bluuebunny/crossref_metadata_2025 huggingface dataset:
Control of intracellular chloride concentration and GABA response polarity in rat retinal ON bipolar cells
Authors: Billups Daniela , Attwell David
Published: November 2002

GABAergic modulation of retinal bipolar cells plays a crucial role in early visual processing. It helps to form centre‐surround receptive fields which filter the visual signal spatially at the bipolar cell dendrites in the outer retina, and it produces temporal filtering at the bipolar cell synaptic terminals in the inner retina. The observed chloride transporter distribution in ON bipolar cells has been predicted to produce an intracellular chloride concentration, [Cl−]i, that is significantly higher in the dendrites than in the synaptic terminals. This would allow dendritic GABA‐gated Cl− channels to generate the depolarization needed for forming the lateral inhibitory surround of the cell's receptive field, while synaptic terminal GABA‐gated Cl− channels generate the hyperpolarization needed for temporal shaping of the light response. In contrast to this idea, we show here that in ON bipolar cells [Cl−]i is only slightly higher in the dendrites than in the synaptic terminals, and that GABA‐gated channels in the dendrites may generate a hyperpolarization rather than a depolarization. We also show that [Cl−]i is controlled by movement of Cl− through ion channels in addition to transporters, that changes of [K+]o alter [Cl−]i and that voltage‐dependent equilibration of [Cl−]i in bipolar cells will produce a time‐dependent adaptation of GABAergic modulation with a time constant of 8 s after illumination‐evoked changes of membrane potential. Time‐dependent adaptation of [Cl−]i to voltage changes in retinal bipolar cells may add a previously unsuspected layer of temporal processing to signals as they pass through the retina.
Passive basilar membrane vibrations in gerbil neonates: mechanical bases of cochlear maturation
Authors: Overstreet Edward H. , Temchin Andrei N. , Ruggero Mario A.
Published: November 2002

Using a laser velocimeter, basilar membrane (BM) responses to tones were measured in neonatal gerbils at a site near the round window of the cochlea. In adult gerbils, ‘active’ BM responses at this site are most sensitive at 34–37 kHz and exhibit a compressive non‐linearity. Postmortem, BM responses in adults become ‘passive’, i.e. linear and insensitive, and the best frequency (BF) shifts downwards by about 0.5 octaves. At 14 and 16 days after birth (DAB), BM responses in neonatal gerbils were passive but otherwise very different from postmortem responses in adult gerbils: BF was more than an octave lower, the steep slopes of the phase vs. frequency curves were shifted downwards in frequency by nearly 1 octave, and the maximum phase lags amounted to only 180 deg relative to stapes. BFs and phase lags increased systematically between 14 and 20 DAB, implying drastic alterations of the passive material properties of cochlear tissues and accounting for a large part of the shift in BF that characterizes maturation of auditory nerve responses during the same period.
Subcellular Ca2+ alternans represents a novel mechanism for the generation of arrhythmogenic Ca2+ waves in cat atrial myocytes
Authors: Kockskämper Jens , Blatter Lothar A.
Published: November 2002

Ca2+ alternans is a potentially arrhythmogenic beat‐to‐beat alternation of the amplitude of the action potential‐induced [Ca2+]i transient in cardiac myocytes. Despite its pathophysiological significance the cellular mechanisms underlying Ca2+ alternans are poorly understood. Recent evidence, however, points to the modulation of Ca2+‐induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) by localized alterations in energy metabolism as an important determinant of Ca2+ alternans. We therefore studied the subcellular properties of Ca2+ alternans in field‐stimulated cat atrial myocytes employing fast two‐dimensional fluorescence confocal microscopy. Ca2+ alternans was elicited by an increase in stimulation frequency or by metabolic interventions targeting glycolysis. Marked subcellular variations in the time of onset, the magnitude, and the phase of alternans were observed. Longitudinal and transverse gradients of Ca2+ alternans were found as well as neighbouring subcellular regions alternating out‐of‐phase. Moreover, focal inhibition of glycolysis resulted in spatially restricted Ca2+ alternans. When two adjacent regions within a myocyte alternated out‐of‐phase, steep [Ca2+]i gradients developed at their border giving rise to delayed propagating Ca2+ waves. The results demonstrate that Ca2+ alternans is a subcellular phenomenon caused by modulation of SR Ca2+ release, which is mediated, at least in part, by local inhibition of energy metabolism. The generation of arrhythmogenic Ca2+ waves by subcellular variations in the phase of Ca2+ alternans represents a novel mechanism for the development of atrial disrhythmias.
Human, rat and chicken small intestinal Na+‐Cl−‐creatine transporter: functional, molecular characterization and localization
Authors: Peral M. J. , García‐Delgado M. , Calonge M. L. , Durán J. M. , De La Horra M. C. , Wallimann T. , Speer O. , Ilundáin A. A.
Published: November 2002

In spite of all the fascinating properties of oral creatine supplementation, the mechanism(s) mediating its intestinal absorption has(have) not been investigated. The purpose of this study was to characterize intestinal creatine transport. [14C]Creatine uptake was measured in chicken enterocytes and rat ileum, and expression of the creatine transporter CRT was examined in human, rat and chicken small intestine by reverse transcription‐polymerase chain reaction, Northern blot, in situ hybridization, immunoblotting and immunohistochemistry. Results show that enterocytes accumulate creatine against its concentration gradient. This accumulation was electrogenic, Na+‐ and Cl−‐dependent, with a probable stoichiometry of 2 Na+: 1 Cl−: 1 creatine, and inhibited by ouabain and iodoacetic acid. The kinetic study revealed a Km for creatine of 29 μm. [14C]Creatine uptake was efficiently antagonized by non‐labelled creatine, guanidinopropionic acid and cyclocreatine. More distant structural analogues of creatine, such as GABA, choline, glycine, β‐alanine, taurine and betaine, had no effect on intestinal creatine uptake, indicating a high substrate specificity of the creatine transporter. Consistent with these functional data, messenger RNA for CRT was detected only in the cells lining the intestinal villus. The sequences of partial clones, and of the full‐length cDNA clone, isolated from human and rat small intestine were identical to previously cloned CRT cDNAs. Immunological analysis revealed that CRT protein was mainly associated with the apical membrane of the enterocytes. This study reports for the first time that mammalian and avian enterocytes express CRT along the villus, where it mediates high‐affinity, Na+‐ and Cl−‐dependent, apical creatine uptake.
Neonatal development of the rat visual cortex: synaptic function of GABAa receptor α subunits
Authors: Bosman Laurens W. J. , Rosahl Thomas W. , Brussaard Arjen B.
Published: November 2002

