Catecholamine Secretion from Bovine Adrenal Chromaffin Cells: The Role of the Na+/Ca2+ Exchanger and the Intracellular Ca2+ Pool

Abstract: The role of the Na⁺/Ca²⁺ exchanger and intracellular nonmitochondrial Ca²⁺ pool in the regulation of cytosolic free calcium concentration ([Ca²⁺]_i) during catecholamine secretion was investigated. Catecholamine secretion and [Ca²⁺]_i were simultaneously monitored in a single chromaffin cell. After high-K⁺ stimulation, control cells and cells in which the Na⁺/Ca²⁺ exchange activity was inhibited showed similar rates of [Ca²⁺]_i elevation. However, the recovery of [Ca²⁺]_i to resting levels was slower in the inhibited cells. Inhibition of the exchanger increased the total catecholamine secretion by prolonging the secretion. Inhibition of the Ca²⁺ pump of the intracellular Ca²⁺ pool with thapsigargin caused a significant delay in the recovery of [Ca²⁺]_i and greatly enhanced the secretory events. These data suggest that both the Na⁺/Ca²⁺ exchanger and the thapsigargin-sensitive Ca²⁺ pool are important in the regulation of [Ca²⁺]_i and, by modulating the time course of secretion, are important in determining the extent of secretion.     

Endothelin-1 Increases Arachidonic Acid Release in C6 Glioma Cells Through a Potassium-Modulated Influx of Calcium

Abstract: Endothelin-1 (Et-1) but not a range of other receptor agonists stimulated the release of arachidonic acid (AA) in C6 glioma. Et-1 activation was concentration dependent and was inhibited by chelation of extracellular calcium. The calcium ionophores A23187 and ionomycin could also stimulate release of AA. Et-1 caused an early increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) followed by a sustained but lower plateau level. The sensitivity of the response to quinacrine, its dependence on Ca²⁺, and the demonstration of an increase in phospholipase A₂ (PLA₂) activity that was insensitive to dithiothreitol suggested that the release of AA was due to activation of cytosolic PLA₂ in the cells. Staurosporine, a protein kinase C (PKC) inhibitor, had no effect on Et-1-induced AA release but abolished that by phorbol 12-myristate 13-acetate, demonstrating that the Et-1 response was PKC independent. Raised levels of extracellular KCI inhibited both AA release and the increase in [Ca²⁺]_i triggered by Et-1, whereas valinomycin, which causes K⁺ efflux, not only caused a rapid rise in [Ca²⁺]_i but also caused AA mobilisation. The results therefore suggest that Et-1 activation of PLA₂ in this cell type requires calcium influx dependent on K⁺ efflux.     

Involvement of Calcium Influx in Muscarinic Cholinergic Regulation of Phospholipase C in Cerebellar Granule Cells

Abstract: Inositol phosphate accumulation on carbachol stimulation of rat cerebellar granule cells shows a marked dependence on factors affecting cytosolic Ca²⁺ concentration ([Ca²⁺]_c). After 5 min, potassium depolarisation caused a modest accumulation of inositol phosphates but augmented the response to carbachol by a factor of 2–3. These effects of potassium were dependent on an extracellular source of calcium and could be partially blocked by specific (nifedipine) and nonspecific (verapamil) calcium channel blockers. Measurements of [Ca²⁺]_c under a range of stimulatory conditions demonstrated a close correlation between the elevation of [Ca²⁺]_c and agonist-stimulated phospholipase C (PLC) activity. The maximal potentiation of carbachol-stimulated inositol phosphate accumulation was achieved using 20 m M KCl, which increased [Ca²⁺]_c from ∼20 to ∼75 n M , indicating the involvement of relatively low threshold Ca²⁺ channels and the high sensitivity of the relevant PLC to small changes in [Ca²⁺]_c. By contrast, increases in [Ca²⁺]_c induced by the Ca²⁺ ionophore ionomycin were associated with more modest and less potent effects on agonist-stimulated PLC. These results demonstrate a cooperative interaction between a receptor/G protein-regulated PLC and voltage-stimulated elevations of [Ca²⁺]_c, which may function to integrate ionotropic and metabotropic signalling mechanisms in cerebellar granule cells.     

