Regulation of cell membrane function and secretion by extracellular fluid viscosity

Plasma viscosity is elevated in various pathological states, due to increased levels of protein and other macromolecules. The possibility that elevation of extracellular fluid viscosity (EFV) affects cellular and biochemical functions was examined in cultured liver cells and in red blood cells. The viscosity was modified by the addition of various macromolecules, which differ in their capacity to increase viscosity and in their chemical nature. It was found that secretion of lipoproteins and lysosomal enzymes by liver cells is inhibited as a function of the medium viscosity. Correspondingly, elevation of plasma viscosity of hyperlipidemic rats reduced lipoprotein levels. In search for the mechanism of this phenomenon we examined the effects of EFV on two cell membrane components which are involved in transmembrane processes: Gangliosides (GMs), and phospholipase A2 (PLA2). It was found that the rate of GMs degradation is decreased with increasing EFV. Of special interest was the finding that the activity of cell membrane PLA2, a key enzyme in secretory processes, is inhibited by increasing EFV. This phenomena was not confined to cell membrane PLA2, as we further found that erythrocyte hemolysis, induced by soluble snake venom PLA2, is inhibited as the EFV is increased. It is proposed that the extracellular fluid viscosity may play an important role in regulation of cellular and biochemical processes in general.     

Medium viscosity regulates the activity of membrane-bound and soluble phospholipase A2

Medium viscosity is a regulator of very low density lipoprotein production by cultured hepatocytes; their secretion and synthesis are inversely proportional to the extracellular fluid viscosity. The possibility that the mechanism of this extracellular effect on cell function involves modulation of cell membrane component(s) was considered. Along with this assumption, we studied the effect of medium viscosity on the activity of phospholipase A2 (PLA2), an enzyme present in the cell surface membrane, and the activity has been correlated with cellular secretion. We have found that culture medium viscosity inhibits the activity of PLA2 in the plasma membrane of cultured liver cells, concomitantly with the inhibition of lysosomal enzyme and lipoprotein secretion. It was also found that the degradation of liposomal phosphatidylcholine by soluble snake venom PLA2 is inversely proportional to the solvent viscosity. The possibility that the effect of medium viscosity on the enzymatic reaction involves the modulation of dynamic properties of membrane phospholipids was then considered. This hypothesis was examined by monitoring the fluorescence depolarization of fluorophores incorporated into phospholipid vesicles. No significant effect of the solvent viscosity on the phospholipid bilayer was observed. It is proposed that the regulation of cellular secretion by extracellular fluid viscosity involves modulation of the cell membrane PLA2 activity.     

Hemolysis of human erythrocytes by hypochlorous acid is modulated by amino acids,antioxidants, oxidants,membrane-perforating agents and by divalent metals

The optimal conditions under which hypochlorous acid (NaOCl) either hemolyzes human RBC or kills monkey kidney epithelial cells (BGM) in culture had been investigated. While in Hank's balanced salt solution (HBSS), micromolar amounts of NaOCl caused full hemolysis and also killed BGM cells, in D-MEM or RPMI media rich in amino acids, 25-40 mM of hypochlorite were needed to induce cell injury. Cells exposed to high amounts of NaOCl became highly refractory to strong detergents. Hemolysis by NaOCl was strongly inhibited by a large variety of antioxidants. RBC treated by subtoxic concentrations either of peroxide, peroxyl radical, NO, cholesterol, PLA2, PLC as well as by N2, argon or by mixture of CO2 (10%) and O2 (90%) became much more susceptible to lysis by NaOCl. On the other hand, while RBC treated by Fe2+, Co2+, and V2+ and to a lesser extent with Cu2+ became highly resistant to NaOCl hemolysis presumably due to NaOCl decomposition, no such effect was found either with Co2+ or by Mn2+. RBC treated by azide to destroy catalase and then incubated with peroxide and with NaOCl failed to undergo hemolysis due to the ability of peroxide to decompose NaOCl. The inhibitory effects of the divalent metals on NaOCl-induced hemolysis were also substantiated by measuring the decrease in pH and by cyclic voltammetry. The findings that like peroxide, NaOCl also synergizes with membrane-perforating agents and with a protease to kill epithelial cells further implicate such "cocktails" in cell injury in inflammatory conditions. Taken together, because of the capacity of many agents to scavenge NaOCl, tissue damage by NaOCl-generated neutrophils can take place primarily if activated neutrophils closely adhere to target cells to avoid the scavenging effects of amino acids and of antioxidants. Therefore, the significance of the data which had tested the cytotoxic effects of NaOCl using cells suspended only in salt solutions, should be reconsidered.     

