Regulation of calpactin I phospholipid binding by calpactin I light-chain binding and phosphorylation by p60v-src.

Calpactins I and II are proteins that bind Ca2+, phospholipids, actin and spectrin; they are also major substrates of oncogene and growth-factor-receptor tyrosine kinases. Since calpactins have been proposed to provide a link between membrane lipids and the cytoskeleton, we examined in detail the interactions between purified calpactin I and phospholipid liposomes. We focused on the Ca2+-dependence, the effects of phosphorylation of calpactin I by p60v-src (the protein kinase coded for by the Rous-sarcoma-virus oncogene), and the effects of the binding of calpactin I light chain to calpactin I heavy chain. Binding of the light chain to the heavy chain increased the affinity of calpactin I for phosphatidylserine (PS) liposomes. The opposite effect was observed for phosphorylation by p60v-src; phosphorylation decreased the affinity of calpactin I for PS liposomes. These two opposite effects appeared to be independent, since phosphorylation did not prevent light-chain binding to the heavy chain. Calpactin I was found, by the use of three different techniques, to bind to phospholipid liposomes at less than 10(-8) M free Ca2+. This result is in contrast with those of previous studies, which indicated that greater than 10(-6) M free Ca2+ was required. Our findings suggest that calpactin I may be bound to phospholipids in vivo at Ca2+ concentrations of about 1.5 x 10(-7) M, typical of resting unstimulated cells, and that this interaction may be modulated by light-chain binding and phosphorylation by p60v-src.     

Disposition of preformed mineral in matrix vesicles. Internal localization and association with alkaline phosphatase

Studies were made on the disposition of mineral ions in matrix vesicles (MV) and their relationship to alkaline phosphatase by treatment of MV-enriched microsomes (MVEM) with graded levels of Ca2+-chelating agents to complex accessible ions, fractionation of MVEM on hypertonic sucrose gradients at two different pH values (7.5 and 8.0) to evaluate for the presence of calcium phosphate mineral, and passage of MVEM through cation-exchange columns to determine the accessibility of the Ca2+. The effect of removal of Ca2+ and Pi on subsequent ability of MVEM to induce mineral formation from synthetic cartilage lymph was also determined. Passage through cation-exchange columns revealed that MV Ca2+ was not freely exchangeable, but coeluted in the void volume with alkaline phosphatase. However, upon incubation in synthetic cartilage lymph, progressively more Ca2+ was retained by the column. These findings indicate that, initially, the majority of Ca2+ in MVEM is internal and not readily exchangeable, but as Ca2+ accumulates, progressively more becomes external. The mineral in MV is labile and readily susceptible to loss; treatment with graded levels of EGTA removed major portions of the original Ca2+ and Pi. 45Ca uptake by these mineral-depleted MV was markedly reduced, even in the presence of alkaline phosphatase substrates. Sucrose gradient fractionation of MVEM caused extensive loss of Pi, but not Ca2+, from the low-density alkaline phosphatase-rich fractions. This reveals that Ca2+ and Pi are not initially coupled together: Pi is largely soluble, whereas Ca2+ must be tightly bound. In the high-density vesicles, large amounts of both Ca2+ and Pi are present. The slightly enhanced recovery at higher pH suggests the presence of a solid mineral phase. During mineralization by MV, Ca2+ became externalized, and concomitantly alkaline phosphatase activity declined. This suggests that a direct association exists between the enzyme and the developing mineral.     

Antibodies to the N-terminus of calpactin II (p35) affect Ca2+ binding and phosphorylation by the epidermal growth factor receptor in vitro

