Neurotransmitters as ion shuttles in regulation of neural activity.

A molecular framework is described within which a single neural cell can modulate its excitability, or the quantity of transmitter released upon stimulation, in relationship to past stimulation. The key elements in this regulating system are complexation in the synaptic area by the transmitter molecule with extracellular ions, interaction of the complexed transmitter with the presynaptic receptor, possibly followed by reuptake of complexed transmitter.A number of mechanisms are suggested by which the transmitter/metal ion complex can regulate cellular function. Calculations are made to estimate the possible degree of change in the interior calcium concentration of a catecholaminergic cell by a calcium ion complex formed in the synapse. Experimental evidence is cited, which(a) documents the existence in the catecholaminergic cell of the necessary machinery for a calcium-ion-regulated cell-use registry device.(b) supports the hypothesis that catecholamines transport metal ions in neural systems, and(c) indicates that the ionic shuttle function of neurotransmitters plays a significant, but not exclusive, role in the transport of calcium. Calcium transported in this manner may be uniquely distinguishable from that derived from other sources of intracellular calcium in its temporal or spatial distribution. The existing evidence is discussed and rationalized with respect to the hypothesis that one of the chief presynaptic functions of many neurotransmitters is to feedback regulate cell function by performance as an ion shuttle.     

Some factors affecting oxygen uptake by red blood cells in the pulmonary capillaries

In this study we investigate the equations governing the transport of oxygen in pulmonary capillaries. We use a mathematical model consisting of a red blood cell completely surrounded by plasma within a cylindrical pulmonary capillary. This model takes account of convection and diffusion of oxygen through plasma, diffusion of oxygen through the red blood cell, and the reaction between oxygen and haemoglobin molecules. The velocity field within the plasma is calculated by solving the slow flow equations. We investigate the effect on the solution of the governing equations of: (i) mixed-venous blood oxygen partial pressure (the initial conditions); (ii) alveolar gas oxygen partial pressure (the boundary conditions); (iii) neglecting the convection term; and (iv) assuming an instantaneous reaction between the oxygen and haemoglobin molecules. It is found that: (a) equilibrium is reached much more rapidly for high values of mixed-venous blood and alveolar gas oxygen partial pressure; (b) the convection term has a negligible effect on the time taken to reach a prescribed degree of equilibrium; and (c) an instantaneous reaction may be assumed. Explanations are given for each of these results.     

Alkaline-shifted pHo sensitivity of AE2c1-mediated anion exchange reveals novel regulatory determinants in the AE2 N-terminal cytoplasmic domain

The mouse anion exchanger AE2/SLC4A2 Cl(-)/HCO(-)(3) exchanger is essential to post-weaning life. AE2 polypeptides regulate pH(i), chloride concentration, cell volume, and transepithelial ion transport in many tissues. Although the AE2a isoform has been extensively studied, the function and regulation of the other AE2 N-terminal variant mRNAs of mouse (AE2b1, AE2b2, AE2c1, and AE2c2) have not been examined. We now present an extended analysis of AE2 variant mRNA tissue distribution and function. We show in Xenopus oocytes that all AE2 variant polypeptides except AE2c2 mediated Cl(-) transport are subject to inhibition by acidic pH(i) and to activation by hypertonicity and NH(+)(4). However, AE2c1 differs from AE2a, AE2b1, and AE2b2 in its alkaline-shifted pH(o)((50)) (7.70 +/- 0.11 versus 6.80 +/- 0.05), suggesting the presence of a novel AE2a pH-sensitive regulatory site between amino acids 99 and 198. Initial N-terminal deletion mutagenesis restricted this site to the region between amino acids 120 and 150. Further analysis identified AE2a residues 127-129, 130-134, and 145-149 as jointly responsible for the difference in pH(o)((50)) between AE2c1 and the longer AE2a, AE2b1, and AE2b2 polypeptides. Thus, AE2c1 exhibits a unique pH(o) sensitivity among the murine AE2 variant polypeptides, in addition to a unique tissue distribution. Physiological coexpression of AE2c1 with other AE2 variant polypeptides in the same cell should extend the range over which changing pH(o) can regulate AE2 transport activity.     

Effects of cellular pharmacology on drug distribution in tissues.

