thapsigargin and Disease-Models--Animal

Acute kidney injury (AKI) is a medical condition characterized by kidney damage with a rapid decline of renal function, which is associated with high mortality and morbidity. Recent research has further established an intimate relationship between AKI and chronic kidney disease. Perturbations of kidney cells in AKI result in the accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER), leading to unfolded protein response (UPR) or ER stress. In this review, we analyze the role and regulation of ER stress in AKI triggered by renal ischemia-reperfusion and cisplatin nephrotoxicity. The balance between the two major components of UPR, the adaptive pathway and the apoptotic pathway, plays a critical role in determining the cell fate in ER stress. The adaptive pathway is evoked to attenuate translation, induce chaperones, maintain protein homeostasis and promote cell survival. Prolonged ER stress activates the apoptotic pathway, resulting in the elimination of dysfunctional cells. Therefore, regulating ER stress in kidney cells may provide a therapeutic target in AKI. KEY MESSAGES Perturbations of kidney cells in acute kidney injury result in the accumulation of unfolded and misfolded proteins in ER, leading to unfolded protein response (UPR) or ER stress. The balance between the adaptive pathway and the apoptotic pathway of UPR plays a critical role in determining the cell fate in ER stress. Modulation of ER stress in kidney cells may provide a therapeutic strategy for acute kidney injury.

Topics: Acute Kidney Injury; Animals; Apoptosis; Autophagy; Cisplatin; Disease Models, Animal; Disease Progression; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Humans; Ischemic Preconditioning; Kidney; Molecular Chaperones; Protein Folding; Renal Insufficiency, Chronic; Reperfusion Injury; Thapsigargin; Treatment Outcome; Tunicamycin; Unfolded Protein Response

Progesterone and adipoQ receptor 9 (PAQR9) is an endoplasmic reticulum (ER)-localized membrane protein that is involved in protein quality control of ER by interacting with BAG6. One of the physiological functions of PAQR9 is regulation of fasting-induced ketogenesis and fatty acid oxidation in the liver via modulating protein degradation of PPARα. However, it is currently unknown whether or not PAQR9 impacts glucose homeostasis. We addressed this question using a Paqr9-deleted mouse model in which type 1 diabetes was induced by streptozotocin injection and type 2 diabetes was induced by high-fat diet (HFD) with streptozotocin injection. Paqr9 deletion improved hyperglycemia and glucose tolerance in both of the diabetic mouse models. In the pancreatic islets, Paqr9 deletion reduced apoptosis of β cells in type 2 diabetic mice. Paqr9 deletion also reduced HFD-induced hepatic steatosis and adiposity of white adipose tissue. In Min6 cells, overexpression of DUF3538 domain of BAG6 to block the interaction of PAQR9 with BAG6 was able to enhance glucose-stimulated insulin secretion upon treatment with inflammatory factors or thapsigargin, an ER stress inducer. Thapsigargin-induced ER stress markers were also reduced by overexpression of DUF3538 domain. Collectively, these results indicate that PAQR9 has a modulatory role in glucose homeostasis, associated with regulation on insulin secretion of β cells in vitro under stress conditions.

Topics: Animals; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Diet, High-Fat; Disease Models, Animal; Endoplasmic Reticulum Stress; Glucose; Homeostasis; Insulin; Insulin Secretion; Insulin-Secreting Cells; Mice; Streptozocin; Thapsigargin

Topics: Aging; Animals; Behavior, Animal; Cells, Cultured; Diet, High-Fat; Disease Models, Animal; Doublecortin Domain Proteins; Endoplasmic Reticulum Stress; Hippocampus; Memory; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurons; Obesity; Thapsigargin

Fibrosis is a common final pathway of chronic kidney disease, which is a major incurable disease. Although fibrosis has an irreversible pathophysiology, the molecular and cellular mechanisms responsible remain unclear and no specific treatment is available to halt the progress of renal fibrosis. Thus, an improved understanding of the cellular mechanism involved and a novel therapeutic approach are urgently required for end-stage renal disease (ESRD). We investigated the role played by interleukin-10 (IL-10, a potent anti-inflammatory cytokine) in kidney fibrosis and the mechanisms involved using

Topics: Animals; Apoptosis; Brefeldin A; Disease Models, Animal; Endoplasmic Reticulum Stress; Fibrosis; Interleukin-10; Kidney; Kidney Diseases; Mice; Renal Insufficiency, Chronic; RNA, Small Interfering; Thapsigargin; Tunicamycin; Ureteral Obstruction

Pancreatic islets are exposed to strong pro-apoptotic stimuli: inflammation and hyperglycemia, during the progression of the autoimmune diabetes (T1D). We found that the. We studied T1D pathogenesis in NOD mice hemideficient for CDK11 (N-HTZ), and, in N-HTZ deficient for Cyclin D3 (K11HTZ-D3KO), in comparison to their respective controls (N-WT and K11WT-D3KO). Moreover, we exposed pancreatic islets to either pro-inflammatory cytokines in the presence of increasing glucose concentrations, or Thapsigargin, an Endoplasmic Reticulum (ER)-stress inducing agent, and assessed apoptotic events. The expression of key ER-stress markers (. N-HTZ mice were significantly protected against T1D, and NS-HTZ pancreatic islets exhibited an impaired sensitivity to cytokine-induced apoptosis, regardless of glucose concentration. However, thapsigargin-induced apoptosis was not altered. Furthermore, CDK11 hemideficiency did not attenuate the exacerbation of T1D caused by Cyclin D3 deficiency.. This study is the first to report that CDK11 is repressed in T1D as a protection mechanism against inflammation-induced apoptosis and suggests that CDK11 lies upstream Cyclin D3 signaling. We unveil the CDK11/Cyclin D3 tandem as a new potential intervention target in T1D.

Topics: Activating Transcription Factor 4; Animals; Apoptosis; Autoimmunity; Blood Glucose; Cyclin D3; Cyclin-Dependent Kinases; Cytokines; Diabetes Mellitus, Type 1; Disease Models, Animal; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Heat-Shock Proteins; Inflammation; Insulin-Secreting Cells; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Knockout; Protein Serine-Threonine Kinases; Thapsigargin; Tissue Culture Techniques; Transcription Factor CHOP

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

There is a major clinical need for new therapies for the treatment of chronic itch. Many of the molecular components involved in itch neurotransmission are known, including the neuropeptide NPPB, a transmitter required for normal itch responses to multiple pruritogens in mice. Here, we investigated the potential for a novel strategy for the treatment of itch that involves the inhibition of the NPPB receptor NPR1 (natriuretic peptide receptor 1). Because there are no available effective human NPR1 (hNPR1) antagonists, we performed a high-throughput cell-based screen and identified 15 small-molecule hNPR1 inhibitors. Using in vitro assays, we demonstrated that these compounds specifically inhibit hNPR1 and murine NPR1 (mNPR1). In vivo, NPR1 antagonism attenuated behavioral responses to both acute itch- and chronic itch-challenged mice. Together, our results suggest that inhibiting NPR1 might be an effective strategy for treating acute and chronic itch.

Topics: Animals; Behavior, Animal; Cell-Free System; Dermatitis, Contact; Disease Models, Animal; Ganglia, Spinal; Humans; Mice, Inbred C57BL; Mice, Knockout; Neurons; Pruritus; Receptors, Atrial Natriuretic Factor; Reproducibility of Results; Signal Transduction; Small Molecule Libraries

Previous studies have shown that pretreatment with thapsigargin (TG), a cellular stress inducer, produced potent protective actions against various pathologic injuries. So far there is no information on the effects of TG on the development of bacterial sepsis. Using lipopolysaccharides- and cecal ligation/puncture-induced sepsis models in mice, we demonstrated that preconditioning with a single bolus administration of TG conferred significant improvements in survival. The beneficial effects of TG were not mediated by ER stress induction or changes in Toll-like receptor 4 signaling. In vivo and in cultured macrophages, we identified that TG reduced the protein production of pro-inflammatory cytokines, but exhibited no significant effects on steady state levels of their transcriptions. Direct measurement on the fraction of polysome-bound mRNAs revealed that TG reduced the translational efficiency of pro-inflammatory cytokines in macrophages. Moreover, we provided evidence suggesting that repression of the mTOR (the mammalian target of rapamycin) signaling pathway, but not activation of the PERK (protein kinase R-like endoplasmic reticulum kinase)-eIF2α (eukaryotic initiation factor 2α) pathway, might be involved in mediating the TG effects on cytokine production. In summary, our results support that pharmacological preconditioning with TG may represent a novel strategy to prevent sepsis-induced mortality and organ injuries.

Topics: Animals; Anti-Inflammatory Agents; Cytokines; Disease Models, Animal; Humans; Male; Mice; Mice, Inbred C57BL; Oxidative Stress; Pre-Exposure Prophylaxis; Protective Agents; RAW 264.7 Cells; Sepsis; Thapsigargin; Toll-Like Receptor 4

Chemical proteasome inhibition has been a valuable animal model of neurodegeneration to uncover roles for the ubiquitin-proteasome system in the central nervous system. However, little is known about the effects of chemical proteasome inhibitors on retinal integrity. Therefore, we characterized the effects of structurally different chemical proteasome inhibitors on the retinal morphology and the mechanisms of their action in the normal adult rat eyes. Intravitreal injection of MG-262 and other proteasome inhibitors led to inner retinal degeneration. MG-262-induced inner retinal degeneration was accompanied by reduced proteasome activity, increased poly-ubiquitinated protein levels, and increased positive immunostaining of ubiquitin, 20S proteasome subunit and GADD153/CHOP in the retina. Its retinal degenerative effect was also associated with reduced retinal neurofilament light chain gene expression, reflecting retinal ganglion cell death. MG-262-induced neurofilament light chain downregulation was largely resistant to pharmacological modulation including endoplasmic reticulum stress, apoptosis or MAP kinase inhibitors. Thus, this study provides further evidence of roles for the ubiquitin-proteasome system in the maintenance of the retinal structural integrity. Chemical proteasome inhibition may be used as a novel animal model of inner retinal degeneration, including retinal ganglion cell loss, which warrants further analysis of the molecular mechanisms underlying its retinal degenerative effect.

Topics: Animals; Apoptosis; Boronic Acids; Disease Models, Animal; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Humans; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Retina; Retinal Degeneration; Retinal Ganglion Cells; Thapsigargin; Tunicamycin

Background Cornu ammonis 3 (CA3) hippocampal neurons are resistant to global ischemia, whereas cornu ammonis (CA1) 1 neurons are vulnerable. Hamartin expression in CA3 neurons mediates this endogenous resistance via productive autophagy. Neurons lacking hamartin demonstrate exacerbated endoplasmic reticulum stress and increased cell death. We investigated endoplasmic reticulum stress responses in CA1 and CA3 regions following global cerebral ischemia, and whether pharmacological modulation of endoplasmic reticulum stress or autophagy altered neuronal viability . Methods In vivo: male Wistar rats underwent sham or 10 min of transient global cerebral ischemia. CA1 and CA3 areas were microdissected and endoplasmic reticulum stress protein expression quantified at 3 h and 12 h of reperfusion. In vitro: primary neuronal cultures (E18 Wistar rat embryos) were exposed to 2 h of oxygen and glucose deprivation or normoxia in the presence of an endoplasmic reticulum stress inducer (thapsigargin or tunicamycin), an endoplasmic reticulum stress inhibitor (salubrinal or 4-phenylbutyric acid), an autophagy inducer ([4'-(N-diethylamino) butyl]-2-chlorophenoxazine (10-NCP)) or autophagy inhibitor (3-methyladenine). Results In vivo, decreased endoplasmic reticulum stress protein expression (phospho-eIF2α and ATF4) was observed at 3 h of reperfusion in CA3 neurons following ischemia, and increased in CA1 neurons at 12 h of reperfusion. In vitro, endoplasmic reticulum stress inducers and high doses of the endoplasmic reticulum stress inhibitors also increased cell death. Both induction and inhibition of autophagy also increased cell death. Conclusion Endoplasmic reticulum stress is associated with neuronal cell death following ischemia. Neither reduction of endoplasmic reticulum stress nor induction of autophagy demonstrated neuroprotection in vitro, highlighting their complex role in neuronal biology following ischemia.

Topics: Animals; Brain Ischemia; CA1 Region, Hippocampal; CA3 Region, Hippocampal; Cell Death; Cells, Cultured; Disease Models, Animal; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Hypoglycemia; Hypoxia; Male; Neurons; Neuroprotective Agents; Rats, Wistar; Thapsigargin; Tuberous Sclerosis Complex 1 Protein; Tunicamycin

Tacrolimus is an immunosuppressive drug that inhibits the release of inflammatory cytokines involved in rheumatoid arthritis development by blocking T cell activation. "Endoplasmic reticulum stress," an imbalance between protein folding load and capacity leading to the accumulation of unfolded proteins in the endoplasmic reticulum lumen, has been implicated in rheumatoid arthritis and other inflammatory and metabolic diseases. We aimed to investigate the effect of tacrolimus on endoplasmic reticulum stress-mediated osteoclastogenesis and inflammation and elucidate the underlying mechanisms. In vitro studies were performed using mouse bone marrow cells that were cultured with or without interleukin-1β, thapsigargin, or tacrolimus to induce osteoclast differentiation. A mouse model of arthritis was established by immunizing mice with bovine type II collagen. Tacrolimus was orally administered to mice from day 20 to 45 following the initial immunization, and histopathological changes and expression of specific biomarkers of endoplasmic reticulum stress-mediated inflammatory signaling pathways were examined. In vitro, tacrolimus inhibited receptor activator of nuclear factor-κB ligand-mediated osteoclast formation augmented by interleukin-1β, thapsigargin, or both. Furthermore, tacrolimus inhibited glucose-regulated protein (GRP78), protein kinase R-like endoplasmic reticulum kinase, inositol-requiring enzyme 1 (IRE 1), and activating transcription factor 6 (ATF6) augmented by interleukin-1β, thapsigargin, or both. Tacrolimus significantly ameliorated osteolysis and endoplasmic reticulum stress intensity in mice. Simultaneously, it reduced inflammatory cell infiltration, osteoclastogenesis, and inflammatory responses by inhibiting GRP78, IRE 1, and ATF6. These findings suggest that tacrolimus exhibits an anti-inflammation effect in rheumatoid arthritis and might inhibit joint damage progression by inhibiting endoplasmic reticulum stress.

