Biomedical subjects

E Wanke

Publications and source records attributed to E Wanke.

At least 37 records · Page 2Linked to original sources

PubMed1998

Sulfonylureas blockade of neural and cardiac HERG channels.

The human ether-a-go-go-related gene (herg) encodes a K+ current (I(HERG)) which plays a fundamental role in heart excitability and in neurons by contributing to action potential repolarization and to spike-frequency adaptation, respectively. In this paper we show that I(HERG), recorded in neuroblastoma cells and guinea-pig ventricular myocytes, was reversibly inhibited by the K(ATP) channel blocker glibenclamide (IC50 = 74 microM). The voltage and use dependence of glibenclamide blockade were also evaluated. Another sulfonylurea, glimepiride, had less effective results in blocking I(HERG). The findings of this study are relevant to the interpretation of glibenclamide effects on cellular electrophysiology and suggest that oral antidiabetic therapy with sulfonylureas may contribute to iatrogenic QT prolongation and related arrhythmias.

Action Potentials↗

PubMed1998

Long-term exposure to retinoic acid induces the expression of IRK1 channels in HERG channel-endowed neuroblastoma cells.

The modulation of inward K+ conductances was studied during neuronal differentiation of human SH-SY5Y neuroblastoma cells. Under standard culture conditions, these cells express the herg gene, and the HERG current is the main inward K+ current regulating their Vrest. After 10-20 days exposure to Retinoic Acid (RA), SH-SY5Y cells showed, in addition to HERG currents, a novel current characterized by inward rectification, dependence on the extracellular K+ concentration, and blockade by Cs+ and Ba2+, the main features of the IRK1 current. The appearance of this current is accompanied by a strong hyperpolarisation of Vrest. RT-PCR experiments confirmed that a transcript of the IRK1 (Kir 2.1) gene actually appears in SH-SY5Y cells treated for 10-20 days with RA. On the whole, data here presented demonstrate that RA-induced neuronal differentiation of neuroblastoma cells is accompanied by the switch from a HERG-driven to a IRK1-driven control of Vrest, similarly to what happens in normal differentiating neurons; however, in tumor cells, this switch does not imply the abolition of HERG channel expression.

Cation Transport Proteins↗

PubMed1998

herg encodes a K+ current highly conserved in tumors of different histogenesis: a selective advantage for cancer cells?

The human ether-a-go-go-related gene (herg) encodes a K+ current (IHERG) that plays a fundamental role in heart excitability by regulating the action potential repolarization (IKr); mutations of this gene are responsible for the chromosome 7-linked long QT syndrome (LQT2). In this report, we show that in a variety (n = 17) of tumor cell lines of different species (human and murine) and distinct histogenesis (neuroblastoma, rhabdomyosarcoma, adenocarcinoma, lung microcytoma, pituitary tumors, insulinoma beta-cells, and monoblastic leukemia), a novel K+ inward-rectifier current (IIR), which is biophysically and pharmacologically similar to IHERG, can be recorded with the patch-clamp technique. Northern blot experiments with a human herg cDNA probe revealed that both in human and murine clones the very high expression of herg transcripts can be quantified in at least three clearly identifiable bands, suggesting an alternative splicing of HERG mRNA. Moreover, we cloned a cDNA encoding for IIR from the SH-SY5Y human neuroblastoma. The sequence of this cDNA result was practically identical to that already reported for herg, indicating a high conservation of this gene in tumors. Consistently, the expression of this clone in Xenopus oocytes showed that the encoded K+ channel had substantially all of the biophysical and pharmacological properties of the native IIR described for tumor cells. In addition, in the tumor clones studied, IIR governs the resting potential, whereas it could not be detected either by the patch clamp or the Northern blot techniques in cells obtained from primary cell cultures of parental tissues (sensory neurons and myotubes), whose resting potential is controlled by the classical K+ anomalous rectifier current. This current substitution had a profound impact on the resting potential, which was markedly depolarized in tumors as compared with normal cells. These results suggest that IIR is normally only expressed during the early stages of cell differentiation frozen by neoplastic transformation, playing an important pathophysiological role in the regulatory mechanisms of neoplastic cell survival. In fact, because of its biophysical features, IIR, besides keeping the resting potential within the depolarized values required for unlimited tumor growth, could also appear suitable to afford a selective advantage in an ischemic environment.

