bluuebunny/crossref_metadata_2025 huggingface dataset:
Albumin elicits calcium signals from astrocytes in brain slices from neonatal rat cortex
Authors: Nadal Angel , Sul Jai‐Yoon , Valdeolmillos Miguel , McNaughton Peter A.
Published: September 2004

Albumin causes calcium signals and mitosis in cultured astrocytes, but it has not been established whether astrocytes in intact brain also respond to albumin. The effect of albumin on intracellular calcium concentration ([Ca2+]i) in single cells was therefore studied in acutely isolated cortical brain slices from the neonatal rat.

Physiological concentrations of albumin from plasma and from serum produced an increase in [Ca2+]i in a subpopulation of cortical cells. Trains of transient elevations in [Ca2+]i (Ca2+ spikes) were seen in 41 % of these cells.

The cells responding to albumin are identified as astrocytes because the neurone‐specific agonist NMDA caused much smaller and slower responses in these cells. On the other hand NMDA‐responsive cells, which are probably neurones, exhibited only small and slow responses to albumin. The residual responses of astrocytes to NMDA and neurones to albumin are likely to be due to crosstalk with adjacent neurones and astrocytes, respectively.

Methanol extraction of albumin removes a polar lipid and abolishes the ability of albumin to increase intracellular calcium.

Astrocyte calcium signalling caused by albumin may have important physiological consequences when the blood‐brain barrier breaks down and allows albumin to enter the CNS.
Identification and characterization of a monocarboxylate transporter (MCT1) in pig and human colon: its potential to transport l‐lactate as well as butyrate
Authors: Ritzhaupt Armin , Wood I. Stuart , Ellis Antony , Hosie Ken B. , Shirazi‐Beechey Soraya P.
Published: September 2004

Oligonucleotide primers based on the human heart monocarboxylate transporter (MCT1) cDNA sequence were used to isolate a 544 bp cDNA product from human colonic RNA by reverse transcription‐polymerase chain reaction (RT‐PCR). The sequence of the RT‐PCR product was identical to that of human heart MCT1. Northern blot analysis using the RT‐PCR product indicated the presence of a single transcript of 3.3 kb in mRNA isolated from both human and pig colonic tissues. Western blot analysis using an antibody to human MCT1 identified a specific protein with an apparent molecular mass of 40 kDa in purified and well‐characterized human and pig colonic lumenal membrane vesicles (LMV).

Properties of the colonic lumenal membrane l‐lactate transporter were studied by the uptake of L‐[U‐14C]lactate into human and pig colonic LMV. l‐lactate uptake was stimulated in the presence of an outward‐directed anion gradient at an extravesicular pH of 5.5. Transport of l‐lactate into anion‐loaded colonic LMV appeared to be via a proton‐activated, anion exchange mechanism.

l‐lactate uptake was inhibited by pyruvate, butyrate, propionate and acetate, but not by Cl− and SO42−. The uptake of l‐lactate was inhibited by phloretin, mercurials and α‐cyano‐4‐hydroxycinnamic acid (4‐CHC), but not by the stilbene anion exchange inhibitors, 4,4′‐diisothiocyanostilbene‐2,2′‐disulphonic acid (DIDS) and 4‐acetamido‐4′‐isothiocyanostilbene‐2,2′‐disulphonic acid (SITS).

The results indicate the presence of a MCT1 protein on the lumenal membrane of the colon that is involved in the transport of l‐lactate as well as butyrate across the colonic lumenal membrane. Western blot analysis showed that the abundance of this protein decreases in lumenal membrane fractions isolated from colonic carcinomas compared with that detected in the normal healthy colonic tissue.
Kainate induces an intracellular Na+‐activated K+ current in cultured embryonic rat hippocampal neurones
Authors: Liu Qi‐Ying , Schaffner Anne E. , Barker Jeffery L.
Published: September 2004

In embryonic rat hippocampal neurones cultured for < 3 days, kainate induced an inward current at negative potentials that recovered to baseline levels immediately upon termination of agonist application. However, in neurones cultured for longer, the kainate‐induced current was often followed by a long‐lasting inward current that slowly recovered to baseline levels. The amplitude of the delayed current (Idelay) triggered by kainate was positively related both to the duration of application at constant agonist concentration and to concentration at constant application duration.

