A direct mass-action mechanism explains capacitative calcium entry in Jurkat and skeletal L6 muscle cells.
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We examined capacitative calcium entry (CCE) in Jurkat and in L6 skeletal muscle cells. We found that extracellular Ca2+ can enter the endoplasmic reticulum (ER) of both cell types even in the presence of thapsigargin, which blocks entry into the ER from the cytosol through the CaATPase. Moreover, extracellular Ca2+ entry into the ER was evident even when intracellular flow of Ca2+ was in the direction of ER to cytosol due to the presence of caffeine. ER Ca2+ content was assessed by two separate means. First, we used the Mag-Fura fluorescent dye, which is sensitive only to the relatively high concentrations of Ca2+ found in the ER. Second, we transiently expressed an ER-targeted derivative of aequorin, which reports Ca2+ by luminescence. In both cases, the Ca2+ concentration in the ER increased in response to extracellular Ca2+ after the ER had been previously depleted despite blockade by thapsigargin. We found two differences between the Jurkat and L6 cells. L6, but not Jurkat cells, inhibited Ca2+ uptake at very high Ca2+ concentrations. Second, ryanodine receptor blockers inhibited the appearance of cytosolic Ca2+ during CCE if added before Ca2+ in both cases, but the L6 cells were much more sensitive to ryanodine. Both of these can be explained by the known difference in ryanodine receptors between these cell types. These findings imply that the origin of cytosolic Ca2+ during CCE is the ER. Furthermore, kinetic data demonstrated that Ca2+ filled the ER before the cytosol during CCE. Our results suggest a plasma membrane Ca2+ channel and an ER Ca2+ channel joined in tandem, allowing Ca2+ to flow directly from the extracellular space to the ER. This explains CCE; any decrease in ER [Ca2+] relative to extracellular [Ca2+] would provide the gradient for refilling the ER through a mass-action mechanism. (+info)
Myoplasmic Mg2+ concentration in Xenopus muscle fibres at rest, during fatigue and during metabolic blockade.
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Intracellular free Mg2+ concentration ([Mg2+]i) was measured in isolated single fibres of Xenopus muscle using the fluorescent Mg2+ indicator furaptra. In resting muscle the [Mg2+]i was 1.7 mM in a Mg(2+)-free Ringer solution. There was no significant change in [Mg2+]i over 2 h in Mg(2+)-free Ringer solution. Elevating extracellular [Mg2+] to 40 mM for 5 min caused a small rise (0.13 mM) in [Mg2+]i. There was no detectable rise in [Mg2+]i after 5 min in Na(+)-free Ringer solution. These results suggest that the membrane is relatively impermeable to Mg2+ and that there was no detectable Na(+)-Mg2+ exchange over 5 min. When muscle fibres were fatigued by repeated tetani continued until force declined to about 40% of control, [Mg2+]i showed characteristic changes. During the early period of fatigue when force first showed a small decline and then became almost stable, [Mg2+]i was unchanged; during the final period of fatigue when force declined more rapidly, [Mg2+]i increased by 0.8 mM. Recovery of [Mg2+]i took about 30 min. Recovery of force was complex: tetanic force first declined (post-contractile depression) and then slowly recovered to control. Since the minimum force occurred at about the time when [Mg2+]i had recovered, it seems unlikely that post-contractile depression is caused by elevated [Mg2+]i. Rigor, produced by inhibiting oxidative phosphorylation and glycolysis, was associated with a larger increase (1.6 mM) in [Mg2+]i than fatigue. The rise in [Mg2+]i during fatigue and metabolic blockade could be explained as release of Mg2+ normally bound to ATP. A model of the metabolic changes and the resulting increase in [Mg2+]i explains our results reasonably well. (+info)
Decreased myofilament responsiveness in myocardial stunning follows transient calcium overload during ischemia and reperfusion.
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The purpose of this study was to test the hypothesis that abnormal intracellular calcium handling characterizes myocardial stunning. Isolated, isovolumic, buffer-perfused ferret hearts were loaded with the bioluminescent calcium indicator aequorin for simultaneous measurement of individual calcium transients and left ventricular pressure. After 15 minutes of global ischemia and 20 minutes of reperfusion, left ventricular developed pressure was significantly reduced (75 +/- 7 versus 93 +/- 6 mm Hg, p < 0.05). During ischemia, [Ca2+]i levels were significantly elevated compared with preischemic levels, both during systole (1.38 +/- 0.31 versus 0.88 +/- 0.2 microM, p < 0.05) and end diastole (0.85 +/- 0.16 versus 0.38 +/- 0.13 microM, p < 0.05). Early during reperfusion, [Ca2+]i was also significantly elevated during systole (1.63 +/- 0.44 versus 0.88 +/- 0.20 microM, p < 0.05) and end diastole (0.75 +/- 0.15 versus 0.38 +/- 0.13 microM, p < 0.05). After 20 minutes of reperfusion, myocardial stunning occurred, but [Ca2+]i was not significantly different from preischemic levels. Thus, myocardial stunning does not result from decreased levels of activator calcium. The force-pCa relation generated by the stunned hearts was shifted downward compared with that generated by the control hearts, consistent with a decrease in maximum calcium-activated force (Fmax). At steady state during tetanus, the decrease in Fmax was confirmed, but there was no significant difference in the slope of the force-pCa relation of the stunned hearts versus controls. Thus, we conclude that stunned myocardium is characterized by decreased Fmax without desensitization of the myofilaments to [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS) (+info)
Calcium transients triggered by planar signals induce the expression of ZIC3 gene during neural induction in Xenopus.
