Biomedical subjects

M Treiman

Publications and source records attributed to M Treiman.

At least 19 recordsLinked to original sources

PubMed2001

Upregulation of the SERCA-type Ca2+ pump activity in response to endoplasmic reticulum stress in PC12 cells.

BACKGROUND: Ca2+-ATPases of endoplasmic reticulum (SERCAs) are responsible for maintenance of the micro- to millimolar Ca2+ ion concentrations within the endoplasmic reticulum (ER) of eukaryotic cells. This intralumenal Ca2+ storage is important for the generation of Ca2+ signals as well as for the correct folding and posttranslational processing of proteins entering ER after synthesis. ER perturbations such as depletion of Ca2+ or abolishing the oxidative potential, inhibition of glycosylation, or block of secretory pathway, activate the Unfolded Protein Response, consisting of an upregulation of a number of ER-resident chaperones/stress proteins in an effort to boost the impaired folding capacity. RESULTS: We show here that in PC12 cells, depletion of ER Ca2+ by EGTA, as well as inhibition of disulphide bridge formation within the ER by dithiotreitol or inhibition of N-glycosylation by tunicamycin, led to a 2- to 3-fold increase of the SERCA-mediated 45Ca2+ transport to microsomes isolated from cells exposed to these stress agents. The time course of this response corresponded to that for transcriptional upregulation of ER stress proteins, as well as to the increase in the SERCA2b mRNA, as we recently observed in an independent study. CONCLUSIONS: These findings provide the first functional evidence for the increase of SERCA pumping capacity in cells subjected to the ER stress. Since at least three different and unrelated mechanisms of eliciting the ER stress response were found to cause this functional upregulation of Ca2+ transport into the ER, these results support the existence of a coupling between the induction of the UPR pathway in general, and the regulation of expression of at least one of the SERCA pump isoforms.

Animals↗

PubMed2000

The sarco/endoplasmic reticulum calcium-ATPase 2b is an endoplasmic reticulum stress-inducible protein.

The sarco/endoplasmic reticulum calcium-ATPase (SERCA) translocates Ca(2+) from the cytosol to the lumen of the endoplasmic reticulum. This Ca(2+) storage is important for cellular processes such as calcium signaling and endoplasmic reticulum (ER)-associated posttranslational protein modifications. We investigated the expression of the SERCA2 and SERCA3 isozymes in PC12 cells exposed to agents interfering with different aspects of the posttranslational protein processing within the ER, thereby activating the ER stress-induced unfolded protein response (UPR). All agents increased the SERCA2b mRNA level 3-4-fold, in parallel with increasing mRNA levels for the ER stress marker proteins BiP/GRP78 and CHOP/GADD153. In contrast, SERCA3 mRNA levels did not change. SERCA2b mRNA stability was not changed, indicating that the mechanism of its up-regulation was transcriptional, in accordance with the presence of ER stress response elements in the promoter region of the SERCA2 gene. SERCA2b was also increased at the protein level upon ER stress treatments. Induction of ER stress by tunicamycin, dithiothreitol, or l-azetidine 2-carboxylic acid did not result in depletion of ER calcium, showing that such depletion was not necessary for up-regulation of SERCA2b expression or UPR activation in general. We conclude that the SERCA2b expression can be controlled by the UPR pathway independently of ER Ca(2+) depletion.

Animals↗

PubMed2000

Effect of nitric oxide on endoplasmic reticulum calcium homeostasis, protein synthesis and energy metabolism.

