Hypericum perforatum L. (St John's wort) extract Ze 117 inhibits dopamine re-uptake in rat striatal brain slices. An implication for use in smoking cessation treatment?

Classic synthetic antidepressant drugs, as well as St John's wort extract (SJW), directly inhibit the re-uptake of norepinephrine (NE) and/or serotonin (5-HT) into pre-synaptic axons. With chronic treatment they induce adaptive changes in a number of neurotransmitter receptors in synaptic membranes. The immediate effects of SJW Ze 117, an extract low in hyperforin content, on the specific dopamine (DA) uptake were studied in rat striatal brain slices and compared with the effects on NE and 5-HT uptake in rat cortical brain slices. Specific DA uptake was inhibited in a dose dependent manner. In contrast to the findings in synaptosomal preparations published so far, the extract showed different inhibitory potencies for the respective transporters. The potencies for the uptake inhibition of NA, DA and 5-HT were 30, 7 and 1, respectively. The results indicate that the SJW Ze 117 extract interferes in three ways with the individual uptakes of the relevant neurotransmitters that are considered to be causal in the development of depression. This observation, the concomitant and potent inhibition of DA re-uptake by SJW extract, may additionally provide a rationale for the treatment of nicotine or drug addiction with SJW.

Differential inhibition of inflammatory effector functions by petasin, isopetasin and neopetasin in human eosinophils.

BACKGROUND: Priming of eosinophils with granulocyte-macrophage colony-stimulating factor (GM-CSF) and subsequent stimulation with platelet-activating factor (PAF) or the anaphylatoxin C5a is associated with a rapid production of leukotrienes (LTs) and release of eosinophil cationic protein (ECP). OBJECTIVE: This study was designed to determine the effects of the sesquiterpene esters petasin, isopetasin and neopetasin on LT generation and ECP release in eosinophils in vitro. METHODS: The model of eosinophil activation described above was used to induce LT production and ECP release. Cells were incubated with petasins and control inhibitors prior to priming and stimulation. To analyse intracellular steps of eosinophil activation and determine potential drug targets, some key signalling events were studied. Activity of cytosolic phospholipase A2 (cPLA(2)) was measured by analysing the generation of arachidonic acid (AA). Translocation of 5-lipoxygenase (5-LO) was observed using immunofluorescence microscopy. Intracellular calcium concentrations [Ca2+]i were measured by a bulk spectrofluorometric assay. RESULTS: Whereas all three compounds inhibited LT synthesis, ECP release from eosinophils was blocked by petasin only, but not isopetasin or neopetasin. Similarly, PAF- or C5a-induced increases in [Ca2+]i were completely abrogated by petasin only, whereas isopetasin and neopetasin had significant lower blocking efficacy. Moreover, only petasin, but not isopetasin or neopetasin, prevented increases in cPLA(2) activity and 5-LO translocation from the cytosolic compartment to the nucleus envelope in calcium ionophore-stimulated eosinophils. CONCLUSION: These data suggest that different petasins may at least partially block different intracellular signalling molecules. To reduce LT synthesis, isopetasin and neopetasin may act at the level of or distal to 5-LO. In contrast, petasin may inhibit inflammatory effector functions in human eosinophils by disrupting signalling events at the level of or proximal to phospholipase Cbeta (PLCbeta), besides its potential inhibitory activity within mitogen-activated protein kinase (MAPK) and LT pathways.

Role of petasin in the potential anti-inflammatory activity of a plant extract of petasites hybridus.

A large production of leukotrienes (LTs) can be induced in human eosinophils or neutrophils by priming with granulocyte-macrophage colony-stimulating factor and subsequent stimulation with platelet-activating factor (PAF) or the anaphylatoxin C5a. Here, we investigated the effects of a plant extract of petasites hybridus (Ze339) and its isolated active sesquiterpene ester petasin in these two in vitro cell models. Zileuton, a 5-lipoxygenase inhibitor, was used as a positive control. All compounds inhibited both cysteinyl-LT synthesis in eosinophils and LTB(4) synthesis in neutrophils. In contrast, only Ze339 and petasin, but not zileuton, abrogated PAF- and C5a-induced increases in intracellular calcium concentrations. These data suggest that Ze339 and petasin may block, compared to zileuton, earlier signalling events initiated by G protein-coupled receptors in granulocytes, perhaps at the level of or proximal to phospholipase C(beta). Taken together, petasin appears to be one major active compound of petasites hybridus extract, since it demonstrates the same inhibitory activities on calcium fluxes and subsequent LT generation in both eosinophils and neutrophils as Ze339 does.

