Extracellular pH Is Required for Rapid Release of Insulin from Zn−Insulin Precipitates in β-Cell Secretory Vesicles during Exocytosis

Kennedy, Robert T.; Huang, Lan; Aspinwall, Craig A.

doi:10.1021/ja953271w

Cited by 114 publications

(63 citation statements)

References 28 publications

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“…In an initial study on postfusion clearance of insulin, we found that exposure to a pH gradient between the vesicle and the extracellular environment is required to achieve rapid release of free insulin from solid Zn 2ϩ -insulin granules (37). In the present study, we extended this investigation to a variety of extracellular pH values.…”

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confidence: 82%

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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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confidence: 82%

“…Accumulation of 5-Hydroxytryptamine into Beta Cells-For experiments requiring measurement of 5-HT secretion, dispersed beta cells were allowed to incubate in G-100 containing 0.5 mM 5-HT and 1 mM 5-hydroxytryptophan for 16 h at 37°C in 5% CO 2 , pH 7.40 (37). 2 Cells were used for secretion experiments immediately after loading.…”

Section: Methodsmentioning

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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“…We have previously shown that increasing H ϩ and Zn 2ϩ in the extracellular medium significantly decreases the extrusion rate of insulin from single secretory vesicles after vesicle fusion leading to a decrease in the concentration of free insulin at the cell surface in cells undergoing exocytosis (33,34). Table I summarizes the amount of 5-HT secretion, measured as a number exocytosis events detected, evoked by 200 M tolbutamide detected from cells incubated in control buffers and buffers containing varying H ϩ and Zn 2ϩ concentrations.…”

Section: Insulin-stimulated Insulin Secretion In Single Betamentioning

confidence: 99%

“…Secreted insulin can be detected directly using a carbon fiber amperometric electrode that is modified with a mixed valent film of cyanoruthenate and ruthenium oxide (32). Alternatively, 5-hydroxytryptamine (5-HT) 1 secretion can be detected as a marker of exocytosis in beta cells (33)(34)(35)(36). In this approach, 5-HT is allowed to accumulate into insulin containing secretory vesicles of beta cells, and 5-HT secretion is detected using an unmodified carbon fiber electrode.…”

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confidence: 99%

Insulin-stimulated Insulin Secretion in Single Pancreatic Beta Cells

Aspinwall

Lakey

Kennedy

1999

Journal of Biological Chemistry

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203

172

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Functional insulin receptors are known to occur in pancreatic beta cells; however, except for a positive feedback on insulin synthesis, their physiological effects are unknown. Amperometric measurements at single, primary pancreatic beta cells reveal that application of exogenous insulin in the presence or absence of nonstimulatory concentrations of glucose evokes exocytosis mediated by the beta cell insulin receptor. Insulin also elicits increases in intracellular Ca 2؉ concentration in beta cells but has minimal effects on membrane potential. Conditions where the insulin receptor is blocked or cell surface concentration of free insulin is reduced during exocytosis diminishes secretion induced by other secretagogues, providing evidence for direct autocrine action of insulin upon secretion from the same cell. These results indicate that the beta cell insulin receptor can mediate positive feedback for insulin secretion. The presence of a positive feedback mechanism for insulin secretion mediated by the insulin receptor provides a potential link between impaired insulin secretion and insulin resistance.

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“…In previous work, Kennedy et al [20] show that a pH change (towards physiological pH) is a requirement for proper disaggregation of the insulin hexamer. In separate studies, Shafqat et al [21] show that C-peptide has the ability to enhance the disaggregation of hexameric insulin into mono- Figure 1.…”

Section: +mentioning

confidence: 99%

A Molecular Level Understanding of Zinc Activation of C-peptide and its Effects on Cellular Communication in the Bloodstream

et al. 2009

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■ AbstractInspired by previous reports, our group has recently demonstrated that C-peptide exerts beneficial effects upon interactions with red blood cells (RBCs). These effects can be measured in RBCs obtained from animal models of both type 1 diabetes and type 2 diabetes, though to different extents. To date, the key metrics that have been measured involving C-peptide and RBCs include an increase in glucose uptake by these cells and a subsequent increase in adenosine triphosphate (ATP) release. Importantly, to date, our group has only been able to elicit these beneficial effects when the C-peptide is prepared in the presence of Zn 2+ . The C-peptide-induced release of ATP is of interest when considering that ATP is a purinergic signaling molecule known to stimulate the production of nitric oxide (NO) in the endothelium and in platelets. This NO production has been shown to participate in smooth muscle relaxation and subsequent vessel dilation. Furthermore, NO is a well-established platelet inhibitor. The objective of this review is to provide information pertaining to C-peptide activity on RBCs. Special attention is paid to the necessity of Zn 2+ activation, and the origin of that activation in vivo. Finally, a mechanism is proposed that explains how C-peptide is exerting its effects on other cells in the bloodstream, particularly on endothelial cells and platelets, via its ability to stimulate the release of ATP from RBCs.

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Extracellular pH Is Required for Rapid Release of Insulin from Zn−Insulin Precipitates in β-Cell Secretory Vesicles during Exocytosis

Cited by 114 publications

References 28 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

Insulin-stimulated Insulin Secretion in Single Pancreatic Beta Cells

A Molecular Level Understanding of Zinc Activation of C-peptide and its Effects on Cellular Communication in the Bloodstream

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