It has been suggested that nitric oxide (NO) may contribute to ischemia-induced cell injury. However, the mechanisms underlying NO toxicity have not yet been fully elucidated. In the present study, we investigated the effect of NO on the level of endoplasmic reticulum (ER) calcium stores, on ER Ca2+ pump activity, on protein synthesis, on concentrations of high-energy phosphates, and on gadd153 mRNA levels. Primary neuronal cells were exposed to the NO-donor (+/-)-S-Nitroso-N-acetylpenicillamine (SNAP) for 1 h, 2 h, 6 h or 24 h. The level of ER calcium stores was evaluated by measuring the increase in cytoplasmic calcium activity induced by exposing cells to thapsigargin, an irreversible inhibitor of ER Ca(2+)-ATPase; the activity of ER Ca(2+)-ATPase was determined by measuring a phosphorylated intermediate; SNAP-induced changes in gadd153 expression were evaluated by quantitative PCR; SNAP-induced changes in protein synthesis were investigated by measuring the incorporation of L-[4,5-3H]leucine into proteins, and changes in the levels of ATP, ADP, AMP were measured by HPLC. Exposing cells to SNAP for 1 h to 2 h induced a marked depletion of ER calcium stores through an inhibition of ER Ca(2+)-ATPase (to 58% of control), and a concentration-dependent suppression of protein synthesis which was reversed in the presence of hemoglobin, suggesting NO-related effects. ATP levels and adenylate energy charge were significantly decreased only when cells were exposed to the highest SNAP concentration for 6 h or 24 h, excluding significant effects of NO on the energy state of cells in the acute state, i.e. when ER calcium stores were already completely depleted and protein synthesis severely suppressed. In light of the regulatory role of ER calcium homeostasis in the control of protein synthesis, the results imply that the suppression of protein synthesis resulted from NO-induced inhibition of ER Ca(2+)-ATPase and depletion of ER calcium stores, and that NO-induced disturbances of energy metabolism are secondary to the effect of NO on ER calcium homeostasis. It is, therefore, concluded that ER calcium stores are a primary target of NO-toxicity.

Adenine Nucleotides↗

PubMed1999

Thapsigargin analogues for targeting programmed death of androgen-independent prostate cancer cells.

A number of analogues of thapsigargin, a selective inhibitor of the sarco-endoplasmic reticulum Ca2+-ATPases have been synthesized. In all of the prepared analogues the butanoyl residue at O-8 has been replaced with a residue containing an aromatic amine. The amine can be used as an anchoring point for attaching a peptide group sensitive to the proteolytic enzyme, prostate specific antigen, secreted by prostate cancer cells. Like thapsigargin, the analogues are capable of elevating the cytoplasmic Ca2+ concentration approximately sevenfold when tested at effective cytotoxic doses. The analogues in which the 8-O-butanoyl group has been replaced with 3-(4-aminophenyl)propanoyl or 4-aminocinnamoyl were found potently to induce programmed cell death of the prostate cancer cells.

Androgens↗

PubMed1999

Recovery of neuronal protein synthesis after irreversible inhibition of the endoplasmic reticulum calcium pump.

In the physiological state, protein synthesis is controlled by calcium homeostasis in the endoplasmic reticulum (ER). Recently, evidence has been presented that dividing cells can adapt to an irreversible inhibition of the ER calcium pump (SERCA), although the mechanisms underlying this adaption have not yet been elucidated. Exposing primary neuronal cells to thapsigargin (Tg, a specific irreversible inhibition of SERCA) resulted in a complete suppression of protein synthesis and disaggregation of polyribosomes indicating inhibition of the initiation step of protein synthesis. Protein synthesis and ribosomal aggregation recovered to 50-70% of control when cells were cultured in medium supplemented with serum for 24 h, but recovery was significantly suppressed in a serum-free medium. Culturing cells in serum-free medium for 24 h already caused an almost 50% suppression of SERCA activity and protein synthesis. SERCA activity did not recover after Tg treatment, and a second exposure of cells to Tg, 24 h after the first, had no effect on protein synthesis. Acute exposure of neurons to Tg induced a depletion of ER calcium stores as indicated by an increase in cytoplasmic calcium activity, but this response was not elicited by the same treatment 24 h later. However, treatments known to deplete ER calcium stores (exposure to the ryanodine receptor agonists caffeine or 2-hydroxycarbazole, or incubating cells in calcium-free medium supplemented with EGTA) caused a second suppression of protein synthesis when applied 24 h after Tg treatment. The results suggest that after Tg exposure, restoration of protein synthesis was induced by recovery of the regulatory link between ER calcium homeostasis and protein synthesis, and not by renewed synthesis of SERCA protein or development of a new regulatory system for the control of protein synthesis. The effect of serum withdrawal on SERCA activity and protein synthesis points to a role of growth factors in maintaining ER calcium homeostasis, and suggests that the ER acts as a mediator of cell damage after interruption of growth factor supplies.

