bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characteristics of nitric oxide‐mediated cholinergic modulation of calcium current in rabbit sino‐atrial node
Authors: Han X. , Kobzik L. , Severson D. , Shimoni Y. 
Published: September 2004

We have previously shown that nitric oxide (NO) production is essential for cholinergic inhibition of the β‐adrenergic stimulated L‐type calcium current (ICa‐L) in rabbit pacemaker (sino‐atrial node (SAN)) cells. The present experiments demonstrate the presence of constitutive nitric oxide synthase (cNOS) in SAN cells, and characterize the NO‐mediated cholinergic response.
Immunohistochemical staining, using an antibody prepared against endothelial cNOS, demonstrated that this enzyme was present in single myocytes obtained from the SAN.
The activation of cNOS is known to be Ca2+ and calmodulin dependent. Strongly buffering intracellular Ca2+ with the membrane‐permeable chelator BAPTA‐AM (10 μM) significantly reduced (and in some cases abolished) the attenuation of ICa‐L by the muscarinic agonist carbamylcholine (CCh). In contrast, the CCh‐induced activation of an outward K+ current, IK,ACh, was unaffected by buffering of [Ca2+]i. The calmodulin inhibitor 48/80 (20 μM) also abolished the attenuation of ICa‐L by CCh, with no change in the activation of IK,ACh.
Neither thapsigargin nor ryanodine (5‐10 μM), agents which deplete intracellular Ca2+ stores, significantly changed the attenuation of ICa‐L by CCh.
Pertussis toxin (PTX) completely abolished both the inhibitory action of CCh on ICa‐L and the activation of IK,ACh. This establishes that a PTX‐sensitive GTP‐binding protein links the muscarinic receptor to NO synthase activation in SAN cells.
Our hypothesis is that NO leads to activation of a cyclic GMP (cGMP)‐activated phosphodiesterase (PDE II) as a mechanism for enhanced cyclic AMP breakdown and ICa‐L attenuation. This was supported by showing that a specific inhibitor of PDE II, erythro‐9‐(2‐hydroxy‐3‐nonyl) adenine (EHNA), blocks the effect of CCh on ICa‐L, but not on IK,ACh. Using reverse transcriptase‐polymerase chain reaction techniques, we have established that PDE II is the dominant cyclic nucleotide phosphodiesterase isoform in SAN cells.
Inhibition of Ca2+‐dependent K+ channels in rat carotid body type I cells by protein kinase C
Authors: Peers C. , Carpenter E. 
Published: September 2004

Whole‐cell patch clamp recordings were used to investigate the effects of protein kinase C (PKC) activation on K+ and Ca2+ currents in type I cells isolated from the rat carotid body.
Pretreatment of cells for 10 min at 37 °C with 4α‐phorbol 12,13‐didecanoate (4α‐PDD, 200 nm), a phorbol ester which does not activate PKC, did not affect K+ current density as compared with cells pretreated with vehicle alone. By contrast, identical pretreatment with 200 nm 12‐O‐teradecanoylphorbol‐13‐acetate (TPA, a PKC activator) dramatically reduced K+ current density in type I cells. This effect was prevented by co‐incubation of cells with the PKC inhibitor bisindolylmaleimide (BIM, 3 μm).
The sensitivity of K+ currents to inhibition by 200 μm Cd2+ (indicative of the presence of Ca2+‐dependent K+ channels) was markedly reduced in TPA‐treated cells as compared with sham‐treated cells, cells treated with 4α‐PDD, and cells treated with both TPA and BIM. Cd2+‐resistant K+ current densities were of similar magnitude in all four groups of cells, as were the input resistances determined over the voltage range −100 mV to −50 mV.
Ca2+ channel current density was not significantly different in type I cells pretreated with 200 nm 4α‐PDD as compared with cells treated with the same concentration of TPA.
The degree of inhibition of K+ currents caused by hypoxia (Po2 15–20 mmHg) was unaltered by pretreatment of cells with 3 μm BIM.
The resting membrane potential of cells pretreated with TPA was depolarized as compared with controls, and the Ca2+‐dependent K+ channel inhibitor iberiotoxin (20 nm) failed to depolarize these cells further.
Our results suggest that activation of PKC causes a marked, selective inhibition of Ca2+‐dependent K+ currents in type I carotid body cells, but that PKC activation is unlikely to account for inhibition of these channels by acute hypoxia.
Na+‐activated K+ channels in small dorsal root ganglion neurones of rat
Authors: Bischoff Ulrike , Vogel Werner , Safronov Boris V. 
Published: September 2004

