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Biomedical subjects

G Lynch

Publications and source records attributed to G Lynch.

At least 199 records · Page 11Linked to original sources
PubMed1996

Ultraviolet radiation, thiol reagents, and solubilization enhance AMPA receptor binding affinity via a common mechanism.

The binding properties of membrane-bound or solubilized AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid)-type glutamate receptors from rat brain were tested following exposure to ultraviolet (UV) radiation or incubation with the thiol reagent p-chloromercuriphenyl-sulfonic acid (PCMBS). Brief exposure to UV radiation (254 nm) increased [3H]AMPA binding to brain membranes, while binding to soluble fractions decreased. The increase in brain membrane binding was caused by an apparent interconversion of low-affinity [3H]AMPA binding sites into a higher-affinity state. Incubation with PCMBS caused a significant increase in [3H]AMPA binding to brain membranes but had no significant effect on [3H]AMPA binding to solubilized receptors. There was an interaction between the PCMBS and UV effects in the brain membranes such that prior exposure to one of the treatments reduced the relative magnitude of the other's effects. The present results suggest that ultraviolet radiation, PCMBS and solubilization all increase AMPA receptor binding affinity via a common mechanism.

4-Chloromercuribenzenesulfonate↗
PubMed1996

Distinct distributions of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits and a related 53,000 M(R) antigen (GR53) in brain tissue.

Polyclonal antibodies against specific carboxy-terminal sequences of known alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits (GluR-4) were used to screen regional homogenates and subcellular fractions from rat brain. Affinity purified anti-GluR1 (against amino acids 877-899), anti-GluR2/3 (850-862), and anti-GluR4a and anti-GluR4b (868-881) labeled distinct subunits with the expected molecular weight of approximately 105,000. These antigens were shown to have distinct distributions in the brain. While GluR2/3 epitopes had a distribution profile similar to that of the presynaptic marker synaptophysin, GluR1 was notable for its abundance in the hippocampus and its relatively low density in neocortical areas, and GluR4 was highly enriched in cerebellar tissue. An additional antigen (glutamate receptor-related, GR53) of lower molecular weight (50,000-59,000) was recognized in rat, human, frog, chick and goldfish brain samples by anti-GluR4a as well as by anti-GluR1 at, an antibody that specifically recognizes the extracellular aminoterminal domain of GluR1 (amino acids 163-188). Both antibodies also labeled antigens of approximately 105,000 mol. wt in brain tissue from all species tested. The approximately 53,000 mol. wt antigen was concentrated 10-20-fold in synaptic membranes vs homogenates across rat brain regions. Both the 105,000 and the 53,000 mol. wt proteins were also concentrated in postsynaptic densities, and neither of the two antigens were evident in seven non-brain tissue samples. These data indicate that AMPA receptors have regionally different subunit combinations and that some AMPA receptor composites include proteins other than the conventional 105,000 mol. wt GluR subunits.

Amino Acid Sequence↗
PubMed1996

Effects of pharmacologically facilitating glutamatergic transmission in the trisynaptic intrahippocampal circuit.

The effects of a recently synthesized benzoyl-piperidine drug that enhances currents mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors were tested on monosynaptic and polysynaptic responses in hippocampal slices of the rat. Stimulation of perforant path inputs to the dentate gyrus evoked extracellular responses in field CA1 that had latencies and laminar profiles indicating that they were relayed through the trisynaptic intrahippocampal circuit. Under control conditions, trisynaptic field excitatory postsynaptic potentials did not show larger paired-pulse facilitation than monosynaptic responses and failed to exhibit frequency facilitation. Low concentrations of picrotoxin greatly enhanced trisynaptic responses and, under these conditions, frequency facilitation was obtained. Benzoyl-piperidine-12 (250 microM) had a three-fold greater effect on the amplitude of trisynaptic responses than on monosynaptic field excitatory postsynaptic potentials, indicating that the drug's effect is amplified across the successive stages of a polysynaptic circuit. The AMPA receptor modulator did not change the frequency characteristics of monosynaptic potentials and had only a modest influence on those of the trisynaptic response. The effect of benzoyl-piperidine-12 on trisynaptic responses was significantly greater when GABAergic inhibition was partially blocked with picrotoxin; the GABA blocker did not alter the effects of benzoyl-piperidine-12 on monosynaptic responses. These results indicate that centrally active AMPA receptor modulators are likely to have a greater influence on brain operations involving long chains of connections than on those mediated by simple reflex-like circuits, and will vary markedly in their effects depending upon the excitability of local interneurons.

