High Concentrations Of A23187 Research Articles

The functional consequences of partial calcineurin inhibition in human peripheral blood mononuclear leucocytes

Calcium-dependent signal transduction is essential to the induction of cytokine expression by stimuli acting through the T cell receptor. In vitro, the immunosuppressant cyclosporine (CyA) blocks this pathway by inhibition of calcineurin (CN) phosphatase activity. But in vivo, patients on CyA have only 50% inhibition of CN and can mount cytokine responses. To simulate this state of partial inhibition, we studied the responses of human peripheral blood mononuclear leucocytes (PBL) in vitro at low CyA concentrations. PBL were challenged in vitro with calcium ionophores or anti-CD3 monoclonal antibody. The induction of IFN-γ (interferon-gamma) and IL-2 (interleukin 2) steady-state mRNA was studied by Northern blotting and reverse transcriptase-polymerase chain reaction. IFN-γ protein was assessed in a radiolabelled antibody binding assay or by ELISA (enzyme-linked immunosorbent assay). CN was assessed by dephosphorylation of a 32P-serine labelled 19 amino acid substrate. CyA inhibited CN with an IC 50 (concentration giving 50% inhibition) of 10 ng/ml (95% confidence interval, CI = 8–13 ng/ml). Likewise, the induction of IFN-γ and IL-2 mRNA by calcium ionophore A23187 was inhibited with IC 50 of 14 ng/ml (95% CI = 8–27 ng/ml) and 32 ng/ml (95% CI = 5–178 ng/ml), respectively, while the IC 50 for inhibition of IFN-γ protein secretion was 8 ng/ml (95% CI = 9–18 ng/ml). Partial inhibition of CN also altered the threshold for IFN-γ induction. CyA 10 ng/ml inhibited IFN-γ induction by anti-CD3 monoclonal antibody OKT3 significantly more at low OKT3 concentrations (10 ng/ml, mean ± SEM = 72 ± 9% inhibition) compared to high OKT3 concentrations (1000 ng/ml, 47 ± 6%, p < 0.01). Similar results were seen using high and low concentrations of A23187. Finally, cells pretreated with CyA recovered the ability to respond to high concentrations of A23187 (5 μM) faster than low concentrations (0.5 μM). We conclude that the principal defect in lymphocytes with partial CN inhibition is a reduction in maximum cytokine output which is closely related to the degree of CN inhibition. In addition, there is significantly greater inhibition of weak stimuli compared to maximal stimuli. These defects may explain why patients on CyA can have a reduction in immune responsiveness but still retain protection from infection.

Induction of calbindin-D 28K gene and protein expression by physiological stimuli but not in calcium-mediated degeneration in rat PC12 pheochromocytoma cells

To understand the role of calbindin-D 28K in neuronal degeneration, we examined its expression in differentiated PC12 cells in response to calcium intoxication, using the ionophore A23187 treatment, that results in cell degeneration and death. We first established that calbindin-D 28K is expressed in PC12 cells. The amounts of calbindin-D 28K mRNA and protein were increased by the differentiation factors, NGF and retinoic acid, but not by vitamin D 3. Calbindin-D 28K expression was also significantly up-regulated by stimuli (depolarization, low concentrations of Ca 2+ ionophore A23187) which increase intracellular calcium levels within the physiological range. In contrast, the calbindin-D 28K mRNA and protein concentrations were not modulated by high concentrations of A23187, which resulted in cell degeneration and death. Experiments with the antisense oligonucleotides showed that, although the calbindin-D 28K protein levels were decreased significantly, the progression of degenerative changes induced by calcium via A23187, was not altered.

