Sodium Transport from Blood to Brain: Inhibition by Furosemide and Amiloride

Brain sodium uptake in vivo was studied using a modified intracarotid bolus injection technique in which the uptake of 22 Na + was compared with that of the relatively impermeable molecule, [ 3 H]l-glucose. At a Na + concentration of 1.4 m M , Na + uptake was 1.74 ± 0.07 times greater than l-glucose uptake. This decreased to 1.34 ± 0.04 at 140 m M Na + , indicating saturable Na + uptake. Relative Na + extraction was not affected by pH but was inhibited by amiloride ( K i = 3 ± 10 −7 M ) and by 1 m M furosemide. The effects of these two inhibitors were additive. Brain uptake of 86 Rb + , a K + analogue, was measured to study interaction of K + with Na + transport systems. Relative 86 Rb + extraction was also inhibited by amiloride; however, it was not inhibited by furosemide. The results suggest the presence of two distinct transport systems that allow Na + to cross the luminal membrane of the brain capillary endothelial cell. These transport systems could play an important role in the movement of Na + from blood to brain.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66216/1/j.1471-4159.1983.tb09066.x.pd

Mechanisms of Sodium Transport at the Blood-Brain Barrier Studied with In Situ Perfusion of Rat Brain

The mechanism of unidirectional transport of sodium from blood to brain in pentobarbital-anesthetized rats was examined using in situ perfusion. Sodium transport followed Michaelis-Menten saturation kinetics with a V max of 50.1 nmol/g/min and a K m of 17.7 m M in the left frontal cortex. The kinetic analysis indicated that, at a physiologic sodium concentration, ∼26% of sodium transport at the blood-brain barrier (BBB) was carrier mediated. Dimethylamiloride (25 µ M ), an inhibitor of Na + /H + exchange, reduced sodium transport by 28%, whereas phenamil (25 µ M ), a sodium channel inhibitor, reduced the transfer constant for sodium by 22%. Bumetanide (250 µ M ) and hydrochlorothiazide (1.5 m M ), inhibitors of Na + -K + -2Cl − /NaCl symport, were ineffective in reducing blood to brain sodium transport. Acetazolamide (0.25 m M ), an inhibitor of carbonic anhydrase, did not change sodium transport at the BBB. Finally, a perfusate pH of 7.0 or 7.8 or a perfusate Pco 2 of 86 mm Hg failed to change sodium transport. These results indicate that 50% of transcellular transport of sodium from blood to brain occurs through Na + /H + exchange and a sodium channel in the luminal membrane of the BBB. We propose that the sodium transport systems at the luminal membrane of the BBB, in conjunction with Cl − /HCO 3 − exchange, lead to net NaCl secretion and obligate water transport into the brain.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65689/1/j.1471-4159.1996.66020756.x.pd

Oxygen Free-Radical Reduction of Brain Capillary Rubidium Uptake

Free radicals are proposed to play a role in the injury following cerebral ischemia in which cerebral edema is a prominent feature. To determine whether free radicals might alter the movement of ions and water across the blood-brain barrier, we examined their effect on brain capillary transport. Rat brain capillaries were isolated, incubated with a system that generates free radicals, and various capillary transport systems were studied. Rubidium uptake was reduced 74% whereas rubidium efflux, glucose transport, and capillary water space were unchanged. The results following the addition of radical scavengers indicated that hydrogen peroxide or a related free radical was the toxic species. These data suggest that free radicals can impair capillary endothelial cell mechanisms that help maintain homeostasis of electrolytes and water in brain.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65815/1/j.1471-4159.1986.tb12981.x.pd

Isolated rat brain capillarie spossess intact, structurally complex, interendothelial tight junctions; freeze-fracture verification of tight junction integrity

Populations of isolated brai capillaries have been proposed as useful models for in vitro studies of the blood-brain barrier. Preliminary investigations of barrier properties using such preparations of brain microvessels have suggested that the tight interendothelial junctions (zonulae occludentes) are intact and retain the impermeability to the protein tracer horseradish peroxidase, exhibited by them in vivo. The endothelial junctions of isolated capillaries are therefore assumed to be functionally `tight' in vitro. In order to determine the precise structural organization of these occluding junctions, including an estimate of their tightness (complexity), and to demonstrate a method for simple but precise assessment of junctional integrity, pellets of isolated rat brain capillaries were freeze-fractured and then replicated with platinum and carbon. The freeze-fracture images of interendothelial zonulae occludentes revealed complex arrays of intramembrane ridges and grooves characteristics of tight junctions. Longitudinal fractures of the cellular lining of capillaries exposed vast expanses of interendothelial plasma membrane interfaces and the junctional complexes situated between the cells. From such arrays, the elaborate and complex architecture of the zonulae occludentes could bre readily appreciated. Situated on the PF fracture faces are 6-8 parallel ridges which display a high degree of anastomosing between adjacent strands. The EF facture face contains grooves complementary to the PF face ridges. The zonulae occludentes of these capillary endothelial cells are similar in complexity to those reported in the literature for reptilian brain capillaries and therefore can be presumed `very tight'. This study demonstrate that freeze-fracture of pellets of brain capillaries alleviates problems inherent in whole tissue preparations and, in addition, demonstrates the usefulness of freeze-fracture as a tool to monitor junction structure during in vitro investigation of the blood-brain barrier.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24599/1/0000007.pd

