Transient Calcium Increase Research Articles

Gene expression is differentially regulated by neurotransmitters in embryonic neuronal cortical culture

Neurotransmitters and their receptors have been involved in both proper brain development and neurodevelopmental disorders. The role that nicotinic receptors play in immature cortical neurons was initially investigated by gene profiling using Affymetrix DNA arrays. Both short (15 min) and prolonged (18 h) treatments with nicotine did not induce modification in gene expression, whereas a significant down-regulation of c-fos protein levels was observed after 18 h treatment. Conversely, a brief treatment with the glutamatergic agonist NMDA triggered up-regulation of immediate early genes and transcription factors, which remained unaffected by pre-treatment for 18 h with nicotine. Calcium imaging studies revealed that NMDA activated a sustained increase in intracellular calcium concentration in the majority of neurons, whereas nicotine evoked only a transient calcium increase in a smaller percentage of neurons, suggesting that the calcium signalling response was correlated with activation of gene expression. Nicotine effects on immature cortical neurons perhaps do not require gene regulation but may be still acting on signalling pathways.

Effect of lysophospholipids on signaling in the Human jurkat T Cell Line

AbstractLysophospholipids have recently been demonstrated to induce activation and proliferation of fibroblasts and other cell lineages by interacting with high affinity cell surface receptors leading to specific intracellular signaling events. Platelet activation, likely at the site of injury or inflammation, results in increased production of lysophospholipids suggesting a possible source of lysophospholipids. We have recently demonstrated that high concentrations of lysophospholipids are present in ascites and plasma from ovarian cancer patients, suggesting that physiologically produced lysophospholipids could interact with cells present in these fluids, including lymphocytes, and alter their function. We demonstrate herein that lysophosphatidic acid (LPA), lysophosphatidylserine (LPS), and sphingosylphosphorylcholine (SPC) activate the Jurkat T cell line. Each of the lysophospholipids induced a transient increase in cytosolic free calcium ([Ca2+]i) in Jurkat cells. Increases in [Ca2+]i were cross‐desensitized by LPA, LPS and SPC, suggesting that the lysophospholipids share the same receptor(s) or that their downstream signaling pathways converge or interact. Lysophosphatidylgycerol (LPG), a competitive inhibitor of the putative LPA receptor, inhibited the calcium releasing activity of LPA, but not that of LPS and SPC, suggesting that these lysophospholipids interact with different receptors and that desensitization is due to interactions in downstream signaling pathways. The ability of the lysophospholipids to induce increases in [Ca2+]i was attenuated, but not completely blocked, by increases in [Ca2+]i induced by activation of the thrombin receptor. In contrast, increases in [Ca2+]i induced by the lysophospholipids and cross‐linking the CD3 component of the T cell receptor complex with the UCHT1 antibody did not undergo heterologous desensitization. Strikingly, LPA is sufficient to stimulate proliferation of Jurkat cells in serum‐free medium or in synergy with low concentrations of fetal bovine serum. In addition, LPA also increased the production of the T cell growth factor, interleukin 2 (IL‐2), by Jurkat cells treated with phorbol esters. LPS, in contrast, inhibited Jurkat proliferation while increasing IL‐2 production and SPC inhibited both processes. Thus, although all three lysophospholipids were sufficient to induce a transient increase in [Ca2+]i in Jurkat cells, they induced markedly different physiological consequences. © 2005 Wiley‐Liss, Inc.

5-Lipoxygenase products regulate basophil functions: 5-Oxo-ETE elicits migration, and leukotriene B 4 induces degranulation

