Aim: 

Insulin secretory granules of pancreatic b-cells contain high concentration of calcium and other divalent cations. Therefore, it is reasonable to assume that dynamic fluctuations in the extracellular [Ca²⁺] ([Ca²⁺]_ext) close to b-cell plasma membrane are likely the result of insulin exocytosis. Here, by using a direct electrophysiological approach we aimed at measuring these local [Ca²⁺]_ext changes and assess their putative role as paracrine signals, able to stimulate the extracellular calcium sensing receptor (CaR).

Methods: 

Rat insulinoma INS-1E cells grown as spheroids were perfused with diverse agonists/antagonists of insulin exocytotic process. Extracellular [Ca²⁺] changes were recorded in real time using Ca²⁺-selective microelectrodes inserted in restricted domains near the plasma membrane.

Results: 

High glucose stimulation (20 mmol/l) elicited a transient, verapamil-sensitive increase in [Ca²⁺]_ext (D[Ca²⁺]_ext: 1.31±0.17 mmol/l, n=4, p<0.01) that was completely blocked by pretreatment with 10 mmol/l norepinephrine, a well known inhibitor of insulin secretion, and 10 mmol/l cytochalasin D, a blocker of actin polymerization. Significant increases in [Ca²⁺]_ext, were also obtained upon stimulation with the ATP-sensitive potassium channel blocker glibenclamide (10 mmol/l, D[Ca²⁺]_ext: 1.92±0.18 mmol/l, n=5, p<0.01) and the Ca²⁺-mediated agonist ATP (100 mmol/l, D[Ca²⁺]_ext: 0.43±0.14 mmol/l, n=6, p<0.05). Importantly, also the activation of the CaR with 2 mmol/l spermine induced a significant increase in the extracellular [Ca²⁺] (D[Ca²⁺]_ext: 0.17±0.03 mmol/l, n=4, p<0.01).

Conclusions: 

Extracellular spaces within INS-1E spheroids represent a restricted domain where Ca²⁺ released during insulin exocytosis may work as an extracellular messenger able to activate the CaR on neighboring cells and thus initiate/coordinate their secretory response.