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.     

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.     

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.     

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.     

Effects of the removal of extracellular Ca on [Ca]i responses to FCCP and acetate in carotid body glomus cells of adult rabbits

The effects of the removal of extracellular Ca²⁺ on the responses of cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) to acidic stimuli, a protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and an organic acid acetate, were examined in clusters of cultured carotid body glomus cells of adult rabbits using fura-2 microfluorometry. Application of FCCP (1 μM) induced an increase in [Ca²⁺]_i (mean±S.E.M., 108±14%). After withdrawal of the protonophore the increased [Ca²⁺]_i returned slowly to a resting level. The [Ca²⁺]_i response was attenuated by an inorganic Ca²⁺ channel antagonist Ni²⁺ (2 mM) by 81±4%, and by an L-type voltage-gated Ca²⁺ channel antagonist D600 (10 μM) by 53±13%. The removal of extracellular Ca²⁺ eliminated the [Ca²⁺]_i response in 71% of the tested cells (n=17), and depressed it by 68±6% in the rest. Recovery following stimulation with FCCP in the absence of Ca²⁺ reversibly produced a rapid and large rise in [Ca²⁺]_i, referred to as a [Ca²⁺]_i rise after Ca²⁺-free/FCCP. The magnitude of a [Ca²⁺]_i rise after Ca²⁺-free/FCCP (285±28%, P<0.05) was larger than that of an increase in [Ca²⁺]_i induced by FCCP in the presence of Ca²⁺ and had a correlation with the intensity of the suppression of the [Ca²⁺]_i response by Ca²⁺ removal. A [Ca²⁺]_i rise after Ca²⁺-free/FCCP was inhibited mostly by D600. Similarly, recovery following exposure to acetate in the absence of Ca²⁺ caused a rise in [Ca²⁺]_i, referred to as a [Ca²⁺]_i rise after Ca²⁺-free/acetate which was sensitive to D600. The magnitude of the [Ca²⁺]_i rise was larger than that of a change in [Ca²⁺]_i caused by acetate in the presence of Ca²⁺. These results suggest that FCCP-induced increase in [Ca²⁺]_i was, in most cells, due to Ca²⁺ influx via L-type voltage-gated Ca²⁺ channels and, in some cells, due to both Ca²⁺ influx and Ca²⁺ release from internal Ca²⁺ pool. The removal of extracellular Ca²⁺ might modify [Ca²⁺]_i responses to acidic stimuli, causing [Ca²⁺]_i rises after Ca²⁺-free/acidic stimuli which involve mostly L-type 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.     

A pharmacological model for studying the role of Na gradients in the modulation of synaptosomal free [Ca]i levels and energy metabolism

Lactate production (J_lac), oxygen consumption rate (Q_O2), plasma membrane potentials (E_m) and cytosolic free calcium levels [Ca²⁺]_i were studied on symaptosomes isolated from rat brains, incubated in presence of high doses of nicardipine (90 μM), diltiazem (0.5 mM) and verapamil (0.25 mM), and submitted to depolarizing stimulation or inhibition of mitochondrial respiration. Nicardipine was able to completely prevent the veratridine-induced stimulation ofJ_lac, Q_O2andE_m depolarization, whereas diltiazem and verapamil were less effective, although the concentrations used were 5 and 3 times higher, respectively, than nicardipine. Diltiazem, verapamil and nicardipine (9 μM) also prevented the veratridine-induced increase in [Ca²⁺]_i, this effect being much less pronounced if the drugs were added after veratridine. Monensin (20 μM) was also able to increase [Ca²⁺]_i but this effect was not affected by verapamil. Synaptosomes were also submitted to an inhibition of respiration of intrasynaptic mitochondria by incubation with rotenone (5 μM); in this condition of mimicked hypoxiaE_m was more positive of about 11 mV; none of the drugs utilized modified this situation. The rotenone-induced 3-fold increase inJ_lac was barely modified by diltiazem and verapamil but it was completely abolished by nicardipine. The possible mechanism of the counteracting action of the drugs towards veratridine stimulation and rotenone inhibition and the involvement of Na⁺/Ca²⁺ exchanger in affecting [Ca²⁺]_i are discussed.     

