Electrochemical Detection of Exocytosis at Single Rat Melanotrophs

Paras, Charina D.; Kennedy, Robert T.

doi:10.1021/ac00116a003

Cited by 56 publications

(41 citation statements)

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“…Single Cell Experiments-Single cell measurements were performed similar to those described elsewhere (19,23,31). All amperometric experiments were performed on a Zeiss Axiovert 100 inverted microscope.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, amperometry with microelectrodes has been applied to the study of exocytosis at a variety of cell types, including adrenal chromaffin cells (16 -19), PC12 cells (20), mast cells (21), neurons (22), and melanotrophs (23,24). Secretion is detected as a series of current spikes that correspond to detection of concentration pulses generated by exocytosis.…”

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confidence: 99%

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Effects of Intravesicular H+ and Extracellular H+ and Zn2+ on Insulin Secretion in Pancreatic Beta Cells

Aspinwall

Brooks

Kennedy

et al. 1997

Journal of Biological Chemistry

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118

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The effects of extracellular Zn 2؉ and pH and intravesicular pH on insulin and 5-hydroxytryptamine (5-HT) secretion from pancreatic beta cells were investigated. Insulin and 5-HT secretion from single cells was detected by amperometry as a series of current spikes corresponding to detection of multimolecular packets secreted by exocytosis. Spike width was used as a measure of the kinetics of clearance from the cell and the area of spikes as a measure of amount released. Changes in extracellular pH from 6.9 to 7.9 caused insulin spikes to become narrower with no change in area, whereas the same treatments had no effect on 5-HT secretion. Treatment of cells with Bafilomycin A 1 or N-ethylmaleimide, both of which are expected to increase intravesicular pH by inhibiting V-type H ؉ -ATPase, had no effect on 5-HT secretion but caused insulin spikes to become more narrow. These results indicate that exposure to high pH, whether intravesicular or extracellular, accelerates release of insulin during exocytosis without affecting the amount of insulin released. Increasing extracellular Zn 2؉ concentration from 0 to 25 M increased the width and decreased the area of insulin spikes without affecting 5-HT secretion. Zn 2؉ effects were likely exerted through a common-ion effect on Zn 2؉ -insulin dissociation. It was concluded that intravesicular storage conditions and extracellular ions can affect free insulin concentration in the vicinity of beta cells during secretion.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Effects of Intravesicular H+ and Extracellular H+ and Zn2+ on Insulin Secretion in Pancreatic Beta Cells

Aspinwall

Brooks

Kennedy

et al. 1997

Journal of Biological Chemistry

Self Cite

118

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show abstract

“…This technique has primarily been used for the detection of easily oxidized substances such as indoleamines 1151 and catecholamines 16,171; however, more recent work has extended this approach to more difficult to oxidize compounds such as insulin 1181 and histamine [19]. We have recently reported on the use of carbon fiber microelectrodes to detect secretion of MSH and related peptides from single melanocytes [9]. Melanocytes synthesize and secrete a collection of peptides derived from proopiocortin (POC).…”

Section: Introductionmentioning

confidence: 99%

Amperometry and cyclic voltammetry of tyrosine and tryptophan‐containing oligopeptides at carbon fiber microelectrodes applied to single cell analysis

Paras

Kennedy

1997

Electroanalysis

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The amperometric and cyclic voltammetric detection of a-melanocyte stimulating hormone (MSH), 0-endorphin, and corticotropin-like intermediate lobe peptide (CLIP), all proopiocortin (POC) derived peptides secreted from melanocytes of the pituitary intermediate lobe, at carbon fiber microelectrodes was investigated. For amperometry, it was found that all of these peptides could be detected; however, fouling of the electrodes reduced the response of the electrode after successive application of the peptide in flow injection experiments. The fouling wa5 apparently due to oxidation of tyrosine in the peptides as similar results were found for tyrosine but not tryptophan. The effect of fouling could be reversed if the electrode was electrochemically treated by scanning from -1 .OV to +1.OV at 300 V/s for 2 min between application of the peptides. Using cyclic voltammtery at 800 V/s, it was possible to distinguish MSH, which had a peak shaped voltammogram, from the other POC peptides, which had relatively flat voltammetric waves at this scan rate. The scan rate dependence of the peak current for MSH revealed that the voltammetry was adsorption controlled. As a result, in a monitoring application, where voltammograms are continuously obtained with a fixed interval between them, decreasing the interval increases the temporal resolution but decreases the sensitivity for MSH. It was found that when monitoring the current in the potential range of 0.90 to 1 .OOV, the temporal response to MSH was dependent upon the potential window used for scanning. Using high scan rates and a potential window of 0 to 1.2 V, it was possible to monitor exocytosis from single melanocytes and use the voltammogram to demonstrate detection of MSH from the cells.

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“…The dynamics of calcium-induced exocytosis in the melanotrope has been well characterized [23, 24, 25, 26, 27, 28, 29, 30, 31]. Three kinetic phases of secretion have been identified, probably representing the release of granules at different stages of docking and priming [30].…”

Section: Discussionmentioning

confidence: 99%

Monensin and Hypo-Osmolar Medium Cause Calcium-Independent β-Endorphin Secretion from Melanotropes

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Soinila²,

Törnquist

2000

Neuroendocrinology

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Monensin has been shown to cause nonexocytotic release of catecholamines from adrenal medullary and PC12 cells. We examined the effect of monensin on peptide secretion with cultured melanotropes from the rat pituitary as a model. 1 µM monensin caused an immediate, transient increase in β-endorphin secretion. The effect was still seen in a calcium-free medium, but was totally abolished in a sodium-free medium. Intracellular calcium concentration was measured with Fura 2: no increase was observed during monensin stimulation. Hypo-osmolar medium mimicked the effect of monensin, causing a 12-fold transient increase in β-endorphin secretion. This effect was not abolished in either calcium-free or sodium-free medium. No increase in the number of exocytotic figures captured by tannic acid incubation was observed during 5 min of incubation with 1 µM monensin or hypo-somolar medium. We thus show that monensin causes β-endorphin secretion from the melanotrope and that this effect is due to sodium influx and resultant cell swelling. The calcium independency and lack of increase of exocytotic figures suggest that swelling-induced secretion is nonexocytotic, possibly via transient exocytotic pore opening.

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Electrochemical Detection of Exocytosis at Single Rat Melanotrophs

Cited by 56 publications

References 0 publications

Effects of Intravesicular H+ and Extracellular H+ and Zn2+ on Insulin Secretion in Pancreatic Beta Cells

Effects of Intravesicular H+ and Extracellular H+ and Zn2+ on Insulin Secretion in Pancreatic Beta Cells

Amperometry and cyclic voltammetry of tyrosine and tryptophan‐containing oligopeptides at carbon fiber microelectrodes applied to single cell analysis

Monensin and Hypo-Osmolar Medium Cause Calcium-Independent β-Endorphin Secretion from Melanotropes

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