Distinct light and clock modulation of cytosolic free Ca2+ oscillations and rhythmic CHLOROPHYLL A/B BINDING PROTEIN2 promoter activity in Arabidopsis

Plants have circadian oscillations in the concentration of cytosolic free calcium ([Ca(2+)](cyt)). To dissect the circadian Ca(2+)-signaling network, we monitored circadian [Ca(2+)](cyt) oscillations under various light/dark conditions (including different spectra) in Arabidopsis thaliana wild type and photoreceptor and circadian clock mutants. Both red and blue light regulate circadian oscillations of [Ca(2+)](cyt). Red light signaling is mediated by PHYTOCHROME B (PHYB). Blue light signaling occurs through the redundant action of CRYPTOCHROME1 (CRY1) and CRY2. Blue light also increases the basal level of [Ca(2+)](cyt), and this response requires PHYB, CRY1, and CRY2. Light input into the oscillator controlling [Ca(2+)](cyt) rhythms is gated by EARLY FLOWERING3. Signals generated in the dark also regulate the circadian behavior of [Ca(2+)](cyt). Oscillations of [Ca(2+)](cyt) and CHLOROPHYLL A/B BINDING PROTEIN2 (CAB2) promoter activity are dependent on the rhythmic expression of LATE ELONGATED HYPOCOTYL and CIRCADIAN CLOCK-ASSOCIATED1, but [Ca(2+)](cyt) and CAB2 promoter activity are uncoupled in the timing of cab1 (toc1-1) mutant but not in toc1-2. We suggest that the circadian oscillations of [Ca(2+)](cyt) and CAB2 promoter activity are regulated by distinct oscillators with similar components that are used in a different manner and that these oscillators may be located in different cell types in Arabidopsis.     

Dichlorodiphenylchloroethylene elevates cytosolic calcium concentrations and oscillations in primary cultures of human granulosa-lutein cells

1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), a metabolite of DDT (1,1-dichlorodiphenyltrichloroethane), is a persistent hormonally active environmental toxicant that has been found in human serum and follicular fluid. The objective of this study was to determine whether DDE can alter free calcium ion concentrations in the cytosol ([Ca(2+)](cyt)) of human granulosa cells. Changes in [Ca(2+)](cyt) in single cells loaded with Fura-2 were studied using a dynamic digital Ca(2+) imaging system. At a concentration of 100 ng/ml, DDE stimulated small elevations of [Ca(2+)](cyt) accompanied by Ca(2+) oscillations. At 1 microg DDE/ml, there was a biphasic Ca(2+) response with marked elevations of [Ca(2+)](cyt) over time. In Ca(2+)-free medium, cells showed an initial small elevation of [Ca(2+)](cyt), which was magnified after addition of Ca(2+) to the medium. Washing the cells after DDE treatment failed to remove the elevated [Ca(2+)](cyt) and oscillations, both of which were eliminated by addition of EGTA. ATP also induced [Ca(2+)](cyt) elevations and oscillations, and these effects were potentiated when DDE was added. FSH induced transient [Ca(2+)](cyt) elevations, whereas hCG caused a prolonged elevation and marked oscillations in [Ca(2+)](cyt). These results suggest that DDE at concentrations normally found in human tissues induces elevations in [Ca(2+)](cyt) in granulosa-lutein cells. Our data therefore highlight a novel mechanism through which DDE can alter endocrine homeostasis and possibly act as an endocrine toxicant.     

Direct cytoplasmic CA(2+) responses to gastrin-releasing peptide in single beta cells

The neuropeptide gastrin releasing peptide (GRP) stimulates insulin secretion and induces oscillations of the cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)) in clonal insulinoma cells. It is not known whether GRP affects [Ca(2+)](cyt) in normal beta cells. We investigated, in single, normal, mouse islet beta cells, the effects of GRP on [Ca(2+)](cyt), by dual wavelength spectrophotofluorometry. Beta cells were identified by their typical response to glucose or tolbutamide. At 15 mM glucose, GRP (100 nM) evoked an immediate [Ca(2+)](cyt) transient to 423 +/- 48 nM compared to 126 +/- 18 nM before GRP (P < 0.001). After the initial transient, [Ca(2+)](cyt) exhibited either a sustained elevation or oscillations. At 3.3 mM glucose, in cells with a non-oscillating [Ca(2+)](cyt), GRP stimulated a prompt increase in [Ca(2+)](cyt) (from 60 +/- 6 to 285 +/- 30 nM, P = 0.024) followed by either a sustained increase in [Ca(2+)](cyt) or [Ca(2+)](cyt) oscillations. We conclude that GRP promptly elevates [Ca(2+)](cyt) by a direct action in normal mouse pancreatic beta cells.     

