Inhibition of glutamate release by bupropion in rat cerebral cortex nerve terminals

Central glutamate neurotransmission has been postulated to play a role in pathophysiology of depression and in the mechanism of antidepressants. The present study was undertaken to elucidate the effect and the possible mechanism of bupropion, an atypical antidepressant, on endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes). Result showed that bupropion exhibited a dose-dependent inhibition of 4-aminopyridine (4-AP)-evoked release of glutamate. The effect of bupropion on the evoked glutamate release was prevented by the chelating the intrasynaptosomal Ca²⁺ ions, and by the vesicular transporter inhibitor, but was insensitive to the glutamate transporter inhibitor. Bupropion decreased depolarization-induced increase in [Ca²⁺]_C, whereas it did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization. The effect of bupropion on evoked glutamate release was abolished by the N-, P- and Q-type Ca²⁺ channel blocker, but not by the ryanodine receptor blocker, or the mitochondrial Na⁺/Ca²⁺ exchanger blocker. In addition, the inhibitory effect of bupropion on evoked glutamate release was prevented by the mitogen-activated/extracellular signal-regulated kinase kinase (MEK) inhibitors. Western blot analyses showed that bupropion significantly decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), and this effect also was blocked by MEK inhibitor. These results are the first to suggest that, in rat cerebrocortical nerve terminals, bupropion suppresses voltage-dependent Ca²⁺ channel and MEK/ERK activity and in so doing inhibits evoked glutamate release. This finding may provide important information regarding the beneficial effects of bupropion in the brain.     

Local anesthetics inhibit glutamate release from rat cerebral cortex synaptosomes

Local anesthetics have been widely used for regional anesthesia and the treatment of cardiac arrhythmias. Recent studies have also demonstrated that low‐dose systemic local anesthetic infusion has neuroprotective properties. Considering the fact that excessive glutamate release can cause neuronal excitotoxicity, we investigated whether local anesthetics might influence glutamate release from rat cerebral cortex nerve terminals (synaptosomes). Results showed that two commonly used local anesthetics, lidocaine and bupivacaine, exhibited a dose‐dependent inhibition of 4‐AP‐evoked release of glutamate. The effects of lidocaine or bupivacaine on the evoked glutamate release were prevented by the chelation of extracellular Ca²⁺ ions and the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor dl ‐threo‐beta‐benzyl‐oxyaspartate did not have any effect on the action of lidocaine or bupivacaine. Both lidocaine and bupivacaine reduced the depolarization‐induced increase in [Ca²⁺]_C but did not alter 4‐AP‐mediated depolarization. Furthermore, the inhibitory effect of lidocaine or bupivacaine on evoked glutamate release was prevented by blocking the Ca_v2.2 (N‐type) and Ca_v2.1 (P/Q‐type) channels, but it was not affected by blocking of the ryanodine receptors or the mitochondrial Na⁺/Ca²⁺ exchange. Inhibition of protein kinase C (PKC) and protein kinase A (PKA) also prevented the action of lidocaine or bupivacaine. These results show that local anesthetics inhibit glutamate release from rat cortical nerve terminals. This effect is linked to a decrease in [Ca²⁺]_C caused by Ca²⁺ entry through presynaptic voltage‐dependent Ca²⁺ channels and the suppression of the PKA and PKC signaling cascades. Synapse 67:568–579, 2013 . © 2013 Wiley Periodicals, Inc.     

Measured increase in intracellular Ca during stimulated release of endogenous glutamate from human cerebrocortical synaptosomes

Presynaptic terminals (synaptosomes) prepared from guinea pig and rat cerebral cortex release endogenous glutamate in a Ca²⁺-dependent manner in response to membrane depolarisation. In the present study, synaptosomes were prepared from human cerebral cortex removed in association with temporal lobe resections in epileptic patients. The cytosolic free Ca²⁺ concentration increased from 474±66 before to 649±89 nM after 2 min depolarisation. The basal level of free cytosolic Ca²⁺ is higher and the increase in response to depolarisation is more pronounced in human synaptosomes than observed in animal experiments. The Ca²⁺-dependent glutamate release, estimated as the difference between total — and the Ca²⁺-independent glutamate release, increased from 0 to 5.4±1.9 nmol/mg protein. The released amount of glutamate is larger than reported in animal models. These results demonstrate that membrane depolarisation of synaptosomes from human brain evokes a rapid rise in cytosolic free Ca²⁺ and a more prolonged rise in synaptic, Ca²⁺-dependent glutamate release.     

