Participation of calcium-permeable AMPA receptors in the regulation of epileptiform activity of hippocampal neurons.

Introduction: Epileptiform activity is the most striking result of hyperexcitation of a group of neurons that can occur in different brain regions and then spread to other sites. Later it was shown that these rhythms have a cellular correlate in vitro called paroxysmal depolarization shift (PDS). In 13-15 DIV neuron-glial cell culture, inhibition of the GABA(A) receptors induces bursts of action potential in the form of clasters PDS and oscillations of intracellular Ca2+ concentration ([Ca2+]i). We demonstrate that GABAergic neurons expressing calcium-permeable AMPA receptors (CP-AMPARs) as well as Kv7-type potassium channels regulate hippocampal glutamatergic neurons' excitability during epileptiform activity in culture. Methods: A combination of whole-cell patch-clamp in current clamp mode and calcium imaging microscopy was used to simultaneously register membrane potential and [Ca2+]i level. To identify GABAergic cell cultures were fixed and stained with antibodies against glutamate decarboxylase GAD 65/67 and neuron-specific enolase (NSE) after vital [Ca2+]i imaging. Results and discussion: It was shown that CP-AMPARs are involved in the regulation of the PDS clusters and [Ca2+]i pulses accompanied them. Activation of CP-AMPARs of GABAergic neurons is thought to cause the release of GABA, which activates the GABA(B) receptors of other GABAergic interneurons. It is assumed that activation of these GABA(B) receptors leads to the release of beta-gamma subunits of Gi protein, which activate potassium channels, resulting in hyperpolarization and inhibition of these interneurons. The latter causes disinhibition of glutamatergic neurons, the targets of these interneurons. In turn, the CP-AMPAR antagonist, NASPM, has the opposite effect. Measurement of membrane potential in GABAergic neurons by the patch-clamp method in whole-cell configuration demonstrated that NASPM suppresses hyperpolarization in clusters and individual PDSs. It is believed that Kv7-type potassium channels are involved in the control of hyperpolarization during epileptiform activity. The blocker of Kv7 channels, XE 991, mimicked the effect of the CP-AMPARs antagonist on PDS clusters. Both drugs increased the duration of the PDS cluster. In turn, the Kv7 activator, retigabine, decreased the duration of the PDS cluster and Ca2+ pulse. In addition, retigabine led to deep posthyperpolarization at the end of the PDS cluster. The Kv7 channel is believed to be involved in the formation of PDS, as the channel blocker reduced the rate of hyperpolarization in the PDS almost three times. Thus, GABAergic neurons expressing CP-AMPARs, regulate the membrane potential of innervated glutamatergic neurons by modulating the activity of postsynaptic potassium channels of other GABAergic neurons.

Improvement of Blood Parameters of Male Rats Exposed to Different Injection Doses of Liquid Chlorophyll.

Introduction Chlorophylls are natural pigments in our everyday diet, especially with customers' rising preference for more natural and healthful habits. The antioxidant capabilities of both classes of lipophilic substances have been researched since disrupting antioxidant equilibrium appears to be linked to the development of several diseases. Methods This research aimed to evaluate the effect of injection with chlorophyll (30 and 60 mg/ml) on enhancing the blood parameters of rats. Twenty-one white male rats were included in this study and divided into three groups: control, 30 mg/ml, and 60 mg/ml.  Results Treatment with liquid chlorophyll significantly increased white blood cells (WBCs), red blood cells (RBCs), granulocytes, lymphocytes, hemoglobin (Hgb), hematocrit (Hct), mean corpuscular Hgb concentration (MCHC), and platelets. However, it nonsignificantly increased mean corpuscular volume (MCV). These results confirm a great increase in important hematological parameters in response to exogenous injectable chlorophyll with concentrations of 30 and 60 mg/ml and at two different time points, 14 and 28 days after injection. The platelet count was significantly (p<0.001) increased after 30 mg/ml and 60 mg/ml. Conclusion These results show a significant increase in important hematological parameters in response to exogenous injectable chlorophyll. The liquid chlorophyll is recommended to increase blood parameters and improve blood characteristics avoiding anemia.

Properties of GABAergic Neurons Containing Calcium-Permeable Kainate and AMPA-Receptors.

