Intracellular Stores Research Articles

Bradykinin-Induced Sensitization of Transient Receptor Potential Channel Melastatin 3 Calcium Responses in Mouse Nociceptive Neurons.

TRPM3 is a calcium-permeable cation channel expressed in a range of sensory neurons that can be activated by heat and the endogenous steroid pregnenolone sulfate (PS). During inflammation, the expression and function of TRPM3 are both augmented in somatosensory nociceptors. However, in isolated dorsal root ganglion (DRG) neurons application of inflammatory mediators like prostaglandins and bradykinin (BK) inhibit TRPM3. Therefore, the aim of this study was to examine the effect of preceding activation of cultured 1 day old mouse DRG neurons by the inflammatory mediator BK on TRPM3-mediated calcium responses. Calcium signals were recorded using the intensity-based dye Fluo-8. We found that TRPM3-mediated calcium responses to PS were enhanced by preceding application of BK in cells that responded to BK with a calcium signal, indicating BK receptor (BKR) expression. The majority of cells that co-expressed TRPM3 and BKRs also expressed TRPV1, however, only a small fraction co-expressed TRPA1, identified by calcium responses to capsaicin and supercinnamaldehyde, respectively. Signaling and trafficking pathways responsible for sensitization of TRPM3 following BK were characterized using inhibitors of second messenger signaling cascades and exocytosis. Pharmacological blockade of protein kinase C, calcium–calmodulin-dependent protein kinase II and diacylglycerol (DAG) lipase did not affect BK-induced sensitization, but inhibition of DAG kinase did. In addition, release of calcium from intracellular stores using thapsigargin also resulted in TRPM3 sensitization. Finally, BK did not sensitize TRPM3 in the presence of exocytosis inhibitors. Collectively, we show that preceding activation of DRG neurons by BK sensitized TRPM3-mediated calcium responses to PS. Our results indicate that BKR-mediated activation of intracellular signaling pathways comprising DAG kinase, calcium and exocytosis may contribute to TRPM3 sensitization during inflammation.

Aging modifies receptor expression but not muscular contractile response to angiotensin II in rat jejunum

The involvement of renin-angiotensin system in the modulation of gut motility and age-related changes in mRNA expression of angiotensin (Ang II) receptors (ATR) are well accepted. We aimed to characterize, in vitro, the contractile responses induced by Ang II, in jejunum from young (3–6 weeks old) and old rats (≥ 1 year old), to evaluate possible functional differences associated to changes in receptor expression. Mechanical responses to Ang II were examined in vitro as changes in isometric tension. ATR expression was assessed by qRT-PCR. Ang II induced a contractile effect, antagonized by losartan, AT1R antagonist, and increased by PD123319, AT2R antagonist, as well by neural blocker ω-conotoxin and by nitric oxide (NO) synthase inhibitor. No difference in the response was observed between young and old groups. AT1 receptor-mediated contractile response was decreased by U-73122, phospholipase C (PLC) inhibitor; or 2-aminoethoxy-diphenylborate (2-APB), inositol triphosphate (IP3) receptor inhibitor; or nifedipine, l-type calcium channel blocker. Age-related changes in the expression of both AT1 receptor subtypes, AT1a and AT1b, and of AT2 receptors were detected. In conclusion, Ang II modulates the spontaneous contractility of rat jejunum via postjunctional AT1 receptors, involving Ca2+ mobilization from intracellular stores, via PLC/IP3 pathway, and Ca2+ influx from extracellular space, via l-type channels. Prejunctional AT2 receptors would counteract AT1 receptor effects, via NO synthesis. The observed age-related differences in the expression of all AT receptor subtypes are not reflected in the muscular contractile response to Ang II.

