bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential up‐regulation of Na+‐Ca2+ exchange by phosphoarginine and ATP in dialysed squid axons
Authors: DiPolo Reinaldo , Beaugé Luis 
Published: September 2004

The aim of this study was to characterize further the two main metabolic pathways of regulation of the Na+‐Ca2+ exchanger in squid axons induced by its two naturally ocurring high‐energy compounds: ATP and phosphoarginine (Pa). [Na+]o‐dependent Ca2+ efflux (forward Na+o‐Ca2+i exchange) and [Ca2+]o‐dependent Ca2+ efflux (Ca2+o‐Ca2+i exchange) were measured in internally dialysed squid axons at 16–17 °C.
Measurements of changes in the apparent affinity of the Na+‐Ca2+ exchanger for transporting (Na+o, Na+i, Ca2+o, Ca2+i) and regulatory (Ca2+i) ions induced by ATP and Pa show marked differences for the two substrates: (i) ATP strongly alters the affinity for Na+o and Na+i, while Pa does not, and (ii) in the absence of Na+i, ATP has no stimulatiory effect; on the other hand, Pa causes a dramatic increase in Na+o‐Ca2+i exchange with little activation of Ca2+o‐Ca2+i exchange.
The MgATP analogue chromium‐ATP (CrATP) completely inhibits MgATP stimulation of the Na+‐Ca2+ exchanger. Nevertheless, even with the effects of the nucleotide blocked, Pa exhibits its usual activation of the [Na+]o‐dependent Ca2+ efflux.
None of the classical serine‐threonine‐tyrosine kinase inhibitors, nor the PP1 and PP2 phosphatase inhibitors, affects either the ATP or the Pa effect. However, intracellular microinjections of an exogenous phosphatase (alkaline phosphatase) completely reverses the stimulation of the Na+‐Ca2+ exchange induced by ATP and Pa.
Prolonged intracellular dialysis with highly permeable porous capillaries (18 kDa molecular weight cut‐off), which normally induces a complete run‐down of the MgATP effect, does not alter the Pa stimulation of the exchanger, even after 6 h of continuous dialysis.
We conclude that the ATP and Pa modulation of Na+‐Ca2+ exchange in an invertebrate nerve fibre are two genuinely different mechanisms, which affect the carrier properties in very different ways. An interesting similarity between ATP and Pa is that a phosphorylation‐dephosphorylation process seems to be a common feature of these two regulation modes.
Novel mechanism for non‐genomic action of 17β‐oestradiol on kainate‐induced currents in isolated rat CA1 hippocampal neurones
Authors: Gu Qin , Moss Robert L. 
Published: September 2004

Using whole‐cell voltage‐clamp recordings of dissociated hippocampal CA1 neurones, we demonstrated that 17β‐oestradiol rapidly potentiates kainate‐induced currents when applied either to the outside or the inside of the neurone. However, when the steroid was conjugated to bovine serum albumin (E2‐BSA), application to either the extracellular plasma membrane (E2‐BSAout) or the cytosolic side of the cell (E2‐BSAin) had no observable effect on kainate‐induced currents. However, when applied stimultaneously to both sides of the plasma membrane, E2‐BSA potentiated kainate‐induced currents.
Application of E2‐BSAout and GTPγSin potentiated kainate‐induced currents. The potentiation of kainate‐induced currents by 17β‐oestradiol was occluded by cholera toxin pretreatment and appeared to be pertussis toxin insensitive.
E2‐BSAin prolonged the effect of 8‐bromoadenosine 3′,5′ cyclic monophosphate (8‐bromo‐cAMP) on kainate‐induced currents. The recovery from the 8‐bromo‐cAMP response was found to be a function of the concentration of E2‐BSAin. The application of ATPγSin occluded the effect of 17β‐oestradiol.
These results suggest that the non‐genomic action of 17β‐oestradiol in the potentiation of kainate‐induced currents is mediated via an action on Gs protein‐coupled receptors. This operates in concert with an internal action of 17β‐oestradiol on a cAMP‐dependent phosphorylation.
Ca2+ influx through carbachol‐activated non‐selective cation channels in guinea‐pig gastric myocytes
Authors: Kim Sung Joon , Koh Eun‐Mi , Kang Tong Mook , Kim Young Chul , So Insuk , Isenberg Gerrit , Kim Ki Whan 
Published: September 2004

