An MspI polymorphism at the D7S599E locus.
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Alleles↗
Biomedical subjects
M J Neal
Publications and source records attributed to M J Neal.
Analysis of differentially expressed genes in retinitis pigmentosa retinas. Altered expression of clusterin mRNA.
The molecular and cellular processes underlying photoreceptor degeneration in retinitis pigmentosa (RP) are unknown. We have investigated gene expression in diseased retinas using differential hybridization screening of a retinal cDNA library with probes derived from normal and RP retinal RNA. Most differential clones detected corresponded to transcripts absent from the dystrophic state, including e.g. opsin. However, one clone was noticeably increased in RP in comparison with the control: partial sequencing showed it encoded clusterin. Increased expression of clusterin has been identified in several cases of tissues undergoing apoptosis (programmed cell death), and our finding suggests that the degenerative changes in advanced RP may represent another example of apoptosis, possibly with common causative mechanisms.
Adult↗
Calcium dependent release of acetylcholine and gamma-aminobutyric acid from the rabbit retina.
The concurrent release of endogenous ACh and GABA from the retina (in the presence of physostigmine) was measured using either an eye-cup preparation in rabbits anaesthetized with urethane or isolated rabbit retinas. There was a spontaneous resting release of ACh and GABA from the dark adapted retina of ca 5 and 160 pmol min-1 respectively. Stimulation of the initially dark adapted retina in vivo with flickering light (0.1-20 Hz) increased the release of ACh by up to 5 times the spontaneous resting release but did not cause a detectable increase in GABA release. The maximum light-evoked release of ACh was about 24 pmol min-1/retina and occurred at a frequency of 10 Hz. However, the maximum release of ACh per flash occurred at 0.1 Hz at which frequency the average ACh release per flash from one amacrine cell was ca 2.35 x 10(-18) mol. Exposure of the retina to the potent inhibitors of GABA uptake, SKF89976A and SKF100330A markedly reduced the resting release of ACh and abolished the light-evoked release of ACh but did not enable a light-evoked release of GABA to be detected. Bicuculline blocked the inhibitory actions of both SKF89976A and SKF100330A on ACh release but the combination of bicuculline and uptake inhibitor did not result in a light-evoked release of GABA. In contrast, KCl (20 mM) applied locally to the retina in vivo resulted in the release of both ACh and GABA (61 and 2.6-fold respectively). KCl (20 mM) also evoked large increases in ACh and GABA release from isolated rabbit retinas in room light (13.5 and 3.4-fold respectively). The K-evoked release of ACh and GABA from the rabbit retina both in vivo and in vitro was calcium dependent. These experiments are the first in which endogenous ACh and GABA release from the retina have been simultaneously measured and suggest that the release mechanisms for these transmitters are fundamentally similar.
Acetylcholine↗
Effect of sulphur containing amino acids on [3H]-acetylcholine release from amacrine cells of the rabbit retina.
1. The effects of the sulphur containing amino acids, homocysteic acid, homocysteine sulphinic acid, cysteic acid and cysteine sulphinic acid on the release of [3H]-acetylcholine ([3H]-ACh) from the cholinergic amacrine cells of the rabbit retina were examined. 2. All the compounds stimulated the spontaneous resting release and abolished the light-evoked release of [3H]-ACh. Except for homocysteine sulphinic acid these actions occurred at concentrations that did not affect the erg b-wave amplitude, indicating a site of action at the inner retina. 3. N-methyl-D-aspartate (in Mg(2+)-containing medium) clearly blocked the effects of homocysteic acid and homocysteine sulphinic acid on the resting release of [3H]-ACh but had no effect on the actions of cysteic acid and cysteine sulphinic acid. 4. Since N-methyl-D-aspartate is an antagonist of the light-evoked endogenous bipolar cell transmitter released onto cholinergic cells, these results are consistent with the suggestion that homocysteic acid or homocysteine sulphinic acid may be a transmitter released from this subpopulation of bipolar cells. 5. The present experiments indicate the existence of excitatory amino acids that have closer pharmacological properties to a bipolar cell transmitter than glutamate but it remains to be seen whether homocysteic acid or homocysteine sulphinic acid occur in these particular bipolar cells.