Neurosteroid allopregnanolone inhibits glutamate release from rat cerebrocortical nerve terminals

Allopregnanolone, an active metabolite of progesterone, has been reported to exhibit neuroprotective activity in several preclinical models. Considering that the excitotoxicity caused by excessive glutamate is implicated in many brain disorders, the effect of allopregnanolone on glutamate release in rat cerebrocortical nerve terminals and possible underlying mechanism were investigated. We observed that allopregnanolone inhibited 4‐aminopyridine (4‐AP)‐evoked glutamate release, and this inhibition was prevented by chelating the extracellular Ca²⁺ ions and the vesicular transporter inhibitor. Allopregnanolone reduced the elevation of 4‐AP‐evoked intrasynaptosomal Ca²⁺ levels, but did not affect the synaptosomal membrane potential. In the presence of N‐, P/Q‐, and R‐type channel blockers, allopregnanolone‐mediated inhibition of 4‐AP‐evoked glutamate release was markedly reduced; however, the intracellular Ca²⁺‐release inhibitors did not affect the allopregnanolone effect. Furthermore, allopregnanolone‐mediated inhibition of 4‐AP‐evoked glutamate release was completely abolished in the synaptosomes pretreated with inhibitors of Ca²⁺/calmodulin, adenylate cyclase, and protein kinase A (PKA), namely calmidazolium, MDL12330A, and H89, respectively. Additionally, the allopregnanolone effect on evoked glutamate release was antagonized by the GABA_A receptor antagonist SR95531. Our data are the first to suggest that allopregnanolone reduce the Ca²⁺ influx through N‐, P/Q‐, and R‐type Ca²⁺ channels, through the activation of GABA_A receptors present on cerebrocortical nerve terminals, subsequently suppressing the Ca²⁺‐calmodulin/PKA cascade and decreasing 4‐AP‐evoked glutamate release.     

Differential effect of a dihydropyridine derivative to Ca entry pathways in neuronal preparations

Nicardipine is one of the 1,4-dihydropyridine derivatives known as blockers for the voltage-dependent Ca²⁺ channels in muscle cells. The effects of nicardipine on the neuronal functions were studied in several neuronal preparations including clonal rat pheochromocytoma (PC12) cells, rat brain synaptosomes and slices. Nicardipine failed to block the Ca²⁺-dependent action potentials and the after-spike hyperpolarizations evoked by intracellularly injected current pulses in rat pheochromocytoma cells, while the high K^+- stimulated Ca²⁺ influx and ATP release were dose-dependently inhibited in the same cells. In rat cerebral synaptosomes and cortical slices, nicardipine showed no blockade on the high K⁺-stimulated Ca²⁺ influx and transmitter releases. It was then suggested that the voltage-dependent Ca²⁺ channels are polymorphic among tissues or even in a single cell from the viewpoint of dihydropyridine susceptibility.     

The effect of 4-aminopyridine on acetylcholine release

Summary The effect of 2-, and 4-aminopyridine (4-APYR) on the release mechanism of acetylcholine (ACh) from the nerve terminals of the Auerbach plexus-longitudinal muscle preparation of the guinea-pig ileum, suspended in eserinized Krebs' solution, was investigated.2- and 4-APYR increased the release of ACh from the nerve terminals at rest and at both low and high frequency stimulation. The enhanced ACh release was found to be due to increased volley output. At lower frequency of stimulation, the potentiation of ACh release was much higher than at higher rate of stimulation.4-APYR was able to increase ACh release in the absence of [Ca²⁺]₀. However, when a Ca-chelating agent, EDTA, was also added to the Ca-free Krebs' solution, 4-APYR was entirely ineffective. The depression of ACh release induced by Mg-excess was completely antagonized by 4-APYR. Tetrodotoxin (TTX) prevented augmentation of ACh release by 4-APYR.It is suggested that 4-APYR lowers the demand of nerve terminals for [Ca²⁺]₀ required for the excitation-secretion coupling process. The presence of a low concentration [Ca²⁺]₀, however, is essential for the action of 4-APYR.     

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.     

Effects of GABA on presumed presynaptic Ca entry in hippocampal slices

Evoked field potentials and changes in [Ca²⁺]_o were measured in the ‘in vitro’ hippocampal slice of the rat. When [Ca] in the perfusion medium was lowered to 0.2 mM synaptic transmission from Schaffer collateral/comissural fibers was blocked. Nevertheless, repetitive stimulation of afferent fibers still resulted in detectable decreases of [Ca²⁺]_o. In contrast to findings in normal medium these decreases in [Ca²⁺]_o could be larger in stratum radiatum than in stratum pyramidale, so mimicking the spatial distribution of activated afferent fibers. These findings suggest, that the loss of extracellular Ca²⁺ in low Ca²⁺ media is predominantly due to entry into presynaptic terminals. This permits to study effects of drugs on presynaptic endings. We found that iontophoretic application of GABA is capable to block this presumed presynaptic Ca²⁺ entry without affecting the electrical activity of the afferent fibers. This suggests, that presynaptic GABA receptors occur also in the Schaffer collateral/commissural fiber system.     

