Lawrence J. Mietus

Transport of Steroid Hormones through the Rat Blood-Brain Barrier: PRIMARY ROLE OF ALBUMIN-BOUND HORMONE

These studies were undertaken to investigate (a) the permeability properties of the blood-brain barrier (BBB) to the major gonadal and adrenal steroid hormones, and (b) the role of the binding proteins of plasma (albumin and specific globulins) in the regulation of BBB steroid hormone transport. The permeability of the BBB to [(3)H]-labeled progesterone, testosterone, estradiol, corticosterone, aldosterone, and cortisol, was measured relative to [(14)C]butanol, a freely diffusable reference, in the barbiturate anesthetized rat using a tissue sampling-single injection technique. The isotopes were rapidly injected in a 200-mul bolus of Ringers solution (0.1 g/dl albumin) via the common carotid artery and the percent extraction of unidirectional influx of hormone was determined after a single pass through brain: progesterone, 83+/-4%; testosterone, 85+/-1%; estradiol, 83+/-3%; corticosterone, 39+/-2%; aldosterone, 3.5+/-0.8%; and cortisol, 1.4+/-0.3%. The selective permeability of the BBB was inversely related to the number of hydrogen bonds each steroid formed in aqueous solution and directly related to the respective 1-octanol/Ringers partition coefficient. When the bolus injection was 67% human serum, >95% of the labeled steroid was bound as determined by equilibrium dialysis. However, the influx of the steroids through the BBB was inhibited by human serum to a much less extent than would be expected if only the free (dialyzable) hormone was transported; progesterone, estradiol, testosterone, and corticosterone transport was inhibited 18, 47, 70, and 85% respectively, or in proportion to the steroid binding to plasma globulins. Rat serum (67%) only inhibited the transport of these four hormones, 0, 13, 12, and 69%, respectively, reflecting the absence of a sex hormone-binding globulin in rat plasma. However, neonatal rat serum (67%) inhibited progesterone, testosterone, and estradiol transport 0, 0, and 91%, respectively, consistent with the presence of an estradiol-binding protein in neonatal rat serum. The binding of steroid hormone to bovine albumin in vitro (as determined by equilibrium dialysis) was compared to albumin binding in vivo (as determined by the single injection technique). The ratio of apparent dissociation constant in vivo, K(D)(app), to the in vitro K(D) was: >>200 for progesterone, >200 for testosterone, 120 for estradiol, and 7.7 for corticosterone. Assuming the steady-state condition, the K(D)(app)/K(D) was found to be proportional to the BBB permeability for each steroid. These data demonstrate (a) the selective permeability properties of the BBB to the major steroid hormones is proportional to the tendency of the steroid to partition in a polar lipid phase and is inversely related to the number of hydrogen bond-forming functional groups on the steroid nucleus; (b) the presence of albumin in serum may bind considerable quantities of steroid hormone, but exerts little inhibitory effects on the transport of steroids into brain, whereas globulin-bound hormone does not appear to be transported into brain to a significant extent. Therefore, the hormone fraction in plasma that is available for transport into brain is not restricted to the free (dialyzable) fraction, but includes the larger albumin-bound moiety.

Palmitate and Cholesterol Transport Through the Blood-Brain Barrier

The transport into brain of the plasma lipids (free fatty acids, cholesterol, triglycerides, and phospholipids) is impeded by the tight binding (more than 99%) of these compounds by plasma proteins. Since proteins do not cross the brain capillary wall (Saunders, 1977), i.e., the blood-brain barrier (BBB), lipid transport through the BBB, if it occurs, must involve a transfer of lipid from the plasma protein to the BBB membrane. While it is known that triglycerides (Dhopeshwarkar and Mead, 1973) and such phospholipids as lecithin or lysolecithin (Pardridge, et al., 1979) are not transported through the rat BBB, the reported data are conflicting in regard to BBB transport of cholesterol and free fatty acids. Regarding cholesterol, it was shown many years ago that labeled cholesterol does not enter the adult (Bloch et al., 1943), fetal, or neonatal rat brain (Morris and Chaikoff, 1961). Subsequently, however. it was reported that dietary labeled cholesterol enters brain, albeit slowly (Serougne et al., 1976), suggesting that cholesterol may cross the BBB. Concerning BBB transport of free fatty acids, it is known that the brain of mature dog (Spitzer, 1973) or man (Owen et al., 1967) does not take up a net amount of free fatty acid from the circulation. However, a measureable influx of labeled palmitate into brain after carotid injection has also been reported (Dhopeshwarkar and Mead, 1973); the studies were not quantitative and, thus, estimates of the rnrrgrzitrtde of blood-brain fatty acid flux are not available. Therefore, the present investigations were designed to study the permeability properties of the BBB to cholesterol and palmitate, with special emphasis on the role of the plasma proteins in limiting BBB transport of these two lipids.

