Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a ...neuromodulator, able to influence the activity of cortical neurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM). To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM) or switched to different glucose concentrations (0.5 or 10 mM). None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX) an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism.
Abstract Astrocytes are responsible for the majority of the clearance of extracellular glutamate released during neuronal activity. dl - threo -β-benzyloxyaspartate (TBOA) is extensively used as ...inhibitor of glutamate transport activity, but suffers from relatively low affinity for the transporter. Here, we characterized the effects of (2 S , 3S)-3-3-4-(trifluoromethyl)benzoylaminobenzyloxyaspartate (TFB-TBOA), a recently developed inhibitor of the glutamate transporter on mouse cortical astrocytes in primary culture. The glial Na+ -glutamate transport system is very efficient and its activation by glutamate causes rapid intracellular Na+ concentration (Na+i ) changes that enable real time monitoring of transporter activity. Na+i was monitored by fluorescence microscopy in single astrocytes using the fluorescent Na+ -sensitive probe sodium-binding benzofuran isophtalate. When applied alone, TFB-TBOA, at a concentration of 1 μM, caused small alterations of Na+i . TFB-TBOA inhibited the Na+i response evoked by 200 μM glutamate in a concentration-dependent manner with IC50 value of 43 ± 9 nM, as measured on the amplitude of the Na+i response. The maximum inhibition of glutamate-evoked Na+i increase by TFB-TBOA was > 80%, but was only partly reversible. The residual response persisted in the presence of the AMPA/kainate receptor antagonist CNQX. TFB-TBOA also efficiently inhibited Na+i elevations caused by the application of d -aspartate, a transporter substrate that does not activate non-NMDA ionotropic receptors. TFB-TBOA was found not to influence the membrane properties of cultured cortical neurons recorded in whole-cell patch clamp. Thus, TFB-TBOA, with its high potency and its apparent lack of neuronal effects, appears to be one of the most useful pharmacological tools available so far for studying glial glutamate transporters.
The ablative potential and toxicity of gemcitabine, administered intravesically in low stage and grade superficial transitional cell carcinoma (TCC), were evaluated.
Patients with a history of ...recurrent Ta-T1, GI-G2 bladder TCC were considered eligible for the study. Gemcitabine was administered intravesically at 40 mg/mL concentration (2000 mg in 50 ml saline) in one weekly instillation for 4 consecutive weeks. Fifteen days after the last instillation, patients were submitted to transurethral resection (TUR).
Twenty-six patients were evaluable for toxicity, and 20 were evaluable for response, 6 patients being excluded due to toxicity. A complete response was achieved by 10 out of 20 patients (50%), whereas no response was documented in the remainder. Toxicity leading to treatment interruption was grade 3 in 1 patient and grade 2 in 5 patients.
Intravesical gemcitabine administered at 40 mg/mL showed the capability of ablating small volume, superficial TCC in 50% of the population under study, with acceptable tolerability.
The P2X(7) receptor (P2X(7)R) is an ATP-gated cation channel whose biophysical properties remain to be unravelled unequivocally. Its activity is modulated by divalent cations and organic messengers ...such as arachidonic acid (AA). In this study, we analysed the differential modulation of magnesium (Mg(2+)) and AA on P2X(7)R by measuring whole-cell currents and intracellular Ca(2+) (Ca(2+)(i)) and Na(+) (Na(+)(i)) dynamics in HEK293 cells stably expressing full-length P2X(7)R and in cells endowed with the P2X(7)R variant lacking the entire C-terminus tail (trP2X(7)R), which is thought to control the pore activation. AA induced a robust potentiation of the P2X(7)R- and trP2X(7)R-mediated Ca(2+)(i) rise but did not affect the ionic currents in both conditions. Extracellular Mg(2+) reduced the P2X7R- and trP2X(7)R-mediated Ca(2+)(i) rise in a dose-dependent manner through a competitive mechanism. The modulation of the magnitude of the P2X(7)R-mediated ionic current and Na(+)(i) rise were strongly dependent on Mg(2+) concentration but occurred in a non-competitive manner. In contrast, in cells expressing the trP2X(7)R, the small ionic currents and Na(+)(i) signals were totally insensitive to Mg(2+). Collectively, these results support the tenet of a functional structure of P2X(7)R possessing at least two distinct conductive pathways one for Ca(2+) and another for monovalent ions, with the latter which depends on the presence of the receptor C-terminus.
