Prediction of Pregnancy-Induced Changes in Secretory and Total Renal Clearance of Drugs Transported by Organic Anion Transporters.

Pregnancy can significantly change the pharmacokinetics of drugs, including those renally secreted by organic anion transporters (OATs). Quantifying these changes in pregnant women is logistically and ethically challenging. Hence, predicting the in vivo plasma renal secretory clearance (CLsec) and renal CL (CLrenal) of OAT drugs in pregnancy is important to design correct dosing regimens of OAT drugs. Here, we first quantified the fold-change in renal OAT activity in pregnant versus nonpregnant individual using available selective OAT probe drug CLrenal data (training dataset; OAT1: tenofovir, OAT2: acyclovir, OAT3: oseltamivir carboxylate). The fold-change in OAT1 activity during the 2nd and 3rd trimester was 2.9 and 1.0 compared with nonpregnant individual, respectively. OAT2 activity increased 3.1-fold during the 3rd trimester. OAT3 activity increased 2.2, 1.7 and 1.3-fold during the 1st, 2nd, and 3rd trimester, respectively. Based on these data, we predicted the CLsec, CLrenal and total clearance ((CLtotal) of drugs in pregnancy, which are secreted by multiple OATs (verification dataset; amoxicillin, pravastatin, cefazolin and ketorolac, R-ketorolac, S-ketorolac). Then, the predicted clearances (CLs) were compared with the observed values. The predicted/observed CLsec, CLrenal, and CLtotal of drugs in pregnancy of all verification drugs were within 0.80-1.25 fold except for CLsec of amoxicillin in the 3rd trimester (0.76-fold) and cefazolin in the 2nd trimester (1.27-fold). Overall, we successfully predicted the CLsec, CLrenal, and CLtotal of drugs in pregnancy that are renally secreted by multiple OATs. This approach could be used in the future to adjust dosing regimens of renally secreted OAT drugs which are administered to pregnant women. SIGNIFICANCE STATEMENT: To the authors' knowledge, this is the first report to successfully predict renal secretory clearance and renal clearance of multiple OAT substrate drugs during pregnancy. The data presented here could be used in the future to adjust dosing regimens of renally secreted OAT drugs in pregnancy. In addition, the mechanistic approach used here could be extended to drugs transported by other renal transporters.

Quantitation of Plasma Membrane Drug Transporters in Kidney Tissue and Cell Lines Using a Novel Proteomic Approach Enabled a Prospective Prediction of Metformin Disposition.

The successful prospective incorporation of in vitro transporter kinetics in physiologically based pharmacokinetic (PBPK) models to describe drug disposition remains challenging. Although determination of scaling factors to extrapolate in vitro to in vivo transporter kinetics has been facilitated by quantitative proteomics, no robust assessment comparing membrane recoveries between different cells/tissues has been made. HEK293 cells overexpressing OCT2, MATE1, and MATE2K or human kidney cortex were homogenized and centrifuged to obtain the total membrane fractions, which were subsequently subjected to liquid-liquid extraction followed by centrifugation and precipitation to isolate plasma membrane fractions. Plasma membrane recoveries determined by quantitation of the marker Na+/K+-ATPase in lysate and plasma membrane fractions were ≤20% but within 3-fold across different cells and tissues. A separate study demonstrated that recoveries are comparable between basolateral and apical membranes of renal proximal tubules, as measured by Na+/K+-ATPase and γ-glutamyl transpeptidase 1, respectively. The plasma membrane expression of OCT2, MATE1, and MATE2K was quantified and relative expression factors (REFs) were determined as the ratio between the tissue and cell concentrations. Corrections using plasma membrane recovery had minimal impact on REF values (<2-fold). In vitro transporter kinetics of metformin were extrapolated to in vivo using the corresponding REFs in a PBPK model. The simulated metformin exposures were within 2-fold of clinical exposure. These results demonstrate that transporter REFs based on plasma membrane expression enable a prediction of transporter-mediated drug disposition. Such REFs may be estimated without the correction of plasma membrane recovery when the same procedure is applied between different matrices. SIGNIFICANCE STATEMENT: Transporter REFs based on plasma membrane expression enable in vitro-in vivo extrapolation of transporter kinetics. Plasma membrane recoveries as determined by the quantification of sodium-potassium adenosine triphosphatase were comparable between the in vitro and in vivo systems used in the present study, and therefore had minimal impact on the transporter REF values.

