bluuebunny/crossref_metadata_2025 huggingface dataset:
Paired‐pulse magnetic stimulation of the human motor cortex: differences among I waves
Authors: Hanajima Ritsuko , Ugawa Yoshikazu , Terao Yasuo , Sakai Katsuyuki , Furubayashi Toshiaki , Machii Katsuyuki , Kanazawa Ichiro
Published: September 2004

In paired‐pulse cortical stimulation experiments, conditioning subthreshold stimuli suppress the electromyographic (EMG) responses of relaxed muscles to suprathreshold magnetic test stimuli at short interstimulus intervals (ISIs) (1‐5 ms) and facilitate them at long ISIs (8‐15 ms).

We made paired‐pulse magnetic stimulation studies on the response of the first dorsal interosseous muscle (FDI) produced by I1 or I3 waves using our previously reported method which preferentially elicits one group of I waves when subjects make a slight voluntary contraction. In some experiments the conditioning and test stimuli were oppositely directed, in the others they were oriented in the same direction. Single motor unit responses were recorded with a concentric needle electrode, and surface EMG responses with cup electrodes.

In post‐stimulus time histograms (PSTHs) of the firing probability of motor units, the peaks produced by I3 waves were decreased by a subthreshold conditioning stimulus that preferentially elicited I1 or I3 waves at an ISI of 4 ms. The amount of decrement depended on the intensity of the conditioning stimulus. The stronger the conditioning stimulus, the greater the suppression. In contrast, the peaks produced by I1 waves were little affected by any type of subthreshold conditioning stimulus, given 4 ms prior to the test stimulus. At an ISI of 10 ms, a subthreshold conditioning stimulus slightly decreased the size of the peak produced by the I3 waves, but did not affect the peaks evoked by I1 waves.

Surface EMGs showed that a subthreshold conditioning stimulus suppressed the responses produced by I3 waves irrespective of its current direction (anterior or posterior). Both the amount and duration of suppression depended on the intensity of the conditioning stimulus, but not on its current direction. Both parameters increased when the intensity increased. At a high intensity conditioning stimulus, suppression was evoked at ISIs of 1‐20 ms, compatible with the duration of GABA‐mediated inhibition found in animal experiments. Responses produced by I1 waves were little affected by any type of subthreshold conditioning stimulus.

We conclude that a subthreshold conditioning stimulus given over the motor cortex moderately suppresses I3 waves but does not affect I1 waves. The duration of suppression of the I3 waves supports the idea that this is an effect of GABAergic inhibition within the motor cortex.
Zn2+ inhibition of recombinant GABAA receptors: an allosteric, state‐dependent mechanism determined by the γ‐subunit
Authors: Gingrich K. J. , Burkat P. M.
Published: September 2004

The γ‐subunit in recombinant γ‐aminobutyric acid (GABAA) receptors reduces the sensitivity of GABA‐triggered Cl− currents to inhibition by Zn2+ and transforms the apparent mechanism of antagonism from non‐competitive to competitive. To investigate underlying receptor function we studied Zn2+ effects on macroscopic and single‐channel currents of recombinant α1β2 and α1β2γ2 receptors expressed heterologously in HEK‐293 cells using the patch‐clamp technique and rapid solution changes.

Zn2+ present for > 60 s (constant) inhibited peak, GABA (5 μM)‐triggered currents of α1β2 receptors in a concentration‐dependent manner (inhibition equation parameters: concentration at half‐amplitude (IC50) = 0.94 μM; slope related to Hill coefficient, S= 0.7) that was unaffected by GABA concentration. The γ2 subunit (α1β2γ2 receptor) reduced Zn2+ sensitivity more than fiftyfold (IC50= 51 μM, S= 0.86); increased GABA concentration (100 μM) antagonized inhibition by reducing apparent affinity (IC50= 322 μM, S= 0.79). Zn2+ slowed macroscopic gating of α1β2 receptors by inducing a novel slow exponential component in the activation time course and suppressing a fast component of control desensitization. For α1β2γ2 receptors, Zn2+ accelerated a fast component of apparent desensitization.

Zn2+ preincubations lasting up to 10 s markedly increased current depression and activation slowing of α1β2 receptors, but had little effect on currents from α1β2γ2 receptors.

Steady‐state fluctuation analysis of macroscopic α1β2γ2 currents (n= 5) resulted in control (2 μM GABA) power density spectra that were fitted by a sum of two Lorentzian functions (relaxation times: 37 ± 5.6 and 1.41 ± 0.15 ms, means ± s.e.m.). Zn2+ (200 μM) reduced the total power almost sixfold and accelerated the slow (23 ± 2.8 ms, P < 0.05) without altering the fast (1.40 ± 0.16 ms) relaxation time. The ratio (fast/slow) of Lorentzian areas was increased by Zn2+ (control, 3.39 ± 0.55; Zn2+, 4.9 ± 0.37, P < 0.05).

Zn2+ (500 μM) depression of previously activated current amplitudes (% control) for α1β2γ2 receptors was independent of GABA concentration (5 μM, 13.2 ± 0.72 %; 100 μM, 12.2 ± 2.9 %, P < 0.8, n= 5). Both onset and offset inhibition time courses were biexponential. Onset rates were enhanced by Zn2+ concentration. Inhibition onset was also biexponential for preactivated α1β2 receptors with current depression more than fourfold less sensitive (5 μM GABA, IC50= 3.8 μM, S= 0.84) relative to that in constant Zn2+.

