Inhibition of NMDA-induced protein kinase C translocation by a Zn chelator: Implication of intracellular Zn

The role of intracellular Zn²⁺ in the translocation of protein kinase C from cytosol to membrane fractions was examined by the [³H]phorbol 12,13-dibutyrate (PDBu) binding method in guinea pig cerebral synaptoneurosomes. N-methyl-d-aspartate (NMDA, 100 μM) and calcium ionophore A23187 (0.3–30 μM) decreased the binding activity in the cytosol with a concomitant increase in the membrane fractions. Pretreatment of synaptoneurosomes with a heavy metal chelator, N,N,N′,N′-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), inhibited the NMDA- and A23187-induced changes of the distribution of [³H]PDBu binding sites in cytosol and membrane fractions. The inhibitory effect of TPEN was negated by a preincubation of TPEN with equimolar Zn²⁺ but not by that with Ca²⁺. The addition of 500 μM Zn²⁺ to the lysate of synaptoneurosomes induced an increase of [³H]PDBu binding activity in the membrane fraction with a concomitant decrease in the cytosol fraction, as did 100 μM Ca²⁺. Low concentrations of Zn²⁺ (10 μM), which alone had no effect on the distribution of the binding, significantly enhanced the effect of 10 μM Ca²⁺ in the lysate. Under those conditions TPEN inhibited the Zn²⁺-potentiated Ca²⁺-dependent changes in the binding. These results suggest that intracellular Zn²⁺ is essential for the agonist-induced translocation of protein kinase C in guinea pig synaptoneurosomes.     

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.     

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.     

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.     

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.     

Acute ethanol alters calcium signals elicited by glutamate receptor agonists and K depolarization in cultured cerebellar Purkinje neurons

The effect of acute ethanol on Ca²⁺ signals evoked by ionotropic (iGluR) and metabotropic (mGluR) glutamate receptor (GluR) activation and K⁺ depolarization was examined in cultured rat cerebellar Purkinje neurons to assess the ethanol sensitivity of these Ca²⁺ signaling pathways. Mature Purkinje neurons 3 weeks in vitro were studied. iGluRs were activated by (RS)-α-amino-3-hydroxyl-5 methyl-4-isoxazolepropionic acid (AMPA; 1 and 5 μM) and domoate (5 μM). mGluRs were activated by (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD; 300 μM) and (R,S)-3,5-dihydroxyphenylglycine (DHPG; 200 μM). These agents and K⁺ (150 mM) were applied from micropipettes by brief (1 s) microperfusion pulses. Ca²⁺ levels were monitored at 2–3 s intervals during pre- and post-stimulus periods using microscopic digital imaging and the Ca²⁺ sensitive dye fura-2. iGluR and mGluR agonists and K⁺ produced abrupt increases in intracellular Ca²⁺ that slowly recovered to baseline resting levels. Acute exposure to ethanol at 33 mM (150 mg%) and 66 mM (300 mg%) significantly reduced the amplitude of the Ca²⁺ signals to iGluR agonists and K⁺ with little or no effect on Ca²⁺ signals to mGluR agonists. In contrast, acute ethanol at 10 mM (45 mg%) had no effect on the Ca²⁺ signals to the iGluR agonist AMPA but significantly enhanced the Ca²⁺ signals to the mGluR agonist DHPG. These results show that ethanol modulates Ca²⁺ signaling linked to GluR activation in a receptor subtype specific manner, and suggest that Ca²⁺ signaling pathways linked to GluR activation and membrane depolarization may be important mechanisms by which ethanol alters the transduction of excitatory synaptic signals at glutamatergic synapses and thereby affects intercellular and intracellular communication in the CNS.     

On the role of calcium ions in the presynaptic alpha-receptor mediated inhibition of [H]noradrenaline release from rat brain cortex synaptosomes

