bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of acetylcholine release by intracellular acidification of developing motoneurons in Xenopus cell cultures
Authors: Chen Yu‐Hwa , Wu Mei‐Lin , Fu Wen‐Mei 
Published: September 2004

The effects of intracellular pH changes on the acetylcholine (ACh) release and cytoplasmic Ca2+ concentration at developing neuromuscular synapses were studied in Xenopus nerve‐muscle co‐cultures.
Spontaneous and evoked ACh release of motoneurons was monitored by using whole‐cell voltage‐clamped myocytes. Intracellular alkalinization with 15 mm NH4Cl slightly reduced the frequency of spontaneous synaptic currents (SSCs). However, cytosolic acidification following withdrawal of extracellular NH4Cl caused a marked and transient increase in spontaneous ACh release.
Another method of cytosolic acidification was used in which NaCl in Ringer solution was replaced with weak organic acids. The increase in spontaneous ACh release paralleled the level of intracellular acidification resulting from addition of these organic acids. Acetate and propionate but not isethionate, methylsulphate and glucuronate, caused an increase in intracellular pH and a marked increase in spontaneous ACh release.
Impulse‐evoked ACh release was slightly augmented by intracellular alkalinization and inhibited by cytosolic acidification.
Cytosolic acidification was accompanied by an elevation in the cytoplasmic Ca2+ concentration ([Ca2+]i), resulting from both external Ca2+ influx and intracellular Ca2+ mobilization. In contrast, the increase in [Ca2+]i induced by high K+ was inhibited by cytosolic acidification.
We conclude that cytosolic acidification regulates spontaneous and evoked ACh release differentially in Xenopus motoneurons, increasing spontaneous ACh release but inhibiting evoked ACh release.
Interaction of memantine and amantadine with agonist‐unbound NMDA‐receptor channels in acutely isolated rat hippocampal neurons
Authors: Sobolevsky Alexander I. , Koshelev Sergey G. , Khodorov Boris I. 
Published: September 2004

Using whole‐cell patch‐clamp techniques, the mechanisms of NMDA channel blockade by amino‐adamantane derivatives (AADs) memantine (3,5‐dimethyl‐aminoadamantane, MEM) and amantadine (1‐aminoadamantane, AM) have been studied in rat hippocampal neurons acutely isolated by the vibrodissociation method. A rapid concentration‐jump technique was used to replace superfusing solutions.
The aspartate (Asp)‐induced channel opening greatly accelerated but was not a prerequisite for the recovery from the block by MEM: it was able to leave the channel without agonist assistance. The co‐agonist (glycine) as well as the competitive NMDA antagonist dl‐2‐amino‐7‐phosphonoheptanoic acid (APV), did not affect this recovery. Membrane depolarization accelerated it, strongly suggesting that this process proceeded via the hydrophilic pathway of the channel.
A comparison of the kinetics of the recovery from the block by AADs in the presence and absence of the agonist prompted a hypothesis that the blocker trapped in the channel increased the probability of its transition to the open state.
Both MEM and AM were able to block NMDA channels not only in the presence but also in the absence of Asp, although in the latter case the effective blocking concentrations were much higher and the rate of the block development was much smaller than in the former case. The extent of the block increased with the duration of the blocker application. Glycine enhanced this block, while APV attenuated it. The MEM‐induced blockade of agonist‐unbound channels was enhanced by membrane hyperpolarization and weakened by external Mg2+. These findings strongly suggested that the blocker reached its binding sites via the same hydrophilic pathway both in the presence and absence of the agonist.
A comparative analysis of the channel unblocking kinetics in the presence of Asp after their blockade with or without the agonist assistance led us to conclude that in the two cases AADs were bound to the same blocking sites in the channel.
The channel opening rate of adult‐ and fetal‐type mouse muscle nicotinic receptors activated by acetylcholine
Authors: Maconochie David J. , Steinbach Joe Henry 
Published: September 2004

