Calcium-mediated Mechanisms Research Articles

ATP and Formyl Peptides Facilitate Chemoattractant Leukotriene-B4 Synthesis and Drive Calcium Fluxes, Which May Contribute to Neutrophil Swarming at Sites of Cell Damage and Pathogens Invasion.

Here, we demonstrate that human neutrophil interaction with the bacterium Salmonella typhimurium fuels leukotriene B4 synthesis induced by the chemoattractant fMLP. In this work, we found that extracellular ATP (eATP), the amount of which increases sharply during tissue damage, can effectively regulate fMLP-induced leukotriene B4 synthesis. The vector of influence strongly depends on the particular stage of sequential stimulation of neutrophils by bacteria and on the stage at which fMLP purinergic signaling occurs. Activation of 5-lipoxygenase (5-LOX), key enzyme of leukotriene biosynthesis, depends on an increase in the cytosolic concentration of Ca2+. We demonstrate that eATP treatment prior to fMLP, by markedly reducing the amplitude of the fMLP-induced Ca2+ transient jump, inhibits leukotriene synthesis. At the same time, when added with or shortly after fMLP, eATP effectively potentiates arachidonic acid metabolism, including by Ca2+ fluxes stimulation. Flufenamic acid, glibenclamide, and calmodulin antagonist R24571, all of which block calcium signaling in different ways, all suppressed 5-LOX product synthesis in our experimental model, indicating the dominance of calcium-mediated mechanisms in eATP regulatory potential. Investigation into the adhesive properties of neutrophils revealed the formation of cell clusters when adding fMLP to neutrophils exposed to the bacterium Salmonella typhimurium. eATP added simultaneously with fMLP supported neutrophil polarization and clustering. A cell-derived chemoattractant such as leukotriene B4 plays a crucial role in the recruitment of additional neutrophils to the foci of tissue damage or pathogen invasion, and eATP, through the dynamics of changes in [Ca2+]i, plays an important decisive role in fMLP-induced leukotrienes synthesis during neutrophil interactions with the bacterium Salmonella typhimurium.

Effects of exogenous calcium on the drought response of the tea plant (Camellia sinensis (L.) Kuntze).

Drought is one of the major factors reducing the yield of many crops worldwide, including the tea crop (Camellia sinensis (L.) Kuntze). Calcium participates in most of cellular signaling processes, and its important role in stress detection and triggering a response has been shown in many crops. The aim of this study was to evaluate possible effects of calcium on the tea plant response to drought. Experiments were conducted using 3-year-old potted tea plants of the best local cultivar Kolkhida. Application of ammonium nitrate (control treatment) or calcium nitrate (Ca treatment) to the soil was performed before drought induction. Next, a 7-day drought was induced in both groups of plants. The following physiological parameters were measured: relative electrical conductivity, pH of cell sap, and concentrations of cations, sugars, and amino acids. In addition, relative expression levels of 40 stress-related and crop quality-related genes were analyzed. Under drought stress, leaf electrolyte leakage differed significantly, indicating greater damage to cell membranes in control plants than in Ca-treated plants. Calcium application resulted in greater pH of cell sap; higher accumulation of tyrosine, methionine, and valine; and a greater Mg2+ content as compared to control plants. Drought stress downregulated most of the quality-related genes in both groups of tea plants. By contrast, significant upregulation of some genes was observed, namely CRK45, NAC26, TPS11, LOX1, LOX6, Hydrolase22, DREB26, SWEET2, GS, ADC, DHN2, GOLS1, GOLS3, and RHL41. Among them, three genes (LOX1, RHL41, and GOLS1) showed 2-3 times greater expression in Ca-treated plants than in control plants. Based on these results, it can be speculated that calcium affects galactinol biosynthesis and participates in the regulation of stomatal aperture not only through activation of abscisic-acid signaling but also through jasmonic-acid pathway activation. These findings clarify calcium-mediated mechanisms of drought defense in tree crops. Thus, calcium improves the drought response in the tea tree.

Glycosphingolipids are mediators of cancer plasticity through independent signaling pathways.

