Quantitation of cytosolic [Ca2+] in whole perfused rat hearts using Indo-1 fluorometry.

Fluorometric determination of cytosolic calcium, [Ca2+]c, using Indo-1 in intact tissue, is limited by problems in obtaining calibration parameters for Indo-1 in vivo. Therefore, the goal of this study was to calibrate Indo-1 using in vitro constants, obtained from protein-containing reference solutions designed to produce similar Indo-1 spectral properties to those in vivo. Due to wavelength-dependent tissue light absorbance, the in vitro constants had to be absorbance-corrected using a novel method. The correction factor was calculated from the relationship between the Indo-1 fluorescence intensities at the two detection wavelengths. A mixture of proteins at approximately 28 mg/ml had a similar Indo-1 isosbestic wavelength (430 nm) to that found in vivo (427 nm), and a similar fluorescence ratio maximum with saturating Ca2+ to that found in vivo (after absorbance correction). Using calibration constants from this protein mixture, calculated [Ca2+]c in a Langendorf perfused rat heart was 187 nM during diastole, and 464 nM in systole. This new calibration method circumvented the considerable experimental problems of previous methods which required measurements with the cytosol fully depleted and fully saturated with Ca2+.     

Three-photon induced fluorescence of the calcium probe Indo-1.

We report the calcium-dependent emission spectral properties of the calcium probe Indo-1 for three-photon excitation. We found that Indo-1 could be readily excited with the femtosecond pulses from a mode-locked Ti:sapphire laser at 885 nm. This wavelength is too long for two-photon excitation, which is expected to occur for wavelengths no longer than twice the longest single-photon absorption wavelength of 400 nm. For excitation at 885 nm the emission intensity was found to depend on the cube of the laser power, as expected for simultaneous interaction with three photons. At wavelengths below 840 nm the emission intensity depends on the square of the laser power, indicating two-photon excitation at shorter wavelengths. The intensity decays of Indo-1 were found to be dependent on Ca2+ and essentially identical for one- and three-photon excitation. The emission anisotropy of Indo-1 was found to be considerably higher for three-photon excitation than for one-photon excitation, consistent with cos6 theta photoselection, as compared with cos2 theta photoselection for one-photon excitation. The high values of the anisotropy are in agreement with those expected for a three-photon process. Calcium-dependent emission spectra were observed for Indo-1 with three-photon excitation, demonstrating that three-photon excitation of Indo-1 can be used for calcium imaging by emission intensity ratio measurements. The calcium-dependent emission spectra indicate a higher three-photon cross-section for the calcium-free form of Indo-1 than for the calcium-bound form. The possible advantages of three-photon excitation include the availability of the appropriate wavelengths with solid-state lasers, enhanced spatial resolution due to a reduced size of the excited volume, absence of light quenching, and possibly high selectivity of the three-photon excitation process.     

Microspectrofluorometry as a tool for investigation of non-calcium interactions of Indo-1.

Indo-1 is a fluorescent calcium probe used to measure intracellular free calcium concentrations. These measurements are often performed by comparing the fluorescence intensities of Indo-1-treated cells at two selected wavelengths corresponding to the maxima of the fluorescence spectra of the calcium-bound and calcium-free forms. In this study, we used an optical multichannel analyser to numerise the fluorescence emitted by a single cell. A computerised resolution of numerised spectra was used on intracellular Indo-1 fluorescence. Calculation of numerical and graphic estimators allows us to evaluate the fit of the resolution. Different sets of characteristic spectra were compared using this method. It appeared that no linear combination of the two known forms of Indo-1 and of the cell autofluorescence can fit with spectra of Indo-1-treated cells. In addition, a study of the physico-chemical properties of Indo-1 shows the existence of two other forms of the molecule: a protonated form (maximum emission at 455 nm) and a form in interaction with proteins (maximum emission at 438 nm). Taking into account the contribution of these two new forms leads to an improved spectral resolution of the fluorescence of Indo-1-treated living cells and, therefore, improves calcium measurements. Moreover, quantification of the amount of the protonated form of Indo-1 allows a measurement of intracellular pH at the same time as calcium determination.     

