Calcium channel involvement in potassium depolarization-induced phosphoinositide hydrolysis in rat cortical slices

The stimulation of production of inositol phosphates in rat cortical slices by KCl depolarization and the effects of calcium channel active drugs were investigated. Elevation of K⁺ in the medium up to 48 mM KCl caused a linear concentration-dependent increase in [³H]inositol phosphate accumulation. The KCl stimulated response was not significantly inhibited in the presence of muscarinic or ₁-adrenergic antagonists. KCl stimulated the production of inositol trisphosphate at 60 min but not 10 min. Addition of peptidase inhibitors did not significantly affect KCl-stimulated PI hydrolysis. The KCl-stimulated response was still observed in the absence of extracellular calcium, although the net accumulation of inositol phosphates was greater in the presence of 0.1 or 0.5 mM calcium. KCl (48 mM) inhibited [³H]inositol uptake into phospholipids of cortical slices. The dihydropyridine calcium channel agonist BAY K 8644 stimulated PI hydrolysis in cortical slices in a concentration dependent manner in the presence of 19 mM KCl. The BAY K 8644-stimulated PI response was partially inhibited by 1M atropine but not by 1M prazosin. Calcium channel blockers nitrendipine, verapamil, flunarizine, and nifedipine slightly inhibited the PI response stimulated by 19 mM KCl in the presence or absence of BAY K 8644. The effects of the calcium channel antagonists were attenuated in the presence of 1 M atropine. The peptide calcium channel blocker -conotoxin did not affect KCl-stimulated PI hydrolysis. These results suggest that endogenous muscarinic or adrenergic neurotransmitters are not involved in KCl-stimulated PI hydrolysis in cortical slices. Although extracellular calcium is necessary for optimal KCl-stimulated PI hydrolysis, it is not required for the expression of the KCl-evoked response suggesting that depolarization is the primary trigger for this stimulant.     

NMDA and non-NMDA receptors stimulation causes differential oxidative stress in rat cortical slices

Glutamate receptor activated neuronal cell death is attributed to a massive influx of Ca(2+) and subsequent formation of reactive oxygen species (ROS) but the relative contribution of NMDA and non-NMDA sub-types of glutamate receptors in excitotoxicity is not known. In the present study, we have examined the role of NMDA and non-NMDA receptors in glutamate-induced neuronal injury in cortical slices from young (20+/-2 day) and adult (80+/-5 day) rats. Treatment of slices with glutamate receptor agonists NMDA, AMPA and KA elicited the formation of reactive oxygen species (ROS) and neuronal cell death. In young slices, NMDA receptor stimulation caused a higher ROS formation and neurotoxicity, but KA was more effective in producing ROS and cell death in adult slices. AMPA exhibited an intermediate effect on ROS formation and toxicity in both the age groups. A significant protection in glutamate mediated ROS formation and neurotoxicity was observed in presence of NMDA or/and non-NMDA receptors antagonists APV and NBQX, respectively. This further confirms the involvement of both NMDA and non-NMDA receptors in glutamate mediated neurotoxicity. In adult slices, we did not find positive correlation between ligand induced neurotoxicity and mitochondrial depolarization. Though, NMDA and KA stimulation produced differential effect on ROS formation and neurotoxicity in young and adult slices, the mitochondrial depolarization was higher and comparable on NMDA stimulation in both the age groups as compared to KA, suggesting that the mitochondrial depolarization may not be a good indicator for neurotoxicity. Our results demonstrate that both NMDA and non-NMDA sub-types of glutamate receptors are involved in glutamate mediated neurotoxicity but their relative contribution is highly dependent on the age of the animal.     