Ca2+ microfluorometry (100 μm K5 fura‐2) and the voltage‐clamp technique were combined to study the effect of carbachol (CCh, 50 μm) in inducing currents (ICCh) through non‐selective cation channels (NSCCCh) and increments in global cytosolic Ca2+ concentration (Δ[Ca2+]c).
In Na+‐containing bath solution, ICCh fell from an initial phasic to a subsequent small (5 %) tonic component; Δ[Ca2+]c fell to zero. Tonic ICCh and [Ca2+]c became prominent after substitution of extracellular 140 mm Na+ by 140 mm Cs+. Tonic ICCh and Δ[Ca2+]c were insensitive to intracellular heparin (3 mg ml−1) and ryanodine (4 μm), i.e. they did not depend on Ca2+ release from sarcoplasmic reticulum (SR).
Single channel currents of NSCCCh could be resolved in whole‐cell recordings. Substitution of Na+ by Cs+ increased NSCCCh activity by one order of magnitude and slope conductance from 22 to 30 pS. Extracellular quinidine (3 μm) reversibly blocked the NSCCCh activity.
Both tonic ICCh and tonic Δ[Ca2+]c (a) followed a similar time course of activation, desensitization and facilitation, (b) were reversibly blocked by 3 μm quinidine, and (c) persisted upon block of SR Ca2+ release.
A Ca2+ fractional current of tonic ICCh (fCa) of 0.009 was calculated by comparing the ratio Δ[Ca2+]c (corrected for simultaneous Ca2+ redistribution) over ICCh with depolarization‐induced *Δ[Ca2+]c (Δ[Ca2+]c calculated from ICa induced by a 400 ms depolarization from −60 to 0 mV at 2 mm[Ca2+]o, 145 mm[Cs+]o) over ICa. fCa was 0.023 at [Ca2+]o= 4 mm.
With 110 mm extracellular CaCl2 and 145 mm intracellular CsCl, ICCh reversed at +19.5 mV suggesting a permeability ratio PCa/PCs of 2.8.
We conclude that Ca2+ influx through NSCCCh under physiological [Ca2+]o could induce Δ[Ca2+]c. The fCa was, however, much smaller than the one calculated from the reversal potential.
Ca2+ permeability of mouse and chick nicotinic acetylcholine receptors expressed in transiently transfected human cells
Authors: Ragozzino Davide , Barabino Benedetta , Fucile Sergio , Eusebi Fabrizio 
Published: September 2004

Combinations of cDNAs encoding mouse and chick nicotinic acetylcholine receptor (nAChR) subunits were transiently transfected into human BOSC 23 cells, and the expressed receptors were studied by simultaneously recording transmembrane currents and fluorescence transients using the whole‐cell patch‐clamp technique, and confocal microscopy with the Ca2+ indicator dye fluo‐3.
The fractional Ca2+ current, Pf, of nAChRs was evaluated as the normalized ratio of nicotine‐evoked fluorescence transient over total charge entering the cell (F/Q ratio). Mouse fetal muscle nAChR channels had a Pf,αβγδ value of 2.1 %. The substitution of the γ subunit with the ɛ subunit resulted in a 2‐fold increase in Pf (4.2 %). The difference in Ca2+ permeability was confirmed by determination of Ca2+/Cs+ permeability ratios.
Among the chick neuronal nAChRs tested, Pf,α3β4 was 4.6 %, while Pf,α4β4 and Pf,α4β2 were 3.0 % and 2.9 %, respectively.
The amplitude of the current elicited by the activation of α3β4 nAChRs increased as the external Ca2+ concentration was raised from 2 to 110 mM, whereas currents flowing through all other nAChRs tested were reduced to various extents.
Our findings indicate that the adult‐type muscle nAChR (αβɛδ) is more permeable to Ca2+ than the fetal‐type (αβγδ), while ganglionic‐like α3β4 nAChR is more permeable to Ca2+ than the examined α4‐containing nAChRs. The functional significance is discussed.
Regulation of murine cystic fibrosis transmembrane conductance regulator Cl− channels expressed in Chinese hamster ovary cells
Authors: Lansdell K. A. , Kidd J. F. , Delaney S. J. , Wainwright B. J. , Sheppard D. N. 
Published: September 2004