Acetylcholine↗
Chronic diazepam treatment in rats causes long-lasting changes in central [3H]-5-hydroxytryptamine and [14C]-gamma-aminobutyric acid release.
The effects of chronic diazepam administration to rats on the central release of [3H]-5-hydroxytryptamine ([3H]-5-HT) and [14C]-gamma-aminobutyric acid ([14C]-GABA, ex vivo) were examined. Chronic (5 and 21 days) administration of diazepam (4 mg kg-1 i.p. daily for 21 days) reduced the K-evoked (20 mM KCl) release of [3H]-5-HT from frontal cortex by approximately 50%. Remarkably, this decrease was still present 1 week after diazepam withdrawal. Chronic diazepam treatment did not significantly affect hippocampal [3H]-5-HT release but after 21 days the K-evoked release of [14C]-GABA was more than doubled and remained elevated 30 h after withdrawal; it returned to control levels after 1 week, and decreased below control levels after 2 weeks. This study indicates that chronic diazepam treatment produces striking changes in transmitter release in rats that persist long after treatment has ceased.
Animals↗
Development of tolerance to the effects of vigabatrin (gamma-vinyl-GABA) on GABA release from rat cerebral cortex, spinal cord and retina.
1. The effects of acute and chronic vigabatrin (gamma-vinyl-GABA) (GVG) administration on gamma-aminobutyric acid (GABA) levels and release in rat cortical slices, spinal cord slices and retinas were studied. 2. GVG (250 mgkg-1 i.p.) administered to rats 18 h before death (acute administration) produced an almost 3 fold increase in GABA levels of the cortex and spinal cord and a 6 fold increase in retinal GABA. The levels of glutamate, aspartate, glycine and taurine were unaffected. 3. When GVG (250 mgkg-1 i.p.) was administered daily for 17 days (chronic administration) a similar (almost 3 fold) increase in cortical GABA occurred but the increases in spinal and retinal GABA were reduced by approximately 40%. 4. Acute administration of GVG strikingly increased the potassium-evoked release (KCl 50 mM) of GABA from all three tissues. This enhanced evoked release was reduced by about 50% in tissues taken from rats that had been chronically treated with GVG. 5. Acute administration of GVG reduced GABA-transaminase (GABA-T) activity by approximately 80% in cortex and cord and by 98% in the retina. Following the chronic administration of GVG, there was a trend for GABA-T activities to recover (significant only in cortex). Acute administration of GVG had no effect on glutamic acid decarboxylase (GAD) activity in cortex or spinal cord. However, chronic treatment resulted in significant decreases in GAD activity in both the cortex and cord (35% and 50% reduction respectively). 6. The K-evoked release of glutamate, aspartate, glycine and taurine from cortical slices and the Kevoked release of glycine from spinal slices and retinas were not affected by either acute or chronic GVG treatment. 7. These experiments indicate that GVG treatment increases specifically the K-evoked release of GABA and that tolerance can develop to this enhancing effect of GVG on central GABA release. This tolerance may result from increased feedback inhibition of GAD with a consequent reduction of presynaptic GABA stores.
4-Aminobutyrate Transaminase↗
L-homocysteic acid--a possible bipolar cell transmitter in the rabbit retina.
