Influence of cell number on the characteristics and synchrony of Ca2+ oscillations in clusters of mouse pancreatic islet cells

Abstract

1. The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in single cells and cell clusters of different sizes prepared from mouse pancreatic islets. 2. During stimulation with 15 mM glucose, 20 % of isolated cells were inert, whereas 80 % showed [Ca2+]i oscillations of variable amplitude, duration and frequency. Spectral analysis identified a major frequency of 0.14 min-1 and a less prominent one of 0.27 min-1. 3. In contrast, practically all clusters (2-50 cells) responded to glucose, and no inert cells were identified within the clusters. As compared to single cells, mean [Ca2+]i was more elevated, [Ca2+]i oscillations were more regular and their major frequency was slightly higher (but reached a plateau at approximately 0.25 min-1). In some cells and clusters, faster oscillations occurred on top of the slow ones, between them or randomly. 4. Image analysis revealed that the regular [Ca2+]i oscillations were well synchronized between all cells of the clusters. Even when the Ca2+ response was irregular, slow and fast [Ca2+]i oscillations induced by glucose were also synchronous in all cells. 5. In contrast, [Ca2+]i oscillations resulting from mobilization of intracellular Ca2+ by acetylcholine were restricted to certain cells only and were not synchronized. 6. Heptanol and 18alpha-glycyrrhetinic acid, two agents widely used to block gap junctions, altered glucose-induced Ca2+ oscillations, but control experiments showed that they also exerted effects other than a selective uncoupling of the cells. 7. The results support theoretical models predicting an increased regularity of glucose-dependent oscillatory events in clusters as compared to isolated islet cells, but contradict the proposal that the frequency of the oscillations increases with the number of coupled cells. Islet cell clusters function better as electrical than biochemical syncytia. This may explain the co-ordination of [Ca2+]i oscillations driven by depolarization-dependent Ca2+ influx during glucose stimulation.

Figure 1. Patterns of [Ca²⁺]_i responses in single cells and clusters of cells from mouse pancreatic islets during continuous stimulation with 15 mM glucose

The categories of small and large clusters correspond to 2–4 and 16–50 cells, respectively. The recordings were obtained after 1, 2 or 4 days of culture as indicated. Note that the figure illustrates the different patterns of [Ca²⁺]_i responses but not their relative incidence, which is shown in Fig. 2.

Figure 2. Analysis of [Ca²⁺]_i responses in single cells and clusters of cells from mouse pancreatic islets during continuous stimulation with 15 mM glucose

A, percentage of single cells and clusters showing oscillations or a sustained elevation of [Ca²⁺]_i. The total number of preparations studied (n) is given for each column. B, frequency of the major [Ca²⁺]_i oscillation identified by spectral analysis; means and s.e.m.C, percentage of single cells and clusters showing [Ca²⁺]_i oscillations that were regular in amplitude only, frequency only or both amplitude and frequency. D, average [Ca²⁺]_i calculated over the 30 min period of stimulation, for all single cells and clusters, or for only those showing [Ca²⁺]_i oscillations; means and s.e.m.

Figure 3. Spectral analysis of [Ca²⁺]_i profiles in single cells and clusters of cells from mouse pancreatic islets during continuous stimulation with 15 mM glucose

The curves show mean spectra for all responsive preparations in each group.

Figure 4. Synchrony of [Ca²⁺]_i oscillations in clusters of cells from mouse pancreatic islets during continuous stimulation with 15 mM glucose

The changes in [Ca²⁺]_i were analysed in the cells or cell regions shown by the numbered shaded areas in the drawings of the whole clusters at the top of each panel. A, cluster of 41 cells (4 days) in which the global response was regular. B and C, cell doublet (2 days), and cluster of 10 cells (2 days) with irregular oscillations. D, single cell (2 days) showing both slow and fast oscillations.

Figure 5. Effects of a putative blocker of gap junctions on [Ca²⁺]_i in a single islet cell

A single islet cell was continuously stimulated with 15 mM glucose (G15), and 75 μM 18α-glycyrrhetinic acid (AGA) was added to the medium for the indicated period. Representative of 6 experiments.

Figure 6. Asynchrony of [Ca²⁺]_i changes resulting from intracellular Ca²⁺ mobilization in clusters of cells from mouse pancreatic islets

A cell doublet (2 days) and a cluster of 4 cells (2 days) were perifused with a medium containing 15 mM glucose (G15) throughout. A, diazoxide (Dz, 250 μM) was present throughout, and acetylcholine (ACh, 0.5 μM) was added as indicated. B, diazoxide and acetylcholine were added as indicated. The recording was interrupted for 6 min after addition of diazoxide. The changes in [Ca²⁺]_i were analysed in the cells or cell regions shown by the numbered shaded areas in the drawings of the clusters above each panel. Representative of 7 experiments.