The efficacy of targeted therapeutics such as immunotoxins is directly related to both the extent of distribution achievable and the degree of drug internalization by individual cells in the tissue of interest. The factors that influence the tissue distribution of such drugs include drug transport; receptor/drug binding; and cellular pharmacology, the processing and routing of the drug within cells. To examine the importance of cellular pharmacology, previously treated only superficially, we have developed a mathematical model for drug transport in tissues that includes drug and receptor internalization, recycling, and degradation, as well as drug diffusion in the extracellular space and binding to cell surface receptors. We have applied this "cellular pharmacology model" to a model drug/cell system, specifically, transferrin and the well-defined transferrin cycle in CHO cells. We compare simulation results to models with extracellular diffusion only or diffusion with binding to cell surface receptors and present a parameter sensitivity analysis. The comparison of models illustrates that inclusion of intracellular trafficking significantly increases the total transferrin concentration throughout much of the tissue while decreasing the penetration depth. Increasing receptor affinity or tissue receptor density reduces permeation of extracellular drug while increasing the peak value of the intracellular drug concentration, resulting in "internal trapping" of transferrin near the source; this could account for heterogeneity of drug distributions observed in experimental systems. Other results indicate that the degree of drug internalization is not predicted by the total drug profile. Hence, when intracellular drug is required for a therapeutic effect, the optimal treatment may not result from conditions that produce the maximal total drug distribution. Examination of models that include cellular pharmacology may help guide rational drug design and provide useful information for whole body pharmacokinetic studies.     

Albumin enhances the diffusion of lipophilic drugs into the membrane bilayer

In earlier work, we reported on the manner with which lipophilic drug molecules interact with the cell membrane in order to (a) enter the bilayer and laterally diffuse to their respective protein sites of action, or (b) penetrate this biological barrier to reach the cell interior. A remaining uncertainty is how lipophilic molecules reach the hydrophobic membrane core after traversing the aqueous medium and membrane polar surface. Here we present preliminary data using deuterium NMR, demonstrating the role of bovine serum albumin in facilitating this process. Our observation allows us to postulate a mechanism by which the passive transport of lipophilic ligands across the membrane can be greatly enhanced through the assistance of carrier proteins.     

Electroporation of Brain Endothelial Cells on Chip toward Permeabilizing the Blood-Brain Barrier

The blood-brain barrier, mainly composed of brain microvascular endothelial cells, poses an obstacle to drug delivery to the brain. Controlled permeabilization of the constituent brain endothelial cells can result in overcoming this barrier and increasing transcellular transport across it. Electroporation is a biophysical phenomenon that has shown potential in permeabilizing and overcoming this barrier. In this study we developed a microengineered in vitro model to characterize the permeabilization of adhered brain endothelial cells to large molecules in response to applied pulsed electric fields. We found the distribution of affected cells by reversible and irreversible electroporation, and quantified the uptaken amount of naturally impermeable molecules into the cells as a result of applied pulse magnitude and number of pulses. We achieved 81 ± 1.7% (N = 6) electroporated cells with 17 ± 8% (N = 5) cell death using an electric-field magnitude of ∼580 V/cm and 10 pulses. Our results provide the proper range for applied electric-field intensity and number of pulses for safe permeabilization without significantly compromising cell viability. Our results demonstrate that it is possible to permeabilize the endothelial cells of the BBB in a controlled manner, therefore lending to the feasibility of using pulsed electric fields to increase drug transport across the BBB through the transcellular pathway.     

A mathematical model of the P-glycoprotein pump as a mediator of multidrug resistance

Cells displaying the classic multidrug resistant (MDR) phenotype possess a transmembrane protein (p170 or P-glycoprotein) which can actively extrude cytotoxic agents from the cytoplasm. A mathematical model of this drug efflux pump has been developed. Outward transport is modeled as a facilitated diffusion process. Since energy-dependent efflux of cytotoxic agents requires that ATP also bind to p170, the model includes a dynamic calculation for efflux rate which considers Michaelis-Menten kinetics for both the substrate agent and ATP. The final system consists of one partial differential equation (PDE) for the facilitated diffusion of substrate agents out of the cell a 2×2 ordinary differential equation (ODE) system for the dynamic calculation of the ATP-ADP pool, and a dynamic algebraic calculation of the efflux rate given substrate levels at the interior cell membrane interface and ATP levels in the cell. A stability analysis of the ATP-ADP pool distribution and a simplistic closed form solution of the linearized PDE are included. Numerical simulations are also provided.     

Electroosmosis in Membranes: Effects of Unstirred Layers and Transport Numbers: II. Experimental

In an earlier paper, it was shown that the differences in transport numbers in membranes and adjacent solutions will result in a depletion and enhancement of the local concentration profiles at the appropriate interfaces. These should, in general, cause both current-induced volume flows and transient changes in membrane potential difference (PD). The predicted concentration changes were measured near an isolated segment of plant cell wall just after a current pulse. The current-induced volume flows observed were separated into a “transport number component” and an instantaneous, electroosmotic one for both cell walls and whole cells. For walls, the electroosmotic component contributed about 53 moles · Faraday^-1 to a total coefficient of 112 moles · Faraday^-1. For whole cells, the average electroosmotic component (for both hyperpolarizing and depolarizing currents) contributed about 38 moles · Faraday^-1 to a total of about 100 moles · Faraday^-1. There was good agreement between the magnitudes and time courses of the flows and membrane PD's predicted from the theory in the previous paper, and those measured in both cell walls and whole cells.     