Animals↗

PubMed1998

ACTA, a fluorescent analogue of thapsigargin, is a potent inhibitor and a conformational probe of skeletal muscle Ca2+-ATPase.

Thapsigargin is a highly potent and selective inhibitor of sarco-endoplasmic reticulum (SERCA) family of Ca2+-ATPases and a useful tool in research concerning the function of intracellular Ca2+ stores. We describe here a novel fluorescent derivative (8-O-(4-aminocinnamoyl)-8-O-debutanoylthapsigargin, termed ACTA) of this compound, acting as a Ca2+-ATPase inhibitor with a potency approaching that of thapsigargin. Binding of ACTA to the skeletal muscle sarcoplasmic reticulum vesicles results in a strong fluorescence enhancement, approximately 66% of which depends on ACTA association with Ca2+-ATPase. This specific component of ACTA fluorescence is sensitive to the E1-E2 conformational equilibrium of the pump. The combined properties of high potency and binding-dependent fluorescence suggest ACTA to be a useful probe for a range of studies involving the SERCA class of ATPases.

Calcium↗

PubMed1998

A tool coming of age: thapsigargin as an inhibitor of sarco-endoplasmic reticulum Ca(2+)-ATPases.

Thapsigargin is the most widely used inhibitor of the ubiquitous sarco-endoplasmic reticulum Ca(2+)-ATPases in mammalian cells. Over the past ten years, this guaianolide compound of plant origin has become a popular tool in a host of studies directed at elucidating the mechanisms of intracellular Ca2+ signalling. Its remarkable potency and selectivity have been instrumental in widening our view of the function of intracellular Ca2+ stores to include such key aspects as store-operated Ca2+ entry or the involvement of the stores in protein synthesis or cell growth. In this article Marek Treiman, Casper Caspersen and Søren Brøgger Christensen review the key pharmacological features of thapsigargin action; they also discuss some of the ways in which its unique properties have shown to be important for obtaining new insights into the biology of Ca2+ stores, and how these properties might encompass a therapeutic potential. In parallel, attention is drawn to some of the limitations and pitfalls encountered when working with thapsigargin.

Animals↗

PubMed1996

Depletion of neuronal endoplasmic reticulum calcium stores by thapsigargin: effect on protein synthesis.

We have used thapsigargin (TG), a specific, irreversible inhibitor of endoplasmic reticulum (ER) Ca(2+)-ATPases, and caffeine, an agonist of the ryanodine receptor, to study the effect of emptying of ER calcium stores on protein synthesis in neuronal cells. TG at 1 microM caused a permanent inhibition of protein synthesis in hippocampal slices from 3-week-old rats but no inhibition in slices prepared from 2-month-old animals. Caffeine at 10 mM caused a reduction of protein synthesis in both 3-week- and 2-month-old rats immediately after exposure, but complete recovery of protein synthesis occurred within 30 min after treatment. In neuronal cells, TG produced an almost complete inhibition of protein synthesis that was only partially reversed over a 24-h recovery period. TG did not significantly affect neuronal ATP levels or energy charge. Fifty percent inhibition of protein synthesis was achieved with approximately 5 nM TG. Recovery of protein synthesis after TG treatment was significantly hindered when serum was omitted from the medium after TG exposure, suggesting that serum promotes recovery of ER calcium homeostasis. It is concluded that TG is a suitable tool for the study of the mechanisms of protein synthesis inhibition after transient cerebral ischemia. The possibility that disturbances in ER calcium homeostasis may contribute to the pathological process of ischemic cell death is discussed.

Animals↗

PubMed1995

Thapsigargin discriminates strongly between Ca(2+)-ATPase phosphorylated intermediates with different subcellular distributions in bovine adrenal chromaffin cells.

