bluuebunny/crossref_metadata_2025_split huggingface dataset:
Divalent ion block of inward rectifier current in human capillary endothelial cells and effects on resting membrane potential
Authors: Jow Flora , Numann Randy 
Published: September 2004

Cultured human capillary endothelial cells (HCEC) contain a large inward rectifier current, IK(IR), that can be abolished by removing external K+ or by adding 50 μm Ba2+.
We show that IK(IR) is responsible for maintaining the hyperpolarized potential (−60.6 ± 0.5 mV, n= 83) of HCEC. Blocking IK(IR) with 50 μm Ba2+ shifts the zero current level and depolarizes HCEC by 36.5 ± 1.3 mV (n= 4).
Increasing external Ca2+ concentration ([Ca2+]o) from 0.5 to 7 mm reduces the magnitude of IK(IR) by 36.5 ± 2.3 % (n= 5) and depolarizes the cells by 10.33 ± 2.4 mV (n= 3), whereas decreasing [Ca2+]o from 1.8 to 0.5 mm increases the amplitude of IK(IR) by 6.9 ± 1.9 % (n= 4). The relationship between [Ca2+]o and the percentage block of IK(IR) gives a Kd value of 5.4 ± 0.6 mm at −120 mV.
I
K(IR) is also blocked by other divalent ions, with Ba2+ >> Sr2+ > Mg2+ > Mn2+= Ca2+, and the block of peak current at −120 mV being 85.3 ± 3.2 % (n= 5) for 50 μm Ba2+, 62.9 ± 2.2 % (n= 5) for 5 mm Sr2+, 40.7 ± 2.5 % (n= 9) for 5 mm Mg2+, 33.4 ± 2.1 % (n= 5) for 5 mm Mn2+ and 32.9 ± 2.1 % (n= 5) for 5 mm Ca2+.
The voltage dependence of Sr2+ block of peak IK(IR) occurred with a Kd value of 1.0 ± 0.09 mm for −140 mV, 1.9 ± 0.16 mm for −130 mV, 3.1 ± 0.28 mm for −120 mV, 4.6 ± 0.34 mm for −110 mV and 6.4 ± 0.5 mm for −100 mV (n= 5), with a calculated electrical distance (δ) of 0.44 from the outside.
Presynaptic inhibition by 5‐HT1B receptors of glutamatergic synaptic inputs onto serotonergic caudal raphe neurones in rat
Authors: Li Yu‐Wen , Bayliss Douglas A. 
Published: September 2004

Autonomous, pacemaker‐like activity of serotonergic raphe neurones and its autoregulation by somatodendritic 5‐HT1A receptors are well described, but little is known of synaptic inputs onto raphe neurones or their modulation. Therefore, we recorded unitary excitatory postsynaptic currents (EPSCs) in caudal raphe neurones (raphe obscurus and pallidus) following local electrical stimulation in a neonatal rat brainstem slice preparation; most neurones (79 %; n= 72/91) recovered following post hoc immunohistochemistry were tryptophan hydroxylase‐immunoreactive, indicating that they were serotonergic.
Evoked EPSCs occurred at relatively constant latency with variable amplitude and apparent ‘failures’ at fixed suprathreshold stimulus intensity. At −60 mV, EPSCs were wholly due to CNQX‐sensitive, non‐NMDA glutamate receptors; at depolarized potentials, a small AP‐5‐sensitive NMDA component was often observed.
EPSCs were potently and reversibly inhibited by 5‐HT with an EC50 of 0.1 μM. This effect was mimicked by 5‐HT1B agonists (CP‐93,129 and anpirtoline), but not by a 5‐HT1A agonist (8‐OH‐DPAT), indicating that 5‐HT1B receptors mediate the inhibition of EPSCs.
Multiple lines of evidence indicate that inhibition of EPSCs by 5‐HT was mediated presynaptically. First, currents evoked by exogenous glutamate application were unaffected by 5‐HT and/or 5‐HT1B agonists. In addition, the frequency of spontaneous glutamatergic miniature EPSCs was diminished by CP‐93,129 and paired‐pulse facilitation of EPSCs was enhanced by 5‐HT. Finally, the 5‐HT1B receptor agonists that blocked synaptic transmission had no effect on resting membrane properties of raphe neurones.
These data indicate that serotonergic caudal raphe neurones receive glutamatergic inputs that are inhibited by presynaptic 5‐HT1B receptors; inhibition of excitatory synapses onto raphe cells may represent a novel mechanism for autoregulation of serotonergic neuronal activity by 5‐HT.
Activation of bovine tracheal chloride channels by amino group‐specific reagents
Authors: Duszyk Marek , Shu Yali , Ho Anthony K. , Man S. F. Paul 
Published: September 2004

