bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stealth ryanodine‐sensitive Ca2+ release contributes to activity of capacitative Ca2+ entry and nitric oxide synthase in bovine endothelial cells
Authors: Paltauf‐Doburzynska Jolanta , Posch Karla , Paltauf Guenther , Graier Wolfgang F. 
Published: September 2004

The involvement of ryanodine‐sensitive Ca2+ release (RsCR) in bradykinin (Bk)‐induced Ca2+ release, capacitative Ca2+ entry (CCE) and nitric oxide synthase (NOS) activation was assessed in freshly isolated bovine coronary artery endothelial cells.
Using deconvolution microscopy fura‐2 was found throughout the whole cytosol, while the cell membrane impermeable dye FFP‐18 was exclusively in the cell membrane. Thus, perinuclear ([Ca2+]pn) and subplasmalemmal Ca2+ concentration ([Ca2+]sp) were monitored using fura‐2 and FFP‐18.
Inhibition of Na+−Ca2+ exchange by lowering extracellular Na+ concentration augmented the Bk‐induced [Ca2+]pn signal in Ca2+‐free solution. This effect was abolished when RsCR was prevented with 25 μmmu;mol l−1 ryanodine, while inhibition of RsCR had no effect on Bk‐induced increase in [Ca2+]pn without inhibition of Na+−Ca2+ exchange.
Initiating RsCR by 200 nmol l−1 ryanodine increased [Ca2+]sp, while [Ca2+]pn remained constant. However, when Na+−Ca2+ exchange was prevented, ryanodine was also able to elevate [Ca2+]pn.
Blockage of RsCR diminished Ca2+ extrusion in response to stimulation with Bk in normal Na+‐containing solution.
Inhibition of RsCR blunted Bk‐activated CCE, while inhibition of Na+−Ca2+ exchange during stimulation enhanced CCE.
Although direct activation of RsCR failed to activate NOS, inhibition of RsCR diminished the effect of ATP and Bk on NOS, while the effect of thapsigargin remained unchanged.
These data suggest that during stimulation subplasmalemmal RsCR occurs, which contributes to the activities of CCE and NOS. Thus, the function of the subplasmalemmal Ca2+ control unit must be extended as a regulator for CCE and NOS.
Local calcium release in mammalian skeletal muscle
Authors: Shirokova Natalia , García Jesús , Ríos Eduardo 
Published: September 2004

Fluo‐3 fluorescence associated with Ca2+ release was recorded with confocal microscopy in single muscle fibres mechanically dissected from fast twitch muscle of rats or frogs, voltage clamped in a two Vaseline‐gap chamber.
Interventions that elicited Ca2+ sparks in frog skeletal muscle (low voltage depolarizations, application of caffeine) generated in rat fibres images consistent with substantial release from triadic regions, but devoid of resolvable discrete events. Ca2+ sparks were never observed in adult rat fibres. In contrast, sparks of standard morphology were abundant in myotubes from embryonic mice.
Depolarization‐induced gradients of fluorescence between triadic and surrounding regions (which are proportional to Ca2+ release flux) peaked at about 20 ms and then decayed to a steady level. Gradients were greater in frog fibres than in rat fibres. The ratio of peak over steady gradient (R) was steeply voltage dependent in frogs, reaching a maximum of 4.8 at −50 mV (n= 7). In rats, R had an essentially voltage‐independent value of 2.3 (n= 5).
Ca2+‐induced Ca2+ release, resulting in concerted opening of several release channels, is thought to underlie Ca2+ sparks and the peak phase of release in frog skeletal muscle. A diffuse ‘small event’ release, similar to that observed in these rats, is also present in frogs and believed to be directly activated by voltage. The present results suggest that in these rat fibres there is little contribution by CICR to Ca2+ release triggered by depolarization, and a lack of concerted channel opening.
Characterization of the putative chloride channel xClC‐5 expressed in Xenopus laevis oocytes and comparison with endogenous chloride currents
Authors: Schmieder S. , Lindenthal S. , Banderali U. , Ehrenfeld J. 
Published: September 2004

