Inhibition Of Active Transport Research Articles

SARS-CoV-2 Orf6 is positioned in the nuclear pore complex by Rae1 to inhibit nucleocytoplasmic transport.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) accessory protein Orf6 works as an interferon antagonist, in part, by inhibiting the nuclear import activated p-STAT1, an activator of interferon-stimulated genes, and the export of the poly(A) RNA. Insight into the transport regulatory function of Orf6 has come from the observation that Orf6 binds to the nuclear pore complex (NPC) components: Rae1 and Nup98. To gain further insight into the mechanism of Orf6-mediated transport inhibition, we examined the role of Rae1 and Nup98. We show that Rae1 alone is not necessary to support p-STAT1 import or nuclear export of poly(A) RNA. Moreover, the loss of Rae1 suppresses the transport inhibitory activity of Orf6. We propose that the Rae1/Nup98 complex strategically positions Orf6 within the NPC where it alters FG-Nup interactions and their ability to support nuclear transport. In addition, we show that Rae1 is required for normal viral protein production during SARS-CoV-2 infection presumably through its role in supporting Orf6 function.

Cross-Linking and Functional Analyses for Dimerization of a Cysteine Mutant of Glycine Transporter 1.

Glycine transporter 1 (GlyT1) is responsible for the reuptake of glycine, which regulates glutamate signaling as a co-agonist with N-methyl-D-aspartic acid (NMDA) receptors in the excitatory synapse and has been proposed to be a potential target in the development of therapies for a broad range of disorders of the central nervous system. Despite significant progress in characterizing structure and transport mechanism of the transporter, the regulation of transport function through oligomerization remains to be understood. In the present work, association of two forms of GlyT1 into dimers and higher order oligomers was detected by coimmunoprecipitation. To investigate functional properties of dimers of a GlyT1 cysteine mutant L288C, we performed oxidative cross-linking of the positioned cysteine residues in extracellular loop 3 (EL3) near the extracellular end of TM6. By analyzing the effect of copper phenanthroline (CuP)-induced dimerization on transport function, cross-linking of L288C was found to inhibit transport activity. In addition, an intramolecular ion pair Lys286-Glu289 was revealed to be critical for stabilizing EL3 in a conformation that modulates CuP-induced dimerization and transport function of the GlyT1 L288C mutant. Furthermore, the influence of transporter conformation on GlyT1 L288C dimerization was investigated. The substrate glycine, in the presence of both Na+ and Cl-, significantly reduced oxidative cross-linking, suggesting a large-scale rotation of the bundle domain during substrate transport impairs interfacial interactions between L288C protomers. The present study provides new insights into structural and functional elements regulating GlyT1 transport activity through its dimerization or oligomerization.

Predicting Hepatic Clearance of Psychotropic Drugs in Isolated Perfused Fish Livers Using a Combination of Two In Vitro Assays.

In vitro biotransformation assays with primary trout hepatocytes (RT-HEP) or liver subcellular fractions (RT-S9) have been proposed as valuable tools to help scientists and regulators better understand the toxicokinetics of chemicals. While both assays have been applied successfully to a diversity of neutral organic chemicals, only the RT-S9 assay has been applied to a large number of ionizable organic chemicals. Here, a combination of an in vitro biotransformation assay with RT-HEP with an active transport assay based on the permanent rainbow trout liver cell line RTL-W1 was used to qualitatively predict the potential hepatic clearance of nine psychotropic drugs with various degrees of ionization. Predictions were compared with rates of clearance measured in isolated perfused rainbow trout livers, and the importance of active transport was verified in the presence of the active transport inhibitor cyclosporin A. For the first time, it was demonstrated that a combination of biotransformation and active transport assays is powerful for the prediction of rates of hepatic clearance of ionizable chemicals. Ultimately, it is expected that this approach will allow for use of fewer animals while at the same time improving our confidence in the use of data from in vitro assays in chemical risk assessment.

