Lactobacillus growth and membrane composition in the presence of linoleic or conjugated linoleic acid

Five Lactobacillus strains of intestinal and food origins were grown in MRS broth or milk containing various concentrations of linoleic acid or conjugated linoleic acid (CLA). The fatty acids had bacteriostatic, bacteriocidal, or no effect depending on bacterial strain, fatty acid concentration, fatty acid type, and growth medium. Both fatty acids displayed dose-dependent inhibition. All strains were inhibited to a greater extent by the fatty acids in broth than in milk. The CLA isomer mixture was less inhibitory than linoleic acid. Lactobacillus reuteri ATCC 55739, a strain capable of isomerizing linoleic acid to CLA, was the most inhibited strain by the presence of linoleic acid in broth or milk. In contrast, a member of the same species, L. reuteri ATCC 23272, was the least inhibited strain by linoleic acid and CLA. All strains increased membrane linoleic acid or CLA levels when grown with exogenous fatty acid. Lactobacillus reuteri ATCC 55739 had substantial CLA in the membrane when the growth medium was supplemented with linoleic acid. No association between level of fatty acid incorporation into the membrane and inhibition by that fatty acid was observed.     

Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport

Long-chain nonesterified (“free”) fatty acids (FFA) can affect the mitochondrial generation of reactive oxygen species (ROS) in two ways: (i) by depolarisation of the inner membrane due to the uncoupling effect and (ii) by partly blocking the respiratory chain. In the present work this dual effect was investigated in rat heart and liver mitochondria under conditions of forward and reverse electron transport. Under conditions of the forward electron transport, i.e. with pyruvate plus malate and with succinate (plus rotenone) as respiratory substrates, polyunsaturated fatty acid, arachidonic, and branched-chain saturated fatty acid, phytanic, increased ROS production in parallel with a partial inhibition of the electron transport in the respiratory chain, most likely at the level of complexes I and III. A linear correlation between stimulation of ROS production and inhibition of complex III was found for rat heart mitochondria. This effect on ROS production was further increased in glutathione-depleted mitochondria. Under conditions of the reverse electron transport, i.e. with succinate (without rotenone), unsaturated fatty acids, arachidonic and oleic, straight-chain saturated palmitic acid and branched-chain saturated phytanic acid strongly inhibited ROS production. This inhibition was partly abolished by the blocker of ATP/ADP transfer, carboxyatractyloside, thus indicating that this effect was related to uncoupling (protonophoric) action of fatty acids. It is concluded that in isolated rat heart and liver mitochondria functioning in the forward electron transport mode, unsaturated fatty acids and phytanic acid increase ROS generation by partly inhibiting the electron transport and, most likely, by changing membrane fluidity. Only under conditions of reverse electron transport, fatty acids decrease ROS generation due to their uncoupling action.     

Fatty acids decrease mitochondrial generation of reactive oxygen species at the reverse electron transport but increase it at the forward transport

Long-chain nonesterified ("free") fatty acids (FFA) can affect the mitochondrial generation of reactive oxygen species (ROS) in two ways: (i) by depolarisation of the inner membrane due to the uncoupling effect and (ii) by partly blocking the respiratory chain. In the present work this dual effect was investigated in rat heart and liver mitochondria under conditions of forward and reverse electron transport. Under conditions of the forward electron transport, i.e. with pyruvate plus malate and with succinate (plus rotenone) as respiratory substrates, polyunsaturated fatty acid, arachidonic, and branched-chain saturated fatty acid, phytanic, increased ROS production in parallel with a partial inhibition of the electron transport in the respiratory chain, most likely at the level of complexes I and III. A linear correlation between stimulation of ROS production and inhibition of complex III was found for rat heart mitochondria. This effect on ROS production was further increased in glutathione-depleted mitochondria. Under conditions of the reverse electron transport, i.e. with succinate (without rotenone), unsaturated fatty acids, arachidonic and oleic, straight-chain saturated palmitic acid and branched-chain saturated phytanic acid strongly inhibited ROS production. This inhibition was partly abolished by the blocker of ATP/ADP transfer, carboxyatractyloside, thus indicating that this effect was related to uncoupling (protonophoric) action of fatty acids. It is concluded that in isolated rat heart and liver mitochondria functioning in the forward electron transport mode, unsaturated fatty acids and phytanic acid increase ROS generation by partly inhibiting the electron transport and, most likely, by changing membrane fluidity. Only under conditions of reverse electron transport, fatty acids decrease ROS generation due to their uncoupling action.     

