Steven W. Leslie

Ethanol inhibits NMDA-induced increases in free intracellular Ca2+ in dissociated brain cells

The effect of N-methyl-D-aspartate (NMDA) on free intracellular Ca2+ concentrations [( Ca2+]i) and the interaction of ethanol on the NMDA-mediated response was examined in freshly dissociated brain cells isolated from newborn rats. NMDA (25 microM) increased [Ca2+]i by approximately 70 nM, measured by fura-2 fluorometry, and this increase could be prevented or reversed by the NMDA antagonists Mg2+ (1.0 mM) and 2-amino-5-phosphonovalerate (AP5, 100 microM). Ethanol (25, 50, 100 mM) added 50 s before NMDA (25 microM) reduced the rise in [Ca2+]i when compared to the 25 microM NMDA response in the absence of ethanol. Thus, ethanol may have direct actions on NMDA-receptor activated increases in [Ca2+]i.

45Ca2+ Uptake into Rat Whole Brain Synaptosomes Unaltered by Dihydropyridine Calcium Antagonists

Abstract: Voltage‐dependent 45Ca2+ uptake into rat whole brain synaptosomes was measured after 3‐s KClinduced depolarization to investigate possible inhibitory effects of calcium antagonists, nitrendipine, nimodipine, and nisoldipine. At a Ca2+ concentration of 1.2 mM, nitrendipine, in concentrations ranging from 0.1 nM to 10 μM, had no effect on 45Ca2+ uptake. When the Ca2+ concentration was lowered to 0.06 and 0.12 μM, nitrendipine, 10 μM, inhibited 45Ca2+ uptake in response to 109 mM KC1 depolarization. However, in a separate concentration response study, nitrendipine, nimodipine, and nisoldipine, 0.1 μM to 10 μM, failed to alter the uptake of 45Ca2+ (0.06 mM Ca2+) into 30 mM KCl‐depolarized synaptosomes. The high concentrations of these agents required to depress 45Ca2+ uptake indicate that the dihydropyridine calcium antagonists are considerably less potent in brain tissue than in peripheral tissue.

Mechanism of Inhibition of N‐Methyl‐d‐Aspartate‐Stimulated Increases in Free Intracellular Ca2+ Concentration by Ethanol

Abstract: Dissociated brain cells were isolated from newborn rat pups and loaded with fura‐2. These cells were sensitive to low N‐methyl‐d‐aspartate (NMDA) concentrations with EC50 values for NMDA‐induced intracellular Ca2+ concentration ([Ca2+]i) increases of approximately 7–16 μM measured in the absence of Mg2+. NMDA‐stimulated [Ca2+]j increases could be observed in buffer with Mg2+ when the cells were predepolarized with 15 mMKQ prior to NMDA addition. Under these predepolarized conditions, 100 mMethanol inhibited 25 μM NMDA responses by approximately 50%, which was similar to the ethanol inhibition observed in buffer without added Mg2+. Ethanol did not alter [Ca2+]i prior to NMDA addition. In the absence of Mg2+, 50 and 100 mM ethanol did not significantly alter the EC50 value for NMDA, but did inhibit NMDA‐induced increases in [Ca2+]i in a concentration‐dependent manner at 4, 16, 64, and 256 μM NMDA. Whereas NMDA‐induced increases in [Ca2+]i were dependent on extracellular Ca2+ and were inhibited by Mg2+, the ability of 100 mM ethanol to inhibit 25 μM NMDA responses was independent of the external Ca2+ or Mg2+ concentrations. Glycine (1, 10, and 100 μM) enhanced 25 μM NMDA‐induced increases in [Ca2+]i by approximately 50%. Glycine (1‐100 μM) prevented the 100 mM ethanol inhibition of NMDA‐stimulated [Ca2+]i observed in the absence of exogenous glycine. MK‐801 (25‐400 μM) inhibited 25 μM NMDA‐stimulated rises in [Ca2+]i in a concentration‐dependent manner. Unlike the additive inhibition observed with Mg2+ plus ethanol, as the concentration of MK‐801 increased above 50 μM, ethanol (at 100 mM) did not produce further inhibition of NMDA responses compared with MK‐801 alone. These results suggest that ethanol may produce a noncompetitive inhibition of NMDA‐stimulated Ca2+ influx in dissociated brain cells, with interactions at the glycine and possibly the phencyclidine site on the NMDA‐receptor complex.

