AIMTo study whether store-operated Ca2+ channel (SOC) is present in rat colonic smooth muscle cells.

METHODSIntracellular Ca2+ ([Ca2+]i) changes induced by thapsigargin- or caffeine-activated SOC channel were measured in enzymatically dissociated rat colonic smooth muscle cells with the fluorescent indicator Fura-2/AM.

RESULTSIn the absence of external Ca2+ , the sarco-endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (1 micromol/L) and ryanodine receptor (RyR) activator caffeine both transiently elevated [Ca2+]i from (68.32 +/- 3.43) nmol/L to (240.85 +/- 12.65 ) nmol/L, (481.25 +/- 34.77) nmol/L. A subsequent reintroduction of Ca2+ into the extracellular solution resulted in [Ca2+]i further elevated to (457.55 +/- 19.80) nmol/L, (1005.93 +/- 54.62) nmol/L; (643.88 +/- 34.65) nmol/L, (920.16 +/- 43.25) nmol/L, respectively. And the elevated response was blocked by lanthanum (1 mmol/L), but was insensitive to L-type voltage calcium channels blocker verapamil and membrane depolarization.

CONCLUSIONSOC activated by store depletion are present in rat colonic smooth muscle cells. And we further prove the existence of such Ca2+ channels in excitable cells.

Animals ; Caffeine ; pharmacology ; Calcium ; metabolism ; Calcium Channels ; physiology ; Colon ; cytology ; Fura-2 ; metabolism ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Rats ; Rats, Wistar ; Thapsigargin ; pharmacology

To explore the apoptotic effect of simvastatin on K562 cells through endoplasmic reticulum stress, morphological change of apoptotic cells was observed by Hoechst33258 fluorescent staining under fluorescent microscope. Apoptosis rate of cells was determined with annexinV-FITC/PI double staining by flow cytometry; Intracellular calcium concentration ([Ca2+]i) was measured by laser scanning confocal microscope (LSCM); The expression levels of glucose regulated protein 78 (GRP78) and calpain gene mRNA were determined by RT-PCR; The expression levels of caspase-3, -6, -7, -9, -12, calpain and GRP78 proteins were evaluated by Western blotting. In this study, K562 cells treated with simvastatin for 72 h exhibited typical morphological change of apoptosis cells. After 72 h exposed to 10, 20, 30 micromol x L(-1) simvastatin, the apoptotic rates of K562 cells were 12.41%, 19.08% and 23.41%, respectively. Simvastatin induced the increase of [Ca2+]i in K562 cells, fluorescent intensities were 43, 54, and 64, respectively. The expression levels of GRP78 and calpain gene mRNA were up-regulated. The cleavage and activation of caspase-3, -6, -7, -9, -12 and upregulation of GRP78 expression were determined by Western blotting. These findings suggest that endoplasmic reticulum is an important pathway of apoptosis in cells and participates simvastatin-induced apoptosis in K562 cells. It is implied that simvastatin may be suitable for clinical usage in the treatment of myeloma patients.
Apoptosis ; drug effects ; Calcium ; metabolism ; Calpain ; genetics ; metabolism ; Caspases ; metabolism ; Endoplasmic Reticulum ; drug effects ; metabolism ; Enzyme Inhibitors ; pharmacology ; Heat-Shock Proteins ; genetics ; metabolism ; Humans ; K562 Cells ; RNA, Messenger ; metabolism ; Simvastatin ; pharmacology ; Thapsigargin ; pharmacology

