Studies on the mechanism of membrane fusion. Role of head-group composition in calcium- and magnesium-induced fusion of mixed phospholipid vesicles

We have investigated the contribution of various phospholipids to membrane fusion induced by divalent cations. Fusion was followed by means of a new fluorescence assay monitoring the mixing of internal aqueous contents of large (0.1 μm diameter) unilamellar liposomes. The rate and extent of fusion induced by Ca²⁺ in mixed phosphatidylserine/phosphatidylcholine vesicles were lower compared to those in pure phosphatidylserine vesicles. The presence of 50% phosphatidylcholine completely inhibited fusion, although the vesicles aggregated upon Ca²⁺ addition. When phosphatidylserine was mixed with phosphatidylethanolamine, however, rapid fusion could be induced by Ca²⁺ even in mixtures that contained only 25% phosphatidylserine. Phosphatidylethanolamine also facilitated fusion by Mg²⁺ which could not fuse pure phosphatidylserine vesicles. In phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine mixtures, in which the phosphatidylcholine content was kept at 25%, phosphatidylethanolamine could not substitute for phosphatidylserine, and the fusogenic capacity of Mg²⁺ was abolished by the presence of merely 10% phosphatidylcholine. The initial rate of release of vesicle contents was slower than the rate of fusion in all the mixtures used. The presence of phosphate effected a considerable decrease in the threshold concentration of Ca²⁺ and also enhanced     

Control of membrane fusion by phospholid head groups I. Phosphatidate/phosphatidylinositol specificity

We have studied the characteristics of fusion of large unilamellar vesicles composed of phosphatidate and phosphatidylinositol alone and in mixtures with other naturally occurring phospholipids. Fusion was induced by the addition of Ca²⁺ or Mg²⁺ and was monitored by detecting the mixing of aqueous vesicle contents. Release of vesicle contents was measured by dequenching of carboxyfluorescein fluorescence. Aggregation was monitored by 90° light scattering. The results indicated striking differences with respect to the fusion capacity of the different vesicles. Phosphatidate vesicles fuse in the presence of both Ca²⁺ and Mg²⁺ at threshold concentration ranges of 0.03–0.1 mM (Ca²⁺) and 0.07–0.15 mM (Mg²⁺) depending on the pH of the medium, 8.5-6.0, respectively. In contrast, phosphatidylinositol vesicles do not fuse with either Ca²⁺ or Mg²⁺ even at 50 mM concentrations, in spite of aggregation induced by both cations in the range of 5–10 mM. A large difference in terms of fusion capacity is retained even when these two phospholipids are mixed with phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in 2 : 2 : 4 : 2 molar ratios. The results are discussed in terms of the molecular mechanism of membrane fusion and the possible role of the metabolic interconversion of phosphatidylinositol to phosphatidate as an on-off control system for membrane fusion phenomena involved in secretion.     

Ca2+-dependent ATPase associated with plasma membrane from a calcareous alga, Serraticardia maxima (Corallinaceae, Rhodophyta)

The plasma membrane was isolated from a calcareous red alga, Serraticardia maxima (Yendo) Silva (Corallinaceae), by aqueous two-phase partitioning. Its purity was examined with marker enzymes, Mg²⁺-dependent ATPase, inosine diphosphatase, cytochrome c oxidase and NADH-cytochrome c reductase, as well as the sensitivity of Mg²⁺-dependent ATPase to vanadate, azide and nitrate. The results showed that the isolated plasma membrane was purified enough to study its functions. Electron microscopic observations on thin tissue sections revealed that most vesicles of the isolated plasma membrane were stained by the plasma membrane specific stain, phosphotungstic acid-chromic acid. Mg²⁺- or Ca²⁺-dependent ATPases were associated with the plasma membrane. Ca²⁺-dependent ATPase was activated at physiological cytoplasmic concentrations of Ca²⁺ (0.1–10 μmol/L). However, calmodulin (0.5 μmol/L) did not affect its activity. The pH optimum was 8.0, in contrast to 7.0 for Mg²⁺-dependent ATPase. The isolated plasma membrane vesicles were mostly right side-out. To test for H⁺-translocation, right side-out vesicles were inverted; 27% of vesicles were inside-out after treatment with Triton X-100. The inside-out plasma membrane vesicles showed reduction of quinacrine fluorescence in the presence of 1 mmol/L ATP and 100 μmol/L Ca²⁺. The reduced fluorescence was recovered with the addition of 10 mmol/L NH₄Cl, or 5 μmol/L nigericin plus 50 mmol/L KCl. UTP and CTP substituted for ATP, but ADP did not. Ca²⁺-dependent ATPase might pump H⁺ out in the physiological state. The acidification by this pump might be coupled with alkalinization at the calcifying sites, which induces calcification.     