Each GABAA receptor consists of two α and three other subunits. The spatial and temporal distribution of different α subunit isomeres expressed by the CNS is highly regulated. Here we study changes in functional contribution of different α subunits during neonatal development in rat visual cortex. First, we characterized postsynaptic α subunit expression in layer II‐III neurons, using subunit‐specific pharmacology combined with electrophysiological recordings in acutely prepared brain slices. This showed clear developmental downregulation of the effects of bretazenil (1 μm) and marked upregulation of the effect of 100 nm of zolpidem on the decay of spontaneous inhibitory postsynaptic currents (sIPSCs). Given the concentrations used we interpret this as downregulation of the synaptic α3 and upregulation of α1 subunit. Furthermore, the effect of furosemide, being indicative of the functional contribution of α4, was increased between postnatal days 6 and 21. Our second aim was to study the effects of plasticity in α subunit expression on decay properties of GABAergic IPSCs. We found that bretazenil‐sensitive IPSCs have the longest decay time constant in juvenile neurons. In mature neurons, zolpidem‐ and furosemide‐sensitive IPSCs have relatively fast decay kinetics, whereas bretazenil‐sensitive IPSCs decay relatively slowly. Analysis of α1 deficient mice and α1 antisense oligonucleotide deletion in rat explants showed similar results to those obtained by zolpidem application. Thus, distinct α subunit contributions create heterogeneity in developmental acceleration of IPSC decay in neocortex.
Biphasic modulation of synaptic transmission by hypertonicity at the embryonic Drosophila neuromuscular junction
Authors: Suzuki Kazuhiro , Okamoto Tomonori , Kidokoro Yoshiaki
Published: November 2002

Puff‐application of hypertonic saline (sucrose added to external saline) causes a transient increase in the frequency of spontaneous miniature synaptic currents (mSCs) at the neuromuscular junctions of Drosophila embryos. The frequency gradually returns to pre‐application levels. External Ca2+ is not needed for this response, but it may modify it. At 50 mm added sucrose, for example, enhanced spontaneous release was observed only in the presence of external Ca2+, suggesting that Ca2+ augments the response. In a high‐K+ solution, in which the basal mSC frequency was elevated, higher sucrose concentrations produced an increase in mSC frequency that was followed (during and after the hypertonic exposure) by depression, with the magnitude of both effects increasing with hypertonicity between 100 and 500 mm. Evoked release by nerve stimulation showed only depression in response to hypertonicity. We do not believe that the depression of spontaneous or evoked release can be explained by the depletion of releasable quanta, however, since the frequency of quantal release did not reach levels compatible with this explanation and the enhancement and depression could be obtained independent of one another. In a mutant lacking neuronal synaptobrevin, only the depression of mSC frequency was induced by hypertonicity. Conversely, only the enhancing effect was observed in wild‐type embryos when the mSC frequency was elevated with forskolin in Ca2+‐free saline. In cultured embryonic Drosophila neurons, Ca2+ signals that were induced by high K+ and detected by Fura‐2, were reduced by hypertonicity, suggesting that the depressing response is due to a direct effect of hypertonicity on Ca2+ influx.
Systemic salt loading decreases body temperature and increases heat‐escape/cold‐seeking behaviour via the central AT1 and V1 receptors in rats
Authors: Konishi Masahiro , Nagashima Kei , Kanosue Kazuyuki
Published: November 2002

Salt loading decreases body core temperature (Tcore) at neutral ambient temperature (26 °C) and increases heat‐escape/cold‐seeking behaviour in desalivated rats. In this study, we tested the hypothesis that brain angiotensin II (AII) and arginine vasopressin (AVP) are associated with these responses. Surgically desalivated rats (n= 28) were administered an injection (s.c., 10 ml kg−1) of either normal saline (154 mm, NS) or hypertonic saline (2500 mm, HS) following an intracerebroventricular injection (10 μl kg−1) of an AII AT1‐receptor antagonist (candesartan, 5 μg μl−1), an AVP V1‐receptor antagonist ((β‐mercapto‐β, β‐cyclopenta‐methylene propionyl1, O‐Me‐Tyr2, Arg8)‐vasopressin, 0.5 μg μl−1), or normal saline (154 mm). Each rat was placed in a behaviour box, first at 26 °C for 1 h to allow the measurement of baseline Tcore and movement. The ambient temperature was then elevated to 40 °C for the next 2 h, during which time the rat was able to trigger a 0 °C air reward for 30 s by moving into a specific area of the box (operant behaviour). The s.c. HS significantly decreased baseline Tcore at 26 °C (36.5 ± 0.1 °C) and increased counts of operant behaviour at 40 °C (57 ± 3) compared with results obtained following s.c. NS injection (37.4 ± 0.1 °C and 42 ± 1, respectively). These responses to s.c. HS were inhibited by the intracerebroventricular injection of AT1 (37.3 ± 0.1 °C and 43 ± 2, respectively; P < 0.05) and V1 antagonists (37.2 ± 0.2 °C and 42 ± 2, respectively; P < 0.05), although administration of both antagonists with s.c. NS had no effect. These results suggest that brain AII and AVP are involved in the decrease in Tcore observed at neutral ambient temperature and the increase in heat‐escape/cold‐seeking behaviour in response to osmotic stimulation, via the central AT1 and V1 receptors, respectively
Interaction with 14‐3‐3 proteins promotes functional expression of the potassium channels TASK‐1 and TASK‐3
Authors: Rajan Sindhu , Preisig‐Müller Regina , Wischmeyer Erhard , Nehring Ralf , Hanley Peter J. , Renigunta Vijay , Musset Boris , Schlichthörl Günter , Derst Christian , Karschin Andreas , Daut Jürgen
Published: November 2002

