Mitochondrial Participation in the Intracellular Ca2+ Network

Abstract

Calcium can activate mitochondrial metabolism, and the possibility that mitochondrial Ca2+ uptake and extrusion modulate free cytosolic [Ca2+] (Cac) now has renewed interest. We use whole-cell and perforated patch clamp methods together with rapid local perfusion to introduce probes and inhibitors to rat chromaffin cells, to evoke Ca2+ entry, and to monitor Ca2+-activated currents that report near-surface [Ca2+]. We show that rapid recovery from elevations of Cac requires both the mitochondrial Ca2+ uniporter and the mitochondrial energization that drives Ca2+ uptake through it. Applying imaging and single-cell photometric methods, we find that the probe rhod-2 selectively localizes to mitochondria and uses its responses to quantify mitochondrial free [Ca2+] (Cam). The indicated resting Cam of 100–200 nM is similar to the resting Cac reported by the probes indo-1 and Calcium Green, or its dextran conjugate in the cytoplasm. Simultaneous monitoring of Cam and Cac at high temporal resolution shows that, although Cam increases less than Cac, mitochondrial sequestration of Ca2+ is fast and has high capacity. We find that mitochondrial Ca2+ uptake limits the rise and underlies the rapid decay of Cac excursions produced by Ca2+ entry or by mobilization of reticular stores. We also find that subsequent export of Ca2+ from mitochondria, seen as declining Cam, prolongs complete Cac recovery and that suppressing export of Ca2+, by inhibition of the mitochondrial Na+/ Ca2+ exchanger, reversibly hastens final recovery of Cac. We conclude that mitochondria are active participants in cellular Ca2+ signaling, whose unique role is determined by their ability to rapidly accumulate and then release large quantities of Ca2+.

Carbonic anhydrase inhibition selectively prevents amyloid β neurovascular mitochondrial toxicity

“…In addition, uneven distribution of the dye within mitochondria could also occur when using this probe. However, rhodamine‐based fluorescence probes have been extensively used in the literature as a method to assay mitochondrial calcium (Abramov & Duchen, 2003; Babcock, Herrington, Goodwin, Park & Hille, 1997; Boitier, Rea & Duchen, 1999), and we conducted all the experiments accurately and using the appropriate controls.…”

Simultaneous Detection of Blood Volume, Oxygenation, and Intracellular Calcium Changes during Cerebral Ischemia and Reperfusionin vivoUsing Diffuse Reflectance and Fluorescence

“…Minta et al (1989) originally described that Rhod2 accumulate, hydrolyzed and trapped in the mitochondrial compartment. Since then several investigators using fluorescence imaging in single cells preloaded at room temperature have shown that Rhod-2 indeed localized in punctuate and filamentous regions around the nucleus consistent with mitochondrial localization (Babcock et al, 1997;Boitier et al, 1999). Further in chromatoffin cells potassium-induced depolarization induced rapid calcium entry into the mitochondria and increased the Rhod-2 fluorescence within the mitochondria (Babcock et al, 1997).…”

Mitochondria and chromaffin cell function

“…κ S values 1,000 have been reported for myocytes [58] and various neuronal types [1,23,46,54]. On the other hand, values of 40-100 have been reported for rat chromaffin cells [11,29] and bovine chromaffin cells [40,60,63].…”

“…2a). The calciumbinding capacity for Ca 2+ (κ S ) inside the mitochondrial matrix seems to be very high, in the 10 3 range [11,17,29,30]. In addition, it has been proposed recently that precipitation of Ca 3 (PO 4 ) 2 into the matrix con account for additional, almost unlimited, buffering [16,57].…”

“…It has been repeatedly reported that mitochondria can clear cytosolic Ca 2+ loads in rat [11,29,57] and bovine chromaffin cells [36,55,60]. We have studied the kinetics of uptake by mitochondria in permeabilized bovine chromaffin cells infected with mitochondria-targeted aequorin probes [36,55].…”