Activation of calcium-ion (Ca²⁺) channels on the plasma membrane and on intracellular Ca²⁺ stores, such as the endoplasmic reticulum, generates local transient increases in the cytosolic Ca²⁺ concentration that induce Ca²⁺ uptake by neighbouring mitochondria. Here, by using mitochondrially targeted aequorin proteins with different Ca²⁺ affinities, we show that half of the chromaffin-cell mitochondria exhibit surprisingly rapid millimolar Ca²⁺ transients upon stimulation of cells with acetylcholine, caffeine or high concentrations of potassium ions. Our results show a tight functional coupling of voltage-dependent Ca²⁺ channels on the plasma membrane, ryanodine receptors on the endoplasmic reticulum, and mitochondria. Cell stimulation generates localized Ca²⁺ transients, with Ca²⁺ concentrations above 20–40 µM, at these functional units. Protonophores abolish mitochondrial Ca²⁺ uptake and increase stimulated secretion of catecholamines by three- to fivefold. These results indicate that mitochondria modulate secretion by controlling the availability of Ca²⁺ for exocytosis.

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.

We have studied the mechanism of the regulation of plasma membrane Ca2+ permeability by the degree of filling of the intracellular Ca2+ stores. Using Mn2+ as a Ca2+ surrogate for plasma membrane Ca2+ channels, we found that Mn2+ uptake by rat thymocytes is inversely related to the degree of filling of the intracellular Ca2+ stores. This store-dependent plasma membrane permeability is inhibited by oxygen scavenging, CO, imidazole antimycotics and other cytochrome P-450 inhibitors. The pattern of inhibition is similar to that reported previously for the inhibition of microsomal cytochrome P-450-mediated aryl hydrocarbon hydroxylase activity of lymphocytes. Several calmodulin antagonists, both phenothiazinic (trifluoperazine, fluphenazine and chlorpromazine) and dibenzodiazepinic (clozapine), accelerate Mn2+ uptake by cells with Ca2(+)-filled stores, and this effect is prevented by imidazole antimycotics. Our results suggest that cytochrome P-450 may be the link between the stores and the plasma membrane Ca2+ pathway. We propose a model in which this cytochrome, sited at the stores, stimulates plasma membrane Ca2+ influx. This stimulatory effect is, in turn, prevented by the presence of Ca2+ inside the stores, possibly via a calmodulin-dependent mechanism.

During cell activation, mitochondria play an important role in Ca²⁺ homoeostasis due to the presence of a fast and specific Ca²⁺ channel in its inner membrane, the mitochondrial Ca²⁺ uniporter. This channel allows mitochondria to buffer local cytosolic [Ca²⁺] changes and controls the intramitochondrial Ca²⁺ levels, thus modulating a variety of phenomena from respiratory rate to apoptosis. We have described recently that SB202190, an inhibitor of p38 MAPK (mitogen-activated protein kinase), strongly activated the uniporter. We show in the present study that a series of natural plant flavonoids, widely distributed in foods, produced also a strong stimulation of the mitochondrial Ca²⁺ uniporter. This effect was of the same magnitude as that induced by SB202190 (an approx. 20-fold increase in the mitochondrial Ca²⁺ uptake rate), developed without measurable delay and was rapidly reversible. In intact cells, the mitochondrial Ca²⁺ peak induced by histamine was also largely increased by the flavonoids. Stimulation of the uniporter by either flavonoids or SB202190 did not require ATP, suggesting a direct effect on the uniporter or an associated protein which is not mediated by protein phosphorylation. The most active compound, kaempferol, increased the rate of mitochondrial Ca²⁺ uptake by 85±15% (mean±S.E.M., n=4) and the histamine-induced mitochondrial Ca²⁺ peak by 139±19% (mean±S.E.M., n=5) at a concentration of 1 μM. Given that flavonoids can reach this concentration range in plasma after ingestion of flavonoid-rich food, these compounds could be modulating the uniporter under physiological conditions.

We have measured the [Ca²⁺] in the endoplasmic reticulum ([Ca²⁺]_er) of intact HeLa cells at both 22 °C and 37 °C using endoplamsic reticulum-targeted, low Ca²⁺ affinity aequorin reconstituted with coelenterazine n. Aequorin consumption was much slower at 22 °C, and this allowed performing a much longer study of the dynamics of [Ca²⁺]_er. The steady-state [Ca²⁺]_er (500–600 μm) was not modified by the temperature, although both the rates of pumping and leak were decreased at 22 °C. The behavior of both [Ca²⁺]_er and cytoplasmic [Ca²⁺] ([Ca²⁺]_c) after the addition of increasing concentrations of agonists and/or Ca²⁺-ATPase inhibitors, or following incubation in Ca²⁺-free medium were compared. We show that agonists induce a fast but relatively small decrease in [Ca²⁺]_er, which is enough to produce a sharp increase in [Ca²⁺]_c. Termination of Ca²⁺ release is controlled by feedback inhibition of the inositol 1,4,5-trisphosphate receptors by [Ca²⁺]_c, a mechanism that appears to be designed to release the minimum amount of Ca²⁺ necessary to produced the required [Ca²⁺]_c signal. We also show that Ca²⁺ release is inhibited progressively when [Ca²⁺]_er decreases below a threshold of about 150 μm, even in the absence of Ca²⁺ pumping or [Ca²⁺]_c increase. This effect is consistent with a regulation of the inositol 1,4,5-trisphosphate-gated channels by [Ca²⁺]_er.

Emptying of the intracellular calcium stores of human neutrophils, by prolonged incubation in Ca(2+)-free medium, by treatment with low concentrations of the Ca2+ inophore ionomycin, or by activation with cell agonists, increased the plasma-membrane permeability to Ca2+ and Mn2+. The chemotactic peptide formylmethionyl-leucyl-phenylalanine and the natural agonists platelet-activating factor and leukotriene B4 released different amounts of calcium from the stores and induced Ca2+ (Mn2+) uptake, the rate of which correlated inversely with the amount of calcium left in the stores. The increased Mn2+ uptake induced by these agonists was persistent in cells incubated in Ca(2+)-free medium, but returned to basal levels in cells incubated in Ca(2+)-containing medium, with the same time course as the refilling of the calcium stores. The calcium-stores-regulated Mn2+ influx, including that induced by agonists, was prevented by cytochrome P-450 inhibitors. We propose that agonist-induced Ca2+ (Mn2+) influx in human neutrophils is secondary to the emptying of the intracellular stores which, in turn, activates plasma-membrane Ca2+ channels by a mechanism involving microsomal cytochrome P-450, similar to that described previously in thymocytes [Alvarez, Montero & Garcia-Sancho (1991) Biochem. J. 274, 193-197].