Saccharomyces cerevisiaeCells Produce Platelet-activating Factor in Response to Calcium Ionophore A23187

Synthesis of platelet-activating factor (PAF) was stimulated by the treatment of cells with the calcium ionophore A23187 in Saccharomyces cerevisiae. The amount of PAF production increased A23187-concentration- and time-dependently. The maximum PAF synthesis was observed when the yeast cells were treated with 2 μm of A23187 for 5 min, and the PAF amount decreased at the higher concentrations of A23187 and/or for the longer incubation time. Treatment of the cells with 1 mm of PMSF (phenylmethyl-sulfonyl fluoride), an inhibitor of PAF catabolism, resulted in transient accumulation of PAF; however, at more than 2 mm of PMSF, PAF production was reduced. Synthesized PAF was not released into the extracellular fraction and was found to be cell-associated. In the absence of A23187 or CaCl2, and in the presence of EGTA, a Ca2+-chelator, no new synthesis of PAF was observed. These results suggested A23187-induced PAF production was Ca2+ dependent, and proceed via PAF remodeling pathway. PAF synthesis in response t...

The ratio of macrophage prostaglandin and leukotriene synthesis is determined by the intracellular free calcium level

The induction of eicosanoid synthesis in various cell types by different physiological stimuli is dependent on an increase in the intracellular calcium level and stimulation of the protein kinase C (PKC). In a model system this can be mimicked by using calcium ionophores and direct PKC activators. In mouse peritoneal macrophages calcium ionophores induced the formation of prostaglandin E 2 (PGE 2) and leukotriene C 4 (LTC 4). A synergistic enhancement of both eicosanoids could be achieved by simultaneous addition of the calcium ionophore A23187 together with a suboptimal dose of the direct protein kinase C activator 12- O-tetradecanoylphorbol 13-acetate (TPA). Low concentrations of the ionophore, resulting in only marginally increased intracellular calcium levels, led to a more than additive prostaglandin E 2 production in combination with TPA. Higher concentrations of A23187 together with TPA favoured LTC 4 synthesis, whereas PGE 2 levels at the same time were even diminished. This observed shift from prostaglandin to leukotriene formation was amplified by simultaneous addition of indomethacin. Manganese as inhibitor of the A23187-induced calcium influx decreased PGE 2 synthesis. On the other hand, in the presence of manganese LTC 4 production was also inhibited at high concentrations of A23187 but elevated in the absence or at low doses of A23187. Our data provide evidence that in macrophages the ratio of cyclooxygenase and lipoxygenase products caused by mediators, acting via the phospholipase C or D/PKC signal transduction pathway, is regulated by the extent of the intracellular calcium increase.

Ionophore A23187 stimulates phosphorylation of the 40, 000dalton protein in human platelets without phospholipase C activation

The Ca 2+ ionophore A23187 (0.2–5 μM) stimulates the phosphorylation of the substrates of protein kinase C (40,000 dalton protein) and myosin light chain kinase (20,000 dalton protein) in the presence or absence of cyclooxygenase inhibitors. In the presence of cyclooxygenase inhibitors or millimolar Ca 2+ there is no stimulation of phospholipase C by A23187. Fingerprints of the 32P-labeled 40,000 dalton protein isolated from platelets that have been stimulated with A23187, thrombin, phorbol 12, 13-dibutyrate and 1,2-didecanoylglycerol were identical. Higher concentrations of A23187 (1–5 μM) induced the loss of polyphosphoinositides through phosphomonoesterase activity.

The role of Ca2+ and Ca2+-activated phospholipid-dependent protein kinase in degranulation of human neutrophils.

The degranulation reactions of human neutrophils induced by 1-oleoyl-2-acetylglycerol (OAG), phorbol 12-myristate 13-acetate (PMA), and calcium ionophore A23187 or their combinations, were studied. OAG in the absence of the Ca2+-ionophore A23187 stimulated the releases of both lysozyme and lactoferrin, constituents of the specific granules, but did not stimulate the release of beta-glucuronidase, an enzyme of the azurophil granules. Electron microscopy revealed a selective decrease in the numbers of the specific granules in this case. The combined effects of A23187 at a concentration higher than 0.1 microM and OAG were essentially additive. W-7, known to be an inhibitor of both Ca2+-activated phospholipid-dependent protein kinase (C-kinase) and calmodulin, inhibited the degranulation induced by OAG or PMA, while it inhibited the reaction induced by A23187 less markedly. The release of lysozyme reached a plateau at about 0.1 microM A23187 and increased again at higher concentrations of A23187. The observations suggest that degranulation can be induced by the activation of the C-kinase, and the degranulation by A23187 at low concentrations may be due to the activation of the C-kinase; the effects of A23187 at high concentrations, however, could not be explained only in terms of the activation of the C-kinase.