Kinetics of Regional Blood–Brain Barrier Transport and Brain Phosphorylation of Glucose and 2-Deoxyglucose in the Barbiturate–Anesthetized Rat

Abstract: Recent studies indicate the lumped constant (LC), which defines the relative rates of brain utilization of glucose and 2‐deoxyglucose (2‐DG), doubles to values > 1.0 under conditions of hypoglycemia. Since changes in the LC should be predictable given the kinetic parameters of blood‐brain barrier (BBB) transport and brain phosphorylation of glucose and 2‐DG, the present studies were designed to measure the necessary kinetic parameters. The carotid injection technique was used to determine cerebral blood flow and the Km, Vmax, and KD of glucose and 2‐DG transport through the BBB in seven brain regions in rats anesthetized with 50 mg/kg i.p. pentobarbital. Regional glucose transport through the BBB was characterized by an average Km= 6.3 mm, average Vmax= 0.53 μmol min−1g−1, and average KD= 0.022 ml min−1g−1. The nonsaturable route of transport of glucose represented on the average 40% of the total glucose influx into brain regions at an arterial glucose concentration of 10 mm. In addition, the rate constants of phosphorylation of glucose and 2‐DG were measured for each region. Substitutions of the measured kinetic parameters for sugar transport and phosphorylation into equations defining the LC confirm the observation that the LC would be expected to vary under extreme conditions such as hypoglycemia and to exceed values of 1.0 under these conditions.

Transport of Albumin-bound Melatonin Through the Blood-Brain Barrier

Melatonin (N-acetyl-5-methoxytryptamine) i s an indole amide secreted by one of the circumventricular organs of brain, viz., the pineal gland (Wurtman and Moskowitz, 1977). Unlike the precursor amine, serotonin (5hydroxytryptamine), which is dot readily exchanged between blood and brain (Oldendorf, 1971), melatonin is known to equilibrate rapidly (Reppert et al., 1979) between blood and cerebrospinal fluid (CSF). However, the CSF melatonin pool does not achieve complete equilibration with plasma; i.e., the CSFiplasma ratio is about 0.4 (Reppert et al., 1979). The relatively low CSF level of melatonin has been attributed to plasma protein binding (Reppert et al., 1979), since about 70% of circulating melatonin is albumin-bound (Cardinali et al., 1972). Recent studies, however, have shown that such albuminbound ligands as tryptophan (Madras et al., 1974), tryptophol (Cornford et al., 1979), triiodothyronine (Pardridge, 1979), the adrenal and gonadal steroid hormones (Pardridge and Mietus, 1979a), and free fatty acid (Pardridge and Mietus, 1980) are readily transported through the brain capillary wall, i.e., the blood-brain barrier (BBB). Therefore, the present studies were designed to investigate the permeability of the BBB to melatonin and to examine the role of plasma protein, if any, in limiting the blood-brain transport of this compound. In addition, the permeability of the BBB to melatonin is compared to the permeability of the liver cell membrane, since recent studies have shown that these two membranes may differ substantially in their respective permeability to some hormonal substances (Pardridge and Mietus, 1979b).

Influx of Thyroid Hormones into Rat Liver In Vivo: DIFFERENTIAL AVAILABILITY OF THYROXINE AND TRIIODOTHYRONINE BOUND BY PLASMA PROTEINS

The transport of [(125)I]thyroxine (T(4)) and [(125)I]triiodothyronine (T(3)) into liver was investigated with a tissue sampling-portal vein injection technique in the anesthetized rat. The method allows the investigation of the effects of plasma proteins in human serum on the unidirectional influx of T(4) or T(3) into liver cells. The percent extraction of unidirectional clearance of T(3) and T(4) was 77+/-2% and 43+/-2%, respectively, after portal injection of a bolus of Ringers solution. Cell membrane transport of T(4) or T(3) was nonsaturable because 50-muM concentrations of unlabeled hormone had no effect on transport. The addition of bovine albumin in concentrations of 1, 5, or 10 g/100 ml bound >98% of T(4) or T(3) in vitro, but had no significant effect on T(3) or T(4) transport in vivo. Conversely, 10% rabbit antisera specific for T(3) or T(4), completely abolished the intracellular distribution of thyroid hormone into liver. In the presence of rat serum, which contains albumin and thyroid hormone binding pre-albumin (TBPA), 18 and 81% of total plasma T(4) and T(3), respectively, were available for transport in vivo. The fraction of hormone available for transport in the presence of normal human serum, which contains albumin, TBPA, and thyroid hormone binding globulin (TBG) was 11% for T(4) and 72% for T(3). The fraction of hormone transported into liver after injection of serum obtained from pregnant or birth control pilltreated volunteers was 4% for T(4) (but this was not significantly different from zero) and 54% for T(3). THESE DATA SUGGEST: (a) The mechanism by which T(4) and T(3) traverse the liver cell membrane is probably free diffusion. (b) Albumin-bound T(4) or T(3) is freely cleared by liver, approximately 50% of TBG-bound T(3) is transported, but little, if any, of TBPA-bound T(4) or TBG-bound T(4) is cleared by liver cells. (c) Although the albumin-bound fraction of T(4) greatly exceeds the free (dialyzable) moiety, the two fractions are both inversely related to the existing TBA or TBG level; therefore, in vitro measurements of free T(4) would be expected to accurately reflect what is available for transport in vivo. Conversely, TBG-bound T(3) is readily transported in vivo; therefore, it is proposed that in vitro measurements of free T(3) do not reliably predict the fraction of T(3) available for transport into liver in vivo.