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delay could last for several minutes and was accompanied by a conductance increase, which closely paralleled current amplitude. Depression of the kainate‐induced current response at receptor level with CNQX or at ionic level with Na+‐free solution eliminated Idelay. However, when applied during Idelay neither CNQX nor Na+‐free solution had any effect on Idelay. Li+ effected the same response as Na+ in mediating kainate‐induced Idelay.

GABA‐activated Cl− current, which was associated with the same amount of inwardly directed charge flow at the same potential as that induced by kainate, did not trigger a long‐lasting delayed current.

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delay depended on the existence of extracellular K+ and its amplitude increased with the increase in K+ concentration. Neither applying Cl−‐ or Ca2+‐free solutions nor increasing intracellular Ca2+ buffering speed and capacity altered Idelay. Exposure to the specific KCa channel blockers apamin and charybdotoxin also failed to alter Idelay. However, Idelay could be blocked by Cs+, Ba2+ and high concentrations of 4‐aminopyridine (4‐AP) and TEA.

Inside‐out excised patch‐clamp recordings revealed that low density or highly clustered Na+‐activated K+ channels were expressed in the cell bodies of cultured embryonic rat hippocampal neurones. These could be the elementary channels underlying Idelay.
Functional expression of the Ile693Thr Na+ channel mutation associated with paramyotonia congenita in a human cell line
Authors: Plassart‐Schiess Emmanuelle , Lhuillier Loïc , George Alfred L. , Fontaine Bertrand , Tabti Nacira
Published: September 2004

The Ile693Thr mutation of the skeletal muscle Na+ channel α‐subunit is associated with an unusual phenotype of paramyotonia congenita characterized by cold‐induced muscle weakness but no stiffness. This mutation occurs in the S4‐S5 linker of domain II, a region that has not been previously implicated in paramyotonia congenita.

The Ile693Thr mutation was introduced into the human skeletal muscle Na+ gene for functional expression in human embryonic kidney (HEK) cells. The currents expressed were recorded with the whole‐cell voltage‐clamp technique.

In comparison with wild‐type currents, Ile693Thr mutant currents showed a clear shift of about −9 mV in the voltage dependence of activation.

In contrast to other mutations of the Na+ channel known to cause paramyotonia congenita, the Ile693Thr mutation did not induce any significant change in the kinetics, nor in the voltage dependence, of fast inactivation.

In conclusion, this study provides further evidence of the involvement of the S4‐S5 linker in the voltage dependence of Na+ channel activation. The negative shift in the voltage dependence found in this mutation must be associated to other defects, plausibly an impairment of the slow inactivation, to account for the long periods of muscle weakness experienced by the patients.
Facilitation of the presynaptic calcium current at an auditory synapse in rat brainstem
Authors: Cuttle Matthew F. , Tsujimoto Tetsuhiro , Forsythe Ian D. , Takahashi Tomoyuki
Published: September 2004

The presynaptic calcium current (IpCa) was recorded from the calyx of Held in rat brainstem slices using the whole‐cell patch clamp technique.

Tetanic activation of IpCa by 1 ms depolarizing voltage steps markedly enhanced the amplitude of IpCa. Using a paired pulse protocol, the second (test) response was facilitated with inter‐pulse intervals of less than 100 ms. The facilitation was greater at shorter intervals and was maximal (about 20 %) at intervals of 5–10 ms.

When the test pulse duration was extended, the facilitation was revealed as an increased rate of IpCa activation. From the current‐voltage relationship measured at 1 ms from onset, facilitation could be described by a shift in the half‐activation voltage of about −4 mV.

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pCa facilitation was not attenuated when guanosine‐5′‐O‐(3‐thiotriphosphate) (GTPγS) or guanosine‐5′‐O‐(2‐thiodiphosphate) (GDPβS) was included in the patch pipette, suggesting that G‐proteins are not involved in this phenomenon.

On reducing [Ca2+]o, the magnitude of facilitation diminished proportionally to the amplitude of IpCa. Replacement of [Ca2+]o by Ba2+ or Na+, or buffering of [Ca2+]i with EGTA or BAPTA attenuated IpCa facilitation.

