multidisciplinary approaches, including electrophysiology, pharmacology, molecular biology, and transgenic mouse models (TRPV1 and TRPV4 KO mice) in vitro and in vivo approaches, we uncover that capsaicin inhibits intestinal Cl- secretion and promotes Na+ absorption predominately by blocking TRPV4 channels to exert its beneficial anti-colitic action. Since epithelial ion transport is a defining physiological process of the intestine, we performed Ussing chamber experiments to examine the effect of capsaicin on epithelial ion transport in the jejunum and distal colon in mice. In the first series of experiments, we tested the effect of capsaicin on jejunal Isc. In the present study, we applied capsaicin at 30 μM (IC50 value on T84 cell monolayer Isc) (15Bouyer P.G. Tang X. Weber C.R. Shen L. Turner J.R. Matthews J.B. Capsaicin induces NKCC1 internalization and inhibits chloride secretion in colonic epithelial cells independently of TRPV1.Am. J. Physiol. Gastrointest. Liver Physiol. 2013; 304: G142-G156Crossref PubMed Scopus (15) Google Scholar) to native intestinal tissues. Both mucosal and serosal additions of capsaicin (30 μM) did not affect basal jejunal Isc (Fig. 1A). However, either mucosal or serosal addition of capsaicin significantly attenuated carbachol (CCh)-induced jejunal Isc, with bilateral additions further attenuating CCh-induced jejunal Isc (Fig. 1, A and B), indicating that capsaicin inhibits CCh-induced Isc from both apical and basolateral sides of the jejunum via Ca2+ signaling because CCh is a well-known cell Ca2+ mobilizer in the epithelia. We also examined if capsaicin affects caffeine (Caf)-induced jejunal Isc via Ca2+ signaling predominately (26Zhang F. Wan H. Yang X. He J. Lu C. Yang S. Tuo B. Dong H. Molecular mechanisms of caffeine-mediated intestinal epithelial ion transports.Br. J. Pharmacol. 2019; 176: 1700-1716Crossref PubMed Scopus (8) Google Scholar). Similarly, capsaicin also inhibited Caf-induced Isc from each side of the jejunum (Fig. 1, A and C). Therefore, capsaicin inhibits the jejunal anion secretion likely triggered by Ca2+ signaling. In the second series of experiments, we tested if there is any regional heterogeneity between the jejunum and distal colon for the inhibitory effect of capsaicin. Like in the jejunum, capsaicin (30 μM) did not affect basal colonic Isc (Fig. 1D). (Z)-capsaicin and dihydrocapsaicin (30 μM), the other two selective activators of TRPV1 channels, did not affect basal colonic Isc neither (data not shown). However, capsaicin significantly attenuated CCh- and Caf-induced Isc after its addition to each side or both sides of the distal colon (Fig. 1, E and F). Thus, capsaicin as a well-known activator of TRPV1 channels inhibited intestinal Isc without altering its baseline. Moreover, there is no regional heterogeneity between the jejunum and distal colon for the inhibitory effect of capsaicin on CCh- or Caf-induced epithelial ion transports. Given these findings, the jejunum and distal colon were used in the subsequent experiments. Since capsaicin can act in both TRPV1-dependent and -independent manners, we utilized pharmacological blockade and genetic KO of TRPV1 channels to study the detailed role of capsaicin on intestinal ion transport. In WT mice, either mucosal or serosal addition of capsaicin still attenuated CCh- or Caf-induced colonic Isc in the presence of SB705498 (5 μM), a selective TRPV1 blocker (Fig. 1, G and H). Similarly, in TRPV1 KO mice, serosal addition of capsaicin also attenuated CCh- or Caf-induced jejunal Isc (Fig. 1, I and J). These data indicate that capsaicin inhibits intestinal anion secretion independently of TRPV1 channels. After excluding the well-known TRPV1-dependent actions of capsaicin, we examined its TRPV1-independent actions on intestinal anion secretion. Since cholinergic signaling can increase Ca2+ entry through TRPV4 channels (27Sonkusare S.K. Bonev A.D. Ledoux J. Liedtke W. Kotlikoff M.I. Heppner T.J. Hill-Eubanks D.C. Nelson M.T. Elementary Ca2+ signals through endothelial TRPV4 channels regulate vascular function.Science. 