Paracellular Transport Pathway Research Articles

Investigating the Urinary Salt Losing Phenotype in R607H and L919X Mutant Mice with Distal Renal Tubular Acidosis

Introduction: The renal collecting duct consists of two cell types: principal and intercalated cells (ICs, alpha- and beta-type). These cells play a crucial role in regulating fluid, electrolyte, and acid-base homeostasis. Specifically, alpha-ICs promote secretion of protons into the urine through an apical proton ATPase while enabling the reabsorption of bicarbonate into the blood via the basolateral protein, kidney anion exchanger 1 (kAE1). Genetic mutations that impact IC cell function can lead to a condition called distal renal tubular acidosis (dRTA). Patients with dRTA typically exhibit alkaline urine, metabolic acidosis, electrolyte imbalances, and urinary ion loss. Study Objective and Hypothesis: Some dRTA patients continue to exhibit impaired conservation of sodium, despite sustained correction of their acidosis. The mechanisms that lead to this impairment need further investigation. Our lab has two dRTA kAE1 knock in (KI) mouse models, L919X (LX) and R607H (RH), that recapitulate classical metabolic acidosis seen in human patients. We hypothesize that these mice serve as an effective model for studying the cause behind the persistent urinary ion loss. The objectives of our work are to 1) determine whether our mouse models accurately reflect the urinary ion loss seen in dRTA patients, 2) comprehensively characterise the previously unpublished mouse line, kAE1 KI L919X, and 3) elucidate the mechanism behind this urinary salt loss. Methods: To test our hypothesis, we fed wild-type (WT) and dRTA mutant mice a low NaCl, acid, or combination (NaCl + acid) diet, to mirror conditions previously studied in human patients. We collected urine and plasma from the mice to measure ion concentrations and pH. We harvested and processed their kidney and gut tissues for analysis through qRT-PCR, immunoblot, and immunofluorescence to investigate acid-base and salt transports in the collecting duct and thick ascending limb. We aimed to identify potential alterations in these transporters between WT and KI mice. Results: On the low NaCl diet, there were little differences between WT and KI mice, either in comparison to each other or relative to their baseline conditions. However, the combination diet revealed a urinary salt losing phenotype in the RH and LX KI mice. These KI mice also displayed hypernatremia, hyperchloremia, and a failure to secrete potassium and to properly concentrate their urine. qRT-PCR analysis of whole kidney tissues revealed a surprising increase in the paracellular transport pathways, including claudin-4 and claudin-10b in the KI compared to WT. Initial immunoblots indicate a concordant upregulation and downregulation of protein expression that aligns with transcriptional changes seen at the mRNA level. Conclusion: Our current findings suggest a defective collecting duct function in the KI mice, thus validating our hypothesis. Additionally, there appears to be a compensatory mechanism at play in earlier nephron segments to rescue ion homeostasis. The results from this research may contribute to improved treatment options for patients that suffer with this severe disease. It will also deepen the understanding of the interconnected and complex functions of the nephron, aiding in treatment of other major conditions like hypertension or end-stage renal disease. Canadian Institutes of Health Research (CIHR), Canada Graduate Scholarship - Master's CIHR, and Kidney Foundation of Canada. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Understanding the Transepithelial Transport and Transbilayer Diffusion of the Antihypertensive Peptide Asn-Cys-Trp: Insights from Caco-2 Cell Monolayers and the DPPC Model Membrane.

Understanding the transport mechanism of the peptide Asn-Cys-Trp (NCW) is crucial to improving its intestinal absorption and bioavailability. This study investigated the absorption of NCW through Caco-2 cell monolayers and its interaction with the DPPC bilayers. Results revealed that after a 3 h incubation, the Papp (AP-BL) and Papp (BL-AP) values of NCW at a concentration of 5 mmol/L were (22.24 ± 4.52) × 10-7 and (6.63 ± 2.31) × 10-7 cm/s, respectively, with the transport rates of 1.59 ± 0.32 and 0.62 ± 0.20%, indicating its moderate absorption. NCW was found to be transported via PepT1 and paracellular transport pathways, as evidenced by the significant impact of Gly-Pro and cytochalasin D on the Papp values. Moreover, NCW upregulated ZO-1 mRNA expression. Further investigation of the ZO-1-mediated interaction between NCW and tight junction proteins will contribute to a better understanding of the paracellular transport mechanism of NCW. The interaction between NCW and the DPPC bilayers was predominantly driven by entropy. NCW permeated the bilayers through electrostatic, hydrogen bonding, and hydrophobic interactions, resulting in increased fluidity, flexibility, and disorder as well as phase transition and phase separation of the bilayers.