The molecular repertoire promoting cancer cell plasticity is not fully elucidated. Here, we propose that glycosphingolipids (GSLs), specifically the globo and ganglio series, correlate and promote the transition between epithelial and mesenchymal cells. The epithelial character of ovarian cancer remains stable throughout disease progression, and spatial glycosphingolipidomics reveals elevated globosides in the tumor compartment compared with the ganglioside-rich stroma. CRISPR-Cas9 knockin mediated truncation of endogenous E-cadherin induces epithelial-to-mesenchymal transition (EMT) and decreases globosides. The transcriptomics analysis identifies the ganglioside-synthesizing enzyme ST8SIA1 to be consistently elevated in mesenchymal-like samples, predicting poor outcome. Subsequent deletion of ST8SIA1 induces epithelial cell features through mTORS2448 phosphorylation, whereas loss of globosides in ΔA4GALT cells, resulting in EMT, is accompanied by increased ERKY202/T204 and AKTS124. The GSL composition dynamics corroborate cancer cell plasticity, and further evidence suggests that mesenchymal cells are maintained through ganglioside-dependent, calcium-mediated mechanisms.

On the Molecular Mechanisms of the Effect of a Zero Magnetic Field on the Production of Reactive Oxygen Species in Inactivated Neutrophils

Abstract—It is shown that the lower intensity of 2,7-dichlorodihydrofluorescein oxidation processes in inactivated neutrophils exposed to hypomagnetic field (the residual static magnetic field 20 nT) is not related to calcium-mediated mechanisms as shown by the absence of the effect of cell-permeant Ca2+ chelators, such as 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester), on the intensity of the process. This decrease is hardly likely to be caused by effects of a hypomagnetic field on phosphorylation of NADPH-oxidase components, because addition of the protein kinase C inhibitor Ro 31-6233 decreases the fluorescence intensity of intracellular dichlorodihydrofluorescein little, if at all. Addition of phospholipase C inhibitor U73122 causes a negligible decrease in ROS production in the control and experiment, almost equally. Different concentrations of apocynin increase ROS production in nonactivated neutrophils and this effect is approximately two times lower under hypomagnetic conditions. The decrease in ROS production is more pronounced in cells treated with a hypomagnetic field with the presence of rotenone, indicating that the mitochondrial electron-transport chain is involved in the mechanism of the effect of hypomagnetism.

Distinguishing mechanisms for alternans in cardiac cells using constant-diastolic-interval pacing.

Alternans, a proarrhythmic dynamical state in which cardiac action potentials alternate between long and short durations despite a constant pacing period, traditionally has been explained at the cellular level using nonlinear dynamics principles under the assumption that the action potential duration (APD) is determined solely by the time elapsed since the end of the previous action potential, called the diastolic interval (DI). In this scenario, APDs at a steady state should be the same provided that the preceding DIs are the same. Nevertheless, experiments attempting to eliminate alternans by dynamically adjusting the timing of pacing stimuli to keep the DI constant showed that alternans persisted, contradicting the traditional theory. It is now widely known that alternans also can arise from a different mechanism associated with intracellular calcium cycling. Our goal is to determine whether intracellular calcium dynamics can explain the experimental findings regarding the persistence of alternans despite a constant DI. For this, we use mathematical models capable of producing alternans through both voltage- and calcium-mediated mechanisms. We show that for voltage-driven alternans, action potentials elicited from a constant-DI protocol are always the same. However, in the case of calcium-driven alternans, the constant-DI protocol can result in alternans. Reducing the strength of the calcium instability progressively reduces and finally eliminates constant-DI alternans. Our findings suggest that screening for the presence of alternans using a constant-DI protocol has the potential for differentiating between voltage-driven and calcium-driven alternans.

Hyperhomocysteinemia selectively alters expression and stoichiometry of intermediate filament and induces glutamate- and calcium-mediated mechanisms in rat brain during development

