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

Abstract

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.

Glucose Acutely Decreases pH of Secretory Granules in Mouse Pancreatic Islets

“…We estimate that granular pH is above 6.0 under these conditions. Bafilomycin has been reported not to affect immediate (Ͻ60 s) exocytosis of insulin granules as measured by amperometry in canine ␤-cells (50) but to inhibit insulin release from permeabilized INS-1 cells (51). In our hands, bafilomycin markedly inhibited glucose-induced insulin secretion by mouse islets but also impaired the glucose-induced [Ca 2ϩ ] c rise (not shown), as in MIN6 cells (52).…”

Roles of Insulin Receptor Substrate-1, Phosphatidylinositol 3-Kinase, and Release of Intracellular Ca2+ Stores in Insulin-stimulated Insulin Secretion in β-Cells

“…The 5-HT method was used instead of direct detection of insulin at a modified electrode (23) because of the relative simplicity of this approach, especially for autocrine studies. The validity of the 5-HT method has been established by previous studies that demonstrated: 1) detection of exocytosis with a variety of insulin secretogogues (24,28,29); 2) that insulin and 5-HT are released exclusively from the same vesicles (30); and 3) that direct detection of insulin and detection of 5-HT give comparable results for insulin stimulation (18). As seen in Fig.…”

“…Amperometric Detection of Exocytosis-Microelectrodes were constructed of carbon fibers sealed in glass micropipettes and were polished to a 45°angle and cleaned prior to use (23,24). Amperometric measurements were made by positioning microelectrodes ϳ1 m from a cell and applying stimulant from a micropipette ϳ 30 m from the cell as described elsewhere (23,24 Amperometry was performed using a battery to apply potential to a sodium-saturated calomel reference electrode as described previously (24).…”

“…Amperometric measurements were made by positioning microelectrodes ϳ1 m from a cell and applying stimulant from a micropipette ϳ 30 m from the cell as described elsewhere (23,24 Amperometry was performed using a battery to apply potential to a sodium-saturated calomel reference electrode as described previously (24). For measurements of 5-hydrotryptamine (5-HT) secretion, dispersed ␤-cells were incubated in tissue culture medium containing 0.5 ] i increases in the interior region (red line) and the edge of the cell (blue line).…”

Arginine vasopressin-induced glucagon release: interaction with glucose and cyclic AMP-dependent protein kinase

“…Amperometry is useful in monitoring secretion from single cells, even at the level of single exocytotic events, because it allows detection of attomole quantities with millisecond temporal resolution (Wightman et al, 1991;Chow et al, 1992). Amperometry has been applied to (3-cells either by using a ruthenium oxide/cyanoruthenate-coated microelectrode to directly detect insulin (Huang et al, 1995;Aspinwall et al, 1997) or by using a carbon fiber microelectrode to detect 5 -hydroxytryptamine that had been allowed to accumulate in the secretory vesicles (Smith et al, 1995;Zhou and Misler, 1996). A limitation of amperometry is that a microelectrode is inherently a single-point sensor, meaning that measurements are made at one location on a cell with a detection area determined by the size of the electrode Recent advances in fluorescence imaging have resulted in new approaches to monitor exocytosis with a non-invasive method.…”