Epithelial transient receptor potential ankyrin 1 (TRPA1)-dependent adrenomedullin upregulates blood flow in rat small intestine

Abstract

The functional roles of transient receptor potential (TRP) channels in the gastrointestinal tract have garnered considerable attention in recent years. We previously reported that daikenchuto (TU-100), a traditional Japanese herbal medicine, increased intestinal blood flow (IBF) via adrenomedullin (ADM) release from intestinal epithelial (IE) cells (Kono T et al. J Crohns Colitis 4: 161-170, 2010). TU-100 contains multiple TRP activators. In the present study, therefore, we examined the involvement of TRP channels in the ADM-mediated vasodilatatory effect of TU-100. Rats were treated intraduodenally with the TRP vanilloid type 1 (TRPV1) agonist capsaicin (CAP), the TRP ankyrin 1 (TRPA1) agonist allyl-isothiocyanate (AITC), or TU-100, and jejunum IBF was evaluated using laser-Doppler blood flowmetry. All three compounds resulted in vasodilatation, and the vasodilatory effect of TU-100 was abolished by a TRPA1 antagonist but not by a TRPV1 antagonist. Vasodilatation induced by AITC and TU-100 was abrogated by anti-ADM antibody treatment. RT-PCR and flow cytometry revealed that an IEC-6 cell line originated from the small intestine and purified IE cells expressed ADM and TRPA1 but not TRPV1. AITC increased ADM release in IEC cells remarkably, while CAP had no effect. TU-100 and its ingredient 6-shogaol (6SG) increased ADM release dose-dependently, and the effects were abrogated by a TRPA1 antagonist. 6SG showed similar TRPA1-dependent vasodilatation in vivo. These results indicate that TRPA1 in IE cells may play an important role in controlling bowel microcirculation via ADM release. Epithelial TRPA1 appears to be a promising target for the development of novel strategies for the treatment of various gastrointestinal disorders.

Fig. 1.

Intraluminal transient receptor potential (TRP) vanilloid type 1 (TRPV1) and TRP ankyrin 1 (TRPA1) agonists increase blood flow in the small intestine. Capsaicin (CAP, 3 mg/kg body wt) or allyl isothiocyanate (AITC, 0.002 mg/kg body wt) was administered intraduodenally, and vascular conductance (VC) in the midjejunum was monitored. A: the TRPV1 antagonist N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide (BCTC) (10 mg/kg) was given intravenously 15 min before CAP administration; N = 3; B: TRPA1 antagonist HC-030031 (1 mg/kg) was administrated intraluminally together with AITC; N = 5–6. *P < 0.05, **P < 0.01 vs. water + vehicle (A) or vehicle (B). #P < 0.05, ##P < 0.01 vs. agonist alone, respectively.

Fig. 2.

TU-100 (daikenchuto) increases blood flow via the TRPA1-adrenomedullin cascade. TU-100 was administered intraduodenally at a dose of 2,700 mg/kg. Blood flow was monitored every 15 min after administration of TU-100. A: BCTC (10 mg/kg) was given intravenously 15 min before TU-100 administration; N = 3–5; B: HC-030031 (1 mg/kg) was administrated intraluminally together with TU-100; N = 6–7. C: antibody to adrenomedullin (ADM) was injected intravenously at a dose of 50 μg/kg 15 min before the administration of TU-100; N = 7–8. D: ADM content in the portal veins was measured using an EIA kit after purification on a C18 Sep-column; N = 16. E: AITC (0.002 mg/kg body wt) was administrated intraduodenally and anti-ADM antibody was injected as described above; N = 4. *P < 0.05, **P < 0.01 vs. water (B), no-antibody control (C), water (D), or no-antibody control (E). #P < 0.05, ##P < 0.01 vs. TU-100 alone, respectively.

Fig. 3.

TRPA1 and ADM expression in intestinal epithelial cells. A: RT-PCR analysis was performed for TRPA1, TRPV1, ADM, and β-actin in rat intestinal epithelial (IE) cells of the small intestine (S-IE), those of the large intestine (L-IE), dorsal root ganglion (DRG) cells, and the rat IE cell line IEC-6. The PCR products were resolved on a 2% agarose gel electrophoresis. B: flow cytometric analysis. a–c ADM images. d–f: TRPA1 images. a and d: S-IE. b and e: L-IE. c and f: IEC-6. Thin solid line: control Ab (rabbit IgG); thick solid line: antigen specific Ab; broken line: antigen specific Ab + epitope peptide. Data shown represent the results of 3 experiments.

Fig. 4.

TRPA1 agonists, TU-100, and individual TU-100 ingredients induce ADM release in IEC-6 cells. IEC-6 cells were incubated for 6 h in Hanks buffer containing 0.1% BSA with test compounds. Various TRP agonists (A), TU-100 (B), and ingredients in the medicinal plants constituting TU-100 (C, D, E) were added at the indicated concentrations. The concentrations of ADM in the incubation were measured by EIA. Among the TU-100 ingredients tested, 6-shogaol (6SG) and hydroxy-α-sanshool (HAS) induced ADM release; N = 3–4. 2-APB, 2-aminoethoxy diphenyl borate; 4α-PDD, 4α-phorbol 12,13-didecanoate; CNA, cinnamaldehyde; HBS, hydroxy-β-sanshool. *P < 0.05, **P < 0.01 vs. control, respectively.

Fig. 5.

AITC and 6SG stimulate TRPA1 to induce ADM release via protein kinase C. ADM release by TU-100 (2,700 μg/ml), AITC (30 μmol/l), and 6SG (30 μmol/l) was abrogated by cotreatment with 100 μmol/l of HC-030031 (A). AITC and 6SG induce calcium influx in T-Rex293 cells stably transfected with rat TRPA1 (B). Among the kinase inhibitors tested, the protein kinase C (PKC) inhibitor calphostin C potently inhibited AITC- and 6SG-induced ADM release (C). The PKC activator phorbol 12-myristate 13-acetate (PMA) induced ADM release (D); N = 3. **P < 0.01 vs. control.

Fig. 6.

Intraluminal 6SG increases blood flow in the small intestine. 6SG was administrated intraduodenally at a dose of 0.07, 0.2, or 0.6 mg/kg body wt and VC in the midjejunum was monitored. HC-030031 (1 mg/kg) was administered intraluminally together with 0.6 mg/kg of 6SG. Quantitation by area under curve (A) and time-dependent changes (B) are shown; N = 4–6, *P < 0.05, **P < 0.01 vs. vehicle, #P < 0.05, ##P < 0.01 vs. 6SG alone, respectively.

Fig. 7.

Gut TRPA1 elicits physiological and pathophysiological responses in 3 ways. TRPA1 activators have 3 potential target cells: intestinal epithelial (IE) cells, enterochromaffin (EC) cells, and TRPA1-positive sensory neurons. As a result of TRPA1 stimulation, TRPA1 agonists stimulate IE cells to release ADM, EC cells to release 5-HT, and sensory neurons to release neuropeptides/neurotransmitters, respectively, resulting in physiological and biodefensive responses in vasodilatation, motility, secretion, and pain signaling.