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1.  Modeling and Simulation of Intracellular Drug Transport and Disposition Pathways with Virtual Cell 
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.
doi:10.13188/2327-204X.1000004
PMCID: PMC3869409  PMID: 24364041
Virtual Cell; Drug Disposition; Simulation and Modeling; Pharmacokinetics; Pharmacology; Computational Biology; Systems Biology; Systems Pharmacology
2.  A CELL-BASED MOLECULAR TRANSPORT SIMULATOR FOR PHARMACOKINETIC PREDICTION AND CHEMINFORMATIC EXPLORATION 
Molecular pharmaceutics  2006;3(6):704-716.
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.
doi:10.1021/mp060046k
PMCID: PMC2710883  PMID: 17140258
Metoprolol; permeability; chemical space; computer aided drug design; virtual screening; chemical genomics; cellular pharmacokinetics; cheminformatics; drug transport; PAMPA; Biopharmaceutics Classification System
3.  Simulation-based cheminformatic analysis of organelle-targeted molecules: lysosomotropic monobasic amines 
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.
doi:10.1007/s10822-008-9194-7
PMCID: PMC2516532  PMID: 18338229
Cheminformatics; Lysosomotropic; Cellular pharmacokinetics; Drug transport; Small molecule permeability; Subcellular localization; Simulation; Rational drug design
4.  In vitro placental model optimization for nanoparticle transport studies 
Background
Advances in biomedical nanotechnology raise hopes in patient populations but may also raise questions regarding biodistribution and biocompatibility, especially during pregnancy. Special consideration must be given to the placenta as a biological barrier because a pregnant woman’s exposure to nanoparticles could have significant effects on the fetus developing in the womb. Therefore, the purpose of this study is to optimize an in vitro model for characterizing the transport of nanoparticles across human placental trophoblast cells.
Methods
The growth of BeWo (clone b30) human placental choriocarcinoma cells for nanoparticle transport studies was characterized in terms of optimized Transwell® insert type and pore size, the investigation of barrier properties by transmission electron microscopy, tight junction staining, transepithelial electrical resistance, and fluorescein sodium transport. Following the determination of nontoxic concentrations of fluorescent polystyrene nanoparticles, the cellular uptake and transport of 50 nm and 100 nm diameter particles was measured using the in vitro BeWo cell model.
Results
Particle size measurements, fluorescence readings, and confocal microscopy indicated both cellular uptake of the fluorescent polystyrene nanoparticles and the transcellular transport of these particles from the apical (maternal) to the basolateral (fetal) compartment. Over the course of 24 hours, the apparent permeability across BeWo cells grown on polycarbonate membranes (3.0 μm pore size) was four times higher for the 50 nm particles compared with the 100 nm particles.
Conclusion
The BeWo cell line has been optimized and shown to be a valid in vitro model for studying the transplacental transport of nanoparticles. Fluorescent polystyrene nanoparticle transport was size-dependent, as smaller particles reached the basal (fetal) compartment at a higher rate.
doi:10.2147/IJN.S26601
PMCID: PMC3273982  PMID: 22334780
nanoparticles; placenta; BeWo cells; transport; model optimization; nanotoxicology
5.  Ionic conductance pathways in the mouse medullary thick ascending limb of Henle. The paracellular pathway and electrogenic Cl- absorption 
The Journal of General Physiology  1986;87(4):567-590.
Net Cl- absorption in the mouse medullary thick ascending limb of Henle (mTALH) involves a furosemide-sensitive Na+:K+:2 Cl- apical membrane symport mechanism for salt entry into cells, which occurs in parallel with a Ba++-sensitive apical K+ conductance. The present studies, using the in vitro microperfused mouse mTALH, assessed the concentration dependence of blockade of this apical membrane K+-conductive pathway by Ba++ to provide estimates of the magnitudes of the transcellular (Gc) and paracellular (Gs) electrical conductances (millisiemens per square centimeter). These studies also evaluated the effects of luminal hypertonicity produced by urea on the paracellular electrical conductance, the electrical Na+/Cl- permselectivity ratio, and the morphology of in vitro mTALH segments exposed to peritubular antidiuretic hormone (ADH). Increasing luminal Ba++ concentrations, in the absence of luminal K+, produced a progressive reduction in the transcellular conductance that was maximal at 20 mM Ba++. The Ba++- sensitive transcellular conductance in the presence of ADH was 61.8 +/- 1.7 mS/cm2, or approximately 65% of the total transepithelial conductance. In phenomenological terms, the luminal Ba++-dependent blockade of the transcellular conductance exhibited negative cooperativity. The transepithelial osmotic gradient produced by luminal urea produced blebs on apical surfaces, a striking increase in shunt conductance, and a decrease in the shunt Na+/Cl- permselectivity (PNa/PCl), which approached that of free solution. The transepithelial conductance obtained with luminal 800 mM urea, 20 mM Ba++, and 0 K+ was 950 +/- 150 mS/cm2 and provided an estimate of the maximal diffusion resistance of intercellular spaces, exclusive of junctional complexes. The calculated range for junctional dilution voltages owing to interspace salt accumulation during ADH-dependent net NaCl absorption was 0.7-1.1 mV. Since the Ve accompanying ADH-dependent net NaCl absorption is 10 mV, lumen positive, virtually all of the spontaneous transepithelial voltage in the mouse mTALH is due to transcellular transport processes. Finally, we developed a series of expressions in which the ratio of net Cl- absorption to paracellular Na+ absorption could be expressed in terms of a series of electrical variables. Specifically, an analysis of paired measurement of PNa/PCl and Gs was in agreement with an electroneutral Na+:K+:2 Cl- apical entry step. Thus, for net NaCl absorption, approximately 50% of Na+ was absorbed via a paracellular route.
