Mayte Montero

Cytochrome P450 may regulate plasma membrane Ca2+ permeability according to the filling state of the intracellular Ca2+ stores.

The filling state of the intracellular Ca2+ stores of rat thymocytes regulates plasma membrane permeability to Mn2+, used here as a Ca2+ surrogate for plasma membrane Ca2+ channels. Emptying of the Ca2+ stores accelerated Mn2+ entry about 10‐fold, and refilling with Ca2+ restored low Mn2+ permeability. The acceleration of Mn2+ entry observed in cells with empty intracellular Ca2+ stores was prevented by cytochrome P450 inhibitors. Imidazole antimycotics, especially econazole and miconazole, were the most potent inhibitors (IC50 ≌ 10–6 m). The inhibitor sensitivity profile was similar to IA‐type cytochrome P450. Calmodulin antagonists increased the plasma membrane permeability to Mn2+ in cells with filled Ca2+ stores, and this effect was also blocked by imidazole antimycotics. On this basis, we propose a model in which activation of a cytochrome P450, situated at the Ca2+ stores, opens a plasma membrane Ca2+ pathway. This activity would be inhibited by Ca2+ inside the stores by a calmodulin‐dependent mechanism.—Alvarez, J.; Montero, M.; Garcia‐Sancho, J. Cytochrome P450 may regulate plasma membrane Ca2+ permeability according to the filling state of the intracellular Ca2+ stores. FASEB J. 6: 786‐792; 1992.

[Ca2+] microdomains control agonist-induced Ca2+ release in intact HeLa cells.

We have monitored specifically the [Ca2+] in the lumen of the endoplasmic reticulum (ER) of intact HeLa cells using an ER‐targeted low‐Ca2+‐affinity aequorin. The steady‐state [Ca2+] in the ER was around 600 μM. Histamine induced a concentration‐dependent decrease in lumenal [Ca2+], whose rate increased near one order of magnitude and became “quantal” when cytosolic [Ca2+] ([Ca2+]c) was clamped with the Ca2+ chelator BAPTA. This effect was not due to decreased Ca2+ pumping because simultaneous addition of a SERCA inhibitor produced only additive effects. Given that inhibition by [Ca2+]c of the inositol 1,4,5‐trisphosphate‐gated channels requires a [Ca2+]c much higher than that observed in the bulk cytosol after histamine addition, we conclude that local [Ca2+]c microdomains at the site of release strongly inhibit agonist‐induced Ca2+ mobilization in intact cells. This effect should play a key role in the mechanism controlling cytosolic [Ca2+] oscillations and waves, and therefore in the generation of spatio‐temporal Ca2+ patterns.—Montero, M., Barrero, M. J., Alvarez, J. [Ca2+] Microdomains control agonist‐induced Ca2+ release in intact HeLa cells. FASEB J. 11, 881–885 (1997)

Comparative effects of cytochrome P-450 inhibitors on Ca2+ and Mn2+ entry induced by agonists or by emptying the Ca2+ stores of human neutrophils

The effects of cytochrome P-450 inhibitors of different chemical structures, including several imidazole antimycotics, SKF525A, 5,8,11,14-eicosatetraynoic acid (ETYA), gossypol and nordihydroguaiaretic acid (NDGA), were tested on the entry of Ca2+ and Mn2+ induced either by emptying the intracellular Ca2+ stores with thapsigargin or by stimulation with platelet activating factor (PAF). Most of the drugs inhibited thapsigargin-induced Ca2+ and Mn2+ entry with the same affinity, with the striking exceptions of econazole and miconazole, which were 5- and 2-fold more potent to inhibit the thapsigargin-induced Mn2+ entry than to inhibit Ca2+ entry, respectively. Additionally, high doses of every drug (3-10-times the Ki) activated a pathway permeable to Mn2+ and Ni2+ but not to Ca2+. These findings indicate that Mn2+ entry data should be interpreted with caution and always be cross-checked with Ca2+ uptake measurements. Most of the drugs inhibited PAF-induced Mn2+ uptake with an affinity similar to that found for thapsigargin-induced Mn2+ uptake. PAF- and thapsigargin-induced Ca2+ uptake were also inhibited similarly by NDGA, SKF525A and gossypol, but PAF-induced Ca(2+)-uptake was inhibited about 5-fold more strongly by econazole and ETYA and two-fold more strongly by miconazole and clotrimazole. These findings suggest that the Ca2+/Mn2+ entry pathway opened by agonists in human neutrophils is the same that activates on emptying the Ca2+ stores and that cytochrome P-450 activity may be involved en the activation of the channels.

The pathway for refilling intracellular Ca2+ stores passes through the cytosol in human leukaemia cells

The pathway for refilling the intracellular Ca2+ stores of HL60 and U937 human leukaemia cells loaded with fura-2 has been investigated. On addition of external Ca2+ to cells with empty stores there was an increase in the cytosolic Ca2+ concentration ([Ca2+]i) which preceded the refilling of the stores. The increase in [Ca2+]i was faster than the refilling, by 3-to 15-fold, depending on the cell type. In measurements in single HL60 cells we found that the refilling of the stores correlated with the extent of the [Ca2+]i increase on addition of external Ca2+. The cells showing no [Ca2+]i increase were unable to refill their stores. The addition of Ni2+ to the extracellular medium prevented both the [Ca2+]i increase and the refilling of the stores. These results indicate that the limiting step for store refilling is the entry of Ca2+ from the extracellular medium to the cytosol. Hence, we conclude that extracellular Ca2+ cannot gain access directly to the intracellular Ca2+ stores in these cells, but must first enter the cytosol and be taken up from there into the stores.

