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.     

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.     

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.     

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.     

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.     

NMDA-induced increase in [Ca]i andCa uptake in acutely dissociated brain cells derived from adult rats

A preparation of acutely dissociated brain cells derived from adult (3-month-old) rat has been developed under conditions preserving the metabolic integrity of the cells and the function of N-methyl-d-aspartate (NMDA) receptors. The effects of glutamate and NMDA on [Ca²⁺]_i measured with fluo3 and⁴⁵Ca²⁺ uptake have been studied on preparations derived from hippocampus and cerebral cortex. Glutamate (100 μM) and N-methyl-dl-aspartate (200 μM) increased [Ca²⁺]_i by 26-12 nM and 23-9 nM after 90 s in cerebral cortex and hippocampus, and stimulated⁴⁵Ca²⁺ uptake about 16–10% in the same regions. The increases in [Ca²⁺]_i and⁴⁵Ca²⁺ uptake were inhibited by 40% in the presence of 1 mM MgCl₂ and by 90–50% in the presence of MK-801. The results indicate (a) that a large fraction of the [Ca²⁺]_i response to glutamate in freshly dissociated brain cells from the adult rat involves NMDA receptors, (b) when compared with results in newborn rats, there is a substantial blunting of the [Ca²⁺]_i increase in adult age.     

Pharmacologic characterization of refilling inositol 1,4,5-trisphosphate-sensitive Ca stores in NG108-15 cells

Following mobilization with the inositol 1,4,5-trisphosphate (IP₃)-generating agonist bradykinin, Ca²⁺ stores in neuroblastoma × glioma hybrid, NG108-15 cells require extracellular Ca²⁺ to refill. The process by which this store refills with Ca²⁺ was characterized by recording bradykinin-induced intracellular free Ca²⁺ concentration transients as an index of the degree of refilling of the store. Cyclopiazonic acid, a microsomal Ca²⁺ ATPase inhibitor, reversibly depleted intracellular Ca²⁺ stores in these cells, but did not recruit detectable Ca²⁺ influx, suggesting that these cells lack substantial capacitative Ca²⁺ entry. The paucity of voltage-sensitive Ca²⁺ channels in undifferentiated NG108-15 cells, suggested that a channel analogous to that proposed to mediate capacitative Ca²⁺ entry in nonexcitable cells might assist refilling IP₃-sensitive Ca²⁺ stores in these cells. The possibility that compounds shown previously to inhibit capacitative Ca²⁺ entry in nonexcitable cells might inhibit the refilling of the IP₃-sensitive store in NG108-15 cells was explored. The IP₃-sensitive store was depleted by exposure to bradykinin, allowed to refill briefly in the presence of the test compound and then challenged again with bradykinin to evaluate the degree of refilling of the store. The imidazole derivatives, econazole (10 μM), L-651582 (10 μM)and SKF 96365 (20 μM), all completely blocked the bradykinin-induced Ca²⁺ response. Calmodulin antagonists, W-7 (100 μM)and trifluoperazine (10 μM), were also effective, although at concentrations well above those required to inhibit calmodulin. Because of the high concentrations required to inhibit bradykinin responses, the possibility that these agents might have additional effects was explored. Compounds were tested in a paradigm in which the store was preloaded with Ca²⁺ before treatment. All of these agents depleted, at least partially, the preloaded store. Econazole was the least effective of the compounds tested for releasing stores, although it was comparable to the other compounds for inhibition of refilling. Although NG108-15 cells refill intracellular Ca²⁺ stores by a plasmalemmal Ca²⁺ leak, this leak shares a pharmacology similar to the capacitative Ca²⁺ entry pathway described for nonexcitable cells.     

Metabolic inhibition potentiates AMPA-induced Ca fluxes and neurotoxicity in rat cerebellar granule cells

The effects of partial metabolic inhibition (induced by 2 h exposure to low concentrations of cyanide (NaCN)) on the glutamate receptor agonist α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-induced excitotoxicity and elevation of free cytoplasmic Ca²⁺ levels ([Ca²⁺]_i) were studied in glucose-deprived primary cultures of cerebellar granule cells. Co-application of AMPA plus NaCN caused a marked increase of cell death, with morphological features of both necrotic and apoptotic cell death as estimated by the capacity of cultured cerebellar granule cells to metabolize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide into formazan (MTT method), and by measuring the amount of DNA fragmentation in neurons using an ELISA test for histone-bound DNA fragments, respectively. Cell morphology was assessed by confocal microscopy of propidium iodide-stained cultures. No toxic effects were observed when AMPA or a low concentration of NaCN (0.1–0.3 mM; in the presence of NMDA receptor antagonist MK-801; 10 μM) were applied alone. The neurotoxic actions induced by AMPA plus NaCN were preceded and accompanied by a significant elevation of [Ca²⁺]_i, as well as by depletion of neuronal ATP stores. The marked enhancement in the functional responsiveness of AMPA receptors in energetically compromised neurons suggests that at least under certain conditions AMPA receptors may play an important role in excitotoxic processes which might be of relevance for the slowly developing neuronal death seen in several neurodegenerative diseases.     