We recently cloned a putative chloride channel (xClC‐5) from the renal cell line A6, which induced the appearance of a Cl− conductance not found in control oocytes after homologous expression in Xenopus oocytes. With the aim of increasing the Xenopus oocyte xClC‐5 expression, we constructed a new plasmid in which the native 5′ and 3′ non‐coding regions of xClC‐5 were replaced by the non‐coding regions of the Xenopusβ‐globin sequence and in which a Kozak consensus site was introduced before the initiator ATG.
We then compared the induced currents Inative (induced by injection of cRNA presenting the native non‐coding regions of xClC‐5) and Iβ‐globin (induced by injection of cRNA presenting the non‐coding regions of the Xenopusβ‐globin sequence) investigating anion selectivity and anion blocker sensitivity. Several differences were found: (1) expression yield and oocyte surviving rate were largely increased by injecting (β) xClC‐5 cRNA, (2) the Iβ‐globin outward rectification score was 2.6 times that of Inative, (3) the anion conductivity sequence was nitrate > bromide > chloride > iodide >> gluconate for Iβ‐globin and iodide > bromide > nitrate > chloride >> gluconate for Inative, (4) 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoic acid (NPPB), anthracene‐9‐carboxylic acid (9‐AC), DIDS, lanthanum ions, cAMP and ionomycin‐induced [Ca2+]i increase inhibited Inative but had no effect on Iβ‐globin, and (5) Inative showed considerable similarity to the previously reported endogenous current appearing after ClC‐6 or pICln cRNA injection.
Comparison of Inative with the endogenous chloride current ICl,swell which develops under hyposmotic conditions demonstrated several similarities in their electrophysiological and pharmacological characteristics but were nevertheless distinguishable.
In vitro translation assays demonstrated that protein synthesis was much greater using the (β) xClC‐5 construct than that of xClC‐5. Furthermore, immunoreactivity of membrane preparations of Xenopus oocytes was only observed with the (β) xClC‐5 construct, its intensity being positively correlated with Iβ‐globin levels.
In addition, the current induced in (β) xClC‐5 cRNA‐injected oocytes presented a very marked pH dependence (inhibition by acid external media) with a pKa value (negative log of the acid dissociation constant) of 5.67.
In conclusion, Iβ‐globin may be due to the presence of xClC‐5 in the oocyte plasma membrane playing a role as an anion channel whereas Inative may represent an endogenous current induced by xClC‐5 cRNA injection. The use of antibodies will facilitate the tissue and subcellular localization of xClC‐5 and the identification of its physiological role.
Comparison of the gating behaviour of human and murine cystic fibrosis transmembrane conductance regulator Cl− channels expressed in mammalian cells
Authors: Lansdell K. A. , Delaney S. J. , Lunn D. P. , Thomson S. A. , Sheppard D. N. , Wainwright B. J. 
Published: September 2004

To investigate the function of the murine cystic fibrosis transmembrane conductance regulator (CFTR), a full‐length cDNA encoding wild‐type murine CFTR was assembled and stably expressed in Chinese hamster ovary (CHO) cells.
Like human CFTR, murine CFTR formed Cl− channels that were regulated by cAMP‐dependent phosphorylation and intracellular ATP. However, murine CFTR Cl− channels had a reduced single‐channel conductance and decreased open probability (Po) compared with those of human CFTR.
Analysis of the dwell time distributions of single channels suggested that the reduced Po of murine CFTR was caused by both decreased residence in the open state and transitions to a new closed state, described by an intermediate closed time constant.
For both human and murine CFTR, ATP and ADP regulated the rate of exit from the long‐lived closed state.
5′‐Adenylylimidodiphosphate (AMP‐PNP) and pyrophosphate, two compounds that disrupt cycles of ATP hydrolysis, stabilized the open state of human CFTR. However, neither agent locked murine CFTR Cl− channels open, although AMP‐PNP increased the Po of murine CFTR.
The data indicate that although human and murine CFTR have many properties in common, some important differences in function are observed. These differences could be exploited in future studies to provide new understanding about CFTR.
Cytosolic and mitochondrial Ca2+ signals in patch clamped mammalian ventricular myocytes
Authors: Zhou Zhuan , Matlib Mohammed A. , Bers Donald M. 
Published: September 2004