We investigated the effect of protein kinases and phosphatases on murine cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels, expressed in Chinese hamster ovary (CHO) cells, using iodide efflux and the excised inside‐out configuration of the patch‐clamp technique.
The protein kinase C (PKC) activator, phorbol dibutyrate, enhanced cAMP‐stimulated iodide efflux. However, PKC did not augment the single‐channel activity of either human or murine CFTR Cl− channels that had previously been activated by protein kinase A.
Fluoride, a non‐specific inhibitor of protein phosphatases, stimulated both human and murine CFTR Cl− channels. However, calyculin A, a potent inhibitor of protein phosphatases 1 and 2A, did not enhance cAMP‐stimulated iodide efflux.
The alkaline phosphatase inhibitor, (−)‐bromotetramisole augmented cAMP‐stimulated iodide efflux and, by itself, stimulated a larger efflux than that evoked by cAMP agonists. However, (+)‐bromotetramisole, the inactive enantiomer, had the same effect. For murine CFTR, neither enantiomer enhanced single‐channel activity. In contrast, both enantiomers increased the open probability (Po) of human CFTR, suggesting that bromotetramisole may promote the opening of human CFTR.
As murine CFTR had a low Po and was refractory to stimulation by activators of human CFTR, we investigated whether murine CFTR may open to a subconductance state. When single‐channel records were filtered at 50 Hz, a very small subconductance state of murine CFTR was observed that had a Po greater than that of human CFTR. The occupancy of this subconductance state may explain the differences in channel regulation observed between human and murine CFTR.
Hypoxia activates ATP‐dependent potassium channels in inspiratory neurones of neonatal mice
Authors: Mironov S. L. , Langohr K. , Haller M. , Richter D. W. 
Published: September 2004

The respiratory centre of neonatal mice (4 to 12 days old) was isolated in 700 μm thick brainstem slices. Whole‐cell K+ currents and single ATP‐dependent potassium (KATP) channels were analysed in inspiratory neurones.
In cell‐attached patches, KATP channels had a conductance of 75 pS and showed inward rectification. Their gating was voltage dependent and channel activity decreased with membrane hyperpolarization. Using Ca2+‐containing pipette solutions the measured conductance was lower (50 pS at 1.5 mM Ca2+), indicating tonic inhibition by extracellular Ca2+.
KATP channel activity was reversibly potentiated during hypoxia. Maximal effects were attained 3‐4 min after oxygen removal from the bath. Hypoxic potentiation of open probability was due to an increase in channel open times and a decrease in channel closed times.
In inside‐out patches and symmetrical K+ concentrations, channel currents reversed at about 0 mV. Channel activity was blocked by ATP (300‐600 μM), glibenclamide (10‐70 μM) and tolbutamide (100‐300 μM).
In the presence of diazoxide (10‐60 μM), the activity of KATP channels was increased both in inside‐out, outside‐out and cell‐attached patches. In outside‐out patches, that remained within the slice after excision, the activity of KATP channels was enhanced by hypoxia, an effect that could be mediated by a release of endogenous neuromodulators.
The whole‐cell K+ current (IK) was inactivated at negative membrane potentials, which resembled the voltage dependence of KATP channel gating. After 3‐4 min of hypoxia, K+ currents at both hyperpolarizing and depolarizing membrane potentials increased. IK was partially blocked by tolbutamide (100‐300 μM) and in its presence, hypoxic potentiation of IK was abolished.
We conclude that KATP channels are involved in the hypoxic depression of medullary respiratory activity.
Unitary IPSPs evoked by interneurons at the stratum radiatum‐stratum lacunosum‐moleculare border in the CA1 area of the rat hippocampus in vitro
Authors: Vida Imre , Halasy Katalin , Szinyei Csaba , Somogyi Peter , Buhl Eberhard H. 
Published: September 2004

