Sodium-binding Benzofuran Isophthalate Research Articles

Lidocaine Increases Intracellular Sodium Concentration Through a Na+-H+ Exchanger in an Identified Lymnaea Neuron

The intracellular sodium concentration ([Na(+)]in) is related to neuron excitability. For [Na(+)]in, a Na(+)-H(+) exchanger plays an important role, which is affected by intracellular pH ([pH]in). However, the effect of lidocaine on [pH]in and a Na(+)-H(+) exchanger is unclear. We used neuron from Lymnaea stagnalis to determine how lidocaine affects [pH]in, Na(+)-H(+) exchanger, and [Na(+)]in. Intracellular sodium imaging by sodium-binding benzofuran isophthalate and intracellular pH imaging by 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein were used to measure [Na(+)]in and [pH]in. Measurements for [Na(+)]in were made in normal, Na(+) free saline, with modified extracellular pH, and a Na(+)-H(+) exchanger antagonist [(5-N-ethyl-N-isopropyl amiloride, N-methylisopropylamiloride, and 5-(N,N-hexamethylene)-amiloride) pretreatment trials. Furthermore, [Na(+)]in and [pH]in were recorded simultaneously. From 0.1 to 10 mM, lidocaine, mepivacaine, bupivacaine, prilocaine, and QX-314 were evaluated. Lidocaine, mepivacaine, and prilocaine increased the [Na(+)]in in a dose-dependent manner. In contrast, QX-314 did not change the [Na(+)]in at each dose. In the Na(+) free saline or in the presence of each Na(+)-H(+) exchanger antagonist, lidocaine failed to increase [Na(+)]in. Lidocaine, mepivacaine, and prilocaine induced a significant decrease in [pH]in below baseline with an increase in [Na(+)]in. In contrast, QX-314 did not change the [pH]in. These results demonstrated that lidocaine increases [Na(+)]in through Na(+)-H(+) exchanger activated by intracellular acidification, which is induced by the proton trapping of lidocaine. This [Na(+)]in increase and [pH]in change induces cell toxicity. Lidocaine increases the [Na(+)] through a Na(+)-H(+) exchanger by proton trapping.

Influence of Lipid-Soluble Gating Modifier Toxins on Sodium Influx in Neocortical Neurons

The electrical signals of neurons are fundamentally dependent on voltage-gated sodium channels (VGSCs), which are responsible for the rising phase of the action potential. An array of naturally occurring and synthetic neurotoxins have been identified that modify the gating properties of VGSCs. Using murine neocortical neurons in primary culture, we have compared the ability of VGSC gating modifiers to evoke Na+ influx. Intracellular sodium concentration ([Na+](i)) was monitored using the Na+-sensitive fluorescent dye, sodium-binding benzofuran isophthalate. All sodium channel gating modifier compounds tested produced a rapid and concentration-dependent elevation in neuronal [Na+](i). The increment in [Na+](i) exceeded 40 mM at high concentrations of brevetoxins, batrachotoxin, and the novel lipopeptide, antillatoxin. The maximal increments in neuronal [Na+](i) produced by neurotoxin site 2 alkaloids, veratridine and aconitine, and the pyrethroid deltamethrin were somewhat lower with maximal [Na+](i) increments of less than 40 mM. The rank order of efficacy of sodium channel gating modifiers was brevetoxin (PbTx)-1 > PbTx-desoxydioxolane > batrachotoxin > antillatoxin > PbTx-2 = PbTx-3 > PbTx-3alpha-naphthoate > veratridine > deltamethrin > aconitine > gambierol. These data demonstrate that the ability of sodium channel gating modifiers to act as partial agonists is shared by compounds acting at both neurotoxin sites 2 and 5. The concentration-dependent increases in [Na+](i) produced by PbTx-2, antillatoxin, veratridine, deltamethrin, aconitine, and gambierol were all abrogated by tetrodotoxin, indicating that VGSCs represent the sole pathway of Na+ entry after exposure to gating modifier neurotoxins.