The importance of N-acetylneuraminic acid and of its interaction with Ca++ in the stability of the erythrocytic membrane

The aim of this paper is to draw information about influence of human red cell N-acetyl-neuraminic acid and its interaction with Ca++ on membrane itself stability. Then, changes of red cell behavior in reply to osmotic stress with and without Ca++ after treatment with neuraminidase has been studied. We noted that the treatment with neuraminidase causes spontaneous hemolysis (about 9%), independently of medium osmolarity. As regards membrane resistance to osmotic stretching, N-acetyl-neuraminic acid has a destabilizing effect on most erythrocytes whereas its interaction with Ca++ don't influences significantly membrane resistance to osmotic stretching. Nevertheless, in extreme conditions of osmolarity (i.e. when hemolysis of younger red cells occurs), destabilizing effect of N-acetyl-neuraminic acid is no longer observable and, on the contrary, when it interacts with Ca++, it increases the osmotic resistance of red cells.     

Mild pressure induces resistance of erythrocytes to hemolysis by snake venom phospholipase A2.

It is generally assumed that mild pressure of a few atmospheres, such as that applied to blood cells during routine centrifugation, does not affect cell function. The results of the present study refute this notion. To explore the effect of mild pressure on cell function we examined its effect on the susceptibility of red blood cells (RBC) to hemolysis by snake venom phospholipase A2 (PLA2). Rat RBC were subjected to pressure of up to five atmospheres, returned to ambient pressure and interacted with PLA2 to induce hemolysis. The hemolysis was markedly decreased with increasing the pressure applied before induction of hemolysis. Application of such a pressure induces the shedding of a chemical factor, as yet uncharacterized, which facilitates the action of PLA2 on RBC.     

Hemolysis of Human Erythrocytes by Hypochlorous Acid is Modulated by Amino Acids,Antioxidants, Oxidants,Membrane-perforating Agents and by Divalent Metals

The optimal conditions under which hypochlorous acid (NaOCl) either hemolyzes human RBC or kills monkey kidney epithelial cells (BGM) in culture had been investigated. While in Hank's balanced salt solution (HBSS), micromolar amounts of NaOCl caused full hemolysis and also killed BGM cells, in D-MEM or RPMI media rich in amino acids, 25-40 mM of hypochlorite were needed to induce cell injury. Cells exposed to high amounts of NaOCl became highly refractory to strong detergents. Hemolysis by NaOCl was strongly inhibited by a large variety of antioxidants. RBC treated by subtoxic concentrations either of peroxide, peroxyl radical, NO, cholesterol, PLA?, PLC as well as by N?, argon or by mixture of CO?, (10%) and 0? (90%) became much more susceptible to lysis by NaOCl. On the other hand, while RBC treated by FE²?, Co²?, and V²? and to a lesser extent with Cu²? became highly resistant to NaOCl hemolysis presumably due to NaOCl decomposition, no such effect was found either with Co²? or by Mn²?. RBC treated by azide to destroy catalase and then incubated with peroxide and with NaOCl failed to undergo hemolysis due to the ability of peroxide to decompose NaOCl. The inhibitory effects of the divalent metals on NaOCl -induced hemolysis were also substantiated by measuring the decrease in pH and by cyclic voltammetry. The findings that like peroxide, NaOCl also synergizes with membrane-perforating agents and with a protease to kill epithelial cells further implicate such "cocktails" in cell injury in inflammatory conditions.

Taken together, because of the capacity of many agents to scavenge NaOCl, tissue damage by NaOCl generated neutrophils can take place primarily if activated neutrophils closely adhere to target cells to avoid the scavenging effects of amino acids and of antioxidants. Therefore, the significance of the data which had tested the cytotoxic effects of NaOCl using cells suspended only in salt solutions, should be considered.     

