Newborn response to cationic amphiphilic drugs

Administration of various cationic amphiphilic drugs in utero results in induction of a phospholipid storage disorder in many tissues, particularly in lungs. In addition to the phospholipidosis in utero, drug exposure results in toxicity to the offspring; newborn rats die within 48 h of birth. Although drug-induced pulmonary pathological changes appear to be involved in the observed mortality, this relationship remains unclear. In contrast to mammals, administration of cationic amphiphilic drugs to the chick embryo seems not to induce phospholipid storage in the tissues examined. Treatment of newborn rats directly with these drugs also induces phospholipidosis in several tissues including lung and kidney; however, mortality does not occur. Concurrent administration of phenobarbital and chlorphentermine reduces or prevents amphiphilic drug-induced phospholipid storage in newborn rat lung and kidney. Modification of chlorphentermine actions by phenobarbital may be caused by alterations in amphiphilic drug excretion, metabolism, and catabolic phospholipase activity. Evidence thus indicates that regardless of age, animals appear susceptible to the effects of cationic amphiphilic drugs; however, species and tissues examined, as well as specific drug administration, play an important role in the observed qualitative and quantitative responses.     

Drug induced phospholipidosis: An acquired lysosomal storage disorder

There is a strong association between lysosome enzyme deficiencies and monogenic disorders resulting in lysosomal storage disease. Of the more than 75 characterized lysosomal proteins, two thirds are directly linked to inherited diseases of metabolism. Only one lysosomal storage disease, Niemann–Pick disease, is associated with impaired phospholipid metabolism. However, other phospholipases are found in the lysosome but remain poorly characterized. A recent exception is lysosomal phospholipase A2 (group XV phospholipase A2). Although no inherited disorder of lysosomal phospholipid metabolism has yet been associated with a loss of function of this lipase, this enzyme may be a target for an acquired form of lysosomal storage, drug induced phospholipidosis. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Comparative effects of aminoglycosides on renal cortical and urinary phospholipids in the rat

We examined the relationship between the nephrotoxicity potential of four aminoglycosides and the capacity of the drugs to induce a renal cortical phospholipidosis. Sprague-Dawley rats were injected subcutaneously with neomycin, gentamicin, tobramycin, or netilmicin, 100 mg/kg per day, for 1 to 4 days, and phospholipid accumulation in the renal cortex and phospholipid excretion in the urine were measured. The rank order of the drug-induced renal cortical phospholipidosis was netilmicin greater than tobramycin greater than gentamicin greater than neomycin. This order is the reverse of the previously established nephrotoxicity potentials of these drugs. Conversely, the rank order according to peak urinary excretion of phospholipids was gentamicin greater than neomycin greater than tobramycin greater than netilmicin. The rank order of the total urinary phospholipid excretion during the 4 days of the study was neomycin greater than or equal to gentamicin greater than tobramycin greater than or equal to netilmicin. Urinary phospholipid excretion may prove to be a sensitive indicator of aminoglycoside nephrotoxicity.     

Regulation of lung surfactant phospholipid synthesis and metabolism

The alveolar type II epithelial (ATII) cell is highly specialised for the synthesis and storage, in intracellular lamellar bodies, of phospholipid destined for secretion as pulmonary surfactant into the alveolus. Regulation of the enzymology of surfactant phospholipid synthesis and metabolism has been extensively characterised at both molecular and functional levels, but understanding of surfactant phospholipid metabolism in vivo in either healthy or, especially, diseased lungs is still relatively poorly understood. This review will integrate recent advances in the enzymology of surfactant phospholipid metabolism with metabolic studies in vivo in both experimental animals and human subjects. It will highlight developments in the application of stable isotope-labelled precursor substrates and mass spectrometry to probe lung phospholipid metabolism in terms of individual molecular lipid species and identify areas where a more comprehensive metabolic model would have considerable potential for direct application to disease states.     