We conclude that repetitive presynaptic activity can facilitate the presynaptic Ca2+ current through a Ca2+‐dependent mechanism. This mechanism would be complementary to the action of residual Ca2+ on the exocytotic machinery in producing activity‐dependent facilitation of synaptic responses.
Functional and molecular expression of volume‐regulated chloride channels in canine vascular smooth muscle cells
Authors: Yamazaki Jun , Duan Dayue , Janiak Robert , Kuenzli Karri , Horowitz Burton , Hume Joseph R.
Published: September 2004

We examined the possibility of functional and molecular expression of volume‐regulated Cl− channels in vascular smooth muscle using the whole‐cell patch‐clamp technique and quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) on cells from canine pulmonary and renal arteries.

Decreasing external osmolarity induced cell swelling, which was accompanied by activation of Cl−‐dependent outward‐rectifying membrane currents with an anion permeability sequence of SCN− > I− > Br− > Cl− > aspartate−. These currents were sensitive to block by DIDS, extracellular ATP and the antioestrogen compound tamoxifen.

Experiments were performed to determine whether the molecular form of the volume‐regulated chloride channel (ClC‐3) is expressed in pulmonary and renal arteries. Quantitative RT‐PCR confirmed expression of ClC‐3 in both types of smooth muscle. ClC‐3 expression was 76.4 % of β‐actin in renal artery and 48.0 % of β‐actin in pulmonary artery.

We conclude that volume‐regulated Cl− channels are expressed in vascular smooth muscle cells and exhibit functional properties similar to those found in other types of cells, presumably contributing to the regulation of cell volume, electrical activity and, possibly, myogenic tone.
Facilitatory effect of Ca2+ on the noradrenaline‐evoked cation current in rabbit portal vein smooth muscle cells
Authors: Helliwell R. M. , Large W. A.
Published: September 2004

The facilitatory effect of external calcium ions (Ca2+o) on the α1 ‐adrenoceptor‐activated non‐selective cation current (Icat) was investigated in rabbit portal vein cells using noise and voltage‐jump relaxation analysis of the whole‐cell macroscopic current.

Micromolar concentrations of Ca2+o potentiated the peak amplitude of Icat at a holding potential (Vh) of −50 mV. The effective [Ca2+]o which produced a 50 % potentiation (EC50) was 3 μm.

From noise analysis the estimated single channel conductance (γ) was approximately 23 pS with [Ca2+]o between 3 and 100 μm, whereas in < 10 nm or 1 μm Ca2+oγ was approximately 10 pS.

The spectral density function of Icat at negative potentials could be described by the sum of two Lorentzians in every [Ca2+]o examined. The time constant of the lower frequency Lorentzian component (τ1) was about 11 ms in < 10 nm Ca2+o and was about 45 ms in micromolar concentrations of Ca2+o (1–100 μm). In contrast, the time constant of the higher frequency component (τ2) was similar in < 10 nm Ca2+o and 100 μm Ca2+o (between 1 and 2 ms).

The lower frequency Lorentzian component was responsible for about half the total current variance in < 10 nm Ca2+o whereas in micromolar concentrations of Ca2+o it was responsible for most of the measured current variance.

In voltage‐jump experiments, on stepping the voltage from −50 to +50 mV the instantaneous current was followed by an exponential decline of Icat. Stepping back to −30 mV produced an exponential inward relaxation (Irelax,‐30 mV) leading to an increase in the steady‐state amplitude of Icat in micromolar concentrations of Ca2+o, but this relaxation was not observed in < 10 nm Ca2+o. The relative amplitude of Irelax,‐30 mV increased in an [Ca2+]o ‐dependent manner (EC50 was 2 μm) although the time constant of this relaxation (τrelax,‐30 mV) remained unchanged (about 60 ms between 2 and 100 μm Ca2+o).

