A case of ochronosis successfully treated with the picosecond laser.

Exogenous ochronosis is a cutaneous condition characterized by blue-black pigmentation resulting as a complication of long-term application of skin-lightening creams containing hydroquinone and other substances such as quinine, phenol and mercury derivatives. We report a case of a 55-year-old woman who developed exogenous ochronosis as a result of prolonged use of topical hydroquinone for 5 years, characterized by greyish hyperpigmented patches on the nose and cheeks. The diagnosis was confirmed histologically. Treatment with picosecond laser resulted in marked clinical improvement together with improvement in overall texture and quality of the skin.

Selective tracking of FFAR3-expressing neurons supports receptor coupling to N-type calcium channels in mouse sympathetic neurons.

Activation of short-chain free fatty acid receptors 3 (FFAR3) has been suggested to promote sympathetic outflow in postganglionic sympathetic neurons or hamper it by a negative coupling to N-type calcium (CaV2.2) channels. Heterogeneity of FFAR3 expression in sympathetic neurons, however, renders single neurons studies extremely time-consuming in wild-type mice. Previous studies demonstrated large variability of the degree of CaV2.2 channel inhibition by FFAR3 in a global population of rat sympathetic neurons. Therefore, we focused on a small subpopulation of mouse sympathetic neurons using an FFAR3 antibody and an Ffar3 reporter mouse to perform immunofluorescent and electrophysiological studies. Whole-cell patch-clamp recordings of identified FFAR3-expressing neurons from reporter mice revealed a 2.5-fold decrease in the CaV2.2-FFAR3 inhibitory coupling variability and 1.5-fold increase in the mean ICa2+ inhibition, when compared with unlabeled neurons from wild-type mice. Further, we found that the ablation of Ffar3 gene expression in two knockout mouse models led to a complete loss-of-function. Subpopulations of sympathetic neurons are associated with discrete functional pathways. However, little is known about the neural pathways of the FFAR3-expressing subpopulation. Our data indicate that FFAR3 is expressed primarily in neurons with a vasoconstrictor phenotype. Thus, fine-tuning of chemically-coded neurotransmitters may accomplish an adequate outcome.

Sodium-calcium exchanger modulates the L-glutamate Ca(i) (2+) signalling in type-1 cerebellar astrocytes.

We have previously demonstrated that rat type-1 cerebellar astrocytes express a very active Na(+)/Ca(2+) exchanger which accounts for most of the total plasma membrane Ca(2+) fluxes and for the clearance of Ca (i) (2+) induced by physiological agonist. In this chapter, we have explored the mechanism by which the reverse Na(+)/Ca(2+) exchange is involved in agonist-induced Ca(2+) signalling in rat cerebellar astrocytes. Laser-scanning confocal microscopy experiments using immunofluorescence labelling of Na(+)/Ca(2+) exchanger and RyRs demonstrated that they are highly co-localized. The most important finding presented in this chapter is that L-glutamate activates the reverse mode of the Na(+)/Ca(2+) exchange by inducing a Na(+) entry through the electrogenic Na(+)-glutamate co-transporter and not through the ionophoric L-glutamate receptors as confirmed by pharmacological experiments with specific blockers of ionophoric L-glutamate receptors, electrogenic glutamate transporters and the Na/Ca exchange.

Regulation of Na+/K+ ATPase transport velocity by RNA editing.

Because firing properties and metabolic rates vary widely, neurons require different transport rates from their Na(+)/K(+) pumps in order to maintain ion homeostasis. In this study we show that Na(+)/K(+) pump activity is tightly regulated by a novel process, RNA editing. Three codons within the squid Na(+)/K(+) ATPase gene can be recoded at the RNA level, and the efficiency of conversion for each varies dramatically, and independently, between tissues. At one site, a highly conserved isoleucine in the seventh transmembrane span can be converted to a valine, a change that shifts the pump's intrinsic voltage dependence. Mechanistically, the removal of a single methyl group specifically targets the process of Na(+) release to the extracellular solution, causing a higher turnover rate at the resting membrane potential.

Structural basis of Na(+)/K(+)-ATPase adaptation to marine environments.

Throughout evolution, enzymes have adapted to perform in different environments. The Na(+)/K(+) pump, an enzyme crucial for maintaining ionic gradients across cell membranes, is strongly influenced by the ionic environment. In vertebrates, the pump sees much less external Na(+) (100-160 mM) than it does in osmoconformers such as squid (450 mM), which live in seawater. If the extracellular architecture of the squid pump were identical to that of vertebrates, then at the resting potential, the pump's function would be severely compromised because the negative voltage would drive Na(+) ions back to their binding sites, practically abolishing forward transport. Here we show that four amino acids that ring the external mouth of the ion translocation pathway are more positive in squid, thereby reducing the pump's sensitivity to external Na(+) and explaining how it can perform optimally in the marine environment.