Calpactins I and II are related 39-kilodalton (kDa) proteins that interact with phospholipids and actin in a calcium-dependent manner and are substrates of tyrosine protein kinases. They contain a short amino-terminal tail attached to a 36-kDa core domain. Monoclonal antibodies (Mabs) were raised to bovine calpactin II and used as site-specific probes of its structure and function. All of the antibodies reacted with native calpactin II and gave rise to a single band of 39 kDa among total cell protein displayed on Western blots. Most of the antibodies (9/14) reacted with determinants on the tail as shown by Western blots and competition with a synthetic tail peptide. Four antibodies reacted with determinants on the core and a 10-kDa tryptic fragment. Antibody-calpactin II complexes were tested for their ability to interact with lipid, actin, and Ca2+ and to serve as substrates of the epidermal growth factor (EGF) receptor tyrosine protein kinase. Whereas none of the antibodies had a detectable effect on actin binding, two anticore antibodies reduced calpactin's affinity for phospholipid. Ca2+-binding sites are known to reside within the core region, yet most antitail antibodies markedly increased the affinity of calpactin II for Ca2+, with four Ca2+-binding sites observed. Antitail antibodies either (i) abolished or (ii) greatly stimulated (10-fold) the phosphorylation of calpactin II by the EGF receptor. These results suggest that the interactions between calpactin II and Ca2+, phospholipid, or the EGF receptor are more complex than previously thought and can be modulated by interactions occurring in the tail.     

Comparative structural aspects of cation binding to phosphatidylserine bilayers

X-ray diffraction data recorded for monovalent and divalent cation complexes of a series of phosphatidylserines (PS) varying in chain length reveal a simple structural pattern. Only two bilayer structural types differing in hydrocarbon chain tilt but with similar polar group conformations are observed for (i) anhydrous acidic PS, (ii) anhydrous K+-PS, and (iii) Li+, Mg2+, Ca2+, Sr2+, Ba2+, and Pr3+ complexes of 'hydrated' PS. The X-ray diffraction data suggest that PS becomes dehydrated on complexing with Li+, Mg2+, Ca2+, and other divalent cations and adopts either the chain untilted (form I) or tilted (form II) bilayer structure.     

Differential fractionation of matrix vesicle proteins. Further characterization of the acidic phospholipid-dependent Ca2(+)-binding proteins

Matrix vesicles (MV) initiate de novo mineralization in a variety of vertebrate-calcifying tissues. In recent studies, a quantitatively major group of MV proteins, the acidic phospholipid-dependent Ca2(+)-binding proteins (APD-CaBP) were found to be immunologically related to the annexin family of proteins that possess phospholipase A2 inhibitory activity. This finding helped explain the enrichment of phosphatidylserine as well as the presence of large amounts of complexed Ca2+ noted previously in these structures. To characterize further these annexin-like proteins, preparations of both collagenase-released MV and MV-enriched microsomes were subjected to a differential fractionation process that led to the isolation and purification to homogeneity of two of the MV APD-CaBP, a 33-kDa protein and a 36-kDa calpactin II-like protein. Polyclonal antibodies raised to each pure protein were found not to cross-react with the other, thus indicating two distinctive proteins. Measurement of the phosphatidylserine-dependent Ca2(+)-binding properties of the proteins revealed apparent Kd values of 2.5 x 10(-7) and 5.0 x 10(-7) M for the 36- and 33-kDa proteins, respectively. Such high affinities indicate that both proteins would be normally bound to the membrane of MV. Immunological studies revealed the presence of both APD-CaBP in cultured growth plate chondrocytes but not in vesicles released into the culture medium. The finding of the 33-kDa but not the 36-kDa protein in vesicles released from the calcifying matrix of the chondrocyte cultures by collagenase digestion may indicate a role for this protein in MV mineralization.     

Mineralization of annexin-5-containing lipid-calcium-phosphate complexes: modulation by varying lipid composition and incubation with cartilage collagens

