Hepatic versus gallbladder bile composition: in vivo transport physiology of the gallbladder in rainbow trout

Ion and water transport across the teleost Oncorhynchus mykiss gallbladder were studied in vivo by comparing flow and composition of hepatic bile, collected by chronic catheter, to volume and composition of terminally collected gallbladder bile. Differences in composition were comparable with those of other vertebrates, whereas bile flow (75 microl. kg(-1). h(-1)) was below values reported for endothermic vertebrates. The gallbladder concentrates bile acids five- to sevenfold and exhibits higher net Cl(-) than Na(+) transport in vivo, in contrast to the 1:1 transport ratio from gallbladders under saline/saline conditions. Transepithelial potential (TEP) in the presence of bile, at the apical surface, was -13 mV (bile side negative) but +1.5 mV in the presence of saline. Bile acid in the apical saline reversed the TEP, presumably by a Donnan effect. We propose that ion transport across the gallbladder in vivo involves backflux of Na(+) from blood to bile resulting in higher net Cl(-) than Na(+) flux. This Na(+) backflux is driven by a bile side negative TEP and low Na(+) activity in bile due to the complexing effects of bile acids.     

Determination of thalidomide in transport buffer for Caco-2 cell monolayers by high-performance liquid chromatography with ultraviolet detection

We report simple validated HPLC methods for the determination of thalidomide in the transport buffer for the human colonic cell line (Caco-2) cell monolayers. An aliquot of 50 microl of the mixture was injected onto a Spherex C(18) column (150 x 4.6 mm; 5 microm) at a flow-rate of 0.5 ml/min of mobile phase consisting of acetonitrile-10 mM ammonium acetate buffer (24:76, v/v, pH 5.5), and thalidomide was detected by ultraviolet detector at a wavelength of 220 nm. Calibration curves for thalidomide were constructed at the concentration range of 0.025-1.0 and 1.0-50 microM in transport buffer. The validated methods were used to determine the transport of thalidomide by Caco-2 monolayers. The transport across the monolayers from the apical (A) to basolateral (B) side was similar to that from B to A side. The apparent permeability coefficient (P(app)) values of thalidomide at 10-300 microM from the A to B and from B to A side was 2-6 x 10(-5) cm/s, with a marked decrease in P(app) values from A to B side at increased thalidomide concentration. The A to B transport appears to be dependent on temperature and sodium ion. Sodium azide, 2,4-dinitrophenol (both ATP inhibitors), 5-fluorouracil, cytidine and glutamic acid significantly inhibited the transport of thalidomide. These results indicate that the transport of thalidomide by Caco-2 monolayers was rapid, which might involve an energy-dependent mechanism.     

Characterization of the blood-brain barrier choline transporter using the in situ rat brain perfusion technique

Choline enters brain by saturable transport at the blood-brain barrier (BBB). In separate studies, both sodium-dependent and passive choline transport systems of differing affinity have been reported at brain capillary endothelial cells. In the present study, we re-examined brain choline uptake using the in situ rat brain perfusion technique. Saturable brain choline uptake from perfusion fluid was best described by a model with a single transporter (V:(max) = 2.4-3.1 nmol/min/g; K(m) = 39-42 microM) with an apparent affinity (1/Km)) for choline five to ten-fold greater than previously reported in vivo, but less than neuronal 'high-affinity' brain choline transport (K(m) = 1-5 microM). BBB choline uptake from a sodium-free perfusion fluid using sucrose for osmotic balance was 50% greater than in the presence of sodium suggesting that sodium is not required for transport. Hemicholinium-3 inhibited brain choline uptake with a K(i) (57 +/- 11 microM) greater than that at the neuronal choline system. In summary, BBB choline transport occurs with greater affinity than previously reported, but does not match the properties of the neuronal choline transporter. The V:(max) of this system is appreciable and may provide a mechanism for delivering cationic drugs to brain.     

