Contributions of Intracellular Compartments to Calcium Dynamics: Implicating an Acidic Store

Abstract

Many cells show a plateau of elevated cytosolic Ca 2+ after a long depolarization, suggesting delayed Ca 2+ release from intracellular compartments such as mitochondria and endoplasmic reticulum (ER). Mouse pancreatic b-cells show a thapsigargin-sensitive plateau ('hump') of Ca 2+ after a 30 s depolarization but not after a 10 s depolarization. Surprisingly, this hump depends primarily on compartments other than the mitochondria or ER. It is reduced by only 22% upon blocking mitochondrial Na + -Ca 2+ exchange and by only 18% upon blocking ryanodine or IP 3 receptors together. Further, the time course of ER Ca 2+ measured by a targeted cameleon does not depend on the duration of depolarizations. Instead, the hump is reduced 35% by treatments with the dipeptide glycylphenylalanine b-napthylamide, a tool often used to lyse lysosomes. We show that this dipeptide does not disturb ER functions, but it lyses acidic compartments and releases Ca 2+ into the cytosol. Moreover, it induces leaks in and possibly lyses insulin granules and stops mobilization of secretory granules to the readily releasable pool in b-cells. We conclude that the dipeptide compromises dense-core secretory granules and that these granules comprise an acidic calcium store in b-cells whose loading and/or release is sensitive to thapsigargin and which releases Ca 2+ after cytosolic Ca 2+ elevation.

Contribution of Different Mechanisms to Pancreatic Beta-cell Hyper-secretion in Non-obese Diabetic (NOD) Mice during Pre-diabetes

“…3H). It has been shown previously that depolarization triggered calcium release from endoplasmic reticulum (ER) calcium store as well as Ca 2ϩ influx in primary beta-cells (33)(34). Severe SERCA inhibition leads to partial depletion of ER calcium stores, and possible changes of ER release pathway during diabetes has also been proposed (35).…”

A computational model of lysosome-ER Ca2+ microdomains

“…GPN is a dipeptide substrate for cathepsin C (Jadot et al, 1984) and has been widely used to investigate Ca 2+ signalling through acidic stores in a range of contexts (Berg et al, 1994;Churchill et al, 2002;Duman et al, 2006;Haller et al, 1996;López-Sanjurjo et al, 2013). Hydrolysis of GPN leads to an increase in luminal osmolarity, followed by the ingress of water into cathepsin-C-containing vesicles down the osmotic gradient.…”

Functional stoichiometry of the unitary calcium-release-activated calcium channel

“…4) or a TIRF microscope, as described in ref. 34. EGFP was excited with a 488-nm laser, and Ϸ6 mW laser power was applied to the rear pupil of an oil objective (Zeiss 100ϫ, N.A.…”