1 Selected abstracts: MODULATION OF CALCIUM ACTIVATED POTASSIUM CHANNELS T. M. WEIGER, A. HERMANN, I. B. LEVITAN Potassium currents play a critical role in action potential repolarization, setting of the resting membrane potential, control of neuronal firing rates, and regulation of neurotransmitter release. The diversity of the potassium channels that generate these currents is nothing less than staggering. This diversity is generated by multiple genes (as many as 100 and perhaps more in some creatures) encoding the pore-forming channel subunits, alternative splicing of channel gene transcripts, formation of heteromultimeric channels, participation of auxiliary (non-pore-forming) and other subunits, and modulation of channel properties by posttranslational modifications and other mechanisms. Prominent among the potassium channels are several families of calcium activated potassium channels, which are highly selective for potassium ions as their charge carrier, and require intracellular calcium for channel gating. The modulation of one of these families, that of the large conductance calcium activated and voltage-dependent potassium channels, has been especially widely studied. In this review we discuss a few selected examples of the modulation of these channels, to illustrate some of the molecular mechanisms and physiological consequences of ion channel modulation. Figure Putative structures of BK channel subunits. The pore-forming subunits of the Slowpoke family of BK channels are large proteins (~1,200 amino acids) that resemble other voltage-dependent potassium channels in having six membrane-spanning domains (S1 - S6), with a pore region between S5 and S6. An additional membrane-spanning domain (S0) places the amino terminal outside the plasma membrane. Most notable is the extended carboxyl terminal tail domain, comprising about two thirds of the subunit protein sequence. It includes a negatively charged region (the so-called calcium bowl) that has been implicated in calcium binding, and is the site of interaction with several channel modulatory proteins including protein kinases. The auxiliary subunits are small proteins (~200 amino acids) with two membrane-spanning domains (T1 and T2)., potential sites for N-linked glycosylation. Modified from Vergara et al., WEIGER, T., HERMANN, A. & LEVITAN, I. B. (2002). Modulation of calcium-activated potassium channels. J. Comp. Physiol. A (invited Review), 188,
2 MOLEKÜLSONDEN ZUR ERFORSCHUNG VON IONENKANÄLEN T. M. WEIGER, T. LANGER, A. HERMANN Ionenkanäle sind wesentliche Bestandteile jeder lebenden Zelle bei Mensch, Tier und Pflanzen. Sie sind verantwortlich für bioelektrische Vorgänge in unserem Körper, wie zum Beispiel für die Entstehung der elektrischen Aktivität bei Sinnes-, Nerven-, und Muskelzellen, die Volumenregulation oder die Steuerung des Salz-/Wasserhaushalts. Ionenkanäle sind winzige Nanoporen die man in allen Zellmembranen, wie der Plasmamembran um Zellen, bei Mitochondrien, dem Endoplasmatischen Reticulum oder in der Membran des Zellkerns, findet, Zelluläre Membranen bestehen aus Doppelschichten von Lipiden (fettähnliche Stoffe) die für die meisten geladenen Teilchen (Ionen) wie Natrium, Kalium, Calcium oder Chlorid unpassierbar sind. Durch Kanäle, die in diese Lipdschicht eingelagert sind, können Ionen ausgetauscht werden. Auf den ersten Blick sind Ionenkanäle einfache, wassergefüllte Poren die Ionen, durch die Membran schleusen. Aber ungleich einfachen Löchern in der Membran sind Ionenkanäle tatsächlich sehr kompliziert gebaute Eiweißmoleküle (Proteine). Mittels elektrophysiologischer und molekulargenetischer Verfahren kann man heute die Funktion und Struktur dieser Kanalproteine erforschen. Ionenkanäle können entweder offen oder geschlossen sein. Sie sind demnach winzige molekulare Schalter die im offenen Zustand Ionen entlang ihrem Konzentrationsgradienten fließen lassen. Zudem sind viele Ionenkanäle hoch selektiv, das heißt, sie lassen nur eine bestimmte Sorte von Ionen durch. Diese Eigenschaft hängt mit der molekularen Struktur der Kanäle zusammen, wobei eine enge Stelle im Kanal, der sog. Selektivitätsfilter eine wichtige Rolle spielt. Große Bedeutung haben Wirkstoffe, die Ionenkanäle blockieren oder modulieren können. In der Medizin werden solche Substanzen zu mannigfaltigen Therapien eingesetzt, wie z. B. bei multipler Sklerose, bei Herzrhythmusstörungen, bei Epilepsie etc. An Hand von einfach gebauten Molekülen und deren Varianten, wie den natürlich vorkommenden Polyaminen und ihren Analoga, den Diaminen, wird gezeigt, wie diese Substanzen quasi als Sonden zur Erforschung von Kaliumkanälen eingesetzt werden können. PUT SPD SP 1,12DD Computersimulation der Hydrathüllen (korbartige Strukturen) die Putrescin (PUT), Spermidine (SPD), Spermin (SP) und 1,12-Diaminododecan (1,12-DD) umgeben. Beachten Sie, daß alle Moleküle mit Ausnahme von 1,12-Diaminododecan vollständig von einer Hydrathülle umschlossen sind. 1,12-Diaminododecan kann daher leichter eine seine beiden, kleinen, endständigen Hydrathüllen abstreifen und in den Ionenkanal gelangen.