Opportunities and Challenges in Tunneling Nanotubes Research: How Far from Clinical Application?

Tunneling nanotubes (TNTs) are recognized long membrane nanotubes connecting distance cells. In the last decade, growing evidence has shown that these subcellular structures mediate the specific transfer of cellular materials, pathogens, and electrical signals between cells. As intercellular bridges, they play a unique role in embryonic development, collective cell migration, injured cell recovery, cancer treatment resistance, and pathogen propagation. Although TNTs have been considered as potential drug targets for treatment, there is still a long way to go to translate the research findings into clinical practice. Herein, we emphasize the heterogeneous nature of TNTs by systemically summarizing the current knowledge on their morphology, structure, and biogenesis in different types of cells. Furthermore, we address the communication efficiency and biological outcomes of TNT-dependent transport related to diseases. Finally, we discuss the opportunities and challenges of TNTs as an exciting therapeutic approach by focusing on the development of efficient and safe drugs targeting TNTs.

Depth- and direction-dependent changes in solute transport following cross-linking with riboflavin and UVA light in ex vivo porcine cornea.

Diffusion is an important mechanism of transport for nutrients and drugs throughout the avascular corneal stroma. The purpose of this study was to investigate the depth- and direction-dependent changes in stromal transport properties and their relationship to changes in collagen structure following ultraviolet A (UVA)-riboflavin induced corneal collagen cross-linking (CXL). After cross-linking in ex vivo porcine eyes, fluorescence recovery after photobleaching (FRAP) was performed to measure fluorescein diffusion in the nasal-temporal (NT) and anterior-posterior (AP) directions at corneal depths of 100, 200, and 300 µm. Second harmonic generation (SHG) imaging was also performed at these three corneal depths to quantify fiber alignment. For additional confirmation, an electrical conductivity method was employed to quantify ion permeability in the AP direction in corneal buttons and immunohistochemistry (IHC) was used to image collagen structure. Cross-linked corneas were compared to a control treatment that received the riboflavin solution without UVA light (SHAM). The results of FRAP revealed that fluorescein diffusivity decreased from 23.39 ± 11.60 µm2/s in the SHAM group to 19.87 ± 10.10 µm2/s in the CXL group. This change was dependent on depth and direction: the decrease was more pronounced in the 100 µm depth (P = 0.0005) and AP direction (P = 0.001) when compared to the effect in deeper locations and in the NT direction, respectively. Conductivity experiments confirmed a decrease in solute transport in the AP direction (P < 0.0001). FRAP also detected diffusional anisotropy in the porcine cornea: the fluorescein diffusivity in the NT direction was higher than the diffusivity in the AP direction. This anisotropy was increased following CXL treatment. Both SHG and IHC revealed a qualitative decrease in collagen crimping following CXL. Analysis of SHG images revealed an increase in coherency in the anterior 200 µm of CXL treated corneas when compared to SHAM treated corneas (P < 0.01). In conclusion, CXL results in a decrease in stromal solute transport, and this decrease is concentrated in the most anterior region and AP direction. Solute transport in the porcine cornea is anisotropic, and an increase in anisotropy with CXL may be explained by a decrease in collagen crimping.

Targeting the Four Pillars of Enterohepatic Bile Salt Cycling; Lessons From Genetics and Pharmacology.