Natural killer cells activate human dermal fibroblasts.

Human dermal fibroblasts (HDF) undergo activation and secrete cytokines when cocultured with T cells. Here, we identify potent activators of HDF among human peripheral CD2(+)-lymphocytes. Populations with strong HDF activating capacity consisted essentially of cells with a natural killer (NK) surface marker phenotype (CD3(-), CD4(-), CD8(-), CD56(+)). Addition of these cells to HDF resulted in rapid increase of intracellular free calcium concentrations as an early rapid cell activation signal. Upregulation of mRNA encoding for the inflammatory cytokines IL-1 beta and IL-6 as well as for chemokines IL-8 and MCP-1 was detected after cells were cocultured. Elevated concentrations of IL-6 and IL-8 were found in coculture supernatants of HDF and NK-cells. Skin-homing NK cells leaving the blood-stream during an inflammatory skin reaction might therefore represent potent activators of local inflammatory cytokine and chemokine production.

Antioxidant and thyroid hormone status in selenium-deficient phenylketonuric and hyperphenylalaninemic patients.

BACKGROUND: Subjects consuming protein-restricted diets, such as patients with phenylketonuria (PKU) or milder hyperphenylalaninemias (HPAs) are at risk of selenium deficiency. Selenium is a cofactor of the antioxidant enzyme glutathione peroxidase and of the thyroid hormone converting enzyme thyroxine deiodinase. OBJECTIVE: Our goal was to investigate the effects of low plasma selenium on antioxidant and thyroid hormone status. DESIGN: We assessed plasma selenium, plasma total antioxidant status and the individual components thereof, erythrocyte antioxidant status, and plasma thyroid hormones in 24 PKU and 10 HPA patients and in 42 age-matched control subjects. RESULTS: Selenium was significantly lower in both PKU and HPA patients than in control subjects and the PKU patients had lower values than did the HPA patients. Total antioxidant status was lower in both patient groups than in the control group, whereas alpha-tocopherol, albumin, and uric acid were not significantly different among groups. Plasma selenium correlated well (r = 0.76) with erythrocyte glutathione peroxidase. PKU patients had lower glutathione peroxidase activity than did HPA patients and control subjects and lower glutathione concentrations than did control subjects. Both patient groups had lower superoxide dismutase activity than did control subjects. Free triiodothyronine was higher in both patient groups than in control subjects, whereas free thyroxine was higher in the PKU patients only. Free thyroxine and reverse triiodothyronine were inversely correlated with selenium. CONCLUSION: Supplementation with selenium seems to be advisable for patients consuming diets low in natural protein.

Soluble IL-1 receptor type I binds to human dermal fibroblasts and induces calcium flux.

Soluble cytokine receptors appear to modify ligand concentrations by stabilizing ligands or by specifically inhibiting interactions of ligands with their membrane-bound receptors. Here we describe a new function of the soluble interleukin-1 receptor type I (IL-1sR I). This receptor induced a transient rise of intracellular free calcium concentration in human dermal fibroblasts in a dose-dependent fashion. Mobilization of calcium by IL-1sR I was abolished in the presence of an equimolar concentration of IL-1 receptor antagonist (IL-1ra). Neutralizing antibodies against IL-1beta also abolished calcium mobilization stimulated with IL-1sR I indicating that IL-1beta is involved. IL-1sR I bound with high affinity (Kd 1-2 nM) to the fibroblasts. In addition, IL-1sR I enhanced expression of IL-6 and IL-8 mRNA. The observation that IL-1sR I can act as a ligand and agonist for membrane IL-1 extends the concept of the ligand-receptor functions of both IL-1 and IL-1sR I and adds a new dimension to the cytokine network.

Weekly versus biweekly lipid removal and effect of statins in severe hypercholesterolemia.

