bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential modulation of the phases of a Ca2+ spike by the store Ca2+‐ATPase in human umbilical vein endothelial cells
Authors: Morgan Anthony J. , Jacob Ron 
Published: September 2004

Histamine‐stimulated cytosolic free Ca2+ ([Ca2+]i) oscillations in human umbilical vein endothelial cells (HUVECs) comprise repetitive spikes generated by pulsatile release from stores. We have investigated the roles of the store Ca2+‐ATPases in regulating both the upstroke and downstroke of a Ca2+ spike.
The sarco‐endoplasmic reticulum Ca2+‐ATPase (SERCA) inhibitor cyclopiazonic acid (CPA) dramatically affected oscillations whereas inhibition of the plasma membrane Ca2+‐ATPase (PMCA) with La3+ had little effect. This and other evidence suggested that the downstroke of a spike is predominantly mediated by SERCA.
Artificial [Ca2+]i spiking generated by repetitive pulsatile application of 0.3 μm histamine in Ca2+‐free medium did not cause net loss of Ca2+ from the cell whereas repetitive pulsatile application of 1 and 10 μm histamine did, with the higher concentration being more effective. We conclude that there is an inverse relationship between stimulus intensity and relative SERCA activity.
For a Ca2+ transient, the initiation of release was suppressed by SERCA during either the lag phase or the interspike period (ISP) since: (i) the ISP was shortened by low CPA concentrations, (ii) higher concentrations of CPA stimulated an explosive Ca2+ release when applied during the ISP but not when applied in the absence of agonist, and (iii) CPA synchronized the initial Ca2+ response to a low histamine dose (even recruiting silent, histamine‐unresponsive cells).
Two aspects of the regenerative upstroke of a spike were differently affected by SERCA inhibition: Ca2+ wave velocity was entirely unaffected by CPA whereas the local rate of rise was increased.
The [Ca2+]i at which a Ca2+ spike terminated depended on SERCA since CPA dose dependently enhanced the peak [Ca2+]i.
We conclude that SERCA plays a powerful and dynamic role in regulating [Ca2+]i oscillations in HUVECs. SERCA differentially modulates the phases of Ca2+ release in addition to bringing about the falling phase of a Ca2+ spike.
ATP‐evoked increases in [Ca2+]i and peptide release from rat isolated neurohypophysial terminals via a P2X2 purinoceptor
Authors: Troadec Jean‐Denis , Thirion Sylvie , Nicaise Ghislain , Lemos José R. , Dayanithi Govindan 
Published: September 2004

The effect of externally applied ATP on cytosolic free Ca2+ concentration ([Ca2+]i) was tested in single isolated rat neurohypophysial nerve terminals by fura‐2 imaging. The release of vasopressin (AVP) and oxytocin (OT) upon ATP stimulation was also studied from a population of terminals using specific radioimmunoassays.
ATP evoked a sustained [Ca2+]i increase, which was dose dependent in the 1‐100 μM range (EC50= 4·8 μM). This effect was observed in only ≈40 % of the terminals.
Interestingly, ATP, in the same range (EC50= 8·6 μM), evoked AVP, but no significant OT, release from these terminals.
Both the [Ca2+]i increase and AVP release induced by ATP were highly and reversibly inhibited by suramin, suggesting the involvement of a P2 purinergic receptor in the ATP‐induced responses. Pyridoxal‐5‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), another P2 purinergic receptor antagonist, strongly reduced the ATP‐induced [Ca2+]i response.
To further characterize the receptor, different agonists were tested, with the following efficacy: ATP = 2‐methylthio‐ATP > ATP‐γ‐S > α,β‐methylene‐ATP > ADP. The compounds adenosine, AMP, β,γ‐methylene‐ATP and UTP were ineffective.
The ATP‐dependent [Ca2+]i increase was dependent on extracellular Ca2+ concentration ([Ca2+]o). Fluorescence‐quenching experiments with Mn2+ showed that externally applied ATP triggered a Mn2+ influx. The ATP‐induced [Ca2+]i increase and AVP release were independent of and additive to a K+‐induced response, in addition to being insensitive to Cd2+. The ATP‐induced [Ca2+]i increase was strongly reduced in the presence of Gd3+. These results suggest that the observed [Ca2+]i increases were elicited by Ca2+ entry through a P2X channel receptor rather than via a voltage‐dependent Ca2+ channel.
We propose that ATP, co‐released with neuropeptides, could act as a paracrine‐autocrine messenger, stimulating, via Ca2+ entry through a P2X2 receptor, the secretion of AVP, in particular, from neurohypophysial nerve terminals.
Facilitation by the β2a subunit of pore openings in cardiac Ca2+ channels
Authors: Costantin James , Noceti Francesca , Qin Ning , Wei Xiangyang , Birnbaumer Lutz , Stefani Enrico 
Published: September 2004