Using Endogenous Biomarkers to Derisk Assessment of Transporter‐Mediated Drug‐Drug Interactions: A Scientific Perspective

Comprehensive characterization of transporter mediated drug-drug interactions (DDIs) is important to formulate clinical management strategies and ensure the safe and effective use of concomitantly administered drugs. The potential of a drug to inhibit transporters is predicted by comparing the ratio of the relevant concentration (depending on the transporter) and the half maximum inhibitory concentration to a predefined "cutoff" value. If the ratio is greater than the cutoff value, modeling approaches such as physiologically based pharmacokinetic modeling or a clinical DDI trial may be recommended. Because false-positive (in vitro data suggest the potential for a DDI, whereas no significant DDI is observed in vivo) and false-negative (in vitro data does not suggest the potential for a DDI, whereas significant DDI is observed in vivo) outcomes have been observed, there is interest in exploring additional approaches to facilitate prediction of transporter-mediated DDIs. The idea of assessing changes in the concentration of endogenous biomarkers (which are substrates of clinically relevant transporters) to gain insight on the potential for a drug to inhibit transporter activity has received widespread attention. This brief report describes how endogenous biomarkers may help to expand the DDI assessment toolkit, highlights some current knowledge gaps, and outlines a conceptual framework that may complement the current paradigm of predicting the potential for transporter-mediated DDIs.

Monolignol export by diffusion down a polymerization-induced concentration gradient.

Lignin, the second most abundant biopolymer, is a promising renewable energy source and chemical feedstock. A key element of lignin biosynthesis is unknown: how do lignin precursors (monolignols) get from inside the cell out to the cell wall where they are polymerized? Modeling indicates that monolignols can passively diffuse through lipid bilayers, but this has not been tested experimentally. We demonstrate significant monolignol diffusion occurs when laccases, which consume monolignols, are present on one side of the membrane. We hypothesize that lignin polymerization could deplete monomers in the wall, creating a concentration gradient driving monolignol diffusion. We developed a two-photon microscopy approach to visualize lignifying Arabidopsis thaliana root cells. Laccase mutants with reduced ability to form lignin polymer in the wall accumulated monolignols inside cells. In contrast, active transport inhibitors did not decrease lignin in the wall and scant intracellular phenolics were observed. Synthetic liposomes were engineered to encapsulate laccases, and monolignols crossed these pure lipid bilayers to form polymer within. A sink-driven diffusion mechanism explains why it has been difficult to identify genes encoding monolignol transporters and why the export of varied phenylpropanoids occurs without specificity. It also highlights an important role for cell wall oxidative enzymes in monolignol export.

Recent Advances in the Pesticide Activities of 2-Cyanoacrylate Derivatives

2-Cyanoacrylates are a unique class of compounds with high photosystem II electron transport inhibitory activity. Through the in-depth study of molecular design, synthesis, biological activity, structure-activity relationship, and action mechanism, a large number of 2-cyanoacrylate derivatives with excellent herbicidal activity were found. In addition, 2-cyanoacrylate derivatives with different substituents have also been found to have good fungicidal or anti-plant virus activities. To further guide the design and development of new pesticide candidates, the representative research work of 2-cyanoacrylate derivatives with herbicidal, fungicidal, and anti-plant virus activities was reviewed.

The Conformationally Sensitive Spatial Distance Between the TM3-4 Loop and Transmembrane Segment 7 in the Glutamate Transporter Revealed by Paired-Cysteine Mutagenesis.

Excitatory amino acid transporters can maintain extracellular glutamate concentrations lower than neurotoxic levels by transferring neurotransmitters from the synaptic cleft into surrounding glial cells and neurons. Previous work regarding the structural studies of GltPh, GltTK, excitatory amino acid transporter 1 (EAAT1), EAAT3 and alanine serine cysteine transporter 2 described the transport mechanism of the glutamate transporter in depth. However, much remains unknown about the role of the loop between transmembrane segment 3 and 4 during transport. To probe the function of this loop in the transport cycle, we engineered a pair of cysteine residues between the TM3-TM4 loop and TM7 in cysteine-less EAAT2. Here, we show that the oxidative cross-linking reagent CuPh inhibits transport activity of the paired mutant L149C/M414C, whereas DTT inhibits the effect of CuPh on transport activity of L149C/M414C. Additionally, we show that the effect of cross-linking in the mutant is due to the formation of the disulfide bond within the molecules of EAAT2. Further, L-glutamate or KCl protect, and D,L-threo-β-benzyloxy-aspartate (TBOA) increases, CuPh-induced inhibition in the L149C/M414 mutant, suggesting that the L149C and M414C cysteines are closer or farther away in the outward- or inward-facing conformations, respectively. Together, our findings provide evidence that the distance between TM3-TM4 loop and TM7 alter when substrates are transported.