Lipid removal using a continuous-flow extracorporeal system is of proven efficacy in severe hypercholesterolemia. Because of the inconveniences and expenses of extracorporeal removal of lipids, the effects of two treatment intervals (weekly versus biweekly) were assessed in two adolescents with circulating cholesterol higher than 20.0 mmol/L. In both patients, circulating levels were largely lower on a weekly lipid removal interval when compared with a biweekly interval. A rapid reaccumulation of cholesterol was noted after lipid removal. Treatment with simvastatin decreased the rapid reappearance of total cholesterol noted during the first 2 days after lipid removal but without any major effect on the subsequent reaccumulation of cholesterol. Aggressive treatment of severe hypercholesterolemia with statins and especially with extracorporeal lipid removal is now possible and of proven efficacy. The minimal practical lipid removal treatment interval should be used.

Autologous versus allogeneic T cell-stimulated IL-6 production by dermal fibroblasts.

T cells adhere to human dermal fibroblasts (HDF). This cellular interaction leads to a pronounced secretion of the proinflammatory cytokines IL-6 and IL-8 via a juxtacrine stimulation induced by HDF-associated IL-1. Upon stimulation, fibroblasts express various surface proteins such as MCH-I molecules, which may interact with corresponding receptors on T cells. The present study was conducted to further investigate the mechanism of this complex interaction with regard to the secretion of IL-6 in cocultures of T cells and HDF. IL-6 was time- and dose-dependently upregulated in such cocultures. Spatial separation of the cells by microporous membranes resulted in a 90% reduction of IL-6 secretion, but when cells had limited cell contact IL-6 secretion was increased again. Allogeneic cocultures of T cells and HDF showed increased capacity of IL-6 stimulation as compared to autologous cultures. Our results suggest that MHC-I/T cell receptor interaction modulates IL-6 secretion in allogeneic and autologous cocultures.

Uptake and intracellular stability of glycosylphosphatidylinositol-specific phospholipase D in neuroblastoma cells.

Glycosylphosphatidylinositol-specific phospholipase D from mammalian serum has been described to be relatively stable towards the action of proteases in vitro, and it has been speculated that the enzyme may only be active on glycosylphosphatidylinositol-anchored substrates after its proteolytic processing in an intracellular compartment following uptake from body fluids. To test this hypothesis, we studied the possible uptake and intracellular processing of purified glycosylphosphatidylinositol-specific phospholipase D into the mouse neuroblastoma cell line N2A. We found that after incubation of neuroblastoma cells with glycosylphosphatidylinositol-specific phospholipase D at 37 degrees C the amount of cell-associated glycosylphosphatidylinositol-specific phospholipase D activity increased in a concentration- and time-dependent way. A similar uptake was also observed with 125I-labeled intact and trypsin-treated form of glycosylphosphatidylinositol-specific phospholipase D. We found that the incorporated radiolabeled proteins were processed intracellularly to distinct low molecular mass products, and that this process was in part inhibited by the presence of chloroquine during incubation.

Juxtacrine stimulation of cytokine production in cocultures of human dermal fibroblasts and T cells.

Adhesion of T cells to fibroblasts activates cells to produce cytokines, either by direct cell contact and/or soluble factors. A cell-associated form of IL-1 beta on fibroblasts might act through a cell contact mediated fashion. To test this hypothesis we analysed the activation of T cells and human dermal fibroblasts (HDF) in coculture experiments. Elevated levels of IL-1 beta, secreted by T cells as well as IL-6 and IL-8, mainly produced by HDF, were found in supernatant fluids of cocultured cells. IL-1 beta mRNA expression was induced in T cells as well as in HDF. While in HDF IL-1 beta remained cell-associated, T cells were activated to produce and secrete soluble IL-1 beta and IL-6. IL-1 beta and possibly other soluble factors increased IL-6 production by fibroblasts. These effects could be mainly attributed to CD8+ T cells. Our results suggest, that IL-1 beta, produced as a cell-associated cytokine by human dermal fibroblasts, acts as a juxtacrine molecule to stimulate T cells. Such a cellular cooperation, could be a powerful mediator in inflammatory response and possibly in wound healing.

Chondrodysplasia punctata with a mild clinical course.

