Perturbation Of Intracellular Ca2 Research Articles

PO-01-217 FUNCTIONAL CHARACTERIZATION OF CALCIUM HANDLING KINETICS IN RE-ENGINEERED CARDIOMYOCYTES FROM A PATIENT WITH TYPE 2 LONG QT SYNDROME

Type 2 long QT syndrome (LQT2) is caused by pathogenic variants in the KCNH2-encoded Kv11.1 K+ channel resulting in prolongation of the action potential duration (APD) at the cellular level and QT prolongation in the patient, predisposing LQT2 patients to potentially lethal ventricular arrhythmias. Limited evidence, namely prolonged Ca2+ transient in human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) and enhanced Ca2+ leak in transgenic LQT2 rabbit model, suggests that KCNH2 pathogenic variants may be associated with perturbations in intracellular Ca2+ handling. To characterize the APD and Ca2+ handling properties of an iPSC-CM model of LQT2. iPSC-CMs were generated from a patient with a pathogenic KCNH2 frame-shift variant (p.Q901fs/71) and an unrelated healthy control. Confocal imaging of FluoVolt voltage dye was used to measure the APD at 90% repolarization (APD90) in iPSC-CMs. Ca2+ kinetics was characterized using Ca2+ sensing dye Fluo4-AM before and after treatment with 100 nM isoproterenol (ISO). Q901fs/71-iPSC-CMs had a significantly prolonged APD90 compared to wild-type (WT) iPSC-CMs (444 ±9 ms vs 631±8 ms, p<0.001). Q901fs/71-iPSC-CMs had a higher Ca2+ amplitude (DF/F0) before (0.423±0.043 vs 0.97±0.07, p=0.001) and after ISO treatment (0.602±0.051 vs 1.056 ±0.06, p<0.001) compared to WT. The Ca2+ transient duration (CTD90) was longer in Q901fs/71-iPSC-CMs than in WT before (1.062±0.041 s vs 1.306±0.038 s, p=0.01), but not after ISO (1.071±0.062 s vs 1.092±0.044 s, p=0.96). Peak to 90% Ca2+ decay time was increased in Q901fs/71-iPSC-CMs before (0.785±0.04 s vs 0.942±0.033 s, p=0.028), but not after ISO (0.748±0.054 s vs 0.767±0.038 s, p=0.95). Upstroke time was longer in Q901fs/71-iPSC-CMs compared to WT before (0.281±0.006 s vs 0.364±0.014 s; p=0.002) but not after ISO (0.327±0.014 s vs 0.344±0.025 s; p=0.88). Upstroke velocity (DF/F0/t) was increased in Q901fs/71-iPSC-CMs before (1.092±0.122 vs 2.278±0.278, p<0.001) and after ISO (1.310±0.117 vs 2.2±0.139, p=0.005). Ca2+ leak from sarcoplasmic reticulum was higher in Q901fs/71-iPSC-CMs before (16% vs 38%) and after ISO (16% vs 55%). We show that iPSC-CMs derived from the patient with a truncating LQT2-causative variant (yielding likely Kv11.1 channel haploinsufficiency) have prolonged APD90 and display abnormal Ca2+ handling properties such as higher amplitude, increase in Ca2+ leak, prolonged transient duration, Ca2+ decay time, and upstroke velocity compared to WT iPSC-CMs.

The roles of K+-dependent Na+/Ca2+ exchanger 2 (NCKX2) in methamphetamine-induced behavioral sensitization and conditioned place preference in mice

Drug addiction, including methamphetamine (METH) addiction, is a significant public health and social issue. Perturbations in intracellular Ca2+ homeostasis are associated with drug addiction. K+-dependent Na+/Ca2+ exchanger 2 (NCKX2) is located on neuronal cell membranes and constitutes a Ca2+ clearance mechanism, with key roles in synaptic plasticity. NCKX2 is associated with motor learning, memory, and cognitive functions. However, the role of NCKX2 in METH addiction remains unclear. In this study, we investigated the expression levels of NCKX2 in four addiction-related brain regions: the prefrontal cortex (PFc), nucleus accumbens (NAc), dorsal striatum (DS), and hippocampus (Hip) in a C57/BL6 mouse model of METH-induced conditioned place preference (CPP) and behavioral sensitization. Levels of NCKX2 were unchanged in these brain regions in mice with METH-induced CPP but were decreased in the PFc and NAc of mice with METH-induced behavioral sensitization. Adeno-associated virus (AAV)-mediated overexpression of NCKX2 in the PFc attenuated the expression phase of METH-induced behavioral sensitization in mice, whereas AAV-mediated knockdown of NCKX2 enhanced the effects of METH. Collectively, our results suggest that NCKX2 is involved in METH-induced behavioral sensitization but does not affect conditioned reward-related memory, highlighting the potential of NCKX2 as a molecular target for studying the mechanisms underscoring METH addiction.