Elucidating the mechanism of cellular uptake of fullerene nanoparticles

Understanding the molecular interactions between biological cells and engineered nanoparticles is a key to evaluating potential toxicities to humans and the environment. This study developed a method to determine the mechanisms by which fullerene aggregates are distributed into a representative cell line, human intestinal Caco-2 cells. First, we determined that the presence of fetal bovine serum (FBS) in the cell culture media changes the particle characteristics and inhibits particle adsorptions onto cell surfaces. Second, significantly lower amounts of fullerene were internalized at 4°C, a temperature at which active transport mechanisms are effectively impeded, than at 37°C. Third, metabolic inhibitors of active transport and a microtubule transport inhibitor decreased fullerene uptake at 37°C. Fourth, cellular uptake of fullerene increased with increasing fullerene concentration, suggesting that passive diffusion into lipid membranes contributed to uptake over the broad concentration range used in this study. Together, these results indicate fullerene transport into cells occurs via two mechanisms: passive diffusion across the lipid bilayer and active transport including microtubule involved endocytosis. The results also suggest that simple physical-chemical partitioning models do not fully describe fullerene uptake, and instead, active transport models are also required to estimate the cellular uptake and toxicity of fullerene.

Insights in the molecular mechanisms of an azole stress adapted laboratory-generated Aspergillus fumigatus strain.

A pan-azole-resistant strain was generated in vitro, in which drug transporter overexpression was a major trait. Analyses suggested a role of the transporter inhibitor milbemycin oxime in inhibiting drug transporters and potentiating azole activity.

Synthesis and biological evaluation of benzhydryl-based antiplasmodial agents possessing Plasmodium falciparum chloroquine resistance transporter (PfCRT) inhibitory activity.

Due to the surge in resistance to common therapies, malaria remains a significant concern to human health worldwide. In chloroquine (CQ)-resistant (CQ-R) strains of Plasmodium falciparum, CQ and related drugs are effluxed from the parasite's digestive vacuole (DV). This process is mediated by mutant isoforms of a protein called CQ resistance transporter (PfCRT). CQ-R strains can be partially re-sensitized to CQ by verapamil (VP), primaquine (PQ) and other compounds, and this has been shown to be due to the ability of these molecules to inhibit drug transport via PfCRT. We have previously developed a series of clotrimazole (CLT)-based antimalarial agents that possess inhibitory activity against PfCRT (4a,b). In our endeavor to develop novel PfCRT inhibitors, and to perform a structure-activity relationship analysis, we synthesized a new library of analogues. When the benzhydryl system was linked to a 4-aminoquinoline group (5a-f) the resulting compounds exhibited good cytotoxicity against both CQ-R and CQ-S strains of P.falciparum. The most potent inhibitory activity against the PfCRT-mediated transport of CQ was obtained with compound 5k. When compared to the reference compound, benzhydryl analogues of PQ (5i,j) showed a similar activity against blood-stage parasites, and a stronger invitro potency against liver-stage parasites. Unfortunately, in the invivo transmission blocking assays, 5i,j were inactive against gametocytes.