Topics: Animals; Arthritis; Arthritis, Experimental; Collagen; Disease Models, Animal; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Inflammation; Interleukin-1beta; Male; Mice; Mice, Inbred DBA; Osteoclasts; Osteogenesis; Signal Transduction; Tacrolimus; Thapsigargin

Mouse models of heart disease are extensively employed. The echocardiographic characterization of contractile function is usually focused on systolic function with fewer studies assessing diastolic function. Furthermore, the applicability of diverse echocardiographic parameters of diastolic function that are commonly used in humans has not been extensively evaluated in different pathophysiological models in mice.. We used high resolution echocardiography to evaluate parameters of diastolic function in mouse models of chronic pressure overload (aortic constriction), volume overload (aorto-caval shunt), heart failure with preserved ejection fraction (HFpEF; DOCA-salt hypertension), and acute sarcoplasmic reticulum dysfunction induced by thapsigargin - all known to exhibit diastolic dysfunction. Left atrial area increased in all three chronic models while mitral E/A was difficult to quantify at high heart rates. Isovolumic relaxation time (IVRT) and Doppler E/E' increased significantly and the peak longitudinal strain rate during early filling (peak reverse longitudinal strain rate) decreased significantly after aortic constriction, with the changes being proportional to the magnitude of hypertrophy. In the HFpEF model, reverse longitudinal strain rate decreased significantly but changes in IVRT and E/E' were non-significant, consistent with less severe dysfunction. With volume overload, there was a significant increase in reverse longitudinal strain rate and decrease in IVRT, indicating a restrictive physiology. Acute thapsigargin treatment caused significant prolongation of IVRT and decrease in reverse longitudinal strain rate.. These results indicate that the combined measurement of left atrial area plus reverse longitudinal strain rate and/or IVRT provide an excellent overall assessment of diastolic function in the diseased mouse heart, allowing distinction between different types of pathophysiology.

Topics: Animals; Cardiomegaly; Diastole; Disease Models, Animal; Echocardiography; Heart Diseases; Heart Failure; Mice, Inbred C57BL; Observer Variation; Pressure; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Stroke Volume; Systole; Thapsigargin

On-target drug delivery remains a challenge in cancer precision medicine; it is difficult to deliver a targeted therapy to cancer cells without incurring toxicity to normal tissues. The SERCA (sarco-endoplasmic reticulum Ca

Topics: Animals; Antineoplastic Agents; Biological Transport; Cell Line, Tumor; Disease Models, Animal; Drug Delivery Systems; Endocytosis; Folate Receptor 2; Folic Acid; Gene Expression; Humans; Leukemia; Mice; Mutation; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Protein Binding; Receptor, Notch1; Signal Transduction; Thapsigargin; Xenograft Model Antitumor Assays

Excessive extracellular matrix degradation and chondrocyte apoptosis are the pathological features of osteoarthritis (OA). The ability of flavonoid compounds isolated from Chinese hawthorn leaves to exert protective effects on several diseases, via inhibition of oxidative stress and inflammation, has been demonstrated in several studies. This study explored the effects of vitexin on chondrocytes, and the underlying mechanisms thereof. Vitexin, an active ingredient in hawthorn leaf extracts, was shown to exert protective effects on chondrocytes, by inhibiting the expression of GRP78 and PDI, and an apoptotic protein (CHOP) induced by interleukin-1β. It also modulated thapsigargin-induced upregulation of GRP78 and PDI and subsequently an apoptotic protein (CHOP). Among rat chondrocytes, both the ER stress-activated nuclear factor kappa B (NF-κB) pathway and the induced expression of inflammatory cytokines (IL-6 and TNF-α) were significantly inhibited by vitexin. Finally, vitexin attenuated the progression of OA in vivo in rats. Taken together, all data demonstrate the relationship of ER stress and inflammation in the progression of OA, the ability of vitexin to protect chondrocytes and thus its therapeutic potential in patients with OA.

Topics: Animals; Apigenin; Apoptosis; Cartilage; Caspase 3; Cell Survival; Chondrocytes; Disease Models, Animal; Endoplasmic Reticulum Stress; Heat-Shock Proteins; Inflammation; Interleukin-1beta; Male; NF-kappa B; Osteoarthritis; Rats; Rats, Sprague-Dawley; Sincalide; Thapsigargin; Transcription Factor CHOP; Tumor Necrosis Factor-alpha; Up-Regulation

Increased levels of circulating sphingosine-1-phosphate (S1P) have been reported in ulcerative colitis. The objective of this study was to examine the effect of S1P on colonic smooth muscle contractility and how is it affected by colitis.. Colonic inflammation was induced by intrarectal administration of trinitrobenzene sulfonic acid. Five days later colon segments were isolated and used for contractility experiments and immunoblotting.. S1P contracted control and inflamed colon segments and the contraction was significantly greater in inflamed colon segments. S1P-induced contraction was mediated by S1PR1 and S1PR2 in control and S1PR2 in inflamed colon segments. S1PR3 did not play a significant role in S1P-induced contractions in control or inflamed colon. S1PR1, S1PR2 and S1PR3 proteins were expressed in colon segments from both groups. The expression of S1PR1 and S1PR2 was significantly enhanced in control and inflamed colon segments, respectively. S1PR3 levels however were not significantly different between the two groups. Nifedipine significantly reduced S1P-induced contraction in control but not inflamed colon segments. Thapsigargin significantly reduced S1P-induced contraction of the inflamed colon. GF 109203X and Y-27632, alone abolished S1P-induced contraction of the control but not inflamed colon segments. Combination of GF 109203X, Y-27632 and thapsigargin abolished S1P-induced contraction of inflamed colon segments.. S1P contracted control colon via S1PR1 and S1PR2 and inflamed colon exclusively via S1PR2. Calcium influx (control) or release (inflamed) and calcium sensitization are involved in S1P-induced contraction. Exacerbated response to S1P in colitic colon segments may explain altered colonic motility reported in patients and experimental models of inflammatory bowel disease.

Topics: Animals; Calcium; Colitis, Ulcerative; Colon; Disease Models, Animal; Humans; Inflammation; Lysophospholipids; Muscle Contraction; Muscle, Smooth; Rats; Receptors, Lysosphingolipid; Sphingosine; Sphingosine-1-Phosphate Receptors; Thapsigargin; Trinitrobenzenesulfonic Acid

Autosomal dominant polycystic kidney disease (ADPKD) is associated with progressive enlargement of multiple renal cysts, often leading to renal failure that cannot be prevented by a current treatment. Two proteins encoded by two genes are associated with ADPKD: PC1 (

Topics: Animals; Calcium; Calcium Chelating Agents; Calcium Signaling; Cell Line; Cyclic AMP; Cysts; Disease Models, Animal; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; Mice; Mice, Knockout; Mice, Transgenic; Polycystic Kidney, Autosomal Dominant; Protein Kinase C; Thapsigargin; TRPP Cation Channels

Major facilitator superfamily domain containing 2a (Mfsd2a) is a member of the major facilitator superfamily. Mfsd2a functions as a transporter for docosahexaenoic acid and also plays a role in the unfolded protein response (UPR) upon tunicamycin (TM) exposure. UPR is involved in the pathogenesis of various human diseases. TM and thapsigargin are representative experimental reagents that induce UPR. To elucidate the detailed function of Mfsd2a in UPR in vivo, we generated Mfsd2a-deficient mice and investigated the role of Mfsd2a during UPR induced by TM or thapsigargin. Phenotypically, Mfsd2a-deficient mice were small and short-lived. No gross anatomical abnormalities in Mfsd2a-deficient mice compared with the wild-type mice were exhibited. Embryonic fibroblasts derived from Mfsd2a-null mice failed to show induction of GRP78 and DDIT3 expressions upon TM exposure but not upon Tg exposure. This phenomenon could not be overcome despite the exposure under high TM concentration. Reconstitution of Mfsd2a in Mfsd2a-null MEF showed hypersensitivity to TM. Furthermore, we examined the physiological role of Mfsd2a against TM using an in vivo mouse model. DDIT3 induction by TM was drastically attenuated in both the liver and brain of Mfsd2a-deficient mice. These results reveal that Mfsd2a plays a critical role in UPR upon TM exposure.

Topics: Animals; Brain; Cells, Cultured; Disease Models, Animal; Endoplasmic Reticulum Chaperone BiP; Fibroblasts; HEK293 Cells; Humans; Liver; Membrane Transport Proteins; Mice; Protein Unfolding; Symporters; Thapsigargin; Transcription Factor CHOP; Tunicamycin

Hyperalgesic priming, a model of pain chronification in the rat, is mediated by ryanodine receptor-dependent calcium release. Although ryanodine induces priming in both sexes, females are 5 orders of magnitude more sensitive, by an estrogen receptor α (EsRα)-dependent mechanism. An inositol 1,4,5-triphosphate (IP

Topics: Animals; Cells, Cultured; Dinoprostone; Disease Models, Animal; Enzyme Inhibitors; Female; Ganglia, Spinal; Hyperalgesia; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Macrocyclic Compounds; Male; Oligodeoxyribonucleotides, Antisense; Oxazoles; Pain Measurement; Pain Threshold; Rats; Rats, Sprague-Dawley; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sensory Receptor Cells; Sex Characteristics; Thapsigargin

In Ca(2+)-overloaded ventricular myocytes, SERCA is crucial to steadily achieve the critical sarcoplasmic reticulum (SR) Ca(2+) level to trigger and sustain Ca(2+) waves, that propagate at constant rate (ʋwave). High luminal Ca(2+) sensitizes RyR2, thereby increasing Ca(2+) sparks frequency, and the larger RyR2-mediated SR Ca(2+) flux (dF/dt) sequentially activates adjacent RyR2 clusters. Recently, it was proposed that rapid SERCA Ca(2+) reuptake, ahead of the wave front, further sensitizes RyR2, increasing ʋwave. Nevertheless, this is controversial because rapid cytosolic Ca(2+) removal could instead impair RyR2 activation. We assessed whether rapid SR Ca(2+) uptake enhances ʋwave by changing SERCA activity (ҡDecay) over a large range (∼175%). We used normal (Ctrl) and hyperthyroid rat (HT; reduced phospholamban by ∼80%) myocytes treated with thapsigargin or isoproterenol (ISO). We found that ʋwave and dF/dt had a non-linear dependency with ҡDecay, while Ca(2+) waves amplitude was largely unaffected. Furthermore, SR Ca(2+) also showed a non-linear dependency with ҡDecay, however, the relationships ʋwave vs. SR Ca(2+) and ʋwave vs. dF/dt were linear, suggesting that high steady state SR Ca(2+) determines ʋwave, while rapid SERCA Ca(2+) uptake does not. Finally, ISO did not increase ʋwave in HT cells, therefore, ISO-enhanced ʋwave in Ctrl depended on high SR Ca(2+).

Topics: Animals; Calcium; Calcium Signaling; Calcium-Binding Proteins; Cytosol; Disease Models, Animal; Heart Ventricles; Hyperthyroidism; Isoproterenol; Male; Myocytes, Cardiac; Rats; Rats, Wistar; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin

The development of atherosclerosis is closely related to excessive endoplasmic reticulum stress (ERs). Equol reportedly protects against cardiovascular disease; however, the underlying mechanism for this protection remains unknown. Herein, the mechanisms contributing to the atheroprotective effect of equol were addressed using apolipoprotein E knockout (apoE-/-) mice fed a high-fat diet (HFD) with or without equol. Equol intervention reduced atherosclerotic lesions in the aorta in HFD-fed apoE-/- mice. Plasma lipid analysis showed that equol intervention reduced triglycerides, total cholesterol and LDL-cholesterol and increased HDL-cholesterol. Additionally, equol administration decreased lipid accumulation in the liver. Simultaneously, equol treatment inhibited cell apoptosis induced by t-BHP and thapsigargin in human umbilical vein endothelial cells (HUVECs). Furthermore, equol treatment attenuated palmitate, t-BHP or thapsigargin-induced upregulation of ER stress markers, including p-PERK, p-eIF2α, GRP78, ATF6 and CHOP proteins expression. The same tendency was also observed in aortic lysates in apoE-/- mice fed with equol plus HFD compared with HFD alone. Moreover, equol treatment dose dependently activated the Nrf2 signaling pathway under oxidative stress. Additionally, elevation of Nrf2 induction was found in aortic lysates in apoE-/- mice fed with a HFD diet containing equol compared with a HFD diet without equol. Importantly, Nrf2 siRNA interference induced CHOP and attenuated the effect of equol to inhibit t-BHP mediated CHOP induction, furthermore, abrogated cell apoptosis induced by t-BHP, suggesting a role for Nrf2 in the protective effect of equol in HUVECs. Collectively, these findings implicate that the improvement of atherosclerosis by equol through attenuation of ER stress is mediated, at least in part, by activating the Nrf2 signaling pathway.

Topics: Activating Transcription Factor 6; Animals; Aorta; Apolipoproteins E; Apoptosis; Atherosclerosis; Cholesterol, HDL; Cholesterol, LDL; Diet, High-Fat; Disease Models, Animal; eIF-2 Kinase; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Equol; Eukaryotic Initiation Factor-2; Gene Expression Regulation; Heat-Shock Proteins; Human Umbilical Vein Endothelial Cells; Humans; Liver; Mice; Mice, Knockout; NF-E2-Related Factor 2; Palmitic Acid; Phytoestrogens; Signal Transduction; Thapsigargin; Transcription Factor CHOP; Triglycerides

Zonisamide has been reported to have protective effects on epilepsy and Parkinson׳s disease and to work via various mechanisms of action, such as inhibition of monoamine oxidase-B and enhancement of tyrosine hydroxylase. Recently, it has been suggested that zonisamide itself shows neuroprotective actions. Therefore, in the present study we investigated the neuroprotective effects of zonisamide against endoplasmic reticulum (ER) stress. We used human neuroblastoma (SH-SY5Y) cells and investigated the protective effects of zonisamide against tunicamycin- and thapsigargin-induced neuronal cell death. In addition, we investigated the effect of zonisamide against 1-methyl-4-phenylpyridinium (MPP⁺)-induced cell death and the mechanism of protection against ER stress. In vivo, we investigated the effect of zonisamide (20 mg/kg, p.o.) in the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced mouse model of Parkinson׳s disease. Zonisamide not only suppressed MPP⁺-induced cell death, but also inhibited ER stress-induced cell death and suppressed the expression of ER stress-related factors such as C/EBO homologous protein (CHOP) in vivo. Furthermore, zonisamide inhibited the activation of caspase-3 in vitro. These results suggest that zonisamide affected ER stress via caspase-3. We think that ER stress, particularly the mechanism via caspase-3, is involved in part of the neuroprotective effect of zonisamide against the experimental models of Parkinson׳s disease.