Bile salts play a pivotal role in lipid homeostasis, are sensed by specialized receptors, and have been implicated in various disorders affecting the gut or liver. They may play a role either as culprit or as potential panacea. Four very efficient transporters mediate most of the hepatic and intestinal bile salt uptake and efflux, and are each essential for the efficient enterohepatic circulation of bile salts. Starting from the intestinal lumen, conjugated bile salts cross the otherwise impermeable lipid bilayer of (primarily terminal ileal) enterocytes through the apical sodium-dependent bile acid transporter (gene SLC10A2) and leave the enterocyte through the basolateral heteromeric organic solute transporter, which consists of an alpha and beta subunit (encoded by SLC51A and SLC51B). The Na+ -taurocholate cotransporting polypeptide (gene SLC10A1) efficiently clears the portal circulation of bile salts, and the apical bile salt export pump (gene ABCB11) pumps the bile salts out of the hepatocyte into primary bile, against a very steep concentration gradient. Recently, individuals lacking either functional Na+ -taurocholate cotransporting polypeptide or organic solute transporter have been described, completing the quartet of bile acid transport deficiencies, as apical sodium-dependent bile acid transporter and bile salt export pump deficiencies were already known for years. Novel pathophysiological insights have been obtained from knockout mice lacking functional expression of these genes and from pharmacological transporter inhibition in mice or humans. Conclusion: We provide a concise overview of the four main bile salt transport pathways and of their status as possible targets of interventions in cholestatic or metabolic disorders.

Transport mechanism of P4 ATPase phosphatidylcholine flippases.

The P4 ATPases use ATP hydrolysis to transport large lipid substrates across lipid bilayers. The structures of the endosome- and Golgi-localized phosphatidylserine flippases-such as the yeast Drs2 and human ATP8A1-have recently been reported. However, a substrate-binding site on the cytosolic side has not been found, and the transport mechanisms of P4 ATPases with other substrates are unknown. Here, we report structures of the S. cerevisiae Dnf1-Lem3 and Dnf2-Lem3 complexes. We captured substrate phosphatidylcholine molecules on both the exoplasmic and cytosolic sides and found that they have similar structures. Unexpectedly, Lem3 contributes to substrate binding. The conformational transitions of these phosphatidylcholine transporters match those of the phosphatidylserine transporters, suggesting a conserved mechanism among P4 ATPases. Dnf1/Dnf2 have a unique P domain helix-turn-helix insertion that is important for function. Therefore, P4 ATPases may have retained an overall transport mechanism while evolving distinct features for different lipid substrates.

A single power stroke by ATP binding drives substrate translocation in a heterodimeric ABC transporter.

ATP-binding cassette (ABC) transporters constitute the largest family of primary active transporters, responsible for many physiological processes and human maladies. However, the mechanism how chemical energy of ATP facilitates translocation of chemically diverse compounds across membranes is poorly understood. Here, we advance the quantitative mechanistic understanding of the heterodimeric ABC transporter TmrAB, a functional homolog of the transporter associated with antigen processing (TAP) by single-turnover analyses at single-liposome resolution. We reveal that a single conformational switch by ATP binding drives unidirectional substrate translocation. After this power stroke, ATP hydrolysis and phosphate release launch the return to the resting state, which facilitates nucleotide exchange and a new round of substrate binding and translocation. In contrast to hitherto existing steady-state assays, our single-turnover approach uncovers the power stroke in substrate translocation and the tight chemomechanical coupling in these molecular machines.

Corneal Endothelial Pump Coupling to Lactic Acid Efflux in the Rabbit and Mouse.

Purpose: Confirm that the corneal endothelial pump uses a lactate-coupled water efflux mechanism. Methods: Corneal thickness, lactate efflux, and stromal [lactate] were measured in de-epithelialized swollen and nonswollen ex vivo-mounted rabbit corneas perfused with bicarbonate-rich and bicarbonate-free Ringers, ouabain, or acetazolamide to determine if the relationships among these parameters were similar to previous data using intact corneas. The role of barrier function was tested by perfusion with calcium-free EGTA. Predictions of [lactate] in endothelial dystrophy were examined in the Slc4a11 knock out mouse. Results: De-epithelialized corneal swelling, lactate efflux, and stromal [lactate] in response to bicarbonate-free Ringers, ouabain, and acetazolamide perfusion had the same relationship as in intact corneas. The absolute amount of lactate efflux and stromal [lactate] in the de-epithelialized corneas was about half of intact corneas. De-epithelialized, swollen corneas deswelled fully with bicarbonate-rich, partially in the presence of acetazolamide, but continued to swell with bicarbonate-free or ouabain. The relationship among corneal thickness, lactate efflux, and [lactate] was the same as with nonswollen de-epithelialized corneas. In intact corneas swollen by perfusion with calcium-free EGTA, the relationship between swelling and lactate flux was the inverse of control corneas. The relationship between corneal swelling and [lactate] of intact corneas exposed to ouabain, but perfused with 7 mM lactate to simulate aqueous humor, was the same as without lactate. Corneal [lactate] in Slc4a11 knock out was twice that of wild type. Conclusions: The corneal endothelial pump works via a lactate efflux mechanism that requires an intact osmotic barrier.