A Ca2+-ATPase and a Mg2+/H+-antiporter are present on tonoplast membranes from roots of Zea mays L.

The primary or secondary energized transport of Ca²⁺, Mg²⁺ and H⁺ into tonoplast membrane vesicles from roots of Zea mays L. seedlings was studied photometrically by using the fluorescent Ca²⁺ indicator Indo 1 and the pH indicator neutral red. The localization of an ATP-dependent, vanadate-sensitive Ca²⁺ pump on tonoplast-type vesicles was demonstrated by the co-migration of the Ca²⁺-pumping and tonoplast H⁺-pyrophosphatase (PP_iase) activity on continuous sucrose density gradients. In ER-membrane fractions, only a low Ca²⁺-pumping activity could be detected. The ATP-dependent Ca²⁺ uptake into tonoplast vesicles (using Ca²⁺ concentrations from 0.8–1 μM) was completely inhibited by the Ca²⁺ ionophore ionomycin (1 μM) whereas the protonophore nigericin (1 μM) which eliminates ATP-dependent intravesicular H⁺ accumulation had no effect. Vanadate (IC₅₀ = 43 μM) and diethylstilbesterol (IC₅₀ = 5.2 μM) were potent inhibitors of this type of Ca²⁺ transport. The nucleotides GTP, UTP, ITP, and ADP gave 27%–50% of the ATP-dependent activity (K _m = 0.41 mM). From these results, it was suggested that this ATP-dependent high-affinity Ca²⁺ transport mechanism is the only functioning Ca²⁺ transporter of the tonoplast under in-vivo conditions i.e. under the low cytosolic Ca²⁺ concentration. In contrast, the secondary energized Ca²⁺-transport mechanism of the tonoplast, the low-affinity Ca²⁺/H⁺-antiporter, which was reported to allow the uptake of Ca²⁺ in exchange for H⁺, functions chiefly as an Mg²⁺ transporter under physiological conditions because cytosolic Mg²⁺ is several orders of magnitude higher than the Ca²⁺ concentration. This conclusion was deduced from experiments showing that Mg²⁺ ions in a concentration range of 0.01 to 1 mM triggered a fast efflux of H⁺ from acid-loaded vesicles. Furthermore, the proton-pumping activity of the tonoplast H⁺-ATPase and H⁺-PP_iase was found to be influenced by Ca²⁺ differently from and independently of the Mg²⁺ concentration. Calcium was a strong inhibitor for the H⁺-PP_iase (IC₅₀ = 18 μM, Hill coefficient nH = 1.7) but a weak one for the H⁺-ATPase (IC₅₀ = 330 μM, nH = 1). From these results it is suggested that at the tonoplast membrane a functional interaction exists between (i) the Ca²⁺-and Mg²⁺-regulated H⁺-PP_iase, (ii) the newly described high-affinity Ca²⁺-AT-Pase, (iii) the low-affinity Mg²⁺(Ca²⁺)/H⁺-antiporter and (iv) the H²⁺-ATPase.     

