Background:Immunodeficiency in ataxia telangiectasia (A-T) is less severe in patients expressing some mutant or normal ATM kinase activity. We, therefore, determined whether expression of residual ATM kinase activity also protected against tumour development in A-T.Methods:From a total of 296 consecutive genetically confirmed A-T patients from the British Isles and the Netherlands, we identified 66 patients who developed a malignant tumour; 47 lymphoid tumours and 19 non-lymphoid tumours were diagnosed. We determined their ATM mutations, and whether cells from these patients expressed any ATM with residual ATM kinase activity.Results:In childhood, total absence of ATM kinase activity was associated, almost exclusively, with development of lymphoid tumours. There was an overwhelming preponderance of tumours in patients <16 years without kinase activity compared with those with some residual activity, consistent with a substantial protective effect of residual ATM kinase activity against tumour development in childhood. In addition, the presence of eight breast cancers in A-T patients, a 30-fold increased risk, establishes breast cancer as part of the A-T phenotype.Conclusion:Overall, a spectrum of tumour types is associated with A-T, consistent with involvement of ATM in different mechanisms of tumour formation. Tumour type was influenced by ATM allelic heterogeneity, residual ATM kinase activity and age.

Three isoforms of the sarcoplasmic/endoplasmic reticulum Ca 2؉ ATPase (SERCA) are known to exist in mammalian cells. This study investigated the effects of thapsigargin and a variety of commonly used hydrophobic inhibitors on these SERCA isoforms (i.e. SERCA1b, SERCA2b, and SERCA3a), which were transiently expressed in COS-7 cells. In addition, the study assessed whether the introduction of the phenylalanine to valine mutation at position 256 (F256V), known to reduce the potency of thapsigargin inhibition in avian SERCA1, affects the other SERCA isoforms in a similar manner and whether this mutation also affects the inhibition by other inhibitors. This study has shown that the sensitivity to thapsigargin is different for the SERCA isoforms (apparent K i values being 0.21, 1.3, and 12 nM for SERCA1b, SERCA2b, and SERCA3a, respectively). The reduction in thapsigargin sensitivity caused by the F256V mutation was also different for the three isoforms, with SERCA2b only being modestly affected by this mutation. Although some of the other inhibitors investigated (i.e. cyclopiazonic acid and curcumin) showed some differences in their sensitivity toward the SERCA isoforms, most were little affected by the F256V mutation, indicating that they inhibit the Ca 2؉ -ATPase by binding to sites on SERCA distinct from that of thapsigargin.

No abstract

The distribution of the secretory pathway Ca2+ -ATPase (SPCA1) was investigated at both the mRNA and protein level in a variety of tissues. The mRNA and the protein for SPCA1 were relatively abundant in rat brain, testis and testicular derived cells (myoid cells, germ cells, primary Sertoli cells and TM4 cells; a mouse Sertoli cell line) and epididymal fat pads. Lower levels were found in aorta (rat and porcine), heart, liver, lung and kidney. SPCA activities from a number of tissues were measured and shown to be particularly high in brain, aorta, heart, fat pads and testis. As the proportion of SPCA activity compared to total Ca2+ ATPase activity in brain, aorta, fat pads and testis were relatively high, this suggests that SPCA1 plays a major role in Ca2+ storage within these tissues. The subcellular localisation of SPCA1 was shown to be predominantly around the Golgi in both human aortic smooth muscle cells and TM4 cells.

The sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors thapsigargin (0.1-1 μM) and cyclopiazonic acid (10 μM), failed to affect resting [Ca2+] in human spermatozoa. Slow progesterone-induced [Ca2+ i]i oscillations in human spermatozoa, which involve cyclic emptying-refilling of an intracellular Ca2+ store were also insensitive to these inhibitors. Non-selective doses of thapsigargin (5-30 μM, 50-300 times the saturating dose for SERCA inhibition), caused elevation of resting [Ca2+]i and partial, dose-dependent disruption of oscillations. A 10-40 μM concentration of bis(2-hydroxy-3-tert-butyl-5-methyl-phenyl)methane (bis-phenol), which inhibits both thapsigargin-sensitive and -insensitive microsomal Ca2+ ATPases, caused elevation of resting [Ca2+]i and inhibition of [Ca2+]i oscillations at doses consistent with inhibition of thapsigargin-resistant, microsomal ATPase and liberation of stored Ca2+. Low doses of bis-phenol had marked effects on [Ca2+]i oscillation kinetics. Application of the drug to cells previously stimulated with progesterone had effects very similar to those observed when it was applied to unstimulated cells, suggesting that the sustained Ca2+ influx induced by progesterone is not mediated via mobilisation of Ca2+ stores. Western blotting for human sperm proteins showed expression of secretory pathway Ca2+ ATPase (SPCA1). Immunolocalisation studies revealed expression of SPCA1 in all cells in an area behind the nucleus, extending into the midpiece. Staining for SERCA, carried out in parallel, detected no expression with either technique. We conclude that: (1) intracellular Ca2+ store(s) and store-dependent [Ca2+]i oscillations in human spermatozoa rely primarily on a thapsigargin/cyclopiazonic acid-insensitive Ca2+ pump, which is not a SERCA as characterised in somatic cells; (2) effects of high-dose thapsigargin on spermatozoa primarily reflect non-specific actions on non-SERCAs and; (3) secretory pathway Ca2+ ATPases contribute at least part of this non-SERCA Ca2+ pump activity.

