Browsing by Person "Hanke, Wolfgang"

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Exzitatorische Pharmakologie der retinalen Spreading Depression
(2010) Sieber, Michaela; Hanke, Wolfgang
The phenomenon of Spreading Depression (SD) is a suppression of neuronal activity, propagating wave-like in the grey matter. This results from a massive ion translocation where potassium ions pour into the cell and sodium-, chlorideand calcium ions pour out of the cell. At the same time there is also a slow negative potential shift up to 30mV. Spreading Depression also occurs in the retina, a part of the central nervous system and can easily be observed there with the naked eye. This dissertation describes the effects of excitatory pharmaceuticals on neuronal tissue, using retinal spreading depression as a model system. By applying different excitatory substances, a reduction of the velocity and an inhibitory effect on the conductivity of the membranes can be observed. In the case of nicotine this may be due to the desensitization of the nicotinic AChreceptors. For caffeine, cocaine, amphetamine and metylphenidate this effect may derive from the hyperpolarisation of the tissue through open cation channels. The latency, a parameter for the excitability of the tissue, increases with the application of any of the investigated substances. This is also caused by the desensitization of the nicotinic ACh-receptors or the hyperpolarisation of the tissue, respectively. A neuroprotective effect reducing excitotoxic cell death induced by activation of NMDA-receptors was successfully verified for all substances. In the case of nicotine, the α7- and α4β2-nACh-receptors are assumed to be involved. The basic mechanism however is still unknown. A possible explanation could be the reduced excitability of the tissue through desensitization of the nACh-receptors. In the case of the remaining substances, the abidance of transmitters in the synaptic cleft suggests a neuroprotective effect through hyperpolarisation. The resulting hyperpolarisation leads to a reduced excitability of the neuronal tissue and most likely prevents over-excitation.
Isolierung und funktionelle Charakterisierung von Einzelkomponenten des Gifts der südamerikanischen Klapperschlange Crotalus durissus terrificus
(2017) Kaltenbach, Stefan; Hanke, Wolfgang
In order to understand the pharmacological activities of proteins and peptides it is important to study the relation of their molecular structure and their mode of action. Snake venoms contain a large number of biologically active compounds. They influence and damage the organism of prey in various manners. Therefore, venoms are an abundant source of substances with biological activity. The isolation of these substances is an important way to search for new, pharmacologically active proteins and peptides. The presented work focused on the isolation and characterization of venom compounds from the South American rattlesnake Crotalus durissus terrificus. Two of its main compounds, crotamine and a mixture of Crotoxin B isoforms, were isolated by RP-HPLC and identified by mass spectrometry. Afterwards, the emphasis was laid on how crotamine interacts with artificial membranes. Crotamine is a small, amphipathic and highly basic polypeptide with a molecular weight of 4.9 kDa and a high positive surface charge. It used to be described as a neurotoxin, but meanwhile many additional properties have been reported. These include myotoxic, analgetic, antimicrobial and antitumor activities. To some extent, these modes of action are based on the interaction of crotamine with the membrane lipids. In this context, the membrane modifying properties of crotamine and its ability to translocate into cells are under discussion. The presented work clearly demonstrates that the lipid composition of membranes influences the membrane modifying properties of crotamine. In lipid monolayers built from asolectin, cholesterol had the effect, that crotamine is integrated into the membrane more slowly. Furthermore, it is obvious that the presence of bivalent cations as well as an increasing lateral pressure inside the monolayer leads to a faster integration of crotamine. Moreover, crotamine altered the membrane fluidity of vesicles. In pure asolectin-vesicles crotamine stiffened the membranes (decreased fluidity), whereas it increased the fluidity in DOPC-vesicles. In vesicles containing a mixture of either asolectin or DOPC and cholesterol, crotamine increased the membrane fluidity, whereby its influence was weakened with rising concentrations of cholesterol. However, the impact of cholesterol was stronger in asolectin:cholesterol-vesicles. Hence, cholesterol reduces the membrane modifying properties of crotamine. It was assumed that this is due to the fact that cholesterol decrease the fluidity of membranes. Also, cholesterol may reduce the possibility of polar lipid head groups interacting with cationic molecules like crotamine. As asolectin contains negatively charged lipids, it was concluded that these lipids could be responsible for the stronger impact crotamine had on the fluidity of membranes. They could lead to a stronger attraction of crotamine, due to the more negatively charged membrane surface. Negatively charged lipids might also increase the possibility of raft building, caused by crotamine. In conclusion cholesterol as well as negatively charged lipids alter the effect crotamine has on membranes. It is also influenced by bivalent cations in the surrounding solution as well as the lateral pressure of the membrane. In addition, it was shown that crotamine also affects the calcium homeostasis of neuronal cells. By enlarging the amount of crotamine the internal free calcium concentration also showed an increase.