Calcium-permeable kainate and AMPA receptors (CP-KARs and CP-AMPARs), as well as NMDARs, play a pivotal role in plasticity and in regulating neurotransmitter release. Here we visualized in the mature hippocampal neuroglial cultures the neurons expressing CP-AMPARs and CP-KARs. These neurons were visualized by a characteristic fast sustained [Ca2+]i increase in response to the agonist of these receptors, domoic acid (DoA), and a selective agonist of GluK1-containing KARs, ATPA. Neurons from both subpopulations are GABAergic. The subpopulation of neurons expressing CP-AMPARs includes a larger percentage of calbindin-positive neurons (39.4 ± 6.0%) than the subpopulation of neurons expressing CP-KARs (14.2 ± 7.5% of CB+ neurons). In addition, we have shown for the first time that NH4Cl-induced depolarization faster induces an [Ca2+]i elevation in GABAergic neurons expressing CP-KARs and CP-AMPARs than in most glutamatergic neurons. CP-AMPARs antagonist, NASPM, increased the amplitude of the DoA-induced Ca2+ response in GABAergic neurons expressing CP-KARs, indicating that neurons expressing CP-AMPARs innervate GABAergic neurons expressing CP-KARs. We assume that CP-KARs in inhibitory neurons are involved in the mechanism of outstripping GABA release upon hyperexcitation.

Curcumin and Carnosic Acid Cooperate to Inhibit Proliferation and Alter Mitochondrial Function of Metastatic Prostate Cancer Cells.

Anticancer activities of plant polyphenols have been demonstrated in various models of neoplasia. However, evidence obtained in numerous in vitro studies indicates that proliferation arrest and/or killing of cancer cells require quite high micromolar concentrations of polyphenols that are difficult to reach in vivo and can also be (geno)toxic to at least some types of normal cells. The ability of certain polyphenols to synergize with one another at low concentrations can be used as a promising strategy to effectively treat human malignancies. We have recently reported that curcumin and carnosic acid applied at non-cytotoxic concentrations synergistically cooperate to induce massive apoptosis in acute myeloid leukemia cells, but not in normal hematopoietic and non-hematopoietic cells, via sustained cytosolic calcium overload. Here, we show that the two polyphenols can also synergistically suppress the growth of DU145 and PC-3 metastatic prostate cancer cell cultures. However, instead of cell killing, the combined treatment induced a marked inhibition of cell proliferation associated with G0/G1 cell cycle arrest. This was preceded by transient elevation of cytosolic calcium levels and prolonged dissipation of the mitochondrial membrane potential, without generating oxidative stress, and was associated with defective oxidative phosphorylation encompassing mitochondrial dysfunction. The above effects were concomitant with a significant downregulation of mRNA and protein expression of the oncogenic kinase SGK1, the mitochondria-hosted mTOR component. In addition, a moderate decrease in SGK1 phosphorylation at Ser422 was observed in polyphenol-treated cells. The mTOR inhibitor rapamycin produced a similar reduction in SGK1 mRNA and protein levels as well as phosphorylation. Collectively, our findings suggest that the combination of curcumin and carnosic acid at potentially bioavailable concentrations may effectively target different types of cancer cells by distinct modes of action. This and similar combinations merit further exploration as an anticancer modality.

Epileptiform activity promotes decreasing of Ca2+ conductivity of NMDARs, AMPARs, KARs, and voltage-gated calcium channels in Mg2+-free model.

NMDA, AMPA, and kainate receptors are the principal excitatory receptors in the brain. These receptors have been considered as the main targets in the treatment of epilepsy in recent years. This work aimed to determine how the Ca2+ conductivity of ionotropic glutamate receptors and voltage-gated Ca2+ channels changes in an in vitro model of epilepsy. For induction of epileptiform activity, hippocampal neurons were exposed to Mg2+-free medium. It has been shown that removal of Mg2+ from the medium not only removes the block from the NMDA receptors but also stimulates the release of glutamate in a way that is independent of the NMDA receptors. Under these conditions, the structure of the bursts significantly differs from the spontaneous bursts arising in mature hippocampal cultures. We have demonstrated that the frequency and amplitude of Mg2+-free medium-induced Ca2+ oscillations decrease after the 60-min exposure. Besides, the Ca2+ conductivity of ionotropic glutamate receptors and voltage-gated calcium channels significantly reduces. Thus, the decrease of Ca2+ conductivity can be considered as one of the mechanisms of adaptation during epilepsy.

Danazol alters mitochondria metabolism of fibrocystic breast Mcf10A cells.

