In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca²⁺) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca²⁺ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca²⁺ indicator such as Fluo-4 permits measurements of Ca²⁺ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca²⁺ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca²⁺ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca²⁺ responses in enteric neurons and glial cells.     

Enhanced Ca2+ storage in sphingosine-1-phosphate lyase-deficient fibroblasts

Sphingosine-1-phosphate (S1P) regulates cell growth and survival, migration and adhesion in many cell types. S1P is generated by sphingosine kinases (SphKs), and dephosphorylated by phosphatases or cleaved by S1P lyase. Extracellular S1P activates specific G protein-coupled receptors while intracellular S1P can mobilize Ca²⁺ from thapsigargin-sensitive stores. Here, we have studied Ca²⁺ signalling in mouse embryonic fibroblasts (MEFs) deficient in S1P lyase. In these cells, S1P and sphingosine concentrations were elevated about 6-fold and 2-fold, respectively, as measured by liquid chromatography/tandem mass spectrometry. Measurements with fura-2-loaded cells in suspension revealed that resting [Ca²⁺]_i was elevated and agonist-induced [Ca²⁺]_i increases were augmented in S1P lyase-deficient MEFs both in the presence and absence of extracellular Ca²⁺. Importantly, [Ca²⁺]_i increases and Ca²⁺ mobilization induced by the SERCA inhibitor, thapsigargin, were augmented, indicating enhanced Ca²⁺ storage in S1P lyase-deficient MEFs. Measurements with single cells expressing the calmodulin-based Ca²⁺ sensor, cameleon, revealed that at least two cell types could be distinguished in both MEF cell populations, one with a rapid and transient [Ca²⁺]_i increase and the other with a slower and prolonged [Ca²⁺]_i elevation upon stimulation with thapsigargin. The area under the time course of thapsigargin-induced [Ca²⁺]_i increases, reflecting overall Ca²⁺ release, was significantly increased by more than 50% in both rapidly and slowly responding S1P lyase-deficient cells. It is concluded that elevated concentrations of S1P and/or sphingosine lead to enhanced Ca²⁺ storage and elevated basal [Ca²⁺]_i. S1P metabolism thus plays a role not only in acute Ca²⁺ mobilization but also in long-term regulation of Ca²⁺ homeostasis.     

Dysfunction of mitochondria Ca uptake in cystic fibrosis airway epithelial cells

In the genetic disease cystic fibrosis (CF), the most common mutation F508del promotes the endoplasmic reticulum (ER) retention of misfolded CF proteins. Furthermore, in homozygous F508del-CFTR airway epithelial cells, the histamine Ca²⁺ mobilization is abnormally increased. Because the uptake of Ca²⁺ by mitochondria during Ca²⁺ influx or Ca²⁺ release from ER stores may be crucial for maintaining a normal Ca²⁺ homeostasis, we compared the mitochondria morphology and distribution by transmission electron microscopy technique and the mitochondria membrane potential variation (ΔΨ_mit) using a fluorescent probe (TMRE) on human CF (CF-KM4) and non-CF (MM39) tracheal serous gland cell lines. Confocal imaging of Rhod-2–AM-loaded or of the mitochondrial targeted cameleon 4mtD3cpv-transfected human CF and non-CF cells, were used to examine the ability of mitochondria to sequester intracellular Ca²⁺. The present study reveals that (i) the mitochondria network is fragmented in F508del-CFTR cells, (ii) the ΔΨ_mit of CF mitochondria is depolarized compared non-CF mitochondria, and (iii) the CF mitochondria Ca²⁺ uptake is reduced compared non-CF cells. We propose that these defects in airway epithelial F508del-CFTR cells are the consequence of mitochondrial membrane depolarization leading to a deficient mitochondrial Ca²⁺ uptake.     