Hippocampal non‐principal neurons at the stratum radiatum‐stratum lacunosum‐moleculare border (R‐LM interneurons) of the CA1 area may constitute several cell classes and have been implicated in the generation of GABAergic unitary IPSPs. Using biocytin‐filled electrodes we recorded R‐LM interneurons intracellularly in vitro and determined their postsynaptic effects in concomitantly recorded pyramidal cells.
Light microscopic analysis revealed four populations of R‐LM interneurons with distinct axons: (1) basket cells (n= 4) with axons predominantly ramifying in the pyramidal cell layer; (2) Schaffer collateral/commissural pathway‐associated interneurons (n= 10) stratifying in stratum radiatum and, to a lesser extent, stratum oriens; (3) perforant pathway‐associated interneurons (n= 6) innervating the perforant path termination zone in stratum lacunosum‐moleculare of the CA1 area as well as equivalent portions of the dentate gyrus and subiculum; and (4) neurogliaform interneurons (n= 2) characterized by their dense, compact axonal and dendritic arbour.
Random electron microscopic sampling of synaptic targets revealed a preponderance of pyramidal neurons as postsynaptic elements. Basket cells had a synaptic target preference for somata and proximal dendrites, whereas the remainder of R‐LM interneurons innervated dendritic shafts and spines. The axon of dendrite‐targeting cells formed up to six putative contacts with individual postsynaptic pyramidal cells.
Anatomically recovered R‐LM interneurons (n= 22) had a mean resting membrane potential of ‐56.7 ± 3.6 mV, a membrane time constant of 12.9 ± 7.7 ms and an input resistance of 86.4 ± 29.2 MΩ. Depolarizing current pulses generally elicited overshooting action potentials (70.8 ± 6.9 mV) which had a mean duration, when measured at half‐amplitude, of 0.7 ± 0.1 ms. In response to prolonged (> 200 ms) depolarizing current pulses all R‐LM interneurons displayed (a varying degree of) spike frequency adaptation.
Basket cells, Schaffer‐associated and neurogliaform interneurons elicited small‐amplitude (< 2 mV), short‐latency IPSPs in postsynaptic pyramids (n= 5, 13 and 1, respectively). Those interactions in which an effect was elicited with the repetitive activation of the presynaptic neuron (n= 13) showed a substantial degree of postsynaptic response summation. Unitary IPSPs had fast kinetics and, whenever tested (n= 5; 1 basket cell and 4 Schaffer‐associated interneurons), were abolished by the GABAA receptor antagonist bicuculline.
Thus, R‐LM interneurons comprise several distinct populations which evoke fast GABAA receptor‐mediated IPSPs. The domain‐specific innervation of postsynaptic pyramidal cells suggests functionally diverse effects on the integration of afferent information in functionally non‐equivalent compartments of pyramidal cells.
The effect of tetracaine on stimulated contractions, sarcoplasmic reticulum Ca2+ content and membrane current in isolated rat ventricular myocytes
Authors: Overend C. L. , O'Neill S. C. , Eisner D. A. 
Published: September 2004

The effects of tetracaine were examined on rat ventricular myocytes. In both field‐stimulated and voltage‐clamped cells tetracaine (100–200 μM) produced an initial decrease of contraction before a recovery towards the control level. Removal of tetracaine produced a transient overshoot of contraction to levels greater than the control.
The transient decrease of contraction produced by tetracaine was accompanied by a small transient increase in the integral of the L‐type Ca2+ current and a larger transient decrease of the Na+‐Ca2+ exchange current on repolarization. These are attributed to decreased systolic release of Ca2+. On removal of tetracaine there was an increase of the Na+‐Ca2+ exchange current. Before the addition of tetracaine, calculated Ca2+ influx and efflux across the sarcolemma were approximately equal. On adding tetracaine, efflux was transiently less than influx and, on removal of tetracaine, efflux was greater than influx.
These changes in Ca2+ fluxes result in an increase of cell Ca2+ during exposure to tetracaine. The calculated magnitude of this increase was equal to that measured directly by applying caffeine (20 mM) to release sarcoplasmic reticulum (SR) Ca2+ and integrating the resulting Na+‐Ca2+ exchange current.
It is concluded that the effects of tetracaine can be accounted for by depression of calcium‐induced Ca2+ release (CICR). The response is transient because the inhibition is compensated for by an increase of SR Ca2+ content such that there is no steady‐state effect on the magnitude of the systolic Ca2+ transient. The consequences of this result for the effects of other modulators of CICR are discussed.
Heterologous expression and coupling of G protein‐gated inwardly rectifying K+ channels in adult rat sympathetic neurons
Authors: Ruiz‐Velasco Victor , Ikeda Stephen R. 
Published: September 2004