Transient Receptor Potential channels (TRP) in GtoPdb v.2022.1

Transient Receptor Potential channels (TRP) in GtoPdb v.2022.1

SARS-CoV-2 infection of airway cells causes intense viral and cell shedding, two spreading mechanisms affected by IL-13

Muco-obstructive lung diseases are typically associated with high risks of COVID-19 severity; however, allergic asthma showed reduced susceptibility. To investigate viral spread, primary human airway epithelial (HAE) cell cultures were infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and host–virus interactions were examined via electron microscopy, immunohistochemistry, RNA in situ hybridization, and gene expression analyses. In HAE cell cultures, angiotensin-converting enzyme 2 (ACE2) expression governed cell tropism and viral load and was up-regulated by infection. Electron microscopy identified intense viral egress from infected ciliated cells and severe cytopathogenesis, culminating in the shedding of ciliated cells packed with virions, providing a large viral reservoir for spread and transmission. Intracellular stores of MUC5AC, a major airway mucin involved in asthma, were rapidly depleted, likely to trap viruses. To mimic asthmatic airways, HAE cells were treated with interleukin-13 (IL-13), which reduced viral titers, viral messenger RNA, and cell shedding, and significantly diminished the number of infected cells. Although mucus hyperproduction played a shielding role, IL-13–treated cells maintained a degree of protection despite the removal of mucus. Using Gene Expression Omnibus databases, bulk RNA-sequencing analyses revealed that IL-13 up-regulated genes controlling glycoprotein synthesis, ion transport, and antiviral processes (albeit not the typical interferon-induced genes) and down-regulated genes involved in cilial function and ribosomal processing. More precisely, we showed that IL-13 reduced ACE2 expression, intracellular viral load, and cell-to-cell transmission while increasing the cilial keratan sulfate coating. In conclusion, intense viral and cell shedding caused by SARS-CoV-2 infection was attenuated by IL-13, which affected viral entry, replication, and spread.

Calcium signaling in prostate cancer cells of increasing malignancy

Calcium signaling controls a large variety of cell functions, including proliferation and apoptosis, and plays a major role in neoplastic transformation. Prostate cancer (PCa) is one of the most common malignancies in men. The transition to castration-resistant prostate cancer (CRPC), a lethal form that is still lacking an effective cure, could be influenced by fine tuning intracellular calcium ([Ca2+]i) homeostasis. This study investigates [Ca2+]i dynamics in metastatic PCa cell lines that mimic the progression of PCa to CRPC: (i) well differentiated LNCaP cells that require androgen for survival, and (ii) poorly differentiated, highly aggressive androgen-insensitive prostate cancer (AIPC) PC3 and DU145 cells. In AIPC cells, ATP induces a fast rise in [Ca2+]i, due to release from intracellular stores and sensitive to phospholipase C inhibitors, while LNCaP cells do not respond to ATP challenge. Moreover, AIPC cells showed a reduced capacity to store Ca2+ in thapsigargin-sensitive stores and limited store-operated calcium entry, with respect to androgen-dependent LNCaP cells. Finally, green tea extract causes [Ca2+]i elevation and inhibits proliferation in PC3 and DU145 cells, but is ineffective in LNCaP cells. The consequences of these differences are discussed and interpreted in this study with reference to previously proposed models for Ca2+ dependence of prostate carcinogenesis.

IP3-dependent Ca2+ signals are tightly controlled by Cavβ3, but not by Cavβ1, 2 and 4

Independent of its function as a subunit of voltage-gated Ca2+ channels, the Cavβ3 subunit desensitizes fibroblasts and pancreatic β-cells to low concentrations of inositol-1,4,5-trisphosphate (IP3). This alters agonist-induced Ca2+ signaling and cellular functions, for example, insulin secretion and wound healing. A total of four Cavβ subunits exist, Cavβ1, Cavβ2, Cavβ3, and Cavβ4. To investigate whether the other Cavβ subunits, like Cavβ3, can desensitize cells to IP3 and thereby modulate Ca2+ signaling, we expressed the cDNAs of Cavβ1, Cavβ2, Cavβ3, and Cavβ4 in COS-7 cells lacking endogenous Cavβ proteins. ATP stimulation of these cells results in the release of Ca2+ from intracellular stores. This receptor-mediated Ca2+ release is significantly decreased by Cavβ3 but not by Cavβ1, Cavβ2, and Cavβ4. Electrophysiological recordings of voltage-dependent Ca2+ currents from fibroblasts show a small Ca2+ current, the amplitude of which is slightly but not significantly smaller in fibroblasts from Cavβ2 gene-deficient animals than in fibroblasts from wild-type animals. Compared with fibroblasts from wild-type animals, Ca2+ release is not significantly increased in Cavβ2-deficient fibroblasts, in contrast to Ca2+ release in Cavβ3-deficient fibroblasts. In summary, our results show that desensitization of cells to low concentrations of IP3 is a specific property of Cavβ3 that is not shared by other Cavβ subunits.

