Javier Alvarez

Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells

Direct monitoring of the free Ca2+ concentration in the lumen of the endoplasmic reticulum (ER) is an important but still unsolved experimental problem. We have shown that a Ca(2+)‐sensitive photoprotein, aequorin, can be addressed to defined subcellular compartments by adding the appropriate targeting sequences. By engineering a new aequorin chimera with reduced Ca2+ affinity, retained in the ER lumen via interaction of its N‐terminus with the endogenous resident protein BiP, we show here that, after emptying the ER, Ca2+ is rapidly re‐accumulated up to concentrations of > 100 microM, thus consuming most of the reporter photoprotein. An estimate of the steady‐state Ca2+ concentration was obtained using Sr2+, a well‐known Ca2+ surrogate which elicits a significantly slower rate of aequorin consumption. Under conditions in which the rate and extent of Sr2+ accumulation in the ER closely mimick those of Ca2+, the steady‐state mean lumenal Sr2+ concentration ([Sr2+]er) was approximately 2 mM. Receptor stimulation causes, in a few seconds, a 3‐fold decrease of the [Sr2+]er, whereas specific inhibition of the ER Ca2+ ATPase leads to an approximately 10‐fold drop in a few minutes.

Redistribution of Ca2+ among cytosol and organella during stimulation of bovine chromaffin cells

Recent results indicate that Ca2+ transport by organella contributes to shaping Ca2+ signals and exocytosis 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. Here we have studied changes in Ca2+ inside the endoplasmic reticulum, mitochondria, and nucleus by imaging aequorins targeted to these compartments in cells stimulated by brief depolarizing pulses with high K+ solutions. We find that Ca2+ entry through voltage‐gated Ca2+ channels generates subplasmalemmal high [Ca2+]c domains adequate for triggering exocytosis. A smaller increase of [Ca2+]c is produced in the cell core, which is adequate for recruitment of the reserve pool of secretory vesicles to the plasma membrane. Most of the Ca2+ load is taken up by a mitochondrial pool, M1, closer to the plasma membrane; the increase of [Ca2+]M stimulates respiration in these mitochondria, providing local support for the exocytotic process. Relaxation of the [Ca2+]c transient is due to Ca2+ extrusion through the plasma membrane. At this stage, mitochondria release Ca2+ to the cytosol through the Na+/Ca2+ exchanger, thus maintaining [Ca2+]c discretely increased, especially at core regions of the cell, for periods that outlast the duration of the stimulus.—Villalobos, C., Nuñez, L., Montero, M., García, A.G., Alonso, M. T., Chamero, P., Alvarez, J., García‐Sancho, J. Redistribution of Ca2+ among cytosol and organella during stimulation of bovine chromaffin cells. FASEB J. 16, 343–353 (2002)

A novel regulatory mechanism of the mitochondrial Ca2+ uniporter revealed by the p38 mitogen-activated protein kinase inhibitor SB202190

It is widely acknowledged that mitochondrial Ca2+ uptake modulates the cytosolic [Ca2+] ([Ca2+]c) acting as a transient Ca2+ buffer. In addition, mitochondrial [Ca2+] ([Ca2+]M) regulates the rate of respiration and may trigger opening of the permeability transition pore and start apoptosis. However, no mechanism for the physiological regulation of mitochondrial Ca2+ uptake has been described. We show here that SB202190, an inhibitor of p38 mitogen‐activated protein (MAP) kinase, strongly stimulates ruthenium red‐sensitive mitochondrial Ca2+ uptake, both in intact and in permeabilized HeLa cells. The [Ca2+]M peak induced by agonists was increased about fourfold in the presence of the inhibitor, with a concomitant reduction in the [Ca2+]c peak. The stimulation occurred fast and was rapidly reversible. In addition, experiments in permeabilized cells perfused with controlled [Ca2+] showed that SB202190 stimulated mitochondrial Ca2+ uptake by more than 10‐fold, but only in the physiological [Ca2+]c range (1–4 μM). Other structurally related p38 MAP kinase inhibitors (SB203580, PD169316, or SB220025) produced little or no effect. Our data suggest that in HeLa cells, a protein kinase sensitive to SB202190 tonically inhibits the mitochondrial Ca2+ uniporter. This novel regulatory mechanism may be of paramount importance to modulate mitochondrial Ca2+ uptake under different physiopathological conditions.

Intravesicular calcium release mediates the motion and exocytosis of secretory organelles. A study with adrenal chromaffin cells

Secretory vesicles of sympathetic neurons and chromaffin granules maintain a pH gradient toward the cytosol (pH 5.5 versus 7.2) promoted by the V-ATPase activity. This gradient of pH is also responsible for the accumulation of amines and Ca2+ because their transporters use H+ as the counter ion. We have recently shown that alkalinization of secretory vesicles slowed down exocytosis, whereas acidification caused the opposite effect. In this paper, we measure the alkalinization of vesicular pH, caused by the V-ATPase inhibitor bafilomycin A1, by total internal reflection fluorescence microscopy in cells overexpressing the enhanced green fluorescent protein-labeled synaptobrevin (VAMP2-EGFP) protein. The disruption of the vesicular gradient of pH caused the leak of Ca2+, measured with fura-2. Fluorimetric measurements, using the dye Oregon green BAPTA-2, showed that bafilomycin directly released Ca2+ from freshly isolated vesicles. The Ca2+ released from vesicles to the cytosol dramatically increased the granule motion of chromaffin- or PC12-derived granules and triggered exocytosis (measured by amperometry). We conclude that the gradient of pH of secretory vesicles might be involved in the homeostatic regulation of cytosolic Ca2+ and in two of the major functions of secretory cells, vesicle motion and exocytosis.

