Inhibition of NMDA-induced protein kinase C translocation by a Zn2+ chelator: implication of intracellular Zn2+.

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 microM) and calcium ionophore A23187 (0.3-30 microM) 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 microM 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 microM Ca2+. Low concentrations of Zn2+ (10 microM), which alone had no effect on the distribution of the binding, significantly enhanced the effect of 10 microM Ca2+ in the lysate. Under those conditions TPEN inhibited the Zn(2+)-potentiated Ca(2+)-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.     

L-type Ca channel-mediated Zn toxicity and modulation by ZnT-1 in PC12 cells

In view of evidence that Zn²⁺ neurotoxicity contributes to some forms of pathological neuronal death, we developed a model of Zn²⁺ neurotoxicity in a cell line amenable to genetic manipulations. Exposure to 500 μM ZnCl₂ for 15 min under depolarizing conditions resulted in modest levels of PC12 cell death, that was reduced by the L-type Ca²⁺ channel antagonist, nimodipine, and increased by the L-type Ca²⁺ channel opener, S(−)-Bay K 8644. At lower insult levels (200 μM Zn²⁺+Bay K 8644), Zn²⁺-induced death appeared apoptotic under electron microscopy and was sensitive to the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-CH₂F (Z-VAD); at higher insult levels (1000 μM+Bay K 8644), cells underwent necrosis insensitive to Z-VAD. To test the hypothesis that the plasma membrane transporter, ZnT-1, modulates Zn²⁺ neurotoxicity, we generated stable PC12 cell lines overexpressing wild type or dominant negative forms of rat ZnT-1 (rZnT-1). Clones T9 and T23 overexpressing wild type rZnT-1 exhibited enhanced Zn²⁺ efflux and reduced vulnerability to Zn²⁺-induced death compared to the parental line, whereas clones D5 and D16 expressing dominant negative rZnT-1 exhibited the opposite characteristics.     

The effects of Zn on long-term potentiation of C fiber-evoked potentials in the rat spinal dorsal horn

Tetanic stimuli of peripheral C fibers produces long-term potentiation (LTP) in the spinal cord, which may contribute to sensitization of spinal pain-sensitive neurons. Zn²⁺ is widely distributed in the central nervous system and has blocked (LTP) in the hippocampus. The present study examined the effects of Zn²⁺ on the induction and maintenance of C fiber-evoked LTP in the deep dorsal horn of spinalized rats in vivo. The sciatic nerve was stimulated by tetanic stimuli for inducing LTP. (1) Topical administration of Zinc chloride (15 μM) to the spinal cord 15 min before tetanic stimulation completely blocked the induction of LTP, but not the baseline C responses. When Zn²⁺ was given 2 h after induction of LTP, no significant effect occurred. (2) Chelation of Zn²⁺ by disodium calcium ethylene diaminetelraacetate (CaEDTA) (500 μM) resulted in no effect on LTP. (3) Coadministration of Zn²⁺ (15 μM) and N-methyl-D-aspartic acid (NMDA) (5 μM) significantly attenuated C fiber-evoked potentials, which was prevented by the NMDA receptor antagonist AP-5 (100 μM). The present results showed that Zn²⁺ may contribute to the modulation of the formation, but not the maintenance, of spinal LTP. NMDA receptors may be involved in Zn²⁺-induced modulation.     

Deramciclane inhibits N-methyl-D-aspartate receptor function

Effects of the novel anxiolytic drug deramciclane on excitatory amino acid release and transmembrane Ca²⁺ ion flux processes were compared in rat cerebrocortical homogenates containing resealed plasmalemma fragments and nerve endings. Deramciclane (10 μM) significantly inhibited [³H]D-aspartate release and transmembrane Ca²⁺ flux to N-methyl-D-aspartate in the absence of Mg²⁺. By contrast, inhibition of [³H]D-aspartate release and transmembrane Ca²⁺ flux evoked by 0.1 mM (S)-α-amino-3-hydroxy-5-methyl-4-isoxazole propionate in the presence of Mg²⁺ and 10 μM cyclothiazide by 10 μM deramciclane was not significant. In the presence of N-methyl-D-aspartate receptor antagonists, deramciclane (10 μM) did not inhibit [³H]D-aspartate release to N-methyl-D-aspartate. These results suggest an involvement of the inhibition of a presynaptic N-methyl-D-aspartate receptor in the anxiolytic properties of deramciclane.     