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(+)/Ca(2+) exchanger, which accounts for most of the total plasma membrane Ca(2+) fluxes and for the clearance of loads induced by physiological agonists. In this work, we have explored the mechanism by which the reverse Na(+)/Ca(2+) exchange is involved in agonist-induced Ca(2+) signaling in rat cerebellar astrocytes. Microspectrofluorometric measurements of Cai(2+) with Fluo-3 demonstrate that the Cai(2+) signals associated long (> 20 s) periods of reverse operation of the Na(+)/Ca(2+) 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(+)/Ca(2+) exchanger and RyRs. Low (< 50 micromol/L) or high (> 500 micromol/L) concentrations of L-glutamate (L-Glu) or L-aspartate causes a rise in which is completely blocked by the Na(+)/Ca(2+) 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(+)/Ca(2+) 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 has intrinsic anti-Trypanosoma cruzi activity and acts synergistically with posaconazole.

There is no effective treatment for the prevalent chronic form of Chagas' disease in Latin America. Its causative agent, the protozoan parasite Trypanosoma cruzi, has an essential requirement for ergosterol, and ergosterol biosynthesis inhibitors, such as the antifungal drug posaconazole, have potent trypanocidal activity. The antiarrhythmic compound amiodarone, frequently prescribed for the symptomatic treatment of Chagas' disease patients, has also recently been shown to have antifungal activity. We now show here for the first time that amiodarone has direct activity against T. cruzi, both in vitro and in vivo, and that it acts synergistically with posaconazole. We found that amiodarone, in addition to disrupting the parasites' Ca(2+) homeostasis, also blocks ergosterol biosynthesis, and that posaconazole also affects Ca(2+) homeostasis. These results provide logical explanations for the synergistic activity of amiodarone with azoles against T. cruzi and open up the possibility of novel, combination therapy approaches to the treatment of Chagas' disease using currently approved drugs.

Ceramide-1-P induces Ca2+ mobilization in Jurkat T-cells by elevation of Ins(1,4,5)-P3 and activation of a store-operated calcium channel.

Sphingolipids comprise a very important class of second messengers involved in cell growth, differentiation, and apoptosis, among other different functions. Recently, these lipids have been implicated in calcium mobilization in different cell lines, including Jurkat T-lymphocytes. However, the effect of each particular sphingolipid appears to be cell-line specific. Among them, the least studied is ceramide-1-P (Cer-1-P). Here, we show that Cer-1-P increased the intracellular Ca(2+) concentration in Jurkat T-cells. Furthermore, laser-scanning confocal microscopy indicated that Ca(2+) is released from the endoplasmic reticulum. An effect on store-operated Ca(2+) channels was evidenced by whole-cell "patch clamp" measurements after Cer-1-P induced Ca(2+) store depletion. The mechanism of action of Cer-1-P resembles that of the Jurkat anti-TCR antibody, but differs from that of ceramide, since Cer-1-P induced an increase in Ins(1,4,5)-P(3).

Ceramide increase cytoplasmic Ca2+ concentration in Jurkat T cells by liberation of calcium from intracellular stores and activation of a store-operated calcium channel.

The effect of ceramide on the cytoplasmic Ca2+ concentration ([Ca2+]i) varies depending on the cell type. We have found that in Jurkat human T cells ceramide increases the [Ca2+]i from a thapsigargin-sensitive calcium pool and the subsequent activation of a capacitative Ca2+ entry. This effect occurs both in the presence and in the absence of extracellular calcium. Addition of ceramine, a non-hydrolysable analogue of ceramide, reproduced its effect on the [Ca2+]i ruling out that this is due to the conversion of ceramide to sphingosine. The effect of ceramide was additive to that obtained by sphingosine, but not to the Jurkat T cells specific antibody OKT3. However, different to the latter, ceramide do not induced an elevation of InsP3. The opening of a store operated Ca2+ channel by ceramide was corroborated by experiments of Fura-2 quenching, using Mn2+ as a surrogate for Ca2+ and confirmed by whole-cell recording patch clamp techniques.

Ceramide and sphingosine have an antagonistic effect on the plasma-membrane Ca2+-ATPase from human erythrocytes.

The plasma-membrane Ca(2+)-ATPase is a key enzyme in the regulation of the intracellular Ca(2+) concentration. On the other hand, sphingolipids have been recognized recently as important second messengers, acting in many systems in combination with Ca(2+). In view of the fact that the Ca(2+)-ATPase is stimulated by ethanol, and since sphingolipids possess free hydroxy groups, we decided to study the possible effect of ceramide and sphingosine on this calcium pump. Here we show that ceramide stimulates the Ca(2+)-ATPase in a dose-dependent manner and additively to the activation observed in the presence of calmodulin or ethanol, when compared with any of these effectors added alone. Ceramide affects both the affinity for Ca(2+) and the V(max) of the enzyme. Furthermore, this second messenger also stimulates Ca(2+) transport in inside-out plasma-membrane vesicles from erythro cytes. Conversely, sphingosine, which is reported to act in many systems antagonistically with ceramide, showed an inhibitory effect on Ca(2+)-ATPase activity. This inhibition was also observed on the calmodulin-stimulated enzyme. These results, taken together, suggest that ceramide and sphingosine act antagonistically on the plasma-membrane Ca(2+)-ATPase. This is in accordance with the frequently reported opposite effect of these sphingolipids on intracellular Ca(2+) concentration.