Single-channel currents from diethylpyrocarbonate-modified NMDA receptors in cultured rat brain cortical neurons

The role of histidine residues in the function of N-methyl-D-aspartate (NMDA)-activated channels was tested with the histidine-modifying reagent diethylpyrocarbonate (DEP) applied to cells and membrane patches from rat brain cortical neurons in culture. Channels in excised outside-out patches that were treated with 3 mM DEP for 15-30 s (pH 6.5) showed an average 3.4-fold potentiation in steady state open probability when exposed to NMDA and glycine. Analysis of the underlying alterations in channel gating revealed no changes in the numbers of kinetic states: distributions of open intervals were fitted with three exponential components, and four components described the shut intervals, in both control and DEP-modified channels. However, the distribution of shut intervals was obviously different after DEP treatment, consistent with the single-channel current record. After modification, the proportion of long shut states was decreased while the time constants were largely unaffected. Burst kinetics reflected these effects with an increase in the average number of openings/burst from 1.5 (control) to 2.2 (DEP), and a decrease in the average interburst interval from 54.1 to 38.2 ms. These effects were most likely due to histidine modification because other reagents (n- acetylimidazole and 2,4,6-trinitrobenzene 1-sulfonic acid) that are specific for residues other than histidine failed to reproduce the effects of DEP, whereas hydroxylamine could restore channel open probability to control levels. In contrast to these effects on channel gating, DEP had no effect on average single-channel conductance or reversal potential under bi-ionic (Na+:Cs+) conditions. Inhibition by zinc was also unaffected by DEP. We propose a channel gating model in which transitions between single- and multi-opening burst modes give rise to the channel activity observed under steady state conditions. When adjusted to account for the effects of DEP, this model suggests that one or more extracellular histidine residues involved in channel gating are associated with a single kinetic state.     

Mechanisms of ammonia-induced cell death in rat cortical neurons: roles of NMDA receptors and glutathione

The occurrence, nature and prevention of ammonia-induced cell death were assayed in cultured primary cortical neurons from newborn rats. Treatment with 1-10 mM ammonium chloride for 24 or 48 h, dose-dependently decreased neuronal survival (MTT assay) and GSH/GSSG ratio in the cultures, whereas total GSH content was significantly reduced only with 10mM ammonia. Treatment with a glutathione synthesis inhibitor, buthionyl sulfoximine (BSO) (10 microM), decreased the GSH content and GSH/GSSG ratio to a degree similar to that of 10 mM ammonia, but it did not decrease cell survival in control cells. This indicates that glutathione depletion per se is not a cause of ammonia-induced neuronal death. However, ammonia-induced decrease of cell viability was attenuated by incubation with glutathione diethyl ester (GEE), which transiently increased the intracellular GSH level in both control and ammonia-treated cells. Neuronal survival in the presence of ammonia was partly improved by the NMDA receptor antagonists MK-801 and APV. Morphological analysis revealed that ammonia treatment causes both apoptotic and non-apoptotic neuronal death, the former not being inhibited by MK-801. Apoptosis was the dominant type of cell death at 10mM ammonia, as concluded both from morphologic examination and the absence of survival improvement in the presence of GABA+nipecotic acid or taurine, model anti-excitotoxic treatments of cortical neurons. The mechanism underlying apoptosis may include inhibition of a survival kinase, Akt, whose activatory phosphorylation at Ser473 is reduced in neurons treated with 10 mM, but not 1 mM ammonia.     

Activation of muscarinic receptors inhibits beta-amyloid peptide-induced signaling in cortical slices

Deposition of fibrillar aggregates of the beta-amyloid peptide (Abeta) is a key pathologic feature during the early stage of Alzheimer's disease. The initial neuronal responses to Abeta in cortical circuits and the regulation of Abeta-induced signaling remain unclear. In this study, we found that exposure of cortical slices to Abeta(1-42) or Abeta(25-35) induced a marked increase in the activation of protein kinase C (PKC) and Ca(2+)/calmodulin-dependent kinase II (CaMKII), two enzymes critically involved in a variety of cellular functions. Activation of M1 muscarinic receptors, but not nicotinic receptors, significantly inhibited the Abeta activation of PKC and CaMKII. Increasing inhibitory transmission mimicked the M1 effect on Abeta, whereas blocking GABA(A) receptors eliminated the M1 action. Moreover, electrophysiological evidence shows that application of Abeta to cortical slices induced action potential firing and enhanced excitatory postsynaptic currents, whereas muscarinic agonists potently increased inhibitory postsynaptic currents. These results suggest that Abeta activates PKC and CaMKII through enhancing excitatory activity in glutamatergic synaptic networks. Activation of M1 receptors inhibits Abeta signaling by enhancing the counteracting GABA(ergic) inhibitory transmission. Thus the muscarinic reversal of the Abeta-induced biochemical and physiological changes provides a potential mechanism for the treatment of Alzheimer's disease with cholinergic enhancers.     