A biologically validated mathematical model for decoding epithelial apical, basolateral, and paracellular electrical properties.

Epithelial tissues form selective barriers to ions, nutrients, waste products, and infectious agents throughout the body. Damage to these barriers is associated with conditions such as celiac disease, cystic fibrosis, diabetes, and age-related macular degeneration. Conventional electrophysiology measurements like transepithelial resistance can quantify epithelial tissue maturity and barrier integrity but are limited in differentiating between apical, basolateral, and paracellular transport pathways. To overcome this limitation, a combination of mathematical modeling, stem cell biology, and cell physiology led to the development of 3 P-EIS, a novel mathematical model and measurement technique. 3 P-EIS employs an intracellular pipette and extracellular electrochemical impedance spectroscopy to accurately measure membrane-specific properties of epithelia, without the constraints of prior models. 3 P-EIS was validated using electronic circuit models of epithelia with known resistances and capacitances, confirming a median error of 19% (interquartile range: 14%-26%) for paracellular and transcellular resistances and capacitances (n = 5). Patient stem cell-derived retinal pigment epithelium tissues were measured using 3 P-EIS, successfully isolating the cellular responses to adenosine triphosphate. 3 P-EIS enhances quality control in epithelial cell therapies and has extensive applicability in drug testing and disease modeling, marking a significant advance in epithelial physiology.NEW & NOTEWORTHY This interdisciplinary paper integrates mathematics, biology, and physiology to measure epithelial tissue's apical, basolateral, and paracellular transport pathways. A key advancement is the inclusion of intracellular voltage recordings using a sharp pipette, enabling precise quantification of relative impedance changes between apical and basolateral membranes. This enhanced electrochemical impedance spectroscopy technique offers insights into epithelial transport dynamics, advancing disease understanding, drug interactions, and cell therapies. Its broad applicability contributes significantly to epithelial physiology research.

Effects of N-succinyl-chitosan coating on properties of astaxanthin-loaded PEG-liposomes: Environmental stability, antioxidant/antibacterial activities, and in vitro release

Astaxanthin (AST) has outstanding antioxidant and anti-inflammation bioactivities, but the low biocompatibility and stability limit its application in foods. In this study, N-succinyl-chitosan (NSC)-coated AST polyethylene glycol (PEG)-liposomes were constructed to enhance the biocompatibility, stability, and intestinal-targeted migration of AST. The AST NSC/PEG-liposomes were uniform in size, had larger particles, greater encapsulation efficiency, and better storage, pH, and temperature stability than the AST PEG-liposomes. AST NSC/PEG-liposomes exerted stronger antibacterial and antioxidant activities against Escherichia coli and Staphylococcus aureus than AST PEG-liposomes. The NSC coating not only protects AST PEG-liposomes from gastric acid but also prolongs the retention and sustained release of AST NSC/PEG-liposomes depending on the intestinal pH. Moreover, caco-2 cellular uptake studies showed that AST NSC/PEG-liposomes had higher cellular uptake efficiency than AST PEG-liposomes. And AST NSC/PEG-liposomes were taken up by caco-2 cells through clathrin mediated endocytic, macrophage pathways and paracellular transport pathway. These results further proved that AST NSC/PEG-liposomes delayed the release and promoted the intestinal absorption of AST. Hence, AST PEG-liposomes coated with NSC could potentially be used as an efficient delivery system for therapeutic AST.

Crossing the Blood-Brain Barrier: A Review on Drug Delivery Strategies for Treatment of the Central Nervous System Diseases.