5-Lipoxygenase (5-LO) products have been strongly implicated in the pathogenesis of allergic diseases. In addition to their physiologic effects on residential cells, 5-LO products are capable of stimulating various eosinophil functions. However, little is known regarding the effects of 5-LO products on basophil functions. This study was designed to elucidate the effects of the main 5-LO products (ie, leukotriene [LT] B(4), LTD(4), and 5-oxo-6,8,11,14-eicosatetraenoic acid [5-oxo-ETE]), as well as their receptor expression on human basophils. We studied the effects of 5-LO products on Ca(2+) mobilization, migration, CD 11b expression, and degranulation of human basophils. Expression of the receptors for LTC(4)/D(4)/E(4) (cysteinyl leukotriene 1 [CysLT(1)] and CysLT(2)), LTB4 (BLT(1) and BLT(2)), and 5-oxo-ETE (oxoeicosanoid [OXE]) was assessed by means of real-time PCR and flow cytometry. At the mRNA level, basophils strongly expressed OXE and predominantly expressed CysLT(1) and BLT(2). The expression level of OXE mRNA in basophils was approximately 20-fold higher than in neutrophils and similar to that in eosinophils. At the protein level, basophils expressed CysLT(1), CysLT(2), BLT(1), and OXE, but not BLT(2). All products elicited a transient increase of cytosolic calcium, with the order of magnitude being LTB(4)>5-oxo-ETE>LTD(4). 5-Oxo-ETE induced a strong basophil migratory response that was almost equivalent to that of prostaglandin D(2). LTB(4) elicited significant degranulation of IL-3-primed basophils. In contrast, no functional significance was observed for LTD(4). Among 5-LO products, 5-oxo-ETE induces a potent basophil migratory response, and LTB(4) elicits degranulation under certain conditions. Our results strongly suggest that 5-oxo-ETE might afford opportunities for therapeutic targeting in allergic inflammation.

Inhibiting MAP Kinase Activity Prevents Calcium Transients and Mitosis Entry in Early Sea Urchin Embryos

A transient calcium increase triggers nuclear envelope breakdown (mitosis entry) in sea urchin embryos. Cdk1/cyclin B kinase activation is also known to be required for mitosis entry. More recently, MAP kinase activity has also been shown to increase during mitosis. In sea urchin embryos, both kinases show a similar activation profile, peaking at the time of mitosis entry. We tested whether the activity of both kinases is required for mitosis entry and whether either kinase controls mitotic calcium signals. We found that reducing the activity of either mitotic kinase prevents nuclear envelope breakdown, despite the presence of a calcium transient, when cdk1/cyclin B kinase activity is alone inhibited. When MAP kinase activity alone was inhibited, the calcium signal was absent, suggesting that MAP kinase activity is required to generate the calcium transient that triggers nuclear envelope breakdown. However, increasing intracellular free calcium by microinjection of calcium buffers or InsP(3) while MAP kinase was inhibited did not itself induce nuclear envelope breakdown, indicating that additional MAP kinase-regulated events are necessary. After MAP kinase inhibition early in the cell cycle, the early events of the cell cycle (pronuclear migration/fusion and DNA synthesis) were unaffected, but chromosome condensation and spindle assembly are prevented. These data indicate that in sea urchin embryos, MAP kinase activity is part of a signaling complex alongside two components previously shown to be essential for entry into mitosis: the calcium transient and the increase in cdk1/cyclinB kinase activity.

Exploring Interactions between Pollutant Emissions and Climatic Variability in Growth of Red Spruce in the Great Smoky Mountains National Park

Concern exists as to the status of red spruce (Picea rubens Sarg.) in the Great Smoky Mountains, with evidence both for and against an unprecedented decline in radial growth during the past century. On the basis of a dendrological record from 1850 to 1998, our analyses support a decline in radial growth starting as early as the 1940s through to the 1970s; in the 1970s there was a reversal of this decline. In comparing trees near ridges (2000 m) with those in draws (1500 m), we found differences in the (a) timing of the decline, (b) rate of decline, and (c) homogeneity of the decline, with trees near ridges showing earlier, faster, and more homogeneous declines than trees in draws. We hypothesized that changes in climatic conditions and/or atmospheric pollutants, both of which changed beyond ranges of natural variability, were related to the observed decline in radial growth. In trees near ridges, up to 67.1% of changes in radial growth could be explained by a combination of climatic conditions (7.6%) and annual emissions of nitric oxides (NOx) and sulfur dioxide (SO2) (an additional 59.5%). In trees from draws, up to 38.3% of the changes in radial growth could be explained by climatic conditions only. A conceptual model is presented, where trees in naturally acidic soils with low base saturation provide a sensitive signal for the changing nature of acidic pollutants, but trees in anthropogenically acidifying soils with an initially higher baser saturation provide a signal that is confounded by a transient increase of calcium (Ca) and magnesium (Mg) in the soil that results in a transient increase in radial growth.

