Rat Brain Capillaries Research Articles

Role of blood-brain barrier organic anion transporter 3 (OAT3) in the efflux of indoxyl sulfate, a uremic toxin: its involvement in neurotransmitter metabolite clearance from the brain.

Renal impairment is associated with CNS dysfunctions and the accumulation of uremic toxins, such as indoxyl sulfate, in blood. To evaluate the relevance of indoxyl sulfate to CNS dysfunctions, we investigated the brain-to-blood transport of indoxyl sulfate at the blood-brain barrier (BBB) using the Brain Efflux Index method. [(3)H]Indoxyl sulfate undergoes efflux transport with an efflux transport rate of 1.08 x 10(-2)/min, and the process is saturable with a Km of 298 microm. This process is inhibited by para-aminohippuric acid, probenecid, benzylpenicillin, cimetidine and uremic toxinins, such as hippuric acid and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid. RT-PCR revealed that an OAT3 mRNA is expressed in conditionally immortalized rat brain capillary endothelial cell lines and rat brain capillary fraction. Xenopus oocytes expressing OAT3 were found to exhibit [(3)H]indoxyl sulfate uptake, which was significantly inhibited by neurotransmitter metabolites, such as homovanillic acid and 3-methoxy-4-hydroxymandelic acid, and by acyclovir, cefazolin, baclofen, 6-mercaptopurine, benzoic acid, and ketoprofen. These results suggest that OAT3 mediates the brain-to-blood transport of indoxyl sulfate, and is also involved in the efflux transport of neurotransmitter metabolites and drugs. Therefore, inhibition of the brain-to-blood transport involving OAT3 would occur in uremia and lead to the accumulation of neurotransmitter metabolites and drugs in the brain.

Functional expression and localization of P-glycoprotein at the blood brain barrier.

Until recently, the blood-brain barrier was viewed as a static lipid membrane barrier. Physical attributes of the cerebral endothelial cells such as the presence of tight junctions, paucity of vesicles or caveolae, and high electrical resistance were believed to be the primary components that provide the membrane selectivity of the blood-brain barrier to a variety of circulating compounds from the periphery. However, results from molecular biology, immunocytochemistry, biochemistry, and transport studies show that the cerebral endothelial cells possess an asymmetrical array of metabolic enzymes (i.e., alkaline phosphatase, cytochrome P450 enzymes, glutathione transferases) and energy-dependent efflux transport proteins (i.e., P-glycoprotein and Multidrug-resistance proteins) that are instrumental to the barrier function. P-glycoprotein, a membrane-associated, energy-dependent, efflux transporter, is expressed in brain parenchyma (i.e., astrocytes and microglia) as well as in blood-brain and blood-cerebrospinal fluid barriers. Its function along the blood-brain barrier is believed to prevent the accumulation of potentially harmful compounds in the brain by actively removing them from the brain into the peripheral circulation. This is a brief review on the expression and activity of P-glycoprotein at the blood-brain barrier, which reports on the localization of the protein in rat brain capillaries in situ as well as in a well-characterized in vitro model of the blood-brain barrier, an immortalized rat brain endothelial cell line, the RBE4. Immunocytochemical analysis employing various P-glycoprotein monoclonal antibodies, demonstrated the presence of the protein along the plasma membrane, in plasmalemmal vesicles and nuclear envelope of rat cerebral endothelial cells, both in situ and in vitro. Western blot analysis revealed a single band with a molecular weight of 170-180 kDa, a size previously reported for P-glycoprotein, in RBE4 cells. In addition, results from functional studies show that the accumulation of the P-glycoprotein substrate digoxin by RBE4 monolayer cells is significantly enhanced in the presence of standard P-glycoprotein inhibitors (verapamil, cyclosporin A, PSC 833), protease inhibitors (saquinavir, ritonavir, indinavir), and the metabolic inhibitor, sodium azide. These results demonstrate the functional expression of P-glycoprotein in the immortalized rat brain endothelial cell line, RBE4. Novel in situ and in vitro intracellular locations of P-glycoprotein in cerebral endothelial cells have been identified suggesting that this transporter may play a significant role in the subcellular distribution of substrates in the brain.