K+-Channel Openers Suppress Epileptiform Activities Induced by 4-Aminopyridine in Cultured Rat Hippocampal Neurons

K+ channels are key modulators of neuronal excitability, and mutations in certain types of these channels are known to cause epileptic seizures. Activation of K+ channels is reported to suppress epileptic discharge; however, the types of K+-channel openers that are most effective as anti-epileptic agents are not well understood. We established a quantitative fluorescence assay using the Na+ indicator sodium-binding benzofuran isophthalate (SBFI) for evaluation of various compounds on epileptiform activities induced by 4-aminopyridine (4-AP) in cultured rat hippocampal neurons. Among the K+-channel openers, the KV7.2/KV7.3-channel openers retigabine and flupirtine and KCa2-channel openers NS309, DCEBIO, and 1-EBIO showed potent anti-epileptic effects similar to conventional antiepileptic drugs (AEDs). In contrast, the KCa1.1-channel openers NS1619, isopimaric acid, and chlorzoxazone demonstrated moderate inhibition. The Kir6-channel openers minoxidil, cromakalim, and pinacidil did not show anti-epileptic effects. We concluded that KV7.2/KV7.3, KCa2, and, to some extent, KCa1.1-channel openers, but not Kir6-channel openers, suppress 4-AP–induced epileptiform activities in hippocampal neurons. These results suggest that the K+-channel openers for this category of K+channels might have therapeutic potential as new classes of antiepileptic drugs.

High [Na+]iin cardiomyocytes from rainbow trout

Intracellular Na(+)-concentration, [Na(+)](i) modulates excitation-contraction coupling of cardiac myocytes via the Na(+)/Ca(2+) exchanger (NCX). In cardiomyocytes from rainbow trout (Oncorhyncus mykiss), whole cell patch-clamp studies have shown that Ca(2+) influx via reverse-mode NCX contributes significantly to contraction when [Na(+)](i) is 16 mM but not 10 mM. However, physiological [Na(+)](i) has never been measured. We recorded [Na(+)](i) using the fluorescent indicator sodium-binding benzofuran isophthalate in freshly isolated atrial and ventricular myocytes from rainbow trout. We examined [Na(+)](i) at rest and during increases in contraction frequency across three temperatures that span those trout experience in nature (7, 14, and 21 degrees C). Surprisingly, we found that [Na(+)](i) was not different between atrial and ventricular cells. Furthermore, acute temperature changes did not affect [Na(+)](i) in resting cells. Thus, we report a resting in vivo [Na(+)](i) of 13.4 mM for rainbow trout cardiomyocytes. [Na(+)](i) increased from rest with increases in contraction frequency by 3.2, 4.7, and 6.5% at 0.2, 0.5, and 0.8 Hz, respectively. This corresponds to an increase of 0.4, 0.6, and 0.9 mM at 0.2, 0.5, and 0.8 Hz, respectively. Acute temperature change did not significantly affect the contraction-induced increase in [Na(+)](i). Our results provide the first measurement of [Na(+)](i) in rainbow trout cardiomyocytes. This surprisingly high [Na(+)](i) is likely to result in physiologically significant Ca(2+) influx via reverse-mode NCX during excitation-contraction coupling. We calculate that this Ca(2+)-source will decrease with the action potential duration as temperature and contraction frequency increases.

Monensin causes transient calcium ion influx into mouse splenic lymphocytes in a sodium ion-independent fashion

Monensin, a Na + ionophore, can increase cytosolic free Ca 2+ concentration ([Ca 2+] i) in many cell types, but no studies have investigated the mechanism underlying a monensin-induced increase in [Ca 2+] i in immune cells. In view of this, we investigated the effect of monensin on [Ca 2+] i and cytosolic free Na + concentration ([Na +] i) in mouse splenic lymphocytes using a fluorescence Ca 2+ indicator, fura-2, and a fluorescence Na + indicator, sodium-binding benzofuran isophthalate (SBFI), respectively. Monensin (1–100 μM) caused transient and sustained increases in [Ca 2+] i and [Na +] i, respectively, in a concentration-dependent manner. The monensin-induced increase in [Ca 2+] i was abolished by the omission of extracellular Ca 2+ or 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF-96365, 100–150 μM), and was largely inhibited by Ni 2+ (2–5 mM). The omission of extracellular Na + failed to inhibit the monensin-induced increases in [Ca 2+] i. Furthermore, tetrodotoxin (1–10 μM), 5-( N, N-dimethyl)-amiloride (DMA, 10–20 μM), 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400, 3–10 μM), verapamil (10–200 μM), nifedipine (10–200 μM), ω-agatoxin IVA (0.2–10 μM), ω-conotoxin GVIA (1–10 μM), ω-conotoxin MVIIC (0.5–10 μM), and nordihydroguaiaretic acid (NDGA, 1–10 μM) had no effect on the increases in [Ca 2+] i. Monensin-induced Mn 2+ influx into splenic lymphocytes. The Mn 2+ influx was completely inhibited by SKF-96365. These results suggest that monensin transiently increases [Ca 2+] i in mouse splenic lymphocytes by stimulating Ca 2+ entry via non-selective cation channels in a Na +-independent manner.

