Functional effects of temperature on pancreatic beta-cell insulin secretion and integrity

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Transient receptor potential melastatin 8 channel involvement in the regulation of vascular tone

The transient receptor potential melastatin 8 (TRPM8) channel has been characterized as a cold and menthol receptor expressed in a subpopulation of sensory neurons but was recently identified in other tissues, including the respiratory tract, urinary system, and vasculature. Thus TRPM8 may play multiple functional roles, likely to be in a tissue- and activation state-dependent manner. We examined the TRPM8 channel presence in large arteries from rats and the functional consequences of their activation. We also aimed to examine whether these channels contribute to control of conscious human skin blood flow. TRPM8 mRNA and protein were detected in rat tail, femoral and mesenteric arteries, and thoracic aorta. This was confirmed in single isolated vascular myocytes by immunocytochemistry. Isometric contraction studies on endothelium-denuded relaxed rat vessels found small contractions on application of the TRPM8-specific agonist menthol (300 μM). However, both menthol and another agonist icilin (50 μM) caused relaxation of vessels precontracted with KCl (60 mM) or the α-adrenoceptor agonist phenylephrine (2 μM) and a reduction in sympathetic nerve-mediated contraction. These effects were antagonized by bromoenol lactone treatment, suggesting the involvement of Ca2+-independent phospholipase A2 activation in TRPM8-mediated vasodilatation. In thoracic aorta with intact endothelium, menthol-induced inhibition of KCl-induced contraction was enhanced. This was unaltered by preincubation with either Nω-nitro-l-arginine methyl ester (l-NAME; 100 nM), a nitric oxide synthase inhibitor, or the ACh receptor antagonist atropine (1 μM). Application of menthol (3% solution, topical application) to skin caused increased blood flow in conscious humans, as measured by laser Doppler fluximetry. Vasodilatation was markedly reduced or abolished by prior application of l-NAME (passive application, 10 mM) or atropine (iontophoretic application, 100 nM, 30 s at 70 μA). We conclude that TRPM8 channels are present in rat artery vascular smooth muscle and on activation cause vasoconstriction or vasodilatation, dependent on previous vasomotor tone. TRPM8 channels may also contribute to human cutaneous vasculature control, likely with the involvement of additional neuronal mechanisms

Src and ADAM-17-Mediated Shedding of Transforming Growth Factor-alpha Is a Mechanism of Acute Resistance to TRAIL

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L) has emerged as a promising anticancer agent. However, resistance to TRAIL is likely to be a major problem, and sensitization of cancer cells to TRAIL may therefore be an important anticancer strategy. In this study, we examined the effect of the epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) gefitinib and a human epidermal receptor 2 (HER2)-TKI (M578440) on the sensitivity of human colorectal cancer (CRC) cell lines to recombinant human TRAIL (rhTRAIL). A synergistic interaction between rhTRAIL and gefitinib and rhTRAIL and M578440 was observed in both rhTRAIL-sensitive and resistant CRC cells. This synergy correlated with an increase in EGFR and HER2 activation after rhTRAIL treatment. Furthermore, treatment of CRC cells with rhTRAIL resulted in activation of the Src family kinases (SFK). Importantly, we found that rhTRAIL treatment induced shedding of transforming growth factor-α (TGF-α) that was dependent on SFK activity and the protease ADAM-17. Moreover, this shedding of TGF-α was critical for rhTRAIL-induced activation of EGFR. In support of this, SFK inhibitors and small interfering RNAs targeting ADAM-17 and TGF-α also sensitized CRC cells to rhTRAIL-mediated apoptosis. Taken together, our findings indicate that both rhTRAIL-sensitive and resistant CRC cells respond to rhTRAIL treatment by activating an EGFR/HER2-mediated survival response and that these cells can be sensitized to rhTRAIL using EGFR/HER2-targeted therapies. Furthermore, this acute response to rhTRAIL is regulated by SFK-mediated and ADAM-17-mediated shedding of TGF-α, such that targeting SFKs or inhibiting ADAM-17, in combination with rhTRAIL, may enhance the response of CRC tumors to rhTRAIL

Transient receptor potential ankyrin 1 channel localized to non-neuronal airway cells promotes non-neurogenic inflammation

BACKGROUND: The transient receptor potential ankyrin 1 (TRPA1) channel, localized to airway sensory nerves, has been proposed to mediate airway inflammation evoked by allergen and cigarette smoke (CS) in rodents, via a neurogenic mechanism. However the limited clinical evidence for the role of neurogenic inflammation in asthma or chronic obstructive pulmonary disease raises an alternative possibility that airway inflammation is promoted by non-neuronal TRPA1. METHODOLOGY/PRINCIPAL FINDINGS: By using Real-Time PCR and calcium imaging, we found that cultured human airway cells, including fibroblasts, epithelial and smooth muscle cells express functional TRPA1 channels. By using immunohistochemistry, TRPA1 staining was observed in airway epithelial and smooth muscle cells in sections taken from human airways and lung, and from airways and lung of wild-type, but not TRPA1-deficient mice. In cultured human airway epithelial and smooth muscle cells and fibroblasts, acrolein and CS extract evoked IL-8 release, a response selectively reduced by TRPA1 antagonists. Capsaicin, agonist of the transient receptor potential vanilloid 1 (TRPV1), a channel co-expressed with TRPA1 by airway sensory nerves, and acrolein or CS (TRPA1 agonists), or the neuropeptide substance P (SP), which is released from sensory nerve terminals by capsaicin, acrolein or CS), produced neurogenic inflammation in mouse airways. However, only acrolein and CS, but not capsaicin or SP, released the keratinocyte chemoattractant (CXCL-1/KC, IL-8 analogue) in bronchoalveolar lavage (BAL) fluid of wild-type mice. This effect of TRPA1 agonists was attenuated by TRPA1 antagonism or in TRPA1-deficient mice, but not by pharmacological ablation of sensory nerves. CONCLUSIONS: Our results demonstrate that, although either TRPV1 or TRPA1 activation causes airway neurogenic inflammation, solely TRPA1 activation orchestrates an additional inflammatory response which is not neurogenic. This finding suggests that non-neuronal TRPA1 in the airways is functional and potentially capable of contributing to inflammatory airway diseases