The results lead us to propose a general model of Zn2+ inhibition of GABAA receptors in which Zn2+ binds to a single extracellular site, induces allosteric receptor inhibition involving two non‐conducting states, site affinity is state‐dependent, and the features of state dependence are determined by the γ‐subunit.
Motoneuronal pre‐compensation for the low‐pass filter characteristics of muscle. A quantitative appraisal in cat muscle units
Authors: Baldissera Fausto , Cavallari Paolo , Cerri Gabriella
Published: September 2004

The relevance of motoneurone dynamic sensitivity in compensating for the low‐pass filter properties of muscle was assessed by stimulating cat muscle units (MUs) with impulse discharges generated by two current‐to‐rate converters: (i) a spinal motoneurone, sensitive to both the input intensity and its first derivative, and (ii) a linear current‐to‐rate converter, i.e. a neurone model with the same static sensitivity as the motoneurone but lacking dynamic sensitivity.

Discharges generated by injection of sine‐wave currents in three motoneurones of the ‘fast’ type and in the three related model versions were applied to the axon of forty‐six MUs. The MU isometric tension was modulated at the frequency of the current sine wave (0.5‐20 Hz). Phase and gain of the current‐to‐force transduction were measured.

When MUs were driven by the model, the force lagged the current by 90 deg at 1 Hz in slow MUs and at around 5 Hz in fast MUs. Under motoneurone drive, the 90 deg phase lag was attained at frequencies about twice as high.

The gain of the transduction (peak‐to‐peak force modulation/peak‐to‐peak current modulation) decayed when the modulation frequency was increased. In all but five units, the cut‐off frequency, Fco (gain attenuated by −3 dB), was higher when the unit was motoneurone driven (FcoCell) then when it was model driven (FcoMod). In both conditions, Fco was inversely correlated with the MU's time‐to‐peak. The advantage conferred by the motoneurone dynamic sensitivity was expressed by the Fco ratio (FcoCell/FcoMod). Across the MU population this ratio ranged from 0.6‐2.8, was inversely correlated with the time‐to peak, and was directly correlated with the half‐tension rate, i.e. the impulse rate at which MUs develop 50 % of their maximal tetanic force. The largest improvement (Fco ratio > 2.0) was found in units with mechanical features similar to those presumably coupled ‘in vivo’ to the motoneurones utilized for stimulation.

This estimate was confirmed in experiments in which trains of pulses, generated by injection of ramp currents in another motoneurone and the related model, were used to activate eight MUs, selected for being similar to that connected ‘in vivo’ to the motoneurone. As expected, for any given current slope the rising phase of isometric tension was steeper when units were motoneurone driven than when they were model driven. The gain (force slope/current slope) was plotted against the ramp slope to identify the cut‐off slope, Sco, at which the gain was attenuated by −3 dB. In this homogeneous MU sample, the ratio expressing the advantage of the motoneurone drive (ScoCell/ScoMod, equivalent to the Fco ratio), ranged from 2.62‐2.97, values comparable with those observed in sine‐wave experiments when the motoneurone and muscle units were properly matched.
Human calf precapillary resistance decreases in response to small cumulative increases in venous congestion pressure
Authors: Gamble J. , Christ F. , Gartside I. B.
Published: September 2004

We studied human lower limbs to test the hypothesis that the application of small cumulative venous congestion pressure steps is associated with a reduction in precapillary resistance.

Strain gauge plethysmography was performed on twenty‐one young subjects (22.7 ± 0.6 years). At each of the small cumulative pressure steps, limb blood flow was estimated from the initial slope of the volume response to transient (10 s duration) elevations of venous congestion pressure to 90 mmHg, after which the congestion pressure was returned to the previous value. The blood flow at each pressure was also expressed as a percentage of the initial control value. Peak tibial arterial blood flux was assessed, in four of the subjects, using colour duplex ultrasonography and the same congestion pressure protocol.

We used Darcy's Law to predict the limb arterial blood flow and blood flux at each venous congestion pressure, assuming that both mean arterial blood pressure and precapillary resistance remained constant.

The mean ± s.e.m. control arterial blood flow at the lowest venous congestion pressure, 4.8 ± 0.1 mmHg, was 2.77 ± 0.18 ml min−1 (100 ml)−1. At the highest venous congestion pressure, 59.2 ± 0.2 mmHg, arterial blood flow was 2.45 ± 0.35 ml min−1 (100 ml)−1 (121.6 ± 16.9 % of the initial value). This did not differ significantly from the initial control value, but was significantly greater than the predicted value of 0.77 ± 0.13 ml min−1 (100 ml)−1 (28.6 ± 2.1 % of the initial value) calculated assuming constant resistance and sustained mean arterial pressure. The tibial arterial peak blood flux at 58.3 mmHg venous congestion pressure was 102.2 ± 2.3 % of the control value, which was significantly greater than the predicted 17.2 ± 1.3 % of control, calculated for this pressure, assuming constant resistance and sustained mean arterial pressure.

Our data show that lower limb arterial blood flow is sustained when venous congestion pressure is raised using small cumulative steps, even at congestion pressures approaching mean arterial blood pressure. These data support the notion that precapillary resistance is influenced by signals generated at the microvascular and post microvascular levels and transmitted via the endothelium.
Damage to human muscle from eccentric exercise after training with concentric exercise
Authors: Whitehead N. P. , Allen T. J. , Morgan D. L. , Proske U.
Published: September 2004

It is known that a period of eccentric exercise provides protection against damage to muscle from subsequent eccentric exercise. Here we ask, does concentric exercise do the opposite, make muscle more prone to damage?

The triceps surae muscle group of one leg in each of eight human subjects was subjected to 30 min of concentric exercise per day, for 5 days. At the end of the training period there was a small but significant increase in passive torque in the exercised muscle (P < 0.05), with no changes in the untrained muscle.

After a single period of eccentric exercise, angle‐torque curves for muscles of both legs shifted in the direction of longer muscle lengths, suggestive of an increase in series compliance. The shift in the concentrically trained muscle was significantly greater over the first 48 h post‐exercise (P < 0.05).

The volume of the trained leg increased significantly more than the untrained leg for five subjects over 72 h post‐exercise (P < 0.05). Peak torque fell, passive stiffness increased and both muscles became sore, but with no significant differences between the two legs.