Plant Lipid Bodies Traffic on Actin to Plasmodesmata Motorized by Myosin XIs.

Late 19th-century cytologists observed tiny oil drops in shoot parenchyma and seeds, but it was discovered only in 1972 that they were bound by a half unit-membrane. Later, it was found that lipid bodies (LBs) arise from the endoplasmic reticulum. Seeds are known to be packed with static LBs, coated with the LB-specific protein OLEOSIN. As shown here, apices of Populus tremula x P. tremuloides also express OLEOSIN genes and produce potentially mobile LBs. In developing buds, PtOLEOSIN (PtOLE) genes were upregulated, especially PtOLE6, concomitant with LB accumulation. To investigate LB mobility and destinations, we transformed Arabidopsis with PtOLE6-eGFP. We found that PtOLE6-eGFP fusion protein co-localized with Nile Red-stained LBs in all cell types. Moreover, PtOLE6-eGFP-tagged LBs targeted plasmodesmata, identified by the callose marker aniline blue. Pharmacological experiments with brefeldin, cytochalasin D, and oryzalin showed that LB-trafficking requires F-actin, implying involvement of myosin motors. In a triple myosin-XI knockout (xi-k/1/2), transformed with PtOLE6-eGFP, trafficking of PtOLE6-eGFP-tagged LBs was severely impaired, confirming that they move on F-actin, motorized by myosin XIs. The data reveal that LBs and OLEOSINs both function in proliferating apices and buds, and that directional trafficking of LBs to plasmodesmata requires the actomyosin system.

Estrogen Regulates Duodenal Calcium Absorption Through Differential Role of Estrogen Receptor on Calcium Transport Proteins.

BACKGROUND AND AIMS: Intestinal calcium absorption from the diet plays important role in maintaining calcium homeostasis in the body. Estrogen exerts wide physiological and pathological effects in the human. Previous studies have shown that estrogen is involved in the intestinal calcium absorption. In this study, we made investigation on the mechanism of estrogen action on duodenal calcium absorption. METHODS: The experiments were performed in mice, human, and human duodenal epithelial cells, SCBN cells. Murine duodenal calcium absorption was measured by using single pass perfusion of the duodenum in vivo. The calcium absorption of SCBN cells was evaluated by calcium imaging system. The expression of calcium transport proteins, transient receptor potential cation channel (TRPV6) and plasma membrane calcium pump (PMCA1b), in the duodenum or SCBN cells were analyzed by western blot. RESULTS: The duodenal calcium absorption in ovariectomized mice was significantly decreased, compared with control female mice, which returned to control level after 17ß-estradiol replacement treatment. Estrogen regulated the expressions of TRPV6 and PMCA1b in murine and human duodenal mucosae and SCBN cells. The further results from SCBN cells showed that 17ß-estradiol regulated calcium influx through the respective effects of estrogen receptor (ER) ɑ and ß on TRPV6 and PMCA1b. CONCLUSION: Estrogen regulates duodenal calcium absorption through differential role of ERɑ and ERß on duodenal epithelial cellular TRPV6 and PMCA1b. The study further elucidates the mechanism of estrogen on the regulation of intestinal calcium absorption.

[Ion transporters in the lungs. Use as therapeutic targets]. / Transportadores de iones en pulmón. Uso como dianas terapéuticas.

