Ca2+-induced Ca2+ Release in Chromaffin Cells Seen from inside the ER with Targeted Aequorin

Abstract

The presence and physiological role of Ca2+-induced Ca2+ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca2+] ([Ca2+]c). Using targeted aequorin, we have directly monitored [Ca2+] changes inside the ER ([Ca2+]ER) in bovine adrenal chromaffin cells. Ca2+ entry induced by cell depolarization triggered a transient Ca2+ release from the ER that was highly dependent on [Ca2+]ER and sensitized by low concentrations of caffeine. Caffeine-induced Ca2+ release was quantal in nature due to modulation by [Ca2+]ER. Whereas caffeine released essentially all the Ca2+ from the ER, inositol 1,4,5-trisphosphate (InsP3)- producing agonists released only 60–80%. Both InsP3 and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect. Ryanodine induced permanent emptying of the Ca2+ stores in a use-dependent manner after activation by caffeine. Fast confocal [Ca2+]c measurements showed that the wave of [Ca2+]c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells. Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca2+ pool that can release Ca2+ both via InsP3 receptors or CICR.

Calcium dynamics in the secretory granules of neuroendocrine cells

“…Both cADPR and caffeine released also Ca 2+ from the secretory granules of insulin-secreting MIN6 cells [20], and caffeine, but not cADPR, released Ca 2+ from secretory granules in chromaffin cells [23]. The lack of effect of cADPR may reflect an insensitivity to this agonist of the ryanodine receptors in these cells, as cADPR was also unable to release Ca 2+ from the ER in chromaffin cells [7]. The novel agonist nicotinic acid adenine dinucleotidephosphate (NAADP) was also effective to release Ca 2+ from zymogen granules [69], platelet acidic stores [41], insulin-secreting MIN6 cells [70] and the acidic Ca 2+ stores of PC12 cells [71].…”

“…Electron microscopy techniques have shown that the total calcium content of the ER from different cell types is in the range of 5-50 mM, with the highest values in the terminal cisternae of the sarcoplasmic reticulum and mean values of 5-10 mM [2,18], that is, 5-10-fold lower than in the secretory granules. The free [Ca 2+ ] in the ER has been also measured with low-Ca 2+ -affinity aequorin and is in the 500-800 M range [7], so that the Ca 2+ -bound/Ca 2+ -free relationship in the ER is about 10. In addition, the total calcium distribution in the ER is heterogeneous, with strongly positive cisternae (including the nuclear envelope) lying in the proximity of or even in direct continuity with other, apparently negative cisternae [13].…”

“…These vesicles are attached to the plasma membrane and are also very close to portions of ER and mitochondria. Thus, it is generally assumed that the Ca 2+ required for exocytosis may enter from the extracellular medium through several kinds of plasma membrane Ca 2+ channels [6] or be released from the endoplasmic reticulum through either inositol trisphosphate (InsP 3 ) or ryanodine receptors [7]. Nearby mitochondria may also contribute to modulate the size of the local [Ca 2+ ] microdomain responsible for vesicle fusion and secretion.…”

Mitochondria and chromaffin cell function

“…Uptake by SERCA increases exponentially with [Ca 2+ ] C and can reach maximal rates 70-80 μmol · l cells [3,55,60]. At the steady state, the ER fills to a level of 500-1,000 μM [Ca 2+ ].…”

“…On the other hand, organelles interrupt physically the diffusion pathways and make them much longer and tortuous. In addition, many organelles avidly take up Ca 2+ and drain the Ca 2+ wave; on the contrary, other organelles amplify the [Ca 2+ ] C signal by releasing Ca 2+ [3,5,7,55]. In this way high [Ca 2+ ] C microdomains (HCMD) are built at the secretion site whereas [Ca 2+ ] C near the resting level is preserved at the cell core.…”