Identification and characterization of human PEIG-1/GPRC5A as a 12-O-tetradecanoyl phorbol-13-acetate (TPA) and PKC-induced gene.

Homo sapiens orphan G protein-coupling receptor PEIG-1 was first cloned and characterized by applying differential display to T84 colonic carcinoma cells incubated in the presence of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (GenBank AF506289.1). Later, Lotan's laboratory found the same gene product in response to retinoic acid analogues, naming it with the symbol RAIG1. Now the official HGNC symbol is GPRC5A. Here, we report the extension of its original cDNA fragment towards the 5' and 3' end. In addition, we show that TPA (100 ng/ml, 162 nM) strongly stimulated GPRC5A mRNA in T84 colonic carcinoma cells, with maximal expression at 4 h and 100 ng/ml (162 nM). Western blots showed several bands between 35 and 50 kDa, responding to TPA stimulation. Confocal microscopy confirmed its TPA upregulation and the location in the plasma membrane. The PKC inhibitor Gö 6983 (10 µM), and the Ca2+ chelator BAPTA-AM (150 µM), strongly inhibited its TPA induced upregulation. The PKA inhibitor H-89 (10 µM), and the MEK1/2 inhibitor U0126 (10 µM), also produced a significant reduction in the TPA response (~50%). The SGK1 inhibitor GSK650394 stimulated GPRC5A basal levels at low doses and inhibit its TPA-induced expression at concentrations ≥10 µM. The IL-1ß autocrine loop and downstream signalling did not affect its expression. In conclusion, RAIG1/RAI3/GPRC5A corresponds to the originally reported PEIG-1/TIG1; the inhibition observed in the presence of Gö 6983, BAPTA and U0126, suggests that its TPA-induced upregulation is mediated through a PKC/Ca2+ →MEK1/2 signalling axis. PKA and SGK1 kinases are also involved in its TPA-induced upregulation.

Ca2+ -independent and voltage-dependent exocytosis in mouse chromaffin cells.

AIM: It is widely accepted that the exocytosis of synaptic and secretory vesicles is triggered by Ca2+ entry through voltage-dependent Ca2+ channels. However, there is evidence of an alternative mode of exocytosis induced by membrane depolarization but lacking Ca2+ current and intracellular Ca2+ increase. In this work we investigated if such a mechanism contributes to secretory vesicle exocytosis in mouse chromaffin cells. METHODS: Exocytosis was evaluated by patch-clamp membrane capacitance measurements, carbon fibre amperometry and TIRF. Cytosolic Ca2+ was estimated using epifluorescence microscopy and fluo-8 (salt form). RESULTS: Cells stimulated by brief depolatizations in absence of extracellular Ca+2 show moderate but consistent exocytosis, even in presence of high cytosolic BAPTA concentration and pharmacological inhibition of Ca+2 release from intracellular stores. This exocytosis is tightly dependent on membrane potential, is inhibited by neurotoxin Bont-B (cleaves the v-SNARE synaptobrevin), is very fast (saturates with time constant <10 ms), it is followed by a fast endocytosis sensitive to the application of an anti-dynamin monoclonal antibody, and recovers after depletion in <5 s. Finally, this exocytosis was inhibited by: (i) ω-agatoxin IVA (blocks P/Q-type Ca2+ channel gating), (ii) in cells from knock-out P/Q-type Ca2+ channel mice, and (iii) transfection of free synprint peptide (interferes in P/Q channel-exocytic proteins association). CONCLUSION: We demonstrated that Ca2+ -independent and voltage-dependent exocytosis is present in chromaffin cells. This process is tightly coupled to membrane depolarization, and is able to support secretion during action potentials at low basal rates. P/Q-type Ca2+ channels can operate as voltage sensors of this process.

Dengue virus induced changes in Ca2+ homeostasis in human hepatic cells that favor the viral replicative cycle.

