Rapid and reversible cell volume changes in response to osmotic stress in yeast.

Saccharomyces cerevisiae has evolved diverse mechanisms to osmotic changes: the cell wall, ion and water transport systems, and signaling cascades. At the present time, little is known about the mechanisms involved in short-term responses of osmotic stress in yeast or their physiological state during this process. We conducted studies of flow cytometry, wet weight measurements, and electron microscopy to evaluate the modifications in cell volume and the cell wall induced by osmotic stress. In response to osmotic challenges, we show very fast and drastic changes in cell volume (up to 60%), which were completed in less than eight seconds. This dramatic change was completely reversible approximately 16 s after returning to an isosmotic solution. Cell volume changes were also accompanied by adaptations in yeast metabolism observed as a reduction by 50% in the respiratory rate, measured as oxygen consumption. This effect was also fully reversible upon returning to an isosmotic solution. It is noteworthy that we observed a significant recovery in oxygen consumption during the first 10 min of the osmotic shock. The rapid adjustment of the cellular volume may represent an evolutionary advantage, allowing greater flexibility for survival.

Ryanodine-sensitive intracellular Ca2+ channels in rat suprachiasmatic nuclei are required for circadian clock control of behavior.

Electrophysiological and calcium mobilization experiments have suggested that the intracellular calcium release channel ryanodine receptors (RyRs) are involved in the circadian rhythmicity of the suprachiasmatic nucleus (SCN). In the present report the authors provide behavioral evidence that RyRs play a specific and major role in the output of the molecular circadian clock in SCN neurons. They measured the circadian rhythm of drinking and locomotor behaviors in dim red light before, during, and after administration of an activator (ryanodine 0.1 microM) or an inhibitor (ryanodine 100 microM) of the RyRs. Drugs were delivered directly into the SCN by cannulas connected to osmotic minipumps. Control treatments included administration of artificial cerebrospinal fluid, KCl (20 mM), tetrodotoxin (1 microM), and anysomicin (5 microg/microl). Activation of RyRs induced a significant shortening of the endogenous period, whereas inhibition of these Ca2+ release channels disrupted the circadian rhythmicity. After the pharmacological treatments the period of rhythmicity returned to basal values and the phase of activity onset was predicted from a line projected from the activity onset of basal recordings. These results indicate that changes in overt rhythms induced by both doses of ryanodine did not involve an alteration in the clock mechanism. The authors conclude that circadian modulation of RyRs is a key element of the output pathway from the molecular circadian clock in SCN neurons in rats.

MCF-7 breast carcinoma cells express ryanodine receptor type 1: functional characterization and subcellular localization.

Breast carcinoma-derived MCF-7 cells are frequently used in biomedical research. However, few reports exist regarding the characterization of signaling mechanisms in these cancerous cells involved in intracellular Ca(2+) dynamics. Consequently, the aim of these experiments was to characterize the ryanodine receptor/Ca(2+) release channel (RyR) present in MCF-7 cells. Ryanodine (100 nM), cADPR (5 microM), and caffeine (10 mM) promoted cytoplasmic Ca(2+) mobilization; in contrast, ryanodine at inhibitory concentration (100 microM) decreased the basal Ca(2+) level. Fluorescent probes demonstrated that RyR is located mainly in endomembranes. Some degree of co-localization with inositol trisphosphate receptor (IP(3)R) was observed, whereas coincidence with thapsigargin-sensitive Ca(2+)-ATPase (SERCA) was more limited. Molecular cloning resulted in the detection exclusively of RyR isoform 1. For the first time, it is shown that MCF-7 cells express functional RyR.

Screening of antiproliferative effect of aqueous extracts of plant foods consumed in México on the breast cancer cell line MCF-7.

We evaluated the antiproliferative effect of aqueous extracts of 14 plant foods consumed in Mexico on the breast cancer cell line MCF-7. The plant foods used were avocado, black sapote, guava, mango, prickly pear cactus stems (called nopal in Mexico, cooked and raw), papaya, pineapple, four different cultivars of prickly pear fruit, grapes and tomato. ß-Carotene, total phenolics and gallic acid contents and the antioxidant capacity, measured by the ferric reducing/antioxidant power and the 2,2-diphenyl-1,1-picrylhydrazyl radical scavenging assays, were analyzed in each aqueous extract. Only the papaya extract had a significant antiproliferative effect measured with the methylthiazolydiphenyl-tetrazolium bromide assay. We did not notice a relationship between the total phenolic content and the antioxidant capacity with antiproliferative effect. It is suggested that each extract of plant food has a unique combination of the quantity and quality of phytochemicals that could determine its biological activity. Besides, papaya represents a very interesting fruit to explore its antineoplastic activities.

Granulosa cells express three inositol 1,4,5-trisphosphate receptor isoforms: cytoplasmic and nuclear Ca2+ mobilization.