Single channel recordings were performed on the cardiac calcium channel (α1C) in order to study the effect of coexpression of the accessory β2a subunit. On‐cell patch clamp recordings were performed after expression of these channels in Xenopus oocytes.
The α1C subunit, when expressed alone, had similar single channel properties to native cardiac channels. Slow transitions between low and high open probability (Po) gating modes were found as well as fast gating transitions between the open and closed states.
Coexpression of the β2a subunit caused changes in the fast gating during high Po mode. In this mode, open time distributions reveal at least three open states and the β2a subunit favours the occupancy of the longest, 10‐15 ms open state. No effect of the β2a subunit was found when the channel was gating in the low Po mode.
Slow gating transitions were also affected by the β2a subunit. The high Po mode was maintained for the duration of the depolarizing pulse in the presence of the β2a subunit; while the α1C channel when expressed alone, frequently switched into and out of the high Po mode during the course of a sweep.
The β2a subunit also affected mode switching that occurred between sweeps. Runs analysis revealed that the α1C subunit has a tendency toward non‐random mode switching. The β2a subunit increased this tendency. A χ2 analysis of contingency tables indicated that the β2a subunit caused the α1C channel to gain ‘intrinsic memory’, meaning that the mode of a given sweep can be non‐independent of the mode of the previous sweep.
We conclude that the β2a subunit causes changes to the α1C channel in both its fast and slow gating behaviour. The β2a subunit alters fast gating by facilitating movement of the channel into an existing open state. Additionally, the β2a subunit decreases the slow switching between low and high Po modes.
K+ channel block‐induced mammalian neuroblastoma cell swelling: a possible mechanism to influence proliferation
Authors: Rouzaire‐Dubois B. , Dubois J. M. 
Published: September 2004

A variety of studies have suggested that K+ channel activity is a key determinant for cell progression through the G1 phase of mitosis. We have previously proposed that K+ channels control the activity of cell cycle‐regulating proteins via regulation of cell volume. In order to test this hypothesis, we measured, with a Coulter counter and under different experimental conditions, the volume and rate of proliferation of neuroblastoma × glioma hybrid NG108‐15 cells.
The K+ channel blockers TEA (1‐10 mM), 4‐aminopyridine (0.2‐2 mM) and Cs+ (2.5‐10 mM) increased the cell volume and decreased the rate of cell proliferation. Proliferation was fully inhibited when cell volume was increased by 25 %.
A 40 % increase in the culture medium osmolarity with NaCl induced a 25 % increase in cell volume and an 82 % decrease in the rate of cell proliferation. A 40 % increase in the culture medium osmolarity with mannitol induced a 9 % increase in cell volume and a 60 % decrease in the rate of cell proliferation.
The Cl− channel blocker NPPB (5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid; 50 μM) induced a 12 % increase in cell volume and a 77 % decrease in the rate of cell proliferation.
A 24 % reduction in the culture medium osmolarity with H2O induced a 21 % decrease in cell volume and a 32 % increase in the rate of cell proliferation.
Under whole‐cell patch‐clamp conditions, antibiotics (penicillin plus streptomycin) decreased the voltage‐dependent K+ current. Omission of antibiotics from the culture medium induced a 10 % decrease in the cell volume and a 32 % increase in the rate of cell proliferation.
These results suggest that the mechanisms controlling cell proliferation are strongly influenced by the factors which determine cell volume. This could take into account the role in mitogenesis of K+ channels and of other ionic pathways involved in cell volume regulation.
Calcium‐calmodulin‐dependent mechanisms accelerate calcium decay in gastric myocytes from Bufo marinus
Authors: McGeown J. G. , McCarron J. G. , Drummond R. M. , Fay F. S. 
Published: September 2004