Modulation of Calcium Entry by Mitochondria

The role of mitochondria in intracellular Ca(2+) signaling relies mainly in its capacity to take up Ca(2+) from the cytosol and thus modulate the cytosolic [Ca(2+)]. Because of the low Ca(2+)-affinity of the mitochondrial Ca(2+)-uptake system, this organelle appears specially adapted to take up Ca(2+) from local high-Ca(2+) microdomains and not from the bulk cytosol. Mitochondria would then act as local Ca(2+) buffers in cellular regions where high-Ca(2+) microdomains form, that is, mainly close to the cytosolic mouth of Ca(2+) channels, both in the plasma membrane and in the endoplasmic reticulum (ER). One of the first targets proposed already in the 1990s to be regulated in this way by mitochondria were the store-operated Ca(2+) channels (SOCE). Mitochondria, by taking up Ca(2+) from the region around the cytosolic mouth of the SOCE channels, would prevent its slow Ca(2+)-dependent inactivation, thus keeping them active for longer. Since then, evidence for this mechanism has accumulated mainly in immunitary cells, where mitochondria actually move towards the immune synapse during T cell activation. However, in many other cell types the available data indicate that the close apposition between plasma and ER membranes occurring during SOCE activation precludes mitochondria from getting close to the Ca(2+)-entry sites. Alternative pathways for mitochondrial modulation of SOCE, both Ca(2+)-dependent and Ca(2+)-independent, have also been proposed, but further work will be required to elucidate the actual mechanisms at work. Hopefully, the recent knowledge of the molecular nature of the mitochondrial Ca(2+) uniporter will allow soon more precise studies on this matter.

Ca2+ Measurement with Luminescent Probes in the Endoplasmic Reticulum

The photoprotein aequorin can be used to measure specifically the [Ca2+] inside the lumen of the endoplasmic reticulum ([Ca2+ ER) of intact cells. The main advantage of this technique is the highly precise targeting of the probe. The main disadvantage relies in the relatively low amount of emitted light, which makes it difficult to perform single-cell imaging studies at the required time resolution. On the technical side, the equipment required for luminescence measurements in cell populations is simple and low-cost, but expression of ER-targeted aequorin requires previous transfection or infection to introduce the appropriate DNA plasmid, or alternatively the use of stable cell clones. Finally, calibration of the luminescent records to obtain [Ca2+] values is relatively easy, but interpretation of the results requires taking into account that aequorin is being rapidly consumed during the experiment due to the high levels of Ca2+ (hundreds of micromolar) present in the ER.

Subcellular Ca2+ Dynamics Measured with Targeted Aequorin in Chromaffin Cells

Abstract: In the last years, intracellular organella have emerged as key components in the generation and transduction of Ca2+ signals in adrenal chromaffin cells. Therefore, accurate measurements of Ca2+ inside cytoplasmic organella are essential for a comprehensive analysis of the Ca2+ redistribution that follows cell stimulation. We have engineered the Ca2+‐sensitive photoprotein aequorin to monitor selectively Ca2+ within the endoplasmic reticulum and the mitochondria. The targeted aequorins were delivered to the appropriate organelles of bovine chromaffin cells by using a herpes simplex virus‐based amplicon vector, which permits efficient gene transfer and high levels of expression in infected cells. We have investigated the relationship between the caffeine and InsP3‐sensitive Ca2+ pools and the presence of the Ca2+‐induced Ca2+ release (CICR) mechanism in chromaffin cells. We find that ER Ca2+ pools responding to caffeine and to InsP3 mostly overlap and that CICR can be induced by Ca2+ entry elicited by high K+ depolarization. Moreover, the activation of Ca2+ channels, either the voltage‐gated Ca2+ channels on the plasma membrane or the channels on the endoplasmic reticulum (ER), generates subplasmalemmal high [Ca2+]c domains that induce Ca2+ uptake by mitochondria. Interestingly, only a subpopulation of mitochondria, the one contained in the pool located close to the plasma membrane and the ryanodine receptors, take up Ca2+ efficiently, and the [Ca2+]M reaches values of 300‐500 μM.

Functional Characterization of Three Concomitant MtDNA LHON Mutations Shows No Synergistic Effect on Mitochondrial Activity

The presence of more than one non-severe pathogenic mutation in the same mitochondrial DNA (mtDNA) molecule is very rare. Moreover, it is unclear whether their co-occurrence results in an additive impact on mitochondrial function relative to single mutation effects. Here we describe the first example of a mtDNA molecule harboring three Lebers hereditary optic neuropathy (LHON)-associated mutations (m.11778G>A, m.14484T>C, m.11253T>C) and the analysis of its genetic, biochemical and molecular characterization in transmitochondrial cells (cybrids). Extensive characterization of cybrid cell lines harboring either the 3 mutations or the single classic m.11778G>A and m.14484T>C mutations revealed no differences in mitochondrial function, demonstrating the absence of a synergistic effect in this model system. These molecular results are in agreement with the ophthalmological characteristics found in the triple mutant patient, which were similar to those carrying single mtDNA LHON mutations.