The two‐pore‐domain potassium channels TASK‐1, TASK‐3 and TASK‐5 possess a conserved C‐terminal motif of five amino acids. Truncation of the C‐terminus of TASK‐1 strongly reduced the currents measured after heterologous expression in Xenopus oocytes or HEK293 cells and decreased surface membrane expression of GFP‐tagged channel proteins. Two‐hybrid analysis showed that the C‐terminal domain of TASK‐1, TASK‐3 and TASK‐5, but not TASK‐4, interacts with isoforms of the adapter protein 14‐3‐3. A pentapeptide motif at the extreme C‐terminus of TASK‐1, RRx(S/T)x, was found to be sufficient for weak but significant interaction with 14‐3‐3, whereas the last 40 amino acids of TASK‐1 were required for strong binding. Deletion of a single amino acid at the C‐terminal end of TASK‐1 or TASK‐3 abolished binding of 14‐3‐3 and strongly reduced the macroscopic currents observed in Xenopus oocytes. TASK‐1 mutants that failed to interact with 14‐3‐3 isoforms (V411*, S410A, S410D) also produced only very weak macroscopic currents. In contrast, the mutant TASK‐1 S409A, which interacts with 14‐3‐3‐like wild‐type channels, displayed normal macroscopic currents. Co‐injection of 14‐3‐3ζ cRNA increased TASK‐1 current in Xenopus oocytes by about 70 %. After co‐transfection in HEK293 cells, TASK‐1 and 14‐3‐3ζ (but not TASK‐1ΔC5 and 14‐3‐3ζ) could be co‐immunoprecipitated. Furthermore, TASK‐1 and 14‐3‐3 could be co‐immunoprecipitated in synaptic membrane extracts and postsynaptic density membranes. Our findings suggest that interaction of 14‐3‐3 with TASK‐1 or TASK‐3 may promote the trafficking of the channels to the surface membrane.
Water permeability of capillaries in the subfornical organ of rats determined by Gd‐DTPA2‐ enhanced 1H magnetic resonance imaging
Authors: Seo Yoshiteru , Takamata Akira , Ogino Takashi , Morita Hironobu , Nakamura Shun , Murakami Masataka
Published: November 2002

The water permeability of capillaries in the subfornical organ (SFO) of rat was measured by a 1H nuclear magnetic resonance method in combination with a venous injection of a relaxation reagent, gadolinium‐diethylene triamine‐N,N,N′,N″,N″‐pentaacetic acid (Gd‐DTPA2‐), which could not pass through the blood‐brain barrier (BBB). Judging from results of Gd‐DTPA2‐ dose dependency in the intact brain and the BBB‐permeabilized brain, Gd‐DTPA2‐ could not have leaked out from the capillaries in the cortex, thalamus or SFO, but it could have been extravasated in the posterior lobe of the pituitary gland. The longitudinal (T1) relaxation time of water in the SFO region was measured by inversion‐recovery magnetic resonance imaging at 4.7 T. The T1 relaxation rates (1/T1) before and after Gd‐DTPA2‐ infusion were 0.70 ± 0.02 s−1 (mean ±s.e.m., n= 9) and 1.53 ± 0.11 s−1 (n= 9), respectively. The rate constant for water influx to the capillaries was estimated to be 0.84 ± 0.11 s−1 (n= 9) which corresponds with a diffusive membrane permeability (Pd) of 3.7 × 10−3 cm s−1. Compared with values found in the literature available on this subject, this Pd value for the capillaries in the SFO was the same order of magnitude as that for transmembrane permeability of water for the vasa recta, and it may be 10–100 times larger than that of the blood‐brain barrier in the cortex. Areas of the cortex and thalamus showed minimal changes in the T1 relaxation rate (ca 0.09 s−1), but these values were not statistically significant and they corresponded to Pd values much smaller than those found in the SFO. From these results, we conclude that the capillaries in the SFO have one of the highest water permeability values among all of the capillaries in the brain. It is also suggested that this magnetic resonance imaging, based on T1 relaxation rate, is a useful method to detect local water permeability in situ.
GFR α2/neurturin signalling regulates noxious heat transduction in isolectin B4‐binding mouse sensory neurons
Authors: Stucky Cheryl L. , Rossi Jari , Airaksinen Matti S. , Lewin Gary R.
Published: November 2002