The chloride channels, sodium and bicarbonate channels, and aquaporin water channels are coordinated to maintain the airway surface liquid that is necessary for mucociliary clearance. The general mechanism for the transport of electrolytes and fluids depends mainly on the differential expression and distribution of ion transporters and pumps. Ions and water move through the paracellular or transcellular pathways. The transcellular route of electrolyte transport requires an active transport (dependent on ATP) or passive (following electrochemical gradients) of ions. The paracellular pathway is a passive process that is ultimately controlled by the predominant transepithelial electrochemical gradients. Cystic fibrosis is a hereditary disease that is produced by mutations in the gene that encode cystic fibrosis transmembrane conductance regulatory protein (CFTR) that acts as a chloride channel and performs functions of hydration of periciliary fluid and maintenance of luminal pH. The dysfunction of the chlorine channel in the respiratory epithelium determines an alteration in the bronchial secretions, with an increase in its viscosity and alteration of the mucociliary clearance and that associated with infectious processes can lead to irreversible lung damage. CFTR dysfunction has also been implicated in the pathogenesis of acute pancreatitis, chronic obstructive pulmonary disease, and bronchial hyperreactivity in asthma. There are drugs that exploit physiological mechanisms in the transport of ions with a therapeutic objective.

Los canales de cloruros, de sodio, de bicarbonato y los de agua (aquaporinas) se coordinan para mantener la cubierta líquido superficial de las vías respiratorias, que es necesaria para el aclaramiento mucociliar. El mecanismo general para el transporte de electrolitos y agua depende principalmente de la expresión diferencial y distribución de los transportadores y bombas de iones. Los iones y el agua se mueven a través de las vía paracelular o transcelular. La ruta transcelular del transporte de electrolitos requiere un transporte activo (dependiente de ATP) o pasivo (siguiendo gradientes electroquímicos) de iones. La ruta paracelular es un proceso pasivo que está controlado, en última instancia, por los gradientes electroquímicos transepiteliales predominantes. La fibrosis quística es una enfermedad hereditaria que se produce por mutaciones en el gen que codifica la proteína reguladora de la conductibilidad transmembrana de la fibrosis quística (CFTR) que actúa como un canal de cloro y cumple funciones de hidratación del líquido periciliar y mantenimiento del pH luminal. La disfunción del canal de cloro en el epitelio respiratorio determina una alteración en las secreciones bronquiales, con aumento de su viscosidad y alteración de la depuración mucociliar y que asociado a procesos infecciosos puede conducir a daño pulmonar irreversible. La disfunción del CFTR, también se ha visto implicado en la patogénesis de la pancreatitis aguda, en la enfermedad pulmonar obstructiva crónica y la hiperreactividad en el asma. Existen fármacos que aprovechan los mecanismos fisiológicos en el transporte de iones, con un objetivo terapéutico.

Transportadores de iones en pulmón: Uso como dianas terapéuticas / Ion transporters in the lungs. Use as therapeutic targets

Los canales de cloruros, de sodio, de bicarbonato y los de agua (aquaporinas) se coordinan para mantener la cubierta líquido superficial de las vías respiratorias, que es necesaria para el aclaramiento mucociliar. El mecanismo general para el transporte de electrolitos y agua depende principalmente de la expresión diferencial y distribución de los transportadores y bombas de iones. Los iones y el agua se mueven a través de las vía paracelular o transcelular. La ruta transcelular del transporte de electrolitos requiere un transporte activo (dependiente de ATP) o pasivo (siguiendo gradientes electroquímicos) de iones. La ruta paracelular es un proceso pasivo que está controlado, en última instancia, por los gradientes electroquímicos transepiteliales predominantes. La fibrosis quística es una enfermedad hereditaria que se produce por mutaciones en el gen que codifica la proteína reguladora de la conductibilidad transmembrana de la fibrosis quística (CFTR) que actúa como un canal de cloro y cumple funciones de hidratación del líquido periciliar y mantenimiento del pH luminal. La disfunción del canal de cloro en el epitelio respiratorio determina una alteración en las secreciones bronquiales, con aumento de su viscosidad y alteración de la depuración mucociliar y que asociado a procesos infecciosos puede conducir a daño pulmonar irreversible. La disfunción del CFTR, también se ha visto implicado en la patogénesis de la pancreatitis aguda, en la enfermedad pulmonar obstructiva crónica y la hiperreactividad en el asma. Existen fármacos que aprovechan los mecanismos fisiológicos en el transporte de iones, con un objetivo terapéutico.

