Endoplasmic reticulum stress and alteration in calcium homeostasis are involved in cadmium-induced apoptosis.

Cadmium, a toxic environmental contaminant, exerts adverse effects on different cellular pathways such as cell proliferation, DNA damage and apoptosis. In particular, the modulation of Ca(2+) homeostasis seems to have an important role during Cd(2+) injury, but the precise assessment of Ca(2+) signalling still remains poorly understood. We used aequorin-based probes specifically directed to intracellular organelles to study Ca(2+) changes during cadmium injury. We observed that cadmium decreased agonist-evoked endoplasmic reticulum (ER) Ca(2+) signals and caused a 40% inhibition of sarcoplasmic-ER calcium ATPases activity. Moreover, time course experiments correlate morphological alterations, processing of xbp-1 mRNA and caspase-12 activation during cadmium administration. Finally, the time response of ER to cadmium injury was compared with that of mitochondria. In conclusion, we highlighted a novel pathway of cadmium-induced cell death triggered by ER stress and involving caspase-12. Mitochondria and ER pathways seemed to share common time courses and a parallel activation of caspase-12 and caspase-9 seemed likely to be involved in acute cadmium toxicity.

Fluorescence- and luminescence-based methods for the determination of affinity and activity of neuropeptide Y2 receptor ligands.

With respect to the discovery and characterization of neuropeptide Y(2) receptor ligands as pharmacological tools or potential drugs, fluorescence- and luminescence-based assays were developed to determine both the affinity and the activity of receptor agonists and antagonists. A flow cytometric binding assay is described for the hY(2) receptor stably expressed in CHO cells using cy5-labeled porcine neuropeptide Y and compared with a radioligand binding assay. Binding of the fluorescent ligand was visualized by confocal microscopy. Stable co-transfection with the chimeric G protein Gq(i5) enabled the establishment of a spectrofluorimetric fura-2 and a flow cytometric fluo-4 calcium assay. Further stable expression of apoaequorin targeted to the mitochondria allowed the establishment of an aequorin assay which could be performed in the 96-well format. The shape of the concentration-response curves of porcine neuropeptide Y in the presence of the Y(2)-selective receptor antagonist BIIE0246, characteristic of either competitive or insurmountable antagonism, depended on the period of incubation with the cells. Functional data of Y(2) receptor agonists and antagonists determined in the fluorescence- and luminescence-based assays were in good agreement.

Isolation and characterization of a leucokinin-like peptide of Drosophila melanogaster.

The leucokinin (LK) family of neuropeptides has been found widely amongst invertebrates. A member of this family was purified from adults of the fruit fly Drosophila melanogaster. The peptide sequence for Drosophila leucokinin (DLK) was determined as Asn-Ser-Val-Val-Leu-Gly-Lys-Lys-Gln-Arg-Phe-His-Ser-Trp-Gly-amide, making it the longest member of the family characterized to date. Synthetic DLK peptide was shown to act to stimulate fluid secretion in D. melanogaster Malpighian (renal) tubules by approximately threefold, with an EC(50) of approximately 10(-)(10 )mol l(-)(1), and a secondary effect at approximately 10(-)(7 )mol l(-)(1). DLK also acted to elevate intracellular [Ca(2+)] in the Malpighian tubules by approximately threefold, with an EC(50) of 10(-)(10) to 10(-)(9 )mol l(-)(1). Responses were detected in stellate cells and occasionally in principal cells, although at no concentration tested did [Ca(2+)] in the principal cell increase significantly above background. In stellate cells, DLK produced a biphasic rise in intracellular [Ca(2+)] from resting levels of 80-100 nmol l(-)(1), with a transient peak being followed by a slower rise that peaked at 200-300 nmol l(-)(1) after 3 s, then decayed over approximately 10 s. The wide range of concentrations over which DLK acts suggests the involvement of more than one receptor. The genomic sequence encoding the DLK peptide has been identified, and the gene has been named pp. The gene resides at cytological location 70E3-70F4 of chromosome 3L. The localisation of this first Drosophila LK gene in a genetic model permits a genetic analysis of the locus.

Nuclear calcium flux in Trypanosoma brucei can be quantified with targeted aequorin.

