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PubMed1982

The kinetics of ligand binding for diverse mammalian myoglobins and the effects of substitutions outside the heme cavity.

1. We report rate constants for oxygen dissociation and for oxygen, carbon monoxide, azide, and cyanide binding to whale, horse, dog, beef and human myoglobins. 2. For azide binding, rate constants can vary by at least a factor of two for substitutions outside the heme cavity. Azide binding may be affected by a substitution at residue 66 in the E helix, a site suggested by Case & Karplus (1979) J. molec. Biol. 132, 343-368, to be on a reactive path to the heme. 3. The oxygen and CO data show that substitutions outside the heme cavity can affect rate constants by at least a factor of 1.5. 4. The oxygen equilibrium constant was correlated with the metabolite rate of the corresponding species, in accord with the Wyman (1966) J. biol. Chem. 241, 115-121, model for facilitated diffusion of oxygen.

Animals↗
PubMed1995

Membrane transport of nucleobases: interaction with inhibitors.

1. The kinetic properties and the mechanism of nucleobase transport and transport inhibition are briefly reviewed. 2. Many purine derivatives even when bearing large substituents on N9 and C6 are inhibitors of nucleobase transport, some are also substrates. 3. Papaverine and other benzyl-isoquinolines are efficient inhibitors of facilitated transport of nucleobases. 4. Papaverine is a noncompetitive inhibitor of nucleobase transport in human erythrocytes. 5. Reduction of the aromatic isoquinoline to the tetrahydro form causes loss of inhibitory activity whereas replacement of methoxy groups by ethoxy groups leads to increased activity. 6. Papaverine also inhibits sodium dependent active nucleobase transport in pig kidney cells. 7. The nucleoside transport inhibitors dipyridamole and dilazep have no effect on facilitated diffusion transport of nucleobases, but inhibit in micromolar concentrations active sodium dependent nucleobase transport in pig kidney cells.

Animals↗
PubMed1987

Morphological basis for nonsynaptic communication within the central nervous system by exocytotic release of secretory material from the egg-laying stimulating neuroendocrine caudodorsal cells of Lymnaea stagnalis.

The fine structure of the axons of the cerebral, egg-laying stimulating caudodorsal cells of the snail Lymnaea stagnalis has been studied with various light and electron microscope techniques. Special attention was paid to exocytotic release of secretory material (demonstrated with the tanic acid method) from nonsynaptic release sites in the cerebral commissure. This phenomenon has been compared with neurohaemal release. The commissure consists of two morphological compartments, separated by a sheath of glial cells. The outer compartment is formed by the neurohaemal area of the caudodorsal cells, the inner consists of thousands of, mainly unidentified, axons. Furthermore, ventral caudodorsal cells send axons through the inner compartment. These give rise to collaterals, which divide into smaller collaterals, forming an extensive network ("collateral system") throughout the inner compartment. Eventually, collaterals end blindly within the inner compartment. They contain the same three morphological types of secretory granule as the neurohaemal axon terminals. The collaterals never form synaptic contacts; exocytotic release of the contents of secretory granules takes place at nonsynaptic release sites. These sites occur rather dispersed and do not face one particular type of neighbouring neural element. As in the neurohaemal area, both single and multiple exocytoses occur. Widened intercellular spaces, filled with flocculent, electron-dense material, occur near highly active nonsynaptic release sites. The spaces are often bordered by glial cells and may facilitate diffusion of released secretory material through the inner compartment. Apparently, a ventral caudodorsal cell releases secretory material in two fashions: from neurohaemal axon terminals into the haemolymph, and nonsynaptically, from the collaterals into the intercellular space of the central nervous system. Possible functions of the glial sheath between the neurohaemal area and the inner compartment are proposed. Most likely, the collateral system enables the caudodorsal cells to communicate with targets within the central nervous system in a nonsynaptic fashion. A possible target is the cerebral Ring Neuron, which sends an axon branch through the inner compartment and, as was previously shown neurophysiologically, is controlled by the caudodorsal cells in a nonsynaptic fashion.

Animals↗
PubMed1987

Adenosine 5'-triphosphate synthesis and metabolism localized in neurites of cultured sympathetic neurons.