Characterization of the cytolytic reaction mechanism of the human natural killer (NK) lymphocyte: resolution into binding, programming, and killer cell-independent steps

Various parameters of the cytolytic reaction mechanisms of the human natural killer (NK) lymphocyte were studied to characterize the lytic cycle. NK cytolysis was determined to occur in three definable steps. 1) Binding of PBL to the NK-sensitive targets Molt-4 or K562 was rapid (less than 1 min), occurred at temperatures below 37 degrees C, was Mg++3-dependent, Ca++3-independent, and was prevented by dispersion of the cells into 10% dextran. 2) Subsequent to binding, programming for lysis as determined by a Ca++ pulse method was more protracted, requiring up to 2 hr to occur and was strictly dependent on Ca++ for cytolysis to proceed. In standard cytotoxicity assays, however, programming for lysis was more rapid occurring in 10 to 30 min. Programming was inhibited by EDTA, EGTA/Mg++ and by temperatures below 37 degrees C. Furthermore, after binding but in the absence of initiation of programming for lysis, the frequency of target binding cells did not change and the NK cell did not lose its lytic potential. 3) Killer cell-independent cytolysis (KCIL) was determined by the addition of EDTA to "programmed" targets and dispersion of these cells into dextran-containing medium, which resulted in virtually 100% dissociation of conjugated cells. KCIL was Ca++ and Mg++-independent and was blocked at reduced temperatures only if the dextran was prechilled to 4 degrees C before addition. The kinetics of 51Cr release during KCIL was rapid and complete 30 min after dispersion. Interferon-activated NK cells expressed an increased rate of cytolysis in Ca++ pulse experiments. This was due to an increased rate of the Ca++-dependent step(s) during the programming events. The rate of the Ca++-independent steps, however, were similar with control and IFN-activated cells.     

Early increase in intracellular calcium during photodynamic permeabilization

The purpose of this study was to determine if there was an early increase in intracellular Ca++ which preceded generalized lysis of thymocytes during photodynamic permeabilization. A method was developed that facilitated the simultaneous measurement in real time of permeabilization of the thymocyte cell membrane to Ca++, Mn++, and ethidium bromide during photodynamic action. Quin-2 loaded cells were illuminated in the presence of erythrosin B and the change in the fluorescence emission of the calcium-quin-2 complex was used to determine how soon and to what extent intracellular Ca++ changed following illumination. In the presence of extracellular manganese, the same system was used to determine how soon the cells became permeable to Mn++ or quin-2. It was determined that the fluorescence emission of the ethidium bromide-DNA complex was strong enough to be measured in the presence of the calcium-quin-2 complex. This enabled the concomitant determination of the elapsed time following illumination before ethidium bromide entered the cell. It was established that increased intracellular Ca++ was an early event in the photodynamic permeabilization of thymocytes that preceded permeabilization of the cell membrane to ethidium bromide, Mn++ or quin-2, or lysis.     

Utilization of membranous lipid substrates by membranous enzymes: activation of the latent sphingomyelinase of hen erythrocyte membrane

Previous studies demonstrated that hen erythrocytes have an inoperative, latent sphingomyelinase which is activated when the cells are hemolyzed in a hypotonic medium. Within minutes after hemolysis about 60-80% of the sphingomyelin (SPM) of the RBC "ghost" membrane was hydrolyzed. In this paper, expression of sphingomyelinase activity was further investigated. The percentage of total SPM hydrolyzed depended on the volume of the hypotonic hemolyzing buffer. Thus, suspending the erythrocytes in 4 vol of the buffer resulted in clumping of the hemolyzed "ghosts" and no hydrolysis of SPM. In comparison, suspension in 19 vol of the hypotonic buffer showed no clumping and sphingomyelinase activity was fully expressed. But centrifugation of the latter or, alternatively, addition of concanavalin A induced clumping and elimination of sphingomyelinase activity. Hen RBC could also be hemolyzed in an isotonic medium in the presence of Triton X-100, mellitin, halothane, and phospholipase C. Activation of the latent sphingomyelinase occurred at concentrations of these reagents which caused cell lysis. Hen RBC were dispersed in an isotonic medium containing glutaraldehyde (0.1%) or formaldehyde (10%). This rendered the cells resistant to hemolysis, even when subsequently dispersed in a hypotonic medium or water. But incubation of the "fixed" cells in a hypotonic or isotonic medium activated the enzyme, resulting in hydrolysis of 60% of the cellular SPM. In contrast, when glutaraldehyde was included in the hypotonic buffer, hemolysis occurred but sphingomyelinase activity was eliminated.     