expressing either the fetal (Q‐F18) or the adult (Q‐A33) complement of nicotinic acetylcholine receptor subunits derived from mouse skeletal muscle. Pulses of ACh were applied to outside‐out patches of cell membrane by means of a fast perfusion system, at concentrations from 100 nM to 10 mM. We obtained current records with intracellular potentials of ‐60 and +40 mV. The goal of this study was to estimate the channel opening rate.
By fitting sums of exponentials to averaged responses, we estimated the rate of development of the current on the application of acetylcholine. The rate constant of the predominant exponential component (the on‐rate) ranges over 3 orders of magnitude, from around 100 s−1 (fetal) at low concentrations of ACh to over 100 000 s−1 (fetal and adult) at the highest concentrations.
We establish that our measurement of the on‐rate is not limited by technical constraints, and can therefore be related to the rate constants of a kinetic scheme. Our observations are consistent with a model having a rate‐limiting channel opening step with a forwards rate constant (β) of 80 000 s−1 on average for adult receptors and 60 000 s−1 for fetal receptors, and a minimum opening to closing ratio (β/α) of around 33 (adult) or 50 (fetal). The channel opening rate, β, varies from around 30 000 s−1 to well over 100 000 s−1 for different patches. The large variation cannot all be ascribed to errors of measurement, but indicates patch to patch variation.
Evidence that mitochondria buffer physiological Ca2+ loads in lizard motor nerve terminals
Authors: David Gavriel , Barrett John N. , Barrett Ellen F. 
Published: September 2004

Changes in cytosolic and mitochondrial [Ca2+] produced by brief trains of action potentials were measured in motor nerve terminals using a rapidly scanning confocal microscope. Cytosolic [Ca2+] was measured using ionophoretically injected Oregon Green BAPTA 5N (OG‐5N). Mitochondrial [Ca2+] was measured using rhod‐2, bath loaded as dihydrorhod‐2.
In response to 100‐250 stimuli at 25‐100 Hz the average cytosolic [Ca2+] showed an initial rapid increase followed by a much slower rate of increase. Mitochondrial [Ca2+] showed no detectable increase during the first fifteen to twenty stimuli, but after this initial delay also showed an initially rapid rise followed by a slower rate of increase. The onset of the increase in mitochondrial [Ca2+] coincided with the slowing of the rate of rise of cytosolic [Ca2+]. The peak levels of cytosolic and mitochondrial [Ca2+] both increased with increasing frequencies of stimulation.
When stimulation terminated, the initial rate of decay of cytosolic [Ca2+] was much more rapid than that of mitochondrial [Ca2+].
After addition of carbonyl cyanide m‐chlorophenyl hydrazone (CCCP, 1‐2 μm) to dissipate the proton electrochemical gradient across the mitochondrial membrane, cytosolic [Ca2+] rose rapidly throughout the stimulus train, reaching levels much higher than normal. CCCP inhibited the increase in mitochondrial [Ca2+].
These results suggest that mitochondrial uptake of Ca2+ contributes importantly to buffering presynaptic cytosolic [Ca2+] during normal neuromuscular transmission.
N‐ and L‐type calcium channel involvement in depolarization‐induced suppression of inhibition in rat hippocampal CA1 cells
Authors: Lenz R. A. , Wagner J. J. , Alger B. E. 
Published: September 2004