Influence of Na+-independent Cl--HCO3- exchange on the slow force response to myocardial stretch.

Previous work demonstrated that the slow force response (SFR) to stretch is due to the increase in calcium transients (Ca2+T) produced by an autocrine-paracrine mechanism of locally produced angiotensin II/endothelin activating Na+-H+ exchange. Although a rise in pHi is presumed to follow stretch, it was observed only in the absence of extracellular bicarbonate, suggesting pHi compensation through the Na+-independent Cl--HCO3- exchange (AE) mechanism. Because available AE inhibitors do not distinguish between different bicarbonate-dependent mechanisms or even between AE isoforms, we developed a functional inhibitory antibody against both the AE3c and AE3fl isoforms (anti-AE3Loop III) that was used to explore if pHi would rise in stretched cat papillary muscles superfused with bicarbonate after AE3 inhibition. In addition, the influence of this potential increase in pHi on the SFR was analyzed. In this study, we present evidence that cancellation of AE3 isoforms activity (either by superfusion with bicarbonate-free buffer or with anti-AE3Loop III) results in pHi increase after stretch and the magnitude of the SFR was larger than when AE was operative, despite of similar increases in [Na+]i and Ca2+T under both conditions. Inhibition of reverse mode Na+-Ca2+ exchange reduced the SFR to the half when the AE was inactive and totally suppressed it when AE3 was active. The difference in the SFR magnitude and response to inhibition of reverse mode Na+-Ca2+ exchange can be ascribed to a pHi-induced increase in myofilament Ca2+ responsiveness.

Evidence for inositol triphosphate as a second messenger for glucose-induced calcium signalling in budding yeast.

The Saccharomyces cerevisiae phospholipase C Plc1 is involved in cytosolic transient glucose-induced calcium increase, which also requires the Gpr1/Gpa2 receptor/G protein complex and glucose hexokinases. Differing from mammalian cells, this increase in cytosolic calcium concentration is mainly due to an influx from the external medium. No inositol triphosphate receptor homologue has been identified in the S. cerevisiae genome; and, therefore, the transduction mechanism from Plc1 activation to calcium flux generation still has to be identified. Inositol triphosphate (IP(3)) in yeast is rapidly transformed into IP(4) and IP(5) by a dual kinase, Arg82. Then another kinase, Ipk1, phosphorylates the IP(5) into IP(6). In mutant cells that do not express either of these kinases, the glucose-induced calcium signal was not only detectable but was even wider than in the wild-type strain. IP(3) accumulation upon glucose addition was completely absent in the plc1Delta strain and was amplified both by deletion of either ARG82 or IPK1 genes and by overexpression of PLC1. These results taken together suggest that Plc1p activation by glucose, leading to cleavage of PIP(2) and generation of IP(3), seems to be sufficient for raising the calcium level in the cytosol. This is the first indication for a physiological role of IP(3) signalling in S. cerevisiae. Many aspects about the signal transduction mechanism and the final effectors require further study.

Mechanism of Group IVA Cytosolic Phospholipase A2 Activation by Phosphorylation

Group IVA cytosolic phospholipase A2 (cPLA2) has been shown to play a critical role in the agonist-induced release of arachidonic acid. To understand the mechanism by which phosphorylation of Ser505 and Ser727 activates cPLA2, we systematically analyzed the effects of S505A, S505E, S727A, S727E, S505A/S727A, S505A/S727E, and S505E/S727E mutations on its enzyme activity and membrane affinity. In vitro membrane binding measurements showed that S505A has lower affinity than the wild type or S505E for phosphatidylcholine membranes, which is exclusively due to faster desorption of the membrane-bound S505A. In contrast, neither S727A nor S727E mutation had a significant effect on the phosphatidylcholine vesicle binding affinity of cPLA2. The difference in in vitro membrane affinity between wild type (or S505E) and S505A increased with the decrease in Ca2+ concentration, reaching >60-fold at 2.5 microm Ca2+. When HEK293 cells transfected with cPLA2 and mutants were stimulated with ionomycin, the wild type and S505E translocated to the perinuclear region and caused the arachidonic acid release at 0.4 microm Ca2+, whereas S505A showed no membrane translocation and little activity to release arachidonic acid. Further mutational analysis of hydrophobic residues in the active site rim (Ile399, Leu400, and Leu552) indicate that a main role of the Ser505 phosphorylation is to promote membrane penetration of these residues, presumably by inducing a conformational change of the protein. These enhanced hydrophobic interactions allow the sustained membrane interaction of cPLA2 in response to transient calcium increases. On the basis of these results, we propose a mechanism for cPLA2 activation by calcium and phosphorylation.

