bluuebunny/crossref_metadata_2025_split huggingface dataset:
Voltage‐dependent binding and calcium channel current inhibition by an anti‐α1D subunit antibody in rat dorsal root ganglion neurones and guinea‐pig myocytes
Authors: Wyatt C. N. , Campbell V. , Brodbeck J. , Brice N. L. , Page K. M. , Berrow N. S. , Brickley K. , Terracciano C. M. N. , Naqvi R. U. , MacLeod K. T. , Dolphin A. C. 
Published: September 2004

The presence of calcium channel α1D subunit mRNA in cultured rat dorsal root ganglion (DRG) neurones and guinea‐pig cardiac myocytes was demonstrated using the reverse transcriptase‐polymerase chain reaction.
An antipeptide antibody targeted at a region of the voltage‐dependent calcium channel α1Dsubunit C‐terminal to the pore‐forming SS1–SS2 loop in domain IV (amino acids 1417–1434) only bound to this exofacial epitope if the DRG neurones and cardiac myocytes were depolarized with 30 mM K+.
Incubation of cells under depolarizing conditions for 2–4 h with the antibody resulted in a maximal inhibition of inward current density of 49% (P < 0.005) for DRGs and 30% (P < 0.05) for cardiac myocytes when compared with controls.
S‐(–)‐Bay K 8644 (1 μM) enhanced calcium channel currents in DRGs by 75 ± 19% (n= 5) in neurones incubated under depolarizing conditions with antibody that had been pre‐adsorbed with its immunizing peptide (100 μg ml−1). This was significantly (P < 0.05) larger than the enhancement by S‐(–)‐Bay K 8644 that was seen with cells incubated under identical conditions but with antibody alone, which was 15 ± 4% (n= 5).
These results demonstrate the presence of calcium channel α1D subunits in rat DRG neurones and guinea‐pig cardiac myocytes. They also show that amino acids 1417–1434 of the α1D subunit are only exposed to the extracellular face of the membrane following depolarization and that the binding of an antibody to these amino acids attenuates calcium channel current and reduces the ability of S‐(–)‐Bay K 8644 to enhance this current, indicating that it is an L‐type current that is attenuated.
A Novel Role for HERG K+ Channels: Spike‐Frequency Adaptation
Authors: Chiesa Nicoletta , Rosati Barbara , Arcangeli Annarosa , Olivotto Massimo , Wanke Enzo 
Published: September 2004

The regular firing of a Hodgkin‐Huxley neurone endowed with fast Na+ and delayed K+ channels can be converted into adapting firing by appending HERG (human eag‐related gene) channels.
The computer model predictions were verified by studying the firing properties of F‐11 DRG neurone x neuroblastoma hybrid cells induced to differentiate by long‐term exposure to retinoic acid. These cells, which express HERG currents (IHERG), show clear spike‐frequency adaptation of their firing when current clamped with long depolarizations.
In agreement with the prediction, the selective blocking of IHERG by class III antiarrhythmic drugs always led to the disappearance of the spike‐frequency adaptation, and the conversion of adapting firing to regular firing.
It is proposed that, in addition to their role in the repolarization of the heart action potential, HERG channels may sustain a process of spike‐frequency adaptation, and hence contribute to the control of burst duration in a way that is similar to that of the K+ currents, IAHP, IC and IM. In addition to the known cardiac arrhythmia syndrome (LQT2), genetic mutations or an altered HERG expression could lead to continuous hyperexcitable states sustained by the inability of nerve or endocrine cells to accommodate to repetitive stimuli. This might help in clarifying the pathogenesis of still undefined idiopathic familial epilepsies.
A Charybdotoxin‐Insensitive Conductance in Human T Lymphocytes: T Cell Membrane Potential is Set by Distinct K+ Channels
Authors: Verheugen Jos A. H. , Korn Henri 
Published: September 2004