Matrix vesicles (MVs) in the growth plate bind to cartilage collagens and initiate mineralization of the extracellular matrix. Native MVs have been shown to contain a nucleational core responsible for mineral formation that is comprised of Mg(2+)-containing amorphous calcium phosphate and lipid-calcium-phosphate complexes (CPLXs) and the lipid-dependent Ca(2+)-binding proteins, especially annexin-5 (Anx-5), which greatly enhances mineral formation. Incorporation of non-Ca(2+)-binding MV lipids impedes mineral formation by phosphatidylserine (PS)-CPLX. In this study, nucleators based on amorphous calcium phosphate (with or without Anx-5) were prepared with PS alone, PS + phosphatidylethanolamine (PE), or PS + PE and other MV lipids. These were incubated in synthetic cartilage lymph containing no collagen or containing type II or type X collagen. Dilution of PS with PE and other MV lipids progressively retarded nucleation. Incorporation of Anx-5 restored nucleational activity to the PS:PE CPLX; thus PS and Anx-5 proved to be critical for nucleation of mineral. Without Anx-5, induction of mineral formation was slow unless high levels of Ca(2+) were used. The presence of type II collagen in synthetic cartilage lymph improved both the rate and amount of mineral formation but did not enhance nucleation. This stimulatory effect required the presence of the nonhelical telopeptides. Although type X collagen slowed induction, it also increased the rate and amount of mineral formation. Both type II and X collagens markedly increased mineral formation by the MV-like CPLX, requiring Anx-5 to do so. Thus, Anx-5 enhances nucleation by the CPLXs and couples this to propagation of mineral formation by the cartilage collagens.     

Isolation of Ca2+ sensitive proteins linked to the membrane fraction of the brain cortex and the spinal cord in pigs

Four Ca2+-sensitive proteins of respective subunit molecular weights 67 kDa, 37 kDa, 36 kDa and 32 kDa were purified from pig brain and spinal cord. Associated to the particulate fraction at millimolar concentrations of free Ca2+, they were solubilized using an EGTA-containing buffer and purified by a selective Ca2+-dependent precipitation. The 36 kDa protein is present in the tissues in a tetrameric form of (2 X 36 kDa + 2 X 13 kDa) and in a monomeric form. These proteins with the 37 kDa protein share the functional properties of the two well-known Ca2+-binding proteins, named calpactin I and calpactin II; they were able to interact with F-actin, brain spectrin (fodrin) and phosphatidylserine-liposomes in a Ca2+-dependent manner. The 67 kDa protein depolymerizes the actin filament in presence of Ca2+, it also binds to tubulin and to the neurofilament subunit NF-70, but not to brain spectrin. The 32 kDa protein does not share any association with F-actin and brain spectrin.     

Twenty-eight-kilodalton phosphorylatable calcium- and lipid-binding proteins purified from Physarum plasmodium.

Two 28-kDa calcium- and lipid-binding proteins were isolated from a detergent-insoluble fraction of the Physarum plasmodium. Both proteins have molecular masses of approximately 28 kDa by SDS-PAGE. The protein designated 28K-I has a slightly lower mobility than that designated 28K-II. The purified 28K-I has a dissociation constant of 1.0 microM for Ca2+ ions, while the 28K-II has two different dissociation constants: one of 0.32 microM and the other of 3.2 mM. The 28K-I binds to liposomes at Ca2+ concentrations higher than 1.0 microM and has a dissociation constant for lipid of 34 micrograms/ml at 10 microM Ca2+. The 28K-II binds to liposomes at concentrations of Ca2+ above the mM range and has a dissociation constant of 36 micrograms/ml for lipid at 2 mM Ca2+. There is no evidence of actin-binding activity by either of the 28-kDa (28K) proteins. The 28K proteins crossreacted with an antiserum against chicken brush border calpactin I. The two proteins have quite different phosphorylation levels between a fraction prepared from the cytosolic endoplasm and a fraction prepared from the whole cell. The 28K proteins may play some role in the membrane structure dynamics of the cortical gel layer.     

Effects of ionophore A23187 and Ba++ ions on labelling of phospholipids in rat pancreatic islets

Ionophore A23187, either in the presence or absence of added Ca2+ or Mg2+, caused a marked accumulation of [32P]-phosphatidic acid in pancreatic islets pre-labelled with 32 Pi. A similar effect was observed following the addition of 4 mM Ba2+ ions in the absence of added Ca2+. Neither agent caused a significant modification of labelling in other lipid fractions, although there was a persistent trend towards reduced labelling of phosphatidylcholine and phosphatidylethanolamine. Ionophore A23187 also potentiated the incorporation of 3H-glycerol into phosphatidic acid and reduced the incorporation of this precursor into phosphatidylcholine. In islets pre-labelled with 3H-glycerol and subsequently exposed to A23187 or Ba2+, no significant changes were observed in label associated with either phospholipids or neutral glycerolipids. These results suggest that ionophore A23187 and Ba2+ ions can divert the synthesis of phospholipids resulting in increased formation of phosphatidic acid at the expense of non-acidic phospholipids, principally phosphatidylcholine. We tentatively suggest that this effect may be the result of inhibition by Ca2+ of the breakdown of phosphatidic acid to diglyceride, an enzymic step which may regulate the relative amounts of acidic and neutral phospholipids.     

