Intracellular pH regulation of osteoblasts is of a great importance in the process of bone formation and resorption, and has been suggested to be mediated via intracellular Ca2+ and cAMP messenger systems. To elucidate the mechanism of modulation of intracellular pH by parathyroid hormone and PMA(Phorbo1-12-myristate-13-acetate), effects of these agonists on the individual transporter system, Na+-H+ antiporter and Cl−-HCO3-(−OH−) exchanger, were investigated. Intracellular pH and Ca2+ were measured by using the fluorescent dye BCECF and fura-2, respectively, in UMR-106 cell monolayer grown on glass coverslip. Addition of tumor promotor, PMA(luM) caused 0.14 unit pH rise of resting intracellular pH(pHi) and 38% increase of the initial rate of pHi recovery after cytosolic acid load. Perfusion of Cl−-free solution resulted in rapid cytosolic alkalinization of which the rate was increased 26% by preincubation of PMA. Ca2+ ionophore, ionomycin (1uM) decreased resting pHi by 0.17 unit, but had no effect on the initial rate of pHi recovery after cytosolic acid load. However, the addition of ionomycin augmented the initial rate of pHi increase after Cl−-depletion outside the cells by 34% over the control. Stimulation of cells with parathyroid hormone(10-8M) caused an initial acidification (0.27 unit) followed by cytosolic alkalinization, with inhibiting effect on the initial rate of pHi recovery after acid load (42%). But parathyroid hormone did not have any significant effect on the rate of pHi increase after Cl−-depletion. PMA caused a sustained increase of intracellular Ca2+, of which the peak depended on the concentration of Ca2+ in extracellular medium. Ionomycin caused a transient increase of Ca2+ but PTH had no significant increase of intracellular Ca2+ in the concentration range of 10-6M to 10-12M tested. 10-8M PTH increased cAMP levels by about 10-fold and 10-10M PTH did by 1.6-fold. PMA, which increased cytosolic Ca2+ concentration, also had an stimulatory effect on cAMP production in the concentration range of 10-5M to 10-6M by 2-fold. These findings suggest that in UMR-106 cells Ca2+ and cAMP can influence pHi by altering the activity of pHi regulatory transporter system, and parathyroid hormones modulate pHi by inhibiting Na+-H+ antiporter via intracellular increase of cAMP, which is probably accounts for the inhibitory effect of parathyroid hormone on the proliferation of osteoblasts.
Cytosol ; Fura-2 ; Glass ; Hydrogen-Ion Concentration ; Ion Transport ; Ionomycin ; Osteoblasts ; Osteogenesis ; Parathyroid Hormone ; Perfusion

The effects of extremely low frequency electromagnetic fields (EMF) on intracellular Ca2+ mobilization and cellular function in RBL 2H3 cells were investigated. Exposure to EMF (60 Hz, 0.1 or 1 mT) for 4 or 16 h did not produce any cytotoxic effects in RBL 2H3 cells. Melittin, ionomycin and thapsigargin each dose-dependently increased the intracellular Ca2+ concentration. The increase of intracellular Ca2+ induced by these three agents was not affected by exposure to EMF (60 Hz, 1 mT) for 4 or 16 h in RBL 2H3 cells. To investigate the effect of EMF on exocytosis, we measured beta-hexosaminidase release in RBL 2H3 cells. Basal release of beta-hexosaminidase was 12.3+/-2.3% in RBL 2H3 cells. Exposure to EMF (60 Hz, 0.1 or 1 mT) for 4 or 16 h did not affect the basal or 1 microM melittin-induced beta-hexosaminidase release in RBL 2H3 cells. This study suggests that exposure to EMF (60 Hz, 0.1 or 1 mT), which is the limit of occupational exposure, has no influence on intracellular Ca2+ mobilization and cellular function in RBL 2H3 cells.
beta-N-Acetylhexosaminidases ; Electromagnetic Fields ; Exocytosis ; Ionomycin ; Melitten ; Occupational Exposure ; Thapsigargin

