Expression of Transient Receptor Potential Ankyrin 1 (TRPA1) and Its Role in Insulin Release from Rat Pancreatic Beta Cells

<div><h3>Objective</h3><p>Several transient receptor potential (TRP) channels are expressed in pancreatic beta cells and have been proposed to be involved in insulin secretion. However, the endogenous ligands for these channels are far from clear. Here, we demonstrate the expression of the transient receptor potential ankyrin 1 (TRPA1) ion channel in the pancreatic beta cells and its role in insulin release. TRPA1 is an attractive candidate for inducing insulin release because it is calcium permeable and is activated by molecules that are produced during oxidative glycolysis.</p> <h3>Methods</h3><p>Immunohistochemistry, RT-PCR, and Western blot techniques were used to determine the expression of TRPA1 channel. Ca²⁺ fluorescence imaging and electrophysiology (voltage- and current-clamp) techniques were used to study the channel properties. TRPA1-mediated insulin release was determined using ELISA.</p> <h3>Results</h3><p>TRPA1 is abundantly expressed in a rat pancreatic beta cell line and freshly isolated rat pancreatic beta cells, but not in pancreatic alpha cells. Activation of TRPA1 by allyl isothiocyanate (AITC), hydrogen peroxide (H₂O₂), 4-hydroxynonenal (4-HNE), and cyclopentenone prostaglandins (PGJ₂) and a novel agonist methylglyoxal (MG) induces membrane current, depolarization, and Ca²⁺ influx leading to generation of action potentials in a pancreatic beta cell line and primary cultured pancreatic beta cells. Activation of TRPA1 by agonists stimulates insulin release in pancreatic beta cells that can be inhibited by TRPA1 antagonists such as HC030031 or AP-18 and by RNA interference. TRPA1-mediated insulin release is also observed in conditions of voltage-gated Na⁺ and Ca²⁺ channel blockade as well as ATP sensitive potassium (K_ATP) channel activation.</p> <h3>Conclusions</h3><p>We propose that endogenous and exogenous ligands of TRPA1 cause Ca²⁺ influx and induce basal insulin release and that TRPA1-mediated depolarization acts synergistically with K_ATP channel blockade to facilitate insulin release.</p> </div

Mechanisms Underlying Chronic Pain

Based on duration, pain is categorized as acute and chronic pain. Acute pain is a short-term phenomenon that resolves after the noxious stimulus is removed or the injury is healed. On the other hand, chronic pain is a long-lasting debilitating condition. In a substantial number of cases, acute pain converts to chronic pain after months or years of the initial injury or damage. The current pain models are designed to study either acute or chronic pain, but not the transition from acute to chronic pain. Also, currently available pain models do not permit a direct comparison of molecular changes occurring in each condition or during the transitory process. Despite decades of research, current treatments seek to alleviate but not resolve chronic pain. Previous studies have shown that prolonged inflammation is a critical factor for initiation and maintenance of chronic pain. On a different platform of research, regenerative medicine strategies have employed Extracellular Matrix (ECM) for wound healing. This goal is achieved over a complex myriad of processes, which mainly involve inhibition of long-term inflammation. Based on these, I hypothesize that neurochemical changes become persistent in chronic pain conditions and components in ECM prevent these changes. The current study addresses two main issues: 1) establishing a pain model that allows the study of transition of acute to chronic pain and 2) using ECM as a possible therapy against chronic pain conditions. In the first aim of the study, low-dose of CFA induced acute pain (a short-term thermal hyperalgesia) and a high-dose of CFA induced chronic pain (a long-term thermal hyperalgesia and mechanical allodynia). In low-dose CFA-treated group, RT-PCR analyses revealed a significant increase in mRNA levels of anti-inflammatory mediators (IL-10, IL-13 and TGF-beta) and a significant decrease in mRNA levels of pro-inflammatory mediators (IL-1beta, IL-6, MCP-1, MMP-9, MMP-12 and TNF-alpha) and these neurochemical changes corresponded with behavior. In high-dose CFA-treated group, chronic pain states corresponded with a significant elevation of mRNA levels of pro-inflammatory mediators, while there was a significant decrease in mRNA levels of anti-inflammatory mediators. These findings were validated with well-established models of acute pain (intraplantar capsaicin or carrageenan) and chronic pain (Chronic Constriction Injury; CCI and Streptozotocin-induced neuropathy). The changes observed in low-dose CFA-treated group were similar to those of capsaicin or carrageenan treatment, whereas changes in the high-dose CFA-treated group resembled to those of CCI and STZ-treatment. TRPV1, a nociceptive ion channel expression and function were significantly increased in both acute and chronic pain models. TRPV1 function in the DRG is more pronounced in acute pain, while there was a greater change in the spinal cord in chronic pain, suggesting a differential role of DRG and spinal cord in acute and chronic pain conditions. The role of TRPV1 was further explored by intrathecal administration of RTX, which attenuated only inflammatory thermal hyperalgesia. These results demonstrate that graded doses of CFA as a possible model to study the transition from acute to chronic pain. Monocyte Chemoattractant Protein (MCP-1) and Matrix Metalloproteinases (MMP-9 and MMP-12) are key molecules; the levels of which increase in acute pain conditions and facilitate the transition to chronic pain conditions. In fact, RTX is in clinical trials as a viable treatment option in treating terminal debilitating cancer pain conditions. In the second aim of the study, high-dose of CFA treatment and CCI were used to mimic chronic pain conditions. Significantly, unlike RTX treatment, intrathecal administration of ECM alleviated both inflammatory thermal hyperalgesia and mechanical allodynia. ECM treatment also reduced the intensity of TRPV1 staining in spinal dorsal horn which, was substantiated by a significant decrease in basal and TRPV1-mediated CGRP release in spinal cord samples. RT-PCR analysis of spinal cord and DRG samples revealed that ECM treatment significantly decreased mRNA levels of several pro-inflammatory cytokines. On the other hand, mRNA levels of anti-inflammatory mediators were significantly increased after ECM treatment in both DRG and spinal cord samples. ECM treatment also decreased intensity of OX-42 (a marker for activated microglia) staining in the spinal dorsal horn. Taken together, these data suggest that ECM treatment restores homeostasis by balancing the levels of pro-inflammatory and anti-inflammatory mediators, which leads to alleviation of chronic pain. Therefore, ECM or components in ECM may provide a novel treatment option for chronic pain

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

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

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