The role of Ca2+ during dengue virus (DENV) replication is unknown; thus, changes in Ca2+ homeostasis in DENV infected human hepatic HepG2 and Huh-7 cells were analyzed. Infected HepG2 cells, but not Huh-7 cells, showed a significant increase in plasma membrane permeability to Ca2+, while both cell lines showed marked reduced levels of Ca2+ stored in the endoplasmic reticulum. While the expression levels of STIM1 and ORAI1 showed no changes, STIM1 and ORAI1 were shown to co-localized in infected cells, indicating activation of the store-operated Ca2+ entry (SOCE) pathway. Finally, manipulation in the infected cells of the intra and extracellular Ca2+ levels by chelators (BAPTA-AM and EGTA), SOC inhibitor (SKF96365), IP3 Receptor antagonist (2APB) or increase of extracellular [Ca2+], significantly reduced DENV yield, but not vesicular stomatitis virus yield, used as a control. These results show that DENV infection alters cell Ca2+ homeostasis and that such changes favor viral replication.

The putative flavin carrier family FlcA-C is important for Aspergillus fumigatus virulence.

Aspergillus fumigatus is an opportunistic fungal pathogen and the most important species causing pulmonary fungal infections. The signaling by calcium is very important for A. fumigatus pathogenicity and it is regulated by the transcription factor CrzA. We have previously used used ChIP-seq (Chromatin Immunoprecipitation DNA sequencing) aiming to identify gene targets regulated by CrzA. We have identified among several genes regulated by calcium stress, the putative flavin transporter, flcA. This transporter belongs to a small protein family composed of FlcA, B, and C. The ΔflcA null mutant showed several phenotypes, such as morphological defects, increased sensitivity to calcium chelating-agent ethylene glycol tetraacetic acid (EGTA), cell wall or oxidative damaging agents and metals, repre-sentative of deficiencies in calcium signaling and iron homeostasis. Increasing calcium concentrations improved significantly the ΔflcA growth and conidiation, indicating that ΔflcA mutant has calcium insufficiency. Finally, ΔflcA-C mutants showed reduced flavin adenine dinucleotide (FAD) and were avirulent in a low dose murine infection model.

Rapid effects of aldosterone in primary cultures of cardiomyocytes - do they suggest the existence of a membrane-bound receptor?

Aldosterone acts on its target tissue through a classical mechanism or through the rapid pathway through a putative membrane-bound receptor. Our goal here was to better understand the molecular and biochemical rapid mechanisms responsible for aldosterone-induced cardiomyocyte hypertrophy. We have evaluated the hypertrophic process through the levels of ANP, which was confirmed by the analysis of the superficial area of cardiomyocytes. Aldosterone increased the levels of ANP and the cellular area of the cardiomyocytes; spironolactone reduced the aldosterone-increased ANP level and cellular area of cardiomyocytes. Aldosterone or spironolactone alone did not increase the level of cyclic 3',5'-adenosine monophosphate (cAMP), but aldosterone plus spironolactone led to increased cAMP level; the treatment with aldosterone + spironolactone + BAPTA-AM reduced the levels of cAMP. These data suggest that aldosterone-induced cAMP increase is independent of mineralocorticoid receptor (MR) and dependent on Ca(2+). Next, we have evaluated the role of A-kinase anchor proteins (AKAP) in the aldosterone-induced hypertrophic response. We have found that St-Ht31 (AKAP inhibitor) reduced the increased level of ANP which was induced by aldosterone; in addition, we have found an increase on protein kinase C (PKC) and extracellular signal-regulated kinase 5 (ERK5) activity when cells were treated with aldosterone alone, spironolactone alone and with a combination of both. Our data suggest that PKC could be responsible for ERK5 aldosterone-induced phosphorylation. Our study suggests that the aldosterone through its rapid effects promotes a hypertrophic response in cardiomyocytes that is controlled by an AKAP, being dependent on ERK5 and PKC, but not on cAMP/cAMP-dependent protein kinase signaling pathways. Lastly, we provide evidence that the targeting of AKAPs could be relevant in patients with aldosterone-induced cardiac hypertrophy and heart failure.