Changes in the membrane potential (Vm) of human T lymphocytes upon K+ channel block were inferred from alterations in K+ current reversal potential in cell‐attached patches. It was found that a high concentration of charybdotoxin (100 nm, CTX), which blocks both voltage‐gated (K(V)) and Ca2+‐activated (K(Ca)) potassium channels in these cells, depolarizes Vm of lymphocytes only partially. Subsequent whole‐cell measurements of the same cells showed that 39 ± 25% of the voltage‐gated current remains in the presence of CTX.
The CTX‐resistant current reverses at potentials between −80 and −90 mV, indicating that it is K+ selective. The current is activated at more depolarized potentials compared with the unblocked −IK(V) current with a threshold between −40 and −20 mV and a half‐maximal activation at +50 mV. Inactivation during prolonged depolarization is slow. Steady‐state inactivation is half‐maximal at ‐45 mV and complete at potentials > − 20 mV. The CTX‐resistant IK(V) is completely blocked by nifedipine and is not sensitive to dendrotoxin.
The effect of nifedipine on the Vm of lymphocytes varies between cells depending on the contribution of the nifedipine‐sensitive current to whole‐cell IK(V) Combined application of CTX and nifedipine completely depolarizes Vm.
The extent to which T cell receptor‐evoked Ca2+ signals of resting cells are inhibited by K+ channel blockers correlates with the magnitude of the depolarization induced by the drugs. Complete suppression of the response is achieved only by combined block of the CTX‐sensitive and ‐insensitive IK(V). The enhanced Ca2+ response of activated cells, which express increased numbers of K(Ca) channels, is in addition subject to modulation by blockers which prevent the hyperpolarization during the Ca2+ rise mediated by these channels.
Pituitary adenylate cyclase‐activating polypeptide (PACAP) is an islet substance serving as an intra‐islet amplifier Of glucose‐induced insulin secretion in rats
Authors: Yada Toshihiko , Sakurada Masaya , Ishihara Hisamitsu , Nakata Masanori , Shioda Seiji , Yaekura Kazuro , Hamakawa Noriaki , Yanagida Kazuhiro , Kikuchi Masatoshi , Oka Yoshitomo 
Published: September 2004

We examined whether pituitary adenylate cyclase‐activating polypeptide with 38 or 27 residues (PACAP‐38 or PACAP‐27) serves as an intra‐islet regulator of glucose‐induced insulin secretion in rats. PACAP antiserum specific for PACAP‐38 and PACAP‐27 was used to neutralize the effect of endogenous PACAP in islets. PACAP release from islets was bioassayed using the response of cytosolic Ca2+ concentration ([Ca2+]i) in single β‐cells, monitored by dual‐wavelength fura‐2 microfluorometry. Expression of PACAP mRNA was studied by reverse transcription‐polymerase chain reaction (RT‐PCR), while expression of PACAP was studied by metabolic labelling and immunoblotting. Localization of PACAP receptors was studied immunohistochemically.
High glucose‐stimulated insulin release from isolated islets was attenuated by PACAP anti‐serum but not by non‐immune sera.
The islet incubation medium with high glucose (Med) possessed a capacity, which was neutralized by PACAP antiserum, to increase [Ca2+]i in β‐cells. PACAP antiserum also neutralized the [Ca2+]iincreasing action of synthetic PACAP‐38 and PACAP‐27, but not that of vasoactive intestinal polypeptide (VIP) and glucagon.
Both Med and synthetic PACAP increased [Ca2+]i in β‐cells only in the presence of stimulatory, but not basal, glucose concentrations. In contrast, ATP, a substance that is known to be released from β‐cells, increased [Ca2+]i in β‐cells at both basal and stimulatory glucose concentrations.
Expression of PACAP mRNA and biosynthesis of PACAP‐38 were detected in islets and a β‐cell line, MIN6.
Immunoreactivity for PACAP‐selective type‐I receptor was observed in islets.
[Ca2+]i measurements combined with immunocytochemistry with insulin antiserum revealed a substantial population of glucose‐unresponsive β‐cells, many of which were recruited by PACAP‐38 into [Ca2+]i responses.
These results indicate that PACAP‐38 is a novel islet substance that is synthesized and released by islet cells and then, in an autocrine and/or paracrine manner, potentiates and arouses β‐cells responses to glucose, thereby amplifying glucose‐induced insulin secretion in islets.
Receptors couple to L‐type calcium channels via distinct Go proteins in rat neuroendocrine cell lines
Authors: Degtiar Vadim E. , Harhammer Rainer , Nürnberg Bernd 
Published: September 2004