Topics: Animals; Anticonvulsants; Antiparkinson Agents; Cell Death; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Endoplasmic Reticulum Stress; Half-Life; Humans; Isoxazoles; Male; Mice, Inbred C57BL; MPTP Poisoning; Neurons; Neuroprotective Agents; Parkinson Disease; Substantia Nigra; Thapsigargin; Tunicamycin; Zonisamide

Sustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release-activated calcium modulator ORAI1 is the most abundant Ca(2+) entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice.. Mouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects.. GSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca(2+) currents after Ca(2+) release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis.. Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed for the treatment of patients with pancreatitis.

Topics: Acinar Cells; Acute Disease; Animals; Benzamides; Bile Acids and Salts; Calcium; Calcium Channels; Cells, Cultured; Disease Models, Animal; HEK293 Cells; Humans; Indoles; Mice; Mice, Inbred C57BL; ORAI1 Protein; Pancreatitis; Pyrazoles; Thapsigargin; Time Factors; Treatment Outcome

Transected axons of the central nervous system fail to regenerate and instead die back away from the lesion site, resulting in permanent disability. Although both intrinsic (eg, microtubule instability, calpain activation) and extrinsic (ie, macrophages) processes are implicated in axonal dieback, the underlying mechanisms remain uncertain. Furthermore, the precise mechanisms that cause delayed "bystander" loss of spinal axons, that is, ones that were not directly damaged by the initial insult, but succumbed to secondary degeneration, remain unclear. Our goal was to evaluate the role of intra-axonal Ca(2+) stores in secondary axonal degeneration following spinal cord injury.. We developed a 2-photon laser-induced spinal cord injury model to follow morphological and Ca(2+) changes in live myelinated spinal axons acutely following injury.. Transected axons "died back" within swollen myelin or underwent synchronous pan-fragmentation associated with robust Ca(2+) increases. Spared fibers underwent delayed secondary bystander degeneration. Reducing Ca(2+) release from axonal stores mediated by ryanodine and inositol triphosphate receptors significantly decreased axonal dieback and bystander injury. Conversely, a gain-of-function ryanodine receptor 2 mutant or pharmacological treatments that promote axonal store Ca(2+) release worsened these events.. Ca(2+) release from intra-axonal Ca(2+) stores, distributed along the length of the axon, contributes significantly to secondary degeneration of axons. This refocuses our approach to protecting spinal white matter tracts, where emphasis has been placed on limiting Ca(2+) entry from the extracellular space across cell membranes, and emphasizes that modulation of axonal Ca(2+) stores may be a key pharmacotherapeutic goal in spinal cord injury.

Topics: Animals; Axons; Bacterial Proteins; Boron Compounds; Caffeine; Calcium; Disease Models, Animal; Endoplasmic Reticulum; Enzyme Inhibitors; Laser Therapy; Luminescent Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Nerve Degeneration; Purinergic P1 Receptor Antagonists; Ryanodine; Ryanodine Receptor Calcium Release Channel; Spinal Cord Injuries; Thapsigargin; Time Factors

Nuclear protein 1 (Nupr1) is a stress-inducible protein that is involved in gene transcription. The present study was undertaken to determine whether chondrocytes express Nupr1 and whether Nupr1 regulates matrix metalloproteinase 13 (MMP-13) expression.. Paraffin-embedded cartilage sections from normal human and osteoarthritic (OA) cartilage were immunostained using anti-Nupr1 antibody. To measure Nupr1 expression, total RNA was isolated from joint tissue obtained 8 weeks after surgery from young (12-week-old) and older (12-month-old) mice that underwent destabilization of the medial meniscus (DMM) to induce OA. Human chondrocytes were stimulated with 1-10 ng/ml interleukin-1β (IL-1β), 25 μM tert-butyl-hydroperoxide (tBHP), or 2 μM thapsigargin, and Nupr1 expression was analyzed by quantitative polymerase chain reaction. In addition, chondrocytes were transfected with small interfering RNA to knock down Nupr1 expression and then stimulated overnight with IL-1β. After incubation, the conditioned medium was collected and MMP levels measured.. Increased Nupr1 immunostaining was noted in human OA cartilage compared to normal cartilage. Expression was also increased in joint tissue from 12-month-old mice that underwent DMM surgery compared to sham-operated controls. Stimulation of chondrocytes with IL-1β induced a 2-fold increase in Nupr1 messenger RNA (mRNA) within 1 hour, with the increase peaking to 4-fold at 6 hours. Treatment of chondrocytes with tBHP to induce oxidative stress increased Nupr1 mRNA expression by >2-fold; treatment with thapsigargin to induce endoplasmic reticulum stress did not produce a similar effect. Knockdown of Nupr1 inhibited IL-1β-mediated induction of MMP-13.. Nupr1 is expressed in cartilage, and its levels are increased in OA. Nupr1 expression is required for IL-1β-mediated expression of MMP-13. These findings provide evidence of a novel pathway for regulation of IL-1β-mediated production of MMPs in chondrocytes.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cartilage, Articular; Cells, Cultured; Chondrocytes; Disease Models, Animal; DNA-Binding Proteins; Humans; Interleukin-1beta; Matrix Metalloproteinase 13; Mice; Neoplasm Proteins; Osteoarthritis; Oxidative Stress; tert-Butylhydroperoxide; Thapsigargin

Spinal and bulbar muscular atrophy is an X-linked degenerative motor neuron disease caused by an abnormal expansion in the polyglutamine encoding CAG repeat of the androgen receptor gene. There is evidence implicating endoplasmic reticulum stress in the development and progression of neurodegenerative disease, including polyglutamine disorders such as Huntington's disease and in motor neuron disease, where cellular stress disrupts functioning of the endoplasmic reticulum, leading to induction of the unfolded protein response. We examined whether endoplasmic reticulum stress is also involved in the pathogenesis of spinal and bulbar muscular atrophy. Spinal and bulbar muscular atrophy mice that carry 100 pathogenic polyglutamine repeats in the androgen receptor, and develop a late-onset neuromuscular phenotype with motor neuron degeneration, were studied. We observed a disturbance in endoplasmic reticulum-associated calcium homeostasis in cultured embryonic motor neurons from spinal and bulbar muscular atrophy mice, which was accompanied by increased endoplasmic reticulum stress. Furthermore, pharmacological inhibition of endoplasmic reticulum stress reduced the endoplasmic reticulum-associated cell death pathway. Examination of spinal cord motor neurons of pathogenic mice at different disease stages revealed elevated expression of markers for endoplasmic reticulum stress, confirming an increase in this stress response in vivo. Importantly, the most significant increase was detected presymptomatically, suggesting that endoplasmic reticulum stress may play an early and possibly causal role in disease pathogenesis. Our results therefore indicate that the endoplasmic reticulum stress pathway could potentially be a therapeutic target for spinal and bulbar muscular atrophy and related polyglutamine diseases.

Topics: Age Factors; Androgens; Animals; Anterior Horn Cells; Apoptosis; Cells, Cultured; Dihydrotestosterone; Disease Models, Animal; Embryo, Mammalian; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Female; Gene Expression Regulation; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muscular Disorders, Atrophic; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Spinal Cord; Thapsigargin

The present study evaluated survival effects of N-acetylcysteine (NAC) on cultured corneal endothelial cells exposed to oxidative and endoplasmic reticulum (ER) stress and in a mouse model of early-onset Fuchs endothelial corneal dystrophy (FECD). Cultured bovine corneal endothelial cell viability against oxidative and ER stress was determined by CellTiter-Glo(®) luminescent reagent. Two-month-old homozygous knock-in Col8a2(L450W/L450W) mutant (L450W) and C57/Bl6 wild-type (WT) animals were divided into two groups of 15 mice. Group I received 7 mg/mL NAC in drinking water and Group II received control water for 7 months. Endothelial cell density and morphology were evaluated with confocal microscopy. Antioxidant gene (iNos) and ER stress/unfolded protein response gene (Grp78 and Chop) mRNA levels and protein expression were measured in corneal endothelium by real time PCR and Western blotting. Cell viability of H2O2 and thapsigargin exposed cells pre-treated with NAC was significantly increased compared to untreated controls (p < 0.01). Corneal endothelial cell density (CD) was higher (p = 0.001) and percent polymegathism was lower (p = 0.04) in NAC treated L450W mice than in untreated L450W mice. NAC treated L450W endothelium showed significant upregulation of iNos, whereas Grp78 and Chop were downregulated compared to untreated L450W endothelium by real time PCR and Western blotting. NAC increases survival in cultured corneal endothelial cells exposed against ER and oxidative stress. Systemic NAC ingestion increases corneal endothelial cell survival which is associated with increased antioxidant and decreased ER stress markers in a mouse model of early-onset FECD. Our study presents in vivo evidence of a novel potential medical treatment for FECD.

Topics: Acetylcysteine; Animals; Blotting, Western; Cell Count; Cell Survival; Cells, Cultured; Disease Models, Animal; Endoplasmic Reticulum Chaperone BiP; Endothelium, Corneal; Free Radical Scavengers; Fuchs' Endothelial Dystrophy; Heat-Shock Proteins; Hydrogen Peroxide; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Confocal; Nitric Oxide Synthase Type II; Oxidative Stress; Polymerase Chain Reaction; RNA, Messenger; Thapsigargin; Transcription Factor CHOP

Endoplasmic reticulum (ER) stress occurs as a result of accumulation of unfolded or misfolded proteins in the ER and is involved in the mechanisms of various diseases, such as cancer and neurodegeneration. The goal of the present study was to clarify the relationship between ER stress and pathological neovascularization in the retina. Proliferation and migration of human retinal microvascular endothelial cells (HRMEC) were assessed in the presence of ER stress inducers, such as tunicamycin and thapsigargin. The expression of ER chaperone immunoglobulin heavy-chain binding protein (BiP), known as Grp78, was evaluated by real time RT-PCR, immunostaining, and Western blotting. Tunicamycin or thapsigargin was injected into the intravitreal body of oxygen-induced retinopathy (OIR) model mice at postnatal day 14 (P14) and retinal neovascularization was quantified at P17. The expression and localization of BiP in the retina was also evaluated in the OIR model. Exposure to tunicamycin and thapsigargin increased the proliferation and migration of HRMEC. Tunicamycin enhanced the expression of BiP in HRMEC at both the mRNA level and at the protein level on the cell surface, and increased the formation of a BiP/T-cadherin immunocomplex. In OIR model mice, retinal neovascularization was accelerated by treatments with ER stress inducers. BiP was particularly observed in the pathological vasculature and retinal microvascular endothelial cells, and the increase of BiP expression was correlated with retinal neovascularization. In conclusion, ER stress may contribute to the formation of abnormal vasculature in the retina via BiP complexation with T-cadherin, which then promotes endothelial cell proliferation and migration.

Topics: Animals; Apoptosis; Cadherins; Cell Membrane; Cell Movement; Cell Proliferation; Cell Shape; Disease Models, Animal; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Endothelial Cells; Heat-Shock Proteins; Humans; Mice; Mice, Inbred C57BL; Microvessels; Oxygen; Retinal Neovascularization; RNA, Messenger; Thapsigargin; Transcription Factor CHOP; Transcription, Genetic; Tunicamycin

Lithium previously has been shown to reduce both endoplasmic reticulum (ER) and oxidative stress in other in vitro and in vivo model systems. We investigated lithium's effects on cultured corneal endothelial cells (CECs) exposed to these types of stress and in a mouse model of Fuchs endothelial corneal dystrophy (FECD).. Viability of cultured bovine CECs was determined by CellTiter-Glo. 2-month-old Col8a2(Q455K/Q455K) mutant (Q455K) and C57/Bl6 wild type animals were divided into two groups of 15 mice. Group I received 0.2% lithium carbonate-containing chow and Group II received control chow for 7 months. Confocal microscopy, transmission electron microscopy, real-time PCR (RT-PCR) and western blot were performed.. Pretreatment with lithium increased viability of cultured CECs after H2O2 and thapsigargin exposure compared with untreated controls (p<0.05). In vivo analysis of mouse corneal endothelium showed the following: endothelial cell density of lithium treated Q455K was higher than for untreated Q455K (p<0.01). transmission electron microscopy of lithium treated Q455K showed normal endothelium with enlarged autophagosomes, but untreated Q455K showed dilated ER and guttae. Compared with untreated Q455K endothelium, lithium treated Q455K showed significant upregulation of P62, Tmem74, Tm9sf1 and Tmem166 by RT-PCR and of Atg5-12 conjugate by western blotting indicating that lithium treatment increased autophagy. Although RT-PCR unexpectedly showed increased levels of lithium response genes, caspase 12, Gsk3β, Arrβ2 and Impa1, western blotting showed the expected downregulation of Arrβ2 and Impa1 proteins in response to lithium treatment.. Lithium increases cultured CEC survival against ER and oxidative stress. Increased autophagy in lithium treated endothelium in a mouse model of FECD suggests autophagy may contribute to increased endothelial cell survival.

