Effects of intracellular Na+, K+ and Cl- on Ca(2+)-regulated exocytosis activated by 10 microM acetylcholine (ACh) were studied in guinea-pig antral mucous cells which are permeabilized by nystatin treatment. Ca(2+)-regulated exocytotic events were modulated by [Na+]i, [K+]i and [Cl-]i via mediation of PTX-sensitive G proteins. Increases in [Na+]i and PTX inhibit G protein (G(Na)), which suppressed the exocytosis. Increases in [K+]i caused the exchange of G proteins (from G(Na) to G(K)) to increase, and GK evoked activation of the exocytosis and was inhibited by PTX. Increases in [Cl-]i and PTX inhibit G protein (G(Cl)), which stimulates exocytotic events. Based on these observations, the exocytosis in antral mucous cells were modulated by intracellular ions, concentration of which were increased or decreased by cell volume changes caused by Ach.
Acetylcholine/pharmacology ; Animal ; Cell Membrane Permeability/drug effects ; Exocytosis/physiology* ; Exocytosis/drug effects ; Gastric Mucosa/metabolism ; Gastric Mucosa/cytology ; Guinea Pigs ; Hypertonic Solutions/pharmacology ; Ionophores/pharmacology ; Nystatin/pharmacology ; Pertussis Toxins/pharmacology ; Potassium/pharmacokinetics* ; Pyloric Antrum/metabolism* ; Pyloric Antrum/cytology ; Sodium Chloride/pharmacokinetics* ; Vasodilator Agents/pharmacology

AIMTo examine the ability of calcium ionophore (CI) to induce tryptase and histamine release from human mast cells and its mechanisms.

METHODSEnzymatically dispersed cells from human colons were challenged with CI, and the cell supernatants after challenge were collected. Tryptase release was determined with a sandwich ELISA procedure and histamine release was measured using a glass fibre-based fluorometric assay.

RESULTSCI was able to induce a concentration dependent release of histamine and tryptase from human colon mast cells following 15 min incubation. The maximum of induced histamine and tryptase release were approximately 5.3 and 2.8 fold more than the levels of spontaneous release, respectively. CI at the concentrations higher than 1.0 micromol/L was able to induce significantly more histamine than tryptase release from mast cells. The time course revealed that the action of CI on mast cells started from 10 s, peaked at 6 min and lasted at least 15 min following incubation. Pertussis toxin and metabolic inhibitors were able to inhibit mast cell response to CI.

CONCLUSIONHuman colon mast cells were able to release tryptase and histamine in response to CI. The process seemed to be associated with the activation of a G-protein coupled receptor on the membrane of mast cells and requires cell energy supply.

Calcium Ionophores ; pharmacology ; Cells, Cultured ; Colon ; cytology ; Histamine ; metabolism ; Humans ; Mast Cells ; drug effects ; metabolism ; secretion ; Tryptases ; metabolism