Adenosine triphosphate synthesis and metabolism in cultured sympathetic neurons was studied after the incorporation of [2-3H]adenine into intact or microdissected neurites to determine whether ATP is provided locally during neurite outgrowth, when and where it is synthesized and how its levels are regulated at rest and following depolarization. Neurites maintained an independent capability for synthesis of ATP at any stage of growth: [3H]ATP levels increased in neurites in direct proportion to neurite length and equivalent amounts of [3H]ATP were synthesized by intact neurites, by neurites separated from cell bodies or by neurites further segmented into sections. Thus, metabolic labelling of cultured neurons with [3H]adenine provides a simple method to measure relative neurite outgrowth. Neurite ATP was maintained mainly by respiration but also by glycolysis and [3H]ATP levels were stable for at least 14 h after adenine withdrawal when cells were at rest. Depolarization overcame respiratory control, causing a quantitative conversion of ATP to adenosine monophosphate (AMP) and inosine monophosphate (IMP) and the release of nucleosides (adenosine and inosine) and nucleotides [adenosine diphosphate (ADP) and adenosine monophosphate (AMP)]. Release of nucleosides, but not of nucleotides or [3H]noradrenaline, was enhanced by NaN3 or 2-deoxyglucose under nondepolarizing conditions and was prevented by the adenosine transport inhibitor p-nitrobenzyl-6-thioinosine. It is concluded that neurites can use local mechanisms for ATP synthesis that do not depend on a functional connection to the cell body. Any metabolic stress which causes ATP breakdown causes these cells to express a transient purinergic phenotype involving release of adenosine and inosine by facilitated diffusion. To promote the release of purine nucleotides, however, more specific stimuli are required.

Adenine↗
PubMed1993

Sugar transport in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae consumes mono- and disaccharides preferentially to any other carbon source. Since sugars do not freely permeate biological membranes, cellular uptake of these compounds requires the action of 'transporters'. The purpose of this review is to summarize the present knowledge on sugar transport in this organism. Yeast cells show two transporters for monosaccharides, the so-called glucose and galactose transporters that act by a facilitated diffusion mechanism. In the case of glucose transport, which also acts upon D-fructose and D-mannose, two components with high- and low-affinity constants have been identified kinetically. Activity of the high-affinity component is dependent on the presence of active kinases whereas activity of the low-affinity component is independent of the presence of these enzymes. Three genes, SNF3, HXT1 and HXT2, encode three different glucose transporters with a high affinity for the substrates and are repressed by high concentrations of glucose in the medium. Kinetic studies suggest that at least one additional gene exists that encodes a transporter with a low affinity and is expressed constitutively. The present view is that there are several additional transporters for glucose that have not yet been identified. Galactose transport has only one natural substrate, D-galactose, and is encoded by the gene GAL2. Expression of this gene is induced by galactose and repressed by glucose. Two transporters for disaccharides have been identified in S. cerevisiae: maltose and alpha-methylglucoside transporters. These transporters are H(+)-symports that depend on the electrochemical proton gradient and are independent of the ATP level. The gene that encodes the maltose transporter is clustered with the other two genes required for maltose utilization in a locus that is found repeated at different chromosomal locations. Its expression is induced by maltose and repressed by glucose. The rate of sugar uptake in yeast cells is controlled by changes in affinity of the corresponding transporters as well as by an irreversible inactivation that affects their Vmax. The mechanisms involved in these regulatory processes are unknown at present.

Biological Transport↗
PubMed1995

Localization of damage induced by reactive oxygen species in cultured cells.

N18-RE-105 neuron-derived hybridoma cells were employed to determine the location and degree of damage induced by each of three reactive oxygen species (ROS) generators: 6-hydroxydopamine (6-OHDA), H2O2, and cumene hydroperoxide. Two readily distinguishable plasma membrane markers were used to assess cell surface damage, namely the active transport of alpha-aminoisobutyric acid (AIB) and the facilitated diffusion of glucose. In addition, staining of mitochondria with a tetrazolium dye, 3[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), was used as an intracellular marker to measure the integrity of the metabolic function of the mitochondria. The dose-response curve of inactivation of transport or of metabolic function varied with the ROS generator used and conformed to one of two patterns of toxicity: either threshold-dependent or single-hit inactivation. We determined that 6-OHDA acts simultaneously on multiple targets and steps in the cells, resulting in a very steep dose-effect curve. Similarly, damage induced by H2O2 to the AIB transporters and to mitochondria is consistent with simultaneous inactivation of multiple steps, but damage to glucose transporters conforms to single-hit inactivation of the transporter. Conversely, treatment with cumene hydroperoxide resulted in single-hit inactivation of the AIB transporter, but inactivation of the glucose transporter conformed to threshold-dependent inactivation. Thus, to evaluate quantitatively damage produced by ROS at the subcellular level, both the type of toxic agent and the target to be evaluated must be considered. Finally, the inactivation of each of the targets observed in this study for all of the ROS generators used conform to one of two simple inactivation models. Fitting the appropriate model to the data allows precise quantitative analysis of the inactivation process and provides insight into the chemistry of the inactivation process.