Transmembrane Ca2+ exchange in depolarized rat myometrium mitochondria

Polarization of the inner membrane is the key factor in maintenance of the physiologically significant cations accumulation, in particular Ca2+, in the mitochondria. It has been well established that mitochondria accumulate calcium through the uniporter, driven by the mitochondrial membrane potential. Nevertheless, it has been shown that depolarized mitochondria also accumulate Ca2+. The aim of this paper is to investigate free Ca level in depolarized myometrium mitochondria. As we have shown previously Ca2+ addition to the incubation medium, that did not contain K-phosphate, ATP and Mg2+, led to inner mitochondrial membrane depolarization. Nevertheless Ca2+ addition to such medium led to the concentration-dependent accumulation of this cation in the matrix. RuR or Mg addition to the incubation medium led to the higher elevation of mitochondrial Ca2+ level in depolarized mitochondria. Mitochondrial Ca2+ level was not affected by 5 microM cyclosporine A. It was suggested that H+/Ca2+ exchanger could provide calcium accumulation in depolarized mitochondria. The elevation of mitochondrial Ca2+ level after addition of Mg2+ and RuR may be due to inhibition of Ca2+- efflux through Ca2+ uniporter.     

Calcium-dependent fusion of the plasma membrane fraction from human neutrophils with liposomes

A cell-free assay monitoring lipid mixing was used to investigate the role of Ca2+ in neutrophil membrane-liposome fusion. Micromolar concentrations of Ca2+ were found to directly stimulate fusion of inside-out neutrophil plasma membrane enriched fractions (from neutrophils subjected to nitrogen cavitation) with liposomes (phosphatidylethanolamine:phosphatidic acid, 4:1 molar ratio). In contrast, right-side-out plasma membranes and granule membranes did not fuse with liposomes in the presence of Ca2+. Similar results were obtained with two different lipid mixing assays. Fusion of the neutrophil plasma membrane-enriched fraction with liposomes was dependent upon the concentration of Ca2+, with threshold and 50% maximal rate of fusion occurring at 2 microM and 50 microM, respectively. Furthermore, the fusion was highly specific for Ca2+; other divalent cations such as Ba2+, Mg2+ and Sr2+ promoted fusion only at millimolar concentrations. Red blood cell (RBC) membranes were used in control studies. Ca2(+)-dependent fusion did not occur between right-side-out or inside-out RBC-vesicles and liposomes. However, if the RBC-vesicles were exposed to conditions which depleted spectrin (i.e., low salt), then Ca2(+)-dependent fusion was detected. Other quantitative differences between neutrophil and RBC membranes were found; fusion of liposomes with RBC membranes was most readily achieved with La3+ while neutrophil membrane-liposome fusion was most readily obtained with Ca2+. Furthermore, GTP gamma S was found to enhance Ca2(+)-dependent fusion between liposomes and neutrophil plasma membranes, but not RBC membranes. These studies show that plasma membranes (enriched fractions) from neutrophils are readily capable of fusing with artificial lipid membranes in the presence of micromolar concentrations of Ca2+.     

Similarity in calcium channel activity of annexin V and matrix vesicles in planar lipid bilayers.

Matrix vesicles (MVs), structures that accumulate Ca2+ during the initiation of mineral formation in growing bone, are rich in annexin V. When MVs are fused with planar phospholipid bilayers, a multiconductance Ca2+ channel is formed, with activity essentially identical to that observed when annexin V is delivered to the bilayer with phosphatidylserine liposomes. Ca2+ currents through this channel, from either MV or annexin V liposomes, are blocked by Zn2+, as is Ca2+ uptake by MV incubated in synthetic cartilage lymph. Blockage by Zn2+ was most effective when applied to the side containing the MV or liposomes. ATP and GTP differentially modulated the activity of this channel: ATP increased the amplitude of the current and the number of conductance states; GTP dramatically reduced the number of events and conductance states, leading to well-defined Ca2+ channel activity from either MV or the annexin V liposomes. In the distinctive effects of ATP, GTP, and Zn2+ on the Ca2+ channel activity observed in both the MV and the liposome systems, the common factor was the presence of annexin V. From this we conclude that Ca2+ entry into MV results from the presence of annexin V in these membrane-enclosed structures.     

