INVOLVEMENT OF Na+-K+-2Cl− COTRANSPORTERS IN HYPERTONICITY-INDUCED RISE IN INTRACELLULAR CALCIUM CONCENTRATION

A hypertonic saline containing propylene glycol facilitates calcium (Ca²⁺) influx through voltage-dependent Ca²⁺ channels. The present study performed experiments to elucidate the mechanism by which Na⁺-K⁺-2Cl^? cotransporters participate in the rise in the intracellular calcium concentration ([Ca²⁺]i) under the hypertonic condition. Both furosemide and ethacryonic acid significantly decreased the [Ca²⁺]i raised by hypertonicity. Similarly, Na⁺-, K⁺-, or Cl^?-free saline also reduced it. Both norepinephrine and dopamine significantly enhanced the rise in [Ca²⁺]i. In conclusion, the findings obtained indicate that the Na⁺-K⁺-2Cl^? cotransporters evoke cell depolarization and that this depolarization raises the [Ca²⁺]i by activating voltage-dependent Ca²⁺ channels.     

Cyclic AMP-induced depolarization measured by bis-oxonol fluorescence in bovine adrenal medullary chromaffin cells

Summary Effects of cyclic AMP on membrane potentials were examined by measuring the changes of bis-oxonol fluorescence in bovine adrenal medullary chromaffin cells. 8-Bromo cyclic AMP (8Br-cAMP) or forskolin caused a gradual and long lasting increase of the fluorescence intensity. The effects of 8br-cAMP was blocked by cyclic AMP-dependent protein kinase inhibitor, adenosine-3, 5-cyclic monophosphothioate, Rp-diastereomer (Rp-cAMPS) and there was no further increase in the fluorescence by 8br-cAMP in the cells depolarized with 56 mM KCl or gramicidin D. Ouabain or the removal of extracellular K⁺ ([K⁺]₀free) which block Na⁺, K⁺-ATPase also increased the fluorescence. The effect of 8br-cAMP on the fluorescence was counteracted by ouabain or [K⁺]₀ free and was blocked in the absence of extracellular Na⁺ but not by tetrodotoxin or the removal of Ca²⁺ from the medium. These results may suggest that cyclic AMP causes the membrane depolarization by accumulating Na⁺ through the inhibition of Na⁺, K⁺-ATPase in adrenal chromaffin cells.     

Effect of the removal of extracellular Ca on the response of cytosolic concentrations of Ca to ouabain in carotid body glomus cells of adult rabbits

Effect of the removal of extracellular Ca²⁺ on the response of cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) to ouabain, an Na⁺/K⁺ exchanger antagonist, was examined in clusters of cultured carotid body glomus cells of adult rabbits using fura-2AM and microfluorometry. Application of ouabain (10 mM) induced a sustained increase in [Ca²⁺]_i (mean±S.E.M.; 38±5% increase, n=16) in 55% of tested cells (n=29). The ouabain-induced [Ca²⁺]_i increase was abolished by the removal of extracellular Na⁺. D600 (50 μM), an L-type voltage-gated Ca²⁺ channel antagonist, inhibited the [Ca²⁺]_i increase by 57±7% (n=4). Removal of extracellular Ca²⁺ eliminated the [Ca²⁺]_i increase, but subsequent washing out of ouabain in Ca²⁺-free solution produced a rise in [Ca²⁺]_i (62±8% increase, n=6, P<0.05), referred to as a [Ca²⁺]_i rise after Ca²⁺-free/ouabain. The magnitude of the [Ca²⁺]_i rise was larger than that of ouabain-induced [Ca²⁺]_i increase. D600 (5 μM) inhibited the [Ca²⁺]_i rise after Ca²⁺-free/ouabain by 83±10% (n=4). These results suggest that ouabain-induced [Ca²⁺]_i increase was due to Ca²⁺ entry involving L-type Ca²⁺ channels which could be activated by cytosolic Na⁺ accumulation. Ca²⁺ removal might modify the [Ca²⁺]_i response, resulting in the occurrence of a rise in [Ca²⁺]_i after Ca²⁺-free/ouabain which mostly involved L-type Ca²⁺ channels.     