PMCID: PMC2215874  PMID: 3701299
6.  Basolateral Mg2+ Extrusion via CNNM4 Mediates Transcellular Mg2+ Transport across Epithelia: A Mouse Model 
PLoS Genetics  2013;9(12):e1003983.
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.
Author Summary
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.
doi:10.1371/journal.pgen.1003983
PMCID: PMC3854942  PMID: 24339795
7.  Cellular NH4+/K+ transport pathways in mouse medullary thick limb of Henle. Regulation by intracellular pH 
The Journal of General Physiology  1992;99(3):435-461.
Fluorescence and electrophysiological methods were used to determine the effects of intracellular pH (pHi) on cellular NH4+/K+ transport pathways in the renal medullary thick ascending limb of Henle (MTAL) from CD1 mice. Studies were performed in suspensions of MTAL tubules (S- MTAL) and in isolated, perfused MTAL segments (IP-MTAL). Steady-state pHi measured using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) averaged 7.42 +/- 0.02 (mean +/- SE) in S-MTAL and 7.26 +/- 0.04 in IP- MTAL. The intrinsic cellular buffering power of MTAL cells was 29.7 +/- 2.4 mM/pHi unit at pHi values between 7.0 and 7.6, but below a pHi of 7.0 the intrinsic buffering power increased linearly to approximately 50 mM/pHi unit at pHi 6.5. In IP-MTAL, NH4+ entered cells across apical membranes via both Ba(2+)-sensitive pathway and furosemide-sensitive Na+:K+(NH4+):2Cl- cotransport mechanisms. The K0.5 and maximal rate for combined apical entry were 0.5 mM and 83.3 mM/min, respectively. The apical Ba(2+)-sensitive cell conductance in IP-MTAL (Gc), which reflects the apical K+ conductance, was sensitive to pHi over a pHi range of 6.0-7.4 with an apparent K0.5 at pHi approximately 6.7. The rate of cellular NH4+ influx in IP-MTAL due to the apical Ba(2+)- sensitive NH4+ transport pathway was sensitive to reduction in cytosolic pH whether pHi was changed by acidifying the basolateral medium or by inhibition of the apical Na+:H+ exchanger with amiloride at a constant pHo of 7.4. The pHi sensitivities of Gc and apical, Ba(2+)-sensitive NH4+ influx in IP-MTAL were virtually identical. The pHi sensitivity of the Ba(2+)-sensitive NH4+ influx in S-MTAL when exposed to (apical+basolateral) NH4Cl was greater than that observed in IP-MTAL where NH4Cl was added only to apical membranes, suggesting an additional effect of intracellular NH4+/NH3 on NH4+ influx. NH4+ entry via apical Na+:K+ (NH4+):2Cl- cotransport in IP-MTAL was somewhat more sensitive to reductions in pHi than the Ba(2+)-sensitive NH4+ influx pathway; NH4+ entry decreased by 52.9 +/- 13.4% on reducing pHi from 7.31 +/- 0.17 to 6.82 +/- 0.14. These results suggest that pHi may provide a negative feedback signal for regulating the rate of apical NH4+ entry, and hence transcellular NH4+ transport, in the MTAL. A model incorporating these results is proposed which illustrates the role of both pHi and basolateral/intracellular NH4+/NH3 in regulating the rate of transcellular N H4+ transport in the MTAL.
PMCID: PMC2216601  PMID: 1588302
8.  Contributions of cellular leak pathways to net NaHCO3 and NaCl absorption. 
Journal of Clinical Investigation  1989;83(6):1859-1867.
Proton and formic acid permeabilities were measured in the in vivo microperfused rat proximal convoluted tubule by examining the effect on intracellular pH when [H] and/or [formic acid] were rapidly changed in the luminal or peritubular fluids. Apical and basolateral membrane H permeabilities were 0.52 +/- 0.07 and 0.67 +/- 0.18 cm/s, respectively. Using these permeabilities we calculate that proton backleak from the luminal fluid to cell does not contribute significantly to net proton secretion in the early proximal tubule, but may contribute in the late proximal tubule. Apical and basolateral membrane formic acid permeabilities measured at extracellular pH 6.62 were 4.6 +/- 0.5 X 10(-2) and 6.8 +/- 1.5 X 10(-2) cm/s, respectively. Control studies demonstrated that the formic acid permeabilities were not underestimated by either the simultaneous movement of formate into the cell or the efflux of formic acid across the opposite membrane. The measured apical membrane formic acid permeability is too small to support all of transcellular NaCl absorption in the rat by a mechanism that involves Na/H-Cl/formate transporters operating in parallel with formic acid nonionic diffusion.
PMCID: PMC303906  PMID: 2542374
9.  Does apical membrane GLUT2 have a role in intestinal glucose uptake? 
F1000Research  2014;3:304.
It has been proposed that the non-saturable component of intestinal glucose absorption, apparent following prolonged exposure to high intraluminal glucose concentrations, is mediated via the low affinity glucose and fructose transporter, GLUT2, upregulated within the small intestinal apical border.
The evidence that the non-saturable transport component is mediated via an apical membrane sugar transporter is that it is inhibited by phloretin, after exposure to phloridzin. Since the other apical membrane sugar transporter, GLUT5, is insensitive to inhibition by either cytochalasin B, or phloretin, GLUT2 was deduced to be the low affinity sugar transport route.
