Paclitaxel Regulates TRPA1 Function and Expression Through PKA and PKC.

Paclitaxel (PTX) is a frequently used anticancer drug that causes peripheral neuropathy. Transient receptor potential ankyrin 1 (TRPA1), a plasma membrane calcium channel, has been associated with PTX toxicity and with other chemotherapy agents such as oxaliplatin and vincristine. However, the effect of PTX on the functional expression and calcium currents of TRPA1 has not been determined. The present study shows the effect of PTX on TRPA1 activity in a neuronal cell line (SH-SY5Y). The effect of PTX on the expression of TRPA1 was assessed through quantitative PCR and Western blot analyses to determine the relative mRNA and protein expression levels. To assess the effect on calcium flux and currents, cells were exposed to PTX; simultaneously, a specific agonist and antagonist of TRPA1 were added to evaluate the differential response in exposed versus control cells. To assess the effect of PKA, PKC and PI3K on PTX-induced TRPA1 increased activity, selective inhibitors were added to these previous experiments. PTX increased the mRNA and protein expression of TRPA1 as well as the TRPA1-mediated Ca2+ currents and intracellular Ca2+ concentrations. This effect was dependent on AITC (a selective specific agonist) and was abolished with HC-030031 (a selective specific antagonist). The inhibition of PKA and PKC reduced the effect of PTX on the functional expression of TRPA1, whereas the inhibition of PI3K had no effects. PTX-induced neuropathy involves TRPA1 activity through an increase in functional expression and is regulated by PKA and PKC signaling. These findings support the role of the TRPA1 channel in the mechanisms altered by PTX, which can be involved in the process that lead to chemotherapy-induced neuropathy.

Decellularization and In Vivo Recellularization of Abdominal Porcine Fascial Tissue.

BACKGROUND: Tissue decellularization has evolved as a promising approach for tissue engineering applications. METHODS: In this study, we harvested fascial tissue from porcine anterior abdominal wall and the samples were decellularized with a combination of agents such as Triton X-100, trypsin and DNAase. Afterwards, we evaluated cell removal by histological analysis and DNA quantification. Mechanical functionality was evaluated by applying a range of hydrostatic pressures. A sample of decellularized fascia was transplanted into a rabbit and after 15 days a biopsy of this tissue was examined; the animal was observed during 6 months after surgery. RESULTS: The extracellular matrix was retained with a complete decellularization as evidenced by histologic examination. The DNA content was significantly reduced. The scaffold preserved its tensile mechanical properties. The graft was incorporated into a full thickness defect made in the rabbit abdominal wall. This tissue was infiltrated by granulation and inflammatory cells and the histologic structure was preserved 15 days after surgery. The animal did not develop hernias, infections or other complications, after a 6-months of follow up. CONCLUSIONS: The protocol of decellularization of fascial tissue employed in this study proved to be efficient. The mechanical test demonstrated that the samples were not damaged and maintained its physical characteristics; clinical evolution of the rabbit, recipient of the decellularized fascia, demonstrated that the graft was effective as a replacement of native tissue.In conclusion, a biological scaffold derived from porcine fascial tissue may be a suitable candidate for tissue engineering applications.

Modulating TRPV4 channels with paclitaxel and lithium.

Transient receptor potential V4 (TRPV4), a plasma membrane calcium channel, is implicated as a contributor to the initiation of chemotherapy-induced peripheral neuropathy (CIPN). Paclitaxel (PTX) is a commonly used anticancer drug that causes CIPN and lithium has been shown to prevent CIPN. However, the direct effect of PTX and lithium on TRPV4 is not clear. This study investigated these actions using biochemical, pharmacological, and electrophysiological approaches using a neuronal cell line (SH-SY5Y). The addition of pharmacologically appropriate levels of PTX increased the expression of TRPV4, TRPV4 currents, and TRPV4-dependent calcium fluxes. Prolonged exposure to PTX amplified the acute effects of TRPV4 expression, currents, and calcium fluxes. Pretreatment with lithium (1 mM) decreased TRPV4 currents and calcium fluxes in the absence and presence of PTX. These findings enhance our understanding of the properties and regulation of TRPV4, the cellular mechanisms of PTX-induced neuropathy, and the mechanism of lithium for prevention of CIPN.

TRPV4 Channels in Human White Adipocytes: Electrophysiological Characterization and Regulation by Insulin.

Intracellular calcium homeostasis in adipocytes is important for the regulation of several functions and is involved in pathological changes in obesity and other associated diseases. Transient Receptor Potential Vanilloid 4 (TRPV4) channels are an important route for calcium entry that operates in a variety of cells and intervenes in a number of functions. In this study, the expression and operation of TRPV4 channels in human cultured adipocytes was evaluated using RT-PCR, Western blotting, the whole-cell patch-clamp technique and fluorescence measurements to characterize these channels and determine intracellular calcium responses. Both the hypoosmolarity and 4alpha-phorbol-didecanoate (4αPDD), a specific TRPV4 agonist, induced a similar HC-067047-sensitive current, which was predominantly inward, and an intracellular Ca(2+) concentration increase, which was exclusively dependent on extracellular calcium, and membrane depolarization. The current had a reverse potential of +31 ± 6 mV and exhibited preferential permeability to Ca(2+) . Insulin, which regulates metabolic homeostasis in adipocytes, attenuated the TRPV4-mediated effects. These results confirm the function of TRPV4 in human cultured adipocytes and its regulation by insulin. J. Cell. Physiol. 231: 954-963, 2016. © 2015 Wiley Periodicals, Inc.