3 Schematische Darstellung des Block eines Kalium-Kanals durch Tetraethylammonium und 1,12- Diaminododecan. Die Abbildung zeigt von links nach rechts einen Kaliumkanal durch den Kaliumionen (rot) ungehindert fliessen können (links), ein Tetraethylammonium Molekül (grün, Mitte) das nur ein kurzes Stück in den Kanal von außen eindringen kann, im Kanal stecken bleibt und ihn somit blockiert, und rechts 1,12-Diaminododecan das die Wasserhülle von einer seiner beiden positiv geladenen Aminogruppen abstreifen kann und in gestreckter Form tief in den Kanal eindringt. Die zweite Wasserhülle von 1,12-Diaminododecan wird wahrscheinlich nicht abgestreift, das Molekül ragt auf Grund seiner Länge bis in den Vorhof des Kanals. (Graphik: Peter Steinbacher). WEIGER, T., LANGER, T., & HERMANN, A. (2002). Molekülsonden zur Erforschung von Ionenkanälen. Biologie i. u. Zeit, 2, COMPLEMENTARY DISTRIBUTION OF NADPH DIAPHORASE AND L ARGININE IN THE SNAIL NERVOUS SYSTEM M. XIE, A. HERMANN, H. H. KERSCHBAUM Since the interneuronal messenger nitric oxide (NO) can not be stored in neurons, the regulation of the NO producing enzyme, nitric oxide synthase (NOS), is crucial. Neuronal NOS metabolizes L - arginine to nitric oxide (NO) and L citrulline in a Ca 2+ dependent manner. Therefore, availability of L arginine to NOS may modulate NO production. Using NADPH diaphorase histochemistry to visualize putative NO producing cells and immunocytochemistry, we showed that the distribution of L arginine immunoreactive neurons correlates well with those of NADPH positive neurons in cerebral ganglia of the pulmonate, Helix pomatia. However, substrate and enzyme were visualized in separate but adjacent neurons. Following elevation of intracellular Ca 2+ by the Ca 2+ ionophore, ionomycin, or by a high K + solution, the number of L - citrulline immunoreactive neurons in mesocerebrum and pedal lobe increased up to ten fold. Preincubation of ganglia with the NOS inhibitor, N G -nitro-l-arginine, prevented ionomycin or high K + solution induced L - citrulline synthesis. Most L - citrulline immunoreactive neurons revealed NADPH diaphorase staining. In conclusion, these
4 experiments indicate a complementary distribution of NOS and L arginine and suggests an unknown signaling pathway between neurons to maintain L arginine and NO homeostasis. XIE, M., HERMANN, A. & KERSCHBAUM, H. H. (2002). Complementary distribution of NADPH-diaphorase and L-arginine in the snail nervous system. Cell Tissue Res., 307, NITRIC OXIDE AND CGMP MEDIATED MODULATION OF CA AND K CA - CONDUCTANCES IN SNAIL NEURONS S. SCHROFNER, A. ZSOMBOK, A. HERMANN, H. H. KERSCHBAUM The mechanism of nitric oxide (NO) - dependent modulation of Ca 2+ - and Ca 2+ - activated potassium (K Ca ) conductance was studied in identified subesophageal neurons of the pulmonate snail, Helix pomatia. K Ca is voltage as well as Ca 2+ - dependent and sensitive to charybdotoxin (CTX), Ba 2+, and tetraethylammonium (TEA), but insensitive to aminopyridine (4 AP). Thus, the K Ca current shows similarities to a large-conductance K Ca channel (BK(Ca)). NO - donors (sodium nitroprusside, SNP; S nitro N acetylpenicillamine, SNAP) slowly decreased the BK current amplitude. Decline of the current amplitude by NO - donors was qualitatively mimicked by a membrane permeable cgmp analogue (dibutyryl cgmp, db- cgmp). Methylene blue, an inhibitor of guanylyl cyclase, or erytho 9 (2 hydroxyl 3 nonyl) adenine (EHNA), an inhibitor of the cgmp stimulated phosphodiesterase 2, inhibited NO - donor induced decrease of BK - current. Amplitude of L type Ca 2+ - current either did not respond to NO donors or increased following application of NO donors. Exposure of neurons to db cgmp increased the current amplitude. In EHNA pretreated neurons, amplitude of L type Ca 2+ - current did not respond to NO or cgmp. The experiments demonstrate that the NO mediated affects on Ca 2+ - and K Ca conductance in snail neurons dependent on generation of cgmp and subsequent cgmp SCHROFNER, S., ZSOMBOK, A., HERMANN, A. & KERSCHBAUM, H.H. (2004). Nitric oxide decreases a calcium activated potassium current via activation of phosphodiesterase 2 in Helix U cells. Brain Res., 999, MODULATION OF CALCIUM CHANNELS PHOSPHORYLATION BY NITRIC OXIDE IN SNAIL NEURONS A. ZSOMBOK, S. SCHROFNER, A. HERMANN, H. H. KERSCHBAUM The different responses of cells to nitric oxide (NO) exposure in many cases depends on the direct interaction of NO with metal or thiol containing proteins, like enzymes or ion channels, which enhances or attenuates the activity of these proteins. Although NO modulates L type calcium channel activity in vertebrate tissue, it appears not to directly affect the L type calcium current in snail neurons. This difference could result from an insensitivity of calcium channels to NO or NO dependent processes or may be caused by a simultaneous phosphorylation and dephosphorylation of calcium channels. We recently reported NO donors to increase the intracellular concentration of cgmp in Helix ganglia (Huang et al., Brain Res. 1998, 780:329) and to increase excitability by depressing a calcium-activated potassium current (Zsombok et al., 2000, Neurosci Lett, 295: 85).