As in its uninhibited state, polarized intestinal glucose absorption depends both on coupled entry of glucose and sodium across the brush border membrane and on the enterocyte cytosolic glucose concentration exceeding that in both luminal and submucosal interstitial fluids, upregulation of GLUT2 within the intestinal brush border will usually stimulate downhill glucose reflux to the intestinal lumen from the enterocytes; thereby reducing, rather than enhancing net glucose absorption across the luminal surface.
These states are simulated with a computer model generating solutions to the differential equations for glucose, Na and water flows between luminal, cell, interstitial and capillary compartments. The model demonstrates that uphill glucose transport via SGLT1 into enterocytes, when short-circuited by any passive glucose carrier in the apical membrane, such as GLUT2, will reduce transcellular glucose absorption and thereby lead to increased paracellular flow. The model also illustrates that apical GLUT2 may usefully act as an osmoregulator to prevent excessive enterocyte volume change with altered luminal glucose concentrations.
doi:10.12688/f1000research.5934.1
PMCID: PMC4309173
10.  Molecular Mechanism of Renal Tubular Secretion of the Antimalarial Drug Chloroquine ▿ 
The antimalarial drug chloroquine is eliminated to a significant extent by renal tubular secretion. The molecular mechanism of renal chloroquine secretion remains unknown. We hypothesized that organic cation transporter 2 (OCT2) and multidrug and toxin extrusion protein 1 (MATE1), localized in the basolateral and luminal membranes of proximal tubule cells, respectively, are involved in chloroquine transport. The interaction of chloroquine with both transporters was investigated using single-transfected human embryonic kidney 293 (HEK293)-MATE1 cells in uptake experiments and single-transfected Madin-Darby canine kidney II (MDCK)-OCT2 and MDCK-MATE1 cells as well as double-transfected MDCK-OCT2-MATE1 cells grown as polarized monolayers on transwell filters. In HEK293-MATE1 cells, chloroquine competitively inhibited MATE1-mediated metformin uptake (Ki = 2.8 μM). Cellular accumulation of chloroquine was significantly lower (P < 0.001) and transcellular chloroquine transport was significantly increased (P < 0.001) in MDCK-MATE1 and MDCK-OCT2-MATE1 cells compared to vector control cells after basal addition of chloroquine (0.1 to 10 μM). In contrast, no difference in cellular accumulation or transcellular transport of chloroquine was observed between MDCK-OCT2 and vector control cells. In line with an oppositely directed proton gradient acting as a driving force for MATE1, basal-to-apical transport of chloroquine by MDCK-OCT2-MATE1 cells increased with decreasing apical pH from 7.8 to 6.0. Transcellular transport of chloroquine by MDCK-OCT2-MATE1 cells was inhibited by cimetidine, trimethoprim, and amitriptyline. Our data demonstrate that chloroquine is a substrate and potent competitive inhibitor of MATE1, whereas OCT2 seems to play no role in chloroquine uptake. Concomitantly administered MATE1 inhibitors are likely to modify the renal secretion of chloroquine.
doi:10.1128/AAC.01835-10
PMCID: PMC3122445  PMID: 21518836
11.  Physiologic upper limits of pore size of different blood capillary types and another perspective on the dual pore theory of microvascular permeability 
Background
Much of our current understanding of microvascular permeability is based on the findings of classic experimental studies of blood capillary permeability to various-sized lipid-insoluble endogenous and non-endogenous macromolecules. According to the classic small pore theory of microvascular permeability, which was formulated on the basis of the findings of studies on the transcapillary flow rates of various-sized systemically or regionally perfused endogenous macromolecules, transcapillary exchange across the capillary wall takes place through a single population of small pores that are approximately 6 nm in diameter; whereas, according to the dual pore theory of microvascular permeability, which was formulated on the basis of the findings of studies on the accumulation of various-sized systemically or regionally perfused non-endogenous macromolecules in the locoregional tissue lymphatic drainages, transcapillary exchange across the capillary wall also takes place through a separate population of large pores, or capillary leaks, that are between 24 and 60 nm in diameter. The classification of blood capillary types on the basis of differences in the physiologic upper limits of pore size to transvascular flow highlights the differences in the transcapillary exchange routes for the transvascular transport of endogenous and non-endogenous macromolecules across the capillary walls of different blood capillary types.
Methods
The findings and published data of studies on capillary wall ultrastructure and capillary microvascular permeability to lipid-insoluble endogenous and non-endogenous molecules from the 1950s to date were reviewed. In this study, the blood capillary types in different tissues and organs were classified on the basis of the physiologic upper limits of pore size to the transvascular flow of lipid-insoluble molecules. Blood capillaries were classified as non-sinusoidal or sinusoidal on the basis of capillary wall basement membrane layer continuity or lack thereof. Non-sinusoidal blood capillaries were further sub-classified as non-fenestrated or fenestrated based on the absence or presence of endothelial cells with fenestrations. The sinusoidal blood capillaries of the liver, myeloid (red) bone marrow, and spleen were sub-classified as reticuloendothelial or non-reticuloendothelial based on the phago-endocytic capacity of the endothelial cells.
Results
The physiologic upper limit of pore size for transvascular flow across capillary walls of non-sinusoidal non-fenestrated blood capillaries is less than 1 nm for those with interendothelial cell clefts lined with zona occludens junctions (i.e. brain and spinal cord), and approximately 5 nm for those with clefts lined with macula occludens junctions (i.e. skeletal muscle). The physiologic upper limit of pore size for transvascular flow across the capillary walls of non-sinusoidal fenestrated blood capillaries with diaphragmed fenestrae ranges between 6 and 12 nm (i.e. exocrine and endocrine glands); whereas, the physiologic upper limit of pore size for transvascular flow across the capillary walls of non-sinusoidal fenestrated capillaries with open 'non-diaphragmed' fenestrae is approximately 15 nm (kidney glomerulus). In the case of the sinusoidal reticuloendothelial blood capillaries of myeloid bone marrow, the transvascular transport of non-endogenous macromolecules larger than 5 nm into the bone marrow interstitial space takes place via reticuloendothelial cell-mediated phago-endocytosis and transvascular release, which is the case for systemic bone marrow imaging agents as large as 60 nm in diameter.