J. Neurochem. (2010) 113, 796-806. The P2X₇ receptor (P2X₇R) is an ATP-gated cation channel whose biophysical properties remain to be unravelled unequivocally. Its activity is modulated by divalent ...cations and organic messengers such as arachidonic acid (AA). In this study, we analysed the differential modulation of magnesium (Mg²⁺) and AA on P2X₇R by measuring whole-cell currents and intracellular Ca²⁺ (Ca²⁺i) and Na⁺ (Na⁺i) dynamics in HEK293 cells stably expressing full-length P2X₇R and in cells endowed with the P2X₇R variant lacking the entire C-terminus tail (trP2X₇R), which is thought to control the pore activation. AA induced a robust potentiation of the P2X₇R- and trP2X₇R-mediated Ca²⁺i rise but did not affect the ionic currents in both conditions. Extracellular Mg²⁺ reduced the P2X7R- and trP2X₇R-mediated Ca²⁺i rise in a dose-dependent manner through a competitive mechanism. The modulation of the magnitude of the P2X₇R-mediated ionic current and Na⁺i rise were strongly dependent on Mg²⁺ concentration but occurred in a non-competitive manner. In contrast, in cells expressing the trP2X₇R, the small ionic currents and Na⁺i signals were totally insensitive to Mg²⁺. Collectively, these results support the tenet of a functional structure of P2X₇R possessing at least two distinct conductive pathways one for Ca²⁺ and another for monovalent ions, with the latter which depends on the presence of the receptor C-terminus.
J. Neurochem.
(2010)
113
, 796–806.
Abstract
The P2X
7
receptor (P2X
7
R) is an ATP‐gated cation channel whose biophysical properties remain to be unravelled unequivocally. Its activity is modulated ...by divalent cations and organic messengers such as arachidonic acid (AA). In this study, we analysed the differential modulation of magnesium (Mg
2+
) and AA on P2X
7
R by measuring whole‐cell currents and intracellular Ca
2+
(Ca
2+
i
) and Na
+
(Na
+
i
) dynamics in HEK293 cells stably expressing full‐length P2X
7
R and in cells endowed with the P2X
7
R variant lacking the entire C‐terminus tail (trP2X
7
R), which is thought to control the pore activation. AA induced a robust potentiation of the P2X
7
R‐ and trP2X
7
R‐mediated Ca
2+
i
rise but did not affect the ionic currents in both conditions. Extracellular Mg
2+
reduced the P2X7R‐ and trP2X
7
R‐mediated Ca
2+
i
rise in a dose‐dependent manner through a competitive mechanism. The modulation of the magnitude of the P2X
7
R‐mediated ionic current and Na
+
i
rise were strongly dependent on Mg
2+
concentration but occurred in a non‐competitive manner. In contrast, in cells expressing the trP2X
7
R, the small ionic currents and Na
+
i
signals were totally insensitive to Mg
2+
. Collectively, these results support the tenet of a functional structure of P2X
7
R possessing at least two distinct conductive pathways one for Ca
2+
and another for monovalent ions, with the latter which depends on the presence of the receptor C‐terminus.
Roles of astrocytes in the modulatory effects of oxytocin (OT) in central nervous system are increasingly considered. Nevertheless, OT effects on gliotransmitter release have been neglected.
In ...purified astrocyte processes from adult rat striatum, we assessed OT receptor (OTR) and adenosine A2A receptor expression by confocal analysis. The effects of receptors activation on glutamate release from the processes were evaluated; A2A-OTR heteromerization was assessed by co-immunoprecipitation and PLA. Structure of the possible heterodimer of A2A and OT receptors was estimated by a bioinformatic approach.
Both A2A and OT receptors were expressed on the same astrocyte processes. Evidence for A2A-OTR receptor-receptor interaction was obtained by measuring the release of glutamate: OT inhibited the evoked glutamate release, while activation of A2A receptors, per se ineffective, abolished the OT effect. Biochemical and biophysical evidence for A2A-OTR heterodimers on striatal astrocytes was also obtained. The residues in the transmembrane domains 4 and 5 of both receptors are predicted to be mainly involved in the heteromerization.
When considering effects of OT in striatum, modulation of glutamate release from the astrocyte processes and of glutamatergic synapse functioning, and the interaction with A2A receptors on the astrocyte processes should be taken into consideration.
The ultraluminous accreting pulsar M82-X2 (NuSTAR J095551+6940.8) offers an unprecedented opportunity to study the disc-magnetosphere interaction in a new regime of supercritical accretion. The ...source X-ray emission has been highly variable during the last 15 yrs. It ranged from a maximum of ∼2 × 1040 erg s−1 through intermediate values ∼ a few × 1039 erg s−1, and down to a minimum below 2 × 1038 erg s−1 that we have determined here, by analysing archival Chandra HRC observations of the source at an epoch at which it was undetected. We interpret the source variability via a magnetically threaded disc model: when at peak luminosity, the neutron star (NS) is close to spin equilibrium, its inner disc edge r
m
∼ 108 cm is approximately half the corotation radius r
co, and radiation pressure dominates the disc out to r
tr ≲ 109 cm. In the radiation-pressure-dominated regime, r
m
grows very slowly as the mass inflow rate drops: as a result, r
m
< r
co remains valid until
$\dot{M} \gtrsim$
$\dot{M}_E$
, the Eddington accretion rate, allowing a wide range of accretion luminosities to the NS. Once
$\dot{M} < \dot{M}_E$
accretion on to the NS is inhibited because r
m
> r
co, and the source luminosity is expected to drop by a large factor. We conclude that a magnetically threaded accretion disc surrounding a highly magnetized NS (B ≲ 1013 G), and transitioning between the radiation-pressure and gas-pressure dominated regimes, offers the best interpretation for all the currently observed properties of NuSTAR J095551+6940.8.
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