Biomedical subjects

O Orwar

Publications and source records attributed to O Orwar.

35 records · Page 2Linked to original sources

PubMed1997

Patch clamp detection in capillary electrophoresis.

We describe a capillary electrophoresis-patch clamp (CE-PC) analysis of biomolecules that activate ligand-gated ion channels. CE-PC offers a powerful means for identifying receptor ligands based on the combination of the characteristic receptor responses they evoke and their differential electrophoretic migration rates. Corner frequencies, membrane reversal potentials, and mean and unitary single-channel receptor responses were calculated from currents recorded with patch clamp detection. This information was then combined with the electrophoretic mobility of the receptor ligand, which is proportional to the charge-to-frictional-drag ratio of that species. We applied CE-PC to separate and detect the endogenous receptor agonists gamma-aminobutyrate and L-glutamate and the synthetic glutamate receptor agonists N-methyl-D-aspartate and kainic acid. We present dose-response data for electrophoretically separated kainic acid and discuss its implications for making the CE-PC detection system quantitative.

Animals↗

PubMed1997

Injection of ultrasmall samples and single molecules into tapered capillaries.

A novel injection scheme is described in which ultrasmall samples in the attoliter (10(-18) L) and low femtoliter (10(-15) L) range, or even single molecules, are controllably introduced into a tapered capillary so that electrophoretic separation can be carried out. To match the dimensions of the capillary inlet with that of the sample, capillary tips are tapered to an inside diameter ranging from hundreds of nanometers to a few micrometers. To inject an ultrasmall sample, optical trapping is used to immobilize and manipulate the sample in order to place it inside or next to the capillary inlet. A small controlled suction results in the loading of the sample into the capillary.

Chemistry Techniques, Analytical↗

PubMed1996

Rapid preparation of giant unilamellar vesicles.

We report here a rapid evaporation method that produces in high yield giant unilamellar vesicles up to 50 microns in diameter. The vesicles are obtained after only 2 min and can be prepared from different phospholipids, including L-alpha-phosphatidylcholine (lecithin), dipalmitoleoyl L-alpha-phosphatidylcholine, and beta-arachidonoyl gamma-palmitoyl L-alpha-phosphatidylcholine. Vesicles can be produced in distilled water and in Hepes, phosphate, and borate buffers in the pH range of 7.0 to 11.5 with ionic strengths up to 50 mM. The short preparation time allows encapsulation of labile molecular targets or enzymes with high catalytic activities. Cell-sized proteoliposomes have been prepared in which gamma-glutamyltransferase (EC 2.3.2.2) was functionally incorporated into the membrane wall.

Drug Stability↗

PubMed1996

Patch-clamp detection of neurotransmitters in capillary electrophoresis.

Gamma-aminobutyrate acid, L-glutamate, and N-methyl-D-aspartate were separated by capillary electrophoresis and detected by the use of whole-cell and outside-out patch-clamp techniques on freshly dissociated rat olfactory interneurons. These neuroactive compounds could be identified from their electrophoretic migration times, unitary channel conductances, and power spectra that yielded corner frequencies and mean single-channel conductances characteristic for each of the different agonist-receptor interactions. This technique has the sensitivity to observe the opening of a single ion channel for agonists separated by capillary electrophoresis.

Animals↗

PubMed1996

Cell-to-cell scanning in capillary electrophoresis.

A widespread limitation in using cell-based biosensors for repetitive chemical analysis is loss of agonist-induced response caused by receptor desensitization. We overcome this problem by scanning an array of immobilized cells underneath a capillary electrophoresis column outlet. In this way, electrophoretically fractionated components that exit the separation capillary are always directed onto cells previously unexposed to receptor agonists. To demonstrate this concept of response recovery using a scanning format, we have chosen the bradykinin B2 receptor system in the NG108-15 cell line, which is known to undergo desensitization. Whereas four subsequent injections of 250 microM bradykinin separated by 120 s are found to reduce the NG108-15 cell response markedly, scanning to new cells can fully restore the response during the separation. Furthermore, by pretesting individual NG108-15 cells for an agonist response and then later scanning back to the same cell, we achieved a 100% success rate in detecting bradykinin in subsequent electrophoretic separations.

Animals↗

PubMed1996

Studies on the identity of the rat optic nerve transmitter.