Intracellular acidification induced by membrane depolarization in rat hippocampal slices: roles of intracellular Ca and glycolysis

To elucidate the mechanism of pH_i changes induced by membrane depolarization, the variations in pH_i and [Ca²⁺]_i induced by a number of depolarizing agents, including high K⁺, veratridine, N-methyl-

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-aspartate (NMDA) and ouabain, were investigated in rat hippocampal slices by the fluorophotometrical technique using BCECF or fura-2. All of these depolarizing agents elicited a decrease in pH_i and an elevation of intracellular calcium ([Ca²⁺]_i) in the CA1 pyramidal cell layer. The increases in [Ca²⁺]_i caused by the depolarizing agents almost completely disappeared in the absence of Ca²⁺ (0 mM Ca²⁺ with 1 mM EGTA). In Ca²⁺ free media, pH_i acid shifts produced by high K⁺, veratridine or NMDA were attenuated by 10–25%, and those produced by ouabain decreased by 50%. Glucose-substitution with equimolar amounts of pyruvate suppressed by two-thirds the pH_i acid shifts induced by both high K⁺ and NMDA. Furthermore, lactate contents were significantly increased in hippocampal slices by exposure to high K⁺, veratridine or NMDA but not by ouabain. These results suggest that the intracellular acidification produced by these depolarizing agents, with the exception of ouabain, is mainly due to lactate accumulation which may occur as a result of accelerated glycolysis mediated by increased Na⁺–K⁺ ATPase activity. A Ca²⁺-dependent process may also contribute to the intracellular acidification induced by membrane depolarization. Since an increase in H⁺ concentration can attenuate neuronal activity, glycolytic acid production induced by membrane depolarization may contribute to the mechanism that prevents excessive neuronal excitation.     

Differential Stimulation of Noradrenaline Release by Reversal of the Na+/Ca2+ Exchanger and Depolarization in Chromaffin Cells

We compared the effectiveness of Ca²⁺ entering by Na⁺/Ca²⁺ exchange with that of Ca²⁺ entering by channels produced by membrane depolarization with K⁺ in inducing catecholamine release from bovine adrenal chromaffin cells. The Ca²⁺ influx through the Na⁺/Ca²⁺ exchanger was promoted by reversing the normal inward gradient of Na⁺ by preincubating the cells with ouabain to increase the intracellular Na⁺ and then removing Na⁺ from the external medium. In this way we were able to increase the cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) by Na⁺/Ca²⁺ exchange to 325 ± 14 nM, which was similar to the rise in [Ca²⁺]_c observed upon depolarization with 35 mM K⁺ of cells not treated with ouabain. After incubating the cells with ouabain, K⁺ depolarization raised the [Ca²⁺]_c to 398 ± 31 nM, and the recovery of [Ca²⁺]_c to resting levels was significantly slower. Reversal of the Na⁺ gradient caused an −6-fold increase in the release of noradrenaline or adrenaline, whereas K⁺ depolarization induced a 12-fold increase in noradrenaline release but only a 9-fold increase in adrenaline release. The ratio of noradrenaline to adrenaline release was 1.24 ± 0.23 upon reversal of the Na⁺/Ca²⁺ exchange, whereas it was 1.83 ± 0.19 for K⁺ depolarization. Reversal of the Na⁺/Ca²⁺ exchange appeared to be as efficient as membrane depolarization in inducing adrenaline release, in that the relation of [Ca²⁺]_c to adrenaline release was the same in both cases. In contrast, we found that for the same average [Ca²⁺]_c, the Ca²⁺ influx through voltage-gated channels was much more efficient than the Ca²⁺ entering through the Na⁺/Ca²⁺ exchanger in inducing noradrenaline release from chromaffin ceils. This greater effectiveness of membrane depolarization in stimulating noradrenaline release suggests that there is a pool of noradrenaline vesicles which is more accessible to Ca²⁺ entering through voltage-gated Ca²⁺ channels than to Ca²⁺ entering through the Na⁺/Ca²⁺ exchanger, whereas the adrenaline vesicles do not distinguish between the source of Ca²⁺.     