Topics: Animals; Apoptosis; Blotting, Western; Cattle; Cell Count; Cell Survival; Cells, Cultured; Disease Models, Animal; Endoplasmic Reticulum Chaperone BiP; Endothelium, Corneal; Fuchs' Endothelial Dystrophy; Gene Expression Regulation; Heat-Shock Proteins; Hydrogen Peroxide; Lithium Carbonate; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Confocal; Microscopy, Electron, Transmission; Oxidative Stress; Real-Time Polymerase Chain Reaction; Thapsigargin; Transcription Factor CHOP

The presence of misfolded proteins elicits cellular responses including an endoplasmic reticulum (ER) stress response that may protect cells against the toxic buildup of misfolded proteins. Accumulation of these proteins in excessive amounts, however, overwhelms the "cellular quality control" system and impairs the protective mechanisms designed to promote correct folding and degrade misfolded proteins, ultimately leading to organelle dysfunction and cell death. Studies from multiple laboratories have identified the roles of several ER stress-induced cell death modulators and effectors. Earlier, we reported the role of the small co-chaperone protein p23 in preventing ER stress-induced cell death. p23 undergoes caspase-dependent cleavage to yield a 19-kD product (p19), and mutation of this caspase cleavage site not only blocks the formation of the 19-kD product but also attenuates the ER stress-induced cell death process triggered by various stressors. Thus, a critical question is whether p23 and/or p19 could serve as an in vivo marker for neurodegenerative diseases featuring misfolded proteins and cellular stress. In the present study, we used an antibody that recognizes both p23 and p19 as well as a specific neo-epitope antibody that detects only the p19 fragment. These antibodies were used to detect the presence of both these proteins in cells, primary neurons, brain samples from a mouse model of Alzheimer's disease (AD), and fixed human AD brain samples. While patients with severe AD did display a consistent reduction in p23 levels, our inability to observe p19 in mouse or human AD brain samples suggests that the usefulness of the p23 neo-epitope antibody is restricted to cells and primary neurons undergoing cellular stress.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antibody Specificity; Apoptosis; Brain; Cell Hypoxia; Cells, Cultured; Cytosol; Disease Models, Animal; Endoplasmic Reticulum Stress; Epitopes; Female; Fibroblasts; HEK293 Cells; Humans; Intramolecular Oxidoreductases; Male; Mice; Mice, Transgenic; Microscopy, Fluorescence; Molecular Sequence Data; Neurons; Prostaglandin-E Synthases; Recombinant Fusion Proteins; Thapsigargin; Transfection

Calcium transient triggered firing (CTTF) is induced by large intracellular calcium (Ca(i)) transient and short action potential duration (APD). We hypothesized that CTTF underlies the mechanisms of early afterdepolarization (EAD) and spontaneous recurrent atrial fibrillation (AF) in transgenic (Tx) mice with overexpression of transforming growth factor β1 (TGF-β1).. MHC-TGFcys(33)ser Tx mice develop atrial fibrosis because of elevated levels of TGF-β1. We studied membrane potential and Ca(i)transients of isolated superfused atria from Tx and wild-type (Wt) littermates. Short APD and persistently elevated Ca(i) transients promoted spontaneous repetitive EADs, triggered activity and spontaneous AF after cessation of burst pacing in Tx but not Wt atria (39% vs. 0%, P=0.008). We were able to map optically 4 episodes of spontaneous AF re-initiation. All first and second beats of spontaneous AF originated from the right atrium (4/4, 100%), which is more severely fibrotic than the left atrium. Ryanodine and thapsigargin inhibited spontaneous re-initiation of AF in all 7 Tx atria tested. Western blotting showed no significant changes of calsequestrin or sarco/endoplasmic reticulum Ca(2+)-ATPase 2a.. Spontaneous AF may occur in the Tx atrium because of CTTF, characterized by APD shortening, prolonged Ca(i) transient, EAD and triggered activity. Inhibition of Ca(2+) release from the sarcoplasmic reticulum suppressed spontaneous AF. Our results indicate that CTTF is an important arrhythmogenic mechanism in TGF-β1 Tx atria.

Topics: Action Potentials; Animals; Atrial Fibrillation; Atrial Function; Blotting, Western; Calcium Signaling; Cardiac Pacing, Artificial; Disease Models, Animal; Electrophysiologic Techniques, Cardiac; Enzyme Inhibitors; Fibrosis; Heart Atria; Heart Conduction System; Mice; Mice, Transgenic; Ryanodine; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Time Factors; Transforming Growth Factor beta1; Up-Regulation

To investigate whether and how the endoplasmic reticulum (ER) stress-induced, CCAAT/enhancer-binding protein-homologous protein (CHOP)-mediated pathway regulates myocardial ischemia/reperfusion injury.. Wild-type and chop-deficient mice underwent 50 minutes of left coronary artery occlusion followed by reperfusion. Expression of chop and spliced x-box binding protein-1 (sxbp1) mRNA was rapidly and significantly increased in reperfused myocardium of wild-type mice. chop-deficient mice exhibited markedly reduced injury size after reperfusion compared with wild-type mice, accompanied by a decreasing number of terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cardiomyocytes. Interestingly, myocardial inflammation, as assessed by expression of inflammatory cytokines and chemokines and numbers of infiltrated inflammatory cells, was also attenuated in chop-deficient mice. Moreover, expression of interleukin-6 mRNA in response to lipopolysaccharide was enhanced by simultaneous stimulation with thapsigargin, a potent ER stressor, in wild-type cardiomyocytes but not in chop-deficient cardiomyocytes. Finally, we found that superoxide was produced in reperfused myocardium and that intravenous administration of edaravone, a free radical scavenger, immediately before reperfusion significantly suppressed the superoxide overproduction and subsequent expression of sxbp1 and chop mRNA, followed by reduced injury size in wild-type mice.. The ER stress-induced, CHOP-mediated pathway, which is activated in part by superoxide overproduction after reperfusion, exacerbates myocardial ischemia/reperfusion injury by inducing cardiomyocyte apoptosis and myocardial inflammation.

Topics: Animals; Antipyrine; Apoptosis; Cells, Cultured; Chemokines; Cytokines; Disease Models, Animal; DNA-Binding Proteins; Edaravone; Endoplasmic Reticulum; Free Radical Scavengers; Gene Expression Regulation; Inflammation; Inflammation Mediators; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardial Reperfusion Injury; Myocardium; Regulatory Factor X Transcription Factors; RNA Splicing; RNA, Messenger; Stress, Physiological; Superoxides; Thapsigargin; Time Factors; Transcription Factor CHOP; Transcription Factors

Mast cells play a pivotal role in IgE-mediated allergic responses. Development of specific inhibitors against FcεRI-associated proximal signaling molecules in mast cells may represent a promising therapeutic strategy for allergic diseases. We examined whether a novel synthetic compound, 3-butyl-1-chloro-8-(2-methoxycarbonyl)phenyl-5H-imidazo[1,5-b]isoquinolin-10-one (U63A05), could suppress antigen-stimulated degranulation and cytokine secretion in mast cells and IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. U63A05 reversibly and dose-dependently inhibited degranulation of rat basophilic leukemia (RBL)-2H3 mast cells and bone marrow-derived mast cells (BMMCs) stimulated by antigen (IC(50) values for RBL-2H3 and BMMCs were 4.1 and 4.8 µM, respectively). The secretion of inflammatory cytokines was also suppressed in antigen-stimulated mast cells. However, degranulation by thapsigargin, a typical calcium inducer, was not inhibited by U63A05. U63A05 exerts its inhibitory effect, to the same extent as in degranulation, on the activating phosphorylation of Syk and downstream signaling molecules, including LAT and SLP-76. Further downstream, the activating phosphorylations of Akt, Erk1/2, p38, and JNK were also inhibited. Finally, antigen-stimulated PCA was dose-dependently suppressed in mice (ED(50), 26.3 mg/kg). Taken together, the results suggest that U63A05 suppresses the activation of mast cells and the mast cell-mediated allergic response through the inhibition of Syk activation in mast cells.

Topics: Anaphylaxis; Animals; Cell Degranulation; Cell Line; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Drug Interactions; Imidazoles; Immunoglobulin E; Intracellular Signaling Peptides and Proteins; Isoquinolines; Male; Mast Cells; Mice; Mice, Inbred BALB C; Passive Cutaneous Anaphylaxis; Phosphorylation; Protein-Tyrosine Kinases; Rats; Signal Transduction; Syk Kinase; Thapsigargin

The aim of this study was to determine the reasons why the intravitreal level of extracellular-superoxide dismutase (EC-SOD) increases in proliferative diabetic retinopathy patients by the investigation of two possibilities: first, change of EC-SOD expression in the retina; and secondly, leakage of EC-SOD through the endothelial monolayer by the treatment with endoplasmic reticulum (ER) stress inducers because ER stress is known to be involved in the vascular impairment in diabetic retinopathy. Intravitreous injection of tunicamycin in mice increased the permeability of tracer dye across retinal blood vessels while the retinal EC-SOD mRNA level was not changed. The leakage of EC-SOD through the retinal endothelial cell layer was elevated by the treatment with thapsigargin or tunicamycin. The expression of claudin-5 was significantly decreased by the treatment with the ER stress inducers. These phenomena were significantly suppressed by the pre-treatment of endothelial cells with a chemical chaperone 4-phenylbutyric acid. Our observations suggest that ER stress leads to the down-regulation of claudin-5 among tight junction proteins and may induce the elevation of endothelial permeability and leakage of EC-SOD into the vitreous body.

Topics: Animals; Cell Line; Cell Membrane Permeability; Claudin-5; Claudins; Diabetic Retinopathy; Disease Models, Animal; Endoplasmic Reticulum; Endothelium; Intravitreal Injections; Male; Mice; Rats; Retina; Superoxide Dismutase; Thapsigargin; Tunicamycin; Vitreous Body

ER stress triggers myocardial contractile dysfunction although the underlying mechanism is still elusive. Given that NADPH oxidase was recently implicated in ER stress-induced tissue injury, this study was designed to examine the role of NADPH oxidase in ER stress-induced cardiac mechanical defects and the impact of Akt activation on ER stress-induced cardiac anomalies. Wild-type and transgenic mice with cardiac-specific overexpression of an active mutant of Akt (MyAkt) were subjected to the ER stress inducer thapsigargin (1 and 3mg/kg, ip, for 48h). Thapsigargin compromised echocardiographic parameters, including elevating LVESD and reducing fractional shortening; suppressed cardiomyocyte contractile function, intracellular Ca(2+) handling, and cell survival; and enhanced carbonyl formation, apoptosis, superoxide production, NADPH oxidase expression, and mitochondrial damage. Interestingly, these anomalies were attenuated or mitigated by chronic Akt activation. Treatment with thapsigargin also dephosphorylated Akt and its downstream signal GSK3β (leading to activation of GSK3β), the effect of which was abrogated in MyAkt hearts. Knockdown of the cytosolic subunit of NADPH oxidase, p47(phox), using siRNA abrogated thapsigargin-induced apoptosis and cell death in H9C2 myoblasts. In vitro exposure to thapsigargin induced murine cardiomyocyte dysfunction reminiscent of the in vivo setting, the effects of which were ablated by the NADPH oxidase inhibitor apocynin and the mitochondrial Ca(2+) uptake inhibitor Ru360. In addition, apocynin abrogated thapsigargin-induced loss of mitochondrial membrane potential and permeability transition pore opening, similar to chronic Akt activation. In summary, these data suggest that ER stress interrupts cardiac contractile and intracellular Ca(2+) homeostasis, cell survival, and mitochondrial integrity through an Akt dephosphorylation- and NADPH oxidase-dependent mechanism.

Topics: Animals; Apoptosis; Calcium; Cell Survival; Cells, Cultured; Disease Models, Animal; Endoplasmic Reticulum Stress; Male; Membrane Potential, Mitochondrial; Mice; Mice, Transgenic; Mitochondria; Myocardial Contraction; Myocytes, Cardiac; NADPH Oxidases; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Superoxides; Thapsigargin

Spinal cord injury (SCI) triggers inflammatory responses that involve neutrophils, macrophages/microglia and astrocytes and molecules that potentially cause secondary tissue damage and functional impairment. Here, we assessed the contribution of the calcium-dependent K⁺ channel KCNN4 (KCa3.1, IK1, SK4) to secondary damage after moderate contusion lesions in the lower thoracic spinal cord of adult mice. Changes in KCNN4 mRNA levels (RT-PCR), KCa3.1 protein expression (Western blots), and cellular expression (immunofluorescence) in the mouse spinal cord were monitored between 1 and 28 d after SCI. KCNN4 mRNA and KCa3.1 protein rapidly increased after SCI; double labeling identified astrocytes as the main cellular source accounting for this upregulation. Locomotor function after SCI, evaluated for 28 d in an open-field test using the Basso Mouse Scale, was improved in a dose-dependent manner by treating mice with a selective inhibitor of KCa3.1 channels, TRAM-34 (triarylmethane-34). Improved locomotor function was accompanied by reduced tissue loss at 28 d and increased neuron and axon sparing. The rescue of tissue by TRAM-34 treatment was preceded by reduced expression of the proinflammatory mediators, tumor necrosis factor-α and interleukin-1β in spinal cord tissue at 12 h after injury, and reduced expression of inducible nitric oxide synthase at 7 d after SCI. In astrocytes in vitro, TRAM-34 inhibited Ca²⁺ signaling in response to metabotropic purinergic receptor stimulation. These results suggest that blocking the KCa3.1 channel could be a potential therapeutic approach for treating secondary damage after spinal cord injury.

Topics: Analysis of Variance; Animals; Animals, Newborn; Calcium; Calcium Signaling; CD11b Antigen; Cells, Cultured; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Intermediate-Conductance Calcium-Activated Potassium Channels; Macrophages; Mice; Mice, Inbred C57BL; Microglia; Motor Activity; Nerve Tissue Proteins; Nitric Oxide Synthase Type II; Potassium Channel Blockers; Pyrazoles; RNA, Messenger; Spinal Cord Injuries; Thapsigargin; Time Factors; Up-Regulation; Uridine Triphosphate

Astrocytes are essential cells for maintaining brain integrity. We have recently shown that the transcription factor C/EBP homologous protein (CHOP), associated with endoplasmic reticulum (ER) stress, plays a key role in the astrocyte death induced by ischemia. Meanwhile, mediators of apoptosis downstream of CHOP in the ER stress-dependent pathway remain to be elucidated. Our aim in this work was to determine whether caspase-11, able to activate apoptotic and proinflammatory pathways, is implicated in ER stress-dependent astrocyte death in ischemic conditions. According to our results, caspase-11 is up-regulated in primary astrocyte cultures following either oxygen and glucose deprivation (OGD) or treatment with the ER-stress inducers thapsigargin and tunicamycin. Moreover, these same stimuli increased caspase-11 mRNA levels and luciferase activity driven by a caspase-11 promoter, indicating that caspase-11 is regulated at the transcriptional level. Our data also illustrate the involvement of ER stress-associated CHOP in caspase-11 regulation, insofar as CHOP overexpression by means of an adenoviral vector caused a significant raise in caspase-11. In turn, caspase-11 suppression with siRNA rescued astrocytes from OGD- and ER stress-induced death, supporting the idea that caspase-11 is responsible for the deleterious effects of ischemia on astrocytes. Finally, inhibition of caspase-1 and caspase-3 significantly reduced astrocyte death, which indicates that these proteases act as death effectors of caspase-11. In conclusion, our work contributes to clarifying the pathways leading to astrocyte death in response to ischemia by defining caspase-11 as a key mediator of the ER stress response acting downstream of CHOP.