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In intact Xenopus embryos, an increase in intracellular Ca(2+) in the dorsal ectoderm is both necessary and sufficient to commit the ectoderm to a neural fate. However, the relationship between this Ca(2+) increase and the expression of early neural genes is as yet unknown. In intact embryos, studying the interaction between Ca(2+) signaling and gene expression during neural induction is complicated by the fact that the dorsal ectoderm receives both planar and vertical signals from the mesoderm. The experimental system may be simplified by using Keller open-face explants where vertical signals are eliminated, thus allowing the interaction between planar signals, Ca(2+) transients, and neural induction to be explored. We have imaged Ca(2+) dynamics during neural induction in open-face explants by using aequorin. Planar signals generated by the mesoderm induced localized Ca(2+) transients in groups of cells in the ectoderm. These transients resulted from the activation of L-type Ca(2+) channels. The accumulated Ca(2+) pattern correlated with the expression of the early neural precursor gene, Zic3. When the transients were blocked with pharmacological agents, the level of Zic3 expression was dramatically reduced. These data indicate that, in open-face explants, planar signals reproduce Ca(2+) -signaling patterns similar to those observed in the dorsal ectoderm of intact embryos and that the accumulated effect of the localized Ca(2+) transients over time may play a role in controlling the expression pattern of Zic3. (+info)
Modulation of histamine-induced Ca2+ release by protein kinase C. Effects on cytosolic and mitochondrial [Ca2+] peaks.
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In HeLa cells, histamine induces production of inositol 1,4,5-trisphosphate (InsP3) and release of Ca2+ from the endoplasmic reticulum (ER). Ca2+ release is typically biphasic, with a fast and brief initial phase, followed by a much slower and prolonged one. In the presence of inhibitors of protein kinase C (PKC), including staurosporine and the specific inhibitors GF109203X and Ro-31-8220, the fast phase continued until the ER became fully empty. On the contrary, treatment with phorbol 12,13-dibutyrate inhibited Ca2+ release. Staurosporine had no effect on InsP3-induced Ca2+ release in permeabilized cells and did not modify either histamine-induced InsP3 production. These data suggest that histamine induces Ca2+ release and with a short lag activates PKC to down-regulate it. Consistently, Ca2+ oscillations induced by histamine were increased in amplitude and decreased in frequency in the presence of PKC inhibitors. We show also that mitochondrial [Ca2+] was much more sensitive to changes in ER-Ca2+ release induced by PKC modulation than cytosolic [Ca2+]. PKC inhibitors increased the histamine-induced mitochondrial [Ca2+] peak by 4-fold but increased the cytosolic [Ca2+] peak only by 20%. On the contrary, PKC activation inhibited the mitochondrial [Ca2+] peak by 90% and the cytosolic one by only 50%. Similarly, the combination of PKC inhibitors with the mitochondrial Ca2+ uniporter activator SB202190 led to dramatic increases in mitochondrial [Ca2+] peaks, with little effect on cytosolic ones. This suggests that activation of ER-Ca2+ release by PKC inhibitors could be involved in apoptosis induced by staurosporine. In addition, these mechanisms allow flexible and independent regulation of cytosolic and mitochondrial [Ca2+] during cell stimulation. (+info)
Primary inhibition of hypocotyl growth and phototropism depend differently on phototropin-mediated increases in cytoplasmic calcium induced by blue light.
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The phototropin photoreceptors transduce blue-light signals into several physiological and developmental responses in plants. A transient rise in cytoplasmic calcium (Ca2+) that begins within seconds of phototropin 1 (phot1) excitation is believed to be an important element in the transduction pathways leading to one or more of the phot1-dependent responses. The goal of the present work was to determine whether the Ca2+ response was necessary for (a). the inhibition of hypocotyl elongation that develops within minutes of the irradiation, and (b). hypocotyl phototropism (curved growth of the stem in response to asymmetric illumination). After determining that pulses of light delivering photon fluences of between 1 and 1000 micromol m-2 induced growth inhibition mediated by phot1 without significant interference from other photosensory pathways, the effect of blocking the Ca2+ rise was assessed. Treatment of seedlings with a Ca2+ chelator prevented the rise in cytoplasmic Ca2+ and prevented phot1-mediated growth inhibition. However, the same chelator treatment did not impair phot1-mediated phototropism. Thus, it appears that the early, transient rise in cytoplasmic Ca2+ is an important intermediary process in at least one but not all phot1-signaling pathways. (+info)
Self-reporting Arabidopsis expressing pH and [Ca2+] indicators unveil ion dynamics in the cytoplasm and in the apoplast under abiotic stress.
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For noninvasive in vivo measurements of intra- and extracellular ion concentrations, we produced transgenic Arabidopsis expressing pH and calcium indicators in the cytoplasm and in the apoplast. Ratiometric pH-sensitive derivatives of the green fluorescent protein (At-pHluorins) were used as pH indicators. For measurements of calcium ([Ca(2+)]), luminescent aequorin variants were expressed in fusion with pHluorins. An Arabidopsis chitinase signal sequence was used to deliver the indicator complex to the apoplast. Responses of pH and [Ca(2+)] in the apoplast and in the cytoplasm were studied under salt and "drought" (mannitol) stress. Results are discussed in the frame of ion flux, regulation, and signaling. They suggest that osmotic stress and salt stress are differently sensed, compiled, and processed in plant cells. (+info)
Circadian and diurnal calcium oscillations encode photoperiodic information in Arabidopsis.
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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. (+info)