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  • Methyllycaconitine citrate sale br Materials and methods br


    Materials and methods
    Discussion In nerve terminals, Na+-dependent glutamate transporters of the plasma membrane are responsible for the maintenance of low (non-toxic) extracellular glutamate concentrations and accomplish uptake of glutamate into the Methyllycaconitine citrate sale of neurons and glial cells. Then, the neurotransmitter is transported into synaptic vesicles by special vesicular glutamate transporters. In regulated exocytosis, synaptic vesicles fuse with the plasma membrane upon stimulation and release their content into the synaptic cleft. However, exocytosis is not the only mode of glutamate release from the presynapse. The neurotransmitter is constantly released from nerve terminals without stimulation by means of spontaneous exocytosis, via swelling-activated anion channels, cystine/glutamate exchange and trans-membrane diffusion (Cavelier and Attwell, 2005, Jabaudon et al., 1999, Rutledge et al., 1998). A decrease in extracellular [Na+] and/or intracellular [K+] as well as an increase in extracellular [K+] and/or intracellular [Na+] and/or intracellular [Glu] thermodynamically favour glutamate transporter reversal. Even in partial ischaemia that accompanied by mild membrane depolarization, glutamate release through reverse transport can be dramatic (Grewer et al., 2008, Rossi et al., 2007). Also, transporters may release significant amounts of glutamate upon receptor-induced depolarization of the plasma membrane, thereby regulating glutamatergic neurotransmission. In this study, we assessed whether non-exocytotic glutamate release (heteroexchange, unstimulated, and transporter-mediated release) was inherent to platelets, as non-excitable cells, which express neuronal and glial glutamate transporters EAAT 1–3 in the plasma membrane and vesicular glutamate transporters VGLUT 1,2 in dense secretory granules. Using the glutamate dehydrogenase assay, radiolabeled glutamate, and the Na+-sensitive fluorescent dye SBFI, we revealed a lack of: (1) unstimulated release of glutamate from platelets (Fig. 1); (2) glutamate transporter reversal during depolarization of the plasma membrane of platelets by elevated [K+] (Fig. 4), even in the presence of ouabain or glutamine synthetase inhibitor methionine sulfoximine; (3) Na+ influx in response to platelet membrane depolarization by elevated [K+]; (4) glutamate release by heteroexchange with the inhibitor of glutamate transporters dl-THA (Fig. 4); (5) transporter-mediated glutamate release during dissipation of the proton electrochemical gradient of secretory granules (Fig. 6D); and vice versa, exocytotic release of glutamate in response to thrombin stimulation was registered in platelets even after dissipation of the proton electrochemical gradient of secretory granules. We suggested that the absence of the transporter-mediated release of glutamate was associated with the minority of the cytosolic pool of glutamate and a lack of depolarization-induced Na+ influx in platelets, i.e., the factors, which determined glutamate transporter reversal. The glutamate binding sites in the transporters are optimized for binding of extracellular, but not intracellular, glutamate. This is because the affinity of the transporters for glutamate, when the substrate binding site faces the extracellular space, is about 50-times higher as compared to the intracellular glutamate binding, thereby promoting dissociation of glutamate into the cytoplasm against a high intracellular glutamate concentration (Watzke and Grewer, 2001). This fact together with the low cytosolic pool of glutamate complicates transporter-mediated glutamate release from platelets. We attempted to increase the cytosolic glutamate concentration in platelets through dissipation of the proton electrochemical gradient of secretory granules with bafilomycin A1 and FCCP in order to stimulate glutamate transporter reversal. However, even in the presence of these reagents, transporter-mediated glutamate release was not registered in the platelets. One of the interesting findings of this paper is the fact that Methyllycaconitine citrate sale platelets retain the ability to release glutamate in response to thrombin application, even after the dissipation of the proton electrochemical gradient of secretory granules (Fig. 8C). In nerve terminals, the treatment with bafilomycin A1 (100nM) significantly decreased, and the application of FCCP (1μM) completely abolished the exocytotic release of glutamate stimulated by depolarization of the plasma membrane. Thus, dissipation of the proton electrochemical gradient of platelet secretory granules by the treatment with bafilomycin A1 and FCCP does not lead to massive release of granular glutamate into the cytoplasm. It is possible that vesicular glutamate transporters of platelets effectively allow for influx only, whereas their reverse function is rather doubtful.