IP3 sensitizes TRPV4 channel to the mechano- and osmotransducing messenger 5'-6'-epoxyeicosatrienoic acid

Mechanical and osmotic sensitivity of the transient receptor potential vanilloid 4 (TRPV4) channel depends on phospholipase A2 (PLA2) activation and the subsequent production of the arachidonic acid metabolites, epoxyeicosatrienoic acid (EET). We show that both high viscous loading and hypotonicity stimuli in native ciliated epithelial cells use PLA2-EET as the primary pathway to activate TRPV4. Under conditions of low PLA2 activation, both also use extracellular ATP-mediated activation of phospholipase C (PLC)-inositol trisphosphate (IP3) signaling to support TRPV4 gating. IP3, without being an agonist itself, sensitizes TRPV4 to EET in epithelial ciliated cells and cells heterologously expressing TRPV4, an effect inhibited by the IP3 receptor antagonist xestospongin C. Coimmunoprecipitation assays indicated a physical interaction between TRPV4 and IP3 receptor 3. Collectively, our study suggests a functional coupling between plasma membrane TRPV4 channels and intracellular store Ca2+ channels required to initiate and maintain the oscillatory Ca2+ signal triggered by high viscosity and hypotonic stimuli that do not reach a threshold level of PLA2 activation.     

Two-dimensional lattice Boltzmann study of red blood cell motion through microvascular bifurcation: cell deformability and suspending viscosity effects

Red blood cell (RBC) motion and trajectories in bifurcated microvessels are simulated using a two-dimensional immersed boundary-lattice Boltzmann method (IB-LBM). A RBC is modeled as a capsule with viscous interior fluid enclosed by a flexible membrane. For the symmetric bifurcation model employed, the critical offset position in the mother branch, which separates the RBC flux toward the two branches, has been calculated. The RBC flux and the hematocrit partitioning between the two daughter branches have also been studied. Effects of the flow-rate ratio, cell deformability and suspending viscosity have been examined. Simulation results indicate that increased cell rigidity and suspending viscosity have counter effects on cell trajectory through a bifurcation: the cell trajectory shifts toward the low flow-rate branch for less deformable cells, and toward the high flow-rate branch for more viscous plasma. These results imply that a higher cell rigidity would reduce the regular phase separation of hematocrit and plasma skimming processes in microcirculation, while an increased viscosity has the opposite effect. This has implications for relevant studies in fundamental biology and biomedical applications.     

A permeabilized cell model for studying cytokinesis using mammalian tissue culture cells

PtK1 cells lysed late in cell division in a medium containing the nonionic detergent Brij 58 and polyethylene glycol with continue to undergo cleavage after lysis. Maintenance of cleavage after lysis is dependent on the composition of the lysis medium; the pH must be around neutrality, MgATP must be present, and the free Ca++ concentration should be 1 microM for optimal constriction to occur. Cleavage can be stopped and reinitiated by raising and lowering the Ca++ levels in the lysis medium. Cleavage in the permeabilized cell is blocked by addition of phalloidin, cytochalasin B, and N-ethylmaleimide-modified myosin subfragment-1 to the lysis medium. This represents the first cell model system for studying cleavage since the pioneering studies of Hoffman- Berling in 1954.     

Hemolysis of rabbit erythrocytes in the presence of copper ions

The hemolysis of red blood cells (RBC) induced by Cu(II) is modified by ceruloplasmin (Cp) and albumin. The time course of hemolysis for rabbit RBC by Cu(II) consisted of two parts, an induction period followed by a catastrophic lysis period. The induction period decreased and the lysis rate increased with increasing Cu(II) concentration. Cp or albumin, modified Cu(II) induced hemolysis, by increasing the duration of the induction period and decreasing the overall rate of hemolysis of RBC. The catastrophic lysis period coincided with a sharp increase in the formation of metHb within the cell and in a rapid uptake of Cu(II). The presence of Cp led to an increase in the induction period prior to the rapid increase in metHb formation and in Cu(II) uptake. Porcine Cp was prepared with either two or three nonprosthetic copper binding sites (sites where Cu(II) is easily removed by passing over Chelex-100). Cp with three nonprosthetic binding sites gave more protection than Cp with two. Likewise, albumin can be prepared with three and five nonprosthetic copper binding sites. The albumin with five sites gave more protection than the albumin with three sites.     

