Chung-Ren Jan

MECHANISM OF LANTHANUM INHIBITION OF EXTRACELLULAR ATP-EVOKED CALCIUM MOBILIZATION IN MDCK CELLS

We have studied the effects of La3+ on ATP-evoked rises in intracellular calcium levels ([Ca2+]i) measured by fura-2 fluorimetry in Madin Darby canine kidney (MDCK) cells. ATP evoked [Ca2+]i rises dose-dependently with an EC50 of 2.5 microM. The trigger for the Ca2+ signal was a release of Ca2+ from the inositol-1,4,5-trisphosphate (IP3)-sensitive stores because the signal was completely blocked by pretreatment with the endoplasmic reticulum (ER) Ca2+ pump inhibitor thapsigargin (TG) or the phospholipase C (PLC) inhibitor U73122. Both the peak height and area under the curve of 10 microM ATP-evoked Ca2+ signal was reduced by approximately 50% by extracellular Ca2+ removal, suggesting that ATP induced capacitative Ca2+ entry. La3+ inhibited the ATP-evoked Ca2+ signal dose-dependently when added before or after ATP. Pretreatment of 0.1 mM La3+ inhibited approximately 90% of the Ca2+ signal induced by 10 microM ATP. The mechanisms underlying the La3+ inhibition appear to involve not only block of capacitative Ca2+ entry but also interference with ATP binding to the ATP receptors.

The value of serial plasma nuclear and mitochondrial DNA levels in patients with acute ischemic stroke

BACKGROUND Elevated circulating cell-free DNA in plasma is reported in several critical diseases. This study hypothesized that since plasma nuclear and mitochondrial DNA substantially increase after acute ischemic stroke and decrease thereafter, their levels can predict treatment outcomes. METHODS Plasma nuclear and mitochondrial DNA levels were serially examined in 50 acute ischemic stroke patients and in 50 at risk control subjects during the study period. RESULTS Levels of plasma nuclear and mitochondrial DNA in patients with acute ischemic stroke were significantly higher than those in the controls (p<0.05). Elevated circulating nuclear DNA in plasma persisted until one month after the acute stroke. Levels of plasma nuclear DNA positively correlated to the clinical severity of stroke as reflected by the National Institutes of Health Stroke Scale. CONCLUSION Levels of plasma nuclear and mitochondrial DNA reflect the severity of cerebral damage after acute cerebral infarction. Assay of plasma DNA levels can be considered a neuro-pathologic marker of patients with acute ischemic stroke.

Mechanism of rise and decay of thapsigargin-evoked calcium signals in MDCK cells

We studied the effect of thapsigargin on intracellular calcium levels ([Ca2+]i) measured by fura-2 fluorimetry in Madin Darby canine kidney (MDCK) cells. Thapsigargin elevated [Ca2+]i dose dependently with an EC50 of approximately 0.15 microM. The Ca2+ signal consisted of a slow rise, a gradual decay and a plateau. Depletion of the endoplasmic reticulum Ca2+ store with thapsigargin for 7 min abolished the [Ca2+]i increases evoked by bradykinin. Removal of extracellular Ca2+ reduced the thapsigargin response by approximately 50%. The Ca2+ signal was initiated by Ca2+ release from the internal store followed by capacitative Ca2+ entry (CCE). The thapsigargin-evoked CCE was abolished by La3 and Gd3+, and was partly inhibited by SKF 96365 and econazole. After depletion of the internal Ca2+ store for 30 min with another inhibitor of the internal Ca2+ pump, cyclopiazonic acid, thapsigargin failed to increase [Ca2+]i, thus suggesting that the thapsigargin-evoked Ca2+ influx was solely due to CCE. We investigated the mechanism of decay of the thapsigargin response. Pretreatment with La3+ (or Gd3+) or alkalization of extracellular medium to pH 8 significantly potentiated the Ca2+ signal; whereas pretreatment with carbonylcyanide m-chlorophynylhydrozone (CCCP) or removal of extracellular Na+ had no effect. Collectively, our results imply that thapsigargin increased [Ca2+]i in MDCK cells by depleting the internal Ca2+ store followed by CCE, with both pathways contributing equally. The decay of the thapsigargin response might be significantly governed by efflux via the plasmalemmal Ca2+ pump.

