A model of insulin distribution and uptake in the perfused rat liver

Indicator dilution curves of 51Cr-EDTA and 125I-insulin injected into perfusate entering the rat liver in vivo are used as a basis for developing a mathematical model of insulin distribution and uptake within the organ. EDTA is not taken up by liver cells and therefore serves as a "volume marker" whose dilution curve reflects the characteristics of perfusate flow through the organ and through the cannulae. These two components are modelled separately but the same approach is used in each case, that is, the minimum number of delayed exponential terms sufficient to reproduce the dilution curves is determined and used as the basis for modelling. This allows the cannula, vascular and sinusoidal volumes to be identified and described as corresponding compartmental configurations. The shape of the insulin dilution curve is additionally influenced by binding to, and uptake by, liver cells. Binding of insulin to receptors on hepatocyte plasma membranes and subsequent internalization of the insulin-receptor complex is modelled by the introduction of additional compartments but this is found to be insufficient unless non-specific binding is also taken into account. The accuracy of determination of the rate constants for insulin-receptor dissociation and for endocytosis is improved by a sudden reduction in the pH of the perfusate about 100 sec after injection of the insulin bolus. This releases any residual receptor bound insulin and is modelled by a sudden shift in the insulin-receptor dissociation rate constant. Matching of the complete model to individual pairs of 51Cr-EDTA and 125I-insulin dilution curves allows vascular and sinusoidal volumes to be determined together with the binding and endocytic rate constants. Use of the model to investigate the effect of substances that modify these rate constants is briefly illustrated in the case where the liver is preperfused with 5 mM indomethacin. The model can also be used to simulate the internal distribution and uptake of insulin with any nominated input function and any set of parameters; this is illustrated by comparing the impulse response in the normal case and that in which indomethacin has been preperfused. Although the present study is confined to insulin, the model and methodology that is developed should be applicable to other ligands for which the hepatocyte carries specific receptors.     

Diffusion through a membrane: Approach to equilibrium

The transfer of solute through a membrane separating two aqueous solutions is studied with the time-dependent diffusion equation for composite media. By introducing new independent and dependent variables it is shown that the differential equations and boundary conditions can be transformed into a dimensionless form which does not explicitly depend on the diffusivities of the media. Laplace transforms are used to derive explicit solutions for the solute concentration as a function of position and time. It is shown that at large time the concentration approaches the equilibrium distribution exponentially. Explicit results are given for the decay time as a function of the parameters of the system. In addition, an accurate and simplified expression is derived for the decay time for the case of small membrane permeability. The accuracy of the analytic solutions for the concentration profiles is tested by comparing them with numerical results obtained by solving the diffusion equations by the method of finite differences. Excellent agreement is found. Research supported in part by a grant from the National Science Foundation.     

Effects of membrane potential and surface potential on the kinetics of solute transport

A theoretical study has been made of the influence of the transmembrane potential difference and the surface potential of living cells on the kinetics of carried-mediated solute transport. It is assumed that the form of the free energy barrier within the membrane may be approximated by one dominant symmetrical peak, and that the electrical field is constant. Both single-ion transport kinetics and cotransport of an ion with a neutral solute are dealt with. Provided that the surface potential and the transmembrane potential are constant, the concentration dependence of the uptake rate is given by the Michaelis-Menten equation. The kinetic parameters, the maximal rate of uptake and the K_m, depend on both the surface potential and the membrane potential in a rather complex way. It is shown that the intuitive notion, that the maximal rate of cation uptake will increase when the cell membrane is hyperpolarized, is wrong in its generallity. Both an increase or a decrease may occur, depending on the characteristics of the transport system involved. If the magnitude of the membrane potential and the surface potential depends on the substrate concentration, marked deviations from Michaelis-Menten kinetics may come to the fore. This may result in either apparent positive or apparent negative homotrope cooperative effects. Enhancement of the uptake rate of the substrate ion may occur on adding another cation, despite the fact that the membrane will become depolarized. The same type of complex transport kinetics as found for Rb⁺ and Na⁺ uptake in yeast cells can be simulated by using a single-site transport model and including effects of the membrane potential.     