We studied the effects of thapsigargin on the formation of the phosphorylated intermediates (E approximately Ps) of endoplasmic reticulum Ca(2+)-ATPases in microsomes from bovine adrenal medulla. When submicrosomal fractions were separated on a sucrose gradient, two components of 100 kDa Ca(2+)-ATPase E approximately P displaying distinct subcellular distributions were resolved. The first component was defined by Ca(2+)-induced protection against thapsigargin inhibition. The second component did not display such protection, with a 3 orders of magnitude difference in thapsigargin inhibitory potency towards the 2 components. In the absence of Ca2+, both E approximately P components were highly sensitive to thapsigargin inhibition, revealing the presence of high-affinity thapsigargin-binding sites characteristic of SERCA ATPases. These data demonstrate a new level of molecular heterogeneity among Ca(2+)-ATPases of endoplasmic reticulum, and provide the first evidence of differential subcellular localization of individual Ca2+ pump subtypes in cells of neural origin.

Adrenal Medulla↗

PubMed1995

Thapsigargin-sensitive Ca(2+)-ATPases account for Ca2+ uptake to inositol 1,4,5-trisphosphate-sensitive and caffeine-sensitive Ca2+ stores in adrenal chromaffin cells.

In chromaffin cells of adrenal medulla, heterogeneity of Ca2+ stores has been suggested with respect to the mechanisms of Ca2+ uptake and release. We have examined Ca(2+)-ATPases responsible for loading of Ca2+ stores in these cells for their sensitivity to thapsigargin, a highly selective inhibitor of the SERCA [sarco(endo)plasmic reticulum calcium ATPase] family of intracellular Ca2+ pumps. Using immunostaining, we studied the distribution of Ca(2+)-ATPases, and of receptors for inositol 1,4,5-trisphosphate (InsP3) and ryanodine, in the density-gradient fractions of microsomes from bovine adrenal medulla. In parallel, we examined distribution profiles of ATP-dependent Ca2+ uptake in the same fractions, along with subcellular markers for plasma membranes and endoplasmic reticulum (ER). Two Ca(2+)-ATPase-like proteins (116 and 100 kDa) were detected, consistent with the presence of SERCA 2b and SERCA 3 isoenzymes of Ca2+ pumps. The distribution of these putative Ca(2+)-ATPase iso-enzymes paralleled that of InsP3 and ryanodine receptors. This distribution of ER Ca(2+)-ATPases, as determined immunologically, was consistent with that of thapsigargin-sensitive, but not of thapsigargin-insensitive, ATP-dependent Ca2+ uptake. In contrast, the distribution profile of the thapsigargin-insensitive Ca2+ uptake was strongly correlated to that of plasma membranes, and co-distributed with plasma membrane Ca(2+)-ATPase detected immunologically. In isolated, permeabilized chromaffin cells, InsP3 and caffeine induced Ca2+ release following an ATP-dependent Ca2+ accumulation to the stores. This accumulation was abolished by thapsigargin. Together, these data strongly indicate that the thapsigargin-sensitive, presumably SERCA-type Ca(2+)-ATPases account for Ca2+ uptake to InsP3-sensitive, as well as to caffeine-sensitive, Ca2+ stores in bovine adrenal chromaffin cells.

Adrenal Medulla↗

PubMed1994

Dihydropyridine ligands influence the evoked release of oxytocin and vasopressin dependent on stimulation conditions.

The effects of dihydropyridine ligands on the electrically evoked release of neurohypophysial hormones from isolated, rat neurointermediate lobes were investigated as a function of all combinations of two pulse widths (0.2 and 2 ms) and three stimulation frequencies (6.5, 13 and 30 Hz). The dihydropyridine agonist (S)-(+)-202-791 potentiated concentration dependently the release of both oxytocin and vasopressin at a pulse width of 2 ms and a frequency of 6.5 Hz. This effect of (S)-(+)-202-791 was abolished by the antagonist (-)-nitrendipine and stereospecifically by (R)-(-)-202-791 (only vasopressin). The antagonist (R)-(-)-202-791 alone inhibited the release of oxytocin at 13 Hz and 2 ms. The results presented show that the effects of the dihydropyridine ligands are dependent on the stimulation conditions, and thus demonstrate that the entry of Ca2+ through the dihydropyridine sensitive L-type Ca2+ channel is associated with electrically evoked release of neurohypophysial hormones under certain conditions.

Animals↗

PubMed1993

Derivatives of thapsigargin as probes of its binding site on endoplasmic reticulum Ca2+ ATPase. Stereoselectivity and important functional groups.

