In the body, cell monolayers serve as permeability barriers, determining transport of drug molecules from one organ or tissue compartment to another. After oral administration, for example, drug transport across the epithelial cell monolayer lining the lumen of the intestine determines the fraction of drug in the gut that is absorbed by the body. By modeling passive transcellular transport properties in the presence of an apical to basolateral concentration gradient, we demonstrate how a computational, cell-based molecular transport simulator can be used to define a physicochemical property space occupied by molecules with desirable permeability and intracellular retention characteristics. Considering extracellular domains of cell surface receptors located on the opposite side of a cell monolayer as a drug’s desired site-of-action, simulation of transcellular transport can be used to define the physicochemical properties of molecules with maximal transcellular permeability but minimal intracellular retention. Arguably, these molecules would possess very desirable features: least likely to exhibit non-specific toxicity, metabolism and side effects associated with high (undesirable) intracellular accumulation; and, most likely to exhibit favorable bioavailability and efficacy associated with maximal rates of transport across cells and minimal intracellular retention, resulting in (desirable) accumulation at the extracellular site-of-action. Calculated permeability predictions showed good correlations with PAMPA, Caco2, and intestinal permeability measurements, without “training” the model and without resorting to statistical regression techniques to “fit” the data. Therefore, cell-based molecular transport simulators could be useful in silico screening tools for chemical genomics and drug discovery.
Metoprolol; permeability; chemical space; computer aided drug design; virtual screening; chemical genomics; cellular pharmacokinetics; cheminformatics; drug transport; PAMPA; Biopharmaceutics Classification System
Cell-based molecular transport simulations are being developed to facilitate exploratory cheminformatic analysis of virtual libraries of small drug-like molecules. For this purpose, mathematical models of single cells are built from equations capturing the transport of small molecules across membranes. In turn, physicochemical properties of small molecules can be used as input to simulate intracellular drug distribution, through time. Here, with mathematical equations and biological parameters adjusted so as to mimic a leukocyte in the blood, simulations were performed to analyze steady state, relative accumulation of small molecules in lysosomes, mitochondria, and cytosol of this target cell, in the presence of a homogenous extracellular drug concentration. Similarly, with equations and parameters set to mimic an intestinal epithelial cell, simulations were also performed to analyze steady state, relative distribution and transcellular permeability in this non-target cell, in the presence of an apical-to-basolateral concentration gradient. With a test set of ninety-nine monobasic amines gathered from the scientific literature, simulation results helped analyze relationships between the chemical diversity of these molecules and their intracellular distributions.
Electronic supplementary material
The online version of this article (doi:10.1007/s10822-008-9194-7) contains supplementary material, which is available to authorized users.
Cheminformatics; Lysosomotropic; Cellular pharmacokinetics; Drug transport; Small molecule permeability; Subcellular localization; Simulation; Rational drug design
The bioavailabilities of orally administered drugs depend to a great extent on their capability of being transported across the intestinal mucosa. In an attempt to develop an in vitro model for studying the intestinal transport of drugs, we used an intestinal epithelial cell line (Caco 2) derived from a human colon adenocarcinoma. A renal epithelial cell line (MDCK) was also used to determine the tissue specificity of drug transport. These cell lines, which were grown on filters, form a monolayer of well-polarized cells coupled by tight junctions and can be used for transcellular transport experiments. We studied the transport of nine antimicrobial agents with different physicochemical and pharmacokinetic characteristics using these epithelial cell monolayers to determine whether this model could be predictive of oral bioavailability. The transepithelial passage was assayed from the apical (AP) to the basolateral (BL) side and in the opposite direction (BL to AP) in both cell lines. Radioactively labeled mannitol was used to monitor the intactness of the cell monolayer during drug passage. The results indicated that all antimicrobial agents tested tended to behave in vitro generally according to their known in vivo absorptive characteristics. In addition, the use of epithelia from different tissues enabled us to divide the drugs into four groups according to their behaviors and suggested the existence of different transport mechanisms. In particular, two antibiotics, gentamicin and teicoplanin, showed no passage in either direction or cell line, in accordance with their very poor in vivo absorbances after oral administration. In contrast, rifapentine, rifampin, and nalidixic acid passed very efficiently at similar rates in both directions and cell lines in a concentration-dependent, nonsaturable manner, which is suggestive of passive diffusion down a concentration gradient. Of the remaining drugs, isoniazid and novobiocin sodium showed some differences in passage between the two cell lines and, given their ionized state at the pH that was used, may use the paracellular route. Finally, trimethoprim and D-cycloserine exhibited differences in passage both with respect to polarity and cell line; in particular, trimethoprim had a faster rate of passage only in Caco 2 cells and in the BL to AP direction, while D-cycloserine was exclusively transported by Caco 2 cells in the AP to BL direction. In both cases it is possible that active transport mechanisms are involved.
