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.     

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     

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.     

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.     

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.     

Ca2+载体A23187对受精失败人类成熟卵母细胞的补救激活作用

目的:观察Ca²⁺载体A23187对受精失败人类成熟卵母细胞的补救激活及激活后的胚胎发育情况。方法:收集常规体外受精(IVF)及卵浆内单精子注射(ICSI)后24h仍无受精征象的成熟卵母细胞作为研究对象,5μmol/LCa²⁺载体A23187中处理5min,观察第二极体排出及原核形成情况,激活后卵母细胞在体外继续培养2d。结果:IVF组及ICSI组卵母细胞激活率分别为64.9%(24/37)和73.2%(30/41)。ICSI组受精失败卵母细胞激活后主要表现为二极体二原核(2PB+2PN)(80%,24/30),而IVF组仅有20%的被激活卵母细胞表现为2PB+2PN,两组间差异具极显著(P＜0.01)。31个2PN卵母细胞继续培养,25个发生分裂,11个发育到2-4细胞,8个发育到4-8细胞,6个发育到8-细胞以上阶段。结论:Ca²⁺载体A23187能够有效地激活ICSI后受精失败卵母细胞恢复受精并继续发育成胚胎。     

Effect of the calcium ionophore A23187 on plasma and mitochondrial potentials of rat brain synaptosomes: Fluorescence study

Laboratory of Biochemistry, Research Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR, G. N. Kryzhanovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 107, No. 6, pp. 678–680, June, 1989.     

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.     

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.     

Effect of the ionophore A23187 on chloride transport across isolated frog cornea.

The ionophore A23187 at a concentration of 10(-7) to 10(-5) M stimulated active transport of Cl across the isolated frog cornea. The ionophore had no effect in a Cl-free medium. Both unidirectional Cl fluxes were increased by A23187. The electrical resistance was decreased, and this can be totally accounted for by the increment in passive Cl fluxes. The effect of A23187 on Cl transport and permeability mimicked the effects of cyclic AMP, isoproterenol, and epinephrine. A23187 had no effect when the corneas were fully stimulated by epinephrine or isoproterenol. A23187 produced normal stimulation of the SCC in corneas pretreated with alpha- and beta-adrenergic blockers. The stimulation of the SCC by A23187 was dependent on the presence of Ca in the Ringer solution. Excess Ca (10 mM) resulted in a reduced response. Increasing the Mg concentration in the medium reduced the stimulation of the SCC with Ca concentrations of 0.1-5 mM, but prevented the relative inhibition of 10 mM Ca. Intracellular Ca concentration seemed to regulate Cl permeability of the cornea.     

Effects of the calcium ionophore A23187 on pancreatic acinar cell membrane potentials and amylase release.

1. The effects of the Ca2+-ionophore A23187 and the non-metabolizable cholinergic agonist bethanechol on acinar cell membrane potentials and amylase release from the superfused mouse pancreas were studied. 2. In the presence of extracellular Ca2+ (2.56 mM), A23187 (10(-5)M) and bethanechol (3 X 10(-5)M) caused an equal increase in the release of amylase. Both stimulants depolarized theacinar cells, A23187 by 6-0 mV and bethanechol by 12-3 mV. 3. When Ca2+ and Mg2+ were removed from the superfusate, the ability of A23187 to increase the rate of amylase release was virtually abolished, while the effect of bethanechol remained unaltered. Similarly, in the absence of these divalent cations, A23187 did not cause depolarization of the acinar cells, while depolarization in response to bethanechol was largely normal. Consequently it is unlikely that cholinergic agonists initiate secretion by activating a Ca2+-ionophore-like mechanism in the cell membrane. 4. When the concentration of Ca2+ in the medium was raised to 10 mM was the only extracellular divalent cation present, the depolarization in response to A23187 was increased to 11-8 mV. When Mg2+ in a concentration of 10 mM was the only extracellular divalent cation, the depolarization was only 2-1 mV. 5. The Ca2+ dependent, A23187-induced depolarization was abolished in the absence of Na+ (Tris substitution). Addition of Na+ to the superfusate caused an immediate depolarization. 6. It is concluded that the Ca2+ dependent depolarization of pancreatic acinar cells induced by A23187 is not directly due to an increased divalent cation conductance. Our findings are consistent with the view that the depolarization is due to an increased influx of Na+ resulting from a Ca2+ mediated increase in Na+ permeability.     

Analysis of isolated and combined effects of calcium ionophore and phorbol ester on T lymphocyte activation

Isolated and combined effects of the calcium ionophore A23187 and of the protein kinase C activator phorbol myristate acetate (PMA) on T cell activation parameters were analysed on unprimed Balb/c lymph node T lymphocytes (LNL). High doses of PMA were mitogenic for resting T cells, but non-mitogenic doses of PMA induced T cell proliferation in combination with A23187, which was non-mitogenic by itself. Mitogenesis induced by a combination of A23187 and PMA (A23187/PMA) showed the following characteristics: it was not abolished after extensive depletion of accessory cells; purified L3T4+, but not Lyt2+ T cells responded in the absence of accessory cells; mitogenesis was completely blocked by a mixture of two monoclonal antibodies directed to the murine interleukin 2 (IL-2) receptor (7D4/3C7mAbs); cyclosporin A, dibutyril cyclic AMP, and T cell K+ channel blockers quinine and verapamil all blocked mitogenesis. A marked synergism between A23187 and PMA was noted in induction of T cell enlargement, IL-2 release, and induction of IL-2 responsiveness. No synergism was noted in IL-2 receptor expression, A23187 and PMA being able to induce IL-2 receptors alone. Calcium ionophore induced IL-2 receptor expression, but failed to induce IL-2 release and IL-2 responsiveness. Addition of A23187/PMA to the IL-2-dependent CTL-L clone did not result in cell proliferation. Addition of A23187/PMA to Con A-activated T cell blasts leads to a vigorous proliferative response. This response is blocked by 7D4/3C7 mAbs, indicating a role for endogenously produced IL-2 in this case. The results indicate that T cell mitogenesis by A23187/PMA is IL-2-dependent, and suggest a critical role for protein kinase C in IL-2 release and induction of IL-2 responsiveness. In addition, the data suggest distinct, but co-operative pathways of IL-2 receptor induction, controlled by elevated Ca2+ alone and by protein kinase C. Subsequent intracellular events of T cell activation by A23187/PMA may be quite similar to those triggered by Con A, since both kinds of stimulation are blocked by agents such as cyclosporin A, dbcAMP and K+ channel blockers.     

