Texas Red transport across rat and dogfish shark (Squalus acanthias) choroid plexus

Abstract

Confocal microscopy and image analysis were used to compare driving forces, specificity, and regulation of transport of the fluorescent organic anion, Texas Red (sulforhodamine 101 free acid; TR), in lateral choroid plexus (CP) isolated from rat and an evolutionarily ancient vertebrate, dogfish shark (Squalus acanthias). CP from both species exhibited concentrative, specific, and metabolism-dependent TR transport from bath to subepithelial/vascular space; at steady state, TR accumulation in vascular/subepithelial space was substantially higher than in epithelial cells. In rat CP, steady-state TR accumulation in subepithelial/vascular spaces was reduced by Na(+)-replacement, but was not affected by a 10-fold increase in buffer K(+). In shark CP, Na(+)-replacement did not alter TR accumulation in either tissue compartment; subepithelial/vascular space levels of TR were reduced in high-K(+) medium. In both species, steady-state TR accumulation was not affected by p-aminohippurate or leukotriene C4, suggesting that neither organic anion transporters (SLC22A family) nor multidrug resistance-associated proteins (ABCC family) contributed. In rat CP, digoxin was without effect, indicating that organic anion transporting polypeptide isoform 2 was not involved. Several organic anions reduced cellular and subepithelial/vascular space TR accumulation in both tissues, including estrone sulfate, taurocholate, and the Mrp1 inhibitor MK571. In rat CP, TR accumulation in subepithelial/vascular spaces increased with PKA activation (forskolin), but was not affected by PKC activation (phorbol ester). In shark, neither PKA nor PKC activation specifically affected TR transport. Thus, rat and dogfish shark CP transport TR but do so using different basic mechanisms that respond to different regulatory signals.

Quantitative fluorescence microscopy provides high resolution imaging of passive diffusion and P-gp mediated efflux at the in vivo blood–brain barrier

“…1) and their fluorescence intensity does not differ in standards prepared from brain over a range of physiological pH values (7.0–7.6) and various Na + /Ca 2+ concentrations [data not shown; but is in agreement with previous reports (Ferguson et al, 1999; Gleva et al, 1990; Reichel et al, 2008)]. Further, TxRd and R123 appeared to have stable fluorescence (within ±5%) with repeat fluorescent exposures (15–1500 ms) within tissue standards, provided that the excitation gain signal was maintained and visualization and focus of the sample was obtained in an alternate channel ( e.g., TxRd fluorescence was visualized with the eGFP channel; excitation filter of 470 ± 40 nm) (Table 1).…”

Glutamate-Mediated Upregulation of the Multidrug Resistance Protein 2 in Porcine and Human Brain Capillaries

“…To measure transport, 150 ml of isolated brain capillaries suspension was transferred to a covered imaging chamber (Ibidi, Martinsried, Germany), and the fluorescent substrate Texas Red was added. Texas Red has been repeatedly used to analyze MRP2-mediated transport in previous studies (Miller et al, 2000;Bauer et al, 2008a;Reichel et al, 2008;Wang et al, 2010). The luminal substrate accumulation was measured 1 hour later.…”

Functional and Genetic Analysis of Choroid Plexus Development in Zebrafish

“…Double-chamber culture models provide a more physiological environment containing blood, epithelial, and CSF layers (Monnot and Zheng, 2013;Spuch and Carro, 2011). In addition, whole-tissue experiments have been done by dissecting the CP from rat or dogfish shark and maintaining the tissue in a CSF-like media (Baehr et al, 2006;Reichel et al, 2008;Villalobos et al, 2002). By incubating the intact CP with fluorescent organic ions, such as fluorescein (Villalobos et al, 2002) and Texas Red (Reichel et al, 2008), these studies have been able to uncover the mechanisms involved in the transport of organic ions; for example, whether it is active or passive and whether it is Na + or K + -dependent (Baehr et al, 2006).…”

“…In addition, whole-tissue experiments have been done by dissecting the CP from rat or dogfish shark and maintaining the tissue in a CSF-like media (Baehr et al, 2006;Reichel et al, 2008;Villalobos et al, 2002). By incubating the intact CP with fluorescent organic ions, such as fluorescein (Villalobos et al, 2002) and Texas Red (Reichel et al, 2008), these studies have been able to uncover the mechanisms involved in the transport of organic ions; for example, whether it is active or passive and whether it is Na + or K + -dependent (Baehr et al, 2006). While in vitro systems provide benefits for understanding the CP's role at the cellular level, they do not provide a suitable developmental model to study how epithelial cells migrate over time to form the mature CP and do not allow for a comprehensive genetic analysis.…”