Topics: Animals; Apoptosis; Astrocytes; Brain Ischemia; Caspase Inhibitors; Caspases; Cells, Cultured; Disease Models, Animal; Endoplasmic Reticulum; Genetic Vectors; Rats; Rats, Sprague-Dawley; RNA Interference; RNA, Messenger; Signal Transduction; Stress, Physiological; Thapsigargin; Transcription Factor CHOP; Transfection; Tunicamycin

Continuous turnover of intracellular components by autophagy is necessary to preserve cellular homeostasis in all tissues. Alterations in macroautophagy, the main process responsible for bulk autophagic degradation, have been proposed to contribute to pathogenesis in Huntington's disease (HD), a genetic neurodegenerative disorder caused by an expanded polyglutamine tract in the huntingtin protein. However, the precise mechanism behind macroautophagy malfunction in HD is poorly understood. In this work, using cellular and mouse models of HD and cells from humans with HD, we have identified a primary defect in the ability of autophagic vacuoles to recognize cytosolic cargo in HD cells. Autophagic vacuoles form at normal or even enhanced rates in HD cells and are adequately eliminated by lysosomes, but they fail to efficiently trap cytosolic cargo in their lumen. We propose that inefficient engulfment of cytosolic components by autophagosomes is responsible for their slower turnover, functional decay and accumulation inside HD cells.

Topics: Animals; Apoptosis; Autophagy; Cells, Cultured; Disease Models, Animal; Enzyme Inhibitors; Hepatocytes; Humans; Huntington Disease; Immunosuppressive Agents; Lysosomes; Mice; Mice, Transgenic; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; Mitochondria; Nerve Tissue Proteins; Neurons; Peptides; Serotonin Plasma Membrane Transport Proteins; Serum; Sirolimus; Subcellular Fractions; Thapsigargin; Time Factors; Vinca Alkaloids

Accumulating evidence has pointed that a variety of lipids could exert their beneficial actions against dementia including Alzheimer disease and age-related cognitive decline via diverse signaling pathways. Endoplasmic reticulum (ER) stress-induced neuronal apoptosis, on the other hand, is a critical factor for pathogenesis of neurodegenerative diseases such as Alzheimer disease and Parkinson disease, senile dementia, and ischemic neuronal damage. The present study examined the effects of 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLPhtEtn), a phospholipid, on ER stress-induced neuronal death and age-related cognitive disorders.. PC-12 cell viability was assayed before and after treatment with amyloid-β(1-40) peptide or thapsigargin in the presence and absence of DLPhtEtn. A series of behavioral tests were performed for senescence-accelerated mouse-prone 8 (SAMP8) mice after 7-month oral administration with polyethylene glycol (PEG) or DLPhtEtn and then, the number of hippocampal neurons was counted.. Amyloid-β(1-40) peptide or thapsigargin is capable of causing ER stress-induced apoptosis. DLPhtEtn (30 μM) significantly inhibited PC-12 cell death induced by amyloid-β(1-40) peptide or thapsigargin. In the water maze test, oral administration with DLPhtEtn (1 mg/kg) for 7 months (three times a week) significantly shortened the prolonged retention latency for SAMP8 mice. In contrast, DLPhtEtn had no effect on the acquisition and retention latencies in both the open field test and the passive avoidance test for SAMP8 mice. Oral administration with DLPhtEtn (1 mg/kg) for 7 months prevented a decrease in the number of hippocampal neurons for SAMP8 mice.. The results of the present study show that DLPhtEtn ameliorates age-related spatial memory decline without affecting motor activities or fear memory, possibly by protecting hippocampal neuronal death. DLPhtEtn, thus, might exert its beneficial action against senile dementia and neurodegenerative diseases such as Alzheimer disease.

Topics: Age Factors; Amyloid beta-Peptides; Animals; Cell Death; Cell Survival; Disease Models, Animal; Drug Administration Schedule; Hippocampus; Male; Memory Disorders; Mice; Mice, Inbred Strains; Neurons; PC12 Cells; Peptide Fragments; Phosphatidylethanolamines; Rats; Thapsigargin

Disturbances in the regulation of cytosolic calcium (Ca(2+)) concentration play a key role in the vascular dysfunction associated with arterial hypertension. Stromal interaction molecules (STIMs) and Orai proteins represent a novel mechanism to control store-operated Ca(2+) entry. Although STIMs act as Ca(2+) sensors for the intracellular Ca(2+) stores, Orai is the putative pore-forming component of Ca(2+) release-activated Ca(2+) channels at the plasma membrane. We hypothesized that augmented activation of Ca(2+) release-activated Ca(2+)/Orai-1, through enhanced activity of STIM-1, plays a role in increased basal tonus and vascular reactivity in hypertensive animals. Endothelium-denuded aortic rings from Wistar-Kyoto and stroke-prone spontaneously hypertensive rats were used to evaluate contractions because of Ca(2+) influx. Depletion of intracellular Ca(2+) stores, which induces Ca(2+) release-activated Ca(2+) activation, was performed by placing arteries in Ca(2+) free-EGTA buffer. The addition of the Ca(2+) regular buffer produced greater contractions in aortas from stroke-prone spontaneously hypertensive rats versus Wistar-Kyoto rats. Thapsigargin (10 micromol/L), an inhibitor of the sarcoplasmic reticulum Ca(2+) ATPase, further increased these contractions, especially in stroke-prone spontaneously hypertensive rat aorta. Addition of the Ca(2+) release-activated Ca(2+) channel inhibitors 2-aminoethoxydiphenyl borate (100 micromol/L) or gadolinium (100 micromol/L), as well as neutralizing antibodies to STIM-1 or Orai-1, abolished thapsigargin-increased contraction and the differences in spontaneous tone between the groups. Expression of Orai-1 and STIM-1 proteins was increased in aorta from stroke-prone spontaneously hypertensive rats when compared with Wistar-Kyoto rats. These results support the hypothesis that both Orai-1 and STIM-1 contribute to abnormal vascular function in hypertension. Augmented activation of STIM-1/Orai-1 may represent the mechanism that leads to impaired control of intracellular Ca(2+) levels in hypertension.

Topics: Animals; Aorta; Blood Pressure; Body Weight; Calcium; Calcium Channels; Cytosol; Disease Models, Animal; Enzyme Inhibitors; Homeostasis; Hypertension; Male; Membrane Glycoproteins; Muscle Contraction; ORAI1 Protein; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Stromal Interaction Molecule 1; Thapsigargin

Hypoxia, ischaemia, and exogenous chemicals can induce extracellular and intracellular acidosis, but it is not clear which of these types of acidosis affects endothelial cell function. The synthesis and release of endothelium-derived relaxing factors (EDRFs) are linked to an increase in cytosolic Ca(2+) concentration, and we therefore examined the effects of extracellular and intracellular acidosis on Ca(2+) responses and EDRF production in cultured porcine aortic endothelial cells.. Cytosolic pH (pH(i)) and Ca(2+) were measured using fluorescent dyes, BCECM/AM (pH-indicator) and fura-2/AM (Ca(2+)-indicator), respectively. EDRFs, nitric oxide (NO) and prostaglandin I(2) (PGI(2)) were assessed using DAF-FM/DA (NO-indicator dye) fluorometry and 6-keto PGF(1alpha) enzyme immunoassay, respectively. HEPES buffers titrated to pH 6.4, 6.9, and 7.4 were used to alter extracellular pH (pH(o)), and propionate (20 mmol/L) was applied to cause intracellular acidosis. Extracellular acidosis strongly suppressed bradykinin (BK, 10 nmol/L)- and thapsigargin (TG, 1 micromol/L)-induced Ca(2+) responses by 30 and 23% at pH(o) 6.9, and by 80 and 97% at pH(o) 6.4, respectively. During the examinations, there were no significant differences in pH(i) among the three groups at pH(o) 7.4, 6.9, and 6.4. Extracellular acidosis also inhibited BK-stimulated PGI(2) production by 55% at pH(o) 6.9 and by 77% at pH(o) 6.4, and NO production by 38% at pH(o) 6.9 and by 91% at pH(o) 6.4. The suppressive effects of extracellular acidosis on Ca(2+) responses and NO production were reversible. Propionate changed pH(i) from 7.3 to 6.9, without altering pH(o) (7.4). Intracellular acidosis had no effect on BK- and TG-induced Ca(2+) responses or NO production.. These results indicate that extracellular, but not intracellular, acidosis causes endothelial dysfunction by inhibiting store-operated Ca(2+) entry, so helping to clarify the vascular pathophysiology of conditions such as ischaemia, hypoxia, acidosis, and ischaemia-reperfusion.

Topics: Acidosis; Animals; Aorta; Bradykinin; Calcium; Cells, Cultured; Disease Models, Animal; Endothelium-Dependent Relaxing Factors; Endothelium, Vascular; Epoprostenol; Hydrogen-Ion Concentration; Ion Channels; Nitric Oxide; Swine; Thapsigargin

Mutations in leukocyte NADPH oxidase genes lead to defective respiratory burst in leukocytes and cause chronic granulomatous diseases (CGD) in humans. The most common form of CGD is caused by mutations in the membrane-bound oxidase component gp91phox, which is encoded by the CYBB gene on the X chromosome. We previously reported on a patient with a CYBB mutation (H338Y) that prevents the intracellular trafficking and expression of gp91phox on leukocytes. The capacity of the leukocytes to produce reactive oxygen species (ROS) was rescued by treatment with thapsigargin and flavin adenine dinucleotide (FAD). The increase in ROS production was not due to the increase in cytoplasmic calcium induced by thapsigargin because the treatment of calcium ionophore did not have the same effect. Protein and cellular analyses on leukocytes and cells transfected with GFP-tagged gp91phox mutant showed that treated cells expressed more Endo H-resistant gp91phox protein on the cell surface and are more effective in killing bacteria. Thapsigargin- and FAD-treated CGD leukocytes had enhanced activity in protecting mice from Staphylococcus-induced peritoneal abscess formation in a mouse model of CGD. These results indicate that thapsigargin-FAD ex vivo treatment is effective in rescuing the ROS-producing activity of leukocytes in selected CGD patients.

Topics: Animals; Cytochrome b Group; Disease Models, Animal; Female; Flavin-Adenine Dinucleotide; Granulomatous Disease, Chronic; Humans; Leukocytes; Membrane Glycoproteins; Mice; Mice, Knockout; NADPH Oxidase 2; NADPH Oxidases; Reactive Oxygen Species; Staphylococcus aureus; Thapsigargin

Disablement of cell death programs in cancer cells contributes to drug resistance and in some cases has been associated with altered translational control. As eukaryotic translation initiation factor 4E (eIF4E) cooperates with c-Myc during lymphomagenesis, induces drug resistance, and is a genetic modifier of the rapamycin response, we have investigated the effect of dysregulation of the ribosome recruitment phase of translation initiation on tumor progression and chemosensitivity. eIF4E is a subunit of eIF4F, a complex that stimulates ribosome recruitment during translation initiation by delivering the DEAD-box RNA helicase eIF4A to the 5' end of mRNAs. eIF4A is thought to prepare a ribosome landing pad on mRNA templates for incoming 40S ribosomes (and associated factors). Using small molecule screening, we found that cyclopenta[b]benzofuran flavaglines, a class of natural products, modulate eIF4A activity and inhibit translation initiation. One member of this class of compounds, silvestrol, was able to enhance chemosensitivity in a mouse lymphoma model in which carcinogenesis is driven by phosphatase and tensin homolog (PTEN) inactivation or elevated eIF4E levels. These results establish that targeting translation initiation can restore drug sensitivity in vivo and provide an approach to modulating chemosensitivity.

Topics: Animals; Apoptosis; Benzofurans; Cell Line; Cell Line, Tumor; Disease Models, Animal; Doxorubicin; Drug Resistance, Neoplasm; Drug Synergism; Eukaryotic Initiation Factor-4A; Eukaryotic Initiation Factor-4E; Female; HeLa Cells; Humans; Lymphoma; Mice; Mice, Inbred C57BL; Peptide Chain Initiation, Translational; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Polyribosomes; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Sirolimus; Thapsigargin; Triterpenes

A strong premature electrical stimulus (S(2)) induces both virtual anodes and virtual cathodes. The effects of virtual electrodes on intracellular Ca(2+) concentration ([Ca(2+)](i)) transients and ventricular fibrillation thresholds (VFTs) are unclear. We studied 16 isolated, Langendorff-perfused rabbit hearts with simultaneous voltage and [Ca(2+)](i) optical mapping and for vulnerable window determination. After baseline pacing (S(1)), a monophasic (10 ms anodal or cathodal) or biphasic (5 ms-5 ms) S(2) was applied to the left ventricular epicardium. Virtual electrode polarizations and [Ca(2+)](i) varied depending on the S(2) polarity. Relative to the level of [Ca(2+)](i) during the S(1) beat, the [Ca(2+)](i) level 40 ms after the onset of monophasic S(2) increased by 36+/-8% at virtual anodes and 20+/-5% at virtual cathodes (P<0.01), compared with 25+/-5% at both virtual cathode-anode and anode-cathode sites for biphasic S(2). The VFT was significantly higher and the vulnerable window significantly narrower for biphasic S(2) than for either anodal or cathodal S(2) (n=7, P<0.01). Treatment with thapsigargin and ryanodine (n=6) significantly prolonged the action potential duration compared with control (255+/-22 vs. 189+/-6 ms, P<0.05) and eliminated the difference in VFT between monophasic and biphasic S(2), although VFT was lower for both cases. We conclude that virtual anodes caused a greater increase in [Ca(2+)](i) than virtual cathodes. Monophasic S(2) is associated with lower VFT than biphasic S(2), but this difference was eliminated by the inhibition of the sarcoplasmic reticulum function and the prolongation of the action potential duration. However, the inhibition of the sarcoplasmic reticulum function also reduced VFT, indicating that the [Ca(2+)](i) dynamics modulate, but are not essential, to ventricular vulnerability.