The identity of the retinal bipolar cell transmitter(s) is unknown although there is much indirect evidence that suggests it may be glutamate or a related compound. Some bipolar cells synapse onto cholinergic amacrine cells and in the rabbit retina acetylcholine (ACh) release is increased by light flashes and by the excitatory amino acids glutamate, aspartate and homocysteic acid (HCA). In the retina, the amino acid agonist N-methyl-D-aspartate (NMDA) is unusual in that it sometimes acts as an antagonist, and in the present experiments it blocked the light-evoked release of ACh by acting as an antagonist of the bipolar cell transmitter. However, NMDA did not block the actions of glutamate or aspartate on amacrine cell ACh release, a result that argues against either of these amino acids being the bipolar cell transmitter. On the other hand, the HCA evoked release of ACh was clearly antagonised by NMDA suggesting that HCA may be the bipolar cell transmitter released onto cholinergic amacrine cells. This suggestion is supported by the finding that the rabbit retina possesses HCA at a concentration of 0.8 nmol/g wet wt.
Acetylcholine↗
Immunocytochemical evidence that vigabatrin in rats causes GABA accumulation in glial cells of the retina.
Vigabatrin (gamma-vinyl-GABA, GVG) is an irreversible inhibitor of GABA-aminotransferase (GABA-T) that is under clinical trial as an antiepileptic drug. Rats were injected (i.p.) with GVG and killed 18 h later. GVG administration reduced retinal GABA-T activity to undetectable levels and increased the GABA content 5-fold. Immunocytochemistry using a GABA antiserum clearly revealed the presence of GABA-IR in the glial Muller cells of retinas from GVG-treated rats but not from controls. This experiment indicates that the administration of drugs which inhibit GABA-T may cause the accumulation of GABA in retinal cells that do not normally possess enough endogenous GABA to be detected by immunocytochemistry.
Aminocaproates↗
Baclofen and phaclofen modulate GABA release from slices of rat cerebral cortex and spinal cord but not from retina.
1. The effects of (-)-baclofen, muscimol and phaclofen on endogenous gamma-aminobutyric acid (GABA) release from rat cortical slices, spinal cord slices and entire retinas were studied. 2. The spontaneous resting release of GABA from the three tissues was 3 to 6 pmol mg-1 wet wt 10 min-1. Depolarization of cortical slices with KCl (50 mM) (high-K) produced an 8 fold increase in GABA release but high-K did not evoke an increased release of GABA from spinal slices or retinas. 3. When rats were injected with gamma-vinyl-GABA (250 mg kg-1 i.p.) (GVG) 18 h before death, the tissue GABA stores were increased 3 to 6 fold and high-K then evoked striking Ca-dependent releases of GABA from all three tissues. Thus, in subsequent experiments, unless otherwise stated, the nervous tissues were taken from GVG-treated rats. 4. (-)-Baclofen (10 microM) significantly reduced the K-evoked release of GABA from cortical and spinal slices but retinal release was not affected, even at a concentration of (+/-)-baclofen of 1 mM. For cortical slices, the IC50 for baclofen was approximately 5.2 microM. The inhibitory effect of baclofen on GABA release from cortical slices also occurred in slices prepared from saline-injected rats, indicating that GVG treatment did not qualitatively affect the results. 5. The inhibitory effect of (-)-baclofen on the K-evoked release of GABA from cortical and spinal slices was antagonised by phaclofen (500 microM), confirming that baclofen was producing its effects by acting at the GABAB-receptor. 6. Phaclofen (500 microM) increased the spontaneous resting release of GABA from cortical slices taken from GVG-treated rats but not from saline-injected rats. Phaclofen did not increase GABA release from spinal slices or retinas taken from GVG-treated rats. 7. Baclofen (10 microM) significantly reduced the K-evoked release from cortical slices of glutamate, aspartate, glycine and taurine. 8. Muscimol (10 microM) and delta-aminolaevulinic acid (10 microM) had no effect on either the resting or Kevoked release of GABA from cortical slices prepared from saline-injected or GVG-treated rats. 9. The results obtained with cortical and spinal slices are consistent with the presence of inhibitory GABAB-autoreceptors. The phaclofen-induced increase in GABA release from cortical slices taken from GVG-treated rats, but not from saline-injected rats, implies that under conditions of high GABA release, considerable feedback inhibition is occurring via activation of the GABAB inhibitory autoreceptors. No evidence was found for GABAB-autoreceptors on retinal GABAergic amacrine cells or for GABAA-autoreceptors in cortical slices or spinal cord slices.
