A critical appraisal of the association between energy charge and cell damage

The association between the energy charge and cellular damage caused by metabolic inhibitors was investigated in a cellular system of quiescent fibroblasts. The cell damage was assessed by the release of lactate dehydrogenase (LDH) which indicates a severe change of membrane integrity. Inhibition of glycolysis resulted in release of LDH when the energy charge decreased below 0.5 at an ATP level of 10% of the original level. If oxidative phosphorylation was inhibited, the energy charge decreased to 0.1-0.35 (dependent on the type of inhibitor) a long time before release of LDH, and no change occurred in the energy charge when release of LDH started. The ATP level was 0.5-2% of the original at this time. Even a decrease of the energy charge to 0.1 could be reversed to a normal level, and at the same time the morphological cellular changes were fully reversed and no release of LDH occurred. The conclusion is that no simple correlation between energy charge and cell survival exists. The different levels of ATP at which release of LDH started after inhibition of glycolysis and oxidative phosphorylation indicate a special role of glycolysis in maintaining the membrane function and integrity. This was emphasized by measuring the potassium loss of the cells which was much more marked after inhibition of glycolysis.     

Modulation of red cell vesiculation by protease inhibitors

Release of vesicles from human red cell membranes was induced either by ATP-depletion or by incubation of the cells in presence of sonicated dimyristoylphosphatidylcholine (DMPC) vesicles. Vesicles released from ATP-depleted red cells but not the DMPC-induced vesicles contained degradation products of band 3 protein. Furthermore, in ATP-depleted erythrocytes proteolytic breakdown products could be demonstrated that were not detected in cells incubated with DMPC. Proteolysis was neither significantly affected by the protease inhibitor N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK) nor by other protease inhibitors tested in this study (diisopropylfluorophosphate, N-ethylmaleimide and phenylmethylsulfonyl fluoride). Both vesiculation processes were inhibited in a concentration dependent way by TLCK while other protease inhibitors did not significantly influence membrane vesiculation. Phase contrast microscopy showed that TLCK diminished the DMPC-induced formation of echinocytes which is known to precede vesicle release. These results suggest that the influence of TLCK on membrane vesiculation is not primarily due to inhibition of proteolysis but to a direct interaction of the inhibitor with the intrinsic domain of the erythrocyte membrane.     

The late stage of human immunodeficiency virus type 1 assembly is an energy-dependent process

Several recent studies have indicated the involvement of host cell factors in human immunodeficiency virus type 1 (HIV-1) assembly. To ascertain whether ATP-dependent factors play a role in this process, we quantified virus-like particle (VLP) production by ATP-depleted cells. Pharmacological ATP depletion abrogated VLP production without affecting cell viability or inducing degradation of HIV-1 Gag protein. This effect occurred even when the ATP-depleting agents were added 1 h into the assembly process, and it was reversed by removal of these agents. ATP depletion did not affect Gag membrane binding or multimerization. Density gradient analysis indicated that HIV-1 assembly intermediates were stalled late in the assembly process. This conclusion was further supported by electron microscopy analysis, which revealed a preponderance of plasma membrane-associated stalk-like structures in the ATP-depleted cells. Since no HIV-1 proteins bind or hydrolyze ATP, these findings indicate that an ATP-requiring cellular factor is an obligatory participant late in the HIV-1 assembly process.     