Many drugs have been designed to treat diseases of the central nervous system (CNS), especially neurodegenerative diseases. However, the presence of tight junctions at the blood-brain barrier has often compromised the efficiency of drug delivery to target sites in the brain. The principles of drug delivery systems across the blood-brain barrier are dependent on substrate-specific (i.e. protein transport and transcytosis) and non-specific (i.e. transcellular and paracellular) transport pathways, which are crucial factors in attempts to design efficient drug delivery strategies. This review describes how the blood-brain barrier presents the main challenge in delivering drugs to treat brain diseases and discusses the advantages and disadvantages of ongoing neurotherapeutic delivery strategies in overcoming this limitation. In addition, we discuss the application of colloidal carrier systems, particularly nanoparticles, as potential tools for therapy for the CNS diseases.

Gastric environment-stable oral nanocarriers for in situ colorectal cancer therapy

Colorectal cancer (CRC) is a prevalent and fatal cancer. Oral administration provided the potential for in situ treatment of the colorectal cancer. However, drugs couldn't be well-absorbed mainly due to its degradation in the gastric area and poor intestinal permeability. In this study, we synthesized deoxycholic acid and hydroxybutyl decorated chitosan nanoparticles (DAHBC NPs) as oral curcumin (CUR) delivery system for colorectal cancer treatment. DAHBC with lower critical solution temperature (LCST) below 37 °C (27–33 °C) was obtained. DAHBC NPs were correspondingly stable in simulated gastric conditions (pH 1.2, 37 °C), due to the offset of size change between pH-responsive expansion and thermo-responsive shrinkage. In simulated intestinal tract (pH 7.0–7.4, 37 °C), DAHBC NPs exhibited burst release of CUR owing to the onefold effect of thermo-responsive shrinkage. DAHBC27 NPs showed the minimum CUR leakage (~10%) in simulated gastric conditions, because a furthest temperature-sensitive shrinkage caused by the lowest LCST offset the expansion in acid environment. DAHBC27 NPs induced ~10-fold increased (P < 0.05) CUR absorption by paracellular transport pathway, compared to the free CUR. Thus, DAHBC NPs stabilized in the gastric environment may be a promising oral drugs delivery system for effective in situ colorectal cancer therapy.

Claudins: a tale of interactions in the thick ascending limb

The claudins are the main proteins composing the tight junctions. The differential expression of claudin isoforms contributes to the specificity of paracellular transport pathways along the kidney tubule. Recent studies focusing on claudin-10 and claudin-16 support the longitudinal specialization of paracellular transport of cations within the thick ascending limb and demonstrate that complex tubular adaptations operate to buffer the consequences of chronic segmental lesions in the kidney.

In Vitro Characterization of the Biomimetic Properties of Poly(dimethylsiloxane) To Simulate Oral Drug Absorption

The potential use of poly(dimethylsiloxane) (PDMS) as an in vitro biomimetic analogue of the passive drug absorption process in the human gastrointestinal tract (GI) is assessed. PDMS is biomimetic because of similarities in small molecule transport, such as mechanism, ionization selectivity, lipophilicity. Nine molecular probes are used to evaluate the transport pathways and properties used to predict human oral absorption rates. The transport pathways through PDMS (bulk/pore) are analogous to transcellular (TCDT) and paracellular (PCDT) drug transport pathways. PDMS PCDT is assessed using positronium annihilation lifetime spectroscopy (PALS) and partition experiments; TCDT using diffusion and partition experiments. PALS determined that PDMS pores were uniform (D ∼ 0.85 nm), isolated, and void volume was unaffected by drug accumulation after equilibrium partitioning. Therefore, there is no PCDT or convective flow through PDMS. A strong linear correlation exists between predicted octanol-water partition coefficients and PDMS partition coefficients (LogKPDMS = 0.736 × LogPO-W - 0.971, R2 = 0.981). The pH-partition hypothesis is confirmed in PDMS using ibuprofen over pH 2-12. Diffusivity through PDMS is a function of lipophilicity and polar surface area K × DPDMS = 4.46 × 10-8 × e2.91×LogKPDMS(R2 = 0.963) and [Formula: see text] (R2 = 0.973). Varying the mass% of curing agent changed the lipophilicity and diffusivity (p < 0.02), but not practically (K × D = 2.23 × 10-5cm2s-1 vs 2.60 × 10-5cm2s-1), and does affect elastic modulus (3.2% = 0.3 MPa to 25% = 3.2 MPa).