Food supplements based on palmitoylethanolamide plus hydroxytyrosol from olive tree or Bacopa monnieri extracts for neurological diseases.

Neurological disorders like Parkinson disease and Alzheimer disease, spinal cord injury and stroke have some recurrent characteristics such as abnormal protein aggregation, oxidative stress induction, apoptosis, excitotoxicity, perturbation of intracellular Ca2+ homeostasis and inflammation. To date, there are few effective treatments available and the drugs currently used to manage the symptoms have important side effects. Therefore, research studies are focusing on natural phytochemicals present in diet as bioactive molecules potentially useful against neurodegenerative diseases. In this review, we will discuss the neuroprotective role of palmitoylethanolamide, hydroxytyrosol, and Bacopa monnieri extracts against neuroinflammation and neurodegeneration, thereby revealing their remarkable potential as novel therapeutic options for the treatment of neurodegenerative disorders. (www.actabiomedica.it)

Porcine Circovirus Type 2 Induces ORF3-Independent Mitochondrial Apoptosis via PERK Activation and Elevation of Cytosolic Calcium.

Our previous studies demonstrated that porcine circovirus type 2 (PCV2) triggers an unfolded protein response (UPR) in porcine kidney PK-15 cells by activating the protein kinase R-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α) pathway of endoplasmic reticulum (ER) stress, which in turn facilitates viral replication (Y. Zhou et al., Viruses 8:e56, 2016, https://doi.org/10.3390/v8020056; Y. Zhou et al., J Zhejiang Univ Sci B 18:316-323, 2017, https://doi.org/10.1631/jzus.B1600208). PCV2 is found to cause oxidative stress and upregulation of cytoplasmic Ca2+ levels. The virus is reported to employ its open reading frame 3 (ORF3) to induce apoptosis. We wondered whether and how PCV2-induced UPR would lead to apoptosis independent of ORF3. Using an ORF3-deficient PCV2 mutant (ΔORF3), apoptotic responses in infected PK-15 and porcine alveolar macrophage (PAM) cells were still apparent, although lower than in the parental PCV2 strain. We hypothesized that apoptosis induced by ΔORF3 might result from the UPR. We found that ΔORF3-induced apoptosis was significantly reduced when the infected cells were treated with the selective PERK blocker GSK2606414 (GSK) or the general ER stress attenuator 4-phenylbutyrate (4-PBA). Such treatments also ameliorated elevation of cytoplasmic Ca2+ and reactive oxygen species (ROS) levels in PK-15 and PAM cells, two predisposing factors for apoptosis via disruption of the ER-mitochondrion units. Treatment of ΔORF3-infected cells with GSK and 4-PBA also decreased the mitochondrial Ca2+ load and increased the mitochondrial membrane potential (MMP). With transient expression of the structural protein capsid (Cap) in combination with PERK silencing, we found that Cap induced MMP collapse and mitochondrial apoptosis could result from the UPR and elevation of Ca2+ and ROS levels, which were inhibitable by downregulation of PERK. We propose that PCV2-driven ER stress is Cap dependent and could lead to mitochondrial apoptotic responses independent of ORF3 via perturbation of intracellular Ca2+ homeostasis and accumulation of ROS.IMPORTANCE PCV2 encodes protein ORF3, a putative protein with proapoptotic activity. Our early studies showed that PCV2 infection triggers ER stress via selective activation of the PERK pathway, a branch of the ER stress pathways, in permissive cells for enhanced replication and infection increased cytosolic Ca2+ and ROS levels. Here we clearly show that PCV2 infection or Cap expression induces ORF3-independent apoptosis via increased cytosolic and mitochondrial Ca2+ levels and cellular ROS levels as a result of activation of the PERK pathway.

