Involvement of phospholipase D in store-operated calcium influx in vascular smooth muscle cells

In non-excitable cells, sustained intracellular Ca2+ increase critically depends on influx of extracellular Ca2+. Such Ca2+ influx is thought to occur by a 'store-operated' mechanism, i.e. the signal for Ca2+ entry is believed to result from the initial release of Ca2+ from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Here we show that the depletion of cellular Ca2+ stores by thapsigargin or bradykinin is functionally linked to a phosphoinositide-specific phospholipase D (PLD) activity in cultured vascular smooth muscle cells (VSMC), and that phosphatidic acid formed via PLD enhances sustained calcium entry in this cell type. These results suggest a regulatory role for PLD in store-operated Ca2+ entry in VSMC.     

Origin of intracellular calcium and quantitation of mobilizable calcium in neutrophils stimulated with chemotactic peptide

The origin and amount of mobilized Ca2+ in chemotactic peptide-stimulated guinea pig neutrophils were examined using biochemical techniques. The total amount of releasable Ca2+ by 20 microM A23187 from the unstimulated intact cells was 0.91 nmol/4 X 10(6) cells, as assessed by change in absorbance of the antipyrylazo III-Ca2+ complex. Two types of internal vesicular Ca2+ pool, mitochondrial and non-mitochondrial pool were identified in the saponin-permeabilized cells. The total amount of releasable Ca2+ was comparable to that accumulated by the non-mitochondrial pool at (1-2) X 10(-7) M of a free Ca2+ concentration. The mitochondrial uncoupler, capable of releasing Ca2+ from the mitochondrial pool, neither modified the basal cytosolic free Ca2+ in quin 2-loaded cells nor caused a Ca2+ efflux from the intact cells. These results suggest that the releasable Ca2+ may be located in the non-mitochondrial pool of unstimulated intact cells, and the mitochondrial pool contains little releasable Ca2+. The addition of fMet-Leu-Phe increased the cytosolic free Ca2+ by two processes: Ca2+ mobilization from internal stores and Ca2+ influx through the surface membrane. The Ca2+ mobilized and effluxed from the intact cells by stimulation with the maximal doses of fMet-Leu-Phe was estimated to be 0.27 nmol/4 X 10(6) cells. Almost equal amounts were released by the maximal doses of inositol 1,4,5-trisphosphate from the non-mitochondrial pool of saponin-treated cells that had accumulated Ca2+ at a free Ca2+ concentration of 1.4 X 10(-7) M. The mechanism related to the Ca2+ influx by fMet-Leu-Phe stimulation was also examined. The addition of nifedipine or phosphatidic acid did not affect the change in the cytosolic free Ca2+ induced by fMet-Leu-Phe, thereby suggesting that the receptor-mediated Ca2+ channel may be involved in the Ca2+ influx.     

Influence of alkali metal cations on the transport of calcium by phosphatidate in multi-phase systems

The effects of alkali metal cations on the rates at which Ca2+ and phosphatidic acid were cotransported from aqueous to hydrocarbon medium were examined. The alkali metal cations remained in the aqueous phase yet specifically influenced the transport of Ca2+ into the hydrocarbon solvent. For the physiological cations, Na+ and K+, there were critical concentration ranges in which small changes in concentration effected sharp changes in transport rates. The maximal rate observed with Na+ was an order of magnitude greater than that with K+; however, unlike Na+, K+ promoted low levels of transport below the critical concentration range. Li+ effected only low levels of transport even at high concentrations, whereas Rb+ and Cs+ induced transport at rates proportional to their concentrations. These results are discussed in terms of a classical ionophore model for the complex composed of a neutral phosphatidic acid dimer bridged by Ca2+.     