Evidence that binding of Indo-1 to cardiac myocyte protein does not markedly change Kd for Ca2+

Quantitative measurement of [Ca2+]i with the fluorescent Ca(2+)-indicators Indo-1 and Fura-2 is complicated by the possibility that the value of the dissociation constant (Kd) may be influenced by binding to intracellular proteins. We investigated this question in cultured chick ventricular myocytes by use of two different Indo-1 calibration methods. First, the Indo-1 fluorescence ratio (R) (400/500 nm) was measured in beating myocytes loaded by exposure to Indo-1/AM. Then, cells were exposed to the Ca2+ ionophore Br A-23187 and fluorescence ratio was measured in the presence of 500 nM Ca2+ (EGTA-Ca2+ buffer). Subsequently cells were permeabilized to Ca2+ by a 1 min exposure to 25 microM digitonin in the presence of 'zero' Ca2+ (10 mM EGTA) and saturating 1 mM Ca2+ to obtain Rmin, Rmax and beta. We then calculated [Ca2+]i from the formula ([Ca2+]i = Kd [( R - Rmin)/(Rmax - R)]beta). With Kd = 250 nM, calculated systolic [Ca2+]i was 750 +/- 44 nM and diastolic 269 +/- 19 nM (means +/- SEM, n = 16). The R value calculated for an assumed [Ca2+]i = 500 nM using the above formula and digitonin derived constants was very similar to the value measured using Br A-23187 (digitonin, 0.67 +/- 0.03: Br A-23187, 0.66 +/- 0.03, ns). As the Br A-23187 method is independent of the value chosen for Kd, we conclude that the Kd of 250 nM for Indo-1 measured in free solutions closely approximates the Kd for intracellular Indo-1 in these cells, and that therefore the Kd of Indo-1 for Ca2+ does not appear to be markedly affected by binding to proteins or other intracellular molecules.     

Intracellular elevations of free calcium induced by activation of histamine H1 receptors in interphase and mitotic HeLa cells: hormone signal transduction is altered during mitosis

A broad range of membrane functions, including endocytosis and exocytosis, are strongly inhibited during mitosis. The underlying mechanisms are unclear, however, but will probably be important in relation to the mitotic cycle and the regulation of surface phenomena generally. A major unanswered question is whether membrane signal transduction is altered during mitosis; suppression of an intracellular calcium [( Ca2+]i) transient could inhibit exocytosis; [Ca2+]i elevation could disassemble the mitotic spindle. Activation of the histamine H1 receptor interphase in HeLa cells is shown here by Indo-1 fluorescence to produce a transient elevation of [Ca2+]i. The [Ca2+]i transient consists of an initial sharp rise that is at least partially dependent on intracellular calcium followed by an elevated plateau that is absolutely dependent on extracellular calcium. The [Ca2+]i transient is completely suppressed by preincubation with the tumor promoter, phorbol myristate acetate, but is unaffected by preincubation with pertussis toxin (islet-activating protein). In mitotic (metaphase- arrested) HeLa cells, the [Ca2+]i transient is largely limited to the initial peak. Measurement of 45Ca2+ uptake shows that it is stimulated by histamine in interphase cells, but not in mitotics. We conclude that the histamine-stimulated generation of the second messenger, [Ca2+]i, in mitotic cells is limited by failure to activate a sustained calcium influx. The initial phase of calcium mobilization from intracellular stores is comparable to that in interphase cells. Hormone signal transduction thus appears to be altered during mitosis.     

Calcium channels in PDGF-stimulated A172 cells open after intracellular calcium release and are not voltage-dependent.