On the role of intravesicular calcium in the motion and exocytosis of secretory organelles

Secretory vesicles of sympathetic neurons and chromaffin granules maintain a pH gradient towards the cytosol (5.5 vs. 7.2) promoted by the V-ATPase activity. This gradient of pH is mainly responsible for the accumulation of amines. The secretory vesicles contain large amounts of total Ca2+, but the free intragranular [Ca2+], the mechanisms for Ca2+ uptake and release from the granules and their physiological relevance regarding exocytosis are still matters of debate. We have recently shown that disruption of the pH gradient of secretory vesicles slowed down exocytosis. Fluorimetric measurements, using the dye Oregon green BAPTA-2, showed that the V-ATPase inhibitor bafilomycin A1 directly released Ca2+ from freshly isolated vesicles. Accordingly, vesicle alkalinization released Ca2+ from the granules to the cytosol, measured with fura-2 in intact chromaffin cells. Using TIRFM in cells over-expressing the EGFP-labeled synaptobrevin (VAMP2-EGFP) protein, we have then shown that the Ca2+ released from the vesicles to the cytosol in the presence of bafilomycin, dramatically increased the granule motion of chromaffin- or PC12-derived granules, and triggered exocytosis (measured by amperometry). We conclude that the gradient of pH of secretory vesicles might be involved in the homeostatic regulation of the local cytosolic Ca2+ around the vesicles and in two of the major functions of secretory cells, vesicle motion and exocytosis.1

Pharynx mitochondrial [Ca 2+ ] dynamics in live C. elegans worms during aging

Progressive decline in mitochondrial function is generally considered one of the hallmarks of aging. We have expressed a Ca2+ sensor in the mitochondrial matrix of C. elegans pharynx cells and we have measured for the first time mitochondrial [Ca2+] ([Ca2+]M) dynamics in the pharynx of live C. elegans worms during aging. Our results show that worms stimulated with serotonin display a pharynx [Ca2+]M oscillatory kinetics that includes both high frequency oscillations (up to about 1Hz) and very prolonged “square-wave” [Ca2+]M increases, indicative of energy depletion of the pharynx cells. Mitochondrial [Ca2+] is therefore able to follow “beat-to-beat” the fast oscillations of cytosolic [Ca2+]. The fast [Ca2+]M oscillations kept steady frequency values during the whole worm life, from 2 to 12 days old, but the height and width of the peaks was progressively reduced. [Ca2+]M oscillations were also present with similar kinetics in respiratory chain complex I nuo-6 mutant worms, although with smaller height and frequency than in the controls, and larger width. In summary, Ca2+ fluxes in and out of the mitochondria are relatively well preserved during the C. elegans life, but there is a clear progressive decrease in their magnitude during aging. Moreover, mitochondrial Ca2+ fluxes were smaller in nuo-6 mutants with respect to the controls at every age and decreased similarly during aging.Progressive decline in mitochondrial function is generally considered one of the hallmarks of aging. We have expressed a Ca2+ sensor in the mitochondrial matrix of C. elegans pharynx cells and we have measured for the first time mitochondrial [Ca2+] ([Ca2+]M) dynamics in the pharynx of live C. elegans worms during aging. Our results show that worms stimulated with serotonin display a pharynx [Ca2+]M oscillatory kinetics that includes both high frequency oscillations (up to about 1Hz) and very prolonged square-wave [Ca2+]M increases, indicative of energy depletion of the pharynx cells. Mitochondrial [Ca2+] is therefore able to follow beat-to-beat the fast oscillations of cytosolic [Ca2+]. The fast [Ca2+]M oscillations kept steady frequency values during the whole worm life, from 2 to 12 days old, but the height and width of the peaks was progressively reduced. [Ca2+]M oscillations were also present with similar kinetics in respiratory chain complex I nuo-6 mutant worms, although with smaller height and frequency than in the controls, and larger width. In summary, Ca2+ fluxes in and out of the mitochondria are relatively well preserved during the C. elegans life, but there is a clear progressive decrease in their magnitude during aging. Moreover, mitochondrial Ca2+ fluxes were smaller in nuo-6 mutants with respect to the controls at every age and decreased similarly during aging.