Amino Acid Sequence↗

PubMed1998

Modalities of distortion of physiological voltage signals by patch-clamp amplifiers: a modeling study.

An extensive evaluation of the possible alterations affecting physiological voltage signals recorded with patch-clamp amplifiers (PCAs) working in the current-clamp (CC) mode was carried out by following a modeling approach. The PCA output voltage and current signals obtained during CC recordings performed under simplified experimental conditions were exploited to determine the equations describing the generation of error currents and voltage distortions by PCAs. The functions thus obtained were used to construct models of PCAs working in the CC mode, which were coupled to numerical simulations of neuronal bioelectrical behavior; this allowed us to evaluate the effects of the same PCAs on different physiological membrane-voltage events. The models revealed that rapid signals such as fast action potentials are preferentially affected, whereas slower events, such as low-threshold spikes, are less altered. Prominent effects of model PCAs on fast action potentials were alterations of their amplitude, duration, depolarization and repolarization speeds, and, most notably, the generation of spurious afterhyperpolarizations. Processes like regular firing and burst firing could also be altered, under particular conditions, by the model PCAs. When a cell consisting of more than one single intracellular compartment was considered, the model PCAs distorted fast equalization transients. Furthermore, the effects of different experimental and cellular parameters (series resistance, cell capacitance, temperature) on PCA-generated artifacts were analyzed. Finally, the simulations indicated that no off-line correction based on manipulations of the error-current signals returned by the PCAs can be successfully performed in the attempt to recover unperturbed voltage signals, because of alterations of the overall current flowing through the cell-PCA system.

Action Potentials↗

PubMed1997

A novel role for HERG K+ channels: spike-frequency adaptation.

1. The regular firing of a Hodgkin-Huxley neurone endowed with fast Na+ and delayed K+ channels can be converted into adapting firing by appending HERG (human eag-related gene) channels. 2. The computer model predictions were verified by studying the firing properties of F-11 DRG neurone x neuroblastoma hybrid cells induced to differentiate by long-term exposure to retinoic acid. These cells, which express HERG currents (IHERG), show clear spike-frequency adaptation of their firing when current clamped with long depolarizations. 3. In agreement with the prediction, the selective blocking of IHERG by class III antiarrhythmic drugs always led to the disappearance of the spike-frequency adaptation, and the conversion of adapting firing to regular firing. 4. It is proposed that, in addition to their role in the repolarization of the heart action potential, HERG channels may sustain a process of spike-frequency adaptation, and hence contribute to the control of burst duration in a way that is similar to that of the K+ currents, IAHP, IC and IM. In addition to the known cardiac arrhythmia syndrome (LQT2), genetic mutations or an altered HERG expression could lead to continuous hyperexcitable states sustained by the inability of nerve or endocrine cells to accommodate to repetitive stimuli. This might help in clarifying the pathogenesis of still undefined idiopathic familial epilepsies.

Adaptation, Physiological↗

PubMed1997

Laminin-alpha2 but not -alpha1-mediated adhesion of human (Duchenne) and murine (mdx) dystrophic myotubes is seriously defective.

It has been suggested that alpha-dystroglycan links the dystrophin-associated protein complex and extracellular matrix and that the absence of dystrophin and alpha-dystroglycan in Duchenne muscular dystrophy (DMD) may lead to the breakdown of this linkage. In the present study, myotubes from DMD patients and murine X-linked muscular dystrophic mice (mdx) were used to measure their adhesive force to the physiological laminin-alpha2 substrate, and it was found that the dystrophic myotubes were selectively unable to sustain adhesion. However, normal and dystrophic myotubes attached equally well to the laminin-alpha1 substrate. As far as we know, this is the first experimental evidence that the absence of dystrophin causes the complete loss of a still unknown laminin-alpha2-dependent adhesion force, therefore suggesting that the primary consequence of Duchenne dystrophy consists of the loss of an authentic mechanical linkage at the level of the alpha-dystroglycan/basal lamina interface.

