Inhibition of NHE1 transport activity and gene transcription in DRG neurons in oxaliplatin-induced painful peripheral neurotoxicity.

Oxaliplatin (OHP)-induced peripheral neurotoxicity (OIPN), one of the major dose-limiting side effects of colorectal cancer treatment, is characterized by both acute and chronic syndromes. Acute exposure to low dose OHP on dorsal root ganglion (DRG) neurons is able to induce an increase in intracellular calcium and proton concentration, thus influencing ion channels activity and neuronal excitability. The Na+/H+ exchanger isoform-1 (NHE1) is a plasma membrane protein that plays a pivotal role in intracellular pH (pHi) homeostasis in many cell types, including nociceptors. Here we show that OHP has early effects on NHE1 activity in cultured mouse DRG neurons: the mean rate of pHi recovery was strongly reduced compared to vehicle-treated controls, reaching levels similar to those obtained in the presence of cariporide (Car), a specific NHE1 antagonist. The effect of OHP on NHE1 activity was sensitive to FK506, a specific calcineurin (CaN) inhibitor. Lastly, molecular analyses revealed transcriptional downregulation of NHE1 both in vitro, in mouse primary DRG neurons, and in vivo, in an OIPN rat model. Altogether, these data suggest that OHP-induced intracellular acidification of DRG neurons largely depends on CaN-mediated NHE1 inhibition, revealing new mechanisms that OHP could exert to alter neuronal excitability, and providing novel druggable targets.

P2X Purinergic Receptors Are Multisensory Detectors for Micro-Environmental Stimuli That Control Migration of Tumoral Endothelium.

The tumoral microenvironment often displays peculiar features, including accumulation of extracellular ATP, hypoxia, low pH-acidosis, as well as an imbalance in zinc (Zn2+) and calcium (Ca2+). We previously reported the ability of some purinergic agonists to exert an anti-migratory activity on tumor-derived human endothelial cells (TEC) only when applied at a high concentration. They also trigger calcium signals associated with release from intracellular stores and calcium entry from the external medium. Here, we provide evidence that high concentrations of BzATP (100 µM), a potent agonist of P2X receptors, decrease migration in TEC from different tumors, but not in normal microvascular ECs (HMEC). The same agonist evokes a calcium increase in TEC from the breast and kidney, as well as in HMEC, but not in TEC from the prostate, suggesting that the intracellular pathways responsible for the P2X-induced impairment of TEC migration could vary among different tumors. The calcium signal is mainly due to a long-lasting calcium entry from outside and is strictly dependent on the presence of the receptor occupancy. Low pH, as well as high extracellular Zn2+ and Ca2+, interfere with the response, a distinctive feature typically found in some P2X purinergic receptors. This study reveals that a BzATP-sensitive pathway impairs the migration of endothelial cells from different tumors through mechanisms finely tuned by environmental factors.

Early Stimulation of TREK Channel Transcription and Activity Induced by Oxaliplatin-Dependent Cytosolic Acidification.

Oxaliplatin-induced peripheral neuropathy is characterized by an acute hyperexcitability syndrome triggered/exacerbated by cold. The mechanisms underlying oxaliplatin-induced peripheral neuropathy are unclear, but the alteration of ion channel expression and activity plays a well-recognized central role. Recently, we found that oxaliplatin leads to cytosolic acidification in dorsal root ganglion (DRG) neurons. Here, we investigated the early impact of oxaliplatin on the proton-sensitive TREK potassium channels. Following a 6-h oxaliplatin treatment, both channels underwent a transcription upregulation that returned to control levels after 42 h. The overexpression of TREK channels was also observed after in vivo treatment in DRG cells from mice exposed to acute treatment with oxaliplatin. Moreover, both intracellular pH and TREK channel transcription were similarly regulated after incubation with amiloride, an inhibitor of the Na+/H+ exchanger. In addition, we studied the role of oxaliplatin-induced acidification on channel behavior, and, as expected, we observed a robust positive modulation of TREK channel activity. Finally, we focused on the impact of this complex modulation on capsaicin-evoked neuronal activity finding a transient decrease in the average firing rate following 6 h of oxaliplatin treatment. In conclusion, the early activation of TREK genes may represent a mechanism of protection against the oxaliplatin-related perturbation of neuronal excitability.

Assessment of a Silicon-Photomultiplier-Based Platform for the Measurement of Intracellular Calcium Dynamics with Targeted Aequorin.