Circadian and diurnal calcium oscillations encode photoperiodic information in Arabidopsis

We have tested the hypothesis that circadian oscillations in the concentration of cytosolic free calcium ([Ca2+]cyt) can encode information. We imaged oscillations of [Ca2+]cyt in the cotyledons and leaves of Arabidopsis (Arabidopsis thaliana) that have a 24-h period in light/dark cycles and also constant light. The amplitude, phase, and shape of the oscillations of [Ca2+]cyt and [Ca2+]cyt at critical daily time points were controlled by the light/dark regimes in which the plants were grown. These data provide evidence that 24-h oscillations in [Ca2+]cyt encode information concerning daylength and light intensity, which are two major regulators of plant growth and development.     

Mitochondria shape hormonally induced cytoplasmic calcium oscillations and modulate exocytosis

Pituitary gonadotropes transduce hormonal input into cytoplasmic calcium ([Ca(2+)](cyt)) oscillations that drive rhythmic exocytosis of gonadotropins. Using Calcium Green-1 and rhod-2 as optical measures of cytoplasmic and mitochondrial free Ca(2+), we show that mitochondria sequester Ca(2+) and tune the frequency of [Ca(2+)](cyt) oscillations in rat gonadotropes. Mitochondria accumulated Ca(2+) rapidly and in phase with elevations of [Ca(2+)](cyt) after GnRH stimulation or membrane depolarization. Inhibiting mitochondrial Ca(2+) uptake by the protonophore CCCP reduced the frequency of GnRH-induced [Ca(2+)](cyt) oscillations or, occasionally, stopped them. Much of the Ca(2+) that entered mitochondria is bound by intramitochondrial Ca(2+) buffering systems. The mitochondrial Ca(2+) binding ratio may be dynamic because [Ca(2+)](mit) appeared to reach a plateau as mitochondrial Ca(2+) accumulation continued. Entry of Ca(2+) into mitochondria was associated with a small drop in the mitochondrial membrane potential. Ca(2+) was extruded from mitochondria more slowly than it entered, and much of this efflux could be blocked by CGP-37157, a selective inhibitor of mitochondrial Na(+)-Ca(2+) exchange. Plasma membrane capacitance changes in response to depolarizing voltage trains were increased when CCCP was added, showing that mitochondria lower the local [Ca(2+)](cyt) near sites that trigger exocytosis. Thus, we demonstrate a central role for mitochondria in a significant physiological response.     

Convergence of calcium signaling pathways of pathogenic elicitors and abscisic acid in Arabidopsis guard cells

A variety of stimuli, such as abscisic acid (ABA), reactive oxygen species (ROS), and elicitors of plant defense reactions, have been shown to induce stomatal closure. Our study addresses commonalities in the signaling pathways that these stimuli trigger. A recent report showed that both ABA and ROS stimulate an NADPH-dependent, hyperpolarization-activated Ca(2+) influx current in Arabidopsis guard cells termed "I(Ca)" (Z.M. Pei, Y. Murata, G. Benning, S. Thomine, B. Klüsener, G.J. Allen, E. Grill, J.I. Schroeder, Nature [2002] 406: 731-734). We found that yeast (Saccharomyces cerevisiae) elicitor and chitosan, both elicitors of plant defense responses, also activate this current and activation requires cytosolic NAD(P)H. These elicitors also induced elevations in the concentration of free cytosolic calcium ([Ca(2+)](cyt)) and stomatal closure in guard cells. ABA and ROS elicited [Ca(2+)](cyt) oscillations in guard cells only when extracellular Ca(2+) was present. In a 5 mM KCl extracellular buffer, 45% of guard cells exhibited spontaneous [Ca(2+)](cyt) oscillations that differed in their kinetic properties from ABA-induced Ca(2+) increases. These spontaneous [Ca(2+)](cyt) oscillations also required the availability of extracellular Ca(2+) and depended on the extracellular potassium concentration. Interestingly, when ABA was applied to spontaneously oscillating cells, ABA caused cessation of [Ca(2+)](cyt) elevations in 62 of 101 cells, revealing a new mode of ABA signaling. These data show that fungal elicitors activate a shared branch with ABA in the stress signal transduction pathway in guard cells that activates plasma membrane I(Ca) channels and support a requirement for extracellular Ca(2+) for elicitor and ABA signaling, as well as for cellular [Ca(2+)](cyt) oscillation maintenance.     