Leukocyte migration across endothelial cell borders (paracellular) and through endothelial cells (transcellular) appear to be distinct processes. During paracellular migration, membrane from a parajunctional reticulum of interconnected vesicles, the endothelial lateral border recycling compartment (LBRC), moves to surround the leukocyte in a kinesin-mediated, microtubule-dependent manner. We show that transcellular migration likewise requires targeted trafficking of LBRC membrane. We show that in addition to platelet/endothelial cell adhesion molecule (PECAM; CD31), CD99 and junctional adhesion molecule A (JAM-A), but apparently not vascular endothelial cell–specific cadherin (cadherin 5, CD144), are components of the LBRC. During transcellular migration, LBRC membrane invests the transmigrating leukocyte. Intracellular adhesion molecule 1 (ICAM-1) on the apical endothelial surface is enriched around adherent leukocytes. Depolymerization of microtubules has no effect on ICAM-1 enrichment but blocks targeted trafficking of LBRC membrane and transcellular migration by >90%. Similar to their effects on paracellular transmigration, antibodies against PECAM or CD99, but not JAM-A, block transcellular migration. We conclude that similar molecular mechanisms promote both para- and transcellular migration.
Transcellular Mg2+ transport across epithelia, involving both apical entry and basolateral extrusion, is essential for magnesium homeostasis, but molecules involved in basolateral extrusion have not yet been identified. Here, we show that CNNM4 is the basolaterally located Mg2+ extrusion molecule. CNNM4 is strongly expressed in intestinal epithelia and localizes to their basolateral membrane. CNNM4-knockout mice showed hypomagnesemia due to the intestinal malabsorption of magnesium, suggesting its role in Mg2+ extrusion to the inner parts of body. Imaging analyses revealed that CNNM4 can extrude Mg2+ by exchanging intracellular Mg2+ with extracellular Na+. Furthermore, CNNM4 mutations cause Jalili syndrome, characterized by recessive amelogenesis imperfecta with cone-rod dystrophy. CNNM4-knockout mice showed defective amelogenesis, and CNNM4 again localizes to the basolateral membrane of ameloblasts, the enamel-forming epithelial cells. Missense point mutations associated with the disease abolish the Mg2+ extrusion activity. These results demonstrate the crucial importance of Mg2+ extrusion by CNNM4 in organismal and topical regulation of magnesium.
Magnesium is an essential element for living organisms. Its absorption occurs at the intestine through the barrier comprised of epithelial cells. In this process, transcellular Mg2+ transport across epithelia, involving both entry from one side and extrusion from the other side, is important. Previous studies have revealed the role of Mg2+-permeable channel protein in Mg2+ entry into the epithelial cells. However, the identity of proteins involved in Mg2+ extrusion to the inner parts of body has remained unknown. Mice genetically engineered not to express CNNM4, which localizes to the epithelial membrane facing to the inner parts of body, show hypomagnesemia due to the defect in magnesium absorption. Functional analyses using culture cells directly reveal that CNNM4 can extrude intracellular Mg2+ to the outside of cells. These results indicate that CNNM4 mediates transcellular Mg2+ transport across the intestinal epithelia. Furthermore, we also show that these CNNM4-lacking mice also have a defect in amelogenesis, which is consistent with the disease symptoms of Jalili syndrome that is known to be caused by mutations in the CNNM4 gene.