Amino Acids↗
GABA release from Xenopus retina does not correlate with horizontal cell membrane potential.
The relationship between horizontal cell membrane potential and the release of GABA was explored in the retina of Xenopus laevis. The intracellularly recorded membrane potential of horizontal cells was monitored while the retina was exposed to different concentrations of depolarizing agents. The dose-response curves obtained revealed a rise from 5 to 95% maximum depolarization in 0.5-1.5 log unit concentration change. The molar concentrations that elicited a 20 mV depolarization were 40 mM (potassium), 0.8 mM (glutamate), 0.8 mM (glycine), 5 microM (kainate) and 1.3 microM (quisqualate). Autoradiography revealed that radiolabel was accumulated almost exclusively by horizontal cells when isolated retinas were incubated in medium containing 1 microM [3H]GABA. Thus, retinal release of radioactivity was used as a measure of [3H]GABA release from horizontal cells. Endogenous GABA released from retinas was measured using high performance liquid chromatography and was taken to reflect both amacrine and horizontal cell GABA pools. The release of both [3H]GABA and endogenous GABA was stimulated by glutamate, kainate and potassium, but not by glycine or quisqualate. Similar dose-response curves for GABA release and for depolarization were obtained in the case of potassium and kainate but not for glutamate. Potassium-evoked release either of endogenous GABA or [3H]GABA was both calcium- and sodium-dependent, whereas kainate- or glutamate-evoked GABA release was sodium-dependent but calcium-independent. The results indicate that depolarization per se is not necessarily associated with transmitter release in Xenopus retinal horizontal cells. It is suggested that the action of a given neurotransmitter upon the efflux of GABA from horizontal cells may depend on the degree to which it modifies the sodium conductance of the horizontal cell.
Animals↗
The effects of adrenergic agonists and age on lipogenesis in avian hepatocytes.
1. Adrenergic inhibition of lipogenesis was examined in vitro using hepatocytes isolated from chickens 2-9 weeks old. 2. Lipogenesis was inhibited by beta 1, beta 2 and alpha 1 agonists. Greatest inhibition occurred when more than one type of receptor was stimulated. 3. Clonidine (alpha 2-agonist) may have stimulated lipogenesis. 4. Responsiveness to the agonists decreased as the chickens got older.
Aging↗
Anxiety in the rat is associated with decreased release of 5-HT and glycine from the hippocampus.
Rats, implanted with dialysis loops in the dorsal hippocampus, were injected with subconvulsant doses of pentylenetetrazole (PTZ 15 and 30 mg/kg) and a 30-min sample collected for high-performance liquid chromatography (HPLC) analysis of amines and amino acids. Immediately after the sampling period they were given a 5-min test in the elevated plus-maze. On the basis of their performance in this test, they were divided into an 'anxious' and a 'non-anxious' group. The anxious group had significantly lower levels of glycine and serotonin (5-HT) release. Although PTZ decreased the release of noradrenaline, this was not correlated with behavioural differences in the elevated plus-maze.
Animals↗
Role of fluorescein glucuronide and its metabolism in vitreous fluorophotometry.
Rabbits were given fluorescein or fluorescein glucuronide intravenously. Fluorescein and fluorescein glucuronide concentrations in plasma and vitreous samples were measured by high-performance liquid chromatography. Vitreous fluorophotometry was performed using the Fluorotron Master to compare scans after administration of fluorescein and fluorescein glucuronide, and for comparison of in vivo fluorescence with in vitro high-performance liquid chromatography analysis. Fluorescein glucuronide was shown to enter the vitreous as early as 1 hr after injection. Fluorescein glucuronide was the dominant molecule in both vitreous and plasma of all rabbits at 6 hr. Because fluorescein glucuronide has a lower fluorescence than fluorescein, the fluorophotometer overestimates the vitreous concentration of fluorescein after its administration. Since fluorescein is metabolized rapidly to fluorescein glucuronide in man, entry of fluorescein glucuronide into the eye should be considered in measurements of blood-ocular barrier permeability by vitreous fluorophotometry.