Ca2+- and phospholipid-binding properties of Torpedo electric organ calelectrin

The Ca2+-regulated lipid-binding properties of the H- and L-forms of calelectrin present in the electric organ of Torpedo marmorata have been compared. Binding of H-calelectrin required Ca2+ in millimolar concentrations, whereas that of L-calelectrin occurred in the micromolar range. Dissociation of H-calelectrin previously bound to lipids in the presence of 2 mM Ca2+ took place only when the Ca2+ concentration was reduced to micromolar concentrations. Binding was most effective to acidic phospholipids such as phosphatidylserine. Both forms of calelectrin promoted the aggregation of membrane vesicles in the presence of Ca2+.Mg2+, Na+ and K+ inhibited the Ca2+-induced binding to phospholipid, decreasing in effectiveness in that order. Binding was also inhibited by high pH. The surface activity and hydrophobicity index showed that H-calelectrin is a hydrophilic molecule. It may represent a less active, more highly phosphorylated "down-regulated" form of L-calelectrin. The role of calcium in H-calelectrin binding to lipid appeared to be consistent with the formation of a ternary complex of the protein, an acidic lipid and Ca2+, rather than with a direct interaction of lipid with hydrophobic sequences in H-calelectrin whose accessibility is Ca2+-regulated.     

The importance of an asymmetric distribution of acidic lipids for synaptotagmin 1 function as a Ca2+ sensor

Syt1 (synaptotagmin 1) is a major Ca2+ sensor for synaptic vesicle fusion. Although Syt1 is known to bind to SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) complexes and to the membrane, the mechanism by which Syt1 regulates vesicle fusion is controversial. In the present study we used in vitro lipid-mixing assays to investigate the Ca2+-dependent Syt1 function in proteoliposome fusion. To study the role of acidic lipids, the concentration of negatively charged DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine) in the vesicle was varied. Syt1 stimulated lipid mixing by 3-10-fold without Ca2+. However, with Ca2+ there was an additional 2-5-fold enhancement. This Ca2+-dependent stimulation was observed only when there was excess PS (phosphatidylserine) on the t-SNARE (target SNARE) side. If there was equal or more PS on the v-SNARE (vesicule SNARE) side the Ca2+-dependent stimulation was not observed. We found that Ca2+ at a concentration between 10 and 50?μM was sufficient to give rise to the maximal enhancement. The single-vesicle-fusion assay indicates that the Ca2+-dependent enhancement was mainly on docking, whereas its effect on lipid mixing was small. Thus for Syt1 to function as a Ca2+ sensor, a charge asymmetry appears to be important and this may play a role in steering Syt1 to productively trans bind to the plasma membrane.     

Correlation between loss of alkaline phosphatase activity and accumulation of calcium during matrix vesicle-mediated mineralization

Activity of the bone/liver/kidney isozyme of alkaline phosphatase (AP) is known to be critical for mineralization in developing bone, although its role is unclear. The work now reported explores changes in the activity of this Zn2+-containing enzyme that occur during Ca2+ accumulation by matrix vesicles (MV). A marked loss (up to 65-70%) in AP activity was found to accompany Ca2+ accumulation by MV. These two events were highly correlated, both temporally and quantitatively. Investigation into possible causes revealed that the decline in AP activity during Ca2+ uptake was not due to action of proteases but rather resulted from interaction with the developing mineral phase, loss of metal ions (Zn2+ and Mg2+) from the active site of the enzyme, and concomitant irreversible denaturation of the enzyme. Protease inhibitors did not protect AP from loss of activity during mineralization; in contrast, protease treatments, which progressively destroyed the ability of MV to accumulate Ca2+ actually reduced loss of AP activity. These findings clearly demonstrate that AP is present at the site of MV mineralization and that its catalytic activity is profoundly reduced by the mineralization process.     