Nuclear translocation of cytosolic phospholipase A2 is induced by ATP depletion

Phospholipase A(2) (PLA(2)) enzymes may play a role in cellular injury due to ATP depletion. Renal Madin-Darby canine kidney cells were subjected to ATP depletion to assess the effects of cellular energy metabolism on cytosolic PLA(2) (cPLA(2)) regulation. ATP depletion results in a decrease in soluble cPLA(2) activity and an increase in membrane-associated activity, which is reversed upon restoration of ATP levels by addition of dextrose. In ATP-depleted cells cPLA(2) mass shifts from cytosol to nuclear fractions. GFP-cPLA(2) is localized at the nuclear membrane of stably transfected ATP-depleted LLC-PK(1) cells under conditions where [Ca(2+)](i) is known to increase. cPLA(2) translocation does not occur if the increase in [Ca(2+)](i) increase is inhibited. If [Ca(2+)](i) is allowed to increase when ATP is depleted and the cells are then lysed, cPLA(2) remains associated with nuclear fractions even if the homogenate [Ca(2+)] is markedly reduced. In contrast, cPLA(2), which becomes associated with the nucleus when [Ca(2+)](i) is increased using ionophore, readily dissociates from the nuclear fractions of ATP-replete cells upon reduction of homogenate [Ca(2+)]. Okadaic acid inhibits the ATP depletion-induced association of cPLA(2) with nuclear fractions. Thus energy deprivation results in [Ca(2+)]-induced nuclear translocation, which is partially prevented by a phosphatase inhibitor.     

Membrane protein organization in ATP-depleted and irreversibly sickled red cells

It has been proposed that the spectrin-actin submembrane network participates in control of red cell shape and deformability. We have examined ATP- and calcium-dependent changes in organization of spectrin in the membrane employing cross-linking of the nearest membrane protein neighbors by spontaneous or catalyzed (CuSO₄, O-phenanthroline) intermolecular disulfide couplings and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Cross-linking of fresh red cells resulted in the formation of spectrin and actin dimers and tetramers. ATP-depleted red cells differed from fresh cells in the presence of an additional reducible polymer of MW > 1 × 10⁶ selectively enriched in spectrin. This polymer formed spontaneously when red cells were depleted of ATP under aerobic conditions. After anaerobic ATP depletion, the polymer formed in ghosts after cross-linking by catalytic oxidation. Polymerization was prevented by maintenance of ATP and coincided with an ATP-dependent discocyte-echinocyte transformation. This suggests that, in ATP-depleted red cells, spectrin is rearranged to establish closer contacts, and that this may contribute to the discocyte-echinocyte transformation. The introduction of greater than 0.5 mM Ca⁺⁺ into ghosts by inclusion in hemolysis buffer or into fresh red cells (but not ATP-depleted red cells) by treatment with ionophore A23187 spontaneously produced a nonreducible polymer which others have attributed to transamidative cross-linking of spectrin, band 3, and other proteins. Spontaneous formation of both polymer types (reducible in aerobically ATP-depleted red cells and nonreducible in fresh, Ca⁺⁺ enriched red cells) resulted in stabilization (“autocatalytic fixation”) of spheroechinocytic shape. Irreversibly sickled cells, which have increased calcium and decreased ATP, and exhibit a permanent membrane deformation, failed to form any of the above polymers. This suggests that in contrast to normal cells depleted of ATP in vitro, fixation of ISC shape in vivo is not related to Ca- and ATP-dependent membrane protein polymerization. However, ISCs had an increased propensity to form the reducible, spectrin-rich polymer during a subsequent metabolic depletion in vitro. This was associated with transformation of ISCs into spheroechinocytes. Similar echinocytic ISCs were found to constitute 5–10% of the densest fractions of freshly separated ISCs. ISCs then exhibit sphero-echniocyte transformation, both in vitro and in vivo. We propose that this is due to spectrin reorganization that presumably results from the progressively increasing calcium and decreasing ATP of ISCs. These data provide evidence of altered spectrin organization in membranes of ATP-depleted, calcium-enriched red cells in vitro and in vivo.     

Changes in phosoholipid susceptibility toward phospholipases induced by ATP depletion in avian and amphibian erythrocyte membranes.