Ca2+/calmodulin-dependent protein kinase II regulates cardiac Na+ channels

In heart failure (HF), Ca(2+)/calmodulin kinase II (CaMKII) expression is increased. Altered Na(+) channel gating is linked to and may promote ventricular tachyarrhythmias (VTs) in HF. Calmodulin regulates Na(+) channel gating, in part perhaps via CaMKII. We investigated effects of adenovirus-mediated (acute) and Tg (chronic) overexpression of cytosolic CaMKIIdelta(C) on Na(+) current (I(Na)) in rabbit and mouse ventricular myocytes, respectively (in whole-cell patch clamp). Both acute and chronic CaMKIIdelta(C) overexpression shifted voltage dependence of Na(+) channel availability by -6 mV (P < 0.05), and the shift was Ca(2+) dependent. CaMKII also enhanced intermediate inactivation and slowed recovery from inactivation (prevented by CaMKII inhibitors autocamtide 2-related inhibitory peptide [AIP] or KN93). CaMKIIdelta(C) markedly increased persistent (late) inward I(Na) and intracellular Na(+) concentration (as measured by the Na(+) indicator sodium-binding benzofuran isophthalate [SBFI]), which was prevented by CaMKII inhibition in the case of acute CaMKIIdelta(C) overexpression. CaMKII coimmunoprecipitates with and phosphorylates Na(+) channels. In vivo, transgenic CaMKIIdelta(C) overexpression prolonged QRS duration and repolarization (QT intervals), decreased effective refractory periods, and increased the propensity to develop VT. We conclude that CaMKII associates with and phosphorylates cardiac Na(+) channels. This alters I(Na) gating to reduce availability at high heart rate, while enhancing late I(Na) (which could prolong action potential duration). In mice, enhanced CaMKIIdelta(C) activity predisposed to VT. Thus, CaMKII-dependent regulation of Na(+) channel function may contribute to arrhythmogenesis in HF.

In situ fluorescence imaging of glutamate‐evoked mitochondrial Na+ responses in astrocytes

Astrocytes can experience large intracellular Na+ changes following the activation of the Na+-coupled glutamate transport. The present study investigated whether cytosolic Na+ changes are transmitted to mitochondria, which could therefore influence their function and contribute to the overall intracellular Na+ regulation. Mitochondrial Na+ (Na+(mit)) changes were monitored using the Na+-sensitive fluorescent probe CoroNa Red (CR) in intact primary cortical astrocytes, as opposed to the classical isolated mitochondria preparation. The mitochondrial localization and Na+ sensitivity of the dye were first verified and indicated that it can be safely used as a selective Na+(mit) indicator. We found by simultaneously monitoring cytosolic and mitochondrial Na+ using sodium-binding benzofuran isophthalate and CR, respectively, that glutamate-evoked cytosolic Na+ elevations are transmitted to mitochondria. The resting Na+(mit) concentration was estimated at 19.0 +/- 0.8 mM, reaching 30.1 +/- 1.2 mM during 200 microM glutamate application. Blockers of conductances potentially mediating Na+ entry (calcium uniporter, monovalent cation conductances, K+(ATP) channels) were not able to prevent the Na+(mit) response to glutamate. However, Ca2+ and its exchange with Na+ appear to play an important role in mediating mitochondrial Na+ entry as chelating intracellular Ca2+ with BAPTA or inhibiting Na+/Ca2+ exchanger with CGP-37157 diminished the Na+(mit) response. Moreover, intracellular Ca2+ increase achieved by photoactivation of caged Ca2+ also induced a Na+(mit) elevation. Inhibition of mitochondrial Na/H antiporter using ethylisopropyl-amiloride caused a steady increase in Na+(mit) without increasing cytosolic Na+, indicating that Na+ extrusion from mitochondria is mediated by these exchangers. Thus, mitochondria in intact astrocytes are equipped to efficiently sense cellular Na+ signals and to dynamically regulate their Na+ content.

Triphenyltin impairs a protein kinase A (PKA)-dependent increase of cytosolic Na+ and Ca2+ and PKA-independent increase of cytosolic Ca2+ associated with insulin secretion in hamster pancreatic β-cells

