kb-r7943 and Hypoxia

Motor nerve terminals are especially sensitive to an ischemia/reperfusion stress. We applied an in vitro model of this stress, oxygen/glucose deprivation (OGD), to mouse neuromuscular preparations to investigate how Ca(2+) contributes to stress-induced motor terminal damage. Measurements using an ionophoretically-injected fluorescent [Ca(2+)] indicator demonstrated an increase in intra-terminal [Ca(2+)] following OGD onset. When OGD was terminated within 20-30min of the increase in resting [Ca(2+)], these changes were sometimes reversible; in other cases [Ca(2+)] remained high and the terminal degenerated. Endplate innervation was assessed morphometrically following 22min OGD and 120min reoxygenation (32.5°C). Stress-induced motor terminal degeneration was Ca(2+)-dependent. Median post-stress endplate occupancy was only 26% when the bath contained the normal 1.8mM Ca(2+), but increased to 81% when Ca(2+) was absent. Removal of Ca(2+) only during OGD was more protective than removal of Ca(2+) only during reoxygenation. Post-stress endplate occupancy was partially preserved by pharmacological inhibition of various routes of Ca(2+) entry into motor terminals, including voltage-dependent Ca(2+) channels (ω-agatoxin-IVA, nimodipine) and the plasma membrane Na(+)/Ca(2+) exchanger (KB-R7943). Inhibition of a Ca(2+)-dependent protease with calpain inhibitor VI was also protective. These results suggest that most of the OGD-induced motor terminal damage is Ca(2+)-dependent, and that inhibition of Ca(2+) entry or Ca(2+)-dependent proteolysis can reduce this damage. There was no significant difference between the response of wild-type and presymptomatic superoxide dismutase 1 G93A mutant terminals to OGD, or in their response to the protective effect of the tested drugs.

Topics: Animals; Bacterial Proteins; Bungarotoxins; Calcium; Disease Models, Animal; Egtazic Acid; Enzyme Inhibitors; Glucose; Humans; Hypoxia; In Vitro Techniques; Luminescent Proteins; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mice, Transgenic; Motor Endplate; Motor Neuron Disease; Motor Neurons; Neuromuscular Junction; Protein Binding; Superoxide Dismutase; Thiourea; Time Factors

Closure of the ductus arteriosus (DA) after birth, essential for postnatal adaptation, is initiated by the transition from hypoxia to normoxia. The current study investigated how hypoxia affects the level of cytosolic calcium ([Ca(2+)](i)) in fetal lamb DA smooth muscle cells (DASMCs) and the role of calcium pumps in this process. The [Ca(2+)](i) variation in response to acute hypoxia was determined spectrofluorometrically with fura-3-AM in cultured fetal DASMCs. Interventions using chemicals or solutions including thapsigargin, vanadate, KB-R7943, alkaline PH9.0 solution, or Na(+)-free medium were administered when samples were exposed to acute hypoxia. The results show that [Ca(2+)](i) decreased dramatically under acute hypoxia. This decrease was not attenuated completely by an inhibitor of sarcoplasmic/endoplasmic reticulum Ca(2+) adenosine triphosphatase (ATPase) (SERCA), a blocker of plasma membrane Ca(2+) ATPase (PMCA), or an inhibitor and activator of the reserve mode of the Na(+)/Ca(2+) exchanger (NCX). In contrast, KT-R9743, an inhibitor of the forward mode of NCX at a high concentration (30 microm), greatly diminished the hypoxia-induced [Ca(2+)](i) decrease in fetal DASMCs. These results suggest that a hypoxia-induced Ca(2+) decrease in fetal DASMCs results from cytosolic Ca(2+) efflux mediated primarily by the forward mode of NCX.

Topics: Analysis of Variance; Animals; Calcium; Cells, Cultured; Cytosol; Ductus Arteriosus; Fetus; Hydrogen-Ion Concentration; Hypoxia; Microscopy, Confocal; Myocytes, Smooth Muscle; Sheep; Sodium-Calcium Exchanger; Thapsigargin; Thiourea; Vanadates

