Sodium-binding Benzofuran Isophthalate Research Articles

Early sodium elevations induced by combined oxygen and glucose deprivation in pyramidal cortical neurons.

We investigated the effects of oxygen (O2)/glucose deprivation on intracellular sodium concentration ([Na+]i) of cortical pyramidal cells in a slice preparation of rat frontal cortex. Intracellular recordings were combined with microfluorometric measurements of [Na+]i using the Na+-sensitive dye sodium-binding benzofuran isophthalate (SBFI). Deprivation of O2/glucose caused an initial membrane hyperpolarization that was followed by a slowly developing large depolarization. Levels of [Na+]i started to increase significantly during the phase of membrane hyperpolarization. Neither tetrodotoxin, a combination of ionotropic and metabotropic glutamate receptor antagonists (D-amino-phosphonovalerate, 6-cyano-7-nitroquinoxaline-2,3-dione plus S-methyl-4-carboxyphenylglycine) nor bepridil, an inhibitor of the Na+/Ca2+-exchanger, affected these responses to O2/ glucose. The present results demonstrate that, in cortical neurons, O2/glucose deprivation induces an early rise in [Na+]i which cannot be ascribed to the activity of voltage gated Na+-channels, glutamate receptors or of the Na+/Ca2+-exchanger.

Abnormal myocyte Ca2+ homeostasis in rabbits with pacing-induced heart failure.

To determine whether there are abnormalities in myocyte excitation-contraction coupling and intracellular Ca2+ concentration ([Ca2+]i) homeostasis in pacing-induced heart failure (PF), we measured L-type Ca2+ current (ICa,L) and Na+/Ca2+ exchanger current (INa/Ca) with voltage clamp and measured intracellular Na+ concentration ([Na+]i) and [Ca2+]i with the use of sodium-binding benzofuran isophthalate (SBFI) and fluo 3 in ventricular myocytes isolated from control and paced rabbits. The peak systolic and diastolic levels and the amplitude of electrically stimulated [Ca2+]i transients (0.25 Hz, extracellular Ca2+ concentration = 1.08 mM) were significantly less in PF myocytes. Also, there was prolongation of the times to peak and decline of [Ca2+]i transients. ICa,L density was markedly decreased in PF myocytes. INa/Ca at -40 mV elicited by rapid exposure to 0 Na+ solution with a rapid solution switcher was significantly reduced in PF myocytes, suggesting that the function of the Na+/Ca2+ exchanger is impaired in these myocytes. In PF myocytes the decline of the [Ca2+]i transient when the Na+/Ca2+ exchanger was abruptly disabled was markedly prolonged compared with the decline in control myocytes, consistent with depressed sarcoplasmic reticulum (SR) Ca2+-ATPase function. RNase protection assay showed decreased levels of Na+/Ca2+ exchanger and SR Ca2+-ATPase mRNA in PF hearts, consistent with the function studies. We conclude that the functions of L-type Ca2+ channels, Na+/Ca2+ exchanger, and SR Ca2+-ATPase are impaired in myocytes from rabbit hearts with failure induced by rapid pacing. These abnormalities result in reduced [Ca2+]i transients and systolic and diastolic dysfunction and appear to account for the abnormal ventricular function observed.

Sodium-sensitive, calcium-independent potassium conductance in the leech giant glial cell.

We have measured membrane current, membrane potential and intracellular Na+ and Ca2+ concentrations, [Na+]i and [Ca2+]i, of the giant glial cell in the nervous system of the leech Hirudo medicinalis using conventional microelectrodes and the fluorescent dyes sodium-binding benzofuran isophthalate (SBFI) and fura-2. When the Na+ was removed from the saline, the membrane conductance increased twofold from 1.29±0.1 µS to 2.57±0.18 µS (mean ± SEM; n=27). The rise in membrane conductance was accompanied by a current, which reversed around –74 mV, and the amplitude of K+-induced depolarizations or currents increased during Na+ removal, suggesting an increase in the K+ conductance of the glial membrane. We also monitored [Ca2+]i when removing external Na+ in the presence and absence of external Ca2+, and during injection of the Ca2+-chelator BAPTA into the cells. Our results indicate that Na+ modulates a K+ conductance of these glial cells, independent of intra- and extracellular Ca2+.

