Effects of 3, 4-Dihydroxyacetophenone on Cytosolic Calcium in Pulmonary Artery Endothelial and Smooth Muscle Cells during Acute Hypoxia

Summary: The effects of 3, 4-Dihydroxyacetophenone (3, 4-DHAP) on cytosolic free calcium [Ca~2+ ]_i in pulmonary artery endothelia (PAECs) and smooth muscle cells (PASMCs) during acute hypoxia were studied. Porcine pulmonary artery endothelial and smooth muscle cells (PASMCs) were cultured primarily, and they were divided into 4 groups: groups incubated under normoxia or hypoxia and those with or without treatment with 3, 4-DHAP. The [Ca~2+ ]_i of both PAECs and PASMCs was measured by determining the fluorescence of fura 2 AM on spetrofluorometer. Our results showed that hypoxia caused significant elevation of [Ca~2+ ]_i, in both PAECs and PASMCs, 3, 4-DHAP could attenuate the hypoxic elevation of [Ca~2+ ]_i only in PASMCs but not in PAECs. It is concluded that 3, 4-DHAP decreases the hypoxic elevation of [Ca~2+ ]_i in PASMCs. This might contribute to its inhibitory effect on hypoxic pulmonary vasoconstriction.

Water Channels Are Involved in Stomatal Oscillations Encoded by Parameter-Specific Cytosolic Calcium Oscillations

Earlier studies have shown that various stimuli can induce specific cytosolic calcium （[Ca^2＋]cyt） oscillations in guard cells and various oscillations in stomatal apertures. Exactly how [Ca^2＋]cyt oscillation signaling functions in stomatal oscillation is not known. In the present study, the epidermis of broad bean （Vicia faba L.） was used and a rapid ion-exchange treatment with two shifting buffers differing in K^＋ and Ca^2＋ concentrations was applied. The treatment for fivetransients at a 10-min transient period induced clear and regular stomatal oscillation. However, for other transient numbers and periods, the treatments induced some Irregular oscillations or even no obvious oscillations in stomatal aperture. The results indicate that stomatal oscillation Is encoded by parameter-specific [Ca^2＋]cyt oscillation： the parameters of [Ca^2＋]cyt oscillation affected the occurrence rate and the parameters of stomatal oscillation. The water channel inhibitor HgCl2 completely Inhibited stomatal oscillation and the inhibitory effect could be partially reversed by β-mercaptoethanol （an agent capable of reversing water channel inhibition by HgCl2）. Other Inhibitory treatments against Ion transport （i.e. the application of LaCIs, EGTA, or tetraethylammonlum chloride （TEACI）） weakly impaired stomatal oscillation when the compounds were added after rapid ion-exchange treatment. If these compounds were added before rapid-ion exchange treatment, the inhibitory effect was much more apparent （except In the case of TEACI）. The results of the present study suggest that water channels are involved In stomatal oscillation as a downstream element of [Ca^2＋]cyt oscillation signaling.

Effect of Lanthanum(Ⅲ) on Cytosolic Free Calcium in Isolated Rabbit Mature Osteoclasts

The effect of lanthanum （ Ⅲ ） （La^3 ＋ ） on cytosolic free calcium （ [ Ca^2 ＋ ] i ） in isolated rabbit mature osteoclasts was studied with the employment of fluo-3/AM as an intracellular calcium-sensitive fluorescent probe by using a confocal laser scanning microscope. La^3＋ does not alter basal [Ca^2＋ ]i levels and cell spread area at the concentration of 1.00 × 10^- 8 mol· L ^- 1. However, La^3 ＋ at higher concentrations （ 1. 00 × 10^ - 5 and 1.00 × 10^- 7 mol· L^- 1 ） decreases [ Ca^2 ＋ ] i levels and cell spread area, and greater decreases are observed for the higher concentrations of La^3 ＋ . Since [Ca^2 ＋ ]i affects cytoskeleton and the adhesion properties of osteoclasts, our results seem to suggest that La^3 ＋ inhibit bone resorption by decreasing [Ca^2＋]i in rabbit mature osteoclasts.

ATP Evokes Calcium Transients in Single Pulmonary Artery Endothelial Cell in

Effects of ATF on cytosclic free calcium ([Ca2+]i) in single porcine pulmonary artery endothelia cell were studied.Using a dual-wavelength excitation on microflurometry.it was found that ATP evoked a rapid transient in[Ca2+]i which was then followed by a maintained elevation of[Ca2+]i.The removal of extracellular Ca2+ abolished the maintained plateform, but exerted no obvious effect on the initial-transient.These results suggest that ATP stimulates both calcium release from intracellular calcium pool(s)and calcium influx across the Plasma membrane from extracellular space.