ATP-binding cassette transporter A1 (ABCA1) deficiency does not attenuate the brain-to-blood efflux transport of human amyloid-beta peptide (1-40) at the blood-brain barrier

ATP-binding cassette transporter A1 (ABCA1) mediates apolipoprotein-dependent cholesterol release from cellular membranes. Recent studies using ABCA1 knockout mice have demonstrated that ABCA1 affects amyloid-beta peptide (A beta) levels in the brain and the production of senile plaque. Cerebral A beta(1-40) was eliminated from the brain to the circulating blood via the blood-brain barrier (BBB), which expresses ABCA1. Therefore, in the present study, we examined whether ABCA1 affects the brain-to-blood efflux transport of human A beta(1-40)(hA beta(1-40)) at the BBB. The apparent uptake of [125I]hA beta(1-40) into ABCA1-expressing HEK293 cells was not significantly different from that into parental HEK293 cells. In addition, the apparent uptake was not significantly affected even in the presence of apolipoprotein A-I as a cholesterol release acceptor. Moreover, [125I]hA beta(1-40) elimination from mouse brain across the BBB was not significantly different between ABCA1-deficient and wild-type mice 60 min after its administration into the cerebrum. These results suggest that ABCA1 does not directly transport hA beta(1-40) and a deficiency of ABCA1 does not attenuate the brain-to-blood efflux transport of hA beta(1-40) across the BBB.     

The l-isomer-selective transport of aspartic acid is mediated by ASCT2 at the blood-brain barrier

Aspartic acid (Asp) undergoes l-isomer-selective efflux transport across the blood-brain barrier (BBB). This transport system appears to play an important role in regulating l- and d-Asp levels in the brain. The purpose of this study was to identify the responsible transporters and elucidate the mechanism for l-isomer-selective Asp transport at the BBB. The l-isomer-selective uptake of Asp by conditionally immortalized mouse brain capillary endothelial cells used as an in vitro model of the BBB took place in an Na+- and pH-dependent manner. This process was inhibited by system ASC substrates such as l-alanine and l-serine, suggesting that system ASC transporters, ASCT1 and ASCT2, are involved in the l-isomer selective transport. Indeed, l-Asp uptake by oocytes injected with either ASCT1 or ASCT2 cRNA took place in a similar manner to that in cultured BBB cells, whereas no significant d-Asp uptake occurred. Although both ASCT1 and ASCT2 mRNA were expressed in the cultured BBB cells, the expression of ASCT2 mRNA was 6.7-fold greater than that of ASCT1. Moreover, immunohistochemical analysis suggests that ASCT2 is localized at the abluminal side of the mouse BBB. These results suggest that ASCT2 plays a key role in l-isomer-selective Asp efflux transport at the BBB.     

Treatment with the NK1 Antagonist Emend Reduces Blood Brain Barrier Dysfunction and Edema Formation in an Experimental Model of Brain Tumors

The neuropeptide substance P (SP) has been implicated in the disruption of the blood-brain barrier (BBB) and development of cerebral edema in acute brain injury. Cerebral edema accumulates rapidly around brain tumors and has been linked to several tumor-associated deficits. Currently, the standard treatment for peritumoral edema is the corticosteroid dexamethasone, prolonged use of which is associated with a number of deleterious side effects. As SP is reported to increase in many cancer types, this study examined whether SP plays a role in the genesis of brain peritumoral edema. A-375 human melanoma cells were injected into the right striatum of male Balb/c nude mice to induce brain tumor growth, with culture medium injected in animals serving as controls. At 2, 3 or 4 weeks following tumor cell inoculation, non-treated animals were perfusion fixed for immunohistochemical detection of Albumin, SP and NK1 receptor. A further subgroup of animals was treated with a daily injection of the NK1 antagonist Emend (3 mg/kg), dexamethasone (8 mg/kg) or saline vehicle at 3 weeks post-inoculation. Animals were sacrificed a week later to determine BBB permeability using Evan''s Blue and brain water content. Non-treated animals demonstrated a significant increase in albumin, SP and NK1 receptor immunoreactivity in the peritumoral area as well as increased perivascular staining in the surrounding brain tissue. Brain water content and BBB permeability was significantly increased in tumor-inoculated animals when compared to controls (p<0.05). Treatment with Emend and dexamethasone reduced BBB permeability and brain water content when compared to vehicle-treated tumor-inoculated mice. The increase in peritumoral staining for both SP and the NK1 receptor, coupled with the reduction in brain water content and BBB permeability seen following treatment with the NK1 antagonist Emend, suggests that SP plays a role in the genesis of peritumoral edema, and thus warrants further investigation as a potential anti-edematous treatment.     

