A sensitive capillary electrophoresis method was developed for the determination of aminothiol (cysteine, homocysteine, and glutathione) total levels in human blood plasma. Analytes were derivatized ...with Ellman’s reagent (5,5′-dithiobis(2-nitrobenzoic acid)) after reduction with dithiothreitol. Liquid–liquid extraction was applied to purify the samples and concentrate the analytes. Total analysis time was 7.5 min using a silica capillary (50 μm i.d.; effective separation length 23.5 cm). Electrophoretic separation was performed using 50 mM citric acid with 20 mM triethanolamine (pH 3) containing 2% Ficoll 400. Detection limit was 0.8 μM for glutathione and 0.3 μM for both cysteine and homocysteine. Accuracy was 94 – 107%, repeatability and reproducibility were ca. 2.7 – 3.5 and 2.5 – 6.5%, respectively.
It was shown previously that inhibition of the ubiquitous α1 isoform of Na+,K+-ATPase by ouabain sharply affects gene expression profile via elevation of intracellular Na+i/K+i ratio. Unlike other ...cells, neurons are abundant in the α3 isoform of Na+,K+-ATPase, whose affinity in rodents to ouabain is 104-fold higher compared to the α1 isoform. With these sharp differences in mind, we compared transcriptomic changes in rat cerebellum granule cells triggered by inhibition of α1- and α3-Na+,K+-ATPase isoforms. Inhibition of α1- and α3-Na+,K+-ATPase isoforms by 1 mM ouabain resulted in dissipation of transmembrane Na+ and K+ gradients and differential expression of 994 transcripts, whereas selective inhibition of α3-Na+,K+-ATPase isoform by 100 nM ouabain affected expression of 144 transcripts without any impact on the Na+i/K+i ratio. The list of genes whose expression was affected by 1 mM ouabain by more than 2-fold was abundant in intermediates of intracellular signaling and transcription regulators, including augmented content of Npas4, Fos, Junb, Atf3, and Klf4 mRNAs, whose upregulated expression was demonstrated in neurons subjected to electrical and glutamatergic stimulation. The role Na+i/K+i-mediated signaling in transcriptomic changes involved in memory formation and storage should be examined further.
Neuronal intracellular chloride (Cl−i) is a key determinant in γ-aminobutyric acid type A (GABA)ergic signaling. γ-Aminobutyric acid type A receptors (GABAARs) mediate both inhibitory and excitatory ...neurotransmission, as the passive fluxes of Cl− and HCO3− via pores can be reversed by changes in the transmembrane concentration gradient of Cl−. The cation–chloride co-transporters (CCCs) are the primary systems for maintaining Cl−i homeostasis. However, despite extensive electrophysiological data obtained in vitro that are supported by a wide range of molecular biological studies on the expression patterns and properties of CCCs, the presence of ontogenetic changes in Cl−i—along with the consequent shift in GABA reversal potential—remain a subject of debate. Recent studies showed that the β3 subunit possesses properties of the P-type ATPase that participates in the ATP-consuming movement of Cl− via the receptor. Moreover, row studies have demonstrated that the β3 subunit is a key player in GABAAR performance and in the appearance of serious neurological disorders. In this review, we discuss the properties and driving forces of CCCs and Cl−, HCO3−ATPase in the maintenance of Cl−i homeostasis after changes in upcoming GABAAR function. Moreover, we discuss the contribution of the β3 subunit in the manifestation of epilepsy, autism, and other syndromes.
Metastatic disease is a major and difficult-to-treat complication of lung cancer. Considering insufficient effectiveness of existing therapies and taking into account the current problem of lung ...cancer chemoresistance, it is necessary to continue the development of new treatments.
Previously, we have demonstrated the antitumor effects of reprogrammed CD8
T-cells (rCD8
T-cells) from the spleen in mice with orthotopic lung carcinoma. Reprogramming was conducted by inhibiting the MAPK/ERK signalling pathway through MEKi and the immune checkpoint PD-1/PD-L1. Concurrently, CD8
T-cells were trained in Lewis lung carcinoma (LLC) cells. We suggested that rCD8
T-cells isolated from the spleen might impede the development of metastatic disease.
The present study has indicated that the reprogramming procedure enhances the survival and cytotoxicity of splenic CD8
T-cells in LLC culture. In an LLC model of spontaneous metastasis, splenic rCD8 + T-cell therapy augmented the numbers of CD8
T-cells and CD4
T-cells in the lungs of mice. These changes can account for the partial reduction of tumors in the lungs and the mitigation of metastatic activity.
