Spatial Organization of Ca2+ Entry and Exocytosis in Mouse Pancreatic β-Cells

doi:10.1006/bbrc.2001.5379

Biochemical and Biophysical Research Communications

Volume 286, Issue 2, 17 August 2001, Pages 315-321

https://doi.org/10.1006/bbrc.2001.5379 Get rights and content

Abstract

Secretion from single pancreatic β-cells was imaged using a novel technique in which Zn²⁺, costored in secretory granules with insulin, was detected by confocal fluorescence microscopy as it was released from the cells. Using this technique, it was observed that secretion from β-cells was limited to an active region that comprised ∼50% of the cell perimeter. Using ratiometric imaging with indo-1, localized increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) evoked by membrane depolarization were also observed. Using sequential measurements of secretion and [Ca²⁺]_i at single cells, colocalization of exocytotic release sites and Ca²⁺ entry was observed when cells were stimulated by glucose or K⁺. Treatment of cells with the Ca²⁺ ionophore 4-Br-A23187 induced large Ca²⁺ influx around the entire cell circumference. Despite the nonlocalized increase in [Ca²⁺]_i, secretion evoked by 4-Br-A23187 was still localized to the same region as that evoked by secretagogues such as glucose. It is concluded that Ca²⁺ channels activated by depolarization are localized to specific membrane domains where exocytotic release also occurs; however, localized secretion is not exclusively regulated by localized increases in [Ca²⁺]_i, but instead involves spatial localization of other components of the exocytotic machinery.

