Biomedical subjects

R M Wightman

Publications and source records attributed to R M Wightman.

At least 73 records · Page 4Linked to original sources

PubMed1996

Comparison of cytosolic Ca2+ and exocytosis responses from single rat and bovine chromaffin cells.

The relationship between cytosolic Ca2+ and catecholamine secretion during stimulus-secretion coupling has been examined at individual chromaffin cells isolated from the cow and rat. Vesicular catecholamine exocytosis was determined via amperometric measurements with carbon fibre microelectrodes and fura-2 was used for simultaneous fluorescent monitoring of cytosolic Ca2+ at the same cell. Individual secretory vesicles in cells from the two species were found to release similar amounts of catecholamine. In addition, the time courses for secretion from individual vesicles was similar with rat and bovine chromaffin cells. The total quantity of catecholamine released and the change in cytosolic Ca2+ were also similar in response to exposure to K+ (60 mM), 1,1-dimethyl-4-phenylpiperazinium (50 microM), and histamine (50 microM), although both responses were more prolonged following 1,1-dimethyl-4-phenylpiperazinium and histamine at bovine cells. Agents that mobilize intracellular Ca(2+)-stores such as methacholine, caffeine and bradykinin resulted in different cytosolic Ca2+ and exocytosis responses at the rat and bovine chromaffin cells. Results indicate a heightened Ca(2+)-store activity or a more filled state in chromaffin cells from the rat. The results of this study clearly show that single-cell techniques can be used to characterize stimulus-secretion coupling. The requirement for lower numbers of cells with these techniques means that chromaffin cells from rodents can be routinely employed. This can be advantageous to minimize biological variability which occurs with organs obtained from slaughter houses and enables the investigation of genetically-altered animals.

Animals↗

PubMed1996

Exocytotic release from individual granules exhibits similar properties at mast and chromaffin cells.

The effects of temperature on granular secretion were studied in individual bovine adrenal chromaffin and rat peritoneal mast cells. It was found that more molecules are released from individual granules at physiological temperature than at room temperature, where such experiments are normally performed. In mast cells, there is also a dramatic decrease in the time required for exocytosis to be complete at 37 degrees C compared to room temperature. In the presence of some cations, the amount released from individual granules at room temperature from both types of cells could be altered. The amount of secretion decreased with the divalent cation zinc but increased with the monovalent cation cesium. These experiments used two electrochemical techniques: cyclic voltammetry and amperometry. With amperometry, the concentration gradient created by the electrode near the cell further increased the amount of release. Similar responses to changes in the extracellular environment in chromaffin and mast cells suggest that the mechanism of extrusion of the granule contents is similar in both cell types.

Animals↗

PubMed1996

Temporally resolved, independent stages of individual exocytotic secretion events.

The stages of the complex events involved in exocytotic secretion after vesicle-cell membrane fusion have been examined at the level of individual vesicles. Catecholamine flux from single bovine adrenal medullary cells was measured with carbon-fiber microelectrodes firmly touching the cell surface. The data reveal that secretion during exocytotic events has three distinct stages: a small increase in catecholamine flux, a rapid, but not instantaneous, rise to a maximum, followed by an exponential decrease in the flux. These stages are interpreted in the following ways. The initial stage corresponds to catecholamine secretion through a fusion pore. The rate of pore expansion appears to control the rise time of the flux to its maximum value. The final exponential stage is consistent with chemical dissociation of the intravesicular matrix or gel.

Adrenal Medulla↗

PubMed1996

Vesicular quantal size measured by amperometry at chromaffin, mast, pheochromocytoma, and pancreatic beta-cells.

Amperometric detection of exocytosis at single chromaffin cells has shown that the distribution of spike areas, or quantal size, is dependent on the volume and catecholamine concentration of individual secretory vesicles. The present work offers an alternate, simplified model to analyze the current spikes due to single exocytotic events. When the cube root of these spike areas is plotted as a histogram, a Gaussian distribution is obtained for chromaffin cells and also mast, pheochromocytoma, and pancreatic beta-cells. It was found that the relative SD of these distributions is similar to that for the vesicular radii, which also have a Gaussian distribution in all four cell types. In addition, this model was used to evaluate conditions where the quantal size of individual events was altered. When chromaffin cells were maintained in culture for < 6 days, spikes of approximately double the quantal size were obtained on repeated exposure to 60 mM K+. The results suggest a heterogeneous distribution of catecholamine-containing vesicles at later days in culture is responsible for this alteration.

Animals↗

PubMed1995

Electrochemical detection of histamine and 5-hydroxytryptamine at isolated mast cells.