Animals↗
PubMed1996

Effects of a centrally active benzoylpyrrolidine drug on AMPA receptor kinetics.

A newly developed benzoylpyrrolidine drug (BDP-20) that increases the size of fast, excitatory synaptic responses was examined for its effects on the kinetic properties of alpha-amino-3-hydroxy-5-methyl-4-isoxalepropionic acid (AMPA)-type glutamate receptors. When long pulses of glutamate were applied to excised hippocampal patches of the rat, the compound BDP-20 caused an approximately 15-fold reduction in the rate at which responses desensitized and a similar size increase in steady-state currents. In experiments using 1-ms glutamate pulses, BDP-20 prolonged response deactivation by a factor of about four and greatly reduced the depression in the second response when two consecutive glutamate pulses were given. Two types of equilibrium binding assays indicated that BDP-20 causes a measurable increase in the affinity of AMPA receptors; the EC50 values for this effect were similar to those obtained in excised patch studies. The actions of BDP-20 on physiology and ligand binding could be adequately reproduced in a receptor model by slowing the rate of desensitization and increasing the affinity of the sensitized states. The biochemical and physiological effects of this benzoylpyrrolidine compound were qualitatively different from those obtained with cyclothiazide, although both types of drug increased AMPA receptor-mediated synaptic responses. Moreover, interactions between the drugs were at most only partially competitive; AMPA receptors may thus have multiple modulatory sites with distinct drug preferences and different effects on receptor kinetics.

Animals↗
PubMed1996

Cytosolic proteolysis of tau by cathepsin D in hippocampus following suppression of cathepsins B and L.

Incubation of cultured hippocampal slices with an inhibitor [N-CBZ-L-phenylalanyl-L-alanine-diazomethyl ketone (ZPAD)] of cathepsins B and L resulted in the degradation of high molecular weight isoforms of tau protein and the production of a 29-kDa tau fragment (tau 29). A tau antibody that is sensitive to the phosphorylated state of its epitopes did not recognize tau proteins or the tau 29 fragment in slices that had been treated with a protein phosphatase inhibitor. This strongly suggests that the tau fragment was located in an extralysosomal compartment accessible to kinases and phosphatases. tau 29 exhibited a significant capacity for binding to microtubules and thus has the potential for interfering with normal tau-tubulin interactions. Three lines of evidence indicated that ZPAD-induced tau proteolysis was mediated by cathepsin D: (a) slices treated with the inhibitor had markedly elevated levels of cathepsin D in both lysosomal and extralysosomal compartments; (b) co-incubation of cathepsin D and tau at neutral pH resulted in a loss of intact tau proteins and production of a 28-kDa fragment; and (c) the lysosomotropic drug chloroquine blocked ZPAD-induced increases in mature cathepsin D, and this was accompanied by a suppression of ZPAD-induced tau proteolysis. Changes in lysosomal hydrolases and cytoskeletal perturbations occur during brain aging. The present results suggest that the enzymatic and structural effects are related and, more specifically, are linked by alterations in the concentration and localization of cathepsin D. The tau fragments with microtubule binding capacity generated by cathepsin D could also be a source for the small polypeptides found in association with age-related pathological features.

Animals↗
PubMed1996

Exertional compartment syndromes of the lower extremity.

Compartment syndromes may be acute or chronic secondary to exertion or exercise. The chronic or exertional type most commonly involves the lower extremity, particularly the anterior compartment of the lower leg, and is the subject of this review. Rarely, an exertional compartment syndrome may become acute. The diagnosis is based on history, physical examination, and compartment pressure measurements. The differential diagnosis of exertional leg pain includes stress fractures, stress reaction, periostitis, claudication, popliteal artery entrapment, and peripheral nerve entrapment. Unusual causes, such as a ganglion of the proximal tibiofibular joint causing an anterior compartment syndrome, have recently been reported.