PURPOSE: In non-excitable cells, which include parotid and pancreatic acinar cells, Ca(2+) entry is triggered via a mechanism known as capacitative Ca(2+) entry, or store-operated Ca(2+) entry. This process is initiated by the perception of the filling state of endoplasmic reticulum (ER) and the depletion of internal Ca(2+) stores, which acts as an important factor triggering Ca(2+) entry. However, both the mechanism of store-mediated Ca(2+) entry and the molecular identity of store-operated Ca(2+) channel (SOCC) remain uncertain. MATERIALS AND METHODS: In the present study we investigated the Ca(2+) entry initiation site evoked by depletion of ER to identify the localization of SOCC in mouse parotid and pancreatic acinar cells with microfluorometeric imaging system. RESULTS: Treatment with thapsigargin (Tg), an inhibitor of sarco/endoplasmic reticulum Ca(2+)-ATPase, in an extracellular Ca(2+) free state, and subsequent exposure to a high external calcium state evoked Ca(2+) entry, while treatment with lanthanum, a non-specific blocker of plasma Ca(2+) channel, completely blocked Tg-induced Ca(2+) entry. Microfluorometric imaging showed that Tg-induced Ca(2+) entry started at a basal membrane, not a apical membrane. CONCLUSION: These results suggest that Ca2+ entry by depletion of the ER initiates at the basal pole in polarized exocrine cells and may help to characterize the nature of SOCC.
Animals ; Calcium/*metabolism ; Calcium Channels/drug effects/metabolism ; Cells, Cultured ; Endoplasmic Reticulum/drug effects/*metabolism ; Mice ; Mice, Inbred ICR ; Microscopy, Fluorescence ; Pancreas/cytology/drug effects/*metabolism ; Parotid Gland/cytology/drug effects/*metabolism ; Thapsigargin/pharmacology

OBJECTIVE: Effects of ATP and acetylcholine (ACh) on intracellular Ca2+ concentrations ([Ca2+]i) and possible mechanism of Ca2+-induced Ca2+ release (CICR) of the isolated outer hair cells (OHCs) in the guinea pig cochlea were studied with confocal microscopy. METHOD: OHCs were isolated from guinea pig cochlea by enzymatic and mechanical methods. The effects of ATP, ACh, Ryanodine + ATP (or ACh) and Thapsigargin + ATP (or ACh) in the presence or absence of extracellular Ca2+ on [Ca2+]i in OHCs were examined by confocal microscopy. RESULT: In the presence of ATP, Ryanodine + ATP, Thapsigargin + ATP, ACh, Ryanodine + ACh and Thapsigargin + ACh increased [Ca2+]i and evoked an evident wave, respectively, the relative magnitude of fluorescence were 1.60 +/- 0.01(ATP), 1.644 +/- 0.005 (Ryanodine + ATP), 1.491 +/- 0.005 (Thapsigargin + ATP), 1.43 +/- 0.01 (ACh), 1.58 +/- 0.02 (Ryanodine + ACh), 1.398 +/- 0.003 (Thapsigargin + ACh) in OHCs in the presence of extracellular Ca2+ respectively. In the absence of extracellular Ca2+, ATP and Ryanodine + ATP induced a gradual and small [Ca2+]i wave, the relative magnitude of fluorescence were 1.341 +/- 0.006 and 1.386 +/- 0.008, however, ACh, Ryanodine + ACh, Thapsigargin + ACh and Thapsigargin + ATP can not induce wave but a gradual [Ca2+]i elevation. ACh can not increase [Ca2+]i. CONCLUSION In the presence of extracellular Ca2+, ATP and ACh increased [Ca2+]i in OHCs not only by Ca2+ influx through ion channel on cell membrane but also a release of Ca2+ from IP3-sensitive calcium reservoir and CICR. In the absence of extracellular Ca2+, ATP activated IP3 sensitive calcium reservoir and Ca2+ release through IP3 sensitive calcium reservoir, in turn CICR was induced. ACh can not activate IP3 sensitive calcium reservoir and CICR in the absence of extracellular Ca2+, therefore, the effect of ACh was dependent of extracellular Ca2+.
Acetylcholine ; pharmacology ; Adenosine Triphosphate ; pharmacology ; Animals ; Calcium ; metabolism ; Calcium Channels ; drug effects ; metabolism ; Cells, Cultured ; Cochlea ; cytology ; metabolism ; Guinea Pigs ; Hair Cells, Auditory, Outer ; metabolism ; Microscopy, Confocal ; Ryanodine ; pharmacology ; Thapsigargin ; pharmacology

OBJECTIVETo investigate the effect of Ca(2+) mobilization on release and activation of matrix metalloproteinases (MMPs) in human hepatocellular carcinoma cells.