Kinetics of Neutral Amino Acid Transport Through the Blood‐Brain Barrier of the Newborn Rabbit

Abstract: Since protein synthesis in the developing brain may, under certain conditions, be limited by amino acid availability, the present studies were undertaken to characterize the kinetics of large neutral amino acid transport through the blood‐brain barrier (BBB) of the newborn rabbit. The Km, Vmax, and KD of the transport of eight amino acids were determined by a nonlinear regression analysis of data obtained with the carotid injection technique. Compared with kinetic parameters observed for the adult rat, the Km, Vmax, and KD of amino acid transport were all two‐ to threefold higher in the newborn. Albumin was found to bind tryptophan actively in vitro, but had no inhibitory effect on tryptophan transport through the newborn BBB. Glutamine was transported through the BBB of the newborn at rates severalfold higher than are seen in the adult rat. However, glutamine transport was not inhibited by high concentrations of N‐methylaminoisobutyric acid (NMAIB), a model amino acid that is specific for the alanine‐preferring or A‐system present in peripheral tissues. In conclusion, these studies show that the BBB neutral amino acid transport system of the newborn rabbit has a lower affinity and higher capacity than does the BBB of the adult rat. Under conditions of high plasma amino acids, the increased capacity of the newborn transport system allows for a higher rate of amino acid transport into brain than would occur via the lower capacity system present in the adult rat brain.

Nomogram for 2-Deoxyglucose Lumped Constant for Rat Brain Cortex

The quantitation of local cerebral metabolic rate of glucose with the 2-deoxyglucose technique of Sokoloff requires the use of a correction factor, or lumped constant. We have shown previously (Pardridge et al., 1982) that a simple model may be formulated to predict changes in the lumped constant that occur due to alterations in the distribution of glucose and 2-deoxyglucose in brain. Given experimentally observed values for brain and plasma glucose concentrations, the 2-deoxyglucose lumped constant may be determined from a nomogram constructed from knowledge of the blood–brain barrier transport constants (KM, Vmax, KD) for glucose and for 2-deoxyglucose. However, the nomogram is constructed from transport constants determined in the barbiturate-anesthetized state. The applicability of the nomogram to other physiologic states was examined in the present studies. Large changes in blood–brain barrier hexose transport constants do not appreciably alter the shape of the nomogram, if the changes in KM or Vmax for glucose or for 2-deoxyglucose are the same. Moreover, glucose and 2-deoxyglucose are both transported by the same hexose carrier, and selective changes in the transport of only one hexose have not been reported. Therefore, it is probable that the nomogram constructed from transport constants measured under barbiturate anesthesia is useful in predicting the lumped constant in a variety of physiologic states.

Antibodies to Blood—Brain Barrier Bind Selectively to Brain Capillary Endothelial Lateral Membranes and to a 46K Protein

To begin elucidating the biochemical basis of the polarized membrane features of the blood–brain barrier (BBB), a series of immunochemical and immunoperoxidase studies were initiated with bovine brain microvessels that make up the BBB in vivo. A rabbit antiserum was prepared against isolated bovine brain BBB plasma membranes. The bovine microvessel plasma membranes were radioiodinated with chloramine-T, and the antiserum selectively immunoprecipitated a 46K protein. The antibodies directed against the 46K protein were quantitatively absorbed with bovine brain capillaries but not with rat kidney or liver powder. Only the capillaries of brain reacted with the rat kidney-absorbed antiserum in immunoperoxidase studies of ethanol-fixed, 8-μm sections of bovine brain cortex, whereas the capillaries in heart, liver, and kidney did not react. This antiserum also strongly illuminated the lateral membranes of isolated bovine brain capillary endothelial cells grown in primary tissue culture. These studies provide evidence for a polarized distribution of a surface antigen in bovine brain capillary endothelial cells that is not present in capillary endothelia of liver, heart, or kidney. The correlation of the immunoperoxidase and immunoprecipitation techniques suggests that a candidate for the asymmetrically distributed surface antigen in the BBB is the 46K protein. The relationship between the 46K protein and the composition of BBB tight junctions remains to be determined.