Whole‐cell Na+‐activated K+ (KNa) channel currents and single KNa channels were studied with the patch‐clamp method in small (20‐25 μm) dorsal root ganglion (DRG) neurones in slices of rat dorsal root ganglia.
The whole‐cell KNa channel current was identified as an additional K+‐selective leakage current which appeared after cell perfusion with internal solutions containing different [Na+]. The concentration for half‐maximal activation of KNa channel current was 39 mM and the Hill coefficient was 3.5. At [Na+]i above 12 mM, KNa channel current dominated the unspecific leakage current. The ratio of maximum KNa channel current to unspecific leakage current was 45.
KNa channel current was not activated by internal Li+. It was suppressed by external 20 mM Cs+ but not by 10 mM tetraethylammonium.
Single KNa channels with a conductance of 142 pS in 155 mM external K+ (K+o)‐85 mM internal K+ (K+i) solutions were observed at a high density of about 2 channels μm−2.
In two‐electrode experiments, a direct correlation was seen between development of whole‐cell KNa channel current and activation of single KNa channels during perfusion of the neurone with Na+‐containing internal solution.
Under current‐clamp conditions, KNa channels did not contribute to the action potential. However, internal perfusion of the neurone with Na+ shifted the resting potential towards the equilibrium potential for K+ (EK). Varying external [K+] indicated that in neurones perfused with Na+‐containing internal solution the resting potential followed the EK values predicted by the Nernst equation over a broader voltage range than in neurones perfused with Na+‐free solution.
It is concluded that the function of KNa channels has no links to firing behaviour but that the channels could be involved in setting or stabilizing the resting potential in small DRG neurones.
A characterization of muscarinic receptor‐mediated intracellular Ca2+ mobilization in cultured rat hippocampal neurones
Authors: Irving Andrew J. , Collingridge Graham L. 
Published: September 2004

The properties of muscarinic receptor‐mediated Ca2+ mobilization were investigated in hippocampal cultures using fluorescent imaging techniques.
Somatic responses to carbachol (1‐10 μm) were observed in 21 % of neurones under control conditions (5.4 mM K+, 1.8 mM Ca2+, 0.5‐1 μm tetrodotoxin). Smaller responses were observed in Ca2+‐free medium.
In cells where responses to carbachol were absent under control conditions, responses were often observed following depolarization with high extracellular K+ (16.2‐25 mM). These responses decreased in magnitude with time after the depolarizing episode. Mobilization of Ca2+ from stores using caffeine (50 mM) exhibited similar properties.
Carbachol responses were greatly facilitated in the presence of moderate elevations in extracellular K+ or Ca2+ levels (2‐ or 3‐fold, respectively). These conditions were usually, but not always, associated with a small increase in cytosolic Ca2+ levels (< 50 nM).
Muscarinic responses in 10.8 mM K+ were inhibited by 80–95 % in the presence of the L‐type voltage‐gated Ca2+ channel antagonists nitrendipine (2‐5 μm) or nifedipine (10 μm). Depletion of intracellular Ca2+ stores with thapsigargin (2‐10 μm) blocked responses.
Oscillatory Ca2+ mobilizing responses were observed in some cells. Their expression was facilitated by moderate cytosolic Ca2+ elevations and by increasing the duration of carbachol exposure.
Ca2+ mobilizing responses were also observed in dendritic regions. These were smaller than somatic responses, but had faster decay kinetics.
In conclusion, muscarinic receptor‐mediated Ca2+ mobilization in cultured hippocampal neurones shows a strong Ca2+ dependence. Moderate intracellular Ca2+ rises greatly facilitate muscarinic responses and uncover, in some cells, oscillatory Ca2+ mobilization. These effects appear to reflect the loading state of intracellular Ca2+ stores.
Dorsal medullary 5‐HT3 receptors and sympathetic premotor neurones in the rat
Authors: Sévoz‐Couche Caroline , Nosjean Anne , Franc Bernard , Hamon Michel , Laguzzi Raul 
Published: September 2004