Magnetic forces enable controlled drug delivery by disrupting endothelial cell-cell junctions

The vascular endothelium presents a major transport barrier to drug delivery by only allowing selective extravasation of solutes and small molecules. Therefore, enhancing drug transport across the endothelial barrier has to rely on leaky vessels arising from disease states such as pathological angiogenesis and inflammatory response. Here we show that the permeability of vascular endothelium can be increased using an external magnetic field to temporarily disrupt endothelial adherens junctions through internalized iron oxide nanoparticles, activating the paracellular transport pathway and facilitating the local extravasation of circulating substances. This approach provides a physically controlled drug delivery method harnessing the biology of endothelial adherens junction and opens a new avenue for drug delivery in a broad range of biomedical research and therapeutic applications.

Ouabain promotes partial epithelial to mesenchymal transition (EMT) changes in human autosomal dominant polycystic kidney disease (ADPKD) cells

The hormone ouabain has been shown to enhance the cystic phenotype of autosomal dominant polycystic kidney disease (ADPKD). Among other characteristics, the ADPKD phenotype includes cell de-differentiation and epithelial to mesenchymal transition (EMT). Here, we determined whether physiological concentrations of ouabain induces EMT in human renal epithelial cells from patients with ADPKD. We found that ADPKD cells respond to ouabain with a decrease in expression of the epithelial marker E-cadherin and increase in the expression of the mesenchymal markers N-cadherin, α smooth muscle actin (αSMA) and collagen-I; and the tight junction protein occludin and claudin-1. Other adhesion molecules, such as ZO-1, β-catenin and vinculin were not significantly modified by ouabain. At the cellular level, ouabain stimulated ADPKD cell migration, reduced cell-cell interaction, and the ability of ADPKD cells to form aggregates. Moreover, ouabain increased the transepithelial electrical resistance of ADPKD cell monolayers, suggesting that the paracellular transport pathway was preserved in the cells. These effects of ouabain were not observed in normal human kidney (NHK) cells. Altogether these results show a novel role for ouabain in ADPKD, inducing changes that lead to a partial EMT phenotype in the cells. These effects further support the key role that ouabain has as a factor that promotes the cystic characteristics of ADPKD cells.

Intestinal absorption and renal reabsorption of calcium throughout postnatal development.

Calcium is vital for many physiological functions including bone mineralization. Postnatal deposition of calcium into bone is greatest in infancy and continues through childhood and adolescence until peek mineral density is reached in early adulthood. Thereafter, bone mineral density remains static until it eventually declines in later life. A positive calcium balance, i.e. more calcium absorbed than excreted, is crucial to bone deposition during growth and thus to peek bone mineral density. Dietary calcium is absorbed from the intestine into the blood. It is then filtered by the renal glomerulus and either reabsorbed by the tubule or excreted in the urine. Calcium can be (re)absorbed across intestinal and renal epithelia via both transcellular and paracellular pathways. Current evidence suggests that significant intestinal and renal calcium transport changes occur throughout development. However, the molecular details of these alterations are incompletely delineated. Here we first briefly review the current model of calcium transport in the intestine and renal tubule in the adult. Then, we describe what is known with regard to calcium handling through postnatal development, and how alterations may aid in mediating a positive calcium balance. The role of transcellular and paracellular calcium transport pathways and the contribution of specific intestinal and tubular segments vary with age. However, the current literature highlights knowledge gaps in how specifically intestinal and renal calcium (re)absorption occurs early in postnatal development. Future research should clarify the specific changes in calcium transport throughout early postnatal development including mediators of these alterations enabling appropriate bone mineralization. Impact statement This mini review outlines the current state of knowledge pertaining to the molecules and mechanisms maintaining a positive calcium balance throughout postnatal development. This process is essential to achieving optimal bone mineral density in early adulthood, thereby lowering the lifetime risk of osteoporosis.