Isolated Schwannoma of the Olfactory Groove: A Case Report

Introduction Schwannoma of the olfactory groove is an extremely rare tumor that can share a differential diagnosis with meningioma or neuroblastoma. Objectives The authors present a case of giant schwannoma involving the anterior cranial fossa and ethmoid sinuses. Case Report The patient presented with a 30-month history of left nasal obstruction, anosmia, and sporadic ipsilateral bleeding. Computed tomography of the paranasal sinuses revealed expansive lesion on the left nasal cavity extending to nasopharynx up to ethmoid and sphenoid sinuses bilaterally with intraorbital and parasellar extension to the skull base. Magnetic resonance imaging scan confirmed the expansive tumor without dural penetration. Biopsy revealed no evidence of malignancy and probable neural cell. Bifrontal craniotomy was performed combined with lateral rhinotomy (Weber-Ferguson approach), and the lesion was totally removed. The tumor measured 8.0 4.3 3.7 cm and microscopically appeared as a schwannoma composed of interwoven bundles of elongated cells (Antoni A regions)mixed with less cellular regions (Antoni B). Immunohistochemical study stained intensively for vimentin and S-100. Conclusion Schwannomas of the olfactory groove are extremely rare, and the findings of origin of this tumor is still uncertain but recent studies point most probably to the meningeal branches of trigeminal nerve or anterior ethmoidal nerves. .

Quinolinic acid induces disrupts cytoskeletal homeostasis in striatal neurons. Protective role of astrocyte-neuron interaction.

Quinolinic acid (QUIN) is an endogenous metabolite of the kynurenine pathway involved in several neurological disorders. Among the several mechanisms involved in QUIN-mediated toxicity, disruption of the cytoskeleton has been demonstrated in striatally injected rats and in striatal slices. The present work searched for the actions of QUIN in primary striatal neurons. Neurons exposed to 10 µM QUIN presented hyperphosphorylated neurofilament (NF) subunits (NFL, NFM, and NFH). Hyperphosphorylation was abrogated in the presence of protein kinase A and protein kinase C inhibitors H89 (20 µM) and staurosporine (10 nM), respectively, as well as by specific antagonists to N-methyl-D-aspartate (50 µM DL-AP5) and metabotropic glutamate receptor 1 (100 µM MPEP). Also, intra- and extracellular Ca(2+) chelators (10 µM BAPTA-AM and 1 mM EGTA, respectively) and Ca(2+) influx through L-type voltage-dependent Ca(2+) channel (10 µM verapamil) are implicated in QUIN-mediated effects. Cells immunostained for the neuronal markers ßIII-tubulin and microtubule-associated protein 2 showed altered neurite/neuron ratios and neurite outgrowth. NF hyperphosphorylation and morphological alterations were totally prevented by conditioned medium from QUIN-treated astrocytes. Cocultured astrocytes and neurons interacted with one another reciprocally, protecting them against QUIN injury. Cocultured cells preserved their cytoskeletal organization and cell morphology together with unaltered activity of the phosphorylating system associated with the cytoskeleton. This article describes cytoskeletal disruption as one of the most relevant actions of QUIN toxicity in striatal neurons in culture with soluble factors secreted by astrocytes, with neuron-astrocyte interaction playing a role in neuroprotection.

Chemical activation in Rhinella arenarum oocytes: effect of dehydroleucodine (DhL) and its hydrogenated derivative (2H-DhL).