Development of a Novel Evaluation System of Tip Growth for Antifungal Compounds with the Use of Candida Albicans

Mycosis is an infection to human caused by fungi, such as mold and yeast. Among mycosis, deep mycosis which infects organs and tissues, has become a particularly serious problem. To treat those deep mycosis, chemotherapy with antifungal agents is used. However, the types of antifungals are few and the treatment options are limited. Since fungi and humans have similar cell structures, existing antifungal agents have also been reported to have side effects on humans. Furthermore, the emergence of resistant fungi has become a problem due to the long-term use of the drug.From those problems, development of an antifungal agent having a new mechanism of action is required. Therefore, our laboratory focused on tip growth, a growth method specific to fungi. Tip growth elongates the cell by repeating the decomposition and synthesis of the cell wall (β-1,3-D-Glucan). It has been reported that the cell wall-degrading enzyme β-1,3-D-Glucanase (BGL2) involved in this tip growth is vesicular transported from the inside of the cell to the growth point. We consider that targeting vesicle transport during tip growth will provide selective toxicity to fungi. The purpose of this study is to develop novel antifungal agents targeting vesicle transport during fungal tip growth. An evaluation system for visualizing vesicle transport was constructed by fusing green fluorescent protein GFP downstream of BGL2 to be transported to the growth point by expression of the protein in Saccharomyces cerevisiae (the results of the localization were shown in Fig.1).Using this evaluation system, vesicle transport inhibitory activity was screened for 100 kind herbal medicine extract. As a result, vesicle transport inhibitory activity was observed in 8 types.In addition, we attempted to construct a similar evaluation system using Candida albicans and aimed to evaluate fungi that form hyphae that cause actual mycosis.Fig.1 Confocal laser scanning microscope image of an evaluation system for antifungal compounds using S. cerevisiae.(a) Localization of BGL2-EmGFP to growth point.(b) Localization of BGL2-EmGFP to the division site.(C) Intracellular distribution of BGL2-EmGFP treated with vesicle transport inhibitor. Figure 1

TMPE Derived from Saffron Natural Monoterpene as Cytotoxic and Multidrug Resistance Reversing Agent in Colon Cancer Cells.

Terpenes constitute one of the largest groups of natural products. They exhibit a wide range of biological activities including antioxidant, anticancer, and drug resistance modulating properties. Saffron extract and its terpene constituents have been demonstrated to be cytotoxic against various types of cancer cells, including breast, liver, lung, pancreatic, and colorectal cancer. In the present work, we have studied anticancer properties of TMPE, a newly synthesized monoterpene derivative of β-cyclocitral—the main volatile produced by the stigmas of unripe crocuses. TMPE presented selective cytotoxic activity to doxorubicin-resistant colon cancer cells and was identified to be an effective MDR modulator in doxorubicin-resistant cancer cells. Synergy between this derivative and doxorubicin was observed. Most probably, TMPE inhibited transport activity of ABCB1 protein without affecting its expression level. Analysis of TMPE physicochemical parameters suggested it was not likely to be transported by ABCB1. Molecular modeling showed TMPE being more reactive molecule than the parental compound—β-cyclocitral. Analysis of electrostatic potential maps of both compounds prompted us to hypothesize that reduced reactivity as well as susceptibility to electrophilic attack were related to the lower general toxicity of β-cyclocitral. All of the above pointed to TMPE as an interesting candidate molecule for MDR reversal in cancer cells.

Method for the Detection of Insulin‐independent Glucose Uptake in Skeletal Muscle

