Calcium ionophore A23187 elevates angiotensin-converting enzyme in cultured bovine endothelial cells

Calcium ionophore A23187 (0.3-0.4 microM) elevated cellular angiotensin-converting enzyme activity (ACE) 2-7-fold after 48 h incubation with bovine pulmonary artery endothelial cells in culture. Cycloheximide (0.1 micrograms/ml) blocked the elevation in ACE produced by A23187. The increase in ACE was inhibited by 0.2 mM EGTA, 50 microM verapamil and 50 microM nifedipine, and was not associated with changes in cellular cAMP. Melittin, a phospholipase A2 activator, or addition of exogenous arachidonic acid failed to reproduce the elevation, and indomethacin only partially blocked the A23187 effect. The elevation of ACE was also inhibited by the calcium-calmodulin inhibitor, calmidazolium. Thus, we postulate that the ionophore A23187 elevates ACE in endothelial cells through a calcium-dependent mechanism other than phospholipase A2 activation. The elevation depends on new protein synthesis and involves calcium-calmodulin-dependent cellular mechanisms.     

Calcium ionophore A23187 calcium-dependent cytolytic degranulation in human eosinophils

The divalent cation ionophore A23187 is frequently used for studies of eosinophil degranulation. Nonetheless, the mechanism whereby A23187 induces degranulation in human eosinophils is still unclear. In the present experiments, A23187 caused human eosinophils to release a granule protein, eosinophil-derived neurotoxin (EDN) and a membrane-associated protein, Charcot-Leyden crystal (CLC) protein in a calcium and a concentration-dependent manner. However, A23187 at a concentration (1 microgram/ml) that caused 15% EDN release and 30% CLC protein release also produced release of the cytoplasmic enzyme lactic dehydrogenase (LDH) and loss of cell viability, both of which were calcium dependent. CLC protein release preceded EDN release and was detectable even at 15 min after the addition of 1 microgram/ml A23187, whereas EDN release occurred after a lag period of 30 min, and coincided with LDH release. At 1 microgram/ml A23187, neither the release of LDH nor the loss of viability occurred with purified neutrophils obtained in the same blood sample as a by-product of eosinophil purification. Electron microscopic examination demonstrated that exposure to A23187 for 15 min resulted in an increase and elongation of microridges on the cell surface, and exposure for 45 min caused cell disruption followed by extrusion of membrane-bound granules through breaks in the plasma membrane. Only once was granule exocytosis observed. These results indicate that A23187 treatment of eosinophils causes an initial release of membrane-associated CLC protein by a noncytolytic mechanism, and causes degranulation as a result of eosinophil lysis.     

Calcium ion-flux across phosphatidylcholine membranes mediated by ionophore A23187

The antibiotic A23187 carries Ca²⁺ across Müller-Rudin membranes made from $\text{1,2-}\text{dierucoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphocholine}$ and $\text{n-}\text{decane}$. The conductance of the membranes is not increased by the Ca²⁺-transport. The flux depends linearly on Ca²⁺ concentration and ionophore concentration (above pH 6). It increases with increasing pH, approximately by a factor of 4–5 between pH 6 and pH 8. Maximal Ca²⁺-fluxes of about $\text{10}{}^{\mathrm{?10}}\text{mol}\text{·}\text{cm}{}^{\mathrm{?2}}\text{·}\text{s}{}^{\mathrm{?1}}$ were found. A counter transport of H⁺ could not be detected.The complex formation between A23187 and Ca²⁺ in egg phosphatidylcholine vesicles was studied spectroscopically. The results are consistent with the formation of a 2 : 1 complex. Optical absorption measurements on single phosphatidylcholine membranes were used to calculate the concentration of membrane-bound ionophore A23187.     

Calcium ionophore A 23187 induces arachidonic acid release from phosphatidylcholine in cultured human endothelial cells

Cultured endothelial cells from human umbilical vein were incubated with (³H)arachidonic acid for 24 hours. The label was incorporated into phospholipids (79.3 %), neutral lipids (15.6 %) and non-esterified fatty acids (4.7 %). Upon challenge with the calcium ionophore A 23187, 5.3 % of the total radioactivity were found in supernatant and corresponded to 6-keto-prostaglandin F_1α (1.6 %) and free arachidonic acid (3.7 %). This release was accompanied by a concomitant and selective decrease of phosphatidylcholine. It is concluded that the entry of calcium promoted by A 23187 activates a phospholipase A₂ regulating the availability of arachidonic acid to the prostacyclin synthetase.     

Morphological changes in cultured mammalian cells: prevention by the calcium ionophore A23187.