A cytokinetic model for heterogeneous mammalian cell populations. I. Cell growth and cell death

A general model is proposed for describing the growth behavior of mammalian cell populations, which features:(a) a cell cycle time distribution function with properties such that mean and variance increase with increasing population size; (b) maturation age and maturation rate functions which constrain the maturational pathways of individual cells; and (c) a death rate function, where cell death is construed as irreparable damage to a cell's reproductive apparatus. The biological implications of the model are discussed, and methods for relating the model to real cell systems by means of commonly used experimental techniques are described. The model is compared with earlier models.     

Evidence for IHA migration during axial rotation of a lumbar spine segment by using a novel high-resolution 6D kinematic tracking system

The biomechanical properties of the lumbar spine have long been studied. However, despite its enormous importance, basic functional and morphological properties have been not well understood and require further experimental analysis since data concerning the spatial instantaneous segmental motions are hardly available. This study describes the theoretical background and the technical properties of an innovative method for tracking the instantaneous 3D motion of human spinal segments in vitro at high spatial resolution. This new acquisition system allows to scrutinise closely the location and alignment of the segmental instantaneous helical axis (IHA) and the respective screw pitch as functions of the absolute rotational angle. The required precision of the measuring device was attained (a) by six highly resolving linear inductive displacement sensors in a special spatially configuration (3-2-1), (b) by a method to apply torque and force independently of each other without counteraction, and (c) by suppression of vibrations. The validity and reliability of the experimental set-up and the numerical method of data analysis were tested by subjects of known mechanical properties. In vitro experiments with a human lumbar segment (L3/L4, autopsy material) demonstrated that (a) the IHA migrated during axial rotation from one segmental articulatio zygapophysialis to the other joint, (b) the IHA tilted medial-laterally, and (c) the pitch of the screw altered linearly as a function of the rotational angle. This phenomenon is traced back to the guidance of the articluationes zygapophysiales. The validation of the method allows to map segments of the entire vertebral column. The results can be used as benchmarks for future models of the human spine.     

Further studies on membrane transport with time delay

A theory of cell membrane transport with a time delay which predicts under certain conditions overshoot or oscillatory permeation (Ohshima and Kondo, Biophys. Chem. 33 (1989) 303), is extended with the introduction of a parameter expressing a fraction of solutes inside the cell interior that suffer time delay. It is found that criterion for oscillation depends strongly on this parameter. Results will also be presented for the case of an exponential-type distribution of the delay time.     

The multivesicular body-localized GTPase ARFA1b/1c is important for callose deposition and ROR2 syntaxin-dependent preinvasive basal defense in barley

Host cell vesicle traffic is essential for the interplay between plants and microbes. ADP-ribosylation factor (ARF) GTPases are required for vesicle budding, and we studied the role of these enzymes to identify important vesicle transport pathways in the plant-powdery mildew interaction. A combination of transient-induced gene silencing and transient expression of inactive forms of ARF GTPases provided evidence that barley (Hordeum vulgare) ARFA1b/1c function is important for preinvasive penetration resistance against powdery mildew, manifested by formation of a cell wall apposition, named a papilla. Mutant studies indicated that the plasma membrane-localized REQUIRED FOR MLO-SPECIFIED RESISTANCE2 (ROR2) syntaxin, also important for penetration resistance, and ARFA1b/1c function in the same vesicle transport pathway. This was substantiated by a requirement of ARFA1b/1c for ROR2 accumulation in the papilla. ARFA1b/1c is localized to multivesicular bodies, providing a functional link between ROR2 and these organelles in penetration resistance. During Blumeria graminis f sp hordei penetration attempts, ARFA1b/1c-positive multivesicular bodies assemble near the penetration site hours prior to the earliest detection of callose in papillae. Moreover, we showed that ARFA1b/1c is required for callose deposition in papillae and that the papilla structure is established independently of ARFA1b/1c. This raises the possibility that callose is loaded into papillae via multivesicular bodies, rather than being synthesized directly into this cell wall apposition.     