Transcellular Pathways in Lymphatic Endothelial Cells Regulate Changes in Solute Transport by Fluid Stress.

The transport of interstitial fluid and solutes into lymphatic vessels is important for maintaining interstitial homeostasis and delivering antigens and soluble factors to the lymph node for immune surveillance. Transendothelial transport across lymphatic endothelial cells (LECs) is commonly considered to occur paracellularly, or between cell-cell junctions, and driven by local pressure and concentration gradients. However, emerging evidence suggests that LECs also play active roles in regulating interstitial solute balance and can scavenge and store antigens, raising the possibility that vesicular or transcellular pathways may be important in lymphatic solute transport. The aim of this study was to determine the relative importance of transcellular (vesicular) versus paracellular transport pathways by LECs and how mechanical stress (ie, fluid flow conditioning) alters either pathway. We demonstrate that transcellular transport mechanisms substantially contribute to lymphatic solute transport and that solute uptake occurs in both caveolae- and clathrin-coated vesicles. In vivo, intracelluar uptake of fluorescently labeled albumin after intradermal injection by LECs was similar to that of dermal dendritic cells. In vitro, we developed a method to differentially quantify intracellular solute uptake versus transendothelial transport by LECs. LECs preconditioned to 1 µm/s transmural flow demonstrated increased uptake and basal-to-apical solute transport, which could be substantially reversed by blocking dynamin-dependent vesicle formation. These findings reveal the importance of intracellular transport in steady-state lymph formation and suggest that LECs use transcellular mechanisms in parallel to the well-described paracellular route to modulate solute transport from the interstitium according to biomechanical cues.

Gintonin absorption in intestinal model systems

BackgroundRecently, we identified a novel ginseng-derived lysophosphatidic acid receptor ligand, called gintonin. We showed that gintonin induces [Ca2+]i transient-mediated morphological changes, proliferation, and migration in cells expressing lysophosphatidic acid receptors and that oral administration of gintonin exhibits anti-Alzheimer disease effects in model mice. However, little is known about the intestinal absorption of gintonin. The aim of this study was to investigate gintonin absorption using two model systems. MethodsGintonin membrane permeation was examined using a parallel artificial membrane permeation assay, and gintonin absorption was evaluated in a mouse everted intestinal sac model. ResultsThe parallel artificial membrane permeation assay showed that gintonin could permeate an artificial membrane in a dose-dependent manner. In the everted sac model, gintonin absorption increased with incubation time (from 0 min to 60 min), followed by a decrease in absorption. Gintonin absorption into everted sacs was also dose dependent, with a nonlinear correlation between gintonin absorption and concentration at 0.1–3 mg/mL and saturation at 3–5 mg/mL. Gintonin absorption was inhibited by the Rho kinase inhibitor Y-27632 and the sodium–glucose transporter inhibitor phloridzin. Moreover, lipid extraction with methanol also attenuated gintonin absorption, suggesting the importance of the lipid portion of gintonin in absorption. This result shows that gintonin might be absorbed through passive diffusion, paracellular, and active transport pathways. ConclusionThe present study shows that gintonin could be absorbed in the intestine through transcellular and paracellular diffusion, and active transport. In addition, the lipid component of gintonin might play a key role in its intestinal absorption.

The kidney tight junction (Review)

The tight junction is an important subcellular organelle which plays a vital role in epithelial barrier function. Claudin, as the integral membrane component of tight junctions, creates a paracellular transport pathway for various ions to be reabsorbed by the kidneys. This review summarizes advances in claudin structure, function and pathophysiology in kidney diseases. Different claudin species confer selective paracellular permeability to each of three major renal tubular segments: the proximal tubule, the thick ascending limb of Henle’s loop and the distal nephron. Defects in claudin function can cause a wide spectrum of kidney diseases, such as hypomagnesemia, hypercalciuria, kidney stones and hypertension. Studies using transgenic mouse models with claudin mutations have recapitulated several of these renal disease phenotypes and have elucidated the underlying biological mechanisms. Modern recording approaches based upon scanning ion conductance microscopy may resolve the biophysical nature of claudin transport function and provide novel insight into tight junction architecture.

