Phenylalanine Transport Across the Blood-Brain Barrier as Studied with the In Situ Brain Perfusion Technique

Unidirectional L-phenylalanine transport into six brain regions of pentobarbital-anesthetized rats was studied using the in situ brain perfusion technique. This technique allows both accurate measurements of cerebrovascular amino acid transport and complete control of perfusate amino acid composition. L-Phenylalanine influx into the brain was sodium independent and could be described by a model with a saturable and a nonsaturable component. Best-fit values for the kinetic constants in the parietal cortex equaled 6.9 X 10(-4) mumol/s/g for Vmax, 0.011 mumol/ml for Km, and 1.8 X 10(-4) ml/s/g for KD during perfusion with fluid that did not contain competing amino acids. D-Phenylalanine competitively inhibited L-phenylalanine transport with a Ki approximately 10-fold greater than the Km for L-phenylalanine. There were no significant regional differences in Km, KD, or Ki, whereas Vmax was significantly greater in the cortical lobes than in the other brain regions. L-Phenylalanine influx during plasma perfusion was only 30% of that predicted in the absence of competing amino acids. Competitive inhibition increased the apparent Km during plasma perfusion by approximately 20-fold, to 0.21 mumol/ml. These data provide accurate new estimates of the kinetic constants that describe L-phenylalanine transport across the blood-brain barrier. In addition, they indicate that the cerebrovascular transfer site affinity (1/Km) for L-phenylalanine is three- to 12-fold greater than previously estimated in either awake or anesthetized animals.     

A comparison of anionic sites in the glomerular basement membranes from different classes of fishes

Summary Cationized ferritin was injected into the circulatory system of teleosts, the sea raven and Atlantic eelpout, and into elasmobranchs, the spiny dogfish and the skate, to determine if the glomerular basement membranes (GBM) from these different groups of fishes possess anionic binding sites similar to those present in the GBM of mammals. The distribution of cationized ferritin was the same in all fishes listed. Cationized ferritin was localized only in the GBM and the mesangial matrix. The regular distribution of cationized ferritin within the laminae rarae (60 nm intervals) was taken as evidence of the presence of anionic binding sites. Cationized ferritin did not bind to the glomerular capillary endothelium, nor was any of it localized at the base of the slit diaphragms of the foot processes of the podocytes. The distribution of binding sites in the GBM of these fishes is similar to that in another teleost, the winter flounder, and in a cyclostome, the hagfish.     

Water and solute transport along developing maize roots

Hydraulic and osmotic properties were measured along developing maize (Zea mays L.) roots at distances between 15 and 465 mm from the root tip to quantify the effects of changes in root structure on the radial and longitudinal movement of water and solutes (ions). Root development generated regions of different hydraulic and osmotic properties. Close to the root tip, passive solute permeability (root permeability coefficient, P_sr) was high and selectivity (root reflection coefficient, _sr) low, indicative of an imperfect semipermeable root structure. Within the apical 100–150 mm, P_sr decreased by an order of magnitude and _sr increased significantly. Root hydraulic conductivity (Lp_r) depended on the nature of the force (hydrostatic and osmotic). Osmotic Lp_r was smaller by an order of magnitude than hydrostatic Lp_r and decreased with increasing distance from the root tip. Throughout the root, responses in turgor of cortical cells and late metaxylem to step changes in xylem pressure applied to the base of excised roots were measured at high spatial resolution. The resulting profiles of radial and longitudinal propagation of pressure showed that the endodermis had become the major hydraulic barrier in older parts of the root, i.e. at distances from the apex ä 150 mm. Other than at the endodermis, no significant radial hydraulic resistance could be detected. The results permit a detailed analysis of the root's composite structure which is important for its function in collecting and translocating water and nutrients.Abbreviations and Symbols CPP cell pressure probe - IT root segments with intact tips; - Lp_r root hydraulic conductivity - Lp_rh hydrostatic hydraulic conductivity of root - Lp_ro osmotic hydraulic conductivity of root - P_app hydrostatic pressure applied to cut end of root - P_c cell turgor - P_{c, cor} turgor of cortical cell - P_c,xyl turgor of late metaxylem vessel - P_ro stationary root pressure - P_r0,seal stationary root pressure of sealed root segment - P_sr solute permeability coefficient of root - RPP root pressure probe - TR root segments with tip removed - _sr reflection coefficient of root Dedicated to Professor Andreas Sievers on the occasion of his retirement     