The predominant Cl− channel in bovine tracheal epithelial cells has a conductance of ∼71 pS and accounts for more than 80 % of the total chloride conductance. We examined the effects of protein‐modifying reagents on channel function and found that amino groups are critically involved in gating.
Patch clamp studies showed that lysine‐specific reagents, such as dimethyl adipimidate (DMA), significantly increased the channel open probability, but not its conductance. This suggests that modified residues are involved in the gating mechanism, but are distant from the channel permeation pathway.
Kinetic analysis of channel activity showed that histograms of open and closed durations could be well fitted by double exponential distributions, suggesting that the channel has at least two open and two closed states. DMA did not change the number of open or closed states, but increased channel mean open time.
Since membrane impermeant reagents were effective only from the extracellular side, we conclude that lysine residues in the extracellular domain of the channel are critically involved in gating. These residues may present an important target for site‐directed mutagenesis and pharmacological activation of Cl− channels in epithelial cells.
Changes in [Ca2+]i and membrane currents during impaired mitochondrial metabolism in dissociated rat hippocampal neurons
Authors: Nowicky Alex V. , Duchen Michael R. 
Published: September 2004

This study was designed to establish the basis for altered membrane excitability during the inhibition of mitochondrial metabolism in central mammalian neurons. Perforated whole‐cell patch clamp and fluorimetric techniques were combined to examine changes in membrane currents, intracellular calcium ([Ca2+]i) and mitochondrial potential (ΔΨm) in neurons dissociated from the CA1 subfield of the hippocampus of young rats.
On application of the mitochondrial inhibitor NaCN, or the uncoupler carbonyl cyanide p‐trifluoromethoxyphenylhydrazone (FCCP), the membrane potential hyperpolarized and membrane conductance increased. Under voltage clamp, an outward current was seen. The reversal potential of the current at −83 mV and its dependence on extracellular [K+] confirmed that this was a K+ conductance.
Simultaneous recordings of [Ca2+]i and current showed a striking correlation between a rise in [Ca2+]i and the developed outward current. Flash photolysis of the caged Ca2+ chelator, diazo‐2, reversed both the rise in [Ca2+]i and the outward current. The current was reduced by 80% by charybdotoxin, was attenuated by 10 mm TEA+ but was unaffected by apamin or by the KATP channel blocker tolbutamide (400 μm−1 mm). These data suggest strongly that the current is carried by Ca2+‐dependent K+ channels.
Simultaneous recordings of membrane current, ΔΨm and [Ca2+]i revealed the sequence of events in response to impaired mitochondrial function (CN, FCCP or anoxia): ΔΨm depolarized, followed rapidly by an increase in [Ca2+]i followed in turn by the outward current. [Ca2+]i and membrane current recovered only after mitochondrial repolarization.
The rise in [Ca2+]i appeared to result from an increased Ca2+ influx through voltage‐gated Ca2+ channels. It was dependent on extracellular Ca2+ and was much reduced by methoxyverapamil (D600). The rate of Mn2+ quench of fura‐2 fluorescence was increased by the inhibitors, and the inhibitors induced a small inward current when K+ channels were blocked that preceded the rise in [Ca2+]i. However, the increase in [Ca2+]i showed no obvious dependence on membrane potential in cells clamped at a range of holding potentials from −90 to −45 mV.
Thus, removal of oxygen, uncoupling mitochondrial oxidative phosphorylation or inhibition of respiration, all lead to mitochondrial depolarization, an increased Ca2+ influx through (voltage‐gated) channels, even at hyperpolarized membrane potentials, raising [Ca2+]i which in turn drives an increased K+ conductance that modulates membrane excitability.
Control of pulsatile 5‐HT/insulin secretion from single mouse pancreatic islets by intracellular calcium dynamics
Authors: Barbosa Rui M. , Silva Amélia M. , Tomé Angelo R. , Stamford Jonathan A. , Santos Rosa M. , Rosário Luís M. 
Published: September 2004

