Christiane Kunert-Keil

Deposition of Alzheimer's β-amyloid is inversely correlated with P-glycoprotein expression in the brains of elderly non-demented humans

Deposition of the beta-amyloid peptide (Abeta) in the brain occurs during normal ageing and is substantially accelerated in patients with Alzheimers disease. Since Abeta is continuously produced in the brain, it has been suggested that a clearance mechanism should exist to prevent its accumulation and subsequent aggregation. Until now, little attention has been paid to the possible role of P-glycoprotein (P-gp), a member of the ATP binding cassette superfamily of transporter proteins, in the pathogenesis of Alzheimers disease. A recent study demonstrated that Abeta40 and Abeta42 interact directly with P-gp. We therefore hypothesized that Abeta accumulation in the brain would correlate inversely with the degree of vascular P-gp expression. To study early pathogenetic factors that influence the deposition of Abeta, at routine autopsies, brain tissue samples were taken from 243 non-demented subjects who died between the ages of 50 and 91 years. Vascular P-gp expression and the number of Abeta40- and Abeta42-positive senile plaques were assessed immunohistochemically in the medial temporal lobe. In addition, the apolipoprotein E (apoE) genotypes, as well as multiple drug resistance gene 1 ( ) polymorphisms (exon 2, G-1A; exon 21, G2677T/A; exon 26, C3436T), were also determined for each case. P-gp expression was not correlated with genotypes, but we found a significant inverse correlation between P-gp expression and the deposition of both Abeta40 and Abeta42 in the medial temporal lobe. Our results provide the first evidence in human brain tissue that the accumulation of Abeta may be influenced by the expression of P-gp in blood vessels, and suggest that P-gp may influence the elimination of Abeta from brain.

Tissue-specific expression of TRP channel genes in the mouse and its variation in three different mouse strains

BackgroundThe purpose of this work was to study the gene expression of transient receptor potential (TRP) channels in the mouse. The application of a standardized and quantitative technique, TaqMan RT-PCR, should give information about the pattern and relative importance of TRP channels for murine tissues and cell types. To verify data sets with an independent method, we studied the occurrence of some of the transcripts by in situ hybridization.ResultsWe have characterized the mRNA expression of 22 TRP channels in the mouse with a focus on nerve and muscle tissues. This is the first study to describe the expression profiles of all channel isoforms of the four related Group 1 subfamilies (TRPC, TRPV, TRPM and TRPA) with a standardized and quantitative technique. Comparisons of transcript abundance showed a consistent dominance of TRPM7 and TRPC3 in most tissues. We further observed characteristic patterns and differences in gene expression of individual channels ranging over three orders of magnitude. The overall level of TRP channel mRNAs was highest in brain areas followed by kidney, lung, reproductive organs and muscle. In brain TRPM3 and TRPM7 dominated and 19 other isoforms were detected. In lung and kidney TRPV4, TRPV5 and TRPM7 were found in highest levels. TRPM7, TRPC3, TRPC6 and TRPM3 mRNAs were characteristically present in all tested muscle tissues. Most data obtained with the C57Bl/10 mouse strain were confirmed with Balb/c and NOD mice. However, TRPC3, C6, TRPM7, M3, TRPV2 and V4 expression showed marked differences in the three tested mouse strains. In situ hybridization revealed co-expression of transcripts on the cellular level and widely confirmed the data obtained with RT-PCR.ConclusionTranscripts coding for members of the TRPC, TRPV, TRPM and TRPA subfamilies of TRP cation channels are present in a broad spectrum of murine tissues. Several channel isoforms often coexist in a specific tissue or cell type. TRP channel expression does not show typical tissue specific dominance of individual members as is known from other ion channel families. Mouse strain specific variations of TRP channel expression indicate that genetic background or physiological requirements considerably influence expression levels.

The Role of P-glycoprotein in Cerebral Amyloid Angiopathy; Implications for the Early Pathogenesis of Alzheimer’s Disease

It has been shown in vitro that beta-amyloid (Abeta) is transported by P-glycoprotein (P-gp). Previously, we demonstrated that Abeta immunoreactivity is significantly elevated in brain tissue of individuals with low expression of P-gp in vascular endothelial cells. These findings led us to hypothesize that P-gp might be involved in the clearance of Abeta in normal aging and particularly in Alzheimers disease (AD). As we were interested in the early pathogenesis of Abeta deposition, we studied the correlation between cerebral amyloid angiopathy (CAA) and P-gp expression in brain tissue samples from 243 non-demented elderly cases (aged 50 to 91 years). We found that endothelial P-gp and vascular Abeta were never colocalized, i.e., vessels with high P-gp expression showed no Abeta deposition in their walls, and vice versa. Abeta deposition occurred first in arterioles where P-gp expression was primarily low, and disappeared completely with the accumulation of Abeta. At this early stage, P-gp was upregulated in capillaries, suggesting a compensatory mechanism to increase Abeta clearance from the brain. Capillaries were usually affected only at later stages of CAA, at which point P-gp was lost even in these vessels. We hypothesize that Abeta clearance may be altered in individuals with diminished P-gp expression due, e.g., to genetic or environmental effects (such as drug administration). The impairment of Abeta clearance could lead to the accumulation and earlier deposition of Abeta, both in the walls of blood vessels and in the brain parenchyma, thus elevating the risk of CAA and AD.