Suppression of Arrhythmia by Enhancing Mitochondrial Ca2+ Uptake in Catecholaminergic Ventricular Tachycardia Models

Cardiovascular disease-related deaths frequently arise from arrhythmias, but treatment options are limited due to perilous side effects of commonly used antiarrhythmic drugs. Cardiac rhythmicity strongly depends on cardiomyocyte Ca2+ handling and prevalent cardiac diseases are causally associated with perturbations in intracellular Ca2+ handling. Therefore, intracellular Ca2+ transporters are lead candidate structures for novel and safer antiarrhythmic therapies. Mitochondria and mitochondrial Ca2+ transport proteins are important regulators of cardiac Ca2+ handling. Here we evaluated the potential of pharmacological activation of mitochondrial Ca2+ uptake for the treatment of cardiac arrhythmia. To this aim,we tested substances that enhance mitochondrial Ca2+ uptake for their ability to suppress arrhythmia in a murine model for ryanodine receptor 2 (RyR2)-mediated catecholaminergic polymorphic ventricular tachycardia (CPVT) in vitro and in vivo and in induced pluripotent stem cell-derived cardiomyocytes from a CPVT patient. In freshly isolated cardiomyocytes of RyR2R4496C/WT mice efsevin, a synthetic agonist of the voltage-dependent anion channel 2 (VDAC2) in the outer mitochondrial membrane, prevented the formation of diastolic Ca2+ waves and spontaneous action potentials. The antiarrhythmic effect of efsevin was abolished by blockade of the mitochondrial Ca2+ uniporter (MCU), but could be reproduced using the natural MCU activator kaempferol. Both mitochondrial Ca2+ uptake enhancers (MiCUps), efsevin and kaempferol, significantly reduced episodes of stress-induced ventricular tachycardia in RyR2R4496C/WT mice in vivo and abolished diastolic, arrhythmogenic Ca2+ events in human iPSC-derived cardiomyocytes.

Gram-negative endotoxin lipopolysaccharide induces cardiac hypertrophy: Detrimental role of Na+–Ca2+ exchanger

Several molecular pathways involved in the development of cardiac hypertrophy are triggered by perturbation of intracellular Ca2+ homeostasis. Within the heart, Na+/Ca2+ exchanger 1 (NCX1) is one of the main determinant in controlling Ca2+ homeostasis. In cardiac hypertrophy and heart failure NCX1 expression and activity have been reported to be altered. It has been shown that chronic bacterial infections (sepsis, endocarditis, and myocarditis) can promote cardiac hypertrophy. Bacterial stressors, such as the Gram-negative endotoxin lipopolysaccharide (LPS), can directly or indirectly affect intracellular Ca2+ homeostasis in the heart and induce the development of cardiac hypertrophy. The present study aimed at evaluating the potential link between the signal pathways activated in LPS-exposed myocytes and NCX1. In the whole rat heart, LPS perfusion induced an early hypertrophy response during which NCX1 expression significantly increased. Notably, all these changes were completely prevented by the NCX inhibitor SN-6. We further dissect the role of NCX1 in the LPS-induced hypertrophic response in an in vitro cardiac model based on two H9c2 cardiomyoblast clones, namely H9c2-WT (lacking endogenous NCX1 expression) and H9c2-NCX1 (stably transfected with a functional NCX1). H9c2-NCX1 were more susceptible than H9c2-WT to develop a hypertrophic phenotype, and they displayed a significant increase in NCX1 expression and function after LPS treatment. SN-6 completely counteracted both hypertrophic response and exchanger alterations induced by LPS in H9c2-NCX1 cells, but it had no effects on H9c2-WT. Collectively, our results suggest that NCX1 plays a critical role in promoting myocardial hypertrophy triggered by LPS.