To clarify the effect of extracellular ATP in cultured C6 glioma cells, ATP-induced cytosolic free calcium ((Ca2+)i) mobilization and cell proliferation were investigated. ATP-induced (Ca2+)i increased in a dose-dependent manner (10-7 M apprx 10-3 M). ATP-induced (Ca2+)i increases were slightly slowed in extracellular calcium-free conditions especially in sustained phase. ATP-induced (Ca2+)i increment was also inhibited by the pretreatment of U73122, a phospholipase C (PLC) inhibitor, in a time-dependent manner. Suramin, a putative P2Y receptor antagonist, dose-dependently weakened ATP-induced (Ca2+)i mobilization. Significant increases in cell proliferation were observed at 2, 3, and 4 days after ATP was added. Stimulated cell proliferation was also observed with adenosine at days 2 and 3. This cell proliferation was significantly inhibited by the treatment with suramin. Ionomycin also stimulated cell proliferation in a concentration-dependent manner. In conclusion, we suggest that extracellular ATP stimulates C6 glioma cell proliferation via intracellular free calcium mobilization mediated by purinoceptor.
Adenosine ; Adenosine Triphosphate* ; Calcium* ; Cell Proliferation* ; Cytosol ; Glioma* ; Ionomycin ; Receptors, Purinergic ; Suramin ; Type C Phospholipases

We established an in vitro experimental system using the following procedure. We first introduced tritium-labeled norepinephrine ([3H]-NE) into PC12 cells. The [3H]-NE incorporated-PC12 cells were stimulated by a high concentration (60 mM) of K+ buffer during 12 minutes. Then, we collected 100 microliter supernatant and counted the amount of [3H]-NE release from PC12 cells with a scintillation counter. After screening fungal, Streptomyces spp. or bacterial product using this experimental sytem, we obtained FS390 from Streptomyces spp. which inhibited [3H]-NE release from PC12 cells. FS390 also inhibits the release of ATP as a neurotransmitter of PC12 cells and rat cortical neurons. The inhibitory effect was seen even when the PC12 cells were treated with low K+ buffer containing ionomycin (1 micrometer) as an ionopore. This result suggests that the inhibitory action of FS390 on neurotransmitter release appeared after the influx of Ca2+.
Adenosine Triphosphate ; Animals ; Exocytosis ; Ionomycin ; Mass Screening* ; Neurons ; Neurotransmitter Agents* ; Norepinephrine ; PC12 Cells* ; Rats ; Scintillation Counting ; Streptomyces

FS390, a novel microbial metabolite from Streptomyces spp. was identified as a small molecular substance and shown a inhibition activities for the release of neurotransmitter from rat hippocampal neuron and PC12 cells. FS390 is an inhibitor of trifiated norepinephrine ([3H]-NE) release in high K+ buffer solution containing ionomycin, indicating that FS390 inhibits neurotransmitter release after the influx of Ca2+ ions. When examined the effect of FS390 on beta-glucuronidase release from guinea pig neurophils, FS390 inhibited beta-glucuronidas release: when treated with 5 microgram/mL of FS390, which was not induced cellular cytotoxicity. The fact that the beta-glucuronidase release in neutrophil and norepinephrine release in neuron was inhibited suggests the similarity in the locations and the mechanisms of FS390 action targets. When treated with 5 microgram/mL of FS390, [3H]-NE release and neurite extension for both rat hippocampal neurons and PC12 cells were prevented. These observations of FS390 functioning as an inhibitor of neurotransmitter release suggest that FS390 has an important role in synaptic transmission in neuron.
Animals ; Exocytosis* ; Glucuronidase ; Guinea Pigs ; Ionomycin ; Ions ; Neurites* ; Neurons* ; Neurotransmitter Agents ; Neutrophils ; Norepinephrine ; PC12 Cells* ; Rats* ; Streptomyces ; Synaptic Transmission