The present study examines the hypothesis of G protein subtype selectivity in receptor‐induced inhibition of calcium channel currents (ICa) in the insulin‐secreting RINm5F and pituitary GH3 rat cell lines. Specificity of receptor coupling to G proteins was studied by infusion of purified Ga isoforms into cells via a patch pipette.
In RINm5F cells, the neuropeptide galanin inhibited dihydropyridine (DHP)‐ and ω‐conotoxin‐sensitive components of ICa and slowed down their activation kinetics. In GH3 cells, DHP‐sensitive ICa was inhibited by galanin, as well as by somatostatin and carbachol. Agonist‐induced ICa inhibition was suppressed by pertussis toxin (PTX) pretreatment of the cells. In PTX‐pretreated cells of either cell line, the response to galanin was restored only by the Gα01 subunit. Following PTX treatment of GH3 cells, only the Gα01 subunit restored carbachol‐induced inhibition of ICa, whereas only the Gαo2 subunit restored somatostatin‐induced inhibition of ICa. Gl subtypes had no effect on ICa inhibition.
Both cell lines expressed two distinct immunoreactive Go proteins. Whereas in RINm5F cell membranes G01 was found to be the predominant isoform, we detected more G02 than G01 in GH3 cell membranes. Nevertheless, all agonists stimulated incorporation of the photoreactive GTP analogue [α‐ 32P]GTP azidoanilide into both Go isoforms.
The results indicate that the same Go subtype, i.e. G01, mediates galanin‐induced inhibition of ICa in both cell lines and that the Go subtype specificity of receptor‐G protein coupling is confined to intact cells.
Diadenosine polyphosphates evoke Ca2+ transients in guinea‐pig brain via receptors distinct from those for ATP
Authors: Pintor Jesús , Puche José A. , Gualix Javier , Hoyle Charles H. V. , Miras‐Portugal Maria Teresa 
Published: September 2004

The ability of diadenosine polyphosphates, namely P1,P2‐di(adenosine) pyrophosphate (Ap2A), P1,P3‐di(adenosine) triphosphate (Ap3A), P1,P4‐di(adenosine) tetraphosphate (Ap4A), P1,P5‐di(adenosine) pentaphosphate (Ap5A) and P1,P6‐di(adenosine) hexaphosphate (Ap6A) to evoke Ca2+ signals in synaptosomes prepared from three different regions of the guinea‐pig brain was examined.
In synaptosomal preparations from the paleocortex (cortex), diencephalon/brainstem (midbrain) and cerebellum all the dinucleotides evoked Ca2+ signals that were concentration dependent over the range 1‐300 μm. ATP and its synthetic analogues,α,β‐methylene ATP, 2‐methylthio ATP and adenosine 5‘‐O‐(2‐thio)diphosphate (all 100 μm) also evoked Ca2+ signals in these preparations.
In the midbrain and cerebellum preparations, responses to ATP and its analogues were attenuated or abolished by the P2 receptor antagonist suramin (100μm) but responses to the dinucleotides were not. Also, desensitization by a dinucleotide blocked responses to dinucleotides but not mononucleotides, and desensitization by a mononucleotide blocked responses to mononucleotides but not dinucleotides.
In cortical preparations, suramin (100μm) blocked responses to both classes of nucleotides. Furthermore, there was mutual cross‐desensitization between the mono‐ and dinucleotides.
The adenosine A1 receptor antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine, did not affect responses evoked by the dinucleotides, nor did the pyrimidine UTP.
It is concluded that there are specific dinucleotide receptors, activated by diadenosine polyphosphates, but not ATP or UTP, on synaptic terminals in guinea‐pig diencephalon/brainstem and cerebellum. These receptors bear a similarity to the dinucleotide receptor (P4 receptor) in rat brain. In guinea‐pig cerebral cortex synaptosomes, diadenosine polyphosphates appear to act via the same receptor as ATP.
The role of the sarcolemmal Ca2+ ‐ATPase in the pH transients associated with contraction in rat smooth muscle
Authors: Naderali E. K. , Buttell N. , Taggart M. J. , Bullock A. J. , Eisner D. A. , Wray Susan 
Published: September 2004