Inositol trisphosphate mediates thyrotropin-releasing hormone mobilization of nonmitochondrial calcium in rat mammotropic pituitary cells

Thyrotropin-releasing hormone (TRH) stimulation of prolactin secretion from GH3 cells, cloned rat pituitary tumor cells, is associated with 1) hydrolysis of phosphatidylinositol 4,5-bisphosphate to yield inositol trisphosphate (InsP3) and 2) elevation of cytoplasmic free Ca2+ concentration [( Ca2+]i), caused in part by mobilization of cellular calcium. We demonstrate, in intact cells, that TRH mobilizes calcium and, in permeabilized cells, that InsP3 releases calcium from a nonmitochondrial pool(s). In intact cells, TRH caused a loss of 16 +/- 2.7% of cell-associated 45Ca which was not inhibited by depleting the mitochondrial calcium pool with uncoupling agents. Similarly, TRH caused an elevation of [Ca2+]i from 127 +/- 6.3 nM to 375 +/- 54 nM, as monitored with Quin 2, which was not inhibited by depleting mitochondrial calcium. Saponin-permeabilized cells accumulated Ca2+ in an ATP-dependent manner into a nonmitochondrial pool, which exhibited a high affinity for Ca2+ and a small capacity, and into a mitochondrial pool which had a lower affinity for Ca2+ but was not saturated under the conditions tested. Permeabilized cells buffered free Ca2+ to 129 +/- 9.2 nM when incubated in a cytosol-like solution initially containing 200 to 1000 nM free Ca2+. InsP3, but not other inositol sugars, released calcium from the nonmitochondrial pool(s); half-maximal effect occurred at approximately 1 microM InsP3. Ca2+ release was followed by reuptake into a nonmitochondrial pool(s). These data suggest that InsP3 serves as an intracellular mediator (or second messenger) of TRH action to mobilize calcium from a nonmitochondrial pool(s) leading to an elevation of [Ca2+]i and then to prolactin secretion.     

Relationship between the "phospholipid effect" and calcium in the thyroid. I. - Effects of calcium ions, E.G.T.A., ionophore A 23187, verapamil and chlorpromazine on resting and stimulate thyroid slices.

The "phospholipid effect" which is the enhanced turnover of the phosphorylinositol group of phosphatidylinositol (PI) occurs in the thyroid of response to thyreostimulin (TSH). The possibility that Ca2+ ions are involved in this stimulation has been investigated with pig thyroid slices. Experiments performed in media without Ca2+ or containing E.G.T.A. (2 mM), indicate that it is not the extracellular Ca2+ which is implied, but rather the intracellular Ca2+. The ionophore A23187 (6.10(-6) M) increases the specific radioactivity of the acid soluble precursors, but has also a specific effect on the PI turnover, which is additive with the effect of a high concentration of TSH (50 mU/ml). Washing and loading of slices with various Ca2+ concentrations show that 0.9 mM restores the TSH phospholipid effect. Verapamil (10(-3) M) and Chlorpromazine (10(-3) M) redirect glycerolipid metabolism by increasing PI and phosphatidic acid (PA) synthesis at the expense of other glycerolipids, as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). These results suggest that the "phospholipid effect" is not a result of Ca2+ entry into the thyroid cells. On the contrary, it seems that this increased turnover of PI in "long term" incubations (3 hr). An additive and acute effect of TSH effect is more pronounced when Ca2+ movements     

Calcium-induced condensation-reorganization phenomena in multilamellar vesicles of phosphatidic acid. pH potentiometric and 31P-NMR, Raman and ESR spectroscopic studies

In biological membranes, the anionic characteristics of the polar headgroup of phosphatidic acids are responsible for structural changes induced by Ca2+ in many cellular processes. The very simple headgroup structure of dipalmitoylphosphatidic acid (DPPA) offers particular advantages as a model to study the interactions between Ca2+ and natural phosphatidic acids such as cardiolipin and phosphatidylserine. The effects of calcium ions on DPPA membranes have been studied as a function of temperature by potentiometry and by Raman, ESR and 31P-NMR spectroscopies. The protons in monosodic DPPA liposomes have been considered as a probe to detect pH variations resulting from introduction of Ca2+ inside the membrane. This method has also allowed us to determine the stoichiometry of this reaction: 2 DPPA(H) + Ca2+----Ca(DPPA)2 + 2H+. 31P-NMR spectroscopy has been used to detect reorganization-condensation phenomena in multilamellar vesicles of DPPA under the influence of calcium and temperature. Furthermore, the temperature profiles obtained from Raman spectra for Ca(DPPA)2 membranes provide conclusive evidence that Ca2+ induces major reorganization of the phosphatidic acid component into a highly ordered phase. Quantitative estimates of the degree of motional restriction of spin-labeled soaps embedded inside membranes composed of DPPA with or without Ca2+ have been made using ESR technique. These results are discussed and compared to those found previously for a natural phosphatidic acids such as phosphatidylserine.     