It is concluded that a period of concentric exercise increases the susceptibility of muscle to changes associated with the damage from eccentric exercise.
Exercise‐induced arterial hypoxaemia in healthy young women
Authors: Harms Craig A. , McClaran Steven R. , Nickele Glenn A. , Pegelow David F. , Nelson William B. , Dempsey Jerome A.
Published: September 2004

We questioned whether exercise‐induced arterial hypoxaemia (EIAH) occurs in healthy active women, who have smaller lungs, reduced lung diffusion, and lower maximal O2 consumption rate (V̇O2,max) than age‐ and height‐matched men.

Twenty‐nine healthy young women with widely varying fitness levels (V̇O2,max, 57 ± 6 ml kg−1 min−1; range, 35‐70 ml kg−1 min−1; or 148 ± 5 %; range, 93‐188 % predicted) and normal resting lung function underwent an incremental treadmill test to VO2,max during the follicular phase of their menstrual cycle. Arterial blood samples were taken at rest and near the end of each workload.

Arterial PO2 (Pa,O2) decreased > 10 mmHg below rest in twenty‐two of twenty‐nine subjects at V̇O2,max (Pa,O2, 77.5 ± 0.9 mmHg; range, 67‐88 mmHg; arterial O2 saturation (Sa,O2), 92.3 ± 0.2 %; range, 87‐94 %). The remaining seven subjects maintained Pa,O2 within 10 mmHg of rest. Pa,O2 at VO2,max was inversely related to the alveolar to arterial O2 difference (A‐aDO2) (r= ‐0.93; 35‐52 mmHg) and to arterial PCO2 (Pa,CO2) (r= ‐0.62; 26‐39 mmHg).

EIAH was inversely related to V̇O2,max (r= ‐0.49); however, there were many exceptions. Almost half of the women with significant EIAH had VO2,max within 15 % of predicted normal values (VO2,max, 40‐55 ml kg−1 min−1); among subjects with very high VO2,max (55‐70 ml kg−1 min−1), the degree of excessive A‐aDO2 and EIAH varied markedly (e.g. A‐aDO2, 30‐50 mmHg; Pa,O2, 68‐91 mmHg).

In the women with EIAH at V̇O2,max, many began to experience an excessive widening of their A‐aDO2 during moderate intensity exercise, which when combined with a weak ventilatory response, led to a progressive hypoxaemia. Inactive, less fit subjects had no EIAH and narrower A‐aDO2 when compared with active, fitter subjects at the same VO2 (40‐50 ml kg−1 min−1).

These data demonstrate that many active healthy young women experience significant EIAH, and at a VO2,max that is substantially less than those in their active male contemporaries. The onset of EIAH during submaximal exercise, and/or its occurrence at a relatively low V̇O2,max, implies that lung structure/function subserving alveolar to arterial O2 transport is abnormally compromised in many of these habitually active subjects.
Dynamic response of human muscle spindle afferents to stretch during voluntary contraction
Authors: Kakuda Naoyuki , Nagaoka Masanori
Published: September 2004

The response of twenty‐eight human muscle spindle afferents from m. extensor carpi radialis brevis to large amplitude ramp stretch and release at the wrist joint was recorded. The dynamic index was calculated as the difference in firing rate between that just before the end of stretch and that during the subsequent static phase of stretch. The value during steady voluntary contraction was compared with that during relaxation.

In twenty‐three primary afferents, the dynamic index increased in eleven and decreased in twelve afferents with a range of −8 to +25 impulses s−1. In five secondary afferents the change was less than 2 impulses s−1.

The primary afferents abruptly stopped firing when the stretch was released in the relaxed muscle. This cessation was prevented during contraction in seventeen primary afferents.

The results suggest the presence of dynamic and static fusimotor actions on the human muscle spindles during voluntary contraction.
Fast to slow transformation of denervated and electrically stimulated rat muscle
Authors: Windisch A. , Gundersen K. , Szabolcs M. J. , Gruber H. , Lømo T.
Published: September 2004

Denervated fast extensor digitorum longus (EDL) muscles of adult rats were stimulated electrically for up to 4 months with a ‘slow’ pattern resembling the activity in soleus (Sol) motor units and examined with antibodies against myosin heavy chains (MHCs).

The normal EDL contained, on average, 45 % type IIB, 29 % type IIX, 23 % type IIA and 3 % type I fibres. All type IIB and almost all type IIX fibres disappeared during the first 3 weeks of stimulation. They were replaced by type IIA and type I fibres, whose percentages increased to about 75 and 15, respectively. Type IIA fibres remained at 75 % for nearly 2 months and were then gradually replaced by type I fibres during the next 2 months. The transformation occurred sequentially in the order IIB/IIX → IIA → I, the first step (IIB/IIX → IIA) occurring after a short delay (2 weeks) and the last step (IIA → I in originally IIB or IIX fibres) after a long delay (> 2 months). During the transformation coexpression of MHCs occurred.

It appears that the transformation to type I fibres occurred in pre‐existing type II fibres since no signs of fibre damage or regeneration were observed.

Normal EDL was also stimulated through an intact nerve with the same pattern for up to 37 days. The effects on fibre type distributions were identical to those observed in the denervated EDL. The result indicated that the Sol‐like pattern of evoked muscle activity, rather than nerve‐derived trophic influences or denervation per se, was primarily responsible for the fast to slow transformation.
Effects of voluntary contraction on descending volleys evoked by transcranial stimulation in conscious humans
Authors: Lazzaro V. Di , Restuccia D. , Oliviero A. , Profice P. , Ferrara L. , Insola A. , Mazzone P. , Tonali P. , Rothwell J. C.
Published: September 2004

The spinal volleys evoked by single transcranial magnetic or electric stimulation over the cerebral motor cortex were recorded from a bipolar electrode inserted into the cervical epidural space of three conscious human subjects. These volleys were termed direct (D) and indirect (I) waves according to their latency.

