TRPA1-mediated Ca²⁺

<p><b>influx in pancreatic beta cells. </b><b>a, b.</b> Application of AITC and MG induce an increase in intracellular Ca²⁺ in RIN cells (size of the bar is 100 µM). <b>c.</b> MG-induced Ca²⁺ influx is inhibited by TRPA1 antagonist HC030031. <b>d.</b> Ca²⁺ influx induced by endogenous ligands PGJ₂, 4-HNE, and AITC in RIN cells. <b>e.</b> Ca²⁺ influx induced by H₂O₂ and AITC in RIN cells. <b>f.</b> AITC-and MG-induced an increase in intracellular Ca²⁺ in rat cultured primary pancreatic beta cells.</p

TRPA1-mediated insulin release is independent of voltage-gated Na⁺, Ca²⁺ and K_ATP channels.

<p><b>a.</b> AITC caused a significant increase in insulin release (n=6, ** p<0.01). The basal insulin release is inhibited by incubation of RIN cells with TTX (1 µM) (TTX, n=6, * p<0.05. When challenged with AITC (200 µM), there is a significant increase in insulin release AITC+TTX, n=6, * p<0.05 as compared to TTX. <b>b.</b> In the presence of Ca²⁺ channel blocker nimodipine (5 µM) basal insulin release is decreased significantly (n=6, * p<0.05), but there is a significant increase when challenged with AITC+nimodipine (n=6,** p<0.01). <b>c.</b> In the presence of K_ATP channel opener, diazoxide (200 µM), basal insulin release is significantly decreased (n=6, * p<0.05), when challenged with AITC, there is a significant increase in insulin release (n=3, ** p<0.01).</p

TRPA1-mediated insulin release in pancreatic beta cell line and primary isolated pancreatic islets.

<p><b>a,b.</b> Dose-dependent increase in insulin release induced by AITC (0.1–1000 µM, <b>n=7</b>) and MG (0.1–1000 µM, <b>n=5</b>) in RIN cells (* p<0.05). <b>c,d.</b> AITC and MG induce a significant increase (AITC, n=11,* p<0.001; MG, n=10 * p=0.004) in insulin release from primary isolated pancreatic beta cell islets that could be blocked by the specific TRPA1 antagonist AP-18 (AITC+AP-18, n=6, ** p<0.001; MG+AP-18, n=6, ** p=0.008). <b>e.</b> 4-HNE (100 µM)-induced insulin release is inhibited by HC030031 (100 µM) (4-HNE, n=6, * p<0.001; 4-HNE+HC030031, n=3, ** p<0.001). <b>f.</b> PGJ₂ (20 µM)-induced insulin release is inhibited by HC030031 (100 µM) (PGJ₂, n=6, * p<0.001; PGJ₂+HC030031, n=3, ** p<0.001).</p

Insulin release induced by different concentrations of glucose.

<p>a. Insulin release induced by different concentrations of glucose (6 mM, n=8, * p<0.001; 25 mM, n=9, *p<0.001) <b>b.</b> Insulin release induced by different concentrations of glucose is inhibited by HC030031 (100 µM) (6 mM, n=4, * p<0.001; 25 mM, n=7, * p<0.001, as compared to control). <b>c.</b> Insulin release induced by AITC (200 µM) in different concentrations of glucose is inhibited by HC030031 (100 µM) (6 mM, AITC, n=4, * p<0.01, AITC+HC030031, n=4, ** p<0.001; 25 mM, AITC, n=4, * p=0.023, AITC+HC030031, n=4, ** p<0.01).</p

Expression of TRPA1 in pancreatic beta cells.

<p><b>a.</b> RT-PCR shows the expression of TRPA1 mRNA in DRG neurons, whole pancreas (Pan), isolated islets (Isl), a pancreatic beta cell line (RIN), but not in a pancreatic alpha cell line (INR). <b>b.</b> Western blots show the expression of TRPA1 protein in RIN cells and HEK cells heterologously expressing TRPA1. c. Immunostaining of insulin (red), TRPA1 (green), and the co-expression (yellow) in the pancreatic islet (top panel). When the slices were incubated with the TRPA1 antibody after preabsorbing with a peptide used for making the antibody, the TRPA1 staining was considerably reduced (lower panel). The nuclei were stained with DAPI (scale bar=100 µM).</p

TRPA1-mediated membrane currents in primary pancreatic beta cells.

<p><b>a.</b> Membrane currents induced by extracellular application of MG and AITC in primary pancreatic beta cells. <b>b.</b> A concentration-response curve of membrane currents induced by MG included in the pipette solution in primary beta cells (EC₅₀=0.59 µM). Lower concentrations (∼1 µM) of MG are sufficient to induce maximal currents when applied intracellulary (inset), but the time to peak with lower concentrations is longer and the desensitization is profound at higher concentrations. <b>c.</b> Currents evoked by intracellular application of MG are reversibly blocked by extracellular application of AP-18. <b>d.</b> Currents elicited by MG and AITC in HEK 293T cells heterologously expressing TRPA1. <b>e.</b> MG-induced currents can be blocked by AP-18. <b>f.</b> Under current clamp conditions, extracellular application of MG depolarizes the membrane and generates action potentials that could be blocked by HC030031. <b>g.</b> Intracellular application of MG causes a robust depolarization and generates action potentials that could be blocked by HC030031.</p