bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium permeability and block at homomeric and heteromeric P2X2and P2X3receptors, and P2X receptors in rat nodose neurones
Authors: Virginio Caterina , North R. A. , Surprenant Annmarie
Published: September 2004

Whole‐cell recordings were made from HEK 293 (human embryonic kidney) cells stably transfected with cDNAs encoding P2X2, P2X3or both receptors (P2X2/3) and from cultured rat nodose neurones. Nodose neurones all showed immunoreactivity for both P2X2and P2X3, but not P2X1, receptors.

Reversal potentials were measured in extracellular sodium,N‐methyl‐D‐glucamine (NMDG) and NMDG containing 5 mM Ca2+; the values were used to compute relative permeabilities (PNMDG/PNaandPCa/PNa).PNMDG/PNawas not different for P2X2, P2X2/3and nodose neurones (0.03) but was significantly higher (0.07) for P2X3receptors.PCa/PNawas not different among P2X3, P2X2/3and nodose neurones (1.2‐1.5) but was significantly higher (2.5) for P2X2receptors.

External Ca2+inhibited purinoceptor currents with half‐maximal concentrations of 5 mM at the P2X2receptor, 89 mM at the P2X3receptor and 15 mM at both the P2X2/3heteromeric receptor and nodose neurones. In each case, the inhibition was voltage independent and was overcome by increasing concentrations of agonist.

These results may indicate that Ca2+permeability of the heteromeric (P2X2/3) channel is dominated by that of the P2X3subunit, while Ca2+block of the receptor involves both P2X2and P2X3subunits. The correspondence in properties between P2X2/3receptors and nodose ganglion neurones further supports the conclusion that the native α,β‐methylene ATP‐sensitive receptor is a P2X2/3heteromultimer.
Voltage change‐induced gating transitions of the rabbit skeletal muscle Ca2+ release channel
Authors: Zahradníková A. , Mészáros L. G.
Published: September 2004

We used the planar lipid bilayer method to study single ryanodine receptor Ca2+ release channels (RyRCs) from fast skeletal muscle of the rabbit. We found that changes in membrane voltage directly induced gating transitions of the RyRC: (i) in the steady state, even at activating Ca2+ concentrations (20 μm), at a constant membrane potential the channels resided in a low open probability (Po) state (inactivated‐, I‐mode), and (ii) upon abrupt changes of voltage, the apparent inactivation of the RyRCs was relieved, resulting in a rapid and transient increase in Po.

The magnitude of the Po increase was a function of both the duration and the amplitude of the applied prepulse, but was independent of the channel activity during the prepulse.

The voltage‐induced Po increase probably involved major conformational changes of the channel, as it resulted in substantial alterations in the gating pattern of the channels: the voltage change‐induced increase in Po was accompanied by the rapid appearance of two types of channel activity (high (H) and low (L) open probability modes).

The response of the RyRC to voltage changes raises the interesting possibility that the activation of RyRC in situ might involve electrical events, i.e. a possible dipole‐dipole coupling between the release channel and the voltage sensor.
Effects of caffeine and adenine nucleotides on Ca2+ release by the sarcoplasmic reticulum in saponin‐permeabilized frog skeletal muscle fibres
Authors: Duke Adrian M. , Steele Derek S.
Published: September 2004

The effect of caffeine and adenine nucleotides on the sarcoplasmic reticulum (SR) Ca2+ release mechanism was investigated in permeabilized frog skeletal muscle fibres. Caffeine was rapidly applied and the resulting release of Ca2+ from the SR detected using fura‐2 fluorescence. Decreasing the [ATP] from 5 to 0.1 mm reduced the caffeine‐induced Ca2+ transient by 89 ± 1.4 % (mean ± s.e.m., n= 16), while SR Ca2+ uptake was unaffected.

The dependence of caffeine‐induced Ca2+ release on cytosolic [ATP] was used to study the relative ability of other structurally related compounds to substitute for, or compete with, ATP at the adenine nucleotide binding site. It was found that AMP, ADP and the non‐hydrolysable analogue adenylyl imidodiphosphate (AMP‐PNP) partially substituted for ATP, although none was as potent in facilitating the Ca2+‐releasing action of caffeine.