Glucose‐induced insulin release from single islets of Langerhans is pulsatile. We have investigated the correlation between changes in cytosolic free calcium concentration ([Ca2+]i) and oscillatory insulin secretion from single mouse islets, in particular examining the basis for differences in secretory responses to intermediate and high glucose concentrations. Insulin release was monitored in real time through the amperometric detection of the surrogate insulin marker 5‐hydroxytryptamine (5‐HT) via carbon fibre microelectrodes. The [Ca2+]i was simultaneously recorded by whole‐islet fura‐2 microfluorometry.
In 82 % of the experiments, exposure to 11 mM glucose evoked regular high‐frequency (average, 3.4 min−1) synchronous oscillations in amperometric current and [Ca2+]i. In the remaining experiments (18 %), 11 mM glucose induced an oscillatory pattern consisting of high‐frequency [Ca2+]i oscillations that were superimposed on low‐frequency (average, 0.32 min−1) [Ca2+]i waves. Intermittent high‐frequency [Ca2+]i oscillations gave rise to a similar pattern of pulsatile 5‐HT release.
Raising the glucose concentration from 11 to 20 mM increased the duration of the steady‐state [Ca2+]i oscillations without increasing their amplitude. In contrast, both the duration and amplitude of the associated 5‐HT transients were increased by glucose stimulation. The amount of 5‐HT released per secretion cycle was linearly related to the duration of the underlying [Ca2+]i oscillations in both 11 and 20 mM glucose. The slopes of the straight lines were identical, indicating that there is no significant difference between the ability of calcium oscillations to elicit 5‐HT/insulin release in 11 and 20 mM glucose.
In situ 5‐HT microamperometry has the potential to resolve the high‐frequency oscillatory component of the second phase of glucose‐induced insulin secretion. This component appears to reflect primarily the duration of the underlying [Ca2+]i oscillations, suggesting that glucose metabolism and/or access to glucose metabolites is not rate limiting to fast pulsatile insulin release.
Evidence for an electrogenic Na+‐HCO3− symport in rat cardiac myocytes
Authors: Aiello Ernesto A. , Petroff Martín G. Vila , Mattiazzi Alicia R. , Cingolani Horacio E. 
Published: September 2004