Aminoisobutyric Acids↗
PubMed2004

Cadmium, zinc and the uptake of calcium by two crabs, Carcinus maenas and Eriocheir sinensis.

The uptake of dissolved cadmium and zinc by crustaceans can usually be explained by the passive process of facilitated diffusion involving a transport protein in the membranes of permeable surfaces. Cadmium ions will also enter via uptake routes for calcium, given the similar size of the two free ions. This study has investigated the interaction of cadmium (and comparatively zinc) and calcium uptake in two crabs that show different permeability responses to changes in salinity, with consequently different effects on the uptake of cadmium and zinc with salinity change. Ca uptake rates in Carcinus maenas decreased in reduced salinity (33-15) with the decreased Ca concentration of the medium and increased if the Ca concentration was increased at salinity 20. It is concluded that Ca uptake over the salinity range 33-15 is via apical Ca channels in gill ionocytes, passively down an electrochemical gradient. The Ca uptake rate of Eriocheir sinensis showed no significant decrease over the salinity range 33-10 (probably because of the small differences in an already low Ca uptake rate in this crab against a background of inter-individual variability), but decreased significantly at salinity 5. Added calcium increased the Ca uptake rate of E. sinensis at salinities 15 and 5, supporting the interpretation that Ca uptake in gills is typically passive via apical Ca channels. Cadmium (but not zinc) inhibited calcium uptake in both crabs at 15 salinity, indicating sharing of Ca channels by Cd, but not at salinity 5 (E. sinensis only) when Ca may be taken up into gill ionocytes by another (active?) physiological process.

Analysis of Variance↗
PubMed2006

Different accumulation of cisplatin, oxaliplatin and JM216 in sensitive and cisplatin-resistant human cervical tumour cells.

The significance of reduced drug accumulation in resistance to cisplatin was investigated by using cisplatin, oxaliplatin and JM216 (hydrophobicity rank: JM216>oxaliplatin>cisplatin) in human squamous cell carcinoma cell line A431 and its cisplatin-resistant counterpart A431/Pt. While cisplatin showed a resistance factor of 2.6, oxaliplatin and JM216 circumvented the resistance. Platinum accumulation after cisplatin exposure was lower (2.4-fold) in A431/Pt than in A431 cells, whereas a similar accumulation was found in the two cell lines when oxaliplatin or JM216 were used, thereby suggesting the capability of the latter drugs to bypass the accumulation defect. In the A431 cell line platinum accumulated to a similar extent after exposure to cisplatin, oxaliplatin or JM216, while in A431/Pt cells, Platinum accumulation depended on the hydrophobicity of the drug, and an increased hydrophobicity favours the uptake. No difference in efflux of cisplatin was found between the two cell lines. The values of platinum-DNA binding in A431 cells were similar for cisplatin and JM216 and higher than those of oxaliplatin. In A431/Pt cells: (i) Pt-DNA binding levels of JM216 remained as in sensitive ones; (ii) Pt-DNA levels of cisplatin and oxaliplatin were very similar and nearly two-fold lower than those of JM216. Such results, in this cell system characterized by a low level of cisplatin resistance, support a model whereby platinum uptake occurs by a mechanism of facilitated diffusion, perhaps involving a gated channel, which can be lost during the selection of the drug-resistant variant(s). The hydrophobicity of the drug can be the key to bypass resistance.

Antineoplastic Agents↗
PubMed2004

5-HT3A receptor subunits in the rat medial nucleus of the solitary tract: subcellular distribution and relation to the serotonin transporter.

