Biomedical subjects

S W Leslie

Publications and source records attributed to S W Leslie.

At least 19 recordsLinked to original sources

PubMed1992

Stimulation of N-methyl-D-aspartate receptor-mediated calcium entry into dissociated neurons by reduced and oxidized glutathione.

The effects of GSH (gamma-glutamylcysteinylglycine) and GSSG on intracellular calcium levels ([Ca2+]i) were investigated using fura-2-loaded dissociated brain cells from newborn rat pups. Both produced concentration-dependent increases in [Ca2+]i (EC50 values of 914.3 +/- 190.5 and 583.0 +/- 97.2 microM for GSH and GSSG, respectively), similar to that observed with N-methyl-D-aspartate (NMDA) and other agonists at the NMDA receptor. Maximum response (expressed as percentage change in [Ca2+]i relative to basal) was significantly greater for GSSG (37.5 +/- 1.6%) than for GSH (25.3 +/- 1.6%). The response to both agents was prevented or reversed by competitive (100 microM) (-)-2-amino-5- phosphonovalerate and noncompetitive (400 nM) MK-801 or 1.0 mM Mg2+ antagonists of NMDA receptor-mediated calcium entry, even at concentrations of GSH and GSSG normally producing maximal response. The idea that these effects are mediated, at least in part, by interaction with the NMDA receptor was supported by the effects of GSH and GSSG on the binding of the NMDA receptor ligand [3H]CGP-39653 to membranes isolated from hippocampal and cortical homogenates. Both GSH and GSSG displaced bound [3H]CGP-39653, with IC50 values of 0.93 +/- 0.18 and 11.02 +/- 1.22 microM, respectively, and produced an increase in the apparent Kd of binding (control, 8.92 +/- 0.83 nM, and GSH, 13.31 +/- 1.19 nM; control, 11.59 +/- 0.35 nM, and GSSG, 18.73 +/- 0.66 nM). However, both also produced modest reductions in Bmax (control, 1265 +/- 69 fmol/mg of protein, and GSH, 901 +/- 73 fmol/mg of protein; control, 1068 +/- 30 fmol/mg of protein, and GSSG, 730 +/- 18 fmol/mg of protein) and Hill slopes (GSH, 0.66 +/- 0.02; GSSG, 0.62 +/- 0.04). This suggests complex kinetics for the interaction of GSH and GSSG with the NMDA receptor. Taken together, the results suggest the potential for modulation of the NMDA receptor complex by GSH and GSSG.

2-Amino-5-phosphonovalerate↗

PubMed1991

The effects of chronic ethanol exposure on N-methyl-D-aspartate-stimulated overflow of [3H]catecholamines from rat brain.

The effects of acute and chronic ethanol administration on N-methyl-D-aspartate-stimulated catecholamine overflow were examined. Three groups of male Sprague-Dawley rats were used. The first group received a chronic liquid diet containing ethanol (37%) for 3 weeks. The second group was pair-fed a liquid diet with dextrin substituted for ethanol isocalorically. The third group received Purina rat lab chow and water ad libitum. N-methyl-D-aspartate-stimulated [3H]catecholamine overflow from brain tissue slices was determined. N-methyl-D-aspartate (50-2000 microM) produced a concentration-dependent increase in [3H]norepinephrine overflow from cortical and hippocampal slices with no significant alteration of the response following chronic ethanol treatment. [3H]Dopamine overflow from striatal slices of the chronic ethanol group was significantly different at 1000 microM N-methyl-D-aspartate. The response of the chronic ethanol-treated group at the 1000 microM N-methyl-D-aspartate concentration was 30% and 40% lower than the pair-fed and ad libitum controls, respectively. Ethanol when added in vitro (30-200 mM) produced a concentration-dependent inhibition of N-methyl-D-aspartate (150 microM) stimulated efflux in all brain regions, and chronic ethanol treatment did not alter the inhibitory response. These results indicate an apparant lack of adaptation in N-methyl-D-aspartate-stimulated transmitter release following chronic ethanol treatment in this particular paradigm.

Animals↗

PubMed1991

Mechanism of inhibition of N-methyl-D-aspartate-stimulated increases in free intracellular Ca2+ concentration by ethanol.