BACKGROUND: Granulosa cells play an important endocrine role in folliculogenesis. They mobilize Ca2+ from intracellular stores by a coordinated action between 1,4,5 inositol trisphosphate and ryanodine receptors (IP3R and RyR). The aim of this study was to explore the isoforms of IP3Rs expressed in mouse C57BL/6 NHsd granulosa cells, characterizing their intranuclear localization and the relation with other Ca2+-handling proteins. METHODS: Ovarian tissue and granulosa cells were analyzed by multiphotonic and confocal microscopy to determine the intracellular presence of IP3R types 1, 2 and 3, RyR, thapsigargin-sensitive Ca2+-ATPase, and endomembranes. Cellular fractionation and Western blot assays were also used to further confirm the nuclear occurrence of the three IP3R isoforms. Free nuclear and cytosolic Ca2+ concentrations were measured using Fluo-4 AM by confocal microscopy. RESULTS: By using antibodies and specific fluorophores, was shown that granulosa cells endomembranes contain three isoforms of IP3R, the RyR, and the thapsigargin-sensitive Ca2+-ATPase (SERCA). Interestingly, all these proteins were also detected in the nuclear envelope and in well-defined intranuclear structures. Microsomal membranes depicted characteristic bands of the 3 types of IP3R, but also variants of lower molecular weight. Analysis of nuclear membranes and nucleoplasmic fraction confirmed the nuclear localization of the IP3R types 1, 2 and 3. We demonstrated ATP-induced Ca2+ transients in the nuclear and cytoplasmic compartments. Remarkably, the inhibitory effect on ATP-induced Ca2+ mobilization of brefeldin A was more accentuated in the cytoplasm than in the nucleus. CONCLUSION: These findings provide evidence that granulosa cells, including nuclei, express the Ca2+-handling proteins that allow Ca2+ mobilization. All three IP3R were also detected in ovarian slices, including the nuclei of granulosa cells, suggesting that these cells use the three IP3R in situ to achieve their physiological responses.

Critical Minireview: The Fate of tRNACys during Oxidative Stress in Bacillus subtilis.

Oxidative stress occurs when cells are exposed to elevated levels of reactive oxygen species that can damage biological molecules. One bacterial response to oxidative stress involves disulfide bond formation either between protein thiols or between protein thiols and low-molecular-weight (LMW) thiols. Bacillithiol was recently identified as a major low-molecular-weight thiol in Bacillus subtilis and related Firmicutes. Four genes (bshA, bshB1, bshB2, and bshC) are involved in bacillithiol biosynthesis. The bshA and bshB1 genes are part of a seven-gene operon (ypjD), which includes the essential gene cca, encoding CCA-tRNA nucleotidyltransferase. The inclusion of cca in the operon containing bacillithiol biosynthetic genes suggests that the integrity of the 3' terminus of tRNAs may also be important in oxidative stress. The addition of the 3' terminal CCA sequence by CCA-tRNA nucleotidyltransferase to give rise to a mature tRNA and functional molecules ready for aminoacylation plays an essential role during translation and expression of the genetic code. Any defects in these processes, such as the accumulation of shorter and defective tRNAs under oxidative stress, might exert a deleterious effect on cells. This review summarizes the physiological link between tRNACys regulation and oxidative stress in Bacillus.

Recognition and activation of ryanodine receptors by purines.

Ryanodine receptor (RyR) is a tetrameric, high molecular weight protein that functions as a calcium release channel. It plays a key role in phenomena such as signal transduction, excitation-contraction and excitation-secretion coupling. Hyperthermia maligna, central core disease and myocardial infarction have been related with RyR dysfunction. RyR is present as three isoforms in vertebrates: RyR 1 mainly localized in skeletal muscle, RyR 2 in cardiac muscle, and RyR 3 in nervous system. RyR is regulated by a number of physiological and pharmacological factors. Main physiological modulators: calcium, kinases and phosphatases, redox state and energy charge. Main pharmacological regulators: caffeine, dantrolene, ruthenium red, heavy metals and ryanodine. Purines have to do with both, physiological and pharmacological regulation of the RyR activity. So far, the mechanisms of RyR activation by ATP and caffeine have been described in detail using [3H]-ryanodine binding assays and unitary channel activity recorded in planar lipid bilayers. However, some questions remain to be addressed and are at present aim of active scrutiny: How many sites for purines are present in the RyR? Is the same site recognized by nucleotides and methylxanthines? What differences exist among the interaction between RyR and purine bases, nucleosides and nucleotides? Are the phosphate groups important for the recognition of nucleotides? Is the sugar moiety important for the recognition of nucleosides? The review article will examine the most recent specialized literature about the mechanism of activation of RyR by purines with emphasis on reports with approaches of structure-function and structure-activation.

16K-prolactin inhibits activation of endothelial nitric oxide synthase, intracellular calcium mobilization, and endothelium-dependent vasorelaxation.