Ceramide increases mitochondrial free calcium levels via caspase 8 and Bid: role in initiation of cell death

We investigated how the mitochondrial phase of ceramide-mediated cell death is initiated in nerve growth factor (NGF)-differentiated PC12 cells. We distinguished three independent effects of ceramide: free radical production; a transient increase in cytosolic free calcium; and a long-lasting increase in mitochondrial free calcium. Only the latter led to cell death, which could be prevented by buffering of mitochondrial calcium with the calcium binding protein calbindin D-28K ectopically expressed in mitochondria. We showed that mitochondrial calcium did not increase as a result of the increase in cytosolic free calcium levels. Rather, it appears to derive from the endoplasmic reticulum (ER) since dantrolene, which inhibits release of calcium from ER into cytosol through ryanodine receptors, prevented the increase in cytosolic free calcium but potentiated the increase in mitochondrial free calcium. This suggests that a transfer of calcium occurs directly, or very locally, between the two organelles. This transfer implicated activation of caspase 8 and cleavage of its substrate Bid, a previously unknown function of these cell death intermediaries. The increase in mitochondrial free calcium was also responsible for the release of cytochrome c into the cytosol, underlining the critical role it plays in ceramide-mediated cell death.

IL-12-dependent nuclear factor-κB activation leads to de novo synthesis and release of IL-8 and TNF-α in human neutrophils

The cytokine interleukin (IL)-12 plays a bridging role between innate and adaptive immunity. Here, we demonstrate that treatment of neutrophils with IL-12 leads to a transient increase in intracellular-free calcium [Ca(+)(+)](i) levels, which is necessary for the production of reactive oxygen metabolites (ROM). This production is associated with the activation and nuclear translocation of the transcription factor nuclear factor (NF)-kappaB and is inhibited in the presence of the intracellular calcium chelator 1,2-bis(O-amminophenoxy) ethane-N,N-N',N'-tetraacetic acid-acetoxymethyl ester and the ROM production inhibitor diphenyl iodonium. We show that IL-12 causes a significant increase in total mRNA levels, which appear dependent on the generated ROM. In addition IL-12 induces the de novo synthesis and production of IL-8 and tumor necrosis factor alpha (TNF-alpha) in a calcium- and ROM-dependent manner. Our data demonstrate a direct role for IL-12 in the activation of human neutrophils and suggest a ROM-dependent interplay between IL-12-induced [Ca(+)(+)](i) transient and the release of IL-8 and TNF-alpha through NF-kappaB activation.

Calcium channel subtypes contributing to acetylcholine release from normal, 4-aminopyridine-treated and myasthenic syndrome auto-antibodies-affected neuromuscular junctions.

1 Acetylcholine release at the neuromuscular junction relies on rapid, local and transient calcium increase at presynaptic active zones, triggered by the ion influx through voltage-dependent calcium channels (VDCCs) clustered on the presynaptic membrane. Pharmacological investigation of the role of different VDCC subtypes (L-, N-, P/Q- and R-type) in spontaneous and evoked acetylcholine (ACh) release was carried out in adult mouse neuromuscular junctions (NMJs) under normal and pathological conditions. 2 omega-Agatoxin IVA (500 nM), a specific P/Q-type VDCC blocker, abolished end plate potentials (EPPs) in normal NMJs. However, when neurotransmitter release was potentiated by the presence of the K(+) channel blocker 4-aminopyridine (4-AP), an omega-agatoxin IVA- and omega-conotoxin MVIIC-resistant component was detected. This resistant component was only partially sensitive to 1 micro M omega-conotoxin GVIA (N-type VDCC blocker), but insensitive to any other known VDCC blockers. Spontaneous release was dependent only on P/Q-type VDCC in normal NMJs. However, in the presence of 4-AP, it relied on L-type VDCCs too. 3 ACh release from normal NMJs was compared with that of NMJs of mice passively injected with IgGs obtained from patients with Lambert-Eaton myasthenic syndrome (LEMS), a disorder characterized by a compromised neurotransmitter release. Differently from normal NMJs, in LEMS IgGs-treated NMJs an omega-agatoxin IVA-resistant EPP component was detected, which was only partially blocked by calciseptine (1 micro M), a specific L-type VDCC blocker. 4 Altogether, these data demonstrate that multiple VDCC subtypes are present at the mouse NMJ and that a resistant component can be identified under 'pharmacological' and/or 'pathological' conditions.

