bluuebunny/crossref_metadata_2025 huggingface dataset:
‘Gebranntes Kind’? W.G. Sebald's ‘Metaphysik Der Geschichte’
Authors: Jackman Graham
Published: September 2004

This essay investigates the relationship between Sebald's Luftkrieg und Literatur and the prose works on which his reputation primarily depends. Sebald's account of the bombing of German cities, particularly of Hamburg in 1943, reveals two significant intellectual affinities: first with Walter Benjamin, whose conception of history in Ursprung des deutschen Trauerspiels is linked through the ruins left by the bombing to Sebald's preoccupation with scenes of decay and dereliction in his fictional works, and secondly with Horkheimer and Adorno's critique of ‘progress’ in Dialektik der Aufklärung. Sebald sees the bombing campaign, along with the Holocaust, as part of the one great catastrophe of the Third Reich in which the history of ‘progress’ culminated – a history analysed at length in Sebald's Austerlitz. The ‘firestorm’ in Hamburg, emblematic of this destructive process, illustrates Sebald's recurrent use of the fire motif in his narrative works and is linked to comments on his own childhood in Nach der Natur. His entire oeuvre is at once an attempt to ‘memorialise’ individual victims of this destructive process and a critique of, and a resistance to, the tendency within post‐war German culture to complete the ‘liquidation’ of the past begun during the wartime bombing.
‘Weil Die Geschichte Nicht Aufhört’: Günter Grass's Im Krebsgang
Authors: Dye Elizabeth
Published: September 2004

W.G. Sebald's 1997 lectures, Luftkrieg und Literatur, sparked intense debate in the German media over the supposed taboo surrounding the literary depiction of German civilian victims of Allied attacks and the potential blurring of the already problematic ‘Täter’/‘Opfer’ complex. On its publication, Grass's most recent work Im Krebsgang either fuelled or became embroiled in this dispute and became front‐ page news. It is contended that such a media frenzy led to literary reviews at the time ignoring the aesthetic merits and failures of the novella and thus overlooking the complexities of its narratological structure, giving rise to over‐simplistic interpretations of Grass's contribution to the ‘Täter’/‘Opfer’ debate. The following article attempts to redress the journalistic imbalance by undertaking a detailed analysis of the various narrative strategies employed within the work and relating this to the generational and familial framework with which Grass addresses the issue of German suffering. This serves to illuminate further the topic of memory and memorialising the past which lies at the heart of Sebald's essay and of Grass's novella.
Kinetics of Ca2+ release by InsP3 in pig single aortic endothelial cells: evidence for an inhibitory role of cytosolic Ca2+ in regulating hormonally evoked Ca2+ spikes
Authors: Carter T. D. , Ogden D.
Published: September 2004

The role of the InsP3 receptor and its interaction with Ca2+ in shaping endothelial Ca2+ spikes was investigated by comparing InsP3‐evoked intracellular Ca2+ release with hormonally evoked Ca2+ spikes in single endothelial cells.

Ins P3 was generated by flash photolysis of intracellular caged InsP3. InsP3 at 0.2 μM or higher released Ca2+ from stores with a time course comprising a well‐defined delay, a fast rise of free [Ca2+] to a peak where net flux into the cytosol is zero, and a slow decline to pre‐flash levels. InsP3‐evoked Ca2+ flux into unit cytosolic volume was measured as the rate of change of free [Ca2+]i during the fast rise, d[Ca2+]i/dt (mol s−1 l −1).

The mean delay decreased from 433 ms at 0.2 μM to 30 ms at 5 μM. At very high InsP3 concentrations, 78 μM, the delay was shorter, < 10 ms. At low InsP3 concentration the delay was reduced by ∼30% by prior elevation of free [Ca2+]i, supporting a co‐operative action of free [Ca2+] and InsP3 in activation.

Both Ca2+ flux and peak free [Ca2+]i increased with InsP3 concentration within each cell. Maximal activation was at > 5 μM, 50% maximum Ca2+ flux was at 1.6 μM InsP3 and the Hill coefficient was between 3.6 and 4.3. A large variation of Ca2+ flux and peak [Ca2+]i was found from cell to cell at the same InsP3 concentration.

Strong inhibition of InsP3‐evoked flux was produced by an immediately preceding response, with complete inhibition at peak free [Ca2+]i due to the first pulse. InsP3 sensitivity returned over 1–2 min, with 50% recovery at ∼25 s. The recovery of InsP3 sensitivity may determine the minimum interval between hormonally evoked spikes.

Ca2+ flux due to a pulse of InsP3 terminated rapidly, in the continued presence of InsP3, producing a well‐defined peak [Ca2+]. A reciprocal relation was found between the duration and the rate of Ca2+ flux, such that high Ca2+ flux was of brief duration. The rate of termination of flux measured as the reciprocal of the 10–90% rise time of free [Ca2+]i showed a linear correlation with Ca2+ flux over a large range in all cells. A systematic deviation from linearity at low InsP3 concentration showed a greater rate of termination at low InsP3 concentration than at high for the same flux.

Elevating cytosolic free [Ca2+] by 0.1–2.5 μ M strongly inhibited Ca2+ release by InsP3, and buffering free [Ca2+] to low levels greatly prolonged Ca2+ release. Both results support the idea that Ca2+ flux quickly produces locally high free [Ca2+] which inhibits the receptor and terminates Ca2+ release.