The aim of the present work was to investigate the actions of a chemically induced chronic hyperhomocysteinemia model on intermediate filaments (IFs) of cortical and hippocampal neural cells and explore signaling mechanisms underlying such effects. Results showed that in hyperhomocysteinemic rats the expression of neural IF subunits was affected. In cerebral cortex, glial fibrillary acidic protein (GFAP) expression was donwregulated while in hippocampus high and middle molecular weight neurofilament subunits (NF-H and NF-M, respectively) were up-regulated. Otherwise, the immunocontent of IF proteins was unaltered in cerebral cortex while in hippocampus the immunocontent of cytoskeletal-associated low molecular weight neurofilament (NF-L) and NF-H subunits suggested a stoichiometric ratio consistent with a decreased amount of core filaments enriched in lateral projections. These effects were not accompanied by an alteration in IF phosphorylation. In vitro results showed that 500μM Hcy-induced protein phosphatases 1-, 2A- and 2B-mediated hypophosphorylation of NF subunits and GFAP in hippocampal slices of 17-day-old rats without affecting the cerebral cortex, showing a window of vulnerability of cytoskeleton in developing hippocampus. Ionotropic and metabotropic glutamate receptors were involved in this action, as well as Ca2+ release from intracellular stores through ryanodine receptors. We propose that the mechanisms observed in the hippocampus of 17-day-old rats could support the neural damage observed in these animals.

Pain in the Brain

Delaney et al. provide new insights into signaling mechanisms that may help explain the integration of pain with stress reactions and with cognitive and emotional processing of pain signals in the brain. The discoveries come from an analysis of neurons in the central amygdala, a brain region where emotional responses to sensory stimuli are derived. Neurons carrying nociceptive information from a region known as the parabranchial nucleus make large synapses on cells of the central amygdala. Neurotransmission at these sites in rat brain slices was inhibited by noradrenaline, which is released by noradrenergic afferent neurons in the region. Electrophysiological experiments showed that noradrenaline acted presynaptically and appeared not to affect the probability of vesicle release but rather to limit the number of active release sites within the synapse. Experiments with inhibitors indicated that the noradrenaline acted at α2-adrenergic receptors coupled to G i - or G o -type heterotrimeric guanine nucleotide-binding proteins (G proteins). Loading of presynaptic terminals with a membrane-permeable peptide that binds to G protein βγ subunits inhibited the effects of noradrenaline, implicating Gβγ subunits in the process. Imaging of intracellular calcium concentrations with fluorescent dye showed that noradrenaline’s effects were independent of calcium concentrations. Thus, the Gβγ subunits appeared not to act by modulating channel activity but possibly by interacting with the vesicle release machinery. The actions of exogenous noradrenaline were also reproduced in experiments where release of noradrenaline from endogenous neurons was electrically stimulated. Tully et al . point out in commentary on the paper that this calcium-independent regulation might be particularly appropriate to reduce painful sensations during stressful situations that cause release of noradrenaline. Repetitive neuronal firing might gradually increase intracellular calcium concentrations sufficiently to overcome calcium-mediated inhibitory mechanisms, whereas the noradrenaline effect should be maintained even in the presence of rapid firing of presynaptic neurons. A. J. Delaney, J. W. Crane, P. Sah, Noradrenaline modulates transmission at a central synapse by a presynaptic mechanism. Neuron 56 , 880-892 (2007). [PubMed] K. Tully, Y. Li, V. Y. Bolshakov, Keeping in check painful synapses in central amygdala. Neuron 56 , 757-759 (2007). [PubMed]

Zebra mussels at the freshwater/sea interface: Ionic and osmotic challenges to oocyte integrity

Summary The zebra mussel, Dreissena polymorpha, is a highly prolific biofouling freshwater mollusk accidentally introduced into North America in the 1980s. Understanding its reproductive mechanisms may give insight into its dispersion, adaptive mechanisms, and possible control methods. The spread of zebra mussels into tidal regions and estuaries could be affected by the responses of zebra mussel gametes to seawater. This study investigated the effect of dilute seawater (Instant Ocean, IO) and several of its components on zebra mussel oocyte viability. Zebra mussels were induced to spawn with serotonin. Oocytes exposed to various concentrations of IO or its component ions began to break down within 3 h, usually after a latency of 1–2 h. The percentages of oocytes fragmenting after 3-h exposure to low IO concentration were: control, 11±3%; 2% IO, 18±2%; 5% IO, 47±4%; and 10% IO, 53±4% (ANOVA, p<0.00l). NaCl or CaCl2 concentrations comparable to amounts present in 5% and 10% IO also induced oocyte breakdown (e.g., after 3 h, control, 12±1%; 50 mM NaCl, 49±1%; 1 mM CaCl2, 52±1%; ANOVA, p<0.001). However, NaCl and calcium-mediated mechanisms may differ because: (a) fragmentation morphology differs; (b) osmotically equivalent NaCl, Na-phosphate, and glucose yield similar results, suggesting that these solutions all work primarily by an osmotic effect; and (c) CaCl2 caused twice the oocyte fragmentation of osmotically equivalent NaCl, suggesting that oocyte breakdown may also depend upon specific Ca-activated mechanisms. Both osmotic and calcium-mediated mechanisms may be involved in the breakdown of oocytes in response to seawater. Structural changes of zebra mussel oocyte fragmentation may resemble breakdown of oocytes described in other organisms in response to aging and oxidative stress that have been suggested to be mediated by apoptosis.

