Inmaculada Cuchillo-Ibáñez

Chromaffin-cell stimulation triggers fast millimolar mitochondrial Ca2+ transients that modulate secretion.

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.

Greater diversity than previously thought of chromaffin cell Ca2+ channels, derived from mRNA identification studies

Using reverse transcription followed by PCR amplification (RT‐PCR), we have identified multiple messenger RNAs encoding for the neuronal pore‐forming Ca2+ channel subunits α1A (P/Q channel), α1B (N channel), α1D (neuronal/endocrine L channel), α1E (R channel), α1G‐H (T channel) and α1S (skeletal muscle L channel) in bovine chromaffin cells. mRNAs for the auxiliary β2, β3, β4, α2/δ and γ2 subunits were also identified. In agreement with these molecular data, perforated patch‐clamp recordings of whole‐cell Ca2+ currents reveal the existence of functional R‐type Ca2+ channels in these cells that were previously undetected with other techniques. Our results provide a molecular frame for a much wider functional diversity of Ca2+ channels in chromaffin cells than that previously established using pharmacological and electrophysiological approaches.

Mitochondrial calcium sequestration and protein kinase C cooperate in the regulation of cortical F-actin disassembly and secretion in bovine chromaffin cells

Mitochondria play an important role in the homeostasis of intracellular Ca2+ and regulate its availability for exocytosis. Inhibitors of mitochondria Ca2+ uptake such as protonophore CCCP potentiate the secretory response to a depolarizing pulse of K+. Exposure of cells to agents that directly (cytochalasin D, latrunculin B) or indirectly (PMA) disrupt cortical F‐actin networks also potentiate the secretory response to high K+. The effects of cytochalasin D and CCCP on secretion were additive whereas those of PMA and CCCP were not; this suggests different mechanisms for cytochalasin D and CCCP and a similar mechanism for PMA and CCCP. Mitochondria were the site of action of CCCP, because the potentiation of secretion by CCCP was observed even after depletion of Ca2+ from the endoplasmic reticulum. CCCP induced a small increase in the cytosolic Ca2+ concentration ([Ca2+]c) that was not modified by the protein kinase C (PKC) inhibitor chelerythrine. Both CCCP and PMA induced cortical F‐actin disassembly, an effect abolished by chelerythrine. In addition, rotenone and oligomycin A, two other mitochondrial inhibitors, also evoked cortical F‐actin disassembly and potentiated secretion; again, these effects were blocked by chelerythrine. CCCP also enhanced the phosphorylation of PKC and myristoylated alanine‐rich C kinase substance (MARCKS), and these were also inhibited by chelerythrine. The results suggest that the rapid sequestration of Ca2+ by mitochondria would protect the cell from an enhanced PKC activation and cortical F‐actin disassembly, thereby limiting the magnitude of the secretory response.

Effect of inositol 1,4,5-trisphosphate receptor stimulation on mitochondrial [Ca2+] and secretion in chromaffin cells

Ca(2+) uptake by mitochondria is a potentially important buffering system able to control cytosolic [Ca(2+)]. In chromaffin cells, we have shown previously that stimulation of either Ca(2+) entry or Ca(2+) release via ryanodine receptors triggers large increases in mitochondrial [Ca(2+)] ([Ca(2+)](M)) approaching the millimolar range, whose blockade dramatically enhances catecholamine secretion [Montero, Alonso, Carnicero, Cuchillo-Ibañez, Albillos, Garcia, Carcia-Sancho and Alvarez (2000) Nat. Cell Biol. 2, 57-61]. In the present study, we have studied the effect of stimulation of inositol 1,4,5-trisphosphate (InsP(3)) receptors using histamine. We find that histamine produces a heterogeneous increase in [Ca(2+)](M), reaching peak levels at approx. 1 microM in 70% of the mitochondrial space to several hundred micromolar in 2-3% of mitochondria. Intermediate levels were found in the rest of the mitochondrial space. Single-cell imaging experiments with aequorin showed that the heterogeneity had both an intercellular and a subcellular origin. Those mitochondria responding to histamine with increases in [Ca(2+)](M) much greater than 1 microM (30%) were the same as those that also responded with large increases in [Ca(2+)](M) following stimulation with either high-K(+) medium or caffeine. Blocking mitochondrial Ca(2+) uptake with protonophores or mitochondrial inhibitors also enhanced catecholamine secretion induced by histamine. These results suggest that some InsP(3) receptors tightly co-localize with ryanodine receptors and voltage-dependent Ca(2+) channels in defined subplasmalemmal functional units designed to control secretion induced by different stimuli.