Animals↗

PubMed1997

HERG- and IRK-like inward rectifier currents are sequentially expressed during neuronal development of neural crest cells and their derivatives.

Quail neural crest cells were cultured in a differentiative medium to study the inward K+ channel profile in neuronal precursors at various stages of maturation. Between 12 and 24 h of culture, neural crest-derived neurons displayed, in addition to the previously described outward depolarization-activated K+ currents, an inward current enhanced in high K+ medium. A biophysical and pharmacological analysis led us to conclude that this inward K+ current is identical to that previously demonstrated in mouse and human neuroblastoma cell lines (I[IR]). This current (quail I[IR] or ql[IR]), which is active at membrane potentials positive to -35 mV, was blocked by Cs+ and by class III antiarrhythmic drugs, thus resembling the K+ current encoded by the human ether-a-gò-gò-related gene (HERG). At later stages of incubation (>48 h), neural crest-derived neurons underwent morphological and biochemical differentiation and expressed fast Na+ currents. At this stage the cells lost qI[IR], displaying instead a classical inward rectifier K+ (IRK) current (quail I[IRK] = qI[IRK]). This substitution was reflected in the resting potential (VREST), which became hyperpolarized by >20 mV compared with the 24 h cells. Neurons were also harvested from peripheral ganglia and other derivatives originating from the migration of neural crest cells, viz. ciliary ganglia, dorsal root ganglia, adrenal medulla and sympathetic chain ganglia. After brief culture following harvesting from young embryos, ganglionic neurons always expressed qI(IR). On the other hand, when ganglia were explanted from older embryos (7-12 days), briefly cultured neurons displayed the IRK-like current. Again, in all the above derivatives the qI(IR) substitution by qI(IRK) was accompanied by a 20 mV hyperpolarization of VREST. Together, these data indicate that the VREST of normal neuronal precursors is sequentially regulated by HERG- and IRK-like currents, suggesting that HERG-like channels mark an immature and transient stage of neuronal differentiation, probably the same stage frozen in neuroblastomas by neoplastic transformation.

Animals↗

PubMed1996

Early cytoplasmic acidification in retinamide-mediated apoptosis of human promyelocytic leukemia cells.

The retinamide-mediated apoptosis of promyelocytic cells was investigated using pHi- and DNA-sensitive fluorescent probes (BCECF and Hoechst 33342). Acidification and apoptosis were observed during prolonged (0-12 h) exposure to retinamide (1 microM), but were absent in a retinamide-resistant clone. The analysis of experiments performed by simultaneous staining with the two dyes showed that acidification and apoptosis are correlated; the half-time for acidification is about 5 h while that for apoptosis is 80% longer. On the whole, these results suggest that apoptosis is preceded by an early mechanism of acidification in human leukemia cells treated by a retinoid of clinical interest in cancer chemo prevention and therapy.

Apoptosis↗

PubMed1996

A TTX-sensitive conductance underlying burst firing in isolated pyramidal neurons from rat neocortex.

Pyramidal neurons were acutely isolated from neocortex slices of 14- to 20-day-old rats and patch-clamped under physiological conditions. Current-clamp recordings revealed firing patterns corresponding to those previously reported in slices as regular spiking (RS) and intrinsically bursting (IB), i.e., single action potentials (AP), trains of regular spikes and bursts with depolarizing after-potentials (DAP). In IB neurons, intracellular perfusion with KF blocked the high-voltage-activated Ca2+ and the Ca(2+)-dependent K+ currents, revealing APs with a 10-30 ms shoulder at -35 mV (shoulder AP), which was the supporting plateau of the intraburst spikes. The use of the A channel blocker, 4-aminopyridine, caused a three-fold reduction in the AP repolarizing rate. A study of the de- and repolarizing rates modulating the spike shape (shoulder AP, burst or single APs) suggested that the percentage of available A channels could play a crucial role in burst formation. Blockade of the residual T-type Ca2+ current by Ni2+ did not inhibit the AP shoulder, whereas it was completely and reversibly inhibited by 30 nM TTX, which did not affect AP amplitude. The AP rising rate was only halved by 100 nM TTX. The data concerning the A channel-mediated burst formation and the role of the TTX-sensitive conductance have been successfully simulated in a model cell. We suggest that bursting is an intrinsic property of the membrane of neocortex neurons, and is sustained by TTX-sensitive slowly inactivating and/or persistent Na+ conductances.