A Critical Role for Phosphoinositide 3-Kinase Upstream of Gab1 and SHP2 in the Activation of Ras and Mitogen-activated Protein Kinases by Epidermal Growth Factor

Although the mechanisms involved in the activation of mitogen-activated protein kinases (MAPK) by receptor tyrosine kinases do not display an obvious role for phosphoinositide 3-kinases (PI3Ks), we have observed in the nontransformed cell line Vero stimulated with epidermal growth factor (EGF) that wortmannin and LY294002 nearly abolished MAPK activation. The effect was observed under strong stimulation and was independent of EGF concentration. In addition, three mutants of class Ia PI3Ks were found to inhibit MAPK activation to an extent similar to their effect on Akt/protein kinase B activation. To determine the importance of PI3K lipid kinase activity in MAPK activation, we have used the phosphatase PTEN and the pleckstrin homology domain of Tec kinase. Overexpression of these proteins, but not control mutants, was found to inhibit MAPK activation, suggesting that the lipid products of class Ia PI3K are necessary for MAPK signaling. We next investigated the location of PI3K in the MAPK cascade. Pharmacological inhibitors and dominant negative forms of PI3K were found to block the activation of Ras induced by EGF. Upstream from Ras, although association of Grb2 with its conventional effectors was independent of PI3K, we have observed that the recruitment of the tyrosine phosphatase SHP2 required PI3K. Because SHP2 was also essential for Ras activation, this suggested the existence of a PI3K/SHP2 pathway leading to the activation of Ras. In addition, we have observed that the docking protein Gab1, which is involved in PI3K activation during EGF stimulation, is also implicated in this pathway downstream of PI3K. Indeed, the association of Gab1 with SHP2 was blocked by PI3K inhibitors, and expression of Gab1 mutant deficient for binding to SHP2 was found to inhibit Ras stimulation without interfering with PI3K activation. These results show that, in addition to Shc and Grb2, a PI3K-dependent pathway involving Gab1 and SHP2 is essential for Ras activation under EGF stimulation.

Endothelin-1 and endothelin-3 stimulate insulin release by isolated rat pancreatic islets.

Endothelins (ETs) are potent vasoconstrictive peptides released from the endothelium and other tissues, which act on target cells by receptorial calcium-mediated mechanisms. ET-1 levels are increased in diabetes, and observations suggest the involvement of ETs in the pathogenesis of diabetic angiopathy. However, it is not possible to exclude that ETs might also influence insulin secretion or function. In vivo infusion of ET-1 in rats induces hypoglycaemia and hyperinsulinemia and in vitro incubation with ET-1 stimulates insulin release by mouse islets. Therefore, ETs might be involved in a circulus vitiosus, resulting in hyperinsulinemia and diabetic angiopathy. The purpose of our study was to verify the effect of ET-1 on rat islets, in both the presence and absence of physiological glucose concentration. Moreover, we tested the effect of another isoform of endothelins, ET-3, and verified the involvement of extracellular calcium in such events. Islets were incubated with increasing ET-1 or ET-3, with or without glucose 5.6 mM. Other samples were prepared using calcium-free medium. Incubation in medium containing ET-1 and ET-3, in the presence of glucose and calcium, induced an increase in insulin release. When ET-1 and ET-3 were incubated without glucose and calcium, insulin release was not modified. Our studies demonstrate that: 1) ET-3, like ET-1, stimulates insulin release by rat isolated islets; 2) direct insulin stimulating effect on islets of both ET-1 and ET-3 is evident with physiological glucose concentrations and is calcium mediated. These results support the hypothesis of ET involvement in the regulation of insulin secretion.

Hyperthermia stimulates energy-proteasome-dependent protein degradation in cultured myotubes.