In the preceding paper (Pesonen M., W. Ansorge, and K. Simons, 1984, J. Cell Biol., 99:796-802), we have shown that transcellular transport of the membrane glycoprotein G of vesicular stomatitis virus implanted into the apical membrane of Madin-Darby canine kidney cells is transcytosed through the endosomal compartment to the basolateral plasma membrane. To determine whether the Golgi complex was involved in this process, G protein lacking sialic acid or all of the terminal sugars was implanted into the apical membrane and allowed to move to the basolateral membrane. Using the criteria of endoglycosidase H sensitivity, binding to Ricinus communis agglutinin and two-dimensional gel electrophoresis, the sugars on the transcytosed G protein were found to be the same as in the starting material. The absence of any involvement of the Golgi complex in transcytosis was supported by subcellular fractionation studies in which transcytosing G protein was never found in fractions containing galactosyl transferase.
For optimizing the local, pulmonary targeting of inhaled medications, it is important to analyze the relationship between the physicochemical properties of small molecules and their absorption, retention and distribution in the various cell types of the airways and alveoli.
A computational, multiscale, cell-based model was constructed to facilitate analysis of pulmonary drug transport and distribution. The relationship between the physicochemical properties and pharmacokinetic profile of monobasic molecules was explored. Experimental absorption data of compounds with diverse structures were used to validate this model. Simulations were performed to evaluate the effect of active transport and organelle sequestration on the absorption kinetics of compounds.
Relating the physicochemical properties to the pharmacokinetic profiles of small molecules reveals how the absorption half-life and distribution of compounds are expected to vary in different cell types and anatomical regions of the lung. Based on logP, pKa and molecular radius, the absorption rate constants (Ka) calculated with the model were consistent with experimental measurements of pulmonary drug absorption.
The cell-based mechanistic model developed herein is an important step towards the rational design of local, lung-targeted medications, facilitating the design and interpretation of experiments aimed at optimizing drug transport properties in lung.
Diapedesis is critical for immune system function and inflammatory responses. This occurs by migration of blood leukocytes either directly through individual microvascular endothelial cells (the “transcellular” route) or between them (the “paracellular” route). Mechanisms for transcellular pore formation in endothelium remain unknown. Here we demonstrate that lymphocytes used podosomes and extended “invasive podosomes” to palpate the surface of, and ultimately form transcellular pores through, the endothelium. In lymphocytes, these structures were dependent on Src kinase and the actin regulatory protein WASP; inhibition of podosome formation selectively blocked the transcellular route of diapedesis. In endothelium, membrane fusion events dependent on the SNARE-containing membrane fusion complex and intracellular calcium were required for efficient transcellular pore formation in response to podosomes. These findings provide insights into basic mechanisms for leukocyte trafficking and the functions of podosomes.
In the present study, we developed novel insulin-loaded hyaluronic acid (HA) nanoparticles for insulin delivery. The insulin-loaded HA nanoparticles were prepared by reverse-emulsion-freeze-drying method. This method led to a homogenous population of small HA nanoparticles with average size of 182.2 nm and achieved high insulin entrapment efficiencies (approximately 95%). The pH-sensitive HA nanoparticles as an oral delivery carrier showed advantages in protecting insulin against the strongly acidic environment of the stomach, and not destroying the junction integrity of epithelial cells which promise long-term safety for chronic insulin treatment. The results of transport experiments suggested that insulin-loaded HA nanoparticles were transported across Caco-2 cell monolayers mainly via transcellular pathway and their apparent permeability coefficient from apical to basolateral had more than twofold increase compared with insulin solution. The efflux ratio of Papp (B to A) to Papp (A to B) less than 1 demonstrated that HA nanoparticle-mediated transport of insulin across Caco-2 cell monolayers underwent active transport. The results of permeability through the rat small intestine confirmed that HA nanoparticles significantly enhanced insulin transport through the duodenum and ileum. Diabetic rats treated with oral insulin-loaded HA nanoparticles also showed stronger hypoglycemic effects than insulin solution. Therefore, these HA nanoparticles could be a promising candidate for oral insulin delivery.