Cholecystokinin increases cytosolic calcium in a subpopulation of cultured vagal afferent neurons.

Imaging fluorescent measurements with fura 2 were used to examine cytosolic calcium signals induced by sulfated CCK octapeptide (CCK-8) in dissociated vagal afferent neurons from adult rat nodose ganglia. We found that 40% (184/465) of the neurons responded to CCK-8 with a transient increase in cytosolic calcium. The threshold concentration of CCK-8 for inducing the response varied from 0.01 to 100 nM. In most neurons (13/16) the response was eliminated by removing extracellular calcium. Depleting intracellular calcium stores with thapsigargin slightly augmented the response. Most neurons were unresponsive to nonsulfated CCK-8. The response was eliminated by the CCK-A receptor antagonist lorglumide. Low concentrations of JMV-180 had no effect; however, high concentrations of JMV-180 reduced responses to CCK-8. These results demonstrate that CCK acts at the low-affinity site of the CCK-A receptor to trigger the entry of extracellular calcium into vagal afferent neurons. Increased cytosolic calcium may participate in acute activation of vagal afferent neurons, or it may initiate long-term changes, which modulate future neuronal responses to sensory stimuli.

Nitric Oxide Signals Parallel Fiber Activity to Bergmann Glial Cells in the Mouse Cerebellar Slice

Stimulation of parallel fibers in the cerebellar cortex triggers a transient calcium increase in Bergmann glial cells, a special form of astrocytes. Using patch-clamping and imaging techniques we have found that this form of neuron–glia interaction is mediated by nitric oxide (NO) since the response is blocked by the NO-synthase inhibitor Nω-nitro-l-arginine and mimicked by NO donors. None of the neurotransmitter receptors of Bergmann glia identified so far participates in or interferes with this signaling cascade. The NO-triggered increases in [Ca2+]i, as studied in Bergmann glial cells in the slice or in cultured astrocytes, are due to Ca2+ influx and not to release from cytoplasmic stores. Thus, NO released from parallel fibers serves as a signaling substance to the neighboring glial elements.

Novel G protein-coupled responses in leukocytes elicited by a chemotactic bacteriophage displaying a cell type-selective binding peptide.

Recently, we identified a neutrophil-binding phage displaying a novel peptide motif, GPNLTGRW. It was determined that this peptide, when displayed on bacteriophage (FGP phage), elicits a transient increase in cytosolic calcium. Here, we show that FGP phage stimulate neutrophil chemotaxis and induce a pertussis toxin-sensitive rise in cytosolic calcium in monocytes as well as in neutrophils. In contrast to the calcium response elicited by classical chemoattractants fMLP and IL-8, the FGP phage-elicited response in neutrophils is dependent on extracellular calcium and is mediated by receptor-activated, divalent cation channels. Consistent with G protein-coupled receptor signaling, FGP phage effect homologous and reciprocal heterologous desensitization with fMLP- and IL-8-stimulated calcium responses. Like non-G protein-coupled responses, the FGP-elicited calcium transient is abolished with phosphoinositide-3-kinase inactivation. Nonetheless, specific binding of GTP to neutrophil membranes follows stimulation with FGP phage, further supporting involvement of G proteins. However, FGP phage neither bind to nor elicit a calcium response from transfectant cells harboring known candidate G protein-coupled receptors. These data together suggest that the elicited responses are mediated by a novel G protein-coupled receptor or represent novel responses of a known receptor.