We report a 7-year-old patient with chondrodysplasia punctata but without rhizomelia. He was born with typical clinical and radiological symptoms of this disease. He developed slowly with considerable psychomotor retardation but improved later, gaining some speech and psychosocial contacts. Joint contractures and bilateral cataracts are still major problems. De novo plasmalogen synthesis in fibroblasts was greatly reduced and DHAP-AT activity was at the lower limit of controls. Peroxisomal thiolase was present in its precursor form only. Membrane fluidity (measured by TMA-DPH fluorescence anisotropy) was increased in erythrocyte ghosts and in lymphocytes. Plasma phytanic acid concentration was elevated 5-fold. The patient represents a mild clinical course of chondrodysplasia punctata, resembling Conradi-Hünermann syndrome, but biochemically he has the typical peroxisomal dysfunction of rhizomelic chondrodysplasia punctata except for a high residual activity of DHAP-AT.

Evidence for lysosomotropism of memantine in cultured human cells: cellular kinetics and effects of memantine on phospholipid content and composition, membrane fluidity and beta-adrenergic transmission.

Memantine, an amantadine derivative, is therapeutically used for the treatment of various neurological and psychiatric disorders such as Parkinson's disease, spasticity, and dementia. Pharmacokinetics of memantine and its effects on phospholipid content and composition, on membrane properties and functions such as fluidity and beta-adrenergic transmission were studied in cultured human fibroblasts and macrophages. The kinetic behaviour of memantine was characteristic for a lysosomotropic drug. Fibroblasts exposed to 14C-memantine in the microM range accumulated the drug up to 200 fold above initial medium concentrations. Lysosomal drug storage was proven by indirect evidence and by analyses of subcellular fractions. Repetitive exposure to memantine resulted in a cumulative uptake. While memantine uptake after single exposure was fully reversible, the rate and extent of release of chronically accumulated drug was reduced but could be enhanced by the addition of unlabelled memantine or ammonium chloride to the medium. Chronic, but not single, exposure to memantine above 10 microM resulted in a concentration dependent phospholipid accumulation and in a shift in the phospholipid composition. There was an overproportionate increase in phosphatidylinositol at the expense of phosphatidylserine and sphingomyelin. Chronic exposure of cultured cells to memantine increased fluidity in the superficial layers of the plasma membrane and reduced the isoproterenol-stimulated cAMP-response without affecting beta-adrenoceptor density. All these findings were compatible with the kinetic behaviour and the effectiveness expected of a weak lysosomotropic drug.

Cellular accumulation of amiodarone and desethylamiodarone in cultured human cells. Consequences of drug accumulation on cellular lipid metabolism and plasma membrane properties of chronically exposed cells.

Amiodarone (AMIO), a potent antiarrhythmic drug, is clinically widely used despite its frequent side effects after chronic administration. These side effects coincide with an intralysosomal accumulation of AMIO and its main metabolite desethylamiodarone (DEA) and may be causally related to the drug-induced intracellular storage of phospholipids (PL). Kinetics of cellular uptake and release of radiolabelled AMIO and DEA were studied following single and multiple exposures of cultured human skin fibroblasts to 5 and 10 microM drug concentrations. AMIO and DEA were efficiently taken up into cultured cells. The rate of uptake was slower than that of other cationic amphiphilic drugs. The intracellular steady state concentrations were in the millimolar range suggesting a lysosomal trapping. Repetitive exposures of cultures resulted in a cumulative and partly saturable drug uptake. The accumulation of DEA was higher than that of AMIO throughout. AMIO and DEA previously taken up into the cells during a 2 hr exposure were completely released into the washing media, suggesting an exchangeable form of the accumulated drugs. Following repetitive exposures only part of the drugs was released. Under chasing conditions using washing media containing non-labelled AMIO and DEA respectively or ammonium chloride the release of the chronically accumulated 14C-labelled drugs was increased. This suggested a drug storage in the form of complexes in acidic compartments. Phospholipid (PL) content as well as individual PL fractions were changed in whole cells and in isolated plasma membranes. PL accumulation is assumed to occur by inhibition of PL degradation due to formation of non-degradable drug-PL complexes or by inhibition of phospholipase activities. Cellular PL accumulation seemed to interfere with PL recycling. Changes in PL composition of purified plasma membranes were in part complementary to the ones in whole cells. The alterations in membrane PL composition may explain the changes in membrane fluidity and the decrease in beta-adrenoceptor density and in isoproterenol-stimulated cAMP formation. The results obtained provide an explanation for the pharmacokinetic, and possibly for the pharmacodynamic and also toxicological behaviour of AMIO and DEA in vivo.

