Stimulation of noradrenaline synthesis by the calcium ionophore A23187 and its modulation by presynaptic receptors

Noradrenaline (NA) synthesis in rat hippocampal synaptosomes is increased by calcium ionophore, A23187, in the presence of calcium. Activation of presynaptic muscarinic, gamma-aminobutyric acidB (GABAB) and alpha 2-adrenergic receptors causes inhibition of the ionophore-stimulated synthesis. The results suggest that different mechanisms mediate the depression by presynaptic receptors of NA release and NA synthesis.     

Interleukin 2 stimulates heart contractility in the presence of exogenous arachidonate or the calcium ionophore A 23187

An increase in the isometric developed tension (IDT) of isolated rat atria was observed shortly after the addition of human interleukin 2 (IL-2) to the organ preparation with subthreshold concentrations of either arachidonate (AA, 1.98 × 10⁻⁶ M) or the calcium ionophore A 23187 (1.9 × 10⁻⁶ M). Both natural purified IL-2 (nIL-2) and yeast recombinant IL-2 (rIL-2) were active in this experimental system. It was determined that this lymphokine was active at 2 × 10⁻¹¹ M, considering as a reference the specific activity of rIL-2. Anti-IL-2 monoclonal antibody (anti-IL-2 MAb) abolished this reaction. Inhibition of atrial phospholipase C activity by nitrocarboxyphenyl N,N-diphenylcarbamate (NCDC, 5 × 10⁻⁶ M) prevented the development of the inotropic positive effect of IL-2 in the presence of either AA or A 23187. The synthetic diacylglyceride 1-oleoyl, 2-acetyl-glycerol (OAG) replaced the IL-2 as stimulatory signal but NCDC had no effect on the reaction.The results suggest that IL-2 can alter the physiologic behaviour of the heart and that its mechanism of action is probably similar to the one proposed for other IL-2 targets (IL-2 receptor-positive T lymphocytes, T cell lines).     

Effect of cyclosporin A on lymphocyte activation by the calcium ionophore A23187

The activation of pig lymphocytes by the divalent cation ionophore A23187 is more sensitive to inhibition by cyclosporin A than is activation by plant lectins such as concanavalin A. Complete inhibition of A23187-induced activation was seen at a cyclosporin A concentration of 0.3 micrograms/ml. The very early stimulation of nucleoside uptake induced by A23187 was not affected by cyclosporin A, but other early metabolic changes occurring during the first few hours after activation were inhibited.     

The mode of action of the calcium ionophore A23187 on T cell proliferation. I. The ionophore does not replace lymphokines but acts via induction of IL-2 production on IL-2 responsive cells

The two compounds, the calcium ionophore A23187 and phorbol myristate acetate (PMA), which are not mitogenic for mouse thymocytes, induce proliferation when added in combination to thymocyte cultures. Short exposure to PMA renders the cells responsive to IL-2, added exogeneously. The cells rendered IL-2-responsive by PMA are enriched in the more mature peanut agglutinin (PNA)-negative population. The ionophore does not render PNA-negative thymocytes IL-2-responsive, but induces proliferation of PMA-pulsed PNA-negative thymocytes. PMA-pulsed PNA-negative thymocytes proliferating in response to either exogeneous IL-2 or ionophore express IL-2 receptors. However, the ionophore does not mimic IL-2 action but acts indirectly by induction of both IL-2 production and of IL-2 receptor expression via IL-2. This view is based on the following findings: 1) IL-2 could be detected in supernatants derived from PMA-preactivated thymocytes incubated with the ionophore; 2) The IL-2-induced proliferation, as well as the ionophore-induced proliferation, was specifically blocked by a monoclonal anti-IL-2-receptor antibody; 3) The proliferation induced by exogeneous IL-2, as well as that induced by the ionophore, could be specifically inhibited by metabolically blocked T lymphoblasts carrying IL-2 receptors competing with the responder cells for the available IL-2 added or produced in the system.     