Selected Contribution: Improved anoxic tolerance in rat diaphragm following intermittent hypoxia.

Intermittent hypoxia (IH), associated with obstructive sleep apnea, initiates adaptive physiological responses in a variety of organs. Little is known about its influence on diaphragm. IH was simulated by exposing rats to alternating 15-s cycles of 5% O2 and 21% O2 for 5 min, 9 sets/h, 8 h/day, for 10 days. Controls did not experience IH. Diaphragms were excised 20-36 h after IH. Diaphragm bundles were studied in vitro or analyzed for myosin heavy chain isoform composition. No differences in maximum tetanic stress were observed between groups. However, peak twitch stress (P < 0.005), twitch half-relaxation time (P < 0.02), and tetanic stress at 20 or 30 Hz (P < 0.05) were elevated in IH. No differences in expression of myosin heavy chain isoforms or susceptibility to fatigue were seen. Contractile function after 30 min of anoxia (95% N2-5% CO2) was markedly preserved at all stimulation frequencies during IH and at low frequencies after 15 min of reoxygenation. Anoxia-induced increases in passive muscle force were eliminated in the IH animals (P < 0.01). These results demonstrate that IH induces adaptive responses in the diaphragm that preserve its function in anoxia.     

Calcium transients in single fibers of low-frequency stimulated fast-twitch muscle of rat

Ca²⁺transients were investigated in single fibers isolated from ratextensor digitorum longus muscles exposed to chronic low-frequency stimulation for different time periods up to 10 days. Approximately 2.5-fold increases in resting Ca²⁺ concentration([Ca²⁺]) were observed 2 h after stimulationonset and persisted throughout the stimulation period. The elevated[Ca²⁺] levels were in the range characteristicof slow-twitch fibers from soleus muscle. In addition, we noticed atransitory elevation of the integral[Ca²⁺] per pulse with a maximum (~5-fold)after 1 day. Steep decreases in rate constant of[Ca²⁺] decay could be explained by an immediateimpairment of Ca²⁺ uptake and, with longer stimulationperiods, by an additional loss of cytosolic Ca²⁺ bindingcapacity resulting from a decay in parvalbumin content. A partialrecovery of the rate constant of [Ca²⁺] decayin 10-day stimulated muscle could be explained by an increasing mitochondrial contribution to Ca²⁺ sequestration.

     

Trimethyltin-induced loss of NMDA and kainate receptors in the rat brain

Summary Adult rats exposed acutely to trimethyltin (TMT) manifest a number of behavioral alterations, in conjunction with neuronal degeneration in the limbic system. In the present study, changes in³H-TCP binding to N-methyl-D-aspartate (NMDA) receptors and³H-kainic acid (KA) binding to kainate receptors were studied by autoradiographic methods following TMT exposure (8 mg/kg, i.p.) in adult Sprague Dawley rats. No significant alterations were found at 4 hours after exposure. An extensive loss of³H-TCP and³H-KA binding was seen in the hilar region of the CA3 field at 2 and 12 weeks after TMT exposure. Also, the³H-TCP binding was decreased in piriform cortex and in striatum. Thus, TMT exposure leads to a major and regional selective loss of NMDA and kainate receptors in the limbic system, alterations that may be involved in the neuropathology and behavioral sequelae of TMT toxicity.Abbreviations TMT trimethyltin - NMDA N-methyl-D-aspartate - KA Kainic acid - TCP N-(1-2-thienylcyclohexyl)-3,4-piperidine     