Hormonally evoked Ca2+ spikes showed a similar reciprocal relation between rise time and Ca2+ flux, seen in the initial Ca2+ spike evoked by extracellular ATP in porcine aortic endothelial cells and by acetylcholine in rat aortic endothelial cells in situ, supporting the idea that the same mechanism of cytosolic Ca2+ inhibition determines the duration of hormonally and InsP3‐evoked Ca2+ spikes.
Relationship of substance P to afferent characteristics of dorsal root ganglion neurones in guinea‐pig
Authors: Lawson S. N. , Crepps B. A. , Perl E. R.
Published: September 2004

The relationship between the afferent properties and substance P‐like immunoreactivity (SP‐LI) of L6 and SI dorsal root ganglion (DRG) neuronal somata was examined in anaesthetized guinea‐pigs. Glass pipette microelectrodes filled with fluorescent dyes were used to make intracellular recordings and to label DRG somata. The dorsal root conduction velocity (CV) and the afferent receptive properties of each unit were categorized according to criteria established in other species. Categories included a variety of low threshold mechanoreceptive classes, innocuous thermoreceptive and several nociceptive classes. Nociceptive units were further subdivided on the basis of CV and the locus of the receptive field (superficial cutaneous, deep cutaneous or subcutaneous).

SP‐LI was determined using the avidin–biotin complex method and the relative staining intensity determined by image analysis. The possible significance of labelling intensity is discussed. Clear SP‐LI appeared in twenty‐nine of 117 dye‐labelled neurones. All SP‐LI positive units with identified receptive properties were nociceptive but not all categories of nociceptors were positive. The intensity of SP‐LI labelling varied, often systematically, in relation to afferent properties. There was a tendency for nociceptive neurones with slower CVs and/or smaller cell bodies to show SP‐LI.

Nineteen of fifty‐one C fibre neurones showed SP‐LI. Fewer than half the C polymodal nociceptors (CPMs) were positive. The most intensely labelled units were the deep cutaneous nociceptors and some of the CPMs in glabrous skin. C low threshold mechanoreceptors and cooling‐sensitive units did not show SP‐LI.

Ten of sixty‐six A fibre neurones exhibited SP‐LI, including eight of sixteen Aδ nociceptors and two of fifteen Aα/β nociceptors. A fibre neurones exhibiting SP‐LI included seven of eight deep cutaneous mechanical nociceptors and some superficial cutaneous mechano‐heat nociceptors of hairy skin. In contrast, none of twenty superficial cutaneous A high threshold mechanoreceptor units or the thirty‐five A fibre low threshold units (D‐hair and other units) showed detectable SP‐LI.

We conclude that SP‐LI labelling in guinea‐pig DRG neurones is related to (a) afferent receptive properties, (b) the tissue in which the peripheral receptive terminals are located, (c) the CV and (d) the soma size.
Glucose‐induced swelling in rat pancreatic β‐cells
Authors: Miley Helen E. , Sheader Elizabeth A. , Brown Peter D. , Best Leonard
Published: September 2004

Changes in relative cell volume in response to hypotonic solutions and glucose were studied in single isolated rat pancreatic β‐cells using a video‐imaging technique, β‐cell electrical activity was recorded under similar conditions using the perforated patch technique.

Exposure of β‐cells to hypotonic solutions (10 and 33% hypotonicity) caused an immediate increase in cell volume to relative values of 1.09 and 1.33, respectively. This was followed by a gradual regulatory volume decrease.

Raising the concentration of glucose from 4 to 20 mm or 12 mm (with substitution of mannitol) increased β‐cell volume by 12 and 10%, respectively. This effect of glucose persisted when Co2+ was added to inhibit insulin release. Glucose‐induced volume increases were sustained for the duration of exposure to elevated hexose concentration. The addition of 16 mm 3‐O‐methylglucose, which is transported into the β‐cell but not metabolized, produced only a transient 5% increase in β‐cell volume.

Exposure of β‐cells to a 15% hypotonic solution resulted in a transient depolarization and electrical activity. Raising the glucose concentration to 20 or 12 mm caused a sustained depolarization and generation of electrical activity. However, the addition of 16 mm 3‐O‐methylglucose had no effect on β‐cell membrane potential. The glucose‐induced increase in volume and induction of electrical activity, when measured in single β‐cells simultaneously, showed comparable kinetics.

The secretion of insulin from intact pancreatic islets was stimulated by exposure to hypotonic solutions (10–33% hypotonicity). A 15% hypotonic solution stimulated insulin release to a peak value comparable to that elicited by raising the glucose concentration from 4 to 20 mm. Whereas hypotonic solutions caused a transient stimulation of insulin release, the effect of glucose was sustained.

It is suggested that glucose increases the volume in rat pancreatic β‐cells by a mechanism dependent upon metabolism of the sugar. The extent of cell swelling evoked by raised glucose concentrations is sufficient to depolarize the cells and induce electrical and secretory activity and may involve activation of a volume‐sensitive anion conductance.
Outward Currents in Smooth Muscle Cells Isolated from Sheep Mesenteric Lymphatics
Authors: Cotton K. D. , Hollywood M. A. , McHale N. G. , Thornbury K. D.
Published: September 2004

The patch‐clamp technique was used to measure membrane currents in isolated smooth muscle cells dispersed from sheep mesenteric lymphatics. Depolarizing steps positive to −30 mV evoked rapid inward currents followed by noisy outward currents.

Nifedipine (1 μM) markedly reduced the outward current, while Bay K 8644 (1 μM) enhanced it. Up to 90% of the outward current was also blocked by iberiotoxin (Kd= 36 nM).

Large conductance (304 ±15 pS, 7 cells), Ca2+‐ and voltage‐sensitive channels were observed during single‐channel recordings on inside‐out patches using symmetrical 140 mM K+ solutions (at 37 °C). The voltage required for half‐maximal activation of the channels (V½) shifted in the hyperpolarizing direction by 146 mV per 10‐fold increase in [Ca2+]i.

In whole‐cell experiments a voltage‐dependent outward current remained when the Ca2+‐activated current was blocked with penitrem A (100 nM). This current activated at potentials positive to −20 mV and demonstrated the phenomenon of voltage‐dependent inactivation (V½=−41 ± 2 mV, slope factor = 18 ± 2 mV, 5 cells).