Vesicular demyelination induced by raised intracellular calcium

Incubation of nerve with high concentrations of the divalent cation ionophore A23187 produces myelin vesiculation (Schlaepfer 1977). This observation has now been extended using segments of rat ventral or dorsal root incubated with high (19 μM, 10 μg/ml) or low (1–1.5 μM) concentrations of A23187, or another divalent ionophore, ionomycin. Low concentrations of A23187 induced no vesiculation within a 2-h period. However, subsequent incubation of these roots in fresh, ionophore-free medium for 20 h, resulted in a prominent vesicular demyelination at the Schmidt-Lanterman incisures and paranodes of many fibres. At this time (22 h) the Schwann cells associated with some demyelinating internodes appeared vital upon ultrastructural examination: the cells also excluded the nuclear dye nigrosin. High concentrations of A23187 induced a similar vesicular demyelination in affected fibres within only 15–20 min. While the Schwann cells continued to exclude nigrosin for a further 4 h, their ultrastructural appearance indicated that they were probably in the early stages of necrosis. Incubation of moribund root with the ionophore produced no myelin vesiculation. At all ionophore concentrations, the myelin vesiculation was dependent upon the presence of extracellular Ca 2+, and could be modulated in severity by varying this concentration. Other divalent cations (Ba 2+, Co 2+, Mg 2+, Mn 2+, Ni 2+, Sr 2+) could not substitute for Ca 2+. The vesiculation induced by A23187 could be entirely prevented by the addition of Zn 2+ (⩾ 1 μM), Ni 2+ (⩾ 1–10 μM), Co 2+ (⩾ 100 μM) or Mn 2+ (⩾ 100 μM) to the bathing medium. A23187 applied to only part of an isolated internode resulted in a localization of the myelin disruption to that region. Ionomycin (⩾ 1 μM), an ionophore with a greater selectivity for Ca 2+ than A23187, also induced a prompt Ca 2+-dependent myelin vesiculation. We conclude that vesicular demyelination can be initiated in vital Schwann cells by a raised intracellular Ca 2+ concentration. Such demyelination does not necessarily lead to Schwann cell death. The possible relevance of the findings to vesicular demyelinating neuropathies is discussed, and a hypothesis regarding the mechanism of demyelination is advanced.

Tetanus toxin inhibits secretion of lysosomal contents from human macrophages.

The mechanism by which tetanus toxin (TT) inhibits the release of neurotransmitters from neurons is unknown. Since secretion of lysosomal contents from monocytes/macrophages (MOs) appears to be similar to the release of synaptosomal contents from neural cells, we examined the effects of TT on MO secretion. Adherent human MOs were cultured overnight with or without TT (1.25 micrograms/ml), washed, and resuspended in TT-free medium. In response to the calcium ionophore A23187, TT-treated MOs secreted significantly less lysozyme (27.2% +/- 3.2%) than did control cells (48.6% +/- 6.8%; 44.1% inhibition, P less than .001). TT inhibition of A23187-stimulated release of lysozyme from MOs was dose dependent, and higher concentrations of A23187 could not overcome the inhibitory effect of TT. MO secretions in response to a second secretagogue, phorbol myristate acetate, was also inhibited by TT. Heated TT (100 C for 1 hr), which has no in vivo toxicity, also failed to inhibit MO secretion. The inhibition of secretion by TT required holotoxin. C- and B-fragments produced no appreciable effect on secretion in response to either secretagogue. These studies indicate that human MOs may be a readily accessible, model system for the study of the biochemical basis of TT intoxication. However, observations in this system must be confirmed in neurons, the natural site of TT intoxication.

A23187 increases calcium permeability of store sites more than of surface membranes in the rabbit mesenteric artery.