We investigated depolarization‐induced suppression of inhibition (DSI) under whole‐cell voltage clamp in CA1 pyramidal neurons of rat hippocampal slices. DSI, a transient reduction in monosynaptic evoked GABAAergic IPSCs lasting for ∼1 min, was induced by depolarizing the pyramidal cell to −10 or 0 mV for 1 or 2 s.
Raising extracellular Ca2+ concentration increased DSI, and varying the DSI‐inducing voltage step showed that the voltage dependence of DSI was like that of high‐voltage‐activated Ca2+ channels.
The P‐ and Q‐type Ca2+ channel blocker ω‐agatoxin TK (200 nm and 1 μm) and the R‐ and T‐type Ca2+ channel blocker Ni2+ (100 μm) reduced IPSCs without reducing DSI.
The specific N‐type Ca2+ channel antagonist ω‐conotoxin GVIA (250 nm) reduced IPSC amplitudes and almost completely abolished DSI.
Blocking L‐type Ca2+ channels with nifedipine (10 μm) had no effect on IPSCs or DSI induced by our standard protocol, but reduced DSI induced by the unclamped Na+‐ and Ca2+‐dependent spikes that occurred when 2(triethylamino)‐N‐(2,6‐dimethylphenyl)acetamide (QX‐314) was omitted from the recording pipette solution.
Although intracellular Ca2+ stores were not measured, DSI was not affected by cyclopiazonic acid (CPA, 20–40 μm), a blocker of Ca2+ uptake into intracellular stores.
We conclude that DSI is initiated by Ca2+ influx through N‐ and, under certain conditions, L‐type Ca2+ channels.
The erg inwardly rectifying K+ current and its modulation by thyrotrophin‐releasing hormone in giant clonal rat anterior pituitary cells
Authors: Bauer Christiane K. 
Published: September 2004

The voltage‐dependent inwardly rectifying K+ current (IK,IR) of clonal rat anterior pituitary cells (GH3/B6) was investigated in solutions with physiological K+ gradient using giant polynuclear cells.
I
K,IR was isolated by the use of the selective erg (ether‐à‐go‐go‐related gene) channel blocker E‐4031. In external 5 mM K+ solution, IK,IR carried steady‐state outward current in the potential range between –60 and 0 mV, with a maximum current amplitude at –40 mV. Negative to the K+ equilibrium potential, EK, large transient inward currents occurred.
A selective pharmacological block of IK,IR induced a sustained depolarization of the membrane potential when Ca2+ action potentials were blocked, confirming the contribution of IK,IR to the resting membrane potential of GH3/B6 cells.
Thyrotrophin‐releasing hormone (TRH) reduced effectively the sustained outward and the transient inward IK,IR. The magnitude of a TRH‐induced depolarization of the membrane potential was consistent with an almost complete reduction of IK,IR.
The results demonstrate that the TRH‐induced reduction of IK,IR is able to mediate the resting potential depolarization, suggesting that the increase in the frequency of action potentials occurring during the second phase of the TRH response in GH cells should be sustained by IK,IR inhibition. Moreover, this is the first evidence of a ligand‐induced physiological modulation of an erg‐mediated current.
Layer‐specific NO dependence of long‐term potentiation and biased NO release in layer V in the rat auditory cortex
Authors: Wakatsuki Hidemitsu , Gomi Hiroshi , Kudoh Masaharu , Kimura Shinji , Takahashi Kota , Takeda Masayuki , Shibuki Katsuei 
Published: September 2004

We investigated the role of nitric oxide (NO) in the induction of long‐term potentiation (LTP) in slices prepared from the rat auditory cortex.
Tetanic stimulation of layer IV elicited LTP of field potentials in layer II–III (LTPII–III) and in layer V (LTPV). The magnitude of LTPII–III measured at 30 min after tetanic stimulation was 171 ± 9 % (n= 15, mean ± s.e.m.) of the control measured before tetanic stimulation, while that of LTPV was 138 ± 3 % (n= 17).
NO synthase (NOS) inhibitors had no apparent effect on LTPII–III, but LTPV was significantly suppressed (P < 0.001). This suppression of LTPV was significantly antagonized by a NO donor (P < 0.001) or a cGMP analogue (P < 0.001).
Small non‐pyramidal neurones in the auditory cortex were stained with an anti‐neuronal NOS antibody. More neurones were stained with the antibody in the deeper cortical layers.
We measured neocortical NO release with electrochemical NO probes. Layer IV stimulation elicited significantly more NO release in layer V than in layer II–III (P < 0.001). The amplitude of the increase in NO concentration elicited by stimulation at 20 Hz for 5 s was 380 ± 14 pM (n= 55) in layer V and 55 ± 8 pM (n= 5) in layer II–III.
NO release in layer V was partially but significantly suppressed by non‐NMDA (P < 0.002) or NMDA (P < 0.002) receptor antagonists. Simultaneous application of the antagonists of the two types blocked NO release almost completely.
These results clearly indicate the NO dependence of the induction of LTPV, and the greater NO release in the deeper layer of the rat auditory cortex.
Characterization of a hyperpolarization‐activated current in dedifferentiated adult rat ventricular cells in primary culture
Authors: Farès Nassim , Bois Patrick , Lenfant Jacques , Potreau Daniel 
Published: September 2004