[Assessment of the nephrotoxicity of amikacin in patients with cystic fibrosis]. / Untersuchung der Nephrotoxizität von Amikacin bei Patienten mit zystischer Fibrose.

Cystic fibrosis patients are at risk for nephrotoxic effects of aminoglycosides. Fifteen cystic fibrosis patients were admitted to hospital with 18 acute exacerbations of pulmonary symptoms associated with the isolation of Pseudomonas aeruginosa from sputum. They were treated intravenously with amikacin and ceftazidime for 14 days. Urinary excretion of N-acetyl-beta-D-glucosaminidase and alpha 1-microglobulin, two markers of tubular damage, and of albumin, a marker of glomerular permeability, was studied before and during treatment. Urinary activity of N-acetyl-beta-D-glucosaminidase and excretion of alpha 1-microglobulin was normal before amikacin treatment in approximately two thirds of patients and pathologically increased at the end of the study in 95%. Urinary albumin excretion was always normal before amikacin treatment and failed to increase consistently during treatment. The pattern of urinary protein excretion observed in the study before and during treatment with amikacin indicates a selective tubular toxicity.

Intralysosomal generation of ammonia from urea by endocytosed urease results in secretion of free lysosomal arylsulfatase-A and increased activity of membrane-bound beta-glucosidase in cultured brain cells.

Hyperammonemia interferes with normal brain function. The effect of ammonia on free and membrane-bound lysosomal enzymes and on mucopolysaccharide metabolism was studied in cultured rat brain cells (ROC-1, hybridoma between C6-astrocytoma and oligodendrocytes). Intralysosomal ammoniagenesis was achieved from urea by endocytosed Jackbean urease followed by incubation of the cultures with urea. The intralysosomal location of urease was evidenced by the protective effects of leupeptin and urea on the stability of intracellular urease. Ammonia formed from urea resulted in an increased secretion of lysosomal arylsulfatase-A (AS-A), but not of the membrane-bound lysosomal beta-glucosidase into the culture medium, thus intralysosomal AS-A activity decreased. Lysosomal, membrane-bound beta-glucosidase activity increased, presumably due to intralysosomal proteolytic protection following an increased lysosomal pH. Intralysosomal ammoniagenesis temporarily impaired 35SO4-glycosaminoglycan degradation of prelabeled cells. The results support the hypothesis that hyperammonemic states may interfere with lysosomal functions in vivo as well in cultured cells.

Effects of culture and incubation conditions on membrane fluidity in monolayers of cultured cells measured as fluorescence anisotropy using trimethylammoniumdiphenylhexatriene (TMA-DPH).

Membrane fluidity of coverslip attached living cells was measured as fluorescence anisotropy using 5 microM trimethylammoniumdiphenylhexatriene (TMA-DPH) as fluorescent probe. Fluorescence anisotropy is inversely related to membrane fluidity. Cells were grown on glass coverslips that were inserted and directly incubated in quarz cuvettes. The coverslips were fixed with special holders at an angle of 30 degrees in respect to the incident light. Effects of incubation temperature, of cell growth and densities and of the ionic and nonionic composition of the incubation medium on membrane fluorescence anisotropy were measured. Membranes of growing cells were more fluid than those of stationary cells, while cell densities had no effect except at very low cell numbers. Calcium concentrations increasing from 0 to 8 mmol/l in the incubation medium proportionally decreased membrane fluidity. Hypotonicity of the incubation media increased membrane fluidity while hypertonicity compared to normotonicity had no effect. Differentiated human fibroblasts from different origins exhibited similar membrane fluidities. They were, however, different from those of rat cells. Membrane fluidity of rat brain tumor cells increased with age in culture while membrane fluidity of primary differentiating rat brain cells decreased in with age in culture. Measurement of fluorescence anisotropy in living cells attached to glass coverslips is a convenient tool to study effects of culture--as well as of environmental--conditions on membrane fluidity.

Acetylcholinesterase in mouse neuroblastoma NB2A cells: analysis of production, secretion, and molecular forms.