The self-incompatibility response in Papaver rhoeas is mediated by cytosolic free calcium

The role of Ca²⁺ signalling during the self-incompatibility (SI) response in Papaver rhoeas L. has been investigated using Ca²⁺-sensitive dyes. Pollen tubes were micro-injected with Calcium Green-1 and cytosolic free calcium ([Ca²⁺]_i) imaged using laser scanning confocal microscopy (LSCM). Addition of incompatible stigmatic S-glycoproteins induced a transient increase in the level of [Ca²⁺]_i in pollen tubes. In contrast, no rise in [Ca²⁺]_i was detectable after addition of either compatible or heat-denatured incompatible stigmatic S-glycoproteins. The elevation of [Ca²⁺]_i was followed by the specific inhibition of pollen tube growth in incompatible reactions. It has been shown previously that gene expression in pollen tubes is switched on during an incompatible reaction. Since the [Ca²⁺]_i transient appeared to originate from the region where the nuclei are located, Ca²⁺ may be involved in locally regulating the expression of these genes. The photoactivation of caged Ca²⁺ to artificially elevate [Ca²⁺]_i resulted in the inhibition of pollen tube growth and thus mimicked the SI response. Taken together, the results provide an important link between a transient rise in [Ca²⁺]_i and the biological phenomenon of inhibition of pollen tube growth and demonstrate, for the first time, direct evidence that the SI response in P. rhoeas is mediated by [Ca²⁺]_i.     

Carbon dioxide induces increases in guard cell cytosolic free calcium

The hypothesis that increases in cytosolic free calcium ([Ca²⁺]_i) are a component of the CO₂ signal transduction pathway in stomatal guard cells of Commelina communis has been investigated. This hypothesis was tested using fura-2 fluorescence ratio photometry to measure changes in guard cell [Ca²⁺]_i in response to challenge with 700 µl l⁻¹ CO₂. Elevated CO₂ induced increases in guard cell [Ca²⁺]_i which were similar to those previously reported in response to abscisic acid. [Ca²⁺]_i returned to resting values following removal of the CO₂ and further application of CO₂ resulted in a second increase in [Ca²⁺]_i. This demonstrated that the CO₂-induced increases in [Ca²⁺]_i were stimulus dependent. Removal of extracellular calcium both prevented the CO₂-induced increase in [Ca²⁺]_i and inhibited the associated reduction in stomatal aperture. These data suggest that Ca²⁺ acts as a second messenger in the CO₂ signal transduction pathway and that an increase in [Ca²⁺]_i may be a requirement for the stomatal response to CO₂.     

Inhibition of Bradykinin-Induced Cytosolic Ca2+ Elevation by Muscarinic Stimulation Without Attenuation of Inositol 1,4,5-Trisphosphate Production in Human Neuroblastoma SK-N-BE(2)C Cells

Abstract: Cross talk between two phospholipase C (PLC)-linked receptor signalings was investigated in SK-N-BE(2)C human neuroblastoma cells. Sequential stimulation with two agonists at 5-min intervals was performed to examine the interaction between muscarinic and bradykinin (BK) receptors. Pretreatment of cells with a maximal effective concentration (5 µ M ) of BK did not affect the subsequent carbachol (CCh)-induced [Ca²⁺]_i rise, but CCh (1 m M ) pretreatment completely abolished the BK-induced [Ca²⁺]_i rise without inhibition of BK-induced inositol 1,4,5-trisphosphate (IP₃) generation. Thapsigargin (1 µ M ) pretreatment abolished the subsequent BK- and CCh-induced [Ca²⁺]_i rise, though it did not affect agonist-induced IP₃ generation. However, the addition of atropine at plateau phases of CCh-induced [Ca²⁺]_i rise and IP₃ production caused a rapid decline to the basal levels and then restored the [Ca²⁺]_i rise by BK. Treatment of cells with both CCh and BK at the same time showed additive effects in IP₃ production. However, the [Ca²⁺]_i rise induced by both agonists in the presence or absence of extracellular Ca²⁺ was the same as the responses triggered by CCh alone. The results suggest that each receptor or receptor-linked PLC activity is not influenced by pretreatment with the other agonist but IP₃-sensitive Ca²⁺ stores are shared by signal pathways from both receptors.     