We measured the size and number of volleys elicited by magnetic stimulation at various intensities with subjects at rest and during 20 or 100 % maximum contraction of the contralateral first dorsal interosseous muscle (FDI). Surface EMG activity was also recorded.

Electrical stimulation evoked a D‐wave volley. Magnetic stimulation at intensities up to about 15 % of stimulator output above threshold evoked only I‐waves. At higher intensities, a D‐wave could be seen in two of the three subjects.

At all intensities tested, voluntary contraction increased the number and size of the I‐waves, particularly during maximum contractions. However, there was only a small effect on the threshold for evoking descending activity. Voluntary contraction produced large changes in the size of EMG responses recorded from FDI.

Because the recorded epidural activity is destined for muscles other than the FDI, it is impossible to say to what extent increased activity contributes to voluntary facilitation of EMG responses. Indeed, our results suggest that the main factor responsible for enhancing EMG responses in the transition from rest to activity is likely to be increased excitability of spinal motoneurones, rather than increases in the corticospinal volley. The latter may be more important in producing EMG facilitation at different levels of voluntary contraction.
4‐Aminomethylbenzoic acid is a non‐translocated competitive inhibitor of the epithelial peptide transporter PepT1
Authors: Meredith D. , Boyd C. A. R. , Bronk J. R. , Bailey P. D. , Morgan K. M. , Collier I. D. , Temple C. S.
Published: September 2004

4‐Aminomethylbenzoic acid, a molecule which mimics the spatial configuration of a dipeptide, competitively inhibits peptide influx in both Xenopus laevis oocytes expressing rabbit PepT1 and through PepT1 in rat renal brush border membrane vesicles.

This molecule is not translocated through PepT1 as measured both by direct HPLC analysis in PepT1‐expressing oocytes and indirectly by its failure to trans‐stimulate labelled peptide efflux through PepT1 in oocytes and in renal membrane vesicles.

However 4‐aminomethylbenzoic acid does reverse trans‐stimulation through expressed PepT1 of labelled peptide efflux induced by unlabelled peptide. Quantitatively this reversal is compatible with 4‐aminomethylbenzoic acid competitively binding to the external surface of PepT1.

4‐Aminomethylbenzoic acid (the first molecule discovered to be a non‐translocated competitive inhibitor of proton‐coupled oligopeptide transport) and its derivatives may thus be particularly useful as experimental tools.
Effect of depletion of glycosaminoglycans and non‐collagenous proteins on interstitial hydraulic permeability in rabbit synovium
Authors: Scott D. , Coleman P. J. , Abiona A. , Ashhurst D. E. , Mason R. M. , Levick J. R.
Published: September 2004

The hydraulic resistance of synovial interstitium helps to retain a lubricating fluid within the joint cavity. The contributions of sulphated glycosaminoglycans to resistance were assessed by selective depletion by chondroitinase ABC, keratanase and heparinases I, II and IIIin vivo. Also, since glycosaminoglycans do not account fully for the resistance, the contribution of non‐collagenous, structural proteins in interstitium was assessed by treatment with chymopapain, a collagen‐sparing protease.

Ringer solution containing enzyme was injected into the synovial cavity of the knee in anaesthetized rabbits. After ≥ 30 min the intra‐articular pressure was raised and the relation between pressure (Pj) and trans‐synovial outflow (Q̇s) determined. The slope dQ̇s/dPjat low pressures, i.e. below yield pressure, represents the hydraulic conductance of the lining, i.e. 1/resistance. The contralateral joint received Ringer solution without enzyme as a control. Action of enzymes on the tissue was confirmed by histochemical and immunohistochemical studies.

Treatment with chondroitinase ABC (5 joints) increased the hydraulic conductance of the lining by 2.3 times (control, 1.34 ± 0.22 μl min−1cmH2O−1; post‐enzyme, 3.11 ± 0.45 μl min−1cmH2O−1). This was significantly less than the effects of leech,Streptomycesand testicular hyaluronidases, which caused an average 4.7 times increase (P< 0.001, ANOVA). Analogous findings were made above yield pressure.

Treatment with keratanase (3 joints) or heparinases I, II and III (3 joints) caused no significant increase in trans‐synovial flows or conductance, even though the concentration of heparan sulphate in synovium is higher than that of chondroitin sulphates or hyaluronan.

Treatment with chymopapain (7 joints) caused the greatest increases in trans‐synovial flow, which exceeded control flow by an order of magnitude in one case. After 0.1 U chymopapain the average conductance was 6.6 times the control conductance below yield pressure. Immunohistochemical studies confirmed that chymopapain treatment removed the synovial proteoglycans.

It is concluded that, despite their similar resistivitiesin vitro, the different glycosaminoglycans do not contribute equally, weight for weight, to interstitial resistancein vivo. Hyaluronan is the dominant glycosaminoglycan governing synovial interstitial resistance. In addition, non‐collagenous structural proteins contribute significantly to interstitial resistance.
Bayliss, myogenic tone and volume‐regulated chloride channels in arterial smooth muscle
Authors: Nelson Mark T.
Published: September 2004

The muscular coat of the arteries reacts, like smooth muscle in other situations, to a stretching force by contraction.’ (Bayliss, 1902)
Voltage‐dependent blockade of HERG channels expressed in Xenopus oocytes by external Ca2+ and Mg2+
Authors: Ho Won‐Kyung , Kim Injune , Lee Chin O. , Earm Yung E.
Published: September 2004

We expressed the human eag‐related gene (HERG), which is known to encode the delayed rectifier K+ current (IKr) in cardiac muscle, in Xenopus oocytes. Using a two‐microelectrode voltage clamp technique, the effect of external Ca2+ and Mg2+ on the HERG current (IHERG) was investigated.