Main subunits of ionotropic glutamate receptors are expressed in isolated rat brain microvessels

Excitatory amino acids are known to modulate blood-brain barrier (BBB) permeability, however, the information on glutamate receptors in cerebral capillaries is inconsistent. In the present study, freshly isolated microvessels obtained from saline-perfused rat brains were used. Gene expression of the main N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptor subunits NMDAR1 and GLUR1, respectively, were investigated by reverse transcription-polymerase chain reaction (RT-PCR). The results confirmed the presence of both NMDAR1 and GLUR1 mRNAs in microvessels of seven brain regions studied. Moreover, specific binding of [3H]glutamate to capillary membranes and its displacement by AMPA, NMDA and metabotropic, but not kainate receptor agonists were observed. These results suggest that rat brain capillaries and/or albuminally adhering astrocyte processes possess functional glutamate receptors. Thus, the effects of glutamate agonists and antagonists in modulation of BBB function might be mediated directly by cerebral microvessels. [Neurol Res 2002; 24: 93-96]

Cloned blood-brain barrier adenosine transporter is identical to the rat concentrative Na+ nucleoside cotransporter CNT2.

Adenosine transport into brain is regulated by the activity of the adenosine transporter located at the brain capillary endothelial wall, which forms the blood-brain barrier (BBB) in vivo. To facilitate cloning of BBB adenosine transporters, poly A+ RNA was purified from isolated rat brain capillaries for production of a rat BBB cDNA library in the pSPORT vector. The cloned RNA (cRNA) generated from in vitro transcription of this library was injected into frog oocytes followed by measurement of [3H]-adenosine uptake. After dilutional cloning, a full-length, 2905-nucleotide adenosine transporter cDNA, designated clone A-11, was isolated. The A-11 clone yielded [3H]-adenosine flux ratios of 400 to 500 after injection of cRNA in oocytes. The adenosine uptake was sodium-dependent and insensitive to inhibition by S-(4-nitrobenzyl)-6-thioinosine (NBTI). The Km and Vmax of adenosine transport in the cRNA-injected oocytes were 23.1 +/- 3.7 micromol/L and 10.8 +/- 0.9 pmol/oocyte. min. The K0.5 for sodium was 2.4 +/- 0.1 mmol/L, with a Hill coefficient (n) of 1.06 +/- 0.07. DNA sequence analysis indicated the rat BBB A-11 adenosine cDNA was identical to rat concentrative nucleoside transporter type 2 (CNT2). The adenosine transporter activity of the rat BBB A-11 CNT2 clone is 50-fold more active than previously reported rat CNT2 clones. In summary, these studies describe the expression cloning of CNT2 from a rat BBB library and show that the pattern of sodium dependency and NBTI insensitivity of the cloned CNT2 are identical to patterns of adenosine transport across the BBB in vivo. These results suggest that BBB adenosine transport in vivo is mediated by CNT2, which would make CNT2 one of the few known sodium-dependent cotransporters that mediate substrate transport across the BBB in the blood to brain direction.

Blood-brain barrier genomics.

The blood-brain barrier (BBB) is formed by the brain microvascular endothelium, and the unique transport properties of the BBB are derived from tissue-specific gene expression within this cell. The current studies developed a gene microarray approach specific for the BBB by purifying the initial mRNA from isolated rat brain capillaries to generate tester cDNA. A polymerase chain reaction-based subtraction cloning method, suppression subtractive hybridization (SSH), was used, and the BBB cDNA was subtracted with driver cDNA produced from mRNA isolated from rat liver and kidney. Screening 5% of the subtracted tester cDNA resulted in identification of 50 gene products and more than 80% of those were selectively expressed at the BBB; these included novel gene sequences not found in existing databases, ESTs, and known genes that were not known to be selectively expressed at the BBB. Genes in the latter category include tissue plasminogen activator, insulin-like growth factor-2, PC-3 gene product, myelin basic protein, regulator of G protein signaling 5, utrophin, IkappaB, connexin-45, the class I major histocompatibility complex, the rat homologue of the transcription factors hbrm or EZH1, and organic anion transporting polypeptide type 2. Knowledge of tissue-specific gene expression at the BBB could lead to new targets for brain drug delivery and could elucidate mechanisms of brain pathology at the microvascular level.