The phospholipase C inhibitor U73122 increases cytosolic calcium in MDCK cells by activating calcium influx and releasing stored calcium

The effects of the phospholipase C (PLC) inhibitor U73122 on intracellular calcium levels ([Ca2+]i) were studied in MDCK cells. U73122 elevated [Ca2+]i dose-dependently. Ca2+ influx contributed to 75% of 20 microM U73122-induced Ca2+ signals. U73122 pretreatment abolished the [Ca2+]i transients evoked by ATP and bradykinin, suggesting that U73122 inhibited PLC. The Ca2+ signals among individual cells varied considerably. The internal Ca2+ source for the U73122 response was the endoplasmic reticulum (ER) since the response was abolished by thapsigargin. The depletion of the ER Ca2+ store triggered a La3+-sensitive capacitative Ca2+ entry. Independently of the internal release and capacitative Ca2 entry, U73122 directly evoked Ca2+ influx through a La3+-insensitive pathway. The U73122 response was augmented by pretreatment of carbonylcyanide m-chlorophynylhydrozone (CCCP), but not by Na+ removal, implicating that mitochondria contributed significantly in buffering the Ca2+ signal, and that efflux via Na+/Ca2+ exchange was insignificant.

Multiple effects of 1-[β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF 96365) on Ca2+ signaling in MDCK cells: depletion of thapsigargin-sensitive Ca2+ store followed by capacitative Ca2+ entry, activation of a direct Ca2+ entry, and inhibition of thapsigargin-induced capacitative Ca2+ entry

The effect of 1-[β-[3-(4-methoxyphenyl)pro- poxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF 96365) on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells was examined. SKF 96365 at 25–100 µM evoked a robust [Ca2+]i transient in a dose-dependent manner, measured by fura-2 fluorimetry. A concentration of 10 µM SKF 96365 did not have an effect. The transient consisted of a slow rise, a gradual decay, and a sustained plateau in physiological Ca2+ medium. Removal of extracellular Ca2+ reduced the Ca2+ signals evoked by 50–100 µM SKF 96365 by nearly half in the area under the curve, suggesting that SKF 96365 induced intracellular Ca2+ release and also extracellular Ca2+ influx. A concentration of 100 µM SKF 96365 caused significant Mn2+ quench of fura-2 fluorescence, which was partly inhibited by La3+ (1 mM) or Gd3+ (0.1 mM), indicating that the SKF 96365-induced Ca2+ influx had two components: one is sensitive to La3+ (1 mM) or Gd3+ (0.1 mM), the other is not. The internal Ca2+ source for the SKF 96365-induced [Ca2+]i transient was the endoplasmic reticulum Ca2+ store because, pretreatment with thapsigargin and cyclopiazonic acid, two inhibitors of the endoplasmic reticulum Ca2+ pump nearly abolished the SKF 96365-induced [Ca2+]i increase in Ca2+-free medium. In contrast, pretreatment with 100 µM SKF 96365 only partly depleted the thapsigargin-sensitive Ca2+ store. Addition of 10 mM Ca2+ induced a significant [Ca2+]i increase after prior incubation with 100 µM SKF 96365 in Ca2+-free medium, demonstrating that SKF 96365 induced capacitative Ca2+ entry. This capacitative Ca2+ entry was about 40% of that induced by 1 µM thapsigargin. Additional to inducing its own capacitative Ca2+ entry, 100 µM SKF 96365 partly inhibited thapsigargin- or uridine trisphos-phate (UTP)-induced capacitative Ca2+ entry. We also investigated the mechanisms underlying the decay of the SKF 96365-induced [Ca2+]i transient. Inhibition of the plasma membrane Ca2+ pump with La3+ or Gd3+, or lowering extracellular Na+ level to 0.35 mM, significantly increased the SKF 96365-induced [Ca2+]i transient. In contrast, the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone had little effect. In Ca2+-free medium, the thapsigargin-induced [Ca2+]i increase was greatly reduced by pretreatment with SKF 96365. Collectively, we have found that besides its well-known inhibitory action on capacitative Ca2+ entry in many cell types, in MDCK cells SKF 96365 exerted multiple and complex effects on Ca2+ signaling. It induced a considerable increase in [Ca2+]i by releasing Ca2+ from the endoplasmic reticulum store followed by capacitative Ca2+ entry. It also caused a direct Ca2+ entry. The decay of the SKF 96365 response was significantly governed by efflux via the plasma membrane Ca2+ pump or Na+/Ca2+ exchange. Sequestration by mitochondria or the endoplasmic reticulum played a minor role. We caution use of SKF 96365 as an inhibitor of capacitative Ca2+ entry.