Tetraethylammonium (TEA; 30 mM) reduced the voltage‐dependent current by 75% (Kd= 3.3 mM, 5 cells) while a maximal concentration of 4‐aminopyridine (4‐AP; 10 mM) blocked only 40% of the current. TEA alone had as much effect as TEA and 4‐AP together, suggesting that there are at least two components to the voltage‐sensitive K+ current.

These results suggest that lymphatic smooth muscle cells generate a Ca2+‐activated current, largely mediated by large conductance Ca2+‐activated K+ channels, and several components of voltage‐dependent outward current which resemble ‘delayed rectifier’ currents in other smooth muscle preparations.
Small event Ca2+ release: a probable precursor of Ca2+ sparks in frog skeletal muscle
Authors: Shirokova Natalia , Ríos Eduardo
Published: September 2004

Fluo‐3 fluorescence associated with Ca2+ release was recorded with confocal microscopy in single muscle fibres. Clamp depolarization to −65 or −60 mV elicited Ca2+ sparks with amplitudes and spatial widths distributed approximately normally, with mean values of 0.79 of resting fluorescence and 0.8 μm (S.D., 0.17 and 0.2 μm; n= 193), respectively. Given these distributions, events of amplitude less than 0.45 or width less than 0.4 μm are unlikely to be sparks.

Low voltage depolarization (−72 mV) elicited only one spark per triad every 6 s, but generated a relative increase in fluorescence at triads of 0.05. This increase must therefore have been due to events smaller than sparks.

The variance/mean ratio of triadic fluorescence gradients averaged 0.11 at low voltages and increased severalfold at the higher voltages at which sparks appeared, indicating the existence of at least two event amplitudes.

Tetracaine (200 μm) reversibly abolished sparks and the early peak of Ca2+ release at all voltages. In its presence, discrete events were smaller than the spark criterion, and triadic gradients had a variance/mean ratio of 0.11.

The phenylalkylamine D600 (2 μm) reduced release at all voltages, abolishing sparks and the peak of Ca2+ release at low but not at high voltages.

The parallel abolition of all sparks and the peak of Ca2+ release indicates that both phenomena are activated by Ca2+. The restoration of sparks by voltage in D600 suggests that release in small events provides the trigger Ca2+ for activation of sparks.
Calcium Sparks and Excitation–Contraction Coupling in Phospholamban‐Deficient Mouse Ventricular Myocytes
Authors: Santana L. F. , Kranias E. G. , Lederer W. J.
Published: September 2004

We examined [Ca2+]i and L‐type Ca2+ channel current (ICa) in single cardiac myocytes to determine how the intracellular protein phospholamban (PLB) influences excitation‐contraction (E–C) coupling in heart. Wild type (WT) and PLB‐deficient (KO) mice were used. Cells were patch clamped in whole–Cell mode while [Ca2+]i was imaged simultaneously using the Ca2+ indicator fluo‐3 and a confocal microscope.

Although ICa was similar in magnitude, the decay of ICa was faster in KO than in WT cells and the [Ca2+]i transient was larger and decayed faster. Furthermore, the E–C coupling ‘gain’ (measured as Δ[Ca2+]i/ICa) was larger in KO cells than in WT cells.

Spontaneous Ca2+ sparks were three times more frequent and larger in KO cells than in WT myocytes but, surprisingly, the time constants of decay were similar.

SR Ca2+ content was significantly greater in KO than in WT cells. When the SR Ca2+ content in KO cells was reduced to that in WT cells, Ca2+ sparks in these ‘modified’ (KO') cells decayed faster. E–C coupling gain, [Ca2+]i transient amplitude and the kinetics of decay of ICa were similar in KO' and WT cells.

We conclude that SR Ca2+ content influences (1) ICa, (2) the amplitude and kinetics of Ca2+ sparks and [Ca2+]i transients, (3) the sensitivity of the RyRs to triggering by [Ca2+]i, (4) the amount of Ca2+ released, (5) the magnitude of the E–C coupling ‘gain’ function, and (6) the rate of Ca2+ re‐uptake by the SR Ca2+‐ATPase. In KO cells, the larger [Ca2+]i transients and Ca2+ sparks speed up ICa inactivation. Finally, we conclude that PLB plays an important regulatory role in E–C coupling by modulating SR Ca2+‐ATPase activity, which establishes the SR Ca2+ content and consequently influences the characteristics of local and global Ca2+ signalling.
The Relationship between Cutaneous C Fibre Type and Antidromic Vasodilatation in the Rabbit and the Rat
Authors: Gee M. D. , Lynn B. , Cotsell B.
Published: September 2004

Skin blood flow was monitored during antidromic stimulation of identified cutaneous C fibres in fine filaments dissected from the saphenous nerve of anaesthetized rabbits and rats. The techniques used to monitor skin blood flow were laser Doppler perfusion imaging and laser Doppler flowmetry.

In the rabbit filaments a total of thirty‐three C fibres were tested for their ability to produce antidromic vasodilatation. The only C fibres found to have vasodilator actions were of the polymodal nociceptor afferent class, and fourteen (50%) of the twenty‐eight polymodal nociceptor units tested were vasoactive. The afferent receptive fields of polymodal nociceptor afferents were mapped carefully using suprathreshold mechanical stimuli, and there was a good correlation between afferent receptive field area and area of vasodilatation.

In the rat, eleven of the fifty‐four C fibres antidromically stimulated had vasodilator actions. All eleven vasoactive C fibres were nociceptive and comprised seven polymodal nociceptor units, two heat nociceptor units and two incompletely classified nociceptor units. The area of increased blood flow was always coincident with the afferent field of the stimulated unit.

In the rat the vasodilator units were not evenly distributed over the saphenous nerve receptive field. Nine of the eleven vasoactive C fibres had receptive fields located on the foot or the digits, and only two were on the ankle or lower leg. Overall, the population of nociceptive C fibres was evenly distributed over the saphenous nerve receptive field.