BACKGROUND AND OBJECTIVES: It has been known that the motility of the outer hair cell controls the physiological characteristics of the organ of Corti. Motility can be divided into two different types: fast and slow motility. Slow motility can be induced by high concentration of KCl and increase of intracellular Ca2+ concentration. In this study, authors aimed to define the effect of acetylcholine, one of the efferent neurotransmitters, on the slow motility of the outer hair cells of guinea pig. MATERIALS AND METHOD: Outer hair cells were isolated from guinea pigs by enzymatic and mechanical dissociation. The length of the hair cells was recorded by CCD camera equipped on an inverted microscope. Slow motility was induced by 10 (micro)M of ionomycin and 150 mM of KCl. Carbamylcholine (1 mM), a non-hydrolyzable derivative of acetylcholine, was used to observe the effect of acetylcholine and choline chloride (1 mM) was used as control. RESULTS: The length of outer hair cell was decreased after adding 150 mM of KCl and increased after adding 10 (micro)M of ionomycin. Stimulation of carbamylcholine (1 mM) did not induce the length change of the outer hair cells. Preincubation of 1 mM of carbamylcholine also did not affect the length change induced by ionomycin or KCl in outer hair cells. CONCLUSION: We could suggest that carbamylcholine does not have an effect on the slow motility of outer hair cell induced by the change of osmotic pressure which was elicited by high potassium, or intracellular Ca2+ increase.
Acetylcholine ; Animals ; Calcium* ; Carbachol ; Choline ; Guinea Pigs ; Hair* ; Ionomycin ; Neurons, Efferent ; Neurotransmitter Agents ; Organ of Corti ; Osmotic Pressure ; Potassium Chloride ; Potassium*

Ion channels in carcinoma and their roles in cell proliferation are drawing attention. Intracellular Ca2+ ([Ca2+]i)-dependent signaling affects the fate of cancer cells. Here we investigate the role of Ca(2+)-activated K+ channel (SK4) in head and neck squamous cell carcinoma cells (HNSCCs) of different cell lines; SNU-1076, OSC-19 and HN5. Treatment with 1 microM ionomycin induced cell death in all the three cell lines. Whole-cell patch clamp study suggested common expressions of Ca(2+)-activated Cl- channels (Ano-1) and Ca(2+)-activated nonselective cation channels (CAN). 1-EBIO, an activator of SK4, induced outward K+ current (ISK4) in SNU-1076 and OSC-19. In HN5, ISK4 was not observed or negligible. The 1-EBIO-induced current was abolished by TRAM-34, a selective SK4 blocker. Interestingly, the ionomycin-induced cell death was effectively prevented by 1-EBIO in SNU-1076 and OSC-19, and the rescue effect was annihilated by combined TRAM-34. Consistent with the lower level of ISK4, the rescue by 1-EBIO was least effective in HN5. The results newly demonstrate the role of SK4 in the fate of HNSCCs under the Ca2+ overloaded condition. Pharmacological modulation of SK4 might provide an intriguing novel tool for the anti-cancer strategy in HNSCC.
Carcinoma, Squamous Cell* ; Cell Death* ; Cell Line ; Cell Proliferation ; Head* ; Ion Channels ; Ionomycin ; Neck* ; Neoplasms, Squamous Cell

Swiprosin-1 exhibits the highest expression in CD8+ T cells and immature B cells and has been proposed to play a role in lymphocyte biology through actin remodeling. However, regulation of swiprosin-1 gene expression is poorly understood. Here we report that swiprosin-1 is up-regulated in T cells by PKC pathway. Targeted inhibition of the specific protein kinase C (PKC) isotypes by siRNA revealed that PKC-theta is involved in the expression of swiprosin-1 in the human T cells. In contrast, down-regulation of swiprosin-1 by A23187 or ionomycin suggests that calcium-signaling plays a negative role. Interestingly, swiprosin-1 expression is only reduced by treatment with NF-kappaB inhibitors but not by NF-AT inhibitor, suggesting that the NF-kappaB pathway is critical for regulation of swiprosin-1 expression. Collectively, these results suggest that swiprosin-1 is a PKC-theta-inducible gene and that it may modulate the late phase of T cell activation after antigen challenge.
Actins ; Biology ; Calcimycin ; Down-Regulation ; Gene Expression ; Humans ; Ionomycin ; Lymphocytes ; NF-kappa B ; Precursor Cells, B-Lymphoid ; Protein Kinase C ; Protein Kinases ; RNA, Small Interfering ; T-Lymphocytes