Ventricular myocytes isolated from ferret or cat were loaded with the acetoxymethyl ester form of indo‐1 (indo‐1 AM) such that ∼75 % of cellular indo‐1 was mitochondrial. The intramitochondrial indo‐1 concentration was 0.5‐2 mm.
Myocytes were also voltage clamped (membrane capacitance, Cm= 100 pF) and a typical wash‐out time constant of cytosolic indo‐1 by a patch pipette was found to be ∼300 s. Depolarizations to +110 mV produced graded and progressive cellular Ca2+ load via Na+‐Ca2+ exchange.
During these relatively slow Ca2+ transients, cell contraction (ΔL) paralleled fluorescence ratio signals (R) such that ΔL could be used as a bioassay of cytosolic [Ca2+] ([Ca2+]c), where [Ca2+]CL is the inferred signal which is delayed by ∼200 ms from true [Ca2+]c.
In myocytes without Mn2+ quench, the kinetics of the total cellular indo‐1 signal, ΔR (including cytosolic and mitochondrial components), match ΔL during stimulations at low basal [Ca2+]i. However, after progressive Ca2+ loading, ΔR kinetics deviate from ΔL dramatically. The deviation can be completely blocked by a potent mitochondrial Ca2+ uniport blocker, Ru360.
When cytosolic indo‐1 is quenched by Mn2+, initial moderate stimulation triggers contractions (ΔL), but no change in indo‐1 signal, indicating both the absence of cytosolic Ca2+‐sensitive indo‐1 and unchanged mitochondrial [Ca2+] (Δ[Ca2+]m). Subsequent stronger stimulation evoked larger ΔL and also ΔR. The threshold [Ca2+]c for mitochondrial Ca2+ uptake was 300‐500 nM, similar to that without Mn2+ quench.
At high Ca2+ loads where Δ[Ca2+]m is detected, the time course of [Ca2+]m was different from that of [Ca2+]c. Peak [Ca2+]m after stimulation has an ∼1 s latency with respect to [Ca2+]c, and [Ca2+]m decline is extremely slow.
Upon a Ca2+ influx which increased [Ca2+]c by 0.4 μm and [Ca2+]m by 0.2 μm, total mitochondrial Ca2+ uptake was ∼13 μmol (l mitochondria)−1.
With Mn2+ quench of cytosolic indo‐1, there was no mitochondrial uptake of Mn2+ until the point at which mitochondrial Ca2+ uptake became apparent. However, after mitochondrial Ca2+ uptake starts, mitochondria continually take up Mn2+ even during relaxation, when [Ca2+]c is low.
It is concluded that mitochondria in intact myocytes do not take up detectable amounts of Ca2+ during individual contractions, unless resting [Ca2+]c exceeds 300‐500 nM. At high cell Ca2+ loads and [Ca2+]c, mitochondrial Ca2+ transients occur during the twitch, but with much slower kinetics than those of [Ca2+]c.
Respiration‐modulated membrane potential and chemosensitivity of locus coeruleus neurones in the in vitro brainstem‐spinal cord of the neonatal rat
Authors: Oyamada Y. , Ballantyne D. , Mückenhoff K. , Scheid P. 
Published: September 2004