Mature oocytes are arrested in metaphase II due to the presence of high levels of active maturation promoting factor (MPF). After fertilization, active MPF levels decline abruptly, enabling oocytes to complete meiosis II. One of the first and universal events of oocyte activation is an increase in cytosolic Ca2+ that would be responsible for MPF inactivation. Mature oocytes can also be activated by parthenogenetic activation. The aims of this work are to test the ability of dehydroleucodine (DhL) and its hydrogenated derivative 11,13-dihydro-dehydroleucodine (2H-DhL) to induce chemical activation in amphibian oocytes and to study the participation of calcium in the process. Results indicated that DhL and 2H-DhL induced oocyte activation in a dose-dependent manner. After 90 min of treatment, DhL 36 µM was able to induce 95% activation, while 2H-DhL 36 µM was less active, with only 40% activation. Our results suggest that DhL induced the inhibition of MPF activity, probably by an increase in intracellular Ca2+ concentration. Extracellular Ca2+ would not be significant, although Ca2+ release from intracellular stores is critical. In this sense, IP3Rs and RyRs were involved in the Ca2+ transient induced by lactones. In this species, RyRs appears to be the largest contributor to Ca2+ release in DhL-induced activation. Although more studies are needed on the mechanism of action through which these lactones induce oocyte activation in Rhinella arenarum, the results of this research provide interesting perspectives for the use of these lactones as chemical activators in in vitro fertilization and cloning.

Characterization of cadmium plasma membrane transport in gills of a mangrove crab Ucides cordatus.

Membrane pathway for intracellular cadmium (Cd(2+)) accumulation is not fully elucidated in many organisms and has not been studied in crab gill cells. To characterize membrane Cd(2+) transport of anterior and posterior gill cells of Ucides cordatus, a hypo-hyper-regulating crab, a change in intracellular Cd(2+) concentration under various experimental conditions was examined by using FluoZin, a fluorescent probe. The membrane Cd(2+) transport was estimated by the augmentation of FluoZin fluorescence induced by extracellular application of CdCl2 and different inhibitors. Addition of extracellular calcium (Ca(2+)) to the cells affected little the fluorescence of FluoZin, confirming that Cd(2+) was the main ion increasing intracellular fluorescence. Ca(2+) channels blockers (nimodipine and verapamil) decreased Cd(2+) influx as well as vanadate, a Ca(2+)-ATPase blocker. Chelating intracellular Ca(2+) (BAPTA) decreased Cd(2+) influx in gill cells, while increasing intracellular Ca(2+) (caffeine) augmented Cd influx. Cd(2+) and ATP added at different temporal conditions were not effective at increasing intracellular Cd(2+) accumulation. Ouabain (Na(+)/K(+)-ATPase inhibitor) increased Cd(2+) influx probably through a change in intracellular Na and/or a change in cell membrane potential. Routes of Cd(2+) influx, a non-essential metal, through the gill cell plasma membrane of crabs are suggested.

Mechanisms underlying midazolam-induced peripheral nerve block and neurotoxicity.

BACKGROUND AND OBJECTIVES: The benzodiazepine midazolam has been reported to facilitate the actions of spinally administrated local anesthetics. Interestingly, despite the lack of convincing evidence for the presence of γ-aminobutyric acid type A (GABAA) receptors along peripheral nerve axons, midazolam also has been shown to have analgesic efficacy when applied alone to peripheral nerves.These observations suggest midazolam-induced nerve block is due to another site of action. Furthermore, because of evidence indicating that midazolam has equal potency at the benzodiazepine site on the GABAA receptor and the 18-kd translocator protein (TSPO), it is possible that at least the nerve-blocking actions of midazolam are mediated by this alternative site of action. METHODS: We used the benzodiazepine receptor antagonist flumazenil, and the TSPO antagonist PK11195, with midazolam on rat sciatic nerves and isolated sensory neurons to determine if either receptor mediates midazolam-induced nerve block and/or neurotoxicity. RESULTS: Midazolam (300 µM)-induced block of nerve conduction was reversed by PK11195 (3 µM), but not flumazenil (30 µM). Midazolam-induced neurotoxicity was blocked by neither PK11195 nor flumazenil. Midazolam also causes the release of Ca from internal stores in sensory neurons, and there was a small but significant attenuation of midazolam-induced neurotoxicity by the Ca chelator, BAPTA. BAPTA (30 µM) significantly attenuated midazolam-induced nerve block. CONCLUSIONS: Our results indicate that processes underlying midazolam-induced nerve block and neurotoxicity are separable, and suggest that selective activation of TSPO may facilitate modality-selective nerve block while minimizing the potential for neurotoxicity.