Myo-inositol transport into endothelial cells derived from nervous system microvessels

Myo-inositol, the precursor in the biosynthesis of inositol phospholipids and inositol phosphates, is found in many tissues at concentrations well above its concentration in the plasma, but the highest concentrations are found in the central nervous system and the neuroretina. We describe an active, sodium gradient-dependent transport of myo-inositol into cultured endothelial cells derived from bovine retinal microvessels. Transport is inhibited by cytochalasin B, and phloridzin > phloretin. Mannitol, sorbitol, and fructose do not inhibit uptake, but -galactose inhibits uptake > -glucose > -glucose. The apparent Km of this transport system is 311 +/- 47 (S.D.) [mu]M and the apparent Vmax is 40.8 +/- 2.8 (S.D.) pmol[middle dot]mg protein-1[middle dot]min-1. This transport system may be a key in the maintenance of high tissue concentrations as it could concentrate myo-inositol from the plasma into the extracellular spaces of the eye and central nervous system.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28672/1/0000489.pd

The role of the coagulation cascade in brain edema formation after intracerebral hemorrhage

The coagulation cascade has a potential role in brain edema formation due to intracerebral hemorrhage. In this study blood and other solutions were injected stereotactically into the right basal ganglia in rats. Twenty-four hours following injection, brain water and ion contents were measured to determine the amount of brain edema. Intracerebral blood resulted in an increase in brain water content. The amount of brain edema surrounding the intracerebral hematoma was reduced by a thrombin inhibitor Na-(2-Naphthalenesulfonylglycyl)-4-amidino-DL-phenylalaninepiperidide, (α-NAPAP) infused into the hematoma after the clot had been allowed to solidify. The inhibitor did not alter the actual size of the clot mass. An artificial clot composed of fibrinogen, thrombin, and styrene microspheres also produced brain edema. A fibrin clot led to edema formation even in the absence of mass effect provided by the microspheres. The single component responsible for production of brain edema in all these models was thrombin. The edema was formed in response to a fibrinogen-independent pathway. These results indicate that the coagulation cascade is involved in brain edema that develops adjacent to an intracerebral hematoma.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41647/1/701_2005_Article_BF01420301.pd

Identification of Hypoxanthine Transport and Xanthine Oxidase Activity in Brain Capillaries

Microvessel segments were isolated from rat brain and used for studies of hypoxanthine transport and metabolism. Compared to an homogenate of cerebral cortex, the isolated microvessels were 3.7-fold enriched in xanthine oxidase. Incubation of the isolated microvessels with labeled hypoxanthine resulted in its rapid uptake followed by the slower accumulation of hypoxanthine metabolites including xanthine and uric acid. The intracellular accumulation of these metabolites was inhibited by the xanthine oxidase inhibitor allopurinol. Hypoxanthine transport into isolated capillaries was inhibited by adenine but not by representative pyrimidines or nucleosides. Similar results were obtained when blood to brain transport of hypoxanthine in vivo was measured using the intracarotid bolus injection technique. Thus, hypoxanthine is transported into brain capillaries by a transport system shared with adenine. Once inside the cell, hypoxanthine can be metabolized to xanthine and uric acid by xanthine oxidase. Since this reaction leads to the release of oxygen radicals, it is suggested that brain capillaries may be susceptible to free radical mediated damage. This would be most likely to occur in conditions where the brain hypoxanthine concentration is increased as following ischemia.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65340/1/j.1471-4159.1985.tb05451.x.pd

Hyperosmotic arabinose solutions open the tight junctions between brain capillary endothelial cells in tissue culture

Tight junctions between bovine brain capillary endothelial cells in primary cell culture are impermeable to horseradish peroxidase (HRP) after 5 min of incubation. However, following 1-5 min of exposure to 1.6 M arabinose, HRP penetrates the extracellular space between successive tight junctions. Endothelial cells in control cultures contain a small number of cytoplasmic pits and vesicles containing HRP which do not increase in number after hyperosmotic treatment.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24782/1/0000206.pd