Transgenic zebrafish for ratiometric imaging of cytosolic and mitochondrial Ca response in teleost embryo

Intracellular Ca²⁺ imaging has widely been used to visualize intracellular signals, but the application in an intact animal is still limited due to difficulty of the indicator loading. In addition, the motion of the living animal produces artifacts. To investigate Ca²⁺ signaling at early embryonic stage, we established transgenic zebrafish line expressing a genetically encoded Ca²⁺ indicator, cameleon YC2.60, driven by a constitutively active promoter, hspa8. Although the embryo dynamically changes its morphology, the motion artifact could be canceled out by taking the advantage of YC2.60 as a ratiometric indicator. The transgenic zebrafish was used to visualize the propagation of cytosolic Ca²⁺ during the early embryonic stage upon fertilization and along cleavage furrow, and the rise in Ca²⁺ in the myocytes contracting spontaneously in the embryo. We also established a transgenic zebrafish line expressing YC2.60 targeted to the mitochondria. The rise in mitochondrial Ca²⁺ was rather sustained (≈2 min), which is consistent with the requirement of ATP refilling since the mitochondrial Ca²⁺ upregulates rate-limiting enzymes of Krebs cycle. This is in contrast with the transient rise in the cytosol Ca²⁺ that directly evokes the muscle contraction. These transgenic zebrafish lines are expected to serve as useful tools further Ca²⁺ imaging in vivo.     

Spatiotemporal Correlations between Cytosolic and Mitochondrial Ca2+ Signals Using a Novel Red-Shifted Mitochondrial Targeted Cameleon

The transfer of Ca²⁺ from the cytosol into the lumen of mitochondria is a crucial process that impacts cell signaling in multiple ways. Cytosolic Ca²⁺ ([Ca²⁺]_cyto) can be excellently quantified with the ratiometric Ca²⁺ probe fura-2, while genetically encoded Förster resonance energy transfer (FRET)-based fluorescent Ca²⁺ sensors, the cameleons, are efficiently used to specifically measure Ca²⁺ within organelles. However, because of a significant overlap of the fura-2 emission with the spectra of the cyan and yellow fluorescent protein of most of the existing cameleons, the measurement of fura-2 and cameleons within one given cell is a complex task. In this study, we introduce a novel approach to simultaneously assess [Ca²⁺]_cyto and mitochondrial Ca²⁺ ([Ca²⁺]_mito) signals at the single cell level. In order to eliminate the spectral overlap we developed a novel red-shifted cameleon, D1GO-Cam, in which the green and orange fluorescent proteins were used as the FRET pair. This ratiometric Ca²⁺ probe could be successfully targeted to mitochondria and was suitable to be used simultaneously with fura-2 to correlate [Ca²⁺]_cyto and [Ca²⁺]_mito within same individual cells. Our data indicate that depending on the kinetics of [Ca²⁺]_cyto rises there is a significant lag between onset of [Ca²⁺]_cyto and [Ca²⁺]_mito signals, pointing to a certain threshold of [Ca²⁺]_cyto necessary to activate mitochondrial Ca²⁺ uptake. The temporal correlation between [Ca²⁺]_mito and [Ca²⁺]_cyto as well as the efficiency of the transfer of Ca²⁺ from the cytosol into mitochondria varies between different cell types. Moreover, slow mitochondrial Ca²⁺ extrusion and a desensitization of mitochondrial Ca²⁺ uptake cause a clear difference in patterns of mitochondrial and cytosolic Ca²⁺ oscillations of pancreatic beta-cells in response to D-glucose.     

FRET-based sensor analysis reveals caveolae are spatially distinct Ca stores in endothelial cells

Ca²⁺-regulating and Ca²⁺-dependent molecules enriched in caveolae are typically shaped as plasmalemmal invaginations or vesicles. Caveolae structure and subcellular distribution are critical for Ca²⁺ release from endoplasmic reticulum Ca²⁺ stores and for Ca²⁺ influx from the extracellular space into the cell. However, Ca²⁺ dynamics inside caveolae have never been directly measured and remain uncharacterized. To target the fluorescence resonance energy transfer (FRET)-based Ca²⁺ sensing protein D1, a mutant of cameleon, to the intra-caveolar space, we made a cDNA construct encoding a chimeric protein of lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) and D1 (LOXD1). Immunofluorescence and immunoelectron microscopy confirmed that a significant portion of LOXD1 was localized with caveolin-1 at morphologically apparent caveolar vesicles in endothelial cells. LOXD1 detected ATP-induced transient Ca²⁺ decreases by confocal FRET imaging in the presence or absence of extracellular Ca²⁺. This ATP-induced Ca²⁺ decrease was abolished following knockdown of caveoin-1, suggesting an association with caveolae. The X-ray spectra obtained by the spot analysis of electron-opaque pyroantimonate precipitates further confirmed that ATP-induced calcium decreases in intra-caveolar vesicles. In conclusion, subplasmalemmal caveolae function as Ca²⁺-releasable Ca²⁺ stores in response to ATP. This intracellular local Ca²⁺ delivery system may contribute to the complex spatiotemporal organization of Ca²⁺ signaling.     