We have investigated the origin of the intracellular acid pH transients that accompany myometrial contraction. Intra‐and extracellular pH were measured with SNARF and intra‐cellular Ca2+ concentration ([Ca2+]i) with indo‐1.
An intracellular acidification accompanied spontaneous contractions and those elicited by KC1 depolarization or the addition of the agonists carbachol or prostaglandin F2x. The size of the acidification increased with the magnitude of the contraction.
The intracellular acidification was accompanied by an extracellular alkalinization, showing that it results from proton movement across the surface membrane. Furthermore, it was decreased either by addition of Cd2+ (20 nm, an inhibitor of the sarcolemmal Ca2+‐ATPase) or by elevating [Ca2+]o.
Extracellular alkalinization increased the magnitude of the rise of [Ca2+]j and force produced by KC1.
An intracellular acidification was also associated with contraction in the portal vein and ureter.
We conclude that the sarcolemmal Ca2+‐ATPase produces a significant intracellular acidification while removing Ca2+. Both the acidification and decrease of [Ca2+]i will promote relaxation. Since Ca2+ and protons have opposite effects on many cellular processes, this dual regulation by these two ions may be of general importance.
Mechanism of Glibenclamide Inhibition of Cystic Fibrosis Transmembrane Conductance Regulator Cl− Channels Expressed in a Murine Cell Line
Authors: Sheppard David N. , Robinson Katherine A. 
Published: September 2004

The sulphonylurea drug glibenclamide is a widely used inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR). To investigate how glibenclamide inhibits CFTR, we studied CFTR Cl− channels using excised inside‐out membrane patches from cells expressing wild‐type human CFTR.
Addition of glibenclamide (10–100 μM) to the intracellular solution caused a concentration‐dependent decrease in the open time of CFTR Cl− channels, but closed times did not change. This suggests that glibenclamide is an open‐channel blocker of CFTR.
Glibenclamide is a weak organic acid. Acidification of the intracellular solution relieved glibenclamide inhibition of CFTR, suggesting that the anionic form of glibenclamide inhibits CFTR.
To begin to identify the glibenclamide binding site in CFTR, we investigated whether glibenclamide competes with either MgATP or Cl− ions for a common binding site. Glibenclamide inhibition of CFTR was unaffected by nucleotide‐dependent stimulation of CFTR, suggesting that glibenclamide and intracellular MgATP interact with CFTR at distinct sites.
Glibenclamide inhibition of CFTR was voltage dependent and enhanced when the external Cl− concentration was decreased. The data suggest that glibenclamide and Cl− ions may compete for a common binding site located within a large intracellular vestibule that is part of the CFTR pore.
Variability in frequency and characteristics of Ca2+ sparks at different release sites in rat ventricular myocytes
Authors: Parker Ian , Gil Wier Withrow 
Published: September 2004

High spatial resolution confocal imaging was used to investigate spontaneous calcium release events (Ca2+ sparks) in isolated rat cardiac myocytes loaded with the fluorescent calcium indicator fluo‐3.
Frequencies of sparks at different release sites varied widely, with a few sites showing sustained activities as great as 50 times the average. Sites with frequent sparks showed more rapid recovery of activity following Ca2+ waves and locally elevated basal [Ca2+].
In addition to transient sparks, some sites showed bursts of rapid flurries of spark‐like events, or apparently sustained Ca2+ liberation. Bursts remained localized to individual Z‐lines, but adjacent sites on the same Z‐line could be ‘driven’ by a bursting site to generate similar activity.
Individual sites showed long‐term (tens of seconds) changes in ‘modes’ of activity, with abrupt transitions in frequencies of sparking, and between transient sparks and sustained bursts. These transitions were not associated with changes in the amplitude of the sparks.
We conclude that spontaneous sparks are not stereotyped events generated with uniform probability at all sites. Instead, the Ca2+ release event is variable, and some sites have a high probability of spark generation. Both factors show long‐term changes at individual sites, raising the possibility that properties of fundamental Ca2+ release units may be subject to modulation.
Muscarinic Ca2+ responses resistant to muscarinic antagonists at perisynaptic schwann cells of the frog neuromuscular junction
Authors: Robitaille Richard , Jahromi Babak S. , Charlton Milton P. 
Published: September 2004

