bluuebunny/crossref_metadata_2025 huggingface dataset:
Control of rat GluR6 glutamate receptor open probability by protein kinase A and calcineurin
Authors: Traynelis Stephen F. , Wahl Philip
Published: September 2004

We have used non‐stationary variance analysis to examine the single channel conductance and the probability of channel opening at the peak of the homomeric GluR6 response (Po,peak) to 100–200 ms application (10–90% exchange time, 0–3 ms) of glutamate onto excised membrane patches from transiently transfected human embryonic kidney cells (HEK 293).

Our determinations of both Po,peak and single channel conductance of simulated current responses are insensitive to system filtering, response rise time, desensitization rate and measured variation in our drug perfusion speed. Isolation of stochastic current fluctuations using the local mean response waveform minimizes problems associated with modest run‐down of response amplitude during the experiment.

The slope conductance calculated from the weighted mean unitary currents for the channels activated in response to glutamate application is 16 pS. Chord conductance between −40 and −80 mV is independent of agonist concentration. Conversion of the codon for glutamine621 to arginine (Q621R) by RNA editing reduces conductance by more than 35‐fold to less than 0.4 pS without changing response time course, desensitization, or Po,peak.

P
o,peak is high at saturating glutamate concentrations (0.65 ± 0.23; mean ±s.d.) and varies with agonist concentration. The half‐maximally effective glutamate concentration (EC50) determined for Po,peak (0.2 mm; Hill slope = 0.6) is similar to that determined for the macroscopic peak current amplitude (0.5 mm; Hill slope = 1.0) in response to rapid agonist application.

Inclusion of the purified catalytic subunit of cAMP‐dependent protein kinase A (PKA) in the patch pipette increases Po,peak to 0.85 ± 0.12 and co‐transfection of cells with a cDNA encoding the catalytic subunit of PKA (Cα‐PKA) increases Po,peak to 0.94 ± 0.09.

Inclusion of purified calcineurin plus its coactivators 200 nm Ca2+ and calmodulin in the patch pipette decreases Po,peak to 0.48 ± 0.10. The calcineurin‐stimulated decrease of Po,peak in cells co‐transfected with Cα‐PKA is blocked by 800 nm deltamethrin, a calcineurin inhibitor. Calmodulin, 200 nm Ca2+ and deltamethrin have no effect on Po,peak in the absence of calcineurin. As predicted from its effects on Po,peak, inclusion of calcineurin in the patch pipette accelerates the run‐down of whole cell GluR6 responses in cells co‐transfected with Cα‐PKA.

The effects of both calcineurin and PKA on Po,peak for GluR6 receptors in excised patches occur without any detectable changes to response time course, desensitization, or chord conductance.

We conclude that the binding of glutamate to homomeric GluR6 receptors is associated with a high probability of channel opening, which is under the control of two signalling systems that are known to be co‐localized at the neuronal membrane: PKA (Po,peak near 1.0) and calcineurin (Po,peak near 0.5).
Stimulation of anion secretion by β‐adrenoceptors in the mouse endometrial epithelium
Authors: Chan H. C. , Fong S. K. , So S. C. , Chung Y. W. , Wong P. Y. D.
Published: September 2004

Regulation of anion secretion by adrenoceptors in primary culture of mouse endometrial epithelium was investigated using the short circuit current (ISC) technique.

Adrenaline stimulated a sustained increase in the ISC in a concentration‐dependent manner. The adrenaline‐induced ISC could be inhibited by pretreatment with diphenylamine 2,2'‐dicarboxylic acid (DPC) or replacement of external Cl− and HCO3−, but not by amiloride or replacement of Na+ in apical solution.

The concentration‐dependent responses of the adrenaline‐induced ISC to the CF channel blockers glibenclamide and DPC were examined and exhibited IC50 values of 380 and 960 μm, respectively.

The effect of various adrenoceptor agonists on the ISC was examined. The order of potency appeared to be isoprenaline > adrenaline > noradrenaline, while no response was elicited by the α‐adrenoceptor agonist methoxamine, indicating a predominant involvement of β‐adrenoceptors.

The β‐adrenoceptor antagonist propranolol was found to be much more effective than the α‐adrenoceptor antagonist phentolamine in inhibiting the ISC responses induced by all adrenoceptor agonists examined.

The effect of adrenaline on the ISC was mimicked by an adenylate cyclase activator, forskolin, but suppressed by the adenylate cyclase inhibitor MDL 12,330A, indicating the involvement of cAMP.