Rat brain cortex synaptosomes, previously labeled by incubation with [³H]noradrenaline ([³H]NA) were continuously superfused with Krebs-Ringer media. Release of [³H]NA was induced by superfusion with medium containing either 15 mM K⁺, 20 μM veratrine or 1 μM of the calcium-ionophore A 23187 and was strongly dependent on the concentration of Ca²⁺ in the medium. Noradrenaline (1μM, in the presence of the uptake inhibitor desipramine) inhibited K⁺-induced [³H]NA release by activation of presynaptic alpha-receptors. When the Ca²⁺-concentration in the medium was reduced, or the Mg²⁺-concentration increased, [³H]NA release appeared to be more susceptible to alpha-receptor mediated inhibition.Noradrenaline (1 μM) inhibited [³H]NA release induced by 15 mM K⁺, in the presence of 0.075 Ca²⁺ and 10 mM Mg²⁺, by 86%. Veratrine-induced release was also inhibited by alpha-receptor activation. However, [³H]NA release induced by the calcium-ionophore was not affected by alpha-receptor agonists. These results strongly support the view that alpha-receptor activation results in a decrease of the availability of Ca²⁺ for stimulus-secretion coupling processes. Presumably this is effected by an inhibition of voltage-sensitive calcium channels in the neuronal membrane associated with neurotransmitter release.     

Docosahexaenoic acid may act as a neuroprotector for methylmercury-induced neurotoxicity in primary neural cell cultures

The ability of docosahexaenoic acid (DHA) to modulate methylmercury (MeHg)-induced neurotoxicity was investigated in primary astrocytes and neurons from the cerebellum. Gas chromatography measurements indicated increased DHA content in both cell types after 24 h supplementation. After individual or combined treatment with MeHg (10 μM) and DHA (30 and 90 μM), the cell-associated MeHg measurements were done using ¹⁴C-labelled MeHg. In addition, mitochondrial activity was evaluated by MTT reduction, glutathione (GSH) content was measured with the fluorescent indicator monochlorobimane (MCB) and reactive oxygen species (ROS) were detected with the fluorescent indicator—chloro methyl derivative of di-chloro di-hydro fluorescein diacetate (CMH₂DCFDA). For all the tested treatments, i.e. DHA, MeHg or DHA + MeHg treatment, the neurons differed significantly (p < 0.001) from astrocytes exhibiting increased ROS production and decreased MTT activity. After MeHg and 30 μM DHA treatment there were no changes in MTT or GSH content but significant decrease (p < 0.001) in ROS was observed in both the cell types when compared to MeHg alone. The cell-associated MeHg measurements indicated reduced MeHg-accumulation in both cell types (p < 0.05) upon 30 μM DHA exposure. Taken together, this study, for the first time establishes that DHA pretreatment effectively reduces cell-associated MeHg and prooxidant response from MeHg in both cerebellar astrocytes and neurons and thus supports the hypothesis that fish-derived nutrients offer possible neuroprotection from MeHg.     

Different pathways by which extracellular Ca promotes calcitonin gene-related peptide release from central terminals of capsaicin-sensitive afferents of guinea pigs: effect of capsaicin, high K and low pH media

Different modes by which Ca²⁺, entering the nerve terminal, promotes transmitter secretion as well as the ability of protons to release neuropeptides, have been shown in peripheral endings of capsaicin-sensitive afferents. We have studied these two aspects in the central endings of these neurons by measuring the release of calcitonin-gene related peptide-like immunoreactivity (CGRP-LI) from slices of the dorsal half of the guinea pig spinal cord. Altough capsaicin (1 μM) released both CGRP-LI and substance P-like immunoreactivity (SP-LI), CGRP-LI was chosen as the sole suitable marker of peptides released from central terminals of capsaicin-sensitive afferents, since after in vitro desensitization to capsaicin (1μM capsaicin for 20 min), high K⁺ (80 mM) failed to evoke CGRP-LI release, whereas SP-LI release was still observed. The capsaicin (1 μM)-evoked CGRP-LI release was entirely dependent on extracellular Ca²⁺. It was unaffected by 0.3 μM tetrodotoxin (TTX), slightly reduced by 0.1 μM ω-conotoxin (CTX) and blocked by 10 μM Ruthenium red (RR). The Ca²⁺-dependent K⁺ (80 mM)-evoked CGRP-LI release was unaffected by TTX, markedly reduced by CTX and only moderatedly inhibited by RR. Low pH (pH 5) produced a remarkable increase in CGRP-LI outflow that was abolished after exposure to capsaicin, reduced by about 50% in Ca²⁺-free medium and unaffected by TTX (0.3 μM). The Ca²⁺-dependent component of the proton-evoked CGRP-LI release was abolished in the presence of RR (10 μM) and slightly inhibited by CTX (0.1 μM). The mode by which capsaicin or high K⁺ promote Ca²⁺ entry into the central endings of capsaicin-sensitive afferents, and hence promote neuropeptide release may be distinguished on a pharmacological basis. Protons release CGRP in the spinal cord by a mechanism that shares a common pathway with that activated by capsaicin.     

