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The ability to permeate accross the blood brain barrier (BBB) is essential for drugs acting on the central nervous system (CNS). Thus, systems that allow rapid and inexpensive screening of the BBB-permeability properties of novel lead compounds are of great importance for speeding up the drug discovery process in the CNS-area. We used immortalized porcine brain microvessel endothelial cells (PBMECICl-2) to develop a model for measurement of blood-brain barrier permeation of CNS active drugs. Investigation of different cell culture conditions showed, that a system using C6 astrocyte glioma conditioned medium and addition of a cyclic AMP analog in combination with a type IV phosphodiesterase inhibitor (R020-1724) leads to cell layers with transendothelial electrical resistance values up to 300 Ω.cm2. Permeability studies with U-[14C]sucroseg ave a permeability coefficient Pe of 3.24 + 0.14 × 10−4 cm/min, which is in good agreement to published values and thus indicates the formation of tight junctions in vitro.
During the past several years, ceramide has emerged as an important second messenger triggering cell responses including proliferation, differentiation, growth arrest and apoptosis. This thesis has focused on the regulation of neutral ceramidase which critically determines, in concert with ceramide generating sphingomyelinases, the intracellular ceramide levels. In the first part it is reported that besides a rapid and transient increase in neutral sphingomyelinase activity a second delayed peak of activation occurs after hours of IL-1beta treatment. This second phase of activation is first detectable after 2 h of treatment, and steadily increases over the next two hours reaching maximal values after 4 h. In parallel, a pronounced increase in neutral ceramidase activity is observed, which accounts for a constant or even decreased level of ceramide after long-term IL-1beta treatment, despite continuous sphingomyelinase activation. The increase in neutral ceramidase activity is due to expressional up-regulation, as detected by an increase in mRNA level and enhanced de novo protein synthesis. The increase of neutral ceramidase protein levels and activity can be blocked dosedependently by the p38- mitogen-activated protein kinase (p38-MAPK) inhibitor, SB 202190, whereas the classical MAPK pathway inhibitor U0126, and the PKC inhibitor Ro 31-8220 were ineffective. Moreover, co-treatment of cells for 24 h with IL-1~ and SB 202190 leads to an increase in ceramide formation. Interestingly, IL-1beta-stimulated neutral ceramidase activation is not reduced in mesangial cells isolated from mice deficient in MAPK-activated protein kinase 2 (MAPKAPK-2), which is one possible downstream substrate of the p38-MAPK, thus suggesting that the p38-MAPK-mediated induction of neutral ceramidase occurs independently of MAPKAPK-2. The results suggest a biphasic regulation of sphingomyelin hydrolysis in cytokine-treated mesangial cells with a delayed de novo synthesis of neutral ceramidase counteracting sphingomyelinase activity and apoptosis. Neutral ceramidase may thus represent a novel cytoprotective enzyme for mesangial cells exposed to inflammatory stress conditions. In a second part, the effect of NO on neutral ceramidase was studied. Ceramide levels are strongly increased in a delayed fashion by stimulation of renal mesangial cells with NO. This effect is due to a dual action of NO, comprising an activation of sphingomyelinases and an inhibition of ceramidase activity. The inhibition of neutral ceramidase activity correlates with the decrease of neutral ceramidase protein. A complete loss of neutral ceramidase protein is obtained after 24h of NO stimUlation. Moreover, the NO-induced degradation is reversed by the protein kinase C (PKC) activator, 12-0-tetradecanoylphorbol-13-acetate (TPA) , but also by the physiological PKC activators platelet-derived growth factor-BB (PDGF-BB), angiotensin II and ATP, resulting in a normalisation of neutral ceramidase protein as well as activity. In vivo phosphorylation studies using 32Pj-labelled mesangial cells, reveal that TPA, PDGF-BB, angiotensin II and ATP trigger an increased phosphorylation of the neutral ceramidase, which is blocked by the broad-spectrum PKC inhibitor Ro-31 8220, but not by CGP 41251, which has a preferential action on Ca2+-dependent PKC isoforms, thus suggesting the involvement of a Ca2+-independent PKC isoenzyme. In vitro phosphorylation assays using recombinant PKC isoenzymes and neutral ceramidase immunoprecipitated from unstimulated mesangial cells, show that particularly the PKC-alpha isoform, and to a lesser extent the PKC-a isoform, are efficient in directly phosphorylating neutral ceramidase. The data show that NO is able to induce degradation of neutral ceramidase thereby promoting accumulation of ceramide in the cell. This effect is reversed by PKC activation, most probably by the PKC-delta isoenzyme, which may directly phosphorylate and thereby, prevent neutral ceramidase degradation. In the third chapter it is demonstrated that the NO-triggered degradation of neutral ceramidase involves activation of the ubiquitin/proteasome complex. The specific proteasome inhibitor, lactacystin, completely reverses the NO-induced degradation of ceramidase protein and neutral ceramidase activity. As a consequence, the cellular amount of ceramide, which drastically increases by NO stimulation, is reduced in the presence of lactacystin. Furthermore, ubiquitinated neutral ceramidase accumulates after NO stimulation. The data clearly show that the ubiquitin/proteasome complex is an important determinant of neutral ceramidase activity and thereby regulates the availability of ceramide. In a last part, the cellular localisation of neutral ceramidase was investigated using green fluorescent protein (GFP) as fusion protein to examine cellular distribution and translocation of neutral ceramidase. Unstimulated HEK 293 cells reveal after transient transfection experiments that neutral ceramidase is preferentially localized in the cytoplasm. PKC activation led to an accumulation of neutral ceramidase at the nuclear membrane. In summary, this work demonstrates that the neutral ceramidase is a fine regulated protein that plays a critical role in regulating intracellular ceramide levels and thereby the cell's fate to undergo apoptosis or survive. Regulation of neutral ceramidase can be achieved on all levels, i.e. on the mRNA level, the protein level or posttranslationally by phosphorylation and subcellular translocation. Future work will reveal whether neutral ceramidase can serve as a therapeutic target in the development of novel antiinflammatory and anti-tumour drugs.
