bluuebunny/crossref_metadata_2025_split huggingface dataset:
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.
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+.