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Modeling Ca2+ currents and buffered diffusion of Ca2+ in human β-cells during voltage clamp experiments
2015, Mathematical Biosciences
Citation Excerpt :
The main signal for the release of the RRP granules is the increase of [Ca2+]i, thus the formation of Ca2+ nano and microdomains near the mouth of the VDCCs is extremely important for an adequate secretory response in β-cells. It has been shown that in rodent β-cells, the secretory sites and the VDCCs are colocalized and that both the entry of Ca2+ and the secretion of insulin are limited to a certain region of the plasma membrane [17,24–26]. In contrast to mice β-cells, in which Ca2+ influx is mediated mainly by the L- and R-type Ca2+ channels, the ionic channels responsible for the entry of calcium to the cytosol in human β-cells are the T-, L-, and P/Q-type Ca2+ channels [27] (for recent reviews regarding the electrophysiology of β-cells see Refs. [11,28–30]).
Macroscopic Ca²⁺ currents of the human β-cells were characterized using the Hodgkin–Huxley formalism. Expressions describing the Ca²⁺-dependent inactivation process of the L-type Ca²⁺ channels in terms of the concentration of Ca²⁺ were obtained. By coupling the modeled Ca²⁺ currents to a three-dimensional model of buffered diffusion of Ca²⁺, we simulated the Ca²⁺ transients formed in the immediate vicinity of the cell membrane during voltage clamp experiments performed in high buffering conditions. Our modeling approach allowed us to consider the distribution of the Ca²⁺ sources over the cell membrane. The effect of exogenous (EGTA) and endogenous Ca²⁺ buffers on the temporal course of the Ca²⁺ transients was evaluated. We show that despite the high Ca²⁺ buffering capacity, nanodomains are formed in the submembrane space, where a peak Ca²⁺ concentration between ∼76 and 143 µM was estimated from our simulations. In addition, the contribution of each Ca²⁺ current to the formation of the Ca²⁺ nanodomains was also addressed. Here we provide a general framework to incorporate the spatial aspects to the models of the pancreatic β-cell, such as a more detailed and realistic description of Ca²⁺ dynamics in response to electrical activity in physiological conditions can be provided by future models.
Disruption and stabilization of β-cell actin microfilaments differently influence insulin secretion triggered by intracellular Ca 2+ mobilization or store-operated Ca 2+ entry
2012, FEBS Letters
Citation Excerpt :
This pool of readily releasable granules is small (50–100 ie <1% of the ∼10 000 granules per β-cell) [4–6]. There is evidence that these granules are docked to the plasma membrane, associated with or at least in close proximity of VGCC [7–9] although recent studies using total internal reflection fluorescence microscopy indicate that slightly more distant (non-docked) granules are also releasable and even preferentially exocytosed when Ca2+ influx through VGCC is induced by glucose [10–12]. To sustain insulin secretion, the readily releasable pool must then be constantly refilled by mobilization of granules from other pools [4–6].
Latrunculin depolymerizes and jasplakinolide polymerizes β-cell actin microfilaments. Both increase insulin secretion when Ca²⁺ enters β-cells during depolarization by glucose, sulfonylureas or potassium. Mouse islets were held hyperpolarized with diazoxide, and stimulated with acetylcholine to test the role of microfilaments in insulin secretion triggered by intracellular Ca²⁺ mobilization and store-operated Ca²⁺ entry (SOCE).
Jasplakinolide slightly attenuated Ca²⁺ mobilization and did not affect SOCE, but consistently inhibited the attending insulin secretion. Latrunculin did not affect Ca²⁺ changes induced by acetylcholine, but consistently increased insulin secretion, its effect being larger in response to Ca²⁺ entry than to Ca²⁺ mobilization. Microfilaments have thus a distinct impact on exocytosis of insulin granules depending on the source of triggering Ca²⁺.
Chapter 4 Combining Microfluidics and Quantitative Fluorescence Microscopy to Examine Pancreatic Islet Molecular Physiology
2008, Methods in Cell Biology
Citation Excerpt :
Our strategy to monitor insulin output will entail fluorescent detection of Zn2+, which is bound to insulin in β‐cell secretory granules and co‐secreted with the hormone (Blundell et al., 1972; Emdin et al., 1980; Foster et al., 1993; Gold and Grodsky, 1984). To date, measurement of Zn2+ release from pancreatic β‐cells via fluorescent indicators has been used to examine the spatial organization of the secretion response (Gee et al., 2002; Michael et al., 2006; Qian and Kennedy, 2001; Qian et al., 2003), but these studies were limited to observing a halo of fluorescence around the secreting cells. Because diffusion and turbulence disturb the observation volume, this solution‐based technique is only semi‐quantitative.
Pancreatic islets are functional micro‐organs involved in maintaining normoglycemia through regulated secretion of insulin and other hormones. Extracellular glucose stimulates insulin secretion from islet β‐cells through an increase in metabolic state, which can be measured using two‐photon NAD(P)H imaging. Extracellular glucose concentrations of >7 mM generate synchronous β‐cell calcium (Ca²⁺) oscillations leading to pulsatile insulin secretion. Our studies have focused on the coupling of cells within the islet using quantitative fluorescence imaging of metabolic (NAD(P)H) and electrical (Ca²⁺) responses. This imaging requires immobilization of the tissue to achieve subcellular spatial and subsecond temporal resolutions. We have developed microfluidic devices to stimulate islets while holding them stationary against a glass coverslip. One device is a dual‐channel microfluidic that allows heterogeneous islet stimulation by introducing a standing gradient in glucose concentration across the islet. The second device is a single channel microfluidic with the general utility to hold islets static. This chapter will describe the fabrication and use of these devices, with specific reference to their demonstrated utility for quantitative and dynamic imaging of living pancreatic islets. We will also highlight the general utility of these devices as a paradigm transferable to the study of other tissues.
Timing of Ca2+ response in pancreatic β-cells is related to mitochondrial mass
2006, Biochemical and Biophysical Research Communications
Citation Excerpt :
There was no correlation between the magnitude of responses and the mitochondrial mass in either lean or ob/ob mouse β-cells (r = 0.15 and r = 0.05, NS). The [Ca2+]i rise occurs as localized [Ca2+]i increases within the β-cell [12,13]. We investigated whether areas of localized [Ca2+]i increases were co-located with mitochondria.
The timing and magnitude of calcium response are cell-specific in individual β-cells. This may indicate that the cells have different roles in the intact islet. It is unknown what mechanisms determine these characteristics. We previously found that the mechanisms setting cell-specific response timing are disturbed in β-cells from hyperglycemic mice and one of the causes is likely to be an altered mitochondrial metabolism. Mitochondria play a key role in the control of nutrient-induced insulin secretion. Here, we used confocal microscopy with the fluorescent probe MitoTracker Red CMXRos and Fluo-3 to study how the amount of active mitochondria is related to the lag-time and the magnitude of calcium response to 20 mM glucose in isolated β-cells and in cells within intact lean and ob/ob mouse islets. Results show that the mitochondrial mass is inversely correlated with the lag-times for calcium response both in lean and ob/ob mouse β-cells (r = −0.73 and r = −0.43, respectively, P < 0.05). Thus, the state of mitochondria may determine the timing of calcium response.
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2016, Analytical Chemistry

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¹: To whom correspondence and reprint requests should be addressed. Fax: 352-392-4582. E-mail: [email protected].

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Regular ArticleSpatial Organization of Ca2+ Entry and Exocytosis in Mouse Pancreatic β-Cells

Abstract

FEBS Lett.

J. Biol. Chem.

Cell Calcium

Biochem. Biophys. Res. Commun.

Adv. Protein Chem.

Biophys. J.

J. Biol. Chem.

J. Biol. Chem.

Biophys. J.

Biophys. J.

Stimulus–secretion coupling in pancreatic β-cells

J. Cell Biochem.

Morphological evidence for pancreatic polarity of beta-cell within islets of Langerhans

Diabetes

Localization of the pancreatic beta cell glucose transporter to specific plasma membrane domains

Science

Evidence for polarization of plasma membrane domains in pancreatic endocrine cells

Nature

Co-localization of L-type Ca2+-channels and insulin-containing secretory granules and its significance for the initiation of exocytosis in mouse pancreatic B-cells

EMBO J.

Localized and compound exocytosis detected by spatially resolved amperometry at single pancreatic β-cells

Cell Biochem. Biophys.

Regular Article
Spatial Organization of Ca²⁺ Entry and Exocytosis in Mouse Pancreatic β-Cells

Co-localization of L-type Ca²⁺-channels and insulin-containing secretory granules and its significance for the initiation of exocytosis in mouse pancreatic B-cells