high entrapment efficiency; hyaluronic acid nanoparticles; insulin; pH sensitive; transcellular delivery
The process of saliva production in the salivary glands requires transepithelial water transfer from the interstitium to the acinar lumen. There are two transepithelial pathways: the transcellular and paracellular. In the transcellular pathway, the aquaporin water channels induce passive water diffusion across the membrane lipid bilayer. It is well known that aquaporin-5 (AQP5) is expressed in the salivary glands, in which it is mainly localized at the apical membrane of the acinar cells. This suggests the physiological importance of AQP5 in transcellular water transfer. Reduced saliva secretion under pilocarpine stimulation in AQP5-null mice compared with normal mice further indicates the importance of AQP5 in this process, at least in stimulated saliva secretion. Questions remain therefore regarding the role and importance of AQP5 in basal saliva secretion. It has been speculated that there would be some short-term regulation of AQP5 such as a trafficking mechanism to regulate saliva secretion. However, no histochemical evidence of AQP5-trafficking has been found, although some of biochemical analyses suggested that it may occur. There are no reports of human disease caused by AQP5 mutations, but some studies have revealed an abnormal subcellular distribution of AQP5 in patients or animals with xerostomia caused by Sjögren’s syndrome and X-irradiation. These findings suggest the possible pathophysiological importance of AQP5 in the salivary glands.
water channel; aquaporin-5; salivary gland; xerostomia
Most models developed to represent transport across epithelia assume that the cell interior constitutes a homogeneous compartment, characterized by a single concentration value of the transported species. This conception differs significantly from the current view, in which the cellular compartment is regarded as a highly crowded media of marked structural heterogeneity. Can the finding of relatively simple dynamic properties of transport processes in epithelia be compatible with this complex structural conception of the cell interior? The purpose of this work is to contribute with one simple theoretical approach to answer this question. For this, the techniques of model reduction are utilized to obtain a two-state reduced model from more complex linear models of transcellular transport with a larger number of intermediate states. In these complex models, each state corresponds to the solute concentration in an intermediate intracellular compartment. In addition, the numerical studies reveal that it is possible to approximate a general two-state model under conditions where strict reduction of the complex models cannot be performed. These results contribute with arguments to reconcile the current conception of the cell interior as a highly complex medium with the finding of relatively simple dynamic properties of transport across epithelial cells.
Diet-derived luminal factors have a major influence on zinc available for uptake across the apical membrane of enterocytes. Malabsorption and possibly intestinal microbiota limit this zinc availability. The transporter ZIP4 is expressed along the entire gastrointestinal tract and acts as a major processor of dietary zinc for loading into enterocytes from the apical membrane. Zip4 and other Zip family genes expressed in the gastrointestinal tract are up-regulated in periods of dietary zinc restriction. This provides for powerful homeostatic control. The transporter ZIP14 is up-regulated along the entire gastrointestinal tract by proinflammatory conditions. Intracellular transporters such as ZnT7, influence the transcellular movement of zinc across the enterocyte. Metallothionein, an intracellular metal buffer, and the transporter ZnT1 at the basolateral membrane, regulate the amount of zinc released to the portal circulation for systemic distribution. Pancreatic release of zinc by acinar cells is through the secretory process and apical membrane and involves transporters ZnT2 and ZnT1, respectively. Expression of both transporters is zinc-responsive. Enterocytes and acinar cells constitutively express Zip5 at the basolateral membrane, where it may serve as a monitor of zinc status.
