Calcium- and barium-dependent exocytosis from the rat insulinoma cell line RINm5F assayed using membrane capacitance measurements and serotonin release

Abstract

Electrophysiological measurements of cell capacitance (Cm) and biochemical assays of [3H] serotonin ([3H]5-hydroxytryptamine or [3H]5-HT) release were combined to study the control of secretion in rat insulinoma RINm5F cells. Depolarizing pulses produced Cm changes (DeltaCm), indicative of exocytosis, with the same voltage and Ca2+ dependency as the inward Ca2+ currents (ICa). Ba2+ was able to substitute for Ca2+ in stimulating exocytosis, but not endocytosis. However, both the relative potency and kinetics of Ca2+-versus Ba2+-triggered exocytosis differed significantly. 5-HT synthesis and uptake were demonstrated in RINm5F cells. This allowed the use of [3H]5-HT to study hormone release from cell populations. [3H]5-HT was released in a depolarization-, Ca2+- and time-dependent manner. Ba2+ also substituted for Ca2+ in depolarization-induced [3H]5-HT release. Thapsigargin, used to deplete Ca2+ stores, had no effects on Ca2+-triggered Cm increases, but Ca2+-triggered [3H]5-HT release was abolished. Ba2+-triggered [3H]5-HT release, however, was only slightly affected by Ca2+ store depletion. Ba2+ was found to act directly as a secretagogue of [3H]5-HT in intact cells, but not in Cm measurements of voltage-clamped cells, suggesting that cell depolarization is a prerequisite for this action.

Noradrenaline inhibition of Ca2+ channels and secretion in single patch‐clamped insulinoma cells

“…Despite a relatively stable Ica (Fig. 1, Bi) there was a gradual depression of the secretory response in control cells, possibly due to depletion of readily releasable secretory granules [21,23,26]. The average values for three repetitive stimulations from 15 cells are shown in Fig.…”

“…Furthermore, there is evidence in both nerve terminals [2] and RINm5F cells [35] that not only the hormonal inhibition of release, but also its stimulation, could be achieved by the modulation of distal steps of the secretory process, downstream from second messenger production. We have recently found that while both exocytosis and endocytosis occur following Ca 2+ influx in RINm5F cells, substitution with Ba 2+ preferentially supports exocytosis [23], suggesting that the divalent ion sensitivities of the proteins involved in these two processes are different. Likewise, the observation at the single cell level that the adrenergic inhibition of exocytosis is not accompanied by an inhibition of endocytosis suggests that the proteins targeted by noradrenaline are specific to the exocytotic pathway.…”

“…Ic~ and Cm were measured in the whole-cell patch-clamp configuration [24], as recently described for RINm5F cells [23]. Briefly, data were acquired with an EPC9 amplifier (Instrutech, NY, USA) using HEKA software run on a Macintosh Centris 650.…”

“…This technique has been applied to many cell types including pancreatic beta cells [20][21][22] and, recently, the insulinoma cell line RINm5F [23].…”

3H‐serotonin as a marker of oscillatory insulin secretion in clonal β‐cells (INS‐1)

“…3 H-epinephrine can be loaded into PC12 cells in order to monitor secretion of 3 H-norepinephrine from permeabilized cells [12]. 3 H-serotonin release has been measured from preloaded clonal insulin-secreting RINm5F cells depolarized with KCl, but no correlation with insulin release was attempted [13]. Here, we show that 3 H-serotonin can be loaded into clonal pancreatic b-cells (INS-1) in order to monitor exocytosis from both intact and permeabilized cells validating the use of serotonin as a marker for insulin secretion.…”