The 5-hydroxytryptamine 3 (5HT3) receptor is a serotonin-gated ion channel implicated in reflex regulation of autonomic functions within the nucleus of the solitary tract (NTS). To determine the relevant sites for 5-HT3 receptor mediated transmission in this region, we used electron microscopic immunocytochemistry to examine the subcellular distribution of the 5HT3 receptor subunit A (5HT3A) in relation to the serotonin transporter (SERT) in the intermediate medial NTS (mNTS) of rat brain. The 5HT3A immunolabeling was detected in many axonal as well as somatodendritic and glial profiles. The axonal profiles included small axons and axon terminals in which the 5HT3A immunoreactivity was localized to membranes of synaptic vesicles and extrasynaptic plasma membranes. In dendrites and glia, the 5HT3A immunoreactivity was located on the plasma membranes or in association with membranous cytoplasmic organelles. The dendritic plasmalemmal 5HT3A labeling was prominent within and near excitatory-type synapses from terminals including those that resemble vagal afferents. The 5HT3A-labeled glial processes apposed 5HT3A-immunoreactive axonal and dendritic profiles, some of which also contained SERT. Terminals containing 5-HT3A and/or SERT were among those providing synaptic input to 5HT3A-labeled dendrites. Thus, 5HT3A has a subcellular distribution consistent with the involvement of 5-HT3 receptors in modulation of both presynaptic release and postsynaptic responses of mNTS neurons, some of which are serotonergic. The results further suggest that the neuronal as well as glial 5HT3 receptors can be activated by release of serotonin from presynaptic terminals or by diffusion facilitated by SERT distribution at a distant from the synapse.

Animals↗
PubMed2004

Role of hsp90 and the hsp90-binding immunophilins in signalling protein movement.

The ubiquitous protein chaperone hsp90 has been shown to regulate more than 100 proteins involved in cellular signalling. These proteins are called 'client proteins' for hsp90, and a multiprotein hsp90/hsp70-based chaperone machinery forms client protein.hsp90 heterocomplexes in the cytoplasm and the nucleus. In the case of signalling proteins that act as transcription factors, the client protein.hsp90 complexes also contain one of several TPR domain immunophilins or immunophilin homologs that bind to a TPR domain binding site on hsp90. Using several intracellular receptors and the tumor suppressor p53 as examples, we review evidence that dynamic assembly of heterocomplexes with hsp90 is required for rapid movement through the cytoplasm to the nucleus along microtubular tracks. The role of the immunophilin in this system is to connect the client protein.hsp90 complex to cytoplasmic dynein, the motor protein for retrograde movement toward the nucleus. Upon arrival at the nuclear pores, the receptor.hsp90.immunophilin complexes are transferred to the nuclear interior by importin-dependent facilitated diffusion. The unliganded receptors then distribute within the nucleus to diffuse patches from which they proceed in a ligand-dependent manner to discrete nuclear foci where chromatin binding occurs. We review evidence that dynamic assembly of heterocomplexes with hsp90 is required for movement to these foci and for the dynamic exchange of transcription factors between chromatin and the nucleoplasm.

Animals↗
PubMed2005

Sustaining nurse-managed practice.

Community-based nurse-managed practice (CBNMP) brings primary health care to local, typically vulnerable, populations. Despite cost-effective, high quality care, a 21% decline in academic CBNMPs was documented in the 1990s. A multiple case study addressed factors that hindered or facilitated diffusion and sustainability of CBNMPs in prevalent practice settings. To promote sustainable practice, CBNMPs should articulate a practice mission, identify the practice as nursing, create a team approach, balance mission with margin, and promote attractive sites. Other recommendations include planning for growth and evolution of health care and applying for awards to increase the visibility and political clout of CBNMPs.

Community Health Centers↗
PubMed2003

Nucleoside transporters in the disposition and targeting of nucleoside analogs in the kidney.

Systemic disposition of nucleosides and nucleoside analogs is dependent on renal handling of these compounds. There are five known, functionally characterized nucleoside transporters with varying substrate specificities for nucleosides: concentrative nucleoside transporters (CNT1-CNT3; Solute Carrier (SLC) 28A1-28A3), which mediate the intracellular flux of nucleosides, and equilibrative nucleoside transporters (ENT1-ENT2; SLC29A1-SLC29A2), which mediate bi-directional facilitated diffusion of nucleosides. All five of these transporters are expressed in the kidney. Concentrative nucleoside transporters primarily localize to the apical membrane of renal epithelial cells while equilibrative nucleoside transporters primarily localize to the basolateral membrane. These transporters work in concert to mediate reabsorptive flux of naturally occurring nucleosides and nucleoside analogs. In addition, equilibrative transporters also participate in secretory flux of some nucleoside analogs. Nucleoside transporters also serve in the targeting of nucleoside analog therapies to renal tumors. This review examines the role that these transporters play in renal disposition of nucleosides and nucleoside analogs in both systemic and kidney-specific therapies.