Effects of Glucose Deprivation, Chemical Hypoxia, and Simulated Ischemia on Na+Homeostasis in Rat Spinal Cord Astrocytes

A steep inwardly directed Na+ gradient is essential for glial functions such as glutamate reuptake and regulation of intracellular ion concentrations. We investigated the effects of glucose deprivation, chemical hypoxia, and simulated ischemia on intracellular Na+ concentration ([Na+]i) in cultured spinal cord astrocytes using fluorescence ratio imaging with sodium-binding benzofuran isophthalate (SBFI) AM. Glucose removal or chemical hypoxia (induced by 10 mM NaN3) for 60 min increased [Na+]i from a baseline of 8.3 to 11 mM. Combined glycolytic and respiratory blockage by NaN3 and 0 glucose saline caused [Na+]i to increase by 20 mM, similar to the [Na+]i increases elicited by blocking the Na+/K+-ATPase with ouabain. Recovery from large [Na+]i increases (>15 mM) induced by the glutamatergic agonist kainate was attenuated during glucose deprivation or NaN3 application and was blocked in NaN3 and 0 glucose. To mimic in vivo ischemia, we exposed astrocytes to NaN3 and 0 glucose saline containing L-lactate and glutamate with increased [K+] and decreased [Na+], [Ca2+], and pH. This induced an [Na+]i decrease followed by an [Na+]i rise and a further [Na+]i increase after reperfusion with standard saline. Similar multiphasic [Na+]i changes were observed after NaN3 and 0 glucose saline with only reduced [Na+]e. Our results suggest that the ability to maintain a low [Na+]i enables spinal cord astrocytes to continue uptake of K+ and/or glutamate at the onset of energy failure. With prolonged energy failure, however, astrocytic [Na+]i rises; with loss of their steep transmembrane Na+ gradient, astrocytes may aggravate metabolic insults by carrier reversal and release of acid, K+, and/or glutamate into the extracellular space.

Hyperpolarization induces a rise in intracellular sodium concentration in dopamine cells of the substantia nigra pars compacta.

We investigated the effect of changes in membrane-voltage on intracellular sodium concentration ([Na+]i) of dopamine-sensitive neurons of the substantia nigra pars compacta in a slice preparation of rat mesencephalon. Whole-cell patch-clamp techniques were combined with microfluorometric measurements of [Na+]i using the Na+-sensitive probe, sodium-binding benzofuran isophthalate (SBFI). Hyperpolarization of spontaneously active dopamine neurons (recorded in current-clamp mode) caused the cessation of action potential firing accompanied by an elevation in [Na+]i. In dopamine neurons voltage-clamped at a holding potential of -60 mV elevations of [Na+]i were induced by long-lasting (45-60 s) voltage jumps to more negative membrane potentials (-90 to -120 mV) but not by corresponding voltage jumps to -30 mV. These hyperpolarization-induced elevations of [Na+]i were depressed during inhibition of I(h), a hyperpolarization-activated inward current, by Cs+. Hyperpolarization-induced elevations in [Na+]i might occur also in other cell types which express a powerful I(h) and might signal lack of postsynaptic activity.

Changes in intracellular Na + concentration evoked by nicotinic receptor activation in the guinea-pig adrenal chromaffin cells

Using the whole-cell voltage clamp technique and microfluorometry with sodium-binding benzofuran isophthalate (SBFI), a nicotine-induced inward current and increase in the intracellular Na + concentration ([Na +] in) were examined simultaneously in guinea-pig adrenal chromaffin cells. The increase in [Na +] in expected from the time-integrated inward current was well correlated with that of [Na +] in measured with SBFI. The ratio of the expected [Na +] in to the measured [Na +] in was 0.64 at −85mV and decreased with increasing holding potentials. The decay time constant of the increased [Na +] in was not affected by ouabain. It is concluded that the Na + entering the cell is diffusable in about 60% cell volume without fast buffering mechanisms and is eliminated by the exchange of Na + between the pipette solution and cell interior under the patch clamp condition.