The chloride channels, sodium and bicarbonate channels, and aquaporin water channels are coordinated to maintain the airway surface liquid that is necessary for mucociliary clearance. The general mechanism for the transport of electrolytes and fluids depends mainly on the differential expression and distribution of ion transporters and pumps. Ions and water move through the paracellular or transcellular pathways. The transcellular route of electrolyte transport requires an active transport (dependent on ATP) or passive (following electrochemical gradients) of ions. The paracellular pathway is a passive process that is ultimately controlled by the predominant transepithelial electrochemical gradients. Cystic fibrosis is a hereditary disease that is produced by mutations in the gene that encode cystic fibrosis transmembrane conductance regulatory protein (CFTR) that acts as a chloride channel and performs functions of hydration of periciliary fluid and maintenance of luminal pH. The dysfunction of the chlorine channel in the respiratory epithelium determines an alteration in the bronchial secretions, with an increase in its viscosity and alteration of the mucociliary clearance and that associated with infectious processes can lead to irreversible lung damage. CFTR dysfunction has also been implicated in the pathogenesis of acute pancreatitis, chronic obstructive pulmonary disease, and bronchial hyperreactivity in asthma. There are drugs that exploit physiological mechanisms in the transport of ions with a therapeutic objective.

Renal Excretion of Dabigatran: The Potential Role of Multidrug and Toxin Extrusion (MATE) Proteins.

Following oral administration, dabigatran etexilate (DABE) is rapidly hydrolyzed to its active form, dabigatran. DABE, but not dabigatran, presents as a P-glycoprotein (P-gp) substrate and has increasingly been used as a probe drug. Therefore, although dosed as DABE, a P-gp drug-drug interaction (DDI) is reported as a dabigatran plasma concentration ratio (perpetrator versus placebo). Because the majority of a DABE dose (80 to 85%) is recovered in urine as unchanged dabigatran (renal active secretion is ∼25% of total clearance), dabigatran was evaluated in vitro as a substrate of various human renal transporters. Active (pyrimethamine-sensitive) dabigatran uptake was observed with human embryonic kidney (HEK) 293 cells expressing multidrug and toxin extrusion protein 1 (MATE1) and 2K (MATE2K), with Michaelis-Menten constant (Km) values of 4.0 and 8.0 µM, respectively. By comparison, no uptake of 2 µM dabigatran (versus mock-transfected HEK293 cells) was evident with HEK293 cells transfected with organic cation transporters (OCT1 and OCT2) and organic anion transporters (OAT1, 2, 3, and 4). The efflux ratios of dabigatran across P-gp- and BCRP (breast cancer resistance protein)-MDCK (Madin-Darby canine kidney) cell monolayers were 1.5 and 2.0 (versus mock-MDCK cell monolayers), suggesting dabigatran is a relatively poor P-gp and BCRP substrate. Three of five drugs (verapamil, ketoconazole, and quinidine) known to interact clinically with dabigatran, as P-gp inhibitors, presented as MATE inhibitors in vitro (IC50 = 1.0 to 25.2 µM). Taken together, although no basolateral transporter was identified for dabigatran, the results suggest that apical MATE1 and MATE2K could play an important role in its renal clearance. MATE-mediated renal secretion of dabigatran needs to be considered when interpreting the results of P-gp DDI studies following DABE administration.

Is Active Transport and Leisure-Time Physical Activity Associated With Inflammatory Markers in US Adults? A Cross-Sectional Analyses From NHANES.

BACKGROUND: To investigate the association between levels of active transport and leisure-time physical activity (LTPA) with C-reactive protein, white blood cell count, body mass index, waist circumference, and lipids in a large representative sample of adults residing in the United States. METHODS: Cross-sectional data from the National Health and Nutrition Examination Survey. Adjusted multinomial logistic regressions were carried out to quantify associations between levels of self-reported active transport (or LTPA) and quintiles of anthropometric measures and serum markers. RESULTS: A total of 3248 adults were included. For serum inflammatory biomarkers, the authors observed a lower likelihood of being in the top quintile groups of circulating C-reactive protein (adjusted odds ratio [aOR]: 0.60; 95% confidence interval [CI], 0.40-0.90) and white blood cell count (aOR: 0.65; 95% CI, 0.44-0.95) with engaging in low to medium levels of active transport but not with high levels of active transport. Higher levels of LTPA were associated with lower likelihood of having high levels of serum inflammatory biomarkers (aOR: 0.60; 95% CI, 0.42-0.86 in the top C-reactive protein group and aOR: 0.58; 95% CI, 0.39-0.87 in top white blood cell group). CONCLUSIONS: Promoting active transport and/or LTPA may be a beneficial strategy to improving some, but not all, cardiometabolic health outcomes.