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 microM measured in the absence of Mg2+. NMDA-stimulated [Ca2+]i increases could be observed in buffer with Mg2+ when the cells were predepolarized with 15 mM KCl prior to NMDA addition. Under these predepolarized conditions, 100 mM ethanol inhibited 25 microM 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 microM 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 microM NMDA responses was independent of the external Ca2+ or Mg2+ concentrations. Glycine (1, 10, and 100 microM) enhanced 25 microM NMDA-induced increases in [Ca2+]i by approximately 50%. Glycine (1-100 microM) prevented the 100 mM ethanol inhibition of NMDA-stimulated [Ca2+]i observed in the absence of exogenous glycine. MK-801 (25-400 nM) inhibited 25 microM NMDA-stimulated rises in [Ca2+]i in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Behavioral sensitization following subchronic apomorphine treatment--possible neurochemical basis.

Subchronic treatment with the dopamine agonist apomorphine produces a sensitization to the stereotypic effects of subsequent apomorphine challenge. The present study investigated the effects of this subchronic treatment on apomorphine induced stereotypic behavior and striatal dopamine synthesis, release, metabolism, and D2 receptor binding. The pretreatment, which enhanced the behavioral response to apomorphine challenge, also elevated basal dopamine synthesis and metabolism, but left the ability of a challenge dose of apomorphine to inhibit dopamine synthesis and metabolism unaltered. Thus, ongoing dopamine synthesis and extracellular levels of metabolites would be higher following apomorphine challenge in animals treated subchronically with the agonist. In contrast, neither synaptosomal dopamine release in response to depolarizing stimuli nor the density of D2 dopamine receptors was altered by the treatment. Overall, the results suggest that, while we did not find evidence of autoreceptor desensitization per se, apomorphine treatment may result in enhanced extracellular dopamine levels following dopamine agonist challenge to provide a greater stimulation of an intact dopamine receptor system.

Animals↗

PubMed1989

Development of tolerance in brain mitochondria for calcium uptake following chronic ethanol ingestion.

Brain mitochondria isolated from rats following 10 weeks of chronic exposure to ethanol were not deficient in respiratory function or in rates of calcium uptake under control conditions. Ethanol (80 mM) in the incubation medium caused significant depression in the respiratory and ATP-dependent rates of calcium uptake in control mitochondria, but did not affect mitochondria from ethanol-tolerant rats. Chronic exposure to ethanol causes mitochondria to take calcium up at a normal rate when challenged acutely by ethanol.

Animals↗

PubMed1989

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.

2-Amino-5-phosphonovalerate↗

PubMed1989

Aging does not alter cytosolic calcium levels of cortical synaptosomes in Fischer 344 rats.

Several lines of experimental evidence support an association between altered Ca2+ regulation and aging. It has been supposed that free cytosolic Ca2+ concentrations ([Ca2+]i) may decrease or increase in aged animals. In this study, both resting and KCl-stimulated [Ca2+]i were measured in purified cortical synaptosomes from young (3 mo.), middle-aged (12 mo.), and old (24 mo.) Fischer 344 rats. Two additional groups of rats were included, one middle-aged and one old which were trained on a treadmill for 6 months prior to experimentation. The [Ca2+]i was determined using the fluorescent Ca2+ chelator fura-2. Net KCl-dependent changes (delta K) in [Ca2+]i were determined by the difference between stimulatory (100 microM Ca2+/60 mM KCl) and resting (100 microM Ca2+/5 mM KCl buffer) conditions among the 3 age groups. Significant increases in [Ca2+]i were observed in each age group upon depolarization with 60 mM KCl. However, there were no significant age-dependent differences in either resting [Ca2+]i or KCl-stimulated [Ca2+]i.

Aging↗

PubMed1989

Aging does not alter the voltage-dependent release of endogenous dopamine from mouse striatal synaptosomes.

Striatal synaptosomes isolated from 3-, 12-, and 24-month-old C57B1/6J mice were exposed to low (5 mM) and high (30 mM) concentrations of potassium of 1, 3, 5, 15 and 30 s. There were no age-related differences in the total intrasynaptosomal content of dopamine among the three age groups of mice. The basal release of endogenous dopamine from synaptosomes isolated from 24-month-old animals was significantly greater than that from synaptosomes isolated from 3-month-old animals at all time points tested. Similarly the potassium stimulated release of dopamine from 24-month-old animals was also higher than that from the 3-month-old group, but this increase was not statistically significant. The net voltage-dependent release of dopamine (stimulated minus basal) was similar for all groups at all time points measured. The increase in basal dopamine release in the 24-month-old animals may reflect an increased fragility or leakiness of the synaptosomal preparation.