Expression of GFRalpha-1, receptor for GDNF, in rat brain capillary during postnatal development of the BBB.

It is well known that the blood-brain barrier (BBB) matures at approximately 2 wk after birth in the rat. Recently, we showed that glial cell line-derived neurotrophic factor (GDNF) enhances the barrier function of porcine endothelial cells forming the BBB in culture. In the present study, we examined the relation between permeability of the BBB, using Evans blue as a tracer, and expression of the GDNF family receptor (GFRalpha-1) during postnatal development of the BBB. Morphometric analysis showed that exudation of Evans blue from capillaries of the cerebral cortex progressively decreased until postnatal day 21. Inversely, immunohistochemical examinations showed expression of GFRalpha-1 in the capillaries at postnatal day 3 and expression that reached the same levels as observed in adult rats by postnatal day 10. However, c-ret, which is thought to mediate a signal evoked by binding of GDNF to GFRalpha-1, was not expressed in the capillaries of the brain cortex in 3-mo-old rats. On the other hand, the tight junction proteins occludin and ZO-1 appeared to be fully expressed at birth. The reciprocal relation between GFRalpha-1 expression and the permeability of the BBB strongly suggests active participation of GDNF in postnatal development of the BBB, although the mechanism(s) involved is still veiled.

Does P-glycoprotein Limit Opioid-induced Analgesia In Vivo ? Thompson et al. (page 1392)

P-glycoprotein, a transmembrane protein, was first identified in tumor cells, but is also present at the luminal borders of other normal tissues, including intestinal and bronchial epithelium, and renal tubules, where it acts to either secrete xenobiotics or to prevent their absorption. P-glycoprotein has also been identified in mouse, rat, bovine, and human brain capillary endothelium, and believed to be a vital component of the blood–brain barrier.The team of Thompson et al. used P-glycoprotein knockout mice and wild-type mice to determine whether P-glycoprotein limits opioid-induced analgesia in vivo . After baseline assessment of thermal analgesia using the animals’ response to the standard hotplate test, each mouse received subcutaneous injections of morphine, morphine-6-glucuronide (M-6-G), methadone, fentanyl, and meperidine. Morphine was studied at doses of 1, 5, 10, and 20 mg/kg in the wild-type mice and 1, 3, and 5 mg/kg in the knockout mice; M-6-G at doses of 1, 3, and 5 mg/kg in both groups of animals; methadone at 5 mg/kg; fentanyl at 50 μg/kg; and meperidine at 50 mg/kg. Hot plate tests were repeated and each animal’s latency to hind paw licking behavior was recorded. The effect of cyclosporine (100 mg/kg), a P-glycoprotein inhibitor, on morphine analgesia in both types of mice was also assessed in separate experiments.Morphine induced greater analgesia in knockout mice than in wild-type mice; morphine brain concentrations were also greater in knockout mice. The authors also verified greater analgesia in knockout mice with methadone and fentanyl, but not with meperidine or M-6-G. Pretreatment with cyclosporine significantly increased analgesia in wild-type mice but had no effect in knockout mice. Results suggest that, at least in mice, P-glycoprotein limits morphine entry into the brain.

Alterations in rat's brain capillaries in a model of focal cerebral necrosis

Focal brain compression causes cerebral tissue damage. In this study we followed alterations in capillary ultrastructure in the rat cortex and neurohypophysis caused by 40 mm Hg compression for 15 minutes. One day after experiment we observed clogging of capillaries, accumulation of collagen fibrills under the basement membrane and necrosis or apoptosis of endothelial cells. Four days after it the basement membrane was multiplicated, blurred and thickened. In the neurohypophysis the formation of vessels lined with the atypical continuous endothelium was seen. There was also evidence for the migration of pericytes through the blurred basement membrane and the differentiation of pericytes into endothelial cells. Thus, vascular injury in the compressed brain is followed by a highly ordered sequence of processes in the basement membrane and perivascular cells leading to capillary repair.

