texas-red and verlukast

Quantitative fluorescent microscopy is an emerging technology that has provided significant insight into cellular dye accumulation, organelle function, and tissue physiology. However, historically dyes have only been used to qualitatively or semi-quantitatively (fold change) determine changes in blood-brain barrier (BBB) integrity. Herein, we present a novel method to calculate the blood to brain transfer rates of the dyes rhodamine 123 and Texas red across the in situ BBB. We observed that rhodamine 123 is subject to p-glycoprotein mediated efflux at the rat BBB and can be increased nearly 20-fold with p-glycoprotein inhibition. However, Texas Red appears to not be subject to MRP2 mediated efflux at the rat BBB, agreeing with literature reports suggesting MRP2 may lack functionality at the normal rat BBB. Lastly, we present data demonstrating that once dyes have crossed the BBB, diffusion of the dye molecule is not as instantaneous as has been previously suggested. We propose that future work can now be completed to (1) match BBB transfer coefficients to interstitial diffusion constants and (2) use dyes with specific affinities to cellular organelles or that have specific properties (e.g., subject to efflux transporters) to more fully understand BBB physiology.

Topics: Algorithms; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Autoradiography; Blood-Brain Barrier; Calcium Channel Blockers; Cells, Cultured; Data Interpretation, Statistical; Fluorescent Dyes; Green Fluorescent Proteins; Image Processing, Computer-Assisted; Kinetics; Linear Models; Male; Microscopy, Fluorescence; Perfusion; Propionates; Quinolines; Rats; Rats, Inbred F344; Reference Standards; Rhodamine 123; Verapamil; Xanthenes

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.

Topics: Animals; Biological Transport; Choroid Plexus; Colforsin; Cyclic AMP-Dependent Protein Kinases; Estrone; Female; In Vitro Techniques; Kinetics; Leukotriene C4; Male; Meglumine; Methotrexate; Models, Biological; Organic Anion Transporters; p-Aminohippuric Acid; Potassium; Propionates; Protein Kinase C; Quinolines; Rats; Rats, Sprague-Dawley; Sodium Cyanide; Squalus acanthias; Taurocholic Acid; Tetradecanoylphorbol Acetate; Xanthenes