Cytoplasmic polyamines block the fast-activating vacuolar cation channel

The fast-activating vacuolar (FV) channel dominates the electrical characteristics of the tonoplast at physiological free Ca²⁺ concentrations. Since polyamines are known to increase in plant cells in response to stress, the regulation of FV channels by polyamines was investigated. Patch-clamp measurements were performed on whole barley ( Hordeum vulgare ) mesophyll vacuoles and on excised tonoplast patches. The trivalent polyamine spermidine and the tetravalent polyamine spermine blocked FV channels with K_d≈ 100 μM and K_d≈ 5 μM, respectively. Increasing cytosolic and vacuolar Ca²⁺ had no effect on putrescine and spermidine binding to FV channels but slightly decreased the affinity for spermine. The inhibition of FV channels by all three polyamines was not voltage-dependent. This points to a different mode of binding compared to inward rectifier K⁺ channels and Ca²⁺-permeable glutamate receptor channels from animal cells, which show rectification due to a voltage-dependent block by polyamines. In plant cells, the common polyamines (putrescine, spermidine and spermine) are likely to mediate a salt stress-induced decrease of ion flux across the vacuolar membrane by blocking FV channels.     

Fusion of neurohypophyseal membranes in vitro

Freeze cleaving electron microscopy has shown that fusion of isolated secretory vesicles from bovine neurohypophyses was induced by Ca²⁺ in micromolar concentrations. Mg²⁺ and Sr²⁺ were ineffective. Mg²⁺ inhibited Ca²⁺-induced fusion.In suspensions containing secretory vesicles as well as sheets of cell membrane, release of vasopressin parallel to intervesicular fusion of secretory vesicles with sheets of cell membrane was observed after exposure to Ca²⁺. Mg²⁺ and Sr²⁺ were ineffective in replacing Ca²⁺ as trigger for fusion or vasopressin release.Intervesicular fusion and exocytotic profiles were observed when isolated neurohypophyses or neurosecretosome were exposed to cold.     

Control of membrane fusion by phospholipid head groups II. The role of phosphatidylethanolamine in mixtures with phosphatidate and phosphatidylinositol

Membrane fusion induced by Ca²⁺ and Mg²⁺ in large unilamellar vesicles composed of mixtures of phosphatidylethanolamine with phosphatidate and phosphatidylinositol was studied by means of a fluorescence assay for the intermixing of internal aqueous contents of the vesicles. The threshold concentrations of Ca²⁺ or Mg²⁺ required for fusion increased only moderately when up to 80 mol% phosphatidylethanolamine was included with phosphatidate at pH 7.4, but no fusion could be detected in vesicles containing 70 mol% phosphatidylcholine even at high concentrations of Ca²⁺ or Mg²⁺. Phosphatidate-phosphatidylethanolamine (1 : 4) vesicles could be induced to fuse by 0.1 mM Ca²⁺ in the presence of a Mg²⁺ concentration which alone was insufficient for fusion. When equimolar amounts of phosphatidylethanolamine was included with phosphatidylinositol, the vesicles were susceptible to fusion by Ca²⁺, although pure phosphatidylinositol vesicles themselves merely aggregate and do not fuse (Sundler, R. and Papahadjopoulos, D. (1981) Biochim. Biophys. Acta 649, 743–750, accompanying paper). The role of phosphatidylethanolamine acyl chains, and hence the possible involvement of the bilayer-hexagonal (H_II) transition in membrane fusion, was examined by the temperature dependence of Ca²⁺-induced fusion in phosphatidylinositol-dimyristoylphosphatidylethanolamine (1 : 1) vesicles. Fusion was strictly dependent on the gel-liquid crystalline transition of the mixture and not on the phase behavior of the phosphatidylethanolamines. Comparable fusion rates were obtained for both egg yolk phosphatidylethanolamine and dimyristoylphosphatidylethanolamine at 50°C. As the dimyristoylphosphatidylethanolamine does not convert to a non-bilayer phase in this temperature range, we conclude that the bilayer-hexagonal transition is not necessary for membrane fusion. We propose that the dehydration characteristics of the phospholipids and their metal ion complexes are the critical factors determining fusion suceptibility of phospholipid membranes.     