Effect of lead on intracellular free calcium ion concentration in a presynaptic neuronal model: F-NMR study of NG108-15 cells

The intracellular free calcium ion concentration ([Ca²⁺]_i) of the neuroblastoma × glioma hybrid cell line, NG108-15, was measured using the ¹⁹F-nuclear magnetic resonance divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (5F-BAPTA). The basal [Ca²⁺]_i was measured to be 106 ± 14 nM. Treatment with 5 μM lead (Pb) for 2 h produced a 2-fold increase in [Ca²⁺]_i to 200 ± 24 nM and a measurable intracellular free Pb²⁺ concentration ([Pb²⁺]_i) of 30 ± 10 pM. Intracellular free Zn²⁺ concentrations ([Zn²⁺]_i) were also observed in the presence of Pb. This represents the first direct demonstration that Pb elevates the [Ca²⁺]_i in neurons, thus providing evidence for a role of [Ca²⁺]_i in mediating the neurotoxicity of Pb.     

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.     

The role of sulfhydryl groups and calcium in the mercuric chloride-induced inhibition of glutamate uptake in rat primary astrocyte cultures

Inhibition by mercuric chloride (MC) of the astrocytic uptake of the excitotoxic neurotransmitterL-glutamate (L-GLU) has been postulated to contribute to MC neurotoxicity. In the present study, we analyzed the ability of two sulfhydryl (SH)-protecting agents: a cell membrane non-penetrating compound-reduced glutathione (GSH), and the membrane permeable dithiothreitol (DTT), to reverse the inhibitory action of MC on the initial rate of uptake of radiolabelled GLU (100 μM) in primary cultures of rat astrocytes. MC at 5 μM concentration reduced the uptake to 46% of control when present in the incubation medium during the 5 min of actual uptake, and to 27% of control when astrocytes were preincubation with MC was partly relieved by the addition of 1 mM DTT during the actual 5 min uptake period. However, this inhibition could not be reversed by 1 mM GSH. Accordingly, it is postulated that the inhibitory effect exerted by MC on GLU uptake is associated with vulnerable SH groups located within, but not on the surface of the cell membrane. Neither 5 μMN-ethylmaleimide (NEM) nor 5 μM or 25 μM iodoacetate (IA) affected GLU uptake, indicating steric hindrance of the access of these two sulfhydryl reagents to the SH groups critical for the uptake. The effect of MC on GLU uptake was not altered by omission of Ca²⁺ or addition of a non-specific calcium channel blocker La³⁺ (10 μM), suggesting that the effect was not subsequent to Hg²⁺ entry into the cell through Ca²⁺ channels.     

Both arachidonic acid and 1-oleoyl-2-acetyl glycerol in low magensium solution induce long-term potentiation in hippocampal CA1 neurons in vitro

The effects of phospholipase blockers on tetanus-induced long-term potentiation (LTP) and of diacylglycerol (DG) and arachidonic acid (AA) on synaptic transmission were studied in CA1 neurons of guinea pig hippocampal slices to evaluate the role of protein kinase C (PKC) and AA on the maintenance of LTP. Tetanus-induced LTP was suppressed by perfusion with neomycin (1 mM) or 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC, 0.1 mM), blockers of phospholipase. 1-Oleoyl-2-acetyl-glycerol (OAG, 100 μg/ml) and AA (100 μM) produced a temporal increase in both the amplitude of the population spike (PS) and the slope of the field excitatory postsynaptic potentials (EPSPs) but failed to produce LTP. Application of OAG or AA in low-Mg²⁺ (0.1 mM) solution induced LTP. OAG- and AA-induced LTP was blocked by -2-amino-phosphopentanoic acid (AP5; 50 μM). The administration of a potent activator of PKC, phorbol-12,13-dibutyrate (PDBu), in low-Mg²⁺ (0.1 mM) solution enhanced the PS and EPSPs for 2 or 3 h but this enhancement did not persist. These results suggest that PKC activation is not as important as AA for the maintenance of LTP and that OAG and AA play important roles in the maintenance of LTP in synergy with the influx of Ca²⁺ through NMDA receptor-coupled channels.     

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.     

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.     

Zn differentially modulates signals from red- and short wavelength-sensitive cones to horizontal cells in carp retina

The effects of Zn²⁺ were studied while recording intracellularly from L-type horizontal cells (LHCs) in the isolated, superfused carp retina. In darkness, 25 μM Zn²⁺ hyperpolarized LHCs and potentiated responses of these cells to 500 nm flashes, but decreased those to 680 nm flashes. Zn²⁺ did not change photopic electroretinographic P III responses. The differential modulation by Zn²⁺ persisted when the Zn²⁺-induced membrane hyperpolarization was compensated by lowering Ca²⁺ concentration in the perfusate, but it was abolished in the presence of background illumination. Furthermore, the differential modulation no longer existed in the presence of bicuculline, suggesting the involvement of γ-aminobutyric acid_A (GABA_A) receptors. We speculate that the differential modulation may be a consequence of multiple changes caused by Zn²⁺. Decreased glutamate release from the cone terminal by Zn²⁺ results in a reduction of cone signals. Zn²⁺ antagonizes GABA receptors on LHCs, leading to cone signal reduction. On the other hand, Zn²⁺ may reduce the strength of the negative feedback from LHCs to cones by downregulating the activity of GABA receptors on the cone terminal, which causes a potentiation of LHC light responses. Cone- or wavelength-relevance of the Zn²⁺-induced feedback strength change may account for the differential modulation.     