Regulation of calcium efflux from isolated rat parotid cells

Calcium efflux from isolated rat parotid acinar cells was studied with 45Ca. Carbachol, phenylephrine, substance P, monobutyryl cyclic AMP and isoproterenol stimulated 45Ca efflux. It is suggested that carbachol, phenylephrine and substance P mobilize the same pool of cellular Ca. This suggestion is based on two observations. Firstly, combinations of any two of these three agonists at saturating concentrations result in no more 45Ca efflux than either agonist alone. Secondly, stimulation of 45Ca efflux by any one of the three agonists prevents further stimulation of 45Ca efflux by the same or one of the other two agonists. The pool of calcium mobilized by isoproterenol or monobutyryl cyclic AMP is different from the pool mobilized by carbachol. This conclusion is based on the observation that stimulation of 45Ca efflux by a saturating concentration of carbachol did not inhibit stimulation of 45Ca efflux by isoproterenol. Furthermore the effect of a saturating concentration of isoproterenol on 45Ca efflux is additive with that caused by a saturating concentration of carbachol. The effect of carbachol, phenylephrine and substance P on 45Ca2+ efflux did not require extracellular Ca2+.     

Lowering extracellular pH evokes inositol polyphosphate formation and calcium mobilization

Changing extracellular pH (pHo) from 7.4 to 6.1 increased [3H]inositol bis- and trisphosphates approximately 10- and 5-fold, respectively, in 15 s in human fibroblasts. [3H]Inositol phosphate increased less rapidly than the polyphosphates. Bradykinin similarly increased [3H]inositol phosphates. Shifting pHo from 7.4 to 6.0 evoked a large spike in cytosolic free Ca2+ [( Ca2+]i) which was primarily caused by the release of stored Ca2+. Changing pHo from 7.4 to 6.0 decreased cytoplasmic pH to approximately 7.0. Moderate decreases in intracellular pH had no effect on [Ca2+]i or 45Ca2+ efflux. Decreasing pHo strikingly increased 45Ca2+ efflux and decreased total cell Ca2+ similarly to bradykinin. Changing pHo from 7.4 to approximately 6.4 produced half-maximal effects on [Ca2+]i, 45Ca2+ efflux, and total Ca2+. Cycling pHo between 7.4 and 6.0 produced repetitive decreases and increases in total Ca2+. Bradykinin released the Ca2+ which was reaccumulated after an acid pulse indicating that Ca2+ had returned to the hormone-sensitive pool. Decreasing pHo also released stored Ca2+ from coronary endothelial, neuroblastoma, and umbilical artery muscle cells, but not from rat aortic smooth muscle or human epidermoid carcinoma (A431) cells. We suggest that lowering pHo stimulates a phosphoinositidase-coupled receptor by protonating a functional group with a pKa near 6.5.     