New substrates of the multispecific bile acid transporter in liver cells: interference of some linear renin inhibiting peptides with transport protein(s) for bile acids

Interactions between some stable linear peptides with renin inhibitory activity and a multispecific transport system in the basolateral plasma membrane of liver cells was studied on cell suspensions. The peptides used in our experiments were taken up by liver cells and subsequently eliminated without any biotransformation (e.g., proteolysis). No degradation products could be detected in the extracellular medium by thin-layer chromatography. All peptides tested inhibited the uptake of physiological and of some foreign substrates of the multispecific bile acid transporter (MT). The phalloidin response of liver cells was also inhibited to a similar degree in a concentration-dependent manner. The potency of inhibition did not correlate with the lipophilic properties of the peptides. On the other hand a tight correlation could be documented between the inhibition of cholate transport and that of the phalloidin response. Transport inhibition of typical substrates of the MT by the above renin inhibitors was competitive. In contrast, the transport of a typical substrate of the bilirubin carrier (rifampicin), of amino acids (alpha-aminoisobutyric acid), long chain fatty acids (oleic acid) and cationic compounds (thiamin hydrochloride) was not inhibited by the same renin inhibitors. These results indicate that linear renin inhibiting peptides are taken up into liver cells by carrier proteins related to the MT.     

On the effects of propionate and other short-chain fatty acids on sodium transport by the toad bladder.

1. Propionate and other unbranched short-chain fatty acids, butyrate, pentanoate, hexanoate and octanoate were found to both stimulate and inhibit active sodium transport by the toad bladder, as measured by the short-circuit current (s.c.c.). 2. Stimulation alone followed addition of low concentrations of fatty acids (0.1-1.0 mM) to either the serosal or mucosal bathing medium; stimulation was also seen after an initial period of inhibition in response to higher concentrations (approx. 5 mM) of some compounds. 3. Inhibition alone followed addition of high concentrations (5-20 mM) of these compounds. The duration and magnitude of the inhibition varied with increasing concentration and chain length of the fatty acid, and was greater following mucosal addition than serosal addition. 4. The inhibitory effect of mucosal propionate increased with decreasing pH of the mucosal bathing medium. 5. Inhibition by the fatty acids was completely reversed upon removing the compound from the bathing medium, and stimulation characteristically followed. 6. In studies designed to evaluate the role of metabolism of the fatty acids in their mucosal inhibitory effects it was found that 14-c-labelled propionate, when added to the mucosal surface of the bladder, was converted to 14-CO2, and mucosal succinate and alpha-oxoglutaric acid at 20 mM inhibited the s.c.c. slightly. However, malonate did not interfere with inhibition by mucosal propionate and two non-metabolizable acids, dimethylpropionate and benzoate, induced inhibition (and no stimulation) of the s.c.c. 7. In the presence of an inhibitory concentration of fatty acid, the ability of the bladder to respond to added pyruvate was reduced in proportion to the reduction in the level of the s.c.c., whereas the natriferic response to vasopressin was largely intact. 8. We conclude that stimulation of sodium transport by propionate and other short-chain fatty acids is due to metabolism of the compounds and provision of energy to the sodium transport mechanism. The basis of the inhibition appears complex. It may in part depend on metabolism of the fatty acids and/or uncoupling of oxidative phosphorylation, with resultant reduction in net ATP production for the sodium transport mechanism. However, the inhibition may also be caused in part by a direct effect on the mucosal entry of sodium into the transporting epithelial cells.     

Effects of acetate and other short-chain fatty acids on sugar and amino acid uptake of Bacillus subtilis

Acetate and other short chain n-fatty acids (C(1)-C(6)) inhibit strongly the uptake of l-serine or other l-amino acids but inhibit only weakly that of alpha-methylglucoside or fructose, whether measured in whole cells of Bacillus subtilis or in membrane vesicles that have been energized with reduced nicotinamide adenine dinucleotide (NADH), l-alpha-glycerol phosphate, or ascorbate plus phenazine methosulfate. The acetate inhibition is noncompetitive, as was shown for l-alpha-aminoisobutyric acid uptake by whole cells and for l-serine uptake by membrane vesicles. In membrane preparations, neither NADH oxidation nor the reduction of cytochromes by NADH are affected by fatty acids. All of these effects are similar to those of 2, 4-dinitrophenol. It is concluded that the fatty acids "uncouple" the amino acid carrier proteins from the cytochrome-linked electron transport system (to which they may be coupled via protein interaction or via a cation gradient).     

