Biomedical subjects

R Mark Wightman

Publications and source records attributed to R Mark Wightman.

44 records · Page 3Linked to original sources

PubMed2002

Temporal separation of vesicle release from vesicle fusion during exocytosis.

During exocytosis, vesicles in secretory cells fuse with the cellular membrane and release their contents in a Ca2+-dependent process. Release occurs initially through a fusion pore, and its rate is limited by the dissociation of the matrix-associated contents. To determine whether this dissociation is promoted by osmotic forces, we have examined the effects of elevated osmotic pressure on release and extrusion from vesicles at mast and chromaffin cells. The identity of the molecules released and the time course of extrusion were measured with fast scan cyclic voltammetry at carbon fiber microelectrodes. In external solutions of high osmolarity, release events following entry of divalent ions (Ba2+ or Ca2+) were less frequent. However, the vesicles appeared to be fused to the membrane without extruding their contents, since the maximal observed concentrations of events were less than 7% of those evoked in isotonic media. Such an isolated, intermediate fusion state, which we term "kiss-and-hold," was confirmed by immunohistochemistry at chromaffin cells. Transient exposure of cells in the kiss and hold state to isotonic solutions evoked massive release. These results demonstrate that an osmotic gradient across the fusion pore is an important driving force for exocytotic extrusion of granule contents from secretory cells following fusion pore formation.

Animals↗

PubMed2002

Response times of carbon fiber microelectrodes to dynamic changes in catecholamine concentration.

The electrode response time and the measured concentrations during dynamic catecholamine changes were compared using constant potential amperometry and fast-scan cyclic voltammetry. The amperometric response to a rectangular pulse of catecholamine is more rectangular than the cyclic voltammetric response; however, the response times are very similar when, during cyclic voltammetry, the temporal lag due to adsorption and desorption of catecholamine to the electrode is removed by deconvolution. Deconvolution of cyclic voltammetry data was applied to stimulated dopamine release in vivo, allowing for modeling of release and uptake kinetics and to measure catecholamine release from single cells, resulting in better resolution of peaks from single vesicles. In vitro postcalibrations were performed to calculate concentrations of catecholamine measured with cyclic voltammetry and amperometry. The addition of 600 microM ascorbic acid to the postcalibration buffer, allowing a catalytic reaction to regenerate dopamine, resulted in similar calculated concentrations for stimulated release of dopamine using amperometry and cyclic voltammetry. Using deconvoluted cyclic voltammetry to remove the response time lag and adding ascorbic acid to the calibration buffer, the shape and concentration of dynamic catecholamine changes are very similar when measured with constant potential amperometry and cyclic voltammetry.

Animals↗

PubMed2002

Electrogenerated chemiluminescence detection in reversed-phase liquid chromatography.

Electrogenerated chemiluminescence (ECL) has been developed as a detection method for liquid chromatography. The radical cation of tri-p-tolylamine (TPTA) is used as a common electron acceptor for the electrogenerated radical anions of a variety of organic analytes. ECL is accomplished with a high-frequency potential pulse program applied to a microelectrode immersed in the column eluent. ECL detection is demonstrated with reversed-phase liquid chromatography. Selectivity at the ECL detector is shown to be tunable based on differing electrochemical conditions and excited-state energetics. Low minimum detection limits in ECL are attributed to the dependence on the photon detector shot noise, allowing a limit of detection of 0.14 nM for perylene in the presence of 0.1 mM TPTA. A derivatization agent useful for ECL detection is demonstrated by the use of naphthalene-2,3-dicarboxaldehyde. This reagent, which does not itself result in ECL, forms ECL candidates following reaction with primary amines.

Journal Article↗

PubMed2002

Neurochemistry and electroanalytical probes.

Electroanalytical techniques have been applied to monitoring chemical events including neurotransmitter release during rodent behaviour and the release of zeptomoles of molecules from single cells. Transgenic mice models have been developed and studied to identify specific cell types in vitro. Characterization and surface modification of electroanalytical probes has enhanced the selectivity and sensitivity of measurements.