Animals↗

PubMed1996

A novel K+ channel blocker isolated from 'hiccup nut' toxin.

Combretastatin B1, a polyhydroxybibenzyl compound extracted from the fruit of Combretum kraussii, known to contain 'hiccup nut' toxin, reversibly increased the duration, but not the peak or the rate of rise, of the action potential in rat sensory neurones by approximately 300%. This effect was only seen when it was applied to the extracellular side of the membrane. No effects on the resting potential were observed. K+ delayed rectifier currents were inhibited in neurones and in human myotubes with an IC50 of about 300 microM; the HERG-type inward rectifier channels in tumour cells were inhibited to a greater degree. Due to its selective action and the similarity of its blockade to that produced by class III antiarrhythmic drugs, the toxin could be the origin of compounds of potentially significant pharmacological interest.

Animals↗

PubMed1996

A HERG-like K+ channel in rat F-11 DRG cell line: pharmacological identification and biophysical characterization.

1. The relationships between the K+ inward rectifier current present in neuroblastoma cells (IIR) and the current encoded by the human ether-á-go-go-related gene (HERG), IHERG, and the rapidly activating repolarizing cardiac current IK(r), were investigated in a rat dorsal root ganglion (DRG) x mouse neuroblastoma hybrid cell line (F-11) using pharmacological and biophysical treatments. 2. IIR shared the pharmacological features described for IK(r), including the sensitivity to the antiarrhythmic drugs E4301 and WAY-123,398, whilst responding to Cs+, Ba2+ and La3+ in a similar way to IHERG. 3. The voltage-dependent gating properties of IIR were similar to those of IK(r) and IHERG, although IIR outward currents were negligible in comparison. 4. In high K+ extracellular solutions devoid of divalent cations, IIR deactivation kinetics were removed resulting in long-lasting currents apparently activated in hyperpolarization, with a marked (2.7-fold) increase in conductance, as recorded from the instantaneous linear current-voltage relationship at -120 mV. Re-addition of Ca2+ restored the original closure of the channel whereas re-addition of Mg2+ reduced the peak current. 5. The IIR described here, the heart IK(r) and the IHERG could be successfully predicted by a unique kinetic model where the voltage dependencies of the activation/inactivation gates were properly voltage shifted. On the whole, IIR seems to be the first example of a HERG-type current constitutively expressed and operating in mammalian cells of the neuronal lineage.

Animals↗

PubMed1996

Multipotential stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor.

It has been established that the adult mouse forebrain contains multipotential (neuronal/glial) progenitor cells that can be induced to proliferate in vitro when epidermal growth factor is provided. These cells are found within the subventricular zone of the lateral ventricles, together with other progenitor cell populations, whose requirements for proliferation remain undefined. Using basic fibroblast growth factor (bFGF), we have isolated multipotential progenitors from adult mouse striatum. These progenitors proliferate and can differentiate into cells displaying the antigenic properties of astrocytes, oligodendrocytes, and neurons. The neuron-like cells possess neuronal features, exhibit neuronal electrophysiological properties, and are immunoreactive for GABA, substance P, choline acetyl-transferase, and glutamate. Clonal analysis confirmed the multipotency of these bFGF-dependent cells. Most significantly, subcloning experiments demonstrated that they were capable of self-renewal, which led to a progressive increase in population size over serial passaging. These results demonstrate that bFGF is mitogenic for multipotential cells from adult mammalian forebrain that possess stem cell properties.