Manipulation of fatty acid composition of membrane phospholipid and its effects on cell growth in mouse LM cells

Fatty acid composition of the phospholipids of mouse LM cells grown in suspension culture in serum-free chemically defined medium was modified by supplementing the medium with various fatty acids bound to bovine serum albumin.Following supplementation with saturated fatty acids of longer than 15 carbons (100 μM) profound inhibition of cell growth occurred; this inhibitory effect was completely abolished when unsaturated fatty acids were added at the same concentration. Supplementing with unsaturated fatty acids such as linoleic acid, linolenic acid or arachidonic acid had no effect on the cell growth.Fatty acid composition of membrane phospholipids could be manipulated by addition of different fatty acids. The normal percentage of unsaturated fatty acids in LM cell membrane phospholipids (63%) was reduced to 35–41% following incorporation of saturated fatty acids longer than 15 carbon atoms and increased to 72–82% after addition of unsaturated fatty acids.A good correlation was found between the unsaturated fatty acid content of membrane phospholipids and cell growth. When incorporated saturated fatty acids reduced the percentage of unsaturated fatty acids in membrane phospholipids to less than 50%, severe inhibition of the cell growth was found. Simultaneous addition of an unsaturated fatty acid completely abolished this effect of saturated fatty acids.     

Effects of long-chain cis-unsaturated fatty acids and their alcohol analogs on aggregation of bovine platelets and their relation with membrane fluidity change

The effects of long-chain cis-unsaturated fatty acids with different alkyl chain lengths and different numbers of double bonds on aggregation of bovine platelets and membrane fluidity were investigated. All the cis-unsaturated fatty acids tested inhibited aggregation and at the same time increased membrane fluidity in accordance with their inhibitory effects. The saturated fatty acids and trans-unsaturated fatty acid tested for comparison had much lower or no effects on aggregation and membrane fluidity. The inhibitory effects of mono cis-unsaturated fatty acids increased with increase of their alkyl chain length. cis-Unsaturated fatty acids with two or more double bonds had more inhibitory effects than mono-unsaturated fatty acids. The position of the double bonds had less influence than the number of double bonds. We also examined the effects of cis-unsaturated fatty acids on membrane fluidity with diphenylhexatriene and anthroyloxy derivatives of fatty acids as probes and observed increased fluidity to be considerable in the membrane. The alcohol analogs of cis-unsaturated fatty acids also inhibited aggregation and increased membrane perturbation. These results suggest that the inhibition of platelet aggregation by cis-unsaturated compounds is due to perturbation of the lipid layer.     

Anomalous uncoupling of photophosphorylation by palmitic acid and by gramicidin D

Palmitic acid and gramicidin D at low concentrations uncouple photophosphorylation in a mechanism that is inconsistent with classical uncoupling in the following properties: (1) delta pH, H+ uptake, or the transmembrane electric potential is not inhibited. (2) O2 evolution is stimulated under nonphosphorylating conditions but slightly inhibited in the presence of adenosine 5'-diphosphate + inorganic phosphate (Pi). (3) Light-triggered adenosine 5'-triphosphate (ATP)-Pi exchange is hardly affected, and ATPase activity is only slightly stimulated. (4) ATP-induced delta pH formation is selectively inhibited. This characteristic uncoupling is observed only when the native coupling sites of the electron transport system are used for energization such as for methylviologen-coupled phosphorylation. With pyocyanine, which creates an artificial coupling site, 1000-fold higher gramicidin D and higher palmitic acid concentrations are required for inhibition, and the inhibition is accompanied by a decrease in delta pH. Moreover, comparison between photosystem 1 and photosystem 2 electron transport and the effects of membrane unstacking suggest that low gramicidin D preferentially inhibits photosystem 2, while palmitic acid inhibits more effectively photosystem 1 coupling sites. The inhibitory capacity of fatty acids significantly drops when the chain length is reduced below 16 hydrocarbons or upon introduction of a single double bond in the hydrocarbon chain. It is suggested that palmitic acid and gramicidin D interfere with a direct H+ transfer between specific electron transport and the ATP synthase complexes, which provides an alternative coupling mechanism in parallel with bulk to bulk delta microH+. The sites of inhibition seem to be located in chloroplast ATP synthase, photosystem 2, and the cytochrome b6f complexes.     