The perforated whole‐cell configuration of patch clamp and the pH fluorescent indicator SNARF were used to determine the electrogenicity of the Na+‐HCO3− cotransport in isolated rat ventricular myocytes.
Switching from Hepes buffer to HCO3− buffer at constant extracellular pH (pHo) hyperpolarized the resting membrane potential (RMP) by 2.9 ± 0.4 mV (n= 9, P < 0.05). In the presence of HCO3−, the anion blocker SITS depolarized RMP by 2.6 ± 0.5 mV (n= 5, P < 0.05). No HCO3−‐induced hyperpolarization was observed in the absence of extracellular Na+. The duration of the action potential measured at 50 % of repolarization time (APD50) was 29.2 ± 6.1 % shorter in the presence of HCO3− than in its absence (n= 6, P < 0.05).
Quasi‐steady‐state currents were evoked by voltage‐clamped ramps ranging from −130 to +30 mV, during 8 s. The development of a novel component of Na+‐dependent and Cl−‐independent steady‐state outward current was observed in the presence of HCO3−. The reversal potential (Erev) of the Na+‐HCO3− cotransport current (INa,Bic) was measured at four different levels of extracellular Na+. A HCO3−:Na+ ratio compatible with a stoichiometry of 2:1 was detected. INa,Bic was also studied in isolation in standard whole‐cell experiments. Under these conditions, INa,Bic reversed at −96.4 ± 1.9 mV (n= 5), being consistent with the influx of 2 HCO3− ions per Na+ ion through the Na+‐HCO3− cotransporter.
In the presence of external HCO3−, after 10 min of depolarizing the membrane potential (Em) with 45 mm extracellular K+, a significant intracellular alkalinization was detected (0.09 ± 0.03 pH units; n= 5, P < 0.05). No changes in pHi were observed when the myocytes were pre‐treated with the anion blocker DIDS (0.001 ± 0.024 pH units; n= 5, n.s.), or when exposed to Na+‐free solutions (0.003 ± 0.037 pH units; n= 6, n.s.).
The above results allow us to conclude that the cardiac Na+‐HCO3− cotransport is electrogenic and has an influence on RMP and APD of rat ventricular cells.
Calcium current during a single action potential in a large presynaptic terminal of the rat brainstem
Authors: Borst J. G. G. , Sakmann B. 
Published: September 2004

The calcium current of a ‘giant’ synaptic terminal (the calyx of Held) was studied using two‐electrode voltage clamp in slices of the rat brainstem.
In terminals with a long axon (length > 100 μm), the passive current transient decayed bi‐exponentially following voltage steps. In terminals with a short axon (length < 30 μm), the slow component was reduced or absent. These terminals also had small slow calcium tail currents following long depolarizing voltage steps, suggesting that these are largely due to axonal calcium channels.
Terminals were voltage clamped with action potential waveform commands. At both 24 and 36°C the calcium current began shortly after the peak of the action potential and ended before the terminal was fully repolarized.
The calcium current during the repolarization phase was 69 ± 1 % (n= 3) of maximal, judged from the increase in this current when a plateau phase was added to the action potential waveform.
A Hodgkin‐Huxley m2 model, based on the measured activation and deactivation of the calcium current, reproduced both the time course and the amplitude increase of the calcium currents during the different action potential waveforms well.
The fast gating of the calcium channels in the terminal ensures that they are effectively opened during the repolarization phase of an action potential. This implies that the distance between open calcium channels is minimized, which is in agreement with the view that multiple calcium channels are needed to release a vesicle in this synapse.
Intracellular Ca2+ regulation by the leech giant glial cell
Authors: Nett Wolfgang , Deitmer Joachim W. 
Published: September 2004