Fertilization induces a transient exposure of phosphatidylserine in mouse eggs.

Phosphatidylserine (PS) is normally localized to the inner leaflet of the plasma membrane and the requirement of PS translocation to the outer leaflet in cellular processes other than apoptosis has been demonstrated recently. In this work we investigated the occurrence of PS mobilization in mouse eggs, which express flippase Atp8a1 and scramblases Plscr1 and 3, as determined by RT-PCR; these enzyme are responsible for PS distribution in cell membranes. We find a dramatic increase in binding of flouresceinated-Annexin-V, which specifically binds to PS, following fertilization or parthenogenetic activation induced by SrCl2 treatment. This increase was not observed when eggs were first treated with BAPTA-AM, indicating that an increase in intracellular Ca(2+) concentration was required for PS exposure. Fluorescence was observed over the entire egg surface with the exception of the regions overlying the meiotic spindle and sperm entry site. PS exposure was also observed in activated eggs obtained from CaMKIIγ null females, which are unable to exit metaphase II arrest despite displaying Ca(2+) spikes. In contrast, PS exposure was not observed in TPEN-activated eggs, which exit metaphase II arrest in the absence of Ca(2+) release. PS exposure was also observed when eggs were activated with ethanol but not with a Ca(2+) ionophore, suggesting that the Ca(2+) source and concentration are relevant for PS exposure. Last, treatment with cytochalasin D, which disrupts microfilaments, or jasplakinolide, which stabilizes microfilaments, prior to egg activation showed that PS externalization is an actin-dependent process. Thus, the Ca(2+) rise during egg activation results in a transient exposure of PS in fertilized eggs that is not associated with apoptosis.

The cytotoxic and proapoptotic activities of hypnophilin are associated with calcium signaling in UACC-62 cells.

Hypnophilin (HNP) is a sesquiterpene that is isolated from Lentinus cf. strigosus and has cytotoxic activities. Here, we studied the calcium signaling and cytotoxic effects of HNP in UACC-62 cells, a human skin melanoma cell line. HNP was able to increase the intracellular calcium concentration in UACC-62 cells, which was blocked in cells stimulated in Ca(2+) -free media. HNP treatment with BAPTA-AM, an intracellular Ca(2+) chelator, caused an increase in calcium signals. HNP showed cytotoxicity against UACC-62 cells in which it induced DNA fragmentation and morphological alterations, including changes in the nuclear chromatin profile and increased cytoplasmatic vacuolization, but it had no effect on the plasma membrane integrity. These data suggest that cytotoxicity in UACC-62 cells, after treatment with HNP, is associated with Ca(2+) influx. Together, these findings suggest that HNP is a relevant tool for the further investigation of new anticancer approaches.

Diacylglycerol kinase epsilon in bovine and rat photoreceptor cells. Light-dependent distribution in photoreceptor cells.

The present study shows the selective light-dependent distribution of 1,2-diacylglycerol kinase epsilon (DAGKɛ) in photoreceptor cells from bovine and albino rat retina. Immunofluorescence microscopy in isolated rod outer segments from bleached bovine retinas (BBROS) revealed a higher DAGKɛ signal than that found in rod outer segments from dark-adapted bovine retinas (BDROS). The light-dependent outer segment localization of DAGKɛ was also observed by immunohistochemistry in retinas from albino rats. DAGK activity, measured in terms of phosphatidic acid formation from a) [(3)H]DAG and ATP in the presence of EGTA and R59022, a type I DAGK inhibitor, or b) [γ-(32)P]ATP and 1-stearoyl, 2-arachidonoylglycerol (SAG), was found to be significantly higher in BBROS than in BDROS. Higher light-dependent DAGK activity (condition b) was also found when ROS were isolated from dark-adapted rat retinas exposed to light. Western blot analysis of isolated ROS proteins from bovine and rat retinas confirmed that illumination increases DAGKɛ content in the outer segments of these two species. Light-dependent DAGKɛ localization in the outer segment was not observed when U73122, a phospholipase C inhibitor, was present prior to the exposure of rat eyecups (in situ model) to light. Furthermore, no increased PA synthesis from [(3)H]DAG and ATP was observed in the presence of neomycin prior to the exposure of bovine eyecups to light. Interestingly, when BBROS were pre-phosphorylated with ATP in the presence of 1,2-dioctanoyl sn-glycerol (di-C8) or phorbol dibutyrate (PDBu) as PKC activation conditions, higher DAGK activity was observed than in dephosphorylated controls. Taken together, our findings suggest that the selective distribution of DAGKɛ in photoreceptor cells is a light-dependent mechanism that promotes increased SAG removal and synthesis of 1-stearoyl, 2-arachidonoyl phosphatidic acid in the sensorial portion of this cell, thus demonstrating a novel mechanism of light-regulated DAGK activity in the photoreceptors of two vertebrate species.