Analysis of Ca2+ fluxes and Ca2+ pools in pancreatic acini

45Ca2+ movements have been analysed in dispersed acini prepared from rat pancreas in a quasi-steady state for 45Ca2+. Carbamyl choline (carbachol; Cch) caused a quick 45Ca2+ release that was followed by a slower 45Ca2+ 'reuptake'. Subsequent addition of atropine resulted in a further transient increase in cellular 45Ca2+. The data suggest the presence of a Cch-sensitive 'trigger' pool, which could be refilled by the antagonist, and one or more intracellular 'storage' pools. Intracellular Ca2+ sequestration was studied in isolated acini pretreated with saponin to disrupt their plasma membranes. In the presence of 45Ca2+ (1 microM), addition of ATP at 5 mM caused a rapid increase in 45Ca2+ uptake exceeding the control by fivefold. Maximal ATP-promoted Ca2+ uptake was obtained at 10 microM Ca2+ (half-maximal at 0.32 microM Ca2+). In the presence of mitochondrial inhibitors it was 0.1 microM (half-maximal at 0.014 microM). 45Ca2+ release could still be induced by Cch but the subsequent reuptake was missing. The latter was restored by ATP and atropine caused further 45Ca2+ uptake. Electron microscopy showed electron-dense precipitates in the rough endoplasmic reticulum of saponin-treated cells in the presence of Ca2+, oxalate and ATP which were absent in intact cells or cells pretreated with A23187. The data suggest the presence of a plasma membrane-bound Cch-sensitive 'trigger' Ca2+ pool and ATP-dependent Ca2+ storage systems in mitochondria and rough endoplasmic reticulum of pancreatic acini. It is assumed that Ca2+ is taken up into these pools after secretagogue-induced Ca2+ release.U     

Evidence for tight coupling of phospholipase activation and Ca2+ influx during acrosome reaction of golden hamster spermatozoa

1. Phospholipases have been proposed to play a key role in sperm acrosome reaction. To examine the activation mechanism of phospholipases and subsequently sperm fertilizing capacity. Ca2+ fluxes and phospholipid turnover (breakdown and synthesis) were investigated in golden hamster spermatozoa during acrosome reaction. 2. Upon exposure of the spermatozoa to 1.7 mM Ca2+, a net uptake by the cells occurred in two distinguishable phases. 3. Depletion of extracellular Ca2+ by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) at a time that an initial Ca2+ uptake was observed to reach almost steady-state, prevented the secondary Ca2+ uptake and acrosome reaction. 4. The time course of an initial Ca2+ uptake seemed to precede that of the acrosome reaction. 5. Incubation of the spermatozoa with Ca2+ in the presence of [3H]glycerol induced a rapid increase in labeling of phosphatidic acid, a key intermediate of phosphinositide turnover initiated by the action of phospholipase C, which appeared to parallel the time course of a first phase of Ca2+. 6. Phospholipase A2 activation, detected by lysophospholipid formation, slightly delayed the initial events of first Ca2+ uptake and phosphatidic acid production. 7. It is concluded that first Ca2+ entry into the cells, associated with phosphatidic acid production, activates a phospholipase A2, leading to the production of substances, like lysophospholipids and fatty acids, which may contribute to acrosome reaction.     

Presence of membrane-associated phosphatidate phosphohydrolase activity in cultured islets and its stimulation by glucose

The cellular location at which exogenous phosphatidic acid is hydrolysed in cultured neonatal rat islets was examined. Phosphatidate phosphohydrolase activity could be demonstrated in both whole cell sonicates and isolated plasma membranes. In the whole cell fraction phosphatidic acid hydrolysis to diacylglycerol was stimulated 43% by the presence of Mg2+. The activity present in isolated membranes was totally dependent on the presence of Mg2+ and was increased in plasma membranes from glucose-stimulated islets. Following exposure of islets to low glucose concentrations, raising the Ca2+ concentration from 150 nM to 40 microM in the presence of Mg2+ did not affect the formation of diacylglycerol in whole cell fractions or plasma membranes. These results indicate the presence within the islet of membrane-bound phosphatidate phosphohydrolase activity and demonstrate its activation by glucose.     

Role of calcium in exocrine pancreatic secretion. I. Calcium movements in the rabbit pancreas.