The data suggest that Ca2+o produces marked changes in the kinetics and single channel conductance of cation channels, which may account for the facilitatory effect of micromolar concentrations of Ca2+o on the peak amplitude of Icat.
Glycinergic synaptic inputs to bipolar cells in the salamander retina
Authors: Maple Bruce R. , Wu Samuel M.
Published: September 2004

Glycine activated strychnine‐sensitive chloride conductances at both the dendrites and the axonal telodendria of most bipolar cells in the salamander retina.

The chloride equilibrium potential of bipolar cells was found to be negative to ‐50 mV, indicating that glycinergic synapses on bipolar cells are inhibitory.

Some bipolar cells exhibited discrete, strychnine‐sensitive, chloride‐mediated inhibitory postsynaptic currents (IPSCs). These were elicited by focal application of glutamate at the inner plexiform layer (IPL). Glycinergic synapses were localized using simultaneous focal application of calcium to retinal slices bathed in calcium‐free media. Both dendritic and telodendritic glycinergic IPSCs were observed.

The decay of the telodendritic IPSCs was well fitted by a single exponential with a time constant of 17.7 ± 8.7 ms. Similar kinetics were observed for dendritic IPSCs in some cells, but in one class of on‐centre bipolar cell the decay of the dendritic IPSCs was better fitted by a sum of two exponentials with time constants 9.9 ± 4.3 and 51.3 ± 24.3 ms.

The dendritic IPSCs were best driven by application of glutamate at the distal IPL (the off sublamina), while the telodendritic IPSCs were driven best by application near the telodendria. These results suggest that bipolar cell dendrites receive inhibitory glycinergic inputs from interplexiform cells that are excited by off‐centre bipolar cells, whereas bipolar cell telodendria receive glycinergic amacrine cell inputs that are antagonistic to the photoreceptor inputs.

Both inputs could be elicited in the presence of tetrodotoxin (TTX), but the dendritic IPSCs were sometimes abolished by TTX, suggesting that sodium‐dependent spikes play an important role in the transmission of interplexiform cell signals to the outer plexiform layer.
Differential roles for NMDA and non‐NMDA receptor subtypes in baroreceptor afferent integration in the nucleus of the solitary tract of the rat
Authors: Zhang Jing , Mifflin Steven W.
Published: September 2004

Microinjection studies have established that both NMDA and non‐NMDA excitatory amino acid (EAA) receptor subtypes are involved in the integration of baroreceptor afferent inputs within the nucleus of the solitary tract (NTS). The present study was undertaken to determine which EAA receptor subtypes are involved in baroreceptor afferent integration by second and higher order NTS neurones.

Experiments utilizing intracellular recordings or extracellular recordings with microiontophoresis were performed in pentobarbitone‐anaesthetized, paralysed and artificially ventilated rats to determine the ionotropic EAA receptor subtypes involved in baroreceptor afferent integration in the NTS. NTS neurones were classified according to their responses to aortic depressor nerve (ADN) stimulation: monosynaptic neurones (MSNs), polysynaptic neurones (PSNs) and ADN‐non‐evoked neurones (NENs).

In the extracellular studies, the ADN‐evoked discharge of most MSNs was selectively reduced by microiontophoretic application of the non‐NMDA receptor antagonists 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; n= 8, P < 0.05) or 1,2,3,4‐tetrahydro‐6‐nitro‐2,3‐dioxo‐benzo(f)quinoxaline‐7‐sulphonamide (NBQX; n= 9, P < 0.01), but not by the NMDA antagonist dl‐2‐amino‐5‐phosphonopentanoic acid (AP‐5; n= 6, P= 0.28). ADN‐evoked responses of PSNs were attenuated by microiontophoretic application of AP‐5 (n= 12, P < 0.001), CNQX (n= 13, P < 0.001) or NBQX (n= 11, P < 0.001). All EAA antagonists inhibited the spontaneous discharge of MSNs/PSNs and NENs (P < 0.01 for each group).

In the intracellular studies, ADN stimulation evoked faster rising and shorter duration excitatory postsynaptic potentials (EPSPs) in MSNs (n= 16) than in PSNs (n= 15) (P < 0.05 for each comparison).