Action Potentials↗

PubMed1996

Electric fields at the plasma membrane level: a neglected element in the mechanisms of cell signalling.

Membrane proteins possess certain features that make them susceptible to the electric fields generated at the level of the plasma membrane. A reappraisal of cell signalling, taking into account the protein interactions with the membrane electrostatic profile, suggests that an electrical dimension is deeply involved in this fundamental aspect of cell biology. At least three types of potentials can contribute to this dimension: (1) the potential across the compact layer of water adherent to membrane surfaces; this potential is affected by classical inducers of cell differentiation, like dimethylsulfoxide and hexamethylenebisacetamide; (2) the potential across the Gouy-Chapman double layer, which accounts for the effects of extracellular cations in the modulation of differentiation; and (3) the resting potential. This last potential and its governing ion currents can be exploited in localised mechanisms of cell signalling centred on the functional association of integrin receptors with ion channels.

Animals↗

PubMed1996

Action potentials recorded with patch-clamp amplifiers: are they genuine?

A growing number of experimental studies have used patch-clamp amplifiers (PCAs) in the current-clamp (CC) mode to investigate classical excitability. In this paper we show that the measurements obtained in this way are affected by errors due to the electronic design of the PCA input section. We present experimental evidence of such errors, and demonstrate that they derive from PCA current absorption. Moreover, we propose a new PCA input-circuit configuration for the CC mode, which is suitable for accurately recording physiological voltage signals and is perfectly compatible with the standard voltage-clamp mode.

Action Potentials↗

PubMed1996

An algorithm for finding maximal common subtopologies in a set of protein structures.

For the comparison and analysis of protein structures, it is of interest to find maximal common substructures in a given set of proteins. This question is also relevant for motif definition and structure classification. In this paper we describe first a new suitable representation of the secondary structure topology of a protein by an undirected labeled graph. Based on this representation we developed a new fast algorithm that finds all common subtopologies in a set of protein structures. Our method is based on the algorithm by Bron and Kerbosch (1973), which enumerates all maximal cliques in a graph. The main improvement of our algorithm is to restrict the search process to cliques that represent connected substructures. This restriction reduces the number of cliques to be considered during the search process and the size of the search tree drastically. Thus we are able to handle large proteins. Experiments show the efficiency and superiority of our algorithm in comparison with other existing algorithms basing on graph-theoretical methods.

Algorithms↗

PubMed1996

Soluble or bound laminin elicit in human neuroblastoma cells short- or long-term potentiation of a K+ inwardly rectifying current: relevance to neuritogenesis.

Changes in the resting potential (VREST) and in the underlying ionic conductances were measured by the patch-clamp technique in SH-SY5Y human neuroblastoma cells exposed to substrate-bound or soluble Laminin (bLN; sLN), as compared to integrin-independent substrates (polylysine (PL); bovine serum albumin (BSA)). While PL and BSA were ineffective, both forms of LN caused an early (5-15 min) activation of a peculiar type of Inwardly Rectifying K+ current (IIR) characterised by a voltage-dependent inactivation in the range of membrane potentials around -50/0 mV. IIR was blocked by Cs+ ions and by the antiarrhythmic drug E-4031, a specific inhibitor of the HERG-codified channels. In cells adherent to bLN, IIR potentiation (85%) persisted for 90-120 min and was accompanied by a similar, but transient, increase in the leakage conductance (GL). Successively, the persistence of a high IIR conductance and the decrease of GL progressively bring VREST from -12 to approximately -30 mV in about 120 min. On the other hand, in cells adherent to PL, exposure to sLN produced a similar but not persistent activation of IIR, without any increase in GL: this caused a rapid, transient hyperpolarisation of VREST. The effects of bLN and sLN were mimicked by antibodies raised against the integrin beta 1 subunit, suggesting a specific integrin-mediated mechanism. In fact, when bound to the culture dishes, these antibodies simultaneously activated the IIR and GL, whereas in soluble form they only activated IIR. Cells adherent to bLN emitted neurites, a process impaired by the block of IIR by E-4031 and Cs+. On the whole data suggest that the integrin-mediated activation of IIR plays a crucial role in the commitment to neuritogenesis of neuroblastoma cells, independently on the effects of this activation on VREST.