ATP-driven Ca2+ transport in sealed plasma membrane vesicles prepared by aqueous two-phase partitioning from leaves of Commelina communis

Sealed plasma membrane vesicles were obtained in high purity from leaves of Commelina communis L. by aqueous two-phase partitioning. Based on the analysis of a range of markers, the preparations (U₃+U₃′ phases) were shown to be devoid of tonoplast, Golgi and thylakoid membranes, and showed only trace mitochondrial contamination. One-third of the vesicles were oriented inside out and exhibited ATP-driven ⁴⁵Ca²⁺ transport [? 15 pkat (mg protein)⁻¹]. Ca²⁺ uptake into the vesicles had a pH optimum of 7.2 and apparent K_m values for Ca²⁺ of 4.4 μM and for Mg-ATP of 300 μM. Ca²⁺ uptake, K⁺, Mg²⁺-ATPase (EC 3.6.1.3) activity as well as glucan synthase II (EC 2.4.1.34) activity were all maximal at the same equilibrium density (1.17 g cm⁻³) on continuous sucrose density gradients. The protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) did not inhibit the ATP-dependent Ca²⁺ transport into the vesicles, excluding a Ca²⁺/H⁺ exchange driven by a proton gradient. ATP-dependent Ca²⁺ uptake was inhibited by erythrosin B (I₅₀= 0.1 μM), ruthenium red (I₅₀= 30 μM), La³⁺ (I₅₀= 10 μM) and vanadate (I₅₀= 500 μM), but not by azide, cyanide and oligomycin. The calmodulin antagonists, trifluoperazine (I₅₀= 70 μM) and W-7 (I₅₀= 100 μM) were also inhibitory, However, this inhibition was not overcome by calmodulin. Trifluoperazine and W-7, on the other hand, stimulated Ca²⁺ efflux from the vesicles rather than inhibit Ca²⁺ uptake. Our results demonstrate the presence of a Ca²⁺-ATPase in the plasma membrane of C. communis. In the intact cell, the enzyme would pump Ca²⁺ out of the cell. Its high affinity for Ca²⁺ makes it a likely component involved in adjusting low cytoplasmic Ca²⁺ levels. No indications for a secondary active Ca²⁺/H⁺ transport mechanism in the plasma membrane of C. communis were obtained. Both, the nucleotide specificity and the sensitivity towards vanadate. distinguish the Ca²⁺-ATPase from the H⁺-translocating K⁺. Mg²⁺-ATPase in C. communis plasma membranes.     

Calcium transport and Ca2+-ATPase activity in ram spermatozoa plasma membrane vesicles

Plasma membrane vesicles, isolated from ejaculated ram sperm, were found to contain Ca²⁺-activated Mg²⁺-ATPase and Ca²⁺ transport activities. Membrane vesicles that were exposed to oxalate as a Ca²⁺-trapping agent accumulated Ca²⁺ in the presence of Mg²⁺ and ATP. The $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ for Ca²⁺ uptake was 33 nmol/mg protein per h, and the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for Ca²⁺ and ATP were 2.5 μM and 45 μM, respectively. 1 μM of the Ca²⁺ ionophore A23187, added initially, completely inhibited net Ca²⁺ uptake and, if added later, caused the release of Ca²⁺ previously accumulated. A Ca²⁺-activated ATPase was present in the same membrane vesicles which had a $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 1.5 μmol/mg protein per h at free Ca²⁺ concentration of 10 μM. This Ca²⁺-ATPase had $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values of 4.5 μM and 110 μM for Ca²⁺ and ATP, respectively. This kinetic parameter was similar to that observed for uptake of Ca²⁺ by the vesicles. The Ca²⁺-ATPase activity was insensitive to ouabain. Both Ca²⁺ transport and Ca²⁺-ATPase activity were inhibited by the flavonoid quercetin. Thus, ram spermatozoa plasma membranes have both a Ca²⁺ transport activity and a Ca²⁺-stimulated ATPase activity with similar substrate affinities and specificities and similar sensitivity to quercetin.     