zinc absorption; zinc transporters; zinc homeostasis
In toad urinary bladder epithelium, inhibition of Na transport with amiloride causes a decrease in the apical (Vmc) and basolateral (Vcs) membrane potentials. In addition to increasing apical membrane resistance (Ra), amiloride also causes an increase in basolateral membrane resistance (Rb), with a time course such that Ra/Rb does not change for 1-2 min. At longer times after amiloride (3-4 min), Ra/Rb rises from its control values to its amiloride steady state values through a secondary decrease in Rb. Analysis of an equivalent electrical circuit of the epithelium shows that the depolarization of Vcs is due to a decrease in basolateral electromotive force (Vb). To see of the changes in Vcs and Rb are correlated with a decrease in Na transport, external current (Ie) was used to clamp Vmc to zero, and the effects of amiloride on the portion of Ie that takes the transcellular pathway were determined. In these studies, Vcs also depolarized, which suggests that the decrease in Vb was due to a decrease in the current output of a rheogenic Na pump. Thus, the basolateral membrane does not behave like an ohmic resistor. In contrast, when transport is inhibited during basolateral membrane voltage clamping, the apical membrane voltage changes are those predicted for a simple, passive (i.e., ohmic) element.
Caveolae or noncoated plasmalemmal vesicles found in a variety of cells have been implicated in a number of important cellular functions including endocytosis, transcytosis, and potocytosis. Their function in transport across endothelium has been especially controversial, at least in part because there has not been any way to selectively inhibit this putative pathway. We now show that the ability of sterol binding agents such as filipin to disassemble endothelial noncoated but not coated plasmalemmal vesicles selectively inhibits caveolae-mediated intracellular and transcellular transport of select macromolecules in endothelium. Filipin significantly reduces the transcellular transport of insulin and albumin across cultured endothelial cell monolayers. Rat lung microvascular permeability to albumin in situ is significantly decreased after filipin perfusion. Conversely, paracellular transport of the small solute inulin is not inhibited in vitro or in situ. In addition, we show that caveolae mediate the scavenger endocytosis of conformationally modified albumins for delivery to endosomes and lysosomes for degradation. This intracellular transport is inhibited by filipin both in vitro and in situ. Other sterol binding agents including nystatin and digitonin also inhibit this degradative process. Conversely, the endocytosis and degradation of activated alpha 2- macroglobulin, a known ligand of the clathrin-dependent pathway, is not affected. Interestingly, filipin appears to inhibit insulin uptake by endothelium for transcytosis, a caveolae-mediated process, but not endocytosis for degradation, apparently mediated by the clathrin-coated pathway. Such selective inhibition of caveolae not only provides critical evidence for the role of caveolae in the intracellular and transcellular transport of select macromolecules in endothelium but also may be useful for distinguishing transport mediated by coated versus noncoated vesicles.
The electrical resistances of the transcellular and paracellular pathways across the toad urinary bladder epithelium (a typical "tight" sodium-transporting epithelium) were determined by two independent sets of electrophysiological measurements: (a) the measurement of the total transepithelial resistance, the ratio of resistance of the apical to the basal cell membrane, and cable analysis of the voltage spread into the epithelium; (b) the measurement of the total transepithelial resistance and the ratio of resistances of both cell membranes before and after replacing all mucosal sodium with potassium (thus, increasing selectively the resistance of the apical membrane). The results obtained with both methods indicate the presence of a finite transepithelial shunt pathway, whose resistance is about 1.8 times the resistance of the transcellular pathway. Appropriate calculations show that the resistance of the shunt pathway is almost exclusively determined by the zonula occludens section of the limiting junctions. The mean resistance of the apical cell membrane is 1.7 times that of the basal cell membrane. The use of nonconducting materials on the mucosal side allowed us to demonstrate that apparently all epithelial cells are electrically coupled, with a mean space constant of 460 µm, and a voltage spread consistent with a thin sheet model.