Compartment Syndromes↗
PubMed1996

Psychological effects of a drug that facilitates brain AMPA receptors.

The effects of 1-(quinoxalin-6-ylcarbonyl)piperidine (CX516), a centrally active compound that facilitates AMPA receptor-mediated synaptic responses, were tested in human subjects. Separate tests of delayed recall were given prior to and nearly 3 h after administration of placebo (n = 12) or drug (n = 36). Control subjects exhibited poorer performance in the second session than in the first while subjects given 600-1200 mg of the drug did not. There were no pre- vs post-treatment differences in immediate recall in either group. The drug did not reliably affect self-assessment scores for any of several psychological variables but did disrupt the normally present correlations for within-subject changes in the variables. These results suggest that AMPA receptor modulators may (1) improve memory under some circumstances and (2) produce psychological effects that are subtle or not related to specific mood states.

Adult↗
PubMed1996

Distribution and metabolism of intravitreal cidofovir and cyclic HPMPC in rabbits.

PURPOSE: This study was designed to evaluate the intraocular distribution and metabolism of the antiviral nucleotide analogs cidofovir and cyclic 1-[(S)-3-hydroxy-2-(phosphonomethoxy) propyl]cytosine (HPMPC) in New Zealand white rabbits following intravitreal administration. METHODS: Male rabbits received either 14C-cidofovir or 14C-cyclic HPMPC by intravitreal injection into both eyes (50 micrograms/eye, 11 microCi/eye). Two animals per group were sacrificed at 24, 48, 72 or 240 h post-dose. Ocular tissues, kidney and liver were oxidized to determine total radioactivity and metabolites were determined by HPLC. RESULTS: At 24 h post-dose, total radioactivity was 9.96 and 5.18 micrograms-equiv/g for cidofovir and cyclic HPMPC, respectively, in vitreous and 20.9 and 3.54 micrograms-equiv/g, respectively, in retina. Although the initial vitreal clearance was 2-fold faster for the cyclic analog, the estimated terminal elimination half-lives in vitreous (42 hr) and in retina (66-77 hr) were similar for both drugs. By 240 h post-dose, radioactivity in all ocular tissues was approximately ten-fold higher for cidofovir. Radioactivity in vitreous at 240 h after intravitreal dosing with either drug contained cidofovir, cyclic HPMPC and cidofovir-phosphocholine. CONCLUSIONS: The long retinal half-life observed presumably reflects formation of phosphorylated cidofovir within retinal cells. Cidofovir achieved a ten-fold higher level of phosphorylated drug in retina than cyclic HPMPC: Therefore, intravitreal cidofovir may be expected to suppress progression of retinitis for a longer period than an equivalent intravitreal dose of cyclic HPMPC: The intravitreal half-life of cidofovir was 20-fold longer than that of ganciclovir in the same animal model.

AIDS-Related Opportunistic Infections↗
PubMed1996

Effect of cyclothiazide on binding properties of AMPA-type glutamate receptors: lack of competition between cyclothiazide and GYKI 52466.

The effects of cyclothiazide on the properties of (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors were studied using equilibrium binding techniques and interactions with other compounds known to modulate the receptors. Cyclothiazide caused a reduction in [3H]AMPA binding in assays carried out in the presence of thiocyanate, a chaotropic ion that markedly increases the affinity of AMPA receptors and accelerates their desensitization. In the absence of thiocyanate, however, cyclothiazide had no reliable effect on the binding of [3H]AMPA or on the affinity for this agonist assessed from the displacement of [3H]CNQX. The interaction of cyclothiazide with the receptor appears not to be changed by the presence of thiocyanate. Analysis of the results with a kinetic model of the AMPA receptor suggests that cyclothiazide does not block receptor desensitization by making the desensitized state inaccessible but rather by stabilizing the active state, i.e., by increasing the affinity of the latter to a point where it becomes energetically more favorable than the desensitized state. GYKI 52466, an atypical benzodiazepine that blocks AMPA receptor-gated currents, did not reverse the changes in binding affinity produced by cyclothiazide in the presence of thiocyanate. Physiological experiments conducted in excised patches collected from hippocampal pyramidal cells indicated that thiocyanate does not block access of GYKI 52466 to AMPA receptors. These results point to the conclusion that cyclothiazide acts at a site on the AMPA receptor different from that for GYKI 52466.