The relevance of physiological immune aging is of great interest with respect to determining disorders with pathologic immune function in aging individuals. In recent years, the relevance of changes in peripheral lymphocytes in age-associated neurologic diseases has become more evident. Due to the lack of immunological studies, covering more than one event after mitogenic activation, we envisaged a new concept in the present study, aiming to investigate several events, starting from T cell receptor (TCR) ligation up to T cell proliferation. In addition, we addressed the question whether changes are present in the subsets (CD4, CD8) with aging. Phosphorylation of tyrosine residues declines with increasing age in CD4+ cells. Fewer levels of CD69 positive cells after 4 h mitogenic activation, altered expression of cytokines (IL2, IFN-gamma and TNF-alpha; 22 h) and lower proliferation (72 h) were determined in aging. Moreover, it could be shown that CD8+ lymphocytes react more effectively to mitogenic stimulation with reference to CD69 expression and proliferation in both age groups (<35 and >60 years old). These data indicate that T cell activation, mediated by TCR engagement, is significantly impaired in aging and both subsets are affected. However, bypassing the TCR does not fully restore T cell function, indicating that there are more mechanisms involved than impaired signal transduction through TCR only. The results will be discussed in relation to their relevance in neurodegenerative and psychiatric disorders.
P2X receptor subunits assemble in the ER of Xenopus oocytes to homomultimeric or heteromultimeric complexes that appear as ATP-gated cation channels at the cell surface. In this work it was intended to investigate the posttranslational modifications such as N-linked glycosylation and disulfide bond formation that is undergone by P2X1 receptors. In addition, the aim of this study was to examine the expression and the quaternary structure of selected P2X receptor isoforms in Xenopus oocytes. The investigation of the quaternary structure of the metabolically or surface labeled His-P2X2 receptor by BN-PAGE revealed that, while the protein complex is only partially assembling in oocytes, the plasma membrane form of the His-P2X2 receptor assembled into trimeric and even hexameric complex as was shown by the BN-PAGE analysis. Besides this finding, it is shown that the His-P2X5 protein that was purified from metabolically or surface labeled oocytes appeared as one single band corresponding to a trimer when analyzed by BN-PAGE. The present study signified that His-P2X6 alone does not reach a defined assembly status and possibly needs the hetero-polymerisation with other P2X subunits to assemble properly for insertion into the plasma membrane. Another finding of this study is that the P2X1 and P2X2 subunits could exist as heteromultimeric protein complexes in the plasma membrane of cells. Purification of surface expressed His-P2X2 subunit allowed the detection of co-injected P2X1 subunit and vice versa in Xenopus oocytes. Incubation with glutardialdehyde led to the cross-linking of P2X2 and P2X1 subunits to dimers and trimers. BN-PAGE analysis of the P2X2/P2X1 complex isolated under nondenaturing conditions from surface-labeled oocytes yielded one distinct band corresponding to a trimeric complex. The analysis of a C-terminally GFP tagged His-P2X1 fusion protein by confocal fluorescence microscopy revealed small clusters of the protein complexes, approximately 4-6 µm in diameter from a diffuse distribution of the protein in the plasma membranes of Xenopus oocytes. The cross-linking or BN-PAGE analysis of the fusion protein resulted in proteins that migrated quantitatively as trimers when purified in digitonin. The analysis of some chimeric constructs confirmed the results of others, which showed that desensitization can be removed from the P2X1 or P2X3 receptor by providing the N-domain from the P2X2 receptor (Werner et al., 1996) The exchange of this domain did not alter the quaternary structure of the chimeras, which showed to be present as trimers when expressed in oocytes. In addition, glycan minus mutants of His-P2X1 receptor were analyzed to examine whether carbohydrate side chains are important for P2X1 subunit assembly, surface expression, or ligand recognition. SDS-PAGE analysis of glycan minus mutants carrying Q instead of N at five individual NXT/S sequons reveals that 284N remains unused because of a proline in the 4 position. The four other sites (153Asn, 184N, 210N, and 300N) carry N-glycans, but solely 300N acquires complex-type carbohydrates. Like parent P2X1 receptor, glycan minus mutants migrate as homotrimers when resolved by blue native PAGE. Recording