Heat shock protects cultured rat astrocytes in a model of reperfusion injury

We have previously found that incubation of cultured rat astrocytes in Ca²⁺-containing medium after exposure to Ca²⁺-free medium caused an increase in intracellular Ca²⁺ ([Ca²⁺_i) followed by delayed cell death. Here, we examined whether thermal stress protects astrocytes from cell death in this model system of reperfusion injury. Cultured astrocytes were preincubated at 40–44°C for 10–20 min in fetal calf serum-free medium, incubated at 37°C for 24 h in serum-containing medium, and subjected to the in vitro reperfusion experiment. Thermal stress attenuated reperfusion-induced cell toxicity. Furthermore, the stress increased cell viability after incubation with serum-free medium containing Ca²⁺. These effects of heat shock required incubation in serum-containing medium for at least 12 h after heat shock, and it was blocked by the protein synthesis inhibitor cycloheximide. Thermal stress increased synthesis of several proteins, and one of the inducible proteins was identified as the 72-kDa heat shock protein by an immunoblot analysis. Neither the increase in [Ca²⁺]_i nor the Na⁺-Ca²⁺ exchange activity in astrocytes induced in this model were affected by thermal stress. These findings suggest that heat shock proteins protect astrocytes from cell death in a model of reperfusion injury and they may affect processes down stream of the increase in [Ca²⁺]_i.     

Dibutyryl cGMP raises cytosolic concentrations of Ca in cultured nodose ganglion neurons of the rabbit

The effect of dibutyryl cGMP (dbcGMP), a membrane permeant cGMP analogue, on cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) was studied in cultured nodose ganglion neurons of the rabbit using fura-2AM and microfluorometry. Application of dbcGMP (10–1000 μM) increased [Ca²⁺]_i in 42% of neurons (n=67). The effect was observed in a dose-dependent fashion. The threshold dose was 100 μM and the increase at 500 μM averaged 117±8%. Removal of extracellular Ca²⁺ abolished the dbcGMP effect. Application of Ni²⁺ (1 mM) or neomycin (50 μM), a non-L-type voltage-gated Ca²⁺ channel (VGCC) antagonist, eliminated the dbcGMP effect. ω-conotoxin GVIA (2 μM), the N-type Ca²⁺ channel antagonist, or L-type Ca²⁺ channel antagonists (D600, 50 μM, or nifedipine, 10 μM) did not alter the dbcGMP effect. Ryanodine (10 μM) did not alter the effect of dbcGMP. Therefore, cGMP could play a part of role of an intracellular messenger in primary sensory neurons of the autonomic nervous system.     

Effect of dantrolene on KCl- or NMDA-induced intracellular Ca2+ changes and spontaneous Ca2+ oscillation in cultured rat frontal cortical neurons

Summary Dantrolene has been known to affect intracellular Ca²⁺ concentration ([Ca²⁺]_i) by inhibiting Ca²⁺ release from intracellular stores in cultured neurons. We were interested in examining this property of dantrolene in influencing the [Ca²⁺]_i affected by the NMDA receptor ligands, KCl, L-type Ca²⁺ channel blocker nifedipine, and two other intracellular Ca²⁺-mobilizing agents caffeine and bradykinin. Effect of dantrolene on the spontaneous oscillation of [Ca²⁺]_i was also examined. Dantrolene in M concentrations dose-dependently inhibited the increase in [Ca²⁺]_i elicited by NMDA and KCl. AP-5, MK-801 (NMDA antagonists), and nifedipine respectively reduced the NMDA and KCl-induced increase in [Ca²⁺]_i. Dantrolene, added to the buffer solution together with the antagonists or nifedipine, caused a further reduction in [Ca²⁺]_i to a degree similar to that seen with dantrolene alone inhibiting the increase in [Ca₂₊]_i caused by NMDA or KCl. At 30 M, dantrolene partially inhibited caffeine-induced increase in [Ca²⁺]_i whereas it has no effect on the bradykinin-induced change in [Ca²⁺]_i. The spontaneous oscillation of [Ca²⁺]_i in frontal cortical neurons was reduced both in amplitude and in base line concentration in the presence of 10 M dantrolene. Our results indicate that dantrolene's mobilizing effects on intracellular Ca²⁺ stores operate independently from the influxed Ca²⁺ and that a component of the apparent increase in [Ca²⁺]_i elicited by NMDA or KCl represents a dantrolene-sensitive Ca₂₊ release from intracellular stores. Results also suggest that dantrolene does not affect the IP₃-gated release of intracellular Ca²⁺ and that the spontaneous Ca²⁺ oscillation is, at least partially, under the control of Ca²⁺ mobilization from internal stores.Abbreviations AP-5 (±)-2-amino-5-phosphonopentanoic acid - AMPA amino-3-hydroxy-5-methyl-isoxazole-4-propionate - BSS balanced salt solution - CNS central nervous system - CICR Ca²⁺-induced Ca²⁺ release - DCKA 5,7-dichlorokynurenate - DNasel deoxyribonuclease I - DMEM Dulbecco's Modified Eagle's Medium - EGTA ethylene glycol-bis(-aminoethyl ether)N,N,N,N,-tetraacetic acid - FCS fetal calf serum - fura-2-AM 1-(2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy-2-ethane-N,N,N,N-te-traacetic acid, pentaacetoxymethyl ester - HEPES N-[2-hydroxyethyl] piperazine-N-[2-ethanesulfonic acid] - [Ca ²⁺] _i intracellular free Ca²⁺ concentration - LTP long-term potantiation - MK-801 (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]-cyclohepten-5,10-imine hydrogen maleate - NMDA N-methyl-D-aspartate     