The GFR α2 receptor is the cognate co‐receptor for the neurotrophic factor neurturin and GFR α2 is selectively expressed by isolectin B4 (IB4)‐binding nociceptive sensory neurons. Here, we used two physiological approaches in combination with mice that have a targeted deletion of the GFR α2 gene (GFR α2 ‐/‐ mice) in order to determine whether GFR α2/neurturin signalling regulates the functional properties or the survival of IB4‐binding nociceptors. Because 50 % of IB4‐binding neurons respond to noxious heat and because patch clamp recordings of isolated dorsal root ganglion sensory neurons allow one to neurochemically identify subpopulations of neurons, we analysed the noxious heat responsiveness of IB4‐positive and ‐negative small‐diameter neurons isolated from adult GFR α2 ‐/‐ and littermate control mice. The percentage of IB4‐positive neurons that had large (> 100 pA) heat‐evoked inward currents was severely reduced in GFR α2 ‐/‐ mice (12 %) compared to wild‐type littermates (47 %), and this loss in large‐magnitude heat currents was accounted for by an increase in neurons with very small (< 100 pA) heat‐evoked currents as well as an increase in neurons with no detectable heat current. Counts of IB4‐positive and ‐negative neurons, as well as counts of unmyelinated axons in the saphenous nerve, confirmed that the loss in neurons with large‐amplitude heat currents was due to a deficit in heat transduction and not a decrease in cell survival. The effect was modality specific for heat because mechanical transduction of all fibre types, including IB4‐positive C fibres, was normal. Our data are the first to indicate a transduction‐function role for GFR α2/neurturin signalling in a specific class of sensory neurons.
Motor fatigue and cognitive task performance in humans
Authors: Lorist Monicque M. , Kernell Daniel , Meijman Theo F. , Zijdewind Inge
Published: November 2002

During fatiguing submaximal contractions a constant force production can be obtained at the cost of an increasing central command intensity. Little is known about the interaction between the underlying central mechanisms driving motor behaviour and cognitive functions. To address this issue, subjects performed four tasks: an auditory choice reaction task (CRT), a CRT simultaneously with a fatiguing or a non‐fatiguing submaximal muscle contraction task, and a fatiguing submaximal contraction task alone. Results showed that performance in the single‐CRT condition was relatively stable. However, in the fatiguing dual‐task condition, performance levels in the cognitive CRT deteriorated drastically with time‐on‐task. Moreover, in the fatiguing dual‐task condition the rise in force variability was significantly larger than during the fatiguing submaximal contraction alone. Thus, our results indicate a mutual interaction between cognitive functions and the central mechanisms driving motor behaviour during fatigue. The precise nature of this interference, and at what level this interaction takes place is still unknown.
Upregulation of proinflammatory cytokines and nerve growth factor by intraplantar injection of capsaicin in rats
Authors: Saadé N. E. , Massaad C. A. , Ochoa‐Chaar C. I. , Jabbur S. J. , Safieh‐Garabedian B. , Atweh S. F.
Published: November 2002

Capsaicin‐sensitive primary afferents (CSPA) are known to be involved in nociception and neurogenic inflammation. Extensive research has been devoted to the sensory role of these fibres but less attention has been paid to their local effector function. This study aimed at gaining more insight into the molecular mechanisms underlying the neurogenic inflammation induced by this special group of afferent fibres. Different groups of rats (n= 5 in each group), either naive or subjected to selective ablation of their CSPA, received individual intraplantar injections of saline, capsaicin, its vehicle or capsaicin preceded by its antagonist, capsazepine. Acute tests for nociception were used to assess the variations of the nociceptive thresholds. Variations of the levels of proinflamamtory cytokines and nerve growth factor (NGF) were measured by enzyme‐linked immunosorbant assay (ELISA). Intraplantar injection of capsaicin (10 μg in 50 μl) produced a sustained thermal and mechanical hyperalgesia that peaked at 3–6 h and disappeared 24 h following the injection. Similar capsaicin injection in further groups of rats produced an early upregulation of the proinflamamtory cytokines and NGF, which peaked at 30–60 min and returned to control levels within 2–5 h. Similar effects were observed following the application of either capsaicin or intense electrical stimulation on the cut end of the distal portion of the sciatic nerve. The effects of capsaicin were abolished in rats subjected to selective ablation of their CSPA. These results demonstrate that CSPA can simultaneously challenge the immune system through the release of proinflammatory mediators and the central nervous system through nociceptive signalling and can therefore serve as a common afferent pathway to both immune and nervous systems.
Voltage‐activated proton currents in human lymphocytes
Authors: Schilling Tom , Gratopp Alexander , DeCoursey Thomas E. , Eder Claudia
Published: November 2002

Voltage‐activated proton currents are reported for the first time in human peripheral blood T and B lymphocytes and in the human leukaemic T cell line Jurkat E6‐1. The properties of H+ currents studied using tight‐seal voltage‐clamp recording techniques were similar in all cells. Changing the pH gradient by one unit caused a 47 mV shift in the reversal potential, demonstrating high selectivity of the channels for protons. H+ current activation upon membrane depolarisation had a sigmoidal time course that could be fitted by a single exponential function after a brief delay. Increasing pHo shifted the activation threshold to more negative potentials, and increased both the H+ current amplitude and the rate of activation. In lymphocytes studied at pHi 6.0, the activation threshold was more negative and the H+ current density was three times larger than at pHi 7.0. Increasing the intracellular Ca2+ concentration to 1 μm did not change H+ current amplitude or kinetics detectably. Extracellularly applied Zn2+ and Cd2+ inhibited proton currents, slowing activation and shifting the voltage‐activation curve to more positive potentials. The H+ current amplitude was 100 times larger in CD19+ B lymphocytes and in Jurkat E6‐1 cells than in CD3+ T lymphocytes. Following stimulation with the phorbol ester PMA, the H+ current density in peripheral blood T lymphocytes and Jurkat T cells increased. In contrast, the H+ current density of phorbol ester (PMA)‐stimulated B lymphocytes was reduced and activation became slower. The pattern of expression of H+ channels in lymphocytes appears well suited to their proposed role of charge compensation during the respiratory burst.
The size and the speed of the working stroke of muscle myosin and its dependence on the force
Authors: Piazzesi Gabriella , Lucii Leonardo , Lombardi Vincenzo
Published: November 2002