Adenosine reversibly inhibited caffeine‐induced Ca2+ release, without affecting SR Ca2+ uptake. Five millimolar adenosine markedly reduced the amplitude of the caffeine‐induced Ca2+ transient by 64 ± 4 % (mean ± s.e.m., n= 11). The degree of inhibition was dependent upon the cytosolic [ATP], suggesting that adenosine may act as a competitive antagonist at the adenine nucleotide binding site.

These data show that (i) the sensitivity of the in situ SR Ca2+ channel to caffeine activation is strongly dependent upon the cytosolic [ATP], (ii) the number of phosphates attached to the 5′ carbon of the ribose ring influences the efficacy of the ligand, and (iii) removal of a single phosphate group transforms AMP from a partial agonist, to adenosine, which acts as a competitive antagonist under these conditions.
Osmosensitive Cl− currents and their relevance to regulatory volume decrease in human intestinal T84 cells: outwardly vs. inwardly rectifying currents
Authors: Bond Tamara D. , Ambikapathy Sujidra , Mohammad Samira , Valverde Miguel A.
Published: September 2004

The swelling‐activated outwardly rectifying Cl− current (ICl(swell)) recorded in T84 human intestinal cells was completely blocked by 10 μM tamoxifen, while 300 μM Cd2+ had no effect.

A ClC‐2‐like, inwardly rectifying Cl− current was activated after strong hyperpolarization in T84 cells. This current was completely inhibited by 300 μM Cd2+, unaffected by 10 μM tamoxifen, and its magnitude increased slightly in response to cell swelling under hyposmotic conditions. However, the swelling‐dependent modulation occurred only after prior activation by hyperpolarizing voltages.

T84 cells behaved initially close to perfect osmometers in response to changes in external osmolalities between +20 and ‐30 %. The cells underwent full regulatory volume decrease (RVD) within 16 min when exposed to 30 or 10 % hyposmotic shocks.

Pharmacological tools were used to determine the anionic pathway(s) involved in RVD in T84 cells. Tamoxifen (10 μM), 1,9‐dideoxyforskolin (DDFSK; 100 μM) and 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid (DIDS; 100 μM) blocked RVD while 300 μM Cd2+ had no effect upon RVD following a 30 % hyposmotic shock. The RVD response was similarly unaffected by Cd2+ when cells were exposed to a smaller (10 %) hyposmotic shock.

In conclusion, these data show that the anionic pathway primarily activated by cell swelling and relevant to RVD in T84 cells is the tamoxifen‐, DDFSK‐ and DIDS‐sensitive ICl(swell) and not the hyperpolarization‐activated, Cd2+‐sensitive Cl− current associated with the ClC‐2 Cl− channel.
Modulation of Ca2+ channel currents of acutely dissociated rat periaqueductal grey neurons
Authors: Connor Mark , Christie MacDonald J.
Published: September 2004

The actions of the neuropeptide nociceptin on the calcium channel currents (IBa) of acutely dissociated rat periaqueductal grey (PAG) neurons were examined using whole‐cell patch clamp techniques. These effects were compared with those of opioid receptor agonists and the GABAB receptor agonist baclofen.

Neurons from young adult rats (23 to 56 days old) expressed predominantly ω‐conotoxin GVIA (N‐type)‐ and ω‐agatoxin IVA (P/Q‐type)‐sensitive IBa, together with smaller amounts of nimodipine‐sensitive current and current resistant to all three blockers. There was proportionately more N‐type IBa in neurons from female rats and proportionately more resistant current in neurons from male rats.