CYP2D6 genotype and induction of intestinal drug transporters by rifampin predict presystemic clearance of carvedilol in healthy subjects

Clinical trials have indicated that the combined β‐ and α‐adrenergic receptor blocker carvedilol improves the survival rate in patients with advanced chronic heart failure. The objective of our study was the identification and quantification of factors that modulate steady‐state serum concentrations of carvedilol and its enantiomers and that may influence therapeutic efficacy and safety.

Expression and Localization of P-glycoprotein in Human Heart: Effects of Cardiomyopathy

ABC-type transport proteins, such as P-glycoprotein (P-gp), modify intracellular concentrations of many substrate compounds. They serve as functional barriers against entry of xenobiotics (e.g., in the gut or the blood-brain barrier) or contribute to drug excretion. Expression of transport proteins in the heart could be an important factor modifying cardiac concentrations of drugs known to be transported by P-gp (e.g., β-blockers, cardiac glycosides, doxorubicin). We therefore investigated the expression and localization of P-gp in human heart. Samples from 15 human hearts (left ventricle; five non-failing, five dilated cardiomyopathy, and five ischemic cardiomyopathy) were analyzed for expression of P-gp using real-time RT-PCR, immunohistochemistry, and in situ hybridization. Immunohistochemistry revealed expression of P-gp in endothelium of both arterioles and capillaries of all heart samples. Although P-gp mRNA was detected in all samples, its expression level was significantly reduced in patients with dilated cardiomyopathy. We describe variable expression of P-gp in human heart and its localization in the endothelial wall. Thus, intracardiac concentrations of various compounds may be modified, depending on the individual P-gp level.

Transient receptor potential cation channels in normal and dystrophic mdx muscle

To investigate the defective calcium regulation of dystrophin-deficient muscle fibres we studied gene expression and localization of non-voltage gated cation channels in normal and mdx mouse skeletal muscle. We found TRPC3, TRPC6, TRPV4, TRPM4 and TRPM7 to be the most abundant isoforms. Immunofluorescent staining of muscle cross-sections with antibodies against TRP proteins showed sarcolemmal localization of TRPC6 and TRPM7, both, for mdx and control. TRPV4 was found only in a fraction of fibres at the sarcolemma and around myonuclei, while TRPC3 staining revealed intracellular patches, preferentially in mdx muscle. Transcripts of low abundance coding for TRPC5, TRPA1 and TRPM1 channels were increased in mdx skeletal muscle at certain stages. The increased Ca(2+)-influx into dystrophin-deficient mdx fibres cannot be explained by increased gene expression of major TRP channels. However, a constant TRP channel expression in combination with the well described weaker Ca(2+)-handling system of mdx fibres may indicate an imbalance between Ca(2+)-influx and cellular Ca(2+)-control.

Expression of calcium channel TRPV6 in ovine epithelial tissue.

Functional studies indicate marked differences in the extent and site of gastrointestinal calcium absorption between monogastric and ruminant species. Given that the epithelial calcium channel TRPV6 has a pivotal role in active intestinal calcium transport, this study investigated TRPV6 expression in ovine duodenal, jejunal, ruminal and renal epithelial cells using RT-PCR, in situ hybridisation, Western blot analysis and immunohistochemistry. The expression patterns suggested that, in contrast to monogastric animals, the jejunum is more important than the duodenum for calcium absorption in sheep. Furthermore, the expression of TRPV6-specific transcripts in ruminal epithelium was much lower than expected and its sub-cellular localisation suggested that this ion channel does not contribute to calcium absorption from the forestomachs.