Dose-dependent effects of angiotensin-(1-7) on the NHE3 exchanger and [Ca(2+)](i) in in vivo proximal tubules.

The acute direct action of angiotensin-(1-7) [ANG-(1-7)] on bicarbonate reabsorption (JHCO(3)(-)) was evaluated by stationary microperfusions on in vivo middle proximal tubules in rats using H ion-sensitive microelectrodes. The control JHCO(3)(-) is 2.82 ± 0.078 nmol·cm(-2)·s(-1) (50). ANG-(1-7) (10(-12) or 10(-9) M) in luminally perfused tubules decreases JHCO(3)(-) (36 or 60%, respectively), but ANG-(1-7) (10(-6) M) increases it (80%). A779 increases JHCO(3)(-) (30%) and prevents both the inhibitory and the stimulatory effects of ANG-(1-7) on it. S3226 decreases JHCO(3)(-) (45%) and changes the stimulatory effect of ANG-(1-7) to an inhibitory effect (30%) but does not affect the inhibitory effect of ANG-(1-7). Our results indicate that in the basal condition endogenous ANG-(1-7) inhibits JHCO(3)(-) and that the biphasic dose-dependent effect of ANG-(1-7) on JHCO(3)(-) is mediated by the Mas receptors via the Na(+)/H(+) exchanger 3 (NHE3). The control value of intracellular Ca(2+) concentration ([Ca(2+)](i)), as monitored using fura-2 AM, is 101 ± 2 nM (6), and ANG-(1-7) (10(-12), 10(-9), or 10(-6)M) transiently (3 min) increases it (by 151, 102, or 52%, respectively). A779 increases the [Ca(2+)](i) (25%) but impairs the stimulatory effect of all doses of ANG-(1-7) on it. The use of BAPTA or thapsigargin suggests a correlation between the ANG-(1-7) dose-dependent effects on [Ca(2+)](i) and JHCO(3)(-). Therefore, the interaction of the opposing dose-dependent effects of ANG II and ANG-(1-7) on [Ca(2+)](i) and JHCO(3)(-) may represent an physiological regulatory mechanism of extracellular volume and/or pH changes. However, whether [Ca(2+)](i) modification is an important direct mechanism for NHE3 activation by these peptides or is a side effect of other signaling pathways will require additional studies.

A study of store dependent Ca²âº influx in frog skeletal muscle.

Ca²âº influx across the plasma membrane upon drastic reduction of the sarcoplasmic reticulum Ca²âº content was studied in voltage clamped frog skeletal muscle fibers. Depletion was produced by the application of 30 µM cyclopiazonic acid (CPA) in Ca²âº-free, [Mg²âº] = 8 mM external salines and produced an increase in resting free myoplasmic [Ca²âº]. Once depletion was attained the external solution was changed to one containing the same concentration of the drug but with Ca²âº instead of Mg²âº. Of 27 fibers studied only nine showed a secondary increase in free myoplasmic [Ca²âº] upon readmitting Ca²âº in the external perfusate. In the presence of CPA the resting myoplasmic [Ca²âº] in Ca²âº-free external saline was 0.08 ± 0.01 µM (Mean ± SEM), and in Ca²âº-containing external saline 0.10 ± 0.02 µM when the intracellular solution contained [EGTA] = 5 mM (n = 18). In cells with lower (0.5 mM) intracellular [EGTA] resting [Ca²âº] went from 0.35 +/- 0.08 µM in Ca²âº-free external solution to 0.42 +/- 0.12 µM upon reapplication of Ca²âº(n = 9). In both cases the differences between means were not statistically significant (paired t test, p = 0.13 in high EGTA and p = 0.25 in low EGTA). In the nine fibers that showed a secondary increase of resting [Ca²âº] the holding current measured at -90 mV did not significantly change. These results suggest the Ca²âº entry secondary to store depletion is a labile mechanism in frog skeletal muscle and when present does not have an obvious electrical manifestation.