A plug-in minielectrode for measurements in stirred photometric cuvettes

A device is described to perform potentiometric measurements with ion-sensitive electrodes in stirred photometric cuvettes. Its design allows to make additions to the reaction medium with microliter syringes during measurements. Originally, this plug-in electrode was designed to register the free Ca²⁺ concentrations in the incubation medium of mitochondrial suspensions during measurements of the free matrix Ca²⁺ concentration with a permeant fluorescent Ca²⁺ indicator. However, numerous other applications can be easily realized, such as the combination of mitochondrial light-scattering measurements and ion-transport measurements, the combination of the permeabilized cell technique with fluorescence measurements of intracellular organelles or simply the calibration of the fluorescence of Ca²⁺ indicators with a Ca²⁺ ion-sensitive minielectrode. Compared with the use of a second fluorescent indicator the use of an electrode has the advantage that the signal can be transformed into ion concentrations already during the measurements.     

In Vivo Epithelial Wound Repair Requires Mobilization of Endogenous Intracellular and Extracellular Calcium

We report that a localized intracellular and extracellular Ca²⁺ mobilization occurs at the site of microscopic epithelial damage in vivo and is required to mediate tissue repair. Intravital confocal/two-photon microscopy continuously imaged the surgically exposed stomach mucosa of anesthetized mice while photodamage of gastric epithelial surface cells created a microscopic lesion that healed within 15 min. Transgenic mice with an intracellular Ca²⁺-sensitive protein (yellow cameleon 3.0) report that intracellular Ca²⁺ selectively increases in restituting gastric epithelial cells adjacent to the damaged cells. Pretreatment with U-73122, indomethacin, 2-aminoethoxydiphenylborane, or verapamil inhibits repair of the damage and also inhibits the intracellular Ca²⁺ increase. Confocal imaging of Fura-Red dye in luminal superfusate shows a localized extracellular Ca²⁺ increase at the gastric surface adjacent to the damage that temporally follows intracellular Ca²⁺ mobilization. Indomethacin and verapamil also inhibit the luminal Ca²⁺ increase. Intracellular Ca²⁺ chelation (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/acetoxymethyl ester, BAPTA/AM) fully inhibits intracellular and luminal Ca²⁺ increases, whereas luminal calcium chelation (N-(2-hydroxyetheyl)-ethylendiamin-N,N,N′-triacetic acid trisodium, HEDTA) blocks the increase of luminal Ca²⁺ and unevenly inhibits late-phase intracellular Ca²⁺ mobilization. Both modes of Ca²⁺ chelation slow gastric repair. In plasma membrane Ca-ATPase 1^+/− mice, but not plasma membrane Ca-ATPase 4^−/− mice, there is slowed epithelial repair and a diminished gastric surface Ca²⁺ increase. We conclude that endogenous Ca²⁺, mobilized by signaling pathways and transmembrane Ca²⁺ transport, causes increased Ca²⁺ levels at the epithelial damage site that are essential to gastric epithelial cell restitution in vivo.     

A comparison of fluorescent Ca2+ indicators for imaging local Ca2+ signals in cultured cells

Localized subcellular changes in Ca²⁺ serve as important cellular signaling elements, regulating processes as diverse as neuronal excitability and gene expression. Studies of cellular Ca²⁺ signaling have been greatly facilitated by the availability of fluorescent Ca²⁺ indicators. The respective merits of different indicators to monitor bulk changes in cellular Ca²⁺ levels have been widely evaluated, but a comprehensive comparison for their use in detecting and analyzing local, subcellular Ca²⁺ signals is lacking. Here, we evaluated several fluorescent Ca²⁺ indicators in the context of local Ca²⁺ signals (puffs) evoked by inositol 1,4,5-trisphosphate (IP₃) in cultured human neuroblastoma SH-SY5Y cells, using high-speed video-microscopy. Altogether, nine synthetic Ca²⁺ dyes (Fluo-4, Fluo-8, Fluo-8 high affinity, Fluo-8 low affinity, Oregon Green BAPTA-1, Cal-520, Rhod-4, Asante Calcium Red, and X-Rhod-1) and three genetically-encoded Ca²⁺-indicators (GCaMP6-slow, -medium and -fast variants) were tested; criteria include the magnitude, kinetics, signal-to-noise ratio and detection efficiency of local Ca²⁺ puffs. Among these, we conclude that Cal-520 is the optimal indicator for detecting and faithfully tracking local events; that Rhod-4 is the red-emitting indicator of choice; and that none of the GCaMP6 variants are well suited for imaging subcellular Ca²⁺ signals.     