The effects of a Ca ionophore, A23187, were investigated on intact and skinned smooth muscle tissues of the rabbit mesenteric artery. A23187 (over 10(-9) M) inhibited, dose dependently, contractions induced by 10(-5) M-noradrenaline (NA) or 10 mM-caffeine in Ca2+-free solution containing 2 mM-EGTA. Procaine (3 mM) led to cessation of the caffeine- or NA-induced contractions in the presence or absence of Ca2+. When A23187 (10(-7) M) was applied, the contractions in the presence of procaine were to some extent restored in Krebs solution. A23187 at a concentration of 10(-7) M did not modify the resting muscle tone, but this concentration did increase the amplitude of the contraction evoked by 20.2 or 128 mM-K+ and markedly inhibited the 10(-7) M-NA or 10 mM-caffeine-induced contraction in Krebs solution. A23187 (10(-7) M) delayed the onset and rising phase of the 10(-5) M-NA-induced contraction with inhibition of the oscillatory contractions. High concentrations of A23187 (over 10(-6) M) produced a large contraction in the presence and a small contraction in the absence of 2.6 mM-Ca2+. These A23187-induced contractions were not inhibited by 10(-7) M-nisoldipine, a Ca2+ antagonist. A23187 (over 10(-6) M) applied for a long period functionally skinned the muscle tissues. However, the Ca2+ sensitivity of the A23187-treated skinned muscles was lower than that of saponin-treated muscles. In saponin-treated skinned muscles, A23187 (below 10(-6) M) had no effect on the pCa-tension relation. After filling the store, A23187 (over 10(-7) M) generated a larger contraction than did caffeine in Ca2+-free solution, in the presence or absence of 5 mM-NaN3. When 10(-7) M-A23187 was applied once for 5 min, subsequently applied caffeine (20 mM), following application of Ca2+, no longer produced contraction of skinned muscle tissues. The present results indicate that low concentrations of A23187 show a selective Ca2+-releasing action on Ca2+ store sites in muscle cells and that high concentrations increase the Ca2+ leakage (influx) and the cell membrane is skinned.

Ca2+-ionophore-induced microvilli and cortical contractions inXenopus eggs. Evidence for involvement of actomyosin

Using scanning electron microscopy, we show that the calcium ionophore A23187 has a profound effect on the surface morphology ofXenopus laevis eggs. The response to ionophore can be interpreted with respect to animal/vegetal polarity and the presence of an asymmetrically organized actomyosin-based contractile system in the egg cortex. When incubated in ionophore, the egg cortex contracts, pigment granules move towards the animal pole, and microvilli increase dramatically in size. While at first overall microvilli density decreases, many additional microvilli appear later in the animal hemisphere but not in the vegetal hemisphere. Eggs incubated in high concentrations of A23187 undergo the same surface changes at a faster rate, and rupture due to a massive cortical contraction. Local application of ionophore to the egg surface results in increased microvilli size and density in that area, with the animal hemisphere showing the greatest response. Since the effects of ionophore are inhibited by the actomyosin probe, N-ethylmaleimide-modified heavy meromyosin, actomyosin is implicated in the ionophore-induced surface changes.

Secretion of Collagenolytic Enzymes by Human Polymorphonuclear Leukocytes

The polymorphonuclear leukocyte contains three proteinases capable of degrading the collagenous components of the connective tissue matrix. These proteinases, gelatinase, interstitial collagenase and elastase, were found to be rapidly secreted by the neutrophil in response to soluble stimuli with maximal accumulation of the gelatinase and interstitial collagenase occurring during a 20 minute incubation. When neutrophils were stimulated with the chemotactic peptide, formyl-met-leu-phe, gelatinase was the predominant collagenolytic enzyme detected. Stimulation of neutrophils with increasing doses of the Ca++ ionophore A23187 lead to a sequential release of collagenolytic proteinases. Gelatinase release was detected at Ca++ ionophore concentrations of .05-.1 μM, while significant release of interstitial collagenase required 0.5-1 μM A23187. Elastolytic activity was detected only at high concentrations of A23187 (5-10 μM). No release of lactic acid dehydrogenase was detected indicating that the enzyme release was not due to cell death. These studies suggest that the neutrophil may modulate its collagenolytic potential by selective release of collagenolytic proteinases.