The human intestinal epithelial cell line Caco-2; pharmacological and pharmacokinetic applications

The gastrointestinal tract remains the most popular and acceptable route of administration for drugs. It offers the great advantage of convenience and many compounds are well absorbed and thereby provide acceptable plasma concentration-time profiles. Currently there is considerable interest from the pharmaceutical industry in development of cell culture systems that would mimic the intestinal mucosa in order to evaluate strategies for investigating and/or enhancing drug absorption. The intestinal epithelial cells of primary interest, from the standpoint of drug absorption and metabolism, are the villus cells, which are fully differentiated cells. Anin vitro cell culture system consisting of a monolayer of viable, polarized and fully differentiated villus cells, similar to that found in the small intestine, would be a valuable tool in the study of drug and nutrient transport and metabolism.The Caco-2 cell line, which exhibits a well-differentiated brush border on the apical surface and tight junctions, and expresses typical small-intestinal microvillus hydrolases and nutrient transporters, has proven to be the most popularin vitro model (a) to rapidly assess the cellular permeability of potential drug candidates, (b) to elucidate pathways of drug transport (e.g., passive versus carrier mediated), (c) to assess formulation strategies designed to enhance membrane permeability, (d) to determine the optimal physicochemical characteristics for passive diffusion of drugs, and (e) to assess potential toxic effects of drug candidates or formulation components on this biological barrier. Since differentiated Caco-2 cells express various cytochrome P450 isoforms and phase II enzymes such as UDP-glucuronosyltransferases, sulfotransferases and glutathione-S-transferases, this model could also allow the study of presystemic drug metabolism.     

药物跨膜转运细胞模型影响因素及应用

本文详细介绍了Caco-2细胞系和MDCK细胞系的特点、跨膜转运细胞模型的建立及其影响因素,包括细胞模型的选择、细胞接种密度、细胞单层的紧密性等细胞因素和Transwell多微孔膜的性质等环境因素。概述了国内外关于利用Caco-2和MDCK细胞系作为模型进行药物筛选、药物相互作用和研究药物吸收转运机制等方面的内容及MDCK细胞模型作为肠道模型、肾脏模型及血脑屏障模型的应用。比较了Caco-2细胞和MDCK细胞在肠道模型方面的差别,MDCK细胞主要用于选择性研究药物在小肠吸收及转运机制,特别用于细胞旁被动转运药物的研究,而Caco-2细胞用于双向转运或能量依赖主动转运研究。MDCK细胞模型可在体外培养条件下平稳转染人类MDR1基因,因此可高表达P-gp基因,可作为可用于评估肾脏药物相互作用、快速进行候选药物筛选及研究药物转运机制的理想模型。     

A requirement for ATP for beta-galactoside transport by Bacillus alcalophilus.

Lactose-grown cells of Bacillus alcalophilus actively transported methylthio-beta, D-galactoside (TMG) in a range of pH values from 7.5 to 10.5 with a pH optimum at 8.5. The TMG was accumulated in a chemically unmodified form, and cell extracts failed to catalyze either ATP or P-enolpyruvate-dependent phosphorylation of TMG. At pH 8.5, the lactose-grown cells exhibited a transmembrane proton gradient (deltapH) of 1.38 units, interior acid, and a transmembrane electrical potential (delta psi) of -132 mV. Accordingly, the total protonmotive force at this pH was very low, -51mV. Several lines of evidence indicate that the protonmotive force or delta psi did not directly energize TMG transport but, rather, that ATP was directly required: (a) in cells treated with arsenate so that the delta psi was unaffected and cellular ATP levels were markedly lowered, TMG transport was inhibited in proportion to the reduction of cellular ATP, while electrogenic alpha-aminoisobutyric acid transport was not; (b) when a valinomycin-induced potassium diffusion potential was established in starved cells, alpha-aminoisobutyric acid transport, but not TMG transport, was stimulated; and (c) in a series of experiments in which the delta psi was rapidly abolished by treatment with gramicidin, ATP levels declined slowly and the rate of TMG transport correlated directly with ATP levels rather than with the delta psi. Consumption of cellular ATP concomitant with TMG transport could be demonstrated.     