Animals↗
PubMed1996

Effects of a memory-enhancing drug on DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor currents and synaptic transmission in hippocampus.

The benzoylpiperidine drug BDP-12 (1-(quinoxalin-6-ylcarbonyl)piperidine) enhances the encoding of transient and stable forms of memory by rats. Results reported here show that the drug increases fast, excitatory (glutamatergic) synaptic responses in hippocampal slices by about 50% with an EC50 of 170 microM. Analyses with polysynaptic responses indicated that the drug has a facilitatory action at concentrations as low as 12.5 microM. BDP-12 at 1 mM did not change the resting membrane potential, input resistance or spiking threshold and it did not alter monosynaptic potentials mediated by gamma-aminobutyric acid (GABA) receptors; it did, however, enhance disynaptic inhibitory responses. In membrane patches excised from hippocampal neurons, BDP-12 at moderate concentrations (50 microM) increased the steady-state currents mediated by DL-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and slowed the rate at which the receptors desensitize, with a much larger effect on the former; the drug delayed the closing of the AMPA receptor channel after 1-msec agonist pulses. BDP-12 had no detectable effect on [3H]AMPA binding affinity. A related and more potent analog produced a different pattern of results in that it had about equal effects on steady-state currents and desensitization rates and significantly increased binding to AMPA receptors. These results indicate that the benzoylpiperidine family of modulators has functionally distinct subclasses. The findings also establish that BDP-12: 1) enhances synaptic responses in the same concentration range at which it alters AMPA receptor kinetics, 2) has a lower apparent threshold for effects on complex network operations than on monosynaptic transmission, 3) does not directly influence inhibitory responses and 4) is likely to modulate AMPA receptors on interneurons as well as on pyramidal neurons.

Animals↗
PubMed1996

Pharmacokinetics, bioavailability, metabolism, and tissue distribution of cidofovir (HPMPC) and cyclic HPMPC in rats.

The pharmacokinetics, distribution, and metabolism of [14C]cidofovir and its cyclic analog {[2-14C]-1-[((S)-2-hydroxy-2-oxo-1,4, 2-dioxaphosphorinan-5-yl)methyl]cytosine; [14C]cyclic HPMPC} were compared after administration to Sprague-Dawley rats. After intravenous (iv) administration of [14C]cidofovir (3 or 5 mg/kg) or [14C]cyclic HPMPC (5 mg/kg), plasma concentrations of total radioactivity declined in a multiexponential manner for both drugs with terminal half-lives of 7-11 hr. No metabolites of cidofovir were observed in plasma. However, iv [14C]cyclic HPMPC was converted in vivo to cidofovir (3.9% of the AUC of total radioactivity). The corrected half-life of cyclic HPMPC was 0.53 +/- 0.05 hr. The clearance of iv cidofovir (0.60-0.79 liter/hr/kg) was significantly less than that of cyclic HPMPC (1.10 +/- 0.12 liter/hr/kg). Cyclic HPMPC was more efficiently secreted by the kidney than cidofovir. The steady-state volume of distribution of cyclic HPMPC (0.32 +/- 0.5 liter/kg) was substantially less than that of cidofovir (0.9-3.0 liter/ kg). Concentrations in kidney at 24 hr after iv administration of 5 mg/kg were approximately 20-fold higher for cidofovir (6.6 micrograms-eq/g) than for cyclic HPMPC, whereas levels of radioactivity in the remaining tissues were similar for both drugs. This is consistent with the improved therapeutic index observed for cyclic HPMPC in animals. After iv administration of [14C]cidofovir (3 mg/kg), concentrations in most tissues declined with half-lives > 12 hr, presumably reflecting the long intracellular half-life of phosphorylated drug. Kidneys and liver of animals given iv [14C]cidofovir (5 mg/kg) contained unchanged cidofovir (35-65%), metabolite I (attributed to cidovofir phosphocholine) (29-60%), and a peak coeluting with cyclic HPMPC on three different HPLC systems (metabolite II) (5-7%). Kidneys, liver, and lung of animals given iv [14C]cyclic HPMPC contained cyclic HPMPC (1-6%), cidofovir (44-50%), and metabolite I (44-54%). The subcutaneous bioavailability of cidofovir was 91.5%. The subcutaneous and oral bioavailabilities of cyclic HPMPC were 82.7% and 3.50 +/- 1.07%, respectively, based on total radioactivity.