Na+ gradient-dependent Mg2+ transport in smooth muscle cells of guinea pig tenia cecum

Thin strips of guinea pig tenia cecum were loaded with the Mg2+ indicator furaptra, and the indicator fluorescence signals measured in Ca2+-free condition were converted to cytoplasmic-free Mg2+ concentration ([Mg2+]i). Lowering the extracellular Na+ concentration ([Na+]o) caused a reversible increase in [Mg2+]i, consistent with the inhibition of Na+ gradient-dependent extrusion of cellular Mg2+ (Na+-Mg2+ exchange). Curve-fitting analysis indicated that the relation between [Na+]o and the rate of rise in [Mg2+], had a Hill coefficient of approximately 3, a [Na+]o at the half-maximal rate of rise of approximately 30 mM, and a maximal rate of 0.16 +/- 0.01 microM/s (mean +/- SE, n = 6). Depolarization with 56 mM K+ shifted the curve slightly toward higher [Na+]o without significantly changing the maximal rate, suggesting that the Na+-Mg2+ exchange was inhibited by depolarization. The maximal rate would correspond to a flux of 0.15-0.4 pmol/cm2/s, if cytoplasmic Mg2+ buffering power (defined as the ratio of the changes in total Mg2+ and free Mg2+ concentrations) is assumed to be 2-5. Ouabain (1-5 microM) increased the intracellular Na+ concentration, as assessed with fluorescence of SBFI (sodium-binding benzofuran isophthalate, a Na+ indicator), and elevated [Mg2+]i. In ouabain-treated preparations, removal of extracellular Na+ rapidly increased [Mg2+]i, with an initial rate of rise roughly proportional to the degree of the Mg2+ load, and, probably, to the Na+ load caused by ouabain. The enhanced rate of rise in [Mg2+]i (up to approximately 1 microM/s) could be attributed to the Mg2+ influx as a result of the reversed Na+-Mg2+ exchange. Our results support the presence of a reversible and possibly electrogenic Na+-Mg2+ exchange in the smooth muscle cells of tenia cecum.

Pharmacological characterization of Na+ influx via voltage-gated Na+ channels in spinal cord astrocytes.

Spinal cord astrocytes display a high density of voltage-gated Na+ channels. To study the contribution of Na+ influx via these channels to Na+ homeostasis in cultured spinal cord astrocytes, we measured intracellular Na+ concentration ([Na+]i) with the fluorescent dye sodium-binding benzofuran isophthalate. Stellate and nonstellate astrocytes, which display Na+ currents with different properties, were differentiated. Baseline [Na+]i was 8.5 mM in these cells and was not altered by 100 microM tetrodotoxin (TTX). Inhibition of Na+ channel inactivation by veratridine (100 microM) evoked a [Na+]i increase of 47.1 mM in 44% of stellate and 9.7 mM in 64% of nonstellate astrocytes. About 30% of cells reacted to veratridine with a [Na+]i decrease of approximately 2 mM. Qualitatively similar [Na+]i changes were caused by aconitine. The effects of veratridine were blocked by TTX, amplified by (alpha-)scorpion toxin and usually were readily reversible. Veratridine-induced [Na+]i increases were reduced upon membrane depolarization with elevated extracellular [K+]. Recovery to baseline [Na+]i was unaltered during blocking of K+ channels with 4-aminopyridine. [Na+]i increases evoked by the ionotropic non-N-methyl--aspartate receptor agonist kainate were not altered by TTX. Our results indicate that influx of Na+ via voltage- gated Na+ channels is not a prerequisite for glial Na+,K+-ATPase activity in spinal cord astrocytes at rest nor does it seem to be involved in [Na+]i increases evoked by kainate. During pharmacological inhibition of Na+ channel inactivation, however, Na+ channels can serve as prominent pathways of Na+ influx and mediate large perturbations in [Na+]i, suggesting that Na+ channel inactivation plays an important functional role in these cells.

Influence of stimulation frequency on [Na+]i and contractile function in Langendorff-perfused rat heart.

To study the relationship between stimulation frequency and intracellular Na+ concentration ([Na+]i), Langendorff-perfused rat hearts were loaded with the Na(+)-sensitive dye sodium-binding benzofuran isophthalate (SBFI). An intracellular calibration procedure allowed SBFI fluorescence to be transformed into [Na+]i. Compartmentation of SBFI was evaluated by permeabilizing sarcolemmal membranes with saponin and subcellular compartments with Triton X-100. Most of the indicator was located in the myoplasm (69%). When stimulation frequency was increased from 0 to 6 Hz, [Na+]i increased from 3.0 to 7.9 mM, whereas pressure amplitude (PA) declined to 49% of the maximum recorded at 2 Hz. Blocking sarcoplasmic reticulum (SR) Ca2+ uptake with 2,5-di(tert-butyl)-1,4-benzohydroquinone (TBQ; 10 microM) at 2 Hz increased [Na+]i from 4.9 to 8.4 mM and decreased PA by 70%. Raising stimulation frequency then resulted in a further increase in [Na+]i and decline in PA. In conclusion, these data indicate that the rat heart is characterized by a negative pressure-frequency relationship associated with increasing [Na+]i at higher heart rates. After inhibition of SR Ca2+ uptake, [Na+]i further increases, whereas PA declines with increasing stimulus frequency. It is suggested that part of the rise of [Na+]i with stimulus frequency and TBQ may be associated with increased Ca2+ extrusion and Na+ influx on the Na+/Ca2+ exchange system.