The presence of a hyperpolarization‐activated pacemaker (If)‐like current was tested in dedifferentiated adult rat ventricular myocytes up to 12 days in primary culture with the whole‐cell patch clamp technique.
An If‐like current was found and characterized on freshly isolated and cultured ventricular cells. Both activation and density of the current varied in relation to the stage of dedifferentiation. The current was activated from ‐92.0 ± 2.5 and ‐63.0 ± 1.0 mV at the beginning (4‐day‐cultured cells) and end of the dedifferentiation process (12 days), respectively. Its density measured at ‐170 mV progressively increased from ‐2.34 ± 0.36 to ‐6.12 ± 0.64 pA pF−1 between the two farthest stages of cellular remodelling. In freshly isolated cells the current was activated at ‐108.0 ± 1.5 mV and its current density measured at ‐170 mV was ‐1.97 ± 0.56 pA pF−1.
The current was blocked by extracellular CsCl (3 mM) in a voltage‐dependent manner. Modification of reversal potentials obtained at various values of [K+]o (5.4 or 25 mM) and [Na+]o (140 or 30 mM) suggests that the current was carried by both K+ and Na+ ions.
It is concluded that the hyperpolarization‐activated inward current, recorded in freshly isolated and in cultured ventricular cells has characteristics similar to those of If. In adult rat ventricular cells it is activated in a non‐physiological potential range, but can be elicited in a more physiological range when the cells are remodelled through a dedifferentiated way. It is suggested that such a current could be implicated in ventricular arrhythmias developed in pathological events.
Heterogeneous presynaptic Ca2+ channel types triggering GABA release onto medial preoptic neurons from rat
Authors: Haage David , Karlsson Urban , Johansson Staffan 
Published: September 2004

Voltage‐dependent Ca2+ channels triggering GABA release onto neurons from the medial preoptic nucleus of rat were investigated. Acutely dissociated neurons with adherent functional synaptic terminals were investigated by tight‐seal whole‐cell recordings from the postsynaptic cells.
Spontaneous current events similar to miniature postsynaptic currents were recorded. They were blocked by bicuculline (100 μM), showed a roughly unimodal amplitude distribution and a reversal potential consistent with a Cl− current, and were therefore attributed to GABAA receptors activated by synaptically released GABA.
Application of 140 mM KCl, expected to depolarize presynaptic terminals, evoked currents that were ascribed to a more massive release of GABA. The KCl‐induced synaptic currents were abolished in Ca2+‐free solutions and showed a roughly hyperbolic relation to external Ca2+ concentration with half‐saturation at 0.15 mm. They further depended on the concentration of applied KCl in a way expected for high‐threshold Ca2+ channels.
The KCl‐evoked synaptic currents were completely blocked by 200 μm Cd2+, but only partially blocked by 200 μm Ni2+.
The KCl‐evoked synaptic currents were insensitive to the L‐type Ca2+ channel blocker nifedipine (10 μm). However, the synaptic currents were sensitive to either 1 μmω‐conotoxin GVIA, 25 nmω‐agatoxin IVA or 1 μmω‐conotoxin MVIIC.
It was concluded that, in many presynaptic terminals, the Ca2+ influx triggering GABA release onto medial preoptic neurons is mainly mediated by one predominant type of high‐ threshold Ca2+ channel that may be either of N‐, P‐ or Q‐type.
It was further concluded that terminals with similar predominant channel types often were clustered on the same postsynaptic cell.
Effect of chronic mitral valve damage on activity of pulmonary rapidly adapting receptors in the rabbit
Authors: Gunawardena S. , Bravo E. , Kappagoda C. T. 
Published: September 2004