Animals↗
Localized occurrence of congenital coronary artery fistula in the southeast United States.
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Coronary Vessel Anomalies↗
Muscarinic cholinergic receptors in rabbit retina.
Binding sites with high affinity and specificity for [3H]-quinuclidinyl benzilate are present in rabbit retinal homogenates. Only one set of binding sites was detected with an apparent dissociation constant of 2.13 X 10(-10) M and a density of 59.2 fmol mg-1 of protein. The pharmacological characteristics of specific binding were similar to those in the goldfish, chicken and cow.
Animals↗
Effect of excitatory amino acids on gamma-aminobutyric acid release from frog horizontal cells.
The effects of excitatory amino acids, analogues and K on [3H]gamma-aminobutyric acid [3H]GABA) release from horizontal cells of the isolated superfused frog retina were studied. Exposure of the retina to medium containing high concentrations (25-100 mM) of KCl increased the release of [3H]GABA to a maximum which was 40 times the spontaneous resting release. The K-evoked release of [3H]GABA was almost abolished in high-Mg/low-Ca medium. Glutamate, aspartate, kainate and quisqualate also stimulated the release of [3H]GABA from horizontal cells, the maximum evoked release being similar to that produced by KCl. The release of [3H]GABA evoked by glutamate, aspartate, kainate and quisqualate was abolished in high-Mg/low-Ca medium and by Na-free medium. The evoked releases of [3H]GABA were not reduced by tetrodotoxin. N-Methyl-D-aspartate (NMDA) at concentrations up to 10 mM had virtually no effect on [3H]GABA release from horizontal cells. In Mg-free medium, NMDA stimulated [3H]GABA release, but the maximum release was only 10% of that produced by other agonists. Mg-free medium did not significantly affect the evoked release of [3H]GABA by other agonists. NMDA apparently possessed affinity for the kainate receptor, because in normal medium it antagonized the effects of kainate but not glutamate, aspartate or quisqualate. The non-selective antagonist of excitatory amino acids, (+/-)-cis-2,3-piperidine dicarboxylic acid (PDA) antagonized the action of glutamate, aspartate, kainate and quisqualate on horizontal cell [3H]GABA release. D(-)-2-Amino-4-phosphonobutyrate (APB) and D-gamma-glutamylglycine (D-gamma-GG) antagonized the actions of kainate on horizontal cell [3H]GABA release at concentrations which had little affect on quisqualate-evoked responses. Approximate estimates of pA2 values (Schild, 1947) showed that the specificity and potency of the antagonists was low. Nevertheless, the retinal 'non-NMDA' receptors can probably be subdivided into kainate and quisqualate types. Glutamate diethylester (GDEE) did not affect the action of any agonist. We conclude that glutamate (and aspartate) probably stimulate the release of [3H]GABA from frog horizontal cells by activating receptors of the non-NMDA type. This activation may trigger the opening of tetrodotoxin-insensitive Na channels, resulting in the depolarization of the cell membrane and an increase in the conductance of voltage-sensitive Ca-channels. An influx of Ca ions would then trigger the release of [3H]GABA. Our results are not consistent with previous suggestions that GABA release from horizontal cells involves an outwardly directed transport process.
Amino Acids↗
Effect of excitatory amino acids and analogues on [3H]acetylcholine release from amacrine cells of the rabbit retina.