Characterization of the Acyl-CoA synthetase activity of purified murine fatty acid transport protein 1

Fatty acid transport protein 1 (FATP1) is an approximately 63-kDa plasma membrane protein that facilitates the influx of fatty acids into adipocytes as well as skeletal and cardiac myocytes. Previous studies with FATP1 expressed in COS1 cell extracts suggested that FATP1 exhibits very long chain acyl-CoA synthetase (ACS) activity and that such activity may be linked to fatty acid transport. To address the enzymatic activity of the isolated protein, murine FATP1 and ACS1 were engineered to contain a C-terminal Myc-His tag expressed in COS1 cells via adenoviral-mediated infection and purified to homogeneity using nickel affinity chromatography. Kinetic analysis of the purified enzymes was carried out for long chain palmitic acid (C16:0) and very long chain lignoceric acid (C24:0) as well as for ATP and CoA. FATP1 exhibited similar substrate specificity for fatty acids 16-24 carbons in length, whereas ACS1 was 10-fold more active on long chain fatty acids relative to very long chain fatty acids. The very long chain acyl-CoA synthetase activity of the two enzymes was comparable as were the Km values for both ATP and coenzyme A. Interestingly, FATP1 was insensitive to inhibition by triacsin C, whereas ACS1 was inhibited by micromolar concentrations of the compound. These data represent the first characterization of purified FATP1 and indicate that the enzyme is a broad substrate specificity acyl-CoA synthetase. These findings are consistent with the hypothesis that that fatty acid uptake into cells is linked to their esterification with coenzyme A.     

Stimulation of chloride transport by fatty acids in corneal epithelium and relation to changes in membrane fluidity.

The effect of altering cell membrane lipids on ion transport across isolated corneas was studied. Corneas mounted in Ussing-type chambers showed a rapid increase in short-circuit current following treatment with a variety of unsaturated fatty acids of varying chain length and unsaturation. Measurements of membrane fluidity which utilize immunofluorescence labelling of membrane proteins showed corneal epithelial cell membranes to be significantly more fluid following linoleic acid treatment. Uptake studies indicate rapid incorporation of [14C]linoleic acid into corneal cell membranes. Highly unsaturated fatty acids were found to have the greatest ability to stimulate chloride transport. Saturated fatty acids were tested and were found to have no effect on chloride transport at any concentration. It is proposed that unsaturated fatty acids activate chloride transport by increasing membrane lipid fluidity. The relationship of these parameters is discussed in terms of a mobile receptor model. We speculate that an increase in membrane lipid fluidity promotes lateral diffusion of membrane receptor proteins and enzymes, increasing protein-protein interactions within the membrane, ultimately resulting in the enhancement of cyclic AMP synthesis.     

Sterol-content lowering action of o-chlorobenzylchloride in yeast

o-Chlorobenzylchloride, a simple aromatic halogen compound, was found to inhibit the growth of Saccharomyces cerevisiae and to lower the contents of sterols and fatty acids. The growth inhibition was considerably alleviated by the presence of sterols such as ergosterol and cholesterol and of unsaturated fatty acids such as oleate and linolenate. Inspection of effect of the inhibitor on the electron transport system related to the biosyntheses of these compounds revealed that the cytochrome contents and some enzyme activities in the system of the inhibited cells were much lower than those of the control cells. The features of the inhibition were similar to those of inhibition for other organisms by the hypocholesterolemic compounds such as triparanol and benzmalecene.     

Effect of cerulenin on the growth and differentiation of Dictyostelium discoideum.

The growth of Dictyostelium discoideum Ax-2 was inhibited completely by cerulenin at a concentration of 5 mug/ml. This inhibition of growth was found to be due to the inhibition of fatty acid synthesis. Acetate incorporation into a long-chain fatty acid was inhibited completely by cerulenin, and the growth inhibition could be reversed by inclusion of certain saturated fatty acids in the medium. Unsaturated fatty acids and sterols failed to reverse the inhibitory effect. The fatty acid and sterol compositions of cerulenin-treated cells were determined to establish whether the drug could be used to manipulate the organism's lipid composition. Only relatively small manipulations were obtained under the conditions employed in this study. Cerulenin inhibited differentiation but only at high concentrations (150 mug/ml). This inhibition could be reversed by palmitic acid, suggesting that the prime cause of the inhibition was an inhibition of fatty acid synthesis. Thus, it appears that continued fatty acid synthesis is required for the cellular process of differentiation in D. discoideum.     