Cholesterol affects divalent cation-induced fusion and isothermal phase transitions of phospholipid membranes

The influence of cholesterol on divalent cation-induced fusion and isothermal phase transitions of large unilamellar vesicles composed of phosphatidylserine (PS) was investigated. Vesicle fusion was monitored by the terbium/dipicolinic acid assay for the intermixing of internal aqueous contents, in the temperature range 10-40 degrees C. The fusogenic activity of the cations decreases in the sequence Ca2+ greater than Ba2+ greater than Sr2+ much greater than Mg2+ for cholesterol concentrations in the range 20-40 mol%, and at all temperatures. Increasing the cholesterol concentration decreases the initial rate of fusion in the presence of Ca2+ and Ba2+ at 25 degrees C, reaching about 50% of the rate for pure PS at a mole fraction of 0.4. From 10 to 25 degrees C, Mg2+ is ineffective in causing fusion at all cholesterol concentrations. However, at 30 degrees C, Mg2+-induced fusion is observed with vesicles containing cholesterol. At 40 degrees C, Mg2+ induces slow fusion of pure PS vesicles, which is enhanced by the presence of cholesterol. Increasing the temperature also causes a monotonic increase in the rate of fusion induced by Ca2+, Ba2+ and Sr2+. The enhancement of the effect of cholesterol at high temperatures suggests that changes in hydrogen bonding and interbilayer hydration forces may be involved in the modulation of fusion by cholesterol. The phase behavior of PS/cholesterol membranes in the presence of Na+ and divalent cations was studied by differential scanning calorimetry. The temperature of the gel-liquid crystalline transition (Tm) in Na+ is lowered as the cholesterol content is increased, and the endotherm is broadened. Addition of divalent cations shifts the Tm upward, with a sequence of effectiveness Ba2+ greater than Sr2+ greater than Mg2+. The Tm of these complexes decreases as the cholesterol content is increased. Although the transition is not detectable for cholesterol concentrations of 40 and 50 mol% in the presence of Na+, Sr2+ or Mg2+, the addition of Ba2+ reveals endotherms with Tm progressively lower than that observed at 30 mol%. Although the presence of cholesterol appears to induce an isothermal gel-liquid crystalline transition by decreasing the Tm, this change in membrane fluidity does not enhance the rate of fusion, but rather decreases it. The effect of cholesterol on the fusion of PS/phosphatidylethanolamine (PE) vesicles was investigated by utilizing a resonance energy transfer assay for lipid mixing. The initial rate of fusion of PS/PE and PS/PE/cholesterol vesicles is saturated at high Mg2+ concentrations. With Ca2+, saturation is not observed for cholesterol-containing vesicles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calcium dependence of hormone-stimulated cAMP accumulation in intact glial tumor cells.

The Ca2+ content of glial tumor (C6) cells was reduced approximately 5-fold by repeated treatment with media containing ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) without loss of cellular viability. The ability of the cells to accumulate cAMP in response to beta-adrenergic agonists was reduced 60 to 70% following Ca2+ depletion. Ca2+ did not affect the apparent KACT for norepinephrine, nor did it change the concentration of propranolol required to produce 50% inhibition of the maximal norepinephrine response. Phentolamine did not alter the Ca2+ dependence of the response. The binding of dihydroalprenolol by intact C6 cells was not influenced by Ca2+. Furthermore, pretreatment with norepinephrine did not affect the Ca2+ dependence of cAMP accumulation. The effects of Ca2+, therefore, appeared to be exerted on components of the adenylate cyclase system other than the catecholamine receptor. Micromolar free Ca2+ concentration in the extracellular medium were sufficient to restore a maximal norepinephrine response to Ca2+-depeleted cells. The effect of Ca2+ on cAMP accumulation in response to hormone was immediate and was rapidly reversible upon the addition of EGTA in excess of the cation. Cells in media containing Ca2+ exhibited a characteristic biphasic time course of cAMP accumulation; with Ca2+-depleted cells cAMP was accumulated more slowly and the subsequent decline in cAMP content was also reduced. Verapamil, an inhibitor of plasmalemmal Ca2+ influx, decreased the Ca2+-dependent component of the cAMP accumulation when added prior to the cation. The effect of Ca2+ on cAMP accumulation was reduced more extensively by pretreatment of cells at 45 degrees C under Ca2+-depleted (80% loss) than under Ca2+-restored (30% loss) conditions. Trifluoperazine at micromolar concentrations decreased the Ca2+-dependent increment in accumulation of cAMP in Ca2+-restored cells. This inhibition was not overcome by increasing concentrations of norepinephrine or of extracellular Ca2+.     