Hypoxia Dramatically Increases the Nonspecific Transport of Blood-Borne Proteins to the Brain

Abstract: Increased cerebrovascular permeability is an important factor for the development of cerebral edema. To investigate the effect of hypoxia on the transport of blood-borne proteins to the brain, we used a cell culture model of the blood-brain barrier (BBB) consisting of a coculture of brain capillary endothelial cells and astrocytes that closely mimics the in vivo situation. The permeability of albumin, a marker of the nonspecific transcellular route, is extremely low in this in vitro model of the BBB. After hypoxia, a huge increase in the permeability of albumin is detected. Despite the opening of the tight junctions already demonstrated after hypoxia, the increase in the permeability of albumin is mainly attributed to an increase of the nonspecific vesicular transport in the cell, attested by the temperature dependence of the phenomenon and the visualization of labeled apotransferrin in the cytoplasm. The increase of this pathway could participate in the development of brain edema during hypoxia.     

The morphological background to imbibition in seeds ofPinus sylvestris L. of different provenances

An examination was made of the structure of the coats of Scots pine (Pinus sylvestris L.) seeds of different provenance and the contribution of this factor to differences in imbibition. The seed coat layers derived from the integument, the sarcotesta, sclerotesta and endotesta did little to restrict imbibition, even though the sclerotesta of the northern provenance seeds was composed of a double multicellular layer and the sarcotesta contained large numbers of pigmented, phenol-bearing cells. In addition to the micropyle, the sclerotesta was found to possess structural openings at the chalazal end and at the ridge joining the two halves of the seed, but being covered by the pigmented cells of the sarcotesta, these did not allow water to enter any more than did the micropyle itself. Imbibition was chiefly regulated by the lipophilic covers surrounding the endosperm, which are mainly of nucellar origin, especially by the megaspore membranes nearest to the endosperm, the outer and inner exine. The nucellar cap covering the micropylar end of the endosperm proved to be impermeable to water, and its edge extended between the exine layers, which further enhanced the importance of the endosperm covers as regulators of imbibition.     

The permeability coefficient of the wall of a villous membrane

Summary The equations hitherto used to correct the permeability coefficient for the unstirred layer influence are valid only for flat membranes. Therefore, appropriate equations for membranes with a villous surface (e.g., small intestine) have been derived. They take into account the non-linear concentration gradient in the intervillous part of the unstirred layer. Quantitative information about the geometry of the villous surface and the unstirred layer thickness are needed to calculate the permeability coefficient of the membrane wall (e.g., intestinal epithelium). The concentration of highly permeable substances drops sharply already in the upper part of the intervillous space, so that the tips of the villi function as effective absorbing area. The intervillous concentration gradient of a substance with a low permeability coefficient is so small, that such a substance is absorbed by the total surface area of the villous membrane. The effective absorbing area of substances with intermediate permeability coefficient lies between the described limits.     