The electrochemical oxidation of histamine has been investigated as an analytical tool. In a physiological buffer, histamine is oxidized at carbon fiber microelectrodes at potentials close to the background in a chemically irreversible process. Cylindrical carbon fiber electrodes were used as amperometric detectors for histamine separated with a reversed-phase capillary column, and detection limits of 240 amol were achieved. Electrodes with beveled tips were used as real-time sensors by monitoring with repetitive cyclic voltammograms at a scan rate of 800 V/s with a 16.7-ms repetition rate, and detection limits of 1.4 microM were achieved. Both techniques were used to probe histamine and 5-hydroxytryptamine (5-HT) stored in rat peritoneal mast cells. The content in single cells was measured by capillary HPLC, and both substances were found in single cells. Although the analysis revealed a large cell-to-cell variation in the amount of histamine and 5-HT, the average amount was 150 and 4 fmol of histamine and 5-HT, respectively. Release of histamine and 5-HT was measured with the electrode placed 1 microm from the cell surface. Release was observed as a series of sharp concentration spikes, consistent with corelease of the two substances from individual vesicles following exocytosis.

Animals↗

PubMed1995

Simultaneous amperometric measurement of ascorbate and catecholamine secretion from individual bovine adrenal medullary cells.

Secretion of ascorbate and catecholamines from single bovine adrenal medullary cells has been detected with amperometry at carbon-fiber microelectrodes. Two carbon-fiber electrodes were employed. One was beveled, and voltammograms at this electrode showed overlap of the responses for ascorbate and the catecholamines. The other was beveled and electrochemically oxidized to shift the ascorbate oxidation wave to potentials more negative than that of the catecholamines. Thus, at an applied potential of 0.05 V vs SSCE, ascorbate was selectively oxidized at the treated electrode and both catecholamines and ascorbate were oxidized at an applied potential of 0.65 V at the untreated electrode. Exocytotic release from the cell was stimulated with K+, nicotine, and digitonin. Nicotine and K+ depolarize the cell membrane and elicit vesicular release. Digitonin is a detergent that reacts with cholesterol in the plasma membrane and causes the formation of pores. Ascorbate efflux from individual cells could be induced by digitonin but not by K+ or nicotine and was observed as a single peak with a full width at half-maximum of 4 s. In contrast, catecholamine release was observed as many rapid, sequential current spikes when the cell was exposed to either digitonin, K+, or nicotine. The two different types of release show that ascorbate and catecholamines are being released from two different cellular compartments. The calcium independence of the digitonin-induced ascorbate release provides additional evidence that ascorbate is released by a nonexocytotic process.

Adrenal Medulla↗

PubMed1995

Colocalization of calcium entry and exocytotic release sites in adrenal chromaffin cells.

"Snapshot" images of localized Ca2+ influx into patch-clamped chromaffin cells were captured by using a recently developed pulsed-laser imaging system. Transient opening of voltage-sensitive Ca2+ channels gave rise to localized elevations of Ca2+ that had the appearance of either "hotspots" or partial rings found immediately beneath the plasma membrane. When the Ca2+ imaging technique was employed in conjunction with flame-etched carbon-fiber electrodes to spatially map the release sites of catecholamines, it was observed that the sites of Ca2+ entry and catecholamine release were colocalized. These results provide functional support for the idea that secretion occurs from "active zone"-like structures in neuroendocrine cells.

Adrenal Medulla↗

PubMed1995

Fast-scan cyclic voltammetry of 5-hydroxytryptamine.

Fast-scan cyclic voltammetry, a demonstrated analytical method for the in vivo detection of dopamine, is extended to the detection of in vitro and in vivo 5-hydroxytryptamine (5-HT) with the use of a specific potential wave form applied at 1000 V/s. The wave form, 0.2 to 1.0 to -0.1 to 0.2 V, is employed to accelerate electrode response times which are significantly slower with other wave forms due to the adsorption of 5-HT. The scan rate of 1000 V/s enables follow-up reactions which lead to the formation of strongly adsorptive products to be outrun. The peak current at a carbon fiber disk microelectrode exposed to 1 microM 5-HT in flow injection experiments is 1 nA, with a half-rise time of less than 200 ms. The peak current of Nafion-coated electrodes exposed to the same concentration of 5-HT is 5 nA, with a half-rise time on the order of 400 ms. The rate of adsorption of 5-HT was determined to be 4.22 +/- 0.33 s-1. Several compounds present in brain tissue as well as the pharmacological agents used to elicit 5-HT release in the caudate of the rat were evaluated. Those which gave a response could be differentiated from 5-HT on the basis of respective oxidative and reductive peak potentials. Nafion-coated electrodes were used to monitor transient increases in both dopamine and exogenous 5-HT in the caudate of the anesthetized rat in response to electrical stimulation. The rate of cellular uptake of 5-HT was shown to be 3-fold slower than dopamine uptake. NS-15841

Animals↗

PubMed1995

Dynamics of 5-hydroxytryptamine released from dopamine neurons in the caudate putamen of the rat.