Polarized glucose transporters and mRNA expression properties in newly developed rat syncytiotrophoblast cell lines,TR-TBTs

In this study, we have established new syncytiotrophoblast cell lines (TR-TBTs) from the recently developed transgenic rat harboring temperature-sensitive simian virus 40 large T-antigen gene (Tg-rat). Four conditionally immortalized syncytiotrophoblast cell lines (TR-TBT 18d-1 approximately 4) were obtained from pregnant Tg-rats at gestational day 18. These cell lines had a syncytium-like morphology, could be prepared as monolayers, expressed cytokeratins and rat syncytiotrophoblast markers, and exhibited apical or basal GLUT1 localizations and apical GLUT3 localizations. TR-TBTs express large T-antigen and grow well at 33 degrees C with a doubling time of about 30 h. TR-TBTs have processes for the uptake of dehydroepiandrosteron-3-sulfate (DHEAS) and these are predominantly located on the basal side, and this is the first report of an in vitro model of blood placental barrier (BPB) able to incorporate DHEAS. Therefore, TR-TBTs are an appropriate in vitro model for investigating carrier-mediated transport functions at the BPB. Moreover, TR-TBTs express betaine/GABA transporter (GAT-2/BGT-1), concentrative nucleoside transporter 2 (CNT2), equilibrative nucleoside transporter 1 (ENT1), and ENT2 and the expression of these transporters has been reported in blood-brain barrier (BBB). Thus, the expression patterns of nucleoside and neurotransmitter transporters examined are quite similar in both the BPB and BBB.     

Evidence for basolateral Cl- channels as modulators of apical Cl- secretion in pulmonary epithelia of Xenopus laevis

Pulmonary epithelia of air-breathing vertebrates are covered by a thin, fluid layer that is essential for immune defense and gas diffusion. The composition of this layer is maintained by ion transport mechanisms, including Cl(-) transport. The present study focuses on the function of basolateral Cl(-) channels in Xenopus pulmonary epithelia, since knowledge concerning this issue is limited. Therefore, Ussing chamber measurements were performed, and transepithelial short-circuit currents (I(SC)) were monitored. Basolateral application of the Cl(-) channel inhibitor N-phenylanthranilic acid (DPC) resulted in an increase of the I(SC), indicating a DPC-sensitive Cl(-) conductance. This observation was confirmed in experiments using an apical-to-basolateral Cl(-) gradient, with and without nystatin (apical side) to permeabilize the epithelia as well as by establishing an iodide gradient. The DPC-sensitive Cl(-) conductance was influenced by procedures interfering with apical Cl(-) secretion. For example, the effect of forskolin was increased when basolateral Cl(-) channels were blocked by the simultaneous application of DPC. Activation of apical Cl(-) secretion by forskolin/IBMX and subsequent DPC application resulted in a significantly reduced DPC effect. Accordingly, DPC led to an increased apical Cl(-) secretion estimated by an increased 5-nitro-2-(3-phenylpropylamino)benzoic acid-sensitive I(SC). Furthermore, inhibition of basolateral anion exchangers responsible for Cl(-) uptake resulted in a decreased DPC-sensitive current. Taken together, we have evidence concerning the function of basolateral Cl(-) channels in Xenopus pulmonary epithelium and that these channels play a significant role in mediating apical Cl(-) secretion involving a novel Cl(-) recycling mechanism across the basolateral membrane.     