Anabolic sialosylation of gangliosides in situ in rat brain cortical slices

Radiolabeling of the sialic acid residues of gangliosides was examined in thin slices of rat brain cerebral cortex incubated under physiologic conditions in the presence of either [14C]N-acetyl-mannosamine (ManNAc) or cytidine 5'-monophosphoryl-[14C]N-acetyl-neuraminic acid (CMP-NeuAc). CMP-NeuAc is the direct donor substrate in the transfer of sialic acid to gangliosides by sialosyl transferases (SATs), including ectosialosyl transferases at the cell surface. ManNAc must be internalized by the neural cells (neuronal or glial) where it serves as an obligate precursor for the biosynthesis of the NeuAc moiety of intracellular CMP-NeuAc, via multiple reactions in the cytosol and nucleus. When exogenous [14C]ManNAc was supplied, there appeared to be a 2-h lag period before label was incorporated measurably into ganglioside sialic acid. That was followed by rapid ganglioside labeling continuing up to 6 h. There was high incorporation into ganglioside GM1. Labeling by ManNAc was inhibited by monensin, a monovalent cationophore that blocks anabolic transport in medial and trans Golgi. Extracellular CMP-NeuAc was not internalized by the cells. CMP-[14C]NeuAc labeling of gangliosides had no lag period, reached a maximum within 2 h, and then began to level. The label distribution among gangliosides was high in GD3, but quite low in GM1. CMP-NeuAc labeling was not inhibited by 10(-7) M monensin. These findings support a model in which ManNAc labels gangliosides by an intracellular route involving monensin-sensitive, Golgi-associated SATs. In this intracellular system, the major labeled products are gangliosides of the gangliotetraosyl series (GM1, GD1a, etc.).(ABSTRACT TRUNCATED AT 250 WORDS)     

Excitotoxic damage of the hippocampus in the rat: involvement of N-methyl-D-aspartate receptors

The intrahippocampal injection of two agonists of excitatory aminoacid (EAA) receptors elicited neuronal damages localized in CA1 and dentate gyrus for N-methyl-D-aspartate (NMDA) (20 nmol) and extended to the various hippocampal areas, except dentate gyrus for kainic acid (KA) (2.5 nmol). The pretreatment of the animals with N-[1-(2-thienyl)cyclohexyl]piperidine (TCP) (20 mg/kg), a noncompetitive NMDA-receptor antagonist, prevented the neuronal injury induced by NMDA and KA in CA1. The distribution of neuronal damages and of TCP-protected areas closely correlated to that of EEA-receptors and of TCP binding sites in the hippocampus.     

Calcium transients in subcompartments of the leech Retzius neuron as induced by single action potentials

Regional Ca(2+) influx into neurons plays an essential role for fast signal processing, yet it is little understood. We have investigated intracellular Ca(2+) transients induced by a single action potential (AP) in Retzius neurons in situ of isolated ganglia of the leech Hirudo medicinalis using confocal laser scanning microscopy in the cell body, in different axonal branches, and in dendrites. In the cell body, a single AP induced a Ca(2+) transient in submembrane regions, while in central regions no fluorescence change was detected. Burst activity evoked a much larger Ca(2+) influx, which elicited Ca(2+) signals in central somatic regions, including the cell nucleus. A single AP induced a Ca(2+) transient in distal branches of the axon and in dendrites that was significantly larger than in the proximal axon and in the cell body (p <.05), and the recovery of the Ca(2+) transient was significantly faster in axonal branches than in dendrites (p <.01). The AP-induced Ca(2+) transient was inhibited by Co(2+) (2 mM). The P/Q-type Ca(2+) channel blocker omega-agatoxin TK (500 nM) and the L-type Ca(2+) channel blocker nifedipine (20 microM) had no effect on the Ca(2+) transient, whereas the L-type Ca(2+) channel blocker methoxyverapamil (D600, 0.5-1 mM) irreversibly reduced the Ca(2+) transient by 37% in axons and by 42% in dendrites. Depletion of intracellular Ca(2+) stores following inhibition of endoplasmic Ca(2+)-ATPases by cyclopiazonic acid (10 microM) decreased the AP-induced Ca(2+) transient in the dendrites by 21% (p <.01), but not in axons, and increased the Ca(2+) recovery time constant (tau) in the axonal branches by 129% (p <.01), but not in dendrites. The results indicate that an AP evokes a voltage-gated Ca(2+) influx into all subcompartments of the Retzius neuron, where it produces a Ca(2+) signal of different size and/or kinetics. This may contribute to the modulation of electrical excitation and propagation of APs, and to different modes of synaptic and nonsynaptic processes.     