5 In the present study, we investigated the effect of the membrane permeant 3,5 cyclic guanosine monophosphate (cgmp) analogue, dibutyryl cgmp (dbcgmp) and the NO donor sodium nitroprusside (SNP) on the L type voltage-activated Ca 2+ current in identified neurons in the subesophageal of the terrestrial snail, Helix pomatia. We observed that application of db cgmp resulted in a transient increase of the calcium current by about 30%, whereas in most neurons SNP had no effect on the current amplitude. Treatment of ganglia with the kinase inhibitor, H-7, abolished the enhancing effect of dbcgmp. Furthermore, pretreatment of ganglia with H-7 decreased the calcium current following application of dbcgmp and the NO donor SNP by about 25%. The phosphatase inhibitors okadaic acid and calyculin increased the Ca 2+ current following application of dbcgmp and SNP by about 30%. Our experiments indicate that NO modulates phosphorylation of calcium channels via activation of a cgmp dependent transduction pathway. ZSOMBOK, A., SCHROFNER, S., HERMANN, A & KERSCHBAUM, H.H. (2005). A cgmpdependent cascade enhances an L-type-like Ca 2+ current in identified snail neurons. Brain Res., 1032, LOCALISATION OF ANNEXINS IN THE RETINA OF RAINBOW TROUT: LIGHT- AND ELECTRON MICROSCOPICAL INVESTIGATIONS ZAUNREITER M., BRANDSTÄTTER R., DONATO, R., A. HERMANN We present a first description of annexin immunoreactivity within neural cells of the teleost retina. Antibodies against annexins V and VI were used in light and electron miscoscopic sections of light and dark adapted retinae. Strong immunoreactivity could be found in retinal layers with high synaptic input, such as the outer and inner plexiform layers and dendritic regions within the inner plexiform layer; in cells that are involved in negative feedback control, such as horizontal and amacrine cells; in the membrane metabolism of photoreceptor outer segments; and in close relation to cytoskeletal components. Our findings suggest that both, annexins V and VI are involved in the regulation of transmitter release, especially of transmitters that are not directly involved in phototransduction, and with structures that support the morphological changes involved in light and dark adaptation. ZAUNREITER, M., BRANDSTÄTTER, R., DONATO, R. & HERMANN, A. (2005). Localisation of annexins in the retina of the rainbow trout - light- and electron microscopical investigations. Brain Res., 1032, ANTIPROLIFERATIVE PROPERTIES OF PADMA LAX AND ITS COMPONENTS GINGER AND ELECAMPANE SEBASTIAN HOFBAUERA, VERENA KAINZ, LEOPOLD GOLSER, MICHAELA KLAPPACHER, TOBIAS KIESSLICH, WILHELM HEIDEGGER, BARBARA KRAMMER, ANTON HERMANN & THOMAS M. WEIGER Padma Lax (PL) is a multi-component herbal laxative, derived from traditional Tibetan medicine. It has been used in the treatment of constipation dominant irritable bowel syndrome. Beyond its purgative and bowel-regulating properties we found it to exhibit antiproliferative properties. C6 tumor cells were incubated with either an ethanolic or aqueous extract of PL. Cell proliferation, cell cycle, percentage of apoptotic cells, caspase-3/-7 activity as well as
6 mitochondrial membrane potential were determined. Ethanolic extracts of PL inhibited cell proliferation in a dose- and time-dependent manner (half max concentration: μg/ml after 48 h of incubation). Aqueous extracts were less effective. Ginger and elecampane were the active components of PL in respect to its antiproliferative action and were found to act synergistically. Supplementing the culture medium with polyamines could not override the cytostatic action of PL. Incubation of C6 cells with PL in the presence of catalase proved that the PL effect was specific and not due to oxidative stress. PL had no effects on the cell cycle at a low dose but arrested cells in G1 at high concentrations. Reduction of cell numbers was found to be due to apoptosis. The caspase-3/-7 pathway was not involved in the PL-induced cell death. However, mitochondrial membrane potential was lost during the course of incubation with PL indicating a mitochondrial- but not caspase-mediated induction of apoptosis. PL exhibits antiproliferative properties which may be beneficial to prevent constipation-related cancer. This study may also contribute to a future development of a new herbal-based antiproliferative treatment. HOFBAUER, S., KAINZ, V., GOLSER, L., KLAPPACHER, M., KIESSLICH, T., HEIDEGGER, W., KRAMMER, B., HERMANN, A. & WEIGER, T.M. (2006). Antiproliferative properties of Padma Lax and ist components ginger and elecampane. Forschende Komplementärmedizin, 13, HYPOTONICITY AND ETHANOL MODULATE BK CHANNEL ACTIVITY AND