Fast-scan cyclic voltammetry has been used for the detection of 5-hydroxytryptamine (5-HT) in vivo. The applied potential waveform was previously optimized to maximize response times to concentration changes. This technique has been used to reinvestigate 5-HT release from striatal dopaminergic terminals after pharmacological pretreatment as reported by Stamford et al. [13]. Our results concur with those set forth by Stamford and further show that the dopamine transporter is responsible for 5-HT uptake in this experiment.

Animals↗

PubMed1995

Correlation of real-time catecholamine release and cytosolic Ca2+ at single bovine chromaffin cells.

Previous investigations of the role of Ca2+ in stimulus-secretion coupling have been undertaken in populations of adrenal chromaffin cells. In the present study, the simultaneous detection of intracellular Ca2+, with the fluorescent probe fura-2, and catecholamine release, using a carbon-fiber microelectrode, are examined at single chromaffin cells in culture. Results from classic depolarizing stimuli, high potassium (30-140 mM) and 1,1-dimethyl-4-phenylpiperazinium (3-50 microM), show a dependence of peak cytosolic Ca2+ concentration and catecholamine release on secretagogue concentration. Catecholamine release induced by transient high K+ stimulation increases logarithmically with K+ concentration. Continuous exposure to veratridine (50 microM) induces oscillations in intracellular Ca2+ and at higher concentrations (100 microM) concomitant fluctuation of cytosolic Ca2+ and catecholamine secretion. Mobilization of both caffeine- and inositol trisphosphate-sensitive intracellular Ca2+ stores is found to elicit secretion with or without extracellular Ca2+. Caffeine-sensitive intracellular Ca2+ stores can be depleted, refilled, and cause exocytosis in medium without Ca2+. Single cell measurement of exocytosis and the increase in cytosolic Ca2+ induced by bradykinin-activated intracellular stores reveal cell to cell variability in exocytotic responses which is masked in populations of cells. Taken together, these results show that exocytosis of catecholamines can be induced by an increase in cytosolic Ca2+ either as a result of transmembrane entry or by release of internal stores.

Adrenal Medulla↗

PubMed1995

Distinct pharmacological regulation of evoked dopamine efflux in the amygdala and striatum of the rat in vivo.

The pharmacological regulation of evoked extracellular dopamine was compared in the basolateral amygdaloid nucleus (BAN) and caudate-putamen (CP) of the urethane-anesthetized rat. The effects of drugs, which alter dopamine uptake, release or degradation, were examined. Dopamine efflux was elicited by electrical stimulation of ascending dopamine fibers and was monitored by fast-scan cyclic voltammetry at Nafion-coated, carbon-fiber microelectrodes. Dopamine uptake inhibitors, nomifensine (25 mg/kg) and cocaine (20 mg/kg), and the dopamine receptor antagonist, haloperidol (0.5 mg/kg), robustly increased evoked extracellular dopamine in the CP. In sharp contrast, these drugs were much less effective in the BAN. The relative potencies of the uptake inhibitors varied between the two regions. Nomifensine was more potent than cocaine in the CP, whereas cocaine was more potent that nomifensine in the BAN. The monoamine oxidase inhibitor, pargyline (75 mg/kg), and the catechol-O-methyltransferase (COMT) inhibitor, Ro 40-7592 (40 mg/kg), had small or negligible effects in either region. No electrochemical evidence was found for the formation of 3-methoxytyramine, the dopamine metabolite formed by the action of COMT on released dopamine, on the time scale of the measurements in control or after pharmacological manipulation of the degradative enzymes for dopamine. The conclusions reached are: (1) potent mechanisms for uptake and autoreceptor inhibition of release, which exist in the CP to tightly control the concentration of extracellular dopamine, are considerably weaker in the BAN; (2) the extracellular clearance of evoked dopamine in the BAN and CP is the result of cellular uptake and not degradation; and (3) these results support the view that the pharmacological regulation of extracellular dopamine is regionally distinct in the brain.

Amygdala↗

PubMed1995

Time course of release of catecholamines from individual vesicles during exocytosis at adrenal medullary cells.