Ca2+signalling in stomatal guard cells

Ca(2+) is a ubiquitous second messenger in the signal transduction pathway(s) by which stomatal guard cells respond to external stimuli. Increases in guard-cell cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) have been observed in response to stimuli that cause both stomatal opening and closure. In addition, several important components of Ca(2+)-based signalling pathways have been identified in guard cells, including the cADP-ribose and phospholipase C/Ins(1, 4,5)P(3)-mediated Ca(2+)-mobilizing pathways. The central role of stimulus-induced increases in [Ca(2+)](cyt) in guard-cell signal transduction has been clearly demonstrated in experiments examining the effects of modulating increases in [Ca(2+)](cyt) on alterations in guard-cell turgor or the activity of ion channels that act as effectors in the guard-cell turgor response. In addition, the paradox that Ca(2+) is involved in the transduction of signals that result in opposite end responses (stomatal opening and closure) might be accounted for by the generation of stimulus-specific Ca(2+) signatures, such that increases in [Ca(2+)](cyt) exhibit unique spatial and temporal characteristics.     

Rapid turnover of calcium in the endoplasmic reticulum during signaling. Studies with cameleon calcium indicators

HEK293 cells expressing the thyrotropin-releasing hormone (TRH) receptor were transfected with cameleon Ca(2+) indicators designed to measure the free Ca(2+) concentration in the cytoplasm, [Ca(2+)](cyt), and the endoplasmic reticulum (ER), [Ca(2+)](er). Basal [Ca(2+)](cyt) was about 50 nm; thyrotropin-releasing hormone (TRH) or other agonists increased [Ca(2+)](cyt) to 1 micrometer or higher. Basal [Ca(2+)](er) averaged 500 micrometer and fell to 50-100 micrometer over 10 min in the presence of thapsigargin. TRH consistently decreased [Ca(2+)](er) to 100 micrometer, independent of extracellular Ca(2+), whereas agonists for endogenous receptors generally caused a smaller decline. When added with thapsigargin, all agonists rapidly decreased [Ca(2+)](er) to 5-10 micrometer, indicating that there is substantial store refilling during signaling. TRH increased [Ca(2+)](cyt) and decreased [Ca(2+)](er) if applied after other agonists, whereas other agonists did not alter [Ca(2+)](cyt) or [Ca(2+)](er) if added after TRH. When Ca(2+) was added back to cells that had been incubated with TRH in Ca(2+)-free medium, [Ca(2+)](cyt) and [Ca(2+)](er) increased rapidly. The increase in [Ca(2+)](er) was only partially blocked by thapsigargin but was completely blocked if cells were loaded with 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. In conclusion, these new Ca(2+) indicators showed that basal [Ca(2+)](er) is approximately 500 micrometer, that [Ca(2+)](er) has to be >100 micrometer to support an increase in [Ca(2+)](cyt) by agonists, and that during signaling, intracellular Ca(2+) stores are continuously refilled with cytoplasmic Ca(2+) by the sarcoendoplasmic reticulum Ca(2+)-ATPase pump.     

Analysis and effects of cytosolic free calcium increases in response to elicitors in Nicotiana plumbaginifolia cells

Cell suspensions obtained from Nicotiana plumbaginifolia plants stably expressing the apoaequorin gene were used to analyze changes in cytosolic free calcium concentrations ([Ca(2+)](cyt)) in response to elicitors of plant defenses, particularly cryptogein and oligogalacturonides. The calcium signatures differ in lag time, peak time, intensity, and duration. The intensities of both signatures depend on elicitor concentration and extracellular calcium concentration. Cryptogein signature is characterized by a long-sustained [Ca(2+)](cyt) increase that should be responsible for sustained mitogen-activated protein kinase activation, microtubule depolymerization, defense gene activation, and cell death. The [Ca(2+)](cyt) increase in elicitor-treated cells first results from a calcium influx, which in turns leads to calcium release from internal stores and additional Ca(2+) influx. H(2)O(2) resulting from the calcium-dependent activation of the NADPH oxidase also participates in [Ca(2+)](cyt) increase and may activate calcium channels from the plasma membrane. Competition assays with different elicitins demonstrate that [Ca(2+)](cyt) increase is mediated by cryptogein-receptor interaction.     