Functional domains of the fatty acid transport proteins: studies using protein chimeras

Fatty acid transport proteins (FATP) function in fatty acid trafficking pathways, several of which have been shown to participate in the transport of exogenous fatty acids into the cell. Members of this protein family also function as acyl CoA synthetases with specificity towards very long chain fatty acids or bile acids. These proteins have two identifying sequence motifs: The ATP/AMP motif, an approximately 100 amino acid segment required for ATP binding and common to members of the adenylate-forming super family of proteins, and the FATP/VLACS motif that consists of approximately 50 amino acid residues and is restricted to members of the FATP family. This latter motif has been implicated in fatty acid transport in the yeast FATP orthologue Fat1p. In the present studies using a yeast strain containing deletions in FAT1 (encoding Fat1p) and FAA1 (encoding the major acyl CoA synthetase (Acsl) Faa1p) as an experimental platform, the phenotypic and functional properties of specific murine FATP1-FATP4 and FATP6-FATP4 protein chimeras were evaluated in order to define elements within these proteins that further distinguish the fatty acid transport and activation functions. As expected from previous work FATP1 and FATP4 were functional in the fatty acid transport pathway, while and FATP6 was not. All three isoforms were able to activate the very long chain fatty acids arachidonate (C(20:4)) and lignocerate (C(24:0)), but with distinguishing activities between saturated and highly unsaturated ligands. A 73 amino acid segment common to FATP1 and FATP4 and between the ATP/AMP and FATP/VLACS motifs was identified by studying the chimeras, which is hypothesized to contribute to the transport function.     

有机碳化合物对湛江等鞭金藻生长的影响

为了探讨有机碳化合物对湛江等鞭金藻的营养效应,实验设置了在f/2培养基中添加葡萄糖、乙酸钠、半乳糖、甘油、乙醇、柠檬酸钠和甘氨酸等7种有机碳化合物的处理,测定了湛江等鞭金藻(Isochrysis zhanjiangensis)的生长情况。结果表明,参试的7种有机碳化合物中,甘氨酸对湛江等鞭金藻细胞生长的促进作用最明显,而乙醇对藻细胞生长的促进效果不明显,其他5种均有不同程度的促进作用。7种有机碳对湛江等鞭金藻胞内蛋白质含量和总脂的积累量具有一定差异性影响。0.5～10g·L^-1的葡萄糖、乙酸钠均可提高胞内蛋白质和总脂的含量。半乳糖对总脂积累量的影响不明显。     

Retinoic acid inhibition of transplasmalemma diferric transferrin reductase

All trans retinoic acid inhibited diferric transferrin reduction by HeLa cells. The NADH diferric transferrin reductase activity of isolated liver plasma membranes was also inhibited by retinoic acid. Retinol and retinyl acetate had very little effect. Transplasma membrane ferricyanide reduction by HeLa cells and NADH ferricyanide reductase of liver plasma membrane was also inhibited by retinoic acid, therefore the inhibition was in the electron transport system and not at the transferrin receptor. Since the transmembrane electron transport has been shown to stimulate cell growth, the growth inhibition by retinoic acid thus may be based on inhibition of the NADH diferric transferrin reductase.     

cis-Unsaturated fatty acids induce the fusion of chromaffin granules aggregated by synexin

When isolated chromaffin granules were aggregated by synexin (a Ca2+-binding protein present in chromaffin and other secretory tissues) and then exposed to cis-unsaturated fatty acids at 37 degrees C, they fused together to form large vesicles. The fusion was monitored by phase and electron microscopy and by turbidity measurements on the granule suspension. Arachidonic acid was the most effective fusogen, whereas trans-unsaturated fatty acids, saturated fatty acids, detergents or lysolecithin were inactive. During fusion some of the epinephrine of the granules was released but the soluble core proteins remained trapped in the resulting vesicles. These vesicles swelled to enclose the maximum volume. Although this swelling could be inhibited by increasing the osmotic strength of the medium, it did not appear to depend on the chemiosmotic properties of the granule membranes as it was not influenced by ATP, a proton ionophore, or an anion transport inhibitor. The regulators of this in vitro fusion--Ca2+, synexin, and free, cis-unsaturated fatty acids--may be present in the cytoplasm of the chromaffin cell when it is stimulated to release epinephrine and granule proteins by exocytosis. Therefore, this fusion event may be the same that occurs between chromaffin granules undergoing compound exocytosis.     