Fatty acid transport protein expression in human brain and potential role in fatty acid transport across human brain microvessel endothelial cells

The blood-brain barrier (BBB), formed by the brain capillary endothelial cells, provides a protective barrier between the systemic blood and the extracellular environment of the CNS. Passage of fatty acids from the blood to the brain may occur either by diffusion or by proteins that facilitate their transport. Currently several protein families have been implicated in fatty acid transport. The focus of the present study was to identify the fatty acid transport proteins (FATPs) expressed in the brain microvessel endothelial cells and characterize their involvement in fatty acid transport across an in vitro BBB model. The major fatty acid transport proteins expressed in human brain microvessel endothelial cells (HBMEC), mouse capillaries and human grey matter were FATP-1, -4 and fatty acid binding protein 5 and fatty acid translocase/CD36. The passage of various radiolabeled fatty acids across confluent HBMEC monolayers was examined over a 30-min period in the presence of fatty acid free albumin in a 1 : 1 molar ratio. The apical to basolateral permeability of radiolabeled fatty acids was dependent upon both saturation and chain length of the fatty acid. Knockdown of various fatty acid transport proteins using siRNA significantly decreased radiolabeled fatty acid transport across the HBMEC monolayer. Our findings indicate that FATP-1 and FATP-4 are the predominant fatty acid transport proteins expressed in the BBB based on human and mouse expression studies. While transport studies in HBMEC monolayers support their involvement in fatty acid permeability, fatty acid translocase/CD36 also appears to play a prominent role in transport of fatty acids across HBMEC.     

Blood-brain barrier is involved in the efflux transport of a neuroactive steroid, dehydroepiandrosterone sulfate, via organic anion transporting polypeptide 2

We have investigated the transport characteristics of dehydroepiandrosterone sulfate (DHEAS), a neuroactive steroid, at the blood-brain barrier (BBB) in a series of functional in vivo and in vitro studies. The apparent BBB efflux rate constant of [(3)H]DHEAS evaluated by the brain efflux index method was 2.68 x 10(-2) min(-1). DHEAS efflux transport was a saturable process with a Michaelis constant (K:(m)) of 32.6 microM: Significant amounts of [(3)H]DHEAS were determined in the jugular venous plasma by HPLC, providing direct evidence that most of the DHEAS is transported in intact form from brain to the circulating blood across the BBB. This efflux transport of [(3)H]DHEAS was significantly inhibited by common rat organic anion-transporting polypeptide (oatp) substrates such as taurocholate, cholate, sulfobromophthalein, and estrone-3-sulfate. Moreover, the apparent efflux clearance of [(3)H]DHEAS across the BBB (118 microl/min-g of brain) was 10.4-fold greater than its influx clearance estimated by the in situ brain perfusion technique (11.4 microl/min-g of brain), suggesting that DHEAS is predominantly transported from the brain to blood across the BBB. In cellular uptake studies using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4), [(3)H]DHEAS uptake by TM-BBB4 cells exhibited a concentration dependence with a K:(m) of 34.4 microM: and was significantly inhibited by the oatp2-specific substrate digoxin. Conversely, [(3)H]digoxin uptake by TM-BBB4 cells was significantly inhibited by DHEAS. Moreover, the net uptake of [(3)H]DHEAS at 30 min was significantly increased under ATP-depleted conditions, suggesting that an energy-dependent efflux process may also be involved in TM-BBB4. RT-PCR and sequence analysis suggest that an oatp2 is expressed in TM-BBB4 cells. In conclusion, DHEAS efflux transport takes place across the BBB, and studies involving in vitro DHEAS uptake and RT-PCR suggest that there is oatp2-mediated DHEAS transport at the BBB.     