Our results demonstrate that anion secretion by the mouse endometrial epithelium is regulated by β‐adrenoceptors and involves a cAMP‐dependent mechanism.
Calcium in sympathetic varicosities of mouse vas deferens during facilitation, augmentation and autoinhibition
Authors: Brain K. L. , Bennett M. R.
Published: September 2004

The sympathetic nerve terminals of the mouse vas deferens were loaded with the calcium indicator Oregon Green 488 BAPTA‐1 by orthograde transport along the postganglionic nerves. Changes in the calcium concentration in the varicosity (Δ[Ca2+]v) were determined following single impulses, and short (5‐impulse) and long (200‐impulse) trains at 5 Hz.

All varicosities showed a significant Δ[Ca2+]v in response to every single impulse. The elevated Δ[Ca2+]v declined in two phases with similar kinetics for all varicosities: a fast phase (time constant, 0.42 ± 0.05 s) and a moderate phase (3.6 ± 0.4 s).

Line scanning confocal microscopy revealed that the Δ[Ca2+] of a single terminal following single impulses was smaller for the intervaricose regions than for the varicosities.

Blockade of the voltage‐sensitive calcium channels with Cd2+ (in calcium‐free solution) completely blocked the Δ[Ca2+]v on stimulation. The addition of either nifedipine (10 μm), ω‐conotoxin GVIA (100 nM) or ω‐agatoxin TK (100 nm) showed that 47 ± 6% of the evoked response was mediated by N‐type calcium channels.

Ryanodine (10 μm) did not significantly change the amplitude of Δ[Ca2+]v in response to short trains.

Spontaneous increases in Δ[Ca2+]v were observed in individual varicosities, with coupling in the increase of Δ[Ca2+]v between varicosities.

The presynaptic α2‐receptor antagonist yohimbine (10 μm) increased the amplitude of Δ[Ca2+]v in response to five impulses (5 Hz) by 54 ± 14%, while the α2‐receptor agonist clonidine (1 μm) decreased the Δ[Ca2+]v by 55 ± 4%.

These results are discussed in terms of the hypotheses that the increased probability for secretion at sympathetic nerve terminals which accompanies facilitation and augmentation is due to the residual Δ[Ca2+]v remaining after the calcium influx following impulses and that noradrenaline acts presynaptically to decrease the probability of secretion by modifying calcium influx.
Modulation of inwardly rectifying potassium channels in cultured bovine pulmonary artery endothelial cells
Authors: Kamouchi M. , Van Den Bremt K. , Eggermont J. , Droogmans G. , Nilius B.
Published: September 2004

We have used the patch‐clamp technique to study modulation of the inwardly rectifying K+ current (IK(IR)) in cultured bovine pulmonary artery endothelial cells (CPAE cells). In whole‐cell mode, IK(IR) was defined as the Ba2+‐sensitive current. In single channel recordings, we observed a strongly inwardly rectifying and K+‐selective channel with a conductance of 31 ± 3 pS.

Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis and functional data suggest that the endothelial IRK is most probably Kir2.1.

Intracellular ATP is required to prevent run‐down of IRK in whole‐cell mode. Single channel activity disappeared in inside‐out patches exposed to ATP‐free solution and in cell‐attached patches on cells exposed to metabolic inhibition (KCN, 2‐deoxyglucose).

The non‐hydrolysable ATP analogues, ATPγS and adenylyl imidodiphosphate (AMP‐PNP), did not prevent run‐down. Run‐down did not occur in the presence of okadaic acid, a phosphatase inhibitor, but was enhanced in the presence of protamine, an activator of phosphatase 2A (PP2A).

GTPγS and A1F4− inhibited IRK, also in the presence of ATP. GTPγS antagonized the GTPγS effect. Pretreatment of the cells with PTX did not affect the GTPγS‐induced inhibition. Okadaic acid, however, slowed this inhibition.

Neither activation of protein kinase A (PKA) nor activation of protein kinase C (PKC) affected IRK. Additionally, neither cytochalasin B nor a high concentration of intracellular Ca2+ affected the time course of IRK run‐down.

We conclude that run‐down of IRK is probably due to dephosphorylation by PP2A. Activation of a PTX‐insensitive G protein inhibits this current by a mechanism that is neither mediated via the PKA and PKC pathways nor by intracellular Ca2+, but supposedly by a G protein‐dependent activation of a phosphatase.
Identification of Regions That Regulate the Expression and Activity of G Protein‐Gated Inward Rectifier K+ Channels in Xenopus Oocytes
Authors: Stevens Edward B. , Woodward Robert , Ho Ivan H. M. , Murrell‐Lagnado Ruth
Published: September 2004

The involvement of the cytoplasmic and core regions of K+ channel Kir3.1 and Kir3.2 subunits in determining the cell surface expression and G protein‐gated activity of homomeric and heteromeric channel complexes was investigated by heterologous expression of chimeric and wild‐type subunits together with the m2 muscarinic receptor in Xenopus oocytes.