Conclusions
The physiologic upper limit of pore size in the capillary walls of most non-sinusoidal blood capillaries to the transcapillary passage of lipid-insoluble endogenous and non-endogenous macromolecules ranges between 5 and 12 nm. Therefore, macromolecules larger than the physiologic upper limits of pore size in the non-sinusoidal blood capillary types generally do not accumulate within the respective tissue interstitial spaces and their lymphatic drainages. In the case of reticuloendothelial sinusoidal blood capillaries of myeloid bone marrow, however, non-endogenous macromolecules as large as 60 nm in diameter can distribute into the bone marrow interstitial space via the phago-endocytic route, and then subsequently accumulate in the locoregional lymphatic drainages of tissues following absorption into the lymphatic drainage of periosteal fibrous tissues, which is the lymphatic drainage of myeloid bone marrow. When the ultrastructural basis for transcapillary exchange across the capillary walls of different capillary types is viewed in this light, it becomes evident that the physiologic evidence for the existence of aqueous large pores ranging between 24 and 60 nm in diameter in the capillary walls of blood capillaries, is circumstantial, at best.
doi:10.1186/2040-2384-2-14
PMCID: PMC2928191  PMID: 20701757
12.  Fitting the Elementary Rate Constants of the P-gp Transporter Network in the hMDR1-MDCK Confluent Cell Monolayer Using a Particle Swarm Algorithm 
PLoS ONE  2011;6(10):e25086.
P-glycoprotein, a human multidrug resistance transporter, has been extensively studied due to its importance to human health and disease. In order to understand transport kinetics via P-gp, confluent cell monolayers overexpressing P-gp are widely used. The purpose of this study is to obtain the mass action elementary rate constants for P-gp's transport and to functionally characterize members of P-gp's network, i.e., other transporters that transport P-gp substrates in hMDR1-MDCKII confluent cell monolayers and are essential to the net substrate flux. Transport of a range of concentrations of amprenavir, loperamide, quinidine and digoxin across the confluent monolayer of cells was measured in both directions, apical to basolateral and basolateral to apical. We developed a global optimization algorithm using the Particle Swarm method that can simultaneously fit all datasets to yield accurate and exhaustive fits of these elementary rate constants. The statistical sensitivity of the fitted values was determined by using 24 identical replicate fits, yielding simple averages and standard deviations for all of the kinetic parameters, including the efflux active P-gp surface density. Digoxin required additional basolateral and apical transporters, while loperamide required just a basolateral tranporter. The data were better fit by assuming bidirectional transporters, rather than active importers, suggesting that they are not MRP or active OATP transporters. The P-gp efflux rate constants for quinidine and digoxin were about 3-fold smaller than reported ATP hydrolysis rate constants from P-gp proteoliposomes. This suggests a roughly 3∶1 stoichiometry between ATP hydrolysis and P-gp transport for these two drugs. The fitted values of the elementary rate constants for these P-gp substrates support the hypotheses that the selective pressures on P-gp are to maintain a broad substrate range and to keep xenobiotics out of the cytosol, but not out of the apical membrane.
doi:10.1371/journal.pone.0025086
PMCID: PMC3196501  PMID: 22028772
13.  Mechanism of apical and basolateral Na(+)-independent Cl-/base exchange in the rabbit superficial proximal straight tubule. 
Journal of Clinical Investigation  1994;94(1):173-183.
The present study was undertaken to determine the magnitude and mechanism of base transport via the apical and basolateral Na(+)-independent Cl-/base exchangers in rabbit isolated perfused superficial S2 proximal tubules. The results demonstrate that there is an apical Na(+)-independent Cl-/base exchanger on both membranes. HCO3- fails to stimulate apical Cl-/base exchange in contrast to the basolateral exchanger. Inhibition of endogenous HCO3- production does not alter the rate of apical Cl-/base exchange in Hepes-buffered solutions. Both exchangers are inhibited by H2DIDS and furosemide; however, the basolateral anion exchanger is more sensitive to these inhibitors. The results indicate that the apical and basolateral Cl-/base exchangers differ in their transport properties and are able to transport base equivalents in the absence of formate. The formate concentration in rabbit arterial serum is approximately 6 microM and in vitro tubule formate production is < 0.6 pmol/min per mm. Formate in the micromolar range stimulates Jv in a dose-dependent manner in the absence of a transepithelial Na+ and Cl- gradient and without a measurable effect on Cl(-)-induced equivalent base flux. Apical formic acid recycling cannot be an important component of any cell model, which accounts for formic acid stimulation of transcellular NaCl transport in the rabbit superficial S2 proximal tubule. We propose that transcellular NaCl transport in this nephron segment is mediated by an apical Na+/H+ exchanger in parallel with a Cl-/OH- exchanger and that the secreted H+ and OH- ions form H2O in the tubule lumen.
PMCID: PMC296295  PMID: 8040258
14.  N-Ethylmaleimide Stimulates and Inhibits Ion Transport in Vestibular Dark Cells of Gerbil 
Auditory neuroscience  1994;1:101-109.