Role of human organic anion transporter 4 in the transport of ochratoxin A

The purpose of this study was to investigate the characteristics of ochratoxin A (OTA) transport by multispecific human organic anion transporter 4 (hOAT4) using mouse proximal tubule cells stably transfected with hOAT4 (S(2) hOAT4). Immunohistochemical analysis revealed that hOAT4 protein was localized to the apical side of the proximal tubule. S(2) hOAT4 expressed hOAT4 protein in the apical side as well as basolateral side and the cells were cultured on the plastic dish for experiments. S(2) hOAT4 exhibited a time- and concentration-dependent, and a saturable increase in OTA uptake, with an apparent K(m) value of 22.9+/-2.44 microM. The OTA uptakes were inhibited by several substrates for the OATs. Probenecid, piroxicam, octanoate and citrinin inhibited OTA uptake by hOAT4 in a competitive manner (K(i)=44.4-336.4 microM), with the following order of potency: probenecid > piroxicam > octanoate >citrinin. The efflux of OTA by S(2) hOAT4 was higher than that by mock. Addition of OTA resulted in slight decrease in viability of S(2) hOAT4 compared with mock. These results indicate that hOAT4 mediates the high-affinity transport of OTA on the apical side of the proximal tubule, whereas the transport characteristics of OTA are distinct from those by basolateral OATs.     

Functional characterization and cloning of amino acid transporter B(0,+) (ATB(0,+)) in primary cultured rat pneumocytes

Cationic amino acid transport in primary cultured rat pneumocytes exhibiting characteristics of alveolar epithelial type I-like cells are described. Asymmetry and activator ion dependency of (3)H-L-arginine uptake were characterized from the apical or basolateral fluid of pneumocytes grown on permeable support. Substrate specificity of transport was evaluated as a function of (3)H-L-arginine uptake inhibition in the presence of other amino acids. Transepithelial transport studies estimated (3)H-L-arginine flux in the apical-to-basolateral and basolateral-to-apical directions. Full length cDNA of rat amino acid transporter B(0,+) (rATB(0,+)) was cloned and its relative expression level studied. Results indicate that uptake of (3)H-L-arginine from apical fluid is dependent on Na(+) and Cl(-). Zwitterionic and cationic amino acids (excluding L-proline and anionic amino acids) inhibited uptake of (3)H-L-arginine from apical, but not basolateral incubation fluid. Apical-to-basolateral transepithelial flux of (3)H-L-arginine was 20x higher than basolateral-to-apical transport. Kinetic studies of (3)H-L-arginine uptake from apical fluid revealed maximal velocity (V(max)) and Michaelis-Menten constants (K(t)) of 33.32 +/- 2.12 pmol/mg protein/15 min and 0.50 +/- 0.11 mM, respectively, in a cooperative process having a coupling ratio of 1.18 +/- 0.16 with Na(+) and 1.11 +/- 0.13 with Cl(-). Expression of rATB(0,+) mRNA was identified by RT-PCR and Northern analysis. Corresponding cloned 3.2 kb rATB(0,+) cDNA sequence exhibits pronounced homology in deduced amino acid sequence to mouse (95% identity and 97% similarity) and human (89% identity and 95% similarity) ATB(0,+) homologues. We conclude that rat pneumocytes express ATB(0,+), which may partly contribute towards recovering cationic and neutral amino acids from alveolar luminal fluid.     