Perturbations in Intracellular Ca2+ Concentrations and DNA Damage are Coupled to the Activation of PARP‐1 During ROS‐induced Necrotic Cell Death

2,3,5‐Tris(glutathion‐S‐yl)hydroquinone (TGHQ) generated reactive oxygen species (ROS), cause an increase in intracellular calcium concentrations (iCa2+), DNA strand breaks, and hyperactivation of poly(ADP‐ribose)polymerase (PARP)‐1, resulting in necrotic cell death. The relationship between Ca2+ channels and PARP‐1 was therefore examined in renal proximal tubule epithelial cells (HK‐2) treated with TGHQ. 2‐APB, a store‐operated Ca2+ channel (SOCs) inhibitor, decreased PAR ribosylation, indicating that activation of Ca2+ channels has a direct effect on PARP‐1 activity in ROS‐induced cell death. siRNA knockdown of poly(ADP‐ribose)glycohydrolase indicated that free ADPR does not activate Ca2+ channels in HK‐2 cells. Pharmacological inhibition of the sarcoplasmic‐endoplasmic reticulum calcium pump (SERCA), and knockdown of STIM1/Orai (CRAC channel) revealed the increase in iCa2+ responsible for crosstalk with PARP‐1 does not originate from the ER. Moreover, Ca2+‐free media experiments excluded extracellular Ca2+ as the culprit. Thus PARP‐1 is not hyperactivated via PARG metabolism, and the Ca2+ sources activating PARP‐1 may include mitochondrial stores. We are using calcium imaging to further investigate the elusive relationship between specific Ca2+ channels and PARP‐1, as well as the mechanism(s) by which PARP‐1 and Ca2+ interact to contribute to necrotic cell death.

Proteome-wide study of endoplasmic reticulum stress induced by thapsigargin in N2a neuroblastoma cells

Disturbances in intraluminal endoplasmic reticulum (ER) Ca2+ concentration leads to the accumulation of unfolded proteins and perturbation of intracellular Ca2+ homeostasis, which has a huge impact on mitochondrial functioning under normal and stress conditions and can trigger cell death. Thapsigargin (TG) is widely used to model cellular ER stress as it is a selective and powerful inhibitor of sarcoplasmic/endoplasmic reticulum Ca2+ ATPases. Here we provide a representative proteome-wide picture of ER stress induced by TG in N2a neuroblastoma cells. Our proteomics study revealed numerous significant protein expression changes in TG-treated N2a cell lysates analysed by two-dimensional electrophoresis followed by mass spectrometric protein identification. The proteomic signature supports the evidence of increased bioenergetic activity of mitochondria as several mitochondrial enzymes with roles in ATP-production, tricarboxylic acid cycle and other mitochondrial metabolic processes were upregulated. In addition, the upregulation of the main ER resident proteins confirmed the onset of ER stress during TG treatment. It has become widely accepted that metabolic activity of mitochondria is induced in the early phases in ER stress, which can trigger mitochondrial collapse and subsequent cell death. Further investigations of this cellular stress response in different neuronal model systems like N2a cells could help to elucidate several neurodegenerative disorders in which ER stress is implicated.

Highlights From The Literature

Highlights From The Literature

Co-expression of MG29 and Ryanodine Receptor Leads to Apoptotic Cell Death: EFFECT MEDIATED BY INTRACELLULAR Ca2+ RELEASE

Perturbation of intracellular Ca2+ homeostasis has been shown to regulate the process of cell proliferation and apoptosis. Our previous studies show that mitsugumin 29 (MG29), a synaptophysin-related protein localized in the triad junction of skeletal muscle, serves an essential role in muscle Ca2+ signaling by regulating the process of store-operated Ca2+ entry. Here we report a functional interaction between MG29 and the ryanodine receptor (RyR)/Ca2+ release channel. The purified MG29 protein enhances activity of the RyR/Ca2+ release channel incorporated into the lipid bilayer membrane. Co-expression of MG29 and RyR in Chinese hamster ovary cells leads to apoptotic cell death resulting from depletion of intracellular Ca2+ stores, despite neither protein expression alone exhibits any significant effect on cell viability. In transient expression studies, the presence of RyR in the endoplasmic reticulum leads to retention of MG29 from the plasma membrane into the intracellular organelles. This functional interaction between MG29 and RyR could have important implications in the Ca2+ signaling processes of muscle cells. Our data also show that perturbation of intracellular Ca2+ homeostasis can serve as a key signal in the initiation of apoptosis.