[Ca2+]i was recorded in voltage‐clamped gastric myocytes from Bufo marinus. Repolarization to ‐110 mV following a 300 ms depolarization to +10 mV led to triphasic [Ca2+]i decay, with a fast‐slow‐fast pattern. After a conditioning train of repetitive depolarizations the duration of the second, slow phase of decay was shortened, while the rate of decay during the third, faster phase was increased by 34 ± 6 % (mean ± s.e.m., n= 21) when compared with unconditioned transients.
[Ca2+]i decay was biphasic in cells injected with the calmodulin‐binding peptide RS20, with a prolonged period of fast decay followed by a slow phase. There was no subsequent increase in decay rate during individual transients and no acceleration of decay following the conditioning train (n= 8). Decline of [Ca2+]i in cells injected with the control peptide NRS20 was triphasic and the decay rate during the third phase was increased by 50 ± 19 % in conditioned transients (n= 6).
Cell injection with CK3AA, a pseudo‐substrate inhibitor of calmodulin‐dependent protein kinase II, prevented the increase in the final rate of decay following the conditioning train (n= 6). In cells injected with an inactive peptide similar to CK3AA, however, there was a 45 ± 17 % increase after the train (n= 5).
Inhibition of Ca2+ uptake by the sarcoplasmic reticulum with cyclopiazonic acid or thapsigargin did not prevent acceleration of decay.
These results demonstrate that [Ca2+]i decay is accelerated by Ca2+‐calmodulin and calmodulin‐dependent protein kinase II. This does not depend on Ca2+ uptake by the sarcoplasmic reticulum but may reflect upregulation of mitochondrial Ca2+ removal.
Mechanisms underlying phosphate‐induced failure of Ca2+ release in single skinned skeletal muscle fibres of the rat
Authors: Posterino Giuseppe S. , Fryer Martin W. 
Published: September 2004

Single mechanically skinned fibres from rat extensor digitorum longus (EDL) muscles were used to investigate the mechanisms underlying inorganic phosphate (Pi) movements between the myoplasm and the sarcoplasmic reticulum (SR). Force transients elicited by caffeine/low Mg2+ application were used to assess the rate of Pi‐induced inhibition of SR Ca2+ release and the subsequent recovery of Ca2+ release following removal of myoplasmic Pi.
Myoplasmic Pi reduced SR Ca2+ release in a concentration‐ and time‐dependent manner. A 10 s exposure to 10, 20 and 50 mm myoplasmic Pi reduced SR Ca2+ release by 12 ± 9, 29 ± 5 and 82 ± 5 %, respectively.
Removal of myoplasmic ATP at the time of Pi exposure significantly increased the rate and extent of SR Ca2+ release inhibition. For example, Ca2+ release was reduced by 86 ± 6 % (n= 6) after 20 s exposure to 20 mm Pi in the absence of ATP compared with only 47 ± 5 % (n= 5) in the presence of ATP.
The half and full recovery times for SR Ca2+ release following washout of myoplasmic Pi were 35 s and ∼7 min, respectively. Recovery of Ca2+ release was unaffected by the absence of ATP during washout of Pi but was prevented when fibres were washed in the presence of high myoplasmic Pi (30 mm). Neither the Pi transporter blocker phenylphosphonic acid (PHPA) nor the anion channel blockers anthracene‐9‐carboxylic acid (9‐AC) and 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid (DIDS) affected the rate of recovery of SR Ca2+ release.
These results show that Pi entry and exit from the SR occur primarily through a passive pathway that is insensitive to well‐known anion channel blockers. Pi inhibition of SR Ca2+ release appears to be a complicated phenomenon influenced by the rate of Pi movement across the SR as well as by the rate, extent and species of Ca2+‐Pi precipitate formation in the SR lumen. The more rapid inhibitory effect of Pi in the absence of myoplasmic ATP suggests that Pi may inhibit SR Ca2+ release more efficiently during the later stages of fatigue.
The role of intracellular Na+ and mitochondria in buffering of kainate‐induced intracellular free Ca2+ changes in rat forebrain neurones
Authors: Hoyt Kari R. , Stout Amy K. , Cardman Jamie M. , Reynolds Ian J. 
Published: September 2004