Kinetics and stoichiometry of light-induced proton release and uptake from purple membrane fragments, Halobacterium halobium cell envelopes, and phospholipid vesicles containing oriented purple membrane

We have used flash spectroscopy and pH indicator dyes to measure the kinetics and stoichiometry of light-induced proton release and uptake by purple membrane in aqueous suspension, in cell envelope vesicles and in lipid vesicles. The preferential orientation of bacteriorhodopsin in opposite directions in the envelope and lipid vesicles allows us to show that uptake of protons occurs on the cytoplasmic side of the purple membrane and release on the exterior side.

In suspensions of isolated purple membrane, approximately one proton per cycling bacteriorhodopsin molecule appears transiently in the aqueous phase with a half-rise time of 0.8 ms and a half-decay time of 5.4 ms at 21 °C.

In cell envelope preparations which consist of vesicles with a preferential orientation of purple membrane, as in whole cells, and which pump protons out, the acidification of the medium has a half-rise time of less than 1.0 ms, which partially relaxes in approx. 10 ms and fully relaxes after many seconds.

Phospholipid vesicles, which contain bacteriorhodopsin preferentially oriented in the opposite direction and pump protons in, show an alkalinization of the medium with a time constant of approximately 10 ms, preceded by a much smaller and faster acidification. The alkalinization relaxes over many seconds.

The initial fast acidification in the lipid vesicles and the fast relaxation in the envelope vesicles are accounted for by the misoriented fractions of bacteriorhodopsin. The time constants of the main effects, acidification in the envelopes and alkalinization in the lipid vesicles correlate with the time constants for the release and uptake of protons in the isolated purple membrane, and therefore show that these must occur on the outer and inner surface respectively. The slow relaxation processes in the time range of several seconds must be attributed to the passive back diffusion of protons through the vesicle membrane.     

Effect of pH and monovalent cations on the ionization state of phosphatidylglycerol in monolayers. An experimental (surface potential) and theoretical (Gouy-Chapman) approach

Ionization of the acidic phospholipid phosphatidylglycerol has been studied by measuring the surface potential of monomolecular films of the lipid as a function of the aqueous subphase pH and the concentration of monovalent cations (Li, Na, Cs). It is shown that the experimental data can be interpreted by means of the Gouy-Chapman theory in its simplest formulation, provided an adsorption of cations at the membrane surface is accounted for. This allows us to predict the ionization state of the lipid for given ionic conditions in the subphase. Above pH 4, for subphase ion concentration higher than 10 mM, or for ion concentrations above 0.1 mM at pH 5.6, phosphatidylglycerol is fully deprotonated. Within the limits of our theoretical approach, association constants of the cations to the lipid lie around 0.1-0.6 M-1.     

Unbiased examination of changes in plasma membrane proteins in drug resistant cancer cells

In this study, an unbiased examination is made of the abundance changes between proteins found in the basolateral plasma membranes of a drug susceptible parental MCF-7 breast cancer cell line and a cell line selected from the parent line for resistance to the anticancer drug mitoxantrone. Plasma membrane proteins were differentially labeled metabolically, enriched using the colloidal silica pellicle method, and characterized by tandem mass spectrometry. Fifteen proteins were identified with significant (>2) changes, including receptors, adhesion proteins, proteins involved in amino acid uptake, and proteins involved in glucose uptake. From 40 mug of membrane proteins, 3227 unique peptides and 540 proteins were identified.     

Influence of sickle hemoglobin polymerization and membrane properties on deformability of sickle erythrocytes in the microcirculation.