Nociceptin (EC50, 5 nm) and baclofen (EC50, 0.8 μm) inhibited IBa in all PAG neurons, while the opioid agonist methionine enkephalin (met‐enkephalin; 300 nm‐10 μm) inhibited IBa in 40 % of neurons. The effects of met‐enkephalin were reversed by the μ‐opioid antagonist CTAP, and mimicked by the μ‐opioid agonist DAMGO (300 nm‐3 μm). The δ‐opioid agonists DPDPE and deltorphin II, and the κ‐opioid agonist U69593, did not affect IBa in any neuron. The actions of nociceptin were not mimicked or blocked by the opioid antagonist naloxone or the nociceptin analogue [desPhe1]‐nociceptin.

The effects of nociceptin and baclofen on IBa were blocked by pretreatment of the neurons with pertussis toxin (500 ng ml−1, 8 h).

Nociceptin predominantly inhibited the N‐type (EC50, 2 nm; maximum inhibition, 50 %) and P/Q‐type (EC50, 7 nm; maximum inhibition, 33 %) IBa while having little effect on the L‐type and R‐type IBa.

These results are consistent with the previously described actions of nociceptin, baclofen and μ‐opioids in PAG slices, whereby they couple to increases in an inwardly rectifying K+ conductance. These agonists thus have the potential to modulate the function of PAG neurons via a number of different cellular effectors.
Role of tyrosine phosphorylation in leptin activation of ATP‐sensitive K+ channels in the rat insulinoma cell line CRI‐G1
Authors: Harvey J. , Ashford M. L. J.
Published: September 2004

Using whole‐cell and cell‐attached recording configurations, the role of phosphorylation in leptin activation of ATP‐sensitive K+ (KATP) channels was examined in the rat CRI‐G1 insulinoma cell line.

Whole‐cell current clamp recordings demonstrated that, following dialysis with the non‐hydrolysable ATP analogue 5′‐adenylylimidodiphosphate (AMP‐PNP; 3‐5 mM), the leptin‐induced hyperpolarization and increase in K+ conductance were completely inhibited.

Under current clamp conditions, application of the broad‐spectrum protein kinase inhibitor H‐7 (10 μM) had no effect on the resting membrane potential or slope conductance of CRI‐G1 insulinoma cells and did not occlude the actions of leptin.

Application of the tyrosine kinase inhibitors genistein (10 μM), tyrphostin B42 (10 μM) and herbimycin A (500 nM) all resulted in activation of KATP channels. In cell‐attached recordings, the presence of tyrphostin B42 (10 μM) in the pipette solution activated tolbutamide‐sensitive KATP channels in CRI‐G1 cells. In contrast, the inactive analogues of genistein and tyrphostin B42 were without effect.

The serine/threonine‐specific protein phosphatase inhibitors okadaic acid (50 nM) and cyclosporin A (1 μM) did not prevent or reverse leptin activation of KATP channels. In contrast, whole‐cell dialysis with the tyrosine phosphatase inhibitor orthovanadate (500 μM) prevented the actions of both leptin and tyrphostin B42.

In conclusion, leptin activation of KATP channels appears to require inhibition of tyrosine kinases and subsequent dephosphorylation. This process is likely to occur prior to activation of phosphoinositide 3‐kinase (PI 3‐kinase) as wortmannin prevented activation of KATP channels by tyrphostin B42.
Long‐term induction of a unique Cl− current by endothelin‐1 in an epithelial cell line from rat lung: evidence for regulation of cytoplasmic calcium
Authors: Mair Norbert , Frick Manfred , Meraner Andreas , Schramek Herbert , Dietl Paul
Published: September 2004

Using conventional microelectrodes, the perforated patch clamp technique and fluorescence microscopy with fura‐2, we investigated the relationship between the cell membrane potential, whole‐cell currents and the free cytoplasmic Ca2+ concentration ([Ca2+]i) in response to 10 nM endothelin‐1 (ET) in a rat respiratory epithelial cell line (L2).

Microelectrode experiments revealed that ET caused an immediate depolarization of the cell membrane potential (Vm) by 25 mV, which was unaffected by Na+ replacement with N‐methyl‐D‐glucamine+ (NMDG+) or by omission of bath Ca2+. In contrast, ET depolarized the cells by 61 mV in the presence of low Cl− (6 mM), resulting in a complete breakdown of Vm.

