Ulf Bickmeyer

Endocytotic uptake of iron oxide nanoparticles by cultured brain microglial cells

Microglia are the phagocytotic cells of the brain that respond rapidly to alterations in brain homeostasis. Since iron oxide nanoparticles (IONPs) are used for diagnostic and therapeutic applications in the brain, the consequences of an exposure of microglial cells to IONPs are of particular interest. To address this topic we have synthesized and characterized fluorescent BODIPY®-labelled IONPs (BP-IONPs). The average hydrodynamic diameter and the ζ-potential of BP-IONPs in water were ∼65 nm and -49 mV, respectively. Both values increased after dispersion of the particles in serum containing incubation medium to ∼130 nm and -8 mV. Exposure of cultured rat microglial cells with BP-IONPs caused a time-, concentration- and temperature-dependent uptake of the particles, as demonstrated by strong increases in cellular iron contents and cellular fluorescence. Incubation for 3h with 150 and 450 μM iron as BP-IONPs increased the cellular iron content from a low basal level of ∼50 nmol iron mg(-1) to 219±52 and 481±28 nmol iron (mg protein)(-1), respectively. These conditions did not affect cell viability, but exposure to higher concentrations of BP-IONPs or for longer incubation periods severely compromised cell viability. The BP-IONP fluorescence in viable microglial cells was co-localized with lysosomes. In addition, BP-IONP accumulation was lowered by 60% in the presence of the endocytosis inhibitors 5-(N-ethyl-N-isopropyl)amiloride, tyrphostin23 and chlorpromazin. These results suggest that the rapid accumulation of BP-IONPs by microglial cells is predominantly mediated by macropinocytosis and clathrin-mediated endocytosis, which direct the accumulated particles into the lysosomal compartment.

Mode of action of membrane-disruptive lytic compounds from the marine dinoflagellate Alexandrium tamarense

Certain allelochemicals of the marine dinoflagellate Alexandrium tamarense cause lysis of a broad spectrum of target protist cells but the lytic mechanism is poorly defined. We first hypothesized that membrane sterols serve as molecular targets of these lytic compounds, and that differences in sterol composition among donor and target cells may cause insensitivity of Alexandrium and sensitivity of targets to lytic compounds. We investigated Ca(2+) influx after application of lytic fractions to a model cell line PC12 derived from a pheochromocytoma of the rat adrenal medulla to establish how the lytic compounds affect ion flux associated with lysis of target membranes. The lytic compounds increased permeability of the cell membrane for Ca(2+) ions even during blockade of Ca(2+) channels with cadmium. Results of a liposome assay suggested that the lytic compounds did not lyse such target membranes non-specifically by means of detergent-like activity. Analysis of sterol composition of isolates of A. tamarense and of five target protistan species showed that both lytic and non-lytic A. tamarense strains contain cholesterol and dinosterol as major sterols, whereas none of the other tested species contain dinosterol. Adding sterols and phosphatidylcholine to a lysis bioassay with the cryptophyte Rhodomonas salina for evaluation of competitive binding indicated that the lytic compounds possessed apparent high affinity for free sterols and phosphatidylcholine. Lysis of protistan target cells was dose-dependently reduced by adding various sterols or phosphatidylcholine. For three tested sterols, the lytic compounds showed highest affinity towards cholesterol followed by ergosterol and brassicasterol. Cholesterol comprised a higher percentage of total sterols in plasma membrane fractions of A. tamarense than in corresponding whole cell fractions. We conclude therefore that although the molecular targets of the lytic compounds are likely to involve sterol components of membranes, A. tamarense must have a complex self-protective mechanism that still needs to be addressed.