Cyclic AMP enhances acetylcholine (ACh)- induced ion fluxes and catecholamine release by inhibiting Na+, K+-ATPase and participates in the responses to ACh in cultured bovine adrenal medullary chromaffin cells

Summary The effects of cyclic AMP (cAMP) on intracellular Na⁺ concentration ([Na⁺]i), membrane depolarization and intracellular Ca²⁺ concentration ([Ca²⁺]i) and the involvement of cAMP in acetylcholine (ACh)-induced such cellular events and catecholamine (CA) release were studied in cultured bovine adrenal medullary chromaffin cells. 8-Bromo-cyclic AMP (8Br-cAMP) and forskolin caused a rise in [Na⁺]i, membrane depolarization and a rise in [Ca²⁺]i and potentiated these responses and CA release to ACh. The effects of 8Br-cAMP or forskolin on ACh-induced changes of but not on basal level of [Na⁺]i, membrane potential and [Ca²⁺]i were blocked by tetrodotoxin (TTX, 1 M). In Na⁺ deprivated medium, forskolin failed to produce an increase in basal [Ca²⁺]i level and to potentiate ACh-induced rise. The similar results as in 8Br-cAMP and forskolin were obtained using ouabain, and 8Br-cAMP or foskolin produced no further effects in the presence of ouabain. Inhibitors of cAMP-dependent protein kinase not only blocked the effects of 8Br-cAMP and forskolin on membrane depolarization, [Ca²⁺]i rise and CA release, but also reduced these responses to ACh. From the similarity between the effects of cAMP and those of ouabain on the cellular events and the counteraction of the effects of cAMP by ouabain, it may be suggested that cAMP produces its effects on ion fluxes and CA release probably via an inhibition of Na⁺, K⁺-ATPase in intact chromaffin and cAMP may participate in the responses to ACh.     

Relationship between resting cytosolic Ca and responses induced byN-methyl-d-aspartate in hippocampal neurons

Cytosolic calcium concentrations ([Ca²⁺]_i) in cultured hippocampal neurons from rat embryos were measured using fura-2. Neurons with higher resting [Ca²⁺]_i showed greater [Ca²⁺]_i responses toN-methyl-d-aspartate (NMDA) and K⁺ depolarization. There was a strong relationship between resting [Ca²⁺]_i and the maximal changes in [Ca²⁺]_i (Δ[Ca²⁺]_i), which fit the our proposed equation to describe this relationship.     

Partial characterization of K-induced increase in [Ca]cyt and GnRH release in GT1-7 neurons