BACKGROUND: Apoptosis is a highly selective form of cell suicide with characteristic morphologieal and biochemical features, including chromatin condensation, formation of apoptotic bodies, and DNA fragmentation by the activation of endonucleases. Various cytokines and physical or chemical factors can provoke apoptotic changes in the skin. OBJECTIVE: We investigated the cytotoxic effects with epidermal cytokines and their combinations, K+ ionophores, protein synthesis inhibitor(emetine), inhibitor of endogenous endonuclease(aurintricarboxylic acid, ATA), sodium azide, and retinoic acid witp human epithelial tumor cell lines(A431 cells) to examine the degree of induction of apoptosis in the epidermal keratinocytes. METHODS: Induction of apoptosis was measured in cultured human keratinocytes, keratinocyte cell lines(A-431, HaCat, KB cells), cultured human melanocytes and malignant melanoma cell lines(SK-28, SK-30) using a mixture of ethidium bromide and acridine orange, DNA agarose gel electrophoresis and TUNEL staining. RESULTS: l. In the A-431 cells, (1 to a certain degree, the combination of IFN-gamma and TNF-alpha could only induce apoptosis. Q2 most of K+ ionophores were observed to induce necrosis rather than apoptosis. Q3 emetine, a protein synthesis blocker, was found to induce apoptosis in a dose-dependent pattern. Q4 sodium azide at a concentration of 1% .induced apoptosis rather than necrosis. Q5 retinoic acid inhibit the beuvericin induced apoptosis. 2. In human keratinocytes, Ql more resistant in the induction of apoptosis than any cultured keratinocyte cell lines p aurintricarboxylic acid(ATA)-an endonuclease inhibitor, could inhibit UV induced apoptosis 3. In human keratinocytes and cultured keratinocyte cell lines, c-PAF inhibit the beauvericin induced apoptosis. 4. Human melanocytes is very resistant for the induction of apoptosis by beauvericin. 5. In the melanocytes and melanoma cell lines, sodium azide and beauvericin induced necrosis rather than apoptosis. CONCLUSIONS: The epidermis is continuously exposed to toxic factors which might induce cell death. With the above results, the induction of appeared to be rather resistant, epidermal cell apoptosis which may reflect the existence of some endogenous protective mechanisms in the epidermis to survive at certain toxic environments; melanocytes showed high expression of bcl-2 protein which could play a role in endogenous defense against toxic environments of the epidermis.
Acridine Orange ; Apoptosis* ; Cell Death ; Cell Line ; Chromatin ; Cytokines ; DNA ; DNA Fragmentation ; Electrophoresis, Agar Gel ; Emetine ; Endonucleases ; Epidermis ; Ethidium ; Humans ; In Situ Nick-End Labeling ; Ionophores ; Keratinocytes ; Melanocytes ; Melanoma ; Necrosis ; Skin ; Sodium Azide ; Suicide ; Tretinoin ; Tumor Necrosis Factor-alpha

We studied medium alkalinization in Salvia miltiorrhiza suspension cultures treated with salicylic acid and the effect of Ca2+ in this process through application of calcium channel antagonists (Verapamil, LaCl3, LiCl, 2-APB) and ionophore A23187. The results show that salicylic acid could induce significant medium alkalinization in S. miltiorrhiza culture. Verapamil and LaCl3 or LiCl and 2-APB, two different groups of calcium channel antagonist, significantly inhibited the medium alkalinization induced by salicylic acid. However, the suppression effect of verapamil or LaCl3 on medium alkalinization induced by salicylic acid was higher than that of LiCl or 2-APB. When two types of calcium channel inhibitor (LaCl3 and 2-APB) were used together, the medium alkalinization induced by salicylic acid was completely suppressed and even reduced the pH in medium. On the other hand, A23187 could promote the medium alkalinization. Based on the results above, we speculated that salicylic acid could induce significant medium alkalinization in S. miltiorrhiza culture, depending on the calcium from both extracell and intracell. Moreover, calcium from extracell plays a more dominant role in this process. Reveal of relationship in this research between Ca2+ and medium alkalinization can provide theory evidence for mechanism of the plant secondary metabolism.
Calcimycin ; pharmacology ; Calcium ; chemistry ; Calcium Channel Blockers ; pharmacology ; Calcium Ionophores ; pharmacology ; Cell Culture Techniques ; Culture Media ; chemistry ; Salicylic Acid ; pharmacology ; Salvia miltiorrhiza ; metabolism ; Verapamil ; pharmacology