The quantification of glucose uptake into tissues, alongside co‐solutes flux, is essential to advance basic knowledge of glucose transport and metabolism. However, comprehensive understanding of glucose metabolism has been elusive. A major player in this crucial process is muscle tissue ‐ its large mass, high catabolic rate and storage capabilities make a major contribution to glucose homeostasis. The traditional insulin‐dependent model has proven insufficient to explain a well‐documented clinical observation: Type II diabetic, insulin resistant patients can lower their blood sugar levels with frequent exercise. One challenge in studying a complex mechanism like glucose uptake in muscles is the lack of experimental tools with multi‐channel capabilities, able to quantify most of, if not all the possible related solute fluxes within current models, in‐vivo or ex‐vivo. Our goal was to develop a method to measure glucose, K+, Na+, Ca2+, Fe, Cu, Zn2+ and Mn flux in biological samples using Inductively Coupled Plasma – Mass Spectroscopy (ICP‐MS). ICP‐MS is well established as a sensitive and versatile tool for direct detection and quantification of specific elements.In this study, we developed a new ratiometric method, based on stable‐isotope dilution, to quantify 13C‐based molecules using the molar ratio of 13C/12C. This, added to the multi‐metal detection capabilities of ICP‐MS was combined for a multi‐channel ion flux measurement in mouse EDL muscle during contraction‐stimulated glucose uptake. The preliminary results demonstrated a correlation of K+ uptake with glucose uptake. We showed involvement of the Na+K+ATPase (NKA) with this process using ouabain. An isolated EDL muscle was either stimulated at 90 Hz for 5 min or left unstimulated in 11mM 13C glucose uptake solution at 32°C. The muscle was then washed at 0°C for 20 minutes to wash away excess glucose on the surface of the muscle and to inhibit transporter activity. The muscle was then digested in 1:1 HCl and H2SO4 to break it down to inorganic forms. Finally, we used ICP‐MS to quantify glucose and co‐solute transport. The transport rate for glucose (nM/g min) for stimulated tissues was 601 as compared to 9 for unstimulated tissues. The stimulated muscles also took up K+, as measured through Rb+ tracer, indicating that NKA was active during contraction‐stimulated glucose uptake. To determine which isoform of Na,K,ATPase (NKA) the glucose was coupled to, we used 0.75uM ouabain. This concentration is enough to maintain muscle contractions as well as inhibit NKA α2 but not α1. Ouabain reduced the average glucose uptake rate by 81 %. These preliminary results show that NKA α2 may be coupled to a possible novel glucose transporter in skeletal muscle tissue or to GLUT‐4 in an undescribed way. The development of a method to measure glucose concentrations in biological samples together with coupled fluxes of other ions will advance knowledge of glucose handling in health and disease. In addition, our method to reproducibly quantify C‐based organic molecules by ICP‐MS will advance applications of ICP‐MS to many other fields of biomedical research.Support or Funding InformationNIH RO1 AR 063710

Inhibition of GLUT2 transporter by geraniol from Cymbopogon martinii: a novel treatment for diabetes mellitus in streptozotocin-induced diabetic rats.

To isolate and identify the bioactive component from Cymbopogon martinii having GLUT2 transporter inhibitory activity - towards development of a novel strategy for treatment of diabetes mellitus. Isolation of bioactive component was carried out using differential solvent extraction, HPTLC and HPLC, and identification was done by GC-MS. In-vitrostudies on intestine, liver, kidney and in-vivo assessment by OGTT and long-term treatment on diabetic rats were carried out. Geraniolwas isolated and identified asbioactivecomponent. Intestinal glucose absorption demonstrated 60.28% inhibition of transport at 648.34μm of geraniol. It was found to inhibit glucose release from liver on adrenaline challenge by 89.82% at 324.17μm/ml. Kidney glycogen content doubled using 648.34μm of geraniol as compared to control. Geraniol demonstrated 2.14 times higher renal glucose output than diabetic control. OGTT demonstrated prevention of postprandial spikes. Prolonged treatment for 60days with 29.37mm/kg B.W. twice a day of geraniol improved the lipid profile, HbA1C levels and renal parameters. In mRNA studies for 10days, over expression of GLUT2 was prevented by geraniol. Inhibition of GLUT2 by geraniol has the potential to reduce hyperglycaemia and prevent secondary complications in diabetes.

'Messy' Processing of χ-conotoxin MrIA Generates Homologues with Reduced hNET Potency.

Integrated venomics techniques have shown that variable processing of conotoxins from Conus marmoreus resulted in a dramatic expansion in the number of expressed conotoxins. One conotoxin from C. marmoreus, the χ-conotoxin MrIA, is a selective inhibitor of human norepinephrine transporters (hNET) and therefore a drug candidate for attenuating chronic neuropathic pain. It has been found that “messy” processing of the MrIA transcripts results in the expression of MrIA analogs with different truncations of the pro-peptide that contains portions of the MrIA molecule. The aim of this study was to investigate if variable processing of the expressed peptides results in modulation of the existing hNET pharmacology or creates new pharmacologies. To this end, a number of MrIA analogs found in C. marmoreus venom were synthesized and evaluated for their activity at hNET receptors. While several of the analogs exhibited norepinephrine transporter inhibitory activity comparable to that of MrIA, none significantly improved on the potency of conotoxin MrIA, and those analogs with disrupted pharmacophores produced greatly reduced NET inhibition, confirming previous structure-activity relationships seen on χ-class conopeptides. Additionally, analogs were screened for new activities on ion channels using calcium influx assays, although no major new pharmacology was revealed.