Mammalian cells require a constant O2 supply to produce adequate energy, and sustained hypoxia can kill cells. Mammals therefore have evolved sophisticated mechanisms to allow their cells to adapt to hypoxia. In this study, we investigated the role of TRP channels and the Na+-Ca2+ exchanger (NCX) in mediating hypoxia-induced [Ca2+]i elevation in a model of the O2-sensing rat pheochromocytoma (PC12) cell line by using Ca2+ imaging and molecular biological approaches. Non-selective cation channels, such as TRPC1, 3 and 6, were found to be functionally expressed in PC12 cells. They mediated Ca2+ entry when cells were exposed to acute hypoxia (PO2 of 15 mmHg), in addition to Ca2+ entry via VGCCs. Blockage of TRPCs by 2APB and SKF96365 could significantly reduce hypoxia-mediated [Ca2+]i elevation. Suramin and U73122 attenuated the hypoxia-induced [Ca2+]i elevation, implying the involvement of the G-protein and PLC pathways in the hypoxic response. In addition to TRPCs and VGCCs, NCX also contributed to the hypoxia-induced [Ca2+]i elevation, and blockade of NCX by KBR7943 could significantly decrease the hypoxia-induced [Ca2+]i elevation. Our results suggest that the activation of TRP by hypoxia could lead to NCX reversal; furthermore, membrane depolarization and TRPCs may play a primary role in mediating the hypoxic response in PC12 cells.

Topics: Animals; Boron Compounds; Calcium; Calcium Channel Blockers; Dose-Response Relationship, Drug; Gene Expression Regulation; Hypoxia; Imidazoles; Models, Biological; PC12 Cells; Potassium Chloride; Rats; Sodium-Calcium Exchanger; Suramin; Thiourea; TRPC Cation Channels; Verapamil

We analyzed the role of the Na+/Ca2+ exchanger (NCX) in endothelin-1-aggravated hypoxia/reoxygenation-induced injury in renal epithelial LLC-PK1 cells. KB-R7943, a selective NCX inhibitor, suppressed hypoxia/reoxygenation-induced cell damage, whereas overexpression of NCX1 into cells enhanced it. Endothelin-1 significantly aggravated hypoxia/reoxygenation-induced injury in parental and NCX1-overexpressing LLC-PK1 cells. Such aggravation by endothelin-1 was not observed in cells overexpressing a deregulated NCX1 mutant, which displays no protein kinase C-dependent activation. These results suggest that Ca2+ overload via NCX plays a critical role in hypoxia/reoxygenation-induced renal tubular injury, and that endothelin-1 aggravates the cell damage through the activation of NCX.

Topics: Animals; Endothelin-1; Epithelial Cells; Hypoxia; Kidney; LLC-PK1 Cells; Oxygen; Swine; Thiourea

Intracellular ATP supply and ion homeostasis determine neuronal survival and degeneration after ischemic stroke. The present study provides a systematic investigation in organotypic hippocampal slice cultures of the influence of experimental ischemia, induced by oxygen-glucose-deprivation (OGD). The pathways controlling intracellular Na(+) and Ca(2+) concentration ([Na(+)](i) and [Ca(2+)](i)) and their inhibition were correlated with delayed cell death or protection. OGD induced a marked decrease in the ATP level and a transient elevation of [Ca(2+)](i) and [Na(+)](i) in cell soma of pyramidal neurons. ATP level, [Na(+)](i) and [Ca(2+)](i) rapidly recovered after reintroduction of oxygen and glucose. Pharmacological analysis showed that the OGD-induced [Ca(2+)](i) elevation in neuronal cell soma resulted from activation of both N-methyl-d-aspartate (NMDA)-glutamate receptors and Na(+)/Ca(2+) exchangers, while the abnormal [Na(+)](i) elevation during OGD was due to Na(+) influx through voltage-dependent Na(+) channels. In hippocampal slices, cellular degeneration occurring 24 h after OGD, selectively affected the pyramidal cell population through apoptotic and non-apoptotic cell death. OGD-induced cell loss was mediated by activation of ionotropic glutamate receptors, voltage-dependent Na(+) channels, and both plasma membrane and mitochondrial Na(+)/Ca(2+) exchangers. Thus, we show that neuroprotection induced by blockade of NMDA receptors and plasma membrane Na(+)/Ca(2+) exchangers is mediated by reduction of Ca(2+) entry into neuronal soma, whereas neuroprotection induced by blockade of AMPA/kainate receptors and mitochondrial Na(+)/Ca(2+) exchangers might result from reduced Na(+) entry at dendrites level.