Calcium Entry, Calcium Redistribution, and Exocytosis

Abstract: At a given cytosolic domain of a chromaffin cell, the rate and amplitude of the Ca2+ concentration, [Ca2+]c, depend on at least three efficient regulatory mechanisms: (1) the plasmalemmal Ca2+ channels; (2) the endoplasmic reticulum (ER); and (3) the mitochondria. High‐voltage activated Ca2+ channels of the L, N, P/Q, and R subtypes are expressed with different densities in various mammalian species; they are regulated by G proteins coupled to purinergic and opiate receptors, as well as by voltage and the local changes of [Ca2+]c. Targeted aequorin and confocal microscopy show that Ca2+ entry through Ca2+ channels can refill the ER to near millimolar concentrations and causes the release of ER Ca2+ (CICR). We have also seen that, depending on its degree of filling, the ER may act as a sink or source of Ca2+ that modulates the release of catecholamine. Targeted aequorins with different Ca2+ affinities show that mitochondria undergo surprisingly rapid millimolar Ca2+ transients ([Ca2+]M) upon stimulation of chromaffin cells with ACh, high K+, or caffeine. Physiological stimuli generate [Ca2+]c microdomains at these functional complexes in which the local subplasmalemmal [Ca2+]c rises abruptly from 0.1 μM to about 50 μM. This triggers CICR, mitochondrial Ca2+ uptake, and exocytosis in nearby secretory active sites. That this is true is shown by the observation that protonophores abolish mitochondrial Ca2+ uptake and drastically increase catecholamine release by 3‐ to 5‐fold. This increase is likely due to acceleration of vesicle transport from a reserve pool to a ready‐release vesicle pool; such transport might be controlled by Ca2+ redistribution to the cytoskeleton, through CICR and/or mitochondrial Ca2+ release.

Control of secretion by mitochondria depends on the size of the local [Ca2+] after chromaffin cell stimulation

In chromaffin cells, plasma membrane calcium (Ca2+) channels and mitochondria constitute defined functional units controlling the availability of Ca2+ nearby exocytotic sites. We show here that, when L‐/N‐type Ca2+ channels were inhibited with nisoldipine and ω‐conotoxin GVIA, cytosolic [Ca2+] ([Ca2+]c) peaks measured in fura‐4F‐loaded cells were reduced by 36%; however, mitochondrial Ca2+ uptake was unaffected and secretion was potentiated by protonophores as in control cells. By contrast, when non L‐type Ca2+ channels were inhibited with ω‐conotoxin MVIIC, [Ca2+]c peaks induced by high K+ were reduced by 73%, mitochondrial Ca2+ uptake was abolished, and secretion was not modified by protonophores. However, if Ca2+ entered only through L‐type channels activated by FPL64176, high K+ stimulation induced fast mitochondrial Ca2+ uptake and catecholamine secretion was strongly increased and potentiated by protonophores. These results confirm the close association of catecholamine secretion to mitochondrial Ca2+ uptake, and indicate the sharp threshold of local [Ca2+]c (about 5 µm) required for triggering fast mitochondrial Ca2+ uptake that is able to modulate secretion. The entry of Ca2+ through L‐type channels generated local [Ca2+]c increases just below that, inducing little mitochondrial Ca2+ uptake unless FPL64176 was present. By contrast, Ca2+ entry through P/Q‐type channels fully activated mitochondrial Ca2+ uptake. Control of secretion by mitochondria therefore depends critically on the ability of the stimulus to create large local [Ca2+]c microdomains.

A preferential pole for exocytosis in cultured chromaffin cells revealed by confocal microscopy

Histological studies suggest that adrenal medulla chromaffin cells in situ are polarised, but functional evidence is lacking. We present here the first demonstration for polarisation of exocytosis in isolated, spherical, bovine chromaffin cells. Cells were stimulated with 70 mM K+ to cause a marked enhancement of catecholamine release, monitored amperometrically. FM1‐43 and dopamine β‐hydroxylase antibodies provided fluorescence confocal pictures that were 2–3‐fold more intense in the bottom of the cells, as compared to equatorial or apex planes. This suggests that the solid phase to which the cell attaches serves as a ‘trophic’ signal for the polarisation of its secretory apparatus.

Altered regulation of calcium channels and exocytosis in single human pheochromocytoma cells

We established primary cultures of human pheochromocytoma chromaffin cells. We then tried to find what mechanism of their secretory apparatus could be altered to produce the massive release of catecholamines into the circulation and the subsequent hypertensive crisis observed in patients suffering this type of tumor. Their whole-cell Ca2+ channel currents could be pharmacologically separated into components similar to those found in normal human adrenal chromaffin cells: 20% L-type, 30% N-type, and 50% P/Q-type Ca2+ channels. However, modulation of the channels by exogenous or endogenous ATP and opioids, via a G-protein membrane-delimited pathway, was deeply altered; some cells having no modulation or very little modulation alternated with others having normal modulation. This may be the cause of the uncontrolled secretory response, measured amperometrically at the single-cell level. Some cells secreted for long time periods and were insensitive to nifedipine (L-type channel blocker) or to ω-conotoxin MVIIC (N/P/Q-type channel blocker), while others were highly sensitive to nifedipine and partially sensitive to ω-conotoxin MVIIC. Alteration of the autocrine/paracrine modulation of Ca2+ channels may lead to indiscriminate Ca2+ entry and exacerbate catecholamine release responses in human pheochromocytoma cells.