The morphological changes induced by butyrate in HeLa cells and by monobutyryl or dibutyryl cAMP in CHO cells are prevented by micromolar concentrations of the divalent cation ionophore A23187. The ionophore is unable to prevent such changes in medium from which calcium is omitted. At slightly higher (but nontoxic) concentrations, the ionophore inhibits the butyrate-mediated induction of the ganglioside biosynthetic enzyme, sialyltransferase, in HeLa. In CHO, sialyltransferase activity is normally high and not altered by any of the compounds tested.     

Le ionophore A23187

The acrosomic status of spermatozoa prepared for IVF has been evaluated by means of immunofluorescence test from Fenichel and Hsi using calcium A 23187 ionophore as inductor of acrosome reaction (AR). The spontaneous AR remains slight, even after 6 hour-incubation in Menezo B2 (6,8+2,7%). The response to ionophore, moderate before (11,2+9%), frankly increases after a 6h-capacitation (28,9+8,3%) in a group of 25 IVF couples (tubal indication, normal semen, positive fertilization). Nevertheless, it remains slight or null in 4 cases of unexplained repeated failure of fertilization. The response to ionophore A 23187 allows to explore the kinetics of capacitation of spermatozoa and their ability to perform AR. Its significance in terms of fecondance remains to be precised.     

Calcium ion-flux across phosphatidylcholine membranes mediated by ionophore A23187.

The antibiotic A23187 carries Ca2+ across Müller-Rudin membranes made from 1,2-dierucoyl-sn-glycero-3-phosphocholine and n-decane. The conductance of the membranes is not increased by the Ca2+-transport. The flux depends linearly on Ca2+ concentration and ionophore concentration (above pH 6). It increases with increasing pH, approximately by a factor of 4-5 between pH 6 and pH 8. Maximal Ca2+-fluxes of about 10(-10) mol-cm-2-s-1 were found. A counter transport of H+ could not be detected. The complex formation between A23187 and Ca2+ in egg phosphotidylcholine vesicles was studied spectroscopically. The results are consistent with the formation of a 2:1 complex. Optical absorption measurements on single phophatidylcholine membranes were used to calculate the concentration of membrane-bound ionophore A23187.     

Calcium ionophore A23187 reveals calcium related cellular stress as "I-Bodies": an old actor in a new role

Calcimycin (A23187) is an ionophore widely used in studies related to calcium dynamics in cells, but its fluorometric potential to reveal intracellular physiology has not been explored. Exploiting the microenvironment-induced changes in its fluorescence, we show that a brief exposure of cells to non-toxic concentrations (≤3 μM) of the ionophore results in the characteristic organization of the ionophore forming brightly fluorescent cytoplasmic bodies termed “I-Bodies”, which are closely related to stress linked disturbances/changes in calcium homeostasis. “I-Bodies” appear to be Ca²⁺ rich intracellular sites formed during stress-induced release of intracellular Ca²⁺, causing dysfunction and aggregation of mitochondria, providing scaffold for high density packing of A23187. Formation of “I-Bodies” in cells exposed to ionizing radiation and certain anticancer drugs suggest their potential in revealing alterations in calcium signaling and mitochondrial function during (related to) macromolecular damage-induced cell death. The absence of “I-Bodies” in non-malignant cells and their varying numbers in malignant cells with 5 fold increase in fluorescence imply that they can be potential biomarkers of cancer. Thus, “I-Bodies” are novel indicators of endogenous and induced stress linked to disturbances in calcium homeostasis in cells, with a potential to serve as biomarker of cancer.     

Calcium and ionophore A23187 induce the sickle cell membrane phosphorylation pattern in normal erythrocytes

Pre-treatment of normal erythrocytes with micromolar Ca²⁺ and ionophore A23187 induces abnormal phosphorylation of membrane polypeptides, as determined by labeling with exogenous ³²P_i. The Ca²⁺-induced effects, which include increased incorporation of ³²P into acid-stable linkages and increased labeling in the Band 3 and 4.5–4.9 regions of SDS gels, are similar to those seen in untreated sickle erythrocytes. Part of the abnormal phosphorylation of sickle cells may be caused by their elevated intracellular Ca²⁺ levels.     

Calcium and ionophore A23187 stimulates deposition of extracellular matrix and acetylcholinesterase release in cultured myotubes

Summary Calcium (Ca²⁺) and calcium-transporting ionophores stimulate protein secretion in many cellular systems. We demonstrate here that increases in intracellular calcium concentration induce a time- and concentration-dependent deposition of extracellular matrix and an increase in acetylcholinesterase secretion. Scanning and transmission electron-microscopy revealed that treatment with the calcium ionophore A23187, or high extracellular Ca²⁺ levels (5 mM to 15 mM) produce significant deposits of extracellular matrix around the myotubes, as well as a marked increase in the acetylcholinesterase reaction-product. Blocking muscle contraction was not necessary for the induction of AChE secretory activity. Sucrose density-gradients of media conditioned by muscle cells revealed 3 separate acetylcholinesterase molecular forms. However, incubation with A23187 increased only the 4.5 S and the 7.2 S molecular forms, whereas the 12.0 S form showed no significant differences from controls. Polyacrylamide gel electrophoresis, and autoradiography using [³H]diisopropyl fluorophosphate revealed a broad band at 65000 daltons. This band was broader than for controls when medium was obtained from A23187-treated cells. Our results show that increasing intracellular Ca²⁺ concentration induces marked deposition of extracellular matrix and increased acetylcholinesterase secretion, with an apparent selectivity for the monomeric and dimeric acetylcholinesterase molecular forms.     