Bay K 8644 stimulation of calcium entry and endogenous dopamine release in rat striatal synaptosomes antagonized by nimodipine

The calcium channel agonist Bay K 8644 (0.1-100 nM) significantly increased the fast-phase entry of calcium and release of endogenous dopamine from rat striatal synaptosomes partially depolarized with 15 mM KCl. This increase was completely blocked by 10 nM nimodipine which had no inhibitory effect on calcium influx and dopamine release in the absence of Bay K 8644. Bay K 8644s agonist effect was attenuated with higher KCl concentrations. These findings suggest that Bay K 8644, in combination with partial KCl depolarization, may expose brain synaptosomal calcium channels which are sensitive to nanomolar concentrations of dihydropyridine calcium channel blockers.

N-methyl-D-aspartate mediated responses decrease with age in Fischer 344 rat brain.

N-Methyl-D-aspartate (NMDA) receptor-mediated responses were studied in hippocampus, cortex, and striatum of Fischer 344 rats of various ages (3-5, 12-14, or 24-28 months old; young, middle-aged, and senescent or old, respectively) to determine whether aging alters the function of NMDA receptors. NMDA-induced inhibition of muscarinic-stimulated phosphoinositide hydrolysis in hippocampus, and NMDA-stimulated release of [3H]norepinephrine (NE) or [3H]dopamine (DA) were used as indices of NMDA receptor function. The muscarinic agonist carbachol (1 mM) stimulated PI hydrolysis in hippocampi from all three age groups with no significant differences between the groups. NMDA inhibited the carbachol-evoked PI response in a concentration-dependent manner (10-100 microM) in all age groups. However, the NMDA-induced (100 microM) inhibition of the carbachol-stimulated response was markedly reduced in an age-dependent manner with losses of 25% and 53% in middle-aged and senescent rats compared to young. Concentration-effect curves for NMDA-stimulated [3H]NE release were determined using hippocampal and cortical slices from rats of the three age groups. In the hippocampus the maximal response for NMDA was significantly decreased from 6.55 fractional [3H]NE release in young to 4.51 and 4.18 in middle-aged and old rats, respectively, with no age-related changes in the potency of NMDA or slope of the curves. In cortical slices the maximal response was significantly reduced in an age-dependent manner by 23% in the senescent rats compared to the young rats. NMDA-stimulated [3H]DA release from striatal slices was significantly lower in the senescent rats at concentrations of NMDA from 500-2000 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of acute and chronic ethanol administration on whole mouse brain synaptosomal calcium influx

Abstract Whole brain synaptosomes, isolated from pair-fed acute in vitro (sucrose-Sustacal diet for 10 days), acute in vivo [4.5g/kg, 20% (w/v) i.p. ethanol pretreatment] and chronic in vivo (ethanol-Sustacal diet for 10 days) female, Swiss-Webster mice, were challenged in vitro with ethanol (80 mM, final concentration) in either an incubation medium (12-min exposure), a depolarizing medium (2-min exposure with 74 mM KCI) or a nondepolarizing medium (2-min exposure with 5 mM KCl). Depolarizing and nondepolarizing media also contained 45 Ca 2+ (2μCi/μmole). The results showed (1) a significant enhancement of 45 Ca 2+ influx when synaptosomes isolated from acute in vitro and acute in vivo mice groups were challenged in vitro by ethanol (80 mM) in the depolarizing medium (74 mM KCl), (2) a significant enhancement by 80 mM ethanol of 45 Ca 2+ accumulation by nondepolarized synaptosomes isolated from the acute in vitro mouse group, (3) a significant increase in 45 Ca 2+ accumulation in synaptosomes from acute in vivo mice as compared with acute in vitro mice without an in vitro ethanol challenge, and (4) a significant decrease in 45 Ca 2+ accumulation by synaptosomes isolated from chronic in vivo mice as compared to acute in vitro synaptosomes. The results presented here demonstrate the ability of ethanol to significantly increase calcium accumulation into whole brain synaptosomes and that tolerance to this phenomenon occurs in parallel with behavioral tolerance to the sedative action of ethanol. These ethanol-induced changes in calcium accumulation may be involved in the production of sedation and tolerance to sedation.