Lipoxygenase pathway in islet endocrine cells. Oxidative metabolism of arachidonic acid promotes insulin release.

Metabolism of arachidonic acid (AA) via the cyclooxygenase pathway reduces glucose-stimulated insulin release. However, metabolism of AA by the lipoxygenase pathway and the consequent effects on insulin secretion have not been simultaneously assessed in the endocrine islet. Both dispersed endocrine cell-enriched pancreatic cells of the neonatal rat, as well as intact islets of the adult rat, metabolized [(3)H]AA not only to cyclooxygenase products (prostaglandins E(2), F(2alpha), and prostacyclin) but also to the lipoxygenase product 12-hydroxyeicosatetraenoic acid (12-HETE). 12-HETE was identified by coelution with authentic tritiated or unlabeled 12-HETE using four high performance liquid chromatographic systems under eight mobile-phase conditions and its identity was confirmed by gas chromatography/mass spectrometry using selected ion monitoring. The predominant effect of exogenous AA (5 mug/ml) was to stimulate insulin release from pancreatic cells grown in monolayer. This effect was concentration- and time-dependent, and reversible. The effect of AA upon insulin release was potentiated by a cyclooxygenase inhibitor (indomethacin) and was prevented by either of two lipoxygenase inhibitors (5,8,11,14-eicosatetraynoic acid [ETYA] and BW755c). In addition, glucose, as well as two structurally dissimilar agents (the calcium ionophore A23187 and bradykinin), which activate phospholipase(s) and thereby release endogenous AA in several cell systems, also stimulated insulin secretion. The effects of glucose, glucagon, bradykinin and high concentrations of A23187 (5 mug/ml) to augment insulin release were blocked or considerably reduced by lipoxygenase inhibitors. However, a lower concentration of the ionophore (0.25 mug/ml), which did not appear to activate phospholipase, was resistant to blockade. Exogenous 12-HETE (up to 2,000 ng/ml) did not alter glucose-induced insulin release. However, the labile intermediate 12-hydroperoxy-ETE increased insulin release. Furthermore, diethylmaleate (which binds intracellular glutathione and thereby impedes conversion of the lipoxygenase intermediates hydroperoxy-ETE and leukotriene A(4) to HETE and leukotriene C(4), respectively) potentiated the effect of glucose and of exogenous AA. Finally, 5,6-epoxy, 8,11,14-eicosatrienoic acid (a relatively stable epoxide analogue of leukotriene A(4)) as well as two other epoxy-analogues, potentiated glucose-induced insulin release. We conclude that dual pathways of AA metabolism exist in islet endocrine cells and have opposing regulatory effects on the beta cell-an inhibitory cyclooxygenase cascade and a stimulatory lipoxygenase cascade. Labile products of the latter pathway may play a pivotal role in stimulus-secretion coupling in the islet.

The activation by Ca 2+ of platelet phospholipase A 2: Effects of dibutyryl cyclic adenosine monophosphate and 8-( N, N-diethylamino)-octyl-3,4,5-trimethoxybenzoate

Thrombin-induced release of arachidonic acid from human platelet phosphatidylcholine is found to be more than 90% impaired by incubation of platelets with 1 mM dibutyryl cyclic adenosine monophosphate (Bt 2 cyclic AMP) or with 0.6 mM 8-( N, N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an intracellular calcium antagonist. Incorporation of arachidonic acid into platelet phospholipids is not enhanced by Bt 2 cyclic AMP. The addition of external Ca 2+ to thrombin-treated platelets incubated with Bt 2 cyclic AMP or TMB-8 does not counteract the observed inhibition. However, when divalent cation ionophore A23187 is employed as an activating agent, much less inhibition is produced by Bt 2 cyclic AMP or TMB-8. The inhibition which does result can be overcome by added Ca 2+. Inhibition of arachidonic acid liberation by Bt 2 cyclic AMP, but not by TMB-8, can be overcome by high concentrations of A23187. When Mg 2+ is substituted for Ca 2+, ionophore-induced release of arachidonic acid from phosphatidylcholine of inhibitor-free controls is depressed and inhibition by Bt 2 cyclic AMP is slightly enhanced. The phospholipase A 2 activity of platelet lysates is increased by the presence of added Ca 2+, however, the addition of either A23187 or Bt 2 cyclic AMP is without effect on this activity. We suggest that Bt 2 cyclic AMP may promote a compartmentalization of Ca 2+, thereby inhibiting phospholipase A activity. The compartmentalization may be overcome by ionophore. By contrast, TMB-8 may immobilize platelet Ca 2+ stores in situ or restrict access of Ca 2+ to phospholipase A in a manner not susceptible to reversal by high concentrations of ionophore.