The ouabain-induced [Ca]i increase in leech Retzius neurones is mediated by voltage-dependent Ca channels

In leech Retzius neurones the inhibition of the Na⁺–K⁺ pump by ouabain causes an increase in the cytosolic free calcium concentration ([Ca²⁺]_i). To elucidate the mechanism of this increase we investigated the changes in [Ca²⁺]_i (measured by Fura-2) and in membrane potential that were induced by inhibiting the Na⁺–K⁺ pump in bathing solutions of different ionic composition. The results show that Na⁺–K⁺ pump inhibition induced a [Ca²⁺]_i increase only if the cells depolarized sufficiently in the presence of extracellular Ca²⁺. Specifically, the relationship between [Ca²⁺]_i and the membrane potential upon Na⁺–K⁺ pump inhibition closely matched the corresponding relationship upon activation of the voltage-dependent Ca²⁺ channels by raising the extracellular K⁺ concentration. It is concluded that the [Ca²⁺]_i increase caused by inhibiting the Na⁺–K⁺ pump in leech Retzius neurones is exclusively due to Ca²⁺ influx through voltage-dependent Ca²⁺ channels.     

Involvement of the glutamate transporter and the sodium–calcium exchanger in the hypoxia-induced increase in intracellular Ca in rat hippocampal slices

Hippocampal slices prepared from adult rats were loaded with fura-2 and the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in the CA1 pyramidal cell layer was measured. Hypoxia (oxygen–glucose deprivation) elicited a gradual increase in [Ca²⁺]_i in normal Krebs solution. At high extracellular sodium concentrations ([Na⁺]_o), the hypoxia-induced response was attenuated. In contrast, hypoxia in low [Na⁺]_o elicited a significantly enhanced response. This exaggerated response to hypoxia at a low [Na⁺]_o was reversed by pre-incubation of the slice at a low [Na⁺]_o prior to the hypoxic insult. The attenuation of the response to hypoxia by high [Na⁺]_o was no longer observed in the presence of antagonist to glutamate transporter. However, antagonist to Na⁺–Ca²⁺ exchanger only slightly influenced the effects of high [Na⁺]_o. These observations suggest that disturbance of the transmembrane gradient of Na⁺ concentrations is an important factor in hypoxia-induced neuronal damage and corroborates the participation of the glutamate transporter in hypoxia-induced neuronal injury. In addition, the excess release of glutamate during hypoxia is due to a reversal of Na⁺-dependent glutamate transporter rather than an exocytotic process.     

In vitro dopamine biosynthesis in the median eminence of rat hypothalamic slices: Involvement of voltage-dependent Ca channels

Equimolar replacement of Na⁺ in medium with choline chloride or sucrose and experimental manipulations known to increase [Na⁺]_i, such as ouabain addition and K⁺ deprivation from medium, caused a marked increase in in vitro DOPA synthesis in the median eminence of rat hypothalamic slices in a Ca²⁺-dependent manner. These results suggest that a Na⁺−Ca²⁺ exchange mechanism is closely involved in the regulation of dopamine biosynthesis in tuberoinfundibular neurons.     

Adenosine A1 receptors inhibit Ca channels coupled to the release of ACh, but not of GABA, in cultured retina cells

We investigated the effect of adenosine A₁ receptors on the release of acetylcholine (ACh) and GABA, and on the intracellular calcium concentration ([Ca²⁺]_i) response in cultured chick amacrine-like neurons, stimulated by KCl depolarization. The KCl-induced release of [³H]ACh, but not the release of [¹⁴C]GABA, was potentiated when adenosine A₁ receptor activation was prevented by perfusing the cells with adenosine deaminase (ADA) or with 1,3-dipropyl-8-cycloentylxanthine (DPCPX). The changes in the [Ca²⁺]_i induced by KCl depolarization, measured in neurite segments of single cultured cells, were also modulated by endogenous adenosine, acting on adenosine A₁ receptors. Our results show that adenosine A₁ receptors inhibit Ca²⁺ entry coupled to ACh release, but not to the release of GABA, suggesting that the synaptic vesicles containing each neurotransmitter are located in different zones of the neurites, containing different VSCC and/or different densities of adenosine A₁ receptors.     