CHLORIDE CURRENTS IN GH4/C1 PITUITARY TUMOUR CELLS M. JAKAB, S. SCHMIDT, M. GRUNDBICHLER, M. PAULMICHL, A. HERMANN, T. WEIGER & M. RITTER Description of the effects of hypotonic cell swelling and ethanol onmaxi Ca2+-activated K+ channel (BK channel) activity and Cl) channel activity in GH4/C1 pituitary tumour cells. Whole cell-, cell attached- and outside-out patch clamp measurements, fluorescence (fluo-3) measurements of intracellular Ca2+ concentration, cell size video monitoring. GH4/C1 pituitary tumour cells respond to both hypotonicity and ethanol with cell swelling which is followed by a regulatory volume decrease (RVD). Tetraethylammonium and 4,4 diisothiocyanatostilbene-2,2 -disulphonic acid (DIDS) induced cell swelling per se and inhibited hypotonicity induced RVD. Ethanol-induced swelling is paralleled by an increase in the intracellular Ca2+ concentration and augmented by DIDS. BK channel activation by hypotonicity and ethanol is demonstrated in patch clamp experiments both in intact cells (cell attached configuration) and a subset of excised membrane patches (outside-out configuration). Cell swelling and addition of ionomycin under isotonic conditions leads to the activation of outwardly rectifying Cl) currents with time dependent activation at positive potentials. In GH4/C1 cells both hypotonicity and ethanol lead to cell swelling, RVD and to activation of BK channels. The hypotonicity-induced BK channel activation can also be observed in cell free outside-out patches. Hypotonicity, but not ethanol leads to the activation of Cl) channels. JAKAB, M., SCHMIDT S., GRUNDBICHLER, M., PAULMICHL, M., HERMANN, A., WEIGER, T. & RITTER, M. (2006). Hypertonicity and ethanol modulate BK channel activity and chloride currents in GH4/CI pitiutary tumor cells. Acta Physiol. 187,
7 POTASSIUM CHANNEL BLOCKERS QUINIDINE AND CAESIUM HALT CELL PROLIFERATION IN C6 GLIOMA CELLSVIA A POLYAMINE-DEPENDENT MECHANISM T.M. WEIGER, S. COLOMBATTO, V. KAINZ, W. HEIDEGGER, M.A. GRILLO & A. HERMANN Potassium (K + ) channels are ubiquitous to cells and serve essential functions in physiology and pathophysiology. K + channel blockers have been shown to block tumour growth by arresting cells at the G0/G1 checkpoint of the cell cycle. We investigated the effect of quinidine and caesium (Cs+) on cell proliferation, LDH (lactate dehydrogenase) release, free internal calcium (Ca 2+ ), membrane potential, polyamine concentration, ODC (ornithine decarboxylase) activity and polyamine uptake in C6 glioma cells. The EC50 for reducing cell proliferation was 112 μm for quinidine, whereas Cs + was less effective with an EC50 of 4.75 mm. LDH release was augmented by quinidine. It caused a transient increase in free internal Ca 2+ but decreased Ca 2+ after a 48 h incubation period. Further 300 μm quinidine depolarized the cell membrane in a similar range as did 30 mm KCl. Quinidine decreased cellular putrescine beyond detection levels while spermidine and spermine remained unaffected. ODC activity was reduced. Addition of putrescine could not override the antiproliferative effect owing to a reduced activity of the polyamine transporter. Our study indicates that the antiproliferative effect of quinidine is not due to a simple membrane depolarization but is caused by a block of ODC activity. WEIGER, T.M., COLOMBATTO, S., KAINZ, V., HEIDEGGER, W., GRILLO, M.A. & HERMANN, A. (2007). Potassium channel blockers quinidine and caesium halt cell proliferation in C6 glioma cells via a polyamine-dependent mechanism. Biochemical Society Transactions, 35-2, MODULATION OF POTASSIUM CHANNELS BY POLYAMINES THOMAS M. WEIGER AND ANTON HERMANN Polyamines modulate a large variety of potassium channels. Not only Kir channels which have received most attention are affected bypolyamines but also calcium activated potassium channels like BK channels, the delayed rectifier, KCNQ channels, and TASK channels are modulated by polyamines. In all cases polyamines block potassium channels. The order of potency is spermine > spermidine> putrescine. In most of these channels polyamines act from the intracellular side of the channels and show no blocking properties when applied from the extracellular side except TASK-3 channels. The polyamine action on potassium channels has important bearings on the discharge activity or secretion in excitable cells. WEIGER, T. & HERMANN, A. (2009). Modulation of potassium channels by polyamines, In: Biological Aspects of Biogenic Amines, Polyamines and Conjugates, Ed. G. Dandrifosse, Transworld Research Network, ETHANOL AND POLYAMINES IN BRAIN THOMAS M. WEIGER AND ANTON HERMANN In this review we discuss the role of the polyamines putrescine, spermidine and spermine which are ubiquitous constituents in all eukaryotic cells, during acute and chronic ethanol exposure as well as after ethanol withdrawal in brain. Ethanol as well as polyamines modulate the same set