In both the rabbit and the rat, a subclass of polymodal nociceptor afferents form the majority of the vasoactive units and will make the main contribution to axon reflex flare and other neurogenic inflammatory responses involving vasodilatation. The vasoactive polymodal nociceptor units tend to have relatively low mechanical sensitivity, although they have typical heat thresholds. In the rat heat nociceptor units also have vasodilator actions. However, such heat nociceptor units form a minor functional class of afferent C fibre in the rat saphenous nerve, and are not found in the rabbit saphenous nerve.

The findings from this study in the rabbit and the rat are compared with the situation in pig skin. The close relationship between afferent receptive field area and spread of flare across species is noted, and the way these measures increase with body size is discussed.
Electrogenic K+ transport by the Na+–K+ pump in rat cardiac ventricular myocytes
Authors: Peluffo R. Daniel , Berlin Joshua R.
Published: September 2004

The involvement of electrogenic reaction steps in K+ transport by the Na+,K+‐ATPase was determined in rat cardiac ventricular myocytes using whole‐cell patch clamp techniques.

Under K+–K+ exchange conditions and in the presence of extracellular K+ or Tl+ at concentrations that stimulated submaximal levels of steady‐state Na+,K+‐ATPase activity, ouabain‐sensitive transient currents were observed during (‘on’) and after (‘off’) step changes in membrane potential (Vm).

The quantity of charge moved during the transient currents depended, in a saturable manner, on the magnitude of the voltage step. Maximal ouabain‐sensitive ‘on’ and ‘off’ charges were calculated to be 9.6 ± 0.9 and 9.1 ± 0.4 fC pF−1 (n= 4), respectively, with an effective valency of 0.48 ± 0.07 (n= 7).

Kinetics of the transient currents were independent of Vm and Tlo+ at positive potentials, but became more rapid at increasingly negative Vm values in an ion concentration‐dependent fashion.

These data demonstrate that electrogenic steps participate in K+ transport by the Na+,K+‐ATPase and that the electrogenic step is extracellular ion binding.

The temperature‐and Vm‐dependent properties of transient charge movements were compared under K+–K+ and Na+–Na+ exchange conditions. The data suggest that extracellular K+ and Na+ binding occur at different sites in the enzyme or to different enzyme conformations. The sum of the effective valencies, 1.14 ± 0.12, demonstrates that the electrogenicity of extracellular ion binding can explain the Vm dependence of ion transport by the Na+,K+‐ATPase.
Opposing effects of phorbol esters on transmitter release and calcium currents at frog motor nerve endings
Authors: Redman R.S. , Searl T.J. , Hirsh J.K. , Silinsky E.M.
Published: September 2004

Phorbol esters activate protein kinase C (PKC) and also increase the secretion of neuro‐transmitter substances by an unknown mechanism. To evaluate whether the stimulatory effects of such agents on acetylcholine (ACh) secretion occur as a consequence of stimulation of Ca2+ entry, we made electrophysiological measurements of ACh secretion (i.e. endplate potentials, EPPs) and the component of the prejunctional perineural voltage change associated with nerve terminal calcium currents (perineural calcium current) at frog neuro‐muscular junctions.

In the first series of experiments, modest concentrations of K+ channel blockers were employed so that simultaneous measurements of EPP amplitudes and perineural calcium currents could be made. In these experiments, 12‐O‐tetradecanoylphorbol 13‐acetate (TPA; 162 nm) and phorbol 12,13‐dibutyrate (PDBu; 100‐200 nm) each increased ACh release but simultaneously decreased the calcium component of the prejunctional perineural current. TPA and PDBu also inhibited perineural calcium currents in the presence of higher concentrations of K+ channel blockers.

Blockade of Ca2+ channels by Cd2+ prevented the action of PKC stimulators on perineural waveforms.

The inactive compound 4‐α‐phorbol 12‐myristate 13‐acetate (150 nm) did not affect EPP amplitudes or perineural currents.

The extracellular [Ca2+]‐ACh release relationship was increased in maximum by PDBu without any change in the potency of Ca2+ to support evoked ACh release.

The results demonstrate that phorbol esters increase neurotransmitter secretion whilst simultaneously decreasing the nerve ending calcium currents that promote evoked release. The results, which suggest that the optimal control point for secretion might not be the calcium channel but rather a component of the secretory apparatus, are discussed in conjunction with the possible target sites for phorbol esters in the nerve ending.
Selective Enhancement of the Slow Component of Delayed Rectifier K+ Current in Guinea‐Pig Atrial Cells by External ATP
Authors: Matsimra Hiroshi , Ehara Tsuguhisa
Published: September 2004

The effects of external ATP on the rapidly and slowly activating components (IKr and IKg, respectively) of the delayed rectifier K+ current (IK) in guinea‐pig atrial myocytes were determined using the whole‐cell configuration of the patch‐clamp technique.

An envelope of tails test was conducted by applying depolarizing pulses to +40 mV from a holding potential of −40 mV for various durations between 50 ms and 2 s under control conditions and during exposure to 50 μM ATP. The ATP‐induced IK, obtained by digital subtraction, exhibited a constant ratio (0.37) of the tail current to time‐dependent current, regardless of the pulse duration. This current ratio was compatible with the predicted ratio of the driving force at +40 and −40 mV for a non‐rectifying K+ conductance, suggesting that the ATP‐induced IK is due primarily to IKs.

The amplitude of IKr isolated from the IK enhanced by ATP, determined as an E‐4031 (5 μM)‐sensitive current, was similar to the control magnitude of IKr, thus showing that external ATP did not cause an increase in IKr.

The voltage‐dependent activation of the ATP‐induced IK during 500 ms depolarizing test pulses could be described by a Boltzmann equation with a half‐activation voltage (V1/2) of 11.5 mV and slope factor (k) of 12.0 mV, which were close to those of IKg(V1/4 of 12.1 mV and k of 12.3 mV), determined as an E‐4031‐resistant IK, under the same isochronal (500 ms) activation conditions.