Using laser image cytometry and Indo-1 fluorescence, we investigated the intracellular free Ca2+ concentration ([Ca2+]i) of confluent A172 human glioblastoma cells stimulated by the BB homodimer of platelet-derived growth factor (PDGF-BB). The shape of the calcium transients and the delay time between stimulation and the beginning of the transient varied considerably. The percentage of responsive cells, the peak [Ca2+]i and the duration of the response were directly related to PDGF-BB dose, while the delay time was inversely related; the maximal response occurred at a PDGF-BB concentration of 20 ng/ml. Studies with EGTA and inorganic calcium-channel blockers (Ni2+, La3+) showed that the increase of [Ca2+]i resulted from initial release of intracellular stores and subsequent calcium influx across the plasma membrane. Opening of calcium channels in the plasma membrane, monitored directly by studying Mn2+ quenching of Indo-1 fluorescence, was stimulated by PDGF-BB and blocked by La3+; the opening occurred 55 +/- 10 s after the initial increase in [Ca2+]i. Therefore, in these tumor cells, intracellular release always occurs before channel opening in the plasma membrane. Depolarization of cells with high extracellular [K+] did not generally induce calcium transients but did decrease calcium influx. L-type calcium-channel blockers (verapamil, nifedipine, and diltiazem) had little or no effect on the calcium influx induced by PDGF-BB. These results indicate that PDGF-BB induces calcium influx by a mechanism independent of voltage-sensitive calcium channels in A172 human glioblastoma cells.     

Investigation of factors affecting fluorometric quantitation of cytosolic [Ca2+] in perfused hearts.

The goal of these studies was to examine the effects of several factors that may artifactually influence quantitation of cytosolic [Ca2+], [Ca2+]c, while using the fluorescent calcium indicator Indo-1. The following factors were investigated: 1) a possible fluorescence contribution from unhydrolized Indo-1/AM (by Mn2+ quenching), 2) Ca2+ buffering by Indo-1 (by varying [Indo-1]), 3) endothelial and mitochondrial Indo-1 loading (by bradykinin stimulation and calculations), and 4) effects of changing tissue fluorescence (predominantly NAD(P)H) on calculated [Ca2+]c during hypoxia (by a new method which allowed simultaneous determination of [Ca2+]c and changes in [NAD(P)H]). No significant contribution of Indo-1/AM was found. With increasing [Indo-1], calculated systolic [Ca2+]c fell significantly. Indo-1 incorporation (< 18%) into endothelial cells, caused a slight underestimation of systolic [Ca2+]c, while mitochondrial Indo-1 loading may cause overestimation of [Ca2+]c. With increased tissue fluorescence, during hypoxia, systolic [Ca2+]c may be underestimated by approximately 27% (for Indo-1 loading factors three to five times original tissue fluorescence). These studies suggest conditions in which experimental artifacts could be minimized to allow reliable quantitation of [Ca2+]c in intact perfused hearts using Indo-1 fluorometry. The major problem of obtaining reliable results depended on the ability to correct for changing NAD(P)H fluorescence while keeping [Indo-1] low.     

High temporal resolution video imaging of intracellular calcium

We have developed a system for imaging intracellular free calcium ion concentration ([Ca2+]i) at the highest rate possible with conventional video equipment. The system is intended to facilitate quantitative study of rapid changes in [Ca2+]i in cells that move. It utilizes intensified video cameras with nearly ideal properties and digital image processing to produce two images that can be ratioed without artifacts. Two dichroic mirrors direct images of cellular Indo-1 fluorescence at two different wavelengths to two synchronized video cameras, each consisting of a fast micro-channel plate image intensifier optically coupled with a tapered fiber optic bundle to a CCD image sensor. The critical technical issues in this dual-image system are: (1) minimization and correction of the small geometric and other types of differences in the images provided by the two cameras; and (2) the signal-to-noise ratio that can be achieved in single frames. We have used this system to obtain images of [Ca2+]i at 16.7 ms intervals in voltage-clamped single cardiac cells perfused internally with Indo-1 (pentapotassium salt). The images indicate that, except for the nuclear regions, [Ca2+]i is uniform during normal excitation-contraction coupling. In contrast, changes in [Ca2+]i propagate in rapid 'waves' during the spontaneous release of Ca2+ that accompanies certain 'Ca2(+)-overload conditions.'     