Inflammation alters regional mitochondrial Ca²⁺ in human airway smooth muscle cells

Regulation of cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in airway smooth muscle (ASM) is a key aspect of airway contractility and can be modulated by inflammation. Mitochondria have tremendous potential for buffering [Ca(2+)](cyt), helping prevent Ca(2+) overload, and modulating other intracellular events. Here, compartmentalization of mitochondria to different cellular regions may subserve different roles. In the present study, we examined the role of Ca(2+) buffering by mitochondria and mitochondrial Ca(2+) transport mechanisms in the regulation of [Ca(2+)](cyt) in enzymatically dissociated human ASM cells upon exposure to the proinflammatory cytokines TNF-α and IL-13. Cells were loaded simultaneously with fluo-3 AM and rhod-2 AM, and [Ca(2+)](cyt) and mitochondrial Ca(2+) concentration ([Ca(2+)](mito)) were measured, respectively, using real-time two-color fluorescence microscopy in both the perinuclear and distal, perimembranous regions of cells. Histamine induced a rapid increase in both [Ca(2+)](cyt) and [Ca(2+)](mito), with a significant delay in the mitochondrial response. Inhibition of the mitochondrial Na(+)/Ca(2+) exchanger (1 μM CGP-37157) increased [Ca(2+)](mito) responses in perinuclear mitochondria but not distal mitochondria. Inhibition of the mitochondrial uniporter (1 μM Ru360) decreased [Ca(2+)](mito) responses in perinuclear and distal mitochondria. CGP-37157 and Ru360 significantly enhanced histamine-induced [Ca(2+)](cyt). TNF-α and IL-13 both increased [Ca(2+)](cyt), which was associated with decreased [Ca(2+)](mito) in the case of TNF-α but not IL-13. The effects of TNF-α on both [Ca(2+)](cyt) and [Ca(2+)](mito) were affected by CGP-37157 but not by Ru360. Overall, these data demonstrate that in human ASM cells, mitochondria buffer [Ca(2+)](cyt) after agonist stimulation and its enhancement by inflammation. The differential regulation of [Ca(2+)](mito) in different parts of ASM cells may serve to locally regulate Ca(2+) fluxes from intracellular sources versus the plasma membrane as well as respond to differential energy demands at these sites. We propose that such differential mitochondrial regulation, and its disruption, may play a role in airway hyperreactivity in diseases such as asthma, where [Ca(2+)](cyt) is increased.     

Low cytoplasmic [Ca(2+)] activates I(CRAC) independently of global Ca(2+) store depletion in RBL-1 cells

Release of Ca(2+) from inositol (1,4,5)-trisphosphate-sensitive Ca(2+) stores causes "capacitative calcium entry," which is mediated by the so-called "Ca(2+) release-activated Ca(2+) current" (I(CRAC)) in RBL-1 cells. Refilling of the Ca(2+) stores or high cytoplasmic [Ca(2+)] ([Ca(2+)](cyt)) inactivate I(CRAC). Here we address the question if also [Ca(2+)](cyt) lower than the resting [Ca(2+)](cyt) influences store-operated channels. We therefore combined patch clamp and mag fura-2 fluorescence methods to determine simultaneously both I(CRAC) and [Ca(2+)] within Ca(2+) stores of RBL-1 cells ([Ca(2+)](store)). We found that low [Ca(2+)](cyt) in the range of 30-50 nM activates I(CRAC) and Ca(2+) influx spontaneously and independently of global Ca(2+) store depletion, while elevation of [Ca(2+)](cyt) to the resting [Ca(2+)](cyt) (100 nM) resulted in store dependence of I(CRAC) activation. We conclude that spontaneous activation of I(CRAC) by low [Ca(2+)](cyt) could serve as a feedback mechanism keeping the resting [Ca(2+)](cyt) constant.     