The possible role of glutamate, aspartate, sulfur-containing excitatory amino acids and gamma-glutamyl peptides as major transmitters in the rat optic nerve was evaluated. Four days following optic nerve lesion the K(+)-evoked Ca(2+)-dependent glutamate release was reduced to 31 +/- 16% (+/- S.D., n = 9) comparing release from slices of the denervated (contralateral to the lesion) and non-denervated (ipsilateral) superior colliculus, indicative of a major transmitter function for glutamate. However, significant decreases in glutamate release could not be detected seven days following the lesion (n = 5). Other studies have shown that optic nerve denervation induce formation of synapses of non-retinal origin and cause other cellular changes which may reduce the effect of deafferentation on glutamate release after 7 days. No significant change was observed in aspartate release following the lesion. The concentrations of cysteine sulfinate, cysteate, homocysteine sulfinate, homocysteate and O-sulfo-serine in the optic layers of the superior colliculus were below 1 nmol/g tissue (n = 6). Theoretical considerations indicate that this level is too low for a function of any of these as a major optic nerve transmitter. All postsynaptic components in the rat superior colliculus response, evoked by electrical optic nerve stimulation, were reduced by kynurenate (1-10 mM), a broad spectrum glutamate-receptor antagonist. The study gives further support for the view that glutamate is a major transmitter in the rat optic nerve.

Amino Acids, Sulfur↗

PubMed1996

Gamma-glutamyl peptides and related amino acids in rat hippocampus in vitro: effect of depolarization and gamma-glutamyl transpeptidase inhibition.

The concentrations of gamma-glutamylglutamate, gamma-glutamylglutamine, gamma-glutamylcysteine, glutamate, aspartate, glutamine, cyst(e)ine and glutathione (including disulfides) were determined by HPLC analysis of both the tissue and the surrounding medium of incubated rat hippocampal slices. High potassium concentrations (50 mM; 2 x 4 min) increased the medium concentration of gamma-glutamylglutamate (maximal net efflux 0.07 +/- 0.06 pmol/mg protein/min; n = 8 +/- SD) with a relative time delay compared to the increase in glutamate (maximal net efflux 264 +/- 88 pmol/mg protein/min). Release of gamma-glutamylcysteine, the glutathione precursor, demonstrated an immediate response and gradually approached prestimulus levels (maximal net efflux 0.36 +/- 0.13 pmol/mg protein/min). Addition of acivicin (0.2 mM), a gamma-glutamyl transpeptidase (EC 2.3.2.2.) blocker, during preincubation for 45 min reduced the tissue concentrations (pmol/mg protein) of gamma-glutamylglutamate (19.4 +/- 8.2 (control) vs. 5.8 +/- 3.6 (+ acivicin)), gamma-glutamylglutamine (40.3 +/- 6.7 vs. 25.7 +/- 4.2 pmol/mg protein), glutamine (9.9 +/- 2.0 vs. 4.6 +/- 1.2 nmol/mg protein) and cysteine (1.0 +/- 0.2 vs. 0.56 +/- 0.18 nmol/mg protein). Incubation with acivicin (0.2 mM) reduced the net efflux of gamma-glutamylglutamine (0.79 +/- 0.19 vs. 0.21 +/- 0.07 pmol/mg protein/min) whereas that of the glutathione was increased (4.7 +/- 1.0 vs. 20 +/- 3 pmol/mg protein/min). The medium concentrations of glutamate in both low and high potassium were unaffected by acivicin, while the high potassium induced increase in gamma-glutamylglutamate was blocked. The results demonstrate differential efflux patterns of gamma-glutamyl dipeptides from brain slices and show that in vitro the activity of gamma-glutamyl transpeptidase regulates extracellular concentrations of glutathione, gamma-glutamylglutamine and gamma-glutamylglutamate.

Amino Acids↗

PubMed1995

Use of 2,3-naphthalenedicarboxaldehyde derivatization for single-cell analysis of glutathione by capillary electrophoresis and histochemical localization by fluorescence microscopy.

We report that 2,3-naphthalenedicarboxaldehyde reacts rapidly with glutathione and its precursor, gamma-glutamylcysteine, to form highly fluorescent derivatives under physiological conditions. In contrast to previous accounts of 2,3-naphthalenedicarboxaldehyde labeling of primary amines, no additional CN- ion or any other additional nucleophile is required. The fluorescence spectral properties of the chromophores (lambda exc max = 472 nm, lambda em max = 528 nm) make these derivatives amenable to excitation and detection by optical instrumentation that is optimized for fluorescein wavelengths. This selective labeling chemistry enabled quantitative determination and histochemical localization of glutathione in neurobiological samples. Intracellular glutathione was labeled by incubating cultured cells or cell suspensions in a 2,3-naphthalenedicarboxaldehyde-supplemented, DMSO-containing physiological buffer (pH = 7.4) for 2-10 min. Applications include imaging of cultured NG 108-15 cells (mouse neuroblastoma x rat glioma) and primary glial and neuronal cell cocultures (rat hippocampus) using epiluminescent and confocal fluorescence microscopy. Quantitative determination of glutathione in single NG 108-15 cells was accomplished using laser-induced fluorescence detection and capillary electrophoresis.