Cryo-EM Structures and Mechanism of Human Multidrug ABC Transporters

Multidrug ABC transporters such as ABCB1 (P-glycoprotein) or ABCG2 (BCRP) are expressed in many tissues and mediate the translocation of a wide array of endogenous or exogenous compounds across cellular membranes. Both transporters have a protective role against xenobiotics and simultaneously affect the pharmacokinetics of commonly used drugs as well as drug-drug interactions. Their overexpression in certain tumor cells correlates with a poor prognosis and treatment outcome.To understand their broad substrate specificity, their ATP-driven transport mechanisms, and their inhibition by small-molecule compounds and externally binding antibodies, we determined high-resolution structures of ABCB1 and ABCG2 using single particle cryo-electron microscopy. The structures revealed distinct architectures and identified hydrophobic cavities that differ in size and shape, which can explain their overlapping, but non-identical substrate specificities. Combined with functional studies, transport mechanisms can be formulated for ABCB1 and ABCG2. In both cases, conformational “clamping” allows externally binding antibodies to inhibit transport activity. Bound inhibitors block the central drug-binding cavities of the transporters and prevent a full closing of the cytoplasmic ends of the transmembrane domains and of the nucleotide-binding domain interface, which would be needed for ATP hydrolysis. Chemical modification of specific compounds allows the SAR of inhibitory compounds to be studied and may provide a rational basis for the future design of ABCG2 inhibitors.

TAS-303, a Novel Selective Norepinephrine Reuptake Inhibitor that Increases Urethral Pressure in Rats, Indicating Its Potential as a Therapeutic Agent for Stress Urinary Incontinence.

Stress urinary incontinence (SUI) is characterized by involuntary leakage associated with exertion, effort, sneezing, coughing, or lifting. Duloxetine, a serotonin norepinephrine reuptake inhibitor, is approved for the treatment of patients with SUI in some European countries, but not in the United States. There is currently no globally approved pharmacological drug for the treatment of patients with SUI. Therefore, a new pharmacological treatment option is required. TAS-303 [4-piperidinyl 2,2-diphenyl-2-(propoxy-1,1,2,2,3,3,3-day7 )acetate hydrochloride] is a novel small-molecule selective norepinephrine reuptake inhibitor that displays significant norepinephrine transporter (NET) inhibitory activity toward the serotonin or dopamine transporters. In this report, we describe the pharmacological properties of TAS-303 and its effects on urethral function, using preclinical in vitro and in vivo studies. Radioligand-binding studies showed that TAS-303 selectively and potently inhibited [3H]norepinephrine binding to the human NET. Oral administration of TAS-303 (3 mg/kg) significantly increased norepinephrine levels in the plasma, whereas it did not significantly affect epinephrine, dopamine, and serotonin levels. TAS-303 (0.3, 1, and 3 mg/kg) dose-dependently increased basal urethral pressure in normal rats and leak point pressure in vaginal distention rats, exhibiting a maximal effect comparable to duloxetine. In the forced swimming test, TAS-303 (100 mg/kg) showed no significant effects on immobility time in rats, raising the possibility that this agent would have minimal central nervous system side effects at an effective dose for urethral function. These results demonstrate that TAS-303 has therapeutic potential for the treatment of patients with SUI.