We have examined the mechanisms by which cultured central neurones from embryonic rat brain buffer intracellular Ca2+ loads following kainate receptor activation using fluorescent indicators of [Ca2+]i and [Na+]i.
Stimulation of cultured forebrain neurones with 100 μm kainate produced a rapid increase in [Ca2+]i that displayed a variable rate of recovery. Kainate also increased [Na+]i with a response that was slightly slower in onset and markedly slower in recovery.
The recovery of [Ca2+]i to baseline was not very sensitive to the [Na+]i. The magnitude of the increase in [Na+]i in response to kainate did not correlate well with the [Ca2+]i recovery time, and experimental manipulations that altered [Na+]i did not have a large impact on the rate of recovery of [Ca2+]i.
The recovery of [Ca2+]i to baseline was accelerated by the mitochondrial Na+‐Ca2+ exchange inhibitor CGP‐37157, suggesting that the recovery rate is influenced by release of Ca2+ from a mitochondrial pool and also that variation in the recovery rate is related to the extent of mitochondrial Ca2+ loading. Kainate did not alter the mitochondrial membrane potential.
These studies reveal that mitochondria have a central role in buffering neuronal [Ca2+]i changes mediated by non‐N‐methyl‐D‐aspartate (NMDA) glutamate receptors, and that the variation in recovery times following kainate receptor activation reflects a variable degree of mitochondrial Ca2+ loading. However, unlike NMDA receptor‐mediated Ca2+ loads, kainate receptor activation has minimal effects on mitochondrial function.
An intercellular regenerative calcium wave in porcine coronary artery endothelial cells in primary culture
Authors: Domenighetti Andrea Alberto , Bény Jean‐Louis , Chabaud Fabienne , Frieden Maud 
Published: September 2004

A regenerative calcium wave is an increase in cytosolic free calcium concentration ([Ca2+]i) which extends beyond the stimulated cells without decrement of amplitude, kinetics of [Ca2+]i increase and speed of propagation.
The aim of the present study was to test the hypothesis that such a wave could be evoked by bradykinin stimulation and by scraping cultured endothelial cells from porcine coronary arteries.
Calcium imaging was performed using the calcium‐sensitive dye fura‐2. A wound or a delivery of bradykinin to two to three cells on growing clusters of ≈300 cells caused an increase in [Ca2+]i which was propagated throughout the cluster in a regenerative manner over distances up to 400 μm. This wave spread through gap junctions since it was inhibited by the cell uncoupler palmitoleic acid.
The same experiments performed in confluent cultures caused a rise in [Ca2+]i which failed to propagate in a regenerative way. The wave propagation probably failed because the confluent cells were less dye coupled than the growing cells. This was confirmed by immunohistology which detected a dramatic decrease in the number of connexin 40 gap junctions in the confluent cultures.
The regenerative propagation of the wave was blocked by inhibitors of calcium‐induced calcium release (CICR) and phospholipase C (PLC), and by suppression of extracellular calcium, but not by clamping the membrane potential with high‐potassium solution.
We conclude that regenerative intercellular calcium waves exist in cultured islets but not in confluent cultures of endothelial cells. An increase in [Ca2+]i is not sufficient to trigger a regenerative propagation. The PLC pathway, CICR and extracellular calcium are all necessary for a fully regenerated propagation.
Endogenous calcium buffering in motoneurones of the nucleus hypoglossus from mouse
Authors: Lips Mario B. , Keller Bernhard U. 
Published: September 2004