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.     

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.     

Inhibitory effect of regucalcin on Ca-dependent protein kinase activity in rat brain cytosol: involvement of endogenous regucalcin

The effect of regucalcin, a Ca²⁺-binding protein, on Ca²⁺-dependent protein kinase activity in the brain cytosol of rats with different ages (5 and 50 weeks old) was investigated. The addition of calmodulin (10 μg/ml) or dioctanoylglycerol (5 μg/ml) in the enzyme reaction mixture caused a significant increase in protein kinase activity in the presence of CaCl₂ (1 mM), indicating that Ca²⁺ calmodulin or protein kinase C is present in the cytosol. Such an increase was completely prevented by the addition of regucalcin (10⁻⁷ M). Moreover, regucalcin (10⁻⁷ M) significantly inhibited cytosolic protein kinase activity without Ca²⁺/calmodulin or dioctanoylglycerol addition. Meanwhile, the presence of anti-regucalcin monoclonal antibody (10–50 ng/ml) in the enzyme reaction mixture caused a significant elevation of protein kinase activity, suggesting an inhibitory effect of endogenous regucalcin. Brain cytosolic protein kinase activity was significantly elevated by increasing age (50-week-old rats). Also, regucalcin (10⁻⁷ M) significantly decreased protein kinase activity without Ca²⁺ addition in the brain cytosol of aged rats. However, the effect of anti-regucalcin monoclonal antibody (50 ng/ml) in elevating protein kinase activity was not seen in the brain cytosol of aged rats. These results suggest that regucalcin has an inhibitory effect on Ca²⁺-dependent protein kinase activity in rat brain cytosol, and that the effect of endogenous regucalcin may be weakened in the brain cytosol of aged rats.     

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.     

Mercury (Hg) decreases voltage-gated calcium channel currents in rat DRG and Aplysia neurons

Inorganic mercury (Hg²⁺) reduced voltage-gated calcium channel currents irreversibility in two different preparations. In cultured rat dorsal root ganglion (DRG) neurons, studied with the whole cell patch clamp technique, a rapid concentration-dependent decrease in the L/N-type currents to a steady state was observed with an IC₅₀ of 1.1 μM and a Hill coefficient of 1.3 T-currents were blocked with Hg²⁺ in the same concentration range (0.5–2 μM). With increasing Hg²⁺ concentrations a slow membrane current was additionally activated most obviously at concentrations over 2 μM Hg²⁺. This current was irreversible and might be due to the opening of other (non-specific) ion channels by Hg²⁺. The current-voltage (I–V) relation of DRG neurons shifted to more positive values, suggesting a binding of Hg²⁺ to the channel protein and/or modifying its gating properties. In neurons of the abdominal ganglion of Aplysia californica, studied with the two electrode voltage clamp technique, a continous decrease of calcium channel currents was seen even with the lowest used concentration of Hg²⁺ (5 μM). A steady state was not reached and the effect was irreversible without any change on resting membrane currents, even with high concentrations (up to 50 μM). No shift of the I–V relation of the calcium channel currents was observed. Effects on voltage-activated calcium channel currents with Hg²⁺ concentrations such low have not been reported before. We conclude that neurotoxic effects of inorganic mercury could be partially due to the irreversible blockade of voltage-activated calcium channels.     

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.     

Modulatory effects of aluminum, calcium, lithium, magnesium, and zinc ions on [ H]MK-801 binding in human cerebral cortex

The independent and combined effects of Ca²⁺, Mg²⁺, Zn²⁺, Al³⁺ and Li⁺ on [ ³H]MK-801 binding in human cerebral cortical membranes were studied to further characterize the modulatory effects of metal ions on the N-methyl-d-aspartate (NMDA) receptor-ionophore. Glycine, in the presence of glutamate, significantly intensified the Mg²⁺ inhibition of [ ³H]MK-801 binding whereas it masked the Ca²⁺ enhancement and slightly diminished the Zn²⁺ inhibition. Both Ca²⁺ and Mg²⁺ reduced the Zn²⁺ inhibitory potency. Aluminum demonstrated a potent, relatively glycine-insensitive inhibition of [ ³H]MK-801 binding as an amorphous Al(OH)₃ polymer rather than as the free ion. Cationic modulation of the NMDA receptor-ionophore appears to be regulated at multiple sites which have significant allosteric interactions.     

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.     

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.