Animals↗
PubMed2006

Bidirectional membrane transport: simulations of transport inhibition in uptake studies explain data obtained with flavonoids.

The purpose of the simulations was to obtain an estimate of concentration-dependent uptake curves when two counteracting transporters are present. On the basis of this experimental data obtained with a pair of ovarian carcinoma cell lines, one of which was not expressing the exsorptive transporter P-glycoprotein and one of which was an MDR1-transfected, P-glycoprotein expressing variant, the kinetics of cellular uptake of the radiolabel (3)H-talinolol were calculated and the inhibitory constants at P-gp were determined for different flavonoids. With respect to the inhibition of P-gp function, among others, naringenin and isoquercitrin were identified as inhibitors, yet estimation of the inhibitory constant was only possible for uptake values corrected for non-P-glycoprotein-mediated processes. It was assumed that an additional inside-directed transporting protein (Carrier B), which is inhibited by the presence of test compounds, uptake of radiolabel was simulated as a function of the concentration of test-compound, with exemplary parameters for the rate constant (k(B)) of the additional Carrier B and the inhibition constants (K(I)-values) for both transporting proteins. The obtained uncorrected experimental data, which showed either inhibition or enhancement of radiolabel uptake as a function of the inhibitor concentration, were appropriately explained by the respective model. The respective model included an exsorptive transporter as well as carrier-mediating facilitated diffusion. It is concluded that flavonoids, such as naringenin and isoquercitrin, inhibit an inside-directed process in addition to their inhibition of P-glycoprotein-mediated exsorption.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Arabidopsis thaliana MTP1 is a Zn transporter in the vacuolar membrane which mediates Zn detoxification and drives leaf Zn accumulation.

The Arabidopsis thaliana metal tolerance protein 1 (MTP1) of the cation diffusion facilitator family of membrane transport proteins can mediate the detoxification of Zn in Arabidopsis and yeast. Xenopus laevis oocytes expressing AtMTP1 accumulate more Zn than oocytes expressing the AtMTP1(D94A) mutant or water-injected oocytes. An AtMTP1-GFP fusion protein localizes to the vacuolar membrane in root and leaf cells. The analysis of Arabidopsis transformed with a promoter-GUS construct suggests that AtMTP1 is not produced throughout the plant, but primarily in the subpopulation of dividing, differentiating and expanding cells. RNA interference-mediated silencing of AtMTP1 causes Zn hypersensitivity and a reduction in Zn concentrations in vegetative plant tissues.

Arabidopsis↗
PubMed2004

Membrane separations using molecularly imprinted polymers.

This review presents an overview on the promising field of molecularly imprinted membranes (MIM). The focus is onto the separation of molecules in liquid mixtures via membrane transport selectivity. First, the status of synthetic membranes and membrane separation technology is briefly summarized, emphasizing the need for novel membranes with higher selectivities. Innovative principles for the preparation of membranes with improved or novel functionality include self-assembly or supramolecular aggregation as well as the use of templates. Based on a detailed analysis of the literature, the main established preparation methods for MIM are outlined: simultaneous membrane formation and imprinting, or preparation of imprinted composite membranes. Then, the separation capability of MIM is discussed for two different types, as a function of their barrier structure. Microporous MIM can continuously separate mixtures based on facilitated diffusion of the template, or they can change their permeability in the presence of the template ("gate effect"). Macroporous MIM can be developed towards molecule-specific membrane adsorbers. Emerging further combinations of molecularly imprinted polymers (MIPs), especially MIP nanoparticles or microgels, with membranes and membrane processes are briefly outlined as well. Finally, the application potential for advanced MIM separation technologies is summarized.

Membranes, Artificial↗
PubMed2006

Proteasomes and their associated ATPases: a destructive combination.

Protein degradation by 20S proteasomes in vivo requires ATP hydrolysis by associated hexameric AAA ATPase complexes such as PAN in archaea and the homologous ATPases in the eukaryotic 26S proteasome. This review discusses recent insights into their multistep mechanisms and the roles of ATP. We have focused on the PAN complex, which offers many advantages for mechanistic and structural studies over the more complex 26S proteasome. By single-particle EM, PAN resembles a "top-hat" capping the ends of the 20S proteasome and resembles densities in the base of the 19S regulatory complex. The binding of ATP promotes formation of the PAN-20S complex, which induces opening of a gate for substrate entry into the 20S. PAN's C-termini, containing a conserved motif, docks into pockets in the 20S's alpha ring and causes gate opening. Surprisingly, once substrates are unfolded, their translocation into the 20S requires ATP-binding but not hydrolysis and can occur by facilitated diffusion through the ATPase in its ATP-bound form. ATP therefore serves multiple functions in proteolysis and the only step that absolutely requires ATP hydrolysis is the unfolding of globular proteins. The 26S proteasome appears to function by similar mechanisms.