Fibrocystic Breast Disease (FBD) or Fibrocystic change (FC) affects about 60% of women at some time during their life. Although usually benign, it is often associated with pain and tenderness (mastalgia). The synthetic steroid danazol has been shown to be effective in reducing the pain associated with FBD, but the cellular and molecular mechanisms for its action have not been elucidated. We investigated the hypothesis that danazol acts by affecting energy metabolism. Effects of danazol on Mcf10A cells homeostasis, including mechanisms of oxidative phosphorylation, cytosolic calcium signaling and oxidative stress, were assessed by high-resolution respirometry and flow cytometry. In addition to fast physiological responses the associated genomic modulations were evaluated by Affimetrix microarray analysis. The alterations of mitochondria membrane potential and respiratory activity, downregulation of energy metabolism transcripts result in suppression of energy homeostasis and arrest of Mcf10A cells growth. The data obtained in this study impacts the recognition of direct control of mitochondria by cellular mechanisms associated with altered energy metabolism genes governing the breast tissue susceptibility and response to medication by danazol.

Cooperative antiproliferative and differentiation-enhancing activity of medicinal plant extracts in acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy with poor prognosis and limited treatment options. Sea buckthorn (Hippophae rhamnoides) berries, dog rose (Rosa canina) rosehips, and garden sage (Salvia officinalis) and oregano (Origanum vulgare) aerial parts are widely used in traditional medicine and exhibit antitumor effects in preclinical models. However, these plants remain scarcely tested for antileukemic activity. Here, we show that their water-ethanol leaf extracts reduced the growth and viability of AML cells and, at non-cytotoxic doses, potentiated cell differentiation induced by a low concentration of 1α,25-dihydroxyvitamin D3, the hormonal form of vitamin D, in a cell type-dependent manner. The latter effect was accompanied by upregulation of the vitamin D receptor protein components and its transcriptional activity. Furthermore, at minimally effective doses the extracts cooperated with one another to produce marked cytostatic effects associated with a partial S-phase arrest and a modest induction of apoptosis. In contrast, these combinations only slightly affected the growth and viability of proliferating normal human peripheral blood mononuclear cells. In addition, the extracts strongly inhibited microsomal lipid peroxidation and protected normal erythrocytes against hypoosmotic shock. Our results suggest that further exploration of the enhanced antileukemic effects of the combinations tested here may lead to the development of alternative therapeutic and preventive approaches against AML.

Mitochondria-Mediated Anticancer Effects of Non-Thermal Atmospheric Plasma.

Non-thermal atmospheric pressure plasma has attracted great interest due to its multiple potential biomedical applications with cancer treatment being among the most urgent. To realize the clinical potential of non-thermal plasma, the exact cellular and molecular mechanisms of plasma effects must be understood. This work aimed at studying the prostate cancer specific mechanisms of non-thermal plasma effects on energy metabolism as a central regulator of cell homeostasis and proliferation. It was found that cancer cells with higher metabolic rate initially are more resistant to plasma treated phosphate-buffered saline (PBS) since the respiratory and calcium sensitive signaling systems were not responsive to plasma exposure. However, dramatic decline of cancer oxidative phosphorylation developed over time resulted in significant progression of cell lethality. The normal prostate cells with low metabolic activity immediately responded to plasma treated PBS by suppression of respiratory functions and sustained elevation of cytosolic calcium. However, over time the normal cells start recovering their mitochondria functions, proliferate and restore the cell population. We found that the non-thermal plasma induced increase in intracellular ROS is of primarily non-mitochondrial origin. The discriminate non-thermal plasma effects hold a promise for clinical cancer intervention.

Tumor microenvironment promotes dicarboxylic acid carrier-mediated transport of succinate to fuel prostate cancer mitochondria.

Prostate cancer cells reprogram their metabolism, so that they support their elevated oxidative phosphorylation and promote a cancer friendly microenvironment. This work aimed to explore the mechanisms that cancer cells employ for fueling themselves with energy rich metabolites available in interstitial fluids. The mitochondria oxidative phosphorylation in metastatic prostate cancer DU145 cells and normal prostate epithelial PrEC cells were studied by high-resolution respirometry. An important finding was that prostate cancer cells at acidic pH 6.8 are capable of consuming exogenous succinate, while physiological pH 7.4 was not favorable for this process. Using specific inhibitors, it was demonstrated that succinate is transported in cancer cells by the mechanism of plasma membrane Na(+)-dependent dycarboxylic acid transporter NaDC3 (SLC13A3 gene). Although the level of expression of SLC13A3 was not significantly altered when maintaining cells in the medium with lower pH, the respirometric activity of cells under acidic condition was elevated in the presence of succinate. In contrast, normal prostate cells while expressing NaDC3 mRNA do not produce NaDC3 protein. The mechanism of succinate influx via NaDC3 in metastatic prostate cancer cells could yield a novel target for anti-cancer therapy and has the potential to be used for imaging-based diagnostics to detect non-glycolytic tumors.