Probable participation of phospholipase A2 reaction in the process of fertilization-induced activation of sea urchin eggs

In sea urchin eggs activated by sperm, A23187 or melittin, BPB (4-bromophenacyl bromide, a phospholipase A₂ inhibitor) blocked fertilization envelope formation and transient CN⁻-insensitive respiration in a concentration-dependent manner. BPB had virtually no effect on the increase in [Ca²⁺]_i, (cytosolic Ca²⁺ level), the activity of phosphorylase a and the rate of protein synthesis, as well as acid production and augmentation of CN⁻-sensitive respiration. BPB also inhibited fertilization envelope formation and augmentation of CN⁻-insensitive respiration induced by melittin. Melittin, known to be an activator of phospholipase A₂, induced the envelope formation, acid production, augmentation of CN⁻-insensitive and sensitive respiration, but did not cause any increase in [Ca²⁺]_i, the phosphorylase a activity and the rate of protein synthesis. An activation of phospholipase A₂ induced by Ca²⁺ or melittin seems to result in cortical vesicle discharge and production of fatty acids, which are to be utilized in CN⁻-insensitive lipid peroxidase reactions. Activation of other examined cell functions in eggs activated by sperm or A23187, probably results from Ca²⁺-triggered sequential reactions other than Ca²⁺-caused activation of phospholipase A₂.     

Inhibition of Bradykinin-Induced Calcium Increase by Phosphatase Inhibitors in Neuroblastoma × Glioma Hybrid NG108-15 Cells

Abstract: Prior treatment of NG108-15 cells with phosphatase inhibitors including okadaic acid and calyculin A inhibited the elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) induced by bradykinin by ∼63%. This inhibition was dependent on the concentration of okadaic acid with an IC₅₀ of 0.15 n M . Okadaic acid treatment only lowered the maximal response of [Ca²⁺]_i increase and had no effect on the EC₅₀ value for bradykinin regardless of the presence of extracellular Ca²⁺. Neither the capacity of ⁴⁵Ca²⁺ accumulation within intracellular nonmitochondrial Ca²⁺ stores nor the magnitude of [Ca²⁺]_i increase induced by thapsigargin was reduced by the treatment of okadaic acid. In contrast, the same phosphatase inhibitor treatment inhibited the bradykinin-evoked inositol 1,4,5-trisphosphate (IP₃) generation, the Mn²⁺ influx, and the capacity of mitochondrial Ca²⁺ accumulation. Furthermore, the sensitivity of IP₃ in the Ca²⁺ release was suppressed by okadaic acid pretreatment. Our results suggest that the reduction of bradykinin-induced [Ca²⁺]_i rise by the promotion of protein phosphorylation was attributed to the reduced activity of phospholipase C, the decreased sensitivity to IP₃, and the slowed rate of Ca²⁺ influx. Thus, phosphorylation plays a role in bradykinin-sensitive Ca²⁺ signaling cascade in NG108-15 cells.     

Enteric Glia Exhibit P2U Receptors that Increase Cytosolic Calcium by a Phospholipase C-Dependent Mechanism

Abstract: Calcium signaling in fura-2 acetoxymethyl ester-loaded enteric glia was investigated in response to neuroligands; responses to ATP were studied in detail. Carbachol (1 m M ), glutamate (100 µ M ), norepinephrine (10 µ M ), and substance P (1 µ M ) did not increase the intracellular calcium concentration ([Ca²⁺]_i) in cultured enteric glia. An increasing percentage of glia responded to serotonin (4%; 100 µ M ), bradykinin (11%; 10 µ M ), and histamine (31%; 100 µ M ), whereas 100% of glia responded to ATP (100 µ M ). ATP-evoked calcium signaling was concentration dependent in terms of the percentage of glia responding and the peak [Ca²⁺]_i achieved; responses were pertussis toxin insensitive. Based on responsiveness of enteric glia to purinergic agonists and peak [Ca²⁺]_i evoked, ATP = UTP > ADP > β,γ-methyleneadenosine 5'-triphosphate ≫ 2-methylthioadenosine 5'-triphosphate = α,β-methyleneadenosine 5'-triphosphate = AMP = adenosine, suggesting a glial P_2U receptor. Depletion of d - myo -inositol 1,4,5-trisphosphate-sensitive calcium stores by thapsigargin (10 µ M ) abolished glial responses to ATP. Similarly, calcium responses were decreased 92% by U-73122 (10 µ M ), an inhibitor of phospholipase C, and 93% by the phorbol ester phorbol 12-myristate 13-acetate (100 n M ), an activator of protein kinase C. Thus, cultured enteric glia can respond to neurotransmitters with increases in [Ca²⁺]_i. Our data suggest that glial responses to ATP are mediated by a P_2U receptor coupled to activation of phospholipase C and release of intracellular calcium stores.     