Ageladine A, a pyrrole-imidazole alkaloid from marine sponges, is a pH sensitive membrane permeable dye

The alkaloid ageladine A, a pyrrole-imidazole alkaloid isolated from marine Agelas sponges shows fluorescence in the blue-green range during excitation with UV light with the highest absorption at 370 nm. The fluorescence of this alkaloid is pH dependent. Highest fluorescence is observed at pH 4, lowest at pH 9 with the largest fluorescence changes between pH 6 and 7. Ageladine A is brominated, which facilitates membrane permeation and therefore allows for easy staining of living cells and even whole transparent animal staining. To calculate the exact pH in solutions, cells, and tissues, the actual concentration of the alkaloid has to be known. A ratiometric measurement at the commonly used excitation wavelengths at 340/380 nm allows pH measurements in living tissues with an attenuated influence of the ageladine A concentration on calculated values. The fluorescence changes report small intracellular pH changes induced by extracellular acidification and alkalization as well as intracellular alkalization induced by ammonium chloride.

Functional neuroanatomy of the rhinophore of Aplysia punctata

BackgroundFor marine snails, olfaction represents a crucial sensory modality for long-distance reception, as auditory and visual information is limited. The posterior tentacle of Aplysia, the rhinophore, is a chemosensory organ and several behavioural studies showed that the rhinophores can detect pheromones, initiate orientation and locomotion toward food. However the functional neuroanatomy of the rhinophore is not yet clear. Here we apply serotonin-immunohistochemistry and fluorescent markers in combination with confocal microscopy as well as optical recording techniques to elucidate the structure and function of the rhinophore of the sea slug Aplysia punctata.ResultsWith anatomical techniques an overview of the neuroanatomical organization of the rhinophore is presented. Labelling with propidium iodide revealed one layer of cell nuclei in the sensory epithelium and densely packed cell nuclei beneath the groove of the rhinophore, which extends to about two third of the total length of the rhinophore. Serotonin immunoreactivity was found within the olfactory glomeruli underneath the epithelium as well as in the rhinophore ganglion. Retrograde tracing from the rhinophore ganglion with 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA) demonstrated the connection of glomeruli with the ganglion. Around 36 glomeruli (mean diameter 49 μm) were counted in a single rhinophore. Fluorimetric measurements of intracellular Ca2+ levels using Fura-2 AM loading revealed Ca2+-responses within the rhinophore ganglion to stimulation with amino acids. Bath application of different amino acids revealed differential responses at different positions within the rhinophore ganglion.ConclusionOur neuroanatomical study revealed the number and position of glomeruli in the rhinophore and the rhinophore ganglion as processing stage of sensory information. Serotonin-immunoreactive processes were found extensively within the rhinophore, but was not detected within any peripheral cell body. Amino acids were used as olfactory stimuli in optical recordings and induced sensory responses in the rhinophore ganglion. The complexity of changes in intracellular Ca2+-levels indicates, that processing of odour information takes place within the rhinophore ganglion. Our neuroanatomical and functional studies of the rhinophore open up a new avenue to analyze the olfactory system in Aplysia.

The physiological response of the marine platyhelminth Macrostomum lignano to different environmental oxygen concentrations

SUMMARY The respiration rate of meiofauna is difficult to measure, and the response to variations in the environmental oxygen concentration has so far been mainly addressed through behavioral investigation. We investigated the effect of different oxygen concentrations on the physiology of the marine platyhelminth Macrostomum lignano. Respiration was measured using batches of 20 animals in a glass microtiter plate equipped with optical oxygen sensor spots. At higher oxygen saturations (>12 kPa), the animals showed a clear oxyconforming behavior. However, below this value, the flatworms kept respiration rates constant at 0.064±0.001 nmol O2 l−1 h−1 individual−1 down to 3 kPa PO2, and this rate was increased by 30% in animals that were reoxygenated after enduring a period of 1.5 h in anoxia. Physiological changes related to tissue oxygenation were assessed using live imaging techniques with different fluorophores in animals maintained in normoxic (21 kPa), hyperoxic (40 kPa) or near-anoxic (~0 kPa) conditions and subjected to anoxia–reoxygenation. The pH-sensitive dyes Ageladine-A and BCECF both indicated that pHi under near-anoxia increases by about 0.07–0.10 units. Mitochondrial membrane potential, Δψm, was higher in anoxic and hyperoxic than in normoxic conditions (JC1 dye data). Staining with ROS-sensitive dyes – DHE for detection of superoxide anion (O2•−) formation and C-H DFFDA for other ROS species aside from O2•− (H2O2, HOO• and ONOO) – showed increased ROS formation following anoxia–reoxygenation treatment. Animals exposed to hyperoxic, normoxic and anoxic treatments displayed no significant differences in O2•− formation, whereas mitochondrial ROS formation as detected by C-H2DFFDA was higher after hyperoxic exposure and lowest under near-anoxia conditions compared with the normoxic control group. Macrostomum lignano seems to be a species that is tolerant of a wide range of oxygen concentrations (being able to maintain aerobic metabolism from extremely low PO2 up to hyperoxic conditions), which is an essential prerequisite for successfully dealing with the drastic environmental oxygen variations that occur within intertidal sediments.