Co‐expression of Kir3.1 and Kir3.2 subunits yielded currents severalfold larger than those elicited by the individual expression of these subunits. Immunofluorescence labelling indicated that Kir3.2 homomeric channels and Kir3.1–Kir3.2 heteromeric channels were expressed at high levels at the cell surface whereas Kir3.1 homomeric complexes were not expressed at the cell surface. Chimeric subunits composed of Kir3.1 and Kir3.2 showed that the presence of either the cytoplasmic tails or the core region of Kir3.1 in all subunits inhibits expression of channels at the plasma membrane.

Substituting the cytoplasmic tails of Kir3.1 for the cytoplasmic tails of Kir3.2, generated a chimeric subunit (121) which displayed dramatically increased acetylcholine‐induced channel activity compared with the wild‐type Kir3.2 homomeric channel. Cell‐attached, single‐channel recordings revealed that chimera 121 channel openings were longer than Kir3.2 openings.

Individually substituting the N‐ and C‐terminal tails of Kir3.1 for those of Kir3.2 showed that the C‐terminal tail of Kir3.1 enhanced the activity of heteromeric channels independently of the N‐terminal or core regions of this subunit.

The chimeric channel, 121, displayed a higher ratio of ACh‐induced to basal activity than the Kir3.1–Kir3.2 or Kir3.2 channels. A smaller proportion of chimera 121 channels appear to be activated by the basal turnover of G proteins, implying that they have a lower affinity for Gβγ. Our results suggest that substituting the Kir3.1 C‐terminal tail for the Kir3.2 tail promotes the opening conformational change of the Gβγ‐bound channel.

The core and C‐terminal regions of Kir3.1 independently conferred time dependence on voltage‐dependent activation. The time constant (τ) was between 5 and 10 ms and varied little over the voltage range −60 to −120 mV.
In vivo downregulation of M2 receptors revealed by measurement of muscarinic K+ current in cultured guinea‐pig atrial myocytes
Authors: Bünemann Moritz , Brandts Bodo , Pott Lutz
Published: September 2004

Muscarinic K+ current (IK(ACh)) elicited by acetylcholine (ACh) was measured in guinea‐pig atrial myocytes, which were either freshly isolated or cultured for up to 8 days.

The half‐time of activation of inward IK(ACh) by a saturating concentration (10 μm) of ACh decreased from ∼400 ms (in freshly isolated cells) to 250 ms after 6 days in culture. This was paralleled by an increase in the fast desensitizing component of IK(ACh). The density of steady‐state currents was not changed. Downregulation of M2 receptors by long‐term treatment of isolated myocytes with carbachol in vitro had opposite effects.

The EC50 of ACh for the activation of steady‐state −K(ACh) was reduced from 5 × 107 M (day 0) to 8 × 108 m (day 6). The shift in EC50 occurred with a half‐time of about 2 days, similar to the recovery from downregulation induced by treating atrial myocytes with carbachol in vitro.

The increase in sensitivity to ACh can be accounted for by an ∼6‐fold increase in the density of M2 receptors.

It is concluded that sensitization in culture reflects recovery from downregulation of M2 receptors due to the tonic vagal input to the heart in the intact animal.
Voltage‐induced slow activation and deactivation of mechanosensitive channels inXenopusoocytes
Authors: Silberberg Shai D. , Magleby Karl L.
Published: September 2004

The relationship between stretch and voltage activation of mechanosensitive (MS) channels from Xenopus oocytes was studied in excised patches of membrane using the patch clamp technique.

As is characteristic of MS channels in oocytes, stretching the membrane by applying negative pressure to the patch pipette at −50 mV activated the MS channels rapidly. The channels then deactivated rapidly when the stretch was removed. The stretch‐activated MS channels entered a main conductance level (45 pS) and one or more subconductance levels in the range of about 75–90% of the main conductance level.

In the absence of stretch, a depolarizing step from −50 to +50 mV activated apparent MS channels after long delays of typically 1–20 s (range, 100 ms to 6 min). Upon repolarization, the channels deactivated slowly with a single exponential (mean time constant of 4 s) or double exponential (mean time constants of 0.8 and 3s) time course.