These results provide evidence to suggest that extracellular ATP selectively potentiates the slow component of IK (IKg), with no measurable effects on IKr, in guinea‐pig atrial myocytes.
Photodynamic triggering of calcium oscillation in the isolated rat pancreatic acini
Authors: Cui Zong Jie , Kanno Tomio
Published: September 2004

Photodynamic agents, due to their photon‐dependent selective activation, can selectively activate a number of physiological processes and may directly modulate signal transduction in a number of cells including pancreatic acinar cells.

Activation of the photodynamic agent sulphonated aluminium phthalocyanine (SALPC) triggered recurrent cytosolic calcium ([Ca2+]i) spiking in pancreatic acinar cells.

The photodynamically triggered calcium spiking could be blocked by phosphatidylinositol‐specific phospholipase C (PI‐PLC) inhibitor U73122, but not by phosphatidylcholine‐specific phospholipase C inhibitor D609.

Removal of extracellular Ca2+ abolished spiking, as did 2‐aminoethoxydiphenylborate (2‐APB), an inhibitory modulator of IP3‐mediated Ca2+ release from intracellular stores.

These data suggest that SALPC photodynamic action may permanently fix PI‐PLC in an active conformation, and this produced recurrent [Ca2+]i spiking.
Developmental changes in isolated rat type I carotid body cell K+ currents and their modulation by hypoxia
Authors: Hatton C. J. , Carpenter E. , Pepper D. R. , Kumar P. , Peers C.
Published: September 2004

Whole‐cell patch‐clamp recordings were used to investigate possible age‐related changes in K+ currents of type I carotid body cells isolated from the rat. K+ current density increased with age, as measured in cells isolated from 4‐day‐old, 10‐day‐old and adult rats (≥ 5 weeks old).

The proportion of current reversibly inhibited by high [Mg2+] (6 mm), low [Ca2+] (0.1 mm) solutions, indicative of the proportion of current attributable to activation of Ca2+‐sensitive K+ (KCa) channels, was significantly smaller in cells of 4‐day‐old rats compared with 10‐dayold rats, despite inward Ca2+ current densities being similar in these two age groups. Inhibition of K+ currents by high [Mg2+], low [Ca2+] solutions was similar in 10‐day‐old and adult type I cells.

Hypoxia (PO2, 16–23 mmHg) caused reversible reductions in type I cells from rats of all age groups. However, reductions seen in cells of 4‐day‐old rats were significantly smaller than those seen in cells of 10‐day‐olds and adults. The degree of hypoxic inhibition in these latter two groups was not significantly different.

In the presence of high [Mg2+], low [Ca2+] solutions, hypoxia (PO2, 16–23 mmHg) was without significant effect on residual K+ currents in cells from all age groups.

These observations indicate that K+ current density increases with postnatal age in the rat. Between days 4 and 10, there appears to be a predominant enhancement of KCa channels, and over the same age range hypoxic sensitivity of K+ currents increases. Our findings demonstrate that this latter observation arises because hypoxia selectively inhibits KCa channels in cells at all ages studied. These results suggest an important role for KCa channels in postnatal maturation of hypoxic chemoreception in the rat carotid body.
Modulation of Ca2+‐activated Cl− currents in rabbit portal vein smooth muscle by an inhibitor of mitochondrial Ca2+ uptake
Authors: Greenwood I. A. , Helliwell R. M. , Large W. A.
Published: September 2004

The effects of carbonyl cyanide m‐chlorophenyl hydrazone (CCCP), an inhibitor of mitochondrial Ca2+ uptake, was investigated on the properties of Ca2+‐activated chloride currents (ICl(Ca)) in rabbit portal vein smooth muscle cells using the perforated patch whole‐cell voltage‐clamp technique to ascertain whether this Ca2+ uptake process influences the time course of the subsarcolemmal Ca2+ signal that activates ICl(Ca).

In cells bathed in either physiological calcium (2 mm Ca2+o) or high calcium (10 mm Ca2+o) external solutions, application of CCCP (1–2 μm) evoked an inward current and prolonged the exponential decay time constant (τ) of Ca2+‐activated Cl−‘tail’ currents (Itall) evoked by Ca2+ influx through voltage‐dependent calcium channels (VDCCs). The effect of CCCP on τ was greater in cells where the amplitude of Itall was relatively large and, in different cells, the effect of CCCP on T was positively correlated with the amplitude of Itall.

CCCP abolished spontaneously occurring transient Ca2+‐activated Cl− currents (STICs), but did not alter their time course before complete block.

Thapsigargin and cyclopiazonic acid (inhibitors of the sarcoplasmic Ca2+‐ATPase) inhibited STICs, but did not affect the decay of Itall or STICs.

In conclusion, when Ca2+ enters the cell through VDCCs, the time course of the consequent Ca2+ signal in the subsarcolemmal domain containing Ca2+‐activated chloride channels appears to be regulated by Ca2+ uptake into mitochondria. In contrast, inhibition of Ca2+ uptake by the sarcoplasmic reticulum ATPase does not seem to influence the time course of ICl(Ca).
Volume‐activated DIDS‐sensitive whole‐cell chloride currents in trout red blood cells
Authors: Egée Stéphane , Harvey† Brian J. , Thomas Serge
Published: September 2004

The nystatin‐perforated whole‐cell recording mode of the patch‐clamp technique was used to investigate the membrane conductance of trout (Oncorhynchus mykiss) red blood cells in the steady state, 5 min after exposure to hyposmotic medium and 10 min after return to normal isosmotic medium.

Whole‐cell I–V relations showed outward rectification when red blood cells were bathed in isosmotic (320 mosmol l−1) saline solution and the patch pipette was filled with 117 mm KCl. The membrane conductance was 2.58 ± 0.59 nS (number of experiments, n=18) between 0 and 100 mV and 1.32 ± 0.19 nS (n=18) between 0 and −100 mV. Removal of Cl− from the extracellular side or incubation with the CL−1 channel blocker DIDS caused a reduction in whole‐cell membrane conductance by more than 50%, indicating that the membrane current was generated by Cl− ions. The remaining conductance was voltage independent and probably due to non‐selective cation conductance.