METHODSCa(2+) and chemicals which can induce or inhibit Ca(2+) mobilization were added into human SMMC-7721 hepatoma cells in vitro. SDS-PAGE protein electrophoresis and gelatin zymography analysis were carried out to detect the changes of release and activation of MMPs in the cell culture supernatant.

RESULTSAddition of CaCl(2) into culture system resulted in an enhanced secretion and activation of MMP-2 and MMP-9 in a dose-dependent manner. At a dose of 0.8 mmol/L CaCl(2), it maintained a stable high level of MMPs, especially of MMP-2 with (109.71 +/- 27.93)% elevation as compared to the cells without CaCl(2) addition (P < 0.001). SDS-PAGE analysis showed that most secreted proteins were MMPs (MMP-2 and MMP-9) when the cells cultured in media without serum. Thapsigargin (Tg, 4 micromol/L), an inducer of intracellular Ca(2+) stores depletion, significantly enhanced the release and activation of MMP-2 and MMP-9, compared to the control with (58.63 +/- 31.04)% elevation (P < 0.05), while the inducing effect of Tg on MMPs release and activation was significantly inhibited by S-nitro-N-acetylpenicillamine (SNAP, 200 micromol/L), an NO donor.

CONCLUSIONIntracellular Ca(2+) regulation pathways may play an important role in the process of release and activation of MMPs.

Calcium ; metabolism ; Carcinoma, Hepatocellular ; enzymology ; pathology ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Humans ; Liver Neoplasms ; enzymology ; pathology ; Matrix Metalloproteinase 2 ; metabolism ; secretion ; Matrix Metalloproteinase 9 ; metabolism ; secretion ; Nitric Oxide Donors ; pharmacology ; Penicillamine ; analogs & derivatives ; pharmacology ; Thapsigargin ; pharmacology

Endoplasmic reticulum (ER) stress occurs in macrophage-rich areas of advanced atherosclerotic lesions and contributes to macrophage apoptosis and subsequent plaque necrosis. The purpose of the present study was to investigate the effects of caveolin-1 (Cav-1) on ER stress-induced apoptosis in cultured macrophages and the underlying mechanisms. RAW264.7 cells were incubated with thapsigargin (TG) to establish ER stress model. And Cav-1 expression was detected by Western blot. After being pretreated with filipin(III), a caveolae inhibitor, RAW264.7 cells were assayed with flow cytometry and confocal laser scanning microscopy to detect cell apoptosis. Moreover, p38 mitogen-activated protein kinase (MAPK) phosphorylation and C/EBP homologous protein (CHOP) expression were detected with Western blot. The results showed that Cav-1 expression was markedly increased at early stage of TG treatment (P < 0.05) and then decreased with prolonged or high dose TG treatments. The increasing of Cav-1 expression induced by TG in RAW264.7 cells was abolished under inhibition of caveolae by filipin(III) (P < 0.05). The effect of TG on apoptosis of RAW264.7 cells was further augmented after pretreatment with filipin(III) (P < 0.05). Western blotting showed that MAPK phosphorylation induced by TG was inhibited by filipin(III) in RAW264.7 cells (P < 0.05), whereas CHOP remained unchanged (P > 0.05). These results suggest that Cav-1 may play a critical role in suppressing ER stress-induced macrophages apoptosis in vitro, and one of the mechanisms may be correlated with the activation of p38 MAPK prosurvival pathway.
Animals ; Apoptosis ; drug effects ; Caveolin 1 ; genetics ; metabolism ; Cell Line ; Endoplasmic Reticulum Stress ; physiology ; Filipin ; pharmacology ; MAP Kinase Signaling System ; Macrophages ; cytology ; drug effects ; Mice ; Thapsigargin ; pharmacology ; Transcription Factor CHOP ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