Our results demonstrate that synaptic inputs from ADN to MSNs have faster rise times and shorter durations than those to PSNs, suggesting that baroreceptor inputs to MSNs and PSNs are mediated by different synaptic mechanisms. These more rapid synaptic events are selectively mediated by non‐NMDA receptors. In addition, synaptic integration of ADN inputs by PSNs is mediated by both NMDA and non‐NMDA receptors. Finally, the ADN‐evoked discharge of some MSNs and PSNs is not attenuated by ionotropic EAA antagonists, suggesting that another receptor or transmitter system may mediate synaptic excitation in these neurones.
Inhibition of oxidative metabolism increases persistent sodium current in rat CA1 hippocampal neurons
Authors: Hammarström A. K. M. , Gage P. W.
Published: September 2004

Whole‐cell patch‐clamp recordings from freshly dissociated rat CA1 neurons revealed a large transient Na+ current (INa,T) and a smaller, inactivation‐resistant persistent Na+ current (INa,P). Both currents could be blocked with TTX.

The average current densities of INa,T and INa,P in thirty cells were 111.0 ± 9.62 and 0.87 ± 0.13 pA pF−1, respectively.

Inhibiting oxidative phosphorylation by adding 5 mM sodium cyanide to the pipette solution significantly increased the amplitude of INa,P but had no significant effect on the amplitude of INa,T.

Exposing CA1 neurons to hypoxia for more than 7 min caused an increase in the amplitude of INa,P. There was also a delayed decrease in the amplitude of INa,T.

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Na,P was more sensitive to the Na+ channel blockers TTX and lidocaine than INa,T. The IC50 for the effect of TTX on INa,P was 9.1 ± 1.2 nM whereas the IC50 for INa,T was 37.1 ± 1.2 nM, approximately 4‐fold higher. Lidocaine (lignocaine; 1 μM) reduced INa,P to 0.24 ± 0.15 of control (n= 4) whereas INa,T was essentially unaffected (0.99 ± 0.11, n= 4).

These results show that INa,P is increased when oxidative metabolism is blocked in CA1 neurons. The persistent influx of Na+ through non‐inactivating Na+ channels can be blocked by concentrations of Na+ channel blockers that do not affect INa,T.
Calcium current activated by potassium ions in voltage‐clamped rat hippocampal pyramidal neurones
Authors: Deák Ferenc , Nagy Gábor , Várnai Péter , Madarász Emília , Spät András
Published: September 2004

Neuronal activity results in local elevation of extracellular K+ concentration ([K+]o).

Using the patch‐clamp technique in the whole‐cell configuration, we investigated whether extracellular K+ activates non‐voltage‐operated Ca2+ channels in pyramidal cells cultured from rat embryonic hippocampi.

K+ (12 mM) reversibly activated a sustained inward current at a holding potential of ‐100 mV. Membrane conductance and variance of noise were significantly increased by K+. This current could be observed at membrane potentials negative to +60 mV.

Inhibitors of inward rectifier K+ channels and hyperpolarization‐induced cation current reduced the current only at potentials negative to ‐50 mV.

The K+‐induced current was activated in Na+‐free but not in Ca2+‐free medium, did not depend on cytosolic [Cl−], and was blocked by Cd2+ but not by organic channel inhibitors.

Half‐maximal activation of the current (at ‐100 mV) was attained at [K+]o≈ 20 mM.

The current is similar to Igl, a K+‐induced Ca2+ current described in glomerulosa cells. It was also present in pyramidal cells from prefrontal cortex but not in hippocampal bipolar and glial cells.

Activation of K+‐induced Ca2+ current may elevate cytoplasmic [Ca2+] at [K+]o levels which are insufficent to activate voltage‐dependent Ca2+ channels.
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.
Electrophysiological properties of sodium current subtypes in small cells from adult rat dorsal root ganglia
Authors: Rush A. M. , Bräu M. E. , Elliott A. A. , Elliott J. R.
Published: September 2004

Whole‐cell and single‐channel Na+ currents were recorded from small (ca. 20 μm diameter) cells isolated from adult rat dorsal root ganglia (DRG). Currents were classified by their sensitivity to 0.3 μm tetrodotoxin (TTX), electrophysiological properties and single‐channel amplitude. Cells were classified according to the types of current recorded from them.