Vestibular dark cell epithelium was isolated from the semicircular canal of gerbils to test the proposal that the sulfhydryl alkylating agent N-ethylmaleimide (NEM) inhibits K+ secretion by this tissue and does so by reacting with a site in or near the apical membrane. Dark cell epithelium was mounted in a micro-Ussing chamber for measurements of transepithelial voltage (Vt) and resistance (Rt) or in a perfused bath on the stage of a microscope for measurement of cell height as an index of cell volume. Perfusion of the apical or basolateral side with 10−3 M NEM caused an increase in Vt superimposed upon a slower decrease of Vt, resulting in a triphasic response. There were only small changes in Rt. Under this condition, Vt is proportional to short circuit current and to K+ secretion. Both the stimulatory and the inhibitory responses of Vt were dose-dependent between 10−6 and 10−3 M NEM and the inhibition was irreversible. The specificity of the reaction of NEM with sulfhydryl groups was confirmed by the use of the reducing agent dithiothreitol (DTT). Perfusion of 5×10−4 M DTT on the apical side caused no significant changes in Vt but completely prevented both stimulation and inhibition of Vt by NEM (10−3 M). The amplitudes of the stimulation and the inhibition of Vt were greater for basolateral than for apical perfusion of NEM. Similarly, the response times for each effect were faster from the basolateral side, suggesting that the primary sites of action are at or near the basolateral membrane. The site of action of NEM was further explored by subjecting the tissue to a membrane-impermeant sulfhydryl reagent, stilbenedisulfonate maleimide (SDM). Apical perfusion of 10−3 M SDM had no effect on Vt or Rt, whereas basolateral perfusion caused a reversible increase of Vt (5.2 ± 0.5 to initially 6.8 ± 0.5 mV which relaxed after 60 s to 5.8 ± 0.5 mV) and to an initial decrease in Rt by 4%. No inhibitory phase was observed. Elevation of basolateral [K+] from 3.6 to 25 mM is known to increase Vt and reduce Rt via direct stimulation of basolateral K+ uptake and indirect stimulation of the apical membrane conductance. Basolateral perfusion of 10−3 M NEM fully inhibited the increase of Vt due to 25 mM K+. Elevation of basolateral [K+] from 3.6 to 25 mM is known to increase reversibly cell volume. NEM was found to inhibit cell swelling in a dose-dependent manner but did not initially affect the rate of shrinking after K+-induced swelling, pointing to action only on basolateral transport pathways. The effects of NEM on K+-induced cell swelling were completely prevented by 5×10−4 M DTT, demonstrating that the inhibitory effect of NEM was on sulfhydryl groups. In contrast to interpretations of NEM action in frog semicircular canal, we have found that NEM appears to stimulate an ion transport process in mammalian dark cells at an extracellular site in the basolateral membrane and inhibits another ion transport process in the basolateral membrane at another site. Inhibition by NEM from the apical side occurs most likely by diffusion of the agent to a site at or near the cytosolic side of the basolateral membrane.
PMCID: PMC3291124  PMID: 22389574
Vestibular labyrinth; epithelial transport; cell volume; dark cells; sulfhydryl reagents
15.  Assembly of Nsp1 Nucleoporins Provides Insight into Nuclear Pore Complex Gating 
PLoS Computational Biology  2014;10(3):e1003488.
Nuclear pore complexes (NPCs) form gateways for material transfer across the nuclear envelope of eukaryotic cells. Disordered proteins, rich in phenylalanine-glycine repeat motifs (FG-nups), form the central transport channel. Understanding how nups are arranged in the interior of the NPC may explain how NPC functions as a selectivity filter for transport of large molecules and a sieve-like filter for diffusion of small molecules (< or ). We employed molecular dynamics to model the structures formed by various assemblies of one kind of nup, namely the 609-aa-long FG domain of Nsp1 (Nsp1-FG). The simulations started from different initial conformations and geometrical arrangements of Nsp1-FGs. In all cases Nsp1-FGs collectively formed brush-like structures with bristles made of bundles of 2–27 nups, however, the bundles being cross-linked through single nups leaving one bundle and joining a nearby one. The degree of cross-linking varies with different initial nup conformations and arrangements. Structural analysis reveals that FG-repeats of the nups not only involve formation of bundle structures, but are abundantly present in cross-linking regions where the epitopes of FG-repeats are highly accessible. Large molecules that are assisted by transport factors (TFs) are selectively transported through NPC apparently by binding to FG-nups through populated FG-binding pockets on the TF surface. Therefore, our finding suggests that TFs bind concertedly to multiple FGs in cross-linking regions and break-up the bundles to create wide pores for themselves and their cargoes to pass. In addition, the cross-linking between Nsp1-FG bundles, arising from simulations, is found to set a molecular size limit of < for passive diffusion of molecules. Our simulations suggest that the NPC central channel, near the periphery where tethering of nups is dominant, features brush-like moderately cross-linked bundles, but in the central region, where tethering loses its effect, features a sieve-like structure of bundles and frequent cross-links.
Author Summary
Cells of higher life forms separate their genomes from the rest of the cell in a nucleus that surrounds the genome by a nuclear envelope. Hundreds of pores, each a complex made of many proteins, assure traffic into and out of the nucleus through highly selective transport: small biomolecules can pass unhindered, whereas large biomolecules need to associate with proteins called transport factors, to pass. Little is known about how the nuclear pore complexes function, a key impediment to observation being their huge size and the disordered nature of the pore interior. We investigated computationally what kind of structure the nuclear pore proteins (nups) form. In the computation we place many nups, each a 600 amino acid-long protein, into arrangements considered representative for the nuclear pore, and simulate the subsequent molecular behavior. We find that the nups form bundles of 2–27 proteins, the bundles being cross-linked when a single nup leaves a bundle and joins an adjacent one. The finding suggests an adaptive molecular mesh arrangement of nups in the nuclear pore and explains how selective transport is accomplished, namely that passage of sufficiently small molecules is unhindered by the cross-linking, but that large molecules need the assistance of transport factors to melt the cross-linking.
doi:10.1371/journal.pcbi.1003488
PMCID: PMC3952814  PMID: 24626154
16.  Osmotic water permeability of Necturus gallbladder epithelium 
The Journal of General Physiology  1989;93(4):649-679.