In perforated patch clamp experiments, the ET‐induced whole‐cell current (IET) exhibited a slight outward rectification with a reversal potential (Vrev) of ‐22·7 mV. IET was reduced by 85 % in low Cl− (6 mM), but was unaffected by Ca2+ removal, Na+ replacement with NMDG+, pipette K+ replacement with Cs+ or 1 mM Ni2+ in the bath.

I
ET was unaffected by (+)‐isradipine (100 nM), a specific L‐type Ca2+ channel (L‐VDCC) blocker. Transient inward Sr2+ currents through L‐VDCCs were blocked by ET.

ET induced a biphasic Ca2+ signal, consisting of a ‘peak’ and a ‘plateau’ elevation of [Ca2+]i. Simultaneous patch clamp and fura‐2 measurements revealed that IET coincided with intracellular Ca2+ release but clearly outlasted the elevation of [Ca2+]i. When the rise of [Ca2+]i was prevented by pretreatment with thapsigargin in a Ca2+‐free bath, both activation time and amplitude of IET were reduced. Under these conditions, ET caused a decrease of [Ca2+]i.

The Cl− channel blocker mefenamic acid (MFA) had a dual, concentration‐dependent effect on both IET and the ET‐induced ‘plateau’ elevation of [Ca2+]i: an increase at 10 μM, but an almost complete block at 100 μM. The effect of MFA on IET preceded the effect on [Ca2+]i.

The ET‐induced ‘plateau’[Ca2+]i fell below control values in a low‐Cl− (6 mM) solution.

These data indicate an amplifying function of intracellular Ca2+ release on an otherwise Ca2+‐independent, unique Cl− current by ET. Moreover, this Cl− current appears to be functionally coupled with dihydropyridine (DHP)‐insensitive Ca2+ entry, suggesting a modulatory role for long‐lasting effects of ET.
Gramicidin‐perforated patch revealed depolarizing effect of GABA in cultured frog melanotrophs
Authors: Le Foll Frank , Castel Hélène , Soriani Olivier , Vaudry Hubert , Cazin Lionel
Published: September 2004

In frog pituitary melanotrophs, GABA induces a transient stimulation followed by prolonged inhibition of hormone secretion. This biphasic effect is inconsistent with the elevation of cytosolic calcium and the inhibition of electrical activity also provoked by GABA in single melanotrophs. In the present study, standard patch‐clamp configurations and gramicidin‐perforated patches were used to investigate the physiological GABAA receptor‐mediated response and intracellular chloride concentration ([Cl−]i) in cultured frog melanotrophs.

In the gramicidin‐perforated patch configuration, 1 μM GABA caused a depolarization associated with an action potential discharge and a slight fall of membrane resistance. In contrast, at a higher concentration (10 μm) GABA elicited a depolarization accompanied by a transient volley of action potentials, followed by a sustained inhibitory plateau and a marked fall of membrane resistance. Isoguvacine mimicked the GABA‐evoked responses, indicating a mediation by GABAA receptors.

In gramicidin‐perforated cells, the depolarizing excitatory effect of 1 μm GABA was converted into a depolarizing inhibitory action when 0.4 μm allopregnanolone was added to the bath solution.

After gaining the whole‐cell configuration, the amplitude and/or direction of the GABA‐evoked current (IGABA) rapidly changed before stabilizing. After stabilization, the reversal potential of IGABA followed the values predicted by the Nernst equation for chloride ions when [Cl−]i was varied.

In gramicidin‐perforated cells, the steady‐state I–V relationships of 10 μm GABA‐ or isoguvacine‐evoked currents yielded reversal potentials of −37.5 ± 1.6 (n= 17) and −38.6 ± 2.0 mV (n= 8), respectively. These values were close to those obtained by using a voltage‐ramp protocol in the presence of Na+, K+ and Ca2+ channel blockers. The current evoked by 1 μm GABA also reversed at these potentials.