Simultaneous patch clamp and rapid microfluorometric calcium measurements were performed on sixty‐five motoneurones in slices of the nucleus hypoglossus in the brainstem of 2‐ to 6‐day‐old mice.
Hypoglossal motoneurones were particularly vulnerable to mechanical or metabolic stress during isolation of in vitro slice preparations. Therefore, experimental conditions were optimized for functional integrity, as judged by spontaneous rhythmic activity of hypoglossal nerves (XII).
Calcium concentrations in the cell soma were monitored with a temporal resolution in the millisecond time domain during depolarizing voltage steps. Ratiometric fluorescence measurements were made using a rapid monochromator (switching τ < 10 ms), a photomultiplier tube and the calcium sensitive dyes fura‐2 and mag‐fura‐5.
Dynamics of somatic calcium transients were investigated as a function of the concentration of calcium indicator dye in the cell. Decays of calcium transients were approximated to a single exponential component and decay time constants showed a linear dependence on dye concentration. The extrapolated decay time in the absence of indicator dye was 0·7 ± 0·2 s, suggesting rapid somatic calcium dynamics under physiological conditions.
By a process of back‐extrapolation, the ‘added buffer’ method, a calcium binding ratio of 41 ± 12 (9 cells) was obtained indicating that 98 % of the calcium ions entering a hypoglossal motoneurone were bound by endogenous buffers.
Endogenous calcium binding ratios in hypoglossal motoneurones were small compared with those of other neurones with comparable size or geometry. Accordingly, our measurements suggest that the selective vulnerability of hypoglossal motoneurones to calcium‐related excitotoxicity might partially result from low concentrations of calcium buffers in these cells.
Differential regulation of potassium currents by FGF‐1 and FGF‐2 in embryonic Xenopus laevis myocytes
Authors: Chauhan‐Patel R. , Spruce A. E. 
Published: September 2004

Fibroblast growth factors (FGFs) are involved in the regulation of many aspects of muscle development. This study investigated their role in regulating voltage‐dependent K+ currents in differentiating Xenopus laevis myocytes. Both FGF‐1 and FGF‐2 are expressed by developing muscle cells, so their actions were compared. Experiments were performed on cultured myocytes isolated from stage 15 embryos.
Long‐term exposure of the embryonic myocytes to FGF‐1 downregulated inward rectifier K+ current (IK(IR)) density as well as both sustained and inactivating voltage‐dependent outward K+ currents (IK,S and IK,I, respectively) and their densities. In contrast, FGF‐2 upregulated these currents, although, because of an increase in capacitance caused by FGF‐2, current density did not change with this factor.
The regulation of IK(IR) by FGF‐1 was prevented by the cytoplasmic tyrosine kinase inhibitor herbimycin A, but that of IK,S and IK,I was unaffected, indicating that FGF‐1 achieves its regulatory effects on electrical development via separate signalling pathways. The receptor tyrosine kinase inhibitor genistein in isolation suppressed K+ currents, but this may have occurred through a channel‐blocking mechanism.
In many cells, IK,S was found to be composed of two components with differing voltage dependencies of activation. The FGFs brought about an alteration in the amount of total IK,S by equal effects on each component. Conversely, herbimycin A increased the proportion of low voltage‐activated current without affecting total current amplitude. Therefore, we suggest that a single species of channel whose voltage dependence is shifted by tyrosine phosphorylation generates IK,S.
In summary, FGF‐1 and FGF‐2 exert opposite effects on voltage‐dependent K+ currents in embryonic myocytes and, furthermore, FGF‐1 achieves its effects on different K+ currents via separate second messenger pathways.
α‐Adrenergic effects on Na+‐K+ pump current in guinea‐pig ventricular myocytes
Authors: Wang Y. , Gao J. , Mathias R. T. , Cohen I. S. , Sun X. , Baldo G. J. 
Published: September 2004