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Boron Compounds; Calcium; Calcium Channel Blockers; Cell Death; Clonazepam; Dantrolene; Dizocilpine Maleate; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Fura-2; Glucose; Hippocampus; Hypoxia; In Situ Nick-End Labeling; Indoles; Intracellular Space; Ion Exchange; Lidocaine; Mibefradil; Nimodipine; Organ Culture Techniques; Quinoxalines; Rats; Rats, Wistar; Sodium; Sodium Channel Blockers; Thiazepines; Thiourea; Time Factors

In the heart, reperfusion following an ischaemic episode can result in a marked increase in [Ca2+]i and cause myocyte dysfunction and death. Although the Na(+)-Ca2+ exchanger has been implicated in this response, the ionic mechanisms that are responsible have not been identified. In this study, the hypothesis that the diastolic membrane potential can influence Na(+)-Ca2+ exchange and Ca2+ homeostasis during chemically induced hypoxia-reoxygenation has been tested using right ventricular myocytes isolated from adult rat hearts. Superfusion with selected [K+]o of 0.5, 2.5, 5, 7, 10 and 15 mM yielded the following resting membrane potentials: -27.6+/-1.63 mV, -102.2+/-1.89, -86.5+/-1.03, -80.1+/-1.25, -73.6+/-1.02 and -66.4+/-1.03, respectively. In a second set of experiments myocytes were subjected to chemically induced hypoxia-reoxygenation at these different [K+]o, while [Ca2+]i was monitored using fura-2. These results demonstrated that after chemically induced hypoxia-reoxygenation had caused a marked increase in [Ca2+]i, hyperpolarization of myocytes with 2.5 mM [K+]o significantly reduced [Ca2+]i (7.5+/-0.32 vs. 16.9+/-0.55%); while depolarization (with either 0.5 or 15 mM [K+]o) significantly increased [Ca2+]i (31.8+/-3.21 and 20.8+/-0.36 vs. 16.9+/-0.55%, respectively). As expected, at depolarized membrane potentials myocyte hypercontracture and death increased in parallel with Ca2+ overload. The involvement of the Na(+)-Ca2+ exchanger in Ca2+ homeostasis was evaluated using the Na(+)-Ca2+ exchanger inhibitor KB-R7943. During reoxygenation KB-R7943 (5 microM) almost completely prevented the increase in [Ca2+]i both in control conditions (in 5 mM [K+]o: 2.2+/-0.40 vs. 10.8+/-0.14%) and in depolarized myocytes (in 15 mM [K+]o: -2.1+/-0.51 vs. 11.3+/-0.05%). These findings demonstrate that the resting membrane potential of ventricular myocytes is a critical determinant of [Ca2+]i during hypoxia-reoxygenation. This appears to be due mainly to an effect of diastolic membrane potential on the Na(+)-Ca2+ exchanger, since at depolarized potentials this exchanger mechanism operates in the reverse mode, causing a significant Ca2+ influx.

Topics: Animals; Calcium; Calcium Channels, L-Type; Calcium Signaling; Electric Stimulation; Enzyme Inhibitors; Heart Arrest, Induced; Heart Ventricles; Hypoxia; In Vitro Techniques; Membrane Potentials; Models, Neurological; Models, Statistical; Muscle Cells; Oxygen Consumption; Rats; Sodium-Calcium Exchanger; Thiourea; Ventricular Function

The effects of a novel agent that is reported to selectively block Ca2+ influx by Na+/Ca2+ exchange (NCX), KB-R7943, on the reoxygenation-induced arrhythmias and the recovery of developed tension after reoxygenation, were investigated in guinea pig papillary muscles. KB-R7943 dose-dependently suppressed the contracture tension during low-sodium (21.9 mM) perfusion (23+/-8% of steady-state developed tension at 10 microM vs. 56+/-11% in control; n = 6, p<0.05), but did not change action potential and contractile parameters. During the reoxygenation period after 60-min substrate-free hypoxia, KB-R7943 (10 microM) significantly decreased the incidence of arrhythmias (44 vs. 100% in control; n = 9, p <0.05) and shortened the duration of arrhythmias (16+/-11 vs. 72+/-14 s; p<0.01). KB-R7943 (10 microM) significantly enhanced the recovery of developed tension after reoxygenation (83+/-4 vs. 69+/-3% in control; p<0.05). We conclude that KB-R7943 (10 microM) selectively inhibits the reverse mode of NCX, and that it attenuates reoxygenation-induced arrhythmic activity and prevents contractile dysfunction in guinea pig papillary muscles. These results suggest that Ca2+ influx by NCX may play a key role in reoxygenation injury.