S-nitrosylation of cytoskeletal proteins.

Nitric oxide has pronounced effects on cellular functions normally associated with the cytoskeleton, including cell motility, shape, contraction, and mitosis. Protein S-nitrosylation, the covalent addition of a NO group to a cysteine sulfur, is a signaling pathway for nitric oxide that acts in parallel to cyclic guanosine monophosphate (cGMP), but is poorly studied compared to the latter. There is growing evidence that S-nitrosylation of cytoskeletal proteins selectively alters their function. We review that evidence, and find that S-nitrosylation of cytoskeletal targets has complementary but distinct effects to cyclic-GMP in motile and contractile cells-promoting cell migration, and biasing muscle contraction toward relaxation. However, the effects of S-nitrosylation on a host of cytoskeletal proteins and functions remains to be explored.

Exchange-mode glutamine transport across CNS cell membranes.

CNS cell membranes possess four transporters capable of exchanging Lglutamine (Gln) for other amino acids: the large neutral amino acid (LNAA) transporters LAT1 and LAT2, the hybrid basic amino acid (L-arginine (Arg), L-leucine (Leu)/LNAA transporter y+LAT2, and the L-alanine/L-serine/L-cysteine transporter 2 (ASCT2). LAT1/LAT2 and y+LAT2 are present in astrocytes, neurons and the blood brain barrier (BBB) - forming cerebral vascular endothelial cells (CVEC), while the location of ASCT2 in the individual cell types is a matter of debate. In the healthy brain, contribution of the exchangers to Gln shuttling from astrocytes to neurons and thus their role in controlling the conversion of Gln to the amino acid neurotransmitters l-glutamate (Glu) and γ-aminobutyric acid (GABA) and Gln flux across the BBB appears negligible as compared to the system A and system N uniporters. Insofar, except for the contribution of LAT1 to the maintenance of Gln homeostasis in the interstitial fluid (ISF), no well-defined CNS-specific function has been established for either of the three transporters in the healthy brain. The Gln-accepting amino acid exchangers appear to gain significance under conditions of excessive brain Gln load (glutaminosis). Excess Gln efflux across the BBB enhances influx into the brain of L-tryptophan (Trp). Excess of Trp is responsible for overloading the brain with neuroactive compounds: serotonin, kynurenic acid, quinolinic acid and/or oxindole, which contribute to neurotransmission imbalance accompanying hyperammonemia. In turn, alterations of y+LAT2-mediated Gln/Arg exchange and Arg uptake in astrocyte, modulate astrocytic nitric oxide synthesis and oxidative/nitrosative stress in ammonia-overexposed brain. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.

The bacterial lipid II flippase MurJ functions by an alternating-access mechanism.

The peptidoglycan (PG) cell wall is an essential extracytoplasmic glycopeptide polymer that safeguards bacteria against osmotic lysis and determines cellular morphology. Bacteria use multiprotein machineries for the synthesis of the PG cell wall during cell division and elongation that can be targeted by antibiotics such as the ß-lactams. Lipid II, the lipid-linked precursor for PG biogenesis, is synthesized in the inner leaflet of the cytoplasmic membrane and then translocated across the bilayer, where it is ultimately polymerized into PG. In Escherichia coli, MurJ, a member of the MOP exporter superfamily, has been recently shown to have lipid II flippase activity that depends on membrane potential. Because of its essentiality, MurJ could potentially be targeted by much needed novel antibiotics. Recent structural information suggests that a central cavity in MurJ alternates between inward- and outward-open conformations to flip lipid II, but how these conformational changes occur are unknown. Here, we utilized structure-guided cysteine cross-linking and proteolysis-coupled gel analysis to probe the conformational changes of MurJ in E. coli cells. We found that paired cysteine substitutions in transmembrane domains 2 and 8 and periplasmic loops of MurJ could be cross-linked with homobifunctional cysteine cross-linkers, indicating that MurJ can adopt both inward- and outward-facing conformations in vivo Furthermore, we show that dissipating the membrane potential with an ionophore decreases the prevalence of the inward-facing, but not the outward-facing state. Our study provides in vivo evidence that MurJ uses an alternating-access mechanism during the lipid II transport cycle.