About half of the sphingomyelin content of fresh and ATP-depleted chicken erythrocytes is hydrolysed by sphingomyelinase. Removal of spingomyelin exposes the rest of the membrane phospholipids to hydrolysis by phospholipase C only in ATP-depleted but not in fresh cells. Addition of both sphinogomyelinase and phospholipase C to ATP-depleted cells causes about 60-70 percent hydrolysis of the total phospholipids accompanied by extensive (90 percent) hemolysis. The phospholipids of toad erythrocytes are partially available to phospholipase C activity in fresh cells (17-25 percent hydrolysis) without prior sphingomyelinase treatment. However, in ATP-depleted toad cells phospholipase C hydrolyses 66 percent of phospholipids and causes extensive lysis. Treatment of either fresh or ATP-depleted toad erythrocytes by sphingomyelinase together with phospholipase C induces hydrolysis of most of the phospholipds with complete lysis. Restoration of ATP to ATP-depleted cells endows them with resistance to the attack of phospholipase C. The correlation between changes in ATP level and membrane organization as revealed by increased susceptibility toward phospholipases is discussed.     

Phospholipase C-evoked glycerol release in energy depleted rat myocardial cells

Summary Preincubation of rat myocardial cells in hypoxic substrate-free Krebs-Ringer bicarbonate buffer (pH 7.4, 37°C) resulted in a substantial decline in high energy phosphates (ATP and CP). Thus, 20 and 60 min preincubation produced a 18 and 72% decline in ATP content, whereas the parallel decline in CP content was 51 and 73%. This energy depletion was accompanied by a change in cell morphology from the initial rod-shaped form to rounded up (hyper-contracted) myocytes. In cells preincubated in substrate-free normoxic buffer, both normal morphology and energy homeostasis were maintained. When energy depleted myocytes later were incubated in the presence of phospholipase C (PLC), this resulted in a substantial release of glycerol, amounting to 92 and 137 nmol/10⁶ cells – 2 h in 20 and 60 min energy depleted myocytes, respectively. In addition, PLC caused an increased leakage of lactate dehydrogenase in energy depleted myocytes. Normal cells, on the other hand, were apparently not affected by PLC. These data suggest that PLC selectively attacks energy depleted and/or structurally damaged myocytes. This could well enhance the breakdown of the natural barrier between the extra- and intracellular compartments and thus augment the cellular damage during ischemia. Moreover, energy depleted myocytes appeared exceptionally sensitive to this enzyme, since the levels required to cause glycerol or lactate dehydrogenase release were several orders of magnitude lower than that required to cause membrane permeation in other cell types.     

Oxidative stress and muscular dystrophy

Oxidative stress may be the fundamental basis of many of the structural, functional and biochemical changes characteristic of the inherited muscular dystrophies in animals and humans. The presence of by-products of oxidative damage, and the compensatory increases in cellular antioxidants, both indicate oxidative stress may be occurring in dystrophic muscle. Changes in the proportions and metabolism of cellular lipids, abnormal functions of cellular membranes, altered activity of membrane-bound enzymes such as the SR Ca2+-ATPase, disturbances in cellular protein turnover and energy production and a variety of other changes all indicate that these inherited muscular dystrophies appear more like the results of oxidative stress to muscle than any other type of underlying muscle disturbance. Particular details of these altered characteristics of dystrophic muscle, in combination with current knowledge on the processes of oxidative damage to cells, may provide some insight into the underlying biochemical defect responsible for the disease, as well as direct research towards the ultimate goal of an effective treatment.     