Long-term monitoring of Ca 2+ dynamics in C. elegans pharynx: an in vivo energy balance sensor

Ca2+ is a key signal transducer for muscle contraction. Continuous in vivo monitoring of intracellular Ca2+-dynamics in C. elegans pharynx muscle revealed surprisingly complex Ca2+ patterns. Despite the age-dependent decline of pharynx pumping, we observed unaltered fast Ca2+ oscillations both in young and old worms. In addition, sporadic prolonged Ca2+ increases lasting many seconds or minutes were often observed in between periods of fast Ca2+ oscillations. We attribute them to the inhibition of ATP-dependent Ca2+-pumps upon energy depletion. Accordingly, food deprivation largely augmented the frequency of prolonged [Ca2+] increases. However, paradoxically, prolonged [Ca2+] increases were more frequently observed in young worms than in older ones, and less frequently observed in energy-deficient mitochondrial respiratory chain nuo-6 mutants than in wild-type controls. We hypothesize that young animals are more susceptible to energy depletion due to their faster energy consumption rate, while nuo-6 mutants may keep better the energy balance by slowing energy consumption. Our data therefore suggest that the metabolic state of the pharynx during feeding stimulation depends mainly on the delicate balance between the instant rates of energy production and consumption. Thus, in vivo monitoring of muscle Ca2+ dynamics can be used as a novel tool to study cellular energy availability.

Transient inhibition of capacitative calcium entry in human neutrophils by a monoclonal antibody directed against a 19-kDa antigen.

P1C3 is a monoclonal antibody that binds p19, a novel neutrophil activation antigen that translocates to the cell surface upon neutrophil activation. We find that P1C3 inhibits capacitative Ca2+ entry, induced by emptying the intracellular Ca2+ stores with thapsigargin. The effect is transient, reaching its maximum at 30–60 s, but becomes permanent upon pretreatment of the cells with the protein phosphatase inhibitor calyculin A, suggesting the involvement of protein phosphorylation. The inhibitory action is similar to the one reported previously for the chemotactic peptide N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP), although the transduction mechanism may be different. Inhibition of Ca2+ entry by fMLP was prevented by pretreatment with pertussis toxin, whereas inhibition by P1C3 was not. Pretreatment with cholera toxin had no effect. This suggests that the effect of P1C3 may not be mediated by a heterotrimeric G protein. Tyrosine kinase inhibitors did not prevent inhibition by either fMLP or P1C3. Phospholipase C activation seems not to be involved as P1C3, contrarily to fMLP, was unable to induce Ca2+ release from the intracellular Ca2+ stores. J. Leukoc. Biol. 60: 323–327; 1996.

Ca2+-Activated Potassium Channels

It was 50 years ago that the presence in the human red-cell membrane of a K+-selective pathway activatable by Pb (Orskov, 1935) or fluoride (Wilbrandt, 1937) was reported. Gardos described a similar pathway that was activated by incubation with iodoacetate and adenosine, and pointed out the absolute requirement of calcium for activation (“Gardos effect,” Gardos, 1956, 1958). The site for the Ca2+ effect was later documented to face towards the cytoplasmic side of the membrane (Whittam, 1968; Lew, 1970; Blum and Hoffman, 1972). During the last 15 yr, K+-selective channels that are activated by the increase of ionized calcium levels of the cytoplasm have also been discovered in a large variety of cells. The participation of these channels in important physiological functions, such as the control of membrane potential of excitable cells, the secretion of hormones or neurotransmitters, and the control of cell volume and transephithelial ion transport, is now widely acknowledged (for reviews, see Meech, 1976, 1978; Putney, 1979; Grinstein et al., 1982; Schwarz and Passow, 1983; Petersen and Maruyama, 1984; Sarkadi and Gardos, 1985; Hoffman, 1985).

Inhibition of Sarco-Endoplasmic Reticulum Ca2+ ATPase Extends the Lifespan in C. elegans Worms

The sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) refills the endoplasmic reticulum (ER) with Ca2+ up to the millimolar range and is therefore the main controller of the ER [Ca2+] level ([Ca2+]ER), which has a key role in the modulation of cytosolic Ca2+ signaling and ER-mitochondria Ca2+ transfer. Given that both cytosolic and mitochondrial Ca2+ dynamics strongly interplay with energy metabolism and nutrient-sensitive pathways, both of them involved in the aging process, we have studied the effect of SERCA inhibitors on lifespan in C. elegans. We have used thapsigargin and 2,5-Di-tert-butylhydroquinone (2,5-BHQ) as SERCA inhibitors, and the inactive analog 2,6-Di-tert-butylhydroquinone (2,6-BHQ) as a control for 2,5-BHQ. Every drug was administered to the worms either directly in the agar or via an inclusion compound with γ-cyclodextrin. The results show that 2,6-BHQ produced a small but significant increase in survival, perhaps because of its antioxidant properties. However, 2,5-BHQ produced in all the conditions a much higher increase in lifespan, and the potent and specific SERCA inhibitor thapsigargin also extended the lifespan. The effects of 2,5-BHQ and thapsigargin had a bell-shaped concentration dependence, with a maximum effect at a certain dose and smaller or even toxic effects at higher concentrations. Our data show therefore that submaximal inhibition of SERCA pumps has a pro-longevity effect, suggesting that Ca2+ signaling plays an important role in the aging process and that it could be a promising novel target pathway to act on aging.