PURPOSE: The alpha-melanocyte-stimulating hormone (alpha-MSH) has been shown to interact with various cells of the immune and inflammatory system and down-regulate either the production or the action of the pro-inflammatory cytokines. In this study, we investigated the potential of alpha-MSH on preventing pancreatic islet cell from death and dysfunction by inflammatory cytokines released from peripheral blood mononuclear cells (PBMCs) in rat. METHODS: Rat pancreatic islets were co-cultured with PBMCs, stimulated by phorbol myrstic acid and ionomycin. alpha-MSH was treated to PBMCs for 2 hours before co-culture. Viability and apoptosis of islets were observed by MTT and FACS. Inflammatory cytokines and nitric oxide (NO) were measured. Insulin release from islet co-cultured with mononuclear cells was checked for the islet function. RESULTS: In comparison to control group, viability of islets with alpha-MSH treated mononuclear cells was increased and apoptosis was reduced significantly. Inflammatory cytokines such as TNF-alpha and IL-1beta were reduced in alpha-MSH-treated group. NO production in alpha-MSH-treated group was decreased. Insulin secretory function of islet was recovered in condition of alpha-MSH treatment. CONCLUSION: This study demonstrates that alpha-MSH protects cell death and preserves the secretory function of pancreatic islet cells from the pro-inflammatory reaction of mononuclear cells, and may have the potential to improve the graft survival in clinical islet transplantation.
alpha-MSH ; Animals ; Apoptosis ; Cell Death ; Coculture Techniques ; Cytokines ; Graft Survival ; Insulin ; Ionomycin ; Islets of Langerhans Transplantation ; Islets of Langerhans* ; Nitric Oxide ; Rats ; Tumor Necrosis Factor-alpha

BACKGROUND: Systemic lupus erythematosus (SLE) is one of chronic autoimmune diseases of which the central pathophysiologic derangement has not been yet established. Recently, it has been suggested that immune-regulatory cells might affect the development of autoimmune diseases such as SLE and RA. NKT cells were reported to be strong candidate for regulatory cells to regulate immune responses in vivo. To elucidate the roles of immune regulatory cells in the pathogenesis of SLE, we investigated the fractional distribution and functional status of NK and NKT cells in peripheral blood mononuclear cells (PBMC) of SLE patients and healthy volunteers. METHODS: Twenty-two SLE patients and 18 age-matched healthy volunteers were included in this study. The analysis for NK and NKT cells fraction in PBMCs of patients and normal controls were performed by flow cytometric analysis. In addition, to explore the functional status of these cells in SLE patients, we stimulated PBMCs using phorbol ester and ionomycin and measured cytoplasmic IL-4 and IFN-gamma by flow cytometry. RESULTS: The number (percentage) of NK cells was lower in SLE patients (CD3-CD56+: 4.93+/-1.30%, CD3-CD94+: 4.03+/-1.00%) than in controls (11.28+/-1.77%, 8.15+/-1.40%; p< 0.01, respectively). Peripheral NK cell numbers negatively correlated with anti-dsDNA Ab levels (r=-0.431, p< 0.05) and ESR (r= -0.475, p< 0.05). However, the percentage of these cells was not correlated with renal activity or corticosteroid doses. SLE patients showed, compared with controls, significantly decreased numbers of NKT cells (CD3+CD56+: 1.79+/-0.42% vs 5.04+/-0.44%, CD3+CD94+: 1.21+/-0.27% vs 4.39+/-0.45%; p< 0.01, respectively). The cytoplasmic expression of IL-4 and IFN-gamma in NK and NKT cells of SLE patients stimulated using phorbol ester and ionomycin were almost similar to those of normal controls, suggesting the NKT cells from SLE patients are functionally intact. CONCLUSION: Our results suggested that the decreased numbers of immune regulatory cells were associated with the immune dysregulation of SLE patients. The cellular replacement of NKT cells may be one of useful therapeutic approaches for autoimmune diseases such as SLE.
Autoimmune Diseases ; Cytoplasm ; Flow Cytometry ; Healthy Volunteers ; Humans ; Interleukin-4 ; Ionomycin ; Killer Cells, Natural* ; Lupus Erythematosus, Systemic* ; Natural Killer T-Cells