Thyroxine Transport in Choroid Plexus*

The role of the choroid plexus in thyroid hormone transport between body and brain, suggested by strong synthesis and secretion of transthyretin in this tissue, was investigated in in vitro and in vivo systems. Rat choroid plexus pieces incubated in vitro were found to accumulate thyroid hormones from surrounding medium in a non-saturable process. At equilibrium, the ratio of thyroid hormone concentration in choroid plexus pieces to that in medium decreased upon in- creasing the concentration of transthyretin in the medium. Fluorescence quenching of fluorophores located at different depths in liposome membranes showed maximal hormone accumulation in the middle of the phospholipid bilayer. Partition coefficients of thyroxine and triiodothyronine between lipid and aqueous phase were about 20,000. After intravenous injection of ‘261-labeled thyroid hormones, choroid plexus and parts of the brain steadily accumulated ‘261-thyroxine, but not [‘2”I]triiodothy- ronine, for many hours. The accumulation of lZ6I-thy-roxine in choroid plexus preceded that in brain. The amount of 1261-thyroxine in non-brain tissues and the {1261]triiodothyronine content of all tissues decreased steadily beginning immediately after injection. A model is proposed for thyroxine transport from the bloodstream into cerebrospinal fluid based on parti- tioning of thyroxine between choroid plexus and surrounding fluids and binding of thyroxine to transthy-

secreted by choroid plexus was found to be transthyretin (12). Apparently, choroid plexus epithelial cells are among the cells most highly specialized for the synthesis of one particular protein. The ratio of the concentration in cerebrospinal fluid to that in serum is higher for transthyretin than for all other plasma proteins, except cystatin C (formerly called gamma trace) (13, 14) and P2-microglobulin (for review see Ref. 15).
With the choroid plexus specializing in the synthesis of transthyretin and being part of the interface between the brain and the rest of the body, we addressed the question of whether the choroid plexus is involved in the transport of thyroid hormones from the blood to cerebrospinal fluid and brain by providing transthyretin. The distribution of L-thyroxine and L-triiodothyronine in rat choroid plexus pieces incubated in vitro and in a model system consisting of liposomes of phospholipid bilayers was studied and compared with the in vivo distribution of injected radioactive L-triiodothyronine and L-thyroxine. A model for the mechanism by which the choroid plexus may contribute to the transport of thyroxine between the bloodstream and cerebrospinal fluid by expression of the gene for a thyroid hormone transport protein is proposed.

L-Thyronine and L-Triiodothyronine Accumulate by a Nonsaturable Mechanism within Choroid Plexus Pieces Incubated
in Vitro-Choroid plexus obtained from the brains of rats were incubated with radioactive thyroxine or triiodothyronine i n vitro for different lengths of time as described under "Experimental Procedures." After separation of choroid plexus pieces and medium, the distribution of radioactivity was determined (Fig. 1). The rates of uptake of both thyroid hormones were highest at time 0 and decreased thereafter. Eventually, an equilibrium was reached with 61% of total thyroxine and 56% of total triiodothyronine found associated with the choroid plexus pieces. The final ratio of thyroxine or triiodothyronine concentration in choroid plexus over that in medium was about 240 and 190, respectively. For this comparison, a homogeneous distribution of thyroxine or triiodothyronine is assumed throughout choroid plexus tissue. Most of choroid plexus tissue is an aqueous phase. As is shown below, thyroxine and triiodothyronine accumulate very strongly in the lipid phase of a two-phase aqueous/lipid sys-Portions of this paper (including "Experimental Procedures," part of "Results," Figs. 5, 6, 8, 10, and 12, and Table 11) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 86M-4086, cite the authors, and include a check or money order for $3.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press. tem. Assuming a lipid content of choroid plexus of about 1% and a predominant location of thyroxine and triiodothyronine in the lipid phase of choroid plexus, the ratios of thyroxine and triiodothyronine concentrations in choroid plexus lipid over those in the medium plus the aqueous phase of choroid plexus could be expected to be 2 orders of magnitude higher than the above values. The uptake of thyroxine, and also that of triiodothyronine, was not inhibited when a 100,000-fold excess of non-radioactive hormone (final concentration 1 PM) had been added to the incubation medium. As shown in Fig. 2 the ratio of the concentration of thyroxine or triiodothyronine in choroid plexus incubated in uitro for 80 min over that in the medium was independent of the total amount of added hormone. The amount of hormone taken up varied in direct proportion to the total amount of hormone added.
Release of Thyroxine and Triiodothyronine from Choroid Plexus Incubated in Vitro Leads to an Equilibrium of Insidel Outside Concentrations of Thyroxine and Triiodothyronine: This Equilibrium Is Similar to That Established after Incubation of Choroid Plexus with Thyroxine or Triiodothyronine Added at the Beginning of the Experiment-Choroid plexus pieces were "loaded with radioactive thyroid hormones by incubation of choroid plexus with '"I-thyroxine or triiodothyronine for 1 h. Choroid plexus pieces and medium were then separated by centrifugation, and, after washing in medium free of thyroxine or triiodothyronine, choroid plexus pieces were incubated for different lengths of time in medium without thyroxine or triiodothyronine added. The release of radioactive thyroxine, or triiodothyronine, from the incubated choroid plexus pieces into the surrounding medium was analyzed as described under "Experimental Procedures." The rates of release were highest at time 0 and decreased thereafter (Fig.  3). Equilibria were reached with 30% of total thyroxine and about 40% of total triiodothyronine outside the choroid plexus pieces. Final ratios of concentrations in choroid plexus over those in medium were about 350 for thyroxine and about 220 for triiodothyronine. Apparently, the distribution of thyroid hormones between choroid plexus and medium approached similar equilibria, independent of whether the hormones were introduced into the system via the choroid plexus pieces or via the medium surrounding choroid plexus. Neither uptake nor release was inhibited when protein synthesis was interrupted by added cycloheximide (final concentration 30 p~) .