1. Calcium movements in the isolated rabbit pancreas and in rabbit pancreas fragments have been studied with the aid of 4 5 Ca2+. 2. Addition of 4 5 Ca2+ to the incubation medium of the isolated rabbit pancreas results in an immediate appearance of isotope in the secreted fluid reaching a constant specific activity in 30 min. The absolute activity in the secreted fluid is 30-40% of that in the incubation medium. 3. Addition of 10(-5) M carbachol after 2 h preincubation with 4 5 Ca2+ results in enzyme secretion accompanied by calcium release. There is also an increase in 4 5 Ca2+ secretion, but this is maximal 10 min after the protein and total calcium peaks. 4. Partial removal of 4 5 Ca2+ from the bathing medium, before stimulation, reduces the increase in 4 5 Ca2+ secretion nearly proportionally. 5. [3H]Mannitol, added to the bathing medium, appears in the secreted fluid and behaves upon carbachol stimulation similarly to 4 5 Ca2+. 6. Upon repeated stimulation with 10(-5) M acetylcholine, a 4 5 Ca2+ peak appears, even in virtual absence of enzyme secretion. In this case the peak coincides with a small total calcium peak. 7. Efflux studies of rabbit pancreas fragments, preloaded with 4 5 Ca2+, show a carbachol-stimulated 4 5 Ca2+ efflux in addition to a release of amylase. 8. These studies indicate that there are three calcium movements in rabbit pancreas which can all be influenced by cholinergic agents: (a) an extracellular route for calcium and other small molecules and ions; (b) a calcium release across the apical membrane along with the enzymes, originating from a pool which does not freely exchange with 4 5 Ca2+ in the bath; (c) a calcium flux across the serosal membrane, which involves calcium exchanging freely with 4 5 Ca2+ from the bath. The third flux is thought to result from an increase in cytoplasmic calcium, which may be involved in the stimulus-secretion coupling of pancreatic enzyme secretion.     

Ca(phosphatidate)2 can traverse liposomal bilayers

Phosphatidic acid can act as Ca2+ cross-membrane ionophore without the necessity of previous autoxidation. The apparent PA-CA2+ dissociation constant is 3 X 10(-3), i.e., in the range of extracellular Ca2+ concentration. There is at least 100-fold preference for Ca2+ over Mg2+. Ca2+ transfer rates are proportional to the square of phosphatidic acid concentration in the bilayer. Removal of the fatty acid ester CO groups reduces the Ca2+ ferrying rate by more than 90 percent. It appears that the cation is held in a cage formed by phosphate and carbonyl oxygens of two PA molecules. In this coordination complex both Ca2+ and the phosphatidic acid headgroups are dehydrated, and the Ca(phosphatide)2 assembly becomes lipid-soluble and can traverse the bilayer.     

Phosphatidic acid and arachidonic acid each interact synergistically with glucagon to stimulate Ca2+ influx in the perfused rat liver.

The administration of phosphatidic acid to rat livers perfused with media containing either 1.3 mM- or 10 microM-Ca2+ was followed by a stimulation of Ca2+ efflux, O2 uptake and glucose output. The responses elicited by 100 microM-phosphatidic acid were similar to those induced by the alpha-adrenergic agonist phenylephrine. Contrary to suggestions that phosphatidic acid acts like a Ca2+-ionophore, no net influx of Ca2+ was detected until the phosphatidic acid was removed. Sequential infusions of phenylephrine and phosphatidic acid indicate that the two agents release Ca2+ from the same intracellular source. The co-administration of glucagon (or cyclic AMP) and phosphatidic acid, and also of glucagon and arachidonic acid, led to a synergistic stimulation of Ca2+ uptake of the liver, a feature similar to that observed after the co-administration of glucagon and other Ca2+-mobilizing hormones [Altin & Bygrave (1986) Biochem. J. 238, 653-661]. A notable difference, however, is that the synergistic stimulation of Ca2+ uptake induced by the co-administration of glucagon and arachidonic acid was inhibited by indomethacin, whereas that induced by glucagon and phosphatidic acid, or glucagon and other Ca2+-mobilizing agents, was not. The results suggest that the synergistic action of glucagon and arachidonic acid in stimulating Ca2+ influx is mediated by prostanoids, but that of glucagon and phosphatidic acid is evoked by a mechanism similar to that of Ca2+-mobilizing agents.     