Topics: Action Potentials; Animals; Calcium Signaling; Disease Models, Animal; Electric Stimulation; Enzyme Inhibitors; Heart Ventricles; Myocytes, Cardiac; Perfusion; Pericardium; Rabbits; Ryanodine; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Time Factors; Ventricular Fibrillation

Returning to normal pH after acidosis, similar to reperfusion after ischemia, is prone to arrhythmias. The type and mechanisms of these arrhythmias have never been explored and were the aim of the present work. Langendorff-perfused rat/mice hearts and rat-isolated myocytes were subjected to respiratory acidosis and then returned to normal pH. Monophasic action potentials and left ventricular developed pressure were recorded. The removal of acidosis provoked ectopic beats that were blunted by 1 muM of the CaMKII inhibitor KN-93, 1 muM thapsigargin, to inhibit sarcoplasmic reticulum (SR) Ca(2+) uptake, and 30 nM ryanodine or 45 muM dantrolene, to inhibit SR Ca(2+) release and were not observed in a transgenic mouse model with inhibition of CaMKII targeted to the SR. Acidosis increased the phosphorylation of Thr(17) site of phospholamban (PT-PLN) and SR Ca(2+) load. Both effects were precluded by KN-93. The return to normal pH was associated with an increase in SR Ca(2+) leak, when compared with that of control or with acidosis at the same SR Ca(2+) content. Ca(2+) leak occurred without changes in the phosphorylation of ryanodine receptors type 2 (RyR2) and was blunted by KN-93. Experiments in planar lipid bilayers confirmed the reversible inhibitory effect of acidosis on RyR2. Ectopic activity was triggered by membrane depolarizations (delayed afterdepolarizations), primarily occurring in epicardium and were prevented by KN-93. The results reveal that arrhythmias after acidosis are dependent on CaMKII activation and are associated with an increase in SR Ca(2+) load, which appears to be mainly due to the increase in PT-PLN.

Topics: Acidosis; Action Potentials; Animals; Arrhythmias, Cardiac; Benzylamines; Calcium; Calcium-Binding Proteins; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Dantrolene; Disease Models, Animal; Enzyme Inhibitors; Hydrogen-Ion Concentration; Male; Mice; Mice, Transgenic; Myocytes, Cardiac; Peptides; Phosphorylation; Rats; Rats, Wistar; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sulfonamides; Thapsigargin; Time Factors; Ventricular Function, Left; Ventricular Pressure

Accumulating evidence suggests a pathophysiologic role of endoplasmic reticulum (ER) stress in kidney disease. This study investigated the potential of therapeutic approaches targeting ER stress in the anti-Thy1 model of mesangioproliferative glomerulonephritis in rats. Immunohistochemistry and Western blotting showed a time-dependent increase in the expression of the ER stress-inducible chaperones glucose-regulated protein 78 (GRP78) and oxygen-related protein 150 in isolated glomeruli, especially in the glomerular epithelial cells and mesangial cells, after induction of anti-Thy1 nephritis. For evaluation of whether preconditioning with ER stress ameliorates the severity of disease, rats were pretreated with a subnephritogenic dose of the ER stress inducer tunicamycin or thapsigargin for 4 d before disease was induced. Although preconditioning with ER stress had no effect on the degree of disease induction, it strongly ameliorated the manifestations of disease, evidenced by marked reductions in microaneurysm formation, mesangial proliferation, and adhesion of Bowman's capsule to the glomerular tuft. This improvement in histologic damage was associated with reduced proteinuria (39.4 +/- 10.5 versus 126.1 +/- 18.1 mg/d; P < 0.01) and with attenuated increases in glucose-regulated protein 78 and oxygen-related protein 150 expression. Of note, pretreatment with tunicamycin or thapsigargin decreased the excessive ER stress-induced intracellular signaling observed in anti-Thy1 nephritis. In conclusion, preconditioning with ER stress ameliorates the severity of disease in rats with anti-Thy1 nephritis. These findings suggest the possibility of therapeutic approaches targeting ER stress in mesangioproliferative glomerulonephritis.

Topics: Animals; Anti-Bacterial Agents; Disease Models, Animal; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Glomerulonephritis, Membranoproliferative; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Ischemic Preconditioning; Isoantibodies; Kidney; Male; Mice; Mice, Inbred BALB C; Molecular Chaperones; Oxidative Stress; Proteins; Rats; Rats, Wistar; Signal Transduction; Thapsigargin; Tunicamycin

Impaired Ca2+ homeostasis and smooth muscle contractility co-exist in acute cholecystitis (AC) leading to gallbladder dysfunction. There is no pharmacological treatment for this pathological condition. Our aim was to evaluate the effects of melatonin treatment on Ca2+ signaling pathways and contractility altered by cholecystitis. [Ca2+]i was determined by epifluorescence microscopy in fura-2 loaded isolated gallbladder smooth muscle cells, and isometric tension was recorded from gallbladder muscle strips. Malondialdehyde (MDA) and reduced glutathione (GSH) contents were determined by spectrophotometry and cycloxygenase-2 (COX-2) expression was quantified by western blot. Melatonin was tested in two experimental groups, one of which underwent common bile duct ligation for 2 days and another that was later de-ligated for 2 days. Inflammation-induced impairment of Ca2+ responses to cholecystokinin and caffeine were recovered by melatonin treatment (30 mg/kg). This treatment also ameliorated the detrimental effects of AC on Ca2+ influx through both L-type and capacitative Ca2+ channels, and it was effective in preserving the pharmacological phenotype of these channels. Despite its effects on Ca2+ homeostasis, melatonin did not improve contractility. After de-ligation, Ca2+ influx and contractility were still impaired, but both were recovered by melatonin. These effects of melatonin were associated to a reduction of MDA levels, an increase in GSH content and a decrease in COX-2 expression. These findings indicate that melatonin restores Ca2+ homeostasis during AC and resolves inflammation. In addition, this indoleamine helps in the subsequent recovery of functionality.

Topics: Acalculous Cholecystitis; Animals; Caffeine; Calcium; Cholecystitis, Acute; Common Bile Duct; Disease Models, Animal; Gallbladder; Guinea Pigs; Homeostasis; Ligation; Lipid Peroxidation; Male; Melatonin; Muscle Contraction; Nitrendipine; Pinacidil; Thapsigargin

Chromium has gained popularity as a nutritional supplement for diabetic patients. This study evaluated the effect of chronic administration of a chromium complex of D-phenylalanine (Cr(D-phe)(3)) on glucose and insulin tolerance in obese mice. The study tested the hypothesis that Cr(D-phe)(3) suppresses endoplasmic reticulum (ER) stress and insulin resistance in these animals.. C57BL lean and ob/ob obese mice were randomly divided to orally receive vehicle or Cr(D-phe)(3) (3.8 mug of elemental chromium/kg/day) for 6 months. Insulin sensitivity was evaluated by glucose and insulin tolerance tests. Protein levels of phosphorylated pancreatic ER kinase (PERK), alpha subunit of translation initiation factor 2 (eIF2alpha) and inositol-requiring enzyme-1 (IRE-1), p-c-Jun, and insulin receptor substrate-1 (IRS-1) phosphoserine-307 were assessed by western blotting. In vitro ER stress was induced by treating cultured muscle cells with thapsigargin in the presence or absence of Cr(D-phe)(3).. ob/ob mice showed poor glucose and insulin tolerance compared to the lean controls, which was attenuated by Cr(D-phe)(3). Markers of insulin resistance (phospho-c-Jun and IRS-1 phosphoserine) and ER stress (p-PERK, p-IRE-1, p-eIF2alpha), which were elevated in ob/ob mice, were attenuated following Cr(D-phe)(3) treatment. Chromium treatment was also associated with a reduction in liver triglyceride levels and lipid accumulation. In cultured myotubes, Cr(D-phe)(3) attenuated ER stress induced by thapsigargin.. Oral Cr(D-phe)(3) treatment reduces glucose intolerance, insulin resistance, and hepatic ER stress in obese, insulin-resistant mice.

Topics: Animals; Blood Glucose; Chromium; Diabetes Mellitus, Type 2; Disease Models, Animal; eIF-2 Kinase; Endoplasmic Reticulum; Glucose Intolerance; Insulin; Insulin Resistance; Leptin; Lipids; Liver; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Obese; Obesity; Protein Serine-Threonine Kinases; Thapsigargin; Trace Elements

The flat or negative force frequency relationship (FFR) is a hallmark of the failing heart. Either decreases in SERCA2a expression, increases in Na(+)/Ca(2+) exchanger (NCX) expression or elevated Na(+)(i) have been independently proposed as mediators of the negative FFR.. To determine whether each one of these mechanisms is sufficient to account for the negative FFR of the failing heart or on the contrary, various mechanisms, acting in concert are required. SERCA2a was pharmacologically inhibited with thapsigargin (TG) or cyclopiazonic acid (CPA) or by using siRNA technology; Na(+)(i) was increased with either ouabain (Oua) or monensin and NCX protein was overexpressed by gene transfer (Ad.NCX), to mimic in nonfailing cat myocytes the phenotype of the failing heart and examine their effect on the FFR. The positive FFR of healthy myocytes remained unaffected after either SERCA2a inhibition, Na(+)(i) elevation, or NCX overexpression. However, the combination of TG + Oua, Oua + Ad.NCX, or TG + Ad.NCX, converted the positive FFR to negative. Moreover, the FFR became negative at lower frequencies, when the 3 interventions were combined.. Ca(2+) handling has to be altered at several levels to explain the negative FFR of the failing heart. These anomalies in Ca(2+) homeostasis acting in synergy have additive effects.

Topics: Animals; Blotting, Western; Calcium; Calcium-Transporting ATPases; Cats; Cells, Cultured; Disease Models, Animal; Enzyme Inhibitors; Fluorescent Dyes; Gene Expression; Heart Failure; Indoles; Intracellular Fluid; Myocardium; Myocytes, Cardiac; RNA, Small Interfering; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sodium; Thapsigargin

In acute respiratory distress syndrome, pulmonary vascular permeability increases, causing intravascular fluid and protein to move into the lung's interstitium. The classic model describing the formation of pulmonary edema suggests that fluid crossing the capillary endothelium is drawn by negative interstitial pressure into the potential space surrounding extra-alveolar vessels and, as interstitial pressure builds, is forced into the alveolar air space. However, the validity of this model is challenged by animal models of acute lung injury in which extra-alveolar vessels are more permeable than capillaries under a variety of conditions. In the current study, we sought to determine whether extravascular fluid accumulation can be produced because of increased permeability of either the capillary or extra-alveolar endothelium, and whether different pathophysiology results from such site-specific increases in permeability.. We perfused isolated lungs with either the plant alkaloid thapsigargin, which increases extra-alveolar endothelial permeability, or with 4alpha-phorbol 12, 13-didecanoate, which increases capillary endothelial permeability.. Both treatments produced equal increases in whole lung vascular permeability, but caused fluid accumulations in separate anatomical compartments. Light microscopy of isolated lungs showed that thapsigargin caused fluid cuffing of large vessels, while 4alpha-phorbol 12, 13-didecanoate caused alveolar flooding. Dynamic compliance was reduced in lungs with cuffing of large vessels, but not in lungs with alveolar flooding.. Phenotypic differences between vascular segments resulted in site-specific increases in permeability, which have different pathophysiological outcomes. Our findings suggest that insults leading to acute respiratory distress syndrome may increase permeability in extra-alveolar or capillary vascular segments, resulting in different pathophysiological sequela.

Topics: Animals; Carcinogens; Cell Membrane Permeability; Disease Models, Animal; Endothelial Cells; Enzyme Inhibitors; Extravascular Lung Water; Lung; Lung Compliance; Male; Phorbol Esters; Pulmonary Alveoli; Pulmonary Edema; Rats; Rats, Sprague-Dawley; Respiratory Distress Syndrome; Respiratory Mechanics; Thapsigargin

Studies were performed to see if alterations in Ca2+ homeostasis underlie the gastrointestinal motility complications seen in many diabetic patients. Experiments were performed on colonic and ileal tissues taken from streptozotocin-induced diabetic and control rats. Diabetes caused alterations in the responses of the tissues to Ca2+ manipulation but these differed between the colon and ileum. In the colon a small but not significant increase in contractile responses to CaCl2 was observed in diabetic tissues, whereas the responses of the ileum were depressed relative to those of the controls. In contrast, responses of the diabetic ileum to the Ca2+ channel agonist Bay K8644 were greater than those of the controls, whilst the agonist failed to contract the colon. Similarly, the Ca2+-ATPase inhibitors, thapsigargin and cyclopiazonic acid, produced contractions which were greater in diabetic ileal tissues. Thus, alterations in the responses of the diabetic gut to Ca2+ manipulation are complex, and also tissue-specific.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcimycin; Calcium Chloride; Calcium Signaling; Calcium-Transporting ATPases; Colon, Ascending; Diabetes Mellitus, Experimental; Disease Models, Animal; Dose-Response Relationship, Drug; Humans; Ileum; Indoles; Injections, Intraperitoneal; Male; Muscle Contraction; Muscle, Smooth; Nifedipine; Rats; Rats, Wistar; Streptozocin; Thapsigargin; Time Factors

In chronic heart failure, the lung endothelial permeability response to angiotensin II or thapsigargin-induced store depletion is ablated, although the mechanisms are not understood.. To determine whether the ablated permeability response to store depletion during heart failure was due to impaired expression of store operated Ca2+ channels in lung endothelium.. Heart failure was induced by aortocaval fistula in rats. Permeability was measured in isolated lungs using the filtration coefficient and a low Ca2+/Ca2+ add-back strategy to identify the component of the permeability response dependent on Ca2+ entry.. In fistulas, right ventricular mass and left ventricular end diastolic pressure were increased and left ventricular shortening fraction decreased compared with shams. Thapsigargin-induced store depletion increased lung endothelial permeability in shams, but not in fistulas. Permeability increased in both groups after the Ca2+ ionophore A23187 or 14,15-epoxyeicosatrienoic acid, independent of store depletion. A diacylglycerol analog had no impact on permeability. Increased distance between the endoplasmic reticulum and the plasmalemmal membrane was ruled out as a mechanism for the loss of the permeability response to store depletion. Endothelial expression of the endoplasmic reticulum Ca2+ ATPase was not altered in fistulas compared with shams, whereas the store-operated canonical transient receptor potential channels 1, 3, and 4 were downregulated in extraalveolar vessel endothelium.. We conclude that the adaptive mechanism limiting store depletion-induced endothelial lung injury in the aortocaval model of heart failure involves downregulation of store-operated Ca2+ channels.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Calcimycin; Calcium Channels; Calcium-Transporting ATPases; Disease Models, Animal; Endothelium; Enzyme Inhibitors; Heart Failure; Ionophores; Lung; Permeability; Rats; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Tissue Culture Techniques; Vasodilator Agents