Acetaminophen, via its reactive metabolite N-acetyl-p-benzo-quinoneimine and transient receptor potential ankyrin-1 stimulation, causes neurogenic inflammation in the airways and other tissues in rodents

Acetaminophen [N-acetyl-p-aminophenol (APAP)] is the most common antipyretic/analgesic medicine worldwide. If APAP is overdosed, its metabolite, N-acetyl-p-benzo-quinoneimine (NAPQI), causes liver damage. However, epidemiological evidence has associated previous use of therapeutic APAP doses with the risk of chronic obstructive pulmonary disease (COPD) and asthma. The transient receptor potential ankyrin-1 (TRPA1) channel is expressed by peptidergic primary sensory neurons. Because NAPQI, like other TRPA1 activators, is an electrophilic molecule, we hypothesized that APAP, via NAPQI, stimulates TRPA1, thus causing airway neurogenic inflammation. NAPQI selectively excites human recombinant and native (neuroblastoma cells) TRPA1. TRPA1 activation by NAPQI releases proinflammatory neuropeptides (substance P and calcitonin gene-related peptide) from sensory nerve terminals in rodent airways, thereby causing neurogenic edema and neutrophilia. Single or repeated administration of therapeutic (15-60 mg/kg) APAP doses to mice produces detectable levels of NAPQI in the lung, and increases neutrophil numbers, myeloperoxidase activity, and cytokine and chemokine levels in the airways or skin. Inflammatory responses evoked by NAPQI and APAP are abated by TRPA1 antagonism or are absent in TRPA1-deficient mice. This novel pathway, distinguished from the tissue-damaging effect of NAPQI, may contribute to the risk of COPD and asthma associated with therapeutic APAP use

Functional TRPA1 is expressed in human airway/lung cells.

<p>Intracellular calcium response was used to assess agonist-induced TRPA1 activation in small airways epithelial cells (SAEC) (<b>A</b>), normal human lung fibroblasts (NHLF) (<b>B</b>) and bronchial smooth muscle cells (HBSMC) (<b>C</b>). Typical traces and pooled data of the concentration-dependent calcium response evoked by the selective TRPA1 agonists, cinnamaldehyde (CNM, typical traces and black circles) and acrolein (ACR, grey circles), in all different cell types in primary culture. Similarly to CNM and ACR, cigarette smoke extract (CSE, black triangles) produces in all the different types of cells a concentration-dependent calcium response. Responses to CNM, ACR and CSE are prevented by selective TRPA1 antagonists, HC-030031 (HC, 10 µM) and AP18 (10 µM). The activating peptide (SLIGKV-NH₂) of the PAR-2 receptor (PAR-2 AP, 100 µM) elicits a calcium response that is not modified by TRPA1 antagonists. Veh is a combination of vehicles of HC and AP18. Values represent mean ± SEM of n>25 cells. ^*<i>P</i><0.05 <i>vs.</i> Veh.</p

Potency (EC₅₀ and CI) of the various TRPA1 agonists in different non-neuronal cell types of the human respiratory tract.

<p>Potency (EC₅₀ and CI) of the various TRPA1 agonists in different non-neuronal cell types of the human respiratory tract.</p

TRPA1 channel expression in non-neuronal cells of the human and mouse respiratory tract.

<p>(<b>A</b>) Total RNAs were extracted from primary small airways epithelial cells (SAEC), human type II alveolar epithelial cells (A549), human primary smooth muscle cells (HBSMC), human embryonic lung fibroblasts (IMR90) and primary normal human lung fibroblasts (NHLF) and relative TRPA1 mRNA amounts were measured by Taqman Real-Time PCR assay. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042454#s2" target="_blank">Results</a> are normalized to the reference gene, β-actin. Each column represents mean ± SEM of n>2 independent experiments. Immunohistochemical analysis of TRPA1 expression in samples taken from human (<b>B</b>) or <i>Trpa1^+/+</i> and <i>Trpa1^−/−</i> mouse airways and lung (<b>C</b>). Representative images of TRPA1 immunostaining show intense staining in epithelial and smooth muscle cells in human tissue. No staining is detected in human samples incubated with the normal serum peptide (Negative control). (<b>C</b>) Incubation with TRPA1 antibody shows a strong staining in epithelial and smooth muscle cells in tissues taken from <i>Trpa1^+/+</i> mice, but not in those from <i>Trpa1^−/−</i> mice. Preadsorption of the TRPA1 antibody with the peptide used for immunization abolished staining (Peptide). Staining for cytokeratin and α-smooth muscle actin (α-SMA) overlaps with the TRPA1 staining in the bronchial epithelium and smooth muscle layer in serial section of human and mice airways/lung tissues (<b>B</b> and <b>C</b>). Scale bar 100 µm.</p