TRPV2 modulates mechanically Induced ATP Release from Human bronchial epithelial cells

Abstract

Repetitive bouts of coughing expose the large airways to significant cycles of shear stress. This leads to the release of alarmins and the tussive agent adenosine triphosphate (ATP) which may be modulated by the activity of ion channels present in the human airway. This study aimed to investigate the role of the transient receptor potential subfamily vanilloid member 2 (TRPV2) channel in mechanically induced ATP release from primary bronchial epithelial cells (PBECs).PBECs were obtained from individuals undergoing bronchoscopy. They were cultured in vitro and exposed to mechanical stress in the form of compressive and fluid shear stress (CFSS) or fluid shear stress (FSS) alone at various intensities. ATP release was measured using a luciferin-luciferase assay. Functional TRPV2 protein expression in human PBECs was investigated by confocal calcium imaging. The role of TRPV2 inhibition on FSS-induced ATP release was investigated using the TRPV2 inhibitor tranilast or siRNA knockdown of TRPV2. TRPV2 protein expression in human lung tissue was also determined by immunohistochemistry.ATP release was significantly increased in PBECs subjected to CFSS compared with control (unstimulated) PBECs (N = 3, ***P < 0.001). PBECs expressed functional TRPV2 channels. TRPV2 protein was also detected in fixed human lung tissue. ATP release from FFS stimulated PBECs was decreased by the TRPV2 inhibitor tranilast (N = 3, **P < 0.01) (vehicle: 159 ± 17.49 nM, tranilast: 25.08 ± 5.1 nM) or by TRPV2 siRNA knockdown (N = 3, *P < 0.05) (vehicle: 197 ± 24.52 nM, siRNA: 119 ± 26.85 nM).In conclusion, TRPV2 is expressed in the human airway and modulates ATP release from mechanically stimulated PBECs.

Keywords: Adenosine triphosphate; Mechanotransduction; Purinergic P2 × 3; Transient receptor potential channels.

Fig. 1

Mechanical Stimulation of PBECs Implemented Using a Seesaw Rocking Shaker or a Reciprocating Shaker. To evoke compressive and fluid shear stress (CFSS) a 6-well culture plate was placed on the platform of a seesaw rocking shaker (Sarstedt, Nümbrecht, Germany). Briefly, 6-well plates were placed on the seesaw rocking shaker and titled at a 30° angle horizontal to the ground. The plate was rotated from left to right every 15 s for 15 min, after which point the direction of the 30° angle of the 6-well plate was alternated and the rotation of the plate from left to right every 15 s recommenced for a further 15 min. Movement of the platform induces a wave of culture medium that settles at the bottom of the well for 15 s before a wave in the other direction occurs. This form of stimulation results in fluid shear stress, compressive stress and liquid surface tension from the thin layer of cell culture medium at the upper side of the well and fluid gravity being applied to the cells at the base of the well (A). To evoke fluid shear stress (FSS) 6-well culture plate was placed on the platform of a reciprocating shaker (Avantor, Pennsylvania, USA). The 6-well plate was stimulated with 0 (control), 25, 50 or 100 cycles per minute (CPM). Movement of the platform induced a wave of culture medium, stimulating cells with fluid shear stress stress and liquid surface tension from the thin layer of cell culture medium at one side of the well and fluid gravity being applied to the cells at the other side of the well (B)

Fig. 2

Measurement of ATP in Mechanically Stimulated Primary Bronchial Epithelial Cells (PBECs). ATP release from control and mechanically stressed PBECs using compressive and fluid shear stress treatment (CFSS) was measured with the ATPlite luciferin-luciferase assay. Mean with SEM, N = 3 independent experiments. Mann Whitney test, ***P < 0.001 (A). ATP release from control (0 CPM) and mechanically stressed PBECs using fluid shear stress (FSS) at 25, 50 and 100 CPM was measured with an ATPlite luciferin-luciferase assay. Mean with SEM, N = 3 independent experiments. Kruskal-Wallis test with Dunn’s multiple comparisons test, ***P < 0.001 (B). PBECs originated from a single donor (PBEC_1) (A and B). ATP release from control (0 CPM) and FSS stimulated PBECs (100 CPM) were measured with an ATPlite luciferin-luciferase assay. Mean with SEM, N = 3 independent experiments. Two-way ANOVA with Tukey’s multiple comparisons post-test, ***P < 0.001. PBECs originated from 4 individual donors (PBEC_1, PBEC_2, PBEC_3 and PBEC_4) with various phenotypes and smoking histories (C)

Fig. 3

TRPV2 Functional Expression in Primary Bronchial Epithelial Cells (PBECs). PBEC_1 were stained with TRPV2 primary antibody (A), or primary antibody omitted controls (B). Scale bars 25 μm. Confocal calcium imaging was carried out on PBEC_1 to investigate the functional expression of TRPV2 channels. Exemplar responses from 3 PBECs are shown with the addition of cannabidiol (CBD) and tranilast (TRN) (black arrow at 25 s) and addition of CBD alone (black arrow at 500 s) (C). Representative pseudo-coloured images showing PBEC responses to CBD ± tranilast (D). Peak responses (E) and area under curve of responses (F) of CBD ± tranilast were analysed. Mean with SEM, N = 91 individual PBECs. Wilcoxon matched-pairs signed rank test, ***P < 0.001

Fig. 4

TRPV2 Expression in Distal Airway Human Lung Sections. Tissue sections were stained with TRPV2 antibody (A & B), rabbit IgG control (C & D), no primary antibody controls (E & F). Scale bars 100 μm (A, C & E) or 50 μm (B, D & F)

Fig. 5

TRPV2 Mediates ATP Release in Fluid Shear Stress Stimulated Primary Bronchial Epithelial Cells (PBECs). ATP release from control (0 CPM) and fluid shear stress (FSS) stimulated PBEC_1 (100 CPM) treated with tranilast (TRN), or vehicle (VEH) was measured by ATPlite assay (A). Results were presented as percentage ATP release normalised to VEH (B). Mean with SEM, N = 3 independent experiments. Mann-Whitney test, ***P < 0.001. (A & B). TRPV2 expression in PBEC_2 transfected with 5 nM TRPV2 siRNA or transfection reagent vehicle (VEH) and exposed to fluid shear stress (FSS) at 100 CPM was measured by western blot 48 h post transfection. GAPDH was used as a loading control on the same membrane (C) (Full length blot available in the online supplement (supplementary Fig. 6a and 6b)). Semiqualitative densitometry analysis of TRPV2 band and GAPDH loading control band on the same membrane (D). ATP release from FSS stimulated PBEC_1 and PBEC_2 (100 CPM) transfected with 5 nM TRPV2 siRNA (siRNA) or transfection reagent vehicle (VEH) was measured 48 h post transfection (E). Results were presented as percentage ATP release normalised to VEH (F). Mean with SEM, N = 3 independent experiments. Mann-Whitney test *P < 0.05 (E) and P = 0.08 (F). ATP release from FSS stimulated PBEC_1 (100 CPM) transfected with 5 nM scrambled control (SCRAM) or TRPV2 siRNA (siRNA) was measured 48 h post transfection. Mean with SEM, N = 2 independent experiments. Kruskal-Wallis test with Dunn’s multiple comparisons test, *P < 0.05 and P = 0.31 (G)