Stimulation of proliferation of murine lymphocytes by the calcium ionophore A 23187 in the absence of serum: the requirement for thiols

The proliferation of mouse spleen cells and T-lymphocytes, initiated by the calcium ionophore A 23187 was studied by a serum-free culture technique. In contrast to Con A, A 23187 was capable of stimulating cells only if 2-mercaptoethanol, cysteine and glutathione (reduced form), respectively, were present in the culture medium. In the absence of one of these compounds a stimulating activity of A 23187 was observed only with high concentrations of cells (i.e., 10(7)/ml). With glutathione present, the cells could be stimulated only at concentrations of A 23187 which were found to be suboptimal in cultures with 2-mercaptoethanol. Human serum, fetal calf serum and bovine serum albumin shifted the active and optimally stimulating concentrations of A 23187 to higher values. A similar effect was observed with sera- and Con A-treated cells. The effect of sera and albumin was paralleled by a protecting effect of cells against high concentrations of A 23187.     

Activation of gastro-intestinal smooth muscle induced by the calcium ionophore A23187

Summary Tension development was recorded in isolated smooth muscle preparations from the guineapig, namely circular strips from the fundus and antrum region of the stomach, and taenia coli. The calcium ionophore A23187 (2·10^–6–2·10^–5 mol/l) induced maximum activity in fundus and taenia coli, and in antrum an activity slightly smaller than that obtained with acetylcholine (ACh) (5·10^–6 mol/l). The ionophore-induced activity could be suppressed by so-called calcium antagonists: D600 (3·10^–6 mol/l) suppressed the ionophore-induced activity of taenia coli completely; phasic and tonic components in the stomach preparations were selectively suppressed by D600 and sodium nitroprusside (10^–6 mol/l), respectively.     

Histamine release from rat mast cells induced by the ionophore A23187 in the absence of extracellular calcium

Conclusions These observations are consistent with the view that A23187 was mobilizing a cellular calcium pool trigger the histamine release mechanism. The effect on mast cell ATP content may be explained by an inhibition of oxidative ATP synthesis, possibly caused by an increased cytosolic calcium concentration.     

Mitogenic action of phorbol ester TPA and calcium ionophore A23187 on human cord and maternal/adult peripheral lymphocytes: regulation by prostaglandin E2

We have investigated the mitogenic action of the phorbol ester 12-O-tetrade-canoylphorbol-13-acetate (TPA) and the calcium ionophore A23187 on peripheral blood mononuclear leucocytes (PBML) from human neonates (cord), their mothers and other unrelated adults. TPA induced similar proliferation among maternal and unrelated adult PBML. In contrast, cord PBML regularly gave lower responses, on average 40-45% at optimal TPA concentration, than either maternal or other adult cells. A23187 had only a weak mitogenic effect on cord and maternal/adult cells. However, A23187 added together with TPA induced a strong proliferative response at low, non-mitogenic concentrations of either agent. Furthermore, TPA and the mitogenic OKT3 antibody showed a marked synergism (5- to 6-fold, on average) among cord PBML, whereas this effect was weaker (up to 2-fold) among maternal/adult cells. Prostaglandin E2 (PGE2), at 1.4 x 10(-6) M, inhibited cord and maternal/adult PBML proliferation induced by A23187 (an average 50% inhibition at optimal ionophore concentration). In contrast, PBML cultures stimulated by TPA or by TPA combined with A23187, OKT3 or PHA were virtually insensitive to PGE2-mediated suppression. Our results suggest that PGE2 can down-regulate ionophore-induced, receptor-mediated lymphocyte responses, whereas post-protein kinase C activation events (induced by TPA) are insensitive to PG-mediated inhibition.     