Experimental and histological investigations of the bone using two different Oscillating Osteotomy techniques compared with conventional rotary osteotomy

Over the past decade, coinciding with the appearance of a number of new ultrasonic surgical devices, there has been a marked increase in interest in the use of ultrasound in oral surgery and implantology as alternative osteotomy method. The aim of this study was the comparison of the effect of osteotomies performed using ultrasonic surgery (Piezosurgery(®)), sonic surgery SONICflex(®) and the conventional bur method on the heat generation within the bone underneath the osteotomy and light-microscopy observations of the bone at different cutting positions in porcine mandibular segments. It was found that the average heat generated by SONICflex(®) sonic device was close to that by conventional rotary bur (1.54-2.29°C), whereas Piezosurgery(®) showed a high generated heat up to 18.17°C. Histological investigations of the bone matrix adjacent to the defect radius showed intact osteocytes with all three instruments and similar wide damage diameter at the bottom region. SONICflex(®) showed smooth cutting surfaces with minimal damage in the upper defect zone. Finally, presented results showed that sonic surgery performed with SONICflex(®) is an alternative osteotomy method and can be used as an alternative to the conventional bur method.

Diacylglycerol analogues activate second messenger‐operated calcium channels exhibiting TRPC‐like properties in cortical neurons

The lipid diacylglycerol (DAG) analogue 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) was used to verify the existence of DAG‐sensitive channels in cortical neurons dissociated from E13 mouse embryos. Calcium imaging experiments showed that OAG increased the cytosolic concentration of Ca2+ ([Ca2+]i) in nearly 35% of the KCl‐responsive cells. These Ca2+ responses disappeared in a Ca2+‐free medium supplemented with EGTA. Mn2+ quench experiments showed that OAG activated Ca2+‐conducting channels that were also permeant to Ba2+. The OAG‐induced Ca2+ responses were unaffected by nifedipine or omega‐conotoxin GVIA (Sigma‐Aldrich, Saint‐Quentin Fallavier, France) but blocked by 1‐[β‐(3‐(4‐Methoxyphenyl)propoxy)‐4‐methoxyphenethyl]‐1H‐imidazole hydrochloride (SKF)‐96365 and Gd3+. Replacing Na+ ions with N‐methyl‐d‐glucamine diminished the amplitude of the OAG‐induced Ca2+ responses showing that the Ca2+ entry was mediated via Na+‐dependent and Na+‐independent mechanisms. Experiments carried out with the fluorescent Na+ indicator CoroNa Green showed that OAG elevated [Na+]i. Like OAG, the DAG lipase inhibitor RHC80267 increased [Ca2+]i but not the protein kinase C activator phorbol 12‐myristate 13‐acetate. Moreover, the OAG‐induced Ca2+ responses were not regulated by protein kinase C activation or inhibition but they were augmented by flufenamic acid which increases currents through C‐type transient receptor potential protein family (TRPC) 6 channels. In addition, application of hyperforin, a specific activator of TRPC6 channels, elevated [Ca2+]i. Whole‐cell patch‐clamp recordings showed that hyperforin activated non‐selective cation channels. They were blocked by SKF‐96365 but potentiated by flufenamic acid. Altogether, our data show the presence of hyperforin‐ and OAG‐sensitive Ca2+‐permeable channels displaying TRPC6‐like properties. This is the first report revealing the existence of second messenger‐operated channels in cortical neurons.

Overexpression of glutathione S‐transferase A1‐1 in ECV 304 cells protects against busulfan mediated G2‐arrest and induces tissue factor expression

The antineoplastic drug busulfan is frequently used in preconditioning regimens for bone marrow transplantation. Pharmacokinetics vary tremendously between patients due to extensive metabolism in the liver via conjugation to glutathione catalysed by glutathione S‐transferase (GST) A1‐1. Since elevated busulfan plasma levels have been reported to be a risk factor for developing veno‐occlusive disease (VOD), metabolism of busulfan may play a pivotal role in the induction of VOD. Therefore, we developed a cell model to investigate the influence of busulfan metabolism on its biological effects. GSTA1‐1 cDNA was transfected into the cell line ECV 304 and protein expression was demonstrated by Western blotting. Enzymatic activity could be detected by formation of tetrahydrothiophene. Additionally, effects of busulfan treatment on cell cycle and expression of tissue factor have been investigated. A busulfan‐induced G2‐arrest was reduced in GSTA1‐1‐transfected cells, which consequently displayed a significantly higher activity of cdc2 kinase (24.1±1.5 AU mg−1 protein) after busulfan treatment compared to controls (14.7±2.3 AU mg−1 protein; P<0.01). Elevated basal expression of tissue factor in GSTA1‐1‐transfected ECV 304 cells could be 4 fold increased by busulfan treatment. These data demonstrate that ECV 304 cells transfected with GSTA1‐1 provide a valuable tool to assess busulfan metabolism in vitro. Furthermore, overexpression of GSTA1‐1 leads to a partial protection against cell cycle effects of busulfan and affects tissue factor expression.