Extracellular Ca2+ influx is crucial for the early embryonic development of the sea urchin Echinometra lucunter.

The involvement of Ca(2+) in the activation of eggs and in the first steps of the embryonic development of several species is a well-known phenomenon. An association between Ca(2+) sources with the fate of the blastopore during embryonic development has been investigated by several authors. Ca(2+) influx mediated by voltage-gated channels and Ca(2+) mobilization from intracellular stores are the major sources of Ca(2+) to egg activation and succeeding cell divisions. Studies on sea urchins embryonic development show that intracellular Ca(2+) stores are responsible for egg activation and early embryogenesis. In the present work we investigated the involvement of extracellular Ca(2+) in the first stages of the embryonic development of the sea urchin Echinometra lucunter. Divalent cation chelators EDTA and EGTA strongly blocked the early embryonic development. Adding to this, we demonstrated the involvement of voltage-gated Ca(2+) channels in E. lucunter embryogenesis since Ca(2+) channel blockers powerfully inhibited the early embryonic development. Our data also revealed that Ca(2+) influx is crucial for embryonic development during only the first 40 min postfertilization. However, intracellular Ca(2+) remains mandatory to embryonic development 40 min postfertilization, seen that both the intracellular Ca(2+) chelator BAPTA-AM and calmodulin antagonists trifluoperazine and chlorpromazine inhibited the first stages of development when added to embryos culture 50 min postfertilization. Our work highlights the crucial role of extracellular Ca(2+) influx through voltage-gated Ca(2+) channels for the early embryonic development of the sea urchin E. lucunter and characterizes an exception in the phylum Echinodermata.

The involvement of calcium carriers and of the vacuole in the glucose-induced calcium signaling and activation of the plasma membrane H(+)-ATPase in Saccharomyces cerevisiae cells.

Previous work from our laboratories demonstrated that the sugar-induced activation of plasma membrane H(+)-ATPase in Saccharomyces cerevisiae is dependent on calcium metabolism with the contribution of calcium influx from external medium. Our results demonstrate that a glucose-induced calcium (GIC) transporter, a new and still unidentified calcium carrier, sensitive to nifedipine and gadolinium and activated by glucose addition, seems to be partially involved in the glucose-induced activation of the plasma membrane H(+)-ATPase. On the other hand, the importance of calcium carriers that can release calcium from internal stores was analyzed in glucose-induced calcium signaling and activation of plasma membrane H(+)-ATPase, in experimental conditions presenting very low external calcium concentrations. Therefore the aim was also to investigate how the vacuole, through the participation of both Ca(2+)-ATPase Pmc1 and the TRP homologue calcium channel Yvc1 (respectively, encoded by the genes PMC1 and YVC1) contributes to control the intracellular calcium availability and the plasma membrane H(+)-ATPase activation in response to glucose. In strains presenting a single deletion in YVC1 gene or a double deletion in YVC1 and PMC1 genes, both glucose-induced calcium signaling and activation of the H(+)-ATPase are nearly abolished. These results suggest that Yvc1 calcium channel is an important component of this signal transduction pathway activated in response to glucose addition. We also found that by a still undefined mechanism Yvc1 activation seems to correlate with the changes in the intracellular level of IP(3). Taken together, these data demonstrate that glucose addition to yeast cells exposed to low external calcium concentrations affects calcium uptake and the activity of the vacuolar calcium channel Yvc1, contributing to the occurrence of calcium signaling connected to plasma membrane H(+)-ATPase activation.