The membrane conductance increased approximately 2‐fold after cell swelling induced by exposure to hyposmotic saline solution (215 mosmol I1). This effect was abolished in Cl−1‐free hyposmotic medium or in the presence of DIDS.

The return to isosmotic solution produced a fall in membrane conductance to, or below, control values.

We conclude that trout red blood cells possess a significant Cl− conductance in the steady state which is reversibly activated during cell swelling and contributes to volume recovery.
Tachykinin‐induced activation of non‐specific cation conductance via nk3 neurokinin receptors in guinea‐pig intracardiac neurones
Authors: Hardwick Jean C. , Mawe Gary M. , Parsons Rodney L.
Published: September 2004

Whole mount preparations from guinea‐pig hearts were used to characterize the receptors and ionic mechanisms mediating the substance P (SP)‐induced depolarization of para‐sympathetic postganglionic neurones of the cardiac ganglion.

Measurement of the amplitude of depolarization in response to superfusion of different tachykinin agonists (neurokinins A (NKA) and B (NKB), SP, and senktide) gave a rank‐order potency of NKB=senktide > NKA > SP, indicating involvement of an NK3 receptor. The use of the selective tachykinin receptor antagonists SR 140333, SR 48986, and SR 142801 demonstrated that only the NK3 receptor antagonist SR 142801 inhibited the SP‐induced depolarization.

The SP‐induced depolarization was not inhibited by Ba2+, TEA, or niflumic acid, or altered by reduced Cl− solutions, but was attenuated in reduced Na+ solutions. Single electrode voltage clamp studies demonstrated that the SP‐induced inward current increased in amplitude at more negative potentials, had a reversal potential of approximately 0 mV, and was reduced in amplitude in reduced Na+ solutions.

We conclude that the SP‐induced depolarization in guinea‐pig postganglionic parasympathetic neurones of the cardiac ganglion is due to NK3‐mediated activation of a non‐selective cation conductance.
Fractional Ca2+ Currents through Capsaicin‐ and Proton‐Activated Ion Channels in Rat Dorsal Root Ganglion Neurones
Authors: Zeilhofer Hanns Ulrich , Kress Michaela , Swandulla Dieter
Published: September 2004

Capsaicin and protons cause excitation and sensitization of primary nociceptive afferents. In a subset of dorsal root ganglion (DRG) neurones, which probably represent nociceptive neurones, both capsaicin and protons induce slowly inactivating non‐selective cation currents. Whole‐cell as well as single channel currents activated by these two stimuli share many biophysical and physiological properties in these neurones. This has lead to the suggestion that protons and capsaicin might activate the same ion channels.

In this study we simultaneously measured fluorescence signals and whole‐cell currents activated by capsaicin or protons in acutely isolated DRG neurones filled with a high concentration (1 mM) of the Ca2+ indicator dye fura‐2. From these measurements the fractional contribution of Ca2+ (Pf; the portion of the whole‐cell current carried by Ca2+) to capsaicin‐ and two types of proton‐induced (fast and slowly inactivating) membrane currents was determined.

Capsaicin‐ and slowly inactivating proton‐induced currents were accompanied by a change in fluorescence that was dependent on the presence of extracellular Ca2+. With 1.6 mM extracellular Ca2+ and at a holding potential of −80 mV Pf of capsaicin‐induced currents (at pH 7.3) was 4.30 ± 0.17% (mean ±s.e.m.; no. of experiments, n= 16) and of slowly inactivating proton‐induced currents (at pH 5.1) was 1.65 ± 0.11% (n= 17). Pf of fast inactivating proton‐induced currents was negligible.

P
f of capsaicin‐ and slowly inactivating proton‐induced currents increased with increasing extracellular Ca2+ concentration (0.5–4.8 mM).

P
f of both current types decreased linearly with decreasing extracellular pH by about 0.7% per pH unit over the pH range investigated. When determined at the same extracellular pH Pf values were significantly different for the two current types at all pH values tested.

In summary, our results provide evidence that capsaicin and protons activate ion channels which are markedly permeable to Ca2+. The fractional contribution of Ca2+, however, was significantly different for capsaicin‐ and slowly inactivating proton‐induced currents. This strongly suggests that the two stimuli activate different populations of ion channels and supports the possibility that Ca2+ influx through these channels may be important for Ca2+‐dependent sensitization of primary nociceptive neurones.
Modulation by adenosine of GABA‐activated current in rat dorsal root ganglion neurons
Authors: Hu Hong‐Zhen , Li Zhi‐Wang
Published: September 2004

The modulation by adenosine of GABA‐activated current (IGABA) was studied in freshly isolated rat dorsal root ganglion (DRG) neurons using the whole‐cell patch‐clamp technique.

In most of the DRG neurons examined (68/90, 75.5%) adenosine (1–100 μm) suppressed Igaba, while in some neurons examined, it potentiated (16/90, 17.8%) Igaba. It exerted no effects on Igaba in a few cells (6/90, 6.7%).

Adenosine shifted the GABA concentration–response curve downward with no significant change of the EC50. The maximal response to GABA was suppressed by 29.6 ± 2.6%. The adenosine‐induced inhibition of Igaba showed no voltage dependence.

8‐Cyclopentyl‐1,3‐dimethylxanthine (DPCPX; 1 μm), a selective A1 adenosine receptor antagonist, partially reversed adenosine inhibition of Igaba and completely blocked N6‐cyclo‐hexyladenosine (CHA; an A1 adenosine receptor agonist) inhibition of IGABA. DPCPX (1 μm) also blocked the suppression of Igaba by 2‐chloroadenosine (CADO). CGS 21680, a selective A2A adenosine receptor agonist, did not inhibit Igaba and DMPX, a selective A2A adenosine receptor antagonist, did not prevent adenosine inhibition of Igaba.

