ATP analogues induce membrane permeabilization in transformed mouse fibroblasts

The mechanism underlying ATP-induced permeabilization of transformed mouse fibroblasts was studied by using nonhydrolyzable analogues of ATP. Incubation of 3T6 cells with 0.6 mM of either ATP, 5′-adenylyl imidodiphosphate (p[NH]ppA) or adenosine 5′-[β,γ-methylene]triphosphate (p[CH₂]ppA) resulted in an increase of 17-, 8- or 5-times, respectively, in the cell membrane permeability, measured by the efflux of normally impermeant metabolites from the cells. The induced cell permeabilization was preceded by a reduction in the membrane potential (Δψ), determined according to the distribution of the cation tetraphenylphosphonium (TPP⁺) between the cells and the medium. Reduction of 26, 18 and 13 mV in Δψ was exerted by 0.6 mM of either ATP, p[NH]ppA or p[CH₂]ppA, respectively. In 3T3 cells the untransformed counterparts of 3T6 cells, neither reduction of Δψ, nor alterations in membrane permeability were exerted by either ATP or by its analogues. The data indicate that the dissociation of the β,γ-phosphate bond is not essential for membrane permeabilization by external ATP, implying that the binding of ATP to the cell surface of transformed cells is sufficient to initiate the permeabilization process. The data also suggest that Δψ is involved in the control of membrane permeability.     

ATP analogues induce membrane permeabilization in transformed mouse fibroblasts

The mechanism underlying ATP-induced permeabilization of transformed mouse fibroblasts was studied by using nonhydrolyzable analogues of ATP. Incubation of 3T6 cells with 0.6 mM of either ATP, 5'-adenylyl imidodiphosphate (p[NH]ppA) or adenosine 5'-[beta, gamma-methylene]triphosphate (p[CH2]ppA) resulted in an increase of 17-, 8- or 5-times, respectively, in the cell membrane permeability, measured by the efflux of normally impermeant metabolites from the cells. The induced cell permeabilization was preceded by a reduction in the membrane potential (delta psi), determined according to the distribution of the cation tetraphenylphosphonium (TPP+) between the cells and the medium. Reduction of 26, 18 and 13 mV in delta psi was exerted by 0.6 mM of either ATP, p[NH]ppA or p[CH2]ppA, respectively. In 3T3 cells the untransformed counterparts of 3T6 cells, neither reduction of delta psi, nor alterations in membrane permeability were exerted by either ATP or by its analogues. The data indicate that the dissociation of the beta, gamma-phosphate bond is not essential for membrane permeabilization by external ATP, implying that the binding of ATP to the cell surface of transformed cells is sufficient to initiate the permeabilization process. The data also suggest that delta psi is involved in the control of membrane permeability.     

Permeability change in transformed mouse fibroblasts caused by ionophores,and its relationship to membrane permeabilization by exogenous ATP

Summary Electrogenic ionophores have been found to induce membrane permeabilization in Swiss mouse 3T3 cells that had undergone spontaneous transformation (3T6 cells). Cells attached to plastic dishes were loaded with [³H] uridine, and then the medium was replaced by buffered salt solution at pH 7.8. The enhancement of membrane permeability was assayed by following the efflux of uridine nucleotides, normally impermeant substances. Titration with electrogenic ionophores, such as carbonylcyanidem-chlorophenylhydrazone (CCCP), SF-6847 and gramicidin D, markedly increased the membrane permeability within a very narrow range of ionophore concentration. Nonelectrogenic ionophores, such as monensin and nigericin, did not affect membrane permeability. Measurements of the distribution of the lipophilic cation tetraphenylphosphonium (TPP⁺) between the cells and their environment implied that the remarkable increase in permeability took place within a narrow range of membrane potential (). The data could be explaine by a threshold value, under which aqueous channels are opened in the plasma membrane. The effects exerted by electrogenic ionophores on the plasma membrane were found to be similar to those induced by exogenous ATP. In both cases rapid efflux of K⁺, influx of Na⁺ and reduction of preceded membrane permeabilization to low molecular weight, charged molecules, such as nucleotides. It is suggested that dissipation of induces conformational alterations in membranal components, and/or topological changes, such as aggregation of protein molecules, to form membranal aqueous channels. Electrogenic ionophores permeabilize both normal (3T3) and transformed (3T6) mouse fibroblasts, whereas ATP effects are specific for transformed cells. Thus, it is postulated that ATP actsvia specific sites on the surface of transformed cells.     