Lytic agents, cell permeability, and monolayer penetrability

Cell lysis induced by lytic agents is the terminal phase of a series of events leading to membrane disorganization and breadkdown with the release of cellular macromolecules. Permeability changes following exposure to lytic systems may range from selective effects on ion fluxes to gross membrane damage and cell leakage. Lysis can be conceived as an interfacial phenomenon, and the action of surface-active agents on erythrocytes has provided a model in which to investigate relationships between hemolysis and chemical structure, ionic charge, surface tension lowering, and ability to penetrate monolayers of membrane lipid components. Evidence suggests that lysis follows the attainment of surface pressures exceeding a "critical collapse" level and could involve membrane cholesterol or phospholipid. Similarities of chemical composition of membranes from various cell types could account for lytic responses observed on interaction with surface-active agents. Cell membranes usually contain about 20–30 % lipid and 50–75 % protein. One or two major phospholipids are present in all cell membranes, but sterols are not detectable in bacterial membranes other than those of the Mycoplasma group. The rigid cell wall in bacteria has an important bearing on their response to treatment with lytic agents. Removal of the wall renders the protoplast membrane sensitive to rapid lysis with surfactants. Isolated membranes of erythrocytes and bacteria are rapidly dissociated by surface-active agents. Products of dissociation of bacterial membranes have uniform behavior in the ultracentrifuge (sedimentation coefficients 2–3S). Dissociation of membrane proteins from lipids and the isolation and characterization of these proteins will provide a basis for investigating the specificity of interaction of lytic agents with biomembranes.     

ATP is required for receptor-mediated endocytosis in intact cells

We have demonstrated a requirement for cellular ATP in the receptor- mediated endocytosis of transferrin. This has been accomplished using a novel assay for endocytosis based on acquisition of resistance to the membrane impermeable reducing agent, glutathione (GSH). Diferric- transferrin was conjugated to biotin via a cleavable disulfide bond and iodinated. Internalization of 125I-biotin-S-S-transferrin (125I-BSST) was quantitated by adsorption to avidin-Sepharose after treatment of cells with GSH. Receptor-mediated endocytosis of 125I-BSST was severely inhibited in ATP-depleted cells. Similar results were obtained when ATP was depleted by incubation of cells either under a N2-atmosphere or in the presence of NaN3 and NaF. The latter treatment, alone, also resulted in a loss of surface transferrin receptors which could not be correlated to reductions in cellular ATP. In contrast to the acquisition of GSH resistance, the apparent internalization of 125I- BSST as assessed by inaccessibility to antitransferrin antibodies reached control levels in ATP-depleted cells. Our biochemical and morphological data suggested that, although ATP is required for receptor-mediated endocytosis, in ATP-depleted cells ligands can become efficiently sequestered into deeply invaginated pits that are inaccessible to large probes such as antibodies, but remain accessible to small molecules such as GSH.     

UV-induced changes in the skin: can they be repaired?

The damaging effects of UVB light have been described previously and include a number of changes to multiple cell types. At previous meetings of this society, we have shown that Langerhans' cells are the most susceptible to UVB induced damage which can be shown as ultrastructural changes in dendrites, nucleus and cytoplasm by transmission electron microscopy. We have also shown that their patterns of migration from skin to regional lymph node and their ability to present antigens to autologous T cells have been profoundly altered by UVB irradiation. The aim of this work was to establish if it was possible to reverse any of the damage done to Langerhans' cells by UVB exposure by topical application of a DNA repair enzyme such as T4N5 endonuclease. These experiments were undertaken in a sheep model that allowed collection of cells as they migrate from the skin. This allowed for a direct examination of the migration characteristics and ultrastructural features of all Langerhans' cells before, during, and for 2 weeks after exposure to a single dose of UVB. Results obtained from this project indicate that treatment by topical application of DNA repair enzyme immediately after UVB irradiation may restore a number of normal immune parameters associated with the structure and function of migrating Langerhans' cells. It appears that there is a dose related correction of the increased tempo of cell migration and some improvements in the number of ultrastructurally damaged Langerhans' cells have also been associated with application of higher doses of DNA repair enzyme. These preliminary findings indicate that some potential therapeutic benefits are associated with the use of such agents in reversing the immunological damage caused by exposure to erythemal doses of UVB light.     

