Glutamate release induced by mild depolarization was studied in astroglial preparations from the adult rat cerebral cortex, that is acutely isolated glial sub-cellular particles (gliosomes), cultured ...adult or neonatal astrocytes, and neuron-conditioned astrocytes. K⁺ (15, 35 mmol/L), 4-aminopyridine (0.1, 1 mmol/L) or veratrine (1, 10 μmol/L) increased endogenous glutamate or ³H d-aspartate release from gliosomes. Neurotransmitter release was partly dependent on external Ca²⁺, suggesting the involvement of exocytotic-like processes, and partly because of the reversal of glutamate transporters. K⁺ increased gliosomal membrane potential, cytosolic Ca²⁺ concentration Ca²⁺i, and vesicle fusion rate. Ca²⁺ entry into gliosomes and glutamate release were independent from voltage-sensitive Ca²⁺ channel opening; they were instead abolished by 2-2-4-(4-nitrobenzyloxy)phenylethylisothiurea (KB-R7943), suggesting a role for the Na⁺/Ca²⁺ exchanger working in reverse mode. K⁺ (15, 35 mmol/L) elicited increase of Ca²⁺i and Ca²⁺-dependent endogenous glutamate release in adult, not in neonatal, astrocytes in culture. Glutamate release was even more marked in in vitro neuron-conditioned adult astrocytes. As seen for gliosomes, K⁺-induced Ca²⁺ influx and glutamate release were abolished by KB-R7943 also in cultured adult astrocytes. To conclude, depolarization triggers in vitro glutamate exocytosis from in situ matured adult astrocytes; an aptitude grounding on Ca²⁺ influx driven by the Na⁺/Ca²⁺ exchanger working in the reverse mode.
P2X7 receptors trigger Ca2+‐dependent exocytotic glutamate release, but also function as a route for non‐exocytotic glutamate release from neurons or astrocytes. To gain an insight into the ...mechanisms involving the P2X7 receptor as a direct pathway for glutamate release, we compared the behavior of a full‐length rat P2X7 receptor, a truncated rat P2X7 receptor in which the carboxyl tail had been deleted, a rat P2X7 receptor with the 18‐amino acid cysteine‐rich motif of the carboxyl tail deleted, and a rat P2X2 receptor, all of which are expressed in HEK293 cells. We found that the P2X7 receptor function as a route for glutamate release was antagonized in a non‐competitive way by extracellular Mg2+, did not require the recruitment of pore‐forming molecules, and was dependent on the carboxyl tail. Indeed, the truncated P2X7 receptor and the P2X7 receptor with the deleted cysteine‐rich motif both lost their function as a pathway for glutamate release, while still evoking intracellular Ca2+ elevation. No glutamate efflux was observed through the P2X2 receptor. Notably, HEK293 cells (lacking the machinery for Ca2+‐dependent exocytosis), when transfected with P2X7 receptors, appear to be a suitable model for investigating the P2X7 receptor as a route for non‐exocytotic glutamate efflux.
P2X7 receptors as a route for non‐exocytotic glutamate efflux
P2X7 receptors trigger Ca2+‐dependent exocytotic glutamate release, but also function as a route for non‐exocytotic glutamate release. The P2X7 receptor's function for non‐exocytotic glutamate efflux was dependent on the receptor C‐tail and involved the juxtamembrane cysteine‐rich motif of the tail. The finding should be relevant to understand the impact of naturally occurring P2X7 variants on glutamatergic transmission in health and disease.
Although growing evidence suggests that extracellular ATP might play roles in the control of astrocyte/neuron crosstalk in the CNS by acting on P2X₇ receptors, it is still unclear whether neuronal ...functions can be attributed to P2X₇ receptors. In the present paper, we investigate the location, pharmacological profile, and function of P2X₇ receptors on cerebrocortical nerve terminals freshly prepared from adult rats, by measuring glutamate release and calcium accumulation. The preparation chosen (purified synaptosomes) ensures negligible contamination of non-neuronal cells and allows exposure of 'nude' release-regulating pre-synaptic receptors. To confirm the results obtained, we also carried out specific experiments on human embryonic kidney 293 cells which had been stably transfected with rat P2X₇ receptors. Together, our findings suggest that (i) P2X₇ receptors are present in a subpopulation of adult rat cerebrocortical nerve terminals; (ii) P2X₇ receptors are localized on glutamatergic nerve terminals; (iii) P2X₇ receptors play a significant role in ATP-evoked glutamate efflux, which involves Ca²⁺-dependent vesicular release; and (iv) the P2X₇ receptor itself constitutes a significant Ca²⁺-independent mode of exit for glutamate.