The mouse neuroblastoma cell line NB2A produces cellular and secreted acetylcholinesterase (AChE). After incubation of the cells for 4 days the ratio between AChE secreted into the medium and AChE in the cells was 1:1. The cell-associated enzyme could be subdivided into soluble AChE (25%) and detergent-soluble AChE (75%). Both extracts contained predominantly monomeric AChE (4.6S) and minor amounts of tetrameric AChE (10.6S), whereas the secreted AChE in the culture supernatant contained only the tetrameric form. All forms were partially purified by affinity chromatography. It could be demonstrated that the secretory and the intracellular soluble tetramers were hydrophilic, whereas the detergent-soluble tetramer was an amphiphilic protein. On the other hand the soluble and the detergent-soluble monomeric forms were amphiphilic and their activity depended on the presence of detergent. By digestion with proteinase K amphiphilic monomeric and tetrameric AChE could be converted to a hydrophilic form that no longer required detergent for catalytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [3H]diisopropylfluorophosphate-labelled AChE gave one band at 64 kilodaltons (kD) under reducing conditions and two additional bands at 120 kD and 140 kD under nonreducing conditions.

Chronic exposure of human cells in culture to the tricyclic antidepressant desipramine reduces the number of beta-adrenoceptors.

The effects of the antidepressant drug desipramine (DMI) on the density of beta-adrenoceptor sites were studied on intact cultured human cells: skin fibroblasts, lung fibroblasts and macrophages. Direct binding studies were performed with the radioligand 3H-CGP 12177, a hydrophilic beta-adrenergic antagonist. The confluent cell cultures were exposed to DMI and all three cell types showed a dose-dependent decrease in the number of beta-adrenergic binding sites. This receptor desensitisation was only seen after chronic exposure of the cells to DMI. The extent of desensitisation was comparable to that seen in brain following chronic treatment of rats with DMI. The affinity of the binding sites to the radioligand was not affected by the antidepressant drug action. From these results we suggest that the in vivo effect of antidepressant drugs on postsynaptic beta-adrenoceptor density, at least in part, reflects a primary drug action and not only an adaptive change to presynaptic events.

An unusual form of arylsulfatase A deficiency combined with sulfatide-excretion and a normal sulfatide-loading.

A 7-year-old girl who showed retarded psychomotor development and generalized hypotonia without any signs of progression is described. Marked deficiency of arylsulfatase A activity in leukocytes and fibroblasts was observed. Both parents showed activity in cultured fibroblasts within the heterozygote-normal range. Cerebroside-sulfatase activity was absent in cultured fibroblasts from the patient. Urinary analyses revealed a pathologically increased sulfatide excretion. Normal sensory nerve conduction velocity was found, but no metachromatic material was found in a sural nerve biopsy. Loading of the patient's fibroblasts with sulfatides resulted in normal uptake and normal degradation.

Changes in beta adrenergic receptors in submaxillary glands of chronically reserpine- or isoproterenol-treated rats.

Submaxillary glands of rats, chronically treated with isoproterenol or reserpine undergo morphological and functional alterations. These changes have been described to resemble those seen in human cystic fibrosis. Since it has been proposed that the beta adrenergic-mediated response is altered in exocrine glands of cystic fibrosis patients, we have examined whether the drug-induced alterations in rat salivary glands were accompanied by changes in the numbers and affinities of beta adrenergic receptor sites. Beta receptor characteristics were determined by means of direct binding studies with the beta adrenergic antagonist [3H]dihydroalprenolol. Compared to controls, specific binding capacities of [3H]dihydroalprenolol per unit of protein increased by 110 +/- 14% after reserpine treatment and decreased by 34 +/- 11% after isoproterenol administration (P less than .001). The difference in the number of receptor sites remained statistically significant whether expressed per gram of fresh weight or per unit of the membrane marker 5'-nucleotidase activity. Dissociation constants of the binding were not significantly different between the treatment groups. The observed changes in the number of beta receptors showed an inverse relationship to the drug-induced presumed changes of catecholamine concentrations at the receptor sites. This suggests the existence of a feedback system which maintains the balance within the autonomous nervous system. We speculate that in cystic fibrosis this adaptive system is genetically abnormal.