The aim was to study the apoptotic induction effect of thapsigargin on leukemia cell line K562 and its possible mechanism. After the treatment of leukemia cell line K562 by thapsigargin, morphological change of apoptotic cells was investigated by AO/EB fluorescent staining under fluorescent microscope; apoptosis rate was determined with annexin V-FITC/PI double staining by flow cytometry; intracellular calcium concentrations ([Ca(2+)]i) were measured by fluorescence spectrophotometer with calcium sensitive fluorescence indicator Fura-2/AM; mitochondrial transmembrance potentials (Delta Psi m) was detected on flow cytometry through staining of Rhodamine (Rh123); the changes of caspase-3, -7, -9, -12, cytochrome C, GRP78 proteins were detected by Western blot. The results showed that K562 cells cultured in 4 micromol/L thapsigargin for 48 hours exhibited typical morphological changes of apoptotic cells under fluorescent microscope, including shrinkage of cell, condensation of chromatin, breakage of nuclear, formation of apoptotic bodies, fluorescence of yellow green and pellet observed in early apoptoyic cells and hyacinth fluorescence of chromatin showed in late apoptotic cells. 24 and 48 hours after exposure to 1, 2, 4, 8 micromol/L thapsigargin, the apoptotic rates of K562 were respectively 7.51%, 11.65%, 23.22%, 30.56% and 12.85%, 20.27%, 31.51%, 44.16%, in dose-dependent manner, and were statistically significant when compared with the controls (P < 0.05). The apoptotic rate of K562 was dose- and time-dependent in experiment range. The enhancement of [Ca(2+)]i and the decrease of the Delta Psi m in K562 cells were induced by thapsigargin and were dose-dependent in experiment range, compared with control, P < 0.05. Western blot results indicated that cleavage and activation of caspase-3, -7, -9, -12, releasing of cytochrome C from mitochondria, upregulation of GRP78 expression at the endoplasmic reticulum were induced in K562 cells after 24 hours exposure of 4 micromol/L thapsigargin. It is concluded that thapsigargin induces endoplasmic reticulum stress-induced apoptosis in K562 cells. Endoplasmic reticulum is a novel important initiatory site of apoptosis in cells; the cleavage and activation of caspase-3, -7, -9, -12 play very important role in endoplasmic reticulum stress-induced apoptosis of K562 cells and is one of the important mechanisms for thapsigargin-induced apoptosis. Thapsigargin-induced apoptosis in K562 cells is associated closely with the disruption of the Delta Psi m and the release of cytochrome C from mitochondria, mitochondria participates in endoplasmic reticulum stress-induced apoptosis in K562 cells.
Apoptosis ; drug effects ; Calcium-Transporting ATPases ; antagonists & inhibitors ; Caspase 3 ; metabolism ; Caspase 7 ; metabolism ; Cytochromes c ; metabolism ; Endoplasmic Reticulum ; drug effects ; enzymology ; Enzyme Inhibitors ; pharmacology ; Heat-Shock Proteins ; metabolism ; Humans ; K562 Cells ; Leukemia ; pathology ; Mitochondria ; drug effects ; Molecular Chaperones ; metabolism ; Thapsigargin ; pharmacology

OBJECTIVEThe 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.

METHODSThe 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.

RESULTSCompared 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).

CONCLUSIONThere 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.

Adrenergic beta-Antagonists ; pharmacology ; Animals ; Animals, Newborn ; Calcium ; metabolism ; Calcium Signaling ; drug effects ; Calcium-Transporting ATPases ; antagonists & inhibitors ; Carbazoles ; pharmacology ; Disease Models, Animal ; Heart Failure ; diagnostic imaging ; drug therapy ; metabolism ; Myocardial Contraction ; drug effects ; Myocytes, Cardiac ; drug effects ; metabolism ; Propanolamines ; pharmacology ; Rats ; Rats, Wistar ; Ryanodine Receptor Calcium Release Channel ; metabolism ; Sarcoplasmic Reticulum ; drug effects ; metabolism ; Spectrometry, Fluorescence ; Thapsigargin ; pharmacology ; Ultrasonography