Regulation of calcium signalling by 1-O-alkylglycerols in human Jurkat T lymphocytes

We studied the role of natural occurring 1-O-alkylglycerols on the calcium signalling in Jurkat T-cells. Alkylglycerols evoked an increase in free intracellular calcium concentration [Ca2+]i, in a dose-dependent manner. When the experiments were performed in calcium-free buffer, the alkylglycerol response on the rise of [Ca2+]i was wholly abolished compared with the one in calcium-containing buffer, suggesting that these etherlipids induce a calcium influx by the opening of Ca2+ channels. We further employed inhibitors of voltage-gated calcium channels. We observed that omega-conotoxin, a blocker of N-type voltage-activated Ca2+ channels, but not verapamil, a blocker of L-type voltage-activated Ca2+ channels, curtailed significantly the calcium rise evoked by the lipid agents. Alkylglycerols also induced plasma membrane depolarisation, known to be involved in the opening of the voltage-gated calcium channels. Our study shows that alkylglycerols increase [Ca2+]i influx in human Jurkat T-cells possibly by modulating the permeability of calcium channels.     

Intracellular calibration of the calcium indicator indo-1 in isolated fibers of Xenopus muscle.

Estimates of the free myoplasmic [Ca2+] ([Ca2+]i) with fluorescent dyes are complicated by the fact that some properties of these dyes are altered in the intracellular environment. In the present study indo-1 was used to measure [Ca2+]i in isolated muscle fibers from Xenopus frogs. Fluorescent ratio signals obtained from indo-1 were converted into [Ca2+]i by means of an intracellular calibration method, which involved microinjection of 0.5 M EGTA and 1 M CaCl2 to get the ratio at very low (Rmin) and high (Rmax) [Ca2+], respectively; ratios at intermediate [Ca2+] were obtained by injection of solutions with different EGTA/Ca(2+)-EGTA proportions. This calibration gave an intracellular Ca2+ dissociation constant of indo-1 of 311 nM and a [Ca2+]i at rest of 52 +/- 4 nM (mean +/- SE; n = 15). Indo-1 records during twitches were compared with records obtained with the much faster indicator mag-indo-1. This analysis suggests a Ca2+ dissociation rate of indo-1 of 52 s-1 (22 degrees C). This makes indo-1 less suitable for measurements of [Ca2+]i during twitches, whereas it is fast enough to follow most aspects of [Ca2+]i during tetani, including the relaxation phase.     

Intracellular Ca2+ measurement with Indo-1 in substrate-attached cells: advantages and special considerations

The dual emission, Ca2+ sensitive fluorescent dye, Indo-1, offers several potential advantages over its dual excitation analogue, Fura-2. Most notable among these advantages are increased speed of measurement using dual wavelength photometry and the absence of a requirement for special quartz optics. Despite these potential advantages, only a tiny fraction of the microscopic studies of intracellular free calcium ([Ca2+]i) on substrate-attached cells has employed Indo-1. Among the reasons for the infrequent use of Indo-1 are the fact that it exhibits somewhat different spectral properties in the cytosol than it does in extracellular buffers, and the notion that it is much more sensitive to photobleaching than Fura-2. We report here that under our experimental conditions, Indo-1 photobleaching is small and does not noticeably affect the measurement of free Ca2+, even after 30 minutes of continuous illumination. We also report a new method for creating in situ standard curves that is easy, reproducible, and yields values for [Ca2+]i that are identical to those obtained with Fura-2. In addition, we have found that Indo-1 is less subject than Fura-2 to compartmentalization within subcellular organelles. These results provide baseline data to take advantage of the significant improvement afforded by Indo-1 in the measurement of rapid [Ca2+]i responses and the avoidance of compartmentalization artifacts during experiments of long duration.     

The B lymphocyte calcium response to anti-Ig is diminished by membrane immunoglobulin cross-linkage to the Fc gamma receptor