Effects of Pb and Cd on acetylcholine release and Ca movements in synaptosomes and subcellular fractions from rat brain and Torpedo electric organ

In this work we examined the effects of Pb²⁺ and Cd²⁺ on (a) [³H]ACh release and voltage-sensitive Ca²⁺ channels in rat brain synaptosomes, and (b)⁴⁵Ca²⁺ binding to isolated brain mitochondria and microsomes, and synaptic vesicles isolated from Torpedo electric organs. Pb²⁺ (K_i ≈ 1.1 μM) and Cd²⁺ (K_i ≈ 2.2) competitively block the K⁺-evoked influx of⁴⁵Ca²⁺ through the ‘fast’ calcium channels in synaptosomes. The K_is obtained with synaptosomes are in good agreement with the K_i values obtained from electrophysiological experiments at the frog neuromuscular junction (K_Pb:0.99 μM, K_Cd: 1.7 μM)⁷. The K_i for the inhibition of ACh release from synaptosomes by Cd²⁺ is 4.5 μM. Pb²⁺ is a less effective inhibitor of transmitter release (K_i ≈ 16 μM) because it secondarily augments spontaneous transmitter efflux. Cd²⁺ has no effect on spontaneous release at concentrations ≤ 100 μM. The enhancing effect of Pb²⁺ on spontaneous release is (a) not abolished by omission of Ca²⁺ from the bathing medium, (b) is delayed by 1–2 min after the beginning of Pb²⁺ exposure, (c) is reversed upon the removal of Pb²⁺. In the presence of physiological concentrations of ATP (1 mM), Mg²⁺ (1 mM) and P_i (2 mM), 1–10 μM Pb²⁺ inhibits calcium uptake but Pb²⁺ > 10μM causes a several-fold stimulation of passive binding of calcium to the organelles. This effect is associated with Pb²⁺-induced enhancement of P_i uptake. Cd²⁺ inhibits Ca²⁺ binding at all concentrations tested (1–50 μM) and reduces the Pb²⁺-induced Ca²⁺-binding to organelles. Neither Pb²⁺ nor Cd²⁺ have any discernible effects on spontaneous loss of calcium from mitochondria or microsomes preloaded with⁴⁵Ca. In summary, these data are consistent with the notion that Pb²⁺ and Cd²⁺ are potent blockers of presynaptic voltage-sensitive Ca²⁺ channels and the evoked release of transmitter which is contingent on Ca²⁺ influx through these channels. Our results are not consistent with the hypothesis that Pb²⁺ augments spontaneous release by interfering with intraterminal Ca²⁺-buffering by mitochondria, endoplasmic reticulum, or synaptic vesicles.     

Chronic amitriptyline exposure reduces 5-HT3 receptor-mediated cyclic GMP formation in NG 108-15 cells

In the present study, we investigated the effects of chronic in vitro administration of amitriptyline, a tricyclic antidepressant, on cyclic GMP formation stimulated by 5-hydroxytryptamine (5-HT) in the neuroblastoma × glioma hybrid cell line, NG 108-15. 5-HT (0.01–100 μM)-stimulated cyclic GMP formation was concentration-dependent and was sensitive to ICS 205-930, a 5-HT₃ receptor antagonist. Exposure of NG 108-15 cells to 5 μM amitriptyline for 3 days significantly reduced 5-HT-stimulated cyclic GMP formation. Acute treatment with amitriptyline had no effect on 5-HT-stimulated cyclic GMP formation. The reduction by chronic amitriptyline exposure of 10 μM 5-HT-stimulated cyclic GMP formation was concentration-dependent over the concentration range examined (0.5 to 10 μM). The IC₅₀ of amitriptyline was 1.9 μM. In contrast, amitriptyline exposure, even at a concentration of 8 μM, failed to modify cyclic GMP formation stimulated by bradykinin, sodium nitroprusside, or atrial natriuretic peptide. Increases in intracellular Ca²⁺ concentration ([Ca²⁺]) evoked by 10 μM 5-HT were attenuated in amitriptyline-exposed cells, while 100 nM bradykinin-induced [Ca²⁺]_i increases were not affected. In addition, chronic exposure to 5 μM amitriptyline caused a decrease in affinity (K_d) of [³H]zacopride specific binding to 5-HT₃ recognition sites. TheB_max for the labelled ligand remained unchanged. These results suggest that chronic amitriptyline exposure reduces 5-HT-stimulated cyclic GMP formation and [Ca²⁺]_i increases, and this may reflect the functional changes of 5-HT₃ receptors.     