An electrophysiological technique that is sensitive to small changes in cell water content and has good temporal resolution was used to determine the hydraulic permeability (Lp) of Necturus gallbladder epithelium. The epithelial cells were loaded with the impermeant cation tetramethylammonium (TMA+) by transient exposure to the pore-forming ionophore nystatin in the presence of bathing solution TMA+. Upon removal of the nystatin a small amount of TMA+ is trapped within the cell. Changes in cell water content result in changes in intracellular TMA+ activity which are measured with intracellular ion-sensitive microelectrodes. We describe a method that allows us to determine the time course for the increase or decrease in the concentration of osmotic solute at the membrane surface, which allows for continuous monitoring of the difference in osmolality across the apical membrane. We also describe a new method for the determination of transepithelial hydraulic permeability (Ltp). Apical and basolateral membrane Lp's were assessed from the initial rates of change in cell water volume in response to anisosmotic mucosal or serosal bathing solutions, respectively. The corresponding values for apical and basolateral membrane Lp's were 0.66 x 10(-3) and 0.38 x 10(-3) cm/s.osmol/kg, respectively. This method underestimates the true Lp values because the nominal osmotic differences (delta II) cannot be imposed instantaneously, and because it is not possible to measure the true initial rate of volume change. A model was developed that allows for the simultaneous determination of both apical and basal membrane Lp's from a unilateral exposure to an anisosmotic bathing solution (mucosal). The estimates of apical and basal Lp with this method were 1.16 x 10(-3) and 0.84 x 10(-3) cm/s.osmol/kg, respectively. The values of Lp for the apical and basal cell membranes are sufficiently large that only a small (less than 3 mosmol/kg) transepithelial difference in osmolality is required to drive the observed rate of spontaneous fluid absorption by the gallbladder. Furthermore, comparison of membrane and transepithelial Lp's suggests that a large fraction of the transepithelial water flow is across the cells rather than across the tight junctions.
PMCID: PMC2216222  PMID: 2732678
17.  MCU encodes the pore conducting mitochondrial calcium currents 
eLife  2013;2:e00704.
Mitochondrial calcium (Ca2+) import is a well-described phenomenon regulating cell survival and ATP production. Of multiple pathways allowing such entry, the mitochondrial Ca2+ uniporter is a highly Ca2+-selective channel complex encoded by several recently-discovered genes. However, the identity of the pore-forming subunit remains to be established, since knockdown of all the candidate uniporter genes inhibit Ca2+ uptake in imaging assays, and reconstitution experiments have been equivocal. To definitively identify the channel, we use whole-mitoplast voltage-clamping, the technique that originally established the uniporter as a Ca2+ channel. We show that RNAi-mediated knockdown of the mitochondrial calcium uniporter (MCU) gene reduces mitochondrial Ca2+ current (IMiCa), whereas overexpression increases it. Additionally, a classic feature of IMiCa, its sensitivity to ruthenium red inhibition, can be abolished by a point mutation in the putative pore domain without altering current magnitude. These analyses establish that MCU encodes the pore-forming subunit of the uniporter channel.
DOI: http://dx.doi.org/10.7554/eLife.00704.001
eLife digest
Mitochondria are tiny organelles, less than a micrometre across, found inside almost all eukaryotic cells. Their main function is to act as the ‘power plant’ of the cell, generating adenosine triphosphate or ATP, which is the source of chemical energy for cellular processes. Beyond generating ATP, mitochondria perform many other functions: they contribute to various signalling pathways; they influence cellular differentiation; and they are involved in processes related to cell death.
Mitochondria are quite distinctive in appearance—they are enclosed by two membranes, a porous outer one and a largely impermeable inner membrane. Most mitochondrial functions involve proteins that control the movement of various molecules and ions across the inner membrane. One particularly important ion that must pass through this membrane is calcium; once inside the mitochondria, these calcium ions regulate cell survival and the generation of ATP.
Although several calcium import mechanisms exist, the best-studied pathway involves a pore-forming protein complex called the mitochondrial calcium uniporter. This ion channel has an exquisite selectivity, allowing only calcium into mitochondria even when other ions outnumber it a million-fold. Previously, researchers had identified several genes that are required for the formation of the uniporter, but it had not been established which of these encodes the central pore through which the calcium ions pass. Now, Chaudhuri et al. have shown that one of these—a gene called mitochondrial calcium uniporter (MCU)—codes for the protein subunit that creates the pore.
Prior studies used optical methods or purified proteins to study genes encoding the uniporter complex, producing controversial results regarding pore identity. This study uses a much more direct assay, namely electrophysiology performed on mitochondrial inner membranes. To access the inner membrane, the authors stripped off the outer membrane from whole mitochondria, and made them expand. By using a technique called voltage-clamping, Chaudhuri et al. were able to precisely measure calcium ion movement through intact or mutated channels. This technique controls confounding factors and minimizes the effect of contaminants that can plague interpretation of data acquired by other methods. They showed that blocking the expression of the MCU gene reduced the flow of calcium ions through the uniporter, whereas increasing MCU expression increased calcium transport.