Alterations in rat alveolar surfactant phospholipids and proteins induced by administration of chlorphentermine

Chlorphentermine is a cationic amphiphilic drug which produces a phospholipid storage disorder in rat lungs. Experiments were carried out to characterize changes in the composition of acellular alveolar lavage materials and to study possible mechanisms by which pulmonary surfactant phospholipidosis is produced by administration of the drug. Following ten daily injections of chlorphentermine (25 mg/kg body weight), there are 12.2- and 13.6-fold increases of pulmonary lavage total phospholipids and disaturated phosphatidylcholines (disaturated PC), respectively. In addition, there is a 2.8-fold increase in total protein and a 12.7-fold increase in the surfactant apoprotein group with molecular weights from 28,000 to 32,000. We measured incorporation of labeled palmitate, choline and glycerol into disaturated PC in type II cells and alveolar macrophages isolated from control and chlorphentermine-treated animals. The drug does not affect the incorporation of labeled substrates into disaturated PC in either cell type. However, in alveolar macrophages there is a decrease in the rate of intracellular degradation of recently synthesized disaturated PC in chlorphentermine-treated animals. The drug also inhibits the phospholipase-induced catabolism of rat surfactant disaturated PC which occurs during incubation of alveolar lavage fluid in vitro at 37 degrees C. When the lavage fluid is divided into subfractions by differential centrifugation, a larger percentage of the phospholipid is distributed in the less sedimentable subfractions in chlorphentermine-treated animals relative to controls, suggesting the accumulation of older surfactant materials. These results suggest that chlorphentermine-induced phospholipidosis of pulmonary surfactant materials is due to decreased rates of phospholipid degradation.     

Autistic disorder and phospholipids: A review

Dysregulated phospholipid metabolism has been proposed as an underlying biological component of neurodevelopmental disorders such as autistic disorder (AD) and attention-deficit/hyperactivity disorder (ADHD). This review provides an overview of fatty acid and phospholipid metabolism and evidence for phospholipid dysregulation with reference to the membrane hypothesis of schizophrenia. While there is evidence that phospholipid metabolism is at least impaired in individuals with AD, it has not been established whether phospholipid metabolism is implicated in causal, mechanistic or epiphenomenological models. More research is needed to ascertain whether breastfeeding, and specifically, the administration of colostrum or an adequate substitute can play a preventative role by supplying the neonate with essential fatty acids (EFAs) at a critical juncture in their development. Regarding treatment, further clinical trials of EFA supplementation are essential to determine the efficacy of EFAs in reducing AD symptomatology and whether supplementation can serve as a cost-effective and readily available intervention.     

Role of phospholipase C in chlorphentermine-induced pulmonary phospholipidosis in rat

Daily, oral administration of chlorphentermine (60 mg/kg) for 5 days to rats produced a significant increase in the concentration of whole lung total phospholipid as well as sphingomyelin, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, and phosphatidylcholine. Similarly, a significant elevation in total and all individual phospholipid components was found in the lysosomal fraction of chlorphentermine-treated rat lung. In contrast, the activities of pulmonary Na+,K+-ATPase and alkaline phosphatase, enzymatic markers of membrane function, were not markedly affected by chlorphentermine treatment. The observed lung phospholipidosis was accompanied by inhibition of phospholipase C activity. Regardless of the phospholipid substrate, chlorphentermine significantly decreased pulmonary phospholipase C to approximately the same extent. Our data show that accumulation of phospholipid in whole lung and lysosomes is associated with an inhibition of phospholipase C activity.     

In vivo toxicity and pulmonary effects of promazine and chlorpromazine in rats

Cationic amphiphilic drugs induce a phospholipid storage disorder known as phospholipidosis. Halogenated analogs of the drugs are more potent inducers of phospholipidosis when compared to nonhalogenated analogs. Two such antipsychotic drugs, promazine and chlorpromazine, are effectively taken up by the lungs and induce lamellar inclusions in vitro. We compared the in vivo toxicity and efficacy of promazine and chlorpromazine to induce phospholipidosis in the lung and in pulmonary alveolar macrophages. Male Sprague-Dawley rats were given promazine or chlorpromazine (25 mg/kg/day, P.O., in water) for 5 weeks. Food intake was decreased in promazine- and chlorpromazine-treated rats, chlorpromazine rats being affected more than promazine rats. To minimize experimental error due to starvation, control rats were pair-fed. The body weight gain was decreased in chlorpromazine rats in comparison to pair-fed controls. Chlorpromazine-treated rats, but not promazine-treated rats, showed increased mortality over the 5-week treatment period. Histopathologic examination of lung revealed loss of alveolar macrophages with no other gross abnormalities in chlorpromazine-treated rats. Quantitative analysis of lung lavage also showed significant reduction in the number of macrophages. This finding is in contrast to other cationic amphiphilic drugs, which induce phospholipidosis as well as accumulation of alveolar macrophages. Phospholipid level increased in alveolar macrophages but not in lavaged lung following chlorpromazine treatment. Acid phosphatase activity in lavaged lung homogenate and macrophages of promazine- and chlorpromazine-treated rats, taken as an index of toxicity to cells, did not differ significantly from control rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence studies on binding of amphiphilic drugs to isolated lamellar bodies: relevance to phospholipidosis