Activation of endothelial nitric oxide synthase (eNOS) and subsequent nitric oxide production (NO) are events that mediate the effect of important angiogenic, vasopermeability, and vasorelaxation factors, including vascular endothelial growth factor (VEGF), bradykinin (BK), and acetylcholine (ACh). The N-terminal 16-kDa fragment of prolactin (16K-PRL) acts on endothelial cells to inhibit angiogenesis both in vivo and in vitro. Here, we show that 16K-PRL inhibits VEGF-induced eNOS activation in endothelial cells. Inhibition of eNOS activation may mediate the antiangiogenic properties of 16K-PRL, because the NO donor (Z)-1-[2-(2-aminoethyl)- N-(2-ammonio-ethyl)amino]diazen-1-ium-1,2-diolate (DETANONOate) prevented 16K-PRL from blocking the VEGF-induced proliferation of endothelial cells. In addition, 16K-PRL inhibited eNOS activation by BK and blocked the BK-evoked transient increase in intracellular Ca(2+) in endothelial cells. This finding suggests that 16K-PRL interferes with the mobilization of intracellular Ca(2+), thereby inhibiting the Ca(2+)-dependent activation of eNOS. Blockage of eNOS activation can lead to inhibition of vasodilation. Consistently, 16K-PRL inhibited BK-induced relaxation of coronary vessels in isolated perfused guinea pig hearts. Moreover, 16K-PRL inhibited eNOS activation induced by ACh, and this action resulted in the inhibition of both ACh-evoked relaxation of coronary vessels in isolated perfused rat hearts and ACh-induced, endothelium-dependent relaxation of rat aortic segments. In conclusion, 16K-PRL can block the Ca(2+)-mediated activation of eNOS by three different vasoactive substances, and this action results in the inhibition of both angiogenesis and vasorelaxation.

Subsurface cistern (SSC) proliferation in Purkinje cells of the rat cerebellum in response to acute and chronic exposure to paint thinner: A light and electron microscopy study.

Intentional inhalation and occupational exposure are two ways humans are exposed to thinner, a widely employed solvent in industry. Inhalation of thinner induces toxic effects in various organs, with the cerebellum being one of the most affected structures of the CNS. The aim of this work was to describe specific structural alterations of cerebellum Purkinje cells in rats following exposure to thinner for 16 weeks. A histological analysis of the cerebellum of solvent-exposed rats revealed swollen Purkinje cell dendrites surrounded by empty space, and electronic microscopy showed an increase in the number of subsurface cisterns (SSCs) within their dendritic processes. After a period of non-exposure, the number of SSCs decreased without reaching normal levels, suggesting a degree of plasticity. Purkinje cell SSCs, which are derived from smooth endoplasmic reticulum, contain inositol trisphosphate receptors (IP3Rs), ryanodine receptors (RR), and a recently identified characteristic cluster of large conductance calcium-activated potassium (BKCa) channels. We found that SSCs in Purkinje cell dendrites were closely associated with mitochondria, and immunofluorescence microscopy showed higher levels of RR and calbindin receptors (CB), in Purkinje cells of exposed than normal rats. These changes are probably related to behavioral manifestations of cerebellar alterations, such as imbalance and ataxia, consistent with the suggested involvement of increases in SSCs in ataxia in rats and humans. This increase in SSCs, taken together with the localization of RR, IP3R and BKCa proteins in this structure, suggests altered intracellular calcium-buffering processes in the Purkinje cells of thinner-exposed rats.

Characterization of the maitotoxin-activated cationic current from human skin fibroblasts.

The maitotoxin (MTX)-induced cationic current (I(mtx)) from human skin fibroblasts was characterized using the patch-clamp technique in whole-cell configuration. Under resting conditions (absence of MTX), the main current observed is produced by an outwardly rectifying K(+) channel which is inhibited by 1 mM TEA. The current reversal potential was -86 mV (n = 12). MTX (500 pM) activated a current with a linear current-voltage relationship and a reversal potential of -10 mV (n = 10). Replacing the extracellular Na(+) and K(+) with N-methyl-D-glucamine (NMDG) caused a shift of the reversal potential to a value below -100 mV, indicating that Na(+) and K(+), but not NMDG, carry I(mtx). Further ion selectivity experiments showed that Ca(2+) carries I(mtx) also. The resulting permeability sequence obtained with the Goldman-Hodgkin-Katz equation yielded Na(+) (1) approximately equal to K(+) (1) > Ca(2+) (0.87). The I(mtx) activation time course reflected the changes in intracellular Ca(2+) and Na(+) measured with the fluorescent indicators fura-2 and SBFI, respectively, suggesting that the activation of I(mtx) brings about an increment in intracellular Ca(2+) and Na(+). Reducing the extracellular Ca(2+) concentration below 1.8 mM prevented the activation of I(mtx) and the increment in intracellular Na(+) induced by MTX. Mn(2+) and Mg(2+) could not replace Ca(2+), but Ba(2+) could replace Ca(2+). MTX activation of current in 10 mM Ba(2+) was approximately 50 % of that induced in the presence of 1.8 mM Ca(2+). When 5 mM of the Ca(2+) chelator BAPTA was included in the patch pipette, MTX either failed to activate the current or induced a small current (less than 15 % of the control), indicating that intracellular Ca(2+) is also required for the activation of I(mtx). Intracellular Ba(2+) can replace Ca(2+) as an activator of I(mtx). However, in the presence of 10 mM Ba(2+) the activation by MTX of the current was 50 % less than the activation with nM concentrations of free intracellular Ca(2+).