Acetylcholine causes a rise of intracellular Ca2+ in perisynaptic Schwann cells (PSCs) of the frog neuromuscular junction. The signalling pathway was characterized using the fluorescent Ca2+ indicator fluo‐3 and fluorescence microscopy.
Nicotinic antagonists had no effect on Ca2+responses evoked by ACh and no Ca2+ responses were evoked with the nicotinic agonist nicotine. The muscarinic agonists muscarine and oxotremorine‐M induced Ca2+ signals in PSCs.
Ca2+ responses remained unchanged when extracellular Ca2+ was removed, indicating that they are due to the release of Ca2+ from internal stores. Incubation with pertussis toxin did not alter the Ca2+ signals induced by muscarine, but did block depression of transmitter release induced by adenosine and prevented Ca2+ responses in PSCs induced by adenosine.
The general muscarinic antagonists atropine, quinuclidinyl benzilate and N‐methyl‐scopolamine failed to block Ca2+ responses to muscarinic agonists. Atropine (at 20000‐fold excess concentration) also failed to reduce the proportion of cells responding to a threshold muscarine concentration sufficient to cause responses in less than 50% of cells. Only the allosteric, non‐specific blocker, gallamine (1–10 μm) was effective in blocking muscarine‐induced Ca2+ responses.
In preparations denervated 7 days prior to experiments, low concentrations of atropine reversibly and completely blocked Ca2+ responses to muscarine.
The lack of blockade by general muscarinic antagonists in innervated, in situ preparations suggests that muscarinic Ca2+ responses at PSCs are not mediated by any of the five known muscarinic receptors or that post‐translational modification prevented antagonist binding.
Role in fast inactivation of the IV/S4–S5 loop of the human muscle Na+ channel probed by cysteine mutagenesis
Authors: Lerche H. , Peter W. , Fleischhauer R. , Pika‐Hartlaub U. , Malina T. , Mitrovic N. , Lehmann‐Horn F. 
Published: September 2004

In order to investigate the role in fast inactivation of the cytoplasmic S4‐S5 loop of the fourth domain (IV/S4–S5) within the α‐subunit of the adult human muscle Na+ channel, every single amino acid from R1469 to G1486 was substituted by a cysteine and the mutants were studied by functional expression in human embryonic kidney cells (tsA201) using whole‐cell patch clamping. Effects following intracellular application of the sulfhydryl reagents MTSET and MTSES on the mutants were investigated.
Sixteen of eighteen mutants resulted in the formation of functional channels. For P1480C and N1484C, no Na+ currents could be detected in transfected cells. In the absence of sulfhydryl reagents, F1473C and A1481C slowed fast Na+ channel inactivation by 2‐ and 1 .5‐fold, respectively, and L1482C induced a steady‐state Na+ current (Iss) of 3% of peak current (I≥peak) (1 % for wild‐type).
Upon application of MTSET and MTSES, changes in fast inactivation gating occurred for most of the mutants. The most dramatic destabilizing effects on fast inactivation were observed for M1476C (9‐fold slowing of inactivation; Iss/Ipeak, 3.6%; + 15 mV shift in steady‐state inactivation; 2‐ to 3‐fold acceleration of recovery from inactivation), A1481C (3‐fold; 14%; +20 mV; no change) and F1473C (2.5‐fold; 2.4%; +8 mV; 1.5‐fold). Less pronounced destabilizing effects were observed for M1477C and L1479C. Strongly stabilizing effects on the inactivated state, that is a 20–30 mV hyperpolarizing shift of the inactivation curve associated with a 3‐ to 4‐fold decrease in the rate of recovery from inactivation, occurred for T1470C, L1471C and A1474C. Almost all effects were independent of the membrane potential; however, A1474C only reacted when cells were depolarized. Significant effects on activation were not observed.
We conclude that the IV/S4–S5 loop plays an important role in fast inactivation of the muscle Na+ channel and may contribute to the formation of a receptor for the putative inactivation particle. The effects of sulfhydryl reagents on the various mutations suggest an α‐helical structure of IV/S4–S5 (up to P1480) with destabilizing effects on inactivation for one cluster of amino acids (1473/76/77/79) and a stabilized inactivation at the opposite side of the helix (1470/71/74).
Tetrahydrobiopterin Regulates Cyclic GMP‐Dependent Electrogenic Cl− Secretion in Mouse Ileum In Vitro
Authors: Rolfe V. E. , Brand M. P. , Heales S. J. R. , Lindley K. J. , Milla P. J. 
Published: September 2004