Endothelin(ET), is the most potent endogenous vasoconstrictor. Myocardial ischemia and chemical stimuli including calcium ionophores are known to release ET-1. Recently, halothane has been shown to block calcium channel. Thus, halothane might attenuate coronary endothelial ET-1 production during myocardial ischemia-reperfusion. To test this hypothesis, we measured plasma ET-1 level continuously in open chest dogs subjected to 15 min of left anterior coronary arterial occlusion and 1 hour of reperfusion during fentanly(n=8) or halothane(n=7) anesthesia. The results were as follows. I) Baseline ET-1 levels of both femoral artery and great cardiac vein in the halothane group were lower than in the fentanly group(NS). 2) ET-1 level of femoral artery and great coronary vein in both halothane and fentanyl group remained unchanged 10 min into ischemia. 3) Coronary blood flow increased by 325, 250% in the halothane group and by 315, 258% in the fentanly group 2, 5 min into reperfusion, respectively. 4) ET-1 production increased from baseline of -2.9+/-1.7 pg/min to 66.0+/-21.5(p<0.05), 20.8+/-5.1 (p<0.01), 13.2+/-6.2(p<0.05) pg/min 5, 15, 30 min into reperfusion, respectively in the fentanyl group, but it remained unchanged from baseline of 0.8+/-3.1 pg/min in the halothane group. These findings suggest that ET-1 production or release is diminished by halothane during myocardial ischemia-reperfusion. Thus, halothane provides an advantage over fentanyl in patients with myocarial ischemic episodes.
Analgesics ; Anesthesia* ; Anesthetics ; Animals ; Calcium Channels ; Calcium Ionophores ; Coronary Vessels ; Dogs* ; Endothelin-1* ; Endothelins ; Femoral Artery ; Fentanyl* ; Halothane* ; Humans ; Ischemia ; Myocardial Ischemia ; Plasma ; Reperfusion ; Thorax ; Veins

The acrosome reaction is a prerequisite for fertilization, and its signaling pathway has been investigated for decades. Regardless of the type of inducers present, the acrosome reaction is ultimately mediated by the elevation of cytosolic calcium. Inositol 1,4,5-trisphosphate-gated calcium channels are important components of the acrosome reaction signaling pathway and have been confirmed by several researchers. In this study, we used a novel permeabilization tool BioPORTER^® and first demonstrated its effectiveness in spermatozoa. The inositol 1,4,5-trisphosphate type-1 receptor antibody was introduced into spermatozoa by BioPORTER^® and significantly reduced the calcium influx and acrosome reaction induced by progesterone, solubilized zona pellucida, and the calcium ionophore A23187. This finding indicates that the inositol 1,4,5-trisphosphate type-1 receptor antibody is a valid inositol 1,4,5-trisphosphate receptor inhibitor and provides evidence of inositol 1,4,5-trisphosphate-gated calcium channel involvement in the acrosome reaction in human spermatozoa. Moreover, we demonstrated that the transfer of 1,4,5-trisphosphate into spermatozoa induced acrosome reactions, which provides more reliable evidence for this process. In addition, by treating the spermatozoa with inositol 1,4,5-trisphosphate/BioPORTER^® in the presence or absence of calcium in the culture medium, we showed that the opening of inositol 1,4,5-trisphosphate-gated calcium channels led to extracellular calcium influx. This particular extracellular calcium influx may be the major process of the final step of the acrosome reaction signaling pathway.
Acrosome Reaction/physiology* ; Calcimycin/pharmacology* ; Calcium/pharmacology* ; Calcium Ionophores/pharmacology* ; Drug Delivery Systems ; Humans ; Inositol 1,4,5-Trisphosphate Receptors/metabolism* ; Male ; Progesterone/pharmacology* ; Spermatozoa/metabolism* ; Zona Pellucida/metabolism*

OBJECTIVETo investigate the pathogenesis of globozoospermia, fertilization ability of round-headed sperm, and the application value of assisted oocyte activation in intracytoplasmic sperm injection (ICSI) for the wives of glohozoospermia men.

METHODSWe collected oocytes from the wives of 2 globozoospermia patients and randomly divided them into two groups after ICSI to receive calcium ionophore A23187-activation and conventional treatment, respectively. We reviewed the relevant literature published at home and abroad, and discussed the etiology of globozoospermia, fertilization ability of round-headed sperm, and treatment options for this disease.

RESULTSQuality embryos were obtained in the A23187-activation group while no fertilized oocytes, oocyte cleavage, quality embryos, or blastular formation were found in the conventional treatment group. Both women achieved pregnancy and gave birth to healthy neonates after transfer of the quality embryos from the A23187-activation group.

CONCLUSIONCalcium ionophore A23187 can be applied to ICSI for the wives of globozoospermia men and bring about desirable clinical outcomes. Meanwhile, attention should be paid to its safety.