Animals↗

PubMed1995

Identification of receptor ligands and receptor subtypes using antagonists in a capillary electrophoresis single-cell biosensor separation system.

A capillary electrophoresis system with single-cell biosensors as a detector has been used to separate and identify ligands in complex biological samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions. The single-cell biosensor was based on the ligand-receptor binding and G-protein-mediated signal transduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion indicator fluo-3-acetoxymethyl ester. Specifically, a mixture of bradykinin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HOE 140), a selective antagonist to the BK B2 receptor subtype (B2BK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Structurally related forms of BK were also identified based on inhibiting B2BK receptors. Applications of this technique include identification of endogenous BK in a lysate of human hepatocellular carcinoma cells (Hep G2) and screening for bioactivity of BK degradation products in human blood plasma. The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of separated components and receptor subtypes.

Animals↗

PubMed1995

Fluorescence, photodestruction, photoionization and thermal degradation of o-phthalaldehyde/beta-mercaptoethanol-labelled aliphatic alpha-oligopeptides.

Photophysical and photochemical properties of o-phthalaldehyde/beta-mercaptoethanol-labelled aliphatic alpha-peptides were investigated. It is found that alpha-peptide derivatives have lower fluorescence quantum yields, higher photodestruction quantum yields and lower yields for formation of solvated electrons as compared to amino acid and simple alkylamine derivatives in aqueous alkaline solution. These properties of the alpha-peptide derivatives sets narrow limits for their utilization in laser-based (high light intensity) detector systems. In contrast, the thermal stability of the peptide derivatives was found to be severalfold higher than for the parent amino acid derivatives. The differential rates of thermal derivative degradation could be utilized in a new approach towards selective determination of peptides. determination of peptides. determination of peptides.

Mercaptoethanol↗

PubMed1994

Liquid chromatographic determination of acidic beta-aspartyl and gamma-glutamyl peptides in extracts of rat brain.

This work describes further development of our previously presented method for determination of acidic sulfur/phosphor-containing amino acids, gamma-glutamyl di/tripeptides, and beta-aspartyl dipeptides. Automated precolumn fluorogenic derivatization was performed with o-phthaldialdehyde/beta-mercaptoethanol and the derivatives were separated by reversed-phase liquid chromatography. The method was optimized for the analysis of brain tissue extracts. Due to the complex sample matrix, three separation schemes with complementary selectivities were developed. Different extraction protocols were evaluated and sonication of frozen tissue powder in methanol-H2O (9:1, v/v) yielded the highest recoveries and precision. beta-Mercaphtoethanol and EDTA were added to the extraction media to inhibit spontaneous oxidation of thiol-containing amino compounds. Analyte identification was based on retention times and recovery of standards added to extracts. The following compounds were identified in rat cerebral cortex (mean tissue concentration +/- SD, n = 6): gamma-glutamylglutamine (38.5 +/- 12.6 microM), gamma-glutamylglutamate (14.4 +/- 6.0 microM), gamma-glutamyltaurine (4.9 +/- 2.2 microM), beta-aspartylglycine (4.0 +/- 0.4 microM), beta-aspartyltaurine (3.7 +/- 0.6 microM), O-phosphoserine (3.2 +/- 0.8 microM), gamma-glutamylcysteine (1.9 +/- 0.3 microM), gamma-glutamylglycine (1.1 +/- 0.1 microM), and gamma-glutamylcysteateglycine (0.8 +/- 0.1 microM). In addition over 15 unidentified components were found. Cysteate, cysteine sulfinate, homocysteate, homocysteine sulfinate, O-Sulfoserine, gamma-glutamylaspartate, gamma-glutamylcysteate, gamma-glutamylhistidine, and beta-aspartylalanine were not present at concentrations above 1 microM.

Animals↗

PubMed1994

Increased intra- and extracellular concentrations of gamma-glutamylglutamate and related dipeptides in the ischemic rat striatum: involvement of glutamyl transpeptidase.