Influence of calcium and ionophore 23187 on tubular phosphate reabsorption

In previous studies it has been demonstrated that a decline of plasma calcium concentration accounts for the decrease of phosphate reabsorption in thyroparathyroidectomized (TPTX) rats undergoing phosphate loading.Microinfusion studies were performed in TPTX rats in order to discriminate between a systemic effect of calcium an a direct renal effect.Thyroparathyroidectomized animals were infused with a phosphate solution continuously. When plasma calcium concentration fell below 1.30 mmol/l, proximal convoluted tubules were microinfused with a phosphate tracer solution for 42 min. After 18 min a calcium chloride-containing solution was applied superficially (superfused) to the area of the microinfused tubule. This elevation of peritubular calcium concentration led to an immediate increase of phosphate reabsorption up to 12% of the microinfused phosphate load within 24 min.In another series of experiments, the calcium specific ionophore A 23187 — a substance which is known to increase intracellular calcium — was superfused on the microinfused tubule. This resulted again in an increase of fractional phosphate reabsorption of about 15% after 24 min. In contrast, when calcium chloride-free as well as ionophore-free solutions were superfused fractional phosphate reabsorption decreased (7%).From these data we conclude that 1. calcium has a direct renal effect on phosphate reabsorption in the absence of parathyroid hormone and 2. intracellular calcium appears to be a major parameter in the regulation of renal phosphate transport under these conditions.This study was supported by Dr. Legerlotz StiftungParts of this study were presented at the fall meeting of the Nephrologische Gesellschaft in Bonn, 1977 and at the spring meeting of the Deutsche Physiologische Gesellschaft in Göttingen, 1978     

Neurotoxicity of calcium ionophore A23187 in immature rat cerebellar slices

The causal role of Ca2+ in neuronal necrosis is controversial and it has been suggested that neuronal Ca2+ uptake is only a secondary effect to cell death. Here, I address this question directly by studying the morphological effects of calcium ionophore A23187 on immature cerebellar slices. Parasagittal slices were prepared and incubated for 30, 90 or 120 min in physiological saline with or without A23187. In some cases Ca2+ was omitted from the incubation medium. Slices were processed for light microscopy. A23187 produced nuclear changes indicative of cell death that encompassed cells of the external granule cell layer at short incubation times (30 min) and more deeply situated cells at longer times (120 min). This indicates that A23187 diffusion is limited in the slice. The histological changes produced by 30 min exposure to the ionophore could not be reversed by incubation for 90 min in normal medium. Necrosis was never observed when slices were exposed to A23187 in Ca2+-free medium. The results demonstrate that influx of excessive amounts of Ca2+ kills cells of the central nervous system.     

Conductance rise induced by the calcium ionophore A23187 in unfertilized eggs of tilapia.

Current-clamp measurements on unfertilized tilapia eggs gave linear current-voltage relations in the range 0 to -120 mV. Eggs had low resting potentials (-22 mV) and high specific membrane resistances (500 k omega cm2) and specific membrane capacitances (0.9 microF cm-2) indicating no marked folding of their plasma membrane. Application of A23187 induced a small depolarization and a conductance rise, the response having a reversal potential of about -16 mV. The results suggest that tilapia eggs have a calcium-gated conductance probably activated at fertilization.     

Role of mast cells in calcium ionophore (A23187)-induced peritoneal inflammation in mice

Several lines of evidence document a critical role for mast cells in immune complex-mediated inflammatory models. However, their role in nonimmune models of acute inflammation is largely unknown. In the present investigation, the role of mast cells was examined in calcium ionophore (A23187)-induced mouse peritoneal inflammation. Intraperitoneal injection of A23187 (20)g/mouse) elicited marked and transient increases in immunoreactive levels of 6-ketoprostaglandin-F₂, leukotrienes B₄, C₄, D₄, E₄, and F₄. There were no discernible differences in levels of these mediators in male Swiss Webster mice, mast cell-deficient mice (WBB6F1-W/W), and age-matched controls (WBB6F1-+/+), suggesting a minimal role of mast cells in eicosanoid biosynthesis in this model. However W/W mice showed smaller increases in levels of myeloperoxidase, a marker for neutrophils, compared to +/+ mice. Both W/W and +/+ mice have lower constitutive levels of peritonealN-acetyl--d-glucosaminidase (NAG), a marker for mononuclear cells. Similar to the changes seen in myeloperoxidase, W/W mice exhibited a blunted NAG response compared to +/+ mice. These results suggest that mast cell products other than eicosanoids may contribute to the changes in cellular trafficking in response to intraperitoneal A23187. These results also suggest that mast cells are required for full expression of inflammatory responses.     

