Calcium release from presynaptic internal stores is required for ethanol to increase spontaneous gammaaminobutyric acid release onto cerebellum Purkinje neurons

ABSTRACT Recent data have demonstrated that ethanol increases ␥-aminobutyric acid (GABA) release in many brain regions, but little is known about the mechanism responsible for this action. Consistent with previous results, ethanol increased miniature inhibitory postsynaptic current (mIPSC) frequency at the interneuron-Purkinje cell synapse in the slice and in mechanically dissociated neurons. These data suggest that ethanol is increasing spontaneous GABA release at this synapse. It is generally accepted that ethanol increases levels of intracellular calcium and that changes in intracellular calcium can alter neurotransmitter release. Therefore, we examined the contribution of calcium-dependent pathways to the effect of ethanol on spontaneous GABA release at the interneuron-Purkinje cell synapse. Ethanol continued to increase mIPSC frequency in a nominally calcium-free extracellular solution and in the presence of a voltage-dependent calcium channel inhibitor, cadmium chloride. These data suggest that influx of extracellular calcium does not play a critical role in the mechanism of ethanol-enhanced spontaneous GABA release. However, a sarco/ endoplasmic-reticulum calcium ATPase pump inhibitor (thapsigargin), an inositol 1,4,5-trisphosphate receptor antagonist (2-aminoethoxydiphenylborate) and a ryanodine receptor antagonist (ryanodine) significantly reduced the ability of ethanol to increase mIPSC frequency. In addition, ethanol was still able to increase mIPSC frequency in the presence of intracellular 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid (BAPTA) and a cannabinoid receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM-251); thus, retrograde messengers are not involved in ethanol-enhanced spontaneous GABA release. Overall, these data suggest that calcium release from presynaptic internal stores plays a vital role in the mechanism of ethanol-enhanced spontaneous GABA release at the interneuronPurkinje cell synapse

Ethanol-enhanced GABA release: A focus on G protein-coupled receptors

While research on the actions of ethanol at the GABAergic synapse has focused on postsynaptic mechanisms, recent data have demonstrated that ethanol also facilitates GABA release from presynaptic terminals in many, but not all, brain regions. The ability of ethanol to increase GABA release can be regulated by different G protein-coupled receptors (GPCRs), such as the cannabinoid-1 receptor, corticotropin-releasing factor 1 receptor, GABAB receptor, and the 5-hydroxytryptamine 2C receptor. The intracellular messengers linked to these GPCRs, including the calcium that is released from internal stores, also play a role in ethanol-enhanced GABA release. Hypotheses are proposed to explain how ethanol interacts with the GPCR pathways to increase GABA release and how this interaction contributes to the brain region specificity of ethanol-enhanced GABA release. Defining the mechanism of ethanol-facilitated GABA release will further our understanding of the GABAergic profile of ethanol and increase our knowledge of how GABAergic neurotransmission may contribute to the intoxicating effects of alcohol and to alcohol dependence

The PLC/IP3R/PKC pathway is required for ethanol-enhanced GABA release

Research on the actions of ethanol at the GABAergic synapse has traditionally focused on postsynaptic mechanisms, but recent data demonstrate that ethanol also increases both evoked and spontaneous GABA release in many brain regions. Using whole-cell voltage-clamp recordings, we previously showed that ethanol increases spontaneous GABA release at the rat interneuron-Purkinje cell synapse. This presynaptic ethanol effect is dependent on calcium release from internal stores, possibly through activation of inositol 1,4,5-trisphosphate receptors (IP3Rs). After confirming that ethanol targets vesicular GABA release, in the present study we used electron microscopic immunohistochemistry to demonstrate that IP3Rs are located in presynaptic terminals of cerebellar interneurons. Activation of IP3Rs requires binding of IP3, generated through activation of phospholipase C (PLC). We find that the PLC antagonist edelfosine prevents ethanol from increasing spontaneous GABA release. Diacylglycerol generated by PLC and calcium released by activation of the IP3R activate protein kinase C (PKC). Ethanol-enhanced GABA release was blocked by two PKC antagonists, chelerythrine and calphostin C. When a membrane impermeable PKC antagonist, PKC (19-36), was delivered intracellularly to the postsynaptic neuron, ethanol continued to increase spontaneous GABA release. Overall, these results suggest that activation of the PLC/IP3R/PKC pathway is necessary for ethanol to increase spontaneous GABA release from presynaptic terminals onto Purkinje cells

Ethanol-enhanced GABA release: A focus on G protein-coupled receptors

While research on the actions of ethanol at the GABAergic synapse has focused on postsynaptic mechanisms, recent data have demonstrated that ethanol also facilitates GABA release from presynaptic terminals in many, but not all, brain regions. The ability of ethanol to increase GABA release can be regulated by different G protein-coupled receptors (GPCRs), such as the cannabinoid-1 receptor, corticotropin-releasing factor 1 receptor, GABA(B) receptor, and the 5-hydroxytryptamine 2C receptor. The intracellular messengers linked to these GPCRs, including the calcium that is released from internal stores, also play a role in ethanol-enhanced GABA release. Hypotheses are proposed to explain how ethanol interacts with the GPCR pathways to increase GABA release and how this interaction contributes to the brain region specificity of ethanol-enhanced GABA release. Defining the mechanism of ethanol-facilitated GABA release will further our understanding of the GABAergic profile of ethanol and increase our knowledge of how GABAergic neurotransmission may contribute to the intoxicating effects of alcohol and to alcohol dependence

Recent Advances in the Development of Non-PIKKs Targeting Small Molecule Inhibitors of DNA Double-Strand Break Repair

The vast majority of cancer patients receive DNA-damaging drugs or ionizing radiation (IR) during their course of treatment, yet the efficacy of these therapies is tempered by DNA repair and DNA damage response (DDR) pathways. Aberrations in DNA repair and the DDR are observed in many cancer subtypes and can promote de novo carcinogenesis, genomic instability, and ensuing resistance to current cancer therapy. Additionally, stalled or collapsed DNA replication forks present a unique challenge to the double-strand DNA break (DSB) repair system. Of the various inducible DNA lesions, DSBs are the most lethal and thus desirable in the setting of cancer treatment. In mammalian cells, DSBs are typically repaired by the error prone non-homologous end joining pathway (NHEJ) or the high-fidelity homology directed repair (HDR) pathway. Targeting DSB repair pathways using small molecular inhibitors offers a promising mechanism to synergize DNA-damaging drugs and IR while selective inhibition of the NHEJ pathway can induce synthetic lethality in HDR-deficient cancer subtypes. Selective inhibitors of the NHEJ pathway and alternative DSB-repair pathways may also see future use in precision genome editing to direct repair of resulting DSBs created by the HDR pathway. In this review, we highlight the recent advances in the development of inhibitors of the non-phosphatidylinositol 3-kinase-related kinases (non-PIKKs) members of the NHEJ, HDR and minor backup SSA and alt-NHEJ DSB-repair pathways. The inhibitors described within this review target the non-PIKKs mediators of DSB repair including Ku70/80, Artemis, DNA Ligase IV, XRCC4, MRN complex, RPA, RAD51, RAD52, ERCC1-XPF, helicases, and DNA polymerase θ. While the DDR PIKKs remain intensely pursued as therapeutic targets, small molecule inhibition of non-PIKKs represents an emerging opportunity in drug discovery that offers considerable potential to impact cancer treatment