The rheological properties of normal erythrocytes appear to be largely determined by those of the red cell membrane. In sickle cell disease, the intracellular polymerization of sickle hemoglobin upon deoxygenation leads to a marked increase in intracellular viscosity and elastic stiffness as well as having indirect effects on the cell membrane. To estimate the components of abnormal cell rheology due to the polymerization process and that due to the membrane abnormalities, we have developed a simple mathematical model of whole cell deformability in narrow vessels. This model uses hydrodynamic lubrication theory to describe the pulsatile flow in the gap between a cell and the vessel wall. The interior of the cell is modeled as a Voigt viscoelastic solid with parameters for the viscous and elastic moduli, while the membrane is assigned an elastic shear modulus. In response to an oscillatory fluid shear stress, the cell--modeled as a cylinder of constant volume and surface area--undergoes a conical deformation which may be calculated. We use published values of normal and sickle cell membrane elastic modulus and of sickle hemoglobin viscous and elastic moduli as a function of oxygen saturation, to estimate normalized tip displacement, d/ho, and relative hydrodynamic resistance, Rr, as a function of polymer fraction of hemoglobin for sickle erythrocytes. These results show the transition from membrane to internal polymer dominance of deformability as oxygen saturation is lowered. More detailed experimental data, including those at other oscillatory frequencies and for cells with higher concentrations of hemoglobin S, are needed to apply fully this approach to understanding the deformability of sickle erythrocytes in the microcirculation. The model should be useful for reconciling the vast and disparate sets of data available on the abnormal properties of sickle cell hemoglobin and sickle erythrocyte membranes, the two main factors that lead to pathology in patients with this disease.     

Reconstitution of respiratory oxidases in membrane nanodiscs for investigation of proton-coupled electron transfer

The function of membrane-bound transporters is commonly affected by the milieu of the hydrophobic, membrane-spanning part of the transmembrane protein. Consequently, functional studies of these proteins often involve incorporation into a native-like bilayer where the lipid components of the membrane can be controlled. The classical approach is to reconstitute the purified protein into liposomes. Even though the use of such liposomes is essential for studies of transmembrane transport processes in general, functional studies of the transporters themselves in liposomes suffer from several disadvantages. For example, transmembrane proteins can adopt two different orientations when reconstituted into liposomes, and one of these populations may be inaccessible to ligands, to changes in pH or ion concentration in the external solution. Furthermore, optical studies of proteins reconstituted in liposomes suffer from significant light scattering, which diminishes the signal-to-noise value of the measurements. One attractive approach to circumvent these problems is to use nanodiscs, which are phospholipid bilayers encircled by a stabilizing amphipathic helical membrane scaffold protein. These membrane nanodiscs are stable, soluble in aqueous solution without detergent and do not scatter light significantly. In the present study, we have developed a protocol for reconstitution of the aa(3)- and ba(3)-type cytochrome c oxidases into nanodiscs. Furthermore, we studied proton-coupled electron-transfer reactions in these enzymes with microsecond time resolution. The data show that the nanodisc membrane environment accelerates proton uptake in both oxidases.     

Targeting of liposomes carrying recombinant fragments of platelet membrane glycoprotein Ibalpha to immobilized von Willebrand factor under flow conditions

Liposomes with covalently bound recombinant fragments of platelet membrane glycoprotein Ibalpha that retain the von Willebrand factor (vWf)-binding function (rGPIbalpha-liposomes) were prepared. Their interactions with an immobilized vWf surface under flow conditions were evaluated with a recirculating flow chamber, mounted on an epifluorescence microscope, which allows real-time visualization of fluorescence-labeled liposomes interacting with the surface. The interaction of rGPIbalpha-liposomes with the vWf surface was directly related to shear rate. At high densities of rGPIbalpha and vWf, rGPIbalpha-liposomes establishing contact with the vWf surface exhibited continuous displacement with decreased velocity relative to the hydrodynamic flow, depending on receptor density and matrix concentration. At lower densities of rGPIbalpha and vWf, rGPIbalpha-liposomes stopped only transiently, in the millisecond range, on the surface. This is the first study to demonstrate that the targeting of rGPIbalpha-liposomes is specific to the vWf surface under flow conditions.     

Active transport membrane concentration amplifiers

Three different active transport membrane configurations are suggested for achieving concentration amplification in a circulating substrate solution. The steady-state characteristics of the structures are investigated on the basis of a linear approximation of the rate of active transport. An analysis of the effects of system parameters and geometry on the concentration gradient along the flow path is presented. It is seen that the concentration gradient may be synthesized by appropriate choice of membrane arrangement, flow and physico-chemical transport parameters.     