Ca2+ is among the most important intracellular second messengers participating in a plethora of biological processes, and the measurement of Ca2+ fluctuations is significant in the phenomenology of the underlying processes. Aequorin-based Ca2+ probes represent an invaluable tool for reliable measurement of Ca2+ concentrations and dynamics in different subcellular compartments. However, their use is limited due to the lack on the market of ready-to-use, cost-effective, and portable devices for the detection and readout of the low-intensity bioluminescence signal produced by these probes. Silicon photomultipliers (SiPMs) are rapidly evolving solid-state sensors for low light detection, with single photon sensitivity and photon number resolving capability, featuring low cost, low voltage, and compact format. Thus, they may represent the sensors of choice for the development of such devices and, more in general, of a new generation of multipurpose bioluminescence detectors suitable for cell biology studies. Ideally, a detector customized for these purposes must combine high dynamic range with high fidelity in reconstructing the light intensity signal temporal profile. In this article, the ability to perform aequorin-based intracellular Ca2+ measurements using a multipurpose, low-cost setup exploiting SiPMs as the sensors is demonstrated. SiPMs turn out to assure performances comparable to those exhibited by a custom-designed photomultiplier tube-based aequorinometer. Moreover, the flexibility of SiPM-based devices might pave the way toward routinely and wide scale application of innovative biophysical protocols.

Deletion of calcineurin from GFAP-expressing astrocytes impairs excitability of cerebellar and hippocampal neurons through astroglial Na+ /K+ ATPase.

Astrocytes perform important housekeeping functions in the nervous system including maintenance of adequate neuronal excitability, although the regulatory mechanisms are currently poorly understood. The astrocytic Ca2+ /calmodulin-activated phosphatase calcineurin (CaN) is implicated in the development of reactive gliosis and neuroinflammation, but its roles, including the control of neuronal excitability, in healthy brain is unknown. We have generated a mouse line with conditional knockout (KO) of CaN B1 (CaNB1) in glial fibrillary acidic protein-expressing astrocytes (astroglial calcineurin KO [ACN-KO]). Here, we report that postnatal and astrocyte-specific ablation of CaNB1 did not alter normal growth and development as well as adult neurogenesis. Yet, we found that specific deletion of astrocytic CaN selectively impairs intrinsic neuronal excitability in hippocampal CA1 pyramidal neurons and cerebellar granule cells (CGCs). This impairment was associated with a decrease in after hyperpolarization in CGC, while passive properties were unchanged, suggesting impairment of K+ homeostasis. Indeed, blockade of Na+ /K+ -ATPase (NKA) with ouabain phenocopied the electrophysiological alterations observed in ACN-KO CGCs. In addition, NKA activity was significantly lower in cerebellar and hippocampal lysates and in pure astrocytic cultures from ACN-KO mice. While no changes were found in protein levels, NKA activity was inhibited by the specific CaN inhibitor FK506 in both cerebellar lysates and primary astroglia from control mice, suggesting that CaN directly modulates NKA activity and in this manner controls neuronal excitability. In summary, our data provide formal evidence for the notion that astroglia is fundamental for controlling basic neuronal functions and place CaN center-stage as an astrocytic Ca2+ -sensitive switch.

The interaction of SiO2 nanoparticles with the neuronal cell membrane: activation of ionic channels and calcium influx.

AIM: To clarify the mechanisms of interaction between SiO2 nanoparticles (NPs) and the plasma membrane of GT1-7 neuroendocrine cells, with focus on the activation of calcium-permeable channels, responsible for the long lasting calcium influx and modulation of the electrical activity in these cells. MATERIALS & METHODS: Nontoxic doses of SiO2 NPs were administered to the cells. Calcium imaging and patch clamp techniques were combined with a pharmacological approach. RESULTS: TRPV4, Cx and Panx-like channels are the major components of the NP-induced inward currents. Preincubation with the antioxidant N-acetyl-L-cysteine strongly reduced the [Ca2+]i increase. CONCLUSION: These findings suggest that SiO2 NPs directly activate a complex set of calcium-permeable channels, possibly by catalyzing free radical production.

Oxaliplatin induces pH acidification in dorsal root ganglia neurons.

Oxaliplatin induced peripheral neurotoxicity is characterized by an acute cold-induced syndrome characterized by cramps, paresthesias/dysesthesias in the distal limbs and perioral region, that develops rapidly and lasts up to one week affecting nearly all the patients as well as by long-lasting symptoms. It has been previously shown that pharmacological or genetic ablation of TRPA1 responses reduces oxaliplatin-induced peripheral neurotoxicity in mouse models. In the present report, we show that treatment with concentrations of oxaliplatin similar to those found in plasma of treated patients leads to an acidification of the cytosol of mouse dorsal root ganglia neurons in culture and this in turn is responsible for sensitization of TRPA1 channels, thereby providing a mechanistic explanation to toxicity of oxaliplatin. Reversal of the acidification indeed leads to a significantly reduced activity of TRPA1 channels. Last, acidification occurs also in vivo after a single injection of therapeutically-relevant doses of oxaliplatin.