We report the cytosolic free calcium, [Ca2+]i, responses of single murine B lymphocytes to whole and F(ab')2 fragments of anti-Ig measured in the flow cytometer with indo-1, a new fluorescent chelator of calcium. The principle advantages of this recording system are these: Indo-1 is highly fluorescent; hence, loading concentrations that introduce artifacts in the reported [Ca2+]i signal may be avoided. The measurement of [Ca2+]i by fluorescence ratio corrects for nonuniform dye uptake, making possible quantitative estimates of [Ca2+]i in single cells and an assessment of the variability of population responses. Baseline recordings of unstimulated lymphocytes indicated a narrow, stable range of [Ca2+]i (75 to 125 nM). The [Ca2+]i rise induced by various anti-Ig preparations exhibited considerable heterogeneity. The initial mean value for F(ab')2 anti-Ig-stimulated cells peaked above 1 microM and was due only to the release of Ca2+ from intracellular stores. A steady state elevation of [Ca2+]i was reached by 5 min and persisted for hours. Cells stimulated with intact anti-Ig reached similar initial peak [Ca2+]i values, but then declined toward baseline. This difference was due to membrane Ig-IgG Fc receptor (mIg-Fc gamma R) cross-linkage, because blocking the Fc gamma R with a monoclonal antibody made the [Ca2+]i responses to F(ab')2 and intact anti-Ig identical. The attenuation of the [Ca2+]i signal by mIg-Fc gamma R cross-linkage is proceeded by a corresponding Fc gamma-mediated reduction in anti-Ig-induced inositol trisphosphate elevation. These findings outline a biochemical basis for mIg- and Fc gamma R-mediated activation and regulation intrinsic to the B cell, and demonstrate the advantages of indo-1 over quin2 for fluorescent measurement of [Ca2+]i in small cells.     

Cytosolic free calcium levels in monolayers of cultured rat aortic smooth muscle cells. Effects of angiotensin II and vasopressin

A rise in cytosolic free calcium ([Ca2+]i) is thought to be the principal mediator in vascular smooth muscle contraction. Quantitative changes of [Ca2+]i in response to two vasoconstrictor peptide hormones, angiotensin II and vasopressin, were directly measured in monolayers of adherent cultured rat aortic smooth muscle cells loaded with the fluorescent calcium indicator Quin 2. Angiotensin II induced rapid, concentration-dependent rises in [Ca2+]i from 1.53 +/- 0.27 X 10(-7) (n = 16) up to 1.2 X 10(-6) M, with ED50 of 0.45 X 10(-9) M, an effect which was blocked by the antagonist analogue [Sar1, Ala8]angiotensin II. Vasopressin also elicited transient rises in [Ca2+]i to peak levels of about 8 X 10(-7) M, with ED50 of 1.05 X 10(-9) M, and this response was completely abolished by a vasopressor antagonist. In calcium-free medium, basal [Ca2+]i levels fell to 0.92 +/- 0.24 X 10(-7) M (n = 4), and both hormones were still able to raise [Ca2+]i, although to a lesser extent. Readdition of extracellular calcium following the [Ca2+]i transient induced a second, slower [Ca2+]i rise. In calcium-containing medium, lanthanum ion (2 X 10(-5) M) reduced peptide-evoked [Ca2+]i rises to the values observed in calcium-free medium. Stimulation with each peptide completely desensitized the smooth muscle cells to a subsequent identical challenge, with little crosstachyphylaxis. Potassium ion (50 mM) only minimally affected [Ca2+]i levels. The calcium channel blocker nifedipine (10(-6) M) did not prevent the [Ca2+]i rises induced by angiotensin II, vasopressin, or potassium. These findings indicate that the two physiologically important vasoconstrictor hormones angiotensin II and vasopressin rapidly raise [Ca2+]i in cultured vascular smooth muscle cells, in part by mobilizing calcium from intracellular pools and in part through activation of receptor-operated calcium channels.     

Indo-1 fluorescence signals elicited by membrane depolarization in enzymatically isolated mouse skeletal muscle fibers.

Indo-1 fluorescence signals were measured from one extremity of enzymatically isolated skeletal muscle fibers of mice. An original and simple method was developed to allow the measurements to be made under voltage-clamp control: the major part of a single fiber was embedded in silicone grease, so that only a short portion of one end of the fiber, from which the fluorescence measurements were taken, was in contact with the external solution. Membrane potential was held and varied by using a patch-clamp amplifier in whole-cell configuration with a single microelectrode, the tip of which was inserted across the silicone grease within the insulated portion of the fiber. In response to 100-ms depolarizing command pulses to voltages more positive than -40 mV (from a holding potential of -80 mV), clear changes in fluorescence were qualitatively observed to feature a time course of rise and decay expected from a change in intracellular calcium concentration ([Ca2+]i) due to voltage-dependent sarcoplasmic reticulum (SR) calcium release. Although the peak [Ca2+]i elicited by a 100-ms depolarization at 0 or +10 mV varied from fiber to fiber, it could clearly reach a value high enough to saturate Indo-1. The overall results show that this method represents an efficient way of measuring depolarization-induced [Ca2+]i changes in enzymatically dissociated skeletal muscle fibers.     