In the brain, arachidonic acid (AA) plays a critical role in the modulation of a broad spectrum of biological responses, including those underlying neuroinflammation. By using microfluorometry, we ...investigated the action of extracellular AA in the modulation of the purinoceptor P2X7-mediated elevation of Ca(2+)(i) in cultured neocortical type-1 astrocytes and P2X7-, P2X2-transfected human embryonic kidney (HEK) 293 cells. We report that in cultured astrocytes, AA-induced Ca(2+)(i) elevation is coupled to depletion of intracellular Ca(2+) stores and to a sustained noncapacitative Ca(2+) entry. AA also induced a robust potentiation of the astrocytic P2X7-mediated Ca(2+)(i) rise evoked by the selective agonist 3'-O-(4-benzoyl)benzoyl-ATP (BzATP). Pharmacological studies demonstrate that the selective P2X7 antagonists oxidized ATP and Brilliant Blue G abrogated the AA-mediated potentiation of BzATP-evoked Ca(2+)(i) elevation. Fluorescent dye uptake experiments showed that the AA-induced increase in Ca(2+)(i) was not due to a switch of the P2X7 receptor from channel to the pore mode of gating. The synergistic effect of AA and BzATP was also observed in HEK293 cells stably expressing rat and human P2X7 but not in rat P2X2. Control HEK293 cells responded to AA exposure only with a transient Ca(2+)(i) elevation, whereas in those expressing the P2X7 receptor, AA elicited a potentiation of the BzATP-induced Ca(2+)(i) rise. Together, these findings indicate that AA mediates a complex regulation of Ca(2+)(i) dynamics also through P2X7-mediated Ca(2+) entry, suggesting that variations in AA production may be relevant to the control of both the temporal and spatial kinetics of Ca(2+)(i) signaling in astroglial cells.
Guanosine (Guo) is an endogenous neuroprotective molecule of the CNS, which has various acute and long‐term effects on both neurones and astroglial cells. Whether Guo also modulates the ...activity/expression of ion channels involved in homeostatic control of extracellular potassium by the astrocytic syncytium is still unknown. Here we provide electrophysiological evidence that chronic exposure (48 h) to Guo (500 μm) promotes the functional expression of an inward rectifier K+ (Kir) conductance in primary cultured rat cortical astrocytes. Molecular screening indicated that Guo promotes the up‐regulation of the Kir4.1 channel, the major component of the Kir current in astroglia in vivo. Furthermore, the properties of astrocytic Kir current overlapped those of the recombinant Kir4.1 channel expressed in a heterologous system, strongly suggesting that the Guo‐induced Kir conductance is mainly gated by Kir4.1. In contrast, the expression levels of two other Kir channel proteins were either unchanged (Kir2.1) or decreased (Kir5.1). Finally, we showed that inhibition of translational process, but not depression of transcription, prevents the Guo‐induced up‐regulation of Kir4.1, indicating that this nucleoside acts through de novo protein synthesis. Because accumulating data indicate that down‐regulation of astroglial Kir current contributes to the pathogenesis of neurodegenerative diseases associated with dysregulation of extracellular K+ homeostasis, these results support the notion that Guo might be a molecule of therapeutic interest for counteracting the detrimental effect of K+‐buffering impairment of the astroglial syncytium that occurs in pathological conditions.
Accumulating evidence indicate that the gap-junction inhibitor carbenoxolone (CBX) regulates neuronal synchronization, depresses epileptiform activity and has a neuroprotective action. These CBX ...effects do not depend solely on its ability to inhibit gap junction channels formed by connexins (Cx), but the underlying mechanisms remain to be elucidated. Here we addressed the questions whether CBX modulates volume-regulated anion channels (VRAC) involved in the regulatory volume decrease and regulates the associated release of excitatory amino acids in cultured rat cortical astrocytes. We found that CBX inhibits VRAC conductance with potency comparable to that able to depress the activity of the most abundant astroglial gap junction protein connexin43 (Cx43). However, the knock down of Cx43 with small interfering RNA (siRNA) oligonucleotides and the use of various pharmacological tools revealed that VRAC inhibition was not mediated by interaction of CBX with astroglial Cx proteins. Comparative experiments in HEK293 cells stably expressing another putative target of CBX, the purinergic ionotropic receptor P2X7, indicate that the presence of this receptor was not necessary for CBX-mediated depression of VRAC. Finally, we show that in COS-7 cells, which are not endowed with pannexin-1 protein, another astroglial plasma membrane interactor of CBX, VRAC current retained its sensitivity to CBX. Complementary analyses indicate that the VRAC-mediated release of excitatory amino acid aspartate was decreased by CBX. Collectively, these findings support the notion that CBX could affect astroglial ability to modulate neuronal activity by suppressing excitatory amino acid release through VRAC. They also provide a possible mechanistic clue for the neuroprotective effect of CBX in vivo.