Na influx through Ca channels can promote striatal GABA efflux in Ca-deficient conditions in response to electrical field depolarization

Electrical field depolarization releases γ-aminobutyric acid (GABA) in rat striatal slices in the absence of external Ca²⁺. ω-Conotoxin GVIA (ω-CgTx; 1–50 nM), a neuronal Ca²⁺ channel blocker, inhibits electrically evoked efflux of newly taken up [³H]GABA in a concentration-dependent manner in either normal or Ca²⁺-free medium. This suggests that ion influx occurs through Ca²⁺ channels in the absence of external Ca²⁺ and contributes to the efflux of GABA. Reducing external Na⁺ concentration to 27.25 mM (low [Na⁺]₀ medium) by equimolarly substituting choline chloride for sodium chloride has differential effects on electrically evoked GABA efflux depending on the external Ca²⁺ concentrations. In normal Ca²⁺ medium, electrically evoked GABA efflux increases whereas, in Ca²⁺-free medium, it is greatly inhibited when [Na⁺]₀ is reduced to 27.25 mM. In low [Na⁺]₀ medium, GABA efflux is largely tetrodotoxin (TTX)-sensitive, however, spike firing evoked by antidromic stimulation of striatal cells is inhibited. In Na⁺-free medium, resting GABA efflux increases 17-fold whereas evoked GABA efflux diminishes. In Ca²⁺-free medium, 70 min of incubation with 1–2-bis-(2-aminophenoxy)ethane-N,N,N′,N′ tetraacetoxy methyl ester (BAPTA-AM, 1 μM), an intracellular calcium chelator, increases both resting GABA efflux and electrically evoked GABA overflow by 100%. These results suggest that: (1) in Ca²⁺-free conditions, Na⁺ permeability of cells increases via Ca²⁺ channels and this profoundly affects GABA efflux. (2) Electrical field depolarization is likely to release GABA by directly depolarizing axon terminals. (3) Ca²⁺-independent GABA efflux is not promoted by an increase in intracellular free Ca²⁺ concentration via Na⁺/Ca²⁺ exchange processes from internal pools.     

Coupling of AMPA receptors with the Na/Ca exchanger in cultured rat astrocytes

Astrocytes exhibit three transmembrane Ca²⁺ influx pathways: voltage-gated Ca²⁺ channels (VGCCs), the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) class of glutamate receptors, and Na⁺/Ca²⁺ exchangers. Each of these pathways is thought to be capable of mediating a significant increase in Ca²⁺ concentration ([Ca²⁺]_i); however, the relative importance of each and their interdependence in the regulation astrocyte [Ca²⁺]_i is not known. We demonstrate here that 100 μM AMPA in the presence of 100 μM cyclothiazide (CTZ) causes an increase in [Ca²⁺]_i in cultured cerebral astrocytes that requires transmembrane Ca²⁺ influx. This increase of [Ca²⁺]_i is blocked by 100 μM benzamil or 0.5 μM U-73122, which inhibit reverse-mode operation of the Na⁺/Ca²⁺ exchanger by independent mechanisms. This response does not require Ca²⁺ influx through VGCCs, nor does it depend upon a significant Ca²⁺ influx through AMPA receptors (AMPARs). Additionally, AMPA in the presence of CTZ causes a depletion of thapsigargin-sensitive intracellular Ca²⁺ stores, although depletion of these Ca²⁺ stores does not decrease the peak [Ca²⁺]_i response to AMPA. We propose that activation of AMPARs in astrocytes can cause [Ca²⁺]_i to increase through the reverse mode operation of the Na⁺/Ca²⁺ exchanger with an associated release of Ca²⁺ from intracellular stores. This proposed mechanism requires neither Ca²⁺-permeant AMPARs nor the activation of VGCCs to be effective.     