Activation of spinal group I metabotropic glutamate receptors (mGluRs) may have antinociceptive or pro-nociceptive effects in different pain models. Pharmacological activation of group I mGluRs leads to long-term depression (LTD) of synaptic strength between Adelta-fibers and neurons in lamina II of spinal dorsal horn of the rat. Here, we studied the signal transduction pathways involved. Synaptic strength between Adelta-fibers and lamina II neurons was assessed by perforated whole-cell patch-clamp recordings in a spinal cord-dorsal root slice preparation of young rats. Bath application of the specific group I mGluR agonist (S)-3,5-dihydroxyphenylglycine [(S)-3,5-DHPG] produced an LTD of Adelta-fiber-evoked responses. LTD induction by (S)-3,5-DHPG was prevented, when intracellular Ca(2+) stores were depleted by thapsigargin or cyclopiazonic acid (CPA). Preincubation with ryanodine to inhibit Ca(2+)-induced Ca(2+) release had no effect on LTD-induction by (S)-3,5-DHPG. In contrast, pretreatment with 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of inositol-1,4,5-trisphosphate (IP(3))-sensitive Ca(2+) stores prevented LTD induction. Preincubation with the specific protein kinase C (PKC) inhibitors bisindolylmaleimide I (BIM) or chelerythrine, respectively, had no effect. Inhibition of L-type VDCCs by verapamil or nifedipine prevented LTD-induction by (S)-3,5-DHPG. The presently identified signal transduction cascade may be relevant to the long-term depression of sensory information in the spinal cord, including nociception.

Topics: Animals; Benzoates; Calcium; Disease Models, Animal; Excitatory Amino Acid Antagonists; Female; Glycine; Intracellular Space; Long-Term Synaptic Depression; Male; Neurons, Afferent; Nociceptors; Patch-Clamp Techniques; Posterior Horn Cells; Protein Kinase C; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; Resorcinols; Signal Transduction; Spinal Cord; Synapses; Thapsigargin

The release of intracellular stores of Ca(2+) occurs virtually in all types of cells by a means of amplifying external signals that modulate intracellular signaling events. In cardiac myocytes, type 2 ryanodine receptor (RyR(2)) is activated during excitation-contraction (E-C) coupling by Ca(2+)-induced Ca(2+) release (CICR) triggered by Ca(2+) influx across the sarcolemma. The hyperadrenergic state of heart failure results in leaky RyR(2) channels attributable to PKA hyperphosphorylation and depletion of the stabilizing FK506 binding protein, FKBP12.6. Dysregulation of sarcoplasmic reticulum (SR) Ca(2+) release via RyR(2) could contribute to defects in Ca(2+) signaling in failing hearts. Researchers tested the hypothesis that improved cardiac muscle function attributable to beta-AR blockade is associated with restoration of normal RyR(2) channel function in patients with heart failure. The authors aimed to observe change of RyR in junior mouse with HF and the effect of beta-adrenoreceptor blocker on RyR in HF in this experiment.. The animal model of congestive heart failure was established by constriction of abdominal aorta. Five weeks old mice were randomly divided into 3 groups: (1) HF group without treatment (n = 20); (2) HF group treated with carvedilol (n = 20); (3) Sham-operated group (n = 20). Carvedilol was administered through direct gastric gavage. After 4 weeks of treatment the high frequency ultrasound was performed. Myocardial SR was fractionated with velocity centrifugation. The time courses of Ca(2+) uptake and leak were determined by fluorescent spectrophotometr.. Compared with the sham-operated group, left ventricular diastolic dimension (LVEDD) (P < 0.05), left ventricular systolic dimension (LVESD), interventricular septal thickness at end-diastole (IVSTd), interventricular septal thickness at end-systole (IVSTs), left ventricular posterior wall thickness at end-diastole (LVPWTd), and left ventricular posterior wall thickness at end-systole (LVPWTs) were all significantly increased (P < 0.01). Ejection fraction (EF) and fractional shortening (FS) were decreased (P < 0.01) in HF group without treatment. LVEDD (P < 0.05), LVESD, IVSTd, IVSTs, LVPWTd and LVPWTs were all prominently decresed (P < 0.01). EF and FS were increased (P < 0.01) in cases of HF treated with carvedilol when compared with HF group without treatment. After adding thapsigargin to the buffer including SR of three groups, there were fewer Ca(2+) leak in sham-operated group and HF group treated with carvedilol than that of HF group without treatment (P < 0.01), while after adding FK506 and thapsigargin together to the buffer including SR of three groups, there were marked Ca(2+) leak in sham-operated group and HF group treated with carvedilol (P < 0.01). However, there was no additional increase in Ca(2+) leak in HF group compared with that of the group where only thapsigargin was added (P > 0.05).. There is more cardiac Ca(2+) leak in HF. Carvedilol can inhibite Ca(2+) leak by restoring the contactation of FKBP12.6 back to RyR in HF to improve cardiac function and prevent left ventricle from remodeling.

Topics: Adrenergic beta-Antagonists; Animals; Animals, Newborn; Calcium; Calcium Signaling; Calcium-Transporting ATPases; Carbazoles; Carvedilol; Disease Models, Animal; Heart Failure; Myocardial Contraction; Myocytes, Cardiac; Propanolamines; Rats; Rats, Wistar; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Spectrometry, Fluorescence; Thapsigargin; Ultrasonography

1. beta-Adrenoceptor (beta-AR)-mediated vasodilation, which plays an important physiological role in the regulation of vascular tone, is decreased in two-kidney, one clip (2K-1C) renal hypertension. In this study, downstream pathways related to vascular beta-AR activation were evaluated in 2K-1C rats. 2. Relaxation responses to isoprenaline, forskolin and 8-Br-cAMP were diminished in aortas without endothelium from 2K-1C when compared to those in normotensive two kidney (2K). Basal adenosine-3',5'-monophosphate (cAMP), as well as isoprenaline-induced increase in cAMP levels, was not different between 2K and 2K-1C aortas. 3. Contractile responses to caffeine, after depletion and reloading of intracellular Ca(2+) stores, were greater in 2K-1C than in 2K. The presence of isoprenaline during the Ca(2+)-reloading period abolished the differences between groups by increasing caffeine contraction in 2K without changing this response in 2K-1C aortas. Inhibition of the sarcolemmal Ca(2+)ATPase with thapsigargin markedly attenuated isoprenaline vasodilation in both 2K and 2K-1C and abolished the differences between groups. 4. Blockade of ATP-sensitive K(+) channels (K(ATP)) channels with glibenclamide significantly decreased isoprenaline vasodilation in 2K-1C without affecting this response in 2K. Both vascular gene and protein expression of protein kinase A (PKA), as well as phosphoserine-containing proteins, were increased in 2K-1C vs 2K rats. 5. In conclusion, decreased isoprenaline vasodilation in 2K-1C hypertensive rats is related to impaired modulation of the sarcolemmal Ca(2+)ATPase activity. Moreover, K(ATP) channels may play a compensatory role on isoprenaline-induced relaxation in renal hypertension. Both Ca(2+)ATPase and K(ATP) channel functional alterations, associated with decreased beta-AR vasodilation, are paralleled by an upregulation of protein kinase A (PKA) and phosphoserine proteins expression.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Aorta, Thoracic; Caffeine; Calcium-Transporting ATPases; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Disease Models, Animal; Gene Expression; Glyburide; Hypertension, Renovascular; Isoproterenol; Kidney; Male; Membrane Proteins; Muscle, Smooth, Vascular; Phenylephrine; Potassium Channels; Rats; Rats, Wistar; Receptors, Adrenergic, beta; RNA, Messenger; Sarcolemma; Signal Transduction; Thapsigargin; Vasoconstriction; Vasodilation

The endoplasmic reticulum (ER) is recognized as an organelle that participates in folding secretory and membrane proteins. The ER responds to stress by upregulating ER chaperones, but prolonged and/or excess ER stress leads to apoptosis. However, the potential role of ER stress in pathophysiological hearts remains unclear.. Mice were subjected to transverse aortic constriction (TAC) or sham operation. Echocardiographic analysis demonstrated that mice 1 and 4 weeks after TAC had cardiac hypertrophy and failure, respectively. Cardiac expression of ER chaperones was significantly increased 1 and 4 weeks after TAC, indicating that pressure overload by TAC induced prolonged ER stress. In addition, the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells increased, and caspase-3 was cleaved in failing hearts. The antagonism of angiotensin II type 1 receptor prevented upregulation of ER chaperones and apoptosis in failing hearts. On the other hand, angiotensin II upregulated ER chaperones and induced apoptosis in cultured adult rat cardiac myocytes. We also investigated possible signaling pathways for ER-initiated apoptosis. The CHOP- (a transcription factor induced by ER stress), but not JNK- or caspase-12-, dependent pathway was activated in failing hearts by TAC. Pharmacological ER stress inducers upregulated ER chaperones and induced apoptosis in cultured cardiac myocytes. Finally, mRNA levels of ER chaperones were markedly increased in failing hearts of patients with elevated brain natriuretic peptide levels.. These findings suggest that pressure overload by TAC induces prolonged ER stress, which may contribute to cardiac myocyte apoptosis during progression from cardiac hypertrophy to failure.

Topics: Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Animals; Aortic Valve Stenosis; Apoptosis; Cardiomegaly; Cells, Cultured; Disease Models, Animal; Disease Progression; Endoplasmic Reticulum; Gene Expression Regulation; Heart Failure; Humans; Imidazoles; Ligation; Male; Mice; Mice, Inbred C57BL; Molecular Chaperones; Myocytes, Cardiac; Natriuretic Peptide, Brain; Olmesartan Medoxomil; Rats; Rats, Inbred WKY; RNA, Messenger; Signal Transduction; Stress, Physiological; Tetrazoles; Thapsigargin; Tunicamycin

No studies exist concerning the ability of the plasma membrane Ca(2+) pump (PMCA), sarcoplasmic reticulum Ca(2+) pump (SERCA) and Na(+)-Ca(2+) exchanger (NCX) to regulate myoplasmic Ca(2+) (Ca(m)) in vascular smooth muscle cells from diabetic individuals with dyslipidemia. We tested the hypothesis that diabetic dyslipidemia would increase vascular smooth muscle cells to buffer Ca(m). Cells were isolated from the coronary artery of male Yucatan pigs treated for 20 weeks with: (1) a low fat diet (control group); (2) a high fat/cholesterol diet (F group); or (3) alloxan-induced diabetic pigs fed the high fat diet (DF group). The maximum Ca(m) response to a depolarizing 80 mM KCl (80 K) solution was evaluated in the absence and presence of thapsigargin (TSG; inhibits SERCA) and low Na (inhibits NCX). In response to 80 K alone, there was no difference in the Ca(m) response between groups. In the presence of TSG, the 80 K response decreased by 43% in the DF group; TSG did not affect the 80 K response in the control and F groups. When exposed to both TSG and low Na, the 80 K response also decreased by 55% in the DF group. This suggests increased Ca(m) buffering by the PMCA and/or mitochondria in the DF group when SERCA and NCX are inhibited. Compared to the control and F groups, low Na alone elicited a 50% lower Ca(m) amplitude in the DF group, which was reversed with TSG treatment; this suggests that SERCA activity is increased in DF pigs. Western blots also indicated a 7-fold increase in the approximately 115 kDa band density of an anti-SERCA2 antibody in DF compared to control pigs. This is the first report to demonstrate increased Ca(2+) buffering, specifically by SERCA, in vascular smooth muscle cells from diabetic individuals with dyslipidemia.

Topics: Analysis of Variance; Animals; Blotting, Western; Buffers; Calcium-Transporting ATPases; Cells, Cultured; Coronary Vessels; Diabetes Mellitus, Experimental; Disease Models, Animal; Enzyme Inhibitors; Hyperlipidemias; Myocytes, Smooth Muscle; Probability; Reference Values; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sensitivity and Specificity; Swine; Thapsigargin

Gangliosides are found at high levels in neuronal tissues where they play a variety of important functions. In the gangliosidoses, gangliosides accumulate because of defective activity of the lysosomal proteins responsible for their degradation, usually resulting in a rapidly progressive neurodegenerative disease. However, the molecular mechanism(s) leading from ganglioside accumulation to neurodegeneration is not known. We now examine the effect of ganglioside GM2 accumulation in a mouse model of Sandhoff disease (one of the GM2 gangliosidoses), the Hexb-/- mouse. Microsomes from Hexb-/- mouse brain showed a significant reduction in the rate of Ca2+-uptake via the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), which was prevented by feeding Hexb-/- mice with N-butyldeoxynojirimycin (NB-DNJ), an inhibitor of glycolipid synthesis that reduces GM2 storage. Changes in SERCA activity were not due to transcriptional regulation but rather because of a decrease in Vmax. Moreover, exogenously added GM2 had a similar effect on SERCA activity. The functional significance of these findings was established by the enhanced sensitivity of neurons cultured from embryonic Hexb-/- mice to cell death induced by thapsigargin, a specific SERCA inhibitor, and by the enhanced sensitivity of Hexb-/- microsomes to calcium-induced calcium release. This study suggests a mechanistic link among GM2 accumulation, reduced SERCA activity, and neuronal cell death, which may be of significance for delineating the neuropathophysiology of Sandhoff disease.