Myosin II is the motor protein that produces force and shortening in muscle by ATP‐driven cyclic interactions of its globular portion, the head, with the actin filament. During each interaction the myosin head undergoes a conformational change, the working stroke, which, depending on the mechanical conditions, can generate a force of several piconewtons or an axial displacement of the actin filament toward the centre of the sarcomere of several nanometres. However, the sizes of the elementary force and length steps and their dependence on the mechanical conditions are still under question. Due to the small fraction of the ATPase cycle time myosin II spends attached to actin, single molecule mechanics failed to produce definitive measurements of the individual events. In intact frog muscle fibres, however, myosin II's working stroke can be synchronised in the few milliseconds following a step reduction in either force or length superimposed on the isometric contraction. Here we show that with 150 μs force steps it is possible to separate the elastic response from the subsequent early rapid component of filament sliding due to the working stroke in the attached myosin heads. In this way we determine how the size and the speed of the working stroke depend on the clamped force. The relation between mechanical energy and force provides a molecular basis for muscle efficiency and an estimate of the isometric force exerted by a myosin head.
Selective muscarinic regulation of functional glutamatergic Schaffer collateral synapses in rat CA1 pyramidal neurons
Authors: de Sevilla David Fernández , Cabezas Carolina , de Prada Amaranta N. Oshima , Sánchez‐Jiménez Abel , Buño Washington
Published: November 2002

Analysis of the cholinergic regulation of glutamatergic neurotransmission is an essential step in understanding the hippocampus because it can influence forms of synaptic plasticity that are thought to underlie learning and memory. We studied in vitro the cholinergic regulation of excitatory postsynaptic currents (EPSCs) evoked in rat CA1 pyramidal neurons by Schaffer collateral (SC) stimulation. Using ‘minimal’ stimulation, which activates one or very few synapses, the cholinergic agonist carbamylcholine (CCh) increased the failure rate of functional more (36 %) than of silent synapses (7 %), without changes in the EPSC amplitude. These effects of CCh were insensitive to manipulations that increased the probability of release, such as paired pulse facilitation, increases in temperature and increases in the extracellular Ca2+ : Mg2+ ratio. Using ‘conventional’ stimulation, which activates a large number of synapses, CCh inhibited more the pharmacologically isolated non‐NMDA (86 %) than the NMDA (47 %) EPSC. The changes in failure rate, EPSC variance and the increased paired pulse facilitation that paralleled the inhibition imply that CCh decreased release probability. Muscarine had similar effects. The inhibition by both CCh and by muscarine was prevented by atropine. We conclude that CCh reduces the non‐NMDA component of SC EPSCs by selectively inhibiting transmitter release at functional synapses via activation of muscarinic receptors. The results suggest that SCs have two types of terminals, one in functional synapses, selectively sensitive to regulation through activation of muscarinic receptors, and the other in silent synapses less sensitive to that regulation. The specific inhibition of functional synapses would favour activity‐dependent plastic phenomena through NMDA receptors at silent synapses without the activation of non‐NMDA receptors and functional synapses.
Short‐interval paired‐pulse inhibition and facilitation of human motor cortex: the dimension of stimulus intensity
Authors: Ilić Tihomir V. , Meintzschel Frank , Cleff Ulrich , Ruge Diane , Kessler Kirn R. , Ziemann Ulf
Published: November 2002

Paired transcranial magnetic stimulation has greatly advanced our understanding of the mechanisms which control excitability in human motor cortex. While it is clear that paired‐pulse excitability depends on the exact interstimulus interval (ISI) between the first (S1) and second stimulus (S2), relatively little is known about the effects of the intensities of S1 and S2, and the effects of manipulating neurotransmission through the GABAA receptor. When recording the motor evoked potential (MEP) from the resting abductor digiti minimi (ADM) muscle, using a fixed ISI of 1.5 ms, and expressing the interaction between S1 and S2 as MEPS1+S2/(MEPS1+ MEPS2), then a systematic variation of the intensities of S1 and S2 revealed short‐interval intracortical facilitation (SICF) if S1 and S2 were approximately equal to MEP threshold (RMT), or if S1 > RMT and S2 < RMT. In contrast, short‐interval intracortical inhibition (SICI) occurred if S1 < RMT and S2 > RMT. Contraction of the ADM left SICI unchanged but reduced SICF. The GABAA receptor agonist diazepam increased SICI and reduced SICF in the resting ADM while diazepam had no effect during ADM contraction. Surface EMG and single motor unit recordings revealed that during ADM contraction SICI onset was at the I3‐wave latency of S2, whereas SICF typically ‘jumped up’ by one I‐wave and started with the I2‐wave latency of S2. Findings suggest that SICI is mediated through a low‐threshold GABAA receptor‐dependent inhibitory pathway and summation of IPSP from S1 and EPSP from S2 at the corticospinal neurone. In contrast, SICF originates through non‐synaptic facilitation at the initial axon segment of interneurones along a high‐threshold excitatory pathway.
ATP release from human airway epithelial cells studied using a capillary cell culture system
Authors: Guyot Annick , Hanrahan John W.
Published: November 2002

Epithelial release of adenosine triphosphate (ATP), an important autocrine and paracrine signalling molecule, is acutely mechanosensitive and therefore difficult to study. We describe here a novel preparation that minimizes mechanical and metabolic perturbations, and use it to examine ATP secretion by epithelial cells. The Calu‐3 cell line derived from human airway sub‐mucosal glands was cultured in a hollow fibre bioreactor on porous capillaries that were perfused by a re‐circulating medium pump. Cells became polarized and cultures were stable for > 5 months, as evidenced by microscopy and lactate production (≈250 μg (108 cells)−1 day−1). Elevating apical flow rate 5‐fold increased ATP secretion from ≈200 to 6618 fmol min−1. Reducing apical osmolarity by 25–43 % also increased ATP secretion, which then declined spontaneously to a plateau rate that persisted as long as hypotonic perfusion was maintained. Release deactivated rapidly after shear and osmotic stresses were terminated, and was not associated with detectable cell lysis. Lowering apical [Ca2+] to increase connexin hemichannel permeability also stimulated ATP release and increased secretion during both hyposmotic and shear stress; however, the connexin 43 blocker flufenamic acid inhibited shear‐induced ATP release only in low‐Ca2+ solution, and therefore another secretory pathway may operate with physiological (i.e. mm) calcium. Regardless of the mechanism, the present results quantify ATP responses to mechanical and osmotic stimuli and demonstrate the usefulness of capillary cultures for studying epithelial secretion.
Elucidating KChiP effects on Kv4.3 inactivation and recovery kinetics with a minimal KChiP2 isoform
Authors: Patel Sangita P. , Campbell Donald L. , Strauss Harold C.
Published: November 2002