2012; 336: 597-601Crossref PubMed Scopus (369) Google Scholar), we tested if capsaicin-induced inhibition of intestinal anion secretion occurs via TRPV4 channels. In WT mice, either mucosal or serosal addition of capsaicin failed to attenuate CCh- or Caf-induced colonic Isc in the presence of HC067047 (10 μM), a selective TRPV4 blocker (Fig. 1, K and L). Similarly, in TRPV4 KO mice, capsaicin lost its inhibitory action on CCh- or Caf-induced colonic Isc (Fig. 1, M–O) and jejunal Isc (Fig. 1, P and Q). Taken together, these data suggest that TRPV4 channels are responsible for capsaicin inhibition of intestinal anion secretion. After demonstrating the involvements of TRPV4 channels in capsaicin-induced inhibition, we focused on regulatory role of these channels in anion secretion. First, the CCh- and Caf-induced colonic Isc was significantly reduced in TRPV4 KO mice compared to WT mice (Fig. 2, A and B). Second, TRPV4 activator RN1747 (40 μM) significantly potentiated the CCh (50 μM)-induced colonic Isc, which was prevented by RN1734 (50 μM) and HC067047 (10 μM), two selective TRPV4 blockers with different chemical structures (Fig. 2C). Similarly, TRPV4 activator RN1747 potentiated the CCh (50 μM)-induced colonic Isc in WT mice, which was attenuated by capsaicin (30 μM) (Fig. 2D). Third, these phenomena occurred in WT mice were abolished in TRPV4 mice (Fig. 2E). Together, these data confirm capsaicin inhibition of intestinal secretion by blocking TRPV4 channels. Although capsaicin inhibits anion secretion by blocking TRPV4 channels, their role in regulating secretion has not been explored. We initially applied RN1747, GSK1016790A, and 4α-PPD, three selective TRPV4 activators with different chemical structures, but all of them did not alter basal colonic Isc in WT mice (data not shown, n = 6). Since TRPV4 channels could be suppressed by phosphatidylinositol 4,5-bisphosphate (PIP2) under the resting state (28Harraz O.F. Longden T.A. Hill-Eubanks D. Nelson M.T. PIP2 depletion promotes TRPV4 channel activity in mouse brain capillary endothelial cells.Elife. 2018; 7e38689Crossref PubMed Scopus (64) Google Scholar), we tested if this also happens in the intestine by hydrolyzing PIP2 via activation of phospholipase C (PLC) and phospholipase A2 (PLA2) during cholinergic signaling (29Kankanamge D. Ubeysinghe S. Tennakoon M. Pantula P.D. Mitra K. Giri L. Karunarathne A. Dissociation of the G protein betagamma from the Gq-PLCbeta complex partially attenuates PIP2 hydrolysis.J. Biol. Chem. 2021; 296: 100702Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar, 30Sulpice J.C. Zachowski A. Devaux P.F. Giraud F. Requirement for phosphatidylinositol 4,5-bisphosphate in the Ca(2+)-induced phospholipid redistribution in the human erythrocyte membrane.J. Biol. Chem. 1994; 269: 6347-6354Abstract Full Text PDF PubMed Google Scholar, 31Rogalski S.L. Chavkin C. Eicosanoids inhibit the G-protein-gated inwardly rectifying potassium channel (Kir3) at the Na+/PIP2 gating site.J. Biol. Chem. 2001; 276: 14855-14860Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The active PLC inhibitor U73122 (30 μM), but not its inactive form U73343 (10 μM), attenuated TRPV4 activator RN1747-potentiated Isc induced by CCh (50 μM) (Fig. 3, A and B). Similar findings were seen with methyl arachidonyl fluorophosphonate (10 μM), a selective PLA2 inhibitor (Fig. 3C), However, arachidonic acid (AA) pretreatment could rescue this process (Fig. 3D). These data suggest that PIP2 suppresses intestinal TRPV4 channels, which can be relieved after hydrolysis of PIP2 by PLC and PLA2. Finally, we tested if cytochrome P450 (CYP450) enzyme is involved since CYP450 metabolites have been considered as the endogenous activators of TRPV4 channels. Indeed, the