Structural changes of isolated hepatocytes during treatment with digitonin

The structural changes accompanying digitonin-induced release of enzymes and metabolites from isolated hepatocytes have been studied by scanning and transmission electron microscopy. In the initial phase, characterized by total release of the cytosolic marker enzyme, lactate dehydrogenase, the plasma membrane was immediately damaged, rapidly followed by extensive damage to the endoplasmic reticulum. The shape of the cell, however, was maintained, and the mitochondria and nucleus remained tightly held together by the cytoskeleton. Mitochondria remained intact initially, whereas the cytosol became less electron dense and the nuclear chromatin was more dispersed. An intermediate phase was characterized by total release of adenylate kinase and most of the glucose-6-phosphatase, marker enzymes for the mitochondrial intermembrane space and the endoplasmic reticulum, respectively. The outer mitochondrial membrane was ruptured, but mitochondria maintained their normal matrix electron density. In the final phase, characterized by the beginning of citrate synthase release from the mitochondrial matrix space, the mitochondria became swollen, and only the nucleus, inner and outer mitochondrial membranes, and the cytoskeleton could be clearly distinguished. Although the plasma membrane could not be readily discerned in electron micrographs after the initial phase, the plasma membrane marker enzyme 5'-nucleotidase remained associated with digitonin-treated hepatocytes. Acetyl-CoA carboxylase was released much more slowly than lactate dehydrogenase, indicating some severe restriction on its release. The release of acetyl-CoA carboxylase closely paralleled the release of glucose-6-phosphatase. The controlled exposure of hepatocytes to digitonin, therefore, leads to the sequential release of soluble, compartmentalized cellular components and some membrane-bound components, but the mitochondrial membrane, cytoskeleton and the nucleoskeleton survive even long-term digitonin treatment.     

Structural changes of isolated hepatocytes during treatment with digitonin

The structural changes accompanying digitonin-induced release of enzymes and metabolites from isolated hepatocytes have been studied by scanning and transmission electron microscopy. In the initial phase, characterized by total release of the cytosolic marker enzyme, lactate dehydrogenase, the plasma membrane was immediately damaged, rapidly followed by extensive damage to the endoplasmic reticulum. The shape of the cell, however, was maintained, and the mitochondria and nucleus remained tightly held together by the cytoskeleton. Mitochondria remained intact initially, whereas the cytosol became less electron dense and the nuclear chromatin was more dispersed. An intermediate phase was characterized by total release of adenylate kinase and most of the glucose-6-phosphatase, marker enzymes for the mitochondrial intermembrane space and the endoplasmic reticulum, respectively. The outer mitochondrial membrane was ruptured, but mitochondria maintained their normal matrix electron density. In the final phase, characterized by the beginning of citrate synthase release from the mitochondrial matrix space, the mitochondria became swollen, and only the nucleus, inner and outer mitochondrial membranes, and the cytoskeleton could be clearly distinguished. Although the plasma membrane could not be readily discerned in electron micrographs after the initial phase, the plasma membrane marker enzyme 5′-nucleotidase remained associated with digitonin-treated hepatocytes. Acetyl-CoA carboxylase was released much more slowly than lactate dehydrogenase, indicating some severe restriction on its release. The release of acetyl-CoA carboxylase closely paralleled the release of glucose-6-phosphatase. The controlled exposure of hepatocytes to digitonin, therefore, leads to the sequential release of soluble, compartmentalized cellular components and some membrane-bound components, but the mitochondrial membrane, cytoskeleton and the nucleoskeleton survive even long-term digitonin treatment.     

Endocytosis in adenosine triphosphate-depleted erythrocytes.

The extent of membrane invagination or endocytosis in intact erythrocytes was quantified by measuring the loss of acetylcholinesterase activity. Primaquine-induced endocytosis was completely inhibited in ATP-depleted cells. However, chlorpromazine and vinblastine were capable of inducing membrane invagination in depleted cells. With both drugs, the loss of enzyme activity was less than that measured in fresh cells. We conclude that drug-induced endocytosis is not necessarily an energy-dependent process.     