The rate of Ca2+ translocation by sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase measured with intravesicular arsenazo III

Release of Ca²⁺ from the (Ca²⁺ + Mg²⁺)-ATPase into the interior of intact sarcoplasmic reticulum vesicles was measured using arsenazo III, a metallochromic indicator of Ca²⁺. Arsenazo III was placed inside the sarcoplasmic reticulum vesicles by making the vesicles transiently leaky with an osmotic gradient in the presence of arsenazo III. External arsenazo III was then removed by centrifugation. Addition of ATP to the (Ca²⁺ + Mg²⁺)-ATPase in the presence of Ca²⁺ causes the rapid phosphorylation of the enzyme at which time the bound Ca²⁺ becomes inaccessible to external EGTA. The release of Ca²⁺ from the (Ca²⁺ + Mg²⁺)-ATPase to the interior of the vesicle measured with intravesicular arsenazo III was much slower indicating that there is an occluded from the Ca²⁺-binding site which precedes the release of Ca²⁺ into the vesicle. The rate of Ca²⁺ accumulation by sarcoplasmic reticulum vesicles is increased by K⁺ (5–100 mM) and ATP (50–1000 μM) but the initial rate of Ca²⁺ translocation measured after the simultaneous addition of ATP and EGTA to vesicles that were preincubated in Ca²⁺ was not influenced by these concentrations of K⁺ and ATP.     

Calcium-induced calcium increase in secretory vesicles of permeabilized rat neurohypophysial nerve terminals

Digitonin-permeabilized isolated neurohypophysial nerve terminals are known to release their secretory vesicle content under calcium challenge. On this preparation, we monitored intra-organelle Ca²⁺ concentration using digital fluorescence microscopy of Fura-2. The superfusion of artificial intracellular solution containing 10 to 50 μM Ca²⁺ induced an intra-organelle [Ca²⁺] increase. Two major organelles are candidates for this increase: secretory vesicles and mitochondria. In an attempt to detect calcium changes in the vesicles, ruthenium red was used to impair mitochondrial calcium uptake. Part of the ruthenium red-insensitive intra-organelle [Ca²⁺] increase was abolished by raising sodium in the solution. Removing sodium boosted the intra-organelle [Ca²⁺] increase. These results taken together suggest the participation of Na/Ca exchange, known to exist in the membrane of these secretory vesicles. In addition to Na/Ca exchange, there would be at least another mechanism of vesicular calcium intake, as suggested by the partial inhibition of intra-organelle [Ca²⁺] increase obtained under acidic compartments: neutralization with NH₄Cl. This mechanism remains to be defined. The main conclusion presented here, that an intravesicular [Ca²⁺] increase takes place at the rate of secretion, was predicted by the hypothesis that intravesicular Ca²⁺ changes would be involved in stimulus-secretion coupling.     

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°C. The fusogenic activity of the cations decreases in the sequence Ca²⁺ > Ba²⁺ > Sr²⁺ 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°C, reaching about 50% of the rate for pure PS at a mole fraction of 0.4. From 10 to 25°C, Mg²⁺ is ineffective in causing fusion at all cholesterol concentrations. However, at 30°C, Mg²⁺-induced fusion is observed with vesicles containing cholesterol. At 40°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 (T_m) in Na⁺ is lowered as the cholesterol content is increased, and the endotherm is broadened. Addition of divalent cations shifts the T_m upward, with a sequence of effectiveness Ba²⁺ > Sr²⁺ > Mg²⁺. The T_m 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 T_m 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 T_m, 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. The highest rate of fusion for both Ca²⁺- and Mg²⁺-induced fusion is observed with vesicles containing 30 mol% cholesterol.     

