Héctor Rojas

Na+ entry via glutamate transporter activates the reverse Na+/Ca2+ exchange and triggers Ca(i)2+-induced Ca2+ release in rat cerebellar Type-1 astrocytes.

We have previously demonstrated that rat cerebellar Type‐1 astrocytes express a very active genistein sensitive Na+/Ca2+ exchanger, which accounts for most of the total plasma membrane Ca2+ fluxes and for the clearance of loads induced by physiological agonists. In this work, we have explored the mechanism by which the reverse Na+/Ca2+ exchange is involved in agonist‐induced Ca2+ signaling in rat cerebellar astrocytes. Microspectrofluorometric measurements of with Fluo‐3 demonstrate that the signals associated long (> 20 s) periods of reverse operation of the Na+/Ca2+ exchange are amplified by a mechanism compatible with calcium‐calcium release, while those associated with short (< 20 s) pulses are not amplified. This was confirmed by pharmacological experiments using ryanodine receptors agonist (4‐chloro‐m‐cresol) and the endoplasmic reticulum ATPase inhibitor (thapsigargin). Confocal microscopy demonstrates a high co‐localization of immunofluorescent labeled Na+/Ca2+ exchanger and RyRs. Low (< 50 μmol/L) or high (> 500 μmol/L) concentrations of l‐glutamate (l‐Glu) or l‐aspartate causes a rise in which is completely blocked by the Na+/Ca2+ exchange inhibitors KB‐R7943 and SEA0400. The most important novel finding presented in this work is that l‐Glu activates the reverse mode of the Na+/Ca2+ exchange by inducing Na+ entry through the electrogenic Na+–Glu‐co‐transporter and not through the ionophoric l‐Glu receptors, as confirmed by pharmacological experiments with specific blockers of the ionophoric l‐Glu receptors and the electrogenic Glu transporter.

Amiodarone Destabilizes Intracellular Ca2+ Homeostasis and Biosynthesis of Sterols in Leishmania mexicana

ABSTRACT Leishmaniasis represents a serious public health problem worldwide. The first line of treatment is based on glucantime and pentostan, which generate toxic effects in treated patients. We have recently shown that amiodarone, frequently used as an antiarrhythmic, possesses activity against Trypanosoma cruzi through the disruption of mitochondrial Ca2+ homeostasis and the inhibition of parasite ergosterol biosynthesis, specifically at the level of oxidosqualene cyclase activity (G. Benaim, J. Sanders, Y. Garcia-Marchan, C. Colina, R. Lira, A. Caldera, G. Payares, C. Sanoja, J. Burgos, A. Leon-Rossell, J. Concepcion, A. Schijman, M. Levin, E. Oldfield, and J. Urbina, J. Med. Chem. 49:892-899, 2006). Here we show that at therapeutic concentrations, amiodarone has a profound effect on the viability of Leishmania mexicana promastigotes. Additionally, its effect on the viability of the parasite was greater against intracellular amastigotes than against promastigotes, and it did not affect the host cell. Using fluorimetric and confocal microscopy techniques, we also demonstrated that the mechanism of action of amiodarone was related to the disruption of intracellular Ca2+ homeostasis through a direct action not only on the mitochondria but also on the acidocalcisomes. On the other hand, analysis of the free sterols in promastigotes incubated with amiodarone showed that this drug also affected the biosynthesis of 5-dehydroepisterol, which results in squalene accumulation, thus suggesting that amiodarone inhibits the squalene epoxidase activity of the parasite. Taken together, the results obtained in the present work point to a more general effect of amiodarone in trypanosomatids, opening potential therapeutic possibilities for this infectious disease.

A novel 13 kDa cytoplasmic soluble protein is required for the nucleotide (MgATP) modulation of the Na/Ca exchange in squid nerve fibers

The Na/Ca exchange is a highly regulated transport mechanism in which MgATP, a powerful modulatory intracellular substrate, has important implications for its function. As occurs with some preparations, in squid axons, nucleotide regulation is lost after membrane vesicle isolation. This has been a significant obstacle in the biochemical characterization of the MgATP effect. An important clue in solving this long‐standing puzzle is presented in this work by showing that prolonged intracellular dialysis of squid axons produces a complete run down of the MgATP effect. Here we report that a soluble cytoplasmic factor isolated from fresh squid axoplasm and brain reconstitutes the MgATP stimulation of the Na‐gradient‐dependent 45Ca uptake in squid optic nerve membrane vesicles. Partial purification of this factor uncovers the presence of a novel 13 kDa soluble cytoplasmic protein (SCPr) which, when microinjected in ATP de‐regulated dialyzed squid axons, completely restores the MgATP stimulation of Nao‐dependent Ca efflux. We propose that in the squid preparation this SCPr constitutes the link between the nucleotide and target effector: the Na/Ca exchanger itself, or other plasma membrane structures which may secondarily interact with the exchanger.