Immunocytochemical localization of Na+-HCO3- cotransporters and carbonic anhydrase dependence of fluid transport in corneal endothelial cells

In corneal endothelium, there is evidence for basolateral entry of HCO(3)(-) into corneal endothelial cells via Na(+)-HCO(3)(-) cotransporter (NBC) proteins and for net HCO(3)(-) flux from the basolateral to the apical side. However, how HCO(3)(-) exits the cells through the apical membrane is unclear. We determined that cultured corneal endothelial cells transport HCO(3)(-) similarly to fresh tissue. In addition, Cl(-) channel inhibitors decreased fluid transport by at most 16%, and inhibition of membrane-bound carbonic anhydrase IV by benzolamide or dextran-bound sulfonamide decreased fluid transport by at most 29%. Therefore, more than half of the fluid transport cannot be accounted for by anion transport through apical Cl(-) channels, CO(2) diffusion across the apical membrane, or a combination of these two mechanisms. However, immunocytochemistry using optical sectioning by confocal microscopy and cryosections revealed the presence of NBC transporters in both the basolateral and apical cell membranes of cultured bovine corneal endothelial cells and freshly isolated rabbit endothelia. This newly detected presence of an apical NBC transporter is consistent with its being the missing mechanism sought. We discuss discrepancies with other reports and provide a model that accounts for the experimental observations by assuming different stoichiometries of the NBC transport proteins at the basolateral and apical sides of the cells. Such functional differences might arise either from the expression of different isoforms or from regulatory factors affecting the stoichiometry of a single isoform.     

Prediction of intestinal absorption and metabolism of pharmacologically active flavones and flavanones

Three glycosilated flavonoids (diosmin, hesperidin and naringin) and respective aglycones were characterized in terms of their apparent ionisation constants and bidirectional permeability using the cellular model Caco-2 as well as the artificial membrane model PAMPA. Ionisation curves were established by capillary electrophoresis. It was confirmed that significant amounts of the aglycones are ionised at physiological pH whereas the glycosides are in the neutral form. Permeation was not detected for the glycosides in either the apical-to-basolateral or basolateral-to-apical directions confirming the need for metabolism before absorption through the intestinal membrane. The aglycones permeated in both directions with apparent permeabilities (P(app)) in the range of 1-8x10(-5) cm/s. The results from both in vitro methods correlated providing some evidence of passive transport; however, the hypothesis of active transport cannot be excluded particularly in the case of diosmetin. Metabolism of the aglycones was detected with the cell model, more extensively when loading in the apical side. Some of the metabolites were identified as glucuronide conjugates by enzymatic hydrolysis.     

Distinct mechanisms of zinc uptake at the apical and basolateral membranes of caco-2 cells.

Zinc uptake mechanisms at the apical and basolateral membrane borders of caco-2 cells were examined. This human-derived cell line possesses many morphological and functional characteristics of absorptive small intestinal cells. By day 14, confluent and well-differentiated monolayers were formed when the cells were grown on porous polycarbonate filters. Labelled zinc was placed on the apical or basal side of the monolayer and its uptake by the cells, as well as its transport across the monolayer, were measured. Zinc uptake by the cells from the apical side was found to be a saturable process (Kt = 41 microM; Vmax = 0.3 nmols/cm2/10 min) with a diffusional term at higher concentrations (1.0 sec/cm). Apical uptake was not affected by metabolic inhibitors or potential zinc ligands. Zinc uptake from the basolateral side was concentration dependent (Kd = 1.3 sec/cm) and was partially inhibited (30%) by ouabain and vanadate, suggesting that the (Na-K)-ATPase on the basolateral membrane is involved in the serosal uptake of zinc by the cell. Transport of zinc across the monolayers from the apical or basolateral compartment was concentration dependent and was not affected by metabolic inhibitors. Zinc transport from the basolateral side was greater than 2-fold greater than apical transport. Hence, separate mechanisms can be distinguished with respect to zinc uptake at the apical and basolateral membranes of caco-2 cells.     