Mechanism of Bradykinin-Induced Ca2+ Mobilization in MG63 Human Osteosarcoma Cells

Background: The effect of bradykinin on intracellular free Ca²⁺ levels ([Ca²⁺]_i) in MG63 human osteosarcoma cells was explored using fura-2 as a Ca²⁺ dye. Methods/Results: Bradykinin (0.1 nM–1 µM) increased [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ value of 0.5 nM. The [Ca²⁺]_i signal comprised an initial peak and a fast decay which returned to baseline in 2 min. Extracellular Ca²⁺ removal inhibited the peak [Ca²⁺]_isignals by 35 ± 3%. Bradykinin (1 nM) failed to increase [Ca²⁺]_i in the absence of extracellular Ca²⁺after cells were pretreated with thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor; 1 µM). Bradykinin (1 nM)-induced intracellular Ca²⁺ release was nearly abolished by inhibiting phospholipase C with 2 µM 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). The [Ca²⁺]_iincrease induced by 1 nM bradykinin in Ca²⁺- free medium was abolished by 1 nM HOE 140 (a B2 bradykinin receptor antagonist) but was not altered by 100 nM Des-Arg-HOE 140 (a B1 bradykinin receptor antagonist). Pretreatment with 1 pM pertussis toxin for 5 h in Ca²⁺ medium inhibited 30 ± 3% of 1 nM bradykinin-induced peak [Ca²⁺]_i increase. Conclusions: Together, this study shows that bradykinin induced [Ca²⁺]_i increases in a concentration-dependent manner, by stimulating B2 bradykinin receptors leading to mobilization of Ca²⁺ from the thapsigargin-sensitive stores in a manner dependent on inositol-1,4,5-trisphosphate, and also by inducing extracellular Ca²⁺ influx. The bradykinin response was partly coupled to a pertussis toxin-sensitive G protein pathway.

Multiple effects of econazole on calcium signaling: depletion of thapsigargin-sensitive calcium store, activation of extracellular calcium influx, and inhibition of capacitative calcium entry

The effect of econazole on intracellular calcium levels ([Ca2+]i) in Madin Darby canine kidney cells was investigated using fura-2 fluorimetry. Econazole increased [Ca2+]i dose-dependently at 5-50 microM. The Ca2+ signal consisted of an initial rise, a gradual decay and a sustained plateau. Extracellular Ca2+ removal partially reduced the econazole response. Mn2+ quench of fura-2 fluorescence confirmed econazole-induced Ca2+ influx. The econazole-sensitive intracellular Ca2+ store overlaps with that sensitive to thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump, because 25 microM econazole depleted the thapsigargin-sensitive store, and conversely, thapsigargin abolished the econazole response. Econazole (25-50 microM) partially inhibited capacitative Ca2+ entry induced by cyclopiazonic acid, another endoplasmic reticulum Ca2+ pump inhibitor, measured by depleting internal Ca2+ store in Ca(2+)-free medium followed by adding 10 mM CaCl2. Econazole induced capacitative Ca2+ entry itself. Pretreatment with La3+ (100 microM) partially inhibited 25 microM econazole-induced Mn2+ quench of fura-2 fluorescence, and La3+ immediately reduced 20 microM econazole-induced Ca2+ signal when added at the peak of the signal, suggesting that econazole induced Ca2+ influx via two separate pathways: one is sensitive to La3+, the other is not. La3+ enlarged 25 microM econazole-induced [Ca2+]i transient during the decay phase. The econazole response was not altered when the cytosolic level of inositol 1,4,5-trisphosphate was inhibited by the phospholipase C inhibitor U73122.