Mechanical activation of dorsal root ganglion cells in vitro: comparison with capsaicin and modulation by κ-opioids

The aim of this study was to characterize plasma membrane pathways involved in the intracellular calcium ([Ca²⁺]_i) response of small DRG neurons to mechanical stimulation and the modulation of these pathways by κ-opioids. [Ca²⁺]_i responses were measured by fluorescence video microscopy of Fura-2 labeled lumbosacral DRG neurons obtained from adult rats in short-term primary culture. Transient focal mechanical stimulation of the soma, or brief superfusion with 300 nM capsaicin, resulted to [Ca²⁺]_i increases which were abolished in Ca²⁺-free solution, but unaffected by lanthanum (25 μM) or tetrodotoxin (10⁻⁶ M). 156 out of 465 neurons tested (34%) showed mechanosensitivity while 55 out of 118 neurons (47%) were capsaicin-sensitive. Ninty percent of capsaicin-sensitive neurons were mechanosensitive. Gadolinium (Gd³⁺; 250 μM) and amiloride (100 μM) abolished the [Ca²⁺]_i transient in response to mechanical stimulation, but had no effect on capsaicin-induced [Ca²⁺]_i transients. The κ-opioid agonists U50,488 and fedotozine showed a dose-dependent inhibition of mechanically stimulated [Ca²⁺]_i transients but had little effect on capsaicin-induced [Ca²⁺]_i transients. The inhibitory effect of U50,488 was abolished by the κ-opioid antagonist nor-Binaltorphimine dihydrochloride (nor-BNI; 100 nM), and by high concentrations of naloxone (30–100 nM), but not by low concentrations of naloxone (3 nM). We conclude that mechanically induced [Ca²⁺]_i transients in small diameter DRG somas are mediated by influx of Ca²⁺ through a Gd³⁺- and amiloride-sensitive plasma membrane pathway that is co-expressed with capsaicin-sensitive channels. Mechanical-, but not capsaicin-mediated, Ca²⁺ transients are sensitive to κ-opioid agonists.     

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.     

Emergence of excitotoxicity in cultured forebrain neurons coincides with larger glutamate-stimulated [Ca]i increases and NMDA receptor mRNA levels

We examined several factors related to the increase in susceptibility to excitotoxicity that occurs in embryonic forebrain neurons over time in culture. Neuronal cultures were resistant to a 5-min exposure to 100 μM glutamate/10 μM glycine at 5 days in vitro (DIV), but became vulnerable to the same stimulus by 14 DIV. We used the fluorescent indicators, fura-2 and magfura-2, which have high and low affinity for Ca²⁺, respectively, to measure changes in [Ca²⁺]_i. Glutamate-stimulated increases in the fura-2 and magfura-2 ratio reached maximum values by 10 DIV. Fura-2 reported similar [Ca²⁺]_i changes with exposure to 3 or 100 μM glutamate for 5 min, whereas magfura-2 reported larger [Ca²⁺]_i increases with 5-min exposure to 100 μM glutamate than with exposure to 3 μM glutamate, 100 μM kainate or 50 mM K⁺ from 10 DIV onward. This suggests that the magnitude of the [Ca²⁺]_i changes correlated with the excitotoxicity potential of a stimulus when magfura-2, but not fura-2, was used to measure Ca²⁺. We also used RNase protection assays to measure NMDA receptor subunit mRNA levels. NR1 and NR2A mRNA increased continuously over time in culture, whereas NR2B mRNA increased dramatically during the first 10 days and subsequently remained stable. The time course of the increase in NR2B mRNA most closely followed the increase in glutamate-stimulated changes in the magfura-2 signal and neuronal injury. Therefore, the increases in the glutamate-stimulated [Ca²⁺]_i responses and NMDA receptor subunit mRNA levels (especially NR2B) are likely involved in the development of susceptibility to excitotoxicity in cultured rat forebrain neurons.     