The effects of valproate on the arachidonic acid metabolism of rat brain microvessels and of platelets

Long-term administration of the antiepileptic drug valproate can induce hematologic, hepatic and endocrine abnormalities and morphologic alterations in the brain capillaries and glial cells. Valproate elicits bone marrow suppression, reducing the number of red blood cells and platelets, and causes platelet functional abnormalities. Various data suggest that more than one mechanism of valproate-associated toxicity may exist, but the pathomechanism of cell function alterations elicited by valproate has not yet been elucidated. The reported ex vivo experiments were designed to investigate the effects of valproate on the arachidonic acid cascade of rat brain capillaries and platelets. Valproate was administered (300 mg/kg body weight/day) in the drinking water to male Wistar rats for 2 weeks. Isolated platelets and brain microvessels were labelled with [ 14C]arachidonic acid and the released [ 14C]eicosanoids were separated by overpressure thin-layer chromatography and determined quantitatively by liquid scintillation counting. Valproate treatment reduced the synthesis of cyclooxygenase and lipoxygenase products in rat platelets. In brain microvessels valproate stimulated the synthesis of lipoxygenase metabolites and attenuated the cyclooxygenase pathway. Modifications of the arachidonate cascade in platelets and brain microvessels may contribute to the cell function alterations caused by valproate.

Genetically engineered brain drug delivery vectors: cloning, expression and in vivo application of an anti-transferrin receptor single chain antibody-streptavidin fusion gene and protein.

A single chain Fv antibody-streptavidin fusion protein was expressed and purified from bacterial inclusion bodies following cloning of the genes encoding the variable region of the heavy chain and light chain of the murine OX26 monoclonal antibody to the rat transferrin receptor. The latter undergoes receptor mediated transcytosis through the brain capillary endothelial wall in vivo, which makes up the blood-brain barrier (BBB); therefore, the OX26 monoclonal antibody and its single chain Fv analog may act as brain drug delivery vectors in vivo. Attachment of biotinylated drugs to the antibody vector is facilitated by production of the streptavidin fusion protein. The bi-functionality of the OX26 single chain Fv antibody-streptavidin fusion protein was retained, as the product both bound biotin and the rat transferrin receptor in vitro and in vivo, based on pharmacokinetic and brain uptake analyses in anesthetized rats. The attachment of biotin-polyethyleneglycol-fluorescein to the OX26 single chain Fv antibody-streptavidin fusion protein resulted in illumination of isolated rat brain capillaries in confocal fluorescent microscopy. In conclusion, these studies demonstrate that genetically engineered single chain Fv antibody-streptavidin fusion proteins may be used for non-invasive neurotherapeutic delivery to the brain using endogenous BBB transport systems such as the transferrin receptor.

Effect of ischemia-reperfusion on xanthine oxidase activity in fetal rat brain capillaries

Objective: The purpose of this study was to investigate whether ischemia and subsequent reperfusion would affect xanthine oxidase activity in fetal rat brain capillaries. Study Design: We used rats on day 19 of pregnancy. Fetal ischemia was induced by bilateral occlusion of the utero-ovarian artery for 20 minutes. Reperfusion was achieved by releasing the occlusion to restore the circulation for 30 minutes. Control rats underwent a sham operation. Fetal brain capillaries were isolated for measurement of concentrations of hypoxanthine, xanthine, and uric acid, as well as of concentrations of thiobarbituric acid–reactive substances. The brain capillaries were incubated with hypoxanthine for 1-5 hours at 25°C. The activity of xanthine oxidase was estimated by measuring the amount of xanthine converted from hypoxanthine. Results: Occlusion for 20 minutes markedly increased the concentration of hypoxanthine but had no effect on levels of xanthine, uric acid, and thiobarbituric acid–reactive substances. However, subsequent reperfusion led to significant increases in the levels of xanthine, uric acid, and thiobarbituric acid–reactive substances. Xanthine oxidase activity, as measured by the amount of xanthine produced, was significantly greater in the animals subjected to both ischemia and ischemia-reperfusion compared with the control group. Conclusion: Ischemic insult led to the accumulation of hypoxanthine and stimulated xanthine oxidase activity in fetal brain capillaries. Subsequent reperfusion enhanced the degradation of hypoxanthine to uric acid, which may induce cerebral lipid peroxidation. (Am J Obstet Gynecol 1999;181:731-5.)