Relation between echinocytosis and erythrocyte calcium content in hemodialyzed uremic patients.

A rise in intracellular calcium concentration in erythrocytes has multiple effects on these cells. The purpose of this study was to determine the changes of calcium content in red blood cells (RBCs) and of echinocyte percentages in uremic patients during hemodialysis sessions. In 30 uremic patients under hemodialysis, the calcium content of RBCs and echinocyte percentages were determined in 3 blood samples collected at 0 min hemodialysis (prehemodialysis), 45 min hemodialysis, and 240 min hemodialysis (end hemodialysis) for a 4 h hemodialysis session. Calcium content of RBCs and echinocytes were also determined in 22 normal subjects (controls). The findings of the present study were that the mean values (+/-SD) of calcium content of RBCs in patients at 0 min hemodialysis, 45 min hemodialysis, and 240 min hemodialysis were 2.00 +/- 1.0, 2.66 +/- 0.87, and 1.62 +/- 0.66 microg/ml respectively and 0.65 +/- 0.07 microg/ml in controls. These values show that the calcium content of RBCs in uremic patients at 0 min hemodialysis, 45 min hemodialysis, and 240 hemodialysis was significantly higher than in controls (p < 0.0001), and that RBC calcium content at 45 min hemodialysis was significantly higher in comparison to that at 0 min hemodialysis (p < 0.001) and to that at 240 min hemodialysis (p < 0.0001), while that at 240 min hemodialysis was significantly lower than at 0 min hemodialysis (p < 0.05). The mean values (+/-SD) of echinocyte percentages in patients at 0 min hemodialysis, 45 min hemodialysis, and 240 hemodialysis were 11.93 +/- 6.18, 17.23 +/- 4.1, and 7.96 +/- 5.67% respectively, and in controls ranged from 0 to 1%. The values in uremic patients show a transient increase of echinocyte percentages at 45 min hemodialysis, which is significant in comparison to that at 0 min hemodialysis (p < 0.001) and to that at 240 min hemodialysis (p < 0.0001). Echinocyte percentages at 240 min hemodialysis were significantly lower to those at 0 min hemodialysis (p < 0.001). Correlation between calcium content of erythrocytes and echinocyte percentages shows a significantly positive relationship at 45 min hemodialysis (r = 0.368, p < 0.05) but no significant relationship at 0 min hemodialysis and 240 min hemodialysis. In conclusion, uremic patients under hemodialysis present with high calcium content in erythrocytes and abnormal erythrocytes like echinocytes. A rapid and transient increase of erythrocyte calcium is also accompanied by transient elevation of echinocytes in the first hour of hemodialysis (45 min hemodialysis), which returns after hemodialysis to lower than prehemodialysis levels.

A moderate decrease in temperature inhibits the calcium signaling mechanism(s) of the regulatory volume decrease in chick embryo cardiomyocytes.

Chick cardiomyocytes, when submitted to hyposmotic swelling, exhibit a partial regulatory volume decrease (RVD). A Ca2+ influx by stretch-activated channels signals a taurine efflux and the RVD at 37 degrees C. We evaluated the cell's performance at room temperature. Cardiomyocytes isolated and cultured from 11-day-old chick embryos were submitted to a hyposmotic solution (180 mOsm/kg H2O) at 37 degrees C and at room temperature (26 degrees C). Under these conditions we measured the changes in cell volume as well as the intracellular free Ca2+ (using fura-2). During hyposmotic swelling, cells at 37 degrees C displayed a peak relative volume of 1.61 +/- 0.03 and recovery to 1.22 +/- 0.04 (N = 14), while cells at 26 degrees C presented a peak swell relative volume of 1.74 +/- 0.06 and did not recover (1.59 +/- 0.09, N = 9). Transient increases in intracellular Ca2+, which are characteristic of the normal RVD, were observed at both temperatures (29.1 +/- 4.5% (N = 8) and 115.2 +/- 42.8% (N = 5) increase at 37 degrees and 26 degrees C (P<0.05), respectively). A delay in the Ca2+ transient increase was also observed when the cells were at 26 degrees C (109 +/- 34 s compared to 38 +/- 9 s at 37 degrees C, P<0.05). At room temperature the RVD does not occur because the calcium transient increase, which is an early event in the signaling of the RVD, is delayed. Also, free calcium is not cleared as in the 37 degrees C RVD. In the normal RVD the free calcium returns to baseline levels. The very high and persistent free calcium levels seen at room temperature can lead to unregulated enzyme activities and may promote irreversible injury and cell death.