Membrane depolarization inhibits thrombin-induced calcium influx and aggregation in human platelets.

The relationship between thrombin-evoked changes in intracellular calcium concentration [( Ca2+]i) and aggregation was examined in Indo-1-loaded human platelets. The stimulus-induced intracellular calcium release and external calcium influx, as well as platelet aggregation, were studied in the same cell preparation. A close correlation between the sustained high [Ca2+]i level, depending on calcium entry, and the aggregation response was found. Gramicidin, at a concentration high enough to induce membrane depolarization, strongly inhibited the calcium influx and aggregation, but did not influence the thrombin-induced intracellular calcium release. We conclude that calcium influx through depolarization-inhibited calcium channels is a prerequisite of thrombin-induced platelet aggregation.     

Con A刺激致T淋巴细胞胞浆游离Ca~(2+)浓度升高

本文分别应用荧光Ca~(2+)指示剂Quin2和Indo-1研究了Con A刺激的T淋巴细胞[Ca~(2+)]i升高过程及其发生机制.结果表明Con A与T淋巴细胞作用可导致细胞[Ca~(2+)]i的迅速升高.这种增加的胞内游离Ca~(2+)不仅来自胞外Ca~(2+)的内流,也来源于胞内钙库的释放.其中Ca~(2+)内流与T细胞钙通道的开放有关.可被钙通道抑制剂戊脉胺抑制,细胞的去极化及钾通道阻断剂四乙胺均不能阻断Ca~(2+)的内流,提示Ca~(2+)内流不是通过电位操纵的钙通道实现的,也与拥通道的开闭无关.Ca~(2+)内流可能是通过Con A受体活化的受体操纵的钙通道而实现的.     

A role for the transient increase of cytoplasmic free calcium in cell rescue after photodynamic treatment.

Chinese hamster ovary (CHO) cells and T24 human bladder transitional carcinoma cells were treated with the photosensitizers aluminum phthalocyanine (AlPc) and hematoporphyrin derivative (HPD), respectively. Exposure of both sensitized cell lines to red light caused an immediate increase of cytoplasmic free calcium, [Ca2+]i, reaching a peak within 5-15 min after exposure and then returning to basal level (approximately 200 nM). The level of the peak [Ca2+]i depended on the light fluence, reaching a maximum of 800-1000 nM at light doses that kill about 90% of the cells. Loading the cells with the intracellular calcium chelators quin2 or BAPTA prior to light exposure enhanced cell killing. This indicates that increased [Ca2+]i after photodynamic therapy (PDT) contributed to survivability of the treated cells by triggering a cellular rescue response. The results of experiments with calcium-free buffer and calcium chelators indicate that both in CHO cells treated with AlPc and with HPD-PDT of T24 cells extracellular Ca2+ influx is mainly responsible for elevated [Ca2+]i. PDT is unique in triggering a cell rescue process via elevated [Ca2+]i. Other cytotoxic agents, e.g., H2O2, produce sustained increase of [Ca2+]i that is involved in the pathological processes leading to cell death.     

Epididymal maturation affects calcium regulation in equine spermatozoa exposed to heparin and glucose