Administration, Cutaneous↗
PubMed1995

Differences in the refractory properties of two distinct inhibitory circuitries in field CA1 of the hippocampus.

Extracellular reflections of IPSPs were examined in two distinct circuitries in field CA1 of the hippocampus. Stimulation in the stratum radiatum in the presence of AMPA receptor antagonists elicited positive potentials in the same stratum that were eliminated by picrotoxin, a blocker of GABAA receptors. Laminar profile analysis revealed that the response was maximal in the stratum radiatum at a point well distal to the pyramidal cell body layer and had a negative reflection in the stratum oriens. These field IPSPs presumably mediate the feedforward inhibition normally activated by the Schaffer-commissural projections to field CA1. Stimulation of the alveus produced an antidromic response followed by a much slower positive potential in recordings collected in the pyramidal cell layer. The latter response was suppressed by AMPA receptor antagonists or picrotoxin, as expected for disynaptic, recurrent (feedback) inhibition. The laminar profile for the feedback field IPSPs had its maximum near the pyramidal cell layer and its negative dipole in the stratum radiatum. Feedforward IPSPs were inhibited by about 50% if they were preceded within 200 ms by a priming pulse while feedback IPSPs were reduced by less than 20% under comparable conditions. The refractory effect was minimally dependent on stimulation intensity but was strongly affected by an antagonist of GABAB receptors. Attempts to modify IPSPs in the s. radiatum with long trains of low frequency stimulation or with theta-burst stimulation were not successful, suggesting that GABAergic synapses do not have the plasticities found in their glutamatergic counterparts. These results indicate that interneurons contacted by the extrinsic afferents of hippocampus form GABAergic synapses that differ in terms of spatial location and functional properties from the synapses generated by interneurons innervated by the recurrent collaterals of the pyramidal cells. The findings also suggest that repetitive afferent activity, while reducing the influence of dendritic IPSPs on excitatory input, will leave feedback suppression of cell spiking largely intact.

2-Amino-5-phosphonovalerate↗
PubMed1995

Translational suppression of calpain I reduces NMDA-induced spectrin proteolysis and pathophysiology in cultured hippocampal slices.

Transfection of cultured hippocampal slices for five days with antisense oligonucleotides directed against mRNA encoding calpain I resulted in an approximately 60% decrease in the amount of caseinolytic activity stimulated by 10 microM calcium. Increases in a single proteolytic fragment of spectrin produced by 10-20 min of NMDA receptor stimulation were substantially (approximately 50%) reduced in antisense treated slices; this effect was not obtained in slices exposed to NMDA for 45 min. Attenuation of NMDA receptor-induced spectrin proteolysis by the antisense oligonucleotides was confirmed in immunoassays using antibodies that recognize multiple spectrin breakdown products and in immunocytochemical experiments with an antibody that detects an individual calpain I-mediated fragment. Translational suppression of calpain I did not detectably affect evoked synaptic responses but markedly improved their recovery from a 15 min infusion of NMDA. These results indicate that spectrin breakdown products provide a useful index of in situ calpain I activity and support the hypothesis that the protease plays a significant role in excitotoxicity.

Animals↗
PubMed1995

Enhanced glutamatergic neurotransmission facilitates classical conditioning in the freely moving rat.