Nitroxide radicals. Controlled release from and transport through biomimetic and hollow fibre membranes

Stable nitroxide radicals have found wide applications in chemistry and biology and they have some potential applications in medicine due to their antioxidant properties. Nitrocellulose filters impregnated with lipid-like substances are used as an imitation of biomembranes and could be used as a controlled drug release vehicle, while experiments with hollow fibres can be useful in the modelling of a drug delivery via blood vessels. This paper describes mechanisms of the nitroxide transport in four different model systems, i.e. a) exit of nitroxide into aqueous solution from porous nitrocellulose filters, impregnated with organic solvents, b) transport of nitroxides through the impregnated membrane from one into another aqueous solution, c) transport of nitroxides from bulk phase of organic solvents through the impregnated membrane into aqueous phase with ascorbic acid, and d) transport of nitroxides from liquid organic phase into aqueous solution through porous hollow fibres. The results are analysed in terms of mass transfer resistance of a membrane, organic and aqueous phase, based on nitroxide diffusion and distribution coefficients. Ascorbic acid reduced nitroxides in water and enhanced the rate of their transfer due to the decrease of transport resistance of unstirred aqueous layers. It is demonstrated that in the case of biomembranes the rate limiting step could be the transport through unstirred aqueous layers and membrane/water interface.     

Nitroxide radicals. Controlled release from and transport through biomimetic and hollow fibre membranes

Stable nitroxide radicals have found wide applications in chemistry and biology and they have some potential applications in medicine due to their antioxidant properties. Nitrocellulose filters impregnated with lipid-like substances are used as an imitation of biomembranes and could be used as a controlled drug release vehicle, while experiments with hollow fibres can be useful in the modelling of a drug delivery via blood vessels. This paper describes mechanisms of the nitroxide transport in four different model systems, i.e. a) exit of nitroxide into aqueous solution from porous nitrocellulose filters, impregnated with organic solvents, b) transport of nitroxides through the impregnated membrane from one into another aqueous solution, c) transport of nitroxides from bulk phase of organic solvents through the impregnated membrane into aqueous phase with ascorbic acid, and d) transport of nitroxides from liquid organic phase into aqueous solution through porous hollow fibres. The results are analysed in terms of mass transfer resistance of a membrane, organic and aqueous phase, based on nitroxide diffusion and distribution coefficients. Ascorbic acid reduced nitroxides in water and enhanced the rate of their transfer due to the decrease of transport resistance of unstirred aqueous layers. It is demonstrated that in the case of biomembranes the rate limiting step could be the transport through unstirred aqueous layers and membrane/water interface.     

Impact of Allee effect on an eco-epidemiological system

In this paper, we propose a general ratio-dependent prey-predator model with disease in predator subject to the strong Allee effect in prey. We obtain the complete dynamics of both models: (a) full model with Allee effect; (b) full model without Allee effect. Model (a) may have more than one interior equilibrium point, but model (b) has only one interior equilibrium point. Numerical results reveal that the coexistence of all the populations at the endemic state is possible for both the models. But for the model with Allee effect, the coexistence can be destroyed by an increased supply of alternative food for the predators. It can also be proved that for the full model with Allee effect, the disease can be suppressed under certain parametric conditions. Also by comparing models (a) and (b), we conclude that Allee effect can create or destroy the interior attractor. Finally, we have studied the disease free-submodel (prey and susceptible predator model) with and without Allee effect. The comparative study between these two submodels leads to the following conclusions: 1) In the presence of Allee effect, the number of interior equilibrium points can change from zero to two whereas the submodel without Allee effect has unique interior equilibrium point; 2) Both with and without Allee effect, initial conditions play an important role on the survival and extinction of prey as well as its corresponding predator; 3) In the presence of Allee effect, bi-stability occurs with stable or periodic coexistence of prey and susceptible predator and the extinction of prey and susceptible predator; 4) Allee effect can generate or destroy the interior equilibrium points.     

The role of electron transport in the defence response of the South African abalone, Haliotis midae

In order to establish health management systems for farmed abalone, it is necessary to understand how the abalone immune system functions and responds to stimulation. Two electron transport system genes, cytochrome b and cytochrome c oxidase III, were found to be upregulated in a cDNA microarray experiment performed on haemocytes from immune-stimulated abalone (Arendze-Bailey, unpublished). The current study sought to elucidate the role of these genes, and thus the electron transport system, in the abalone immune response by specifically inhibiting cytochrome b with antimycin A and measuring haemocyte immune parameters in vivo. Antimycin A did not decrease haemocyte cell viability, but halved cellular ATP from 4 x 10(12) nM/cell to 2 x 10(12) nM/cell (p < 0.05, unpaired t-test). Inhibition of electron transport resulted in a 0.6 fold increase in cellular superoxide levels (p < 0.05, unpaired t-test), while phagocytosis dropped by nearly 50% (p < 0.05, ANOVA) and the ability of haemocytes to kill bacteria was also reduced. Since cytochrome b and cytochrome c oxidase III expression is upregulated in immune-stimulated abalone, and inhibition of electron transport resulted in a decreased immune response in vivo, we conclude that the abalone immune response is dependent on electron transport and that oxidative phosphorylation plays a role in the immune response following stimulation.