Oral administration of triphenyltin chloride (TPT) (60 mg/kg body weight) inhibits the insulin secretion by decreasing the cytoplasmic Ca2+ concentration ([Ca2+]i) induced by glucose-dependent insulinotropic polypeptide (GIP) in pancreatic beta-cells of the hamster. To test the possibility that the abnormal level of [Ca2+]i induced by TPT administration could be due to a defect in the cAMP-dependent cytoplasmic Na+ concentration ([Na+]i) in the beta-cells, we investigated the effects of TPT administration on the changes of [Na+]i induced by GIP, glucagon-like peptide-1 (GLP-1), or forskolin, an activator of adenylyl cyclase, and on the changes of [Na+]i or [Ca2+]i induced by 6-Bnz-cAMP, an activator of protein kinase A (PKA), and 8-pCPT-2'-O-Me-cAMP, an activator of Epac. The [Na+]i and [Ca2+]i were measured in islet cells loaded with sodium-binding benzofuran isophthalate (SBFI) and fura-2, respectively. In the presence of 135 mM Na+, TPT administration significantly reduced the rise in [Na+]i by 10 nM GLP-1, 10 microM forskolin, and 50 microM 6-Bnz-cAMP, but had not effect in a Na+-free medium. In the presence of 135 mM Na+, TPT administration also reduced the rise in [Ca2+]i by 8-pCPT-2'-O-Me-cAMP plus10 microM H-89, a inhibitor of PKA, and 6-Bnz-cAMP. Moreover, TPT administration significantly reduced the insulin secretion by 2 mM db-cAMP, GLP-1, GIP, and 8-pCPT-2'-O-Me-cAMP with and without H-89, and that by 6-Bnz-cAMP and forskolin. Our study suggested that TPT has inhibitory effects on the cellular Ca2+ response due to a reduced Na+ permeability through PKA-dependent mechanisms in hamster islet cells. Also TPT has the reduction of [Ca2+]i related to Na+-dependent insulin secretion after an activation of Epac.

Properties of the new fluorescent Na+ indicator CoroNa Green: Comparison with SBFI and confocal Na+ imaging

Neuronal activity causes substantial Na+ transients in fine cellular processes such as dendrites and spines. The physiological consequences of such Na+ transients are still largely unknown. High-resolution Na+ imaging is pivotal to study these questions, and, up to now, two-photon imaging with the fluorescent Na+ indicator sodium-binding benzofuran isophthalate (SBFI) has been the primary method of choice. Recently, a new Na+ indicator dye, CoroNa Green (CoroNa), that has its absorbance maximum at 492nm, has become available. In the present study, we have compared the properties of SBFI with those of CoroNa by performing Na+ measurements in neurons of hippocampal slices. We show that CoroNa is suitable for measurement of Na+ transients using non-confocal wide-field imaging with a CCD camera. However, substantial transmembrane dye leakage and lower Na+ sensitivity are clearly disadvantages when compared to SBFI. We also tested CoroNa for its suitability for high-resolution imaging of Na+ transients using a confocal laser scanning system. We demonstrate that CoroNa, in contrast to SBFI, can be employed for confocal imaging using a conventional argon laser and report the first Na+ measurements in dendrites using this dye. In conclusion, CoroNa may prove to be a valuable tool for confocal Na+ imaging in fine cellular processes.

Enhancement of Veratridine-Induced Sodium Dynamics in NG108-15 Cells during Differentiation

Developmental changes in dynamics of Na+ were studied in neuroblastomaxglioma hybrid NG108-15 cells during differentiation which was induced by dibutyryl cAMP (Bt2cAMP). Ratiometric Na+ imaging with a Na+-sensitive fluorescent dye SBFI (sodium-binding benzofuran isophthalate) revealed that the intracellular Na+ concentration ([Na+]i) was not affected by the application of high K+ (60 mM) solution to either control or differentiated cells. When cells were exposed to 50 microM veratridine (Vtd), an agonist of voltage-sensitive sodium channels (VSSCs), a significant increase in [Na+]i was observed in differentiated but not in undifferentiated cells. Calculated mean [Na+]i value increased from the basal 10.4 to 44.1 mM in response to 50 microM Vtd. This Vtd response was reversibly inhibited by tetrodotoxin (TTX), a specific blocker for VSSCs, in a dose-dependent manner (IC50 = 1 nM). It is suggested that VSSCs in NG108-15 cells are sensitive to TTX and Vtd and that the number of VSSCs increases during differentiation.