CADPR Does Not Activate TRPM2.

cADPR is a second messenger that releases Ca2+ from intracellular stores via the ryanodine receptor. Over more than 15 years, it has been controversially discussed whether cADPR also contributes to the activation of the nucleotide-gated cation channel TRPM2. While some groups have observed activation of TRPM2 by cADPR alone or in synergy with ADPR, sometimes only at 37 °C, others have argued that this is due to the contamination of cADPR by ADPR. The identification of a novel nucleotide-binding site in the N-terminus of TRPM2 that binds ADPR in a horseshoe-like conformation resembling cADPR as well as the cADPR antagonist 8-Br-cADPR, and another report that demonstrates activation of TRPM2 by binding of cADPR to the NUDT9H domain raised the question again and led us to revisit the topic. Here we show that (i) the N-terminal MHR1/2 domain and the C-terminal NUDT9H domain are required for activation of human TRPM2 by ADPR and 2′-deoxy-ADPR (2dADPR), (ii) that pure cADPR does not activate TRPM2 under a variety of conditions that have previously been shown to result in channel activation, (iii) the cADPR antagonist 8-Br-cADPR also inhibits activation of TRPM2 by ADPR, and (iv) cADPR does not bind to the MHR1/2 domain of TRPM2 while ADPR does.

Homeostasis of Mitochondrial Ca2+ Stores Is Critical for Signal Amplification in Drosophila melanogaster Olfactory Sensory Neurons.

Simple SummaryThe evolution of flight imposed new challenges on insects when locating and identifying food sources, mates, or enemies. As an adaptation, flying insects developed a remarkably sensitive olfactory system to detect faint odor traces. This ability is linked to the olfactory receptor class of odorant receptors, which are found in insect olfactory sensory neurons. In a subgroup of these neurons, sensitivity can be further enhanced through a process called sensitization. Extracellular calcium ions, calmodulin, and protein kinase C are known to be key factors in this process. While manipulation of mitochondrial calcium im- and export has been shown to influence odor responses in general, the connection of intracellular calcium stores to sensitization has so far been only speculative. Using two pharmacological approaches, we disrupted mitochondrial calcium management in order to explore its importance to sensitization. Overall, our findings reveal that mitochondrial calcium stores are important players in the complex intracellular signaling pathways required for sensitization.Insects detect volatile chemosignals with olfactory sensory neurons (OSNs) that express olfactory receptors. Among them, the most sensitive receptors are the odorant receptors (ORs), which form cation channels passing Ca2+. OSNs expressing different groups of ORs show varying optimal odor concentration ranges according to environmental needs. Certain types of OSNs, usually attuned to high odor concentrations, allow for the detection of even low signals through the process of sensitization. By increasing the sensitivity of OSNs upon repetitive subthreshold odor stimulation, Drosophila melanogaster can detect even faint and turbulent odor traces during flight. While the influx of extracellular Ca2+ has been previously shown to be a cue for sensitization, our study investigates the importance of intracellular Ca2+ management. Using an open antenna preparation that allows observation and pharmacological manipulation of OSNs, we performed Ca2+ imaging to determine the role of Ca2+ storage in mitochondria. By disturbing the mitochondrial resting potential and induction of the mitochondrial permeability transition pore (mPTP), we show that effective storage of Ca2+ in the mitochondria is vital for sensitization to occur, and release of Ca2+ from the mitochondria to the cytoplasm promptly abolishes sensitization. Our study shows the importance of cellular Ca2+ management for sensitization in an effort to better understand the underlying mechanics of OSN modulation.