Gap junctions equalize intracellular Na+ concentration in astrocytes.

Gap junctions between glial cells allow intercellular exchange of ions and small molecules. We have investigated the influence of gap junction coupling on regulation of intracellular Na+ concentration ([Na+]i) in cultured rat hippocampal astrocytes, using fluorescence ratio imaging with the Na+ indicator dye SBFI (sodium-binding benzofuran isophthalate). The [Na+]i in neighboring astrocytes was very similar (12.0 +/- 3.3 mM) and did not fluctuate under resting conditions. During uncoupling of gap junctions with octanol (0.5 mM), baseline [Na+]i was unaltered in 24%, increased in 54%, and decreased in 22% of cells. Qualitatively similar results were obtained with two other uncoupling agents, heptanol and alpha-glycyrrhetinic acid (AGA). Octanol did not alter the recovery from intracellular Na+ load induced by removal of extracellular K+, indicating that octanol's effects on baseline [Na+]i were not due to inhibition of Na+, K+-ATPase activity. Under control conditions, increasing [K+]o from 3 to 8 mM caused similar decreases in [Na+]i in groups of astrocytes, presumably by stimulating Na+, K+-ATPase. During octanol application, [K+]o-induced [Na+]i decreases were amplified in cells with increased baseline [Na+]i, and reduced in cells with decreased baseline [Na+]i. This suggests that baseline [Na+]i in astrocytes "sets" the responsiveness of Na+, K+-ATPase to increases in [K]o. Our results indicate that individual hippocampal astrocytes in culture rapidly develop different levels of baseline [Na+]i when they are isolated from one another by uncoupling agents. In astrocytes, therefore, an apparent function of coupling is the intercellular exchange of Na+ ions to equalize baseline [Na+]i, which serves to coordinate physiological responses that depend on the intracellular concentration of this ion.

Sodium kinetics of Na,K-ATPase alpha isoforms in intact transfected HeLa cells.

By participating in the regulation of ion and voltage gradients, the Na-K pump (i.e., Na,K-ATPase) influences many aspects of cellular physiology. Of the four alpha isoforms of the pump, alpha1 is ubiquitous, alpha2 is predominant in skeletal muscle, and alpha3 is found in neurons and the cardiac conduction system. To determine whether the isoforms have different intracellular Na+ affinities, we used the Na+-sensitive dye sodium-binding benzofuran isophthalate (SBFI) to measure pump-mediated Na+ efflux as a function of [Na+]i in human HeLa cells stably transfected with rat Na-K pump isoforms. We Na+-loaded the cells, and then monitored the time course of the decrease in [Na+]i after removing external Na+. All transfected rat alpha subunits were highly ouabain resistant: the alpha1 isoform is naturally resistant, whereas the alpha2 and alpha3 isoforms had been mutagenized to render them resistant. Thus, the Na+ efflux mediated by endogenous and transfected pumps could be separated by studying the cells at low (1 microM) and high (4 mM) ouabain concentrations. We found that the apparent Km for Na+ efflux attributable to the native human alpha1 isoform was 12 mM, which was similar to the Km of rat alpha1. The alpha2 and alpha3 isoforms had apparent Km's of 22 and 33 mM, respectively. The cells expressing alpha3 had a high resting [Na+]i. The maximal activity of native alpha1 in the alpha3-transfected cells was only approximately 56% of native alpha1 activity in untransfected HeLa cells, suggesting that transfection with alpha3 led to a compensatory decrease in endogenous alpha1 pumps. We conclude that the apparent Km(Na+) for rat Na-K pump isoforms increases in the sequence alpha1 < alpha2 < alpha3. The alpha3 isoform may be suited for handling large Na+ loads in electrically active cells.