The relation between active transepithelial Na transport across rabbit ileum and 42K exchange from the serosal solution across the basolateral membranes has been explored. Although 42K influx across the basolateral membranes is inhibited by ouabain and by complete depletion of cell Na, it is not affected when transepithelial Na transport is abolished (i.e. in the presence of an Na-free mucosal solution) or stimulated (i.e. when glucose or alanine is added to the mucosal solution). We are unable to detect any relation between the ouabain-sensitive Na-K exchange mechanism responsible for the maintenance of intracellular Na and K concentrations and active transcellular Na transport. In addition, the maintenance of cell volume (water content) does not appear to be dependent upon transepithelial Na transport or the ouabain- sensitive Na-K exchange pump. Although the results of these studies cannot be considered conclusive, they raise serious questions regarding the role of the Na-K exchange pump, located at the basolateral membranes, in active transepithelial Na transport and the maintenance of cell volume.
The blood-brain barrier (BBB) is a highly specialized structural and biochemical barrier that regulates the entry of blood-borne molecules into brain, and preserves ionic homeostasis within the brain microenvironment. BBB properties are primarily determined by junctional complexes between the cerebral endothelial cells. These complexes are comprised of tight and adherens junctions. Such restrictive angioarchitecture at the BBB reduces paracellular diffusion, while minimal vesicle transport activity in brain endothelial cells limits transcellular transport. Under normal conditions, this largely prevents the extravasation of large and small solutes (unless specific transporters are present) and prevents migration of any type of blood-borne cell. However, this is changed in many pathological conditions. There, BBB disruption (“opening”) can lead to increased paracellular permeability, allowing entry of leukocytes into brain tissue, but also contributing to edema formation. In parallel, there are changes in the endothelial pinocytotic vesicular system resulting in the uptake and transfer of fluid and macromolecules into brain parenchyma. This review highlights the route and possible factors involved in BBB disruption in a variety of neuropathological disorders (e.g. CNS inflammation, Alzheimer’s disease, Parkinson’s disease, epilepsy). It also summarizes proposed signal transduction pathways that may be involved in BBB “opening”.
Although bile acid transport by bile duct epithelial cells, or cholangiocytes, has been postulated, the details of this process remain unclear. Thus, we performed transport studies with [3H]taurocholate in confluent polarized monolayers of normal rat cholangiocytes (NRC). We observed unidirectional (i.e., apical to basolateral) Na+-dependent transcellular transport of [3H]taurocholate. Kinetic studies in purified vesicles derived from the apical domain of NRC disclosed saturable Na+-dependent uptake of [3H]taurocholate, with apparent Km and Vmax values of 209+/-45 microM and 1.23+/-0.14 nmol/mg/10 s, respectively. Reverse transcriptase PCR (RT-PCR) using degenerate primers for both the rat liver Na+-dependent taurocholate-cotransporting polypeptide and rat ileal apical Na+-dependent bile acid transporter, designated Ntcp and ASBT, respectively, revealed a 206-bp product in NRC whose sequence was identical to the ASBT. Northern blot analysis demonstrated that the size of the ASBT transcript was identical in NRC, freshly isolated cholangiocytes, and terminal ileum. In situ RT-PCR on normal rat liver showed that the message for ASBT was present only in cholangiocytes. Immunoblots using a well-characterized antibody for the ASBT demonstrated a 48-kD protein present only in apical membranes. Indirect immunohistochemistry revealed apical localization of ASBT in cholangiocytes in normal rat liver. The data provide direct evidence that conjugated bile acids are taken up at the apical domain of cholangiocytes via the ASBT, and are consistent with the notion that cholangiocyte physiology may be directly influenced by bile acids.