Neuronale Modulation : der Einfluss von Agonisten und inverser Agonisten auf das Cannabinoidsystem einer hippocampalen Primärkultur
(2007) Klink, Oliver; Hanke, Wolfgang
The aim of this thesis was to investigate the effect of inverse agonists on the CB1-receptor with an established complex neuronal-/ glia- co culture obtained from hippocampi of embryonic rats. To provide evidence of the expression of the CB1-receptors in the established culture immunocytochemical studies have been used and showed a sufficient expression level of the CB1-receptors. The neuronal culture was further tested on various electrophysiological parameters to verify an in vitro assay that resembles in vivo characteristics. Thus the exposure of TTX to neurons lead to reduced spike activity which refers to the blocking of voltage gated sodium channels. Also the inhibition of AMPA receptors using CNQX showed a reduction of spike activity in respect of the reduced synaptic activity. Analyses of the kinetic and spike frequencies of the generated actionpotentials as well as the kinetics and frequencies of spontaneous AMPA- and NMDA epscs are to a large extent comparable to published data of in vitro and in vivo assays. To reduce the intrinsic variability of the established cannabinoid assay the method of induced burst activity under low magnesium conditions has been used. This method results indeed in a lower variability of the assay but also the analysis of the effect of cannabinoid agonists and inverse agonists on the evoked burst activity showed interesting inverse modulations of burst durations, event intervals and inter-event intervals compared to alterations of the spike frequencies. On the basis of the obtained data by the analyzed spike-frequencies an agonistic effect of nanomolar concentrations of the investigated inverse agonists could be shown for the first time. This observation could lead to the conclusion that this might be a specific interaction of the investigated inverse agonist with an other receptor. The inhibition of the adenylatcyclase, a key-enzyme of the CB1 signal transduction, neutralizes the agonistic effect, although the inverse agonistic effect dissapeard. In addition the interaction of the opioid system in respect to the observed agonistic effect of rimonabant has been investigated. However, non of the published interaction of this two systems in respect to the agonistic effect of rimonabant could be observed.