Previous studies suggest that elevated temperature stimulates protein degradation in skeletal muscle, but the intracellular mechanisms are not fully understood. We tested the role of different proteolytic pathways in temperature-dependent degradation of long- and short-lived proteins in cultured L6 myotubes. When cells were cultured at different temperatures from 37 to 43 degrees C, the degradation of both classes of proteins increased, with a maximal effect noted at 41 degrees C. The effect of high temperature was more pronounced on long-lived than on short-lived protein degradation. By using blockers of individual proteolytic pathways, we found evidence that the increased degradation of both long-lived and short-lived proteins at high temperature was independent of lysosomal and calcium-mediated mechanisms but reflected energy-proteasome-dependent degradation. mRNA levels for enzymes and other components of different proteolytic pathways were not influenced by high temperature. The results suggest that hyperthermia stimulates the degradation of muscle proteins and that this effect of temperature is regulated by similar mechanisms for short- and long-lived proteins. Elevated temperature may contribute to the catabolic response in skeletal muscle typically seen in sepsis and severe infection.

Calcium-mediated mechanisms of eicosapentaenoic acid-induced relaxation in hypertensive rat aorta

We have previously demonstrated the vasorelaxant properties of the omega-3 fatty acid, eicosapentaenoic acid (EPA), in normotensive and spontaneously hypertensive rat (SHR) aorta, although the mechanism(s) of action are not fully understood. Because endothelial dysfunction and increased intracellular free calcium concentration ([Ca 2+] i) are seen in hypertensive rat aorta, we investigated the potential role of Ca 2+ signaling, endothelium and derived factors, and the opening of potassium (K +) channels in EPA-induced relaxation. In the presence of extracellular Ca 2+, EPA induced significant relaxations at > 10 μmol/L ( P < .01) in norepinephrine (NE) (10 −6 mol/L)-contracted aortic rings and at 30 μmol/L ( P <. 001) in high K + (80 mmol/L)-contracted aortic rings. In the absence of extracellular Ca 2+, EPA (10 to 30 μmol/L) inhibits the tonic component of NE-induced contraction ( P < .0001). The relaxant properties of EPA in SHR aorta appear specific to Ca 2+ release from an internal storage site associated with NE-induced tonic contraction. Further studies with the use of fura-2 to measure [Ca 2+] i in cultured vascular smooth muscle (VSM) cells from SHR aorta indicated that EPA (30 μmol/L)-pretreatment attenuated angiotensin II (50 nmol/L)-induced Ca 2+ transient by 95%, suggesting that an inhibitory effect on the Ca 2+ signaling may underlie EPA-induced relaxation of the vessel preparation. In addition, EPA per se induced an increase in [Ca 2+] i with a duration of approximately 20 min in VSM cells, and the effect was not altered by removal of extracellular Ca 2+. There was no increase in the level of inositol-1,4,5-trisphosphate in response to EPA (30 μmol/L). The actions of EPA are independent of endothelium-derived factors, cyclooxygenase metabolites, and activation of K + channels since endothelium removal, N ω-nitro- l-arginine methyl ester hydrochloride, (L-NAME, 100 μmol/L), indomethacin (10 μmol/L), tetraethylammonium (1 mmol/L), and glibenclamide (10 μmol/L) did not affect EPA-induced vasodilation in NE-precontracted aortic rings. These results suggest that EPA directly modulates intracellular Ca 2+ signaling in VSM cells, and that this may contribute to the vasorelaxant effect and, at least in part, the blood pressure-lowering effect of fish oil.

A chronological study of the expression of glial fibrillary acidic protein and calbindin-D 28 k by reactive astrocytes in the electrically lesioned rat brain

