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      Loop Diuretics Inhibit Ischemia-Induced Intracellular Ca 2+ Overload in Neurons via the Inhibition of Voltage-Gated Ca 2+ and Na + Channels

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          Abstract

          One consequence of ischemic stroke is disruption of intracellular ionic homeostasis. Intracellular overload of both Na + and Ca 2+ has been linked to neuronal death in this pathophysiological state. The etiology of ionic imbalances resulting from stroke-induced ischemia and acidosis includes the dysregulation of multiple plasma membrane transport proteins, such as increased activity of sodium-potassium-chloride cotransporter-1 (NKCC-1). Experiments using NKCC1 antagonists, bumetanide (BMN) and ethacrynic acid (EA), were carried out to determine if inhibition of this cotransporter affects Na + and Ca 2+ overload observed following in vitro ischemia-acidosis. Fluorometric Ca 2+ and Na + measurements were performed using cultured cortical neurons, and measurements of whole-cell membrane currents were used to determine target(s) of BMN and EA, other than the electroneutral NKCC-1. Both BMN and EA depressed ischemia-acidosis induced [Ca 2+] i overload without appreciably reducing [Na +] i increases. Voltage-gated Ca 2+ channels were inhibited by both BMN and EA with half-maximal inhibitory concentration (IC 50) values of 4 and 36 μM, respectively. Similarly, voltage-gated Na + channels were blocked by BMN and EA with IC 50 values of 13 and 30 μM, respectively. However, neither BMN nor EA affected currents mediated by acid-sensing ion channels or ionotropic glutamatergic receptors, both of which are known to produce [Ca 2+] i overload following ischemia. Data suggest that loop diuretics effectively inhibit voltage-gated Ca 2+ and Na + channels at clinically relevant concentrations, and block of these channels by these compounds likely contributes to their clinical effects. Importantly, inhibition of these channels, and not NKCC1, by loop diuretics reduces [Ca 2+] i overload in neurons during ischemia-acidosis, and thus BMN and EA could potentially be used therapeutically to lessen injury following ischemic stroke.

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          Most cited references43

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          Voltage-Gated Sodium Channels: Structure, Function, Pharmacology, and Clinical Indications.

          The tremendous therapeutic potential of voltage-gated sodium channels (Na(v)s) has been the subject of many studies in the past and is of intense interest today. Na(v)1.7 channels in particular have received much attention recently because of strong genetic validation of their involvement in nociception. Here we summarize the current status of research in the Na(v) field and present the most relevant recent developments with respect to the molecular structure, general physiology, and pharmacology of distinct Na(v) channel subtypes. We discuss Na(v) channel ligands such as small molecules, toxins isolated from animal venoms, and the recently identified Na(v)1.7-selective antibody. Furthermore, we review eight characterized ligand binding sites on the Na(v) channel α subunit. Finally, we examine possible therapeutic applications of Na(v) ligands and provide an update on current clinical studies.
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            Low access resistance perforated patch recordings using amphotericin B.

            We present experimental procedures describing the creation of perforated patches by use of amphotericin B. In 13 different cellular preparations, access resistances below 10 M omega were achieved and with blunt electrode tips, access resistances of 3-4 M omega were possible. In addition to using the techniques to measure whole cell currents, we have used them to measure single channel currents in a new "outside-out patch" preparation and we have utilized them to measure the resting voltage of epithelial monolayers. We conclude that these new approaches can provide a substantial increase in versatility and quality for many kinds of electrophysiological measurements.
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              Evidence for a role of Nav1.6 in facilitating increases in neuronal hyperexcitability during epileptogenesis.

              During epileptogenesis a series of molecular and cellular events occur, culminating in an increase in neuronal excitability, leading to seizure initiation. The entorhinal cortex has been implicated in the generation of epileptic seizures in both humans and animal models of temporal lobe epilepsy. This hyperexcitability is due, in part, to proexcitatory changes in ion channel activity. Sodium channels play an important role in controlling neuronal excitability, and alterations in their activity could facilitate seizure initiation. We sought to investigate whether medial entorhinal cortex (mEC) layer II neurons become hyperexcitable and display proexcitatory behavior of Na channels during epileptogenesis. Experiments were conducted 7 days after electrical induction of status epilepticus (SE), a time point during the latent period of epileptogenesis and before the onset of seizures. mEC layer II stellate neurons from post-SE animals were hyperexcitable, eliciting action potentials at higher frequencies compared with control neurons. Na channel currents recorded from post-SE neurons revealed increases in Na current amplitudes, particularly persistent and resurgent currents, as well as depolarized shifts in inactivation parameters. Immunocytochemical studies revealed increases in voltage-gated Na (Nav) 1.6 isoform levels. The toxin 4,9-anhydro-tetrodotoxin, which has greater selectivity for Nav1.6 over other Na channel isoforms, suppressed neuronal hyperexcitability, reduced macroscopic Na currents, persistent and resurgent Na current densities, and abolished depolarized shifts in inactivation parameters in post-SE neurons. These studies support a potential role for Nav1.6 in facilitating the hyperexcitability of mEC layer II neurons during epileptogenesis.
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                Author and article information

                Contributors
                Journal
                Front Pharmacol
                Front Pharmacol
                Front. Pharmacol.
                Frontiers in Pharmacology
                Frontiers Media S.A.
                1663-9812
                15 September 2021
                2021
                : 12
                : 732922
                Affiliations
                Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, FL, United States.
                Author notes

                Edited by: Sarel Francois Malan, University of the Western Cape, South Africa

                Reviewed by: Xiangping Chu, University of Missouri–Kansas City, United States

                Jinwei Zhang, University of Exeter, United Kingdom

                *Correspondence: Javier Cuevas, jcuevas@ 123456usf.edu

                This article was submitted to Pharmacology of Ion Channels and Channelopathies, a section of the journal Frontiers in Pharmacology

                Article
                732922
                10.3389/fphar.2021.732922
                8479115
                34603048
                c6141217-1e2b-49c0-8a74-39a96b60e3b5
                Copyright © 2021 Katnik and Cuevas.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 29 June 2021
                : 10 August 2021
                Categories
                Pharmacology
                Original Research

                Pharmacology & Pharmaceutical medicine
                bumetanide,ethacrynic acid,voltage-gated channels,sodium,calcium,neurons,ischemia,acidosis

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