Intracellular and Extracellular Concentrations of Na+ Modulate Mg2+ Transport in Rat Ventricular Myocytes

Apparent free cytoplasmic concentrations of Mg2+ ([Mg2+]i) and Na+ ([Na+]i) were estimated in rat ventricular myocytes using fluorescent indicators, furaptra (mag-fura-2) for Mg2+ and sodium-binding benzofuran isophthalate for Na+, at 25°C in Ca2+-free conditions. Analysis included corrections for the influence of Na+ on furaptra fluorescence found in vitro and in vivo. The myocytes were loaded with Mg2+ in a solution containing 24mM Mg2+ either in the presence of 106mM Na+ plus 1mM ouabain (Na+ loading) or in the presence of only 1.6mM Na+ to deplete the cells of Na+ (Na+ depletion). The initial rate of decrease in [Mg2+]i from the Mg2+-loaded cells was estimated in the presence of 140mM Na+ and 1mM Mg2+ as an index of the rate of extracellular Na+-dependent Mg2+ efflux. Average [Na+]i, when estimated from sodium-binding benzofuran isophthalate fluorescence in separate experiments, increased from 12 to 31mM and 47mM after Na+ loading for 1 and 3h, respectively, and decreased to ∼0mM after 3h of Na+ depletion. The intracellular Na+ loading significantly reduced the initial rate of decrease in [Mg2+]i, on average, by 40% at 1h and by 64% at 3h, suggesting that the Mg2+ efflux was inhibited by intracellular Na+ with 50% inhibition at ∼40mM. A reduction of the rate of Mg2+ efflux was also observed when Na+ was introduced into the cells through the amphotericin B-perforated cell membrane (perforated patch-clamp technique) via a patch pipette that contained 130mM Na+. When the cells were heavily loaded with Na+ with ouabain in combination with intracellular perfusion from the patch pipette containing 130mM Na+, removal of extracellular Na+ caused an increase in [Mg2+]i, albeit at a very limited rate, which could be interpreted as reversal of the Mg2+ transport, i.e., Mg2+ influx driven by reversed Na+ gradient. Extracellular Na+ dependence of the rate of Mg2+ efflux revealed that the Mg2+ efflux was activated by extracellular Na+ with half-maximal activation at 55mM. These results contribute to a quantitative characterization of the Na+-Mg2+ exchange in cardiac myocytes.

RHO-ASSOCIATED KINASE MODULATES MYOCARDIAL INFLAMMATORY CYTOKINE RESPONSES

Rho, a Ser-Thr kinase identified as a member of the RAS GTPase super family, is highly expressed in the heart, and has been implicated in the development of heart failure. GTPase Rho is located downstream of Gq, and Rho and the associated kinase (Rho kinase) regulate myofibril organization, apoptosis, and myofibrillar sensitivity to calcium. Myocardial injury and dysfunction occur after major burn injury, and this phenomenon has been linked to cardiac myocyte synthesis and the secretion of proinflammatory cytokines. Whether Rho-associated kinase modulates any aspect of cardiomyocyte synthesis of inflammatory mediators, contributing to myocardial dysfunction, has not been studied and was the focus of this study. Hearts were collected at several times postburn to determine if an acute injury such as thermal trauma altered myocardial Rho kinase expression. In addition, cardiomyocytes were isolated (collagenase digestion) from adult control Sprague Dawley rats, plated (5 x 10 cells/microtiter well), incubated with medium alone or in the presence of burn serum (collected 24 h after burn over 40% total body surface area in rats) in a CO2 incubator at 37 degrees C in the presence/absence of specific Rho-kinase inhibitors (HA1077, 10 microM or Y27632, 10 microM). After 18 h, supernatants were collected to measure secreted cytokines (enzyme-linked immunoabsorbant assay), cells were loaded with Fura-2AM (2 microg) or sodium-binding benzofuran isophthalate (2 microg) for 45 min at 37 degrees C, and fluorescence was measured with an InCyt IM2 fluorescence imaging system to measure myocyte calcium and sodium. In parallel studies, cells were examined to determine if burn serum challenge increased Rho kinase in this cell population. In vivo burn injury or in vitro burn serum challenge of isolated myocytes increased Rho-kinase expression and promoted cardiomyocyte secretion of tumor necrosis factor-alpha, interleukin 1beta, and interleukin 6, and increased cardiomyocyte calcium and sodium levels compared with values measured when myocytes were incubated in medium alone (P < 0.05). Pretreating cardiomyocytes with Rho-kinase inhibitor (HA1077 or Y27632) prevented burn serum-related upregulation of Rho-kinase and attenuated the associated inflammatory cytokine responses, and attenuated myocyte calcium and sodium loading. Our data suggest that the Rho-kinase pathway is one potential upstream regulator of cardiac inflammatory response to burn injury.