Reactive oxygen species and permeability transition pore in rat liver and kidney mitoplasts.

Mitochondrial permeability transition is typically characterized by Ca(2+) and oxidative stress-induced opening of a nonselective proteinaceous membrane pore sensitive to cyclosporin A, known as the permeability transition pore (PTP). Data from our laboratory provide evidence that the PTP is formed when inner membrane proteins aggregate as a result of disulfide cross-linking caused by thiol oxidation. Here we compared the redox properties between PTP in intact mitochondria and mitoplasts. The rat liver mitoplasts retained less than 5% and 10% of the original outer membrane markers monoamine oxidase and VDAC, respectively. Kidney mitoplasts also showed a partial depletion of hexokinase. In line with the redox nature of the PTP, mitoplasts that were more susceptible to PTP opening than intact mitochondria showed higher rates of H(2)O(2) generation and decreased matrix NADPH-dependent antioxidant activity. Mitoplast PTP was also sensitive to the permeability transition inducer tert-butyl hydroperoxide and to the inhibitors cyclosporin A, EGTA, ADP, dithiothreitol and catalase. Taken together, these data indicate that, in mitoplasts, PTP exhibits redox regulatory characteristics similar to those described for intact mitochondria.

Short-term synaptic plasticity regulates the level of olivocochlear inhibition to auditory hair cells.

In the mammalian inner ear, the gain control of auditory inputs is exerted by medial olivocochlear (MOC) neurons that innervate cochlear outer hair cells (OHCs). OHCs mechanically amplify the incoming sound waves by virtue of their electromotile properties while the MOC system reduces the gain of auditory inputs by inhibiting OHC function. How this process is orchestrated at the synaptic level remains unknown. In the present study, MOC firing was evoked by electrical stimulation in an isolated mouse cochlear preparation, while OHCs postsynaptic responses were monitored by whole-cell recordings. These recordings confirmed that electrically evoked IPSCs (eIPSCs) are mediated solely by α9α10 nAChRs functionally coupled to calcium-activated SK2 channels. Synaptic release occurred with low probability when MOC-OHC synapses were stimulated at 1 Hz. However, as the stimulation frequency was raised, the reliability of release increased due to presynaptic facilitation. In addition, the relatively slow decay of eIPSCs gave rise to temporal summation at stimulation frequencies >10 Hz. The combined effect of facilitation and summation resulted in a frequency-dependent increase in the average amplitude of inhibitory currents in OHCs. Thus, we have demonstrated that short-term plasticity is responsible for shaping MOC inhibition and, therefore, encodes the transfer function from efferent firing frequency to the gain of the cochlear amplifier.

Custom-made modification of a commercial confocal microscope to photolyze caged compounds using the conventional illumination module and its application to the observation of Inositol 1,4,5-trisphosphate-mediated calcium signals.

The flash photolysis of "caged" compounds is a powerful experimental technique for producing rapid changes in concentrations of bioactive signaling molecules. These caged compounds are inactive and become active when illuminated with ultraviolet light. This paper describes an inexpensive adaptation of an Olympus confocal microscope that uses as source of ultraviolet light the mercury lamp that comes with the microscope for conventional fluorescence microscopy. The ultraviolet illumination from the lamp (350 - 400 nm) enters through an optical fiber that is coupled to a nonconventional port of the microscope. The modification allows to perform the photolysis of caged compounds over wide areas (∼ 200 µm) and obtain confocal fluorescence images simultaneously. By controlling the ultraviolet illumination exposure time and intensity it is possible to regulate the amount of photolyzed compounds. In the paper we characterize the properties of the system and show its capabilities with experiments done in aqueous solution and in Xenopus Laevis oocytes. The latter demonstrate its applicability for the study of Inositol 1,4,5-trisphosphate-mediated intracellular calcium signals.