Our proposed reprogramming method enhances the antitumor activity of CD8
T-cells isolated from the spleen and could be valuable in formulating an approach to treating metastatic disease in patients with lung cancer.
Zinc ions (Zn2+) are concentrated in various brain regions and can act as a neuromodulator, targeting a wide spectrum of postsynaptic receptors and enzymes. Zn2+ inhibits the GABAARs, and its potency ...is profoundly affected by the subunit composition and neuronal developmental stage. Although the extracellular amino acid residues of the receptor’s hetero-oligomeric structure are preferred for Zn2+ binding, there are intracellular sites that, in principle, could coordinate its potency. However, their role in modulating the receptor function during postembryonic development remains unclear. The GABAAR possesses an intracellular ATPase that enables the energy-dependent anion transport via a pore. Here, we propose a mechanistic and molecular basis for the inhibition of intracellular GABAAR/ATPase function by Zn2+ in neonatal and adult rats. The enzymes within the scope of GABAAR performance as Cl−ATPase and then as Cl−, HCO3−ATPase form during the first week of postnatal rat development. In addition, we have shown that the Cl−ATPase form belongs to the β1 subunit, whereas the β3 subunit preferably possesses the Cl−, HCO3−ATPase activity. We demonstrated that a Zn2+ with variable efficacy inhibits the GABAAR as well as the ATPase activities of immature or mature neurons. Using fluorescence recording in the cortical synaptoneurosomes (SNs), we showed a competitive association between Zn2+ and NEM in parallel changes both in the ATPase activity and the GABAAR-mediated Cl− and HCO3− fluxes. Finally, by site-directed mutagenesis, we identified in the M3 domain of β subunits the cysteine residue (C313) that is essential for the manifestation of Zn2+ potency.
γ-Aminobutyric acid type A receptors (GABAARs) mediate primarily inhibitory synaptic transmission in the central nervous system. Following fast-paced activation, which provides the selective flow of ...mainly chloride (Cl−) and less bicarbonate (HCO3−) ions via the pore, these receptors undergo desensitization that is paradoxically prevented by the process of their recovery, referred to as resensitization. To clarify the mechanism of resensitization, we used the cortical synaptoneurosomes from the rat brain and HEK 293FT cells. Here, we describe the effect of γ-phosphate analogues (γPAs) that mimic various states of ATP hydrolysis on GABAAR-mediated Cl− and HCO3− fluxes in response to the first and repeated application of the agonist. We found that depending on the presence of bicarbonate, opened and desensitized states of the wild or chimeric GABAARs had different sensitivities to γPAs. This study presents the evidence that recovery of neuronal Cl− and HCO3− concentrations after desensitization is accompanied by a change in the intracellular ATP concentration via ATPase performance. The transition between the desensitization and resensitization states was linked to changes in both conformation and phosphorylation. In addition, the chimeric β3 isoform did not exhibit the desensitization of the GABAAR-mediated Cl− influx but only the resensitization. These observations lend a new physiological significance to the β3 subunit in the manifestation of GABAAR resensitization.
γ-Aminobutyric acid type A receptors (GABA
A
Rs) are members of the pentameric ligand-gated ion channel (pLGIC) family, which are widespread throughout the invertebrate and vertebrate central nervous ...system. GABA
A
Rs are engaged in short-term changes of the neuronal concentrations of chloride (Cl
−
) and bicarbonate (HCO
3
−
) ions by their passive permeability through the ion channel pore. GABA
A
Rs are regulated by various structurally diverse phenolic substances ranging from simple phenols to complex polyphenols. The wide chemical and structural variability of phenols suggest similar and different binding sites on GABA
A
Rs, allowing them to manifest themselves as activators, inhibitors, or allosteric ligands of GABA
A
R function. Interest in phenols is associated with their great potential for GABA
A
R modulation, but also with their subsequent negative or positive role in neurological and psychiatric disorders. This review focuses on the GABAergic deficit hypotheses during neurological and psychiatric disorders induced by various phenols. We summarize the structure–activity relationship of general phenol groups concerning their differential roles in the manifestation of neuropsychiatric symptoms. We describe and analyze the role of GABA
A
R subunits in manifesting various neuropathologies and the molecular mechanisms underlying their modulation by phenols. Finally, we discuss how phenol drugs can modulate GABA
A
R activity via desensitization and resensitization. We also demonstrate a novel pharmacological approach to treat neuropsychiatric disorders via regulation of receptor phosphorylation/dephosphorylation.