We have measured the intracellular Ca2+ concentration, [Ca2+]i, and the intracellular Na+ concentration, [Na+]i, with the fluorescent dyes fura‐2 (for Ca2+) and SBFI (for Na+) in situ in giant glial cells of the central nervous system of the leech Hirudo medicinalis.
The basal [Ca2+]i was 79 ± 35 nm (n= 27) in cells voltage clamped at −70 to −80 mV, and 75 ± 29 nm (mean ±s.d., n= 82) in unclamped cells at a mean membrane potential of −67 ± 6 mV.
Removal of external Na+ evoked a small reversible [Ca2+]i increase of 29 ± 21 nm (n= 27) in cells voltage clamped at −70 to −80 mV, and of 35 ± 18 nm (n= 37) in unclamped cells. This [Ca2+]i increase, and the time constant of the subsequent [Ca2+]i recovery after Na+ re‐addition, did not change significantly with the holding potential between −110 and −60 mV.
The basal [Na+]i was 5.6 ± 1.3 mm (n= 18). Increasing [Na+]i by inhibiting the Na+‐K+ pump with 100 μm ouabain had no effect on the [Ca2+]i rise upon removal of external Na+.
The time course of recovery from a [Ca2+]i load mediated by voltage‐dependent Ca2+ influx during depolarization in high K+ was unaffected by the removal of external Na+.
Cyclopiazonic acid (10 μm), an inhibitor of the endoplasmic reticulum Ca2+‐ATPase, caused a transient increase in [Ca2+]i of 28 ± 11 nm (n= 5), and significantly slowed the recovery from imposed [Ca2+]i loads.
Iontophoretic injection of orthovanadate, an inhibitor of P‐type ATPases including the plasma membrane Ca2+‐ATPase, caused a persistent increase in the basal [Ca2+]i of 163 ± 101 nm (n= 5) in standard saline, and of 427 ± 338 nm in Na+‐free saline (n= 5). Vanadate injection significantly slowed the recovery from [Ca2+]i loads. Removal of external Na+ during vanadate injection induced an additional, reversible [Ca2+]i increase of 254 ± 64 nm (n= 3).
The results suggest that the low basal [Ca2+]i in these glial cells is predominantly maintained by a Ca2+‐ATPase in the plasma membrane. This ATPase is also the main Ca2+ extruder after an intracellular Ca2+ load, while intracellular stores appear to contribute little to this recovery. A Na+‐Ca2+ exchanger seems to play a minor role in the maintenance of basal [Ca2+]i in these cells, but becomes prominent when the plasma membrane Ca2+‐ATPase is blocked.
Vagally evoked synaptic currents in the immature rat nucleus tractus solitarii in an intact in vitro preparation
Authors: Smith Bret N. , Dou Ping , Barber William D. , Dudek F. Edward 
Published: September 2004

Whole‐cell voltage‐clamp recordings in an in vitro brainstem‐cranial nerve explant preparation were used to assess the local circuitry activated by vagal input to nucleus tractus solitarii (NTS) neurones in immature rats.
All neurones that responded to vagal stimulation displayed EPSCs of relatively constant latency. Approximately 50 % of these also demonstrated variable‐latency IPSCs, and ∼31 % also displayed variable‐latency EPSCs to vagal stimulation. All neurones also had spontaneous EPSCs and IPSCs.
Evoked and spontaneous EPSCs reversed near 0 mV and were blocked by the glutamate AMPA/kainate receptor antagonists 6,7‐nitroquinoxaline‐2,3‐dione (DNQX) or 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) at rest. Evoked EPSCs had rapid rise times (< 1 s) and decayed monoexponentially (τ= 2.04 ± 0.03 ms) at potentials near rest.
At holding potentials positive to ∼−50 mV, a slow EPSC could be evoked in the presence of DNQX or CNQX. This current peaked at holding potentials near −25 mV and was blocked by the NMDA receptor antagonist dl‐2‐amino‐5‐phosphonovaleric acid (AP5). It was therefore probably due to activation of NMDA receptors by vagal afferent fibres.
Fast IPSCs reversed near −70 mV and were blocked by the GABAA receptor antagonist bicuculline. In addition, bicuculline enhanced excitatory responses to vagal stimulation and increased spontaneous EPSC frequency. Antagonists to AMPA/kainate receptors reversibly blocked stimulus‐associated IPSCs and also decreased the frequency of spontaneous IPSCs.
These findings suggest that glutamate mediates synaptic transmission from the vagus nerve to neurones in the immature NTS by acting at non‐NMDA and NMDA receptors. NTS neurones may also receive glutamatergic and GABAergic synaptic input from local neurones that can be activated by vagal input and/or regulated by amino acid inputs from other brainstem neurones.1. Whole‐cell voltage‐clamp recordings in an in vitro brainstem‐cranial nerve explant preparation were used to assess the local circuitry activated by vagal input to nucleus tractus solitarii (NTS) neurones in immature rats.
Regulation of Na+ channels by luminal Na+ in rat cortical collecting tubule
Authors: Palmer Lawrence G. , Sackin Henry , Frindt Gustavo 
Published: September 2004