One unique feature of the mitochondrial calcium uniporter is that it can be blocked by a dye called ruthenium red. Chaudhuri et al. used this property to confirm that the MCU gene encodes the pore-forming subunit of the channel complex—they identified a single point mutation in MCU that did not affect the channel’s ability to transport calcium ions, but did abolish its sensitivity to ruthenium red. Together, these results show that the MCU gene encodes the pore of the mitochondrial calcium uniporter, and should lead to further research into the physiology and structure of this channel.
DOI: http://dx.doi.org/10.7554/eLife.00704.002
doi:10.7554/eLife.00704
PMCID: PMC3673318  PMID: 23755363
mitoplast; MICU1; ruthenium red; calcium channel; electrophysiology; MCUR1; Human
18.  Transcellular water flow modulates water channel exocytosis and endocytosis in kidney collecting tubule. 
Journal of Clinical Investigation  1991;88(2):423-429.
The regulation of osmotic water permeability (Pf) by vasopressin (VP) in kidney collecting tubule involves the exocytic-endocytic trafficking of vesicles containing water channels between an intracellular compartment and apical plasma membrane. To examine effects of transcellular water flow on vesicle movement, Pf was measured with 1-s time resolution in the isolated perfused rabbit cortical collecting tubule in response to addition and removal of VP (250 microU/ml) in the presence of bath greater than lumen (B greater than L), lumen greater than bath (L greater than B), and lumen = bath (L = B) osmolalities. With VP addition, Pf increased from 12 to 240-270 x 10(-4) cm/s (37 degrees C) in 10 min. At 1 min, Pf was approximately 70 x 10(-4) cm/s for B greater than L, L greater than B, and L = B conditions. At later times, Pf increased fastest for L greater than B and slowest for B greater than L osmolalities; at 5 min, Pf was 250 x 10(-4) cm/s (L greater than B) and 158 x 10(-4) cm/s (B greater than L). With VP removal, Pf returned to pre-VP levels at the fastest rate for B greater than L and the slowest rate for L greater than B osmolalities; at 30 min, Pf was 65 x 10(-4) cm/s (B greater than L) and 183 x 10(-4) cm/s (L greater than B). For a series of osmotic gradients of different magnitudes and directions, the rates of Pf increase and decrease were dependent upon the magnitude of transcellular volume flow; control studies showed that paracellular water flux, asymmetric transcellular water pathways, or changes in cell volume could not account for the data. VP-dependent endocytosis was measured by apical uptake of rhodamine-dextran; in paired studies where the same tubule was used for + and - gradients, B greater than L and L greater than B osmolalities gave 168% and 82% of uptake measured with no gradient. In contrast, endocytosis in proximal tubule was not dependent on gradient direction. These data provide evidence that transcellular volume flow modulates the vasopressin-dependent cycling of vesicles containing water channels, suggesting a novel driving mechanism to aid or oppose the targeted, hormonally directed movement of subcellular membranes.
Images
PMCID: PMC295348  PMID: 1864956
19.  Conformational Changes during Pore Formation by the Perforin-Related Protein Pleurotolysin 
PLoS Biology  2015;13(2):e1002049.
Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ∼70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function.
Author Summary
Animals, plants, fungi, and bacteria all use pore-forming proteins of the membrane attack complex-perforin (MACPF) family as lethal, cell-killing weapons. These proteins are able to insert into the plasma membranes of target cells, creating large pores that short circuit the natural separation between the intracellular and extracellular milieu, with catastrophic results. However, the pore-forming proteins must undergo a substantial transformation from soluble precursors to a large barrel-shaped transmembrane complex as they punch their way into cells. Using a combination of X-ray crystallography and cryo electron microscopy, we have visualized, for the first time, the mechanism of action of one of these pore-forming proteins—pleurotolysin, a MACPF protein from the edible oyster mushroom. This enabled us to propose a model of the pleurotolysin pore by fitting the crystallographic structures of the pore proteins into a three-dimensional map of the pore obtained by cryo electron microscopy. We then designed a set of double mutants that allowed us to chemically trap intermediate states along the trajectory of the pore formation process, and to determine their structures too. By combining these data we proposed a detailed molecular mechanism for pore formation. The pleurotolysin first assembles into rings of 13 subunits, each of which then opens up by about 70° during pore formation. This process is accompanied by refolding and extrusion of two compact regions from each subunit into long hairpins that then zipper together to form an 80-Å wide barrel-shaped channel through the membrane.
A combination of structural methods reveals the complex process by which the perforin-like fungal toxin Pleurotolysin rearranges its structure to form a pore that punches a hole in target cell membranes.
doi:10.1371/journal.pbio.1002049
PMCID: PMC4318580  PMID: 25654333
20.  Transcellular migration of leukocytes is mediated by the endothelial lateral border recycling compartment 
The Journal of Experimental Medicine  2009;206(12):2795-2808.
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.
doi:10.1084/jem.20082745
PMCID: PMC2806621  PMID: 19887395
21.  Filipin-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules 
The Journal of Cell Biology  1994;127(5):1217-1232.
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.
PMCID: PMC2120262  PMID: 7525606
22.  Gastrointestinal Factors Influencing Zinc Absorption and Homeostasis 
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.
doi:10.1024/0300-9831/a000030
PMCID: PMC3777256  PMID: 21462106
zinc absorption; zinc transporters; zinc homeostasis
23.  Apical membrane limits urea permeation across the rat inner medullary collecting duct. 
Journal of Clinical Investigation  1990;86(4):1172-1178.