Plasma Ca2+ concentration limits melatonin night production in two fish species

In freshwater (FW) rainbow trout Oncorhynchus mykiss of spontaneously low plasma calcium concentrations ([Ca²⁺]_pl), plasma melatonin at night was significantly lower than that measured in FW fish with the highest [Ca²⁺]_pl. In brackish water adapted rainbow trout with originally high [Ca²⁺]_pl, plasma melatonin concentration at night was elevated. In cannulated flounder Platichthys flesus , night plasma melatonin increases (ΔMel) corresponded to [Ca²⁺]_pl. It is postulated that in physiological steady-state conditions, melatonin synthesis capacity is coupled to free calcium concentration in plasma of O. mykiss and P. flesus .     

Direct Observation of Serotonin 5-HT3 Receptor-Induced Increases in Calcium Levels in Individual Brain Nerve Terminals

Abstract: Confocal microscopy was used to assess internal calcium level changes in response to presynaptic receptor activation in individual, isolated nerve terminals (synaptosomes) from rat corpus striatum, focusing, in particular, on the serotonin 5-HT₃ receptor, a ligand-gated ion channel. The 5-HT₃ receptor agonist-induced calcium level changes in individual synaptosomes were compared with responses evoked by K⁺ depolarization. Using the fluorescent dye fluo-3 to measure relative changes in internal free Ca²⁺ concentration ([Ca²⁺]_i), K⁺-induced depolarization resulted in variable but rapid increases in apparent [Ca²⁺]_i among the individual terminals, with some synaptosomes displaying large transient [Ca²⁺]_i peaks of varying size (two- to 12-fold over basal levels) followed by an apparent plateau phase, whereas others displayed only a rise to a sustained plateau level of [Ca²⁺]_i (two- to 2.5-fold over basal levels). Agonist activation of 5-HT₃ receptors induced slow increases in [Ca²⁺]_i (rise time, 15–20 s) in a subset (∼5%) of corpus striatal synaptosomes, with the increases (averaging 2.2-fold over basal) being dependent on Ca²⁺ entry and inhibited by millimolar external Mg²⁺. We conclude that significant increases in brain nerve terminal Ca²⁺, rivaling that found in response to excitation by depolarization but having distinct kinetic properties, can therefore result from the activation of presynaptic ligand-gated ion channels.     

Lysophosphatidic Acid Induces a Sustained Elevation of Neuronal Intracellular Calcium

Abstract: Lysophosphatidic acid (LPA) is a lipid biomediator enriched in the brain. A novel LPA-induced response in rat hippocampal neurons is described herein, namely, a rapid and sustained elevation in the concentration of free intracellular calcium ([Ca²⁺]_i). This increase is specific, in that the related lipids phosphatidic acid and lysophosphatidylcholine did not induce an alteration in [Ca²⁺]_i. Moreover, consistent with a receptor-mediated process, there was no further increase in [Ca²⁺]_i after a second addition of LPA. The LPA-induced increase in [Ca²⁺]_i required extracellular calcium. However, studies with Cd²⁺, Ni²⁺, and nifedipine and nystatin-perforated patch clamp analyses did not indicate involvement of voltage-gated calcium channels in the LPA-induced response. In contrast, glutamate appears to have a significant role in the LPA-induced increase in [Ca²⁺]_i, because this increase was inhibited by NMDA receptor antagonists and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonists. Thus, LPA treatment may result in an increased extracellular glutamate concentration that could stimulate AMPA/kainate receptors and thereby alleviate the Mg²⁺ block of the NMDA receptors and lead to glutamate stimulation of an influx of calcium via NMDA receptors.     