Possible modulatory role of voltage-activated Ca currents determining the membrane properties of isolated pyramidal neurones of the rat dorsal cochlear nucleus

Voltage-activated Ca²⁺ currents have been studied in pyramidal cells isolated enzymatically from the dorsal cochlear nuclei of 6–11-day-old Wistar rats, using whole-cell voltage-clamp. From hyperpolarized membrane potentials, the neurones exhibited a T-type Ca²⁺ current on depolarizations positive to −90 mV (the maximum occurred at about −40 mV). The magnitude of the T-current varied considerably from cell to cell (−56 to −852 pA) while its steady-state inactivation was consistent (E₅₀=−88.2±1.7 mV, s=−6.0±0.4 mV). The maximum of high-voltage activated (HVA) Ca²⁺ currents was observed at about −15 mV. At a membrane potential of −10 mV the L-type Ca²⁺ channel blocker nifedipine (10 μM) inhibited approximately 60% of the HVA current, the N-type channel inhibitor ω-Conotoxin GVIA (2 μM) reduced the current by 25% while the P/Q-type channel blocker ω-Agatoxin IVA (200 nM) blocked a further 10%. The presence of the N- and P/Q-type Ca²⁺ channels was confirmed by immunochemical methods. The metabotropic glutamate receptor agonist (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (200 μM) depressed the HVA current in every cell studied (a block of approximately 7% on an average). The GABA_B receptor agonist baclofen (100 μM) reversibly inhibited 25% of the HVA current. Simultaneous application of ω-Conotoxin GVIA and baclofen suggested that this inhibition could be attributed to the nearly complete blockade of the N-type channels. Possible physiological functions of the voltage-activated Ca²⁺ currents reported in this work are discussed.     

The calcium-dependent [H]acetylcholine release from synaptosomes of brown trout (Salmo trutta) optic tectum is inhibited by adenosine A1 receptors: Effects of enucleation on A1 receptor density and cholinergic markers

Presynaptic inhibition is one of the major control mechanisms in the CNS. Previously we reported that A₁ adenosine receptors are highly concentrated in the brain, including optic tectum, of trout and that they inhibited the release of glutamate. The optic tectum is heavily innervated by cholinergic nerve terminals. We have investigated whether A₁ receptors inhibit the presynaptic release of acetylcholine and whether the inhibition is triggered by calcium. The release of [³H]ACh evoked by 30 mM KCl was Ca²⁺ dependent and it was dose-dependently inhibited by the A₁ adenosine receptor agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA) ranging between 10 nM to 100 μM. The maximum of inhibition was reached at 10 μM. The A₁ receptor antagonist 8-cyclopentyltheopylline (CPT, 10 μM), reversed almost completely the inhibition induced by CCPA 10 μM. In Fura-2/AM loaded synaptosomes, K⁺ depolarization raised [Ca²⁺]_i by about 64%. CCPA (10 μM) reduced the K⁺-evoked Ca²⁺ influx increase by about 48% and this effect was completely antagonised by CPT 10 μM. Synaptosome pretreatment with different Ca²⁺ channel blockers differently affected K⁺-evoked Ca²⁺ influx. This was not significantly modified by nifedipine (1 μM, L-type blocker) nor by ω-agatoxin IVA (0.3 μM, P/Q-type blocker), whereas about 50% reduction was shown by 0.5 μM ω-conotoxin GVIA (N-type blocker). Neurochemical parameters associated with cholinergic transmission and the density of A₁ adenosine receptors were measured in the trout optic tectum 12 days after unilateral eye ablation. A significant drop of both acetylcholinesterase (AChE) activity (24%) and choline acetyltransferase (CAT) activity (32%) was observed in deafferentated optic tectum, whereas the high affinity choline uptake did not parallel the decrease in enzyme activity. Eye ablation caused a marked decrease (43%) of A₁ receptor density without changing the affinity. The K⁺-evoked release of [³H]ACh from synaptosomes of deafferentated was not modify as well as the efficacy of 10 μM CCPA in decreasing [³H]ACh release was not apparently modified.     