Semi-automated analysis of organelle movement and membrane content: understanding rab-motor complex transport function

Organelle motility is an essential cellular function that is regulated by molecular motors, and their adaptors and activators. Here we established a new method that allows more direct investigation of the function of these peripheral membrane proteins in organelle motility than is possible by analysis of the organelle movement alone. This method uses multi-channel time-lapse microscopy to record the movement of organelles and associated fluorescent proteins, and automatic organelle tracking, to compare organelle movement parameters with the association of membrane proteins. This approach allowed large-scale, unbiased analysis of the contribution of organelle-associated proteins and cytoskeleton tracks in motility. Using this strategy, we addressed the role of membrane recruitment of Rab GTPases and effectors in organelle dynamics, using the melanosome as a model. We found that Rab27a and Rab32/38 were mainly recruited to sub-populations of slow-moving/static and fast-moving melanosomes, respectively. The correlation of Rab27a recruitment with slow movement/docking was dependent on the effector melanophilin. Meanwhile, using cytoskeleton-disrupting drugs, we observed that this speed:Rab content relationship corresponded to a decreased frequency of microtubule (MT)-based transport and an increased frequency of actin-dependent slow movement/docking. Overall, our data indicate the ability of Rab27a and effector recruitment to switch melanosomes from MT- to actin-based tethering and suggest that a network of Rab signalling may integrate melanosome biogenesis and transport.     

Particulate enhanced membrane uptake of 1,2-benzanthracene observed by fluorescence spectroscopy: A possible role in co-carcinogenesis

In recognition of the co-carcinogenic effect of particulate matter and chemical carcinogens, we investigated the effect of particulate silica on the rates of membrane uptake of 1,2-benzanthracene. The fluorescence emission spectra and the apparent quantum yields of benzathracene are dependent upon adsorption to silica and upon the surface density of benzathracene on the silica. The fluorescence spectral shifts which occur upon transfer of benzathracene from silica surface to phospholipid vesicles provided a convenient means to quantitate the membrane uptake of benzanthracene from particulates.The rate of benzathracene uptake by dipalmitoyl-L-α-phosphatidylcholine vesicles was independent of the concentration of lipid, indicating that the rate-limiting step may involve its sulubilization in the aqueous phase. These uptake rates were also independent of the surface density of benzanthracene on the silica, indicating that the benzanthracene molecules are dispersed uniformly on the silica surface.Rates of membrane uptake of benzanthracene from the crystalline, microcrystalline, and the silica-absorbed states were compared, and are greatly enhanced by a reduction in crystal size. Silica-adsorbed benzanthracene had the most rapid rate of membrane uptake. Silica did not cause disruption of the lipid vesicles.These results indicate that particulates can enhance the cellular availability of chemical carcinogens.     

Particulate enhanced membrane uptake of 1,2-benzanthracene observed by fluorescence spectroscopy: a possible role in co-carcinogenesis.

In recognition of the co-carcinogenic effects of particulate matter and chemical carcinogens, we investigated the effect of particulate silica on the rates of membrane uptake of 1,2-benzanthracene. The fluorescence emission spectra and the apparent quantum yields of benzanthracene and dependent upon adsorption to silica and upon the surface density of benzanthracene on the silica. The fluorescence spectral shifts which occur upon transfer of benzanthracene from the silica surface to phospholipid vesicles provided a convenient means to quantitate the membrane uptake of benzanthracene from particulates. The rate of benzanthracene uptake by dipalmitoyl-L-alpha-phosphatidylcholine vesicles was independent of the concentration of lipid, indicating that the rate-limiting step may involve its solubilization in the aqueous phase. These uptake rates were also independent of the surface density of benzanthracene on the silica, indicating that the benzathracene molecules are dispersed uniformly on the silica surface. Rates of membrane uptake of benzanthracene from the crystalline, microcrystalline, and the silica-absorbed states were compared, and are greatly enhanced by a reduction in crystal size. Silica-adsorbed benzanthracene had the most rapid rate of membrane uptake. Silica did not cause disruption of the lipid vesicles. These results indicate that particulates can enhance the cellular availability of the chemical carcinogens.     