Urea diffuses across the terminal inner medullary collecting duct (IMCD) via a facilitated transport pathway. To examine the mechanism of transcellular urea transport, membrane-apparent urea (Purea) and osmotic water (Pf) permeabilities of IMCD cells were measured by quantitative light microscopy in isolated IMCD-2 tubules perfused in the absence of vasopressin. Basolateral membrane Pf, determined by addition of raffinose to the bath, was 69 microns/s. Basolateral membrane Purea, determined by substituting urea for raffinose without change in osmolality, was 14 X 10(-5) cm/s. Bath phloretin inhibited basolateral Purea by 85% without a significant effect on Pf. The basolateral reflection coefficient for urea, determined by addition of urea in the presence of phloretin, was 1.0. These results indicate that urea crosses the basolateral membrane by diffusion, and not by solvent drag. In perfused tubules, the rate of cell swelling following substitution of urea for mannitol was significantly greater with bath than lumen changes. After correcting for membrane surface area, the basolateral membrane was twofold more permeable than the apical membrane. Conclusions: (a) in the absence of vasopressin, urea permeation across the IMCD cell is limited by the apical membrane; (b) the basolateral membrane contains a phloretin-sensitive urea transporter; (c) transepithelial urea transport occurs by movement of urea through the IMCD cell.
PMCID: PMC296847  PMID: 2212006
24.  Transcellular migration of neutrophil granulocytes through the blood-cerebrospinal fluid barrier after infection with Streptococcus suis 
Background
A critical point during the course of bacterial meningitis is the excessive influx of polymorphnuclear neutrophils (PMNs) from the blood into the brain. Both paracellular and transcellular routes of leukocyte transmigration through the blood-brain barrier have been described in CNS diseases so far. Thus, we investigated the mechanism of PMN transmigration through the blood-CSF barrier under inflammatory conditions.
Methods
In an "inverted" Transwell culture model of the blood-CSF barrier, the zoonotic agent Streptococcus suis (S. suis) was used to stimulate porcine choroid plexus epithelial cells (PCPECs) specifically from the physiologically relevant basolateral side. Barrier function was analyzed by measuring TEER and TR-dextran-flux, and tight junction morphology was investigated by immunofluorescence. Route and mechanism of PMN transmigration were determined by immunofluorescence, electron microscopy and FACS analysis. Quantitative real time-PCR was used to determine expression levels of ICAM-1 and VCAM-1.
Results
Here, we show that the transmigration of PMNs through PCPECs was significantly higher after stimulation with TNFα or infection with S. suis strain 10 compared to its non-encapsulated mutant. Barrier function was not significantly affected by PMN migration alone, but in combination with S. suis infection. Tight junction and cytoskeletal actin reorganisation were also observed after stimulation with S. suis or TNFα. Most strikingly, PMNs preferentially migrated across PCPECs via the transcellular route. Extensive sequential analyses of the PMN transmigration process with Apotome®-imaging and electron microscopy revealed that paracellular migrating PMNs stop just before tight junctions. Interestingly, PMNs subsequently appeared to proceed by transcellular migration via funnel-like structures developing from the apical membrane. It is noteworthy that some PMNs contained bacteria during the transmigration process. Flow cytometric and transmigration inhibition studies with integrin-specific antibodies showed that PMN traversal is dependent on CD11b/CD18. Analysis of cell adhesion molecules in PCPECs revealed a significant increase of ICAM-1 and VCAM-1 expression after TNFα and S. suis stimulation.
Conclusion
Our data underline the relevance of the blood-CSF barrier as a gate for leukocyte entry into the CNS and suggest a novel transcellular migration step during the pathogenesis of bacterial meningitis.
doi:10.1186/1742-2094-8-51
PMCID: PMC3120695  PMID: 21592385
25.  Expression of macrophage-lymphocyte Fc receptors in Madin-Darby canine kidney cells: polarity and transcytosis differ for isoforms with or without coated pit localization domains 
The Journal of Cell Biology  1989;109(6):3291-3302.
Many cells of the immune system and certain epithelia express receptors for the Fc domain of IgG (FcR). On mouse macrophages and lymphocytes, two distinct receptor isoforms have been identified, designated FcRII- B1 and FcRII-B2. The isoforms are identical except for an in-frame insertion of 47 amino acids in the cytoplasmic tail of FcRII-B1 that blocks its ability to be internalized by clathrin-coated pits. We have recently found that at least one IgG-transporting epithelium, namely placental syncytial trophoblasts, expresses transcripts encoding a receptor similar or identical to macrophage-lymphocyte FcRII. To determine whether FcRII of hematopoietic cells might also function as a transcytotic receptor if expressed in epithelial cells, FcRII-B1 and - B2 were transfected into Madin-Darby canine kidney (MDCK) cells and grown on permeable filter units. The two FcRII isoforms exhibited different patterns of polarized expression: FcRII-B1 was localized mainly to the apical plasma membrane domain, whereas FcRII-B2 was found predominantly on the basolateral surface. As expected for FcR in placenta, FcRII-B2 and to a lesser extent FcRII-B1 mediated transcellular transport of IgG-complexes from the apical to the basolateral plasma membrane. Neither receptor mediated transcytosis in the opposite direction, although FcRII-B2 also delivered ligand to lysosomes when internalized from either the basolateral or apical domains. Furthermore, FcRII-B2 was capable of transporting monovalent antireceptor antibody Fab fragments across the cell, suggesting that transcytosis was not dependent on receptor cross-linking. These findings suggest the possibility that FcRII can mediate transepithelial IgG transport when expressed in placental syncytial trophoblasts in addition to its "classical" endocytic and signaling activities when expressed in macrophages. Because FcRII-B1 and -B2 are expressed with distinct polarities, the results also suggest that interactions with clathrin-coated pits may play a role in generating the polarized distribution of at least some plasma membrane proteins in MDCK cells.
PMCID: PMC2115896  PMID: 2574723

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