Transcellular transport of a variety of ligands may be an important mechanism by which regulatory substances reach their site of action. We have studied the transcellular transport of two 6,000-mol-wt proteins, epidermal growth factor (EGF) and insulin, across polarized Madin-Darby canine kidney (MDCK) cells grown on dual-sided chambers on a nitrocellulose filter substrate. When grown on these chambers, MDCK cells are polarized and express distinct basal and apical surfaces. MDCK cells are capable of unidirectional transport of EGF from the basal-to-apical direction, 50% of bound EGF transported in 2 h. Transport was inhibited by the addition of unlabeled EGF in a dose- dependent manner. Anti-EGF receptor Ab, which inhibited binding, also inhibited transport. No transport in the apical-to-basal direction is noted. Insulin transport is not observed in either direction. Transport correlates with the presence of ligand-specific receptors on the cell surface. Hence, EGF receptors (Ro = 48,000, Kd = 3.5 X 10(-10) M) are found only on the basal surface of the MDCK cells and neither surface expresses insulin receptors. Characterization of the EGF receptors on MDCK cells, as assessed by affinity, molecular mass, and anti-receptor antibody binding reveals that this receptor has similar characteristics to EGF receptors previously described on a variety of cells. Hence, the EGF receptor can function as a transporter of EGF across an epithelial cell barrier.
A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains while selectively allowing transport of proteins and lipids from one pole to the opposite by transcytosis. The small GTPase, rab17, a member of the rab family of regulators of intracellular transport, is specifically induced during cell polarization in the developing kidney. We here examined its intracellular distribution and function in both nonpolarized and polarized cells. By confocal immunofluorescence microscopy, rab17 colocalized with internalized transferrin in the perinuclear recycling endosome of BHK-21 cells. In polarized Eph4 cells, rab17 associated with the apical recycling endosome that has been implicated in recycling and transcytosis. The localization of rab17, therefore, strengthens the proposed homology between this compartment and the recycling endosome of nonpolarized cells. Basolateral to apical transport of two membrane-bound markers, the transferrin receptor and the FcLR 5-27 chimeric receptor, was specifically increased in Eph4 cells expressing rab17 mutants defective in either GTP binding or hydrolysis. Furthermore, the mutant proteins stimulated apical recycling of FcLR 5-27. These results support a role for rab17 in regulating traffic through the apical recycling endosome, suggesting a function in polarized sorting in epithelial cells.
The development of computational approaches for modeling the spatiotemporal dynamics of intracellular, small molecule drug concentrations has become an increasingly important area of pharmaceutical research. For systems pharmacology, the system dynamics of subcellular transport can be coupled to downstream pharmacological effects on biochemical pathways that impact cell structure and function. Here, we demonstrate how a widely used systems biology modeling package – Virtual Cell – can also be used to model the intracellular, passive transport pathways of small druglike molecules. Using differential equations to represent passive drug transport across cellular membranes, spatiotemporal changes in the intracellular distribution and concentrations of exogenous chemical agents in specific subcellular organelles were simulated for weakly acidic, neutral, and basic molecules, as a function of the molecules’ lipophilicity and ionization potentials. In addition, we simulated the transport properties of small molecule chemical agents in the presence of a homogenous extracellular concentration or a transcellular concentration gradient. We also simulated the effects of cell type-dependent variations in the intracellular microenvironments on the distribution and accumulation of small molecule chemical agents in different organelles over time, under influx and efflux conditions. Lastly, we simulated the transcellular transport of small molecule chemical agents, in the presence of different apical and basolateral microenvironments. By incorporating existing models of drug permeation and subcellular distribution, our results indicate that Virtual Cell can provide a user-friendly, open, online computational modeling platform for systems pharmacology and biopharmaceutics research, making mathematical models and simulation results accessible to a broad community of users, without requiring advanced computer programming knowledge.
Virtual Cell; Drug Disposition; Simulation and Modeling; Pharmacokinetics; Pharmacology; Computational Biology; Systems Biology; Systems Pharmacology
The aim of this study was to investigate the transport properties of carnosine in kidney using SKPT cell cultures as a model of proximal tubular transport, and to isolate the functional activities of renal apical and basolateral transporters in this process.
The membrane transport kinetics of 10 µM [3H]carnosine was studied in SKPT cells as a function of time, pH, potential inhibitors and substrate concentration. A cellular compartment model was constructed in which the influx, efflux and transepithelial clearances of carnosine were determined. Peptide transporter expression was probed by RT-PCR.