8 p(open) of ion channels with the NMDA receptor being a main target. Ethanol exposure or withdrawal is responsible for changes in polyamine content via modulation of ornithine decarboxylase, the rate limiting enzyme for polyamine synthesis. Alterations of polyamine concentrations in turn affect ion channels interacting with polyamines in addition to the direct effect of ethanol on these ion channels. Any increase or decrease in ion channel activity impacts electrical signalling in the brain and subsequently behaviour. A better understanding of these mechanisms concerning ethanol, polyamines and ion channel activity may generate improved treatment options for alcoholics. Not only ethanol but also acetaldehyde, the first by-product of ethanol metabolism, may be involved in the modulation of polyamines as well as ion channels, a field that is largely ignored by recent research. WEIGER, T.M. & HERMANN, A. (2009). Ethanol and polyamines in brain. In: Biologically Active Amines and Related Enzymes: Biochemical, Physiological and Clinical Aspects, Ed. A. Toninello, Transworld Research Network, HYDROGEN SULFIDE INCREASES CALCIUM-ACTIVATED POTASSIUM (BK) CHANNEL ACTIVITY OF RAT PITUITARY TUMOR CELLS GUZEL F. SITDIKOVA, THOMAS M. WEIGER & ANTON HERMANN Hydrogen sulfide (H2S) is the third gasotransmitter found to be produced endogenously in living cells to exert physiological functions. Large conductance (maxi) calcium-activated potassium channels (BK) which play an important role in the regulation of electrical activity in many cells are targets of gasotransmitters. We examined the modulating action of H2S on BK channels from rat GH3 pituitary tumor cells using patch clamp techniques. Application of sodium hydrogen sulfide as H2S donor to the bath solution in whole cell experiments caused an increase of calcium-activated potassium outward currents. In single channel recordings H2S increased BK channel activity in a concentration-dependent manner. H2S induced a reversible increase in channel open probability in a voltage-dependent, but calcium independent manner. The reducing agent dithiothreitol prevented the increase of open probability by H2S whereas the oxidizing agent thimerosal increased channel open probability in the presence of H2S. Our data show that H2S augments BK channel activity and this effect can be linked to its reducing action on sulfhydryl groups of the channel protein. SITDIKOVA, G.F., WEIGER, T.M. & HERMANN, A. (2010). Hydrogen sulphide increases calcium-activated potassium (BK) channel activity of rat pituitary tumor cells. Pflügers Arch - Eur J Physiol 459, ms control NaHS 300 µm wash 10 pa sec
9 GASOTRANSMITTER - GASE ALS ZELLULÄRE SIGNALMOLEKÜLE ANTON HERMANN, GUZEL F. SITDIKOVA, UND THOMAS M. WEIGER Gase, wie Stickoxid (NO), Kohlenmonoxid (CO) oder Schwefelwasserstoff (H 2 S) sind als umweltbelastende Industrieabfallstoffe und als sehr giftige Substanzen bekannt. Dass Gase mit physiologischer Wirksamkeit in unseren Körper vorkommen sollten, war zunächst schwer vorstellbar. Nun wurde aber gefunden, dass diese Gase in allen Organismen von Bakterien bis zum Menschen vorkommen, in deren Zellen produziert werden und wichtige Funktionen erfüllen. HERMANN, A., SITDIKOVA, G.F. & WEIGER, T.M. (2010).Gase als zelluläre Signalstoffe. Biologie i.u. Zeit, 3, GIFTIGE GEISTER VON ANTON HERMANN, GUZEL F. SITDIKOVA UND THOMAS M. WEIGER Stickstoffmonoxid, Kohlenmonoxid und Schwefelwasserstoff gelten als hochtoxisch. Umso erstaunlicher ist, dass der menschliche Körper diese Gase selbst produziert: Sie dienen im Organismus als universelle Botenmoleküle. Die Biologen Anton Hermann, Guzel F. Sitdikova und Thomas M. Weiger erforschen die vielfältigen Wirkungen der Gasotransmitter etwa beim Lernen oder bei neurodegenerativen Erkrankungen. Ein»animalischer Geist«durchströmt nach Ansicht der alten Griechen unsere Adern. AlsLebenskraft wirke dieses Pneuma vor allem im Gehirn, im Herzen und in der Leber. Heute wissen wir es besser und lehnen solche Vorstellungen als esoterisch ab. Vielleicht etwas vorschnell denn geisterhaft wirkende Substanzen konnten Wissenschaftler in den letzten Jahrzehnten tatsächlich aufspüren: Die Gase Stickstoffmonoxid, Kohlenmonoxid und Schwefelwasserstoff kontrollieren wichtige Stoffwechselprozesse in Zellen und Organen unseres Körpers. HERMANN, A., SITDIKOVA, G.F. & WEIGER, T.M. (2011). Giftige Geister. Gehirn & Geist 5, 28-35, Spektrum Verlag.