Synergistic action of calcium ionophore A23187 and protein kinase C activator bryostatin 1 on human B cell activation and proliferation.

In this study we have examined the immunostimulatory effects of the macrocyclic lactone bryostatin 1 on various aspects of B cell activation and proliferation using human tonsillar B cells. Bryostatin 1 is an activator of protein kinase C (PKC) and its properties were compared to those of the classical PKC activator phorbol 12-myristate 13-acetate (PMA), a phorbol ester. Time-course kinetics and dose-response curves of RNA and DNA synthesis induced by bryostatin 1 or PMA were comparable, albeit the phorbol ester was significantly more potent. The responses triggered by both bryostatin 1 and PMA could be blocked by the PKC inhibitor H7. Bryostatin 1 and PMA mediated similar effects with regard to the activation parameters, increase in cell size, expression of activation-associated antigens and hyperexpression of major histocompatibility complex class II antigens. Addition of the calcium ionophore A23187 to bryostatin 1-treated cultures resulted in synergistically enhanced activation and proliferation responses, and this potentiation by A23187 could be inhibited by cyclosporin A. Bryostatin 1 antagonized the effects of PMA-triggered stimulation in a time- and dose-dependent manner. The basis for this modulation of PMA-induced effects and the reason for the difference in the abilities of the two agents to stimulate B cells is unclear; possibly, bryostatin 1 and PMA activate different isoforms of PKC and elicit different signals on intracellular biochemical pathways. Bryostatin 1 lacks the tumor-promoting activity of PMA and is a potent anti-neoplastic substance. These features together with its immunomodulatory properties qualify bryostatin 1 as a candidate for in vivo use as a biological response modifier.     

Calcium inhibits urinary acidification: Effect of the ionophore A23187 on the turtle bladder

The role of intracellular calcium in urinary acidification was studied in the turtle bladder with the use of the ionophore A23187. In the presence of calcium acidification was significantly inhibited in the hemibladders treated with the ionophore as compared to control hemibladders treated with dimethylsulfoxide (the vehicle used to dissolve the inophore). In the absence of calcium both the ionophore and dimethylsulfoxide failed to cause any change in acidification. The apparent proton motive force and active conductance of H⁺ were unchanged in dimethylsulfoxide treated hemibladders. In the presence of the ionophore and calcium, the proton motive force and the conductance were significantly decreased when the H⁺ current was low (less than 30% of control values); when the H⁺ current was decreased, but not less than 30%, the proton motive force was unchanged. These data provide evidence for an important role of intracellular calcium in the regulation of urinary acidification.     

Studies on the role of arachidonic acid metabolites in airways contraction inducedin vitro by antigen and calcium ionophore A23187

The effects of inhibitors of the cyclooxygenase and lipoxygenase pathways of arachidonic acid (AA) metabolism were studied on the contractions of guinea pig tracheal spirals and lung parenchymal strips induced by antigen and calcium ionophore A23187. Inhibition of the cyclooxygenase pathway with indomethacin results in enhancement of antigen- and A23187-induced contraction of trachea which is a result of diversion of AA into the lipoxygenase pathway and inhibition of modulatory cyclooxygenase products. Indomethacin does not enhance parenchymal contractions but partly inhibits contraction induced by a strong stimulus (5.7 μM A23187 or 100 μg/ml ovalbumin). Parenchymal contraction is inhibited by agents that block the lipoxygenase pathway of AA metabolism, phenidone and nordihydroguaiaretic acid. These agents inhibit the prolonged phase of antigen-induced tracheal contraction but in some cases enhance the early phase of tracheal contraction induced by antigen or A23187. The results suggest that contraction of parenchyma induced by antigen or A23187 are the result of a bronchoconstrictor lipoxygenase product and only have a cyclooxygenase bronchoconstrictor component following a strong stimulus. In contrast, contraction of trachea also appears to be the result of a bronchoconstrictor lipoxygenase product which may not be identical to that contracting parenchyma, and is modulated by cyclooxygenase products. The results emphasize the importance of comparing small and large airways to further our understanding of asthmatic bronchoconstriction.     

Stimulation of thymus cells by the calcium ionophore A23187 in the presence of LPS and 2-ME: on the mode of action of A23187 and 2-ME

The stimulation of murine spleen and thymus cells by the calcium ionophore A23187 was studied using a serum-free culture technique. In cultures of spleen cells, rabbit IgG was capable of replacing A23187 in bringing about the stimulation of cells. Low concentrations of A23187 were inhibitory to proliferating B lymphocytes. In cultures of thymus cells a stimulating activity of A23187 was observed only if LPS was present in the medium.