Histamine causes Ca entry via both a storemoperated and a store-independent pathway in bovine adrenal chromaffin cells

The characteristics and properties of the increase in cytosolic [Ca²⁺] that occurs in bovine adrenal medullary chromaffin cells on exposure to histamine have been investigated. Specifically, these experiments were conducted to determine how much external Ca²⁺ enters the cell through a (capacitative) Ca²⁺ entry pathway activated as a consequence of intracellular Ca²⁺ store mobilization, relative to that which enters independently of store depletion via other channels activated by histamine. In Fura-2 loaded cells continued exposure to histamine (10 μM) caused a rapid but transient increase in cytosolic [Ca²⁺] followed by a lower plateau that was sustained as long as external Ca²⁺ was present. In the absence of external Ca²⁺ only the initial brief transient was observed. In cells previously treated with thapsigargin (100 nM) in Ca²⁺-free medium to deplete the internal Ca²⁺ stores, histamine caused no increase in cytosolic [Ca²⁺] when external Ca²⁺ was absent. Re-introduction of external Ca²⁺ to thapsigargin-treated store-depleted cells caused a sustained increase in cytosolic [Ca²⁺] that was further increased (P < 0.0002) upon exposure to histamine. The histamine-evoked increase was prevented by the H₁-receptor antagonist, mepyramine (2 μM). A comparison was made between store-dependent Ca²⁺ entry consequent upon store mobilization with histamine in Ca²⁺-free medium and plateau phase Ca²⁺ entry resulting from stimulation with histamine in Ca²⁺-containing medium. The latter was found to be approximately 3 times greater in magnitude than the former (P ? 0.0001) at the same concentration of histamine (10 μM). It is concluded that histamine causes Ca²⁺ entry not only via a capacitative entry pathway secondary to internal store mobilization, but also causes substantial Ca²⁺ entry through other pathways.     

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca²⁺ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca²⁺ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca²⁺ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca²⁺ indicator and a hydrophilic fluorescent dye/Ca²⁺ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F₀.     

Differential Ca2+ mobilization and mast cell degranulation by FcεRI- and GPCR-mediated signaling

Mast cells play a primary role in allergic diseases. During an allergic reaction, mast cell activation is initiated by cross-linking IgE-FcεRI complex by multivalent antigen resulting in degranulation. Additionally, G protein-coupled receptors also induce degranulation upon activation. However, the spatio-temporal relationship between Ca²⁺ mobilization and mast cell degranulation is not well understood. We investigated the relationship between oscillations in Ca²⁺ level and mast cell degranulation upon stimulation in rat RBL-2H3 cells. Nile red and Fluo-4 were used as probes for monitoring histamine and intracellular Ca²⁺ levels, respectively. Histamine release and Ca²⁺ oscillations in real-time were monitored using total internal reflection fluorescence microscopy (TIRFM). Mast cell degranulation followed immediately after FcεRI and GPCR-mediated Ca²⁺ increase. FcεRI-induced Ca²⁺ increase was higher and more sustained than that induced by GPCRs. However, no significant difference in mast cell degranulation rates was observed. Although intracellular Ca²⁺ release was both necessary and sufficient for mast cell degranulation, extracellular Ca²⁺ influx enhanced the process. Furthermore, cytosolic Ca²⁺ levels and mast cell degranulation were significantly decreased by downregulation of store-operated Ca²⁺ entry (SOCE) via Orai1 knockdown, 2-aminoethyl diphenylborinate (2-APB) or tubastatin A (TSA) treatment. Collectively, this study has demonstrated the role of Ca²⁺ signaling in regulating histamine degranulation.     