Lysosomal phospholipid storage disorder in lung tissue was observed during chronic treatment with amphiphilic amine drugs. The prevailing and widely accepted mechanism of phospholipidosis is that amphiphilic drugs bind to phospholipids and make the phospholipids unsuitable substrates for the action of phospholipases. We investigated hydrophobic and hydrophilic binding of fifteen drugs to the phospholipid storage organelle, lung lamellar bodies, isolated from male Sprague-Dawley rats. Hydrophobic interactions were studied using 1,6-diphenyl-1,3,5-hexatriene as a fluorescent probe and hydrophilic binding was studied using 1-anilino-8-naphthalene sulfonate as a fluorescent probe. The binding parameters were calculated using Scatchard equations. Of the fifteen drugs used, nine drugs bound to the hydrophobic moiety of lamellar bodies. The order of binding capacities was promethazine greater than chloramphenicol greater than amiodarone = desethylamiodarone greater than promazine greater than chlorpromazine greater than trimipramine greater than propranolol greater than imipramine much greater than chlorphentermine, phentermine, chloroquine, chlorimipramine, cyclizine and chlorcyclizine. Two binding affinities were calculated for all the bound drugs. Binding affinities to hydrophilic sites of lamellar bodies were calculated in terms of emission coefficients for 1-anilino-8-naphthalene sulfonate in the presence of drugs. Hydrophilic binding was in the order chlorpromazine greater than chlorimipramine greater than promazine greater than trimipramine greater than imipramine greater than chlorcyclizine greater than propranolol greater than promethazine greater than chlorphentermine greater than cyclizine greater than phentermine greater than chloroquine much greater than chloramphenicol, amiodarone and desethylamiodarone. The binding affinities of chlorinated analogs were stronger to hydrophilic sites when compared to the parent compound. Amiodarone, which is known to induce pulmonary phospholipidosis and its major non-polar metabolite, desethylamiodarone, bound strongly to lamellar bodies. These two drugs also inhibit phospholipases in vitro. The drugs with weak phospholipidosis-inducing capacity and extensive in vivo metabolism, namely, imipramine, chlorpromazine and promazine, also bound strongly to lamellar bodies with hydrophilic as well as hydrophobic interactions. On the other hand, chloroquine, which is known to induce phospholipidosis and to inhibit phospholipases, did not bind to lamellar bodies. Two major conclusions could be drawn from this study: one is that the drug interactions with isolated lamellar bodies could be studied using membrane fluorescence probes, 1,6-diphenyl-1,3,5-hexatriene and 1-anilino-8-naphthalene sulfonate; second is that the amphiphilic drugs bind to lamellar bodies, as reported for phospholipid vesicles, and the binding of drugs to lamellar bodies could be correlated with their phospholipidosis-inducing capacity only if     

Dynamics of arachidonic acid mobilization by inflammatory cells

The development of mass spectrometry-based techniques is opening new insights into the understanding of arachidonic acid (AA) metabolism. AA incorporation, remodeling and release are collectively controlled by acyltransferases, phospholipases and transacylases that exquisitely regulate the distribution of AA between the different glycerophospholipid species and its mobilization during cellular stimulation. Traditionally, studies involving phospholipid AA metabolism were conducted by using radioactive precursors and scintillation counting from thin layer chromatography separations that provided only information about lipid classes. Today, the input of lipidomic approaches offers the possibility of characterizing and quantifying specific molecular species with great accuracy and within a biological context associated to protein and/or gene expression in a temporal frame. This review summarizes recent results applying mass spectrometry-based lipidomic approaches to the identification of AA-containing glycerophospholipids, phospholipid AA remodeling and synthesis of oxygenated metabolites.     