Oszillation und Musterbildung in reinen und Polypeptid-dotierten Bilayern
(2017) Gaugele, Sebastian; Hanke, Wolfgang
The question of how lipids and proteins interact in bilayers and membranes and why cells and organelles have different lipid compositions in their membranes has been in focus of research for decades. Which role these different lipid compositions play for interaction between integrated and associated proteins has not yet been clarified until today. This thesis with the title "Oszillation und Musterbildung in reinen und Polypeptid-dotierten Bilayern" focuses on forces that influence the interaction of lipids and proteins. In both, living cells as well as in bilayer experiments, the protein-lipid interaction is a complex process involving a huge number of molecules. Different test series with temperature and amperage have been carried out using electrophysiological experiments with bilayers and fluorescence in vesicles. The results were used to determine similarities and differences that indicate certain characteristics of the setup and the interaction among the molecules. Since the question of the interaction between proteins and lipids is a very complex process, the already well-known pore former alamethicin has been used for this work. Alamethicin was used in the both types of experiments, bilayer and vesicles, with different lipid compositions consisting of DOPC, DOPE, DOPS and DMPC. The lipids were solved in ethanol first and then added with certain concentrations of alamethicin in two test chambers which were connected to each other by a small hole. In this hole the bilayer was formed by the addition of aqueous KCl solution. After that the current-clamp-method with different currents was used and subjected to the bilayer. The resulting voltage profiles were observed for oscillation patterns. Subsequently, the obtained data were subjected to the method of Fourier-Transformation to separate the results from noise and interference as well as overlays which resulted from the multi-channel system of alamethicin. The Fourier-Transformation has shown a large number of heterogeneous oscillation patterns. Due to the great heterogeneity of the results that did not allow direct comparisons of individual data with each other the average time-period of oscillation was calculated. This data were used to form data clouds where regression lines were generated. In addition the slopes of the lines were calculated for comparison. The results show that the orientation and slope angle of the slopes directly dependent on the concentrations of alamethicin and KCl. It was found that the addition of a small amount of negatively charged lipid like DOPS to a mixture of DOPC and DOPE has a big impact on the results and can change the orientation of the slopes of the average time-period of oscillation. Similar results were found while using asolectin bilayer containing inositol that is negatively charged, too. As a result, it was concluded that contrary to previous assumptions of some earlier work groups, bilayers are not pure hydrophobic barrier layers between two aqueous phases but highly complex structures that affect the gating of its integrated pores in different ways. The insert and operation of alamethicin strongly depends on the used lipids. Moreover a major dependency from the used salt concentration of the alamethicin pores and bilayer stability was found. The experiments with DMPC and temperature showed that the chain length of the lipids play an important role for integrated proteins on the one hand. On the other hand it could be confirmed that the phase transition of lipids can comprise a wide range of temperature of several degree Celsius depending on the experimental conditions. It could also be confirmed that alamethicin remains active in the liquid-crystal domains during phase transition of a bilayer. In addition it was found that temperature dependent changes appear in voltage oscillation patterns that affect both the average time-period and its resulting slopes in angle and orientation.
Patch clamp experiments with human neuron-like cells under different gravity conditions
(2010) Kohn, Florian Peter Michael; Hanke, Wolfgang
Gravitation influences many physical, chemical and biological processes. Cells and their behaviour are no exception. The gravitational impact on the activity of neuronal cells is very important with the perspective of manned space missions. Previous experiments with vertebrates showed that the velocity of neuronal impulses (action potentials) in nerve fibres is decreasing under zero gravity and is increasing at high gravity. There are many theories about the changed properties of the nervous systems under zero gravity, but the molecular principles are mostly unexplored. For this dissertation hardware for patch clamp experiments under microgravity was developed. The patch clamp technique is a common used tool in investigating the electrophysiological properties of cells and single ion channels. Since the conditions during parabolic flights render classic patch clamp nearly impossible, advanced chip-based planar patch-clamp hardware, the Port-a-Patch from Nanion Technologies was integrated into a setup. This setup had to comply with the mandatory design and safety regulations to be used in parabolic flights. Two parabolic flight campaigns were used to validate the hardware, adapt the patch clamp procedures to the