Effects of serotonin on extracellular potassium concentration in the rat hippocampal slice

The effects of serotonin (5-HT) on extracellular potassium concentration ([K⁺]₀) were measured with ion-selective microelectrodes in rat hippocampal slices. Electrical stimulation of an excitatory afferent system, the Schaffer collateral commissural pathway, caused a 2–4 mM rise in [K⁺]₀ in the stratum pyramidale of area CA1. 5-HT caused a 0.6–1.1 mM rise in [K⁺]₀. This rise was associated with hyperpolarization of neurons and cessation of their spontaneous spike discharge. Methysergide, a 5-HT antagonist, reduced the 5-HT effect. The change in [K⁺]₀ was highest in stratum moleculare and lowest in stratum pyramidale, the opposite gradient to that found with excitatory electrical stimulation. The 5-HT-induced [K⁺]₀ changes were maximal in CA1 stratum moleculare, intermediate in the dentate stratum granulare and almost non-existent in the CA3 stratum pyramidale.GABA, but not norepinephrine, produced a small (up to 0.5 mM) rise in [K⁺]₀ in stratum pyramidale. Extracellular calcium concentration measured with a Ca²⁺-sensitive microelectrode was reduced by electrical stimulation but unchanged by 5-HT or norepinephrine. It is suggested that 5-HT hyperpolarizes hippocampal cells by activation of sodium- and calcium-independent potassium channels, which cause a rise in [K⁺]₀.     

K-current modulated by PO2 in type I cells in rat carotid body is not a chemosensor

According to the membrane channel hypothesis of carotid body O₂ chemoreception, hypoxia suppresses K⁺ currents leading to cell depolarization, [Ca²⁺]_i rise, neurosecretion, increased neural discharge from the carotid body. We show here that tetraethylammonium (TEA) plus 4-aminopyridine (4-AP) which suppressed the Ca²⁺ sensitive and other K⁺ currents in rat carotid body type I cells, with and without low [Ca²⁺]_o plus high [Mg²⁺]_o, did not essentially influence low

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effects on [Ca²⁺]_i and chemosensory discharge. Thus, hypoxia may suppress the K⁺ currents in glomus cells but K⁺ current suppression of itself does not lead to chemosensory excitation. Therefore, the hypothesis that K⁺–O₂ current is linked to events in chemoreception is not substantiated. K⁺–O₂ current is an epiphemenon which is not directly linked with O₂ chemoreception.     

Release of γ-[H]aminobutyric acid from synaptosomes: effect of external cations and of ouabain

In the present study we have investigated the effect of cations and ouabain on Ca²⁺-independent and Ca²⁺-dependent release of γ-[³H]aminobutyric acid ([³H]GABA) from sheep brain synaptosomes. The presence of Na⁺ in the external medium is essential for the Ca²⁺-independent release induced by K⁺ or ouabain. Thus, in the absence of Ca²⁺, ouabain of K⁺ causes the release of [³H]GABA provided that Na⁺ is present in the external medium. Under K⁺-depolarizing conditions, in a Na⁺ medium, either ouabain or Ca²⁺ further increases the [³H]GABA release induced by depolarization, but their effects are not additive. The presence of external Na⁺ is not required for the Ca²⁺-dependent release of [³H]GABA due to K⁺ depolarization, and this release, which occurs in a choline medium, is not modified by ouabain. Under these conditions (choline medium) K⁺-depolarization dependent release is absolutely dependent on external Ca²⁺, which suggests that this release of [³H]GABA occurs only by exocytosis, without the carrier-mediated efflux which normally co-exists with exocytosis due to K⁺-depolarization in a Na⁺ medium. It is likely that the release induced by ouabain or K⁺ involves the membrane carrier which responds to changes in membrane potential.     

Possible occurrence of Na-dependent Ca influx mechanism in isolated bovine chromaffin cells

Chromaffin cells were isolated from bovine adrenals and the effects of experimental manipulations which alter the level of internal Na⁺ on the release of catecholamines and⁴⁵Ca uptake by these cells were investigated. In response to Na⁺ deprivation both parameters were increased or decreased when internal Na⁺ was raised or reduced, respectively. The results suggest the existence of Na⁺-dependent Ca²⁺ influx mechanism in these cells.     