The present work relates to the possibility that the ATP-independent enzyme gamma-glutamyl transpeptidase (EC 2.3.2.2), which has been postulated to be part of an amino acid uptake system, is active during cerebral ischemia. This was evaluated in the ischemic rat striatum by determination of intra- and extracellular concentrations of gamma-glutamyl dipeptides (the products of the transpeptidation) and glutathione (the physiological gamma-glutamyl donor). An ischemic period (0-30 and 31-60 min) resulted in prominent increases in the respective concentration of extracellular gamma-glutamylglutamate (24- and 67-fold), gamma-glutamyltaurine + gamma-glutamylglycine (5.8- and 19-fold), and gamma-glutamylglutamine (2.6- and 6.8-fold) as revealed using in vivo microdialysis. The changes coincided with increased respective extracellular concentrations of glutamate (83- and 115-fold), taurine (17- and 25-fold), glycine (4.6- and 6.1-fold), and glutamine (1.7- and 2.1-fold). Furthermore, under anoxic conditions in vitro (0-30 and 0-60 min), respective striatal tissue concentrations were increased for gamma-glutamylglutamate (20- and 17-fold), gamma-glutamyltaurine (6.7- and 11-fold), gamma-glutamylglutamine (1.7- and 1.2-fold), and gamma-glutamylglycine (14- and 18-fold), whereas glutathione levels were, on an average, decreased by approximately 350 microM. In summary, gamma-glutamyl transpeptidase is involved in de novo dipeptide synthesis in the mammalian brain during anoxic conditions, indicating transport of amino acids such as glutamate.

Amino Acids↗

PubMed1993

Cysteine sulphinate and cysteate: mediators of cysteine toxicity in the neonatal rat brain?

Excitotoxic amino acids contain two acidic groups, but cysteine represents an exception to this rule. The hypothesis that cysteine toxicity is mediated by the oxidized and diacidic metabolites cysteine sulphinate and/or cysteate was tested in the present study. The issue was approached in three different ways. Firstly, the distribution of brain injury after subcutaneous administration of cysteine (1 mg/g) to 4-day-old rats was compared with that caused by cysteine sulphinate (3 mg/g). Secondly, the effects of excitatory amino acid receptor antagonists on cysteine and cysteine sulphinate toxicity were investigated. Thirdly, the cerebral concentrations of cysteine sulphinate were determined after cysteine administration and compared with those obtained after cysteine sulphinate injection. The cerebral cortex was the region most vulnerable to cysteine toxicity, followed by the hippocampus (especially the medial subicular neurons), amygdala, caudoputamen, cerebellum and septum. Pronounced extravasation of red blood cells was observed in lesioned areas. One day after cysteine administration, the injury was infarction-like and sharply demarcated. Cysteine sulphinate-induced damage resembled cysteine-induced lesions in some respects: the anterior cingulate and retrosplenial cortices, as well as medial subicular cells, were quite vulnerable. However, the differences prevailed. Cysteine sulphinate, but not cysteine, killed neurons of the superficial part of the tectum, the medial habenula, the ventromedial hypothalamus and the arcuate nucleus. Further, while cysteine toxicity was prominent in deep cortical layers, cysteine sulphinate preferentially damaged superficial cortical neurons. Cysteine toxicity was abolished by pretreatment with MK-801, a selective NMDA antagonist, but not by 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline, a selective AMPA receptor blocker. In contrast, the considerably smaller lesion seen after cysteine sulphinate administration was only partially prevented by MK-801. Large (19-fold) increases in cortical cysteine sulphinate concentration were noted after injection of a toxic dose of cysteine. This corresponds to 90 nmol cysteine sulphinate/g protein. The cysteate concentration was not increased above the detection limit. Injection of a toxic dose of cysteine sulphinate elevated cysteine sulphinate concentration in the frontomedial cortex (a region consistently injured by cysteine sulphinate) almost three orders of magnitude more than that observed after cysteine administration. Taken together, these results strongly suggest that neither cysteine sulphinate nor cysteate alone mediate cysteine toxicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗

PubMed1993

Gamma-L-glutamyl-L-glutamate is an endogenous dipeptide in the rat olfactory bulb which activates N-methyl-D-aspartate receptors.