Calcium transport in and out of brain nerve endings in vitro—the role of synaptosomal plasma membrane Ca-ATPase in Ca-extrusion

The effects of cellular cations and ATP on calcium transport in and out of the nerve endings (synaptosomes) of mice brain were studied. The synaptosomes accumulated⁴⁵Ca time-dependently in the absence of ATP or other additions for at least 10 min. When ATP was present, the overall⁴⁵Ca accumulation was decreased and was maximal at about 4 min, after which it started to decline. Studies on the effects of cations with or without ATP at 4 min revealed selective activities for different cations. Mg²⁺ inhibited⁴⁵Ca accumulation in the absence of ATP but increased⁴⁵Ca accumulation when ATP was present. Similarly, ATP increased⁴⁵Ca accumulation only when Mg²⁺ was present. Na⁺, on the other hand, inhibited⁴⁵Ca accumulation both in the presence and absence of ATP and/or Mg²⁺. K⁺ increased⁴⁵Ca accumulation in the presence of ATP with or without Mg²⁺; however, K⁺-stimulation was not noted in the presence of 100 mM Na⁺, and in fact, K⁺ became inhibitory. The ATP-stimulated⁴⁵Ca accumulation in the presence of Mg²⁺ peaked within 4–6 min and then declined, suggesting release of⁴⁵Ca. Compatible with this notion, in⁴⁵Ca-loaded synaptosomes, ATP evoked⁴⁵Ca release which was accompanied by the appearance of P_i in the medium. Although ATP-activated⁴⁵Ca-release can occur in the presence of Mg²⁺, Mg²⁺ is not required and, infact, is inhibitory. Rapid release of⁴⁵Ca was also noted when⁴⁵Ca-loaded synaptosomes were incubated in the presence of Na⁺ without ATP. It is concluded that Mg²⁺, Na⁺,⁺K and ATP each has a specific role in regulating Ca²⁺ permeability of the plasma membrane, calcium binding and calcium extrusion.     

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.     

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     

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.     

Fluoxetine depresses glutamate exocytosis in the rat cerebrocortical nerve terminals (synaptosomes) via inhibition of P/Q-type Ca2+ channels

Fluoxetine, an antidepressant that is used clinically in the treatment of mood disorders, is a selective serotonin reuptake inhibitor. In the present study we investigated the effects of fluoxetine on 4-aminopyridine (4AP)-evoked glutamate release in cerebrocortical nerve terminals (synaptosomes). Fluoxetine suppressed the release of glutamate evoked by 4AP in a concentration-dependent manner. This effect was associated with a reduction in the depolarization-evoked increase in cytosolic free calcium levels in the absence of significant effect on the synaptosomal membrane potential. In addition, both ionomycin- and sucrose-evoked glutamate releases were not affected by fluoxetine, indicating that fluoxetine-mediated inhibition of glutamate release is not a direct effect on the exocytotic machinery. Furthermore, the inhibitory action of fluoxetine was completely abolished in synaptosomes pretreated with P/Q type Ca(2+) channel blocker omega-agatoxin IVA (omega-AgTX IVA) or protein kinase C (PKC) stimulator 4beta-phorbol 12, 13-dibutyrate (PDBu). These results suggest that, in cerebrocortical nerve terminals, fluoxetine inhibits glutamate release through the suppression of P/Q type Ca(2+) channel activity. The presynaptic action of fluoxetine is mediated by a PKC-sensitive signaling pathway. Synapse 48:170-177, 2003.     

Glutamate selectively increases the high-threshold Ca channel current in sensory and hippocampal neurons

Previous studies resulted in conflicting conclusions that glutamate application either decreases or increases the activity of Ca²⁺ channels in hippocampal neurons. We studied whole-cell Ca²⁺ currents (I_Ca) in chick dorsal root ganglion neurons and rat hippocampal cells. For both cell types glutamate (1–30 μM) increased high-threshold Ca²⁺ current. It was independent of the charge carriers, Ca²⁺ or Ba²⁺. Low-threshold Ca²⁺ channel current and the fast sodium current were not changed with glutamate application. The effect developed within 1–2 min and then further facilitated after washout of the agonist. A second application of glutamate produced no additional increase in _ICa. No changes in the time-course of whole-cell currents were observed, suggesting that glutamate recruits ‘sleepy’ Ca²⁺ channels. Whatever its mechanism, overlasting increase of I_Ca by glutamate may be important in neuronal plasticity.     