Ionophorous functions of phosphatidic acid in the plant cell

Effects of phosphatidic acid (PA), a product of phospholipase D activity, on Ca²⁺ and H⁺ transport were investigated in membrane vesicles obtained from roots and coleoptiles of maize (Zea mays L.). Calcium flows were measured with fluorescent probes indo-1 and chlorotetracycline loaded into the vesicles and added to the incubation medium, respectively. Phosphatidic acid (50–500 μM) was found to induce downhill flow of Ca²⁺ along the concentration gradient into the plasma membrane vesicles and endomembrane vesicles (tonoplast and endoplasmic reticulum). Protonophorous functions of PA were probed with acridine orange. First, the ionic H⁺ gradient was created on the tonoplast vesicles by means of H⁺-ATPase activation with Mg-ATP addition. Then, the vesicles were treated with 25–100 μM PA, which induced the release of protons from tonoplast vesicles and dissipation of the proton gradient. Thus, PA could function as an ionophore and was able to transfer Ca²⁺ and H⁺ across plant cell membranes along concentration gradients of these ions. The role of PA in mechanisms of intracellular signaling in plants is discussed.     

α-Adrenergically mediated changes in membrane lipid fluidity and Ca2+ binding in isolated rat liver plasma membranes

Noradrenaline (0.1–5 μM, in the presence of 5 μM propranolol to block β-receptors), ATP (100 μM) and angiotensin II (0.1 μM), which are thought to increase cytosolic Ca²⁺ concentration by mobilizing Ca²⁺ from internal stores, increased the lipid fluidity as measured by diphenylhexatriene fluorescence polarization in plasma membranes isolated from rat liver. The effect of noradrenaline was dose-dependent and blocked by the α-antagonists phenoxybenzamine (50 μM) and phentolamine (1 μM). The response to a maximal dose of noradrenaline (5 μM) and that to ATP (100 μM) were not cumulative, suggesting that both agents use a common mechanism to alter the membrane lipid fluidity. In contrast, the addition of noradrenaline (5 μM) along with the foreign amphiphile Na⁺-oleate (1–30 μM) resulted in an increase in membrane lipid fluidity which was equivalent to the sum of individual responses to the two agents. In the absence of Mg²⁺, reducing free Ca²⁺ concentration by adding EGTA increased membrane lipid fluidity and abolished the effect of noradrenaline, suggesting that Ca²⁺ is involved in the mechanism by which the hormone exerts its effect on plasma membranes. Noradrenaline (5 μM) and angiotensin II (0.1 μM) also promoted a small release of ⁴⁵Ca²⁺ (16 pmol/mg membrane proteins) from prelabelled plasma membranes. The effect of noradrenaline was suppressed by the α-antagonist phentolamine (5 μM). It is proposed that noradrenaline, via α-adrenergic receptors and other Ca²⁺-mobilizing hormones, increases membrane lipid fluidity by displacing a small pool of Ca²⁺ bound to phospholipids, removing thus the mechanical constraints brought about by this ion.     

Effects of polyamine biosynthesis inhibitors on the progesterone-initiated increase in intracellular free Ca2+ and acrosome reactions in human sperm