Tetraspanins regulate the protrusive activities of cell membrane

Tetraspanins have gained increased attention due to their functional versatility. But the universal cellular mechanism that governs such versatility remains unknown. Herein we present the evidence that tetraspanins CD81 and CD82 regulate the formation and/or development of cell membrane protrusions. We analyzed the ultrastructure of the cells in which a tetraspanin is either overexpressed or ablated using transmission electron microscopy. The numbers of microvilli on the cell surface were counted, and the radii of microvillar tips and the lengths of microvilli were measured. We found that tetraspanin CD81 promotes the microvillus formation and/or extension while tetraspanin CD82 inhibits these events. In addition, CD81 enhances the outward bending of the plasma membrane while CD82 inhibits it. We also found that CD81 and CD82 proteins are localized at microvilli using immunofluorescence. CD82 regulates microvillus morphogenesis likely by altering the plasma membrane curvature and/or the cortical actin cytoskeletal organization. We predict that membrane protrusions embody a common morphological phenotype and cellular mechanism for, at least some if not all, tetraspanins. The differential effects of tetraspanins on microvilli likely lead to the functional diversification of tetraspanins and appear to correlate with their functional propensity.     

Metabolically labeled cell membrane proteins in spontaneously and in SV40 virus transformed mouse fibroblasts.

A family of mouse fibroblast cell lines in exponential phase of growth were compared in protein constitution of their cell membranes. In preparations from these cells enriched in cell-surface membrane we observed one protein component (apparent molecular weight about 250 000) consistently to be reduced or absent in an SV40 virus transformed cell line, when compared with the normal cell line. No such compositional difference was observed in a spontaneously transformed tumorigenic clonal derivative cell line, or in subclones of such a derivative cell line, with or without SV40 virus infection. However, in metabolic labeling experiments with 14C-labeled mixed amino acids, a consistent decrease also was demonstrated in the biosynthesis of the same protein in the SV40 virus infected subclone, as compared to an uninfected sister subclone, during exponential growth. This specific difference in biosynthesis is apparently related to the presence and functioning of the SV40 gene, and correlates with the ability of these cells to grow in viscous medium, but not with cellular tumorigenicity.     

Growth inhibition of transformed mouse fibroblasts by adenine nucleotides occurs via generation of extracellular adenosine

The growth of transformed mouse fibroblasts (3T6 cells) in medium containing 5% fetal bovine serum was inhibited after treatment with concentrations greater than 50 microM ATP, ADP, or AMP. Adenosine, the common catabolite of the nucleotides, had no effect on cell growth at concentrations below 1 mM. However, the following results indicate that the toxicity of ATP, ADP, and AMP is mediated by serum- and cell-associated hydrolysis of the nucleotides to adenosine. 1) ADP and AMP, but not ATP, were toxic to 3T6 cells grown in serum-free medium or medium in which phosphohydrolase activity of serum was inactivated. Under these conditions, the cells exhibited cell-associated ADPase and 5'-nucleotidase activity, but little ecto-ATPase activity. 2) Inhibition of adenosine transport in 3T6 cells by dipyridamole or S-(p-nitrobenzyl)-6-thioinosine prevented the toxicity of ATP in serum-containing medium and of ADP and AMP in serum-free medium. 3) A 16-24-h exposure to 125 microM AMP or ATP was needed to inhibit cell growth under conditions where serum- and cell-associated hydrolysis of the nucleotides generated adenosine in the medium continuously over the same time period. In contrast, 125 microM adenosine was completely degraded to inosine and hypoxanthine within 8-10 h. Furthermore, multiple doses of adenosine added to the cells at regular intervals over a 16-h period were significantly more toxic than an equivalent amount of adenosine added in one dose. Treatment of 3T6 cells with AMP elevated intracellular ATP and ADP levels and reduced intracellular UTP levels, effects which were inhibited by extracellular uridine. Uridine also prevented growth inhibition by ATP, ADP, and AMP. These and other results indicate that serum- and cell-associated hydrolysis of adenine nucleotides to adenosine suppresses growth by adenosine-dependent pyrimidine starvation.     

Autocrine signaling involved in cell volume regulation: the role of released transmitters and plasma membrane receptors