Aging↗

PubMed1989

Protein kinase C activation enhances K+-stimulated endogenous dopamine release from rat striatal synaptosomes in the absence of an increase in cytosolic Ca2+.

The possibility that protein kinase C modulates neurotransmitter release in brain was investigated by examining the effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on Ca2+ transport and endogenous dopamine release from rat striatal synaptosomes. TPA (0.16 and 1.6 microM) significantly increased dopamine release by 24 and 33%, respectively, after a 20-min preincubation with TPA followed by 60 s of depolarization with 30 mM KCl. Depolarization-induced 45Ca2+ uptake, measured simultaneously with dopamine release, was not significantly increased by TPA. Neither 45Ca2+ uptake nor dopamine release was altered under resting conditions. When the time course of K+-stimulated 45Ca2+ uptake and dopamine release was examined, TPA (1.6 microM) enhanced dopamine release after 15, 30, and 60 s, but not 1, 3, or 5 s, of depolarization. A slight increase in 45Ca2+ uptake after 60 s of depolarization was also seen. The addition of 30 mM KCl to synaptosomes which had been preloaded with the Ca2+-sensitive fluorophore fura-2 increased the cytosolic free Ca2+ concentration ([Ca2+]i) from 445 nM to 506 nM after 10 s of depolarization and remained elevated after 60 s. TPA had no effect on [Ca2+]i under depolarizing or resting conditions. Replacing extracellular Ca2+ with 100 microM EGTA reduced K+-stimulated (60 s) endogenous dopamine release by 53% and decreased [Ca2+]i to 120 nM. In Ca2+-free medium, 30 mM KCl did not produce an increase in the [Ca2+]i. TPA (1.6 microM) did not alter the [Ca2+]i under resting or depolarizing conditions, but did increase K+-stimulated dopamine release in Ca2+-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Outpatient metabolic evaluation of patients with recurrent kidney stones.

We have designed an outpatient, comprehensive metabolic evaluation program for patients with recurrent kidney stone disease. This protocol can be expected to detect and identify specific biochemical abnormalities contributing to kidney stone formation in about 95% of these patients. Individualized therapy can then be started based on the particular metabolic defect discovered in each patient. This diagnostic and treatment program will stop new kidney stone formation in these high-risk patients virtually completely. For each stone-related hospitalization eliminated, the average savings are at least $2,700. The total potential savings in our small community alone are over $460,000! Our metabolic stone evaluation protocol is the only one with which we are familiar that was designed and implemented exclusively at the community hospital level. It is readily adaptable to other medical facilities.

Ambulatory Care↗

PubMed1988

Effects of pentobarbital on behavioral and synaptic plasticities in crayfish.

Intra-abdominal injections of 90 mg/kg (3.5 x 10(-4) M estimated body concentration) sodium pentobarbital (PB) eliminated the cheliped closing response and eyestalk withdrawal response in crayfish (Procambarus clarkii). Repeated injections produced tolerance to both of these behavioral measures within several days. At glutamatergic synapses of the opener muscle, PB at dosages from 10(-7) to 10(-4) M had no significant effect on non-facilitated transmitter release evoked by 1 Hz stimuli. Facilitated transmitter release at 10 Hz stimuli was significantly decreased by 10(-3) M PB but was not significantly affected at lower concentrations. At 10(-4) M and 10(-3) M, PB significantly reduced the ratio of excitatory postsynaptic potential (EPSP) amplitudes at 10 Hz to those at 1 Hz. The frequency of spontaneous miniature EPSPs (MEPSPs) was reduced by 10(-4) M PB to about half of the control level, while MEPSP amplitudes and time constants were not significantly affected. These results suggest that the ability of PB to depress various crayfish behaviors is due, at least in part, to a presynaptic ability of the drug to depress facilitation of transmitter release at glutamatergic synapses.

Action Potentials↗

PubMed1988

Differential sensitivity of synaptosomal calcium entry and endogenous dopamine release to omega-conotoxin.