Arabidopsis transcriptional response to extracellular Ca^(2+) depletion involves a transient rise in cytosolic Ca^(2+)

Ecological evidence indicates a worldwide trend of dramatically decreased soil Ca2＋ levels caused by increased acid deposition and massive timber harvesting. Little is known about the genetic and cellular mechanism of plants＇ responses to Ca2＋ depletion. In this study, transcriptional profiling analysis helped identify multiple extracellular Ca2＋ （[Ca2＋]ext） depletion-responsive genes in Arabidopsis thaliana L., many of which are involved in response to other environmental stresses. Interestingly, a group of genes encoding putative cytosolic Ca2＋ （[Ca2＋]cyt） sensors were significantly upregulated, implying that [Ca2＋]cyt has a role in sensing [Ca2＋]ext depletion. Consistent with this observation, [Ca2＋]ext depletion stimulated a transient rise in [Ca2＋]cyt that was negatively influenced by [K＋]ext, suggesting the involvement of a membrane potential-sensitive component. The [Ca2＋]cyt response to [Ca2＋]ext depletion was significantly desensitized after the initial treatment, which is typical of a receptor-mediated signaling event. The response was insensitive to an animal Ca2＋ sensor antagonist, but was suppressed by neomycin, an inhibitor of phospholipase C. Gd3＋, an inhibitor of Ca2＋ channels, suppressed the [Ca2＋]ext-triggered rise in [Ca2＋]cyt and downstream changes in gene expression. Taken together, this study demonstrates that [Ca2＋]cyt plays an important role in the putative receptor-mediated cellular and transcriptional response to [Ca2＋]ext depletion of plant cells.

Comparative Detection of Calcium Fluctuations in Single Female Sex Cells of Tobacco to Distinguish Calcium Signals Triggered by in vitro Fertilization

Double fertilization is a key process of sexual reproduction in higher plants. The role of calcium in the activation of female sex cells through fertilization has recently received a great deal of attention. The establishment of a Ca^2＋-imaging technique for living, single, female sex cells is a difficult but necessary prerequisite for evaluating the role of Ca^2＋ in the transduction of external stimuli, including the fusion with the sperm cell, to internal cellular processes. The present study describes the use of Fluo-3 for reporting the Ca^2＋ signal in isolated, single, female sex cells, egg cells and central cells, of tobacco plants. A suitable loading protocol was optimized by loading the cells at pH 5.6 with 2 μM Fluo-3 for 30 min at 30 ℃. Under these conditions, several key factors related to in vitro fertilization were also investigated in order to test their possible effects on the [Ca^2＋]cyt of the female sex cells. The results indicated that the bovine serum albumin-fusion system was superior to the polyethlene glycol-fusion system for detecting calcium fluctuations in female sex cells during fertilization. The central cell was fertilized with the sperm cell in bovine serum albumin; however, no evident calcium dynamic was detected, implying that a transient calcium rise might be a specific signal for egg cell fertilization.

Mutation of the Arabidopsis Calmodulin-Like Protein CML37 Deregulates the Jasmonate Pathway and Enhances Susceptibility to Herbivory

Throughout their life, plants are challenged by various abiotic and biotic stress factors. Among those are attacks from herbivorous insects. The molecular mechanisms underlying the detection of herbivores and the subsequent signal transduction are not well understood. As a second messenger, fluxes in intracellular Ca2＋ levels play a key role in mediating stress response pathways. Ca2＋ signals are decoded by Ca2＋ sensor proteins such as calmodulin-like proteins （CMLs）. Here, we demonstrate that recombinant CML37 behaves like a Ca2＋ sensor in vitro and, in Arabidopsis, AtCML37 is induced by mechanical wounding as well as by infestation with larvae of the generalist lepidopteran herbivore Spodoptera littoralis. Loss of function of CML37 led to a better feeding performance of larvae suggesting that CML37 is a positive defense regulator. No herbivory-induced changes in secondary metabolites such as glucosinolates or flavonoids were detected in cml37 plants, although a significant reduction in the accumulation of jasmonates was observed, due to reduced expression of JAR1 mRNA and cellular enzyme activity. Consequently, the expression of jasmonate-responsive genes was reduced as well. Summarizing, our results suggest that the Ca2＋ sensor protein, CML37, functions as a positive regulator in Ca2＋ signaling during herbivory, connecting Ca2＋ and jasmonate signaling.

Dihydrosphingosine-lnduced Programmed Cell Death in Tobacco BY-2 Cells Is Independent of H2O2 Production

Sphinganine or dihydrosphingosine （d18：0, DHS）, one of the most abundant free sphingoid Long Chain Base （LCB） in plants, has been recently shown to induce both cytosolic and nuclear calcium transient increases and a correlated Programmed Cell Death （PCD） in tobacco BY-2 cells. In this study, in order to get deeper insight into the LCB signaling pathway leading to cell death, the putative role of Reactive Oxygen Species （ROS） has been investigated. We show that DHS triggers a rapid dose-dependent production of H2O2 that is blocked by diphenyleniodonium （DPI）, indicating the involvement of NADPH oxidase（s） in the process. In addition, while DPI does not block DHS-induced calcium increases, the ROS production is inhibited by the broad spectrum calcium channel blocker lanthanum （La^3＋）. Therefore, ROS production occurs downstream of DHS-induced Ca^2＋ transients. Interestingly, DHS activates expression of defense-related genes that is inhibited by both La^3＋ and DPI. Since DPI does not prevent DHS-induced cell death, these results strongly indicate that DHS-induced H2O2 production is not implicated in PCD mechanisms but rather would be associated to basal cell defense mechanisms.