Measurement of intracellular Na + concentration by a Na +-sensitive fluorescent dye, sodium-binding benzofuran isophthalate, in porcine adrenal chromaffin cells—usage of palytoxin as a Na + ionophore

Palytoxin was found to equilibrate sodium ions (Na +) across the cell membrane much faster than dose gramicidin, which has been frequently used to calibrate the intracellular Na + concentration ([Na +]in), in cells treated with a Na +-sensitive fluorescent dye, sodium-binding benzofuran isophthalate (SBFI). Palytoxin was capable of equilibrating Na + in cells treated with SBFI-acetoxymethyl ester (SBFI-AM) and in voltage-clamped cells loaded with SBFI through a patch pipette. Nicotine caused a dose-dependent increase in [Na +]in in porcine adrenal chromaffin cells treated with SBFI-AM and caused a simultaneous increase in [Na +]in and inward current in the voltage-clamped cells loaded with SBFI. Palytoxin has an advantage of calibrating [Na +]in in a shorter time than dose gramicidin because of its powerful ionophoretic activity.

Veratridine delays apoptotic neuronal death induced by NGF deprivation through a Na+-dependent mechanism in cultured rat sympathetic neurons

Superior-cervical ganglion (SCG) cells dissociated from newborn rats depend on nerve growth factor (NGF) for survival. Membrane depolarization with elevated K+ is known to prevent neuronal death following NGF deprivation and/or to promote survival via a Ca2+-dependent mechanism. Here we have exploited the possibility of whether or not a Na+-dependent pathway for neuronal survival is present in these cells. Veratridine (ec50=40 nM), a voltage-dependent Na+ channel activator, significantly delayed the onset of apoptotic cell death in NGF-deprived SCG neurons that had been cultured for 7 days in the presence of NGF. This effect was blocked completely by Na+ channel blockers including tetrodotoxin (TTX, 1 μM), benzamil (25 μM) and flunarizine (1 μM), but was not attenuated by nimodipine (1 μM), an L-type Ca2+ channel blocker. The saving effect of veratridine on cultured neurons was observed even in low Ca2+ media (0–1.0 mM), but was completely abolished in a low Na+ medium (38 mM). Sodium-binding benzofuran isophthalate was employed as a fluorescent probe for monitoring the level of cytoplasmic free Na+, which revealed a sustained increase in its level (12.9 mM, 307% of that of control) in response to veratridine (0.75 μM). The TTX or flunarizine completely blocked veratridine-induced Na+ influx in these cultured neurons. Moreover, no appreciable increase in intracellular Ca2+ was detected under these conditions. Though Na+ channels were effectual in SCG neurons which were freshly isolated from newborn rats, the Na+-dependent saving effect of veratridine was not observed in these young neurons. These lines of evidence suggest that the death-suppressing effect of veratridine on cultured SCG neurons depends on the Na+ influx via voltage-dependent Na+ channels, and suggests the presence of Na+-dependent regulatory mechanism(s) in neuronal survival.

Regulation of intracellular sodium in cultured rat hippocampal neurones.

1. We studied regulation of intracellular Na+ concentration ([Na+]i) in cultured rat hippocampal neurones using fluorescence ratio imaging of the Na+ indicator dye SBFI (sodium-binding benzofuran isophthalate). 2. In standard CO2/HCO3(-)-buffered saline with 3 mM K+, neurones had a baseline [Na+]i of 8.9 +/- 3.8 mM (mean +/- S.D.). Spontaneous, transient [Na+]i increases of 5 mM were observed in neurones on 27% of the coverslips studied. These [Na+]i increases were often synchronized among nearby neurones and were blocked reversibly by 1 microM tetrodotoxin (TTX) or by saline containing 10 mM Mg2+, suggesting that they were caused by periodic bursting activity of synaptically coupled cells. Opening of voltage-gated Na+ channels by application of 50 microM veratridine caused a TTX-sensitive [Na+]i increase of 25 mM. 3. Removing extracellular Na+ caused an exponential decline in [Na+]i to values close to zero within 10 min. Inhibition of Na+,K(+)-ATPase by removal of extracellular K+ or ouabain application evoked a [Na+]i increase of 5 mM min-1. Baseline [Na+]i was similar in the presence or absence of CO2/HCO3-; switching from CO2/HCO3(-)-free to CO2/HCO3(-)-buffered saline, however, increased [Na+]i transiently by 3 mM, indicating activation of Na(+)-dependent Cl(-)-HCO3- exchange. Inhibition of Na(+)-K(+)-2Cl- cotransport by bumetanide had no effect on [Na+]i. 4. Brief, small changes in extracellular K+ concentration ([K+]o) influenced neuronal [Na+]i only weakly. Virtually no change in [Na+]i was observed with elevation or reduction of [K+]o by 1 mM. Only 30% of cells reacted to 3 min [K+]o elevations of up to 5 mM. In contrast, long [K+]o alterations (> or = 10 min) to 6 mM or greater slowly changed steady-state [Na+]i in the majority of cells. 5. Our results indicate several differences between [Na+]i regulation in cultured hippocampal neurones and astrocytes. Baseline [Na+]i is lower in neurones compared with astrocytes and is mainly determined by Na+,K(+)-ATPase, whereas Na(+)-dependent Cl(-)-HCO3- exchange, Na(+)-HCO3- cotransport or Na(+)-K(+)-2Cl- cotransport do not play a significant role. In contrast to glial cells, [Na+]i of neurones changes only weakly with small alterations in bath [K+]o, suggesting that activity-induced [K+]o changes in the brain might not significantly influence neuronal Na+,K(+)-ATPase activity.