Potassium-efflux channels in extensor and flexor cells of the motor organ of Samanea saman are not identical. Effects of cytosolic calcium

Leaflet movements in the mimosa-family tree Samanea saman stem from coordinated volume changes of cells in the leaf motor organs in the adaxial and abaxial motor cells ("flexors" and "extensors"). Shrinking, initiated by dissimilar light signals in extensors and in flexors, depends in both cell types on K(+) efflux via depolarization-dependent potassium (K(D)) channels. To compare between flexor and extensor K(D) channels and to test for a possible interaction of these channels with the Ca(2+)-mobilizing phosphoinositide cascade evoked in these motor cells by the "shrinking signals," we probed the channels with varying (5 nM-3 mM) cytosolic free-Ca(2+) concentration ([Ca(2+)](cyt)) in patch-clamped inside-out excised membrane patches. Ca(2+) was not required for K(D) channel activation. [Ca(2+)](cyt) of 600 nM decreased the mean number of open K(D) channels in flexors, as monitored at -30 mV. Detailed analysis revealed that in flexors millimolar [Ca(2+)](cyt) decreased the maximum number of open channels, but simultaneously increased K(D) channel opening probability by negatively shifting the half-maximum-activation voltage by 40 to 50 mV. Thus, the promoting and the inhibitory effects at millimolar [Ca(2+)](cyt) practically cancelled-out. In contrast to flexors, none of the gating parameters of the extensor K(D) channels were affected by [Ca(2+)](cyt). Irrespective of [Ca(2+)](cyt), the steady-state gating of extensor K(D) channels was slightly but significantly more voltage sensitive than that of flexors. The unitary conductances of flexor and extensor K(D) channels were similar and decreased by approximately 20% at millimolar [Ca(2+)](cyt). It is intriguing that the extensor K(D) channels were significantly less K(+) selective than those in flexors.     

Receptor-mediated increase in cytoplasmic free calcium required for activation of pathogen defense in parsley

Transient influx of Ca(2+) constitutes an early element of signaling cascades triggering pathogen defense responses in plant cells. Treatment with the Phytophthora sojae-derived oligopeptide elicitor, Pep-13, of parsley cells stably expressing apoaequorin revealed a rapid increase in cytoplasmic free calcium ([Ca(2+)](cyt)), which peaked at approximately 1 microM and subsequently declined to sustained values of 300 nM. Activation of this biphasic [Ca(2+)](cyt) signature was achieved by elicitor concentrations sufficient to stimulate Ca(2+) influx across the plasma membrane, oxidative burst, and phytoalexin production. Sustained concentrations of [Ca(2+)](cyt) but not the rapidly induced [Ca(2+)](cyt) transient peak are required for activation of defense-associated responses. Modulation by pharmacological effectors of Ca(2+) influx across the plasma membrane or of Ca(2+) release from internal stores suggests that the elicitor-induced sustained increase of [Ca(2+)](cyt) predominantly results from the influx of extracellular Ca(2+). Identical structural features of Pep-13 were found to be essential for receptor binding, increases in [Ca(2+)](cyt), and activation of defense-associated responses. Thus, a receptor-mediated increase in [Ca(2+)](cyt) is causally involved in signaling the activation of pathogen defense in parsley.     

Potassium-efflux channels in extensor and flexor cells of the motor organ of Samanea saman are not identical. Effects of cytosolic calcium

Leaflet movements in the mimosa-family tree Samanea saman stem from coordinated volume changes of cells in the leaf motor organs in the adaxial and abaxial motor cells ("flexors" and "extensors"). Shrinking, initiated by dissimilar light signals in extensors and in flexors, depends in both cell types on K(+) efflux via depolarization-dependent potassium (K(D)) channels. To compare between flexor and extensor K(D) channels and to test for a possible interaction of these channels with the Ca(2+)-mobilizing phosphoinositide cascade evoked in these motor cells by the "shrinking signals," we probed the channels with varying (5 nM-3 mM) cytosolic free-Ca(2+) concentration ([Ca(2+)](cyt)) in patch-clamped inside-out excised membrane patches. Ca(2+) was not required for K(D) channel activation. [Ca(2+)](cyt) of 600 nM decreased the mean number of open K(D) channels in flexors, as monitored at -30 mV. Detailed analysis revealed that in flexors millimolar [Ca(2+)](cyt) decreased the maximum number of open channels, but simultaneously increased K(D) channel opening probability by negatively shifting the half-maximum-activation voltage by 40 to 50 mV. Thus, the promoting and the inhibitory effects at millimolar [Ca(2+)](cyt) practically cancelled-out. In contrast to flexors, none of the gating parameters of the extensor K(D) channels were affected by [Ca(2+)](cyt). Irrespective of [Ca(2+)](cyt), the steady-state gating of extensor K(D) channels was slightly but significantly more voltage sensitive than that of flexors. The unitary conductances of flexor and extensor K(D) channels were similar and decreased by approximately 20% at millimolar [Ca(2+)](cyt). It is intriguing that the extensor K(D) channels were significantly less K(+) selective than those in flexors.     