Measuring Ca2+-signalling at fertilization in the sea urchin Psammechinus miliaris: alterations of this Ca2+-signal by copper and 2,4,6-tribromophenol.

During fertilization, eggs undergo a temporary rise in the intracellular concentration of free Ca(2+) ions. Using the membrane permeable acetoxymethylester of the fluorescent calcium indicator dye Fura-2, Fura-2 AM, the Ca(2+)-signal at fertilization was not detectable in eggs of the sea urchin Psammechinus miliaris. However, after treatment of the eggs with Fura-2 AM in combination with MK571, an inhibitor for multidrug resistance associated proteins, clear Ca(2+)-signals at fertilization could be measured without microinjection of the dye. We used this methodology to detect possible alterations of Ca(2+)-signalling at fertilization by exposure of eggs to environmental pollutants. For this purpose, the heavy metal copper, the bromophenol 2,4,6-tribromophenol, the organic compound bisphenol A and the polycyclic aromatic hydrocarbon phenanthrene were tested for their potential to inhibit fertilization success of P. miliaris. Copper and 2,4,6-tribromophenol showed a dose-dependent effect on fertilization rates of P. miliaris and significantly inhibited fertilization at 6.3 microM Cu(2+) and 1 microM 2,4,6-tribromphenol. Bisphenol A significantly inhibited fertilization success at 438 microM while phenanthrene had no effect up to 56 microM. 6.3 microM copper and 100 microM 2,4,6-tribromophenol significantly increased the Ca(2+)-signal at fertilization. This alteration may contribute to the reduced fertilization rates of P. miliaris after exposure to copper and 2,4,6-tribromophenol.

Inhibition of multidrug/xenobiotic resistance transporter by MK571 improves dye (Fura 2) accumulation in crustacean tissues from lobster, shrimp, and isopod

Multidrug/xenobiotic resistance transporters are present in living organisms as a first line defence system against small, potentially harmful molecules from the environment or from internal metabolic reactions. Multidrug resistance associated proteins (MRP) are one type of ATP-Binding-Cassette (ABC) transporters, which also transport dyes such as Fura 2, a calcium chelating fluorescence indicator. The specific MRP inhibitor MK571 was used to investigate the fluorescence intensity of cells in tissues of the brain and the midgut gland of the crustaceans Homarus gammarus (lobster), Crangon crangon (brown shrimp) and Idotea emarginata (isopod) during incubation with Fura 2AM (1 microM). In the presence of the inhibitor MK571 (50 microM), the fluorescence of brain tissue significantly increased in all of the three species. The midgut gland of H. gammarus showed a significant increase of fluorescence, whereas there was no effect in the midgut glands of C. crangon and I. baltica. The half maximal concentration of MK571 was 50 microM as measured in the midgut gland of H. gammarus. In conclusion, MRP transporters are present in the three investigated crustacean nervous systems. Using the midgut glands of the three species, only in H. gammarus MK571 inhibited dye extrusion, indicating species-specific differences of transporter systems, their specificity, or tissue specific expression.