Asphyxia induced by umbilical cord occlusion alters glutamatergic and GABAergic synaptic transmission in neurons of the superior colliculus in fetal rats

Using optical recordings, we studied the effects of asphyxia on intracellular Cl⁻ and Ca²⁺ concentrations ([Cl⁻]i; [Ca²⁺]i) in the superior colliculus of fetal rats, which were connected via the umbilical cord to the dam. Acute asphyxia was induced by umbilical cord occlusion. The number of fetal superior colliculus neurons showing GABA-mediated increases in [Cl⁻]i (leading to hyperpolarization) following local synaptic electrical stimulation had decreased by 3 h post-asphyxiation, while the number showing GABA-mediated decreases in [Cl⁻]i (leading to depolarization) increased. [Ca²⁺]i rise, which occurred after acute asphyxiation, was antagonized by both non-NMDA and NMDA receptor antagonists. The increase in [Ca²⁺]i following focal superior colliculus stimulation was markedly attenuated at 3 h post-asphyxiation.     

Depolarization of mouse forebrain minces with veratridine and high K: Failure to stimulate the Ca independent, spontaneous release of acetycholine from the cytoplasm due to hydrolysis of the acetylcholine stored there

Both high K⁺ and veratridine, depolarizing agents with different mechanisms of action, lowered the ACh content of the cytoplasmic (S₃) fraction of mouse forebrain minces incubated in a Ca²⁺-free Krebs solution, without stimulating ACh release or altering the level of ACh in the vesicle-bound (P₃) fraction. Veratridine increased the level of choline in the P₃ fraction by the same amount as it reduced the level of ACh in the S₃ fraction, and these changes did not occur in the presence of tetrodotoxin (TTX). Pretreatment of minces in normal Krebs increased the ACh but not the choline content of the S₃ fraction. Following this expansion of the S₃ ACh content, veratridine caused an eve greater loss of S₃ ACh, and increased the Ca²⁺-independent release of ACh slightly. Under these conditions, veratridine also stimulated the Ca²⁺ independent release of choline, and this increase exceeded that obtained for the Ca²⁺-independent release of ACh. Preincubation in normal Krebs with paraoxon did not alter the S₃ ACh content after 5 min, but raised it by 78% after 30 min. Under the latter conditions of pretreatment, veratridine then stimulated tha Ca²⁺-independent release of ACh even more, but did not stimulate the release of choline. These results suggest that depolarization of brain tissue does not facilitate the Ca²⁺-independent release of ACh from the cytoplasm because a portion of ACh stored there is hydrolyzed. When the cytoplasmic level of ACh is sufficiently elevated prior to depolarization, then some ACh escapes hydrolysis and is released independently of Ca²⁺. It is suggested that the depolarization-induced hydrolysis of cytoplasmic ACh may be mediated by an intraterminal form of AChE and may, in addition to the hydrolysis of extracellular ACh, provide substrate for the formation and release of ACh by the vesicle-bound fraction.     

The action of valproate on spontaneous epileptiform activity in the absence of synaptic transmission and on evoked changes in [Ca]0 and [K]0 in the hippocampal slice

The effects of valproate (VPA) on neuronal excitability and on changes in extracellular potassium ([K⁺]₀) and calcium ([Ca²⁺]₀) were investigated with ion selective-reference electrode pairs in area CA₁ of rat hippocampal slices. Field potential responses to single ortho- and antidromic stimuli were unaltered by VPA (1–5 mM). The afferent volley evoked in the Schaffer-commissural fibers was also unaffected. In contrast, VPA (1 mM) depressed frequency potentiation and paired pulse facilitation markedly. Decreases in [Ca²⁺]₀ induced either by repetitive stimulation or by application of the excitatory amino acids N-methyl-d-aspartate and quisqualate were reduced, and the latter results suggest that VPA interferes with postsynaptic Ca²⁺ entry. When synaptic transmission was blocked by lowering [Ca²⁺]₀ (0.2 mM) and elevating [Mg²⁺]₀ (7 mM), prolonged afterdischarges elicited by antidromic stimulation were blocked by VPA. VPA also suppressed the spontaneous epileptiform activity seen when [Ca²⁺]₀ was lowered to 0.2 mM, without elevating [Mg²⁺]₀. The amplitudes of the rises in [K⁺]₀ induced by repetitive orthodromic stimulation were only slightly depressed and those elicited by antidromic stimulation were generally unaltered by VPA, as were laminar profiles of stimulus-evoked [K⁺]₀ signals. These results indicate that VPA has membrane actions in addition to known effects on excitatory and inhibitory transmitter pools.     