Beta-adrenoceptor-mediated control of apical membrane conductive properties in fetal distal lung epithelia

Distal lung epithelial cells isolated from fetal rats were cultured (48 h) on permeable supports so that transepithelial ion transport could be quantified electrometrically. Unstimulated cells generated a short-circuit current (I(sc)) that was inhibited (~80%) by apical amiloride. The current is thus due, predominantly, to the absorption of Na(+) from the apical solution. Isoprenaline increased the amiloride-sensitive I(sc) about twofold. Experiments in which apical membrane Na(+) currents were monitored in basolaterally permeabilized cells showed that this was accompanied by a rise in apical Na(+) conductance (G(Na(+))). Isoprenaline also increased apical Cl- conductance (G(Cl-)) by activating an anion channel species sensitive to glibenclamide but unaffected by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). The isoprenaline-evoked changes in G(Na(+)) and G(Cl(minus sign)) could account for the changes in I(sc) observed in intact cells. Glibenclamide had no effect upon the isoprenaline-evoked stimulation of I(sc) or G(Na(+)) demonstrating that the rise in G(Cl-) is not essential to the stimulation of Na(+) transport.     

ABCA1 and scavenger receptor class B,type I,are modulators of reverse sterol transport at an in vitro blood-brain barrier constituted of porcine brain capillary endothelial cells

The objective of the present study was to investigate the involvement of key players in reverse cholesterol/24(S)OH-cholesterol transport in primary porcine brain capillary endothelial cells (pBCEC) that constitute the BBB. We identified that, in addition to scavenger receptor class B, type I (SR-BI), pBCEC express ABCA1 and apolipoprotein A-I (apoA-I) mRNA and protein. Studies on the regulation of ABCA1 by the liver X receptor agonist 24(S)OH-cholesterol revealed increased ABCA1 expression and apoA-I-dependent [3H]cholesterol efflux from pBCEC. In unpolarized pBCEC, high density lipoprotein, subclass 3 (HDL3)-dependent [3H]cholesterol efflux, was unaffected by 24(S)OH-cholesterol treatment but was enhanced 5-fold in SR-BI overexpressing pBCEC. Efflux of cellular 24(S)-[3H]OH-cholesterol was highly efficient, independent of ABCA1, and correlated with SR-BI expression. Polarized pBCEC were cultured on porous membrane filters that allow separate access to the apical and the basolateral compartment. Addition of cholesterol acceptors to the apical compartment resulted in preferential [3H]cholesterol efflux to the basolateral compartment. HDL3 was a better promoter of basolateral [3H]cholesterol efflux than lipid-free apoA-I. Basolateral pretreatment with 24(S)OH-cholesterol enhanced apoA-I-dependent basolateral cholesterol efflux up to 2-fold along with the induction of ABCA1 at the basolateral membrane. Secretion of apoA-I also occurred preferentially to the basolateral compartment, where the majority of apoA-I was recovered in an HDL-like density range. In contrast, 24(S)-[3H]OH-cholesterol was mobilized efficiently to the apical compartment of the in vitro BBB by HDL3, low density lipoprotein, and serum. These results suggest the existence of an autoregulatory mechanism for removal of potentially neurotoxic 24(S)OH-cholesterol. In conclusion, the apoA-I/ABCA1- and HDL/SR-BI-dependent pathways modulate polarized sterol mobilization at the BBB.     

Acute upregulation of blood-brain barrier glucose transporter activity in seizures