Our aim was to determine whether the cardiovascular neurones in the rostro‐ventrolateral medulla (CV‐RVLM neurones) were involved in the sympathoexcitation induced by stimulation of 5‐HT3 receptors in the region of the nucleus tractus solitarii (NTS). Experiments were performed in pentobarbitone‐anaesthetized rats, artificially ventilated and paralysed with pancuronium bromide.
Using extracellular recordings, different types of RVLM neurones were characterized: cardiovascular (CV), ventilation‐related and baroreflex‐insensitive (unidentified) neurones. The CV‐RVLM cells were further subdivided into three populations according to their axonal conduction velocities: A (1.2 ± 0.1 m s−1), B (2.5 ± 0.2 m s−1) and C (6.8 ± 1.1 m s−1).
Only the CV‐RVLM neurones of the A and B categories were partially inhibited (‐30 %) by a hypotensive dose (2.5 μg kg−1 i.v.) of clonidine.
Microinjections into the region of the commissural NTS of 1‐(m‐chlorophenyl)‐biguanide (CPBG, 2 nmol), a selective 5‐HT3 receptor agonist, elicited an increase in both lumbar sympathetic nerve discharge (SND) and arterial pressure. In addition, this treatment produced a marked excitation of CV‐RVLM neurones of the A and B categories, without affecting those of the C type, as well as ventilation‐related and unidentified RVLM cells.
The activity of the CV neurones in the caudo‐ventrolateral part of the medulla oblongata (CV‐CVLM) was not modified by 5‐HT3 receptor stimulation in the NTS.
Prior intra‐NTS microinjections of ondansetron (300 pmol, a selective 5‐HT3 receptor antagonist) into the region of the commissural NTS prevented the excitation of A and B CV‐RVLM neurones induced by CPBG.
Intracarotid administration of saline saturated with CO2 (chemoreceptor activation) elicited both an increase in the SND and an excitation of the clonidine‐insensitive CV‐RVLM neurones of the C type, without affecting A and B neurones.
In conclusion, the sympathoexcitation elicited following 5‐HT3 receptor stimulation in the region of the commissural NTS of pentobarbitone‐anaesthetized rats seems to result from the excitation of two different pools of clonidine‐sensitive CV‐RVLM neurones. These neurones are apparently not involved in the sympathetic component of the chemoreceptor reflex.
A repolarization‐induced transient increase in the outward current of the inward rectifier K+ channel in guinea‐pig cardiac myocytes
Authors: Ishihara Keiko , Ehara Tsuguhisa 
Published: September 2004