Early decrease of P-glycoprotein in the endothelium of the rat brain capillaries after moderate dose of irradiation

In this study, we examined the degree of disruption of blood-brain barrier (BBB) in irradiated rat brains using P-glycoprotein, one of the functional molecules of BBB, as a marker. In the animal experiments, disruption of BBB has been mainly studied at the acute stage of brain edema caused by a lethal dose of irradiation. However, they do not mimic the clinical situation of radiotherapy for malignant brain tumors. Therefore, we examined effects of a clinically compatible dose of radiation on BBB. The rat hemisphere received a single application of 25 Gy of X-rays, and P-glycoprotein was analyzed 5 days later by immunohistochemistry and Western blotting. Immunoreactivity of P-glycoprotein was found to be strong in endothelial cells of the brain of the nonirridiated rat as well as in the nonirradiated hemisphere of the irradiated rat. In contrast, very weak or no immunoreactivity was observed in the majority of endothelial cells in the irradiated hemisphere. Western blotting quantitatively showed that P-glycoprotein in the irradiated hemisphere decreased to nearly 60% that of the controls. The present study indicated that even a clinically applicable dose of radiation causes early disruption of BBB in the rat model. [Neural Res 1999; 21: 209-215]

Octanol-Water Partition: Searching for Predictive Models

The log n-octanol/water partition coefficient (log Po/w) still represents one of the most informative physicochemical parameters available to medicinal chemists. In the present work, principles, methodologies, and parameters are briefly reviewed for a variety of models developed to predict this parameter based on molecular structure. To include the developments of recent years, a total of more than 40 different approaches are mentioned with relevant bibliography within four major categories: group contribution methods, atomic contribution methods, molecular methods, and other physicochemical methods. To underscore once more the utility of this partition coefficient, a comprehensive and reevaluated correlation between log Po/w and in vivo permeability data of rat brain capillaries is included. Most deviants that fell below the trendline are those that have been recently found to be substrates for P-glycoprotein, a multidrug transporter that actively removes them from the brain. Accurate predictions of log Po/w may necessitate many parameters, but there is mounting evidence that molecular size and hydrogen bonding ability can account for a major part of the variance. Our recently developed, molecular size-based approach is reviewed, and it is argued that introduction of three-dimensionality allows the elimination of many empirically derived fragment constants without a significant deterioration of the predictive accuracy. A comparison of predictive power for six different methods on 145 molecules of interest for medicinal chemists is also included.

Examination of Blood‐Brain Barrier Transferrin Receptor by Confocal Fluorescent Microscopy of Unfixed Isolated Rat Brain Capillaries

Isolated rat brain capillaries were analyzed by confocal microscopy. Fluorescent immunoliposomes bearing the OX26 anti-transferrin receptor monoclonal antibody were synthesized and incubated with freshly isolated unfixed microvessels to visualize binding to luminal and abluminal membranes of the endothelium. Intactness of the endothelial structure was demonstrated by computer-aided reconstruction of a series of consecutive optical sections. These results indicate that analysis of unfixed brain capillaries by confocal microscopy offers the possibility of assigning the presence of membrane receptors to either the luminal or the basolateral plasma membrane domain.

Regulation of the Genes Encoding Interleukin‐6, Its Receptor, and gp130 in the Rat Brain in Response to the Immune Activator Lipopolysaccharide and the Proinflammatory Cytokine Interleukin‐1β