Kinetics of Uptake and Release of Thyroxine and Triwdothyronine into and from Choroid Plexus Incubated in Vitro-
The uptake and release of hormones could be described by the equations shown in Figs. 1 and 3. These curves represent first-order reactions (for derivation, see Miniprint). Rate constants, kin and k,,,, and initial reaction rates, dX,/dt and dX,,,/dt, were calculated from the equations. These rate constants are independent of concentrations, Xmed, xcp, x,,, (concentration in medium, in choroid plexus, and total, respectively). The initial reaction rates are proportional to X,, or Xmed at time 0 and to the rate constants. The values obtained are summarized in Table I. Although total concentrations of hormones at the beginning of the "release experiments" were only about 60% of those in the "uptake experiments," the values for kin and kOut were identical within experimental variation. The initial rate of uptake of thyroxine into choroid plexus, i.e. 10 pmol s-' liter-', gives an uptake of 350 fmol min"/choroid plexus.
Partitioning between Aqueous and Lipid Phases Leads to Accumulation of Thyroid Hormones in Lipid Bilayer Membranes-Both thyroxine and triiodothyronine were found to quench the fluorescence of 9-anthroyloxy fatty acids in arti-  in Choroid Plexus between thyroxine and triiodothyronine). Therefore, thyroid hormones accumulate greatly in phospholipid bilayers suspended in an aqueous medium. Above pH 7 the quenching of the fluorescence of 16-(9anthroyloxy) palmitic acid (i.e. a compound with the fluorophore group located near the middle of the bilayer) by either thyroxine or triiodothyronine became strongly dependent on Ihe pH (Fig. 5, Miniprint).
Transthyretin Shifts Partitioning Equilibrium between Choroid Plexus and Medium toward the Aqueous Phase-The average ratio of the concentration of thyroid hormone inside the choroid plexus to that in the medium, established when about 4 mg of choroid plexus was incubated for 40 min in 0.6 ml of medium, was 292 for thyroxine and 208 for triiodothyronine. The medium used in this incubation did not contain transthyretin. However, when pieces of choroid plexus were loaded with radioactive thyroid hormones and then incubated in transthyretin containing medium, the equilibrium distribution was shifted to a higher proportion of hormones being present in the medium. The distribution of thyroxine, or triiodothyronine, between choroid plexus and medium and the concentration of transthyretin in the incubation medium were related (shown for thyroxine in Fig. 6, Miniprint). The ratio of the concentration inside the choroid plexus to that in the medium decreased to 76 for either thyroxine or triiodothyronine for a concentration of 40 pg of transthyretin/ml of medium, and to 20 for thyroxine in the presence of 160 pg of transthyretin/ml of medium, measured after 40 min.
When transthyretin (40 pg/ml) to be added to the release incubation medium was saturated first by an excess amount of thyroxine (10 p~) , the shift in the distribution ratio for radioactive thyroxine was prevented. The concentrations of transthyretin used in the experiments were comparable to those reported by others for cerebrospinal fluid (27).
Tissue Distribution of lZ5I-Thyroxine and Triiodothyronine 10 Min after Intravenous Injection-The distribution of intravenously injected 9-labeled thyroxine and triiodothyronine in various tissues after 10 min was determined as described under "Experimental Procedures." After correction for radioactivity contributed by thyroxine or triiodothyronine in blood, a typical pattern was obtained for the accumulation of thyroxine (Fig. 7) and triiodothyronine (Fig. 8 in various tissues. Choroid plexus was found to be the tissue with the strongest accumulation of thyroxine. After subtracting the amount of thyroxine contributed by the blood, choroid plexus contained about 2.7 times more thyroxine in 1 mg of tissue than in 1 pl of blood. The only other organ in which thyroxine accumulated over the concentration in blood was the liver, with about 1.9 times the level in blood. Both choroid plexus and liver are the only two of the tissues shown in Fig.  7 which synthesize transthyretin.