Spatiotemporal gradients of intra-axonal [Na+] after transection and resealing in lizard peripheral myelinated axons.

1. Post-transection changes in intracellular Na+ ([Na+]i) were measured in lizard peripheral axons ionophoretically injected with the Na(+)-sensitive ratiometric dye, sodium-binding benzofuran isophthalate (SBFI). 2. Following axonal transection in physiological saline [Na+]i increased to more than 100 mM in a region that quickly extended hundreds of micrometers from the transection site. This post-transection increase in [Na+]i was similar when the bath contained 5 microM tetrodotoxin, but was absent in Na(+)-free solution. Depolarization of uncut axons in 50 mM K+ produced little or no elevation of [Na+]i until veratridine was added. These results suggest that the post-transection increase in [Na+]i was due mainly to Na+ entry via the cut end, rather than via depolarization-activated Na+ channels. 3. The spatiotemporal profile of the post-transection increase in [Na+]i could be accounted for by movement of Na+ from the cut end with an apparent diffusion coefficient of 1.3 x 10(-5) cm2 s-1. 4. [Na+]i began to decline toward resting levels by 20 +/- 15 min (mean +/- S.D.) post-transection, except in regions of the axon within 160 +/- 85 microns of the transection site, where [Na+]i remained high. The boundary between axonal regions in which [Na+]i did or did not recover probably defines a locus of resealing of the axonal membrane. 5. [Na+]i returned to resting values within about 1 h after resealing, even in axonal regions where the normal transmembrane [Na+] gradient had completely dissipated. The recovery of [Na+]i was faster and reached lower levels than expected by diffusional redistribution of Na+ along the axon. Partial recovery occurred even in an isolated internode, indicating that the internodal axolemma can actively extrude Na+.

Spatial distribution of synaptically activated sodium concentration changes in cerebellar Purkinje neurons.

The spatial distribution of Na(+)-dependent events in guinea pig Purkinje cells was studied with a combination of high-speed imaging and simultaneous intracellular recording. Individual Purkinje cells in sagittal cerebellar slices were loaded with either fura-2 or the Na+ indicator sodium binding benzofuran isophthalate (SBFI) with sharp electrodes or patch electrodes on the soma or dendrites. [Na+]i changes were detected in response to climbing fiber and parallel fiber stimulation. These changes were located both at the anatomically expected sites of synaptic contact in the dendrites and in the somatic region. The variation in time course of these [Na+]i changes in different locations implies that Na+ enters at the synapse and diffuses rapidly to locations of lower initial [Na+]i. The synaptically activated somatic [Na+]i changes probably reflect Na+ entry through voltage-sensitive Na+ channels because they were detected only when regenerative potentials were recorded in the soma. [Na+]i changes in response to antidromically or intrasomatically evoked Na+ action potentials also were confined to the cell body. These observations are in agreement with other evidence that Na+ spikes are generated in the somatic region of the Purkinje neuron and spread passively into the dendrites. Plateau potentials, evoked by depolarizing pulses to the soma or dendrites, caused [Na+]i changes only in the soma, indicating that the noninactivating Na+ channels contributing to this potential also were concentrated in this region. The climbing fiber-activated [Na+]i changes were blocked by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, indicating that these changes were not due to direct stimulation of the Purkinje neuron or activation of metabotropic receptors. Direct depolarization of the soma or dendrites never caused dendritic [Na+]i increases, suggesting that the climbing fiber-activated [Na+]i changes in the dendrites are due to Na+ entry through ligand-gated channels. A climbing fiber-like regenerative potential could be recorded in the soma after anode break stimulation, parallel fiber activation, or depolarizing pulses to the soma. The [Na+]i changes evoked by all of these potentials were confined to the cell body region of the Purkinje cell. [Ca2+]i changes in the dendrites evoked by the anode break potential were small relative to climbing fiber-activated changes, suggesting that a Ca2+ spike was not evoked by this response. The anode break and directly responses were blocked by tetrodotoxin. These results suggest that the somatically recorded climbing fiber response is predominantly a Na(+)-dependent event, consisting of a few fast action potentials and a slower regenerative response activating the same channels as the Na+ plateau potential.