Vinpocetine blockade of sodium channels inhibits the rise in sodium and calcium induced by 4-aminopyridine in synaptosomes

The objective of this study was to get a more understandable picture of the mechanism underlying the anticonvulsant action of vinpocetine. The question of how the cerebral excitability is affected was investigated by determining the effect of vinpocetine on the changes on the internal concentrations of Na + (Na i ) and Ca 2+ (Ca i ) induced by different concentrations of the convulsing agent 4-aminopyridine (4-AP) in striatal isolated nerve endings. The cytosolic concentrations of Na i and Ca i were detected fluorimetrically with sodium-binding benzofuran isophthalate (SBFI) and fura-2, respectively. Vinpocetine, like the Na + channel blocker, tetrodotoxin, abolished the increase in Na i induced by 0.1 mM 4-AP and only inhibited in 30% the rise in Na i induced by 1 mM 4-AP. In contrast with the different sensitivity of the rise in Na i induced by 0.1 and 1 mM 4-AP to vinpocetine and tetrodotoxin, the rise in Ca i induced by the two concentrations of 4-AP was markedly inhibited by vinpocetine (and tetrodotoxin), indicating that only the voltage-sensitive sodium channels (VSSC)-mediated fraction of the rise in Na i induced by 4-AP is linked with the activation of pre-synaptic Ca 2+ channels. The elevation of Ca 2+ induced by high K + (30 mM) does not require a Na + gradient and is vinpocetine and tetrodotoxin insensitive. In contrast, the elevation of Ca i induced by 4-AP, requires a physiological (out/in) Na + gradient and is vinpocetine and tetrodotoxin-sensitive. It is concluded that by blocking the tetrodotoxin-sensitive fraction of the rise in Na i induced by 4-AP, vinpocetine inhibits the concomitant rise in Ca i induced by 4-AP. The inhibitory effect of vinpocetine on pre-synaptic voltage-sensitive sodium channels may underlie the in vivo anticonvulsant action of vinpocetine.

Mechanical strain injury increases intracellular sodium and reverses Na+/Ca2+ exchange in cortical astrocytes

Traditionally, astrocytes have been considered less susceptible to injury than neurons. Yet, we have recently shown that astrocyte death precedes neuronal death in a rat model of traumatic brain injury (TBI) (Zhao et al.: Glia 44:140-152, 2003). A main mechanism hypothesized to contribute to cellular injury and death after TBI is elevated intracellular calcium ([Ca2+]i). Since calcium regulation is also influenced by regulation of intracellular sodium ([Na+]i), we used an in vitro model of strain-induced traumatic injury and live-cell fluorescent digital imaging to investigate alterations in [Na+]i in cortical astrocytes after injury. Changes in [Na+]i, or [Ca2+]i were monitored after mechanical injury or L-glutamate exposure by ratiometric imaging of sodium-binding benzofuran isophthalate (SBFI-AM), or Fura-2-AM, respectively. Mechanical strain injury or exogenous glutamate application produced increases in [Na+]i that were dependent on the severity of injury or concentration. Injury-induced increases in [Na+]i were significantly reduced, but not completely eliminated, by inhibition of glutamate uptake by DL-threo-beta-benzyloxyaspartate (TBOA). Blockade of sodium-dependent calcium influx through the sodium-calcium exchanger with 2-[2-[4-(4-Nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate (KB-R7943) reduced [Ca2+]i after injury. KB-R7943 also reduced astrocyte death after injury. These findings suggest that in astrocytes subjected to mechanical injury or glutamate excitotoxicity, increases in intracellular Na+ may be a critical component in the injury cascade and a therapeutic target for reduction of lasting deficits after traumatic brain injury.

Sodium Influx Pathways during and after Anoxia in Rat Hippocampal Neurons

Mechanisms that contribute to Na+ influx during and immediately after 5 min anoxia were investigated in cultured rat hippocampal neurons loaded with the Na+-sensitive fluorophore sodium-binding benzofuran isophthalate. During anoxia, an influx of Na+ in the face of reduced Na+,K+-ATPase activity caused a rise in [Na+]i. After the return to normoxia, Na+,K+-ATPase activity mediated the recovery of [Na+]i despite continued Na+ entry. Sodium influx during and after anoxia occurred through multiple pathways and increased the longer neurons were maintained in culture. Under the experimental conditions used, Na+ entry during anoxia did not reflect the activation of ionotropic glutamate receptors, TTX- or lidocaine-sensitive Na+ channels, plasmalemmal Na+/Ca2+ exchange, Na+/H+ exchange, or HCO3--dependent mechanisms; rather, contributions were received from a Gd3+-sensitive pathway activated by reactive oxygen species and Na+/K+/2Cl- cotransport in neurons maintained for 6-10 and 11-14 d in vitro (DIV), respectively. Sodium entry immediately after anoxia was not attributable to the activation of ionotropic glutamate receptors, voltage-activated Na+ channels, or Na+/K+/2Cl- cotransport; rather, it occurred via Na+/Ca2+ exchange, Na+/H+ exchange, and a Gd3+-sensitive pathway similar to that observed during anoxia; 11-14 DIV neurons received an additional contribution from an -dependent mechanism(s). The results provide insight into the intrinsic mechanisms that contribute to disturbed internal Na+ homeostasis during and immediately after anoxia in rat hippocampal neurons and, in this way, may play a role in the pathogenesis of anoxic or ischemic cell injury.