Kv channel interacting proteins (KChIPs) are Ca2+‐binding proteins with four EF‐hands. KChIPs modulate Kv4 channel gating by slowing inactivation kinetics and accelerating recovery kinetics. Thus, KChIPs are believed to be important regulators of Kv4 channels underlying transient outward K+ currents in many excitable cell types. We have cloned a structurally minimal KChIP2 isoform (KChIP2d) from ferret heart. KChIP2d corresponds to the final 70 C‐terminal amino acids of other KChIPs and has only one EF‐hand. We demonstrate that KChIP2d is a functional KChIP that both accelerates recovery and slows inactivation kinetics of Kv4.3, indicating that the minimal C‐terminus can maintain KChIP regulatory properties. We utilize KChIP2d to further demonstrate that: (i) the EF‐hand modulates effects on Kv4.3 inactivation but not recovery; (ii) Ca2+‐dependent effects on Kv4.3 inactivation are mediated through a mechanism reflected in the slow time constant of inactivation; and (iii) a short stretch of amino acids exclusive of the EF‐hand partially mediates Ca2+‐independent effects on recovery. Our results demonstrate that distinct regions of a KChIP molecule are involved in modulating inactivation and recovery. The potential ability of KChIP EF‐hands to sense intracellular Ca2+ levels and transduce these changes to alterations in Kv4 channel inactivation kinetics may serve as a mechanism allowing intracellular Ca2+ transients to modulate repolarization. KChIP2d is a valuable tool for elucidating structural domains of KChIPs involved in Kv4 channel regulation.
Drug Susceptibilities of Yeast Cells Are Affected by Membrane Lipid Composition
Authors: Mukhopadhyay Kasturi , Kohli Avmeet , Prasad Rajendra
Published: November 2002

In the present study we have exploited isogenic
erg
mutants of
Saccharomyces cerevisiae
to examine the contribution of an altered lipid environment on drug susceptibilities of yeast cells. It is observed that
erg
mutants, which possess high levels of membrane fluidity, were hypersensitive to the drugs tested, i.e., cycloheximide (CYH),
o
-phenanthroline, sulfomethuron methyl, 4-nitroquinoline oxide, and methotrexate. Most of the
erg
mutants except mutant
erg4
were, however, resistant to fluconazole (FLC). By using the fluorophore rhodamine-6G and radiolabeled FLC to monitor the passive diffusion, it was observed that
erg
mutant cells elicited enhanced diffusion. The addition of a membrane fluidizer, benzyl alcohol (BA), to
S. cerevisiae
wild-type cells led to enhanced membrane fluidity. However, a 10 to 12% increase in BA-induced membrane fluidity did not alter the drug susceptibilities of the
S. cerevisiae
wild-type cells. The enhanced diffusion observed in
erg
mutants did not seem to be solely responsible for the observed hypersensitivity of
erg
mutants. In order to ascertain the functioning of drug extrusion pumps encoding the genes
CDR1
(ATP-binding cassette family) and
CaMDR1
(MFS family) of
Candida albicans
in a different lipid environment, they were independently expressed in an
S. cerevisiae erg
mutant background. While the fold change in drug resistance mediated by
CaMDR1
remained the same or increased in
erg
mutants, susceptibility to FLC and CYH mediated by
CDR1
was increased (decrease in fold resistance). Our results demonstrate that between the two drug extrusion pumps, Cdr1p appeared to be more adversely affected by the fluctuations in the membrane lipid environment (particularly to ergosterol). By using 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino-hexanoyl] sphingosyl phosphocholine (a fluorescent analogue of sphingomyelin), a close interaction between membrane ergosterol and sphingomyelin which appears to be disrupted in
erg
mutants is demonstrated. Taken together it appears that multidrug resistance in yeast is closely linked to the status of membrane lipids, wherein the overall drug susceptibility phenotype of a cell appears to be an interplay among drug diffusion, extrusion pumps, and the membrane lipid environment.
In Vivo Efficacy of Continuous Infusion versus Intermittent Dosing of Linezolid Compared to Vancomycin in a Methicillin-Resistant
Staphylococcus aureus
Rabbit Endocarditis Model
Authors: Jacqueline Cédric , Batard Eric , Perez Lucia , Boutoille David , Hamel Antoine , Caillon Jocelyne , Kergueris Marie-France , Potel Gilles , Bugnon Denis
Published: November 2002