Immunoreactivity of neuronal and glial marker proteins of reactive astrocytes around the electrically damaged pyramidal layer and stratum radiatum of the hippocampal CA1 region and corpus callosum was chronologically studied in electrically lesioned rat brains. A monoclonal antibody against calbindin-D 28 k (CD28-Ab) and a polyclonal antibody against glial fibrillary acidic protein (GFAP-Ab) were used for immunostaining. Immunoreactivity of CD28 and GFAP in the reactive astrocytes was detected in brains 1–6 weeks post-lesion but not in non-lesioned brains. The number of immunohistochemically stained reactive astrocytes around the electrically damaged areas were counted and then compared with the number of those in the same areas of non-lesioned brains. The number of CD28- and GFAP-immunoreactive astrocytes began to increase around the lesion from 1–3 weeks following lesion in the pyramidal layer of the hippocampal CA1 region and from 1–4 weeks following lesion in the stratum radiatum of the hippocampal CA1 region and corpus callosum. These immunoreactive astrocytes could be observed for 6 weeks (the maximum survival time studied) in all areas of the lesioned brains studied. The increase in the number of reactive astrocytes might have been induced by the stimulatory effects of neurotrophic factors, or growth factors, produced around the lesioned site. The constancy in the number of reactive astrocytes after 3 and 4 weeks in the lesioned areas may have been due to the termination of the initial phase of the repair process, i.e. space-filling. Reactive astrocytes which were stained by GFAP-Ab were separated into two groups, based on the presence of CD28, i.e. CD28-positive and CD28-negative reactive astrocytes. The presence of CD28 might confer certain functions via calcium-mediated mechanisms on CD28-positive astrocytes in addition to the constructive role mediated by GFAP.

Norepinephrine modulates excitability of neonatal rat optic nerves through calcium-mediated mechanisms.

We report that norepinephrine markedly increases excitability of neonatal rat optic nerves. To investigate the mechanisms of the norepinephrine-induced excitability increase, we studied isolated optic nerves from 42 neonatal (< three days old) and five adult (> three months old) Long-Evan's hooded rats. Norepinephrine (10(-6), 10(-5) and 10(-4) M) rapidly and reversibly increased the amplitude (mean +/- S.D.: 3.5 +/- 1.7%, 12.1 +/- 2.8% and 35.6 +/- 8.4%) of compound action potentials elicited by submaximal stimulation of neonatal optic nerves. The beta-1 adrenoceptor antagonist atenolol (10(-5) M) blocked the norepinephrine-induced increase in excitability but the alpha antagonist phentolamine (10(-5) M) did not. The beta agonist isoproterenol (10(-5) and 10(-4) M) increased response amplitudes (8.7 +/- 4.1% and 25.8 +/- 4.6%) but the alpha-1 agonist methoxamine and alpha-2 agonist clonidine did not. The beta antagonist propranolol blocked the isoproterenol effect. Replacing Ca2+ with Mg2+ or adding 0.8 mM of Cd2+ reversibly blocked the norepinephrine effects. Extracellular K+ concentrations did not change in optic nerves during norepinephrine application. Blockade of K+ channels with apamin (10(-6) M) or tetraethylammonium (10(-3) M) did not prevent the excitatory effects of norepinephrine. Adult rat optic nerves were insensitive to both norepinephrine (10(-4) M) and isoproterenol (10(-4) M). Our results indicate that norepinephrine increases neonatal optic axonal excitability through Ca(2+)-dependent mechanisms. The data suggest that the adrenoceptors are situated on the axons, that the excitability changes are not due to changes in extracellular K+ concentration or K+ channels sensitive to apamin or tetraethylammonium. The sensitivity of rat optic nerves to norepinephrine declined with age. Axonal adrenoceptors may play a role in optic axonal development and injury.

Influence of drugs on blood pressure variability

To study the causes of blood pressure variability by using antihypertensive drugs. Survey of published studies. The results are sometimes unclear and, for some drugs, contradictory. beta-Adrenoceptor antagonists appear to have no effect on blood pressure variability, whereas alpha-adrenoceptor antagonists and atropine can lessen the amplitude of the variations. Similarly, there is a decrease after the administration of clonidine. Guanfacine, however, which acts on the same central receptors, does not lessen variability. Calcium antagonists do not influence blood pressure variability and the results with angiotensin converting enzyme inhibitors are contradictory. From these results we conclude that blood pressure variability is not dependent on beta-receptor activity or calcium-mediated mechanisms. It is most likely that both alpha-adrenergic tone and the vagal system have significant effects on blood pressure variability.

Calcium-mediated mechanisms in chemically induced cell death.

Biochemical mechanisms involved in toxic cell injury have attracted in­ creased interest during recent years and have been the subject of several reviews (1-6). In this chapter we discuss one particular aspect of chemically induced cell death, i.e. the role of the calcium ion in the early development of damage. We summarize current evidence for an association between a per­ turbation of intracellular Ca2+ homeostasis and cytotoxicity and discuss various Ca2+ -mediated mechanisms that may contribute to the development of cell damage.