Effects of ionophore A23187 on calcium fluxes from cultured adrenal cells

The effect of the calcium ionophore A23128 on calcium fluxes from Y-1 adrenal cortical cells was investigated. Conditions were chosen which are known to result in an inhibition of steroidogenesis (6 . 10(-6) M ionophore and 3 . 10(-4) M extracellular calcium). Calcium efflux from Y-1 cells exhibited two distinct phases. A fast phase which was insensitive to the mitochondrial poison sodium azide and a slow, azide-sensitive phase. The ionophore brought about a rapid increase in the rate of calcium efflux and an 84% reduction in the size of the calcium pool which was associated with the slow efflux phase as well as a reduction in its rate constant. A decrease in the size of the rapidly exchanging calcium pool was also detected. Ethanol, the solvent which was used for the ionophore, slightly increased the rate constant of the rapidly exchanging pool. Conditions which resulted in diminished steroidogenic capacity also brought about a reduction in the size of an energy dependent, intracellular pool. The data is interpreted as being consistent with a hypothesis that the ionophore-induced inhibition of steroidogenesis may be causatively related to the loss of intracellular calcium or to the mechanism which brings about the loss.     

Nitrite is a signaling molecule and regulator of gene expression in mammalian tissues

Mammalian tissues produce nitric oxide (NO) to modify proteins at heme and sulfhydryl sites, thereby regulating vital cell functions. The majority of NO produced is widely assumed to be neutralized into supposedly inert oxidation products including nitrite (NO2(-)). Here we show that nitrite, also ubiquitous in dietary sources, is remarkably efficient at modifying the same protein sites, and that physiological nitrite concentrations account for the basal levels of these modifications in vivo. We further find that nitrite readily affects cyclic GMP production, cytochrome P450 activities, and heat shock protein 70 and heme oxygenase-1 expression in a variety of tissues. These cellular activities of nitrite, combined with its stability and abundance in vivo, suggest that this anion has a distinct and important signaling role in mammalian biology, perhaps by serving as an endocrine messenger and synchronizing agent. Thus, nitrite homeostasis may be of great importance to NO biology.     

Structural and functional changes in root cells induced by calcium ionophore A23187

The shifts of Ca²⁺, K⁺ and proton homeostasis of wheat (Triticum aestivum L. M. cv Ljuba) root cells induced by the Ca²⁺-ionophore A23187 caused different responses, depending on the time of exposure to the ionophore. Oxygen consumption and heat production by roots were increased when the Ca²⁺-specific effect of A23187 was expressed. Ultrastructural re-organization of cell organelles was found to follow the ion shifts. The endoplasmic reticulum, Golgi apparatus and mitochondria rearranged their membranes following treatment. The increased ion permeability of root cell membranes is proposed to cause an excessive energy expenditure for the restoration of ion homeostasis.     

Stepwise activation of T cells. Role of the calcium ionophore A23187

The calcium ionophore A23187, at a concentration of 1 microgram/ml, is able to stimulate proliferation of freshly isolated peripheral blood lymphocytes, CD4+-enriched cells, or CD8+-enriched cells as measured by [3H]thymidine incorporation. This proliferation is accompanied by an increase in interleukin 2 (IL-2) receptor expression but not by a detectable up-regulation in (IL-2) production or the development of cytotoxicity. Proliferation can be blocked by anti-CD3, CD4, or CD8 monoclonal antibodies, but not by anti-Tac. If CD8+-enriched cells are activated for 3 days with A23187 and the blasts present on day 3 are sorted and returned to culture, they rapidly develop cytolytic activity in the presence of recombinant IL-2 but not recombinant interferon-gamma. CD4+ enriched cells, after activation with A23187, do not become cytotoxic in the presence of either recombinant IL-2 or recombinant interferon-gamma. These findings permit study of the stepwise maturation of T cells in this alternative pathway by using "minimal signals" that do not, by themselves and as used in these studies, stimulate precursor Tc to mature to full effector cytotoxic function. These findings are consistent with the model that A23187 drives T cells only part way along a pathway of maturation and that an additional second signal must be given to effect maturation of cytotoxic status.