Semiconducting Carbon Nanotube-Based Nanodevices for Monitoring the Effects of Chlorphenamine on the Activities of Intracellular Ca2+ Stores.

We report a flexible and noninvasive method based on field-effect transistors hybridizing semiconducting single-walled carbon nanotubes for monitoring the effects of histamine on Ca2+ release from the intracellular stores of a nonexcitable cell. These nanodevices allowed us to evaluate the real-time electrophysiological activities of HeLa cells under the stimulation of histamine via the recording of the conductance changes of the devices. These changes resulted from the binding of histamine to its receptor type 1 on the HeLa cell membrane. Moreover, the effects of chlorphenamine, an antihistamine, on the electrophysiological activities of a single HeLa cell were also evaluated, indicating that the pretreatment of the cell with chlorpheniramine decreased intracellular Ca2+ release. Significantly, we only utilized a single nanodevice to perform the measurements for multiple cells pretreated with various concentrations of chlorphenamine. This enabled the statistically meaningful analysis of drug effects on cells without errors from device variations. Obtained results indicated the novel advantages of our method such as real-time monitoring and quantitative capability. Our devices, therefore, can be efficient tools for biomedical applications such as electrophysiology research and drug screening.

CRACking the Molecular Regulatory Mechanism of SOCE during Platelet Activation in Thrombo-Occlusive Diseases.

Thrombo-occlusive diseases such as myocardial infarction, ischemic stroke and deep vein thrombosis with subsequent pulmonary embolism still represent a major health burden worldwide. Besides the cells of the vasculature or other hematopoietic cells, platelets are primarily responsible for the development and progression of an occluding thrombus. The activation and function of platelets crucially depend on free cytosolic calcium (Ca2+) as second messenger, which modulates platelet secretion, aggregation and thrombus formation. Ca2+ is elevated upon platelet activation by release of Ca2+ from intracellular stores thus triggering of the subsequent store-operated Ca2+ entry (SOCE), which is facilitated by Ca2+ release-activated channels (CRACs). In general, CRACs are assembled by the pore-forming unit Orai in the plasma membrane and the Ca2+-sensing stromal interaction molecule (STIM) in the endoplasmic reticulum after the depletion of internal Ca2+ stores. In the last few years, there is a growing body of the literature demonstrating the importance of STIM and Orai-mediated mechanism in thrombo-occlusive disorders. Thus, this review provides an overview of the recent understanding of STIM and Orai signaling in platelet function and its implication in the development and progression of ischemic thrombo-occlusive disorders. Moreover, potential pharmacological implications of STIM and Orai signaling in platelets are anticipated and discussed in the end.

Coronin 1A facilitates calcium mobilization and promotes astrocyte reactivity in HIV-1 neuropathogenesis.

Astrocyte reactivity, a phenomenon observed in a variety of neurodegenerative disorders, can have both beneficial and detrimental manifestations which significantly affect neuronal physiology. In neuroAIDS, reactive astrocytes have been observed to severely affect the neuronal population present in their vicinity. Calcium signaling plays a central role in mediating astrocyte reactivity. Coronin 1A, an actin‐binding protein, majorly reported in hematopoietic cells, regulates cell activity in a calcium‐dependent manner, but its role in astrocyte physiology and reactivity is largely unknown. Using a well‐characterized primary culture of human astroglia and neurons, we explored the roles of coronin 1A in astrocyte physiology and its involvement in facilitating astrocyte reactivity. In this study, we report coronin 1A expression in human primary astrocytes and autopsy brain sections, and that it plays activity‐dependent roles by facilitating calcium mobilization from the intracellular stores. HIV‐1 Tat, a potent neurotoxicant that turns astrocytes reactive, augments coronin 1A expression, apart from affecting GFAP and pro‐inflammatory molecules. Also, the autopsy brain tissue of HIV‐1 infected individuals has a higher expression of coronin 1A. Downregulation of coronin 1A attenuated the HIV‐1 Tat‐induced deleterious effects of reactive astrocytes, measured as the upregulated expression of GFAP, pro‐inflammatory molecules, and enhanced release of IL‐6, and hence reduced astrocyte‐mediated neurodegeneration. Our findings also suggest that out of a pool of dysregulated miRNAs studied by us, hsa‐miR‐92b‐5p regulates coronin 1A expression under the effect of HIV‐1 Tat. These findings highlight the novel roles of coronin 1A in regulating astrocyte activity in stimulated conditions and astrocyte reactivity observed in HIV‐1 neuropathogenesis.