ATP generation during reduced inorganic sulfur compound oxidation by<Emphasis Type="Italic"> Acidithiobacillus caldus</Emphasis> is exclusively due to electron transport phosphorylation

The synthesis of adenosine 5-triphosphate (ATP) (increase in phosphorylation potential) during the oxidation of reduced inorganic sulfur compounds was studied in the moderately thermophilic acidophileAcidithiobacillus caldus (strain KU) (formerly Thiohacillus caldus). The phosphorylation potential increased during the oxidation of all reduced inorganic sulfur compounds tested compared with resting cells. The generation of ATP in whole cells was inhibited by the F0F1 ATPase inhibitor oligomycin, electron transport chain inhibitors, valinomycin and potassium ions. There was no increase in the phosphorylation potential, nor synthesis of ATP. in the absence of electron transport. An apparent lack of substrate-level phosphorylation was indicated by the lack of adenosine 5-phosphosulfate reductase in tetrathionate-grown At. caldus. Studies were also performed on the synthesis of ATP by membrane vesicles of At. caldus when presented with an artificial proton gradient. Complete inhibition of ATP synthesis in these vesicles occurred when they were loaded with N,N-dicyclohexylcarbodiimide (DCCD), but not when they were loaded with oligomycin, vanadate or electron transport chain inhibitors. The data presented here suggest that during the oxidation of reduced inorganic sulfur compounds by At. caldus, all ATP is synthesized by oxidative phosphorylation via a membrane-bound F0F1 ATPase driven by a proton gradient.     

Labeling of adipocyte membranes by sulfo-N-succinimidyl derivatives of long-chain fatty acids: Inhibition of fatty acid transport

Summary Sulfo-N-succinimidyl derivatives of the long-chain fatty acids, oleic and myristic, were synthesized and covalently reacted with isolated rat adipocytes. The plasma membrane proteins labeled by these compounds and the effect of labeling on the transport of long-chain fatty acids were investigated. Sulfo-N-succinimidyl oleate (SSO) and myristate (SSM) inhibited the transport of fatty acids (by about 70%). Inhibition of fatty acid transport was not a result of alterations in cell integrity, as intracellular water volume was not changed. It did not reflect effects on fatty acid metabolism, since it was observed under conditions where greater than 90% of the fatty acid taken up was recovered in the free form. The inhibitory effect was specific to the fatty acid transport system, as the transport of glucose and the permeation of retinoic acid, a substance with structural similarities to long-chain fatty acids, were unaffected. Sulfosuccinimidyl oleate reacted exclusively with a plasma membrane protein with an apparent size of 85 kDa while sulfosuccinimidyl myristate also labeled a 75-kDa while sulfosuccinimidyl myristate also labeled a 75-kDa protein. These proteins were among the ones labeled by diisothiocyanodisulfonic acid (DIDS) which also inhibits fatty acid transport irreversibly. The data suggest that the 85-kDa protein, which is the only one labeled by all three inhibitors is involved in facilitating membrane permeation of long-chain fatty acids.     

Ionic dependence of the extracellular ATP-induced permeabilization of transformed mouse fibroblasts: role of plasma membrane activities that regulate cell volume

Extracellular ATP rendered the plasma membrane of transformed mouse fibroblasts permeable to normally impermeant molecules. This permeability change was prevented by increasing the ionic strength of the isotonic medium with NaCl. Conversely, the cells exhibited increased sensitivity to ATP when the NaCl concentration was decreased below isotonicity, when the KCl concentration was increased above 5 mM while maintaining isotonicity, and when the pH of the medium was raised above 7.0. These conditions as well as the addition of ATP itself caused cell swelling. However, the effect of ATP was independent of cell volume and dependent upon the ionic strength and not the osmolarity of the medium since 1) addition of sucrose to isotonic medium did not prevent permeabilization although media made hypertonic with either sucrose or NaCl caused a decrease in cell volume; and 2) addition of sucrose or NaCl to hypotonic media caused a decrease in cell volume, but only NaCl addition decreased the response to ATP. Conditions that have been shown to inhibit plasma membrane proteins that play a reciprocal role in cell volume regulation had reciprocal effects on the permeabilization process, even though the effect of ATP was independent of cell volume. For example, inhibition of the Na+,K+-ATPase by ouabain increased sensitivity of cells to ATP while conditions which inhibit Na+,K+,Cl- -cotransporter activity, such as treatment of the cells with the diuretics furosemide or bumetanide or replacement of sodium chloride in the medium with sodium nitrate or thiocyanate, inhibited permeabilization. The furosemide concentration that inhibited permeabilization was greater than the concentration that inhibited Na+,K+,Cl- -cotransporter-mediated 86Rb+ (K+) uptake, suggesting that the effect of furosemide on the permeabilization process may not be specific for the Na+,K+,Cl- -cotransporter.