Burn trauma alters calcium transporter protein expression in the heart.

We have shown previously that burn trauma produces significant cardiac dysfunction, which is first evident 8 h postburn and is maximal 24 h postburn. Because calcium handling by the cardiomyocyte is essential for cardiac function, one mechanism by which burn injury may cause cardiac abnormalities is via calcium dyshomeostasis. We hypothesized that major burn injury alters cardiomyocyte calcium handling through changes in calcium transporter expression. Sprague-Dawley rats were given either burn injury or no burn injury (controls). Cardiomyocyte intracellular calcium and sodium were quantified at various times postburn by fura 2-AM or sodium-binding benzofuran isophthalate fluorescent indicators, respectively. In addition, hearts freeze-clamped at various times postburn (2, 4, 8, and 24 h) were used for Western blot analysis using antibodies against the sarcoplasmic reticulum calcium-ATPase (SERCA), the L-type calcium-channel, the ryanodine receptor, the sodium/calcium exchanger, or the sodium-potassium-ATPase. Intracellular calcium levels were elevated significantly 8-24 h postburn, and intracellular sodium was increased significantly 4 through 24 h postburn. Expression of SERCA was significantly reduced 1-8 h postburn, whereas L-type calcium-channel expression was diminished 1 and 2 h postburn (P < 0.05) but returned toward control levels 4 h postburn. Ryanodine receptor protein was significantly reduced at 1 and 2 h postburn, returning to baseline by 4 h postburn. Sodium/calcium exchanger expression was significantly elevated 2 h postburn but was significantly reduced 24 h postburn. An increase in sodium-potassium-ATPase expression occurred 2-24 h postburn. These data confirm that burn trauma alters calcium transporter expression, likely contributing to cardiomyocyte calcium loading and cardiac contractile dysfunction.

Mechanism of Collagen Activation in Human Platelets

The mechanism of collagen-induced human platelet activation was examined using Ca2+, Na+, and the pH-sensitive fluorescent dyes calcium green/fura red, sodium-binding benzofuran isophthalate, and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Administration of a moderate dose of collagen (10 microg/ml) to human platelets resulted in an increase in [Ca2+](i) and platelet aggregation. The majority of this increase in [Ca2+](i) resulted from the influx of calcium from the extracellular milieu via the Na+/Ca2+ exchanger (NCX) functioning in the reverse mode and was reduced in a dose-dependent manner by the NCX inhibitors 5-(4-chlorobenzyl)-2',4'-dimethylbenzamil (KD(50) = 4.7 +/- 1.1 microm) and KB-R7943 (KD(50) = 35.1 +/- 4.8 microm). Collagen-induced platelet aggregation was dependent on an increase in [Ca2+](i) and could be inhibited by chelation of intra- and extracellular calcium through the administration of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) and EGTA, respectively, or via the administration of BAPTA-AM to platelets suspended in no-Na+/HEPES buffer. Collagen induced an increase in [Ca2+](i) (23.2 +/- 7.6 mm) via the actions of thromboxane A(2) and, to a lesser extent, of the Na+/H+ exchanger. This study demonstrates that the collagen-induced increase in [Ca2+](i) is dependent on the concentration of Na+ in the extracellular milieu, indicating that the collagen-induced increase in [Ca2+](i) causes the reversal of the NCX, ultimately resulting in an increase in [Ca2+](i) and platelet aggregation.

Myocardial inflammatory responses to sepsis complicated by previous burn injury.