10 S100 CALCIUM-BINDING PROTEINS AND ION CHANNELS ANTON HERMANN 1*, ROSARIO DONATO 2, THOMAS M. WEIGER 1 AND WALTER J. CHAZIN 3 S100 Ca 2+ -binding proteins have been associated with a multitude of intracellular Ca 2+ - mediated functions including regulation of the cell cycle, cell differentiation, cell motility and apoptosis, modulation of membrane-cytoskeletal interactions, transduction of intracellular Ca 2+ signals, and in mediating learning and memory. S100 proteins are fine tuned to read the intracellular free Ca 2+ concentration and affect protein phosphorylation, which makes them candidates to modulate certain ion channels and neuronal electrical behaviour. Certain S100s are secreted from cells and are found in extracellular fluids where they exert unique extracellular functions. In addition to their neurotrophic activity, certain S100 proteins modulate neuronal electrical discharge activity and appear to act directly on ion channels. The first reports regarding these effects suggested S100-mediated alterations in Ca 2+ fluxes, K + currents and neuronal discharge activity. Recent reports revealed direct and indirect interactions with Ca 2+, K +, Cl - and ligand activated channels. This review focuses on studies of the physical and functional interactions of S100 proteins and ion channels. HERMANN, A., DONATO, R., WEIGER, T.M. &.CHAZIN, W.J. (2012). S100 Calcium Binding Proteins and Ion Channels. Front. Pharmacol. 3 (67), p. 1-10, doi: /fpharm , Review. Online:http://www.frontiersin.org/Journal/Abstract.aspx?s=762&name=neuropharmacology& ART_DOI= /fphar BK CHANNELS - FOCUS ON POLYAMINES, ETHANOL/ACETALDEHYDE AND HYDROGEN SULFIDE (H2S) HERMANN ANTON, SITDIKOVA GUZEL F. & WEIGER THOMAS M. Calcium-activated potassium channels are expressed in a great variety of tissues from bacteria to men. The channels constitute a link between cellular activity, calcium signalling and metabolism. The importance of the channels results from their unique adaptability, modulating capacity and versatile physiology. To study these channels many scientists have been attracted to this field who created a great wealth of detailed insight into structure and function of these channels. In this chapter we will describe techniques to record maxi calcium-activated potassium (BK) channels, delineate properties and functions of BK channels and we will focus on some aspects of channel modulation by polyamines, ethanol /acetaldehyde and by the gasotransmitter - hydrogen sulfide. HERMANN, A., SITDIKOVA, G.F. & WEIGER, T.M. (2012). BK Channels Focus on Polyamines, Ethanol/Acetaldehyde and Hydrogen Sulfide (H2S), In Patch Clamp Technique, InTech, Ed. F. S. Kaneez, p , ISBN: , Review. Online:
11 GASOTRANSMITTERS: PHYSIOLOGY AND PATHOPHYSIOLOGY ANTON HERMANN, GUZEL F. SITDIKOVA, THOMAS M. WEIGER - EDITORS Since the epochal discovery of the radical and highly toxic gas nitric oxide (NO) as signaling molecule two other not less toxic gases carbon monoxide (CO) and hydrogen sulfide (H 2 S) - have been found to be also involved in a plethora of physiological and pathophysiological functions. The gases termed Gasotransmitters play an increasingly important role in understanding how signalling into cells and between cells is modulated and fine tuned The advent of gasotransmitters has profoundly changed our way of thinking about biosynthesis, liberation, storage and action mechanisms by cellular signaling. In recent years an impressive amount of new data distributed in the literature has been generated. For this book we have asked distinguished colleagues in the field to summarize and review important biological, pharmacological and medical functions and their implications as well as methods for the detection of gasotransmitters. MODULATED BY GASOTRANSMITTERS - BK CHANNELS HERMANN ANTON, SITDIKOVA GUZEL F. & WEIGER THOMAS M. Calcium-activated potassium BK channels interconnect cellular activity, calcium signaling and cell metabolism. Major virtues of these channels are their adaptability to different functions, their versatile physiology and their capacity being modulated. The channels are present in a large variety of cells and organs in different forms of life from bacteria to men. Scientists attracted to these channels have produced a great wealth of information regarding their structure and function. Mutations at channels proteins are involved in a number of diseases (channelopathies), like diabetes, epilepsy or heart failure. The gasotransmitters NO, CO and H 2 S all act on BK channels directly or indirectly via signaling pathways. In this chapter we will briefly summarize some of the basic properties of BK channels and focus on aspects of BK channel modulation by gasotransmitters and their implications in physiology and pathophyiology. HERMANN, A., SITDIKOVA, G.F. & WEIGER, T.M. (2012). Gasotransmitters: Physiology and Pathophysiology (book), Eds. A. Hermann. G. Sitdikova & T. Weiger, Springer Press, Heidelberg, Germany, ISBN: e-book citation: HERMANN, A., SITDIKOVA, G.F. & WEIGER, T.M. (2012). Modulated by Gasotransmitters BK channels, In Gasotransmitters: Physiology and Pathophysiology, Eds. A. Hermann et al., Springer Press, Heidelberg, Germany, p , Review.
