Evidence suggests that neuronal dysfunction in Huntington's disease (HD) striatum involves deficits in mitochondrial function and in Ca²⁺handling. However, the relationship between mitochondria and Ca²⁺handling has been incompletely studied in intact HD striatal cells. Treatment with histone deacetylase (HDAC) inhibitors reduces cell death in HD models, but the effects of this promising therapy on cellular function are mostly unknown. Here, we use real-time functional imaging of intracellular Ca²⁺and mitochondrial membrane potential to explore the role ofin situHD mitochondria in Ca²⁺handling. Immortalized striatal (STHdh) cells and striatal neurons from transgenic mice, expressing full-length mutant huntingtin (Htt), were used to model HD. We show that (1) active glycolysis in STHdhcells occludes the mitochondrial role in Ca²⁺handling as well as the effects of mitochondrial inhibitors, (2) STHdhcells and striatal neurons in the absence of glycolysis are critically dependent on oxidative phosphorylation for energy-dependent Ca²⁺handling, (3) expression of full-length mutant Htt is associated with deficits in mitochondrial-dependent Ca²⁺handling that can be ameliorated by treatment with HDAC inhibitors (treatment with trichostatin A or sodium butyrate decreases the proportion of STHdhcells losing Ca²⁺homeostasis after Ca²⁺-ionophore challenging, and accelerates the restoration of intracellular Ca²⁺in striatal neurons challenged with NMDA), and (4) neurons with different response patterns to NMDA receptor activation exhibit different average somatic areas and are differentially affected by treatment with HDAC inhibitors, suggesting subpopulation or functional state specificity. These findings indicate that neuroprotection induced by HDAC inhibitors involves more efficient Ca²⁺handling, thus improving the neuronal ability to cope with excitotoxic stimuli.

Vancomycin is a fundamental antibiotic in the management of severe Gram‐positive infections. Inappropriate vancomycin dosing is associated with therapeutic failure, bacterial resistance and toxicity. Therapeutic drug monitoring (TDM) is acknowledged as an important part of the vancomycin therapy management, at least in specific patient subpopulations, but implementation in clinical practice has been difficult because there are no consensus and agglutinator documents. The aims of the present work are to present an overview of the current knowledge on vancomycinTDMand population pharmacokinetic (PPK) models relevant to specific patient subpopulations. Based on three published international guidelines (American, Japanese and Chinese) on vancomycinTDMand a bibliographic review on availablePPKmodels for vancomycin in distinct subpopulations, an analysis of evidence was carried out and the current knowledge on this topic was summarized. The results of this work can be useful to redirect research efforts to address the detected knowledge gaps. Currently,TDMof vancomycin presents a moderate level of evidence and practical recommendations with great robustness in neonates, pediatric and patients with renal impairment. However, it is important to investigate in other subpopulations known to present altered vancomycin pharmacokinetics (eg neurosurgical, oncological and cystic fibrosis patients), where evidence is still unsufficient.

During cerebral inflammation uracil nucleotides leak to the extracellular medium and activate glial pyrimidine receptors contributing to the development of a reactive phenotype. Chronically activated microglia acquire an anti-inflammatory phenotype that favors neuronal differentiation, but the impact of these microglia on astrogliosis is unknown. We investigated the contribution of pyrimidine receptors to microglia-astrocyte signaling in a chronic model of inflammation and its impact on astrogliosis. Co-cultures of astrocytes and microglia were chronically treated with lipopolysaccharide (LPS) and incubated with uracil nucleotides for 48 h. The effect of nucleotides was evaluated in methyl-[3H]-thymidine incorporation. Western blot and immunofluorescence was performed to detect the expression of P2Y6 receptors and the inducible form of nitric oxide synthase (iNOS). Nitric oxide (NO) release was quantified through Griess reaction. Cell death was also investigated by the LDH assay and by the TUNEL assay or Hoechst 33258 staining. UTP, UDP (0.001 to 1 mM) or PSB 0474 (0.01 to 10 μM) inhibited cell proliferation up to 43 ± 2% (n = 10, P <0.05), an effect prevented by the selective P2Y6 receptor antagonist MRS 2578 (1 μM). UTP was rapidly metabolized into UDP, which had a longer half-life. The inhibitory effect of UDP (1 mM) was abolished by phospholipase C (PLC), protein kinase C (PKC) and nitric oxide synthase (NOS) inhibitors. Both UDP (1 mM) and PSB 0474 (10 μM) increased NO release up to 199 ± 20% (n = 4, P <0.05), an effect dependent on P2Y6 receptors-PLC-PKC pathway activation, indicating that this pathway mediates NO release. Western blot and immunocytochemistry analysis indicated that P2Y6 receptors were expressed in the cultures being mainly localized in microglia. Moreover, the expression of iNOS was mainly observed in microglia and was upregulated by UDP (1 mM) or PSB 0474 (10 μM). UDP-mediated NO release induced apoptosis in astrocytes, but not in microglia. In LPS treated co-cultures of astrocytes and microglia, UTP is rapidly converted into UDP, which activates P2Y6 receptors inducing the release of NO by microglia that causes astrocyte apoptosis, thus controlling their rate of proliferation and preventing an excessive astrogliosis.