Centrally active drugs that enhance alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor-gated currents by increasing the amplitude and duration of fast, excitatory synaptic responses in the hippocampus have recently been described. The effects of the compound 1-(1,3-benzodioxol-5-ylcarbonyl) perperidine (BDP) on associative and non-associative aspects of the classically conditioned eyeblink response in the freely moving rat were examined. Rats injected with BDP blinked significantly more to an acoustic stimulus of 85 dB than did vehicle controls, indicating that the drug enhances alpha responding to discrete auditory cues. Using a less intense stimulus of 80 dB, rats injected with BDP did not exhibit alpha responding or pseudo-conditioning, yet learned the conditioned response at a facilitated rate. These results suggest that increasing excitatory neurotransmission enhances the processing of sensory information and may contribute to subsequent contingency detection.

Acoustic Stimulation↗
PubMed1995

Proteolysis of spectrin by calpain accompanies theta-burst stimulation in cultured hippocampal slices.

Tests were carried out to determine if repetitive bursts of afferent stimulation activate calpain, a calcium-dependent protease hypothesized to be involved in the production of long-term potentiation. Antibodies against a stable breakdown product that results from proteolysis of spectrin by calpain were used to identify sites of enzyme activation in cultured hippocampal slices. Slices in which theta-burst stimulation was applied to the Schaffer collateral fibers had pronounced accumulations of breakdown product that were restricted to field CA1, the zone innervated by the stimulated axons. Labelling occurred in the form of scattered puncta and was also present in dendritic processes. The extent of these effects was correlated (r = 0.73) with the amount of theta-burst stimulation delivered. Control slices or those receiving low frequency stimulation had variable, but uniformly lower, amounts of breakdown product and were clearly distinguishable from those given theta bursts. Statistical analyses using a six point rating scheme confirmed this point (P < 0.001). These results satisfy an essential prediction of the hypothesis that calpain plays an important role in the induction of long-term potentiation.

Afferent Pathways↗
PubMed1995

Comparison of long-term potentiation in the proximal versus distal stratum radiatum of hippocampal field CA1.

Recent studies indicate that long-term potentiation is accompanied by changes in the waveform of field excitatory postsynaptic potentials in the CA1 field of disinhibited hippocampal slices, suggesting that long-term potentiation alters the kinetics of the glutamate receptor channels that mediate excitatory synaptic responses. The present study examined the effects of stimulating and recording location within stratum radiatum on the magnitude of long-term potentiation and the associated waveform changes. Stimulation of stratum radiatum proximal to the cell body layer evoked field excitatory postsynaptic potentials in proximal stratum radiatum that had decay phases well-fit by single exponentials; long-term potentiation reduced the decay time constant of these potentials, as reported previously. Stimulation of distal stratum radiatum evoked field excitatory postsynaptic potentials in distal stratum radiatum that were contaminated by positive after-potentials; meaningful estimates of decay time constants for these responses could not be obtained. Long-term potentiation of distal responses tended to be smaller than that obtained proximally. Comparisons were also made between responses recorded distally and proximally for either distal or proximal stimulation. For both stimulation loci, the distally-recorded responses had positive after-potentials and the proximally-recorded responses did not. The decay time constants for proximally-recorded responses to distal stimulation decreased significantly after long-term potentiation. Proximal and distal stimulation both produced greater long-term potentiation recorded proximally than distally and the difference was significantly greater for proximal stimulation. When long-term potentiation was induced by stimulation of proximal and distal sites simultaneously, the difference between proximally- and distally-recorded long-term potentiation was significantly reduced. Paired-pulse facilitation was greater when recorded proximally but the variation in facilitation with stimulation and recording position was different from that obtained for long-term potentiation. Paired-pulse facilitation of response amplitude was slightly reduced after long-term potentiation, but the change did not depend on the stimulation-recording arrangement; facilitation of response slope was not reliably affected by long-term potentiation. These results indicate that the shape of the dendritic field excitatory postsynaptic potential is influenced by the relative position of recording electrodes in stratum radiatum; when the position is such that after-potentials are minimized, long-term potentiation produces a decrease in the decay time constant of the synaptic field potential.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