The idea that luminal Na+ can regulate epithelial Na+ channels was tested in the cortical collecting tubule of the rat using whole‐cell and single‐channel recordings. Here we report results consistent with the idea of Na+ self‐inhibition.
Macroscopic amiloride‐sensitive currents (INa) were measured by conventional whole‐cell clamp. INa was a saturable function of external Na+ concentration ([Na+]o) with an apparent Km of 9 mm. Single channel currents (iNa) were measured in cell‐attached patches. iNa increased with pipette Na+ concentration with an apparent Km of 48 mm. Since INa= (iNa)NPo, the different Km values imply that the channel density (N) and/or open probability (Po) increase as [Na+]o decreases. Reduction of [Na+]o after increasing intracellular Na+ concentration also increased the outward amiloride‐sensitive conductance, consistent with activation of the Na+ channels.
The underlying mechanism was studied by changing pipette Na+ concentration while recording from cell‐attached patches. No increase in NPo was observed, suggesting that the effect is not a direct interaction between [Na+]o and the channel.
[Na+]o was varied outside the patch‐clamp pipette while recording from cell‐attached patches. When amiloride was in the bath to prevent Na+ entry, no change in NPo was observed.
Activation of the channels by hyperpolarization was observed with 140 mm Na+o but not with 14 mm Na+o.
The results are consistent with the concept of self‐inhibition of Na+ channels by luminal Na+. Activation of the channels by lowering [Na+]o is not additive with that achieved by hyperpolarization.
Detection and functional characterization of ryanodine receptors from sea urchin eggs
Authors: Lokuta Andrew J. , Darszon Alberto , Beltrán Carmen , Valdivia Hector H. 
Published: September 2004

Immunoblot analysis, [3H]ryanodine binding, and planar lipid bilayer techniques were used to identify and characterize the functional properties of ryanodine receptors (RyRs) from Lytechinus pictus and Strongylocentrotus purpuratus sea urchin eggs.
An antibody against mammalian skeletal RyRs identified an ≈400 kDa band in the cortical microsomes of sea urchin eggs while a cardiac‐specific RyR antibody failed to recognize this protein. [3H]Ryanodine binding to cortical microsomes revealed the presence of a high‐affinity (Kd= 13 nM), saturable (maximal density of receptor sites, Bmax= 1.56 pmol (mg protein)−1) binding site that exhibited a biphasic response to Ca2+.
Upon reconstitution of cortical microsomes into lipid bilayers, only sparse and unstable openings of a high‐conductance cation channel were detected. Addition of crude sea urchin egg homogenate to the cytosolic (cis side) of the channel increased the frequency of openings and stabilized channel activity. The homogenate‐activated channels were Ca2+ sensitive, selective for Ca2+ over Cs+, and driven by ryanodine into a long‐lived subconductance state that represented ≈40 % of the full conductance level. Homogenate dialysed in membranes with a molecular weight cut‐off <= 2000 lacked the capacity to increase the frequency of RyR openings and to stabilize channel activity.
Direct application of cyclic adenosine diphosphoribose (cADPR) or photolysis of NPE‐cADPR (‘caged’ cADPR) by ultraviolet laser pulses produced transient activation of sea urchin egg RyRs. Calmodulin (CaM) failed to activate reconstituted RyRs; however, channel activity was inhibited by the CaM blocker trifluoroperazine, suggesting that CaM was necessary but not sufficient to sustain RyR activity.
These findings suggest that a functional Ca2+ release unit in sea urchin eggs is a complex of several molecules, one of which corresponds to a protein functionally similar to mammalian RyRs.