Effects of Hg and CH3Hg on Ca fluxes in rat brain microsomes

A permanent increase in cytosolic Ca²⁺ levels seems to be associated with various pathological situations which may result in cell death. Hg²⁺ and CH₃Hg⁺ are potent neurotoxic agents, but the precise molecular mechanism(s) underlying their effects are not sufficiently understood. In the present study we investigated the potential role of Ca²⁺-ATPase located in the endoplasmic reticulum as a molecular target for mercury. Hg²⁺ and CH₃Hg⁺ inhibited Ca²⁺-ATPase and Ca²⁺ uptake by brain microsomes with similar potencies. However, the inhibitory potency of Hg²⁺ was higher than that of CH3Hg+, probably reflecting differences in the affinity for the sulfhydryl groups of these compounds. Passive or unidirectional Ca²⁺ efflux (measured in the absences of Ca²⁺-ATPase ligands) was increased significantly by CH₃Hg⁺ and Hg²⁺. Again, the potency of Hg²⁺ was higher than that of CH₃Hg⁺. Blockers of Ca²⁺ channels (ruthenium red, procaine, heparin) did not affect the increase in passive Ca t+ efflux induced by mercury compounds, possibly indicating that Ca²⁺ release occurs through Ca²⁺-ATPase. Addition of physiological concentrations of glutathione (GSH) simultaneously with mercury abolished the inhibitory effects of both forms of Hg on Ca Z+-transport. However, if the enzyme was first inhibited with Hg²⁺ or CH₃Hg⁺ and subsequently treated with GSH, the reversal of inhibition was about 50%, suggesting that part of the cysteinyl residues involved in the inhibitory actions of mercury in Ca t+-transport bind to mercury with an extremely high affinity.     

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.     

Spontaneous miniature hyperpolarizations of presynaptic nerve terminals in the chick ciliary ganglion

Intracellular recordings from presynaptic nerve terminals in the chick ciliary ganlion revealed the presence of spontaneous miniature hyperpolarizations in virtually all (86%) nerve terminals examined. These spontaneous events appeared as small, brief hyperpolarizations at resting potential and were observed to increase or decrease as the membrane potential was depolarized or hyperpolarized from rest, respectively. The hyperpolarizing potentials were sensitive to blockade by tetraethylammonium and Ba²⁺, while caffeine increased then abolished these events. The voltage fluctuations were unaffected by tetrodotoxin, low Ca²⁺ external solution or the synaptic blockers, picrotoxin and strychnine. These spontaneous, transient, miniature hyperpolarizations may be due to the brief and co-ordinated activation of between 15–60 Ca²⁺-dependent K⁺ channels following the release of Ca²⁺ from internal stores.     

Relation of exocytosis and Ca-activated K current during Ca release from intracellular stores in individual rat chromaffin cells

Measurement of the change in cell membrane capacitance (C_m) along with the change in I_K(Ca) was used to investigate the effects of bradykinin and caffeine on the secretory process in rat adrenal chromaffin cells. In a Ca²⁺-free external solution, bradykinin (100 nM) caused a transient increase in C_m with a concurrent change in I_K(Ca). Extracellular application of neomycin as an inhibitor of phospholipase C activity reversibly inhibited the bradykinin-activated event, implying an IP₃-mediated increase of submembrane-free Ca²⁺. The increases in C_m and I_K(Ca) caused by bradykinin were transient even with the sustained application of bradykinin. Caffeine also caused exocytosis in the Ca²⁺-free solution, and this was irreversibly blocked by ryanodine (1 μM) in a use-dependent manner. Caffeine-sensitive intracellular Ca²⁺ stores were also depleted in several seconds and recovered by an influx of external Ca²⁺. The sequential application of bradykinin and caffeine showed that these are likely to activate Ca²⁺ release from the same or distinct but rapidly equilibrating intracellular Ca²⁺ stores. The single cell assay of exocytosis and the increase in I_K(Ca) revealed cell-to-cell variability in bradykinin- and caffeine-induced exocytotic response. Our results suggest that Ca²⁺ release from intracellular stores potentially increases submembrane Ca²⁺ concentration and modulates simultaneously two submembrane Ca²⁺-dependent processes, exocytosis and I_K(Ca), in rat adrenal chromaffin cells.     

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.     

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.