Delayed activation with depolarization and slow deactivation upon repolarization were also observed for apparent MS channels in on‐cell patches.

The voltage‐activated channels were cation selective and had the same selectivity and conductance levels as the stretch‐activated MS channels. Applying stretch during voltage‐induced channel activity did not activate any additional channels, and the same maximal number of channels were typically activated by either stretch or by voltage. These observations suggest that voltage activates the same MS channels that are activated by stretch.

The opening of MS channels following steps to +50 mV occurred in an apparently co‐operative manner in 70% of the excised patches containing multiple MS channels.

In the absence of stretch, the opening frequency and open probability of MS channels increased with depolarization in the examined voltage range of −60 to −20 mV.

Applying a brief stretch during the delay to activation at +50 mV activated the MS channels rapidly, which then remained active when the stretch was removed. In contrast, applying a brief stretch during the slow deactivation induced by stepping from +50 to −50 mV abruptly terminated the voltage‐induced channel activity upon release of the stretch and inhibited subsequent depolarization‐induced activity.

Depolarizing steps from −50 to +50 mV inhibited any spontaneous channel activity that was present before the depolarizing step. If the potential was stepped back to −50 mV before the channels activated at +50 mV, a delayed activation could occur at −50 mV, followed by normal deactivation, indicating that the depolarizing step initiated activation processes that were initially masked by inhibition.

These observations suggest that voltage and stretch can induce different functional gating configurations of MS channels with associated structures, and that these different gating configurations can interconvert.
A swelling‐activated chloride current in rat sympathetic neurones
Authors: Leaney J. L. , Marsh S. J. , Brown D. A.
Published: September 2004

We have tested whether neurones show a swelling‐induced Cl− current following hypotonic shock, by recording membrane current responses and cell volume changes in voltage clamped isolated rat sympathetic neurones during application of hypotonic solutions.

Using both whole‐cell and perforated patch recording methods, hypotonic solution caused cell swelling and the activation of an inward Cl− current at –60 mV. This current showed weak outward rectification with no obvious time dependence. It was inhibited by SITS (0.3–1 mm), NPPB (30–300 μm and niflumic acid (50–200 μm) but not by tamoxifen (10 μm)

Hypotonic solution did not cause a rise in intracellular Ca2+ concentration as measured by simultaneous indo‐1 fluorescence. Also, neither the volume change nor Cl− current were affected by the removal of external Ca2+ or internal Ca2+ buffering to ≤ 1 nm with EGTA.

The Cl− current was unaffected by an inhibitor of protein kinase C (PKC; GF109203X, 3 μm or by omission of ATP from the pipette solution.

Cells exhibited a regulatory volume decrease during sustained exposure to hypotonic solution. This was completely inhibited by 0'5 mm niflumic acid.

It is concluded that osmotic swelling induces an outwardly rectifying, Ca2+‐ and PKC‐independent Cl− current in these nerve cells. It is suggested that this current may be involved in volume regulatory mechanisms.
A rapidly activating sustained K+ current modulates repolarization and excitation–contraction coupling in adult mouse ventricle
Authors: Fiset C. , Clark R. B. , Larsen T. S. , Giles W. R.
Published: September 2004

The K+ currents which control repolarization in adult mouse ventricle, and the effects of changes in action potential duration on excitation–contraction coupling in this tissue, have been studied with electrophysiological methods using single cell preparations and by recording mechanical parameters from an in vitro working heart preparation.

Under conditions where Ca2+‐dependent currents were eliminated by buffering intracellular Ca2+ with EGTA, depolarizing voltage steps elicited two rapidly activating outward K+ currents: (i) a transient outward current, and (ii) a slowly inactivating or ‘sustained’ delayed rectifier.

These two currents were separated pharmacologically by the K+ channel blocker 4‐amino‐pyridine (4–AP). 4–AP at concentrations between 3 and 200 μm resulted in (i) a marked increase in action potential duration and a large decrease in the sustained K+ current at plateau potentials, as well as (ii) a significant increase in left ventricular systolic pressure in the working heart preparation.

The current‐voltage (I–V) relation, kinetics, and block by low concentrations of 4–AP strongly suggest that the rapid delayed rectifier in adult mouse ventricle is the same K+ current (Kv1.5) that has been characterized in detail in human and canine atria.