2-Aminoethoxydiphenyl Borate (2-APB) has been extensively used recently as a membrane permeable modulator of inositol-1,4,5-trisphosphate-sensitive Ca 2+ channels and store-operated Ca 2+ entry. Here, we report that 2-APB is also an inhibitor of sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) Ca 2+ pumps, and additionally increases ion leakage across the phospholipid bilayer. Therefore, we advise caution in the interpretation of results when used in Ca 2+ signalling experiments. The inhibition of 2-APB on the SERCA Ca 2+ pumps is isoform-dependent, with SERCA 2B being more sensitive than SERCA 1A (IC 50 values for inhibition being 325 and 725 lM, respectively, measured at pH 7.2). The Ca 2+ -ATPase is also more potently inhibited at lower pH (IC 50 ¼ 70 lM for SERCA 1A at pH 6). 2-APB decreases the affinity for Ca 2+ binding to the ATPase by more than 20-fold, and also inhibits phosphoryl transfer from ATP (by 35%), without inhibiting nucleotide binding. Activity studies performed using mutant Ca 2+ -ATPases show that Tyr837 is critical for the inhibition of activity by 2-APB. Molecular modeling studies of 2-APB binding to the Ca 2+ ATPase identified two potential binding sites close to this residue, near or between transmembrane helices M3, M4, M5 and M7. The binding of 2-APB to these sites could influence the movement of the loop between M6 and M7 (L6-7), and reduce access of Ca 2+ to their binding sites.Keywords: 2-APB; Ca 2+ -ATPase; Inhibition; SERCA.Ca 2+ plays a very important role in a number of signalling pathways, both within and between cells. The modulation of its levels in the cytosol is crucial to the viability and survival of the cell. Prolonged exposure to Ca 2+ can result in apoptosis, whereas a lack of rise in cytosolic [Ca 2+ ] may lead to the failure of signal transduction [1]. Specific pharmacological agents have been of great use as probes to aid our understanding of Ca 2+ signalling processes [2][3][4]. One such agent, 2-aminoethoxydiphenylborate (2-APB), has been reported to be a membrane permeable inhibitor of the inositol-1,4,5-trisphosphate (InsP 3 )-sensitive Ca 2+ channel with an IC 50 value of 42 lM (in the presence of 100 nM InsP 3 ) [5]. However, the effectiveness of 2-APB as a modulator of the InsP 3 receptor (InsP 3 R) has recently been questioned. We have recently shown that 2-APB is a lower affinity inhibitor of the type 1 InsP 3 R than was originally reported [6]. Our results show that the potency of 2-APB to inhibit InsP 3 -induced Ca 2+ release is dependent upon InsP 3 concentration used. At 0.25 lM InsP 3 , an IC 50 value of 220 lM was observed, while at 10 lM InsP 3 , the concentration of 2-APB required to half maximally inhibit Ca 2+ release is 1 mM.2-APB and xestospongin C (another cell permeant InsP 3 receptor inhibitor) have been used to characterize the mechanism of store-operated Ca 2+ entry, whereby Ca 2+ influx from the extracellular matrix is triggered by the emptying of Ca 2+ stores [7][8][9][10]. The concentrations of 2-APB used in these studies were in the ...

The pharmacology of macrocyclic lactones is varied, with many beneficial effects in treating disease processes. FK-506, rapamycin and ascomycin have been utilized as immunosuppressant agents. Ivermectin is typically used to treat parasitic worm infections in mammals. Another immunosuppressant, cyclosporin A, is a cyclic oligotide that has similar immunosuppressant properties to those exerted by macrocyclic lactones. Here we report on the inhibition by these compounds of sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase (SERCA) Ca(2+) pumps. Ivermectin, cyclosporin A and rapamycin all inhibited the skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1). In addition, although ivermectin inhibited brain microsomal endoplasmic reticulum (type 2b) Ca(2+)-ATPase, cyclosporin A and rapamycin did not. As cyclosporin A also did not inhibit cardiac Ca(2+)-ATPase activity, this would suggest that it could be an isoform-specific inhibitor. Ivermectin was shown to be the most potent Ca(2+)-ATPase inhibitor of the macrocyclic lactones (IC(50)=7 microM). It appears to show a 'competitive' inhibition with respect to high concentrations of ATP by increasing the regulatory binding site K(m) but without affecting the catalytic site K(m). In addition, ivermectin stabilizes the ATPase in an E1 conformational state, and inhibits Ca(2+) release from the enzyme during turnover. This would suggest that ivermectin inhibits Ca(2+) release from the luminal binding sites of the phosphoenzyme intermediate, a step that is known to be accelerated by high [ATP].

No abstract