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First published online 13 July 2004
doi: 10.1242/jcs.01209

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Glycine, GABA and their transporters in pancreatic islets of Langerhans: evidence for a paracrine transmitter interplay

¹ Anatomical Institute and Centre for Molecular Biology and Neuroscience, University of Oslo, POB 1105 Blindern, N-0317 Oslo, Norway
² Department of Anatomy and Cell Biology, and Locus on Neuroscience, University of Bergen, Årstadveien 19, 5009 Bergen, Norway
³ Centro de Biologia Molecular Severo Ochoa, Facultad de Ciencias, Universidad Autonoma de Madrid, Consejo Superior de Investigaciones Cientificas, Madrid 28049, Spain
⁴ Department of Neurology and Department of Physiology, UCSF School of Medicine, 600 16th Street N272B, San Francisco, CA 94143-2140, USA

View larger version (25K): [in a new window]   Fig. 1. Criteria for recording of immunogold particles as associated with synaptic-like micro vesicles (SLMVs), or secretory granule (SG) limiting membranes (thick contours). Gold particles (and grid points for area determination) with centres within 30 nm of the limiting membranes of SLMVs or SGs were included (grey areas), omitting ones closer to SG dense cores than 15 nm. Particles within 30 nm of the surface of mitochondria (or other, rarer organelles) were excluded from recording (see text).

View larger version (60K): [in a new window]   Fig. 2. GABA (red A,C) is in core cells containing insulin (green B), i.e. B-cells. GABA occurs, at lower concentrations, also in mantle cells (C, arrowheads) co-localized with glucagon (green D, arrowheads), i.e. in A-cells, as shown by double labelling immunofluorescence confocal microscopy. Note lack of labelling (black) of exocrine pancreas. Electron microscopic post-embedding immunolabelling (E) shows a higher concentration of GABA in a B-cell (E top) than in an A-cell (E below). Insets: high magnification examples of SLMVs labelled for GABA, from the two cell types. Quantification (F) of immunogold particles shows the distribution of GABA intracellularly in A- and B-cells (presented as gold particles representing GABA per µm2; background over tissue-free plastic, ca 2 per µm2, subtracted). The SGs and residual cytoplasm (mitochondria etc. excluded) contain GABA at about 6 and 11 times higher concentration in B-cells than in A-cells, respectively (P=0.01, n=3) (F). Further analysis shows (G,H) that SLMVs in the residual cytoplasm also have about 10 times higher particle densities in B-cells than in A-cells (P<0.001, n=4). SLMVs have similar net densities of GABA particles as cytosol both in A-cells and in B-cells (the GABA particle densitiy over cytosol was subtracted from that over SLMVs; cf. Fig. 1 and Materials and Methods). However, as the area of the SLMVs (average diameter 34.1 nm) is much smaller (ca 1/8) than the area over which SLMV associated immunogold particles are distributed, we estimate that the internal SLMV concentration of GABA is about 8 times higher than indicated by the observed gold particles (A-cells P=0.001, B-cells P=0.01, for gross particle densities of SLMVs versus cytosol, n=4). Hence, the GABA concentration inside the SLMVs is nearly an order of magnitude higher in the cytosol in both A- and B-cells (H). Scale bar E: 200 nm (scale for insets: 15 nm gold particles).

View larger version (78K): [in a new window]   Fig. 3. Glycine (red) is found both in core and in mantle cells of the islet (A,B) and is co-localized with glucagon (green) in mantle cells (C). Electron microscopic immungold analysis (D) shows glycine (15 nm gold particles, red arrows) distribution in a B-cell (left) and an A-cell (right, double labelled with 10 nm gold particles representing glucagon, short arrows). Insets: high magnification examples of SLMVs (arrowheads) labelled for glycine in the two cell types. Quantification of immunogold particles (E-G) shows the intracellular distribution of glycine. Note that the numbers cannot be directly compared with those for GABA (Fig. 2), as the labelling efficiencies are not the same. The densities (number of 15 nm gold particles per µm2, background over tissue-free plastic, ca 2 per µm2, subtracted) of glycine immunoreactivity over SGs and residual cytoplasm (free of mitochondria and SGs), show that glycine occurs at higher concentrations in SGs than in the residual cytoplasm (P=0.05, n=5) both in A- and in B-cells (E). (F) In A-cells, SLMVs have higher net densities of glycine particles than cytosol, whereas in B-cells, cytosol has a higher net labelling density than SLMVs (subtraction as in Fig. 2G; P=0.005 for gross particle densities, n=4, for both A- and B-cells). However, owing to the small diameter of the SLMVs compared with the diameter in which the glycine gold particles are observed, the concentration of glycine inside A-cell SLMVs is about 15 times higher than in cytosol, whereas the concentration inside B-cell SLMVs is about 5 times higher than in cytosol (A greater than B at P=0.002) (G). Scale bar D: 200 nm (scale for insets: 15 nm gold particles).

View larger version (74K): [in a new window]   Fig. 4. VGAT (red A,B,D,F) co-localizes with glucagon (green C) in A-cells. Insulin-positive (green A) and GABA-positive (green D) core cells, are very slightly labelled for VGAT (red D). Synaptophysin (green E) is in both A- and B-cells. Electron microscopic immunogold labelling with three different VGAT antibodies (G,H,I) shows strong signal in the SGs of A-cells (G,H,I) but not B-cells (G). The identity of the A-cell SGs is confirmed by double labelling (H) with 10 nm gold particles for glucagon and 15 nm particles (red arrows) for VGAT. Insets: higher power photographs of SLMVs (arrowheads) labelled for VGAT, from the two cell types. Electronmicroscopic quantification (J) shows that VGAT immunogold density over A-cell SGs is >10-fold higher than that over B-cell SGs, or those over mitochondria or cytoplasm of either A- or B-cells (P<0.01). Further analysis (K,L) of the distribution of VGAT was conducted as shown in Fig. 1 (see also Materials and Methods). The density of VGAT immunogold associated with the membrane of SGs (K) is only slightly higher in A-cells than in the B-cells, but is significantly higher than cytosol background (P=0.01 in both A- and B-cells, n=5). Analysis of the distribution of VGAT in the residual cytoplasm (free of SGs and mitochondria) shows a many times higher density of VGAT immunogold associated with SLMVs than over cytosol (L) (P<0.001 in A-cells, P=0.007 in B-cells, n=5), which is close to the background noise over tissue-free plastic (2-3 part/µm2, not subtracted). SLMV-associated VGAT relative to cytosol background is slightly higher in A-cells than in B-cells (P=0.008, n=5). Scale bars G-I: 200 nm (scale for insets: 15 nm gold particles).

View larger version (82K): [in a new window]   Fig. 5. The GABA transporter GAT3 (red A) is expressed at similar levels in core B-cells and mantle A-cells, identified by double labelling for glucagon (green B). GAT3 is concentrated in intracellular compartments.

View larger version (85K): [in a new window]   Fig. 6. The glycine transporter GLYT2 (red B) co-localizes with glucagon (green A, with blue nuclei) in A-cells of islets of Langerhans. There is also a low signal in core B-cells, whereas exocrine cells (blue nuclei, e.g. at upper corners of frame) have hardly any GLYT2 signal.