Outward currents of the inwardly rectifying K+ current (IKir) in guinea‐pig ventricular myocytes were studied in the presence of 1 mM intracellular free Mg2+ using the whole‐cell patch‐clamp technique.
During repolarizing voltage steps following a large depolarizing pulse (> 0 mV), outward IKir increased transiently at voltages positive to the K+ equilibrium potential (EK, ‐84 mV for 5.4 mM extracellular [K+]). The rising phase was almost instantaneous, while the decay was exponential. The decay rate was faster at voltages closer to EK (time constants, 33.9 ± 9.8 and 4.8 ± 1.4 ms at ‐30 and ‐50 mV, respectively).
The transient outward IKir was absent when the preceding depolarization was applied from ‐40 mV. Larger transient currents developed as the voltage before the depolarization was shifted to more hyperpolarized levels.
Shift of the depolarizing voltage from > 0 mV to more negative ranges diminished the amplitudes of transient outward IKir and instantaneous inward IKir during the subsequent repolarizing steps positive and negative to EK, respectively. Since blockage of IKir by internal Mg2+ occurs upon large depolarization, and the block is instantaneously relieved at voltages negative to EK, the rising phase of the transient outward IKir was attributed to the relief of Mg2+ block at voltages positive to EK. Transient outward IKir was absent when intracellular [Mg2+] was reduced to 10 μM or lower.
Prolongation of the repolarizing voltage step increased the amplitude of time‐dependent inward IKir during the subsequent hyperpolarization, indicating the progress of a gating process (presumably the channel block by intracellular polyamine) during the decaying phase of outward IKir.
Progressive prolongation of the depolarizing pulse (> 0 mV) from 100 to 460 ms decreased the transient outward IKir amplitude during the subsequent repolarizing step due to slow progress of the gating (polyamine block) at > 0 mV.
Current‐voltage relations measured using repolarizing ramp pulses (‐3.4 mV ms−1) showed an outward hump at around ‐50 mV, the magnitude of which increased as the voltage before the conditioning depolarization (10 mV) was shifted to more negative levels. With slower ramp speeds (‐1.5 and ‐0.6 mV ms−1), the hump was depressed at voltages near EK.
Our study suggests that the relief of Mg2+ block may increase outward IKir during repolarization of cardiac action potentials, and that the resting potential, the level/duration of action potential plateau and the speed of repolarization influence the outward IKir amplitude.
A kinetic model incorporating a competition between polyamine block and Mg2+ block was able to account for the time dependence of outward IKir.
Requirement of rapid Ca2+ entry and synaptic activation of metabotropic glutamate receptors for the induction of long‐term depression in adult rat hippocampus
Authors: Otani Satoru , Connor John A. 
Published: September 2004

During block of γ‐aminobutyric acid‐A‐mediated inhibition, low‐frequency stimulation (2 Hz, 900 pulses) to Schaffer collateral‐CA1 neuron synapses of adult rat hippocampus induced an N‐methyl‐D‐aspartate receptor‐independent, postsynaptic Ca2+‐dependent depression of synaptic strength (long‐term depression; LTD).
Ratio imaging with fura‐2 revealed moderate dendritic [Ca2+] increases (≈500 nM) during only the initial ≈30 s of the 7.5 min stimulation period. Conditioning for 30 s was, however, insufficient to induce LTD.
The [Ca2+] changes were insensitive to the metabotropic glutamate receptor (mGluR) antagonist (+)‐α‐methyl‐4‐carboxyphenylglycine (MCPG). MCPG, however, completely blocked LTD when present during conditioning.
The [Ca2+] changes were abolished by postsynaptic hyperpolarization (‐110 mV at the soma). Hyperpolarizing neurons to ‐110 mV during conditioning significantly attenuated LTD induction.
LTD induction was also blocked by the postsynaptic presence of the protein kinase C inhibitor peptide PKC(19‐36).
These results suggest that LTD induction in adult hippocampus by prolonged low‐frequency stimulation depends on both a rapid Ca2+ influx through voltage‐sensitive channels and synaptic stimulation of mGluRs which may be coupled to phospholipase C.
Activation of cAMP‐dependent Cl− currents in guinea‐pig Paneth cells without relevant evidence for CFTR expression
Authors: Tsumura Takehiko , Hazama Akihiro , Miyoshi Takashi , Ueda Shunji , Okada Yasunobu 
Published: September 2004

