In the presence of high glucose or pyruvate, INS-1 832/13 insulinoma cells undergo stochastic oscillations in plasma membrane potential (Δψp) leading to associated fluctuations in cytosolic free Ca(2+) concentration ([Ca(2+)]c). Oscillations are not driven by upstream metabolic fluctuations, but rather by autonomous ionic mechanisms, the details of which are unclear. We have investigated the nature of the oscillator, with simultaneous fluorescence monitoring of Δψp, [Ca(2+)]c and exocytosis at single-cell resolution, combined with analysis of the occurrence, frequency and amplitude of Δψp oscillations. Oscillations were closely coupled to exocytosis, indicated by coincident synaptopHluorin fluorescence enhancement. L-type Ca(2+) channel inhibitors enhanced Δψp and [Ca(2+)]c oscillation frequency in the presence of pyruvate, but abolished the sustained [Ca(2+)]c response following KCl depolarization. The L-type Ca(2+) channel inhibitor isradipine did not inhibit oscillation-linked exocytosis. The T-type Ca(2+) channel inhibitor NNC 55-0396 inhibited Δψp and [Ca(2+)]c oscillations, implying that T-type Ca(2+) channels trigger oscillations and consequent exocytosis. Since distinct ion channels operate in oscillating and non-oscillating cells, quantitative analysis of Δψp and [Ca(2+)]c oscillations in a β-cell population may help to improve our understanding of the link between metabolism and insulin secretion.
Keywords: bioenergetics; calcium; exocytosis; fluorescence; membrane transport.
© 2015 Authors; published by Portland Press Limited.