Dysregulated Ryanodine Receptors Mediate Cellular Toxicity: RESTORATION OF NORMAL PHENOTYPE BY FKBP12.6

Ca2+ homeostasis is a vital cellular control mechanism in which Ca2+ release from intracellular stores plays a central role. Ryanodine receptor (RyR)-mediated Ca2+ release is a key modulator of Ca2+ homeostasis, and the defective regulation of RyR is pathogenic. However, the molecular events underlying RyR-mediated pathology remain undefined. Cells stably expressing recombinant human RyR2 (Chinese hamster ovary cells, CHOhRyR2) had similar resting cytoplasmic Ca2+ levels ([Ca2+]c) to wild-type CHO cells (CHOWT) but exhibited increased cytoplasmic Ca2+ flux associated with decreased cell viability and proliferation. Intracellular Ca2+ flux increased with human RyR2 (hRyR2) expression levels and determined the extent of phenotypic modulation. Co-expression of FKBP12.6, but not FKBP12, or incubation of cells with ryanodine suppressed intracellular Ca2+ flux and restored normal cell viability and proliferation. Restoration of normal phenotype was independent of the status of resting [Ca2+]c or ER Ca2+ load. Heparin inhibition of endogenous inositol trisphosphate receptors (IP3R) had little effect on intracellular Ca2+ handling or viability. However, purinergic stimulation of endogenous IP3R resulted in apoptotic cell death mediated by hRyR2 suggesting functional interaction occurred between IP3R and hRyR2 Ca2+ release channels. These data demonstrate that defective regulation of RyR causes altered cellular phenotype via profound perturbations in intracellular Ca2+ signaling and highlight a key modulatory role of FKBP12.6 in hRyR2 Ca2+ channel function.

Regulation of cell death: the calcium-apoptosis link.

To live or to die? This crucial question eloquently reflects the dual role of Ca2+ in living organisms--survival factor or ruthless killer. It has long been known that Ca2+ signals govern a host of vital cell functions and so are necessary for cell survival. However, more recently it has become clear that cellular Ca2+ overload, or perturbation of intracellular Ca2+ compartmentalization, can cause cytotoxicity and trigger either apoptotic or necrotic cell death.

Oxidative glutamate toxicity involves mitochondrial dysfunction and perturbation of intracellular Ca2+ homeostasis.

Glutamate toxicity on PC12 cells is mediated by oxidative stress as a consequence of the inhibition of a cystine uptake system with depletion of GSH. In this study we report that glutamate decreases PC12 cell viability, inhibiting the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). This decrease was prevented by the antioxidants vitamin E, idebenone and L-deprenyl, which were also shown to be effective in reducing the accumulation of reactive oxygen species (ROS) in cells exposed to glutamate, decreasing the fluorescence of 2',7'-dichlorofluorescein (DCF). Incubation of PC12 cells with high glutamate concentrations induced mitochondrial dysfunction, leading to the loss of mitochondrial transmembrane potential, evaluated as a decrease in rhodamine 123 (Rh123) retention by mitochondria, and to the decrease of intracellular ATP levels. The mitochondrial dysfunction, induced by glutamate, can be involved in the observed increase of [Ca2+]i. The elevation of [Ca2+]i occurred after GSH depletion, suggesting that oxidative stress is involved in the disturbances of intracellular calcium homeostasis. In conclusion, our data indicate that glutamate, at concentrations which block cystine uptake in PC12 cells leading to GSH depletion and inducing oxidative stress, increases ROS accumulation and decreases cell survival by a mechanism involving mitochondrial dysfunction and impairment of Ca2+ homeostasis.

Alzheimer's presenilin-1 mutation potentiates inositol 1,4,5-trisphosphate-mediated calcium signaling in Xenopus oocytes.

Perturbations in intracellular Ca2+ signaling may represent one mechanism underlying Alzheimer's disease (AD). The presenilin-1 gene (PS1), associated with the majority of early onset familial AD cases, has been implicated in this signaling pathway. Here we used the Xenopus oocyte expression system to investigate in greater detail the role of PS1 in intracellular Ca2+ signaling pathways. Treatment of cells expressing wild-type PS1 with a cell surface receptor agonist to stimulate the phosphoinositide second messenger pathway evoked Ca2+-activated Cl- currents that were significantly potentiated relative to controls. To determine which elements of the signal transduction pathway are responsible for the potentiation, we used photolysis of caged inositol 1,4,5-trisphosphate (IP3) and fluorescent Ca2+ imaging to demonstrate that PS1 potentiates IP3-mediated release of Ca2+ from internal stores. We show that an AD-linked mutation produces a potentiation in Ca2+ signaling that is significantly greater than that observed for wild-type PS1 and that cannot be attributed to differences in protein expression levels. Our findings support a role for PS1 in modulating IP3-mediated Ca2+ liberation and suggest that one pathophysiological mechanism by which PS1 mutations contribute to AD neurodegeneration may involve perturbations of this function.