Cytoplasmic free Ca2+ dynamics in single tomato (Lycopersicon esculentum) protoplasts subjected to chilling temperatures

Protoplasts were isolated from leaves of tomato seedlings ( Lycopersicon esculentum Mill., cv. Marmande) at the 2nd to 4th true leaf stage and were loaded with the calcium binding tetra[acetoxymethyl⁺] ester of the fluorescent stilbene chromophore, Fura 2. Although the loading efficiency of the dye in these protoplasts was low, many protoplasts loaded only in the cytosol were always obtained. Changes in the cytosolic calcium concentration ([Ca²⁺]_cyt) were determined in single protoplasts in a temperature-controlled perfusion chamber by use of fluorescence photometry microscopy after excitation at 340 and 380 nm. When the protoplasts were subjected to chilling temperatures (10–15°C) by a circulating solution, the [Ca²⁺]_cyt increased in 64% of the analysed protoplasts. Depending on the initial resting level of [Ca²⁺]_cyt, three main types of kinetics were obtained in these protoplasts: (1) In 21% of the protoplasts, [Ca²⁺]_cyt increased to a maximum within 10–20 s from the start of temperature decrease, followed by a fast decrease; (2) in 11% of the protoplasts, the [Ca²⁺]_cyt both increased and decreased somewhat slower; and (3) in 32% a constant increase of [Ca²⁺]_cyt was obtained 1 min after the start of temperature decrease.     

The Metabotropic Glutamate Receptor mGluR5 Induces Calcium Oscillations in Cultured Astrocytes via Protein Kinase C Phosphorylation

Abstract: The metabotropic glutamate receptor mGluR5, but not the closely related mGluR1, is expressed in cultured astrocytes, and this expression is up-regulated by specific growth factors. We investigated the capability and underlying mechanisms of mGluR5 to induce oscillatory responses of intracellular calcium concentration ([Ca²⁺]_i) in cultured rat astrocytes. Single-cell [Ca²⁺]_i recordings indicated that an mGluR-selective agonist, (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylate (1 S ,3 R -ACPD), elicits [Ca²⁺]_i oscillations in good agreement with the growth factor-induced up-regulation of mGluR5 in cultured astrocytes. A protein kinase C (PKC) inhibitor, bisindolylmaleimide I, converted a 1 S ,3 R -ACPD-mediated oscillatory response into a nonoscillatory response. In addition, the PKC activator phorbol 12-myristate 13-acetate completely abolished the [Ca²⁺]_i increase. These and other pharmacological properties of 1 S ,3 R -ACPD-induced [Ca²⁺]_i oscillations correlate well with those of the cloned mGluR5 characterized in heterologous expression systems. Furthermore, the potential involvement of protein phosphatases in [Ca²⁺]_i oscillations is suggested. The present study demonstrates that mGluR5 is capable of inducing [Ca²⁺]_i oscillations in cultured astrocytes and that phosphorylation/dephosphorylation of mGluR5 is critical in [Ca²⁺]_i oscillations, analogous to the cloned mGluR5 expressed in heterologous cell lines.     

Actin Dynamics Regulates Voltage-Dependent Calcium-Permeable Channels of the Vicia faba Guard Cell Plasma Membrane