Cell volume regulation is a basic homeostatic mechanism transcendental for the normal physiology and function of cells. It is mediated principally by the activation of osmolyte transport pathways that result in net changes in solute concentration that counteract cell volume challenges in its constancy. This process has been described to be regulated by a complex assortment of intracellular signal transduction cascades. Recently, several studies have demonstrated that alterations in cell volume induce the release of a wide variety of transmitters including hormones, ATP and neurotransmitters, which have been proposed to act as extracellular signals that regulate the activation of cell volume regulatory mechanisms. In addition, changes in cell volume have also been reported to activate plasma membrane receptors (including tyrosine kinase receptors, G-protein coupled receptors and integrins) that have been demonstrated to participate in the regulatory process of cell volume. In this review, we summarize recent studies about the role of changes in cell volume in the regulation of transmitter release as well as in the activation of plasma membrane receptors and their further implications in the regulation of the signaling machinery that regulates the activation of osmolyte flux pathways. We propose that the autocrine regulation of Ca2+-dependent and tyrosine phosphorylation-dependent signaling pathways by the activation of plasma membrane receptors and swelling-induced transmitter release is necessary for the activation/regulation of osmolyte efflux pathways and cell volume recovery. Furthermore, we emphasize the importance of studying these extrinsic signals because of their significance in the understanding of the physiology of cell volume regulation and its role in cell biology in vivo, where the constraint of the extracellular space might enhance the autocrine or even paracrine signaling induced by these released transmitters.     

On the role of protein phosphorylation in the ATP-dependent permeabilization of transformed cells

Incubation of transformed mouse fibroblasts with external ATP in alkaline medium low in divalent cations causes an increase in the permeability of the plasma membrane to nucleotides and other small molecules. Previous suggestions that the phosphorylation of a 44,000 dalton membrane protein is involved in this permeabilization process have been pursued. Fractionation of cells that had been incubated with [γ-³²P] ATP revealed that the labeled 44K phosphoprotein was found in both the membrane and mitochondrial fractions. Incubation of fractions isolated from unlabeled cells with [γ-³²P] ATP resulted in substantial formation of ³²P-44K in the mitochondrial fraction and less incorporation in the membrane fraction. The 44,000 dalton protein was identified as the α-subunit of mitochondrial pyruvate dehydrogenase by partial proteolytic mapping and immunological cross-reactivity with antibodies prepared against bovine pyruvate dehydrogenase. The phosphorylation of this protein in whole cells by externally added ATP is suppressed by inclusion in the incubation medium of carboxyatractyloside (CAT) and EDTA. These substances have no effect on ATP-dependent permeabilization, indicating that the phosphorylation of pyruvate dehydrogenase is not involved in this process.     

Induction of unspecific permeabilization of mitochondrial membrane and its role in cell death

Mitochondria participate in various vital cellular processes. Violation of their functions can lead to the development of cardiovascular and neurodegenerative diseases and malignancies. One of the key events responsible for mitochondrial damage—induction of Ca²⁺-dependent mitochondrial permeability transition, due to the opening of a nonspecific pore in the inner mitochondrial membrane. Despite active studies of pore components, its detailed structure has not yet been established. This review analyzes possible constituents and regulators of the pore, the role of the pore in various pathologies, and hypotheses that explain the organization of the pores. Elucidation of these questions can help developing strategies for the treatment of a wide range of pathologies—from Alzheimer and Parkinson’s disease to cancer.     

Non-lytic, non-ionic detergent extractions of plasma membrane constituents from normal and transformed fibroblasts

A technique has been developed for the selective extraction of plasma membrane protein constituents from normal and transformed cells employing non-ionic detergents. The extraction procedure does not damage cells as judged by cell viability, ⁵¹Cr release, and trypan blue staining. Lactoperoxidase-catalyzed iodina- tion followed by detergent extraction permits demonstration of a 100 000 dalton protein which is found on the surface of normal but not transformed hamster and mouse fibroblasts.     

Hyperpolarization of mouse skeletal muscle plasma membrane induced by extracellular NADH

Extracellularly applied NADH, but not NAD or NADPH, increases the resting membrane potential from -74.1 to -76.6 mV in freshly isolated muscles in the presence of K+ in the incubation medium and from -64.6 to -72.9 mV in muscles equilibrated for 4-6 h in a K+-free solution. The NADH-induced hyperpolarization is blocked by pretreatment of muscles with ouabain, and the inhibitors of plasma membrane NADH dehydrogenase (adriamycin, azide, PCMB, atebrine, DIDS and bleomycin). The effect of NADH is accompanied by the disappearance of NADH from the incubation medium and by decreased membrane resistance. We conclude that NADH hyperpolarization is due to the enhancement of passive membrane permeability, apparently for K+, which might result from the conformational changes in the plasma membrane during the NADH dehydrogenase reaction. The possibility is discussed that NADH dehydrogenase mediates transport of K+ out from the cell using a pathway connected with the transmembrane Na+/K+ pump.     

Lateral diffusion of concanavalin a receptors in the plasma membrane of mouse fibroblasts

Lateral diffusion of receptors binding fluorescein labeled concanavalin A and its succinylated derivative has been measured by bleaching portions of the labeled surface and following return of fluorescence to the bleached spot. Binding of either concanavalin A or its succinylated derivative causes restriction of mobility of the surface receptors for this lectin. The degree of restriction is a function of time after binding the lectin.     

