Fig. 5.

Equipping beta cells with the human V1b receptor allows for specific [Ca²⁺]_i signalling in pseudoislets in vitro. (a) Mouse islet cells were transduced for 7 days with an adenovirus encoding for the human V1b receptor (hV1bR) during reaggregation into pseudoislets. After fixation, pseudoislets were co-stained by immunohistochemistry to visualize beta cells and localization of the synthetically expressed receptor. Representative confocal images show an overview of two pseudoislets as maximum intensity projection, and as single optical plane at higher magnification (corresponding region marked with yellow square). The receptor was expressed in beta cells, with a main localization at the plasma membrane. (b–e) Functional responses of non-fixed pseudoislets to AVP stimulation. (b) Non-transduced pseudoislets have a low background level of vasopressin-induced [Ca²⁺]_i increase. (c) The overexpression of hV1bR in pseudoislet beta cells leads to AVP-induced activation of the receptor and rapid [Ca²⁺]_i increase. (d) Quantitative comparison of responses to AVP in terms of [Ca²⁺]_i mobilization demonstrates an increased activation of the hV1bR signalling pathway in transduced as compared to non-transduced pseudoislets (n = 5–7). (e) Groups of pseudoislets were incubated under static conditions in a buffered solution containing 3 mM glucose with or without AVP. Pseudoislets overexpressing the hV1bR responded to AVP with an increase in insulin release, while control pseudoislets showed no response (n = 3, groups of 20 islets). Scale bars = 50 μm and 10 μm for maximum intensity projection and single plane images, respectively. Data presented as mean ± S.E. **p < 0·01; ***p < 0·001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)