Raman and fluorescence imaging of phospholipidosis induced by cationic amphiphilic drugs in endothelial cells

Cationic amphiphilic drugs (CADs) are known from lysosomotropism, drug-induced phospholipidosis (DIPL), activation of autophagy, and decreased cell viability, but the relationship between these events is not clear and little is known about DIPL in the endothelium. In this work, the effects of fluoxetine, amiodarone, clozapine, and risperidone on human microvascular endothelial cells (HMEC-1) were studied using a combined methodology of label-free Raman imaging and fluorescence staining. Raman spectroscopy was applied to characterize biochemical changes in lipid profile and their distribution in the cellular compartments, while fluorescence staining (LysoTracker, LipidTOX, LC3B, and JC-1) was used to analyze lysosome volume expansion, activation of autophagy, lipid accumulation, and mitochondrial membrane depolarization. We demonstrated that fluoxetine, amiodarone, and clozapine, but not risperidone, at non-toxic concentrations induced lipid accumulations in the perinuclear and cytoplasmic regions of endothelial cells. Spectroscopic markers of DIPL included a robust increase in the ratio (lipid/(protein + lipid)), an increase in choline-containing lipid, fatty acids, and the presence of cholesterol esters, while starvation-induced activated autophagy revealed a spectroscopic signature associated with subtle changes in the lipid profile only. Interestingly, lysosomal volume expansion, occurrence of DIPL, and activation of autophagy induced by selected CADs all depended on drug-accumulation in acidic pH of lysosome cellular compartments whereas reduced endothelial viability did not, and was attributed to mitochondrial mechanisms as evidenced by a decreased mitochondrial transmembrane potential. In conclusion, drug-induced phospholipidosis in the endothelium did not reduce endothelial viability per se and can be efficiently assayed by Raman imaging.     

Dynamics of arachidonic acid mobilization by inflammatory cells

The development of mass spectrometry-based techniques is opening new insights into the understanding of arachidonic acid (AA) metabolism. AA incorporation, remodeling and release are collectively controlled by acyltransferases, phospholipases and transacylases that exquisitely regulate the distribution of AA between the different glycerophospholipid species and its mobilization during cellular stimulation. Traditionally, studies involving phospholipid AA metabolism were conducted by using radioactive precursors and scintillation counting from thin layer chromatography separations that provided only information about lipid classes. Today, the input of lipidomic approaches offers the possibility of characterizing and quantifying specific molecular species with great accuracy and within a biological context associated to protein and/or gene expression in a temporal frame. This review summarizes recent results applying mass spectrometry-based lipidomic approaches to the identification of AA-containing glycerophospholipids, phospholipid AA remodeling and synthesis of oxygenated metabolites.     

Checks and balances in membrane phospholipid class and acyl chain homeostasis,the yeast perspective

Glycerophospholipids are the most abundant membrane lipid constituents in most eukaryotic cells. As a consequence, phospholipid class and acyl chain homeostasis are crucial for maintaining optimal physical properties of membranes that in turn are crucial for membrane function. The topic of this review is our current understanding of membrane phospholipid homeostasis in the reference eukaryote Saccharomyces cerevisiae. After introducing the physical parameters of the membrane that are kept in optimal range, the properties of the major membrane phospholipids and their contributions to membrane structure and dynamics are summarized. Phospholipid metabolism and known mechanisms of regulation are discussed, including potential sensors for monitoring membrane physical properties. Special attention is paid to processes that maintain the phospholipid class specific molecular species profiles, and to the interplay between phospholipid class and acyl chain composition when yeast membrane lipid homeostasis is challenged. Based on the reviewed studies, molecular species selectivity of the lipid metabolic enzymes, and mass action in acyl-CoA metabolism are put forward as important intrinsic contributors to membrane lipid homeostasis.     

Hydrolysis of phosphatidylcholine by cerium(IV) releases significant amounts of choline and inorganic phosphate at lysosomal pH