The pharmacology of cholinergic amacrine cells has been further studied by examining the effects of excitatory amino acids and antagonists on [3H]acetylcholine (ACh) release from the retinas of anaesthetized rabbits. Exposure of the retina to glutamate (5 mM), aspartate (5 mM), kainate (8 microM) and quisqualate (8 microM) abolished the light-evoked release of [3H]ACh but increased the spontaneous resting release four- to fivefold. N-methyl-D-aspartate (NMDA) (5 mM) in normal Krebs bicarbonate medium abolished the light-evoked release of [3H]ACh but did not affect the resting release. However, in Mg-free medium, NMDA (0.5 mM) abolished the light-evoked release of [3H]ACh and increased the resting release fivefold. The effects of other agonists were not altered in Mg-free medium. The amplitude of the electroretinogram (e.r.g.) b-wave was not significantly reduced by glutamate, aspartate or NMDA (in normal or Mg-free medium). Kainate and quisqualate reduced the b-wave amplitude to approximately 50 and 30% of controls respectively. The general excitatory amino acid antagonist, cis-2,3-piperidine dicarboxylic acid (PDA) (2 mM) blocked the light-evoked release of [3H]ACh, but had no significant effect on the e.r.g. b-wave amplitude or on the resting release of [3H]ACh. L(+)-2-amino-4-phosphonobutyrate (L(+)-APB) decreased the light-evoked release of [3H]ACh and the amplitude of the e.r.g. b-wave in parallel (correlation coefficient 0.995). D(-)-APB had similar effects but was fifteen times less potent. Since the L(+)-compound is known to mimic the photoreceptor transmitter on the depolarizing, but not hyperpolarizing, bipolar cells, these results strongly suggest that the [3H]ACh released in response to light originates mainly from the 'on' (displaced) cholinergic amacrine cells. Our experiments give no information on the origin of the spontaneously released [3H]ACh. PDA (2-5 mM) blocked the effects of glutamate, aspartate, kainate, quisqualate and NMDA on the resting release of [3H]ACh. In contrast, D(-)-APB (5 mM), which is a relatively non-specific excitatory amino acid antagonist in the spinal cord, blocked only the actions of kainate and had no blocking effect on the actions of glutamate or aspartate (the putative bipolar cell transmitters) or NMDA. D(-)-2-amino-5-phosphonovalerate (APV) which is a relatively selective NMDA antagonist in the spinal cord failed to discriminate between the effects of kainate and NMDA on the resting release of [3H]ACh. D-alpha-aminoadipate at concentrations up to 5 mM had no effect on any of the agonists.(ABSTRACT TRUNCATED AT 400 WORDS)
2-Amino-5-phosphonovalerate↗
Metabolism of fluorescein after intravenous administration.
The measurement of plasma unbound (free) fluorescein is important in the study of blood-ocular barrier kinetics. The authors became concerned about the quantitative significance of the presumed glucuronide metabolite of fluorescein to the measurement of plasma fluorescence in diabetic and normal subjects. Fluorescein was given intravenously (14 mg/kg) to seven normal subjects and eight diabetic subjects. Plasma samples taken during 60 min were subjected to microfiltration, from which aliquots of ultrafiltrate were incubated with beta-glucuronidase. Samples were subjected to high-performance liquid chromatography, and fluorescence activity was measured in the eluent. All subjects showed an additional fluorescence peak to that of fluorescein in plasma and ultrafiltrate 5 min after fluorescein administration and increased thereafter. This additional peak was abolished by incubation of ultrafiltrate with beta-glucuronidase and resulted in a marked increase in fluorescence due to the liberation of fluorescein from its presumed glucuronide. There were no pharmacokinetic differences between normal and diabetic subjects in plasma-free fluorescein and fluorescein glucuronide pharmacokinetics or in their respective binding to plasma proteins. The glucuronide had only 4.5% of the fluorescence of fluorescein, but because more of the glucuronide was unbound (32%) compared with fluorescein (10%) and its concentration increased while that of fluorescein decreased, it constituted an increasing proportion of the fluorescence in the ultrafiltrate. At 60 min, 80% of the fluorescein was present as glucuronide and contributed 20% of the total fluorescence in the ultrafiltrate. Fluorescein-glucuronide is a potential source of variability in studies on blood-ocular barrier kinetics.
Adult↗