12 CELL PROLIFERATION, POTASSIUM CHANNELS, POLYAMINES AND THEIR INTERACTIONS WEIGER THOMAS M. & HERMANN A. Polyamines, which are obligatory molecules involved in cell cycling and proliferation are subject to a change in their free intracellular concentrations during the cell cycle. Potassium (K + ) channels are also considered, but less well recognized, to be necessary for cell proliferation by either hyperpolarizing or depolarizing cells during the cell cycle. A block of polyamine synthesis as well as block or knockout of K + channels can halt cell proliferation. K + channels like BK (maxi calcium (Ca 2+ ) activated K + ), Kir (inward rectifier), M-type K + - and TASK (two-pore domain K + ) channels or the delayed rectifier K + channels are modulated in their electricalproperties by polyamines. Polyamines are most effective in blocking these channels when applied from the intracellular faces of these channels except for TAKS channels where they act from the extracellular side. Quinidine a general K + channel blocker was found to reduce putrescine concentrations, to block the ornithine decarboxylase and to halt cell proliferation. From these results the question arises if there is an interaction between polyamines, K + channels and proliferation. It might be speculated that a decrease of intracellular polyamines allows more K + channels to be active thus inducing hyperpolarization while an increase of the polyamine concentration may block K + channel activity leading to depolarization of the membrane potential. On the other hand, a block or a deletion of K + channels may cause a decrease of the polyamine concentration in cells. More research is needed to test these hypotheses. WEIGER, T.M. & HERMANN, A. (2013). Cell proliferation, potassium channels, polyamines and their interactions. Amino Acids. DOI /s Link:http://www.springer.com/home?SGWID= &aqId= &download=1&checkval=cf85b2afe38a972b9efeb3232c1cefb1 SCREENING OF ANALGESIC AND ANTI-INFLAMMATORY ACTIVITIES FOR TWO LIBYAN MEDICINAL PLANTS: HELIANTHEMUM LIPPII AND LAUNAEA RESIDIFOLIA NOURI B. ERMELI, SAMI G. ALSABRI, SALAH M. BENSABER, SALAH B. MOHAMED, ABDULMOTTALEB A. ZETRINI, KHALED M. ABURAS, SAFA R. FITOURI, MOUSA I. JAEDA, IBRAHIM A. MREMA, A. HERMANN & ABDUL M. GBAJ1 Natural products are often a source for bioactive compounds which have great potential for developing novel therapeutic agents. In this study, two Libyan medicinal plants Helianthemum lippii (H. lippii) and Launaea residifolia (L. residifolia) were collected from El-Jabel El- Garbi (Gharian) in the Spring season (2010). They were extracted successively by using microwave technique with three different solvents of different polarities. The analgesic activity of these plant extracts was evaluated using the hot-plate method and the anti-inflammatory activity was evaluated using Carrageenen-induced paw edema method. The methanol and chloroform extracts exhibited significant analgesic activity at the doses tested while the petroleum ether extracts of both plants did not show any significant effect. In addition, the anti-inflammatory activities of various extracts showed a significant percentage inhibition of paw edema for H. lippii extracts in methanol and chloroform but not in petroleum ether. Moreover, the results exhibit different percentage inhibitions of paw edema for L. residifolia extracts in methanol, chloroform and petroleum ether. The analgesic and anti-inflammatory effects produced by the
13 extracts may be attributed individually or collectively to the flavonoids and tannins. H. lippii and L. residifolia can be introduced as new plant sources for analgesics and anti inflammatory agents. The methanolic and chloroform extracts of both plants showed a significant analgesic activity due to an increase in the reaction time (p<0.05) in comparison to controls, where codeine was used as standard analgesic drug. The petroleum ether extracts did not show any activity as analgesic. The anti-inflammatory activity was also evaluated using the Carrageenaninduced paw edema method, the methanolic extract of H. lippii (53.42%) exhibit activity comparable to that of aspirin standard antiinflammatory drug (62.28%) with no significant difference, while the petroleum ether extract of L. residifolia (31.65%) exhibited a moderate anti-inflammatory activity (p<0.01) in comparison with aspirin as a standard. The chloroform extract of H. lippii, the methanol and chloroform extracts of L. residifolia exhibited a weak inhibitory effect on paw edema volume with percentage inhibition of (23.60%, p<0.05), (16.45%, p>0.05) and (14.56%, p>0.05) respectively compared to control. ERMELI, N.B., ALSABRI, S.G., BENSABER, S.M., MOHAMED, S.B., ZETRINI, A.A., ABURAS, K.M., FITOURI, S.R., JAEDA, M.I., MREMA, I.A., HERMANN, A.