Transport of phosphate in membrane vesicles from mouse fibroblasts transformed by simian virus 40.

Membrane vesicles were prepared from mouse fibroblasts transformed by SV40 virus (SV3T3). Following disruption of the cells by nitrogen cavitation, the membrane vesicles were obtained by differential centrifugation. As measured by enzyme markers, they consist mainly of membrane from the plasma membrane and smooth and rough endoplasmic reticulum. The vesicles transport Pi by two separate, mediated systems: one is independent of Na+, and the other is secondary active transport driven by a Na+ gradient. Electrical and chemical energy can be provided by a Na+ gradient to drive the concentrative uptake of Pi by the vesicles, one or both forces being used to energize transport. Evidence is provided that both the electrical and chemical potentials produced by the asymmetric distribution of Na+ across the membrane of SV3T3 membrane vesicles are utilized to concentrate phosphate in the vesicles. Phosphate transport by the vesicles cannot be accounted for by a small contamination of this fraction with mitochondria (1 to 4%). The Pi transport properties of the membrane vesicles differ from those of the fraction enriched in mitochondria in the following respects: their kinetic properties, and their responses to a Na+ gradient, N-ethylmaleimide, mersalyl, and succinate/acetate. However, the membrane vesicles share some properties of Pi transport with mitochondria. Cyanide, azide, oligomycin, 2,4-dinitrophenol, and carbonyl cyanide m-cholophenylhydrazone, inhibitors of Pi transport by mitochondria, also inhibit membrane vesicle, Pi transport. The vesicles retain all the features of Pi transport by SV3T3 cells that have been examined. They provide a simplified system for a determination of the details of the mechanism of Pi transport under conditions where transport is dissociated from intracellular reactions and in the presence of a defined electrochemical driving force.     

Mitochondrial membrane permeabilization: the sine qua non for cell death

Mitochondria are essential for maintaining cell life but they also play a role in regulating cell death, which occurs when their membranes become permeabilized. Mitochondria possess two distinct membrane systems including an outer membrane in close communication with the cytosol and an inner membrane involved in energy transduction. Outer membrane permeabilization is regulated by Bcl-2 family proteins, which control the release of proteins from the mitochondrial intermembrane space; these proteins then activate apoptosis. Inner membrane permeabilization is regulated by the mitochondrial permeability transition (MPT), which is activated by calcium and oxidative stress and leads to bioenergetic failure and necrosis. The purpose of this review is to discuss the biochemical mechanisms regulating mitochondrial membrane permeabilization; this is crucial to our understanding of the role of cell death in diseases such as cancer and the neurodegenerative diseases.     

Non-lytic, non-ionic detergent extraction of plasma membrane constituents from normal and transformed fibroblasts.

A technique has been developed for the selective extraction of plasma membrane protein constituents from normal and transformed cell employing non-ionic detergents. The extraction procedure does not damage cells as judged by cell viability, 51Cr release, and trypan blue staining. Lactoperoxidase-catalyzed iodination followed by detergent extraction permits demonstration of a 100,000 dalton protein which is found on the surface of normal but not transformed hamster and mouse fibroblasts.     

Temperature dependence of ultrasound-induced cell killing: The role of membrane fluidity

Chinese hamster cells in suspension were exposed to 20 kHz ultrasound (US) at 54 W/cm² and various temperatures between 2 and 44 °C. Activation energies were 2.6 and 24 kcal/mole below and above 35 °C, respectively. Procaine, a local anaesthetic drug known to increase membrane fluidity, enhanced cellular inactivation by US above 41 °C, increasing the activation energy to 62 kcal/mole. The inactivation of the bacterium Salmonella typhimurium by US was also dependent on the exposure temperature, with an activation energy of 2.9 kcal/mole between 2 and 44 °C. These data are most simply explained by the hypothesis that membranes are a major target for cellular inactivation by US and that the fluidity of the membranes is important in this respect.     

Distinguishable ATPase activities of cell wall and plasma membrane

Basal and Na⁺-K⁺ stimulated ATPase (ATP phosphohydrolase, E.C. 3.6.1.3) are both present in isolated preparations of purified cell wall and plasma membrane from cotyledon tissue of Phaseolus vulgaris. A comparison of the enzymes in the two fractions has revealed that the specific activities of basal and cation-sensitive ATPase are markedly higher in isolated cell wall than in the plasma membrane fraction. In addition, enrichments of both enzymes calculated on a protein basis relative to corresponding homogenates were considerably higher for cell wall than for plasma membrane. Thus, while part of the ATP-hydrolyzing activity of the wall may be attributable to the enzymatic properties of imbedded plasma membrane, there must also be additional non-membranous ATPase in the protein complement of the wall itself.