When [Ca2+]o was increased, the amplitude of outward IHERG elicited by depolarization decreased, and the rate of current onset slowed. The rate of current decay observed on repolarization was greatly accelerated. The threshold and fully activated potential of IHERG shifted to a more positive potential. On the other hand, the inactivation property represented by the negative slope of the I‐V curve and the instantaneous conductance of IHERG were little affected by [Ca2+]o.

The effect of [Ca2+]o on IHERG can be interpreted using the channel blockade model. The blockade is voltage dependent; smaller dissociation constants (KM) at more negative potentials indicate that block is facilitated by hyperpolarization. KM changes e‐fold for 14.5 mV and the fractional electrical distance of the binding site calculated from this value is 0.86.

Blockade by a low concentration of Ca2+ (0.5 mM) was inhibited by increasing [K+]o (from 2 to 20 mM), whereas blockade by a high concentration of Ca2+ (5 mM) was not affected by varying [K+]o, indicating that there is competition between permeating ions and blocking ions.

The effect of [Mg2+]o on IHERG was qualitatively similar to that of [Ca2+]o, but the potency was lower.

These results suggest that external Ca2+ and Mg2+ block the HERG channel in a voltage‐ and time‐dependent manner, resulting in a voltage dependence which has been regarded as a property of the activation gate.
Endogenous bradykinin activates ischaemically sensitive cardiac visceral afferents through kinin B2 receptors in cats
Authors: Tjen‐A‐Looi Stephanie C. , Pan Hui‐Lin , Longhurst John C.
Published: September 2004

Activity of ischaemically sensitive cardiac visceral afferents during myocardial ischaemia induces both angina and cardiovascular reflexes. Increased production of bradykinin (BK) and cyclo‐oxygenase products (i.e. prostaglandins (PGs)) occurs during myocardial ischaemia. However, the role of these agents in activation of ischaemically sensitive cardiac afferents has not been established. The present study tested the hypothesis that BK produced during ischaemia activates cardiac afferents through kinin B2 receptors.

Single‐unit activity of cardiac afferents innervating the left ventricle was recorded from the left thoracic sympathetic chain (T1‐T4) of anaesthetized cats. Ischaemically sensitive cardiac afferents were identified according to their response to 5 min of myocardial ischaemia. The mechanism of BK in activation of ischaemically sensitive cardiac afferents was determined by injection of BK (1 μg kg−1 i.a.), des‐Arg9‐BK (1 μg kg−1 i.a., a specific kinin B1 receptor agonist), kinin B2 receptor antagonists: HOE140 (30 μg kg−1 i.v.) and NPC‐17731 (40 μg kg−1 i.v.), cyclo‐oxygenase inhibition with indomethacin (5 mg kg−1 i.v.) and NPC‐17731 (40 μg kg−1 i.v.) after pretreatment with indomethacin (5 mg kg−1 i.v.).

We observed that BK increased the discharge rate of all eleven ischaemically sensitive cardiac afferents from 0.39 ± 0.12 to 1.47 ± 0.37 impulses s−1 (P < 0.05). Conversely, des‐Arg9‐BK did not significantly increase the activity of eleven ischaemically sensitive fibres (0.58 ± 0.02 vs. 0.50 ± 0.18 impulses s−1). HOE140 significantly attenuated the response of twelve afferents to ischaemia (0.61 ± 0.22 to 1.85 ± 0.5 vs. 0.53 ± 0.16 to 1.09 ± 0.4 impulses s−1). NPC‐17731, another kinin B2 receptor antagonist, had similar inhibitory effects on six other ischaemically sensitive cardiac afferents (0.35 ± 0.14 to 1.19 ± 0.29 vs. 0.22 ± 0.08 to 0.23 ± 0.07 impulses s−1). Indomethacin significantly reduced the responses of seven afferents to ischaemia (0.35 ± 0.13 to 1.89 ± 0.48 vs. 0.40 ± 0.10 to 0.76 ± 0.24 impulses s−1). Indomethacin also significantly reduced the responses of six ischaemically sensitive cardiac afferents to BK (2.65 ± 1.23 to 1.2 ± 0.51 impulses s−1). In six cats pretreated with indomethacin, NPC‐17731 attenuated the impulse activity of six ischaemically sensitive cardiac afferents (0.39 ± 0.12 to 1.0 ± 0.3 vs. 0.26 ± 0.14 to 0.48 ± 0.20 impulses s−1).

This study demonstrates that BK produced during ischaemia contributes to stimulation of ischaemically sensitive cardiac visceral afferents through activation of kinin B2 receptors. Furthermore, BK stimulates ischaemically sensitive cardiac visceral afferents through a mechanism that is, at least in part, independent of cyclo‐oxygenase activation.
Enhancement of spontaneous transmitter release at neonatal mouse neuromuscular junctions by the glial cell line‐derived neurotrophic factor (GDNF)
Authors: Ribchester Richard R. , Thomson Derek , Haddow Lewis J. , Ushkaryov Yuri A.
Published: September 2004

The acute effects of neurotrophic factors on the frequency of spontaneous transmitter release (miniature endplate potentials (MEPPs)) from motor nerve terminals has been examined in skeletal muscles of neonatal mice aged between 9 and 20 days. The following factors were tested at a concentration of 50 ng ml−1: brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3), neurotrophin‐4 (NT‐4), ciliary neuronotrophic factor (CNTF), leukaemia inhibitory factor (LIF), insulin‐like growth factors 1 and 2 (IGF‐1 and IGF‐2), and glial cell line‐derived neurotrophic factor (GDNF). In some experiments, the responses to 2 μm LaCl3 and 10 mm K+, or to 2–5 nm purified α‐latrotoxin (α‐LTX) were also measured.