Niemann-Pick disease and drug-induced phospholipidosis are examples of lysosomal storage disorders in which serious respiratory infections are brought on by high levels of the phospholipid phosphatidylcholine in the acidic lamellar bodies and lysosomes of pulmonary cells. One approach to developing an effective therapeutic agent could involve the use of a metal to preferentially hydrolyze phospholipid phosphate ester bonds at mildly acidic, lysosomal pH values (~ pH 4.8). Towards this end, here we have investigated phosphatidylcholine hydrolysis by twelve metal ion salts at 60 °C. Using a malachite green/molybdate-based colorimetric assay to detect inorganic phosphate released upon metal-assisted phosphate ester bond hydrolysis, Ce(IV) was shown to possess outstanding reactivity in comparison to the eleven other metals. We then utilized cerium(IV) to hydrolyze phosphatidylcholine at normal, core body temperature (37 °C). The malachite green/molybdate assay was used to quantitate free phosphate and an Amplex® Red-based colorimetric assay and matrix-assisted laser desorption ionization time-of-flight mass spectrometry were employed to detect choline. Ce(IV) hydrolyzed phosphatidylcholine more efficiently at lysosomal pH: i.e., at a Triton X-100:phosphatidylcholine molar mixing ratio of 1.57, yields of choline and phosphate were 51 ± 4% and 40 ± 4% at ~ pH 4.8, compared to 28 ± 4% and 27 ± 5% at ~ pH 7.2.     

A cell-based approach for the early assessment of the phospholipidogenic potential in pharmaceutical research and drug development

Phospholipidosis is a term commonly used to indicate a phospholipid storage disorder; in affected cells, phospholipids accumulate in lysosomes that acquire a multilamellar morphological appearance. Cationic amphiphilic drugs (CADs) are suggested to induce phospholipidosis by direct interaction of xenobiotics with intracellular phospholipids or by the action of xenobiotics on the synthesis and metabolism of phospholipids. To date, electron microscopy (EM) represents the most reliable and the preferred method for the demonstration of phospholipidotic cell damage. Nevertheless, EM has a low throughput, it is expensive, and it is not suitable for screening purposes.We discuss here the assessment of the the phospholipidogenic potential of drugs using a cell culture-based model. In this test, intracellular phospholipids of treated U-937 cells (a human monocyte-derived cell line) were measured using the fluorescent probe Nile red. Eleven CADs reported to induce phospholipidosisin vivo and eight nonphospholipidogenic drugs were tested. Results obtained with the U-937 model confirmed the phospholipidogenic potential of drugs tested as described in the literature. Results have also been correlated with data obtained with a physical-chemical model (chromatographic hydrophobicity index measurement). Good correlation was obtained, confirming that the physical-chemical properties of CADs play a crucial role in the development of phospholipidosis.This work demonstrates that the U-937 model is a rapid and sensitive method for the determination of phospholipidosis-mediated cell damage. The specificity and the predictive potency observed make this method suitable for screening purposes in pharmaceutical development.     

Reduction of acid sphingomyelinase activity in human fibroblasts induced by AY-9944 and other cationic amphiphilic drugs

AY-9944 (trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane dihydrochloride), a cationic amphiphilic drug, caused a rapid, irreversible and dose-dependent reduction of acid sphingomyelinase activity in normal human fibroblasts without changing the activities of other lysosomal hydrolases tested. Examinations of activities against synthetic substrates and of the pH-dependency of sphingomyelinase in the drug-treated cells also suggested that the reduction of activity was specific to acid sphingomyelinase. Such a specific reduction was also found with 12 other cationic amphiphilic drugs, most of which have been shown to be inducers of experimental phospholipidosis in animals and/or cultured cells. These results strongly suggest that acid sphingomyelinase is involved in the process of drug-induced lipidosis. The reduction of acid sphingomyelinase seemed not to be due to direct inhibition by these drugs, a specific loss of the enzyme into the culture medium, the presence of inhibitor in the drug-treated cells, or impaired synthesis of the enzyme. There was no indication that changes in the catalytic properties of the enzyme, or changes in the requirement of detergents for its activity occurred in the cell. These results suggest that AY-9944 and other cationic amphiphilic drugs may cause the reduction of acid sphingomyelinase activity by inducing an increased rate of degradation of the enzyme or by causing an irreversible inactivation via some undetected factor.     

Drug-induced phospholipidosis: are there functional consequences?

Phospholipidosis induced by drugs with a cationic amphiphilic structure is a generalized condition in humans and animals that is characterized by an intracellular accumulation of phospholipids and the concurrent development of concentric lamellar bodies. The primary mechanism responsible for the development of phospholipidosis is an inhibition of lysosomal phospholipase activity by the drugs. While the biochemical and ultrastructural features of the condition have been well characterized, much less effort has been directed toward understanding whether the condition has adverse effects on the organism. While there are a few cationic amphiphilic drugs that have been reported to cause phospholipidosis in humans, the principal concern with this condition is in the pharmaceutical industry during preclinical testing. While this class of drugs should technically be referred to as cationic lipophilic, the term cationic amphiphilic is widely used and recognized in this field, and for this reason, the terminology cationic amphiphilic drugs (CADs) will be employed in this Minireview. The aim of this Minireview is to provide an evaluation of the state of knowledge on the functional consequences of CAD-induced phospholipidosis.     