Adenosine Triphosphatases↗
PubMed2005

Functional and pharmacological mechanisms of nucleoside transport across the basolateral membrane of rabbit tracheal epithelial cells.

The role of basolateral membrane nucleoside transport in primary cultured rabbit tracheal epithelial cells (RTEC) was studied. Primary cultured RTEC were grown on permeable support at an air-interface. Transport studies were conducted in the uptake, efflux, and transepithelial transport configurations using (3)H-uridine as a model substrate. Time, temperature and concentration dependency of (3)H-uridine transport were evaluated in parallel to the metabolism of this substrate using scintillation counting and thin layer chromatography. Inhibition of (3)H-uridine uptake from basolateral fluid was estimated in presence of all unlabeled natural nucleosides as well as analogs and nucleobases. Functional modulation pathways of (3)H-uridine uptake were studied after treatment of RTEC with pharmacological levels of A23187, forskolin, tamoxifen, H89 and colchicine. The basolateral aspect has a low-affinity and high-capacity transport system that exhibits characteristics of bi-directionality, temperature/concentration dependency, and broad specificity towards purines and pyrimidines without requiring Na(+). Basolateral equilibrative-sensitive/insensitive (es/ei) type transport machinery manifested as a biphasic dose response to nitro-benzyl-mercapto-purine-ribose (NBMPR) inhibition. In addition, a number of therapeutically relevant nucleoside analogs appeared to compete with the uptake of uridine from basolateral fluid. Short-term pre-incubation of primary cultured RTEC with the calcium ionophore A23187 inhibited basolateral uridine uptake without affecting the J(max) and K(m). The inhibitory effect was not reversible with a protein kinase C (PKC) antagonist, tamoxifen. In contrast, basolateral uridine uptake was increased by adenylyl cyclase activator forskolin (reversible with protein kinase A (PKA) inhibitor H89), resulting in a decreased K(m), but a lower J(max). Uridine exit across the basolateral membrane of primary cultured RTEC occurs via a facilitative diffusion carrier, which can be modulated by intracellular Ca(2+) levels and PKA. Information about these carriers will help improve the transportability of antitumor and antiviral nucleoside analogs in the pulmonary setting.

Animals↗
PubMed2005

Anandamide transport: a critical review.

Anandamide (AEA) uptake has been described over the last decade to occur by facilitated diffusion, but a protein has yet to be isolated. In some cell types, it has recently been suggested that AEA, an uncharged hydrophobic molecule, passively diffuses through the plasma membrane in a process that is not protein-mediated. Since that observation, recent kinetics studies (using varying assay conditions) have both supported and denied the presence of an AEA transporter. In this review, we analyze the current literature exploring the mechanism of AEA uptake and endeavor to explain the reasons for the divergent views. One of the main variables among laboratories is the incubation time of the cells with AEA. Initial kinetics (at time points <1 min depending upon the cell type) isolate events that occur at the plasma membrane and are most useful to study saturability of uptake and effects of purported transport inhibitors upon uptake. Results with longer incubation times reflect events not only at the plasma membrane but also interactions at intracellular sites that may include enzyme(s), other proteins, or specialized lipid-binding domains. Furthermore, at long incubation times, antagonists to AEA receptors reduce AEA uptake. Another complicating factor in AEA transport studies is the nonspecific binding to plastic culture dishes. The magnitude of this effect may exceed AEA uptake into cells. Likewise, AEA may be released from plastic culture dishes (without cells) in such a manner as to mimic efflux from cells. AEA transport protocols using BSA, similar to the method used for fatty acid uptake studies, are gaining acceptance. This may improve AEA solution stability and minimize binding to plastic, although some groups report that BSA interferes with uptake. In response to criticisms that many transport inhibitors also inhibit the fatty acid amide hydrolase (FAAH), new compounds have recently been synthesized. Following their characterization in FAAH+/+ and FAAH-/- cells and transgenic mice, several inhibitors have been shown to have physiological activity in FAAH-/- mice. Their targets are now being characterized with the possibility that a protein transporter for AEA may be characterized.

Amidohydrolases↗