Kinetics of Uptake of Thyroxine and Triiodothyronine into Choroid Pkxus and Other Tissues in
Vivo-The distribution of radiolabeled thyroxine in tissues was determined at various times after an intravenous injection of the labeled hormone. The results are presented in Fig. 9. In choroid plexus the level of thyroxine increased with time, beginning with an initial rate of uptake of 38 fmol min"/choroid plexus, reaching a half-maximal value after about 1 h and leveling off after about 5.5 h. Five hours after injection of '251-thyroxine the amount of labeled hormone in 1 mg of choroid plexus was about 20 times that in 1 pl of blood. In contrast, the level of radiolabeled thyroxine in the liver increased only very slightly between 10 and 20 min and decreased thereafter. Radioactive material recovered from liver and choroid plexus was shown to be thyroxine by thin layer chromatography and comparison with authentic thyroxine (Fig. 10, Miniprint). The level of labeled thyroxine in kidney and pituitary did not change between 10 min and 5.5 h of incubation. In contrast to choroid plexus, uptake of '251-thyroxine into striatum, cortex, and cerebellum was initially relatively slow, and half-maximal values were reached only after 3 h or later (Fig. 11).
The kinetics of uptake of ['251]triiodothyronine into tissues differed characteristically from those for lZ5I-thyroxine (Fig.  12, Miniprint). In contrast to the high rate of uptake of 1251thyroxine, the level of labeled triiodothyronine in the choroid plexus decreased with time beginning immediately after injection. This decrease was not specific for the choroid plexus, as  FIG. 11. Kinetics of uptake of intravenously injected lzaIthyroxine into various parts of the brain. The level of 12' 1thyroxine in striatum, cortex, and cerebellum at various times after an intravenous injection of '251-thyroxine was determined as described under "Experimental Procedures." Each point is the mean for three

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the levels in liver and kidney also decreased. The level of [lZ5I] triiodothyronine in the pituitary stayed high during the incubation.

