bluuebunny/crossref_metadata_2025_split huggingface dataset:
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.