Neither BDNF, NT‐3, NT‐4, LIF, IGF‐1 or IGF‐2 ‐ singly or in combination ‐ caused any significant change in MEPP frequency. GDNF, however, produced a highly significant, 2‐fold increase in neurotransmitter release that was reproduced in fourteen muscles.

Potentiation of MEPP frequency in GDNF was of the same order as that induced by tetanic stimulation or substitution of the bathing medium with hypertonic saline; but substantially less than that induced either by lanthanum ions or α‐latrotoxin.

The data suggest that concentrations of GDNF that produce maximal enhancement of motoneurone survival in vitro and in vivo also produce acute, non‐saturating enhancement in transmitter release at immature mammalian neuromuscular synapses. Taken together with other reports, these findings suggest that GDNF may mediate both functional and structural plasticity of neonatal neuromuscular junctions.
An analysis of philanthotoxin block for recombinant rat GluR6(Q) glutamate receptor channels
Authors: Bähring Robert , Mayer Mark L.
Published: September 2004

The action of philanthotoxin 343 (PhTX) on rat homomeric GluR6(Q) recombinant glutamate receptor channels was analysed using concentration‐jump techniques and outside‐out patches from HEK 293 cells. Both onset and recovery from block by external PhTX were dependent on the presence of agonist, indicating that channels must open for PhTX to bind and that channel closure can trap PhTX.

Block by external PhTX developed with double‐exponential kinetics. The rate of onset of the fast component of block showed an exponential increase per 27 mV hyperpolarization over the range ‐40 to ‐100 mV. The rate of onset of the slow component of block showed a non‐linear concentration dependence indicating a rate‐limiting step in the blocking mechanism.

The extent of block by 1 μM external PhTX was maximal at ‐40 mV and did not increase with further hyperpolarization; the rate of recovery from block by external PhTX increased 6‐fold on hyperpolarization from ‐40 to ‐100 mV suggesting that PhTX permeates at negative membrane potentials.

Apparent Kd values for block by external PhTX estimated from dose‐inhibition experiments decreased 300‐fold on hyperpolarization from +40 mV (Kd, 19.6 μM) to ‐40 mV (Kd, 69 nM); there was little further increase in affinity with hyperpolarization to ‐80 mV (Kd, 56 nM), consistent with permeation of PhTX at negative membrane potentials.

Block by internal PhTX showed complex kinetics and voltage dependence. Analysis with voltage ramps from ‐120 to +120 mV indicated a Kd at 0 mV of 20 μM, decreasing e‐fold per 16 mV depolarization. However, at +90 mV the extent of block by 1 and 10 μM internal PhTX (73 % and 95 %, respectively) reached a maximum and did not increase with further depolarization.

Voltage‐jump analysis of block by 100 μM internal PhTX revealed partial trapping. With 100 ms jumps from ‐100 to ‐40 mV, onset and recovery from block were complete within 5 ms. With jumps of longer duration the extent of block increased, with a time constant of 8.1 s, reaching 84 % at 30 s. On repolarization to ‐100 mV, recovery from block showed fast and slow components.

The amplitude of the slow component of block by internal PhTX showed a biphasic voltage dependence, first increasing then decreasing with progressive depolarization. Maximum block was obtained at 0 mV.

Our results suggest that PhTX acts as an open channel blocker; however, provided that the toxin remains bound to the channel, an allosteric mechanism destabilizes the open state, inducing channel closing and trapping PhTX. Strong depolarization for internal PhTX, or strong hyperpolarization for external PhTX, forces the toxin to permeate before it triggers entry into closed blocked states.
Heterogeneity of homomeric GluR5 kainate receptor desensitization expressed in HEK293 cells
Authors: Swanson Geoffrey T. , Heinemann Stephen F.
Published: September 2004

Kainate receptors with pharmacological properties similar to those of the GluR5 subunit have been shown to modulate inhibitory synaptic transmission in the CA1 region of the hippocampus. The kinetic properties of currents gated by GluR5 receptors have not been examined in detail. Here we describe several biophysical features of recombinant GluR5 receptors expressed in HEK293 cells.

We found that homomeric GluR5 receptors can exhibit striking inter‐cell variability in channel kinetics in response to the agonists kainate and glutamate. Desensitization rates in response to kainate varied between individual cells by nearly 1000‐fold (range, 1.5 ms to 1.5 s), while glutamate desensitization rates differed by 9‐fold (range, 1.0 to 9.0 ms).

The time course of recovery from desensitization in response to glutamate also showed inter‐cell variation. The majority of glutamate currents in GluR5‐expressing cells recovered from desensitization with two widely separated exponential components: 50 ± 10 ms and 5.1 ± 1.0 s (contributing 37.6 % and 62.4 % of the sum of the exponential fits, respectively). In contrast, currents with the fastest desensitization kinetics had a recovery time course of 4.8 ± 0.3 s.

Kainate receptors in murine dorsal root ganglion neurons are likely to be composed of homomeric GluR5 subunits. These receptor currents recovered from glutamate desensitization with a biexponential time course of 36 ± 4 ms and 4.7 ± 0.7 s.

These results suggest that aspects of GluR5 kainate receptor function are modulated by intracellular mechanism(s). At synapses such mechanisms could regulate the frequency‐ response relationship of synaptic kainate receptors by altering their rate of entry into and recovery from desensitization.
Interaction of H+ and Zn2+ on recombinant and native rat neuronal GABAA receptors
Authors: Krishek Belinda J. , Moss Stephen J. , Smart Trevor G.
Published: September 2004

The interaction of Zn2+ and H+ ions with GABAA receptors was examined using Xenopus laevis oocytes expressing recombinant GABAA receptors composed of subunits selected from α1, β1, γ2S and δ types, and by using cultured rat cerebellar granule neurones.

The potency of Zn2+ as a non‐competitive antagonist of GABA‐activated responses on α1β1 receptors was reduced by lowering the external pH from 7.4 to 5.4, increasing the Zn2+ IC50 value from 1.2 to 58.3 μM. Zinc‐induced inhibition was largely unaffected by alkaline pH up to pH 9.4.