Effect of internal and external K+ on Na+−Ca2+ exchange in dialyzed squid axons under voltage clamp conditions

The effect of external and internal K+ on Na+o-dependent Ca2+ efflux was studied in dialyzed squid axons under constant membrane potential. With axons clamped at their resting potentials, external K+ (up to 70 mM) has no effect on Na+-Ca2+ exchange. Removal of Ki+ causes a marked inhibition in the Na+o-dependent Ca2+ efflux component. Internal K+ activates the Na+-Ca2+ exchange with low affinity (K 1/2 = 90 mM). Activation by Ki+ is similar in the presence or in the absence of Na+i, thus ruling out a displacement of Na+i from its inhibitory site. Axons dialyzed with ATP also show a dependency of Ca2+ efflux on Ki+. The present results demonstrate that Ki+ is an important cofactor (partially required) for the proper functioning of the forward Na+-Ca2+ exchange.

Measurements of intracellular ionized calcium in squid giant axons using calcium-selective electrodes.

Ca2+-selective electrodes have been used to measure free intracellular Ca2+ concentrations in squid giant axons. Electrodes made of glass cannulas of about 20 microns in diameter, plugged with a poly(vinyl chloride) gelled sensor were used to impale the axons axially. They showed a Nernstian response to Ca2+ down to about 3 microM in solutions containing 0.3 M K+ and 0.025 M Na+. Sub-Nernstian but useful responses were obtained up to pCa 8. The electrodes showed adequate selectivity to Ca2+ over Mg2+, H+, K+ and Na+. To calibrate them properly, a set of standard solutions were prepared using different Ca2+ buffers (EGTA, HEEDTA, nitrilotriacetic acid) after carefully characterizing their apparent Ca2+ association constants under conditions resembling the axoplasmic environment. In fresh axons incubated in artificial seawater containing 4 mM Ca2+, the mean resting intracellular ionized calcium concentration was 0.106 microM (n = 15). The Ca2+-electrodes were used to investigate effects of different experimental procedures on the [Ca2+]i. The main conclusions are: (i) intact axons can extrude calcium ions at low [Ca2+]i levels by a process independent of external Na+; (ii) poisoned axons can extrude calcium ions at high levels of [Ca2+]i by an external Na+-dependent process. The level of free intracellular Ca attained at these latter conditions is about an order to magnitude greater than the resting physiological value.

A Nerve Cytosolic Factor Is Required for MgATP Stimulation of a Na+ Gradient-Dependent Ca2+ Uptake in Plasma Membrane Vesicles from Squid Optic Nervea

As early as 1979’ and 19822 we collected evidence that in dialyzed axons Ca2t1 was required for CaZt influx through the Nat-Caz+ exchanger. This need for Caz’ binding to nontransport internal sites was unambiguously shown in 1986 by following Na+ efflux through the reversal exchange; we named that effect CaZti regulation, and the intracellular site was called the Ca2 + regulatory site.3 In addition, and in the same paper, we presented data indicating that (i) ATP also stimulated reversal exchange and (ii) there seemed to be a relationship (or interaction) between Ca2+, and ATP stimulation. Later work demonstrated that ATP acted precisely by increasing the apparent affinity of the intracellular regulatory site for calcium ions4 In the work that followed (see Ref. 4 for references), always in dialyzed squid giant axons, we accumulated evidence that consistently suggested the effect of ATP was a consequence of a phosphorylation process that involved the interplay of a coupled kinase(s)-phosphatase(s) system(s). The main features of the ATP stimulation of the Nat-Ca2+ exchanger that led to this conclusion are:

In squid nerve fibers monovalent activating cations are not cotransported during Na+/Ca2+ exchange

Squid axons display a high activity of Na+/Ca2+ exchange which is largely increased by the presence of external K+, Li+, Rb+ and NH+4. In this work we have investigated whether this effect is associated with the cotransport of the monovalent cation along with Ca2+ ions. 86Rb+ influx and efflux have been measured in dialyzed squid axons during the activation (presence of Ca2+i) of Ca2+o/Na+i and Ca2+i/Ca2+o exchanges, while 86Rb+ uptake was determined in squid optic nerve membrane vesicles under equilibrium Ca2+/Ca2+ exchange conditions. Our results show that although K+o significantly increases Na+i-dependent Ca2+ influx (reverse Na+/Ca2+ exchange) and Rb+i stimulates Ca2+o-dependent Ca2+ efflux (Ca2+/Ca2+ exchange), no sizable transport of rubidium ions is coupled to calcium movement through the exchanger. Moreover, in the isolated membrane preparation no 86Rb+ uptake was associated with Ca2+/Ca2+ exchange. We conclude that in squid axons although monovalent cations activate the Na+/Ca2+ exchange they are not cotransported.

