Fluorescein-methotrexate transport in dogfish shark (Squalus acanthias) choroid plexus

Abstract

The vertebrate choroid plexus removes potentially toxic metabolites and xenobiotics from cerebrospinal fluid (CSF) to blood for subsequent excretion in urine and bile. We used confocal microscopy and quantitative image analysis to characterize the mechanisms driving transport of the large organic anion, fluorescein-methotrexate (FL-MTX), from bath (CSF-side) to blood vessels in intact lateral choroid plexus from dogfish shark, Squalus acanthias, an evolutionarily ancient vertebrate. With 2 microM FL-MTX in the bath, steady-state fluorescence in the subepithelium/vascular space exceeded bath levels by 5- to 10-fold, and fluorescence in the epithelial cells was slightly below bath levels. FL-MTX accumulation in both tissue compartments was reduced by NaCN, Na removal, and ouabain, but not by a 10-fold increase in medium K. Certain organic anions, e.g., probenecid, MTX, and taurocholate, reduced FL-MTX accumulation in both tissue compartments; p-aminohippurate and estrone sulfate reduced subepithelial/vascular accumulation, but not cellular accumulation. At low concentrations, digoxin, leukotriene C4, and MK-571 reduced fluorescence in the subepithelium/vascular space while increasing cellular fluorescence, indicating preferential inhibition of efflux over uptake. In the presence of 10 microM digoxin (reduced efflux, enhanced cellular accumulation), cellular FL-MTX accumulation was specific, concentrative, and Na dependent. Thus transepithelial FL-MTX transport involved the following two carrier-mediated steps: electroneutral, Na-dependent uptake at the apical membrane and electroneutral efflux at the basolateral membrane. Finally, FL-MTX accumulation in both tissue compartments was reduced by phorbol ester and increased by forskolin, indicating antagonistic modulation by protein kinase C and protein kinase A.

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

“…This also appears to be the case for shark CP, since uptake was not inhibited by high concentrations of PAH or ES (2). In rat CP, TR uptake was Na ϩ -independent and insensitive to inhibition by high concentrations of PAH, 2,4-D, and cimetidine (present study).…”

“…Our recent experiments have shown that transport of FL-MTX in shark CP is under control of protein kinases, increasing when PKA is activated and decreasing when PKC is activated (2). Figure 10 shows differential effects of PKA activator on TR transport in rat and shark CP.…”

“…Our previous imaging-based studies of organic anion transport in rat and shark CP demonstrated three patterns of inhibition by nonfluorescent organic anions (2,4,5,20). First, parallel reductions in cellular and subepithelial/vascular space accumulation indicate inhibition of uptake at the apical membrane, but does not provide information about possible effects at the basolateral membrane.…”

“…In shark CP, where TR uptake is also Na ϩ -independent (present study), an Oatp family member could be responsible for TR uptake. As in rat, this shark uptake transporter is clearly different from those responsible for FL and FL-MTX uptake, which are Na ϩ -dependent (2,20). Mdr1 appears to be expressed in rat CP (8), and the question arises, whether Mdr1 is able to establish a barrier against MDR1 substrates used, e.g., MTX.…”

“…This permits conclusions to be drawn with regard to the mechanisms contributing to both apical uptake and basolateral efflux. To date, we have applied this approach to two fluorescent organic anions in mouse, rat, and shark CP: one small, FL [325 kDa;(5,9,20)], and the second large, FL-methotrexate (MTX) [960 kDa;(2,15,18)]. In the present study, we compare the transport mechanisms in rat and shark CP for an organic anion of intermediate mass, Texas Red (sulforhodamine 101 free acid, 606 Da; TR).…”