DISCUSSION
The in vitro studies showed clearly that the choroid plexus could accumulate thyroid hormones to a very high extent. If choroid plexus was first loaded with thyroid hormones, then these hormones were subsequently released into the medium. The lack of saturation of the uptake process suggested that partitioning of the hormones into lipid could be a mechanism of uptake. This is consistent with the accumulation of thyroid hormones in phospholipid vesicles and the high partition coefficient for thyroid hormones between lipid and aqueous phases. In uptake studies of 16 tissues i n vivo the choroid plexus accumulated '251-thyr~~ine to the greatest extent. Accumulation occurred over a longer time scale in choroid plexus than in any of the other tissues. These typical characteristics of uptake into the choroid plexus were observed only for thyroxine but not for triiodothyronine. After an initial increase in the amount of ['251]triiodothyronine in choroid plexus the level of ['251]triiodothyronine decreased with time when the level of '251-thyroxine in the choroid plexus was still increasing very strongly. Isolation and characterization of the labeled material in the choroid plexus after the in vivo incubation with lZ5I-thyroxine showed that it was thyroxine which was being taken up and not any impurity such as iodine.
The demonstration of thyroxine uptake in a tissue that forms a part of the barrier between the blood and the central nervous system suggested that the choroid plexus could be involved in the transport of thyroxine into the brain. If thyroxine is released from the choroid plexus into the cerebrospinal fluid then there is no further molecular barrier stopping the entry of thyroxine into the brain tissue from the ventricles (28).
Hagen and Solberg (29) reported a similar time course in dogs for the transfer of thyroid hormones from blood to cerebrospinal fluid as described here for the uptake of lZ5Ithyroxine into choroid plexus in rats. In addition, Hagen and Solberg (29) noted that the transfer constant for the transport of 1311-thyroxine from blood to cerebrospinal fluid was much higher than the transfer constant for the transport of [1311] triiodothyronine. These observations are consistent with the suggestion that the choroid plexus is involved in the uptake and secretion of thyroxine (but not triiodothyronine) into the cerebrospinal fluid and then the brain tissue.
It has been suggested that the uptake of thyroid hormones into the brain occurs via the bloodbrain barrier (30), based on data for the fast ( 4 8 s) uptake of thyroid hormones into the brain from a bolus injection. The relatively slow rate of uptake of thyroxine into the choroid plexus could not be seen using the bolus method.
We After intravenous injection of lZ5I-thyroxine, the increase in the level of labeled hormone in the striatum, cortex, and cerebellum was delayed with respect to that in choroid plexus. This would be consistent with uptake of lZ5I-thyroxine via the choroid plexus and the cerebrospinal fluid. The choroid plexus takes up thyroxine, but not the more biologically active triiodothyronine. However, thyroxine taken up into the brain can be deiodinated to triiodothyronine. Dratman and Crutchfield (33) observed the uptake of '251-thyroxine into synaptosomes in rat brain and its conversion to triiodothyronine.
The cellular compartment of the central nervous system (Fig. 13).
Partitioning of thyroxine occurs between blood plasma and choroid plexus. Newly synthesized transthyretin is secreted by the choroid plexus into cerebrospinal fluid. This transthyretin would bind thyroxine. The precise site for this binding (intracellular, near membranes, or extracellular) has still to be identified. The transthyretin-thyroxine complex is swept away from the choroid plexus surface by the unidirectional flow of cerebrospinal fluid from the ventricles to the spinal canal and the subarachnoidal space. The proposed hypothesis does not postulate that the choroid plexus is the exclusive site for the transport of thyroxine from the blood to all parts of the brain. Thyroxine would also partition between lipid and aqueous phases elsewhere at the bloodbrain barrier. However, in the absence of strong local synthesis and secretion of apotransthyretin, the very large partition coefficient would mean that much of the thyroxine remains in the lipid phase.
Thyroxine in the cerebrospinal fluid would not interfere with the regulatory feedback of free thyroid hormones on the stimulation of thyrotropin-releasing hormone on thyrotropin release by the pituitary gland because this gland is located outside the bloodbrain barrier.
Partitioning between two phases is not a very specific feature. However, the expression of the transthyretin gene in the epithelial cells of the choroid plexus is a very specific phenomenon. The combination of partitioning and expression of the gene for a secreted, specific transport protein could be an alternative system to a receptor-mediated transport system. The driving forces for the transfer of thyroxine from blood to brain through choroid plexus could be the synthesis and secretion of transthyretin and the unidirectional flow of the cerebrospinal fluid.
The fact that predominantly thyroxine but not triiodothyronine is transported through choroid plexus has two explanations. First, the molar concentration of free thyroxine in plasma is much higher than that of free triiodothyronine (7fold in the rat (36)). Second, the affinity of transthyretin for thyroxine is much greater than that for triiodothyronine (by a factor of 12.5 in the human; data for the rat are not known).
In the bloodstream, the levels of free thyroid hormones are kept relatively constant, whereas the level of total (free plus protein-bound) hormone changes can vary considerably (the so-called "free thyroid hypothesis"). Previously, transcellular transport involving vesicles has been discussed for the transfer of serum proteins from blood to cerebrospinal fluid (37, 38). Such a transport mechanism would supply thyroid hormones to the cerebrospinal fluid in amounts varying with total hormone levels in blood. The mechanism proposed in this paper would lead to establishing a pool of thyroid hormones in choroid plexus related in amount to the more stable level of free hormones in the bloodstream. The amount of hormones provided to the brain would then depend predominantly on the rate of transthyretin synthesis and secretion by choroid plexus. This has been found to be regulated independently from the expression of the transthyretin gene elsewhere in the body (12).

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t h y r o x i n e and tri10dOthyrOnlne was determined ar described under "Experimental Procedvrer" and The pH dependence of the quenching of fluorescence Of 16-(9-anthroyloxyl palmitic acid by fluorescence quenchlng could be appraxlmated by a curve d e r c n b ? n g t h e changer Of fluorercence i s s h o w i n F i g .

5.
The curves d e s c r i b i n g t h e q w n t i t l t l v e r e l a t i o n s h i p between pH and quenching caused by a one-proton transition but not by d curve describing changer of fluorescence quenching due to two-proton tranr;tionr.