Measurements of mitochondrial calcium in vivo

Mitochondria play a pivotal role in intracellular Ca²⁺ signalling by taking up and releasing the ion upon specific conditions. In order to do so, mitochondria depend on a number of factors, such as the mitochondrial membrane potential and spatio-temporal constraints. Whereas most of the basic principles underlying mitochondrial Ca²⁺ handling have been successfully deciphered over the last 50 years using assays based on in vitro preparations of mitochondria or cultured cells, we have only just started to understand the actual physiological relevance of these processes in the whole animal. Recent advancements in imaging and genetically encoded sensor technologies have allowed us to visualise mitochondrial Ca²⁺ transients in live mice. These studies used either two-photon microscopy or bioluminescence imaging of cameleon or aequorin-GFP Ca²⁺ sensors, respectively. Both methods revealed a consistent picture of Ca²⁺ uptake into mitochondria under physiological conditions even during very short-lasting elevations of cytosolic Ca²⁺ levels. The big future challenge is to understand the functional impact of such Ca²⁺ signals on the physiology of the observed tissue as well as of the whole organism. To that end, the development of multiparametric in vivo approaches will be mandatory.     

Calcium wave for cytoplasmic streaming of Physarum polycephalum

The plasmodium Physarum polycepharum exhibits periodic cycles of cytoplasmic streaming in association with those of contraction and relaxation movement. In the present study, we injected Calcium Green dextran as a fluorescent Ca²⁺ indicator into the thin‐spread living plasmodium. We found changes in the [Ca²⁺]i (intracellular concentration of Ca²⁺), which propagated in a wave‐like form in its cytoplasm. The Ca²⁺ waves were also detected when we used Fura dextran which detected [Ca²⁺]i by the ratio of two wavelengths. We prepared the plasmodial fragment from the thin‐spread and found that the cycles of the contraction–relaxation movement was so synchronized that the measurement of its area provided an indication of the movement. We observed that [Ca²⁺]i also synchronized in the entire fragment and that the relaxation ensued upon the reduction in [Ca²⁺]i. We suggest that the Ca²⁺ wave generated periodically is one of the major factors playing a crucial role in the relaxation of P. polycepharum.     

Rapid Elevation of Calcium Concentration in Cultured Dorsal Spinal Cord Astrocytes by Corticosterone

In addition to the classic genomic effects, increasing evidence suggests that GC can generate multiple rapid effects on many tissues and cells through nongenomic pathway. In the present study, the effects of corticosterone (CORT) on the intracellular calcium concentration ([Ca²⁺]i) in cultured dorsal spinal cord astrocytes were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator that could monitor real-time alterations of [Ca²⁺]i. CORT (0.01–10 μM) caused a rapid increase in [Ca²⁺]i with a dose-dependent manner in cultured dorsal spinal cord astrocytes. The action of CORT on astrocytic [Ca²⁺]i was blocked by pertussis toxin (a blocker of G protein activation, 100 ng/ml), but was unaffected by RU38486 (glucocorticoid receptor antagonist, 10 μM). In addition, cycloheximide (protein-synthesis inhibitor, 10 μg/ml) pretreatment could not impair the CORT-evoked [Ca²⁺]i elevation. Furthermore, Ca²⁺ mobilization induced by CORT was abolished by chelerythrine chloride (protein kinase C inhibitor, 10 μM), but was not impaired by H89 (protein kinase A inhibitor, 10 μM). These observations suggest that a nongenomic pathways might be involved in the effect of CORT on [Ca²⁺]i in cultured dorsal spinal cord astrocytes. In addition, our results also raise a possibility that a putative pertussis toxin-sensitive mGCR (G-protein-coupled membrane-bound glucocorticoid receptor) and the downstream activation of protein kinase C may be responsible for CORT-induced Ca²⁺ mobilization in cultured dorsal spinal cord astrocytes.     

K252a-sensitive protein kinases but not okadaic acid-sensitive protein phosphatases regulate methyl jasmonate-induced cytosolic Ca2+ oscillation in guard cells of Arabidopsis thaliana