Effect of ionophore A23187 upon membrane function and ion movement in human and toad erythrocytes

Addition of 0.1-0.3 micronM A23187, a divalent cation ionophore, to human erythrocytes suspended in a 1.0 mM 45Ca2+ -containing buffer results in a small (approximately two fold) increase in [Ca2+]i, a significant decrease in osmotic fragility, and a decrease in intracellular K+ (100 mmoles/liter of cells to 70 mmoles/liter cells) without significant alteration of intracellular [Na+]. This decrease in [K+]i is associated with a significant decrease in packed cell volume and correlates directly with the observed alteration is osmotic fragility. Increasing extracellular K+ to 125 mM prevents the A23187-induced changes in osmotic fragility, K+ content and cell volume, but does not prevent the ionophore-induced uptake of 45Ca2+. Addition of 0.1-0.3 micronM A23187 to toad erythrocytes leads to an increase in 45Ca2+ uptake comparable to that observed in human erythrocytes, but does not alter osmotic fragility, cell volume or K+ content. Higher concentrations of ionophore (3.0-10.0 micronM) cause a 30- to 50-fold increase in 45Ca2+ uptake and concomitant change in K+ content, cell volume and osmotic fragility. These changes in cell properties can be prevented by increasing extracellular [K+] to 90 mM. The difference in sensitivity of the two cell types to A23187 is attributed to the presence of additional intracellular calcium pools within toad erythrocytes that prevent an increase in cytoplasmic Ca2+ until Ca2+ uptake is increased substantially at the higher concentrations of A23187.

Metabolic aspects of the secretion of stored compounds from blood platelets. V. Effect of ionophore A23187 on washed platelets.

1. The A23187-induced secretion of preabsorbed serotonin from human blood platelets at 37 degrees C is studied. Preincubation at the same temperature before the addition of ionophore is necessary for maximal release induction. When total incubation time is kept constant, longer time with ionophore results in a smaller decrease in the level of metabolic ATP and increase in metabolic ATP and increase in metabolic IMP. This coincides with the reduction in secretion, but statistical treatment of the results suggests that the reduced secretion only partially explains the reduced drop in metabolic ATP, and that therefore a resynthesis of metabolic ATP from IMP may have taken place. 2. In some experiments induction of secretion takes place over a very narrow range of ionophore concentration. 3. When K+ substitutes for Na+ in the extracellular medium, the need for preincubation for maximal secretion becomes less evident, and at times is abolished, while there is still a significant increase in the metabolic ATP level by prolonged incubation with ionophore. 4. A reduction in secretion is observed with metabolic blockers when the ionophore is added after preincubation, but to a much less degree than when secretion is induced by thrombin, in spite of a great reduction in the level of metabolic ATP. This may partly be explained by the increase in secretion induction by A23187 in the presence of inhibitors when the ionophore is added in the cold, suggesting that the inhibitor may cause "weakening" of the platelets' "resistance" to induction of secretion by ionophore. 5. When the effect of Ca2+ and of Mg2+ on the level of intermediates of the TP leads to hypoxanthine conversion is studied, it is evident that the addition of Ca2+ causes enhanced IMP accumulation and a reduction in the level of inosine plus hypoxanthine, while Mg2+ has the opposite effect. This suggests that the two metals affect the enzymes of the IMP leads to hypoxanthine conversion differently. 6. Indomethacin inhibits secretion induced by A23187, suggesting that prostaglandin intermediates may amplify the ionophore-induced release. The adenine nucleotide metabolism is not affected. 7. The results indicate that there is an indirect, rather than direct, link between the major metabolic changes and the secretion induced by A23187, but that the ionophore may cause intracellular changes which are not connected to its effect as release inducer.

