Transient receptor potential vanilloid 4 (TRPV4)-dependent calcium influx and ATP release in mouse oesophageal keratinocytes

Abstract

Gastro-oesophageal reflux disease (GERD) is a multi-factorial disease that may involve oesophageal hypersensitivity to mechanical or heat stimulus as well as acids. Intraganglionic laminar endings (IGLEs) are the most prominent terminal structures of oesophageal vagal mechanosensitive afferents and may modulate mechanotransduction via purinergic receptors. Transient receptor potential channel vanilloid 4 (TRPV4) can detect various stimuli such as warm temperature, stretch and some chemicals, including 4α-phorbol 12,13-didecanoate (4α-PDD) and GSK1016790A. TRPV4 is expressed in many tissues, including renal epithelium, skin keratinocytes and urinary bladder epithelium, but its expression and function in the oesophagus is poorly understood. Here, we show anatomical and functional TRPV4 expression in mouse oesophagus and its involvement in ATP release. TRPV4 mRNA and protein were detected in oesophageal keratinocytes. Several known TRPV4 activators (chemicals, heat and stretch stimulus) increased cytosolic Ca2+ concentrations in cultured WT keratinocytes but not in TRPV4 knockout (KO) cells. Moreover, the TRPV4 agonist GSK1016790A and heat stimulus evoked TRPV4-like current responses in isolated WT keratinocytes, but not in TRPV4KO cells. GSK1016790A and heat stimulus also significantly increased ATP release from WT oesophageal keratinocytes compared to TRPV4KO cells. The vesicle-trafficking inhibitor brefeldin A (BFA) inhibited the ATP release. This ATP release could be mediated by the newly identified vesicle ATP transporter, VNUT, which is expressed by oesophageal keratinocytes at the mRNA and protein levels. In conclusion, in response to heat, chemical and possibly mechanical stimuli, TRPV4 contributes to ATP release in the oesophagus. Thus, TRPV4 could be involved in oesophageal mechano- and heat hypersensitivity.

Figure 1. Expression of TRPV4 and VNUT mRNAs in whole oesophagus and cultured oesophageal keratinocytes

Expression of mRNA for MHC2, CK14, TRPV2, TRPV3, TRPV4, VNUT (vesicular nucleotide transporter) and GAPDH was examined with (+) and without (–) RT reaction. Expected sizes of the amplified fragments were 562, 523, 552, 421, 404, 466 and 545 bp, respectively. TRPV2, TRPV3, TRPV4 and VNUT mRNAs were also detected in cultured oesophageal keratinocytes. MHC2. skeletal muscle marker; CK14, keratinocyte marker.

Figure 2. TRPV4 immunoreactivity in WT and TRPV4KO mouse oesophagus

TRPV4 immunoreactivity was observed in WT but not TRPV4KO oesophageal mucosal layers. Scale bars indicate 50 μm. CK14, keratinocyte marker; L, lumen; M, mucosa; Ms, muscle.

Figure 3. TRPV4-mediated cytosolic Ca²⁺ increase in primary oesophageal keratinocytes

A, traces for [Ca²⁺]_i changes (340/380 nm ratio) in the response to the TRPV4-specific agonist 4α-PDD, in WT and TRPV4KO (V4KO) keratinocytes (mean ± SEM). Bars indicate the duration of chemical application. B, 4α-PDD and another TRPV4-specific agonist GSK1016790A (GSK), increased [Ca²⁺]_i in WT oesophageal keratinocytes, but not in TRPV4KO cells. The increases were significantly inhibited by a broad TRP channel blocker, ruthenium red (RR), or in the absence of extracellular Ca²⁺ (Ca²⁺(–)) (†, *P < 0.05 vs. WT). Changes in ratio (Δ) were calculated by subtracting mean basal from peak values. C, traces for [Ca²⁺]_i changes in response to heat stimuli (25 to 40°C) in WT (in the presence or absence of extracellular Ca²⁺) or TRPV4KO keratinocytes (mean ± SEM). Bars indicate the duration of heat stimuli or a chemical application. D, [Ca²⁺]_i increase was larger in WT than in TRPV4KO keratinocytes and inhibited by RR or in the Ca²⁺(–) condition (†P < 0.05 vs. WT). [Ca²⁺]_i increase in TRPV3KO keratinocytes was similar to that for WT. E, representative pseudocolour images and traces for [Ca²⁺]_i changes in response to mechanical stimuli (140%) in WT and TRPV4KO keratinocytes cultured on silicon chambers (mean ± SEM). Images were obtained at points a, b and c indicated on the traces. A mechanical stimulus was applied between the blue boxes (black bar). F, [Ca²⁺]_i increase was significantly smaller in TRPV4KO than in WT keratinocytes and was inhibited significantly in the absence of Ca²⁺ (Ca²⁺(–), †P < 0.05 vs. WT).

Figure 4. TRPV4 agonist- and heat-induced current responses in primary oesophageal keratinocytes

A, GSK1016790A (GSK, 300 nm) evoked current responses in WT, but not in TRPV4KO cells. The currents were blocked by ruthenium red (RR; 10 μm). B, currents in response to ramp pulses at points a, b and c shown in A show a strongly outwardly rectifying I–V relationship. C, significantly larger inward currents at – 100 mV were obtained from WT keratinocytes compared with TRPV4KO cells (†P < 0.05). D, heat stimuli (25 to 45°C) evoked inward currents at –60 mV in WT and TRPV3KO (V3KO) keratinocytes, but not in TRPV4KO cells. E, currents in response to ramp pulses at points a and b shown in D show outwardly rectifying I–V relationship in WT and TRPV3KO cells. F, significantly larger inward currents at −60mV were obtained from WT keratinocytes compared with TRPV4KO (†P < 0.05 vs. WT or TRPV3KO).

Figure 5. VNUT immunoreactivity in the oesophagus and cultured keratinocytes

A, VNUT immunoreactivity in WT oesophagus mucosal layer. B, a punctate pattern of VNUT immunoreactivity was detected in cultured keratinocytes. Scale bars indicate 50 μm.

Figure 6. TRPV4 activators increase ATP release from oesophageal keratinocytes via exocytosis

A, the TRPV4 agonist GSK1016790A (GSK) increased ATP release from WT cultured oesophageal keratinocytes, but not from TRPV4KO cells. The increase was abolished in the absence of extracellular Ca²⁺ or by ruthenium red (RR) treatment in WT cells (†P < 0.05 vs. control). B, heat stimulus (from 25 to 38.5°C and kept at 38.5°C for 15 min) significantly increased ATP release from WT cultured keratinocytes compared with WT cells incubated at 37°C for 15 min (control) (†P < 0.05 vs. control). The increase was significantly smaller in TRPV4KO cells compared with WT or TRPV3KO cells (*P < 0.05). C, an exocytic stimulator, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), induced ATP release from TRPV4KO keratinocytes similar to that from WT cells (†, *P < 0.05 vs. control). D, the vesicle-trafficking inhibitor brefeldin A (BFA, 10 μm) significantly inhibited GSK-induced ATP release (†P < 0.05).