Methyl jasmonate (MeJA) induces stomatal closure similar to abscisic acid (ABA), and MeJA signaling in guard cells shares some signal components with ABA signaling. As part of this process, MeJA as well as ABA induce the elevation and oscillation of cytosolic free-calcium concentrations ([Ca²⁺]_cyt) in guard cells. While abscisic acid-induced [Ca²⁺]_cyt oscillation has been extensively studied, MeJA-induced [Ca²⁺]_cyt oscillation is less well understood. In this study, we investigated the effects of K252a (a broad-range protein kinase inhibitor) and okadaic acid (OA, a protein phosphatase 1 and 2A inhibitor) on MeJA-induced [Ca²⁺]_cyt oscillation in guard cells of Arabidopsis thaliana ecotype Columbia expressing the Ca²⁺ reporter yellow cameleon 3.6. The protein kinase inhibitor K252a abolished MeJA-induced stomatal closure and reduced MeJA-elicited [Ca²⁺]_cyt oscillation. The protein phosphatase inhibitor OA, on the other hand, did not inhibit these processes. These results suggest that MeJA signaling involves activation of K252a-sensitive protein kinases upstream of [Ca²⁺]_cyt oscillation but not activation of an OA-sensitive protein phosphatase in guard cells of A. thaliana ecotype Columbia.     

Measuring intracellular ca levels in plant cells using the fluorescent probes, indo-1 and fura-2 : progress and prospects

Recent advances in the development of methods for measuring cytoplasmic Ca²⁺ levels in higher plant cells are discussed. Emphasis is placed on the new generation of Ca²⁺-sensitive fluorescent dyes particularly fura-2 and indo-1. These dyes offer many advantages for the measurement of cytosolic Ca²⁺ levels. They can be introduced into cells in a nonintrusive manner, their K_d for Ca²⁺ matches plant cell cytoplasmic Ca²⁺ levels, and shifts in their emission (indo-1) or excitation (fura-2) spectra following Ca²⁺ binding permit accurate quantitation of Ca²⁺ activities. Examples of cytoplasmic Ca²⁺ levels measured in plants with fura-2 and indo-1 are presented, and the prospects for applying more advanced technologies to fluorescent dye measurement are discussed.     

Structure of Avian Thymic Hormone, a High-Affinity Avian β-Parvalbumin, in the Ca-Free and Ca-Bound States

Originally isolated on the basis of its capacity to stimulate T-cell maturation and proliferation, avian thymic hormone (ATH) is nevertheless a parvalbumin, one of two β-lineage isoforms expressed in birds. We recently learned that addition of Ca²⁺-free ATH to a solution of 8-anilinonaphthalene-1-sulfonate (ANS) markedly increases ANS emission. This behavior, not observed in the presence of Ca²⁺, suggests that apolar surface area buried in the Ca²⁺-bound state becomes solvent accessible upon Ca²⁺ removal. In order to elucidate the conformational alterations that accompany Ca²⁺ binding, we have obtained the solution structure of the Ca²⁺-free protein using NMR spectroscopy and compared it to the Ca²⁺-loaded protein, solved by X-ray crystallography. Although the metal-ion-binding (CD-EF) domains are largely coincident in the superimposed structures, a major difference is observed in the AB domains. The tight association of helix B with the E and F helices in the Ca²⁺-bound state is lost upon removal of Ca²⁺, producing a deep hydrophobic cavity. The B helix also undergoes substantial rotation, exposing the side chains of F24, Y26, F29, and F30 to solvent. Presumably, the increase in ANS emission observed in the presence of unliganded ATH reflects the interaction of these hydrophobic residues with the fluorescent probe. The increased solvent exposure of apolar surface area in the Ca²⁺-free protein is consistent with previously collected scanning calorimetry data, which indicated an unusually low change in heat capacity upon thermal denaturation. The Ca²⁺-free structure also provides added insight into the magnitude of ligation-linked conformational alteration compatible with a high-affinity metal-ion-binding signature. The exposure of substantial apolar surface area suggests the intriguing possibility that ATH could function as a reverse Ca²⁺ sensor.     

Activation of a capacitative Ca entry pathway by store depletion in cultured hippocampal neurones

Intracellular Ca²⁺ ([Ca²⁺]_i) changes were measured in cell bodies of cultured rat hippocampal neurones with the fluorescent indicator Fluo-3. In the absence of external Ca²⁺, the cholinergic agonist carbachol (200 μM) and the sarco-endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin (0.4 μM) both transiently elevated [Ca²⁺]_i. A subsequent addition of Ca²⁺ into the bathing medium caused a second [Ca²⁺]_i change which was blocked by lanthanum (50 μM). Taken together, these experiments indicate that stores depletion can activate a capacitative Ca²⁺ entry pathway in cultured hippocampal neurones and further demonstrate the existence of such a Ca²⁺ entry in excitable cells.