The role of Fe2+-induced lipid peroxidation in the initiation of the mitochondrial permeability transition

Iron and iron complexes stimulate lipid peroxidation and formation of malondialdehyde (MDA). We have studied the effects of Fe2+ and ascorbate on mitochondrial permeability transition induced by phosphate and Ca2+. Iron is necessary for detectable MDA formation, but only Ca2+ and phosphate are necessary for the induction of membrane potential loss (Deltapsi) and Ca2+ release. Keeping the iron at a constant concentration and varying the Ca2+ level changed the mitochondrial Ca2+ retention times, but not the amount of MDA formation. The antioxidant butylated hydroxytoluene at low concentrations prevented MDA formation, but not mitochondrial Ca2+ release. Preincubation of mitochondria with Fe2+ decreased Ca2+ retention time in a concentration-dependent manner and facilitated Ca2+-stimulated MDA accumulation. Thus, Ca2+ phosphate-induced mitochondrial permeability transition (MPT) can be separated mechanistically from MDA accumulation. Lipid peroxidation products do not appear to participate in the initial phase of the permeability transition, but sensitize mitochondria toward MPT.     

Effects of calcium and phosphatidyl serine in rat mast cell reaction to dextran.

Histamine release from Sprague-Dawley rat mast cells by dextran was completely inhibited by the absence of exogenous Ca2+ (in contrast to release from the same cells by antigen). Also, spontaneous leakage of histamine from the cells increased in the absence of Ca2+, and cell responsiveness was not completely restored by readding Ca2+. We found no effective substitute for Ca2+ in the release reaction. Ca2+ was not maximally effective immediately when added back to Ca-deficient cells, but almost the full effect of diluting Ca2+ in the medium (which decreased release) and of adding PS (which increased release) were very rapidly established, suggesting that both Ca2+ and PS might act (in part) at superficial cell sites. Release from activated cells could be stopped short by adding glucose or by diluting the cell-dextran mixture with normal buffer, as well as by adding EDTA, which deserves further study.     

Fusogenic capacities of divalent cations and effect of liposome size

The initial kinetics of divalent cation (Ca2+, Ba2+, Sr2+) induced fusion of phosphatidylserine (PS) liposomes, LUV, is examined to obtain the fusion rate constant, f11, for two apposed liposomes as a function of bound divalent cation. The aggregation of dimers is rendered very rapid by having Mg2+ in the electrolyte, so that their subsequent fusion is rate limiting to the overall reaction. In this way the fusion kinetics are observed directly. The bound Mg2+, which by itself is unable to induce the PS LUV to fuse, is shown to affect only the aggregation kinetics when the other divalent cations are present. There is a threshold amount of bound divalent cation below which the fusion rate constant f11 is small and above which it rapidly increases with bound divalent cation. These threshold amounts increase in the sequence Ca2+ less than Ba2+ less than Sr2+, which is the same as found previously for sonicated PS liposomes, SUV. While Mg2+ cannot induce fusion of the LUV and much more bound Sr2+ is required to reach the fusion threshold, for Ca2+ and Ba2+ the threshold is the same for PS SUV and LUV. The fusion rate constant for PS liposomes clearly depends upon the amount and identity of bound divalent cation and the size of the liposomes. However, for Ca2+ and Ba2+, this size dependence manifests itself only in the rate of increase of f11 with bound divalent cation, rather than in any greater intrinsic instability of the PS SUV. The destabilization of PS LUV by Mn2+ and Ni2+ is shown to be qualitatively distinct from that induced by the alkaline earth metals.