For α1β1δ subunits, concentration‐response curves for GABA were displaced laterally by Zn2+ in accordance with a novel mixed/competitive‐type inhibition. The Zn2+ IC50 at pH 7.4 was 16.3 μM. Acidification of Ringer solution resulted in a reduced antagonism by Zn2+ (IC50, 49.0 μM) without affecting the type of inhibition. At pH 9.4, Zn2+ inhibition remained unaffected.

The addition of the γ2S subunit to the α1β1δ construct caused a marked reduction in the potency of Zn2+ (IC50, 615 μM), comparable to that observed with α1β1γ2S receptors (IC50 639 μM). GABA concentration‐response curves were depressed in a mixed/non‐competitive fashion.

In cultured cerebellar granule neurones, Zn2+ inhibited responses to GABA in a concentration‐dependent manner. Lowering external pH from 7.4 to 6.4 increased the IC50 from 139 to 253 μM.

The type of inhibition exhibited by Zn2+ on cerebellar granule neurones, previously grown in high K+‐containing culture media, was complex, with the GABA concentration‐response curves shifting laterally with reduced slopes and similar maxima. The Zn2+‐induced shift in the GABA EC50 values was reduced by lowering the external pH from 7.4 to 6.4.

The interaction of H+ and Zn2+ ions on GABAA receptors suggests that they share either a common regulatory pathway or coincident binding sites on the receptor protein. The apparent competitive mode of block induced by Zn2+ on α1β1δ receptors is shared by GABAA receptors on cerebellar granule neurones, which are known to express δ‐subunit‐containing receptors. This novel mechanism is masked when a γ2 subunit is incorporated into the receptor complex, revealing further diversity in the response of native GABAA receptors to endogenous cations.
Cloning and functional expression of rat ether‐à‐go‐go‐like K+ channel genes
Authors: Engeland Birgit , Neu Axel , Ludwig Jost , Roeper Jochen , Pongs Olaf
Published: September 2004

Screening of rat cortex cDNA resulted in cloning of two complete and one partial orthologue of the Drosophilaether‐à‐go‐go‐like K+ channel (elk).

Northern blot and reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis revealed predominant expression of rat elk mRNAs in brain. Each rat elk mRNA showed a distinct, but overlapping expression pattern in different rat brain areas.

Transient transfection of Chinese hamster ovary (CHO) cells with rat elk1 or rat elk2 cDNA gave rise to voltage‐activated K+ channels with novel properties.

RELK1 channels mediated slowly activating sustained potassium currents. The threshold for activation was at −90 mV. Currents were insensitive to tetraethylammonium (TEA) and 4‐aminopyridine (4‐AP), but were blocked by micromolar concentrations of Ba2+. RELK1 activation kinetics were not dependent on prepulse potential like REAG‐mediated currents.

RELK2 channels produced currents with a fast inactivation component and HERG‐like tail currents. RELK2 currents were not sensitive to the HERG channel blocker E4031.
Lanthanum‐mediated modification of GABAA receptor deactivation, desensitization and inhibitory synaptic currents in rat cerebellar neurons
Authors: Zhu Wei Jian , Wang Jian Feng , Corsi Lorenzo , Vicini Stefano
Published: September 2004

We investigated La3+ effects on recombinant and native γ‐aminobutyric acid A (GABAA) receptors using rapid agonist applications and on inhibitory synaptic currents (IPSCs) in granule and stellate neurons of rat cerebellar slices.

Rapid desensitization of currents elicited by 200 ms pulses of 1 mM GABA to small lifted cells transfected with α1β3γ2 cDNAs was greatly decreased by the coapplication of 100 μm LaCl3.

GABA responses were unaffected when coapplication lasted only 2 ms. In contrast, with LaCl3 pre‐perfusion, a significant slowing of deactivation in response to 2 ms applications was observed. LaCl3 pre‐perfusion also prolonged the duration of responses to 20 mM taurine.

Outside‐out patches excised from cells transfected with α1β3γ2 subunit cDNAs were briefly exposed to a saturating concentration of GABA, eliciting a transient activation of single channel currents with a main conductance of 30 pS. Opening and burst durations increased by pre‐equilibration of patches with LaCl3.

LaCl3 depressed the peak amplitude without affecting the slow deactivation and desensitization of GABA responses in cells transfected with α6β3γ2 and α6β3δ cDNAs. No significant difference in La3+ modulation of GABA‐gated currents was observed between α1β3γ2 and α1β3δ receptors.

The effects of LaCl3 on deactivation and desensitization of GABA responses observed in nucleated patches excised from rat cerebellar granule and stellate neurons were comparable to those in the cells transfected with α1β3γ2 cDNAs. In addition, La3+ clearly prolonged the spontaneous IPSC time course without changing the amplitude.

Our results indicate that La3+ has a dual action on GABA‐gated currents: it decreases desensitization and increases channel opening duration. These actions depend on receptor subunit composition and contribute to the prolongation of IPSCs.
Structure and function of voltage‐gated sodium channels
Authors: Marban Eduardo , Yamagishi Toshio , Tomaselli Gordon F.
Published: September 2004

Sodium channels mediate fast depolarization and conduct electrical impulses throughout nerve, muscle and heart. This paper reviews the links between sodium channel structure and function.

Sodium channels have a modular architecture, with distinct regions for the pore and the gates. The separation is far from absolute, however, with extensive interaction among the various parts of the channel.

At a molecular level, sodium channels are not static: they move extensively in the course of gating and ion translocation.

Sodium channels bind local anaesthetics and various toxins. In some cases, the relevant sites have been partially identified.