Reconstitution of the basal calcium transport in resealed human red blood cell ghosts

The (45)Ca(2+) influx into right-side-out resealed ghosts (RG) prepared from human red blood cells (RBC) was measured. The (45)Ca(2+) equilibration occurred with t(1/2)=2.5 min and the steady-state was reached after 17 min with the level of 22+/-2 micromol/L(packed cells) at 37 degrees C. The rate of the influx was 97+/-17 micromol/L(packed cells)h. The (45)Ca(2+) influx was saturated with [Ca(2+)](0) at 4 mmol/L and was optimal at pH 6.5 and 30 degrees C. Divalent cations (10(-4)-10(-6)mol/L), nifedipine (10(-5)-10(-4)mol/L), DIDS (up to 10(-4)mol/L), and quinidine (10(-4)-10(-3)mol/L), inhibited the (45)Ca(2+) influx while uncoupler (10(-6)-10(-5)mol/L) stimulated it. In contrast to intact RBC, vanadate inhibited the (45)Ca(2+) influx when added to the external medium, however, the stimulation was observed when vanadate was present in media during both lysis and resealing. PMA had no effect under conditions found to stimulate the Ca(2+) influx in intact RBC. The results show that the Ca(2+) influx into RG is a carrier-mediated process but without control by protein kinase C and that the influx and efflux of Ca(2+) are coupled via the H(+) homeostasis similarly as in intact RBC but with modified mechanism.     

Studies on the mechanism of action of guinea pig lymphotoxin. II. Increase of calcium uptake rate in LT-damaged target cells.

In the previous paper it was suggested that the primary action of guinea pig lymphotoxin (LT) involved creation of ionic imbalances within the target L cells. The nature of these ionic disturbances is explored in this paper. The exogenous addition of CaCl2, but not KCl or NaCl, inhibited the cytotoxic action of LT. Cellular uptake rates of 45Ca++, but not 86Rb+, increased in LT-damaged L cells. The factor responsible for increasing the 45Ca++ uptake rate cochromatographed on a hydroxyapatite column with the cytotoxic activity of LT. Ouabain prevented the LT-mediated lysis and, concomitantly, depressed the LT-induced increase of 45Ca++ uptake rate. The LT-damaged L cells excluded trypan blue to the same extent as the normal cells. The addition of LT to and LT-resistant L cell mutant affected neither the 45Ca++ uptake rate nor the viability. From these observations, damage to the calcium transport system in the L cell plasma membrane is proposed as a mechanism of LT action.     

Modulation of the susceptibility of human erythrocytes to snake venom myotoxic phospholipases A(2): role of negatively charged phospholipids as potential membrane binding sites.

Cerrophidion (Bothrops) godmani myotoxins I (CGMT-I) and II (CGMT-II), Asp-49 and Lys-49 phospholipases A(2) (PLA2s), which drastically differ in enzymatic activity, were devoid of direct hemolytic effects on erythrocytes (RBC) from different species despite the fact that enzymatically active CGMT-I was able to hydrolyze RBC membrane phospholipids and disrupt liposomes prepared from RBC lipids. Human RBC did not become susceptible to the toxins after treatment with neuraminidase or after altering membrane fluidity with cholesterol or sublytic concentrations of detergent. Unlike normal RBC, significant hemolysis was induced by CGMT-II and another similar Lys-49 isoform, B. asper MT-II (BAMT-II), in RBC enriched with phosphatidylserine (PS). Hemolysis was greater in RBC preincubated with pyridyldithioethylamine (PDA), a potent inhibitor of aminophospholipid transport. RBC enriched with phosphatidic acid (PA) also became susceptible to the myotoxins but was unaffected by PDA. Cells enriched with phosphatidylcholine (PC) remained resistant to the action of the toxins. BAMT-II also induced damage in black lipid membranes prepared with PS but not PC alone. When RBC binding of BAMT-II was measured by enzyme-linked immunosorbent assay, it was observed that PS- and PA-enriched erythrocytes were always able to capture more toxin than normal and PC-enriched RBC. This effect was significantly improved by PDA (in the case of PS) and it was observed either in the presence or in the absence of calcium in the medium. These data suggest that negatively charged lipids in the outer leaflet of cell membranes constitute myotoxic PLA2 binding sites. The scarcity of anionic phospholipids in the outer leaflet of RBC could explain their resistance to the action of these PLA2s.     

An in vitro model of nephrocalcinosis using rabbit inner medullary collecting tubular cell culture

In order to investigate the pathogenesis of medullary nephrocalcinosis, rabbit inner medullary collecting duct cells were grown in media containing different Ca++, PTH and pH levels. It was found that high Ca++ (7.8mM) only reduced growth slightly and that crystalline deposits were found under the cells. This suggests that high Ca++ is not severely toxic to the cells but can lead to deposition of calcium beneath the basement membrane. PTH did not effect cell growth even in the presence of high Ca++ implying that it has an indirect effect on tubular cells in medullary nephrocalcinosis associated with hyperparathyroidism. In renal tubular acidosis these cells are subjected to a persistently high urinary pH and low interstitial pH. Raising the pH reduced the cell growth in normal Ca++ medium whereas lowering the pH increased cell growth in vitro. Our results show that nephrocalcinosis is not due to the direct effect of raised pericellular Ca++ or PTH alone and that persistently alkaline tubular fluid may play a role.     