Topics: Action Potentials; Animals; Arrhythmias, Cardiac; Calcium; Electrophysiology; Guinea Pigs; Heart; Hypoxia; In Vitro Techniques; Male; Myocardial Contraction; Myocardium; Oxygen Consumption; Papillary Muscles; Sodium-Calcium Exchanger; Thiourea

A prominent feature of cerebral ischemia is the excessive intracellular accumulation of both Na(+) and Ca(2+), which results in subsequent cell death. A large number of studies have focused on pathways involved in the increase of the intracellular Ca(2+) concentration [Ca(2+)](i), whereas the elevation of intracellular Na(+) has received less attention. In the present study we investigated the effects of inhibitors of different Na(+) channels and of the Na(+)/Ca(2+) exchanger, which couples the Na(+) to the Ca(2+) gradient, on ischemic damage in organotypic hippocampal slice cultures. The synaptically evoked population spike in the CA1 region was taken as a functional measure of neuronal integrity. Neuronal cell death was assessed by propidium iodide staining. The Na(+) channel blocker tetrodotoxin, and the NMDA receptor blocker MK 801, but not the AMPA/kainate receptor blocker NBQX prevented ischemic cell death. The novel Na(+)/Ca(2+) exchange inhibitor 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943), which preferentially acts on the reverse mode of the exchanger, leading to Ca(2+) accumulation, also reduced neuronal damage. At higher concentrations, KB-R7943 also inhibits Ca(2+) extrusion by the forward mode of the exchanger and exaggerates neuronal cell death. Neuroprotection by KB-R7943 may be due to reducing the [Ca(2+)](i) increase caused by the exchanger.

Topics: Animals; Brain Ischemia; Cell Death; Culture Techniques; Dizocilpine Maleate; Electrophysiology; Hippocampus; Homeostasis; Hypoglycemia; Hypoxia; Neurons; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Sodium; Sodium Channel Blockers; Sodium Channels; Sodium-Calcium Exchanger; Tetrodotoxin; Thiourea

Spinal cord injury is a devastating condition in which most of the clinical disability results from dysfunction of white matter tracts. Excessive cellular Ca(2+) accumulation is a common phenomenon after anoxia/ischemia or mechanical trauma to white matter, leading to irreversible injury because of overactivation of multiple Ca(2+)-dependent biochemical pathways. In the present study, we examined the role of Na(+)-Ca(2+) exchange, a ubiquitous Ca(2+) transport mechanism, in anoxic and traumatic injury to rat spinal dorsal columns in vitro. Excised tissue was maintained in a recording chamber at 37 degrees C and injured by exposure to an anoxic atmosphere for 60 min or locally compressed with a force of 2 g for 15 s. Mean compound action potential amplitude recovered to approximately 25% of control after anoxia and to approximately 30% after trauma. Inhibitors of Na(+)-Ca(2+) exchange (50 microM bepridil or 10 microM KB-R7943) improved functional recovery to approximately 60% after anoxia and approximately 70% after traumatic compression. These inhibitors also prevented the increase in calpain-mediated spectrin breakdown products induced by anoxia. We conclude that, at physiological temperature, reverse Na(+)-Ca(2+) exchange plays an important role in cellular Ca(2+) overload and irreversible damage after anoxic and traumatic injury to dorsal column white matter tracts.

Topics: Animals; Bepridil; Blotting, Western; Calcium Channel Blockers; Hypoxia; In Vitro Techniques; Male; Nerve Fibers; Rats; Rats, Long-Evans; Sodium-Calcium Exchanger; Spinal Cord; Spinal Cord Injuries; Temperature; Thiourea

The novel inhibitor of the reverse mode of the Na+/Ca2+ exchanger (NCE) KB-R7943 (KB) was tested in isolated rat cardiomyocytes exposed to 80 min of simulated ischemia [substrate-free anoxia, extracellular pH (pHo) of 6.4] and 15 min of reoxygenation (pHo 7.4). At pHo 6.4, 20 micromol/l KB was required for complete inhibition of the reverse mode of NCE. Treatment with 20 micromol/l KB only during anoxia did not influence the onset of rigor contracture and intracellular pH (pHi) (monitored with 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein) but significantly reduced the cytosolic accumulation of Ca2+ (monitored with fura 2) and Na+ (monitored with sodium-binding benzofuran isophthalate). During reoxygenation, cardiomyocytes developed hypercontracture. This was significantly reduced by anoxic KB treatment. To investigate this protection against reoxygenation-induced injury in the whole heart, we exposed Langendorff-perfused rat hearts to 110 min of anoxia (pHo 6.4) and 50 min of reoxygenation (pHo 7.4). Application of 20 micromol/l KB during anoxia significantly reduced the reoxygenation-induced enzyme release. We conclude that KB offers significant protection of cardiomyocytes against Ca2+ and Na+ overload during anoxia and hypercontracture or enzyme release on reoxygenation.

Topics: Animals; Calcium; Cytosol; Dose-Response Relationship, Drug; Heart; Hydrogen-Ion Concentration; Hypoxia; In Vitro Techniques; Magnesium; Male; Myocardial Contraction; Myocardial Ischemia; Myocardial Reperfusion Injury; Osmolar Concentration; Oxygen; Rats; Rats, Wistar; Sodium-Calcium Exchanger; Thiourea; Time Factors