The activity of locus coeruleus (LC) neurones (n= 126) was examined in whole‐cell (conventional and amphotericin B‐perforated patch) recordings, and the relationship of this activity to the respiratory discharge recorded on the C4 or C5 phrenic nerve roots was determined at different CO2 concentrations (2 and 8 %; bath pH 7.8 and 7.2) in the in vitro brainstem‐spinal cord preparation of the neonatal rat (1–5 days old).
In most neurones (n= 105) ongoing activity was modulated at respiratory frequency. Typically, this consisted of a phase of depolarization and increased discharge frequency synchronous with the phrenic burst, followed by a phase of hyperpolarization and inhibition of discharge (n= 94 of 105). The incidence of respiratory modulation decreased from 91 % on P1 to 57 % on P5.
Bath application of the non‐nmDA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 5 μmmu;m) or the nmDA receptor antagonist DL‐2‐amino‐5‐phosphonovaleric acid (APV; 100 μmmu;m) abolished both phases of respiratory modulation. The hyperpolarizing phase alone was abolished by the adrenoceptor antagonists idazoxan (5 μmmu;m) or phentolamine (0.8 μmmu;m). These results indicate that excitatory amino acid pathways are involved in the transmission of both the excitatory and inhibitory components and that the latter involves in addition an α2‐adrenoceptor‐mediated pathway.
Increasing the CO2 concentration from 2 to 8 % resulted in a shortening of expiratory duration and weakening or loss of respiratory‐phased inhibition; this was accompanied by depolarization, increased discharge frequency and, in those neurones where they were initially present (60 %), an increase in the frequency of subthreshold membrane potential oscillations. The depolarizing response was retained in the presence of tetrodotoxin (TTX, 0.2–1.0 μmmu;m).
These results indicate that in this neonatal preparation LC neurones form part of the synaptically connected brainstem respiratory network, and that the LC constitutes a site of CO2‐ or pH‐dependent chemoreception.
Developmental changes in the expression of low‐voltage‐activated Ca2+ channels in rat visual cortical neurones
Authors: Tarasenko A. N. , Isaev D. S. , Eremin A. V. , Kostyuk P. G. 
Published: September 2004

The functional properties of low‐voltage‐activated (LVA) Ca2+ channels were studied in pyramidal neurones from different rat visual cortical layers in order to investigate changes in their properties during early postnatal development. Ca2+ currents were recorded in brain slices using the whole‐cell patch‐clamp technique in rats from three age groups: 2, 3 and 12 days old (postnatal day (P) 2, P3 and P12).
It was demonstrated that LVA Ca2+ currents are present in neurones from superficial (I‐II) and deep (V‐VI) visual cortex layers of P2 and P3 rats. No LVA Ca2+ currents were observed in neurones from the middle (III‐IV) layers of these rats. The LVA Ca2+ currents observed in P2 and P3 neurones from both superficial and deep layers could be completely blocked by nifedipine (100 μM) and were insensitive to Ni2+ (25 μM).
The density of LVA Ca2+ currents decreased rapidly during the early stages of postnatal development, while the density of high‐voltage‐activated (HVA) Ca2+ currents progressively increased up to the twelfth postnatal day. No LVA Ca2+ currents were found in P12 neurones from any of the layers. Only HVA Ca2+ currents with high sensitivity to F− applied through the patch pipette were observed.
The kinetics of LVA Ca2+ currents could be well approximated by the m2h Hodgkin‐Huxley equation with an inactivation time constant of 24 ± 6 ms. The steady‐state inactivation curve fitted by a Boltzmann function had the following parameters: membrane potential at half‐inactivation, ‐86.9 mV; steepness coefficient,3.4 mV.
It is concluded that, in visual cortical neurones, LVA Ca2+ channels are expressed only in the neurones of deep and superficial layers over a short period during the earliest postnatal stages. These channels are nifedipine sensitive and similar in functional properties to those in the laterodorsal (LD) thalamic nucleus. However, the cortical neurones do not express another (‘slow’) type of LVA Ca2+ channel, which is permanently present in LD thalamic neurones after the second postnatal week, indicating that the developmental time course of cortical and thalamic cellsdifferent.
Encoding properties induced by a persistent voltage‐gated muscarinic sodium current in rabbit sympathetic neurones
Authors: Gola Maurice , Delmas Patrick , Chagneux Hélène 
Published: September 2004

