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      Regulated Exocytosis of GABA-containing Synaptic-like Microvesicles in Pancreatic β-cells

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          We have explored whether γ-aminobutyric acid (GABA) is released by regulated exocytosis of GABA-containing synaptic-like microvesicles (SLMVs) in insulin-releasing rat pancreatic β-cells. To this end, β-cells were engineered to express GABA A-receptor Cl -channels at high density using adenoviral infection. Electron microscopy indicated that the average diameter of the SLMVs is 90 nm, that every β-cell contains ∼3,500 such vesicles, and that insulin-containing large dense core vesicles exclude GABA. Quantal release of GABA, seen as rapidly activating and deactivating Cl -currents, was observed during membrane depolarizations from −70 mV to voltages beyond −40 mV or when Ca 2+ was dialysed into the cell interior. Depolarization-evoked GABA release was suppressed when Ca 2+ entry was inhibited using Cd 2+. Analysis of the kinetics of GABA release revealed that GABA-containing vesicles can be divided into a readily releasable pool and a reserve pool. Simultaneous measurements of GABA release and cell capacitance indicated that exocytosis of SLMVs contributes ∼1% of the capacitance signal. Mathematical analysis of the release events suggests that every SLMV contains 0.36 amol of GABA. We conclude that there are two parallel pathways of exocytosis in pancreatic β-cells and that release of GABA may accordingly be temporally and spatially separated from insulin secretion. This provides a basis for paracrine GABAergic signaling within the islet.

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          Most cited references 58

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          Correction for liquid junction potentials in patch clamp experiments.

           E Neher (1991)
          This chapter describes corrections that have to be applied to measured membrane potentials in patch clamp experiments. Some of them [Eqs. (1)-(3)] are required regardless of the nature of the reference electrode (in the Ringer's solution bath) whenever the pipette-filling solution is different from the bath solution. They represent the liquid junction potentials that are present at the pipette tip before patch formation. In addition, corrections have to be applied when the bath solution is being changed during a measurement (i.e., after seal formation). In that case the following rules apply. (1) The new solution should never get into contact with the bare silver/silver chloride wire of the reference electrode. This requirement is best met by using a salt bridge. (2) The "best" salt bridge is a 3 M KCl bridge with an abrupt KCl-bath fluid boundary at its tip (see above). This bridge does not require any additional potential corrections, but it may lead to KCl poisoning of the bath or become contaminated by solutions used previously. (3) Local solution changes (microperfusion by puffer pipette, U tool or sewer pipe arrangements) as well as recessed KCl bridges require additional corrections, which (together with the simple liquid junction potential correction) are approximately given by Eqs. (6)-(8). It should be stressed that all equations given here represent approximate corrections, since liquid junction potentials are thermodynamically ill-defined. This is particularly relevant for Eqs. (6) and (7) where the sum of two liquid junction potentials appears.
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            Glucose-inhibition of glucagon secretion involves activation of GABAA-receptor chloride channels.

            The endocrine part of the pancreas plays a central role in blood-glucose regulation. It is well established that an elevation of glucose concentration reduces secretion of the hyperglycaemia-associated hormone glucagon from pancreatic alpha 2 cells. The mechanisms involved, however, remain unknown. Electrophysiological studies have demonstrated that alpha 2 cells generate Ca2+-dependent action potentials. The frequency of these action potentials, which increases under conditions that stimulate glucagon release, is not affected by glucose or insulin. The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is present in the endocrine part of the pancreas at concentrations comparable to those encountered in the central nervous system, and co-localizes with insulin in pancreatic beta cells. We now describe a mechanism whereby GABA, co-secreted with insulin from beta cells, may mediate part of the inhibitory action of glucose on glucagon secretion by activating GABAA-receptor Cl- channels in alpha 2 cells. These observations provide a model for feedback regulation of glucagon release, which may be of significance for the understanding of the hypersecretion of glucagon frequently associated with diabetes.
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              Islet beta-cell secretion determines glucagon release from neighbouring alpha-cells.

              Homeostasis of blood glucose is maintained by hormone secretion from the pancreatic islets of Langerhans. Glucose stimulates insulin secretion from beta-cells but suppresses the release of glucagon, a hormone that raises blood glucose, from alpha-cells. The mechanism by which nutrients stimulate insulin secretion has been studied extensively: ATP has been identified as the main messenger and the ATP-sensitive potassium channel as an essential transducer in this process. By contrast, much less is known about the mechanisms by which nutrients modulate glucagon secretion. Here we use conventional pancreas perfusion and a transcriptional targeting strategy to analyse cell-type-specific signal transduction and the relationship between islet alpha- and beta-cells. We find that pyruvate, a glycolytic intermediate and principal substrate of mitochondria, stimulates glucagon secretion. Our analyses indicate that, although alpha-cells, like beta-cells, possess the inherent capacity to respond to nutrients, secretion from alpha-cells is normally suppressed by the simultaneous activation of beta-cells. Zinc released from beta-cells may be implicated in this suppression. Our results define the fundamental mechanisms of differential responses to identical stimuli between cells in a microorgan.

                Author and article information

                J Gen Physiol
                The Journal of General Physiology
                The Rockefeller University Press
                March 2004
                : 123
                : 3
                : 191-204
                [1 ]Department of Physiological Sciences, Lund University, BMC B11, SE-22184 Lund, Sweden
                [2 ]Department of Cell and Molecular Biology, Lund University, BMC B11, SE-22184 Lund, Sweden
                [3 ]Lilly Research Laboratories, Lilly Forschung GmbH, D-22419 Hamburg, Germany
                Author notes

                Address correspondence to Matthias Braun, Department of Physiological Sciences, Lund University, BMC B11 SE-22184 Lund, Sweden. Fax: (46) 46-222-7763; email: matthias.braun@

                Copyright © 2004, The Rockefeller University Press

                Anatomy & Physiology

                pancreatic islets, gaba, slmv, gad65, paracrine communication


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