Type A cells expressed essentially pure TTX‐sensitive (TTX‐S) currents. Availability experiments with prepulse durations between 50 ms and 1 s gave a half‐available voltage (Vh) of around ‐65 mV but the availability curves often had a complex shape, consistent with multiple inactivation processes. Measured inactivation time constants ranged from less than 1 ms to over 100 s, depending on the protocol used.

Cell types B and C each had, in addition to TTX‐S currents, substantial and different TTX‐resistant (TTX‐R) currents that we have designated TTX‐R1 and TTX‐R2, respectively. TTX‐R1 currents had a 1 s Vh of ‐29 mV, showed little 1 Hz use dependence at ‐67 mV and recovered from the inactivation induced by a 60 ms depolarizing pulse with time constants of 1.6 ms (91 %) and 908 ms. They also exhibited slow inactivation processes with component time constants around 10 and 100 s. TTX‐R2 currents activated and inactivated at more negative potentials (1 s Vh= ‐46 mV), showed substantial 1 Hz use dependence and had inactivation (60 ms pulse) recovery time constants at ‐67 mV of 3.3 ms (58 %) and 902 ms.

Type D cells had little or no current in 0.3 μm TTX at a holding potential of ‐67 mV. Current amplitude increased on changing the holding potential to ‐107 mV. Type D cell currents had more hyperpolarized availability and I‐V curves than even TTX‐R2 currents and suggest the existence of TTX‐R3 channels.

In outside‐out patches with 250 mM external NaCl, the single‐channel conductance (γ) of TTX‐S channels was 19.5 pS and the potential for half‐maximal activation (Va) was ‐45 mV. One population of TTX‐R channels had a γ of 9.2 pS and a Va of ‐27 mV. A second population had a γ of 16.5 pS and a more negative Va of ‐42 mV. The latter population may underlie the type D cell current.

Small DRG cells express multiple Na+ currents with varied time constants and voltage dependences of activation and inactivation. Nociceptive cells still fire when chronically depolarized by an increased external K+ concentration. TTX‐R1 and TTX‐R2 Na+ channels may support that firing, while the range of inactivation time constants described here would increase the repertoire of DRG cell burst firing behaviour generally.
Cat cerebral arterial smooth muscle cells express cytochrome P450 4A2 enzyme and produce the vasoconstrictor 20‐HETE which enhances L‐type Ca2+ current
Authors: Gebremedhin Debebe , Lange Andrew R. , Narayanan Jayashree , Aebly Mikael R. , Jacobs Elizabeth R. , Harder David R.
Published: September 2004

Cerebral arteries express cytochrome P450 4A enzymes (P450 4A) and produce 20‐ hydroxyeicosatetraenoic acid (20‐HETE), a potent constrictor of pial arterioles. It is not known which cell type in the vessel wall is responsible for the formation of 20‐HETE. We examined whether freshly isolated cerebral arterial muscle cells (VSMCs) express P450 4A and produce 20‐HETE. We also studied the effect of 20‐HETE on pressurized cerebral arteries and on whole‐cell L‐type Ca2+current (ICa) recorded in cat cerebral VSMCs.

Cat cerebral VSMCs incubated with [14C]arachidonic acid ([14C]AA) produced 20‐HETE (3.9 ± 1.1 pmol min−1 (mg protein)−1).

Reverse transcription‐polymerase chain reaction studies revealed that cat cerebral VSMCs express mRNA for P450 4A which metabolizes AA to 20‐HETE. Cloning and sequencing of the cDNA amplified from mRNA isolated from VSMCs showed > 96 % amino acid homology to the rat and human P450 4A2 and 4A3.

20‐HETE (1–300 nM) induced a concentration‐dependent constriction of cat cerebral arteries, which was inhibited by nifedipine.

Addition of 10 and 100 nM 20‐HETE to the bath increased peak ICa by 50 ± 3 and 100 ± 10 %, respectively. This effect was not influenced by altering the frequency of depolarization. 20‐HETE (100 nM) failed to increase ICa in the presence of nifedipine.

These results demonstrate that cat cerebral VSMCs express P450 4A enzyme, and produce 20‐HETE which activates L‐type Ca2+ channel current to promote cerebral vasoconstriction.