Animals↗

PubMed2002

Transient changes in mesolimbic dopamine and their association with 'reward'.

Mesolimbic dopaminergic neurons modulate complex circuitry in the ventral forebrain involved in reward processing, although the precise function of the dopaminergic input is debated. Electrophysiological measurements have revealed that mesolimbic dopaminergic neurons can fire in either tonic or phasic modes, and that phasic firing accompanies the alerting or anticipatory phases of reward. However, the neurochemical relevance of this rapid neuronal discharge within the reward processing circuitry is not yet clear, in part because of difficulty in interpretation of extracellular dopamine measurements. Herein, the nature of the information provided by different neurochemical techniques is critically discussed. Classical methods of monitoring dopamine reveal changes in extracellular dopamine resulting from tonic neuronal activity, but do not have the temporal resolution to distinguish concentration transients. However, recent advances in dopamine sensors now enable transient dopamine concentrations resulting from phasic firing to be positively identified and followed on a physiologically relevant timescale. This has enabled demonstrations of discrete, phasic dopamine signals accompanying rewarding or alerting stimuli. Thus, enhanced dopamine release at terminals appears to be coincident with phasic electrical activity at cell bodies. These accumulating data promise to help unravel the precise role of phasic dopamine transmission in reward processing.

Animals↗

PubMed2002

Separating vesicle fusion and exocytosis in hypertonic conditions.

The existence of an osmotic gradient between the vesicular contents of chromaffin and mast cells and the extracellular fluid plays a key role in the exocytotic process. Altering this gradient by elevating the external buffer osmolarity allows for the inhibition of exocytosis from cells and isolation and identification of prespike current events upon stimulation using cyclic voltammetry. Subsequent replacement of the osmotic gradient leads to release of the contents from fused vesicles.

Animals↗

PubMed2002

The association of vesicular contents and its effects on release.

The oxidation of catecholamines with a carbon-fiber electrode can be used to monitor exocytosis at the single cell level at a variety of different types of cells. These measurements allow release to be followed from individual vesicles and have revealed several unique aspects concerning the coupling between release and storage. The strong association of the vesicular components in chromaffin cells dictates the time course of extrusion of the vesicle contents. Furthermore, liberation of the Ca(2+) normally stored within the vesicles can promote exocytosis without an external Ca(2+) source.

Animals↗

PubMed2002

Functional selectivity of dopamine receptor agonists. I. Selective activation of postsynaptic dopamine D2 receptors linked to adenylate cyclase.

Dihydrexidine (DHX), the first high-affinity D(1) dopamine receptor full agonist, is only 10-fold selective for D(1) versus D(2) receptors, having D(2) affinity similar to the prototypical agonist quinpirole. The D(2) functional properties of DHX and its more D(2) selective analog N-n-propyl-dihydrexidine (PrDHX) were explored in rat brain and pituitary. DHX and PrDHX had binding characteristics to D(2) receptors in rat striatum typical of D(2) agonists, binding to both high- and low-affinity sites and being sensitive to guanine-nucleotides. Consistent with these binding data, both DHX and PrDHX inhibited forskolin-stimulated cAMP synthesis in striatum with a potency and intrinsic activity equivalent to that of quinpirole. Unexpectedly, however, DHX and PrDHX had little functional effect at D(2) receptors expressed on dopaminergic neurons that mediate inhibition of cell firing, dopamine release, or dopamine synthesis. Quantitative receptor competition autoradiography demonstrated that DHX bound to D(2) receptors in striatum (predominantly postsynaptic receptor sites) with equal affinity as D(2) sites in the substantia nigra (autoreceptor sites). The data from these experiments, coupled with what is known about the location of specific dopamine receptor isoforms, lead to the hypothesis that DHX, after binding to D(2L) and D(2S) receptors, causes agonist-typical functional changes only at some of these receptors. This phenomenon (herein termed "functional selectivity") suggests that drugs may be targeted not only at specific receptor isoforms but also at separate functions mediated by a single isoform, yielding novel approaches to drug discovery.

Adenylyl Cyclases↗