Topics: 1-Deoxynojirimycin; Adenosine Triphosphate; Animals; Blotting, Western; Brain; Calcium; Calcium-Transporting ATPases; Cell Death; Disease Models, Animal; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum; Enzyme Inhibitors; G(M2) Ganglioside; Gangliosides; Genotype; Glycolipids; Hippocampus; Kinetics; Lipid Metabolism; Mice; Mice, Transgenic; Microsomes; Neurons; Reverse Transcriptase Polymerase Chain Reaction; Sandhoff Disease; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Spectrophotometry; Thapsigargin; Time Factors

Standard anti-proliferative chemotherapy is relatively ineffective against slowly proliferating androgen-independent prostate cancer cells within metastatic sites. In contrast, the lipophilic cytotoxin thapsigargin, which causes apoptosis by disrupting intracellular free Ca2+ levels, is effective against both proliferative and quiescent (i.e., G0-arrested) cells. However, thapsigargin's mechanism of action indicates that it is unlikely to be selective for cancer cells or prostate cells.. We coupled a chemically modified form of thapsigargin, L12ADT, to a peptide carrier that is a substrate for the prostate-specific antigen (PSA) protease to produce a soluble, cell-impermeant latent prodrug that is specifically activated extracellularly within metastatic prostate cancer sites by PSA. We analyzed the kinetics of PSA hydrolysis of the prodrug, the in vitro cytotoxicity of the prodrug against PSA-producing LNCaP human prostate cancer and PSA non-producing HCT-116 human colon cancer cells, and the in vivo pharmacokinetics of the prodrug in mice. We also analyzed antitumor efficacy of the prodrug in nude mice xenograft models of prostate cancer (using LNCaP cells) and renal carcinoma (using human SN12C cells).. The L12ADT peptide prodrug was hydrolyzed efficiently by PSA, was selectively toxic to PSA-producing prostate cancer cells in vitro, and was stable in human plasma. A single dose of 7 mg/kg resulted in a peak serum prodrug concentration of 15.4 +/- 1.1 microM and a half-life of approximately 2.8 hours. Over 24 hours, less than 0.5% of free L12ADT was observed in plasma. Levels of prodrug and liberated L12ADT in prostate cancer xenograft tumors were approximately eightfold and sixfold, respectively, higher than the in vitro LD50s. Prostate cancer xenograft tumors in mice treated with prodrug by intravenous administration were growth-inhibited without substantial host toxicity. Continuous subcutaneous prodrug administration in mice produced complete growth inhibition of established PSA-producing prostate cancer xenograft tumors but had no effect on PSA non-producing renal carcinoma xenograft tumors.. Further development of PSA-activated thapsigargin prodrugs as therapy for metastatic prostate cancer is warranted.

Topics: Animals; Antineoplastic Agents; Disease Models, Animal; Humans; Hydrolysis; Male; Mice; Mice, Nude; Prodrugs; Prostate-Specific Antigen; Prostatic Neoplasms; Thapsigargin; Transplantation, Heterologous; Treatment Outcome

Transient global cerebral ischemia triggers suppression of the initiation step of protein synthesis, a process which is controlled by endoplasmic reticulum (ER) function. ER function has been shown to be disturbed after transient cerebral ischemia, as indicated by an activation of the ER-resident eIF2alpha kinase PERK. In this study, we investigated ischemia-induced changes in protein levels and phosphorylation states of the initiation factors eIF2alpha, eIF2B epsilon, and eIF4G1 and of p70 S6 kinase, proteins playing a central role in the control of the initiation of translation. Transient focal cerebral ischemia was induced in mice by occlusion of the left middle cerebral artery. Transient ischemia caused a long-lasting suppression of global protein synthesis. eIF2alpha was transiently phosphorylated after ischemia, peaking at 1-3 h of recovery. eIF2B epsilon and p70 S6 kinase were completely dephosphorylated during ischemia and phosphorylation did not recover completely following reperfusion. In addition, eIF2B epsilon, eIF4G1, and p70 S6 kinase protein levels decreased progressively with increasing recirculation time. Thus, several different processes contributed to ischemia-induced suppression of the initiation of protein synthesis: a long-lasting dephosphorylation of eIF2B epsilon and p70 S6K starting during ischemia, a transient phosphorylation of eIF2alpha during early reperfusion, and a marked decrease of eIF2B epsilon, eIF4G1, and p70 S6K protein levels starting during vascular occlusion (eIF4G1). Study of the mechanisms underlying ischemia-induced suppression of the initiation step of translation will help to elucidate the role of protein synthesis inhibition in the development of neuronal cell injury triggered by transient cerebral ischemia.

Topics: Animals; Blotting, Western; Brain; Brain Chemistry; Cells, Cultured; Disease Models, Animal; Disease Progression; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Eukaryotic Initiation Factor-2; Eukaryotic Initiation Factor-2B; Eukaryotic Initiation Factor-4G; Ischemic Attack, Transient; Isoenzymes; Male; Mice; Mice, Inbred C57BL; Neurons; Peptide Chain Initiation, Translational; Phosphorylation; Protein Subunits; Proteins; Ribosomal Protein S6 Kinases, 70-kDa; Thapsigargin

Calcium and calcium-dependent processes have been hypothesized to be involved in the induction of epilepsy. It has been shown that epileptic neurons have altered calcium homeostatic mechanisms following epileptogenesis in the hippocampal neuronal culture (HNC) and pilocarpine models of epilepsy. To investigate the mechanisms causing these alterations in [Ca2+]i homeostatic processes following epileptogenesis, we utilized the HNC model of in vitro 'epilepsy' which produces spontaneous recurrent epileptiform discharges (SREDs). Using [Ca2+]i imaging, studies were initiated to evaluate the mechanisms mediating these changes in [Ca2+]i homeostasis. 'Epileptic' neurons required much longer to restore a glutamate induced [Ca2+]i load to baseline levels than control neurons. Inhibition of Ca2+ entry through voltage and receptor gated Ca2+ channels and stretch activated Ca2+ channels had no effect on the prolonged glutamate induced increase in [Ca2+]i in epileptic neurons. Employing thapsigargin, an inhibitor of the sarco/endoplasmic reticulum calcium ATPase (SERCA), it was shown that thapsigargin inhibited sequestration of [Ca2+]i by SERCA was significantly decreased in 'epileptic' neurons. Using Ca2+ induced Ca2+ release (CICR) cell permeable inhibitors for the ryanodine receptor (dantrolene) and the IP3 receptor (2-amino-ethoxydiphenylborate, 2APB) mediated CICR, we demonstrated that CICR was significantly augmented in the 'epileptic' neurons, and determined that the IP3 receptor mediated CICR was the major release mechanism altered in epileptogenesis. These data indicate that both inhibition of SERCA and augmentation of CICR activity contribute to the alterations accounting for the impaired calcium homeostatic processes observed in 'epileptic' neurons. The results suggest that persistent changes in [Ca2+]i levels following epileptogenesis may contribute to the long-term plasticity changes manifested in epilepsy and that understanding the basic mechanisms mediating these changes may provide an insight into the development of novel therapeutic approaches to treat epilepsy and prevent or reverse epileptogenesis.

Topics: Animals; Boron Compounds; Calcium; Calcium-Transporting ATPases; Cells, Cultured; Disease Models, Animal; Endoplasmic Reticulum; Epilepsy; Glutamic Acid; Hippocampus; Homeostasis; Microscopy, Fluorescence; Neuronal Plasticity; Neurons; Rats; Rats, Sprague-Dawley; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin

The cellular mechanisms of abnormal calcium regulation and excitation-contraction coupling in relation to glucose metabolism in the hypertrophied heart are not well understood. The present study evaluated the myocardial mechanics of 6-7-week-old pressure overload hypertrophied rabbit hearts in response to dobutamine by (1) serial echocardiograms in vivo and (2) isolated Langendorff perfusion. Cytosolic Ca2+([Ca2+]i) and sarcoplasmic reticulum Ca2+-ATPase (SERCA2) expression were measured by fluorescence spectroscopy and Western immunoblotting, respectively. The effect of glycolytic inhibition by 2-deoxy-D-glucose +/- pyruvate was also evaluated. Both systolic and diastolic [Ca2+]i tended to be higher and diastolic calcium removal (tauCa) significantly slower in the hypertrophied heart. The myocardial response to dobutamine was blunted and dobutamine insignificantly improved tauCa. The SERCA2 protein level was higher in early hypertrophy, but was significantly reduced by 6 weeks of age, with progressive contractile failure. Inhibition of glycolysis or SERCA2 caused an increase in [Ca2+]i as well as a slower tauCa. Pyruvate completely preserved myocardial function and [Ca2+]i handling during glycolytic inhibition. It was concluded that in this model of advanced pressure overload hypertrophy, contractile failure and inotrope insensitivity are associated with increased [Ca2+]i, slower tauCa and reduced sensitivity of the contractile proteins to Ca2+. These changes occur in association with downregulation of the SERCA2, probably caused by impaired glucose metabolism.

Topics: Animals; Blotting, Western; Calcium; Calcium-Transporting ATPases; Disease Models, Animal; Echocardiography; Glucose; Heart; Hypertrophy, Left Ventricular; Models, Cardiovascular; Myocardium; Rabbits; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin

Defective excitation-contraction coupling in heart failure is generally associated with both a reduction in sarcoplasmic reticulum (SR) Ca(2+) uptake and a greater dependence on transsarcolemmal Na(+)-Ca(2+) exchange (NCX) for Ca(2+) removal. Although a relative increase in NCX is expected when SR function is impaired, few and contradictory studies have addressed whether there is an absolute increase in NCX activity. The present study examines in detail NCX density and function in left ventricular midmyocardial myocytes isolated from normal or tachycardic pacing-induced failing canine hearts. No change of NCX current density was evident in myocytes from failing hearts when intracellular Ca(2+) ([Ca(2+)](i)) was buffered to 200 nmol/L. However, when [Ca(2+)](i) was minimally buffered with 50 micromol/L indo-1, Ca(2+) extrusion via NCX during caffeine application was doubled in failing versus normal cells. In other voltage-clamp experiments in which SR uptake was blocked with thapsigargin, both reverse-mode and forward-mode NCX currents and Ca(2+) transport were increased >2-fold in failing cells. These results suggest that, in addition to a relative increase in NCX function as a consequence of defective SR Ca(2+) uptake, there is an absolute increase in NCX function that depends on [Ca(2+)](i) in the failing heart.

Topics: Animals; Buffers; Caffeine; Calcium; Cardiac Pacing, Artificial; Cells, Cultured; Disease Models, Animal; Dogs; Electric Stimulation; Enzyme Inhibitors; Female; Heart Failure; Ion Transport; Male; Membrane Potentials; Myocardium; Nickel; Phosphodiesterase Inhibitors; Sarcoplasmic Reticulum; Sodium-Calcium Exchanger; Thapsigargin

Sarcoplasmic reticulum (SR) Ca2+-ATPase gene expression is reduced in the failing myocardium. However, the functional relevance of these changes to myocardial contractility is not yet established. We assessed myocardial contractile function by analyzing sarcomere motion of isolated myocytes and also quantified SR Ca2+ regulatory protein gene expression by Northern blot analysis in the same hearts obtained from 10 dogs with pacing-induced heart failure (HF; 240 beats/min, 4 wk) and 7 control dogs. Sarcomere-shortening velocity was depressed in HF myocytes, accompanied by the prolongation of intracellular Ca2+ concentration ([Ca2+]i) transient measured by indo 1 fluorescence ratio. SR Ca2+-ATPase mRNA levels (normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA) were significantly depressed in HF, and calsequestrin mRNA was increased. For control and HF dogs, sarcomere-shortening velocity correlated positively with Ca2+-ATPase mRNA levels (r = 0.73, n = 17, P < 0.01) but not with calsequestrin mRNA. Ca2+-ATPase mRNA levels were correlated with 45Ca2+ uptake rate by SR, which was also reduced in HF. Moreover, the inhibition of SR Ca2+-ATPase with thapsigargin or cyclopiazonic acid reproduced in normal myocytes the abnormalities observed in HF myocytes, such as depressed contractility and the prolonged [Ca2+]i transient duration. A downregulation of Ca2+-ATPase gene expression and a resultant decrease in Ca2+ uptake by SR may be responsible for the contractile dysfunction and the alterations of [Ca2+]i transient in HF.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Calsequestrin; Cells, Cultured; Disease Models, Animal; Dogs; Echocardiography; Heart; Heart Failure; Myocardial Contraction; Myocardium; RNA, Messenger; Sarcomeres; Sarcoplasmic Reticulum; Tachycardia; Thapsigargin; Time Factors; Transcription, Genetic; Ventricular Function, Left

This study addressed the controversy of whether endothelium-derived nitric oxide (NO) activity is increased or decreased in the hypertensive pulmonary vasculature of chronically hypoxic rats. Thapsigargin, a receptor-independent Ca2+ agonist and stimulator of endothelial NO production, was used to compare NO-mediated vasodilation in perfused lungs and conduit pulmonary artery rings isolated from adult male rats either kept at Denver's altitude of 5,280 ft (control pulmonary normotensive rats) or exposed for 4-5 wk to the simulated altitude of 17,000 ft (chronically hypoxic pulmonary hypertensive rats). Under baseline conditions, thapsigargin (10(-9)-10(-7) M) caused vasodilation in hypertensive lungs and vasoconstriction in normotensive lungs. Whereas the sustained vasodilation in hypertensive lungs was reversed to vasoconstriction by the inhibitor of NO synthase N(omega)-nitro-L-arginine (L-NNA; 10(-4) M), a transient vasodilation to thapsigargin in acutely vasoconstricted normotensive lungs was potentiated. As measured by a chemiluminescence assay, the recirculated perfusate of hypertensive lungs accumulated considerably higher levels of NO-containing compounds that did normotensive lungs, and thapsigargin-induced stimulation of NO-containing compounds accumulation was greater in hypertensive than in normotensive lungs. Similarly, low concentrations of thapsigargin (10(-10)-10(-9) M) caused greater endothelium-dependent L-NNA-reversible relaxation of hypertensive than of normotensive pulmonary artery rings. The increased sensitivity of hypertensive arteries to thapsigargin-induced relaxation was eliminated in nominally Ca(2+)-free medium and was not mimicked by ryanodine, a releaser of intracellular Ca2+. These results with thapsigargin, which acts on endothelial cells to stimulate Ca2+ influx and a sustained rise in intracellular Ca2+ concentration, support the idea that pulmonary vascular endothelium-derived NO activity is increased rather than decreased in chronic hypoxia-induced pulmonary hypertension in rats.

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hypertension; Hypoxia; Male; Nitric Oxide; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Thapsigargin; Time Factors