Stimulus-secretion coupling in pancreatic acinar cells: influences of external sodium and calcium on responses to cholecystokinin-pancreozymin and ionophore A23187

1. The roles of Na and Ca ions in stimulus-secretion coupling were analysed in the isolated and perfused rat pancreas.2. Partial replacement of NaCl with LiCl produced a diminution in both amylase output and pancreatic juice flow which were induced by 5 m-u. CCK-PZ/ml., and almost normal responses were usually regained immediately after the reintroduction of a standard concentration of NaCl. Nearly total replacement of NaCl with LiCl caused an almost complete inhibition of the responses, although 25 mM-NaHCO₃ and 1 mM-NaH₂PO₄ were still present, and only partial recovery was obtained after the re-introduction of a standard concentration of NaCl.3. A quantitative relationship was found between the amount of amylase released by CCK-PZ and the [Na⁺]_o over the range 26-157 mM in the presence of 2·5 mM-Ca. A similar relationship was also observed when [Ca²⁺]_o was decreased to 1·0 mM, but the responses were reduced to about one half of those observed with 2·5 mM-Ca.4. The most satisfactory theory which explains the cellular mechanism of CCK-PZ-induced amylase output, and which fits the experimental data, requires the dominant activity of a complex composed of a carrier molecule bearing one Ca and four Na molecules, if there is no interaction between Na⁺ and Li⁺.5. A quantitative relationship was also found between the amount of pancreatic juice flow stimulated by CCK-PZ and [Na⁺]_o, over the range 26-157 mM, in the presence of 1·0 or 2·5 mM-Ca.6. A similar quantitative relationship was found between the amount of amylase released by Ca-ionophore A23187 and [Na⁺]_o in the presence of 2·5 or 5·0 mM-Ca. The most satisfactory theory which fits this experimental data also requires the dominant activity of a complex composed of a carrier molecule bearing one Ca and four Na molecules, if there is no interaction between Na⁺ and Li⁺.     

Phorbol myristate acetate and calcium ionophore A23187-stimulated human T cells do not express high-affinity IL-2 receptors

Phorbol myristate acetage (PMA) and Ca2+ ionophore A23187 mimic early signal transduction pathways and activate purified human T cells to secrete large quantities of interleukin-2 (IL-2) and to proliferate. Despite producing 50-100-fold more IL-2 than phytohaemagglutinin (PHA)-activated peripheral blood lymphocytes (PBL), PMA/A23187-stimulated human T cells proliferate less than cells activated by PHA. Washing the cells to remove PMA/A23187 was found to increase cellular proliferation two to five-fold. High-affinity IL-2R (HA-IL-2R) were found to be expressed by human T cells that had been washed 24 hr after PMA/A23187 stimulation and recultured without stimulus for an additional 48 hr, but not by T cells constantly exposed to PMA/A23187 for 72 hr. Radioligand binding studies with [125I]IL-2 demonstrated that while the alpha (p55) and beta (p70-75) subunits of HA-IL-2R were both present on the constant PMA/A23187-stimulated T cells, they did not appear to associate to form functional HA-IL-2R. This defect in the expression of bio-active HA-IL-2R on constant PMA/A23187-stimulated human T cells seems to account for their low proliferative response.     