Possible mechanisms underlying the dynamic assembly of calcium entry units: the role of temperature and pH

Skeletal muscle function is regulated by intracellular Ca2+ levels. Two main mechanisms control movements of Ca2+ ions from intracellular stores (i.e. the sarcoplasmic reticulum, SR) and from extracellular space: i) excitation-contraction (EC) coupling; and ii) store-operated Ca2+ entry (SOCE). SOCE allows recovery of extracellular Ca2+ during prolonged muscle activity, when the SR undergoes depletion. We recently discovered that prolonged exercise leads to formation of Calcium Entry Units (CEUs), intracellular junctions located at the I band that are formed by two distinct elements: SR-stacks and transverse tubules (TTs).

Aberrations in T cell calcium signaling and mitochondrial function predict the presence of the gain-of-function mutation in the ryanodine receptor type 1

Gain-of-function RyR1-p.R163C mutation in ryanodine receptors type 1 (RyR1) deregulates Ca2+ signaling in skeletal muscle and causes malignant hyperthermia in humans under triggering conditions. T lymphocytes from heterozygous RyR1-p.R163C knock-in mutant mice (HET T cells) display measurable aberrations in resting cytosolic Ca2+ concentration ([Ca2+]i), Ca2+ release from the intracellular store, and store-operated Ca2+ entry (SOCE), compared with WT T cells. Pretreatment of HET T cells with ryanodine receptor inhibitors ryanodine or dantrolene reduced disparities between HET and WT T cells in the resting [Ca2+]i and the ability of thapsigargin, an inhibitor of the sarco-endoplasmic reticulum Ca2+ ATPase, to mobilize Ca2+ from the store.

Sequestration of membrane micropeptides in hetero-oligomers decreases their regulatory interaction with the SERCA calcium pump

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) is an ion transporter that creates and maintains intracellular calcium stores. Cellular calcium handling is disrupted in diverse diseases, focusing attention on SERCA as a potential target of future therapeutic strategies. SERCA function is inhibited or stimulated by several membrane micropeptides including endoregulin (ELN), another-regulin (ALN), sarcolipin (SLN), phospholamban (PLB), and dwarf open reading frame (DWORF). We investigated hetero-oligomeric interactions of these micropeptides using automated fluorescence resonance energy transfer (FRET) microscopy.

PaOctβ2R: Identification and Functional Characterization of an Octopamine Receptor Activating Adenylyl Cyclase Activity in the American Cockroach Periplaneta americana.

Biogenic amines constitute an important group of neuroactive substances that control and modulate various neural circuits. These small organic compounds engage members of the guanine nucleotide-binding protein coupled receptor (GPCR) superfamily to evoke specific cellular responses. In addition to dopamine- and 5-hydroxytryptamine (serotonin) receptors, arthropods express receptors that are activated exclusively by tyramine and octopamine. These phenolamines functionally substitute the noradrenergic system of vertebrates Octopamine receptors that are the focus of this study are classified as either α- or β-adrenergic-like. Knowledge on these receptors is scarce for the American cockroach (Periplaneta americana). So far, only an α–adrenergic-like octopamine receptor that primarily causes Ca2+ release from intracellular stores has been studied from the cockroach (PaOctα1R). Here we succeeded in cloning a gene from cockroach brain tissue that encodes a β-adrenergic-like receptor and leads to cAMP production upon activation. Notably, the receptor is 100-fold more selective for octopamine than for tyramine. A series of synthetic antagonists selectively block receptor activity with epinastine being the most potent. Bioinformatics allowed us to identify a total of 19 receptor sequences that build the framework of the biogenic amine receptor clade in the American cockroach. Phylogenetic analyses using these sequences and receptor sequences from model organisms showed that the newly cloned gene is an β2-adrenergic-like octopamine receptor. The functional characterization of PaOctβ2R and the bioinformatics data uncovered that the monoaminergic receptor family in the hemimetabolic P. americana is similarly complex as in holometabolic model insects like Drosophila melanogaster and the honeybee, Apis mellifera. Thus, investigating these receptors in detail may contribute to a better understanding of monoaminergic signaling in insect behavior and physiology.

LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

Lysosomes function not only as degradatory compartments but also as dynamic intracellular calcium ion stores. The transient receptor potential mucolipin 1 (TRPML1) channel mediates lysosomal Ca2+ release, thereby participating in multiple cellular functions. The pentameric Ragulator complex, which plays a critical role in the activation of mTORC1, is also involved in lysosomal trafficking and is anchored to lysosomes through its LAMTOR1 subunit. Here, we report that the Ragulator restricts lysosomal trafficking in dendrites of hippocampal neurons via LAMTOR1‐mediated tonic inhibition of TRPML1 activity, independently of mTORC1. LAMTOR1 directly interacts with TRPML1 through its N‐terminal domain. Eliminating this inhibition in hippocampal neurons by LAMTOR1 deletion or by disrupting LAMTOR1‐TRPML1 binding increases TRPML1‐mediated Ca2+ release and facilitates dendritic lysosomal trafficking powered by dynein. LAMTOR1 deletion in the hippocampal CA1 region of adult mice results in alterations in synaptic plasticity, and in impaired object‐recognition memory and contextual fear conditioning, due to TRPML1 activation. Mechanistically, changes in synaptic plasticity are associated with increased GluA1 dephosphorylation by calcineurin and lysosomal degradation. Thus, LAMTOR1‐mediated inhibition of TRPML1 is critical for regulating dendritic lysosomal motility, synaptic plasticity, and learning.

Off‐target inhibition of NGLY1 by the polycaspase inhibitor Z‐VAD‐fmk induces cellular autophagy

The polycaspase inhibitor Z‐VAD‐fmk acts as an inhibitor of peptide: N‐glycanase (NGLY1), an endoglycosidase which cleaves N‐linked glycans from glycoproteins exported from the endoplasmic reticulum (ER) during ER‐associated degradation (ERAD). Both pharmacological N‐glycanase inhibition by Z‐VAD‐fmk and siRNA‐mediated knockdown (KD) of NGLY1 induce GFP‐LC3‐positive puncta in HEK 293 cells. The activation of ER stress markers or induction of reactive oxygen species (ROS) is not observed under either condition. Moreover, Ca2+ handling is unaffected when observing release from intracellular stores. Under conditions of pharmacological NGLY1 inhibition or NGLY1 KD, upregulation of autophagosome formation without impairment of autophagic flux is observed. Enrichment of autophagosomes by immunoprecipitation (IP) and mass spectrometry‐based proteomic analysis reveals comparable autophagosomal protein content. Gene ontology analysis of proteins enriched in autophagosome IPs shows overrepresentation of factors involved in protein translation, localization and targeting, RNA degradation and protein complex disassembly. Upregulation of autophagy represents a cellular adaptation to NGLY1 inhibition or KD, and ATG13‐deficient mouse embryonic fibroblasts (MEFs) show reduced viability under these conditions. In contrast, treatment with pan‐caspase inhibitor, Q‐VD‐OPh, does not induce cellular autophagy. Therefore, experiments with Z‐VAD‐fmk are complicated by the effects of NGLY1 inhibition, including induction of autophagy, and Q‐VD‐OPh represents an alternative caspase inhibitor free from this limitation.EnzymesPeptide:N‐glycanase1, Peptide‐N(4)‐(N‐acetyl‐beta‐glucosaminyl)asparagine amidase [EC:3.5.1.52].