Depolymerisation and rearrangement of actin filaments during exocytosis in rat peritoneal mast cells: involvement of ryanodine-sensitive calcium stores.

Cytoskeletal F-actin associated with synaptic vesicles and granules plays an important role during Ca2+-mediated exocytosis. In the present work, we have used amperometry and confocal fluorescence to study the role of internal Ca2+ in the rearrangement of F-actin (visualised with phalloidin-Alexa 546) during exocytosis in rat mast cells. The F-actin-depolymerising drug, latrunculin A, and the ryanodine receptor agonists ryanodine and caffeine that, per se did not induce exocytosis, enhanced the exocytotic responses elicited by compound 48/80 (C48/80). They also induced cortical actin depolymerisation in the presence or absence of external Ca2+. Degranulation induced by C48/80 was accompanied by the formation of a cytoplasmic F-actin network. Depletion of internal Ca2+ with cyclopiazonic acid inhibited latrunculin potentiation of C48/80-stimulated exocytosis and completely blocked the formation of the cytoplasmic F-actin network. This indicates that the mobilisation of Ca2+ from ryanodine-sensitive intracellular stores plays an important role in the depolymerisation of the cortical F-actin barrier and possibly in the formation of the internal F-actin network during exocytotic activation of peritoneal mast cells.

The Activity of the Na+/Ca2+ Exchanger Largely Modulates the Ca2+i Signal Induced by Hypo-Osmotic Stress in Rat Cerebellar Astrocytes. The Effect of Osmolarity on Exchange Activity

We recently demonstrated that rat cerebellar Type-1 astrocytes express a very active Na(+)/Ca(2+) exchanger highly colocalized with ryanodine receptors (RyRs), which in turn play a key role in glutamate-induced Ca(2+) signaling through a calcium-induced calcium release (CICR) mechanism. In this work we have explored whether the Na(+)/Ca(2+) exchanger has any role in the Ca(2+)(i) signal induced by hypo-osmotic stress in these cells, using microspectrofluorometric measurements with Fura-2, pharmacological tools, and confocal microscopy image analysis. We present evidence for the first time that the increase in [Ca(2+)](i) in rat cerebellar Type-1 astrocytes, resulting from moderate hypotonic shock, is mediated by Ca(2+) release from ryanodine-operated Ca(2+)(i) stores, and that the magnitude of the intracellular Ca(2+) signal induced by hypotonicity in the short term (up to 240 s) is small and controlled by the activity of the Na(+)/Ca(2+) exchanger operating in its extrusion mode. With longer times in the hypotonic medium, intracellular Ca(2+) store depletion leads to Ca(2+) entry through store-operated Ca(2+) channels. We found it interesting that the activity of the Na(+)/Ca(2+) exchanger measured during this reverse mode operation (Ca(2+) entry in exchange for internal Na(+)) was found to be greatly increased in hypotonic solutions and decreased in hypertonic ones. The buffering of the [Ca(2+)](i) rise induced by hypo-osmotic stress may prevent excessive increases in [Ca(2+)](i), which otherwise might impair the normal function of this glial cell.

In dialyzed squid axons Cai2+ activates Cao2+Nai+ and Nao+Nai+ exchanges in the absence of Ca chelating agents

Abstract We used internally dialyzed squid axons to explore whether the reported activatory effect of Ca i 2+ on the partial reactions of the Na + Ca 2+ exchange (essential activator) is secondary to the presence of Ca 2+ chelating agents in the internal medium. The effect of Ca i 2+ pulses on both the reverse (Ca o 2+ -dependent Na + efflux) and Na + Na + exchange (Na o + -dependent Na + efflux) modes of the Na + Ca 2+ exchange was studied in axons dialyzed without EGTA. For these experiments a substantial inhibition of the Ca 2+ buffer capacity of the axoplasm was achieved by the use of Ruthenium red (10–20 μM), cyanide (1 mM) and vanadate (1 mM) in the dialysis solution. Our results indicate that the Ca i 2+ requirement of the reverse and Na + Na + exchange can not be explained by a direct inhibition of the Na + Ca 2+ exchanger by EGTA. In fact, both modes of operation of the exchanger can be activated by internal Ca 2+ ions in the complete absence of Ca 2+ chelating agents thus indicating that the ‘catalytic’ effect of Ca i 2+ on the Na + Ca 2+ exchanger is a real phenomenon.