Interleukin-6 (IL-6) is a pleiotropic cytokine believed to play key roles in the neuroimmune interactions. This molecule may act on the nervous system by interacting with its specific receptor subunit (IL-6R) and the signal transducer gp130. The purposes of the present study were to describe the central distribution of IL-6, IL-6R, and gp130 mRNAs under basal conditions and to verify the influence of the immune activator lipopolysaccharide (LPS) and the proinflammatory cytokine interleukin-1beta (IL-1beta) on the expression of IL-6 and its related genes throughout the rat brain. Rats were killed at multiple times after intraperitoneal injection of the bacterial endotoxin and intravenous administration of the recombinant rat IL-1beta (rrIL-1beta), and their brains were cut into 30-microm coronal sections from the olfactory bulb to the end of the medulla. Each transcript was localized by in situ hybridization histochemistry using 35S-labeled rat riboprobes. The results show that IL-6 mRNA was undetectable in the brain under basal conditions and following the injection of rrIL-1beta. Injection of LPS rapidly stimulated transcription of this gene in the choroid plexus and the sensorial circumventricular organs (CVOs), including the organum vasculosum laminae terminalis (OVLT), subfornical organ, median eminence, and area postrema. Conversely, IL-6R and gp130 mRNAs were heterogeneously distributed throughout the brain under basal conditions. The injection of LPS stimulated the biosynthesis of IL-6R in the CVOs, medial preoptic area, bed nucleus stria terminalis, central nucleus of the amygdala, hippocampus, hypothalamic paraventricular nucleus, cerebral cortex, and blood vessels. Increased levels of IL-6R mRNA were also observed in the microvasculature following rrIL-1beta injection. Finally, gp130 mRNA expression was increased in the OVLT and throughout the endothelium of brain capillaries of LPS-treated rats but remained unchanged after administration of rrIL-1beta. These results demonstrate that expression of the genes encoding IL-6, IL-6R, and gp130 can be up-regulated in selective regions of the brain in response to the bacterial endotoxin LPS and the proinflammatory cytokine IL-1beta (only for IL-6R expression). This fine genetic regulation might be of great importance in the neuroimmune interplay and provides the evidence that sensorial CVOs and microvasculature are in a privileged position to mediate the action of IL-6 of central and/or systemic origin in the brain of immune-challenged animals.

Agrin accumulates in the brain microvascular basal lamina during development of the blood-brain barrier.

The blood-brain barrier (BBB) is an essential feature of the microvasculature in neural tissues. Agrin, a synapse organizing molecule at the neuromuscular junction, also accumulates on brain microvasculature and may be involved in BBB formation and function. We investigated the developmental expression of agrin at rat and chick brain capillaries by immunohistochemistry and immunoblotting. Anti-agrin immunoreactivity (IR) completely ensheathes all observed microvessels labeled with anti-von Willebrand factor in adult brains of both species. Brain microvascular agrin IR codistributes with anti-laminin IR, consistent with agrin localization in the microvessel basal lamina. On microvessels in testis and thymus, tissues which also contain blood-tissue barriers, the pattern of IR is indistinguishable from brain microvessels. In contrast, little or no agrin IR is observed on capillaries in muscle and other tissues. During chick and rat development, agrin accumulates on brain microvessels around the time the vasculature becomes impermeable. Differential staining and electroblotting suggest that the agrin isoforms expressed on brain microvessels lack the 8- and 11-amino acid sequences that confer on agrin high potency in acetylcholine receptor clustering. Taken in context with the organizing role of agrin in synaptogenesis, these results indicate that agrins may function as important players in the formation and maintenance of cerebral microvascular impermeability.

Agrin accumulates in the brain microvascular basal lamina during development of the blood-brain barrier

The blood-brain barrier (BBB) is an essential feature of the microvasculature in neural tissues. Agrin, a synapse organizing molecule at the neuromuscular junction, also accumulates on brain microvasculature and may be involved in BBB formation and function. We investigated the developmental expression of agrin at rat and chick brain capillaries by immunohistochemistry and immunoblotting. Anti-agrin immunoreactivity (IR) completely ensheathes all observed microvessels labeled with anti-von Willebrand factor in adult brains of both species. Brain microvascular agrin IR codistributes with anti-laminin IR, consistent with agrin localization in the microvessel basal lamina. On microvessels in testis and thymus, tissues which also contain blood-tissue barriers, the pattern of IR is indistinguishable from brain microvessels. In contrast, little or no agrin IR is observed on capillaries in muscle and other tissues. During chick and rat development, agrin accumulates on brain microvessels around the time the vasculature becomes impermeable. Differential staining and electroblotting suggest that the agrin isoforms expressed on brain microvessels lack the 8- and 11-amino acid sequences that confer on agrin high potency in acetylcholine receptor clustering. Taken in context with the organizing role of agrin in synaptogenesis, these results indicate that agrins may function as important players in the formation and maintenance of cerebral microvascular impermeability.