Secretion of pituitary gonadotropins is regulated centrally by the hypothalamic decapeptide gonadotropin releasing hormone (GnRH). Using the immortalized hypothalamic GT1-7 neuron, we characterized pharmacologically the dynamics of cytosolic Ca²⁺ and GnRH release in response to K⁺-induced depolarization of GT1-7 neurons. Our results showed that K⁺ concentrations from 7.5 to 60 mM increased [Ca²⁺]_cyt in a concentration-dependent manner. Resting [Ca²⁺]_cyt in GT1-7 cells was determined to be 69.7 ± 4.0 nM (mean ± S.E.M.; N = 69). K⁺-induced increases in [Ca²⁺]_cyt ranged from 58.2 nM at 7.5 mM [K⁺] to 347 nM at 60 mM [K⁺]. K⁺-induced GnRH release ranged from about 10 pg/ml at 7.5 mM [K⁺] to about 60 pg/ml at 45 mM [K⁺]. K⁺-induced increases in [Ca²⁺]_cyt and GnRH release were enhanced by 1 μM BayK 8644, an L-type Ca²⁺ channel agonist. The BayK enhancement was completely inhibited by 1 μM nimodipine, an L-type Ca²⁺ channel antagonist. Nimodipine (1 μM) alone partially inhibited K⁺-induced increases in [Ca²⁺]_cyt and GnRH release. Conotoxin (1 μM) alone had no effect on K⁺-induced GnRH release or [Ca²⁺]_cyt, but the combination of conotoxin (1 μM) and nimodipine (1 μM) inhibited K⁺-induced increase in [Ca²⁺]_cyt significantly more (p < 0.02) than nimodipine alone, suggesting that N-type Ca²⁺ channels exist in GT1-7 neurons and may be part of the response to K⁺. The response of [Ca²⁺]_cyt to K⁺ was linear with increasing [K⁺] whereas the response of GnRH release to increasing [K⁺] appeared to be saturable. K⁺-induced increase in [Ca²⁺]_cyt and GnRH release required extracellular [Ca²⁺]. These experiments suggest that voltage dependent N- and L-type Ca²⁺ channels are present in immortalized GT1-7 neurons and that GnRH release is, at least in part, dependent on these channels for release of GnRH.     

Measurement of intracellular Ca in the bullfrog sympathetic ganglion cells using fura-2 fluorescence

The intracellular free ([Ca²⁺]_i) of the bullfrog sympathetic ganglion cell was measured with fura-2 fluorescence under various conditions, and compared with changes in membrane potential recorded with an intracellular electrode. The [Ca²⁺]_i was 109 nM on average under the resting condition and increased by raising the extracellular K⁺, stimulating repetitively the pre- or post-ganglionic nerve, or by applying acetylcholine or muscarine. Since all these procedures depolarized the cell membrane, most of the rise in [Ca²⁺]_i could be the result of opening of voltage-dependent Ca²⁺ channels. However, Ca²⁺ entries through nicotinic acetylcholine receptor channels and the channel activated by the muscarinic acetylcholine receptor were also indicated by considering the threshold for the opening of voltage-dependent Ca²⁺ channels (for both entries) or a limited number of the cells showing the latter response.     

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.     

Inhibition by neuroprotective drug NS-7 of nicotine-induced Na influx, Ca influx and catecholamine secretion in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells, NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride], a newly-synthesized neuroprotective drug, inhibited nicotine-induced ²²Na⁺ influx via nicotinic receptors (IC₅₀=15.5 μM); the suppression by NS-7 was observed in the presence of ouabain, an inhibitor of Na⁺,K⁺-ATPase, and was not attenuated upon the washout of NS-7. NS-7 decreased nicotine-induced maximum influx of ²²Na⁺ without altering the EC₅₀ value of nicotine. Also, NS-7 diminished nicotine-induced ⁴⁵Ca²⁺ influx via nicotinic receptors and voltage-dependent Ca²⁺ channels (IC₅₀=14.1 μM) and catecholamine secretion (IC₅₀=19.5 μM). These results suggest that NS-7 produces noncompetitive and long-lasting inhibitory effects on neuronal nicotinic receptors in adrenal chromaffin cells, and interferes with the stimulus-secretion coupling.     

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.     

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.     

Warm cells revealed by microfluorimetry of Ca in cultured dorsal root ganglion neurons

Warm cells were identified by Fura-PE3-based microfluorimetry of Ca²⁺ in cultured dorsal root ganglion (DRG) neurons. In response to a physiologically relevant stimulus temperature (43°C), a subpopulation of small DRG neurons from new born rats increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i). Seven percent of the cells responded to the warm stimulus. The stimulus evoked elevation in [Ca²⁺]_i from 52.5±9.5 nM (mean±S.D., n=18) to 171.0±15.6 nM in cells between 15 and 25 μm in diameter. The depletion of extracellular Ca²⁺ diminished the Ca²⁺ elevation. The Na⁺-free condition also diminished the response. We concluded that the heat stimulation opens nonselective cation channels in putative warm cells from DRG neurons.     

Activation of ATP receptor increases the cytosolic Ca concentration in nucleus accumbens neurons of rat brain

ATP increased the cytosolic Ca²⁺ concentration ([Ca]_i) in nucleus accumbens neurons acutely dissociated from rat brain. The ATP response was dependent on external Ca²⁺ and Na⁺, and was blocked by voltage-dependent Ca²⁺ channel blockers. The results suggest that the ATP-induced depolarization increases Ca²⁺ influx resulting in the increase in [Ca]_i.