We studied the effects of the extract of the terrestrial part of
Aconitum baicalense
in BALB/c female mice at the early stages after the injection of N-methyl-N-nitrosourea (MNU). The extract reduced ...inflammatory activity and tumor growth in the mammary gland. The antitumor and anti-inflammatory effects of the extract are based on the inhibition of cancer stem cells, hematopoietic stem cells, and hematopoietic progenitor cells that promote inflammation. The extract of
A. baicalense
disrupted the recruitment of epithelial progenitor cells and angiogenesis precursors to the mammary gland preventing neovascularization and transformation of epithelial cells into tumor cells.
Neuronal intracellular chloride concentration (Cl−i) is a crucial determinant of transmission mediated by the γ‐aminobutyric acid type A receptor (GABAAR), which subserves synaptic and extrasynaptic ...inhibition as well as excitation. The Cl− ion is the main carrier of charge through the GABAAR; however, bicarbonate ions (
HCO3-
) flowing in the opposite direction can also contribute to the net current. The direction of Cl− and
HCO3-
fluxes is determined by the underlying electrochemical gradient, which is controlled by Cl− transporters and channels. Accumulating evidence suggests that active mechanisms of chloride transport across the GABAAR pore can underlie the regulation of Cl−i. Measurement of Cl−/
HCO3-
‐ATPase activity and Cl− transport in HEK 293FT cells expressing homomeric or heteromeric GABAAR ensembles (α2, β3, or γ2) with fluorescent dye for chloride demonstrated that receptor subtypes containing the β3 subunit show enzymatic activity and participate in GABA‐mediated or ATP‐dependent Cl− transport. GABA‐mediated flow of Cl− ions into and out of the cells occurred for a short time period but then rapidly declined. However, Cl− ion flux was stabilized for a long time period in the presence of
HCO3-
ions. The reconstituted β3 subunit isoform, purified as a fusion protein, confirmed that β3 is critical for ATPase; however, only the triplet variant showed the full receptor function. The high sensitivity of the enzyme to γ‐phosphate inhibitors led us to postulate that the β3 subunit is catalytic. Our discovery of a GABAAR type that requires ATP consumption for chloride movement provides new insight into the molecular mechanisms of inhibitory signaling.
Measurement of Cl−/
HCO3-
‐ATPase activity and Cl− transport in HEK 293FT cells expressing the homomeric or heteromeric ectopic GABAAR ensembles (α2, β3, or γ2 subunits) demonstrated that β3‐containing subtypes manifest enzyme activity and participate in GABA‐mediated or ATP‐dependent Cl− transport in the presence of
HCO3-
. Our work highlights the β3 subunit as fundamental for Cl−/
HCO3-
‐ATPase activity and identifies it as catalytic.
Endothelial dysfunction and destruction of the pulmonary microcirculation are important pathogenic factors in chronic obstructive pulmonary disease (COPD). In COPD, bronchial obstruction is ...associated with endothelial dysfunction. Thus, new pharmacological treatment options aimed at restoring the pulmonary endothelium represent a clinical need in COPD therapy. Notch1 has been shown to protect cells against apoptosis, inflammation, and oxidative stress caused by cigarette smoke extract (CSE). Therefore, drug which effect on Notch1 may be a potential therapeutic target for COPD in the future.
In this study, we assessed the potential of spiperone to mediate regeneration of pulmonary endothelium in model of pulmonary emphysema induced by a CSE and lipopolysaccharide (LPS) in female C57BL/6 mice.
Spiperone increased the number of capillaries as well as the expression of the CD31 in the alveolar tissue compared to the controls. Moreover, application of spiperone prevented alveolar wall destruction (DI), and reduced the area of emphysema. Lastly, we demonstrated that spiperone positively influenced mobilization and migration of endothelial progenitor cells (EPC, CD45
CD34
CD31
), CD309
-endothelial cells, and angiogenesis precursors (CD45
CD117
CD309
) into the lung. Spiperone administration significantly reduced the number Notch1 positive CD309
-endothelial cells and Notch1+ EPCs.
Overall, our results suggest that spiperone mediates endothelial regeneration in an animal model of COPD. Thus, it could represent a novel therapeutic approach for treatment of emphysema associated with COPD.
PDF
