BackgroundRecent evidence suggests that the inducible kinin B1 receptor (B1R) contributes to pathogenic neuroinflammation induced by amyloid-beta (Aβ) peptide. The present study aims at identifying the cellular distribution and potentially detrimental role of B1R on cognitive and cerebrovascular functions in a mouse model of Alzheimer’s disease (AD).MethodsTransgenic mice overexpressing a mutated form of the human amyloid precursor protein (APPSwe,Ind, line J20) were treated with a selective and brain penetrant B1R antagonist (SSR240612, 10 mg/kg/day for 5 or 10 weeks) or vehicle. The impact of B1R blockade was measured on i) spatial learning and memory performance in the Morris water maze, ii) cerebral blood flow (CBF) responses to sensory stimulation using laser Doppler flowmetry, and iii) reactivity of isolated cerebral arteries using online videomicroscopy. Aβ burden was quantified by ELISA and immunostaining, while other AD landmarks were measured by western blot and immunohistochemistry.ResultsB1R protein levels were increased in APP mouse hippocampus and, prominently, in reactive astrocytes surrounding Aβ plaques. In APP mice, B1R antagonism with SSR240612 improved spatial learning, memory and normalized protein levels of the memory-related early gene Egr-1 in the dentate gyrus of the hippocampus. B1R antagonism restored sensory-evoked CBF responses, endothelium-dependent dilations, and normalized cerebrovascular protein levels of endothelial nitric oxide synthase and B2R. In addition, SSR240612 reduced (approximately 50%) microglial, but not astroglial, activation, brain levels of soluble Aβ1-42, diffuse and dense-core Aβ plaques, and it increased protein levels of the Aβ brain efflux transporter lipoprotein receptor-related protein-1 in cerebral microvessels.ConclusionThese findings show a selective upregulation of astroglial B1R in the APP mouse brain, and the capacity of the B1R antagonist to abrogate amyloidosis, cerebrovascular and memory deficits. Collectively, these findings provide convincing evidence for a role of B1R in AD pathogenesis.

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Maternofetal transport of l-carnitine, a molecule that shuttles long-chain fatty acids to the mitochondria for oxidation, is thought to be important in preparing the fetus for its lipid-rich postnatal milk diet. Using brush-border membrane (BBM) vesicles from human term placentas, we showed that l-carnitine uptake was sodium and temperature dependent, showed high affinity for carnitine (apparent K(m) = 11.09 +/- 1.32 microM; V(max) = 41.75 +/- 0.94 pmol.mg protein(-1).min(-1)), and was unchanged over the pH range from 5.5 to 8.5. l-Carnitine uptake was inhibited in BBM vesicles by valproate, verapamil, tetraethylammonium, and pyrilamine and by structural analogs of l-carnitine, including d-carnitine, acetyl-d,l-carnitine, and propionyl-, butyryl-, octanoyl-, isovaleryl-, and palmitoyl-l-carnitine. Western blot analysis revealed that OCTN2, a high-affinity, Na(+)-dependent carnitine transporter, was present in placental BBM but not in isolated basal plasma membrane vesicles. The reported properties of OCTN2 resemble those observed for l-carnitine uptake in placental BBM vesicles, suggesting that OCTN2 may mediate most maternofetal carnitine transport in humans.

Over the past two decades, Xenopus laevis oocytes have been widely used as an expression system to investigate both physiological and pathological properties of membrane proteins such as channels and transporters. Past studies have clearly shown the key implications of mistargeting in relation to the pathogenesis of these proteins. To unambiguously determine the plasma membrane targeting of a protein, a thorough purification technique becomes essential. Unfortunately, available techniques are either too cumbersome, technically demanding, or require large amounts of material, all of which are not adequate when using oocytes individually injected with cRNA or DNA. In this article, we present a new technique that permits excellent purification of plasma membranes from X. laevis oocytes. This technique is fast, does not require particular skills such as peeling of vitelline membrane, and permits purification of multiple samples from as few as 10 and up to >100 oocytes. The procedure combines partial digestion of the vitelline membrane, polymerization of the plasma membrane, and low-speed centrifugations. We have validated this technique essentially with Western blot assays on three plasma membrane proteins [aquaporin (AQP)2, Na(+)-glucose cotransporter (SGLT)1, and transient receptor potential vanilloid (TRPV)5], using both wild-type and mistargeted forms of the proteins. Purified plasma membrane fractions were easily collected, and samples were found to be adequate for Western blot identification.