of ATP-gated currents revealed that elimination of 153N or 210N diminishes or increases functional expression levels, respectively. In addition, elimination of 210N causes a 3-fold reduction of the potency for ATP. If three or all four N-glycosylation sites are simultaneously eliminated, formation of P2X1 receptors is severely impaired or abolished, respectively. It is concluded that at least one N-glycan per subunit of either position is absolutely required for the formation of P2X1 receptors. The SDS-PAGE analysis of surface-labeled His-P2X2 and His-P2X5 receptors revealed that, while the His-P2X2 subunit acquires three complex-type carbohydrates, in case of His-P2X5 polypeptide, only two of the three N-glycans could obtain complex-type carbohydrates during transit of the Golgi apparatus. Furthermore, it was shown that DTT treatment blocked the appearance of newly made His-P2X1 at the plasma membranes of Xenopus oocytes. Also, it was revealed that the effects of DTT on His-P2X1 biogenesis are fully reversible. Removal of the reducing agent leads to subsequent folding and assembly into His-P2X1 receptor complex, followed by transport to the cell surface. The characterization of cysteine minus mutants by SDS PAGE and BN-PAGE demonstrated that, the cysteine substitution in the first cysteine rich domain (C1 - C6) does not have a major effect on assembly for the mutant receptors. In contrast, the replacement of the four cysteine residues (C7 - C10) from the second cysteine rich domain demonstrate a critical importance of this domain for the functional surface expression of P2X1 receptor. The investigations of several double cysteine mutants revealed that according to a similarity in the sensitivity to ATP, the C1 and C6, as well as C2 and C4 and finally C3 and C5 are pairs forming two disulfide bonds in each P2X1 subunit.
More than 70 years ago, the effects of extracellular adenosine 5'-triphosphate (ATP), a newly identified and purified biomolecule at that time (Fiske and Subbarow, 1925; Lohmann, 1929) were observed by Drury and Szent-Györgyi (1929). Since then, many pharmacological studies were carried out with extracellular adenine nucleotides in various intact organ systems, isolated tissues, and purified cell preparations. Yet it was not until 1972 that Burnstock introduced the concept of "purinergic nerves" and suggested that ATP might fulfil the criteria generally regarded as necessary for establishing a substance as a neurotransmitter, summarised by Eccles (1964):
• synthesis and storage of transmitter in nerve terminals
Strips of guinea-pig taenia coli (GPTC) were shown to take up large amounts of tritium-labelled adenosine when incubated with tritium-labelled adenosine, adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP) and ATP. The nucleoside was rapidly converted into and retained largely as [ 3 H]-ATP (Su et al., 1971).
• release of transmitter during nerve stimulation
Spontaneous relaxation of GPTC as well as relaxations induced by nerve stimulation or nicotine, respectively, in the presence of compounds which block adrenergic and cholinergic responses were accompanied by a remarkable increase in release of tritium-labelled material from taenia coli incubated in [ 3 H]-adenosine (Su et al., 1971).
• postjunctional responses to exogenous transmitters that mimic responses to nerve stimulation
Burnstock et al. (1966) characterised ATP and ADP as the most potent inhibitory purine compounds in the gut and observed that the effects of ATP mimic more closely the inhibitory response of the taenia to non-adrenergic nerve-stimulation than to adrenergic nerve stimulation (Burnstock et al., 1970).
enzymes that inactivate the transmitter and/or uptake systems for the transmitter or its breakdown products
When ATP was added to a perfusion fluid recycled through the vasculature of the stomach, very little ATP remained, but the perfusate contained substantially increased amounts of adenosine and inosine, as well as some ADP and AMP (Burnstock et al., 1970).
• drugs that can produce parallel blocking of potentiating effects on the responses of both exogenous transmitter and nerve stimulation
Tachyphylaxis to ATP produced in the rabbit ileum resulted in a consistent depression of responses to non-adrenergic inhibitory nerve stimulation, whereas responses to adrenergic nerve stimulation remained unaffected (Burnstock et al., 1970). Lower concentrations of quinidine reduced and finally abolished relaxation of GPTC induced by noradrenaline (NA) and by adrenergic nerve stimulation. Using higher concentrations of the compound, relaxant responses of GPTC to ATP as well as to non-adrenergic inhibitory nerve stimulation were abolished (Burnstock et al., 1970). ...