A time‐ and voltage‐dependent Na+‐selective current termed INa,M is activated by muscarinic agonists or splanchnic nerve stimulation in sympathetic neurones of rabbit coeliac and superior mesenteric ganglia. The firing patterns induced by INa,M were investigated in patch‐clamped neurones within intact ganglia, and compared with those generated by a neuronal model including INa,M.
I
Na,M was characterized by voltage‐dependent low‐threshold activation and high‐threshold inactivation functions. The overlapping functions produced a persistent U‐shaped current between −100 and −20 mV, which peaked at the cell resting potential. The activation and inactivation kinetics were fitted to single exponentials with time constants of ≈100 and 400 ms, respectively.
Activating INa,M with muscarinic agonists or nerve stimulation depolarized and fired the neurones. The depolarization was paralleled by an apparent increase in input membrane resistance. The model showed that this paradox resulted from the turning off of INa,M during resistance tests, which also accounted for the all‐or‐none slow hyperpolarizing responses to current pulses.
I
Na,M gave the neurones an N‐shaped I‐V relationship capable of producing complex firing patterns. Under given conditions, carbachol‐treated neurones could either fire regularly or remain silent at ≈‐80 mV, i.e. they displayed bistability. Transitions from one state to the other were triggered with short current pulses. The transitions resulted from the turning on and off of INa,M.
Firing reduced INa,M, an effect abolished by blocking Ca2+ channels or adding BAPTA (40 mM) to the pipette. The Ca2+‐related negative regulation of INa,M may have mediated endogenous bursting activity. Burst firing was generated by the model upon introducing Ca2+ regulation of INa,M.
The results demonstrate that INa,M gives prevertebral sympathetic neurones a wide repertoire of firing patterns: pacemaker‐like properties, bistability and burst firing capability. They suggest that the INa,M‐related encoding properties may provide sympathetic neurotransmission with new potentialities.
The open duration of fetal ACh receptor‐channel changes during mouse muscle development
Authors: Grassi Francesca , Epifano Olga , Mileo Anna Maria , Barabino Benedetta , Eusebi Fabrizio 
Published: September 2004

We performed an RNase protection assay on cultured C2C12 mouse myotubes, demonstrating that the γ subunit of the fetal muscle acetylcholine receptor (AChR) exists as two splice variants, which differ in the presence of the amino terminal exon 5.
We studied unitary ACh‐evoked events in fibres acutely dissociated from the hindlimb flexor digitorum brevis muscle of BALB/C mice aged between embryonic day 16 (E16) and postnatal day 6 (P6).
At all ages, the channel conductance was about 30 pS, typical of the fetal form of the AChR. The mean open time increased significantly from 6 ms at E16 to 9 ms at E19, then decreased to about 5 ms during the first postnatal week. The lengthening of the open time was considerably delayed in hypothyroid mice. Data were recorded at 24‐26 °C.
On the basis of previously reported experiments in heterologous expression systems, we suggest that the modulation of channel open time is related to the expression of the AChR incorporating the γs subunit. These events might be coupled to the crucial modifications in muscle innervation that take place during the same developmental period.
Cytoplasmic terminus domains of Kir6.x confer different nucleotide‐dependent gating on the ATP‐sensitive K+ channel
Authors: Takano Makoto , Xie Lai‐Hua , Otani Hideo , Horie Minoru 
Published: September 2004