The whole‐cell patch clamp was employed to study Na+‐K+ pump current (Ip) in acutely isolated myocytes. α‐Adrenergic receptors were activated with noradrenaline (NA) after blocking β‐adrenergic receptors with propranolol. Ip was measured as the current blocked by strophanthidin (Str).
Activation of α‐receptors by NA increased Ip in a concentration‐dependent manner. The K0.5 depended on intracellular calcium ([Ca2+]i), however maximal stimulation did not. At 15 nm[Ca2+]i the K0.5 was 219 nm NA whereas at 1.4 μm [Ca2+]i it was 3 nm.
The voltage dependence of Ip was not shifted by NA at either high or low [Ca2+]i. At each voltage, maximal stimulation of Ip was 14‐15 %.
Staurosporine (St), an inhibitor of protein kinase C (PKC), eliminated the α‐receptor‐mediated stimulation of Ip at either high or low[Ca2+]i.
The stimulation of Ip was independent of changes in intracellular sodium or external potassium concentrations, and did not reflect a change in affinity for Str.
Phenylephrine, methoxamine and metaraminol, three selective α1‐adrenergic agonists, stimulate Ip in a similar manner to NA. Stimulation of Ip by NA was eliminated by prazosin, an α1‐antagonist, but was unaffected by yohimbine, an α2‐antagonist.
We conclude noradrenaline activates ventricular α1‐receptors, which are specifically coupled via PKC to increase Na+‐K+ pump current. The sensitivity of the coupling is [Ca2+]i dependent, however the maximal increase in pump current is [Ca2+]i and voltage independent.
Cholinergic activation and tonic excitation induce persistent gamma oscillations in mouse somatosensory cortex in vitro
Authors: Buhl Eberhard H. , Tamás Gabor , Fisahn André 
Published: September 2004

Concomitant application of the cholinergic agonist carbachol and nanomolar doses of kainate can elicit persistent gamma frequency oscillations in all layers of the mouse somatosensory cortex in vitro. Receptor pharmacology with bath‐applied antagonists indicated that oscillatory network activity depended crucially on the participation of cholinergic muscarinic, (S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA)/kainate and GABAA receptors.
The timing of action potentials and the occurrence of excitatory as well as inhibitory postsynaptic events was highly correlated with the phasic change of extracellularly recorded population activity. Firing probability was lowest during the peak negativity of IPSPs and gradually increased during their ensuing decay. In conjunction with the effect of a barbiturate to decrease the frequency of gamma oscillations, this suggests a crucial role of IPSPs in phasing the suprathreshold activity of principal neurons.
At nearby (< 1 mm) sites contained within any given cortical layer, oscillatory extra‐ and intracellular activity was highly synchronous with no apparent phase lag. However, interlaminar mapping experiments demonstrated a phase reversal of both extra‐ and intracellularly recorded activity near the lower border of thalamo‐recipient layer 4, thus corroborating findings that have been obtained in vivo.
In conclusion, a modest increase of tonic excitatory drive in conjunction with the activation of cholinergic muscarinic receptors can elicit persistent gamma frequency network oscillations in the rodent somatosensory cortex. These findings (re)emphasize the role of the cholinergic ascending system in the cortical processing of sensory information.