Induction of exocytosis from glomus cells by incubation of the carotid body of the rat with calcium and ionophore A23187

Carotid bodies from adult rats were electron microscopically studied after incubation in glucose-containing salt solutions containing calcium and/or ionophore A23187 or neither. In the absence of the ionophore, adding or omitting calcium had no effect on the fine structure of theglomus cells. Incubation in the medium containing both 1 mM calcium and the ionophore caused the appearance of exocytotic membrane profiles in several glomus cells. Exocytosis was not seen when only A23187 and endogenous calcium was present. For exocytosis to occur, calcium appeared to be essential and the event seemed to be due to a rise in the intracellular calcium concentration caused by the ionophore.     

Evidence for two calcium-dependent steps and a sodium-dependent step in the mechanism of cell killing by calcium ions in the presence of ionophore A23187

Elevated intracellular Ca2+ appears to play an important role in the mechanism of cell killing in certain pathologic states such as ischemia. The authors have examined aspects of the biochemical mechanism of cell killing by elevated intracellular Ca2+ using as a model system cultured fibroblasts treated with ionophore A23187 in Ca2+-containing medium. Evidence has been obtained for two Ca2+-mediated steps and a Na+-mediated step in the cell killing process. The first Ca2+-mediated step occurs in low extracellular Ca2+ concentrations (1-100 microM) and exhibits a variety of characteristics in common with the arachidonic acid release response stimulated under the same conditions. These results are consistent with the arachidonic acid release response constituting or closely monitoring the initial injury process. The second Ca2+-mediated process is achieved at near physiologic extracellular Ca2+ concentrations in the absence of A23187. Killing of cells injured by the two Ca2+-dependent steps requires extracellular Na+ ions at half or more the physiologic concentration.     

Endogenous arachidonic acid metabolism by calcium ionophore A23187-stimulated lamb lungs: effect of hypoxia

We have determined eicosanoid production from endogenous arachidonic acid by neonatal lamb lungs stimulated with calcium ionophore A23187 during normoxia and hypoxia. Lungs of lambs 19 to 25 d of age were isolated and perfused with cell-free Krebs' bicarbonate buffer at a flow rate of 15 to 20 ml/kg/min. After 30 min of equilibration in a recirculating system, A23187 was added to the perfusate in a 5-microM concentration and perfusion continued for 15 min more. Eicosanoids were measured in perfusate and lung homogenate supernatant. Cyclooxygenase metabolites prostaglandin (PG) E2, thromboxane A2, and PGI2, were measured by radioimmunoassay, and 5-lipoxygenase metabolites leukotrienes (LT) B4, C4, D4, and E4 by high performance liquid chromatography. During normoxia, all three cyclooxygenase metabolites were present in perfusate, but only PGI2 and thromboxane A2 were present in lung homogenate supernatant. Prostacyclin constituted 50% of all the cyclooxygenase products measured. LTC4 and LTD4 were detected in both perfusate and lung homogenate supernatant with little production of LTE4 and LTB4. During hypoxia, the profile of cyclooxygenase products was unchanged and prostacyclin production was not increased. However, the profile of leukotriene metabolites was altered. LTC4 synthesis was markedly reduced. The synthesis of LTE4 and LTB4 was increased 10-fold, with most of the leukotrienes being retained in lung tissue. We conclude that hypoxia significantly alters leukotriene metabolism of endogenous arachidonic acid by calcium ionophore-stimulated lungs. The increased production by stimulated lungs during hypoxia of LTE4, a substance that may increase lung capillary permeability, and that of LTB4, a powerful chemoattractant, may be important contributing factors to lung injury.     

Induction of exocytosis from glomus cells by incubation of the carotid body of the rat with calcium and ionophore A23187.

Carotid bodies from adult rats were electron microscopically studied after incubation in glucose-containing salt solutions containing calcium and/or ionophore A23187 or neither. In the absence of the ionophore, adding or omitting calcium had no effect on the fine structure of the glomus cells. Incubation in the medium containing both 1 mM calcium and the ionophore caused the appearance of exocytotic membrane profiles in several glomus cells. Exocytosis was not seen when only A23187 and endogenous calcium was present. For exocytosis to occur, calcium appeared to be essential and the event seemed to be due to a rise in the intracellular calcium concentration caused by the ionophore.