Water permeability of Betula periderm

The water permeability of periderm membranes stripped from mature trees of Betula pendula Roth was investigated. The diffusion of water was studied using the system water/membrane/water, and transpiration was measured using the system water/membrane/water vapor. Betula periderm consists of successive periderm layers each made up of about 5 heavily suberized cell layers and a varying number of cell layers that are little suberized, if at all. It is shown that these layers act as resistances in series. The permeability coefficient of the diffusion of water (P _d) can be predicted with 79% accuracy from the reciprocal of the membrane weight (x in mg cm^-2) by means of the linear equation P _d=14.69·10^-7 x-0.73·10^-7. For example, the P _d of a periderm membrane having a weight of 10 mg cm^-2 (approx. 250 m thick) is 7.4·10^-8 cm s^-1, which is comparable to the permeability of cuticles. This comparison shows that on a basis of unit thickness, Betula periderm is quite permeable to water as cuticles have the same resistance with a thickness of only 0.5 to 3 m. It is argued that this comparatively high water permeability of birch periderm is due to the fact that middle lamellae and the primary walls of periderm cells are not at all, or only incompletely suberized and, therefore, form a hydrophilic network within which the water can flow. This conclusion is based on the following observations: (1) Middle lamellae and primary walls stain strongly with toluidine blue, which shows them to be polar. (2) If silver ions are added as tracer for the flow of water, they are found only in the middle lamellae, primary walls, and in plasmodesmata, while no silver can be detected in the suberized walls. (3) Permeability coefficients of transpiration strongly depend on water activity. This shows conclusively that water flows across Betula periderm via a polar pathway. It is further argued that liquid continuity is likely to be maintained under all physiological conditions in the network formed by middle lamellae and primary walls. On the other hand, the lumina of periderm cells, intercellular air spaces in the lenticels, and even the pores in the suberized walls (remainders of plasmodesmata) will drain at a humidity of 95% and below. Due to the presence of intercellulars the permeability coefficient of lenticels is much greater than that of the periderm. A substantial amount of the total water, therefore, flows as vapor through lenticels even though they cover only 3% of the surface.Abbreviations PM perideron membrane - P _d permeability coefficient for diffusion of water - P _tt permeability coefficient of transpiration - MES (N-morpholino)ethane sulfonic acid     

Permeability of submandibular salivary glands in dogs to blood-borne horseradish peroxidase (HRP)

Summary Horseradish peroxidase (HRP) was administered to the submandibular glands of dogs by close-arterial bolus-type injections, and its localisation was examined histochemically by light and electron microscopy. The HRP became widespread in the interstices of the glands and reached many central acinar lumina via scattered localised parts of their tight junctional complexes. Reaction product was less often found in the lumina of demilunes, which suggested that the intercellular junctions there were less leaky. HRP was often found in sizeable spaces between myoepithelial cells and the underlying parenchymal cells; such large spaces have not been observed in this situation in other species. The possibility that permeability pathways may arise intermittently at different sites in the adhering mechanisms between the acinar cells is discussed.It is concluded that potential paracellular permeability pathways for macromolecules exist in these glands and, if the concentration gradient is sufficiently high, molecules even as large as those of HRP can to some extent permeate passively from the interstices to the saliva. In resting glands the principal permeability site is between the central acinar cells.Supported by Grants from the M.R.C. and the V.R.T. King's College HospitalWe wish to acknowledge the technical help of Mr. K.J. Davies and Mr. P.S.A. Rowley     

Linoleic acid-induced endothelial cell injury: Role of membrane-bound enzyme activities and lipid oxidation

High plasma levels of linoleic acid (18:2) may injure endothelial cells, resulting in decreased barrier function of the vascular endothelium. The effects of linoleic acid on endothelial barrier function (transendothelial movement of albumin), membrane-bound enzyme activities, and possible autooxidation of linoleic acid under experimental conditions were studied. The exposure of endothelial monolayers to 18:2 for 24 hr at 60, 90, and 120 μM. fatty acid concentrations caused a significant increase in transendothelial movement of albumin, with maximum albumin transfer at 90 μM. Fatty acid treatment resulted in the increased appearance of cytosolic lipid droplets. Activities of the membrane-bound enzymes, angiotensin-converting enzyme (ACE), and Ca²⁺-ATPase increased steadily with increasing time of cell exposure to 90 μM 18:2, reaching significance at 24 hr. Treatment of endothelial cultures with up to 120 μM 18:2 did not cause cytotoxicity, as evidenced by a nonsignificant change in cellular release of [₃H]-adenine. Incubation of 18:2-supplemented serum-containing culture media with 1000 μM 18:2 at 37°C for up to 48 hr did not result in formation of autooxidation products. These results suggest that 18:2 itself, and not its oxidation products, plays a major role in disrupting endothelial barrier function.