Abstract
Connexin36 (Cx36) plays an important role in insulin secretion by controlling the intercellular synchronization of Ca2+ transients induced during stimulation. The lack of drugs acting on Cx36 channels is a major limitation in further unraveling the molecular mechanism underlying this effect. To screen for such drugs, we have developed an assay allowing for a semi-automatic, fluorimetric quantification of Ca2+ transients in large populations of MIN6 cells. Here, we show that (1) compared to control cells, MIN6 cells with reduced Cx36 expression or function showed decreased synchrony of glucose-induced Ca2+ oscillations; (2) glibenclamide, a sulphonylurea which promotes Cx36 junctions and coupling, increased the number of synchronous MIN6 cells, whereas quinine, an antimalarial drug which inhibits Cx36-dependent coupling, decreased this proportion; (3) several drugs were identified that altered the intercellular Ca2+ synchronization, cell coupling and distribution of Cx36; (4) some of them also affected insulin content. The data indicate that the intercellular synchronization of Ca2+ oscillations provides a reliable and non-invasive measurement of Cx36-dependent coupling, which is useful to identify novel drugs affecting the function of β-cells, neurons, and neuron-related cells that express Cx36.
Highlights
In vertebrate tissues, several mechanisms cross talk in a complex network to ensure intercellular coordination
By stable transfection of an antisense mCx36-cDNA, we generated a clone of MIN6 cells (AS MIN6) which expresses significantly reduced levels (,20% of controls) of Cx36 protein (Fig. 1 A and B), and normal distribution of the GLUT-2, a transporter essential for glucose uptake during the insulin secretion process (Fig. 1 B)
We observed that, in contrast to wild type controls, which show synchronous Ca2+ oscillations when stimulated by 20 mM glucose and 15 mM TEA, most AS MIN6 cells featured either asynchronous or nil Ca2+ oscillations (Fig. S1)
Summary
Several mechanisms cross talk in a complex network to ensure intercellular coordination. The b-cells of transgenic mice lacking Cx36 are sensitized to the effects of streptozotocin, alloxan and cytokines that are implicated in the development of type 1 diabetes, whereas transgenic mice overexpressing this connexin are protected against the same conditions [11,12]. These findings indicate that Cx36-dependent signaling plays a physiologically relevant role in the regulation of b-cell function/survival, which may be involved in the development and/or maintenance of the b-cell dysfunctions leading to diabetes
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