The cycad neurotoxic amino acid, ß-N-methylamino-l-alanine (BMAA), elevates intracellular calcium levels in dissociated rat brain cells

Seeds of the Guam cycad Cycas micronesica K.D. Hill (Cycadaceae), which contain ss-methylamino-L-alanine (BMAA), have been implicated in the etiology of the devastating neurodisease ALS-PDC that is found among the native Chamorros on Guam. The disease also occurs in the native populations on Irian Jaya and the Kii Peninsula of Japan, and in all three areas the cycad seeds are used either dietarily or medically. ALS-PDC is a complex of amyotrophic lateral sclerosis and parkinsonism dementia complex with additional symptoms of Alzheimers. It is well known that Ca(2+) elevations in brain cells can lead to cell death and neurodiseases. Therefore, we evaluated the ability of the cycad toxin BMAA to elevate the intracellular calcium concentration ([Ca(2+)](i)) in dissociated newborn rat brain cells loaded with fura-2 dye. BMAA produced an increase in intracellular calcium levels in a concentration-dependent manner. The increases were dependent not only on extracellular calcium concentrations, but also significantly on the presence of bicarbonate ion. Increasing concentrations of sodium bicarbonate resulted in a potentiation of the BMAA-induced [Ca(2+)](i) elevation. The bicarbonate dependence did not result from the increased sodium concentration or alkalinization of the buffer. Our results support the hypothesis that the neurotoxicity of BMAA is due to an excitotoxic mechanism, involving elevated intracellular calcium levels and bicarbonate. Furthermore, since BMAA alone produced no increase in Ca(2+) levels, these results suggest the involvement of a product of BMAA and CO(2), namely a beta-carbamate, which has a structure similar to other excitatory amino acids (EAA) such as glutamate; thus, the causative agent for ALS-PDC on Guam and elsewhere may be the beta-carbamate of BMAA. These findings support the theory that some forms of other neurodiseases may also involve environmental toxins.

Correlation of rates of calcium entry and endogenous dopamine release in mouse striatal synaptosomes

The time course of simultaneous Ca2+ entry and endogenous dopamine release was examined in mouse striatal synaptosomes depolarized by 30 mM KCl. Ca2+ entry and endogenous dopamine release exhibited fast and slow phase processes. The fastest rates occurred between 0 and 1 s. Ca2+ uptake and dopamine release dropped off quickly with 5-15 s rates at 13 and 10%, respectively, of the 0-1 s rate. Both processes were maintained at relatively high rates at the 1-3 and 3-5 s intervals suggesting mixed fast and slow phase processes. Uptake of Ca2+ and release of dopamine occurred in parallel over the entire 30 s measurement period; however, approximately 70% of the Ca2+ uptake and dopamine release occurred within the first 5 s following depolarization. A calculated ratio of Ca2+ entry versus dopamine release showed that approximately 1-2 Ca2+ ions were required to cause the release of one dopamine molecule. This ratio remained constant from 1 to 15 s following depolarization. Our results suggest that Ca2+ entry is coupled to endogenous dopamine release for both the fast and slow phase process.

Acute and chronic effects of barbiturates on depolarization-induced calcium influx into rat synaptosomes

Depolarization-induced 45Ca2+ influx into synaptosomes isolated from nontreated control and acutely treated rats (given 60 mg/kg phenobarbital i.p.) was significantly depressed (54 and 37%, respectively) by an in vitro challenge with pentobarbital, 0.3 mM (final concentration). However, depolarization-induced 45Ca2+ influx into synaptosomes isolated from tolerant rats (received dietary phenobarbital, 2.5 mg/g of diet, for 13 days) was not significantly altered when the synaptosomes were challenged with 0.3 mM pentobarbital. This suggests that synaptosomal membranes adapt during chronic exposure to barbiturates to allow for an enhanced Ca2+ influx subsequent to depolarization. Our data suggest that sedation may, at least in part, occur as a result of depressed stimulus-secretion coupling and that behavioral tolerance to sedation may occur because of the development of membrane tolerance to allow enhanced calcium influx.

Ethanol and Neuronal Calcium Channels

Ethanol alters voltage-dependent calcium channels and receptor-operated channels which transport calcium [e.g., the ion channels associated with N-methyl-D-aspartate (NMDA) receptors]. However, the question which still remain unanswered is whether these modifications have any physiological significance. This review will focus on the effects of ethanol on various calcium-related parameters with an emphasis on the possible relevance to the behavioral effects elicited by ethanol.