CCh (50 μM)-induced Isc potentiated by TRPV4 activator RN1747 was attenuated by miconazole (10 μM), the CYP450 inhibitor (Fig. 3E); however, this process could be rescued by AA, the substrate for CYP450 enzyme (Fig. 3F). Therefore, we reveal a novel role of TRPV4 channels in cholinergic signaling–mediated intestinal secretion, in which PLC, PLA2, and CYP450 enzymes are involved in the relief of TRPV4 channels suppressed by PIP2. After verifying capsaicin blockade of the Ca2+-permeable TRPV4 channels in native intestinal tissues, we applied single cell Ca2+ imaging to further study if capsaicin indeed blocks Ca2+ entry via TRPV4 channels in intestinal epithelial cell (IEC)-6 cells. First, GSK1016790A (10 nM), a selective TRPV4 agonist, significantly stimulated cytosolic Ca2+ concentrations ([Ca2+]cyt) signaling in Ca2+-containing solutions (Fig. 4A), but not in Ca2+-free solutions (Fig. 4C). Moreover, GSK-induced [Ca2+]cyt signaling in Ca2+-containing solutions could be abolished by a selective TRPV4 antagonist HC067047 (5 μM) (Fig. 4B). Figure 4D summarizes the GSK-induced changes in [Ca2+]cyt peak in the absence or the presence of Ca2+ or HC067047, which is consistent with previous reports on the functional expression of TRPV4 channels in IEC-6 (19Yamawaki H. Mihara H. Suzuki N. Nishizono H. Uchida K. Watanabe S. Tominaga M. Sugiyama T. Role of transient receptor potential vanilloid 4 activation in indomethacin-induced intestinal damage.Am. J. Physiol. Gastrointest. Liver Physiol. 2014; 307: G33-G40Crossref PubMed Scopus (25) Google Scholar). Second, capsaicin as a well-known TRPV1 agonist at high concentrations of 10 to 50 μM did not alter basal [Ca2+]cyt signaling in IEC-6 (Fig. 4, E–G), indicating undetectable activity of TRPV1 channels in these cells. However, capsaicin dose-dependently blocked Ca2+ entry via TRPV4 channels (Fig. 4, E–H). Capsaicin at 50 μM totally abolished GSK-induced [Ca2+]cyt signaling without altering 5 mM Ca2+-induced [Ca2+]cyt signaling, suggesting its selectivity for TRPV4 channels. Moreover, after shTRPV4 was applied to successfully knock down the protein expression of TRPV4 in IEC-6 cells (Fig. 4O), GSK-induced [Ca2+]cyt signaling in Ca2+-containing solutions was almost abolished as well (Fig. 4,I–L). These data obtained from IEC-6 cells verify our previous notion that capsaicin blocks Ca2+ entry via epithelial TRPV4 channels predominately. To identify the protein expression of TRPV4 channels in IEC-6 cells, we first performed immunofluorescence to stain for TRPV4 channels. As shown in Figure 4M, TRPV4 proteins were expressed in the cells, however, the immunofluorescence staining was not observed without the primary antibodies against TRPV4 in the control, indicating specific staining on these proteins in IEC-6 cells. Afterward, we applied Western blot to further confirm the protein expression of TRPV4 channels in these cells (Fig. 4N). Finally, we applied immunohistological analysis on TRPV4 protein expression in jejunal and colonic tissues from TRPV4 KO and WT mice. As shown in Figure 4P, TRPV4 expression was slightly detected in the tissues from WT mice (the left panels) but not detected in the tissues from TRPV4 KO mice with (the middle panels) or without primary antibody against TRPV4 (the right panels), indicating the specificity of TRPV4 antibody. Since intestinal Na+ absorption is also important for GI physiology and the reduced Na+ absorption would cause diarrhea, we further examined if capsaicin affects intestinal Na+ absorption. Because Na+-glucose cotransporter Na+-glucose cotransporter is critical for intestinal Na+ absorption, we tested the effect of capsaicin on jejunal Na+-glucose cotransporter. Glucose (5 mM) induced jejunal Isc in WT mice, which was significantly enhanced by either serosal addition of capsaicin (30 μM) (Fig. 5A) or both side additions (Fig. 5B). However, SB705498 (5 μM), a selective