Stochastic modeling reveals how motor protein and filament properties affect intermediate filament transport.

Intermediate filaments are a key component of the cytoskeleton. Their transport along microtubules plays an essential role in the control of the shape and structural organization of cells. To identify the key parameters responsible for the control of intermediate filament transport, we generated a model of elastic filament transport by microtubule-associated dynein and kinesin. The model is also applicable to the transport of any elastically-coupled cargoes. We investigate the effect of filament properties such as number of motor binding sites, length, and elasticity on motion of filaments. Additionally, we consider the effect of motor properties, i.e. off rates, on filament transport. When one motor has a catch bond off rate it dictates the motion, whereas when motors have the same type of off rate filaments can alternate between retrograde and anterograde motions. The elasticity of filaments optimizes the filament transport and the coordination of motors along the length of the filament.

Expression of myo-inositol cotransporters in the sciatic nerve and dorsal root ganglia in experimental diabetes

The transport of myo-inositol is the main mechanism for the maintenance of its high intracellular levels. We aimed to measure the mRNA and protein levels of myo-inositol cotransporters in the sciatic nerve (SN) and dorsal root ganglia (DRG) during experimental diabetes. Streptozotocin-induced (STZ; 4, 8, and 12 weeks; 65 mg/kg; ip) diabetic rats (DB) and age-matched euglycemic (E) rats were used for the analysis of mRNA and protein levels of sodium myo-inositol cotransporters 1, 2 (SMIT1, SMIT2) or H+/myo-inositol cotransporter (HMIT). There was a significant reduction in the mRNA levels for SMIT1 in the SN and DRG (by 36.9 and 31.0%) in the 4-week DB (DB4) group compared to the E group. SMIT2 was not expressed in SN. The mRNA level for SMIT2 was up-regulated only in the DRG in the DB4 group. On the other hand, the protein level of SMIT1 decreased by 42.5, 41.3, and 44.8% in the SN after 4, 8, and 12 weeks of diabetes, respectively. In addition, there was a decrease of 64.3 and 58.0% of HMIT in membrane and cytosolic fractions, respectively, in the SN of the DB4 group. In the DRG, there was an increase of 230 and 86.3% for SMIT1 and HMIT, respectively, in the DB12 group. The levels of the main inositol transporters, SMIT1 and HMIT, were greatly reduced in the SN but not in the DRG. SMIT-1 was selectively reduced in the sciatic nerve during experimental STZ-induced diabetes.

The scaffolding protein ZO-1 coordinates actomyosin and epithelial apical specializations in vitro and in vivo.

Polarized epithelia assemble into sheets that compartmentalize organs and generate tissue barriers by integrating apical surfaces into a single, unified structure. This tissue organization is shared across organs, species, and developmental stages. The processes that regulate development and maintenance of apical epithelial surfaces are, however, undefined. Here, using an intestinal epithelial-specific knockout (KO) mouse and cultured epithelial cells, we show that the tight junction scaffolding protein zonula occludens-1 (ZO-1) is essential for development of unified apical surfaces in vivo and in vitro We found that U5 and GuK domains of ZO-1 are necessary for proper apical surface assembly, including organization of microvilli and cortical F-actin; however, direct interactions with F-actin through the ZO-1 actin-binding region (ABR) are not required. ZO-1 lacking the PDZ1 domain, which binds claudins, rescued apical structure in ZO-1-deficient epithelia, but not in cells lacking both ZO-1 and ZO-2, suggesting that heterodimerization with ZO-2 restores PDZ1-dependent ZO-1 interactions that are vital to apical surface organization. Pharmacologic F-actin disruption, myosin II motor inhibition, or dynamin inactivation restored apical epithelial structure in vitro and in vivo, indicating that ZO-1 directs epithelial organization by regulating actomyosin contraction and membrane traffic. We conclude that multiple ZO-1-mediated interactions contribute to coordination of epithelial actomyosin function and genesis of unified apical surfaces.