Measurement of membrane potential inBacillus subtilis: A comparison of lipophilic cations,rubidium ion,and a cyanine dye as probes

Summary Two of the commonly used probes for measuring membrane potential—lipophilic cations and the cyanine dye diS-C₃(5)—indicated nominally opposite results when tetraphenylarsonium ion was added as a drug to suspensions of metabolizingBacillus subtilis cells. [³H]-Triphenylmethylphosphonium uptake was enhanced by the addition, indicating hyperpolarization, yet fluorescence of diS-C₃(5) was also enhanced, indicating depolarization. Evidence is presented that both effects are artifactual, and can occur without any change in membrane potential, as estimated by⁸⁶Rb⁺ uptake in the presence of valinomycin. The fluorescence studies suggest that tetraphenylarsonium ion displaces the cyanine dye from the cell envelope, or other binding site, into the aqueous phase.The uptake characteristics of the radiolabeled lipophilic cations were quite unusual: At low concentrations (e.g., less than 10 m for triphenylmethylphosphonium) there was potential-dependent uptake of the label to a stable level, but subsequent addition of nonradioactive lipophilic cation caused further uptake of label to a new stable level. Labeled triphenylmethylphosphonium ion taken up to the first stable level could be displaced by 10mm magnesium ion, whereas⁸⁶Rb⁺ uptake was unperturbed. Association of the lipophilic cations with the surface of de-energized cells was concentration-dependent, but there was no evidence for cooperative binding. This phenomenon of stimulated uptake inB. subtilis (which was not seen inEscherichia coli cells or vesicles) is consistent with a two-compartment model with access to the second compartment only being possible above a critical cation concentration. We tentatively propose such a model, in which these compartments are the cell surface and the cytoplasm, respectively.Triphenylmethylphosphonium up to 0.5mm exhibited linear binding to de-energized cells; binding of tetraphenylphosphonium and tetraphenylarsonium was nonlinear but was not saturated at the highest concentration tested (1mm). The usual assumption, that association of the cation with cell surfaces is saturated and so can be estimated on de-energized cells, therefore leads to undercorrected estimates of cytoplasmic uptake inB. subtilis, and hence to overestimates of membrane potential. We describe a more realistic procedure, in which the estimate of extent of binding is based on a mean aqueous concentration related both to the external concentration and to the much higher internal concentration that exists in energized cells. Using this procedure we estimate the membrane potential inB. subtilis to be 120 mV, inside-negative. The procedure is of general applicability, and should yield more accurate estimates of membrane potential in any system where there is significant potential-dependent binding.Work performed while on sabbatical leave from Department of Biology, Ben-Gurion University of the Negev, Beer-Sheva, Israel.     

Changes in the surface membrane during myoblast fusion.

1. During fusion of chick-embryo myoblasts in culture, the surface membrane is affected as follows. Uptake of 2-aminoisobutyrate and 2-deoxyglucose, each of which is concentrated 20-fold relative to its concentration in the medium, is unaltered; uptake of alpha-methyl glucoside and choline (15 mM), each of which equilibrates relative to its concentration in the medium, approximately doubles. An approximate doubling also occurs in iodinatable surface protein (and in total protein) and in cell surface area as judged by light-microscopy. Adenylate cyclase (in the absence or the presence of fluoride) increases by more than 2-fold. 2. It is concluded that, during myoblast fusion cells increase in size, and this is reflected in an increased rate of simple diffusion; the rate of facilitated processes such as the uptake of amino acids and sugars, on the other hand, remains unaltered, though the activity of certain enzymes is increased. These results indicate that specific changes in the function of surface membrane occur during myoblast fusion in vitro.     

Simultaneous extraction and concentration of penicillin G by hollow fiber renewal liquid membrane

In this article, hollow fiber renewal liquid membrane (HFRLM) technique was used for recovery of penicillin G from aqueous solution. The organic solution of 7 vol % di‐n‐octylamine (DOA) + 30 vol % iso‐octanol + kerosene was used as liquid membrane phase, and Na₂CO₃ aqueous solution was used as stripping phase. Experiments were performed as a function of carrier concentration in the organic phase, organic/aqueous volume ratio, pH, and initial penicillin G concentration in the feed phase, pH in the stripping phase, flow rates, etc. The results showed that the HFRLM process was stable and could carry out simultaneous extraction and concentration of penicillin G from aqueous solutions. As a carrier facilitated transport process, the addition of DOA in organic phase could greatly enhance the mass transfer rate; and there was a favorable organic/aqueous volume ratio of 1:20 to 1:30 for this system. The mass transfer flux and overall mass transfer coefficient increased with decreasing pH in the feed phase and increasing pH in the stripping phase, because of variation of the mass transfer driving force caused by pH gradient and distribution equilibrium. The flow rate of the shell side had significant influence on the mass transfer performance, whereas the effect of flow rate of lumen side on the mass transfer performance was slight because of the mass transfer intensification of renewal effect in the lumen side. The results indicated that the HFRLM process was a promising method for the recovery of penicillin G from aqueous solutions. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Cross-flow membrane microfiltration of a bacteriol fermentation broth