Free cytosolic Ca²⁺ ([Ca²⁺]_cyt) is an ubiquitous second messenger in plant cell signaling, and [Ca²⁺]_cyt elevation is associated with Ca²⁺-permeable channels in the plasma membrane and endomembranes regulated by a wide range of stimuli. However, knowledge regarding Ca²⁺ channels and their regulation remains limited in planta . A type of voltage-dependent Ca²⁺-permeable channel was identified and characterized for the Vicia faba L. guard cell plasma membrane by using patch-clamp techniques. These channels are permeable to both Ba²⁺ and Ca²⁺, and their activities can be inhibited by micromolar Gd³⁺. The unitary conductance and the reversal potential of the channels depend on the Ca²⁺ or Ba²⁺ gradients across the plasma membrane. The inward whole-cell Ca²⁺ (Ba²⁺) current, as well as the unitary current amplitude and NP_o of the single Ca²⁺ channel, increase along with the membrane hyperpolarization. Pharmacological experiments suggest that actin dynamics may serve as an upstream regulator of this type of calcium channel of the guard cell plasma membrane. Cytochalasin D, an actin polymerization blocker, activated the NPo of these channels at the single channel level and increased the current amplitude at the whole-cell level. But these channel activations and current increments could be restrained by pretreatment with an F-actin stabilizer, phalloidin. The potential physiological significance of this regulatory mechanism is also discussed.     

Systemin triggers an increase of cytoplasmic calcium in tomato mesophyll cells: Ca2+ mobilization from intra- and extracellular compartments

We show here that, within 1–2 min of application, systemin triggers a transient increase of cytoplasmic free calcium concentration ([Ca²⁺]_c) in cells from Lycopersicon esculentum mesophyll. The systemin-induced Ca²⁺ increase was slightly but not significantly reduced by L-type Ca²⁺ channel blockers (nifedipine, verapamil and diltiazem) and the Ca²⁺ chelator [ethylene glycol tetraacetic acid (EGTA)], whereas inorganic Ca²⁺ channel blockers (LaCl₃, CdCl₂ and GdCl₃) and compounds affecting the release of intracellular Ca²⁺ from the vacuole (ruthenium red, LiCl, neomycin) strongly reduced the systemin-induced [Ca²⁺]_c increase. By contrast, no inhibitory effect was seen with the potassium and chloride channel blockers tested. Unlike systemin, other inducers of proteinase inhibitor (PI) and of wound-induced protein synthesis, such as jasmonic acid (JA) and bestatin, did not trigger an increase of cytoplasmic Ca²⁺. The systemin-induced elevation of cytoplasmic Ca²⁺ which might be an early step in the systemin signalling pathway, appears to involve an influx of extracellular Ca²⁺ simultaneously through several types of Ca²⁺ permeable channels, and a release of Ca²⁺ from intracellular stores sensitive to blockers of inositol 1,4,5-triphosphate (IP₃)- and cyclic adenasine 5'-diphosphoribose (cADPR)-mediated Ca²⁺ release.     

Glutamate Receptor-Mediated Calcium Surges in Neurons Derived from P19 Cells

Abstract: Retinoic acid-treated murine P19 embryonal carcinoma cells differentiate into cells with neuronal morphology that display typical neuronal markers. In this study, the presence of glutamate receptors linked to Ca²⁺-signaling mechanisms on these neurons was demonstrated by testing the effects of glutamate agonists and antagonists on the intracellular calcium ion concentration ([Ca²⁺]_i). Glutamate (1 m M ) induced either sustained or transient increases in [Ca²⁺]_i. The sustained glutamate-induced increase in [Ca²⁺]_i was mimicked by NMDA (40 µ M ). The NMDA-triggered [Ca²⁺]_i response was abolished by incubating the cells in Ca²⁺-free medium or by pretreating them with Mg²⁺ (2 m M ) or MK-801 (0.1 µ M ). These responses were unaffected by the non-NMDA antagonist CNQX (10 µ M ), but they required glycine (3–30 µ M ). Kainate (40 µ M ) and AMPA (40 µ M ) did not affect [Ca²⁺]_i. Without external Ca²⁺, glutamate triggered transient, sometimes oscillating, increases in [Ca²⁺]_i. These responses were mimicked by the metabotropic agonist trans -(1 S ,3 R )-1-amino-1,3-cyclopentanedicarboxylic acid (300 µ M ). These results suggest that neurons derived from P19 embryonal carcinoma cells have NMDA and metabotropic, but not AMPA/kainate receptors, which are linked to Ca²⁺-signaling mechanisms. These cells could provide a consistent and reproducible model with which to study neuronal differentiation, neurotoxicity, and glutamate receptor-signaling mechanisms.     