Hyposmotic activation hyperpolarizes outer hair cells of guinea pig cochlea

The electrophysiological responses of isolated guinea pig outer hair cells (OHCs) to hyposmotic activation were studied using the whole-cell patch-clamp technique. The cell swelling by hyposmotic activation hyperpolarized OHCs by 6.6 ± 2.3 mV from the resting membrane potential of −58.5 ± 5.9 mV (n = 48). This hyperpolarization was associated with an outward current ( 97.7 ± 22.2, pA, n = 15). The hyperpolarization was inhibited by 300 μM quinine, 5 mN Ba²⁺ and increasing the extracellular K⁺ to 30 mM from 5 mM. In the absence of extracellular Ca²⁺ (1 mM EGTA), the hyperpolarization during hyposmotic activation was also abolished while the following depolarization was preserved. 50 μM GdCl₃, which is known to block strecch-activated non-specific cation channels, inhibited the hyperpolarization reversibly. 50 μM GdCl₃ also inhibited [Ca²⁺]_i increase during hyposmotic activation as shown by the calcium-sensitive dye fura-2. Simultaneously, the [Ca²⁺]_i increase and the hyperpolarization during hyposmotic activation could be observed using the combined method of whole-cell patch clamp and fura-2 technique. It is concluded that the cell swelling by hyposmotic activation may activate the stretch-activated non-specific cation channels in the OHCs which allow a Ca²⁺ influx. In turn, this [Ca²⁺]_i increase leads to an activation of the Ca²⁺-activated K⁺ channels at the basolateral membrane of OHCs which results finally in a reversible hyperpolarization of OHCs by K⁺ efflux.     

The calcium channel antagonist, ω-conotoxin, and electric organ nerve terminals: binding and inhibition of transmitter release and calcium influx

We have previously shown that the calcium channel antagonist ω-conotoxin M-VII-A blocks neurotransmitter release from isolated nerve terminals (synaptosomes) from the electric organ of the electric ray (Yeager et al., J. Neurosci., 7 (1987) 2390–2396). We now demonstrate that a related but more readily available peptide, ω-conotoxin G-VI-A (CgTx), also blocks the release of transmitter from these terminals and, in addition, inhibits depolarization-dependent uptake of Ca²⁺ into these terminals. The half-maximal inhibitory concentration (IC₅₀ for block of depolarization-evoked release and for depolarization-dependent uptake of Ca²⁺ are approximately 3 and 2 μM, respectively. These results suggest the inhibitory effects of CgTx are due to inhibition of Ca²⁺ entry into synaptosomes through voltage-sensitive calcium channels. Assays of radioiodinated CgTx binding to electric organ synaptosomal membranes and synaptosomes appear to show a single binding site with a apparent dissociation constant (K_d of 3–5 μM and toxin receptor densities of 290 and 52 pmol/mg protein, respectively. These CgTx receptor densities are equivalent to 6% of the total synaptosomal membrane protein and 1% of the total synaptosomal protein (assuming a molecular weight of 200 kDa for the toxin receptor). If the observed CgTx receptor densities reflect the actual densities of voltage-sensitive calcium channels in electric organ synaptosomal membranes and synaptosomes, these preparations would be the richest source of these channels yet described.     

Differential effects of calcium entry blockers on pre- and postsynaptic influx of calcium in the rat hippocampus in vitro

A decrease in extracellular free Ca ([Ca²⁺]₀) in response to stimulation of Schaffer collaterals could be recorded in or near the stratum pyramidale even when synaptic transmission was completely blocked. Under the same conditions, alvear stimulation also evoked a decrease in [Ca²⁺]₀ at the same site. We attributed the former to influx of Ca²⁺ into presynaptic terminals and the latter to influx postsynaptic (pyramidal) cells. Both pre- and postsynaptic Ca²⁺ influx were completely blocked by Ni²⁺ (2.5 mM). Nifedipide (5–10 μM), verapamil (50–100 μM) and fendiline (100–200 μM) reduced the posysnaptic influx of Ca²⁺ but did not alter Ca²⁺ loss from the extracellular space into presynaptic terminals. The calcium channel activators, BAY-K 8644 and CGP 28,392, had no consistent effect on either pre- or postsynaptic influx. Occasional enhancement of both pre- and postsynaptic responses was seen. In most studies the agents were without effect and on occasions a reduction in both responses was seen. The results could indicate that Ca-channels at pre- and postsynaptic sites in CA₁ may be of different types.     

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.