We conclude that, in frog pituitary melanotrophs, chloride is the exclusive charge carrier of IGABA. In intact cells, the reversal potential of IGABA is positive to the resting potential because of a relatively high [Cl−]i (26.5 mm). Under these conditions, GABA induces a chloride efflux responsible for a depolarization triggering action potentials. However, GABA at a high concentration or in the presence of the potentiating steroid allopregnanolone exerts a concomitant shunting effect leading to a rapid inhibition of the spontaneous firing.
Properties and functions of calcium‐activated K+ channels in small neurones of rat dorsal root ganglion studied in a thin slice preparation
Authors: Scholz Andreas , Gruß Marco , Vogel Werner
Published: September 2004

Properties, kinetics and functions of large conductance calcium‐activated K+ channels (BKCa) were investigated by the patch‐clamp technique in small neurones (Aδ‐ and C‐type) of a dorsal root ganglion (DRG) thin slice preparation without enzymatic treatment.

Unitary conductance of BKCa channels measured in symmetrical high K+ solutions (155 mm) was 200 pS for inward currents, and chord conductance in control solution was 72 pS. Potentials of half‐maximum activation (V1/2) of the channels were linearly shifted by 43 mV per log10[Ca2+]i unit (pCa) in the range of −28 mV (pCa 4) to +100 mV (pCa 7). Open probabilities increased e‐times per 15–32 mV depolarization of potential.

In mean open probability, fast changes with time were mainly observed at pCa > 6 and at potentials > +20 mV, without obvious changes in the experimental conditions.

BKCa channels were half‐maximally blocked by 0.4 mm TEA, measured by apparent amplitude reductions. They were completely blocked by 100 nm charybdotoxin and 50 nm iberiotoxin by reduction of open probability.

Two subtypes of small DRG neurones could be distinguished by the presence (type I) or absence (type II) of BKCa channels. In addition, less than 10 % of small neurones showed fast (∼135 V s−1) and short (∼0.8 ms) action potentials (AP).

The main functions of BKCa channels were found to be shortening of AP duration, increasing of the speed of repolarization and contribution to the fast after‐hyperpolarization. As a consequence, BKCa channels may reduce the amount of calcium entering a neurone during an AP.

BKCa channel currents suppressed a subsequent AP and prolonged the refractory period, which might lead to a reduced repetitive activity. We suggest that the BKCa current is a possible mechanism of the reported conduction failure during repetitive stimulation in DRG neurones.
Preferential regulation of rabbit cardiac L‐type Ca2+ current by glycolytic derived ATP via a direct allosteric pathway
Authors: Losito Vito A. , Tsushima Robert G. , Diaz Roberto J. , Wilson Gregory J. , Backx Peter H.
Published: September 2004

The activity of Ca2+ channels is regulated by a number of mechanisms including direct allosteric modulation by intracellular ATP. Since ATP derived from glycolysis is preferentially used for membrane function, we hypothesized that glycolytic ATP also preferentially regulates cardiac L‐type Ca2+ channels.

To test this hypothesis, peak L‐type Ca2+ currents (ICa) were measured in voltage‐clamped rabbit cardiomyocytes during glycolytic inhibition (2‐deoxyglucose + pyruvate), oxidative inhibition (cyanide + glucose) or both (full metabolic inhibition; FMI).

A 10 min period of FMI resulted in a 40·0 % decrease in peak ICa at +10 mV (‐5·1 ± 0·6 versus ‐3·1 ± 0·4 pA pF−1; n= 5, P < 0·01). Similar decreases in peak ICa were observed during glycolytic inhibition using 2‐deoxyglucose (‐6·2 ± 0·2 versus ‐3·7 ± 0·2 pA pF−1; n= 5, P < 0·01) or iodoacetamide (‐6·7 ± 0·3 versus ‐3·7 ± 0·2 pA pF−1; n= 7, P < 0·01), but not following oxidative inhibition (‐6·2 ± 0·4 versus ‐6·4 ± 0·3 pA pF−1; n= 5, n.s.). The reduction in ICa following glycolytic inhibition was not mediated by phosphate sequestration by 2‐deoxyglucose or changes in intracellular pH.

