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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).
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