Effects of hippocampal IP3R inhibition on contextual fear memory consolidation, retrieval, reconsolidation and extinction

Intracellular calcium stores (ICS) play a dynamic role in neuronal calcium (Ca2+) homeostasis both by buffering Ca2+ excess in the cytoplasm or providing an additional source of Ca2+ when concentration increase is needed. However, in spite of the large body of evidence showing Ca2+ as an essential second messenger in many signaling cascades underlying synaptic plasticity, the direct involvement of the intracellular Ca2+-release channels (ICRCs) in memory processing has been highly overlooked. Here we investigated the role of the ICRC inositol 1,4,5-trisphosphate receptor (IP3R) activity during different memory phases using pharmacological inhibition in the dorsal hippocampus during contextual fear conditioning. We first found that post-training administration of the IP3R antagonist 2-aminoethyl diphenylborinate (2-APB) impaired memory consolidation in a dose and time-dependent manner. Inhibiting IP3Rs also disrupted memory retrieval. Contextual fear memory reconsolidation or extinction, however, were not sensitive to IP3R blockade. Taken together, our results indicate that hippocampal IP3Rs play an important role in contextual fear memory consolidation and retrieval.

Clinical Pharmacology of Levetiracetam in Infants and Children

Levetiracetam inhibits focal and secondary generalized tonic-clonic seizures. The mechanism of levetiracetam action is not fully understood, however the correlation between binding affinity of levetiracetam and its analogues and their potency toward audiogenic seizures suggest that the synaptic vesicle glycoprotein 2A mediates the anticonvulsant effects of levetiracetam. The neural function of the synaptic vesicle 2A protein is not fully understood, but binding of levetiracetam to synaptic vesicle glycoprotein 2A might affect neuronal excitability by modifying the release of glutamate GABA through an action on vesicular function. Synaptic vesicle glycoprotein 2A may plain a role in vesicle recycling following exocytosis of neurotransmitter. In addition, levetiracetam inhibits N-type Ca2+ channels and Ca2+ release from intracellular stores. Levetiracetam may be administered intravenously or orally to infants and children and in children the levetiracetam dose varies according to the child age and body-weight. Levetiracetam is almost completed absorbed after oral administration and levetiracetam is found efficacy and safe in infants and children but it may induce adverse-effects. The levetiracetam elimination half-life is about 6 hours in infants and children, and in children the renal clearance is similar to the non-renal clearance. The prophylaxis, treatment, and trials with levetiracetam have been extensively studied in infants and children. Levetiracetam freely crosses the human placenta and freely migrates into the breast-milk. The aim of this study is to review the levetiracetam dosing, efficacy, safety, adverse-effects, pharmacokinetics, prophylaxis, treatment, and trials and transfer of levetiracetam across the human placenta and levetiracetam migration into the breast-milk.

Effects of Long-Term Lead Exposure on Antioxidant Enzyme Defense System in Organs of Brown Hare (Lepus europaeus Pallas) as a Bioindicator of Environmental Pollution in Croatia.

In Croatia, Podravina is a well-known lead-polluted region due to the intensive exploitation of natural gas, a highly developed agricultural industry, and a traffic hub with several heavily traveled roads. It represents a natural environment with a great variety of wildlife, especially hares (Lepus europaeus Pallas), which may serve as an indicator for environmental quality assessment. This study was conducted to estimate the bioaccumulation of lead in hare liver, kidney, muscle, and brain during long-term exposure and its impact on the oxidative status of the organism and to investigate a possible lead exchange ionic mechanism in the brain. In the organs of two hare groups (experimental from polluted area and control from the island of Krk), Ca, Fe, Mg, Na, lead concentrations, and antioxidant enzyme defense system were analyzed. The accumulation of lead was highest in the brain (3.7-fold higher compared to the control group) and lowest in the liver (1.6-fold higher compared to the control group). Kendall-Tau and multiple regression analysis showed that the increased lead content caused a stronger exchange of Ca and Na ions in the brain. We proposed that lead either mobilizes intracellular cation stores or causes competitive displacement of Ca from the binding site in intoxicated cells. A linear predictive model for cell intoxication by lead was proposed, where GPx and SOD were predominantly influenced by long-term lead exposure. The presented results showed that long-term lead exposure in hares negatively affected their oxidative status and caused the strongest toxicity in the brain and muscles, making their survival and/or population vulnerable.