Cholesterol oxidation reduces Ca + Mg-ATPase activity, interdigitation, and increases fluidity of brain synaptic plasma membranes

These experiments examined effects of cholesterol oxidation on Ca²⁺+Mg²⁺-ATPase activity, Na⁺+K⁺-ATPase activity, and membrane structure of brain synaptic plasma membranes (SPM). Cholesterol oxidase [E.C.1.1.3.6 fromBrevibacterium sp.] was used to oxidize cholesterol. Two cholesterol pools were identified in synaptosomal membranes based on their accessibility to cholesterol oxidase. A rapidly oxidized cholesterol pool was observed with a¹t₁₂ of 1.19±0.09 min and a second pool with a²t₁₂ of 38.30±4.16 min. Activity of Ca²⁺+Mg²⁺-ATPase was inhibited by low levels of cholesterol oxidation. Ten percent cholesterol oxidation, for example, resulted in approximately 35% percent inhibition of Ca²⁺+Mg²⁺-ATPase activity. After 13% cholesterol oxidation, further inhibition of Ca²⁺+Mg²⁺-ATPase activity was not observed. Activity of Na⁺+K⁺-ATPase was not affected by different levels of cholesterol oxidation (5%–40%). SPM interdigitation was significantly reduced and fluidity was significantly increased by cholesterol oxidation. The relatiobship observed between SPM interdigitation and Ca²⁺+Mg²⁺-ATPase activity was consistent with studies using model membranes [7]. Brain SPM function and structure were altered by relatively low levels of cholesterol oxidation and is a new approach to understanding cholesterol dynamics and neuronal function. The sensitivity of brain SPM to cholesterol oxidation may be important with respect to the proposed association between oxygen free radicals and certain neurodegenerative diseases.     

Transport pathways for lithium ions in neuroblastoma × glioma hybrid cells at ‘therapeutic’ concentrations of Li

The pathways of Li⁺ transport in neuroblastoma × glioma hybrid cells were studied at 2 mM external Li⁺. Five components of Li⁺ transport were identified. (1) A Na⁺-dependent Li⁺ countertransport system mediating Li⁺ transport in both directions across the plasma membrane. This transport pathway is insensitive to ouabain or external K⁺. It shows trans-stimulation (i.e. acceleration of Li⁺ extrusion by external Na⁺ and stimulation of Li⁺ uptake by internal Na⁺) and cis-inhibition (i.e. reduction of Li⁺ uptake by external Na⁺). (2) The Na⁺K⁺ pump mediates Li⁺ uptake but not Li⁺ release in cells with physiological Na⁺ and K⁺ content. Li⁺ uptake by the pump in choline media is inhibited by both external Na⁺ and K⁺. In Na⁺ media, external K⁺ exhibits a biphasic effect: in concentrations up to about 1 mM, K⁺ accelerates, and at higher concentrations, K⁺ inhibits, Li⁺ uptake by the pump. (3) Li⁺ can enter the voltage-dependent Na⁺ channel. Li⁺ uptake through this pathway is stimulated by veratridine and scorpion toxin, the stimulation being blocked by tetrodotoxin. Residual pathways comprise (4) a saturable component, which is comparable to basal Na⁺ uptake, and (5) a ouabain-resistant component promoting Li⁺ extrusion against an electrochemical gradient in choline media. The mechanisms for Li⁺ extrusion described here possibly explain how neuronal cells maintain the steady-state ratio of internal to external Li⁺ below 1 during chronic exposure to 1–2 mM external Li⁺.