Showing papers by "Rosalinde Masereeuw published in 1994"

In Vitro and in Vivo Transport of Zidovudine (AZT) Across the Blood–Brain Barrier and the Effect of Transport Inhibitors

Abstract: The transport of the antiviral nucleoside analogue zidovudine (3′-azido-3′-deoxythymidine; AZT) into the central nervous system (CNS) was characterized in vitro and in vivo. The in vitro model consisted of primary cultures of isolated bovine capillary endothelial cells. The transport rate of AZT across the monolayer, expressed as endothelial permeability P, was determined following luminal and abluminal administration. P did not differ between the two administration sites (luminal, 1.65 ± 0.44 cm/min/103; abluminal, 1.63 ± 0.28 cm/min/103). The transport of AZT across the endothelial cell monolayer was found to be concentration independent in the range between 0.4 and 50 µg/mL. AZT transport was not affected by pre-treatment of the cells with either metabolic inhibitors (DODG and DODG/NaN3) or probenecid. This suggests that AZT passes the monolayer mainly by passive diffusion. The in vivo transport of AZT across the blood–brain barrier and the blood–CSF barrier was studied in male Wistar rats after coadministration of potential inhibitors of active transport of AZT: probenecid (organic anion transport) and thymidine (nucleoside transport). Intracerebroventricular and intravenous coadministration of probenecid caused a significant (P < 0.001) increase in the CSF/plasma concentration ratio compared to the control phase, indicating that the organic anion carrier is involved in AZT transport from CSF to blood. Since there was no effect of probenecid on the transport of AZT in vitro, it is suggested that this carrier is located at the choroid plexus. Coadministration of thymidine did not affect the CSF/plasma concentration ratio, suggesting that a nucleoside carrier system is not involved in AZT transport into or out of the CNS.

Quantification and visualization of the transport of octreotide, a somatostatin analogue, across monolayers of cerebrovascular endothelial cells.

Abstract: Confocal laser scanning microscopy (CLSM) was used to quantify and visualize the transport of the octapeptide and somatostatin analogue, octreotide (SMS 201-995, Sandostatin), across monolayers of bovine cerebrovascular endothelial cells, an in vitro model of the blood–brain barrier. The concentrations of octreotide and its conjugates in the cell culture medium were determined by radioimmunoassay (RIA). Two fluorescent conjugates of octreotide (FITC- and NBD-octreotide) were used to obtain CLSM images. The peptides did not undergo significant degradation in the presence of brain endothelial cell monolayers. The transport rate of octreotide expressed as clearance (Cl) and endothelial permeability (Pe) did not depend on either the initial concentration (between 10 nM and 1 µM) or the site of administration (luminal or abluminal side of the mono-layer), indicating the absence of saturable and/or asymmetrical transport mechanisms. The Pe of octreotide and that of the paracellular permeability marker fluorescein correlated well. Although the conjugates are more lipophilic than octreotide itself, they exhibited lower Cl and Pe, values probably because of their larger molecular size. On the CLSM images, FITC-octreotide was present only in the intercellular space, while the cells did not exhibit detectable fluorescence. Transport studies and CLSM images suggest that octreotide passes the endothelial monolayer primarily via the paracellular route without significant contribution of carrier-mediated transport.

Characterization of fluorescein transport in isolated proximal tubular cells of the rat: evidence for mitochondrial accumulation.

Abstract: The uptake and accumulation of the organic anion fluorescein-sodium (Flu-Na) was investigated in freshly isolated proximal tubular cells (PTC) of the rat kidney. Furthermore, the influence of other organic anions on Flu-Na uptake was studied in order to characterize Flu-Na transport in PTC. Flu-Na showed concentration-dependent, saturable and probenecid-sensitive transport. Comparing the transport parameters with para-aminohippurate (PAH), Flu-Na exhibited a higher affinity, but lower capacity to the organic anion transport system. The apparent Km for Flu-Na transport was 59 +/- 15 microM with a Vmax of 186 +/- 26 pmol/mg of protein/min, and for PAH 207 +/- 11 microM and 740 +/- 46 pmol/mg of protein/min, respectively. Dose-dependent inhibition of Flu-Na uptake with PAH resulted in an apparent inhibition constant Ki of 249 microM. This is in good agreement with the apparent Km of PAH, indicating that Flu-Na uptake is regulated by the PAH transport system. It is suggested that cellular uptake of both organic anions is mediated by a carrier at the basolateral membrane. However, after incubating cells with different concentrations of phenol red the percentage of maximum inhibition was 84%, which was significantly different from the 32% with PAH, suggesting that another transport system may be involved in Flu-Na uptake. Experiments with confocal laser scanning microscopy showed cellular uptake of Flu-Na and accumulation in subcellular structures. After superfusion of PTC with rhodamine 123 these structures were identified as mitochondria.