The effects of acute pulmonary venous congestion on the activity of rapidly adapting receptors (RARs) were determined in intact (control and sham‐operated) rabbits and in rabbits 6 and 12 weeks after surgical destruction of the mitral valve.
Destruction of the mitral valve increased the mean left atrial pressure (LAP) by approximately 2·6 and 3·8 mmHg, 6 and 12 weeks after surgery, respectively. These changes were accompanied by significant increases in left ventricular weight. The effect of acute increments in LAP on RAR activity was examined against this background of chronic pulmonary venous congestion.
In intact control and sham‐operated animals RAR activity increased from 48·8 ± 0·9 to 83·5 ± 3·6 and 121·1 ± 4·7 action potentials min−1 when the LAP was raised by 5 and 10 mmHg, respectively, above control values. Six weeks after surgery only 40 % of RARs were activated in this way.
In animals maintained for 12 weeks after surgery, RAR activity at LAPs of 6·6 ± 1·2 (control), 11·6 ± 1·2 and 16·6 ± 1·2 (mmHg) were 35·5 ± 2·3, 33·8 ± 14·4 and 34·0 ± 3·4 action potentials min−1, respectively. These changes were statistically not significant.
Slowly adapting receptors (SARs) in the lung showed a small but statistically significant increase in activity when the left atrial pressure was acutely elevated in both intact and mitral valve damaged animals .
It is concluded that chronic pulmonary venous congestion resulting from destruction of the mitral valve attenuates the ability of RARs to respond to acute moderate elevations of LAP.
Activation and co‐ordination of InsP3‐mediated elementary Ca2+ events during global Ca2+ signals in Xenopus oocytes
Authors: Callamaras Nick , Marchant Jonathan S. , Sun Xiao‐Ping , Parker Ian 
Published: September 2004

The activation of elementary calcium release events (‘puffs’) and their co‐ordination to generate calcium waves was studied in Xenopus oocytes by confocal linescan imaging together with photorelease of inositol 1,4,5‐trisphosphate (InsP3) from a caged precursor.
Weak photolysis flashes evoked no responses or isolated calcium puffs, whereas flashes of increasing strength evoked more frequent puffs, often occurring in flurries as abortive waves, and then a near‐simultaneous calcium liberation originating at multiple sites. The numbers of sites activated increased initially as about the fourth power of photoreleased [InsP3].
Following repeated, identical photolysis flashes, puffs arose after stochastically varying latencies of a few hundred milliseconds to several seconds. The cumulative number of events initially increased as about the third power of time. No rise in free [Ca2+] was detected preceding the puffs, suggesting that this co‐operativity arises through binding of multiple InsP3 molecules, rather than through calcium feedback.
The mean latency to onset of calcium liberation shortened as about the square of the flash strength, and the dispersion in latencies between events reduced correspondingly.
Weak stimuli often evoked coupled puffs involving adjacent sites, and stronger flashes evoked saltatory calcium waves, propagating with non‐constant velocity. During waves, [Ca2+] rose slowly between puff sites, but more abruptly at active sites following an initial diffusive rise in calcium.
Initial rates of rise of local [Ca2+] at release sites were similar during puffs and release induced by much (> 10‐fold) greater [InsP3]. In contrast, macroscopic calcium measurements averaged over the scan line showed a graded dependence of rate of calcium liberation upon [InsP3], due to recruitment of additional sites and decreasing dispersion in activation latencies.
We conclude that the initiation of calcium liberation depends co‐operatively upon [InsP3] whereas the subsequent regenerative increase in calcium flux depends upon local calcium feedback and is largely independent of [InsP3]. Wave propagation is consistent with the diffusive spread of calcium evoking regenerative liberation at heterogeneous discrete sites, the sensitivity of which is primed by InsP3.
cGMP inhibits IP3‐induced Ca2+ release in intact rat megakaryocytes via cGMP‐ and cAMP‐dependent protein kinases
Authors: Tertyshnikova Svetlana , Yan Xiongwei , Fein Alan 
Published: September 2004