Spermatozoal function is affected by the ability to regulate intracellular calcium concentrations ([Ca2+]i), and may be influenced by epididymal maturation as well as environmental components. Regulation of [Ca2+]i in ejaculated and epididymal stallion spermatozoa was monitored over time in various media. Spermatozoa from each of 5 pony stallions (3 ejaculate samples and 1 caput and cauda sample) were labeled with the fluorescent calcium indicator probe Indo-1 in a calcium-free modified Tyrode's buffer. Fluorescent emissions were monitored by a dual wavelength spectrofluorometer over 5 h. Calcium (1 mM) was added at T = 15 min, and heparin (HEP; 10 micrograms/ml) or heparin plus glucose (hGLUC; 5 mM in 10 micrograms/ml heparin) was added at T = 30 min. Spermatozoal Ca2+ content and regulation differed among males (P = 0.0066). Relative initial [Ca2+]i differed significantly among all stages of maturity (0.84 +/- 0.104, 0.76 +/- 0.023, 1.20 +/- 0.036 LSM of relative Ca2+ units for caput, cauda and ejaculate spermatozoa respectively; P = 0.001). Rate of Ca2+ uptake was similar for ejaculate and cauda spermatozoa (0.021 +/- 0.005 and 0.026 +/- 0.002 relative Ca2+ units/sec) but slower for caput spermatozoa (0.012 +/- 0.001; P = 0.0006). There was no immediate effect of HEP or hGLUC in any stage (P > 0.05), and caput spermatozoa did not differ from cauda spermatozoa for any treatment or time period. A significant increase in [Ca2+]i was seen in ejaculate spermatozoa treated with HEP from 2 h on (P < 0.05). This study demonstrates that both the absolute Ca2+ concentration and the rate of Ca2+ internalization in equine spermatozoa is dependent on the stage of maturation. Ejaculate spermatozoa respond to heparin through increased [Ca2+]i, which may play a role in the fertilizing ability of ejaculate spermatozoa.     

Sphingosylphosphorylcholine enhances calcium entry in thyroid FRO cells by a mechanism dependent on protein kinase C

Several sphingolipid derivatives, including sphingosylphosphorylcholine (SPC), regulate a multitude of biological processes. In the present study we show that both human thyroid cancer cells (FRO cells) and normal human thyroid cells express G protein-coupled receptor 4 (GPR4) and ovarian cancer G protein-coupled receptor 1 (OGR1), putative SPC-specific receptors. In FRO cells SPC evoked a concentration-dependent increase in intracellular free calcium concentration ([Ca2+]i) in a calcium containing, but not in a calcium-free buffer. Sphingosine 1-phosphate (S1P) evoked an increase in [Ca2+]i in both a calcium containing and a calcium-free buffer. The phospholipase C (PLC) inhibitor U 73122 potently attenuated the effect of SPC, suggesting that effects of SPC were mediated by a G protein coupled receptor. Overnight pretreatment of the cells with pertussis toxin did not affect the SPC-evoked response. Interestingly, SPC did not evoke an increase in inositol phosphates, although S1P did so. Furthermore, in cells pretreated with thapsigargin to deplete intracellular calcium stores, SPC still evoked an increase in [Ca2+]i, suggesting that SPC mainly evoked entry of extracellular calcium. When the cells were pretreated with the protein kinase C (PKC) inhibitor GF 109203X, or when the cells were pretreated with PMA for 24 h, the SPC-evoked calcium entry was attenuated. Thus, the SPC-evoked calcium entry was apparently dependent on PKC. In sharp contrast, the increase in [Ca2+]i evoked by S1P was not sensitive to GF 109203X. Furthermore, the calcium entry evoked by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol was not inhibited by GF 109203X. In addition, SPC decreased the incorporation of 3H-thymidine in a concentration-dependent manner in FRO cells. Taken together, SPC may be an important factor regulating thyroid cancer cell function.     

Calcium Channel Activity during Pollen Tube Growth and Reorientation

We have shown previously that the inhibition of pollen tube growth and its subsequent reorientation in Agapanthus umbellatus are preceded by an increase in cytosolic free calcium ([Ca2+]c), suggesting a role for Ca2+ in signaling these processes. In this study, a novel procedure was used to measure Ca2+ channel activity in living pollen tubes subjected to various growth reorienting treatments (electrical fields and ionophoretic microinjection). The method involves adding extracellular Mn2+ to quench the fluorescence of intracellular Indo-1 at its ca2+-insensitive wavelength (isosbestic point). The spatial and temporal kinetics of Ca2+ channel activity correlated well with measurements of [Ca2+]c dynamics obtained by fluorescence ratio imaging of Indo-1. Tip-focused gradients in Ca2+ channel activity and [Ca2+]c were observed and quantified in growing pollen tubes and in swollen pollen tubes before reoriented growth. In nongrowing pollen tubes, Ca2+ channel activity was very low and [Ca2+]c gradients were absent. Measurements of membrane potential indicated that the growth reorienting treatments induced a depolarization of the plasma membrane, suggesting that voltage-gated Ca2+ channels might be activated.