Cell Adhesion↗

PubMed1995

A novel inward-rectifying K+ current with a cell-cycle dependence governs the resting potential of mammalian neuroblastoma cells.

1. Human and murine neuroblastoma cell lines were used to investigate, by the whole-cell patch-clamp technique, the properties of a novel inward-rectifying K+ current (IIR) in the adjustment of cell resting potential (Vrest), which was in the range -40 to -20 mV. 2. When elicited from a holding potential of 0 mV, IIR was completely inactivated with time constants ranging from 13 ms at -140 mV to 4.5 s at -50 mV. The steady-state inactivation curve (h(V)) was found to be independent of [Na+]o and [K+]o (2-80 mM) and could be fitted to a Boltzmann curve with a steep slope factor of 5-6, and a V1/2 around Vrest. Divalent ion-free extracellular solutions shifted h(V) to the left by about 28 mV. 3. Peak chord conductance, whose maximal value was approximately proportional to the square root of [K+]o, could be fitted to a Boltzmann curve independently of [K+]o, with a V1/2 value around -48 mV and a slope factor of 18. Extracellular Cs+ and Ba2+ blocked the IIR in a concentration- and voltage-dependent manner, but Ba2+ was less effective than it is on classical inward-rectifier channels. 4. Under control culture conditions the values of Vrest and V1/2 of h(V) varied widely among cells. The knowledge of V1/2 proved crucial or the theoretical prediction of Vrest. After cell synchronization in the G0-G1 phase of the cell cycle, or at the G1-S boundaries, the cells reduced their variability of h(V). The same occurred after cell synchronization in G1 by treatment with retinoic acid. 5. The experimental data could be fitted to a classical model of an inward rectifier, after removing the dependence of conductance activation on (V-EK), and incorporating an inactivation with an intrinsic voltage dependence. Moreover, the model predicts, for this novel inward rectifier and in contrast with the classical inward rectifier, the incapacity of maintaining, in physiological media, a Vrest more negative than -35 to -40 mV, which is an important feature of cancer cells.

Cells, Cultured↗

PubMed1995

Ionic mechanisms underlying burst firing in pyramidal neurons: intracellular study in rat sensorimotor cortex.

In in vitro slices prepared from rat sensorimotor cortex, intracellular recordings were obtained from 107 layer V pyramidal neurons, subsequently injected with biocytin for morphological reconstruction. Of the 107 neurons, 59 (55.1%) were identified as adapting (45) or non-adapting (13) regular spiking neurons (RS), and 48 (44.9%) as intrinsically bursting (IB) neurons discharging with an initial cluster of action potentials, which tended to recur rhythmically in a subset of 19 cells. The block of IAR by extracellular Cs+ did not affect burst generation, but enhanced the tendency to reburst in IB neurons. A similar effect was induced by other procedures affecting K(+)-dependent post-burst hyperpolarization. In IB neurons Ca2+ spikes had a longer decay time than in RS neurons, however selective blockers of both low and high threshold Ca2+ conductances failed to impair bursting activity. On the contrary, the perfusion of the slices with 0.5-1 microM TTX suppressed bursting behaviour in a critical time interval preceding the complete block of Na(+)-dependent action potentials. It is concluded that the persistent Na+ current INAP is the most important intrinsic factor for the typical firing properties of IB neurons, while Ca2+ and K+ conductances appear to contribute towards shaping bursts and controlling their recurrence rate. The morphology, connectivity and physiological properties of adapting and non-adapting RS neurons are particularly suited to the processing of respectively phasic and tonic inputs, whereas the properties of IB neurons are consistent with their suggested role in cortical rhythmogenesis and in the pathophysiological synchronized activities underlying epileptogenesis.

Action Potentials↗