It is generally accepted that an initial injury such as burn trauma alters immune function such that a second insult increases the morbidity and mortality over that observed with each individual insult. We have shown previously that either burn trauma or sepsis promotes cardiomyocyte secretion of TNF-alpha and IL-1beta, cytokines that have been shown to produce myocardial contractile dysfunction. This study determined whether a previous burn injury (given eight days prior to sepsis) (1) provides a preconditioning phenomenon, decreasing inflammatory responses to a second insult or (2) exacerbates inflammatory response observed with either injury alone. Anesthetized Sprague-Dawley rats were given either burn injury over 40% total body surface area, sepsis alone (intratracheal S. pneumoniae, 4 x 10(6) colony forming units) or sepsis eight days after burn; all rats received lactated Ringer's solution. Hearts harvested 24 h after onset of sepsis alone or sepsis plus eight-day burn were used to (1) isolate cardiomyocytes (collagenase) or (2) assess contractile function (Langendorff). Cardiomyocytes loaded with 2 microg/mL Fura-2AM or sodium-binding benzofuran isophthalate were used to measure intracellular calcium and sodium concentrations (Nikon inverted microscope, Grooney optics, InCyt Im2 Fluorescence Imaging System). Additional cardiomyocytes were used to measure myocyte-secreted TNFalpha, IL-1, IL-6, IL-10 (pg/ml, ELISA). Either burn trauma alone or sepsis alone promoted TNF-alpha, IL-1beta, nitric oxide, IL6 and IL-10 secretion by cardiomyocytes (p < 0.05). Producing aspiration-related pneumonia eight days postburn produced myocardial pro- and anti-inflammatory responses and increased myocyte Ca2+/Na+ concentrations to a significantly greater degree than the responses observed after either insult alone. A previous burn injury alters myocardial inflammatory responses, predisposing the burn-injured subject to exaggerated inflammation, which correlates with greater myocardial dysfunction.

Glucagon-like peptide-1 induces a cAMP-dependent increase of [Na+]i associated with insulin secretion in pancreatic beta-cells.

Glucagon-like peptide-1 (GLP-1) elevates the intracellular free calcium concentration ([Ca2+]i) and insulin secretion in a Na+-dependent manner. To investigate a possible role of Na ion in the action of GLP-1 on pancreatic islet cells, we measured the glucose-and GLP-1-induced intracellular Na+ concentration ([Na+]i), [Ca2+]i, and insulin secretion in hamster islet cells in various concentrations of Na+. The [Na+]i and [Ca2+]i were monitored in islet cells loaded with sodium-binding benzofuran isophthalate and fura 2, respectively. In the presence of 135 mM Na+ and 8 mM glucose, GLP-1 (10 nM) strongly increased the [Na+]i, [Ca2+]i, and insulin secretion. In the presence of 13.5 mM Na+, both glucose and GLP-1 increased neither the [Na+]i nor the [Ca2+]i. In a Na+-free medium, GLP-1 and glucose did not increase the [Na+]i. SQ-22536, an inhibitor of adenylate cyclase, and H-89, an inhibitor of PKA, incompletely inhibited the response. In the presence of both 8 mM glucose and H-89, 8-pCPT-2'-O-Me-cAMP, a PKA-independent cAMP analog, increased the insulin secretion and the [Na+]i. Therefore, we conclude that GLP-1 increases the cAMP level via activation of adenylate cyclase, which augments the membrane Na+ permeability through PKA-dependent and PKA-independent mechanisms, thereby increasing the [Ca2+]i and promoting insulin secretion from hamster islet cells.

Neuronal nitric oxide synthase: its role and regulation in macula densa cells.

Macula densa (MD) cells detect changes in distal tubular sodium chloride concentration ([NaCl](L)), at least in part, through an apical Na:2Cl:K co-transporter. This co-transporter may be a site for regulation of tubuloglomerular feedback (TGF), and recently angiotensin II (Ang II) was shown to regulate the MD Na:2Cl:K co-transporter. In addition, nitric oxide (NO) produced via neuronal NO synthase (nNOS) in MD cells attenuates MD-TGF signaling. This study investigated [NaCl](L)-dependent MD-NO production, the regulation of co-transporter activity by NO, and the possible interaction of NO with Ang II. MD cell Na(+) concentration ([Na(+)](i)) and NO production were measured using sodium-binding benzofuran isophthalate and 4-amino-5-methylamino-2',7'-difluorescein diacetate, respectively, using fluorescence microscopy. Na:2Cl:K co-transport activity was assessed as the initial rate of increase in [Na(+)](i) when [NaCl](L) was elevated from 25 to 150 mM. 10(-4) M 7-nitroindazole, a specific nNOS blocker, significantly increased by twofold the initial rate of rise in [Na(+)](i) when [NaCl](L) was increased from 25 to 150 mM, indicating co-transporter stimulation. There was no evidence for an interaction between the stimulatory effect of Ang II and the inhibitory effect of NO on co-transport activity, and, furthermore, Ang II failed to alter MD-NO production. NO production was sensitive to [NaCl](L) but increased only when [NaCl](L) was elevated from 60 to 150 mM. These studies indicate that MD-NO directly inhibits Na:2Cl:K co-transport and that NO and Ang II independently alter co-transporter activity. In addition, generation of MD-NO seems to occur only at markedly elevated [NaCl](L), suggesting that NO may serve as a buffer against high rates of MD cell transport and excessive TGF-mediated vasoconstriction.