Structural effects of covalent inhibition of phospholipase A2 suggest allosteric coupling between membrane binding and catalytic sites

Phospholipase A(2) (PLA(2)) binds to membranes and catalyzes phospholipid hydrolysis, thus initiating the biosynthesis of lipid-derived mediators of inflammation. A snake-venom PLA(2) was completely inhibited by covalent modification of the catalytic histidine 48 by p-bromophenacyl bromide. Moreover, His(48) modification affected PLA(2) structure, its membrane-binding affinity, and the effects of PLA(2) on the membrane structure. The native PLA(2) increased the order parameter of fluid membranes, whereas the opposite effect was observed for gel-state membranes. The data suggest membrane dehydration by PLA(2) and the formation of PLA(2)-membrane hydrogen bonding. The inhibited PLA(2) had lower membrane-binding affinity and exerted weaker effects on membrane hydration and on the lipid-order parameter. Although membrane binding resulted in formation of more flexible alpha-helices in the native PLA(2), which corresponds to faster amide hydrogen exchange, the modified enzyme was more resistant to hydrogen exchange and experienced little structural change upon membrane binding. The data suggest that 1), modification of a catalytic residue of PLA(2) induces conformational changes that propagate to the membrane-binding surface through an allosteric mechanism; 2), the native PLA(2) acquires more dynamic properties during interfacial activation via membrane binding; and 3), the global conformation of the inhibited PLA(2), including the alpha-helices, is less stable and is not influenced by membrane binding. These findings provide further evidence for an allosteric coupling between the membrane-binding (regulatory) site and the catalytic center of PLA(2), which contributes to the interfacial activation of the enzyme.     

Histochemical and cytochemical demonstration of Ca++-ATPase activity in the stellate cells of the adenohypophysis of the guinea pig

The histo- and cytochemical localization of Ca++-ATPase activity in the adenohypophysis of the guinea pig was studied utilizing a newly developed method (Ando et al. 1981). An intense reaction was observed in the wall of the blood vessels and between non-secretory cells (stellate cells) and endocrine cells of the pars distalis. Under the electron microscope the Ca++-ATPase reaction product was located extracellularly in relation to the plasmalemma of the stellate cells. This reaction was dependent on Ca++ and the substrate, ATP, and reduced by the addition of 0,1 mM quercetin to the standard incubation medium. Preheating of the sections before incubation completely inhibited the enzyme activity. When Mg++ in different concentrations were substituted for Ca++ in the incubation medium the reaction was always reduced. Both Ca++ and Mg++ in the incubation medium also reduced the reaction. The plasmalemma of the endocrine cells contains no demonstrable amount of Ca++-ATPase activity. The function of the Ca++-ATPase activity is discussed in relation to the regulation of the extracellular Ca++ concentration which seems to be important with respect not only to the secretory process of the endocrine cells but also to the metabolism of the adenohypophysis.     

Characterization of Brain Synaptic Vesicle Phospholipase A2 Activity and Its Modulation by Calmodulin, Prostaglandin E2, Prostaglandin F2α, Cyclic AMP, and ATP

Brain synaptic vesicle phospholipase A2 (PLA2) activity was characterized. It is Ca2+-dependent and has a pH optimum of 9.0. The enzyme has a Km of 60 microM and a Vmax of 2.0 nmol/mg/h. Calmodulin, prostaglandin F2 alpha, and cAMP, and ATP all increased the Vmax of the enzyme. Prostaglandin E2 inhibited the Vmax in the presence or absence of calmodulin. Light-scattering techniques in conjunction with phase-contrast and electron microscopy demonstrated that an increase in Vmax of PLA2 was correlated with synaptic vesicle aggregation, lysis, and possible fusion. In vitro synaptic vesicle-vesicle association that was stimulated by conditions that increased PLA2 activity could be diminished when synaptic vesicles were preincubated with PLA2 inhibitors. It is suggested that endogenous synaptic vesicle PLA2 activity may be an important mechanism underlying Ca2+-mediated neurotransmitter release.