Cell Shrinkage Activates a Cation Conductance in Principal Cells of Rat Cortical Collecting Duct

We examined whether a nonselective cation (NSC) conductance is activated in the freshly isolated rat cortical collecting duct (CCD) by osmotically induced cell shrinkage. Membrane voltages (Vm), whole cell conductances (Gc) and single-channel currents were measured with the patch-clamp method. Cellular Ca2+ ([Ca2+]i) and Na+ ([Na+]i) activities were estimated fluorimetrically with fura-2 and sodium-binding benzofuran isophthalate. Increasing extracellular osmolality by 50 or 100 mosm/1 depolarized Vm by 6 ± 1 (n = 26) and 11 ± 3 mV (n = 12), respectively. Gc was increased under these conditions by 10 ± 7 (n = 9) and 46 ± 17% (n = 6), respectively. The depolarization was partly inhibited by either amiloride (n = 6) or Gd3+ (n = 4, each 10 µmol/l) and completely inhibited by amiloride plus Gd3+ (n = 13) or removal of Na+ (n = 6). Cell-attached NSC channels could neither be observed under control conditions nor after increasing osmolality. [Ca2+]i did not significantly change upon increasing osmolality by 50 mosm/1 (n = 11) and decreased slightly by 15 ± 3 nmol/l (n = 11) when the osmolality was increased by 100 mosm/1. [Na+]i was significantly increased upon increasing osmolality (+100 mosm/1) by 14 ± 2 mmol/l (n = 23). This increase in [Na+]i was reduced to 8 ± 2 mmol/l (n = 9) in the presence of amiloride (10 µmol/ 1), to 11 ± 3 mmol/l (n = 8) in the presence of Gd3+ (10 µmol/l) and to 6 ± 1 mmoI/1 (n = 7) with both inhibitors. These data indicate that osmotically induced cell shrinkage activates a NSC conductance in rat CCD cells.

Anticonvulsive action of (±)-kavain estimated from its properties on stimulated synaptosomes and Na + channel receptor sites

Kava pyrones are constituents of the intoxicating pepper ( Piper methysticum Forst.), which has been shown to be anticonvulsive. The question of how the excitability of neurons is affected was investigated by determining the interaction of (±)-kavain with epitopes (site 1, site 2) of voltage-dependent Na + channels and the action of (±)-kavain on 4-aminopyridine-stimulated synaptosomes as model of repetitive firing neurons. [ 3H]Saxitoxin and [ 3H]batrachotoxin were used for radioligand-binding assays performed with synaptosomal membranes. Glutamate released from 4-aminopyridine-stimulated cerebrocortical synaptosomes and the cytosolic concentrations of Na + and Ca 2+ ([Na +] i, [Ca 2+] i) were detected fluorometrically by using an enzyme-linked assay, sodium-binding benzofuranisophthalate (SBFI) and Fura-2, respectively. (±)-Kavain failed to compete with [ 3H]saxitoxin up to 400 μmol/l but dose-dependently suppressed binding of [ 3H]batrachotoxin with an IC 50 value of 88 μmol/l ( K i = 72 μmol/l) although displacement of [ 3H]batrachotoxin was restricted to 33% of control at 400 μmol/l (±)-kavain. In stimulated synaptosomes, 5 mmol/1 4-aminopyridine provoked an increase in [Na +] i and [Ca 2+] i by 9 mmol/l Na + and 235 nmol/l Ca 2+. Comparable to the reduction in [ 3H]batrachotoxin binding, 400 μmol/l (±)-kavain suppressed the increase in [Na +] i and [Ca 2+] i to 38 and 29% of control, respectively. Consistent with the increase in [Na +] i and [Ca 2+] i, 5 mmol/l 4-aminopyridine provoked glutamate release (rate: 38 pmol/s∗mg protein) which was dose-dependently diminished to 60% of control by 400 μmol/l (±)-kavain. KCl depolarization (40 mmol/l) provoked an increase in [Ca 2+] i and glutamate release almost identical to the responses elicited by 4-aminopyridine but 400 μmol/l (±)-kavain suppressed only the rate of glutamate release by 9% of control. The data suggest an interaction of (±)-kavain with voltage-dependent Na + and Ca 2+ channels, thereby suppressing the 4-aminopyridine-induced increase in [Na +] i, [Ca 2+] i and the release of endogenous glutamate.