Alzheimer's Presenilin‐1 Mutation Potentiates Inositol 1,4,5‐Trisphosphate‐Mediated Calcium Signaling in Xenopus

Abstract : Perturbations in intracellular Ca2+ signaling may represent one mechanism underlying Alzheimer's disease (AD). The presenilin‐1 gene (PS1), associated with the majority of early onset familial AD cases, has been implicated in this signaling pathway. Here we used the Xenopus oocyte expression system to investigate in greater detail the role of PS1 in intracellular Ca2+ signaling pathways. Treatment of cells expressing wild‐type PS1 with a cell surface receptor agonist to stimulate the phosphoinositide second messenger pathway evoked Ca2+ ‐activated Cl‐ currents that were significantly potentiated relative to controls. To determine which elements of the signal transduction pathway are responsible for the potentiation, we used photolysis of caged inositol 1,4,5‐triphosphate (IP3) and fluorescent Ca2+ imaging to demonstrate that PS1 potentiates IP3‐mediated release of Ca2+ from internal stores. We show that an AD‐linked mutation produces a potentiation in Ca2+ signaling that is significantly greater than that observed for wild‐type PS1 and that cannot be attributed to differences in protein expression levels. Our findings support a role for PS1 in modulating IP3 ‐mediated Ca2+ liberation and suggest that one pathophysiological mechanism by which PS1 mutations contribute to AD neurodegeneration may involve perturbations of this function.

Rapid reduction in ryanodine binding of hippocampus CA1 in cerebral ischemia.

Ryanodine receptors located on the sarcoplasmic or endoplasmic reticulum, play an important role in the regulation of the intracellular Ca2+ level via the mechanism of Ca(2+)-induced Ca2+ release (CICR). Perturbation of intracellular Ca2+ regulation has been considered to be one of the most important mechanisms underlying acute ischemic neuronal damage. The ryanodine binding, an indicator of intracellular channels of CICR, and local cerebral blood flow (LCBF) were therefore examined at 15 min post-ischemia in the gerbil brain. The autoradiographic method developed in our laboratory enabled us to determine both parameters within the same brain. Severe hemispheric cerebral ischemia was induced by occluding the right common carotid artery. LCBF was measured at the end of the experiment using [14C]iodoantipyrine method. The ryanodine binding was evaluated autoradiographically in vitro using [3H] ryanodine. A group of gerbils who underwent a sham procedure served as controls. LCBF was found to be significantly decreased in most cerebral regions on the occluded side. In contrast, a significant reduction in ryanodine binding was noted only in the hippocampus CA1 on the occluded side. Taken together, these findings indicate that the CICR in the hippocampus CA1 may be especially susceptible to acute ischemic stress, and be closely associated with the pathophysiological mechanisms of the selective vulnerability of this region.

Induction of calreticulin expression in HeLa cells by depletion of the endoplasmic reticulum Ca2+ store and inhibition of N-linked glycosylation

Calreticulin is now considered to be a multifunctional Ca(2+)-binding protein. Its primary role is as a Ca2+ storage protein within the lumen of the endoplasmic reticulum (ER), where it also seems to assist in the correct folding and assembly of proteins. We have investigated whether agents that affect these processes can alter calreticulin expression in HeLa cells. Perturbation of intracellular Ca2+ levels by prolonged exposure to either thapsigargin or ionomycin induced calreticulin mRNA, both in the presence and absence of extracellular Ca2+, consistent with the proposal that sustained depletion of the ER Ca2+ store can trigger these increases. The mechanism underlying the induction seems to be transcriptional up-regulation as both agents increased calreticulin promoter-driven firefly luciferase expression in transfected cells to the same degree as the observed increases in calreticulin mRNA. Experiments with a truncated promoter construct showed that the sequences that confer this inducibility reside within the 225 bp immediately upstream of the putative major transcriptional start site. We also examined the effect of tunicamycin, which inhibits N-linked glycosylation in the ER thereby interfering with protein processing. This caused increases in calreticulin mRNA greater than those with either thapsigargin or ionomycin, but failed to transactivate the calreticulin promoter. Thus either additional cis sequences that reside outside our promoter region are necessary for transcriptional activation by tunicamycin, or the increases in calreticulin mRNA occur post-transcriptionally. This suggests that there are probably different mechanisms by which calreticulin expression can be induced in response to agents that affect normal ER functioning.