& GBAJ, A.M. (2012). Screening of analgesic and anti-inflammatory activities for two Libyan medicinal plants: Helianthemum lippii and Launaea residifolia. Journal of Chemical and Pharmaceutical Research, 4(9): BRCA1 MUTATION DETECTION USING FLUORESCENT HYBRIDIZATION PROBES AND MELTING CURVES SAFA R. FITOURI, NOURI B. ERMELI, SALAH M. BENSABER, MOUSA I. JAEDA, IBRAHIM A. MREMA, ANTON HERMANN & ABDUL M. GBAJ The BRCA1 (Breast Cancer Anti-estrogen resistance-1), early-onset gene is expressed in cells of breast and other tissue and helps to repair damaged DNA or destroy cells in cases DNA cannot be repaired. When the BRCA1 gene is damaged, then the DNA is not repaired appropriately and this enhances the risk for cancer. Fluorescence and UV-visible thermal studies were performed for WT (wild type) and MT (mutant type targets) full systems. The target DNAs used were in the form of short oligonucleotides, genomic DNA. The probe system was used for detection of WT and SNP alleles of human BRCA1 [( , G T) and ( , G T)]. The Cy5 dye attached to a probe oligonucleotide (10-mer) undergoes a fluorescence intensity change on hybridisation of the probe to the WT compared to MT targets. Our results indicate that the system consisting of the target sequence and the one probe oligonucleotides bearing the Cy5 dye assemble correctly at the specified target. Once the full system (probe and target) is arranged under suitable conditions, a red-shift emission and change in fluorescence intensity are seen at a suitable wavelength. Thermal studies also showed significant differences in Tm between WT and MT. The results suggest that the differences in the fluorescence intensity at 665 nm and the spectrophotometric Tm(s) for the WT and MT can be attributed to the type of binding of the probe to the target. The systems were sensitive to single nucleotide polymorphisms and this may help in high throughput applications in genetic testing and molecular diagnostics. FITOURI, S.R., ERMELI, N.B., BENSABER, S.M., JAEDA M.I., MREMA, I.A., HERMANN, A. & GBAJ, A.M. (2013). BRCA1 Mutation Detection Using Fluorescent Hybridization Probes and Melting Curves. Journal of Life Sciences, USA, 7-3, ISSN
14 CHEMICAL SYNTHESIS, MOLECULAR MODELLING AND EVALUATION OF ANTICANCER ACTIVITY OF SOME PYRAZOLIDONE SCHIFF'S BASE DERIVATIVES S. M. BENSABER, H. A. ALLAFE, Z. S. ABOOD,, N. B. ERMELI, S. B. MOHAMED, A. EL- ZITRINI, S. G. ALSABRI, I. A. MREMA, M. ERHUMA, ANTON HERMANN, M. I. JOAED & A. M. GBAJ A series of novel 3-pyrazolinone Schiff`s base derivatives (MCS 02-13) were designed and synthesized by microwave chemical synthesis. Their purity was confirmed by melting point and HPLC and their chemical structures were determined by FT-IR, UV, 1H and 13C-NMR spectroscopic techniques. In silico the synthesized compounds have been docked to in the thymidine phosphorylase active site using molecular modeling programs. The compounds were tested in vitro on calf thymus DNA to study the interaction with DNA using a Cary 5000 UV\VIS\NIR spectrophotometer. Some of these compounds showed close match interaction with DNA. The compounds were also studied on angiogenic enzyme thymidine DNA phosphorylase (TP, E.C ), carcinoma cell lines including both human breast (MCF-7) and human lung cell lines (A549). The lead compound in the series, MCS 2, exhibited inhibition of thymidine phosphorylase in the micro molar range (IC50 of 28 ± 2 µm) and was able to retard growing of breast carcinoma cells. Our results indicate that 3-pyrazolinone Schiff`s base derivatives are promising lead compounds for the development of more active antitumor agents and exhibit their highest cytotoxic effect on breast carcinoma cell line. BENSABER, S.M., ALLAFE, H.A., ABOOD, Z.S., ERMELI, N.B., MOHAMED, S.B., EL- ZITRINI, A., ALSABRI, S.G., MREMA, I.A., ERHUMA, M., HERMANN, A., JOAED, M.I. & GBAJ, A.M. (2013). Chemical synthesis, molecular modelling and evaluation of anticancer activity of some pyrazolidone Schiff's base derivatives. (in press).
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