Data demonstrating the challenges of determining the kinetic parameters of P-gp mediated transport of low-water soluble substrates

The presented data are related to the research article entitled “Characterization of the IPEC-J2 MDR1 (iP-gp) cell line as a tool for identification of P-gp substrates” by Ozgur et al. (2017) [1]. This data report describes the challenges of investigating the concentration-dependent transport of P-glycoprotein (P-gp) substrates with relatively low aqueous solubility. Thus, we provide solubility data on two prototypical P-gp substrates, digoxin and rhodamine 123, representing P-gp substrates with a relatively low- and high-aqueous solubility, respectively. We present a hypothetical Michaelis-Menten curve of the P-gp mediated transport of digoxin to demonstrate that the maximal donor concentration, which can be reached in the experimental transport buffer, is too low to yield transport data in the saturable range of the Michaelis-Menten relationship. Furthermore, we present data on the bidirectional transport of digoxin and rhodamine 123 across cell monolayers of the MDCK II MDR1 cell line and iP-pg cell line in the presence of the selective P-gp inhibitor, zosuquidar/LY335979

Effects of oxygen-glucose deprivation (OGD) on barrier properties and mRNA transcript levels of selected marker proteins in brain endothelial cells/astrocyte co-cultures

Ischemic stroke has been shown to induce breakdown of the blood-brain barrier, although these changes are not fully characterized. Oxygen-glucose deprivation (OGD) has been used to investigate the effects of ischemia in cultured brain capillary endothelial cells, however this involves a change of medium which in itself may affect the cells. The aim of the present study was to investigate the effect of OGD and simple medium exchange followed by 48 h of reperfusion on barrier properties of primary bovine endothelial cells co-cultured with rat astrocytes. Barrier properties were evaluated by transendothelial electrical resistance measurements, passive permeability of flux markers, RT-qPCR and immunocytochemistry. Both OGD and simple medium exchange caused an increase in endothelial monolayer permeability. This correlated with reduced transcript levels of a number of tight junction and tight junction-associated proteins (claudin-1, claudin-5, occludin, ZO-1, tricellulin, marveld3 and PECAM-1), as well as with altered transcript level of several transporters and receptors (GLUT-1, HB-EGF, InsR, TfR, two members of the low density lipoprotein receptor family, LDLR and LRP-1, and the efflux transporter BCRP). In contrast, effects induced specifically by OGD were transient de-localization of claudin-5 from the junction zone, increased InsR localization at the plasma membrane and transient downregulation of MRP-1 and P-gp transcript levels. In conclusion, OGD caused changes in claudin-5 and InsR localization, as well as in MRP-1 and P-gp transcript levels. Our results however also indicated that medium exchange alone caused changes in functional barrier properties and expression levels of wide range of proteins

Serotonin transporter protein (SERT) and P-glycoprotein (P-gp) binding activity of montanine and coccinine from three species of <em>Haemanthus</em> L. (Amaryllidaceae)

AbstractThe alkaloid rich extracts from an acid/base extraction of bulb material of Haemanthus coccineus L., H. montanus Baker and H. sanguineus Jacq. revealed that two montanine type Amaryllidaceae alkaloids, montanine (1) and coccinine (2) were the major alkaloid constituents. Together these two alkaloids constituted 88, 91 and 98% of the total alkaloid extract from each species respectively. GC–MS analysis revealed that H. coccineus and H. sanguineus had a relative abundance of coccinine (74 and 91% respectively) to montanine (14 and 7% respectively); whereas H. montanus had 20% coccinine and 71% montanine. The three extracts and two isolated alkaloids were evaluated for binding to the serotonin transporter protein (SERT) in vitro. Affinity to SERT was highest in H. coccineus (IC50=2.0±1.1μg/ml) followed by H. montanus (IC50=6.8±1.0 μg/ml) and H. sanguineus (IC50=28.7±1.1μg/ml). Montanine (IC50=121.3±3.6μM or 36.56±1.14μg/ml; Ki=66.01μM) was more active than coccinine (IC50=196.3±3.8μM or 59.15±1.08μg/ml; Ki=106.8μM), both of which were less active than the total alkaloid extracts of each species investigated. The possible synergistic effects of two coccinine/montanine mixtures (80:20 and 20:80) were investigated, however the mixtures gave similar activities as the pure compounds and did not show any increase in activity or activity similar to the total alkaloid extracts. Thus the considerably higher activity observed in the total alkaloid extracts is not correlated to the relative proportions of coccinine and montanine in the extracts and thus are likely to be due to more potent unidentified minor constituents. Both alkaloids exhibited low binding affinity to P-glycoprotein (P-gp) as demonstrated by low inhibition of calcein-AM efflux in the MDCK-MDR1 cell line. This indicates that P-gp efflux will not be limiting for blood–brain-barrier passage of the alkaloids