Discovery of DSP-1053, a novel benzylpiperidine derivative with potent serotonin transporter inhibitory activity and partial 5-HT1A receptor agonistic activity

We have previously shown that SMP-304, a serotonin uptake inhibitor with weak 5-HT1A partial agonistic activity, may act under high serotonin levels as a 5-HT1A antagonist that improves the onset of paroxetine in the rat swimming test. However, SMP-304 is mostly metabolized by CYP2D6, indicating limited efficacy among individuals and increased side effects. To reduce CYP2D6 metabolic contribution and enhance SERT/5-HT1A binding affinity, we carried out a series of substitutions at the bromine atom in the left part of the benzene ring of SMP-304 and replaced the right part of SMP-304 with a chroman-4-one. This optimization work led to the identification of the antidepressant candidate DSP-1053 as a potent SERT inhibitor with partial 5-HT1A receptor agonistic activity. DSP-1053 showed low CYP2D6 metabolic contribution and a robust increase in serotonin levels in the rat frontal cortex.

The effects of elevated environmental CO2 on nitrite uptake in the air-breathing clown knifefish, Chitala ornata

Nitrite and carbon dioxide are common environmental contaminants in the intensive aquaculture ponds used to farm clown knifefish (Chitala ornata) in the Mekong delta, Vietnam. Here we tested the hypothesis that hypercapnia reduces nitrite uptake across the gills, because pH regulation will reduce chloride uptake and hence nitrite uptake as the two ions compete for the same transport route via the branchial HCO3−/Cl− exchanger. Fish fitted with arterial catheters were exposed to normocapnic/normoxic water (control), nitrite (1 mM), hypercapnia (21 mmHg CO2), or combined hypercapnia (acclimated hypercapnia) and nitrite for 96 h. Blood was sampled to measure acid-base status, haemoglobin derivatives and plasma ions. Plasma nitrite increased for 48 h, but levels stayed below the exposure concentration, and subsequently decreased as a result of nitrite detoxification to nitrate. The total uptake of nitrite (evaluated as [NO2−] + [NO3−]) was significantly decreased in hypercapnia, in accordance with the hypothesis. Methemoglobin and nitrosylhemoglobin levels were similarly lower during hypercapnic compared to normocapnic nitrite exposure. The respiratory acidosis induced by hypercapnia was half-compensated by bicarbonate accumulation in 96 h, which was mainly chloride-mediated (i.e. reduced Cl− influx via the branchial HCO3−/Cl− exchanger). Plasma osmolality and main ions (Na+, Cl−) were significantly decreased by hypercapnia and by nitrite exposure, consistent with inhibition of active transport. We conclude that hypercapnia induces a long-lasting, and mainly chloride-mediated acid-base regulation that reduces the uptake of nitrite across the gills.

Borneol, a messenger agent, improves central nervous system drug delivery through enhancing blood–brain barrier permeability: a preclinical systematic review and meta-analysis

To achieve sufficient blood–brain barrier (BBB), penetration is one of the biggest challenges in the development of diagnostic and therapeutic for central nervous system (CNS) disorders. Here, we conducted a systematic review and meta-analysis to assess the preclinical evidence and possible mechanisms of borneol for improving co-administration of CNS drug delivery in animal models. The electronic literature search was conducted in six databases. Fifty-eight studies with 63 comparisons involved 1137 animals were included. Among 47 studies reporting the assessments of CNS drug concentration, 45 studies showed the significant effects of borneol for improving CNS drug delivery (p<.05), whereas 2 studies showed no difference (p>.05). Nineteen comparisons showed borneol up-regulated BBB permeability (p<.05) using brain EB content (n = 8), Rh 123 content (n = 4), brain imaging agent content (n = 2), brain water content (n = 1) and observing ultrastructure of BBB (n = 4), whereas three studies showed no difference or unclear results. Seven studies reported the safety, in which one study showed borneol was reversible changes in the BBB penetration; six studies showed borneol did not increase co-administration of blood drugs concentration of peripheral tissues (p > .05). Effects of borneol are closely associated with inhibition of efflux protein function, releasement of tight junction protein, increasement of vasodilatory neurotransmitters, and inhibition of active transport by ion channels. In conclusion, borneol is a promising candidate for CNS drug delivery, mainly through mediating a multi-targeted BBB permeability.