Reductions in ICa were still observed when inorganic phosphate and creatine were included in the pipette, confirming a critical role for glycolysis in ICa regulation.

With 5 mM MgATP in the pipette during FMI, peak ICa decreased by only 18·4 % (‐6·8 ± 0·6 versus ‐5·5 ± 0·3 pA pF−1; n= 4, P < 0·05), while inclusion of 5 mM MgAMP‐PCP (β,γ‐methyleneadenosine 5′‐triphosphate, Mg2+ salt) completely prevented the decrease in peak ICa (‐6·9 ± 0·3 versus ‐6·5 ± 0·3 pA pF−1; n= 5, n.s.).

Together, these results suggest that ICa is regulated by intracellular ATP derived from glycolysis and does not require hydrolysis of ATP. This regulation is expected to be energy conserving during periods of metabolic stress and myocardial ischaemia.
A continuum of InsP3‐mediated elementary Ca2+ signalling events in Xenopus oocytes
Authors: Sun Xiao‐Ping , Callamaras Nick , Marchant Jonathan S. , Parker Ian
Published: September 2004

The elementary release events underlying inositol 1,4,5‐trisphosphate (InsP3)‐mediated calcium signalling were investigated in Xenopus oocytes by means of high‐resolution confocal linescan imaging together with flash photolysis of caged InsP3.

Weak photolysis flashes evoked localized, transient calcium signals that arose at specific sites following random latencies of up to several seconds. The duration, spatial spread and amplitude of these elementary events varied widely. Event durations (at half‐maximal amplitude) were distributed exponentially between about 100 and 600 ms. Fluorescence magnitudes (F/F0 of Oregon Green 488 BAPTA‐1) showed a skewed distribution with a peak at about 1.5 and a tail extending as high as 3.5.

Individual release sites exhibited both small events (blips) and large events (puffs). The spatiotemporal distribution of calcium signals during puffs was consistent with calcium diffusion from a point source (< a few hundred nanometres), rather than with propagation of a microscopic calcium wave.

Estimates of the calcium flux associated with individual events were made by integrating fluorescence profiles along the scan line in three dimensions to derive the ‘signal mass’ at each time point. The smallest resolved events corresponded to liberation of < 2 × 10−20 mol Ca2+, and large events to about 2 × 10−18 mol Ca2+. The rise of signal mass was more prolonged than that of the fluorescence intensity, suggesting that calcium liberation persists even while the fluorescence begins to decline. Rates of rise of signal mass corresponded to Ca2+ currents of 0.4‐2.5 pA.

Measurements of signal mass from different events showed a continuous, exponential distribution, arising through variability in magnitude and duration of calcium flux.

We conclude that localized calcium transients in the oocyte represent a continuum of events involving widely varying amounts of calcium liberation, rather than falling into separate populations of ‘fundamental’ and ‘elementary’ events (blips and puffs) involving, respectively, single and multiple InsP3 receptor channels. This variability probably arises through stochastic variation in both the number of channels recruited and the duration of channel opening.
Endomorphins inhibit high‐threshold Ca2+ channel currents in rodent NG108‐15 cells overexpressing μ‐opioid receptors
Authors: Higashida Haruhiro , Hoshi Naoto , Knijnik Rimma , Zadina James E. , Kastin Abba J.
Published: September 2004

Extracellular application of the novel brain peptides endomorphin 1 (EM1) and endomorphin 2 (EM2) inhibited high‐threshold Ca2+ channel currents in NGMO‐251 cells, a daughter clone of NG108‐15 mouse neuroblastoma × rat glioma hybrid cells, in which μ‐opioid receptors are overexpressed.

In contrast, EM1 and EM2 did not induce this inhibition in the parental NG108‐15 cells that predominantly express endogenous δ‐receptors.

The IC50 for EM1 and EM2 was 7.7 and 23.1 nm, respectively.