The naturally occurring sesquiterpene lactone thapsigargin is a potent and selective inhibitor of SERCA ATPases, a family of Ca(2+)-pumping ATPases present in the endoplasmic reticulum of all mammalian cells. We have studied some of the molecular features of thapsigargin responsible for its inhibitory action towards these Ca2+ ATPases. A series of thapsigargin analogues were synthesised and their inhibitory potencies determined using the uptake of 45Ca2+ in bovine cerebellar microsomes as a sensitive marker of Ca2+ ATPase activity. An attenuation of the inhibitory potency relative to the parent compound was found ranging from slight to over 3 orders of magnitude. The inhibitory activity showed a very strong configuration dependence, a major contribution from the ester groups at C3 and C10, and an apparently minor contribution from the lactone ring substituents. The data are consistent with thapsigargin fitting to a sterically discriminating cleft involving the hydrophobic transmembrane region of the ATPase, and is compatible with available kinetic evidence of thapsigargin-mediated inhibition.

Animals↗

PubMed1993

Isolation of chromaffin cell thapsigargin-sensitive Ca2+ store in light microsomes from bovine adrenal medulla.

1. A subcellular fractionation procedure for bovine adrenal glands was designed with the aim to study the biochemical properties of Ca2+ stores in chromaffin cells. 2. The thapsigargin-sensitive compartment of Ca2+ stores was found to be highly enriched in a light microsomal fraction (LMF) on a 15-30% linear sucrose gradient, and was found to be essentially devoid of contamination by plasma, mitochondrial or secretory granule membranes. 3. A Ca(2+)-pumping ATPase was identified in this LMF as a 97 kDa protein forming an acid-stable, Ca(2+)-dependent, thapsigargin-sensitive phosphorylated intermediate upon incubation with [gamma-32P]ATP, suggesting this protein to represent a SERCA-3 isoform of Ca2+ ATPases. 4. A major 162 kDa protein, previously demonstrated in the isolated chromaffin cells, was enriched in the LMF, distributing on sucrose gradients in parallel with the thapsigargin-sensitive Ca2+ uptake. 5. LMF appears to represent a part of the thapsigargin-sensitive Ca2+ store of chromaffin cells, and should be useful for further studies of the store properties at the subcellular and molecular level.

Adrenal Medulla↗

PubMed1991

Parallel expression of synaptophysin and evoked neurotransmitter release during development of cultured neurons.

Primary cultures of GABAergic cerebral cortex neurons and glutamatergic cerebellar granule cells were used to study the expression of synaptophysin, a synaptic vesicle marker protein, along with the ability of each cell type to release neurotransmitter upon stimulation. The synaptophysin expression and neurotransmitter release were measured in each of the culture types as a function of development for up to 8 days in vitro, using the same batch of cells for both sets of measurements to obtain optimal comparisons. The content and the distribution of synaptophysin in the developing cells were assessed by quantitative immunoblotting and light microscope immunocytochemistry, respectively. In both cell types, a close parallelism was found between the temporal pattern of development in synaptophysin expression and neurotransmitter release. This temporal pattern differed between the two types of neurons. The cerebral cortex neurons showed a biphasic time course of increase in synaptophysin content, paralleled by a biphasic pattern of development in their ability to release [3H]GABA in response to depolarization by glutamate or elevated K+ concentrations. In contrast, a monophasic, approximately linear increase in the synaptophysin content and stimulated [3H]D-aspartate release was found in the cerebellar granule cells. These results, particularly regarding the GABAergic neurons, offer correlative evidence in support of the notion that a vesicular pool of these amino acid neurotransmitters may be intimately involved in their release, subsequent to depolarization stimuli.

Animals↗

PubMed1990

Chronic osmotic stimulation reduces vasopressin but not synaptophysin content in rat neurohypophysis.