Inhibition of inositol 1,4,5‐trisphosphate (IP3) receptor‐mediated Ca2+ release by cGMP was examined in intact rat megakaryocytes, by using a combination of single cell fluorescence microscopy to monitor intracellular free calcium ([Ca2+]i) and flash photolysis of caged second messengers.
Sodium nitroprusside (SNP), a nitric oxide (NO) donor, and the hydrolysis‐resistant cGMP analogue 8‐(4‐chlorophenylthio)guanosine 3′,5′‐cyclic monophosphate (pCPT‐cGMP) inhibited Ca2+ release induced by photolysis of caged IP3. Neither of them affected the rate of Ca2+ removal from the cytoplasm following photolysis of caged Ca2+.
Photolysis of the caged NO donor 3‐morpholinosydnonimine (SIN‐1) during agonist‐induced [Ca2+]i oscillations inhibited Ca2+ release without affecting the rate of Ca2+ uptake and/or extrusion.
We conclude that the inhibition of IP3‐induced Ca2+ release is the principal mechanism of NO‐cGMP‐dependent inhibition of [Ca2+]i mobilization.
IPG, a specific peptide inhibitor of cGMP‐dependent protein kinase (cGMP‐PK), blocked the inhibitory effect of pCPT‐cGMP, indicating that the inhibition of IP3‐induced Ca2+ release by pCPT‐cGMP is mediated by cGMP‐PK. However, the simultaneous application of both IPG and IP20, a specific peptide inhibitor of cAMP‐dependent protein kinase (cAMP‐PK), was required to block the inhibitory effect of SNP. These data strongly suggest that NO‐cGMP‐dependent inhibition of [Ca2+]i mobilization is mediated via the activation of both cGMP‐PK and cAMP‐PK.
Activation of Ca2+‐activated K+ channels by an increase in intracellular Ca2+ induced by depolarization of mouse skeletal muscle fibres
Authors: Jacquemond Vincent , Allard Bruno 
Published: September 2004

Ionic currents were simultaneously recorded at macroscopic and unitary level using the whole‐cell and cell‐attached patch‐clamp procedures together on the same portion of isolated mouse skeletal muscle fibres.
In the presence of Tyrode solution in the patch pipette and Tyrode‐TTX solution in the bath, macroscopic and unitary currents through delayed rectifier K+ channels were simultaneously recorded in response to depolarizing pulses of 1 s duration.
In five fibres, successive long‐lasting incremental depolarizing levels induced, at ‐40 mV or ‐30 mV, the opening of a high conductance channel carrying an outward current superimposed on delayed rectifier K+ channel activity. Opening of this high conductance channel was not observed when the depolarization steps were applied in the patch pipette.
Using the same depolarizing protocol, activation of a high conductance channel was also observed in two fibres in the presence of a K+‐rich solution in the pipette (145 mm K+).
With either Tyrode or K+‐rich solution in the pipette, unitary current amplitudes of the high conductance channel matched well with the values obtained for Ca2+‐activated K+ (KCa) channels in inside‐out patches under similar ionic conditions.
Indo‐1 fluorescence measurements showed that the stimulation protocol that led to KCa channel opening induced stepwise increases in intracellular [Ca2+] in the submicromolar range.
Our results provide evidence that activation of sarcolemmal KCa channels can be induced by a rise in intracellular [Ca2+] following voltage‐activated sarcoplasmic reticulum Ca2+ release.