Characteristics of intracellular calcium changes required for augmentation of phorbol ester-stimulated pancreatic enzyme secretion

Previous studies demonstrated that Ca2+ ionophores augment the pancreatic enzyme secretion caused by phorbol esters. The present study was performed to determine the nature of the cellular Ca2+ effects responsible for the augmentation. Relatively low concentrations (0.3-1.0 microM) of the nonfluorescent Ca2+ ionophore, 4-bromo-A23187 (Br-A23187), did not measurably increase free cytosolic Ca2+ ([Ca2+]i) and caused little or no enzyme release from guinea pig pancreatic acini. However, these concentrations of Br-A23187 augmented the amylase release caused by the phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA). This augmentation occurred in the absence of extracellular Ca2+ as long as the intracellular agonist-sensitive pool contained Ca2+. Greater concentrations of Br-A23187 (3-10 microM) alone caused transient increases in [Ca2+]i and transient increases in amylase release. Although not resulting in an increase in [Ca2+]i, the low concentrations of Br-A23187 caused release of Ca2+ from the intracellular agonist-sensitive pool. These results suggest that Ca2+ mediates enzyme release by two distinct mechanisms in the pancreatic acinar cell. First, an increase in [Ca2+]i alone mediates enzyme release. Second, Ca2+ release from the agonist-sensitive pool not resulting in a measurable increase in [Ca2+]i augments enzyme release stimulated by a phorbol ester. The second effect of Ca2+ may be due to a small localized change in cell Ca2+ or an induction of cytosolic Ca2+ oscillations.     

Fusion of phospholipid vesicles induced by muscle glyceraldehyde-3-phosphate dehydrogenase in the absence of calcium

Ca2+-induced fusion of phospholipid vesicles (phosphatidylcholine/phosphatidic acid, 9:1 mol/mol) prepared by ethanolic injection was followed by five different procedures: resonance energy transfer, light scattering, electron microscopy, intermixing of aqueous content, and gel filtration through Sepharose 4-B. The five methods gave concordant results, showing that vesicles containing only 10% phosphatidic acid can be induced to fuse by millimolar concentrations of Ca2+. When the fusing capability of several soluble proteins was assayed, it was found that concanavalin A, bovine serum albumin, ribonuclease, and protease were inactive. On the other hand, lysozyme, L-lactic dehydrogenase, and muscle and yeast glyceraldehyde-3-phosphate dehydrogenase were capable of inducing vesicle fusion. Glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle, the most extensively studied protein, proved to be very effective: 0.1 microM was enough to induce complete intermixing of bilayer phospholipid vesicles. Under conditions used in this work, fusion was accompanied by leakage of internal contents. The fusing capability of glyceraldehyde-3-phosphate dehydrogenase was not affected by 5 mM ethylenediaminetetraacetic acid. The Ca2+ concentration in the medium, as determined by atomic absorption spectroscopy, was 5 ppm. Heat-denatured enzyme was incapable of inducing fusion. We conclude that glyceraldehyde-3-phosphate dehydrogenase is a soluble protein inherently endowed with the capability of fusing phospholipid vesicles.     

Ca2+ translocation across liposomal membranes enhanced by unsaturated long-chain fatty acids

The putative ionophoretic action of phosphatidic acid or arachidonic acid metabolites for Ca2+ has offered an attractive explanation for stimulation-coupled mobilization of cytoplasmic Ca2+. We have examined the effects of Ca2+ ionophore and long-chain unsaturated fatty acids on the translocation of Ca2+ across the liposomal membrane by using Quin II-entrapped liposomes, a sensitive assay system for ionophoresis of Ca2+. A23187 increased Quin II fluorescence intensity corresponding to the translocation of Ca2+ into liposomes. Similar translocation was observed with unsaturated long-chain fatty acids but not with saturated fatty acids. Thus, when phospholipases of cell membrane are activated by certain stimuli, unsaturated long-chain fatty acids are liberated and might mediate the mobilization of cytoplasmic Ca2+.     