The presynaptic neurotoxin omega-conotoxin (omega-CgTx) was tested for its ability to inhibit voltage-dependent calcium flux and transmitter release in rat brain synaptosomes. Conotoxin (0.001-10 microM) had no effect on calcium uptake or endogenous dopamine release from rat striatal synaptosomes in the absence of potassium depolarization. Fast-phase potassium stimulated calcium influx was only partially (20-30%) inhibited by conotoxin at concentrations between 1 nM and 10 microM. The fast-phase release of endogenous dopamine from the same synaptosomal preparation was inhibited by approximately 25% at 0.01 microM and by 60% at 10 microM. These results suggest that a subgroup of high affinity omega-CgTx-sensitive calcium channels may be involved in regulating the release of endogenous dopamine from brain synaptosomes.

Animals↗

PubMed1988

Calcium-dependent and -independent release of endogenous dopamine from rat striatal synaptosomes.

We examined the role of calcium in the stimulus-secretion coupling process of brain neurons by measuring the potassium-stimulated release of endogenous dopamine from striatal synaptosomes in the presence and absence of extracellular calcium. Intracellular free calcium levels were also monitored under these conditions using the fluorescent calcium chelator, fura-2. The fast-phase (less than 3 s) of potassium-stimulated dopamine release was completely blocked by removing calcium from the external medium. Elimination of calcium from the medium with EGTA only partially blocked the slow phase (60 s) of K+-stimulated dopamine release. Depolarization of synaptosomes in the presence of extracellular calcium significantly increased intracellular calcium levels as measured by fura-2. No changes in intracellular calcium were observed during depolarization in calcium free-medium. Reductions in the sodium concentration of the extracellular medium produced a significant increase in the basal release of dopamine under calcium-free conditions. Depolarization of synaptosomes under these conditions markedly enhanced the release of dopamine. These results suggest that the slow-phase of dopamine release from synaptosomes does not require calcium but may be mediated via the reversal of the sodium-linked dopamine transport system.

Animals↗

PubMed1988

Beta-adrenergic receptor characteristics of postnatal rat myocardial cell preparations.

Primary myocardial cell cultures and freshly isolated cardiac cells in suspension represent two isolated, whole cell models for investigating cellular transsarcolemmal 45Ca++ exchange in response to a receptor-coupled stimulus. Studies were performed to characterize beta-adrenergic receptor binding, beta-adrenergic receptor mediated cellular calcium (45Ca++) exchange, and viability in purified primary myocardial cell cultures and freshly isolated cardiac cells in suspension obtained from 3- to 5-d-old Sprague-Dawley rats. In addition, beta-adrenergic receptor binding was characterized in whole-heart crude membrane preparations. All three preparations had saturable beta-adrenergic binding sites with the antagonist [125I]iodopindolol [( 125I]IPIN). The suspensions had a significantly lower Bmax (42 +/- 6 fmol/mg protein) than the membranes and cultures (77 +/- 8 and 95 +/- 10 fmol/mg protein, respectively). The KD of the cultures (218 +/- 2.0 pM) was significantly higher than that for the suspensions (107 +/- 1.3 pM) and membranes (93 +/- 1.3 pM). Viability was significantly lower in the suspensions (57%) when compared to 94% viability in myocardial cell cultures after 3 h of incubation in Kreb's Henseleit buffer. Incubation of the cultures with 5.0 X 10(-7) M isoproterenol resulted in a significant increase in 45Ca++ exchange as early as 15 s. In contrast, 45Ca++ exchange into the suspensions was not increased. Although both primary cell cultures and cardiac cells in suspension possess saturable beta-adrenergic receptors, only the monolayer cultures exhibited functional beta-adrenergic receptor-mediated 45Ca++ exchange. Of the two intact cell models investigated, these data suggest that primary myocardial cell cultures are more suitable than cell suspensions for investigating beta-adrenergic receptor binding and functions in the postnatal rat heart.

Animals↗

PubMed1988

Characterization of dihydropyridine-sensitive calcium channels in rat brain synaptosomes.