Effects of chronic amiodarone treatment on cat myocardial phospholipid content and on in vitro phospholipid catabolism

Amiodarone is used extensively for the chronic treatment of life-threatening arrhythmias caused by ischemic heart disease. However, chronic therapy with this agent results in phospholipidosis in various tissues and it has been suggested that the inhibition of lysosomal phospholipase A by this drug contributes to this abnormality. Exogenous amiodarone has been shown to inhibit purified rat liver lysosomal phospholipase A₁, as well as acid phospholipase activities of alveolar macrophage homogenates and those of snake venom phospholipase A₂ and bacterial phospholipase C. The effects of drug treatment on heart have not been explored. The results described here demonstrate that amiodarone also significantly increases (37%, p < 0.001) phospholipid content in cat hearts. This increase is proportionately distributed to all major phospholipid classes, with the exception of sphingomyelin which appears to increase more than the others. In addition, the data also show that following amiodarone treatment, the endogenous drug levels in the heart were sufficient to reduce in vitro losses of membrane phospholipid at 37°C by inhibiting a variety of endogenous phospholipases at physiological (7.4), ischemic (6.2) and acidic (5.0) pH values. This protection is more pronounced at acidic pH values than at physiological pH. Endogenous amiodarone also affects myocardial phospholipase activities towards exogenous phosphatidylcholine and again the extent of inhibition is more at acidic pH. These results suggest that amiodarone induces phospholipidosis in the heart by inhibiting phospholipid catabolism and that its antiarrhythmic properties may reside in its ability to modulate alkaline, neutral and acid phospholipase activities in ischemia. To what extent amiodarone metabolites (desethylamiodarone and bis-desethylamiodarone) are involved in these actions remains to be determined.     

Tafazzin-dependent cardiolipin composition in C6 glioma cells correlates with changes in mitochondrial and cellular functions,and cellular proliferation

The mitochondrial phospholipid cardiolipin (CL) has been implicated with mitochondrial morphology, function and, more recently, with cellular proliferation. Tafazzin, an acyltransferase with key functions in CL remodeling determining actual CL composition, affects mitochondrial oxidative phosphorylation. Here, we show that the CRISPR-Cas9 mediated knock-out of tafazzin (Taz) is associated with substantial alterations of various mitochondrial and cellular characteristics in C6 glioma cells. The knock-out of tafazzin substantially changed the profile of fatty acids incorporated in CL and the distribution of molecular CL species. Taz knock-out was further associated with decreased capacity of oxidative phosphorylation that mainly originates from impaired complex I associated energy metabolism in C6 glioma cells. The lack of tafazzin switched energy metabolism from oxidative phosphorylation to glycolysis indicated by lower respiration rates, membrane potential and higher levels of mitochondria-derived reactive oxygen species but keeping the cellular ATP content unchanged. The impact of tafazzin on mitochondria was also indicated by altered morphology and arrangement in tafazzin deficient C6 glioma cells. In the cells we observed tafazzin-dependent changes in the distribution of cellular fatty acids as an indication of altered lipid metabolism as well as in stability/morphology. Most impressive is the dramatic reduction in cell proliferation in tafazzin deficient C6 glioma cells that is not mediated by reactive oxygen species. Our data clearly indicate that defects in CL phospholipid remodeling trigger a cascade of events including modifications in CL linked to subsequent alterations in mitochondrial and cellular functions.     

ARF proteins: roles in membrane traffic and beyond

The ADP-ribosylation factor (ARF) small GTPases regulate vesicular traffic and organelle structure by recruiting coat proteins, regulating phospholipid metabolism and modulating the structure of actin at membrane surfaces. Recent advances in our understanding of the signalling pathways that are regulated by ARF1 and ARF6, two of the best characterized ARF proteins, provide a molecular context for ARF protein function in fundamental biological processes, such as secretion, endocytosis, phagocytosis, cytokinesis, cell adhesion and tumour-cell invasion.