Ablation of MMP9 Gene Ameliorates Paracellular Permeability and Fibrinogen–Amyloid Beta Complex Formation during Hyperhomocysteinemia

Increased blood level of homocysteine (Hcy), called hyperhomocysteinemia (HHcy) accompanies many cognitive disorders including Alzheimer's disease. We hypothesized that HHcy-enhanced cerebrovascular permeability occurs via activation of matrix metalloproteinase-9 (MMP9) and leads to an increased formation of fibrinogen-β-amyloid (Fg-Aβ) complex. Cerebrovascular permeability changes were assessed in C57BL/6J (wild type, WT), cystathionine-β-synthase heterozygote (Cbs+/-, a genetic model of HHcy), MMP9 gene knockout (Mmp9-/-), and Cbs and Mmp9 double knockout (Cbs+/-/Mmp9-/-) mice using a dual-tracer probing method. Expression of vascular endothelial cadherin (VE-cadherin) and Fg-Aβ complex formation was assessed in mouse brain cryosections by immunohistochemistry. Short-term memory of mice was assessed with a novel object recognition test. The cerebrovascular permeability in Cbs+/- mice was increased via mainly the paracellular transport pathway. VE-cadherin expression was the lowest and Fg-Aβ complex formation was the highest along with the diminished short-term memory in Cbs+/- mice. These effects of HHcy were ameliorated in Cbs+/-/Mmp9-/- mice. Thus, HHcy causes activation of MMP9 increasing cerebrovascular permeability by downregulation of VE-cadherin resulting in an enhanced formation of Fg-Aβ complex that can be associated with loss of memory. These data may lead to the identification of new targets for therapeutic intervention that can modulate HHcy-induced cerebrovascular permeability and resultant pathologies.

Sphingolipids affect fibrinogen-induced caveolar transcytosis and cerebrovascular permeability.

Inflammation-induced vascular endothelial dysfunction can allow plasma proteins to cross the vascular wall, causing edema. Proteins may traverse the vascular wall through two main pathways, the paracellular and transcellular transport pathways. Paracellular transport involves changes in endothelial cell junction proteins, while transcellular transport involves caveolar transcytosis. Since both processes are associated with filamentous actin formation, the two pathways are interconnected. Therefore, it is difficult to differentiate the prevailing role of one or the other pathway during various pathologies causing an increase in vascular permeability. Using a newly developed dual-tracer probing method, we differentiated transcellular from paracellular transport during hyperfibrinogenemia (HFg), an increase in fibrinogen (Fg) content. Roles of cholesterol and sphingolipids in formation of functional caveolae were assessed using a cholesterol chelator, methyl-β-cyclodextrin, and the de novo sphingolipid synthesis inhibitor myriocin. Fg-induced formation of functional caveolae was defined by association and colocalization of Na+-K+-ATPase and plasmalemmal vesicle-associated protein-1 with use of Förster resonance energy transfer and total internal reflection fluorescence microscopy, respectively. HFg increased permeability of the endothelial cell layer mainly through the transcellular pathway. While MβCD blocked Fg-increased transcellular and paracellular transport, myriocin affected only transcellular transport. Less pial venular leakage of albumin was observed in myriocin-treated HFg mice. HFg induced greater formation of functional caveolae, as indicated by colocalization of Na+-K+-ATPase with plasmalemmal vesicle-associated protein-1 by Förster resonance energy transfer and total internal reflection fluorescence microscopy. Our results suggest that elevated blood levels of Fg alter cerebrovascular permeability mainly by affecting caveolae-mediated transcytosis through modulation of de novo sphingolipid synthesis.