Action of ionophore A23187 at the cellular level. Separation of effects at the plasma and mitochondrial membranes.

Bovine epididymal spermatozoa incubated aerobically in vitro in the presence of 0.1 to 0.2 mM CaCl2 accumulate 25 to 50 nmol of calcium/10(8) cells. The addition of low concentrations of the ionophore A23187 (0.01 to 0.5 nmol/mg of sperm protein) induces efflux of this accumulated calcium. At high ionophore concentrations (0.5 to 5.0 nmol/mg of sperm protein), calcium release is followed by an influx of up to 25 nmol of calcium/10(8) cells that is not dependent on mitochondrial energization. A selective increase in the permeability of the sperm plasma membrane produced by treatment with the polyene antibiotic, filipin, results in the release of that calcium which is accumulated in the presence of high concentrations of A23187. Sperm first treated with filipin possess the ability to accumulate and retain calcium (in the presence of an oxidizable substrate) but release Ca2+ without subsequent reaccumulation after the addition of 3 nmol of A23187/mg of protein. These observations are explained by the existence of competing calcium pumps operating within the mitochondrial and plasma membranes of the spermatozoan. Treatment with high concentrations of A23187 allows calcium influx into a non-mitochondrial compartment of the sperm cell as a consequence of the equilibration of this cation across both mitochondrial and plasma membranes. The amount of calcium uptake and its sensitivity to filipin indicate that calcium binding to soluble, intracellular components is also involved. The ability of low concentrations of A23187 to induce calcium efflux is explained as a result of the continued operation of the plasma membrane pump coincident with ionophore-induced decay of the concentration gradient across the mitochondrial membrane. This hypothetical action of low levels of the ionophore on the mitochondria is supported by the observation of net movements of calcium with filipin-treated cells and the respiratory responses and movements of phosphate and membrane-associated calcium with intact sperm. It is suggested that the basis of this apparent selectivity of ionophore action lies in the relative activities and kinetic properties of the competing calcium pumps in the plasma and mitochondrial membranes of these cells. Ionophore-induced influx of calcium into the extramitochondrial space results in a stimulation of respiration and kinetic activity of the sperm. This activation of motility is observed also with cells made entirely dependent upon glycolysis (by treatment with respiratory inhibitors) and suggests a direct involvement of calcium in the regulation of flagellar function.

Metabolic aspects of the secretion of stored compounds from blood platelets IV. Effects of ionophore X537A on washed platelets

1. 1. X537A at concentrations below 10 μM can liberate platelet serotonin from washed human platelets without inducing the platelet release reaction. Up to 100% of serotonin preabsorbed by the platelets can be liberated before initiation of the release reaction. 2. 2. Concentrations of X537A above 10 μM initiate the platelet release reaction, with a maximum release of adenine nucleotides and platelet factor 4 antigen comparable to that obtained with 1.25 units thrombin/ml. 3. 3. The changes in ATP metabolism at the concentration necessary for X537A-induced release are more profound than those in platelets exposed to concentrations of thrombin or A23187 giving the same degree of release, and approach those seen with high concentrations of A23187. At concentrations where serotonin is liberated but no adenine nucleotide or platelet factor 4 antigen is released, short time incubation causes no change in the level of metabolic ATP. 4. 4. In Na + medium the release increases with prolonged incubation to a level which seems to be characteristic for a particular ionophore concentration. Replacing Na + with K + in the incubation medium produces the maximum release for a particular concentration after short-time incubation, and no further increase is see. 5. 5. When platelets are incubated with antimycin and glucose so that anaerobic metabolism is predominant and then exposed to X537A, a marked sparing effect on ATP utilization can be observed in K + medium, and an increased ATP breakdown in Na + medium. The shift in monovalent cation does not affect the amount liberated and released. 6. 6. We conclude that the increased speed of release induction and of liberation of serotonin reflects a direct effect of the ion change on the ionophore action, possibly by increasing the speed by which X537A is accumulated in the platelet membrane.