Fetal rabbit pulmonary artery smooth muscle cell response to ryanodine is developmentally regulated.

To study developmental changes in intracellular calcium handling in pulmonary artery smooth muscle cells (PASMCs), cells were isolated from distal and proximal pulmonary arteries from rabbits at different developmental stages: juvenile (4-6 wk old), newborn (<48 h), and full-term fetal. Isolated PASMCs were studied using the calcium-sensitive dye fura 2. Cells from each age group responded to caffeine with an increase in calcium; however, ryanodine (50 microM) only increased calcium in fetal distal PASMCs. The ryanodine-induced increase was due to influx of extracellular calcium because it was blocked by removal of extracellular calcium or by diltiazem. The calcium-sensitive potassium (K(Ca)) channel blocker iberiotoxin produced a transient increase in calcium in the fetal distal PASMCs, which could be inhibited by prior application of ryanodine. Conversely, the ryanodine response was inhibited if iberiotoxin was given first. With the use of electrophysiology and confocal microscopy, fetal PASMCs were shown to exhibit spontaneous transient outward currents and calcium sparks, respectively. These observations suggest that ryanodine-sensitive release of calcium from the sarcoplasmic reticulum and K(Ca) channels act together to control intracellular calcium only in fetal distal PASMCs.

Methods for analysis of Ca 2+/H + antiport activity in synaptic vesicles isolated from sheep brain cortex

The involvement of Ca 2+-storage organelles in the modulation of synaptic transmission is well-established [M.K. Bennett, Ca 2+ and the regulation of neurotransmitter secretion, Curr. Opin. Neurobiol. 7 (1997) 316–322 [1]; M.J. Berridge, Neuronal calcium signaling, Neuron 21 (1998) 13–26 [2]; Ph. Fossier, L. Tauc, G. Baux, Calcium transients and neurotransmitter release at an identified synapse, Trends Neurosci. 22 (1999) 161–166 [7]]. Various Ca 2+ sequestering reservoirs (mitochondria, endoplasmic reticulum and synaptic vesicles) have been reported at the level of brain nerve terminals [P. Kostyuk, A. Verkhratsky, Calcium stores in neurons and glia, Neuroscience 63 (1994) 381–404 [18]; V. Mizuhira, H. Hasegawa, Microwave fixation and localization of calcium in synaptic terminals using X-ray microanalysis and electron energy loss spectroscopy imaging, Brain Res. Bull. 43 (1997) 53–58 [21]; A. Parducz, Y. Dunant, Transient increase of calcium in synaptic vesicles after stimulation, Neuroscience 52 (1993) 27–33 [23]; O.H. Petersen, Can Ca 2+ be released from secretory granules or synaptic vesicles?, Trends Neurosci. 19 (1996) 411–413 [24]]. However, the knowledge of the specific contribution of each compartment for spatial and temporal control of the cytoplasmic Ca 2+ concentration requires detailed characterization of the Ca 2+ uptake and Ca 2+ release mechanisms by the distinct intracellular stores. In this work, we described rapid and simple experimental procedures for analysis of a Ca 2+/H + antiport system that transport Ca 2+ into synaptic vesicles at expenses of the energy of a ΔpH generated either by activity of the proton pump or by a pH jumping of the vesicles passively loaded with protons. This secondary active Ca 2+ transport system requires high Ca 2+ concentrations (>100 μM) for activation, it is dependent on the chemical component (ΔpH) of the proton electrochemical gradient across the synaptic vesicle membrane and its selectivity is essentially determined by the size of the transported cation [P.P. Gonçalves, S.M. Meireles, C. Gravato, M.G.P. Vale, Ca 2+–H +-Antiport activity in synaptic vesicles isolated from sheep brain cortex, Neurosci. Lett. 247 (1998) 87–90 [10]; P.P. Gonçalves, S.M. Meireles, P. Neves, M.G.P. Vale, Ionic selectivity of the Ca 2+/H + antiport in synaptic vesicles of sheep brain cortex, Mol. Brain Res. 67 (1999) 283–291 [11]; P.P. Gonçalves, S.M. Meireles, P. Neves, M.G.P. Vale, Synaptic vesicle Ca 2+/H + antiport: dependence on the proton electrochemical gradient, Mol. Brain Res. 71 (1999) 178–184 [12]]. The protocols described here allow to ascertain the characteristics of the Ca 2+/H + antiport in synaptic vesicles and, therefore, may be useful for clarification of the physiological role of synaptic vesicles in fast buffering of Ca 2+ at various sites of the neurotransmission machinery.Theme: Excitable membranes and synaptic transmission.Topic: Presynaptic mechanisms.