The time course of extrusion of the vesicular contents during exocytosis has been examined at adrenal medullary cells with carbon-fiber microelectrodes. Two electrochemical techniques were used: cyclic voltammetry and amperometry. Spikes obtained by amperometry had a faster time course than those measured by cyclic voltammetry, consistent with the different concentration profiles established by each technique. However, the experimental data obtained with both techniques were temporally broadened with respect to dispersion of an instantaneous point source by diffusion. Measurements with the electrode firmly pressed against the cell surface established that the temporal broadening is a result of a rate-limiting kinetic step associated with extrusion of the vesicular contents at the cell surface. The data do not support a rate-limiting process due to restricted efflux from a small pore. When combined with previous results, the data suggest that the rate-limiting step for chemical secretion from adrenal medullary cells during exocytosis is the dissociation of catecholamines from the vesicular matrix at the surface of the cell.

Adrenal Medulla↗

PubMed1995

Comparison of dopamine uptake in the basolateral amygdaloid nucleus, caudate-putamen, and nucleus accumbens of the rat.

Regional differences in the kinetics and pharmacological inhibition of dopamine uptake were investigated with fast-scan cyclic voltammetry in both the intact rat brain and a brain slice preparation. The regions compared were the basolateral amygdaloid nucleus, caudate-putamen, and nucleus accumbens. The frequency dependence of dopamine efflux evoked in vivo by electrical stimulation of the medial forebrain bundle was evaluated by nonlinear curve fitting with a Michaelis-Menten-based kinetic model. The Km for dopamine uptake was found to be significantly higher in the basolateral amygdala (0.6 microM) than in the other two regions (0.2 microM), whereas the Vmax value for dopamine uptake in the basolateral amygdala was significantly lower (0.49 microM/s vs. 3.8 and 2.4 microM/s in the caudate and accumbens, respectively). Similar kinetics were also obtained in brain slices. Addition of a dopamine uptake inhibitor, cocaine or nomifensine (10 microM), to the perfusion buffer increased the apparent Km value > 25-fold in slices of both the caudate-putamen and nucleus accumbens. In contrast, neither uptake inhibitor had an observable effect in the basolateral amygdaloid nucleus. Thus, dopamine uptake in the rat brain is regionally distinct with regard to rate, affinity, and sensitivity to competitive inhibition.

Amygdala↗

PubMed1995

Different effects of cocaine and nomifensine on dopamine uptake in the caudate-putamen and nucleus accumbens.

The effects of cocaine and nomifensine on the uptake of dopamine have been compared in the caudate-putamen and nucleus accumbens of rat brain slices. Electrical stimulation of brain slices was used to evoke dopamine efflux and the changes in dopamine concentration in the extracellular fluid were monitored with fast-scan cyclic voltammetry with Nafion-coated, carbon-fiber electrodes. The disappearance of extracellular dopamine after the stimulation fit Michaelis-Menten kinetics in both regions. Cocaine and nomifensine were found to competitively inhibit dopamine uptake in both regions. The competitive mechanism of action was apparent in the primary data because the initial uptake rates were unchanged by low doses of inhibitor, but dopamine uptake was slowed at concentrations near the Km value. In both regions, the apparent Km value increased with higher concentrations of cocaine (0.01-60 microM) or nomifensine (0.01-30 microM) in the perfusion buffer. The apparent Km values were used to obtain inhibition constants (Ki values) for the uptake inhibitors in each region. This analysis showed that cocaine had a Ki of 0.29 microM in both regions. Nomifensine, however, had a significantly higher potency in the caudate-putamen (Ki = 0.09 microM) than in the nucleus accumbens (Ki = 0.21 microM). These results show that there are differential effects of uptake inhibitors in different brain regions, and suggest that the dopamine transporter exhibits cell-specific regulation.

Animals↗

PubMed1994

In vivo voltammetric measurement of evoked extracellular dopamine in the rat basolateral amygdaloid nucleus.

1. The in vivo measurement of evoked extracellular dopamine was established in the basolateral amygdaloid nucleus (BAN) using fast-scan cyclic voltammetry at carbon-fibre microelectrodes. 2. The identification of evoked extracellular dopamine in the BAN was based on anatomical, electrochemical and pharmacological criteria. Electrochemical and pharmacological evidence indicated that the species was a catecholamine. Mesencephalic sites eliciting overflow and amygdaloid sites supporting overflow correlated well with the mesoamygdaloid dopamine innervation. 3. Marked differences in the dynamics and magnitude of evoked dopamine overflow were observed in the BAN, caudate-putamen and amygdalo-striatal transition area. The results underscore the importance of making spatially resolved measurements of extracellular dopamine in the amygdala. 4. Mesoamygdaloid dopamine neurons have similar release characteristics as mesostriatal dopamine neurons but share with mesoprefrontal cortical dopamine neurons the ability to use a greater percentage of intraneuronal dopamine stores for release.