Carnosine uptake was 15-fold greater from the apical than basolateral surface of SKPT cells. However, the apical-to-basolateral transepithelial transport of carnosine was severely rate-limited by its cellular efflux across the basolateral membrane. The high-affinity, proton-dependence, concentration-dependence and inhibitor specificity of carnosine supports the contention that PEPT2 is responsible for its apical uptake. In contrast, the basolateral transporter is saturable, inhibited by PEPT2 substrates but non-concentrative, thereby, suggesting a facilitative carrier.
Carnosine is expected to have a substantial cellular accumulation in kidney but minimal tubular reabsorption in blood because of its high influx clearance across apical membranes by PEPT2 and very low efflux clearance across basolateral membranes.
PEPT2; SKPT; carnosine; transport mechanisms; cellular kinetics
Gamma-protocadherins (Pcdh-γs) are good candidates to mediate specificity in synaptogenesis but their role in cell-cell interactions is a matter of debate. We proposed that Pcdh-γs modify preformed synapses via trafficking of Pcdh-γs-containing organelles, insertion into synaptic membranes and homophilic transcellular interaction. Here we provide evidence in support of this model. We show for the first time that Pcdh-γs have homophilic properties and that they accumulate at dendrodendritic and axo-dendritic interfaces during neuronal development. Pcdh-γs are maintained in a substantial mobile intracellular pool in dendrites and cytoplasmic deletion shifts the molecule to the surface and reduces the number and velocity of the mobile packets. We monitored Pcdh-γ temporal and spatial dynamics in transport organelles. Pcdh-γ organelles bud and fuse with stationary clusters near synapses. These results suggest that Pcdh-γ-mediated cell-cell interactions in synapse development or maintenance are tightly regulated by control of intracellular trafficking via the cytoplasmic domain.
Adhesion; synaptogenesis; cadherin; live-cell imaging; FRAP; trafficking
The existing consensus on coexistence of transbilayer diffusion and carrier-mediated transport as two main mechanisms for drugs crossing biological membranes was recently challenged by a systems biology group. Their transporters-only hypothesis is examined in this article using published experimental evidence. The main focus is on the key claim of their hypothesis, stating that ‘the drug molecules cross pure phospholipid bilayers through transient pores that cannot form in the bilayers of cell membranes, and thus transbilayer drug transport does not exist in cells’. The analysis shows that the prior consensus remains a valid scientific view of the membrane transport of drugs.
The nuclear pore complex (NPC) is the proteinaceous nanopore that solely mediates molecular transport across the nuclear envelope between the nucleus and cytoplasm of a eukaryotic cell. Small molecules (<40 kDa) diffuse through the large pore of this multiprotein complex. A passively impermeable macromolecule tagged with a signal peptide is chaperoned through the nanopore by nuclear transport receptors (e.g., importins) owing to their interactions with barrier-forming proteins. Presently, this bimodal transport mechanism is not well understood and is described by controversial models. Herein, we report on a dynamic and spatially resolved mechanism for NPC-mediated molecular transport through nanoscale central and peripheral routes with distinct permeabilities. Specifically, we develop a nanogap-based approach of scanning electrochemical microscopy to precisely measure the extremely high permeability of the nuclear envelope to a small probe molecule, (ferrocenylmethyl)trimethylammonium. Effective medium theories indicate that the passive permeability of 5.9 × 10−2 cm/s corresponds to the free diffusion of the probe molecule through ~22 nanopores with a radius of 24 nm and a length of 35 nm. Peripheral routes are blocked by wheat germ agglutinin to yield two-fold lower permeability for 17 nm-radius central routes. This lectin is also used in fluorescence assays to find that importins facilitate the transport of signal-tagged albumin mainly through the 7 nm-thick peripheral route rather than through the sufficiently large central route. We propose that this spatial selectivity is regulated by the conformational changes in barrier-forming proteins that transiently and locally expand the impermeably thin peripheral route while blocking the central route.