special conditions during a parabolic flight (as time pressure and vibrations) and to choose a suitable cell line from the available cell lines. As the laboratory conditions on ground were insufficient at the beginning of the project, the main focus had to lie on robust cells. The SH-SY5Y cell line was chosen for their robustness (they already have been used successfully by other teams) and their origin from the human brain (glioblastoma). During two subsequent parabolic flight campaigns patch clamp experiments were performed and whole cell currents of SH-SY5Y cells were recorded with increasing success rate. Pulse protocols were used to create current-voltage (I-V) characteristics. To obtain 20 seconds of microgravity, two phases of hypergravity, each lasting 20 seconds, had to be endured by the passengers. This fact allowed the subsequent recording of whole cell currents of the same cell during normal Gravity (1g), hypergravity (1.8g) and microgravity (approx. 10-3g) to compare the I-V characteristics of the different gravity conditions. For SH-SY5Y cells it was shown that a gravity dependence of the whole cell currents exists. The micro- and hypergravity whole cell currents were changed compared to 1g flight controls. At -20 and -10mV, the hypergravity current was significantly decreased compared to 1g, by 13.5% at -20mV and by 7.4% at -10mV. A significant 1.8g current < 0g current relation could be observed at potentials between -20 and +10mV. At -20mV the microgravity whole cell currents were increased by 13.5%, at -10mV by 7.4%, at 0mV by 6.2% and at +10mV by 3.9%. The duration and complexity of the used pulse protocols were limited by the time of each gravity phase (20-22 seconds). In a last parabolic flight campaign, therefore the passive electrophysiological properties of the cell membrane were investigated. As the laboratory conditions at the site were greatly improved in the meantime, especially for cell culture, new cell lines could be tested for their usability. SNB19 cells, also originating from the human brain (astrocytoma) were chosen due to their good sealing quality and stability compared to SH-SY5Y cells and constant pulse protocols near the estimated resting potential (-80 and -60mV) were performed. At constant -80mV and -60mV, it was shown that the whole cell currents during the variable gravity conditions are significantly increased compared to the 1g in-flight controls. The hypergravity current of SNB19 was increased by 2.2% at -80V and by 8.2% at -60mV. The microgravity current was increased by 4.1% at -80mV and 3.7% at -60mV. The acquired I-V characteristic of SNB19 differs from the I-V characteristic of SH-SY5Y. These findings show that the planar patch clamp technique can be used in parabolic flights to investigate the electrophysiological properties of single. Furthermore the findings suggest that the electrophysiological properties of single cells originating from the human brain exist are gravity dependent.
Vergleichende Untersuchungen zur Musterbildung in erregbaren Medien mit Vermerken zum Einfluss schwacher magnetischer Felder - Schwerpunkt: Belousov-Zhabotinsky-Reaktion
(2007) Dolzmann, Kerstin; Hanke, Wolfgang
In this work we did some research on the influence of a weak external magnetic field (MF) on the creation of patterns in excitable media (duration field (DC) and alternating field (AC)). As examples we chose the well known Belousov-Zhabotinsky reaction (BZR) and ferrofluids. If ferrofluids are stimulated mechanically by vertical vibration they show changes of phases in the building of patterns while raising the induced energy (here by different hights of amplitudes). The viscosity of the magnetic fluid is increasing in a DC-field. Because of this the changing of the phase is different from the ones without an external force. A non-stationary stirred BZR shows a periodic change of colour between yellow and colourless ? or red and blue, if ferroin is added as a catalyst. This oscillation is described as a simple curve in literature. We, however, found a much more complex behaviour in the experiment. The intrinsic optical signals (IOS) of a ferroin-catalysed, stirred BZR show a double-peak at the beginning of the reaction, which is fading after a few further oscillations. This behaviour depends on the concentration of ferroin and resembles very much electrical and optical signals known from neuronal processes (e.g. retinal spreading depression). This basic similarity makes the BZR an ideal model for a variety of neurophysiological signals, even if the underlying mechanisms are completely different. If this system is put to the influence of a weak external magnetic DC-field a further inner oscillation is added to the double-peak behaviour of the IOS. Also the double-peak itself looks different from the one without external field. If further components are added to the system it gradually changes to chaotic behaviour. This could be the induced little currents in an AC-field. Each expansion of the system is followed by further bifurcation culminating in a transition from pattern to chaos. BRZ gels show bright propagating concentric rings or spirals as a pattern. With the used materials and methods of measurement we were not able to record changes in the formation of patterns if the system was expanded. But one can assume that the behaviour of the gel is changing in the DC-field: all in all it seems to get faster.