This laboratory has previously reported that progesterone can initiate a rapid transient increase in the concentration of intracellular free Ca²⁺([Ca²⁺]_i) and an increase in a Ca²⁺-requiring exocytotic event, the acrosome reaction (AR) in human sperm. Rapid increases in Ca²⁺ fluxes of some mammalian cells caused by another steroid, testosterone, require polyamine biosynthesis. Herein, we tested two polyamine biosynthesis suicide inhibitors for their effects on the progesterone-initiated increase in [Ca²⁺]_i and AR in capacitated human sperm in vitro: DL-α-(difluoromethyl)ornithine hydrochloride (DFMO), an inhibitor of putrescine synthesis by ornithine decarboxylase and (5′-{[(Z))-4-amino-2-butenyl]methylamino}-5′-deoxyadenosine (MDL 73811), an inhibitor of S-adenosylmethionine decarboxylase (required for spermidine and spermine synthesis). Sperm were capacitated in vitro and preincubated 10 min with 4.9 mM DFMO or 9.8 μM MDL 73811 with or without various polyamines (245 μM). Progesterone (3.09 μM final concentration) or progesterone solvent (ethanol, 0.1% final concentration) was then added, sperm fixed 1 min after additions and AR assayed by indirect immunofluorescence or with fluorescein-labeled Con A lectin. DFMO strongly inhibited the AR but putrescine (product of ornithine decarboxylase and precursor of spermidine and spermine) reversed that inhibition. Preincubation for 25 min with DMFO + spermidine also reversed DFMO inhibition. MDL 73811 inhibited the progesterone-initiated AR, and a 10 min preincubation with spermidine, but not putrescine or spermine, reversed that inhibition. Preincubations with putrescine alone or with spermidine alone followed by addition of the progesterone solvent did not initiate the AR, and such preincubations followed by progesterone addition did not increase the AR more than progesterone alone. MDL 73811 and DFMO partially inhibited the rapid progesterone-initiated increase in [Ca²⁺]_i (assayed with fura-2), and those inhibitions were partially reversed by putrescine and spermidine, respectively. Putrescine or spermidine alone did not increase [Ca²⁺]_i nor did preincubation with either polyamine followed by progesterone addition increase [Ca²⁺]_i more than progesterone alone. Neither inhibitor was able to inhibit the AR initiated by the calcium ionophore, ionomycin. Our results suggest that human sperm polyamine biosynthesis is necessary for the progesterone-initiated rapid increase in [Ca²⁺]_i and subsequent membrane events of the AR. © 1993 Wiley-Liss, Inc.     

Studies on membrane fusion. III. The role of calcium-induced phase changes

The interaction of phosphatidylserine vesicles with Ca²⁺ and Mg²⁺ has been examined by several techniques to study the mechanism of membrane fusion. Data are presented on the effects of Ca²⁺ and Mg²⁺ on vesicle permeability, thermotropic phase transitions and morphology determined by differential scanning calorimetry, X-ray diffraction, and freeze-fracture electron microscopy. These data are discussed in relation to information concerning Ca²⁺ binding, charge neutralization, molecular packing, vesicle aggregation, phase transitions, phase separations and vesicle fusion.The results indicate that at Ca²⁺ concentrations of 1.0–2.0 mM, a highly cooperative phenomenon occurs which results in increased vesicle permeability, aggregation and fusion of the vesicles. Under these conditions the hydrocarbon chains of the lipid bilayers undergo a phase change from a fluid to a crystalline state. The aggregation of vesicles that is observed during fusion is not sufficient in itself to induce fusion without a concomitant phase change. Mg²⁺ in the range of 2.0–5.0 mM induces aggregation of phosphatidylserine vesicles but no significant fusion nor a phase change.From the effect of variations in pH, temperature, Ca²⁺ and Mg²⁺ concentration on the fusion of vesicles, it is concluded that the key event leading to vesicle membrane fusion is the isothermic phase change induced by the bivalent metals. It is proposed that this phase change induces a transient destabilization of the bilayer membranes that become susceptible to fusion at domain boundaries.     

Fusion of phospholipid vesicles arrested by quick-freezing. The question of lipidic particles as intermediates in membrane fusion

We have examined the early events in Ca²⁺-induced fusion of large (0.2 μm diameter) unilamellar cardiolipin/phosphatidylcholine and phosphatidylserine/phosphatidylethanolamine vesicles by quick-freezing freeze-fracture electron microscopy, eliminating the necessity of using glycerol as a cryoprotectant. Freeze-fracture replicas of vesicle suspensions frozen after 1–2 s of stimulation revealed that the majority of vesicles had already undergone membrane fusion, as evidenced by dumbbell-shaped structures and large vesicles. In the absence of glycerol, lipidic particles or the hexagonal H_II phase, which have been proposed to be intermediate structures in membrane fusion, were not observed at the sites of fusion. Lipidic particles were evident in less than 5% of the cardiolipin/phosphatidylcholine vesicles after long-term incubation with Ca²⁺, and the addition of glycerol produced more vesicles displaying the particles. We have also shown that rapid fusion occurred within seconds of Ca²⁺ addition by the time-course of fluorescence emission produced by the intermixing of aqueous contents of two separate vesicle populations. These studies therefore have produced no evidence that lipidic particles are necessary intermediates for membrane fusion. On the contrary, they indicate that lipidic particles are structures obtained at equilibrium long after fusion has occurred and they become particularly prevalent in the presence of glycerol.     