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     

Chronic lead exposure alters presynaptic calcium regulation and synaptic facilitation in Drosophila larvae

Prolonged exposure to inorganic lead (Pb²⁺) during development has been shown to influence activity-dependent synaptic plasticity in the mammalian brain, possibly by altering the regulation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). To explore this possibility, we studied the effect of Pb²⁺ exposure on [Ca²⁺]_i regulation and synaptic facilitation at the neuromuscular junction of larval Drosophila. Wild-type Drosophila (CS) were raised from egg stages through the third larval instar in media containing either 0 μM, 100 μM or 250 μM Pb²⁺ and identified motor terminals were examined in late third-instar larvae. To compare resting [Ca²⁺]_i and the changes in [Ca²⁺]_i produced by impulse activity, the motor terminals were loaded with a Ca²⁺ indicator, either Oregon Green 488 BAPTA-1 (OGB-1) or fura-2 conjugated to a dextran. We found that rearing in Pb²⁺ did not significantly change the resting [Ca²⁺]_i nor the Ca²⁺ transient produced in synaptic boutons by single action potentials (APs); however, the Ca²⁺ transients produced by 10 Hz and 20 Hz AP trains were larger in Pb²⁺-exposed boutons and decayed more slowly. For larvae raised in 250 μM Pb²⁺, the increase in [Ca²⁺]_i during an AP train (20 Hz) was 29% greater than in control larvae and the [Ca²⁺]_i decay τ was 69% greater. These differences appear to result from reduced activity of the plasma membrane Ca²⁺ ATPase (PMCA), which extrudes Ca²⁺ from these synaptic terminals. These findings are consistent with studies in mammals showing a Pb²⁺-dependent reduction in PMCA activity. We also observed a Pb²⁺-dependent enhancement of synaptic facilitation at these larval neuromuscular synapses. Facilitation of EPSP amplitude during AP trains (20 Hz) was 55% greater in Pb²⁺-reared larvae than in controls. These results showed that Pb²⁺ exposure produced changes in the regulation of [Ca²⁺]_i during impulse activity, which could affect various aspects of nervous system development. At the mature synapse, this altered [Ca²⁺]_i regulation produced changes in synaptic facilitation that are likely to influence the function of neural networks.     

Cultured postnatal rat septohippocampal neurons change intracellular calcium in response to ethanol and nerve growth factor

Ethanol exposure affects cellular mechanisms involved in the regulation of calcium (Ca²⁺) homeostasis. Neurotrophins, such as nerve growth factor (NGF), stabilize intracellular Ca²⁺([Ca²⁺]_i) during a variety of neurotoxic insults. In this study, changes in [Ca²⁺]_i during treatment with ethanol and NGF were measured at the cell body of neurons using the Ca²⁺ indicator indo-1. Cultured postnatal day-of-birth (P0) septohippocampal (SH) neurons that were labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI), increased [Ca²⁺]_i in response to ethanol. This response was dose-related. P0 SH neurons treated with NGF had lower [Ca²⁺]_i than neurons withdrawn from NGF, implying that NGF may modulate Ca²⁺ homeostasis in these neurons. NGF also prevented the dose-related increase in [Ca²⁺]_i in ethanol-treated SH neurons. The SH neurons increased [Ca²⁺]_i when they were stimulated with 30 mM potassium chloride (KCl). Ethanol inhibited the potassium-stimulated change in [Ca²⁺]_i but the combination of ethanol and NGF caused [Ca²⁺]_i to increase with 100 mg% and 400 mg% ethanol and to decrease to a lower level with 200 mg% ethanol. These data were compared to data from previously published similar aged medial septal (MS) neurons (B. Webb, S.S. Suarez, M.B. Heaton, D.W. Walker, Clin. Exp. Res. 20 (1996) 1385–1394) and with embryonic gestational day 21 (E21) SH neurons (B. Webb, S.S. Suarez, M.B. Heaton, D.W. Walker, Brain Res. 729 (1996) 176–189). Differences in [Ca²⁺]_i responses were observed in ethanol and NGF-treated postnatal SH neurons compared with P0 MS neurons and E21 SH neurons. Of these differences, most occurred during the combined treatment with ethanol and NGF compared with either treatment alone.     

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.     