Intracellular application of H‐7 (20 μm; a protein kinase C inhibitor) reversed adenosine inhibition of Igaba while inclusion of cAMP (1 mm), H‐9 (20 μm; a protein kinase A inhibitor) and BAPTA (10 mm; a chelator of calcium ions) in the recording pipette did not affect the depression of Igaba by adenosine. Igaba was also suppressed by internal perfusion of PMA, a protein kinase C activator.

The results suggest that adenosine, as a neuromodulator, exerts a modulatory effect on the GABA‐induced presynaptic inhibition in primary sensory transmission.
Chloride conductance in mouse muscle is subject to post‐transcriptional compensation of the functional Cl− channel 1 gene dosage
Authors: Chen Mei‐fang , Niggeweg Ricarda , Iaizzo Paul A. , Lehmann‐Horn Frank , Jockusch Harald
Published: September 2004

In mature mammalian muscle, the muscular chloride channel ClC‐1 contributes about 75% of the sarcolemmal resting conductance (Gm). In mice carrying two defective alleles of the corresponding Clc1 gene, chloride conductance (GCl) is reduced to less than 10% of that of wild‐type, and this causes hyperexcitability, the salient feature of the disease myotonia. Potassium conductance (GK) values in myotonic mouse muscle fibres are lowered by about 60% compared with wild‐type.

The defective Clcadr allele causes loss of the 4.5 kb ClC‐1 mRNA. Mice heterozygous for the defective Clc1adr allele contain about 50% functional mRNA in their muscles compared with homozygous wild‐type mice.

Despite a halved functional gene dosage, heterozygous muscles display an average GCl which is not significantly different from that of homozygous wild‐type animals. The GK values in heterozygotes are also indistinguishable from homozygous wild‐type animals.

These results indicate that a regulatory mechanism acting at the post‐transcriptional level limits the density of ClC‐1 channels. GK is probably indirectly regulated by muscle activity.
ATP‐dependent desensitization of the muscarinic K+ channel in rat atrial cells
Authors: Shui Z. , Boyett M. R. , Zang W.‐J.
Published: September 2004

Fast desensitization of the muscarinic K+ channel has been studied in excised patches from rat atrial cells.

In inside‐out patches, ACh was present in the pipette and GTP was applied via the bath to activate the channel. In outside‐out patches, GTP was present in the pipette and ACh was applied via the bath to activate the channel. In both cases, during a 30 s exposure to GTP or ACh there was a decline in channel activity as a result of fast desensitization if ATP was present.

In inside‐out patches, fast desensitization was still observed if the muscarinic ACh receptor was bypassed and the channel was activated by GTPγS. This suggests that fast desensitization is a result of a modification of the channel (or the connecting G protein) and not the receptor.

In both inside‐out and outside‐out patches, channel activity was depressed and fast desensitization was reduced or absent, if ATP was not present.

The non‐hydrolysable analogue of ATP, AMP‐PNP, did not substitute for ATP in its effects on the channel.

The results are consistent with the hypothesis that fast desensitization of the muscarinic K+ channel is the result of a dephosphorylation of the channel.
Lack of Regulation by Intracellular Ca2+ of the Hyper Polarization‐Activated Cation Current in Rat Thalamic Neurones
Authors: Budde Thomas , Biella Gerardo , Munsch Thomas , Pape Hans‐Christian
Published: September 2004

The regulation of the hyperpolarization‐activated cation current, Ih, in thalamocortical neurones by intracellular calcium ions has been implemented in a number of mathematical models on the waxing and waning behaviour of synchronized rhythmic activity in thalamocortical circuits. In the present study, the Ca2+ dependence of Ih in thalamocortical neurones was experimentally investigated by combining Ca2+ imaging and patch‐clamp techniques in the ventrobasal thalamic complex (VB) in vitro.

Properties of Ih were analysed before and during rhythmic stimulation of Ca2+ entry by trains of depolarizing voltage pulses. Despite a significant increase in intracellular Ca2+ concentration ([Ca2+]i) from resting levels of 74 ± 23 nM to 251 ± 78 nM upon rhythmic stimulation, significant differences in the voltage dependence of Ih activation did not occur (half‐maximal activation at −86.4 ± l.3 mV vs.−85.2 ± 2.9 mV; slope of the activation curve, 11.2 ± 2.4 mV vs. 12.5 ± 2.5 mV). Recording of Ih with predefined values of [Ca2+]i (13.2 nM or 10.01 μm in the patch pipette) revealed no significant differences in the activation curve or the fully activated I–V relationship of Ih.

In comparison, stimulation of the intracellular cyclic adenosine monophosphate (cAMP) pathway induced a significantly positive shift in Ih voltage dependence of +5.1 ± l.9 mV, with no alteration in the fully activated I–V relationship.

These data argue against a direct regulation of Ih by intracellular Ca2+, and particularly do not support a primary role of Ca2+‐dependent modulation of the Ih channels in the waxing and waning of sleep spindle oscillations in thalamocortical neurones.
Stimulation‐induced changes in [ca2+] in lizard motor nerve terminals
Authors: David Gavriel , Barrett John N. , Barrett Ellen F.
Published: September 2004

Motor axons were injected ionophoretically with one of five Ca2+‐sensitive dyes (fluo‐3, Calcium Green‐2, Calcium Green‐5N, fluo‐3FF and Oregon Green BAPTA‐5N). Changes in fluorescence (ΔF/Frest) within motor terminal boutons following a single action potential and brief stimulus trains were monitored with high temporal resolution using a confocal microscope.

Stimulation‐induced increases in ΔF/Frest were confined primarily to boutons, with roughly uniform increases in all the boutons of a terminal. The increase in ΔF/Frest began prior to, and decayed more slowly than, the endplate potential (EPP) recorded in the underlying muscle fibre. ΔF/Frest was graded with bath [Ca2+]. Both ΔF/Frest and the EPP were reduced, but not eliminated, by ω‐conotoxin GVIA (5–10 μm).