In order to investigate the structural basis for the nucleotide‐dependent gating of ATP‐sensitive K+ channels (KATP), Kir6.1 (uKATP‐1), Kir6.2 (BIR1) and chimeric channels were co‐expressed with a common subtype of sulphonylurea receptor, SUR1, in COS7 cells. Representing the amino terminal domain‐transmembrane domain‐carboxyl‐terminal domain of Kir6.1 as 1–1–1 and of Kir6.2 as 2‐2‐2, chimeric Kir6.x channels were constructed by swapping the amino and/or carboxyl terminal domains between Kir6.1 and Kir6.2 to give the chimeric x‐1‐x channels 1‐1‐2, 2‐1‐1 and 2‐1‐2, and the chimeric x‐2‐x channels 2‐2‐1, 1‐2‐2 and 1‐2‐1.
Inside‐out patch clamp experiments revealed that both wild‐type Kir6.1 and Kir6.2 formed inwardly rectifying K+ channels. Single‐channel conductances were 36.3 and 66.1 pS, respectively. Chimeric x‐1‐x channels, whose transmembrane domain was that of Kir6.1, showed similar ion‐pore properties to wild‐type Kir6.1. Likewise, chimeric x‐2‐x channels had similar ion‐pore properties to wild‐type Kir6.2.
Wild‐type Kir6.1 and Kir6.2 possessed distinct gating properties towards intracellular nucleotides. The activity of Kir6.1 was entirely dependent on Mg2+ and nucleotide diphosphates (NDPs) such as UDP. In contrast, Kir6.2 was activated upon excision of patch membrane. When Kir6.2 underwent rundown, UDP reactivated the channel.
In order to eliminate UDP dependence from Kir6.1, it was necessary to replace both N‐ and C‐termini; chimera 2‐1‐2 opened in UDP‐free conditions. With Kir6.2, substitution of the N‐terminus with that of Kir6.1 conferred UDP dependence on chimeras 1‐2‐2 and 1‐2‐1. Chimera 2‐2‐1 opened in UDP‐free conditions, but UDP potentiated the channel activity by > 20‐fold.
The kinetics of UDP‐dependent activation were significantly different between Kir6.1 and Kir6.2. Kir6.1 maximally activated by UDP was sensitive to intracellular ATP, although its ATP sensitivity was significantly lower than that of Kir6.2 measured in identical conditions. The kinetics of UDP‐dependent activation and ATP sensitivity could be transferred between Kir6.1 and Kir6.2 only when both N‐ and C‐termini were replaced. We therefore concluded that nucleotide‐dependent gating was regulated by the N‐ and C‐terminal domains irrespective of the transmembrane domains.
Hemispheric asymmetry of macroscopic and elementary calcium signals mediated by InsP3 in Xenopus oocytes
Authors: Callamaras Nick , Sun Xiao‐Ping , Ivorra Isabel , Parker Ian 
Published: September 2004

The mechanisms underlying hemispheric asymmetry of the inositol 1,4,5‐trisphosphate (InsP3)‐calcium signalling pathway in Xenopus oocytes were examined by fluorescence imaging of calcium signals and recording calcium‐activated Cl− currents (ICl,Ca) evoked by intracellular calcium injections and photorelease of InsP3.
The maximal ICl,Ca evoked by strong photorelease of InsP3 was 8 times greater in the animal than the vegetal hemisphere, but the average threshold amounts of InsP3 required to evoke detectable currents were similar in each hemisphere.
Currents evoked by injections of calcium were about 2.5 times greater near the animal pole than near the vegetal pole, whereas fluorescence signals evoked by injections were similar in each hemisphere.
Calcium waves were evoked by photolysis flashes of similar strengths in both hemispheres of albino oocytes, but peak calcium levels evoked by supramaximal stimuli were 70 % greater in the animal hemisphere.
Elementary calcium release events (puffs) in the animal hemisphere had amplitudes about double that in the vegetal hemisphere, and more often involved coupled release from adjacent sites. Calcium release sites were more closely packed in the animal hemisphere, with a mean spacing of about 1.5 μm compared with 2.25 μm in the vegetal hemisphere.
The larger amplitude of currents mediated by InsP3 in the animal hemisphere, therefore, involves an increased flux of calcium at individual release units, a more dense packing of release units and a higher density of Cl− channels.