EM‐induced Ca2+ channel current inhibition was blocked by treatment or pretreatment of the cells with 100 μmN‐methylmaleimide or 100 ng ml−1 pertussis toxin.

These results show that a decrease in conductance of Ca2+ channels results following interaction of EMs with cloned μ‐receptors, which couple via Gi/Go‐type G proteins, and that EMs fulfill one of the necessary synaptic conditions for them to be identified as neurotransmitters.
Ca2+‐inhibited non‐inactivating K+ channels in cultured rat hippocampal pyramidal neurones
Authors: Selyanko A. A. , Sim J. A.
Published: September 2004

Whole‐cell perforated‐patch recording from cultured CA1‐CA3 pyramidal neurones from neonatal rat hippocampus (20‐22 °C; [K+]o= 2.5 mM) revealed two previously recorded non‐inactivating (sustained) K+ outward currents: a voltage‐independent ‘leak’ current (Ileak) operating at all negative potentials, and, at potentials ≥−60 mV, a time‐ and voltage‐dependent ‘M‐current’ (IK(M)). Both were inhibited by 1 mM Ba2+ or 10 μM oxotremorine‐M (Oxo‐M). In ruptured‐patch recording using Ca2+‐free pipette solution, Ileak was strongly enhanced, and was inhibited by 1 mM Ba2+ but unaffected by 0.5 mM 4‐aminopyridine (4‐AP), 1 mM tetraethylammonium (TEA) or 1‐10 nM margatoxin.

Single channels underlying these currents were sought in cell‐attached patch recordings. A single class of channels of conductance ≈7 pS showing sustained activity at resting potential and above was identified. These normally had a very low open probability (Po < 0.1), which, however, showed a dramatic and reversible increase (to about 0.9 at ≈0 mV) following the removal of Ca2+ from the bath. Under these (Ca2+‐free) conditions, single‐channel Po showed both voltage‐dependent and voltage‐independent components on patch depolarization from resting potential. The mean activation curve was fitted by a modified Boltzmann equation. When tested, all channels were reversibly inhibited by addition of 10 μM Oxo‐M to the bath solution.

The channels maintained their high Po in patches excised in inside‐out mode into a Ca2+‐free internal solution and were strongly inhibited by application of Ca2+ to the inner face of the membrane (IC50= 122 nM); this inhibition was observed in the absence of MgATP, and therefore was direct and unrelated to channel phosphorylation/dephosphorylation.

Channels in patches excised in outside‐out mode were blocked by 1 mM Ba2+ but were unaffected by 4‐AP or TEA.

Channels in cell‐attached patches were inhibited after single spikes, yielding inward ensemble currents lasting several hundred milliseconds. This was prevented in Ca2+‐free solution, implying that it was due to Ca2+ entry.

The properties of these channels (block by internal Ca2+ and external Oxo‐M and Ba2+, and the presence of both voltage‐dependent and voltage‐independent components in their Po/V relationship) show points of resemblance to those expected for channels associated with both Ileak and IK(M) components of the sustained macroscopic currents. For this reason we designate them Ksust (‘sustained current’) channels. Inhibition of these channels by Ca2+ entry during an action potential may account for some forms of Ca2+‐dependent after‐depolarization. Their high sensitivity to internal Ca2+ may provide a new, positive feedback mechanism for cell excitation operating at low (near‐resting) [Ca2+]i.
L‐type Ca2+ channels in inspiratory neurones of mice and their modulation by hypoxia
Authors: Mironov S. L. , Richter D. W.
Published: September 2004

Whole‐cell (ICa) and single Ca2+ channel currents were measured in inspiratory neurones of neonatal mice (4–12 days old). During whole‐cell recordings, ICa slowly declined and disappeared within 10–20 min. The run‐down was delayed during hypoxia, indicating ICa potentiation.