Upon glucose elevation, pancreatic beta-cells secrete insulin in a Ca(2+)-dependent manner. In diabetic animal models, different aspects of the calcium signaling pathway in beta-cells are altered, but there is no consensus regarding their relative contributions to the development of beta-cell dysfunction. In this study, we compared the increase in cytosolic Ca(2+) ([Ca(2+)]i) via Ca(2+) influx, Ca(2+) mobilization from endoplasmic reticulum (ER) calcium stores, and the removal of Ca(2+) via multiple mechanisms in beta-cells from both diabetic db/db mice and non-diabetic C57BL/6J mice. We refined our previous quantitative model to describe the slow [Ca(2+)]i recovery after depolarization in beta-cells from db/db mice. According to the model, the activity levels of the two subtypes of the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) pump, SERCA2 and SERCA3, were severely down-regulated in diabetic cells to 65% and 0% of the levels in normal cells. This down-regulation may lead to a reduction in the Ca(2+) concentration in the ER, a compensatory up-regulation of the plasma membrane Na(+)/Ca(2+) exchanger (NCX) and a reduction in depolarization-evoked Ca(2+) influx. As a result, the patterns of glucose-stimulated calcium oscillations were significantly different in db/db diabetic beta-cells compared with normal cells. Overall, quantifying the changes in the calcium signaling pathway in db/db diabetic beta-cells will aid in the development of a disease model that could provide insight into the adaptive transformations of beta-cell function during diabetes development.
Animals ; Calcium ; metabolism ; Calcium Signaling ; drug effects ; Cell Membrane Permeability ; drug effects ; Cells, Cultured ; Down-Regulation ; drug effects ; Endoplasmic Reticulum ; metabolism ; Glucose ; pharmacology ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Obese ; Potassium Chloride ; pharmacology ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Sodium-Calcium Exchanger ; metabolism ; Thapsigargin ; pharmacology ; Up-Regulation ; drug effects

Baicalein is one of the major flavonoids in Scutellaria baicalensis Georgi and possesses various effects, including cytoprotection and anti-inflammation. Because endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and cerebral ischemia, we investigated the effects of baicalein on apoptotic death of HT22 mouse hippocampal neuronal cells induced by thapsigargin (TG) and brefeldin A (BFA), two representative ER stress inducers. Apoptosis, reactive oxygen species (ROS) production, and mitochondrial membrane potential (MMP) were measured by flow cytometry. Expression level and phosphorylation status of ER stress-associated proteins and activation and cleavage of apoptosis-associated proteins were analyzed by Western blot. Baicalein reduced TG- and BFA-induced apoptosis of HT22 cells and activation and cleavage of apoptosis-associated proteins, such as caspase-12 and -3 and poly(ADP-ribose) polymerase. Baicalein also reduced the TG- and BFA-induced expression of ER stress-associated proteins, including C/EBP homologous protein (CHOP) and glucose-regulated protein 78, the cleavage of X-box binding protein-1 and activating transcription factor 6alpha, and the phosphorylation of eukaryotic initiation factor-2alpha and mitogen-activated protein kinases, such as p38, JNK, and ERK. Knock-down of CHOP expression by siRNA transfection and specific inhibitors of p38 (SB203580), JNK (SP600125), and ERK (PD98059) as well as anti-oxidant (N-acetylcysteine) reduced TG- or BFA-induced cell death. Baicalein also reduced TG- and BFA-induced ROS accumulation and MMP reduction. Taken together, these results suggest that baicalein could protect HT22 neuronal cells against ER stress-induced apoptosis by reducing CHOP induction as well as ROS accumulation and mitochondrial damage.
Animals ; *Apoptosis ; Brefeldin A/pharmacology ; Cell Line ; Cytoprotection ; DNA-Binding Proteins/metabolism ; Endoplasmic Reticulum/drug effects/*physiology ; Flavanones/*pharmacology ; Heat-Shock Proteins/biosynthesis ; Hippocampus/cytology ; Membrane Potential, Mitochondrial/drug effects ; Mice ; Mitogen-Activated Protein Kinases/metabolism ; Neurons/*drug effects/physiology ; Reactive Oxygen Species/*metabolism ; Signal Transduction/drug effects ; Thapsigargin/pharmacology ; Transcription Factor CHOP/*biosynthesis ; Transcription Factors/metabolism ; Unfolded Protein Response/drug effects