Basal electrogenic Cl− secretion, measured as the short‐circuit current (Isc), was variable in ileum removed from tetrahydrobiopterin (BH4)‐deficient hph‐1 mice and wild‐type controls in vitro, although values were not significantly different.
The basal nitrite release and mucosal cyclic guanosine 3′,5′‐monophosphate (cyclic GMP) production were similar in control and BH4‐deficient ileum.
Mucosally added Escherichia coli heat‐stable toxin (STa, 55 ng ml−1) increased the nitrite release, cyclic GMP levels and the Isc in control ileum, but its secretory actions were reduced in BH4‐deficient ileum.
L‐Arginine (1 mM) increased the nitrite release, cyclic GMP production and the Isc in control ileum, but the actions were reduced in BH4‐deficient ileum.
Serosal carbachol (1 mM) stimulated maximum short‐circuit currents of similar magnitude in both control and BH4‐deficient ileum, whilst nitrite release and cyclic GMP production were minimal.
E. coli STa and L‐arginine increased electrogenic Cl− secretion across intact mouse ileum in vitro by releasing nitric oxide and elevating mucosal cyclic GMP. The inhibition of these processes in the hph‐1 mouse ileum suggests that BH4 may be a target for the modulation of electrogenic transport, and highlight the complexity of the interactions between nitric oxide and cyclic GMP in the gut.
Endothelium‐dependent frequency modulation of Ca2+ signalling in individual vascular smooth muscle cells of the rat
Authors: Kasai Yasuyo , Yamazawa Toshiko , Sakurai Takashi , Taketani Yuji , Iino Masamitsu 
Published: September 2004

We visualized intracellular Ca2+ concentration ([Ca2+]i) changes, using fluo‐3 as an indicator, of individual vascular smooth muscle cells and endothelial cells within intact rat tail arteries by confocal microscopy.
Using a piezo‐driven objective, we focused on endothelial and smooth muscle cell layers alternately to obtain Ca2+ images of their cells. In the presence of 1 μm acetylcholine (ACh), individual endothelial cells responded with intermittent increases in the [Ca2+]i (Ca2+ oscillations). At the same time, the frequency of Ca2+ oscillations in smooth muscle cells induced by electrical stimulation of the perivascular sympathetic nerve was greatly decreased.
A [Ca2+]i rise during the oscillations in the endothelial cells propagated in the form of a wave along the long axis of the cells.
In the presence of a NO synthase inhibitor, no significant inhibitory effect of ACh on the Ca2+ signalling in the vascular smooth muscle cells was detected, although the Ca2+ oscillations in the endothelial cells persisted.
The inhibitory effect of ACh on the frequency of Ca2+ oscillations in the vascular smooth muscle cells was mimicked by 1 μm sodium nitroprusside, a NO donor.
These results indicate that Ca2+ waves and oscillations in vascular endothelial cells regulate NO production, which modulates vascular tone by decreasing the frequency of Ca2+ oscillations in smooth muscle cells activated by sympathetic agonists.