Inhibition of translational initiation in eukaryotic cells by calcium ionophore

Ca2+ has been recently reported to be required for high rates of translational initiation in GH3 pituitary cells (Chin, K.-V., Cade, C., Brostrom, C.O., Galuska, E.M., and Brostrom, M.A. (1987) J. Biol. Chem. 262, 16509-16514). In the present investigation low concentrations of the Ca2+ ionophores, A23187 and ionomycin, were found to rapidly suppress the Ca2+-dependent component of protein synthesis in GH3 cells. More ionophore was required to inhibit amino acid incorporation into protein as extracellular Ca2+ was increased. Pre-existing inhibitions of protein synthesis produced by low concentrations of ionophore at low extracellular Ca2+ concentrations were reversed by adjustment to high extracellular Ca2+. Treatment with ionophore reduced the cellular contents of polysomes and 43 S preinitiation complex to values equivalent to those found for Ca2+-depleted cells. Average ribosomal transit times were unaffected by ionophore, and treated cells retained the ability to accumulate polysomes when incubated with cycloheximide. Cell types, such as HeLa and Chinese hamster ovary, that normally display only a modest Ca2+-dependent component of protein synthesis, manifested a strong underlying Ca2+ dependence in amino acid incorporation and polysome formation following treatment with low concentrations of ionophore. Protein synthesis in GH3 or HeLa cells during recovery from heat shock and arsenite treatment was not affected by cellular Ca2+ depletion or ionophore treatment. On the basis of these results, Ca2+ ionophore is proposed to inhibit Ca2+-dependent translational initiation through facilitating the mobilization of sequestered intracellular Ca2+.     

Mitochondria and calcium signaling

The kinetic properties for the uptake, storage and release of Ca2+ from isolated mitochondria accurately predict the behaviour of the organelles within the intact cell. While the steady-state cycling of Ca2+ across the inner membrane between independent uptake and efflux pathways seems at first sight to be symmetrical, the distinctive kinetics of the uniporter, which is highly dependent on external free Ca2+ concentration and the efflux pathway, whose activity is clamped over a wide range of total matrix Ca2+ by the solubility of the calcium phosphate complex provide a mechanism whereby mitochondria reversibly sequester transient elevations in cytoplasmic Ca2+. Under non-stimulated conditions, the same transport processes can regulate matrix Ca2+ concentrations and hence citric acid cycle activity.     

Evidence for the calcium-dependent activation of phospholipase D in thrombin-stimulated human erythroleukaemia cells.

Human erythroleukaemia (HEL) cells were exposed to thrombin and other platelet-activating stimuli, and changes in radiolabelled phospholipid metabolism were measured. Thrombin caused a transient fall in PtdInsP and PtdInsP2 levels, accompanied by a rise in diacylglycerol and phosphatidic acid, indicative of a classical phospholipase C/diacylglycerol kinase pathway. However, the rise in phosphatidic acid preceded that of diacylglycerol, which is inconsistent with phospholipase C/diacylglycerol kinase being the sole source of phosphatidic acid. In the presence of ethanol, thrombin and other agonists (platelet-activating factor, adrenaline and ADP, as well as fetal-calf serum) stimulated the appearance of phosphatidylethanol, an indicator of phospholipase D activity. The Ca2+ ionophore A23187 and the protein kinase C activator phorbol myristate acetate (PMA) also elicited phosphatidylethanol formation, although A23187 was at least 5-fold more effective than PMA. Phosphatidylethanol production stimulated by agonists or A23187 was Ca2(+)-dependent, whereas that with PMA was not. These result suggest that phosphatidic acid is generated in agonist-stimulated HEL cells by two routes: phospholipase C/diacylglycerol kinase and phospholipase D. Activation of the HEL-cell phospholipase D in response to agonists may be mediated by a rise in intracellular Ca2+.