Linoleamide, a brain lipid that induces sleep, increases cytosolic Ca2+ levels in MDCK renal tubular cells

Linoleamide is an endogenous lipid that has been shown to induce sleep in cats, rats and humans. However, its physiological function remains unclear. In this study the effect of linoleamide on cytosolic free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) tubular cells was examined, by using fura-2 as a Ca2+ probe. In a concentration-dependent manner, linoleamide induced increases in [Ca2+]i between 10-500 microM with an EC50 of 20 microM. The signal comprised a slow rise and a persistent phase, and was a result of internal Ca2+ release and external Ca2+ influx because it was partly inhibited by external Ca2+ removal. In Ca2+-free medium, depletion of the endoplasmic reticulum Ca2+ store with 1 microM thapsigargin abolished 100 microM linoleamide-induced internal Ca2+ release, and conversely, pretreatment with linoleamide prevented thapsigargin from releasing internal Ca2+. This demonstrates that the internal source of linoleamide-induced [Ca2+]i increase is located in the endoplasmic reticulum. This discharge of internal Ca2+ caused capacitative Ca2+ entry because after incubation with 100 microM linoleamide in Ca2+-free medium for 8 min readmission of 3 mM CaCl2 induced increases in [Ca2+]i. After the formation of inositol-1,4,5-trisphosphate (IP3) was blocked by the phospholipase C inhibitor U73122 (1 microM), linoleamide still induced an increase in [Ca2+]i but the shape of the increase was altered. Similar results were found for another sleep-inducing lipid 9,10-octadecenoamide. Together, the present study shows that the endogenous sleep-inducing lipid linoleamide was able to cause significant increases in [Ca2+]i in renal tubular cells, by releasing the endoplasmic reticulum Ca2+ store and triggering capacitative Ca2+ entry in a manner independent of IP3.

Levels and value of platelet activation markers in different subtypes of acute non-cardio-embolic ischemic stroke

INTRODUCTION Platelet activation and its interaction with leukocytes are important in the pathophysiology of ischemic stroke. This study aimed to evaluate the value of platelet activation and platelet-leukocyte interaction in different subtypes of acute, non-cardio-embolic ischemic stroke. METHODS Fifty-four patients with acute, non-cardio-embolic ischemic stroke, including 32 small-vessel and 22 large-vessel diseases, were evaluated. Platelet activation markers (CD62P, CD63, and CD40L) and platelet-leukocyte interaction were measured by flow cytometry at different time points (<48 hours and Days 7, 30, and 90 post-ischemic stroke). Markers were also evaluated in 28 other stroke patients in the convalescent stage (3 to 9 months after acute stroke) and in 28 control subjects. RESULTS Patients with ischemic stroke had significantly increased circulating CD62P, CD63, platelet-monocyte interaction, and platelet-lymphocyte interaction in the acute stage compared with the convalescent stage and control groups. Levels of CD62P and CD63 were significantly higher in the large-vessel disease group than in the small-vessel disease group, and differences in CD62P were significant even at one month. The CD40L level in the poor outcome group was significantly higher than that in the good outcome group. Stroke patients with diabetes mellitus and large-vessel disease were associated with poor outcome. CONCLUSIONS Patients with large-vessel cerebral infarction elicit higher platelet activation and platelet-leukocyte interaction compared to small-vessel infarction. Further large scale trials are warranted to evaluate the relationship between platelet activation markers and outcome in stroke patients under different anti-platelet therapies, and to clarify optimal treatment.

Desipramine-Induced Apoptosis in Human Pc3 Prostate Cancer Cells: Activation of Jnk Kinase and Caspase-3 Pathways and a Protective Role of Ca2+ (I) Elevation

The antidepressant desipramine has been shown to induce a rise in cytosolic Ca2+ levels ([Ca2+]i) and cytotoxicity in human PC3 prostate cancer cells, but the mechanisms underlying its cytotoxic effect is unclear. Cell viability was examined by WST-1 assays. Apoptosis was assessed by propidium iodide staining and an increase in caspase-3 activation. Phosphorylation of protein kinases was analyzed by immunoblotting. Desipramine caused cell death via apoptosis in a concentration-dependent manner. Immunoblotting data revealed that desipramine activated the phosphorylation of c-Jun NH2-terminal kinase (JNK), but not extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). SP600125 (a selective JNK inhibitor) partially prevented cells from apoptosis. Pretreatment with BAPTA/AM, a Ca2+ chelator, to prevent desipramine-induced [Ca2+]i rises worsened desipramine-induced cytotoxicity. Immunoblotting data suggest that BAPTA/AM pretreatment enhanced desipramine-evoked JNK phosphorylation and caspase-3 cleavage. The results suggest that in PC3 cells, desipramine caused apoptosis via inducing JNK-associated caspase-3 activation, and [Ca2+]i rises may slow down or alleviate desipramine-induced cytotoxicity.