Mechanisms of H+and Na+Changes Induced by Glutamate, Kainate, and d-Aspartate in Rat Hippocampal Astrocytes

The excitatory transmitter glutamate (Glu), and its analogs kainate (KA), and D-aspartate (D-Asp) produce significant pH changes in glial cells. Transmitter-induced pH changes in glial cells, generating changes in extracellular pH, may represent a special form of neuronal-glial interaction. We investigated the mechanisms underlying these changes in intracellular H+ concentration ([H+]i) in cultured rat hippocampal astrocytes and studied their correlation with increases in intracellular Na+ concentration ([Na+]i), using fluorescence ratio imaging with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) or sodium-binding benzofuran isophthalate (SBFI). Glu, KA, or D-Asp evoked increases in [Na+]i; Glu or D-Asp produced parallel acidifications. KA, in contrast, evoked biphasic changes in [H+]i, alkaline followed by acid shifts, which were unaltered after Ca2+ removal and persisted in 0 CI(-)-saline, but were greatly reduced in CO2/HCO3(-)-free or Na(+)-free saline, or during 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS) application. The non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) blocked KA-evoked changes in [H+]i and [Na+]i, indicating that they were receptor-ionophore mediated. In contrast, CNQX increased the [H+]i change and decreased the [Na+]i change induced by Glu. D-Asp, which is transported but does not act at Glu receptors, induced [H+]i and [Na+]i changes that were virtually unaltered by CNQX. Our study indicates that [Na+]i increases are not primarily responsible for Glu- or KA-induced acidifications in astrocytes. Instead, intracellular acidifications evoked by Glu or D-Asp are mainly caused by transmembrane movement of acid equivalents associated with Glu/Asp-uptake into astrocytes. KA-evoked biphasic [H+]i changes, in contrast, are probably attributable to transmembrane ion movements mediated by inward, followed by outward, electrogenic Na+/HCO3- cotransport, reflecting KA-induced biphasic membrane potential changes.

Accumulation of intracellular HCO3- by Na(+)-HCO3- cotransport in interlobular ducts from guinea-pig pancreas.

1. Short segments of interlobular duct were microdissected from guinea-pig pancreas following enzymatic digestion. After overnight culture, intracellular pH (pH1) and Na+ concentration ([Na+]i) were measured by microfluorometry in duct cells loaded with either the pH-sensitive fluoroprobe 2'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) or the sodium-binding benzofuran isophthalate (SBFI). 2. The transporters responsible for maintaining pHi above equilibrium were investigated by using the NH4Cl pulse technique to acid load the cells. In the absence of HCO3-/CO2, the recovery of pH1 was Na+ dependent, abolished by 0.2 mM amiloride and by 10 microM N-methyl-N-isobutylamiloride and was therefore attributed to Na(+)-H+ exchange. 3. In the presence of HCO3-/CO2, amiloride only partially inhibited the recovery from acid loading. The amiloride-insensitive component was abolished by 0.5 mM H2DIDS and unaffected by depletion of intracellular Cl- and was therefore attributed to Na(+)-HCO3- cotransport. 4. Stimulation with 10 nM secretin did not cause a significant change in pH1 despite a significant increase in HCO3- efflux. However, in the presence of secretin, addition of 0.5 mM H2DIDS caused a decline in pH1 that was three times more rapid than that obtained with 0.2 mM amiloride. 5. In secretin-stimulated ducts, Na+ uptake increased when HCO3-/CO2 was added to the bath and this increase was strongly inhibited by 0.5 mM H2DIDS. 6. We conclude that Na(+)-HCO3- cotransport contributes approximately 75% of the HCO3- taken up by guinea-pig pancreatic duct cells during stimulation with secretin. It is proposed that electrical coupling between HCO3- efflux at the luminal membrane and electrogenic Na(+)-HCO3- cotransport at the basolateral membrane explains why secretin causes little change in pH1.