NMDA receptors mediate peripheral chemoreceptor afferent input in the conscious rat

N-methyl-D-aspartate(NMDA) glutamate receptors mediate critical components ofcardiorespiratory control in anesthetized animals. The role of NMDAreceptors in the ventilatory responses to peripheral and centralchemoreceptor stimulation was investigated in conscious, freelybehaving rats. Minute ventilation(E)responses to 10% O₂, 5%CO₂, and increasing intravenousdoses of sodium cyanide were measured in intact rats before and afterintravenous administration of the NMDA receptor antagonist MK-801 (3 mg/kg). After MK-801, eupcapnic tidal volume(VT) decreased while frequencyincreased, resulting in a modest reduction inE.Inspiratory time (TI) decreased, whereas expiratory time remained unchanged. TheE responsesto hypercapnia were qualitatively similar in control and MK-801conditions, with slight reductions in respiratory drive (VT/TI)after MK-801. In contrast, responses to hypoxia were markedly attenuated after MK-801 and were primarily due to reduced frequency changes, whereas VT wasunaffected. Sodium cyanide doses associated with significantEincreases were 5 and 50 µg/kg before and after MK-801,respectively. Thus 1-log shift to the right of individual dose-responsecurves occurred with MK-801. Selective carotid body denervation reducedE duringhypoxia by 70%, and residual hypoxic ventilatory responses wereabolished after MK-801. These findings suggest that, in conscious rats,carotid and other peripheral chemoreceptor-mediated hypoxic ventilatoryresponses are critically dependent on NMDA receptor activation and thatNMDA receptor mechanisms are only modestly involved during hypercapnia.

     

Developmental expression of NMDA receptors in primary cortical cell culture: relationship to function and excitotoxicity

Over‐activation of the N‐methyl‐d ‐aspartate (NMDA) receptor results in a Ca²⁺‐dependent neurotoxicity termed excitotoxicity. Primary neuronal cell cultures are often used to study the mechanisms of excitotoxicity. While the expression of the NMDA receptor (NR) subunits and their relationship to Ca²⁺ entry/accumulation and excitotoxicity has been studied extensively, all three parameters have not been examined concurrently. To determine unequivocally whether developmental expression of NR protein and function do indeed coincide with the appearance of excitotoxicity, we examined the temporal relationship between NR subunit expression, NMDA‐induced Ca²⁺ accumulation, and NMDA‐mediated excitotoxicity simultaneously using sister plates derived from the same mixed cortical cell culture preparations. Western Blot analysis of total protein isolated from cells cultured for 1, 4, 7, 10 and 14 days revealed a time‐dependent increase in NR1, NR2A and NR2B subunit expression, which surprisingly did not correlate with NMDA receptor function, as assessed by measurement of NMDA‐induced ⁴⁵Ca²⁺ accumulation. However, when only NR subunit surface expression was quantified, a correlation between expression and ⁴⁵Ca²⁺ accumulation did indeed exist. To our surprise, the emergence of excitotoxicity did not show a direct relationship to ⁴⁵Ca²⁺ accumulation as has been reported previously. Thus, it appears that other factors besides total Ca²⁺ accumulation must contribute to the emergence of excitotoxicity in mixed murine cortical cell cultures. Acknowledgements: Supported by a grant from The Patrick and Catherine Weldon Donaghue Medical Foundation.     

Ionotropic NMDA and P2X1/5 receptors mediate synaptically induced Ca2+ signalling in cortical astrocytes

Local, global and propagating calcium (Ca(2+)) signals provide the substrate for glial excitability. Here we analyse Ca(2+) permeability of NMDA and P2X(1/5) receptors expressed in cortical astrocytes and provide evidence that activation of these receptors trigger astroglial Ca(2+) signals when stimulated by either endogenous agonists or by synaptic release of neurotransmitters. The Ca(2+) permeability of the ionotropic receptors was determined by reversal potential shift analysis; the permeability ratio P(Ca)/P(K) was 3.1 for NMDA receptors and 2.2 for P2X(1/5) receptors. Selective stimulation of ionotropic receptors (with NMDA and α,β-methyleneATP) in freshly isolated cortical astrocytes induced ion currents associated with transient increases in cytosolic Ca(2+) concentration ([Ca(2+)](i)). Stimulation of neuronal afferents in cortical slices triggered glial synaptic currents and [Ca(2+)](i) responses, which were partially blocked by selective antagonists of NMDA (D-AP5 and UBP141) and P2X(1/5) (NF449) receptors. We conclude that ionotropic receptors contribute to astroglial Ca(2+) signalling and may provide a specific mechanism for fast neuronal-glial signalling at the synaptic level.     