Protein Phosphorylation and Calcium Uptake into Rat Forebrain Synaptosomes: Modulation by the σ Ligand, 1,3-Ditolylguanidine

Abstract: The σ ligand 1,3-di- O -tolylguanidine (DTG) increased basal dynamin and decreased depolarization-stimulated phosphorylation of the synaptosomal protein synapsin Ib without having direct effects on protein kinases or protein phosphatases. DTG dose-dependently decreased the basal cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and blocked the depolarization-dependent increases in [Ca²⁺]_i. These effects were inhibited by the σ antagonists rimcazole and BMY14802. The nitric oxide donors sodium nitroprusside (SNP) and 8-( p -chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate decreased basal [Ca²⁺]_i and the KCl-evoked rise in [Ca²⁺]_i to an extent similar to DTG. SNP, but not DTG, produced a rise in cyclic GMP levels, suggesting that the effect of DTG on [Ca²⁺]_i was not mediated via downstream regulation of cyclic GMP levels. DTG increased ⁴⁵Ca²⁺ uptake and efflux under basal conditions and inhibited the ⁴⁵Ca²⁺ uptake induced by depolarization with KCl. The KCl-evoked rise in [Ca²⁺]_i was inhibited by ω-conotoxin (ω-CgTx)-GVIA and -MVIIC but not nifedipine and ω-agatoxin-IVA. The effect of DTG on decreasing the KCl-evoked rise in [Ca²⁺]_i was additive with ω-CgTx-MVIIC but not with ω-CgTx-GVIA. These data suggest that DTG was producing some of its effects on synapsin I and dynamin phosphorylation and intrasynaptosomal Ca²⁺ levels via inhibition of N-type Ca²⁺ channels.     

NaCl-induced changes in cytosolic free Ca2+ in Arabidopsis thaliana are heterogeneous and modified by external ionic composition

Increases in cytosolic free Ca²⁺ ([Ca²⁺]_cyt) are common to many stress-activated signalling pathways, including the response to saline environments. We have investigated the nature of NaCl-induced [Ca²⁺]_cyt signals in whole Arabidopsis thaliana seedlings using aequorin. We found that NaCl-induced increases in [Ca²⁺]_cyt are heterogeneous and mainly restricted to the root. Both the concentration of NaCl and the composition of the solution bathing the root have profound effects on the magnitude and dynamics of NaCl-induced increases in [Ca²⁺]_cyt. Alteration of external K⁺ concentration caused changes in the temporal and spatial pattern of [Ca²⁺]_cyt increase, providing evidence for Na⁺-induced Ca²⁺ influx across the plasma membrane. The effects of various pharmacological agents on NaCl-induced increases in [Ca²⁺]_cyt indicate that NaCl may induce influx of Ca²⁺ through both plasma membrane and intracellular Ca²⁺-permeable channels. Analysis of spatiotemporal [Ca²⁺]_cyt dynamics using photon-counting imaging revealed additional levels of complexity in the [Ca²⁺]_cyt signal that may reflect the oscillatory nature of NaCl-induced changes in single cells.     

Intracellular water volume and internal pH of Streptomyces antibioticus spores

Abstract In the present study we compared the intracellular level of free calcium ([Ca²⁺]_i) and monomeric (G)/total (G + F) actin ratio in HeLa cells infected with diffuse (DAEC) and localised adherent Escherichia coli (LAEC). The level of [Ca²⁺]_i was increased in both DAEC- and LAEC-infected HeLa cells. However, studies with EGTA- and dantrolene-treated cells and also suspension of cells in Ca²⁺-free buffer suggested that the rise of [Ca²⁺]_i in DAEC-infected cells was due to the influx of Ca²⁺ from extracellular medium, whereas Ca²⁺ mobilisation from the intracellular stores was responsible for the enhancement of [Ca²⁺]_i in LAEC-infected cells. It was also evident that the infection of HeLa cells with DAEC and LAEC caused alteration of G / G + F actin ratio as compared to that of control cells. The ratio was much lower in LAEC-infected cells than that of DAEC-infected ones. Moreover, cytochalasin B inhibited both DAEC and LAEC invasion to HeLa cells, suggesting further the role of microfilaments in the invasion process.