The last few decades have seen the marketing of hundreds of new pesticide products with a forecasted expansion of the global agrochemical industry. As several pesticides directly target nervous ...tissue as their mechanism of toxicity, alternative methods to routine in vivo animal testing, such as the Multi Electrode Array (MEAs)-based approach, have been proposed as an in vitro tool to perform sensitive, quick and low cost neuro-toxicological screening. Here, we examined the effects of a training set of eleven active substances known to have neuronal or non-neuronal targets, contained in the most commonly used agrochemicals, on the spontaneous electrical activity of cortical neuronal networks grown on MEAs. A multiparametric characterisation of neuronal network firing and bursting was performed with the aim of investigating how this can contribute to the efficient evaluation of in vitro chemical-induced neurotoxicity. The analysis of MFR, MBR, MBD, MISI_B and % Spikes_B parameters identified four different groups of chemicals: one wherein only inhibition is observed (chlorpyrifos, deltamethrin, orysastrobin, dimoxystrobin); a second one in which all parameters, except the MISI_B, are inhibited (carbaryl, quinmerac); a third in which increases at low chemical concentration are followed by decreases at high concentration, with exception of MISI_B that only decreased (fipronil); a fourth in which no effects are observed (paraquat, glyphosate, imidacloprid, mepiquat). The overall results demonstrated that the multiparametric description of the neuronal networks activity makes MEA-based screening platform an accurate and consistent tool for the evaluation of the toxic potential of chemicals. In particular, among the bursting parameters the MISI_B was the best that correlates with potency and may help to better define chemical toxicity when MFR is affected only at relatively high concentration.
Objectives
This multicenter study assessed the extent of pancreatic fatty replacement and its correlation with demographics, iron overload, glucose metabolism, and cardiac complications in a cohort ...of well-treated patients with thalassemia major (TM).
Methods
We considered 308 TM patients (median age: 39.79 years; 182 females) consecutively enrolled in the Extension-Myocardial Iron Overload in Thalassemia Network. Magnetic resonance imaging was used to quantify iron overload (IO) and pancreatic fat fraction (FF) by T2* technique, cardiac function by cine images, and to detect replacement myocardial fibrosis by late gadolinium enhancement technique. The glucose metabolism was assessed by the oral glucose tolerance test.
Results
Pancreatic FF was associated with age, body mass index, and history of hepatitis C virus infection. Patients with normal glucose metabolism showed a significantly lower pancreatic FF than patients with impaired fasting glucose (
p
= 0.030), impaired glucose tolerance (
p
< 0.0001), and diabetes (
p
< 0.0001). A normal pancreatic FF (< 6.6%) showed a negative predictive value of 100% for abnormal glucose metabolism. A pancreatic FF > 15.33% predicted the presence of abnormal glucose metabolism. Pancreas FF was inversely correlated with global pancreas and heart T2* values. A normal pancreatic FF showed a negative predictive value of 100% for cardiac iron. Pancreatic FF was significantly higher in patients with myocardial fibrosis (
p
= 0.002). All patients with cardiac complications had fatty replacement, and they showed a significantly higher pancreatic FF than complications-free patients (
p
= 0.002).
Conclusion
Pancreatic FF is a risk marker not only for alterations of glucose metabolism, but also for cardiac iron and complications, further supporting the close link between pancreatic and cardiac disease.
Key Points
• In thalassemia major, pancreatic fatty replacement by MRI is a frequent clinical entity, predicted by a pancreas T2* < 20.81 ms and associated with a higher risk of alterations in glucose metabolism.
• In thalassemia major, pancreatic fatty replacement is a strong risk marker for cardiac iron, replacement fibrosis, and complications, highlighting a deep connection between pancreatic and cardiac impairment.
P2X7-type purinergic receptors are distributed throughout the nervous system where they contribute to physiological and pathological functions. In the retina, this receptor is found in both inner and ...outer cells including microglia modulating signaling and health of retinal cells. It is involved in retinal neurodegenerative disorders such as retinitis pigmentosa and age-related macular degeneration (AMD). Experimental studies demonstrated that saffron protects photoreceptors from light-induced damage preserving both retinal morphology and visual function and improves retinal flicker sensitivity in AMD patients. To evaluate a possible interaction between saffron and P2X7 receptors (P2X7Rs), different cellular models and experimental approaches were used. We found that saffron positively influences the viability of mouse primary retinal cells and photoreceptor-derived 661W cells exposed to ATP, and reduced the ATP-induced intracellular calcium increase in 661W cells. Similar results were obtained on HEK cells transfected with recombinant rat P2X7R but not on cells transfected with rat P2X2R. Finally, patch-clamp experiments showed that saffron inhibited cationic currents in HEK-P2X7R cells. These results point out a novel mechanism through which saffron may exert its protective role in neurodegeneration and support the idea that P2X7-mediated calcium signaling may be a crucial therapeutic target in the treatment of neurodegenerative diseases.
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