For dyes with lower affinity for Ca2+ (e.g. Oregon Green BAPTA‐5N, Kd≈ mm) stimulation‐induced increases in ΔF/Frest were measured in the presence of the K+ channel blocker 3,4‐diaminopyridine (3,4‐DAP, 100 mm). During brief stimulus trains (4 at 50 Hz) in 3,4‐DAP, the EPP exhibited profound depression, but the fluorescence increase associated with each stimulus showed little decrement, suggesting that depression was not mediated by a reduction in Ca2+ entry.

For dyes with a higher affinity for Ca2+ (e.g. fluo‐3, Kd≈ 0.5–1 μm) stimulation‐induced increases in ΔF/Frest could also be measured in normal physiological saline. Increases in ΔF/Frest were much greater with 3,4‐DAP present, but the amplitude decreased with successive stimuli due to partial dye saturation.

Calculations suggested that following a single action potential the average [Ca2+] within a bouton increased by up to 150 nm in normal saline and 940 nM in 3,4‐DAP. With low affinity dyes the ΔF/Frest measured near the membrane had a higher peak amplitude and a faster early decay than that measured in the centre of the bouton, suggesting that substantial spatial [Ca2+] gradients exist within boutons for at least 15 ms following stimulation.
Neurotensin Inhibition of the Hyperpolarization‐Activated Cation Current (Ih) in the Rat Substantia Nigra Pars Compacta Implicates the Protein Kinase C Pathway
Authors: Cathala L. , Paupardin‐Tritsch D.
Published: September 2004

Whole‐cell patch‐clamp recording was performed from principal neurones of the substantia nigra pars compacta (SNc). In 66% of these neurones, neurotensin (NT) induced, at −60 mV, an inward current associated with an increase in conductance.

Principal neurones displayed, in response to hyperpolarizing voltage steps, the voltage‐dependent inward cationic current, Ih. This current activated at potentials more negative than −65 mV and reached a maximum at −106 ± 4 mV, with a half‐activation potential of −86 ± 3 mV. Its estimated reversal potential was −43 ± 7 mV and its activation curve was fitted with two exponentials.

In 41 % of neurones showing the inward current, NT (0.5 μM) also reversibly reduced the amplitude of Ih. The diminution was 48.5 ± 12% when voltage steps were made from −60 to −95 mV. The decrease in Ih resulted from a reduction in the maximal current with no change in the voltage dependence of activation.

Forskolin (10 μM), an activator of adenylate cyclase, increased Ih by shifting its activation range to more positive potentials, but it did not alter the NT inhibition of Ih.

The effect of NT was blocked by staurosporine (0.5 μM) and by PKC‐(19–31) (0.5 μM), a specific protein kinase C (PKC) inhibitor, but was unaffected by Walsh's peptide (100 μM), a specific inhibitor of protein kinase A. The reduction of Ih was mimicked by 1‐oleoyl‐2‐acetyl‐sn‐glycerol (0.5–10 μM), an analogue of diacylglycerol, an endogenous PKC activator.

These results suggest that the inhibition of Ih by NT involves a phosphorylation mechanism that implies activation of PKC.
Reduced outward K+ conductances generate depolarizing after–potentials in rat supraoptic nucleus neurones
Authors: Li Zhenhuii , Hatton Glenn I.
Published: September 2004

Whole‐cell patch clamp recordings were obtained from sixty‐five rat supraoptic nucleus (SON) neurones in brain slices to investigate ionic mechanisms underlying depolarizing after‐potentials (DAPs). When cells were voltage clamped around ‐58 mV, slow inward currents mediating DAPs (Idap), evoked by three brief depolarizing pulses, had a peak of 17 ± 1 pA (mean ± s.e.m.) and lasted for 2.8 ± 0.1 s.

No significant differences in the amplitude and duration were observed when one to three preceding depolarizing pulses were applied, although there was a tendency for twin pulses to evoke larger Idap than a single pulse. The Idap was absent when membrane potentials were more negative than ‐70 mV. In the range ‐70 to ‐50 mV, Idap amplitudes and durations increased as the membrane became more depolarized, with an activation threshold of ‐65.7 ± 0.7 mV.

I

dap
with normal amplitude and duration could be evoked during the decay of a preceding Idap. As frequencies of depolarizing pulses rose from 2 to 20 Hz, the times to peak Idap amplitude were reduced but the amplitudes and durations did not change.

A consistent reduction in membrane conductance during the Idap was observed in all SON neurones tested, and averaged 34.6 ± 3.3%. Small hyperpolarizing pulses used to measure membrane conductances appeared not to disturb major ionic mechanisms underlying Idap, since the slope and duration of Idap with and without test pulses were similar.

The Idap had an averaged reversal potential of ‐87.4 ± 1.6 mV, which was close to the K+ equilibrium potential. An elevation in [K+]O reduced or abolished the Idap, and shifted its reversal potential toward more positive levels. Perifusion of slices with 7.5–10 mm TEA, a K+ channel blocker, reversibly suppressed the Idap.

Both Na+ and Ca2+ currents failed to induce an Idap‐like current during perifusion of slices with media containing high [K+]o or TEA. However, the Idap was abolished by replacing external Ca2+ with Co2+, or replacing 82% of external Na+ with choline or Li+. Perifusion of slices with media containing 1–2 μm TTX also reduced Idap by 55.5 ± 9.0 %.

These results suggest that the generation of DAPs in SON neurones mainly involves a reduction in outward K+ current(s), which probably has little or no inactivation and can be inhibited by [Ca2+]i transients, due to Ca2+ influx during action potentials and Ca2+ release from internal stores. Na+ influx might provide a permissive influence for Ca2+‐induced reduction of K+ conductances and/or help to raise [Ca2+]i via reverse‐mode Ca2+‐Na+ exchange. Other conductances, making minor contributions to the Idap, may also be involved.