Inhibition of Neutrophil Superoxide Secretion By of Preservative, Methylhydroxybenzoate: Effects Mediated By Perturbation of Intracellular Ca2+?

The preservative, methylhydroxybenzoate inhibited O2- secretion from human neutrophils activated by both the chemotactic peptide fMet-Leu-Phe and phorbol myristate acetate (PMA): the low level of oxidant secretion activated by the ionophore A23187 was similarly reduced in preservative-treated suspensions. Oxidant secretion was similarly reduced in fMet-Leu-Phe and A23187 treated suspensions in which intracellular Ca2+ was buffered by loading with Quin-2, indicating that methylhydroxybenzoate may exert its effects by perturbation of intracellular Ca2(+)-dependent processes. Methylhydroxybenzoate could mimic EGTA in preventing the Ca2+ dependent enhancement of trypsin activity and could also bind this cation in experiments using a Ca2+ electrode, although the preservative bound Ca2+ more slowly and had a lower affinity than EGTA. These data indicate that methylhydroxybenzoate may exert its effects on neutrophils by perturbation of Ca2(+)-dependent activation pathways and this phenomenon may also explain its other known pharmacological effects. Furthermore, these observations provide an insight into the mechanisms by which intracellular Ca2+ may regulate oxidant secretion.

Interleukin-1 inhibits glucose-induced Ca2+ uptake by islets of Langerhans

Recombinant interleukin-1 (rIL-1) is known to inhibit glucose-induced insulin secretion by islets of Langerhans, a novel target tissue of cytokine. We have investigated whether rIL-1 pretreatment affects biochemical mechanisms known to be involved in the regulation of Ca2+ homeostasis during glucose-induced insulin secretion. Glucose-induced Ca2+ uptake by intact islets through the plasma membrane was dramatically inhibited (96%) by rIL-1 (2 nM). rIL-1, however, did not affect Ca2+ uptake by, or Ins 1,4,5-P3-induced Ca2+ efflux from, the endoplasmic reticulum in digitonin-permeabilized islets, although glucose-induced accumulation of inositol trisphosphates was inhibited (38%). These results suggest that perturbation of intracellular Ca2+ homeostasis in islets is involved in inhibition of insulin secretion by rIL-1.

Calcium channel antagonists delay regression of androgen‐dependent tissues and suppress gene activity associated with cell death

Androgen deprivation subsequent to castration of an adult male rat results in the regression of sexual accessory tissues. Regression of these tissues involves the massive death of androgen-dependent cells. Using the rat ventral prostate gland as a model to study androgen-programed cell death, we have characterized a series of molecular events that accompany its regression. This analysis has shown that there was a sequential induction of specific gene transcripts in the ventral prostate gland following castration. The first event in this cascade was an abrupt induction of transcripts encoding c-fos. Since c-fos expression has been linked to perturbations in intracellular Ca2+ levels, we investigated whether membrane-mediated Ca2+ flux might be an early physiological step involved in the death of prostatic cells. To test this, rats were treated simultaneously upon castration with either verapamil or nifedipine, two different calcium channel antagonist drugs. Compared to the ventral prostate glands of untreated castrated rats, the glands of the calcium channel antagonist-treated rats showed a significant delay in all parameters associated with regression (loss of wet weight and DNA content and delay in histological changes associated with prostatic regression). The seminal vesicle glands of treated rats also showed signs of delayed regression. Furthermore, calcium channel antagonists suppressed the induction of transcripts encoding both c-fos and testosterone-repressed prostate message-2 (TRPM-2), a gene expressed exclusively by dying cells, during the first 62 hr following castration. These findings further support a role for calcium ion influx in the pathway leading to hormonally programed prostate cell death, and suggest the intriguing possibility that modulation of this activity can alter the process by which cells die.