The concentration of gamma-L-glutamyl-L-glutamate (gamma-glu-glu), a potent displacer of excitatory amino acid receptor binding, and other structurally related dipeptides were determined in extracts of the rat olfactory bulb by a novel liquid chromatographic method. Whole-cell patch clamp recordings of currents from freshly isolated neurons showed that gamma-glu-glu produced inward currents at negative holding potentials, provided microM concentrations of glycine were added and no Mg(2+)-ions were present, indicating activation of N-methyl-D-aspartate receptors. Consistently, geometry optimization of gamma-glu-glu using molecular mechanics calculations, suggested a bent conformer with structural features supporting this view.

Animals↗

PubMed1991

Automated determination of neuroactive acidic sulphur-containing amino acids and gamma-glutamyl peptides using liquid chromatography with fluorescence and electrochemical detection.

A column liquid chromatographic method is presented for the determination of trace levels of acidic sulphur-containing amino acids and gamma-glutamyl di- and tripeptides in microdialysates sampled from rat brain in vivo. Automated precolumn derivatization was performed with o-phthaldialdehyde-beta-mercaptoethanol. The derivatives were separated by reversed-phase liquid chromatography with electrochemical and fluorescence detection. The mean relative standard deviation (n = 10) was 1.03 and 4.59% for retention times and peak heights, respectively. The mean correlation coefficient of linearity (r) was 0.9982 in the range 4.5-450 pmol (n = 15), and the lowest detectable amount was 200 fmol for the homocysteinesulphinic acid derivative, (k' = 5.4, at a signal-to-noise ratio of 3). A microcolumn electrochemical detection method, developed for volume-limited samples, produced a fifteen-fold increase in mass sensitivity. Neurochemical applications using microdialysis in vivo are presented.

Amino Acids↗

PubMed1991

Changes in extracellular amino acids and spontaneous neuronal activity during ischemia and extended reflow in the CA1 of the rat hippocampus.

This study addresses the possible involvement of an agonist-induced postischemic hyperactivity in the delayed neuronal death of the CA1 hippocampus in the rat. In two sets of experiments, dialytrodes were implanted into the CA1 either acutely or chronically (24 h of recovery). During 20 min of cerebral ischemia (four-vessel occlusion model) and 8 h of reflow, we followed extracellular amino acids and multiple-unit activity. Multiple-unit activity ceased within 20 sec of ischemia and remained zero during the ischemic insult and for the following 1 h of reflow. During ischemia, extracellular aspartate, glutamate, taurine, and gamma-aminobutyric acid increased in both acute and chronic experiments (seven- to 26-fold). Multiple-unit activity recovered to preischemic levels following 4-6 h of reflow. In the group with dialytrodes implanted acutely, the continuous increase in multiple-unit activity reached 110% of basal at 8 h of reflow. In the group with dialytrodes implanted chronically, multiple-unit activity recovered faster and reached 140% of control at 8 h, paralleled by an increase in extracellular aspartate (5.5-fold) and glutamate (twofold). In conclusion, the postischemic increase of excitatory amino acids and the recovery of the neuronal activity may stress the CA1 pyramidal cells, which could be detrimental in combination with, e.g., postsynaptic impairments.

Action Potentials↗

PubMed1991

Extracellular acidic sulfur-containing amino acids and gamma-glutamyl peptides in global ischemia: postischemic recovery of neuronal activity is paralleled by a tetrodotoxin-sensitive increase in cysteine sulfinate in the CA1 of the rat hippocampus.

An excessive activation of the excitatory amino acid system has been proposed as one possible mediator of the ischemia-induced delayed death of CA1 pyramidal cells in the hippocampus. Using dialytrodes in the CA1 of the rat, we have investigated multiple-unit activity and extracellular changes in acidic sulfur-containing amino acids and gamma-glutamyl peptides during ischemia (20-min, four-vessel occlusion) and during 8 h of reflow. Multiple-unit activity was abolished during ischemia and for the following 1 h, but then recovered, gradually reaching preischemic levels after 8 h of reflow. Extracellular cysteate, cysteine sulfinate, and gamma-glutamyltaurine increased (1.5- to threefold) during ischemia, and extracellular glutathione and gamma-glutamylaspartate plus gamma-glutamylglutamine increased during early reflow (two- to threefold). The recovery of neuronal activity at 4-8 h was paralleled by an increase in extracellular cysteine sulfinate (2.5-fold at 8 h of reflow). Perfusion with 10 microM tetrodotoxin at 8 h of reflow abolished the multiple-unit activity and reduced extracellular cysteine sulfinate. Considering the glutamate-like properties of cysteine sulfinate, the observed postischemic increase may be involved in the development of the delayed neuronal death.

Acids↗