Amygdala↗

PubMed1994

Zones of exocytotic release on bovine adrenal medullary cells in culture.

Secretion of catecholamines from individual bovine adrenal medullary cells in culture was examined by amperometry with 1-micron radius carbon-fiber electrodes. Vesicular secretion is observed as a series of current spikes upon exposure to a secretagogue. The small size of the electrodes was exploited to map exocytotic release sites on the surface of bovine adrenal medullary cells. These studies reveal for the first time that release sites are spatially localized on endocrine cells in culture for a time scale of at least 15 min. Fluorescent monitoring by confocal microscopy of deposition of dopamine-beta-hydroxylase from the vesicular membrane into the plasma membrane during exocytosis confirms the existence of zones of exocytotic inactivity on the surface of the cell. Measurement of coincident spikes with two adjacent electrodes (2.5-microns radius) has allowed the spatial resolution for measurement of exocytosis to be defined as 2 microns from the projected circumference of the electrode on the surface of the cell. In this small domain, point-source release and diffusional broadening would result in narrow spikes (Schroeder, T. J., Jankowski, J. A., Kawagoe, K. T., Wightman, R. M., Lefrou, C., and Amatore, C. (1992) Anal. Chem. 64, 3077-3083), a feature not seen in the data. Thus, for adrenal medullary cells, release following vesicular fusion is not instantaneous, but is a prolonged event occurring over several milliseconds.

Adrenal Medulla↗

PubMed1994

Quantitation of epinephrine and norepinephrine secretion from individual adrenal medullary cells by microcolumn high-performance liquid chromatography.

The cellular response of individual adrenal medullary cells to a prolonged stimulation with carbamylcholine (carbachol) was studied using microcolumn high-performance liquid chromatography (MHPLC) with electrochemical detection. Chromaffin cells will not release all of their catecholamine stores when continuously exposed to a secretagogue. The process of desensitization prevents the continued release of catecholamines after a certain percentage of chemical stores have been depleted. The percentage of a cell's chemical stores released upon stimulation was determined, and whether there is a difference in release between the cell types was examined. A single cell was exposed to a stimulation solution for 5.0 min at 25 degrees C. Approximately 80% of the solution was removed and analyzed on a microcolumn reversed-phase chromatography column. A lysing solution was immediately added to the miniaturized vial which still contained the cell, and this solution was also analyzed chromatographically. The first run was used to determine the amount of catecholamine the cell released upon stimulation. The second run determined the amount of catecholamine that remained in the cell. From these values the percentage of catecholamine released was calculated. Twenty-seven individual cells were studied by this method, and 22 could be classified as norepinephrine or epinephrine dominant. There was no statistical difference in the amount of catecholamine the norepinephrine dominant cells released (14 +/- 5%, n = 11) as compared to the epinephrine dominant cells (12 +/- 4%, n = 11).

Adrenal Medulla↗

PubMed1994

Interference by pH and Ca2+ ions during measurements of catecholamine release in slices of rat amygdala with fast-scan cyclic voltammetry.

Fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes was used to investigate catecholamine release and uptake induced by local electrical stimulation of rat brain slices containing the basolateral amygdaloid nucleus. The amygdala contains less catecholamine than the striatum, and the observed release is proportionately smaller. Stimulus trains of long duration were required to obtain a well-resolved concentration change in the basolateral amygdala. Voltammetric detection of catecholamines under these conditions was complicated by interference from two extracellular ions, H+ and Ca2+. Ion-selective microelectrodes were used in conjunction with carbon-fiber microelectrodes to monitor pH and Ca2+. The magnitude of the pH changes was correlated with stimulation length and followed the pattern of a brief alkaline shift followed by a longer acidic shift. Extracellular Ca2+ concentration decreased during stimulation and returned fairly rapidly to baseline after the stimulation was over. Because it was not possible to account for all of the ionic interferences using information in the voltammograms, other strategies were employed. Exposure of amygdala slices to L-DOPA or DA increased electrically evoked release of catecholamine, but the effect was transient, and uptake rates decreased during continued exposure to these agents. The most successful approach to remove the interferences was to subtract the response obtained after exposure of the slice to the catecholamine depleter, Ro 4-1284. This agent eliminates the catecholamine response but does not appear to alter the ionic changes.

Amygdala↗