G protein‐gated inwardly rectifying K+ (GIRK) channels were heterologously expressed in rat superior cervical ganglion (SCG) neurons by intranuclear microinjection. The properties of GIRK channels and their coupling to native receptors were characterized using the whole‐cell patch‐clamp technique.
Following coinjection of either GIRK1–2 or GIRK1–4 cDNA, application of noradrenaline (NA) produced large inwardly rectifying K+ currents. Injection of cDNA encoding individual GIRK subunits produced only small and inconsistent NA‐activated inward currents. Current arising from the native expression of GIRK channels in SCG neurons was not observed.
NA‐mediated activation of GIRK channels was abolished by pertussis toxin (PTX) pretreatment, indicating coupling via G proteins of the Gi/Go subfamily. Conversely, vasoactive intestinal peptide (VIP) activated GIRK channel currents via a cholera toxin‐sensitive pathway suggesting coupling through Gαs. Pretreatment of neurons with PTX caused a significant increase in amplitude of the VIP‐mediated GIRK channel currents when compared with untreated cells.
Application of adenosine, prostaglandin E2 and somatostatin resulted in activation of GIRK channel currents. Activation of m1 muscarinic acetylcholine receptors (i.e. application of oxotremorine m to PTX‐treated neurons) failed to elicit overt GIRK channel currents.
GIRK channel overexpression decreased basal Ca2+ channel facilitation significantly when compared with uninjected neurons. Furthermore, the NA‐mediated inhibition of Ca2+ channels was significantly attenuated.
In summary, the ability to heterologously express GIRK channels in adult sympathetic neurons allows the experimental alteration of receptor‐G protein‐effector stoichiometry. Such studies may increase our understanding of the mechanisms underlying ion channel modulation by G proteins in a neuronal environment.
The ion selectivity of a membrane conductance inactivated by extracellular calcium in Xenopus oocytes
Authors: Zhang Yong , McBride Don W. , Hamill Owen P. 
Published: September 2004

The ion selectivity of a membrane ion conductance that is inactivated by extracellular calcium (Ca2+o) in Xenopus oocytes has been studied using the voltage‐clamp technique.
The reversal potential of the Ca2+o‐sensitive current (Ic) was measured using voltage ramps (‐80 to +40 mV) as a function of the external concentration (12‐240 mM) of NaCl or KCl. The direction and amplitude of the shifts in reversal potentials are consistent with permeability ratios of 1:0.99:0.24 for K+:Na+:Cl−.
Current‐voltage (I‐V) relations of Ic, determined during either voltage ramps of 0.5 s duration or at steady state, displayed pronounced rectification at both hyperpolarized and depolarized potentials. However, instantaneous I‐V relations showed less rectification and could be fitted by the constant field equation assuming the above K+:Na+:Cl− permeability ratios.
Ion substitution experiments indicated that relatively large organic monovalent cations and anions are permeant through Ic channels with the permeability ratios K+:NMDG+:TEA+:TPA+:TBA+:Gluc−= 1:0.45:0.35:0.2:0.2:0.2.
External amiloride (200 μM), gentamicin (220 μM), flufenamic acid (40 μM), niflumic acid (100 μM), Gd3+ (0.3 μM) or Ca2+ (200 μM) caused reversible block of Ic without changing its reversal potential.
Preinjection of oocytes with antisense oligonucleotide against connexin 38, the Xenopus hemi‐gap‐junctional protein, inhibited Ic by 80 % without affecting its ion selectivity, thus confirming and extending the recent suggestion of Ebihara that Ic represents current carried through hemi‐gap‐junctional channels.
In vitro and in vivo maturation of oocytes resulted in a significant decrease in Ic conductance to 7 % and 2 % of control values, respectively. This developmental downregulation of Ic minimizes any toxic effect Ic activation would have when the mature egg is released into Ca2+o‐free pond water.
The results of this study are discussed in relation to other Ca2+o‐inactivated conductances seen in a wide variety of cell types and which have previously been interpreted as arising either from Ca2+o‐masked channels or from changes in the ion selectivity of voltage‐gated Ca2+ or K+ channels.
Uneven distribution of K+ channels in soma, axon and dendrites of rat spinal neurones: functional role of the soma in generation of action potentials
Authors: Wolff Matthias , Vogel Werner , Safronov Boris V. 
Published: September 2004

A novel method of ‘entire soma isolation’ was used to describe the distribution of voltage‐gated K+ channels between soma, axon and dendrites of dorsal horn neurones identified in spinal cord slices of newborn rat.
The soma contained 36 % of total inactivating (KA) current but only 15 % of delayed rectifier (KDR) current. The axon initial segment possessed almost half (47 %) of the total KA current and 38 % of KDR current. In contrast, dendrites contained a small portion (17 %) of KA but 47 % of KDR current.
Under current‐clamp conditions, the soma isolated from axon and dendrites was not able to generate action potentials. It passively conducted weak (≤ ‐50 mV) and amplified pronounced (‐50 to 0 mV) depolarizations but inhibited strong (≤ 0 mV) depolarizations.
It is concluded that the soma plays a complex role in the excitability of spinal dorsal horn neurones. It conducts passively or amplifies excitatory postsynaptic potentials on their way from dendrites and soma to the axon initial segment but it inhibits back‐propagation of the action potential from the axon to the dendrites.