TRPV1 antagonist, affected neither glucose-induced Isc (Fig. 5C) nor capsaicin-enhanced Isc (Fig. 5D), excluding the involvement of TRPV1 channels. In contrast, capsaicin-enhanced jejunal Na+ absorption disappeared in TRPV4 KO mice after its addition to either serosal side (Fig. 5E) or both sides (Fig. 5F), indicating the role of TRPV4 channels in capsaicin-enhanced Na+ absorption. Surprisingly, glucose-induced jejunal Isc was about 2-fold greater in TRPV4 KO mice than in WT mice (Fig. 5G), revealing the TRPV4 suppression on jejunal Na+ absorption. Finally, capsaicin-enhanced Na+ absorption was comparable between TRPV4 KO and WT mice after its addition to either serosal side (Fig. 5H) or both sides (Fig. 5I), suggesting that capsaicin enhances Na+ absorption by blocking TRPV4 channels to relieve its suppression on the absorption in WT mice. The loss of TRPV4 channels results in the failure of capsaicin to enhance Na+ absorption in KO mice. Taken together, these data reveal a novel suppressive role of TRPV4 channels in jejunal Na+ absorption, which can be relieved by capsaicin blockade of the channels. Since the therapeutic actions of capsaicin on colitis are still elusive, we examined the effect of capsaicin in dextran sodium sulfate (DSS)-induced mouse colitis, a commonly used experimental model of UC. First, after mice were treated with DSS for 7 days, their body weight and colon length were reduced (Fig. 6, A and B), but the stool score and myeloperoxidase (MPO) were increased (Fig. 6, C and D), indicating successful establishment of colitis. Second, capsaicin (intragastrically 10 mg/kg, once per day for 7 days) rescued not only the DSS-induced decrease in body weight and colon length (Fig. 6, A and B), but also the increase in stool score and MPO (Fig. 6, C and D). Third, histological examination showed that compared to normal colon, the colitic colon had significant epithelial damage, inflammatory cell infiltration, all of which were rescued by capsaicin (Fig. 6, E and F). Finally, immunohistological analysis revealed that compared to normal colon, TRPV4 expression was upregulated in colitic colon, which is consistent with a previous report on the contribution of TRPV4 to UC exacerbation (18Matsumoto K. Yamaba R. Inoue K. Utsumi D. Tsukahara T. Amagase K. Tominaga M. Kato S. Transient receptor potential vanilloid 4 channel regulates vascular endothelial permeability during colonic inflammation in dextran sulphate sodium-induced murine colitis.Br. J. Pharmacol. 2018; 175: 84-99Crossref PubMed Scopus (26) Google Scholar). Likewise, capsaicin attenuated TRPV4 upregulation in mouse colitis (Fig. 6, E and G). These data reveal that capsaicin ameliorates UC likely via TRPV4 suppression, consistently with the previous reports (17D'Aldebert E. Cenac N. Rousset P. Martin L. Rolland C. Chapman K. Selves J. Alric L. Vinel J.P. Vergnolle N. Transient receptor potential vanilloid 4 activated inflammatory signals by intestinal epithelial cells and colitis in mice.Gastroenterology. 2011; 140: 275-285Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 18Matsumoto K. Yamaba R. Inoue K. Utsumi D. Tsukahara T. Amagase K. Tominaga M. Kato S. Transient receptor potential vanilloid 4 channel regulates vascular endothelial permeability during colonic inflammation in dextran sulphate sodium-induced murine colitis.Br. J. Pharmacol. 2018; 175: 84-99Crossref PubMed Scopus (26) Google Scholar, 19Yamawaki H. Mihara H. Suzuki N. Nishizono H. Uchida K. Watanabe S. Tominaga M. Sugiyama T. Role of transient receptor potential vanilloid 4 activation in indomethacin-induced intestinal damage.Am. J. Physiol. Gastrointest. Liver Physiol. 2014; 307: G33-G40Crossref PubMed Scopus (25) Google Scholar). Since the enhanced expression and activity of TRPV4 channels play pathogenesis role during colitis (17D'Aldebert E. Cenac N. Rousset P.
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