Light-induced apoptosis involves a defined sequence of cytoplasmic and nuclear calcium release in AlPcS4-photosensitized rat bladder RR 1022 epithelial cells.

Oxidative stress induced by light activation of photosensitizers is regarded to have a role in triggering cell death pathways during photodynamic therapy (PDT). Reactive oxygen species have been proposed to act as signal transduction molecules activating downstream reactions that lead to apoptosis. Mainly debated is the cooperating role of other signaling systems like calcium or pH. The present work contributes to this discussion by studying PDT effects in cell cultures of rat bladder epithelial cells for the hydrophilic tetrasulfonated aluminum phthalocyanine (AlPcS4). Cells were coincubated with the photosensitizer and the calcium-sensitive probe Fluo-3. The light-induced reactions were analyzed with a confocal laser scanning microscope. The dynamics of the process during light activation was observed with subcellular resolution. A transient calcium elevation during the irradiation process was detected, especially in the cell's nuclei, followed by a more sustained increase. The evaluation of the energy-dose-dependent phototoxicity after an incubation time with the photosensitizer of 1 and 24 h, showed enhanced phototoxicity when the drug was present for 24 h. Surprisingly, stimulation of cell proliferation was observed at very low light doses (at 0.2 J/cm2) when the drug was incubated for 24 h (cell viability 160%). Induction of apoptosis could be observed after irradiation with fluences between 1 and 3 J/cm2. Apoptotic cells were identified with fluorescein isothiocyanate-labeled Annexin V, which binds to phosphatidylserine after its translocation to the outer plasma membrane. In the presence of the antioxidant pyrrolidinedithiocarbamate the transient calcium elevation was totally inhibited, as was the subsequent translocation of PS. In contrast, N-acetyl-L-cysteine did not suppress the transient calcium increase. Our data might be consistent with calcium regulated processes during AlPcS4-PDT and the involvement of oxygen radicals.

Are there inositol 1,4,5-triphosphate (IP3) receptors in human sperm?

Calcium signaling plays important roles in mammalian fertilization, such as the calcium wave in egg and the acrosome reaction (AR) in sperm. The calcium wave is accompanied by a transient increase of cytosolic calcium, which is mediated by inositol 1, 4, 5-triphosphate (IP3) induced calcium release (IICR). During AR, it is commonly recognized that influx of extracellular calcium causes a dramatic increase of cytosolic calcium. Participation of IICR in the phenomena, however, remains unclear. To investigate whether IICR also participates in calcium mobilization during AR, the present study examined the existence of the IP3 receptor family (IP3R type 1, 2 and 3) in human sperm and their changes during the reaction. Immunoblot analyses showed the existence of IP3R types 1 and 3 in human sperm, but IP3R type 2 was undetectable. The expression of IP3R type 1 was diminished after AR and was also detected in the vesiculated membrane fragment which was released by the fusion of the plasma membrane and the outer acrosomal membrane during AR. In contrast, the expression of IP3R type 3 on the blot was similar before and after the reaction. Immunohistochemical observations suggested that IP3R type 1 localized in the anterior portion of the sperm head and the expression was decreased after AR. IP3R type 3 was observed in the posterior portion of the sperm head, midpiece, and tail, and little change was found even after AR. The [ 3H]-IP3 binding assay suggested stoichiometric interactions between IP3 and IP3Rs of nonacrosome reacted and acrosome reacted human sperm.