To determine whether Paneth cells exhibit functional expression of cAMP‐activated Cl− currents and molecular expression of the cystic fibrosis transmembrane conductance regulator (CFTR), we applied whole‐cell patch clamp and single‐cell mRNA analysis by reverse transcription (RT) followed by polymerase chain reaction (PCR) amplification to single Paneth cells in crypts isolated from the guinea‐pig small intestine.
Prominent activation of Cl− currents was consistently observed after stimulation with dibutyryl cAMP and forskolin or with vasoactive intestinal polypeptide (VIP). The cAMP‐activated Cl− current was inhibited by removal of intracellular ATP or administration of an inhibitor of protein kinase A.
Many of the biophysical and pharmacological properties of the currents were phenotypically similar to those of the CFTR Cl− channel, such as the ohmic current‐voltage relationship, the anion selectivity with a Type III sequence (Br− > Cl− > I−≫ F−≥ gluconate−), I−‐induced blockage, insensitivity to a stilbene‐derivative Cl− channel blocker, and sensitivity to a carboxylate analogue Cl− channel blocker. The sensitivity of the current to glibenclamide was, however, much weaker than that reported for the CFTR Cl− channel current. In contrast to the time independence of CFTR currents, the inward component of the Paneth cell Cl− currents exhibited inactivation kinetics.
Expression of CFTR mRNA could not be detected by RT‐PCR analysis in almost all single Paneth cells, although its expression was consistently detected at the whole‐crypt level. The presence of a small number of CFTR‐expressing epithelial cells, which were scattered both in villi and crypts but not at the crypt base where Paneth cells were located, was demonstrated by immunocytochemistry.
Taken together, it appears that guinea‐pig Paneth cells functionally express cAMP‐activated Cl− conductance without relevant evidence for molecular expression of CFTR. Functional expression of VIP receptors in the Paneth cells was also demonstrated.
Electrophysiological and morphological heterogeneity of neurons in slices of rat suprachiasmatic nucleus
Authors: Pennartz C. M. A. , De Jeu M. T. G. , Geurtsen A. M. S. , Sluiter A. A. , Hermes M. L. H. J. 
Published: September 2004

Whole cell patch clamp recordings of neurons in slices of the suprachiasmatic nucleus (SCN) were made in order to assess their electrophysiological and morphological heterogeneity. This assessment was accomplished by (i) quantification of intrinsic membrane properties recorded in current clamp mode, (ii) studying frequency distributions of these properties, (iii) grouping of cells based on visual inspection of data records, and (iv) use of cluster analysis methods.
Marked heterogeneity was found in the resting membrane potential, input resistance, time constant, rate of frequency adaptation, size of rebound depolarization (low‐threshold Ca2+ potential) and regularity of firing. The frequency distribution of these membrane properties deviated significantly from a normal distribution. Other parameters, including spike amplitude and width, amplitude and rising slope of the spike after‐hyperpolarization (AHP) and amplitude of the spike train AHP, showed considerable variability as well but generally obeyed a normal distribution.
Visual inspection of the data led to partitioning of cells into three clusters, viz. cluster I characterized by monophasic spike AHPs and irregular firing in the frequency range from 1.5 to 5.0 Hz; cluster II with biphasic spike AHPs and regular firing in the same range; and cluster III with large rebound depolarizations and biphasic spike AHPs. In a post hoc analysis, these clusters also appeared to differ in other membrane properties. This grouping was confirmed by hierarchical tree clustering and multidimensional scaling.
The light microscopic properties of recorded neurons were studied by biocytin labelling. Neurons had monopolar, bipolar or multipolar branching patterns and were often varicose. Axons sometimes originated from distal dendritic segments and usually branched into multiple collaterals. Many cells with extra‐SCN projections also possessed intranuclear axon collaterals. We found no morphological differences between clusters except that cluster III neurons possessed more axon collaterals than cluster I or II cells.
These results suggest that SCN neurons are heterogeneous in some basic as well as active membrane properties and can be partitioned into at least three clusters. Cluster I and II cells fire spontaneously in a regular and irregular mode, respectively, and sustain prolonged spike trains. In contrast, cluster III cells have low firing rates but may adopt a burst‐like firing mode when receiving appropriate input. While all clusters transmit output to target cells within and outside SCN, cluster III cells in particular are suggested to affect excitability of large numbers of SCN neurons by their extensive local network of axon collaterals.
Autaptic inhibitory currents recorded from interneurones in rat cerebellar slices
Authors: Pouzat Christophe , Marty Alain 
Published: September 2004