Ca2+ channels were recorded in cell‐attached patches using pipettes which contained 110 mm Ba2+. L‐type Ca2+ channels exhibited a non‐ohmic I–V relationship. The slope conductance was 24 pS below and 50 pS above their null potential. The open probability of the channels increased during oxygen depletion, reaching a maximum 2 min after the onset of hypoxia. Restoration of the oxygen supply brought the channel activity back to initial levels.

The channel activity was enhanced by 3–30 μmS(–)Bay K 8644, an agonist of L‐type Ca2+ channels. The open probability was increased about 3‐fold and the activation curve was shifted by 20 mV in the hyperpolarizing direction. In the presence of the agonist, channel open time increased and long openings appeared. Agonist‐modulated channels were also potentiated during oxygen depletion. The effect was due to an increase in open time and a decrease in closed time. The channels were inhibited by bath application of nifedipine (10 μm) and nitrendipine (20 μm).

Weak bases such as NH4Cl and TMA increased and weak acids such as sodium acetate and propionate decreased activity of the channels, indicating that they are modulated by intracellular pH. Bath application of 1 μm forskolin enhanced the channel activity, whereas 500 μm NaF suppressed it.

L‐type Ca2+ channels were modulated by an agonist for mGluR1/5 receptors, (S)‐3,5‐dihydrophenylglycine (DHPG, 5 μm). In its presence, the hypoxic facilitation of channels was abolished.

After blockade of L‐type Ca2+ channels, the respiratory response to hypoxia was modified. The transient enhancement of the respiratory rhythm (augmentation) was no longer evident and the secondary depression occurred earlier.

We suggest that L‐type Ca2+ channels contribute to the early hypoxic response of the respiratory centre. Glutamate release during hypoxia stimulates postsynaptic metabotropic glutamate receptors, which activate the Ca2+ channels.
Clostridium difficile toxin B inhibits carbachol‐induced force and myosin light chain phosphorylation in guinea‐pig smooth muscle: role of Rho proteins
Authors: Lucius Claudia , Arner Anders , Steusloff Annette , Troschka Monika , Hofmann Fred , Aktories Klaus , Pfitzer Gabriele
Published: September 2004

Clostridium difficiletoxin B glucosylates the Ras‐related low molecular mass GTPases of the Rho subfamily thereby inactivating them. In the present report, toxin B was applied as a tool to test whether Rho proteins participate in the carbachol‐induced increase in the Ca2+ sensitivity of force and myosin light chain (MLC) phosphorylation in intactintestinal smooth muscle.

Small strips of the longitudinal muscle of guinea‐pig small intestine were incubated in toxin B (40 ng ml−1) overnight. Carbachol‐induced force and intracellular [Ca2+], and, in a separate series, force and MLC phosphorylation, were determined.

Carbachol induced a biphasic contraction: an initial rapid increase in force (peak 1) followed by a partial relaxation and a second delayed increase in force (peak 2). The peak of the Ca2+ signal measured with fura‐2 preceded peak 1 of force and then declined to a lower suprabasal steady‐state level. Peak 2 was not associated with a significant increase in [Ca2+]. Toxin B nearly completely inhibited peak 2 while peak 1 was not significantly inhibited. Toxin B had no effect on the Ca2+ transient.

In control strips, MLC phosphorylation at peak 2 was 27.7 %, which was significantly higher than the resting value (18.6 %). The inhibition of the second, delayed, rise in force induced by toxin B was associated with complete inhibition of the increase in MLC phosphorylation. The resting MLC phosphorylation was not significantly different from that of the control strips.

The initial increase in MLC phosphorylation determined 3 s after exposure to carbachol was 54 % in the control strips. Toxin B also inhibited this initial phosphorylation peak despite the fact that the Ca2+ transient and the initial increase in force were not inhibited by toxin B. This suggests that Rho proteins play an important role in setting the balance between MLC phosphorylation and dephosphorylation reactions even at high levels of intracellular Ca2+.

These findings are consistent with the hypothesis that the delayed rise in force elicited by carbachol is due to an increase in the Ca2+ sensitivity of MLC phosphorylation mediated by Rho proteins.
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.