The following study was undertaken to determine if calcium ions move from the plasma membrane to the nucleus of Trypanosoma brucei. Nuclear and cytosolic calcium flux was measured with the calcium sensitive photoprotein, aequorin which was targeted to various locations in stably transformed procyclic cells. Immunoblots revealed that the recombinant proteins, CYT-AEQ and NUC-AEQ were translated in transformants, and that CYT-AEQ was contained in a soluble fraction. Immunolocalization demonstrated that NUC-AEQ was contained within the trypanosome nucleus. To evaluate calcium movement from the plasma membrane to the nucleus in live trypanosomes, aequorin was reconstituted in vivo with coelenterazine and luminescence was recorded. The resting levels of [Ca2+]cyt and [Ca2+]nuc were similar (314 +/- 43 and 287 +/- 28 nM, respectively). When calcium influx across the plasma membrane was initiated with 2 microM ionomycin, [Ca2+]cyt and [Ca2+]nuc each became elevated in parallel to a new steady state which was approximately 2-fold above the resting level. Compound 48/80 initiated a calcium flux across the plasma membrane by a different mechanism from ionomycin, and in a manner that was inhibited by the calcium channel antagonist, La3+. Compound 48/80 (8 micrograms/ml) transiently elevated [Ca2+]cyt to 1.73 +/- 0.3 microM over the course of 20 s, and also generated a transient rise in [Ca2+]nuc which peaked at 1.32 + 0.29 microM over the same time course. Overall, these data demonstrate that calcium moves into and out of the trypanosome nucleus in a manner which closely parallels changes in [Ca2+]cyt. A small calcium ion gradient between nucleus and cytoplasm was also observed.

Luminescence of aequorin is triggered by the binding of two calcium ions.

The photoprotein aequorin, capable of emitting light in the presence of a trace amount of Ca2+, is a useful indicator for studying intracellular calcium. The primary structure of aequorin indicated the presence of three Ca(2+)-binding sites, whereas log-log plots of the luminescence intensity versus Ca2+ concentration gave slopes ranging from 2 to 3 depending on the conditions used, suggesting the involvement of two or three Ca2+ ions in the luminescence reaction. Accurate information on the stoichiometry of Ca2+ is essential in interpreting the assay results obtained with aequorin. This study clearly shows that aequorin luminescence is triggered by the binding of two Ca2+ ions, based on the results of titrating aequorin with Ca2+.

A significant portion of the aequorin luminescent signal from stimulated human and rabbit platelets is due to exposure of the aequorin to calcium in the suspending medium.

We have examined in unstimulated and thrombin-stimulated human and rabbit platelets the localization and behavior of aequorin loaded by a variety of published methods. When platelets were suspended at 37 degrees C in Tyrode-albumin medium containing 2 mM Ca2+ and apyrase, we found with all preparations that total aequorin revealed by addition of Triton X-100 decreased by more than 50% over one hour. Incubation in the presence of 5 mM EGTA followed by addition of Ca2+ to restore the concentration to 2 mM showed that some aequorin had entered the medium; subsequent addition of Triton X-100 showed that the increase in aequorin in the medium matched the decrease in aequorin in the platelets, such that total aequorin remained unchanged. However, comparison of aequorin in platelets incubated in media with and without Ca2+ showed a larger decrease in platelets incubated in the presence of Ca2+; this finding may indicate the presence of an intracellular pool of Ca2+ which is more dependent on external Ca2+. Stimulation of platelets with thrombin in the presence of EGTA resulted in a smaller luminescent signal than in the presence of Ca2+. Subsequent addition of Ca2+ to 2 mM in the platelet suspension that originally contained EGTA or to its supernate (after centrifugation of the platelet suspension), resulted in a larger luminescent signal compared with controls, indicating that stimulation of the platelets had increased loss of the aequorin into the medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Light-emitting properties of recombinant semi-synthetic aequorins and recombinant fluorescein-conjugated aequorin for measuring cellular calcium.

15 kinds of recombinant semi-synthetic aequorins and a recombinant fluorescein-conjugated aequorin were prepared and their properties in Ca(2+)-triggered luminescence were studied. The semi-synthetic aequorins showed a wide range of Ca(2+)-sensitivity. The luminescence intensity of a high-sensitivity type (hcp-aequorin) was greater than 10(4)-times that of a low-sensitivity type (n-aequorin) at pCa 6.0-6.5. The fluorescein-conjugated aequorin exhibited fluorescence in addition to the Ca(2+)-triggered luminescence, thus it can be used to visualize the diffusion and distribution of aequorin in cells. The data obtained, particularly the Ca(2+)-sensitivity curves, are useful in selecting a suitable semi-synthetic aequorin for an experiment.

Preparation and initial characterization of crystals of the photoprotein aequorin from Aequorea victoria.

Crystals of recombinant aequorin, the photoprotein from the jellyfish Aequorea victoria, have been grown from solutions containing sodium phosphate. The crystals grow as thin plates which diffract to beyond 2.2 A resolution. The crystals are orthorhombic, space group P2(1)2(1)2(1); the axes are a = 89.1(1), b = 88.4(1), and c = 52.7(1) A. The asymmetric unit contains two molecules. Crystals exposed to calcium ion solutions emit a steady glow and slowly deteriorate, confirming that the crystals consist of a charged, competent photoprotein. This represents the first successful preparation of single crystals of a photoprotein suitable for diffraction analysis.