Linezolid is the first drug issued from the oxazolidinones, a novel class of antimicrobial agents with potent activity against gram-positive pathogens. A rabbit endocarditis model was used to compare the in vivo activities of different linezolid regimens mimicking intermittent dosing of 10 mg/kg of body weight every 12 h for 5 days or continuous (constant-rate) infusion of a daily dose of 20 mg/kg (for 5 days) or 40 mg/kg (for 3 and 5 days) and the activities of intermittent dosing and continuous infusion of vancomycin (for 5 days). The in vivo activities of these regimens were tested against three strains of methicillin-resistant
Staphylococcus aureus
. A human-like pharmacokinetic simulation was used for linezolid in order to improve the extrapolation of the results to human therapy. All linezolid regimens significantly reduced the numbers of
S. aureus
cells in aortic valve vegetations compared to the numbers in the control groups. Linezolid intermittent dosing had an in vivo bacteriostatic effect. Switching from intermittent dosing to continuous infusion (at the same dose) led to in vivo bactericidal activity, with a decrease of at least 3 log
10
CFU/g of vegetation compared to the counts for the controls. After 5 days of treatment, continuous infusion of linezolid (corresponding to a daily dose of 40 mg/kg in humans) seemed to be at least as effective as vancomycin against the three strains. No resistant variant was isolated from the vegetations during any of the treatments. These data suggest that continuous infusion of linezolid could be an appropriate alternative to the use of glycopeptides for the treatment of severe methicillin-resistant
S. aureus
infections.
Effect of a Single Percutaneous Abscess Drainage Puncture and Imipenem Therapy, Alone or in Combination, in Treatment of Mixed-Infection Abscesses in Mice
Authors: Stearne Lorna E. T. , Buijk Steven L. , Mouton Johan W. , Gyssens Inge C.
Published: November 2002

The importance of supplementary imipenem therapy after a single percutaneous abscess drainage puncture was studied in a mouse model of established mixed-infection abscesses. Animals were treated for 3 days with daily dosing regimens of 384 to 1,536 mg/kg of body weight that took into account the short half-life of this antibiotic in mice. Imipenem therapy in conjunction with abscess drainage was significantly better than drainage alone in reducing theEscherichia coliandBacteroides fragiliscounts in the mixed infections. Furthermore, the killing ofB. fragilisby the combination of imipenem therapy and abscess drainage was significantly better than that by imipenem treatment alone. The maximum reductions inE. coliandB. fragiliscounts were 1.1 and 2.2 log10CFU/abscess, respectively. In contrast, the in vitro activity of imipenem was significantly better (maximum reduction, ≥6.2 log10CFU/ml) against mixed cultures of the same strains even when bacterial numbers similar to those found in the abscesses were used. Comparable in vivo activity was achieved only when treatment was started 30 min before inoculation (reduction for both strains, ≥6.1 log10CFU/abscess), but this killing was significantly diminished if the start of treatment was delayed until ≥12 h after inoculation. Imipenem concentrations in abscess tissue reached levels above the MIC forE. colifor >60% of the dosing interval. Possible reasons for the reduced activity of imipenem in vivo are discussed, and we conclude that standard susceptibility tests overestimate the efficacy of this antibiotic against the organisms present in these abscesses.
Ocular Distribution of Intravenously Administered Lipid Formulations of Amphotericin B in a Rabbit Model
Authors: Goldblum David , Rohrer Kaspar , Frueh Beatrice E. , Theurillat Regula , Thormann Wolfgang , Zimmerli Stefan
Published: November 2002

Little is known about the ocular penetration of amphotericin B (AMB) and its lipid formulations, the current drug of choice in fungal endophthalmitis. The ocular distribution of AMB lipid complex (ABLC), liposomal AMB (L-AMB), and AMB deoxycholate (D-AMB) was studied in a rabbit model. D-AMB (1 mg/kg of body weight/day), ABLC (5 mg/kg/day), or L-AMB (5 mg/kg/day) was given intravenously to rabbits as a single dose or as repeated daily doses on 7 consecutive days after induction of unilateral uveitis by intravitreal injection of endotoxin. AMB concentrations in aqueous humor, vitreous humor, and plasma were determined by high-pressure liquid chromatography 16 h after administration of a single dose or 24 h after the last of seven doses. After single-dose administration, L-AMB achieved at least eightfold-higher AMB concentrations in the aqueous of inflamed eyes than ABLC or D-AMB (1.21 ± 0.58 μg/ml versus 0.14 ± 0.04 and 0.11 ± 0.09 μg/ml, respectively). At that time point no drug was detectable in the vitreous. After 7 days of treatment, the concentration of AMB in the vitreous was higher after treatment with L-AMB (0.47 ± 0.21 μg/ml) than after treatment with ABLC (0.27 ± 0.18 μg/ml) and D-AMB (0.16 ± 0.04 μg/ml). Similarly, AMB concentration in the aqueous was higher after repeated doses of L-AMB (0.73 ± 0.43 μg/ml) than after repeated doses of ABLC (0.03 ± 0.02 μg/ml) or D-AMB (0.13 ± 0.06 μg/ml). No AMB was detected in noninflamed eyes. Following systemic administration, AMB distribution to the eye is inflammation dependent and occurs sequentially, first to the aqueous and then to the vitreous. Compared to D-AMB and ABLC, L-AMB reaches higher drug concentrations in both ocular compartments.
Target Site Concentrations of Ciprofloxacin after Single Intravenous and Oral Doses
Authors: Brunner Martin , Staβ Heino , Möller Jan-Georg , Schrolnberger Claudia , Erovic Boban , Hollenstein Ursula , Zeitlinger Markus , Eichler Hans Georg , Müller Markus
Published: November 2002