Calcium-Mediated Mechanisms In Chemically Induced Cell Death

Over the past two decades, deadly coronaviruses, with the most recent being the severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) 2019 pandemic, have majorly challenged public health. The path for virus invasion into humans and other ...Read More

Restoration of Normal Growth Hormone Responsiveness to GHRH in Normal Aged Men by Infusion of Low Amounts of Theophylline

In order to establish whether the age-related reduced growth hormone (GH) responsiveness to GH-releasing hormone (GHRH) could be improved by treatment with low amounts of theophylline, a drug mainly acting through cyclic adenosine 3', 5'-monophosphate (cAMP), and calcium-mediated mechanisms, eight adult subjects (aged 20-37) and eight elderly men (aged 64-79) were tested with GHRH (50 micrograms in an iv bolus), theophylline (1.67 mg/min for 3 h), or the combination of both drugs. The GH response to GHRH was significantly lower in the older than in the younger group (mean peaks were 4.5 and 7.5 times higher than baseline, respectively). Theophylline did not change basal GH levels in any subjects and GHRH-induced GH rise in the younger group, whereas it restored normal GH responses to GHRH in the older subjects (mean peak responses were eight times higher than baseline in both groups). These data show that in elderly subjects the mechanism underlying the GHRH-induced GH secretion may be fully activated by GHRH during treatment with low amounts of theophylline.

Multiple calcium-mediated mechanisms regulate c-myc expression in HL-60 cells.

Treatment of human promyelocytic leukemia cells (HL-60) with the calcium ionophore A23187 induced a biphasic change in the expression of the c-myc protooncogene. Within 1 h of exposure to a single 2.5-microM dose of A23187, steady state levels of c-myc mRNA increased to 170% of control. After this early increase, c-myc mRNA expression underwent an abrupt decline over the next 2 to 4 h. Between 6 to 18 h of treatment with A23107, steady state levels of c-myc mRNA fell gradually toward zero. The early increase in expression of c-myc mRNA was mediated by both enhanced initiation of c-myc transcription and posttranscriptional stabilization of c-myc mRNA. Stimulation of HL-60 cells with A23187 for 30 min increased transcription of both exons 1 and 3 of c-myc by 125%. After 1 h of treatment with A23187, the half-life of c-myc mRNA increased to 150% of control. The late decrease in c-myc expression induced by A23187 was primarily mediated by interruption of elongation of c-myc nascent mRNA beyond exon 1. The combination of calcium ionophore A23187 and phorbol 12,13-dibutyrate acted synergistically to inhibit expression of c-myc mRNA.

Calcium Channel Blockers for Kidney Protection

Acute postischemic renal failure (ARF) is a major complication in surgery and in particular in renal transplantation. Calcium channel blockers (CCBs) are able to prevent or ameliorate ARF in different experimental models when given before ischemic injury. Because ARF, which is observed in 20-60% of graft recipients, carries the risk of undetected rejections and nephrotoxic injury and is also expensive, we tested the hypothesis, suggested by animal experiments, that there is also a beneficial effect of CCBs when given after ischemic injury. A total of 134 recipients of first or second cadaver grafts were randomly assigned to a diltiazem pretreatment (DZ) or control (C) group. Kidney grafts were pretreated immediately prior to transplantation by reperfusion with 500 ml Euro-Collins (preservation fluid), with or without DZ. The DZ patients also received a 74-h infusion of DZ (0.12 mg/kg/h), starting 2 h prior to surgery. DZ was continued (90 mg b.i.d. p.o.) until day 30. Immunosuppression consisted of low-dose steroids and cyclosporine A (CSA) (10 mg/kg p.o.). CSA was instituted 6 h after surgery and later adjusted to achieve whole-blood trough levels of 300-600 ng/ml [by polyclonal radioimmunoassay (RIA)]. A total of 129 patients were available for efficacy analysis. There were no significant differences concerning donor demographics, human lymphocyte antigen (HLA) match, or ischemic times. ARF was defined as a need for dialysis in the first week. Our data show a significant reduction in ARF in grafts pretreated with DZ.(ABSTRACT TRUNCATED AT 250 WORDS)