Calcimycin ; therapeutic use ; Calcium Ionophores ; therapeutic use ; Female ; Humans ; Infertility, Male ; drug therapy ; Male ; Oocytes ; Pregnancy ; Sperm Injections, Intracytoplasmic ; Spermatozoa ; abnormalities

Calcium Ionophores ; metabolism ; Female ; Fertilization in Vitro ; adverse effects ; Humans ; Infant, Newborn ; Karyotype ; Male ; Oocytes ; cytology ; metabolism ; Sperm Injections, Intracytoplasmic ; adverse effects

Toxoplasma gondii is an obligate intracellular protozoan parasite, which invades a wide range of hosts including humans. The exact mechanisms involved in its invasion are not fully understood. This study focused on the roles of Ca2+ in host cell invasion and in T. gondii replication. We examined the invasion and replication of T. gondii pretreated with several calcium modulators, the conoid extrusion of tachyzoites. Calmodulin localization in T. gondii were observed using the immunogold method, and Ca2+ levels in tachyzoites by confocal microscopy. In light microscopic observation, tachyzoites co-treated with A23187 and EGTA showed that host cell invasion and intracellular replication were decreased. The invasion of tachyzoites was slightly inhibited by the Ca2+ channel blockers, bepridil and verapamil, and by the calmodulin antagonist, calmidazolium. We observed that calcium saline containing A23187 induced the extrusion of tachyzoite conoid. By immunoelectron microscopy, gold particles bound to anti-calmodulin or anti-actin mAb, were found to be localized on the anterior portion of tachyzoites. Remarkably reduced intracellular Ca2+ was observed in tachyzoites treated with BAPTA/AM by confocal microscopy. These results suggest that host cell invasion and the intracellular replication of T. gondii tachyzoites are inhibited by the calcium ionophore, A23187, and by the extracellular calcium chelator, EGTA.
Animals ; Calcium/*physiology ; Calcium Channel Blockers/pharmacology ; Calmodulin/antagonists & inhibitors ; Chelating Agents/pharmacology ; Hela Cells ; Host-Parasite Relations ; Humans ; Ionophores/pharmacology ; Research Support, Non-U.S. Gov't ; Toxoplasma/drug effects/pathogenicity/*physiology

The aim of this study was to investigate the ability of calcium ionophore (CI ) to induce the differentiation of CML cells into dendritic cells (DC), to analyze the P210 expression in DCs and to evaluate the stimulatory effect of CML-DC on production of cytotoxic activity against CML cells via activating the autologous T cells. The mononuclear cells were isolated from bone marrow of CML patients whose WBC counts were more than 30x10(9)/L when samples were collected, then the lymphocytes and monocytes were discarded by pouring out supernatant twice at different culture time point. Slightly adherent cells were cultured in RPMI 1640 containing 10% FCS, with or without CI (375 ng/ml) and GM-CSF (200 ng/ml) at 37 degrees C, 5% CO2, fully humidified atmosphere for 96 hours. The cell morphology was observed under the inverted microscope and electron microscope; the expression of CD antigens was analyzed with flow cytometry; the P210 expression was measured with Western blot. LDH assay was used to evaluate the effect of cultured CML cells (CML-DC) generating cytotoxic T lymphocyte (CTL) activity against CML cells. The results indicated that after treatment with calcium ionophore and GM-CSF for 96 hours, CML cells showed DC morphological characteristics under inverted microscope and electron microscope. The expression of CD83, CD86, CD40, CD80 and HLA-DR increased remarkably. P210 was expressed in the CML-DC, but the expression level was lower than that in CML cells without CI and GM-CSF treatment. LDH assay showed that the CTL activity against CML was found greater in autologous T cells activated by CML-DC than that by CML cells. It is concluded that the CML cells can be induced to quickly differentiate into DC when cultured with CI and GM-CSF. CML-DC expresses P210, but the expression level is lower than that in CML cells. CML-DC can stimulate autologous T cells to produce CTL against CML.
Antigens, CD ; metabolism ; Bone Marrow Cells ; cytology ; Calcium ; pharmacology ; Cell Differentiation ; Dendritic Cells ; cytology ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Ionophores ; pharmacology ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; pathology ; Male ; Monocytes ; cytology ; Tumor Cells, Cultured