To characterize the potential of ciprofloxacin penetration into human soft tissues following intravenous (i.v.) and oral (p.o.) administration, we measured the free ciprofloxacin concentrations in interstitial space fluid of skeletal muscle and subcutaneous adipose tissue by microdialysis. In addition, ciprofloxacin concentrations were measured in cantharis-induced skin blisters, saliva, and capillary plasma and were compared to the total concentrations in venous plasma. Furthermore, a pharmacodynamic in vitro model was used to simulate in vivo pharmacokinetics in bacterial culture. Eight healthy volunteers received ciprofloxacin in an open randomized crossover fashion either as a single i.v. infusion of 400 mg over 60 min or as a single p.o. dose of 500 mg. For both tissues the mean areas under the concentration-time curves (AUCs) for interstitial space fluid (AUC
interstitial fluid
s) were significantly lower than the corresponding AUC
plasma
s, with AUC
interstitial fluid
/AUC
plasma
ratios ranging from 0.38 to 0.68. For skeletal muscle, the AUC
interstitial fluid
was significantly higher after administration of 400 mg i.v. than after administration of 500 mg p.o., with a ratio of the AUC after p.o. administration/AUC after i.v. administration of 0.64. The ratio of the concentration in skeletal muscle/concentration in plasma increased over the entire observation period, implying that ciprofloxacin concentrations were not at steady state. The ratio of the concentration in skin blister fluid/concentration in plasma reached values above 4, indicating a preferential penetration of ciprofloxacin into inflamed lesions. The concentrations in saliva and capillary blood were similar to the corresponding total levels in plasma. In vitro both in vivo ciprofloxacin concentration-time profiles were equally effective against select bacterial strains. In conclusion, single-dose administration of two bioequivalent dosage forms of ciprofloxacin might lead to differences in target site pharmacokinetics. These differences, however, are not related to a difference in target site pharmacodynamics.
Resistance to Pentamidine in
Leishmania mexicana
Involves Exclusion of the Drug from the Mitochondrion
Authors: Basselin Mireille , Denise Hubert , Coombs Graham H. , Barrett Michael P.
Published: November 2002

The uptake of [
3
H]pentamidine into wild-type and drug-resistant strains of
Leishmania mexicana
was compared. Uptake was carrier mediated. Pentamidine-resistant parasites showed cross-resistance to other toxic diamidine derivatives. A substantial decrease in accumulation of the drug accompanied the resistance phenotype, although the apparent affinity for pentamidine by its carrier was not altered when initial uptake velocity was measured. The apparent
V
max
, however, was reduced. An efflux of pentamidine could be measured in both wild-type and resistant cells. Only a relatively small proportion of the total accumulated pentamidine was available for efflux in wild-type cells, while in resistant cells the majority of loaded pentamidine was available for release. Pharmacological reagents which diminish the mitochondrial membrane potential reduced pentamidine uptake in wild-type parasites, and the mitochondrial membrane potential was shown to be reduced in resistant cells. A fluorescent analogue of pentamidine, 4′,6′-diamidino-2-phenylindole, accumulated in the kinetoplast of wild-type but not resistant parasites. These data together indicate that diamidine drugs accumulate in the
Leishmania
mitochondrion and that the development of the resistance phenotype is accompanied by lack of mitochondrial accumulation of the drug and its exclusion from the parasites.
Distribution of Extended-Spectrum β-Lactamases in Clinical Isolates of
Enterobacteriaceae
in Vietnam
Authors: Cao Van , Lambert Thierry , Nhu Duong Quynh , Loan Huynh Kim , Hoang Nguyen Kim , Arlet Guillaume , Courvalin Patrice
Published: November 2002

Among 730
Escherichia coli
, 438
Klebsiella pneumoniae
, and 141
Proteus mirabilis
isolates obtained between September 2000 and September 2001 in seven hospitals in Ho Chi Minh City, Vietnam, 26.6% were resistant to ceftazidime, 30% were resistant to cefotaxime, 31.5% were resistant to ceftriaxone, 15.9% were resistant to cefoperazone, and 6% were resistant to cefepime. Resistance to imipenem was found in 5.6% of the isolates. In 55 strains producing extended-spectrum β-lactamases (32
E. coli
isolates, 13
K. pneumoniae
isolates, and 10
P. mirabilis
isolates), structural genes for VEB-1 (25.5%), CTX-M (25.5%), SHV (38.1%), and TEM (76.3%) enzymes were detected alone or in combination. Sequencing of the PCR products obtained from the
K. pneumoniae
isolates revealed the presence of
bla
VEB-1
,
bla
CTX-M-14
,
bla
CTX-M-17
,
bla
SHV-2
, and
bla
TEM-1
. Molecular typing of the strains with a similar resistance phenotype to broad-spectrum cephalosporins indicated polyclonal spread. IS
Ecp1
was presumably responsible for dissemination of the
bla
CTX-M-like
gene.
Mosaic-Like Structure of Penicillin-Binding Protein 2 Gene (
penA
) in Clinical Isolates of
Neisseria gonorrhoeae
with Reduced Susceptibility to Cefixime
Authors: Ameyama Satoshi , Onodera Shoichi , Takahata Masahiro , Minami Shinzaburo , Maki Nobuko , Endo Katsuhisa , Goto Hirokazu , Suzuki Hiroo , Oishi Yukihiko
Published: November 2002

Neisseria gonorrhoeae
strains with reduced susceptibility to cefixime (MICs, 0.25 to 0.5 μg/ml) were isolated from male urethritis patients in Tokyo, Japan, in 2000 and 2001. The resistance to cephems including cefixime and penicillin was transferred to a susceptible recipient,
N. gonorrhoeae
ATCC 19424, by transformation of the penicillin-binding protein 2 gene (
penA
) that had been amplified by PCR from a strain with reduced susceptibility to cefixime (MIC, 0.5 μg/ml). The sequences of
penA
in the strains with reduced susceptibilities to cefixime were different from those of other susceptible isolates and did not correspond to the reported
N. gonorrhoeae penA
gene sequences. Some regions in the transpeptidase-encoding domain in this
penA
gene were similar to those in the
penA
genes of
Neisseria perflava
(
N. sicca
),
Neisseria cinerea
,
Neisseria flavescens
, and
Neisseria meningitidis
. These results showed that a mosaic-like structure in the
penA
gene conferred reductions in the levels of susceptibility of
N. gonorrhoeae
to cephems and penicillin in a manner similar to that found for
N. meningitidis
and
Streptococcus pneumoniae
.