Although cross-flow membrane filtration is a very attractive option for harvesting cells and recovering enzymes from cell homogenates, the process is not without its problems. Foremost of these is the deposit of dissolved and suspended solutes onto the membrane surface during operation. The formation of these dense and sometimes compressive sublayers (often called cakes) offers additional resistance to axial and permeate flows and often affects the retention characteristics of the process. In view of the complex nature of the sublayer formation process and its sensitivity to cross-flow velocity, this investigation was undertaken to determine the main factors responsible for the decline in performance during the harvesting of B. polymyxa broth by membrane microfiltration. System parameters varied include axial flow rate, concentration of cells, proteins and other components in the feed, membrane materials (ceramic, polypropylene, and stainless steel), and cleaning methods. To help explain the observed results, a new mass transport model-the solids flux model-based on the assumptions that back migration of particles from the sublayer or membrane surface is negligible and that particles that reach the solid-solution interface attach (stick) completely, is tested. Using a variety of diagnostic methods, magnesium ammonium phosphate precipitate is formed during steam sterilization of the medium and is implicated as the major foulant in this study.     

Two-dimensional crystals: a powerful approach to assess structure, function and dynamics of membrane proteins

Electron crystallography and atomic force microscopy allow the study of two-dimensional membrane protein crystals. While electron crystallography provides atomic scale three-dimensional density maps, atomic force microscopy gives insight into the surface structure and dynamics at sub-nanometer resolution. Importantly, the membrane protein studied is in its native environment and its function can be assessed directly. The approach allows both the atomic structure of the membrane protein and the dynamics of its surface to be analyzed. In this way, the function-related conformational changes can be assessed, thus providing a detailed insight on the molecular mechanisms of essential biological processes.     

Computer modelling of glycolipids at membrane surfaces.

Interactions of membrane anchored molecules such as glycolipids with a membrane surface are important in determining headgroup conformation. It is therefore essential to represent these membrane surface interactions in molecular modeling studies of glycolipids and other membrane bound molecules. We introduce here an energy term that represents the interaction of molecules with a membrane bilayer. This membrane interaction energy term has been added to the potential energy function of a molecular dynamics and mechanics program and has been parameterized using partition coefficients between an aqueous solution and a vesicular membrane for two model glycolipids.     

An essential ligand-binding domain in the membrane receptor for retinol-binding protein revealed by large-scale mutagenesis and a human polymorphism

Plasma retinol-binding protein (RBP), the principal carrier of vitamin A in the blood, delivers vitamin A from liver, the site of storage, to distant organs that need vitamin A, such as the eye, brain, placenta, and testis. STRA6 is a high-affinity membrane receptor for RBP and mediates vitamin A uptake in these target organs. STRA6 is a 74-kDa multi-transmembrane domain protein that represents a new class of membrane transport protein. In this study, we used an unbiased strategy by analyzing >900 random mutants of STRA6 to study its structure and function, and we identified an essential RBP-binding domain in STRA6. Mutations in any of the three essential residues in this domain can almost completely abolish binding of STRA6 to RBP and its vitamin A uptake activity from holo-RBP without affecting its cell surface expression. We have also functionally characterized the mutations in human STRA6 that cause severe birth defects as well as several human polymorphisms. All STRA6 mutants associated with severe birth defects have largely abolished vitamin A uptake activity, consistent with the severe clinical phenotypes. In addition, we have identified a human polymorphism that significantly reduces the vitamin A uptake activity of STRA6. Interestingly, the residue affected by this polymorphism is located in the RBP-binding domain we identified, and the polymorphism causes decreased vitamin A uptake by reducing RBP binding. This study identifies an essential functional domain in STRA6 and a human polymorphism in this domain that leads to reduced vitamin A uptake activity.