While the presence of autapses in the brain is indicated by a large body of morphological evidence, the functional role of these structures has remained unclear. To probe for autaptic currents, we have recorded current responses following short somatic depolarizing pulses in Cl−‐loaded interneurones (stellate and basket cells) from rat cerebellar slices (animals aged 27‐39 days).
In ≈20 % of the recordings, fluctuating inward current transients were obtained with a latency of 1.15‐2.45 ms (measured from the peak of the depolarization‐induced Na+ current; n= 10).
These transients were blocked by bicuculline and were sensitive to the extracellular Ca2+ concentration.
Assuming low release probability, as suggested by the high failure rate (0.65‐0.92, n= 10), quantal sizes ranging from 21 to 178 pA (‐70 mV; n= 10) were calculated from a variance analysis of autaptic current amplitudes.
We conclude that ≈20 % of interneurones have a functional autapse. Autaptic currents may inhibit firing of interneurones during high frequency discharges.
Overexpression of nerve growth factor in the heart alters ion channel activity and β‐adrenergic signalling in an adult transgenic mouse
Authors: Heath B. M. , Xia J. , Dong E. , An R. H. , Brooks A. , Liang C.‐S. , Federoff H. J. , Kass R. S. 
Published: September 2004

The electrophysiological and pharmacological properties of cardiac myocytes from the hearts of adult transgenic mice engineered to overexpress nerve growth factor (NGF) in the heart were studied.
There was a 12 % increase in the ventricular myocyte capacitance in NGF myocytes consistent with cardiac hypertrophy, and action potential duration at 90 % repolarization (APD90) was prolonged by 142 % compared with wild‐type (WT) myocytes. This was due, at least in part, to a decrease in the density of two K+ currents, Ito and IK(ur), which were significantly reduced in NGF mice with no change in their electrophysiological characteristics. We found no change in the current density or electrophysiological properties of the L‐type Ca2+ current.
The effect on Ito and IK(ur) of TEA and 4‐aminopyridine (4‐AP) was not different in cells isolated from WT and NGF mice. The prolongation of APD observed in NGF cells was mimicked in WT cells by exposure to 1 mm 4‐AP, which partially blocked Ito, completely blocked IK(ur) and increased APD90 by 157 %.
The isoprenaline‐induced increase in ICa was significantly smaller in NGF myocytes than in WT myocytes. This was not due to a decrease in β‐adrenergic receptor (β‐AR) density, as this was increased in NGF tissue by 55 %. Analysis of β‐AR subtypes showed that this increase was entirely due to an increase in β2‐AR density with no change in β1‐ARs.
The response of the β‐AR‐coupled adenylyl cyclase system to isoprenaline, Gpp(NH)p and forskolin was studied by measuring cAMP production. In NGF tissue, isoprenaline elicited a significantly smaller response than in WT myoyctes and this was not due to reduced adenylyl cyclase activity as the responses of NGF tissue to guanylylimidodiphosphate (Gpp(NH)p) and forskolin were unaffected.
In conclusion, the overexpression of NGF in the mouse heart resulted in a decrease in the current density of two K+ channels, which contributed to the prolongation of the cardiac action potential. Despite an increase in β2‐AR density in the hearts of the NGF mice, the response to isoprenaline was diminished, and this was due to an uncoupling of the β‐ARs from the intracellular signalling cascade. These potentially pathological changes may be involved in the occurrence of ventricular arrhythmias in cardiac hypertrophy and failure, and this mouse provides a novel model in which to study such changes.