Treatment with oxidizing agents damages the inner membrane of spores of Bacillus subtilis and sensitizes spores to subsequent stress

AIMS: To determine if treatment of Bacillus subtilis spores with a variety of oxidizing agents causes damage to the spore's inner membrane. METHODS AND RESULTS: Spores of B. subtilis were killed 80-99% with wet heat or a variety of oxidizing agents, including betadine, chlorine dioxide, cumene hydroperoxide, hydrogen peroxide, Oxone, ozone, sodium hypochlorite and t-butylhydroperoxide, and the agents neutralized and/or removed. Survivors of spores pretreated with oxidizing agents exhibited increased sensitivity to killing by a normally minimal lethal heat treatment, while spores pretreated with wet heat did not. In addition, spores treated with wet heat or the oxidizing agents, except sodium hypochlorite, were more sensitive to high NaCl in plating media than were untreated spores. The core region of spores treated with at least two oxidizing agents was also penetrated much more readily by methylamine than was the core of untreated spores, and spores treated with oxidizing agents but not wet heat germinated faster with dodecylamine than did untreated spores. Spores of strains with very different levels of unsaturated fatty acids in their inner membrane exhibited essentially identical resistance to oxidizing agents. CONCLUSIONS: Treatment of spores with oxidizing agents has been suggested to cause damage to the spore's inner membrane, a membrane whose integrity is essential for spore viability. The sensitization of spores to killing by heat and to high salt after pretreatment with oxidizing agents is consistent with and supports this suggestion. Presumably mild pretreatment with oxidizing agents causes some damage to the spore's inner membrane. While this damage may not be lethal under normal conditions, the damaged inner membrane may be less able to maintain its integrity, when dormant spores are exposed to high temperature or when germinated spores are faced with osmotic stress. Triggering of spore germination by dodecylamine likely involves action by this agent on the spore's inner membrane allowing release of the spore core's depot of dipicolinic acid. Presumably dodecylamine more readily alters the permeability of a damaged inner membrane and thus more readily triggers germination of spores pretreated with oxidizing agents. Damage to the inner spore membrane by oxidizing agents is also consistent with the more rapid penetration of methylamine into the core of treated spores, as the inner membrane is likely the crucial permeability barrier to methylamine entry into the spore core. As spores of strains with very different levels of unsaturated fatty acids in their inner membrane exhibited essentially identical resistance to oxidizing agents, it is not through oxidation of unsaturated fatty acids that oxidizing agents kill and/or damage spores. Perhaps these agents work by causing oxidative damage to key proteins in the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: The more rapid heat killing and germination with dodecylamine, the greater permeability of the spore core and the osmotic stress sensitivity in outgrowth of spores pretreated with oxidizing agents is consistent with such agents causing damage to the spore's inner membrane, even if this damage is not lethal under normal conditions. It may be possible to take advantage of this phenomenon to devise improved, less costly regimens for spore inactivation.     

gamma-Glutamyl transpeptidase (gamma-GT) and maintenance of thiol pools in tumor cells resistant to alkylating agents

Previous studies from this laboratory have established that acquired resistance of murine L1210 leukemia cells to L-phenylalanine mustard (L-PAM) and other alkylating agents is accompanied by a two-to threefold elevation in their glutathione (GSH) concentration (Biochem. Pharm. 31:121). In an attempt to gain insight into the mechanism by which resistant tumor cells maintain their increased GSH content, we have assessed the possible role of gamma-glutamyl transpeptidase (gamma-GT), a membrane bound enzyme involved in GSH metabolism. These results indicate that the enzyme is present in both sensitive and resistant murine L1210 leukemia cells but that the cellular content of gamma-GT is elevated two-to threefold in L-PAM resistant cells as compared to their sensitive counterparts. This elevation in enzymatic activity correlates well with the increased cellular GSH content in resistant cells. The results of a detailed kinetic analysis of gamma-GT activity indicate that there is no difference, between cell types, in the apparent Km of the enzyme for the gamma-glutamyl donor (L-gamma-glutamyl-p-nitroanilide) or the acceptor (glycylglycine). However, the apparent Vmax is increased two-to threefold in L-PAM resistant tumor cells. Investigation into the role of gamma-GT in the extracellular metabolism of GSH indicates that resistant tumor cells metabolize two-fold more GSH than do sensitive cells and that such metabolism results in a similar difference in the intracellular concentration of cysteine. Results of studies with cellular lysates also indicate a role for the enzyme in the supply of cysteine to the glutathione precursor pool of the tumor cell and in the maintenance of elevated GSH concentrations in cells resistant to alkylating agents.     