Lithium protection against glutamate excitotoxicity in rat cerebral cortical neurons: involvement of NMDA receptor inhibition possibly by decreasing NR2B tyrosine phosphorylation

The therapeutic mechanisms of lithium for treating bipolar mood disorder remain poorly understood. Recent studies demonstrate that lithium has neuroprotective actions against a variety of insults. Here, we studied neuroprotective effects of lithium against excitotoxicity in cultured cerebral cortical neurons. Glutamate-induced excitotoxicity in cortical neurons was exclusively mediated by NMDA receptors. Pre-treatment of cortical neurons with LiCl time-dependently suppressed excitotoxicity with maximal protection after 6 days of pre-treatment. Significant protection was observed at the therapeutic and subtherapeutic concentration of 0.2-1.6 mm LiCl with almost complete protection at 1 mM. Neuroprotection was also elicited by valproate, another major mood-stabilizer. The neuroprotective effects of lithium coincided with inhibition of NMDA receptor-mediated calcium influx. Lithium pre-treatment did not alter total protein levels of NR1, NR2A and NR2B subunits of NMDA receptors. However, it did markedly reduce the level of NR2B phosphorylation at Tyr1472 and this was temporally associated with its neuroprotective effect. Because NR2B tyrosine phosphorylation has been positively correlated with NMDA receptor-mediated synaptic activity and excitotoxicity, the suppression of NR2B phosphorylation by lithium is likely to result in the inactivation of NMDA receptors and contributes to neuroprotection against excitotoxicity. This action could also be relevant to its clinical efficacy for bipolar patients.     

Effect of pyrithiamine treatment of potassium ion fluxes in rat cortical slices

The effect of thiamine deficiency on energy-requiring processes in brain tissue was studied by comparing cortical slices prepared from control and pyrithiamine-treated rats. Veratridine was used to stimulate energy metabolism by opening voltage-sensitive sodium channels resulting in enchanced Na⁺/K⁺ pumping; subsequent tetrodotoxin addition closed the sodium channels. Pyrithiamine-treated slices showed both lower basal and veratridine-stimulated respiration rates compared to control slices. K⁺ was released from the tissue upon addition of veratridine and was taken up again upon addition of tetrodotoxin. The movement of K⁺ was monitored directly with a K⁺-sensitive electrode as well as by measuring the rubidium diffusion potential. There was no difference between control and pyrithiamine-treated slices in K⁺ fluxes in response to veratridine and tetrodotoxin. The extent of reuptake of K⁺ upon tetrodotoxin addition was inversely related to the extracellular Ca²⁺ concentration and to the incubation temperature. Veratridine resulted in a marked decrease in tissue levels of ATP and creatine phosphate; these levels remained quite low upon tetrodotoxin addition. Despite the different respiration rates, control and pyrithiamine-treated slices showed the same ATP and creatine phosphate levels in response to veratridine and tetrodotoxin.     

Pharmacologic characterization of cirazoline-activated inositol phospholipid hydrolysis in rat brain cortical slices

Interaction of cirazoline, an imidazoline derivative, with alpha 1-adrenoceptor coupled inositol phospholipid hydrolysis was characterized in rat brain cortical slices. Norepinephrine, a full alpha 1-agonist, and phenylephrine, a partial alpha 1-agonist, on inositol phospholipid hydrolysis were included for comparison. Norepinephrine produced a fourfold stimulation of inositol phospholipid hydrolysis, whereas cirazoline and phenylephrine caused only submaximal responses (40-60%) when compared with norepinephrine. The stimulation of inositol phospholipid hydrolysis by cirazoline was completely blocked by the alpha 1-adrenoceptor antagonist prazosin, but not by selective alpha 2- or beta-adrenoceptor antagonists. Furthermore, the norepinephrine dose-response curve was shifted to the right in the presence of cirazoline, without affecting the maximal response. These results suggest that cirazoline behaves as a partial agonist at brain alpha 1-adrenoceptors linked to inositol phospholipid hydrolysis.