Significant water absorption goes paracellular in kidney proximal tubules

it had been a fundamental dispute for several decades, whether water passes epithelia such as kidney proximal tubule or small intestine along the transcellular, paracellular, or both pathways. By discovery of the transmembranal aquaporin water channels (AQP), the molecular basis of the

Ablation of MMP‐9 gene ameliorates paracellular permeability and fibrinogen‐amyloid beta plaque formation during hyperhomocysteinemia

Many inflammatory and cognitive disorders including Alzheimer's disease are accompanied by hyperhomocysteinemia (HHcy). We tasted the hypothesis that HHcy increases microvascular permeability affecting mainly paracellular transport via activation of matrix metalloproteinase‐9 (MMP‐9) leading to an increased formation of fibrinogen (Fg) and β‐amyloid (Aβ) complex.Using a dual‐tracer probing method, changes in cerebrovascular permeability were assessed in male wild type (WT, C57BL/6J), cystathionine β‐synthase heterozygote (CBS+/−, a genetic model of HHcy), MMP‐9 gene knockout (MMP9−/−), and CBS and MMP‐9 double‐knockout (CBS+/−/MMP9−/−) mice. Expression of vascular endothelial cadherin (VE‐cadherin) and Fg‐Aβ plaque formation was assessed in mouse brain cryo‐sections.We found that cerebrovascular permeability in CBS+/− mice was mainly via paracellular transport pathway and it was greater than that in other groups. Expression of VE‐cadherin was lowest and Fg‐Aβ plaque formation was greatest in CBS+/− mice. Absence of MMP‐9 activity in MMP9−/− mice ameliorated these effects.Thus, HHcy increases cerebrovascular permeability by downregulating VE‐cadherin through MMP‐9 activation and enhances Fg‐Aβ plaque formation. These data may lead to identification of new targets for therapeutic intervention that can modulate inflammation‐induced cerebrovascular permeability.

Roles of PKC and phospho‑adducin in transepithelial fluid secretion by Malpighian tubules of the yellow fever mosquito

The diuretic hormone aedeskinin‑III is known to increase the paracellular Cl- conductance in Malpighian (renal) tubules of the mosquito Aedes aegypti via a G protein-coupled receptor. The increase serves the blood-meal-initiated diuresis and is associated with elevated levels of Ca2+ and phosphorylated adducin in the cytosol of tubule. In the present study we have cloned adducin in Aedes Malpighian tubules and investigated its physiological roles. Immunolabeling experiments are consistent with the association of adducin with the cortical cytoskeleton, especially near the apical brush border of the tubule. An antibody against phosphorylated adducin revealed the transient phosphorylation of adducin 2 min after stimulating tubules with aedeskinin‑III. The PKC inhibitor bisindolylmaleimide‑I blocked the phosphorylation of adducin as well as the electrophysiological and diuretic effects of aedeskinin‑III. Bisindolylmaleimide‑I also inhibited fluid secretion in control tubules. Phorbol 12‑myristate 13‑acetate increased phosphorylated adducin levels in Malpighian tubules, but it inhibited fluid secretion. Thus, the phosphorylation of adducin by PKC alone is insufficient to trigger diuretic rates of fluid secretion; elevated levels of intracellular Ca2+ may also be required. The above results suggest that the phosphorylation of adducin, which is known to destabilize the cytoskeleton, may (1) facilitate the traffic of transporters into the apical brush border supporting diuretic rates of cation secretion and (2) destabilize proteins in the septate junction thereby enabling paracellular anion (Cl‑) secretion at diuretic rates. Moreover, PKC and the phosphorylation of adducin play a central role in control and diuretic tubules, consistent with the dynamic behavior of both transcellular and paracellular transport pathways.

Acetaminophen changes paracellular transport activity through regulation of the tight junction protein in an intestinal barrier model

permeability of the small molecules Lucifer Yellow (ca. 520 Da) and FITC-dextran (3– 5k Da) but not of large molecules (40 kDa FITC-dextran). The tight junction protein ZO-1 and its tyrosine phosphorylation were upregulated after 24 h treatment with APAP. Conclusions Tight junction proteins play an important role in maintaining the intestinal barrier function. We assume that APAP affects the paracellular transport pathway through disassembly of the tight junction. We suppose that APAP leads to remodelling of the tight junction protein ZO-1 through changes in the level of protein expression and in tyrosine phosphorylation. APAP may reduce the bioavailability of co-administered substances through remodelling the tight junction by regulating ZO-1 and its tyrosine phosphorylation. As a result, the paracellular transport activity for small molecules is decreased as is net intestinal absorption.