Brain Capillary Tissue Plasminogen Activator in a Diabetes Stroke Model

Tissue plasminogen activator (TPA) is normally expressed in rat brain capillaries. This study examines the expression of TPA in brain capillaries of diabetic rats in relation to focal ischemic brain injury. Diabetes type 1 was induced by streptozotocin for 7 days. Acute hyperglycemia was induced by 50% dextrose. Expression of TPA in brain capillaries was determined by Western blot and reverse transcription-polymerase chain reaction analyses. Focal stroke was produced by 1 hour of reversible middle cerebral artery occlusion. Physiological variables and cerebral blood flow were monitored during occlusion and within 1 hour of reperfusion. Neurological and neuropathologic examinations were performed after 24 hours of reperfusion. All rats developed comparable hyperglycemia (approximately 15 mmol/L). A complete depletion of TPA protein and 6.5-fold decrease in TPA mRNA were found in brain capillaries of diabetic rats, in contrast to normal TPA capillary levels in hyperglycemic rats. The blood flow in the periphery of the ischemic core was significantly reduced during reperfusion by 52% to 62% (P<.001) in diabetic rats and by 23% to 25% (P<.05) in hyperglycemic rats. The neurological score was worsened by 3.2-fold (P<.0003) by diabetes and by 24% by hyperglycemia only. Significant 41% (P<.007) and 29% (P<.05) increases in infarct volume and 163% (P<.007) and 60% increases in edema volume were found in diabetic rats relative to control and hyperglycemic rats, respectively. Diabetes type 1, but not acute hyperglycemia, produces downregulation of TPA in rat brain capillaries. This TPA reduction is associated with impaired restoration of blood flow after an ischemic insult, poor neurological outcome, and enhanced ischemic brain injury.

CDNA Cloning and Functional Characterization of Rat Intestinal Monocarboxylate Transporter

A cDNA clone which encodes a monocarboxylate transporter (ratMCT1) was isolated from a rat small intestinal cDNA library, which was screened by using full-length MCT1 cDNA of Chinese hamster ovary cells. The ratMCT1 cDNA was sequenced and predicted a protein of 494 amino acids with twelve potential transmembrane domains. The amino acid sequence showed 93.1% and 84.6% identity to the hamster and human monocarboxylate transporters, respectively. When expressed in Xenopus laevis oocytes, the ratMCT1 cRNA caused a significant increase in the uptake of radiolabeled lactic acid. Poly(A)+ RNA transcripts hybridizing to the ratMCT1 cDNA were detected in rat brain, heart, kidney, lung, muscle and brain capillaries. These results indicate that MCT1 contributes to pH-dependent and carrier-mediated transport of monocarboxylic acids in many tissues, not just in the small intestine.

Endothelin 1 stimulates Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport through ETA receptors and protein kinase C-dependent pathway in cerebral capillary endothelium.

The effect of endothelins (ET-1 and ET-3) on 86Rb+ uptake as a measure of K+ uptake was investigated in cultured rat brain capillary endothelium. ET-1 or ET-3 dose-dependently enhanced K+ uptake (EC50 = 0.60 +/- 0.15 and 21.5 +/- 4.1 nM, respectively), which was inhibited by the selective ETA receptor antagonist BQ 123 (cyclo-D-Trp-D-Asp-Pro-D-Val-Leu). Neither the selective ETB agonists IRL 1620 [N-succinyl-(Glu9,-Ala11,15)-ET-1] and sarafotoxin S6c, nor the ETB receptor antagonist IRL 1038 [(Cys11,Cys15)-ET-1] had any effect on K+ uptake. Ouabain (inhibitor of Na+,K(+)-ATPase) and bumetanide (inhibitor of Na(+)-K(+)-Cl- cotransport) reduced (up to 40% and up to 70%, respectively) the ET-1-stimulated K+ uptake. Complete inhibition was seen with both agents. Phorbol 12-myristate 13-acetate (PMA), activator of protein kinase C (PKC), stimulated Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport. ET-1- but not PMA-stimulated K+ uptake was inhibited by 5-(N-ethyl-N-isopropyl)amiloride (inhibitor of Na+/H+ exchange system), suggesting a linkage of Na+/H+ exchange with ET-1-stimulated Na+,K(+)-ATPase and Na(+)-K(+)-Cl- cotransport activity that is not mediated by PKC.