The content of synaptophysin, a vesicular integral membrane protein of neurons and endocrine cells, and that of vasopressin was measured in neurohypophyses of rats during chronic osmotic stimulation. The animals received 2% NaCl in their drinking water for up to 4 days. Synaptophysin content of neurohypophyses was determined using quantitative immunoblotting, vasopressin content was measured by radioimmunoassay. Salt loading caused a decrease in the content of vasopressin to about 15% of that of control animals, whether expressed per neurohypophysis or relative to the total tissue protein. In contrast, no change was found in the synaptophysin content. Taken together with published evidence of changes in the relative numbers of the hormone-containing neurosecretory granules (NSGs) and the microvesicles (MVs) under the conditions of chronic osmotic stimulation, these results strongly indicate the surface density of synaptophysin on NSGs to be significantly lower than its surface density on MVs.

Animals↗

PubMed1990

Ca2+ and vasopressin release in isolated rat neurohypophysis: differential effects of four classes of Ca2+ channel ligands.

In order to study the role of different types of voltage-sensitive Ca2+ channels (VSCC) in stimulus-secretion coupling in peptidergic neurons, effects of 4 major classes of pharmacological agents have been examined on evoked vasopressin release from isolated rat neurohypophyses. omega-Conotoxin GVIA (omega-CgTX), a potent blocker of N- and L-type Ca2+ channels, inhibited vasopressin release evoked electrically as well as by high K+. With maximal inhibition, release was decreased to 50% and 75% of control for electrical and 100 mM K+ stimulation, respectively. This stimulation mode-related difference in release sensitivity to omega-CgTX paralleled its stimulation mode-related sensitivity to tetrodotoxin, suggesting that the omega-CgTX-sensitive Ca2+ entry played a larger role when release was activated by action potentials invading nerve terminals. These data, and the characteristics of [125I]omega-CgTX binding to plasma membranes from bovine neurohypophyses, are consistent with N-type Ca2+ channels being responsible for the omega-CgTX-sensitive component of vasopressin release. Verapamil and diltiazem (phenylalkylamine and benzothiazepine, respectively) inhibited secretion in a pattern suggesting non-identical sets of action sites, and in a manner partly additive with inhibition by omega-CgTX. This inhibition by verapamil and diltiazem appeared at least in part to involve sites different from L channels. Several dihydropyridines known to act as agonists or antagonists at L channels did not affect vasopressin release (evoked either electrically or by high K+) in a specific manner. A significant component of neuropeptide release may depend on Ca2+ entry through omega-CgTX- and dihydropyridine-insensitive routes.

Animals↗

PubMed1989

Facilitation by forskolin of electrically evoked vasopressin release in vitro requires bursting pattern of stimulation.

Electrically stimulated release of vasopressin from isolated rat neurohypophysis was measured in the presence and absence of forskolin. Forskolin inhibited the release of vasopressin evoked by continuous stimulation at constant frequency, and enhanced the release evoked by trains of pulses separated by silent intervals. It is suggested that this biphasic effect of forskolin is consistent with an involvement of cyclic adenosine monophosphate (cAMP) in the regulation of cytosolic Ca2+ in the neurosecretory terminals. These results may partly explain some of the discrepant effects of increasing tissue cAMP observed between those studies using electrical pulses, and those using high K+, for stimulation of vasopressin release.

Animals↗

PubMed1989

Two classes of [3H]spiperone binding sites in bovine neurohypophysis: D-2 receptors and putative 5-HT2 receptors.

Binding of [3H]spiperone was studied in membranes obtained from bovine neurohypophyses devoid of intermediate lobe tissue. Non-linear Scatchard plot suggested the presence of more than a single class of binding sites. Competition experiments using ketanserin, a ligand selective for 5-HT2 receptors, were carried out to ascertain whether serotonergic, in addition to dopaminergic receptors, might be responsible for the heterogeneity of [3H]spiperone binding. Computer-assisted modeling suggested the presence of two classes of binding sites for ketanserin (Kd = 1.6 +/- 0.2 and 366.7 +/- 20.5 nM, respectively). The Kd value for ketanserin binding to the high-affinity sites, as well as the Kd of [3H]spiperone for these sites suggested by the 2 sites model indicate that they represent serotonin 5-HT2 receptors. The [3H]spiperone Kd at the ketanserin low-affinity sites (65 +/- 7 pM) and the rank order of inhibitory potencies for several antagonists show that the low-affinity sites represent dopamine D-2 receptors.

Animals↗