Coding efficiency and information rates in transmembrane signaling

A variety of cell types responds to hormonal stimuli by repetitive spikes in the intracellular concentration of calcium ([Ca(2+)](i)) which have been demonstrated to encode information in their frequency, amplitude, and duration. These [Ca(2+)](i)-spike trains are able to specifically regulate distinct cellular functions. Using a mathematical model for receptor-controlled [Ca(2+)](i) oscillations in hepatocytes we investigate the encoding of fluctuating hormonal signals in [Ca(2+)](i)-spike trains. The transmembrane information transfer is quantified by using an information-theoretic reverse-engineering approach which allows to reconstruct the dynamic hormonal stimulus from the [Ca(2+)](i)-spike trains. This approach allows to estimate the accuracy of coding as well as the rate of transmembrane information transfer. We found that up to 87% of the dynamic stimulus information can be encoded in the [Ca(2+)](i)-spike train at a maximum information transfer rate of 1.1 bit per [Ca(2+)](i)-spike. These numerical results for humoral information transfer are in the same order as in a number of sensory neuronal systems despite several orders of magnitude different time scales of operation suggesting a universal principle of information processing in both biological systems.     

Mg2+ blocks myogenic tone but not K+-induced constriction: role for SOCs in small arteries

The effects of Mg(2+) and nifedipine (Nif) on vasoconstriction and Ca(2+) transients were studied in intact, pressurized rat mesenteric arteries with myogenic tone. Changes in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) were measured with confocal microscopy in fluo 4-AM loaded, individual myocytes. Myogenic tone was abolished by 10 mM Mg(2+) or 0.3 microM Nif. Contractions induced by 75 mM K(+) depolarization were blocked by 0.3 microM Nif, but not by 10 mM Mg(2+). Phenylephrine (PE; 5 microM) evoked sustained [Ca(2+)](cyt) elevation and vasoconstriction with superimposed Ca(2+) oscillations and vasomotion. The subsequent addition of 10 mM Mg(2+) or 0.3 microM Nif reduced [Ca(2+)](cyt) and abolished plateau vasoconstriction. When added before PE, both Mg(2+) and Nif abolished the PE-evoked Ca(2+) oscillations and vasomotion. Mg(2+) dilated the PE-constricted arteries after a brief (< or =180-240 s) vasoconstriction, but Nif did not. Both agents also abolished the vasoconstriction attributed to Ca(2+) entry through store-operated channels (SOCs) during internal Ca(2+) store refilling that followed store depletion. The data suggest that Ca(2+) entry through SOCs helps maintain both myogenic tone and alpha(1)-adrenoceptor-induced tonic vasoconstriction.     

Calcium-sensing receptor modulates extracellular Ca(2+) entry via TRPC-encoded receptor-operated channels in human aortic smooth muscle cells

Ca-sensing receptor (CaSR), a member of the G protein-coupled receptor family, regulates the synthesis of parathyroid hormone in response to changes in serum Ca(2+) concentrations. The functions of CaSR in human vascular smooth muscle cells are largely unknown. Here we sought to study CaSR activation and the underlying molecular mechanisms in human aortic smooth muscle cells (HASMC). Extracellular Ca(2+) ([Ca(2+)](o)) dose-dependently increased free cytosolic Ca(2+) ([Ca(2+)](cyt)) in HASMC, with a half-maximal response (EC(50)) of 0.52 mM and a Hill coefficient of 5.50. CaSR was expressed in HASMC, and the [Ca(2+)](o)-induced [Ca(2+)](cyt) rise was abolished by dominant negative mutants of CaSR. The CaSR-mediated increase in [Ca(2+)](cyt) was also significantly inhibited by pertussis toxin, the phospholipase C inhibitor U-73122, or the general protein kinase C (PKC) inhibitor chelerythrine, but not by the conventional PKC inhibitor, G?6976. Depletion of membrane cholesterol by pretreatment with methyl-β-cyclodextrin markedly decreased CaSR-induced increase in [Ca(2+)](cyt). Blockade of TRPC channels with 2-aminoethoxydiphenyl borate, SKF-96365, or La(3) significantly inhibited [Ca(2+)](o) entry, whereas activation of TRPC6 channels with flufenamic acid potentiated [Ca(2+)](o) entry. Neither cyclopiazonic acid nor caffeine or ionomycin had any effect on [Ca(2+)](cyt) in [Ca(2+)](o)-free solutions. TRPC6 and PKCε mRNA and proteins were detected in HASMC, and [Ca(2+)](o) induced PKCε phosphorylation, which could be prevented by chelerythrine. Our data suggest that CaSR activation mediates [Ca(2+)](o) entry, likely through TRPC6-encoded receptor-operated channels that are regulated by a PLC/PKCε cascade. Our study therefore provides evidence not only for functional expression of CaSR, but also for a novel pathway whereby it regulates [Ca(2+)](o) entry in HASMC.     