The function of poly (ADP-ribosylation) in DNA breakage and rejoining

Poly (ADP-ribose) polymerase has an obligatory requirement for DNA strand-breaks in order to show full enzyme activity. Exposure of cells to DNA damaging agents activates this enzyme presumably through the production of DNA strand-breaks, either directly or via cellular enzymes. Recent evidence from manipulations of the cloned cDNA of this enzyme confirm the earlier evidence, obtained using enzyme inhibitors, that this enzyme is involved in DNA excision repair, probably at or near the ligation step. A very unusual human genetic disease has provided direct evidence for a link between the enzyme activities of poly (ADP-ribose) polymerase and of DNA ligase I. There is also some evidence that this enzyme may be involved in other cases of DNA breakage and rejoining, such as homologous and non-homologous DNA recombination, for example, in sister chromatid exchanges, in DNA transfection, in the intergration of retroviral proviral DNA and in variable antigen switching in African trypanosomes.     

MECHANISMS OF LYSOSOMAL ENZYME RELEASE FROM HUMAN LEUKOCYTES : I. Effect of Cyclic Nucleotides and Colchicine

In order to study mechanisms underlying selective enzyme release from human leukocytes during phagocytosis, the effects were studied of compounds which affect microtubule integrity or the accumulation of cyclic nucleotides. Human leukocytes selectively extrude lysosomal enzymes (β-glucuronidase) from viable cells during phagocytosis of zymosan or immune complexes, or upon encounter with immune complexes dispersed along a non-phagocytosable surface such as a millipore filter. In each circumstance, lysosomal enzyme release was reduced by previous treatment of cells with pharmacological doses of drugs which disrupt microtubules (e.g. 10^-3–10^-5 M colchicine) or with agents which affect accumulation of adenosine 3'5'-monophosphate (cAMP) (e.g. 10^-3 M cyclic nucleotides and 2.8 x 10^-4–2.8 x 10^-6 M prostaglandin E (PGE) and A (PGA) compounds). Preincubation of cells with 5 µg/ml cytochalasin B resulted in complete inhibition of zymosan ingestion, but not of adherence of zymosan particles to plasma membranes or selective enzyme release. In this system, in which enzyme release was independent of particle uptake, preincubation of cells with colchicine, vinblastine, dibutyryl cAMP, or PGE₁ also reduced extrusion of lysosomal enzymes. When cell suspensions were incubated with membrane-lytic crystals of monosodium urate (MSU), cytoplasmic as well as lysosomal enzymes were released with subsequent death of the cells. However, enzyme release followed phagocytosis of crystals (as measured by enhanced C-1 oxidation of glucose) and was due to "perforation from within" of the lysosomal membrane, rather than lysis by crystals of the plasma membrane. Enzyme release after MSU ingestion was also reduced when cells were treated with pharmacological doses of the test agents. When cells were killed by Triton X-100, acting on the plasma membrane, C-1 oxidation of glucose was abolished and enzyme release could not be inhibited pharmacologically. These observations suggest that lysosomal enzyme release from human phagocytes can be an active process which accompanies plasma membrane stimulation, is independent of cell death, and may be controlled by cyclic nucleotides and agents which affect microtubules.