Sodium channels are subject to regulation at the levels of transcription, subunit interaction and post‐translational modification (notably glycosylation and phosphorylation).
Na+−Ca2+ exchange and sarcoplasmic reticular Ca2+ regulation in ventricular myocytes from transgenic mice overexpressing the Na+−Ca2+ exchanger
Authors: Terracciano Cesare M. N. , De Souza Ayesha I. , Philipson Kenneth D. , MacLeod Kenneth T.
Published: September 2004

The contribution of the sarcoplasmic reticulum (SR) and Na+−Ca2+ exchanger to intracellular Ca2+ regulation in mouse cardiac myocytes was investigated by measuring contraction after variable rest intervals, rapid cooling contractures (RCCs) and fast application of caffeine. The results obtained showed differences from other species in the roles played by the SR and the Na+−Ca2+ exchanger. They suggest that in mouse ventricular myocytes there is significant Ca2+ entry via the exchanger during rest and during the latter part of the Ca2+ transient.

In cardiac myocytes isolated from transgenic mice overexpressing the cardiac Na+−Ca2+ exchanger the time to peak and relaxation of twitches and RCCs were faster than in control littermates. The decline of Ca2+, assessed by indo‐1 fluorescence, was faster in transgenic myocytes even in the absence of Na+ and Ca2+ in the superfusing solution. This suggests that SR Ca2+ uptake is faster in these myocytes. However, no difference in the expression of SERCA2a, phospholamban or calsequestrin measured with Western blotting could be found in the two groups.

We measured SR Ca2+ content by integrating the caffeine‐induced transient inward current. The amount of Ca2+ stored in the SR of transgenic mouse myocytes was 69 % greater than in non‐transgenic littermates. The increased SR Ca2+ content may be responsible for the faster rate of SR Ca2+ release and uptake in cells from transgenic mice.

We performed experiments to assess whether the reversal potential of the Na+−Ca2+ exchanger (ENa‐Ca) was different in transgenic cardiac cells. We measured a Ni2+‐sensitive current elicited by voltage ramps in non‐dialysed myocytes. The current‐voltage relationship showed no difference in the reversal potential of the Na+−Ca2+ exchanger in transgenic and control myocytes. This suggests that the effects on the SR Ca2+ content in transgenic cardiac myocytes can be ascribed to the overexpression of the exchanger and are not secondary to changes in intracellular diastolic Ca2+ and Na+.
Repetitive hypoxia rapidly depresses arousal from active sleep in newborn lambs
Authors: Johnston Renea V. , Grant Daniel A. , Wilkinson Malcolm H. , Walker Adrian M.
Published: September 2004

Arousal from sleep is an important protective mechanism that is depressed by repeated episodes of hypoxia. We aimed to determine how rapidly arousal depression occurs during repeated hypoxia and to determine if the depression is sleep state specific.

Three successive 12 h overnight sleep recordings were performed in six newborn lambs instrumented to record sleep state, blood pressure, heart rate and blood gases. The first (control) and third (recovery) nights were baseline studies (inspired oxygen fraction, FI,O2 = 0.21) to determine the spontaneous arousal probability. During the second (test) study night, lambs were exposed to a 60 s episode of isocapnic hypoxia (FI,O2 = 0.10; inspired carbon dioxide fraction, FI,CO2 = 0.03) during every epoch of sleep.

During quiet sleep (QS), the probability of arousing to hypoxia (56 %) remained significantly higher than the probability of arousing spontaneously (18 %) throughout the repeated hypoxic exposures (χ2= 81.5, P < 0.001). By contrast, during active sleep (AS) arousal rapidly became depressed with repetition of the hypoxic stimulus; the probability of arousal in hypoxia (52 %) was significantly higher than the probability of spontaneous arousal (12 %) during the first ten hypoxic exposures (χ2= 18.2, P < 0.001), but there was no difference thereafter.

We conclude that, when repeated, moderate hypoxia very rapidly becomes ineffective as an arousing stimulus in AS, but not in QS. These results suggest that the arousal mechanism is particularly vulnerable to failure during AS.
Ion permeation and conduction in a human recombinant 5‐HT3 receptor subunit (h5‐HT3A)
Authors: Brown A. M. , Hope A. G. , Lambert J. J. , Peters J. A.
Published: September 2004

A human recombinant homo‐oligomeric 5‐HT3 receptor (h5‐HT3A) expressed in a human embryonic kidney cell line (HEK 293) was characterized using the whole‐cell recording configuration of the patch clamp technique.

5‐HT evoked transient inward currents (EC50= 3.4 μM; Hill coefficient = 1.8) that were blocked by the 5‐HT3 receptor antagonist ondansetron (IC50= 103 pM) and by the non‐selective agents metoclopramide (IC50= 69 nM), cocaine (IC50= 459 nM) and (+)‐tubocurarine (IC50= 2.8 μM).

5‐HT‐induced currents rectified inwardly and reversed in sign (E5‐HT) at a potential of −2.2 mV. N‐Methyl‐D‐glucamine was finitely permeant. Permeability ratios PNa/PCs and PNMDG/PCs were 0.90 and 0.083, respectively.

Permeability towards divalent cations was assessed from measurements of E5‐HT in media where Ca2+ and Mg2+ replaced Na+. PCa/PCs and PMg/PCs were calculated to be 1.00 and 0.61, respectively.

Single channel chord conductance (γ) estimated from fluctuation analysis of macroscopic currents increased with membrane hyperpolarization from 243 fS at −40 mV to 742 fS at −100 mV.

Reducing [Ca2+]o from 2 to 0.1 mM caused an increase in the whole‐cell current evoked by 5‐HT. A concomitant reduction in [Mg2+]o produced further potentiation. Fluctuation analysis indicates that a voltage‐independent augmentation of γ contributes to this phenomenon.

The data indicate that homo‐oligomeric receptors composed of h5‐HT3A subunits form inwardly rectifying cation‐selective ion channels of low conductance that are permeable to Ca2+ and Mg2+.