Re-evaluation of acute neurotoxic effects of Cd on mesencephalic trigeminal neurons of the adult rat

The mechanism of Cd²⁺ neurotoxicity, which is considered to be secondary to changes in blood vessels, was re-evaluated in dissociated mesencephalic trigeminal (Me5) neurons of the adult rat. Cd²⁺ induced morphological changes in Me5 neurons at 0.1 and 1 mM but not at 0.01 mM. The changes appeared predominantly in the cytoplasm: destruction of the cytoplasmic organelles, swelling and vacuolization of the cell body, and finally resulted in cell lysis. These observations indicate necrosis rather than apoptosis, and no sign of degraded nuclear DNA, characteristic to apoptosis, was detected by the TUNEL technique. Using a Ca²⁺-sensitive dye Indo-1, Cd²⁺ was found to elevate the intracellular Ca²⁺ concentration [Ca²⁺]_i (both in the cytoplasm and the nucleus). Both the elevation in [Ca²⁺]_i and the morphological alteration were inhibited either by removing Ca²⁺ from the bathing medium or by the application of BAPTA/AM (10 μM), a membrane-permeable intracellular Ca²⁺ chelator. Furthermore, neither morphological changes nor elevation in [Ca²⁺]_i by Cd²⁺ occurred in the presence of Zn²⁺. It is concluded that (1) Cd²⁺ can directly affect nerve cells, (2) toxicity of Cd²⁺ on Me5 neurons is mediated by continuous elevation in [Ca²⁺]_i, (3) Cd²⁺ induces necrotic cell death, and (4) Cd²⁺ neurotoxicity can be antagonized by Zn²⁺.     

Activation of central muscarinic receptors inhibit Ca/Mg ATPase and ATP-dependent Ca transport in synaptic membranes

Preparations of lysed synaptosomes exhibit a high affinity Ca²⁺/Mg²⁺ ATPase and ATP-dependent Ca²⁺ accumulation activity, with aK_m forCa²⁺ 0.5 μM, close to the cytosolic concentration of Ca²⁺. When these membrane suspensions were incubated with cholinergic agonists muscarine or oxotremorine (1–20 μM), both Ca²⁺/Mg²⁺ ATPase and ATP-dependent Ca²⁺ uptake were inhibited in a concentration-dependent fashion. Atropine alone (0.5–1.0 μM) had no effect on either enzyme or uptake activity, but significantly inhibited the actions of both muscarine and oxotremorine. No significant effects by cholinergic agonists or antagonists were seen on fast or slow phase voltage-dependent Ca²⁺ channels or Na⁺-Ca²⁺ exchange. These results suggest that activation of presynaptic muscarinic receptors produce inhibition of two processes required for the buffering of optimal free Ca²⁺ by the nerve terminal. Activation of presynaptic muscarinic receptors have been reported to reduce the release of ACh from nerve terminals. Alterations in intracellular free Ca²⁺ may contribute to a reduction in transmitter (ACh) release seen following activation of cholinergic receptors.     

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.     

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.     

Inflammation alters AMPA‐stimulated calcium responses in dorsal striatal D2 but not D1 spiny projection neurons

Neuroinflammation precedes neuronal loss in striatal neurodegenerative diseases and can be exacerbated by the release of proinflammatory molecules by microglia. These molecules can affect trafficking of AMPARs. The preferential trafficking of calcium‐permeable versus impermeable AMPARs can result in disruptions of [Ca²⁺]_i and alter cellular functions. In striatal neurodegenerative diseases, changes in [Ca²⁺]_i and L‐type voltage‐gated calcium channels (VGCCs) have been reported. Therefore, this study sought to determine whether a proinflammatory environment alters AMPA‐stimulated [Ca²⁺]_i through calcium‐permeable AMPARs and/or L‐type VGCCs in dopamine‐2‐ and dopamine‐1‐expressing striatal spiny projection neurons (D2 and D1 SPNs) in the dorsal striatum. Mice expressing the calcium indicator protein, GCaMP in D2 or D1 SPNs, were utilized for calcium imaging. Microglial activation was assessed by morphology analyses. To induce inflammation, acute mouse striatal slices were incubated with lipopolysaccharide (LPS). Here we report that LPS treatment potentiated AMPA responses only in D2 SPNs. When a nonspecific VGCC blocker was included, we observed a decrease of AMPA‐stimulated calcium fluorescence in D2 but not D1 SPNs. The remaining agonist‐induced [Ca²⁺]_i was mediated by calcium‐permeable AMPARs because the responses were completely blocked by a selective calcium‐permeable AMPAR antagonist. We used isradipine, the highly selective L‐type VGCC antagonist to determine the role of L‐type VGCCs in SPNs treated with LPS. Isradipine decreased AMPA‐stimulated responses selectively in D2 SPNs after LPS treatment. Our findings suggest that dorsal striatal D2 SPNs are specifically targeted in proinflammatory conditions and that L‐type VGCCs and calcium‐permeable AMPARs are important mediators of this effect.