Thrombin-mediated increases in cytosolic [Ca2+] involve different mechanisms in human pulmonary artery smooth muscle and endothelial cells

Thrombin is a procoagulant inflammatory agonist that can disrupt the endothelium-lumen barrier in the lung by causing contraction of endothelial cells and promote pulmonary cell proliferation. Both contraction and proliferation require increases in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)). In this study, we compared the effect of thrombin on Ca(2+) signaling in human pulmonary artery smooth muscle (PASMC) and endothelial (PAEC) cells. Thrombin increased the [Ca(2+)](cyt) in both cell types; however, the transient response was significantly higher and recovered quicker in the PASMC, suggesting different mechanisms may contribute to thrombin-mediated increases in [Ca(2+)](cyt) in these cell types. Depletion of intracellular stores with cyclopiazonic acid (CPA) in the absence of extracellular Ca(2+) induced calcium transients representative of those observed in response to thrombin in both cell types. Interestingly, CPA pretreatment significantly attenuated thrombin-induced Ca(2+) release in PASMC; this attenuation was not apparent in PAEC, indicating that a PAEC-specific mechanism was targeted by thrombin. Treatment with a combination of CPA, caffeine, and ryanodine also failed to abolish the thrombin-induced Ca(2+) transient in PAEC. Notably, thrombin-induced receptor-mediated calcium influx was still observed in PASMC after CPA pretreatment in the presence of extracellular Ca(2+). Ca(2+) oscillations were triggered by thrombin in PASMC resulting from a balance of extracellular Ca(2+) influx and Ca(2+) reuptake by the sarcoplasmic reticulum. The data show that thrombin induces increases in intracellular calcium in PASMC and PAEC with a distinct CPA-, caffeine-, and ryanodine-insensitive release existing only in PAEC. Furthermore, a dynamic balance between Ca(2+) influx, intracellular Ca(2+) release, and reuptake underlie the Ca(2+) transients evoked by thrombin in some PASMC. Understanding of such mechanisms will provide an important insight into thrombin-mediated vascular injury during hypertension.     

Mechanisms of nitric-oxide-induced increase of free cytosolic Ca2+ concentration in Nicotiana plumbaginifolia cells

In this study, we investigated a role for nitric oxide (NO) in mediating the elevation of the free cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in plants using Nicotiana plumbaginifolia cells expressing the Ca(2+) reporter apoaequorin. Hyperosmotic stress induced a fast increase of [Ca(2+)](cyt) which was strongly reduced by pretreating cell suspensions with the NO scavenger carboxy PTIO, indicating that NO mediates [Ca(2+)](cyt) changes in plant cells challenged by abiotic stress. Accordingly, treatment of transgenic N. plumbaginifolia cells with the NO donor diethylamine NONOate was followed by a transient increase of [Ca(2+)](cyt) sensitive to plasma membrane Ca(2+) channel inhibitors and antagonist of cyclic ADP ribose. We provided evidence that NO might activate plasma membrane Ca(2+) channels by inducing a rapid and transient plasma membrane depolarization. Furthermore, NO-induced elevation of [Ca(2+)](cyt) was suppressed by the kinase inhibitor staurosporine, suggesting that NO enhances [Ca(2+)](cyt) by promoting phosphorylation-dependent events. This result was further supported by the demonstration that the NO donor induced the activation of a 42-kDa protein kinase which belongs to SnRK2 families and corresponds to Nicotiana tabacum osmotic-stress-activated protein kinase (NtOSAK). Interestingly, NtOSAK was activated in response to hyperosmotic stress through a NO-dependent process, supporting the hypothesis that NO also promotes protein kinase activation during physiological processes.