Effect of Mg2+ and spermine on the kinetics of Ca2+ transport in rat-liver mitochondria

Plots relating the initial rate of mitochondrial Ca²⁺ transport to the Ca²⁺ concentration (kinetic plots) have a hyperbolic shape in a Ca²⁺ concentration range of 2.5–100 µM as measured in sucrose or KCl media. In the presence of Mg²⁺ or a polyamine spermine, which both are competitive inhibitors of Ca²⁺ binding to low affinity sites at the membrane surface, the shape of the plots becomes sigmoidal. At higher concentrations of these agents linear kinetic plots are obtained as measured in a sucrose medium. In a KCl medium the sigmoidality of the kinetic plots is enhanced by an increase in the Mg²⁺ or spermine concentration. It is suggested that Mg²⁺ and spermine affect the kinetics of Ca²⁺ transport by interfering with Ca²⁺ binding to low affinity sites of the membrane surface and that the binding of Ca²⁺ to these sites is the first step of the mitochondrial Ca²⁺ transport.     

Differential effects of spermine on phosphatidylinositol 3-kinase and phosphatidylinositol phosphate 5-kinase

The metabolism of phosphoinositides plays an important role in the signal transduction pathways. We report here that naturally occuring polyamines affect the activities of phosphatidylinositol (PI) 3-kinase and PI 4-phosphate (PIP) 5-kinase differently. While polyamines inhibited the PI 3-kinase activity, they stimulated the activity of PIP 5-kinase in the order of spermine > spermidine > putrescine. Spermine inhibited the PI 3-kinase activity in a concentration-dependent manner with an IC₅₀ of 100 μM. On the other hand, spermine (5 mH) stimulated the activity of PIP 5-kinase 2–3 fold. Kinetic studies of spermine-mediated inhibition of PI 3-kinase revealed that it was noncompetitive with respect to ATP. The effect of Mg²⁺ and PIP, concentration on kinase activity was sigmoidal, with spermine inhibiting PI 3-kinase activity at all PIP₂ concentrations. While 1 mH calcium stimulated PI 3-kinase activity at submaximal concentrations of Mg²⁺ (1.25 mH), inhibition was observed at optimal concentration of Mg²⁺(2 mM). We propose that spermine may modulate the cellular signal by virtue of its differential effects on phosphoinositide kinases.     

Fusion of secretory vesicles isolated from rat liver

Summary Secretory vesicles isolated from rat liver were found to fuse after exposure to Ca²⁺. Vescle fusion is characterized by the occurrence of twinned vesicles with a continuous cleavage plane between two vesicles in freeze-fracture electron microscopy. The number of fused vesicles increases with increasing Ca²⁺-concentrations and is half maximal around 10^–6 m. Other divalent cations (Ba²⁺, Sr²⁺, and Mg²⁺) were ineffective. Mg²⁺ inhibits Ca²⁺-induced fusion. Therefore, the fusion of secretory vesiclesin vitro is Ca²⁺ specific and exhibits properties similar to the exocytotic process of various secretory cells.Various substances affecting secretionin vivo (microtubular inhibitors, local anethetics, ionophores) were tested for their effect on membrane fusion in our system.The fusion of isolated secretory vesicles from liver was found to differ from that of pure phospholipid membranes in its temperature dependence, in its much lower requirement for Ca²⁺, and in its Ca²⁺-specificity. Chemical and enzymatic modifications of the vesicle membrane indicate that glycoproteins may account for these differences.