A secondary metabolite, 4,5-dibromopyrrole-2-carboxylic acid, from marine sponges of the genus Agelas alters cellular calcium signals

A secondary metabolite from sponges of the genus Agelas, 4,5-dibromopyrrole-2-carboxylic acid, which is well known as feeding deterrent, was investigated for effects on the cellular calcium homeostasis in PC12 cells. 4,5-Dibromopyrrole-2-carboxylic acid did not change intracellular calcium levels if applied alone without cell depolarization. During depolarization of the cellular membrane using high potassium solution, a dose dependent reduction of intracellular calcium elevation was revealed utilizing Fura II as calcium indicator. Significant reduction was seen at concentrations higher than 30μM in a series of experiments, but in single experiments a concentration of 300nM was still reversible effective. In the same concentration range, the onset of depolarization induced calcium elevations was significantly delayed by 4,5-dibromopyrrole-2-carboxylic acid. Dose dependent reduction and delay of depolarization evoked calcium elevations are probably due to a reduction of calcium entry via voltage operated calcium channels. One cellular mode of action of the feeding deterrent potential of 4,5-dibromopyrrole-2-carboxylic acid to fishes may be an interaction with the cellular calcium homeostasis of exposed cells.

Incorporated nematocysts in Aeolidiella stephanieae (Gastropoda, Opisthobranchia, Aeolidoidea) mature by acidification shown by the pH sensitive fluorescing alkaloid Ageladine A

The sequestration of nematocysts (a special group of cnidocysts) from cnidarian prey with subsequent use in defence is described for few metazoan phyla. Members of the taxon Aeolidoidea (Nudibranchia, Gastropoda) are well-known for this. Questions regarding the reasons some nematocysts do not discharge when the gastropod feeds and how these same nematocysts can be transported along the digestive tract into specialized morphological structures called cnidosacs, remain unanswered. Within the cnidosac, nematocysts are incorporated in cells and finally be used for defence against predators. The most plausible explanation for this phenomenon suggests there are immature and therefore non-functional nematocysts in the food. A recent study by Berking and Herrmann (2005) on cnidarians suggested that the nematocysts mature by acidification via proton transfer into the nematocyst capsule. According to this hypothesis only immature nematocysts are transported into the cnidosac where they are then made functional through an accumulation of protons. In this study we present a fluorescence staining method that tests the hypothesis by Berking and Herrmann (2005) and detects changes in the pH values of incorporated nematocysts, interpreted as changes in maturation stages. This marker, the fluorescent dye Ageladine A, stains nematocyst capsules according to their pH values. With Ageladine A we were able to show that kleptocnides indeed change their pH value after incorporation into the aeolidoidean cnidosac.

Siphonodictyal B1 from a Marine Sponge Increases Intracellular Calcium Levels Comparable to the Ca2+-ATPase (SERCA) Inhibitor Thapsigargin

Siphonodictyal B1 is a sesquiterpene-hydroquinone isolated from the Caribbean coral reef bioeroding sponge Siphonodictyon coralliphagum. Siphonodictyal B1 increased intracellular calcium levels in neuroendocrine cells (PC12) in the presence and absence of extracellular calcium using Fura-2 as a calcium-sensitive dye. The calcium rise was comparable in amplitude and timing to the application of the sarco-endoplasmic reticulum calcium-ATPase (SERCA) inhibitor thapsigargin from the terrestrial plant Thapsia garganica. The effects of thapsigargin and siphonodictyal B1 on intracellular calcium levels were not distinguishable in pharmacological experiments conducted with caffeine, ryanodine, muscarine, and thapsigargin in calcium-free and calcium-containing buffer, although thapsigargin was effective at lower concentrations. Thapsigargin is a sesquiterpene-lactone and has no structural similarities to siphonodictyal B1. We conclude that thapsigargin and siphonodictyal B1 share SERCAs as cellular targets. Siphonodictyal B1 may be involved in the process of bioeroding the calcium carbonate endoskeleton of the scleractinian corals attacked by S. coralliphagum.