Brain extraction of (18)F-labeled 2-fluoro-2-deoxy-D-glucose (FDG) was significantly higher in pentylene tetrazole (PTZ)-treated rats (32 +/- 4%) than controls (25 +/- 4%). The FDG permeability-surface area product (PS) was also significantly higher with PTZ treatment (0.36 +/- 0.05 ml. min(-1). g(-1)) than in controls (0.20 +/- 0.06 ml. min(-1). g(-1)). Cerebral blood flow rates were also elevated by 50% in seizures. The internal carotid artery perfusion technique indicated mean [(14)C]glucose clearance (and extraction) was increased with PTZ treatment, and seizures increased the PS by 37 +/- 16% (P < 0.05) in cortical regions. Because kinetic analyses suggested the glucose transporter half-saturation constant (K(m)) was unchanged by PTZ, we derived estimates of 1) treated and 2) control maximal transporter velocities (V(max)) and 3) a single K(m). In cortex, the glucose transporter V(max) was 42 +/- 11% higher (P < 0.05) in PTZ-treated animals (2.46 +/- 0.34 micromol. min(-1). g(-1)) than in control animals (1.74 +/- 0.26 micromol. min(-1). g(-1)), and the K(m) = 9.5 +/- 1.6 mM. Blood-brain barrier (BBB) V(max) was 31 +/- 10% greater (P < 0.05) in PTZ-treated (2.36 +/- 0. 30 micromol. min(-1). g(-1)) than control subcortex (1.80 +/- 0.25 micromol. min(-1). g(-1)). We conclude acute upregulation of BBB glucose transport occurs within 3 min of an initial seizure. Transporter V(max) and BBB glucose permeability increase by 30-40%.     

Requirement of potassium for the action of anti-diuretic hormone (ADH) on frog skin

The aim of the present study is to study whether the presence of K(+) in bathing media is required for the action of ADH to the ionic transport across the skin in the frog species Rana hexadactyla. lonic transport was measured as transepithelial potential difference (TEPD) and short circuit current (SCC) by using an indigenously developed computer based voltage-clamp technique. Addition of ADH (40 nM) on the serosal side significantly increased the TEPD and SCC with Normal Ringer (NR) on both sides. ADH had no effect subsequent to amiloride (100 μM) pre-treatment, which confirmed the ADH-induced Na(+) transport. Chloride also has a significant role in the development of TEPD. To determine the role of K(+), Potassium-free Ringer (KFR) was placed on both sides; addition of ADH had no effect consequently. Further experiments were carried out to find out which side of K(+) was required for the action of ADH. There was a lack of ADH effect with apical NR and serosal KFR, demonstrating that serosal K(+) is essential to activate Na(+), K(+)- ATPase. Similarly, the ADH effect was lacking with apical KFR and serosal NR that was the novel finding of this study. Due to the concentration gradient, the K(+) was secreted from serosal side to apical side through barium (1 mM) blockable K(+) channel. This study provides evidence that serosal as well as apical K(+) are necessary for the action of ADH.     

Mannitol derivate used as a marker for voltammetrically monitored transport across the blood-brain barrier under condition of locus coeruleus stimulation

1-Deoxy-1-nitro-D-mannitol (DN-Man) was used (femoral vein injection, approximate concentration in the blood 30 mmol.l-1) in pentobarbital anaesthetized rats as a promising marker detectable by differential pulse voltammetry (DPV) to study its transport across the blood-brain barrier (BBB) to the extra-cellular space of the frontoparietal cortex. DN-Man detection limit in in vitro calibrations (saline, blood) using DPV and carbon fiber microelectrodes was 0.5 mmol.l-1 with a good linearity (r = = 0.996) over the entire tested range (up to 30 mmol.l-1). The slow time-course of the rise of DN-Man signal (y = 106/(1 + (17.8/t)3)) in the cortex confirmed the functional BBB state. Electrical stimulation of the locus coeruleus (LC) (300 rectangular pulses at a frequency of 100 Hz, 1 mA, pulse duration 0.2 ms) elevated significantly DN-Man current in the cortex (to 168 +/- 59% of the control, mean +/- S.D., n = 8). The evoked permeation increase of the BBB to DN-Man was short-lasting (minutes), and the second LC stimulation (repeated 5 min after the first one) was ineffective. This fact was probably due to the reduction of DN-Man levels in blood and/or an altered response of microvessels to neurotransmitters. It was shown here that, under carefully controlled surgical and experimental conditions, DPV and DN-Man might be useful for the monitoring of the regional dynamics of BBB transport changes. The presented results also support the view that BBB transport can be influenced by LC neuronal activity.