The role of ATP on the modulation of noradrenaline release elicited by electrical stimulation (100 pulses/8 Hz) was studied in the prostatic portion of rat vas deferens preincubated with [³H]noradrenaline. In the presence of P1 antagonists, the nucleotides 2-methylthioadenosine-5′-triphosphate (2-MeSATP), 2-methylthioadenosine 5′-diphosphate (2-MeSADP), ADP, and ATP decreased electrically evoked tritium overflow up to 44%, with the following order of potency: 2-MeSATP > 2-MeSADP > ADP ≥ ATP. The P2Y antagonists reactive blue 2 (RB2) and 2-methylthioadenosine 5′-monophosphate (2-MeSAMP) increased, whereas the P2X antagonist pyridoxal-5′-phosphate-6-(2′-naphthylazo-6′-nitro-4′,8′-disulfonate) (PPNDS) decreased evoked tritium overflow. The inhibitory effect of 2-MeSATP was antagonized by RB2 (10 μM) and by 2-MeSAMP (10 μM) but not by the selective P2Y1 receptor antagonist 2′-deoxy-N⁶-methyladenosine 3′,5′-bisphosphate (MRS 2179; 10 μM). When, besides P1 receptors, inhibitory P2Y receptors were blocked with RB2, α,β-methyleneadenosine 5′-triphosphate (α,β-meATP), β,γ-imidoadenosine 5′-triphosphate (β,γ-imidoATP), β,γ-methyleneadenosine 5′-triphosphate (β,γ-meATP), 2-MeSATP, and ATP enhanced tritium overflow up to 140%, with the following order of potency: α,β-meATP > 2-MeSATP = ATP = β,γ-meATP ≥ β,γ-imidoATP. The facilitatory effects of α,β-MeATP and β,γ-imidoATP were prevented by PPNDS. Under the same conditions, apyrase attenuated, whereas the ectonucleotidase inhibitor 6-N,N-diethyl-d-β,γ-dibromomethylene 5′-triphosphate enhanced tritium overflow, an effect that was prevented by PPNDS. In the prostatic portion of the rat vas deferens, endogenous ATP exerts a dual and opposite modulation of noradrenaline release: an inhibition through activation of P2Y receptors with a pharmacological profile similar to that of the P2Y12 and P2Y13 receptors and a facilitation through activation of P2X receptors with a pharmacological profile similar to that of P2X1 and P2X3, or PX2/P2X3 receptors.

1 The effects of analogues of adenosine and ATP on noradrenaline release elicited by electrical stimulation (5 Hz, 2700 pulses) were studied in superfused preparations of rat tail artery. The effects of purinoceptor antagonists, of adenosine deaminase and of adenosine uptake blockade were also examined. Noradrenaline was measured by h.p.l.c. electrochemical detection. 2 The A₁-adenosine receptor agonist, N⁶-cyclopentyladenosine (CPA; 0.1 − 100 nM) reduced, whereas the A_2A-receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS 21680; 3 − 30 nM) increased evoked noradrenaline overflow. These effects were antagonized by the A₁-adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 20 nM) and the A₂-adenosine receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX; 100 nM), respectively. The P_2Y-purinoceptor agonist, 2-methylthio-ATP (1 − 100 μm) reduced noradrenaline overflow, an effect prevented by the P₂-purinoceptor antagonist, cibacron blue 3GA (100 μm) and suramin (100 μm). 3 Adenosine deaminase (2 u ml⁻¹), DMPX (100 nM) and inhibition of adenosine uptake with S-(p-nitrobenzyl)-6-thioinosine (NBTI; 50 nM) decreased evoked noradrenaline overflow. DPCPX alone did not change noradrenaline overflow but prevented the inhibition caused by NBTI. The P_2Y-purinoceptor antagonist, cibacron blue 3GA (100 μm) increased evoked noradrenaline overflow as did suramin, a non-selective P₂-antagonist. 4 It is concluded that, in rat tail artery, inhibitory (A₁ and P_2Y) and facilitatory (A_2A) purinoceptors are present and modulate noradrenaline release evoked by electrical stimulation. Endogenous purines tonically modulate noradrenaline release through activation of inhibitory P_2Y and facilitatory A_2A purinoceptors, whereas a tonic activation of inhibitory A₁ purinoceptors seems to be prevented by adenosine uptake.