We examined the effects of dihydropyridine Ca2+-channel agonists on synaptosomal voltage-dependent Ca2+ entry and endogenous dopamine release. The (-) isomer of Bay K 8644 and the (+) isomer of Sandoz compound 202-791 were 100-1000 times more potent than their respective opposite enantiomers in enhancing Ca2+ uptake and dopamine release from striatal synaptosomes. The active isomer of each of these compounds increased Ca2+ entry and dopamine release to the same extent at a concentration of 1 nM. Fast-phase Ca2+ entry into synaptosomes isolated from cerebellum, cortex, and hippocampus was sensitive to nanomolar concentrations of Bay K 8644. No effect of Bay K 8644 was observed in synaptosomes isolated from brainstem. Bay K 8644 increased synaptosomal Ca2+ uptake and endogenous dopamine release from striatal synaptosomes only during the initial seconds of KCl-induced depolarization. The greatest increase was observed during the first second of depolarization. No effect was observed after greater than or equal to 5 sec of depolarization. Bay K 8644 did not alter Ca2+ uptake or dopamine release under resting conditions (5 mM KCl) or in response to KCl at greater than 15 mM. The activity of Bay K 8644 was also attenuated by lowering the concentrations of divalent cations in the incubation medium. Agonist activity was observed at Mg2+ concentrations greater than 500 microM (Ca2+ held at 100 microM) and Ca2+ concentrations greater than 100 microM (Mg2+ held at 1000 microM). These results suggest that the Ca2+ channels present in synaptosomes are sensitive to nanomolar concentrations of dihydropyridine agonists under a narrow range of experimental conditions.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1987

Calcium channels. Interactions with ethanol and other sedative-hypnotic drugs.

Biochemical and electrophysiological studies have shown that ethanol potently inhibits voltage-dependent calcium channels in presynaptic nerve terminals in brain. The ability of ethanol to inhibit calcium channels appears to vary from one brain region to another. Chronic ethanol administration results in adaptation of the calcium channels in certain brain regions such that they become tolerant to the inhibitory actions of ethanol. Other sedative-hypnotic drugs, such as barbiturates and benzodiazepines, also inhibit calcium channels but may differ from ethanol in their brain regional potency. These studies suggest that the pharmacological actions of ethanol and other sedative-hypnotic drugs may be linked with alterations in calcium channel function.

Animals↗

PubMed1986

5-(2-Cyclohexylideneethyl)-5-ethyl barbituric acid (CHEB): correlation of hypnotic and convulsant properties with alterations of synaptosomal 45Ca2+ influx.

Male ICR mice (20-35 g) were given either 5-(2-cyclohexylideneethyl)-5-ethyl barbituric acid (CHEB) alone (10-15 mg/kg i.p.) or CHEB (25-75 mg/kg i.p.) after a 1 h pretreatment with phenobarbital (75 mg/kg i.p.). CHEB alone (10 mg/kg) produced excitatory behavior but not convulsive seizures. Higher doses (11-15 mg/kg) produced convulsive seizures resulting in death. Pretreatment with phenobarbital prevented seizure activity. Following phenobarbital pretreatment, CHEB in doses of 50 and 75, but not 25 mg/kg, resulted in hypnosis of 53 +/- 16 and 64 +/- 9 min duration, respectively. In vitro, CHEB (10-200 microM) significantly inhibited 'fast-phase' (3 s) K+-stimulated 45Ca2+ uptake into cerebrocortical synaptosomes. CHEB (10 and 100 microM) also significantly increased basal 45Ca2+ uptake. The addition of CHEB (50 and 100 microM) or pentobarbital (100 microM) to striatal synaptosomes inhibited 'fast-phase' K+-stimulated 45Ca2+ uptake and endogenous dopamine release. CHEB (10-200 microM), but not pentobarbital (100 microM), produced a time- and dose-dependent increase in the resting release of endogenous dopamine from striatal synaptosomes. The results of this study show that CHEB possesses hypnotic activity if its lethal convulsant actions are blocked. The hypnotic actions of CHEB appear to correlate with inhibition of voltage-dependent calcium channels in brain synaptosomes.

Animals↗

PubMed1986

Correlation of the hypnotic potency of benzodiazepines with inhibition of voltage-dependent calcium uptake into mouse brain synaptosomes.

Nine benzodiazepines were tested for their ability to inhibit 45Ca2+ uptake into mouse whole brain synaptosomes and for hypnotic activity as indicated by their ability to produce loss of the righting reflex. Eight of the benzodiazepines significantly inhibited fast-phase voltage-dependent 45Ca2+ uptake and five exhibited hypnotic activity. There was a direct correlation between the hypnotic potency of these five benzodiazepines and their ability to inhibit 45Ca2+ uptake. There does not appear to be a correlation between the anticonvulsant potency of the benzodiazepines and their potency for inhibiting 45Ca2+ uptake. These results support previous findings with other sedative/hypnotic drugs and suggest that inhibition of presynaptic calcium uptake may be linked with hypnotic but not anticonvulsant actions of benzodiazepines.

Animals↗