These results show that the 4–AP–sensitive rapid delayed rectifier is a very important repolarizing current in mouse ventricle. The enhanced contractility produced by 4–AP (50 μm) in the working heart preparation demonstrates that modulation of the action potential duration, by blocking a K+ current, is a very significant inotropic variable.
Different G proteins mediate somatostatin‐induced inward rectifier K+ currents in murine brain and endocrine cells
Authors: Takano Koji , Yasufuku‐Takano Junko , Kozasa Tohru , Nakajima Shigehiro , Nakajima Yasuko
Published: September 2004

Types of G proteins (G protein α‐subunit subtypes) which mediate the activation of inward rectifier K+ currents by somatostatin (somatotrophin release‐inhibiting factor, SRIF) were determined in cultured locus coeruleus neurones from newborn rats and in AtT‐20 cells (a mouse pituitary cell line).

The whole‐cell patch clamp technique was used together with injection of antibodies against pertussis toxin (PTX)‐sensitive G protein α‐subunits or with injection of antisense (or sense) oligonucleotides against these G proteins.

In locus coeruleus neurones, the SRIF‐induced activation of inward rectifier K+ currents was inhibited by anti‐Gαi1/Gαi2 antibody injection, but not by anti‐Gαi3 or by anti‐Gαo/Gαi3 antibody injection, suggesting that the SRIF response is mediated through Gαi1 and/or Gαi2.

The SRIF‐induced activation of the inward rectifier was suppressed in locus coeruleus neurones after injection of antisense oligonucleotides against Gαi2, but not by injection of sense oligonucleotides against Gαi2. Injection of antisense (or sense) oligonucleotides against Gαi1, Gαi3 and Gαo(common) had no effect. These results suggest that Gαi2 is involved in this SRIF response.

In AtT‐20 cells, the SRIF‐induced activation of inward rectifier K+ currents was suppressed by injection of anti‐Gαi3 antibody, but not by injection of anti‐Gαi1/Gαi2 antibody.

The above results indicate that Gi mediates the SRIF effects on inward rectifier K+ currents. However, different subtypes of Gi are involved in the brain neurones and in the endocrine cells: Gi2 in locus coeruleus neurones and Gi3 in AtT‐20 cells.
Charge Movement in Cut Twitch Fibres of Rana Temporaries Containing 0.1 mm EGTA
Authors: Hui Chin Shuen , Chen Wei
Published: September 2004

Charge movement was studied in the cut twitch fibres of Rana temporaria with a double Vaseline‐gap voltage‐clamp technique.

In fibres containing 0.1 mm EGTA, the Iγ hump component of charge movement was either unresolvable or had a brief duration at all potentials. In the steady‐state charge‐voltage (Q–V) plots that were separable into the less voltage dependent (Qβ) and more voltage dependent (Qγ) components, the amount of Qγ was 0.9 ± 0.1 nC μF−1 (mean ±s.e.m., n= 18).

Fibres containing 20 mm EGTA showed broad Iγ humps at about −45 mV. The addition of 1.8 mm total Ca2+ to the end‐pools accelerated the rising phase and abbreviated the duration of the hump.

I
γ humps in fibres containing 0.1 mm EGTA resembled those in intact fibres when recorded at similar low temperatures.

These results explain the controversy concerning the variable appearance of Iγ humps in cut fibres.
Calcium‐induced release of strontium ions from the sarcoplasmic reticulum of rat cardiac ventricular myocytes
Authors: Spencer C. Ian , Berlin Joshua R.
Published: September 2004

The effects of strontium ions, Sr2+, on Ca2+‐dependent feedback mechanisms during excitation‐contraction coupling were examined in voltage‐clamped rat ventricular myocytes in which intracellular [Ca2+] and [Sr2+] were monitored with the fluorescent indicator, indo‐1.

Voltage clamp depolarizations and caffeine applications during superfusion in Ca2+‐free, Sr2+‐containing solutions were employed to exchange intracellular Ca2+ with Sr2+. Myocytes were loaded with Sr2+ by applying voltage clamp depolarizations during superfusion in Na+‐free, Sr2+‐containing solutions.

Caffeine applications produced large fluorescence transients in Sr2+‐loaded cells. Thus, Sr2+ could be sequestered and released from the sarcoplasmic reticulum.

Ca2+ influx, but not Sr2+ influx, via sarcolemmal Ca2+ channels evoked ryanodine‐sensitive fluorescence transients in Sr2+‐loaded cells. These results demonstrated that Ca2+ influx‐induced Sr2+ release (CISR) from the sarcoplasmic reticulum occurred in these experiments, even though Sr2+ influx‐induced Sr2+ release was not observed.

The amplitude of the Ca2+ influx‐induced fluorescence transient was 17 ± 1% of the caffeine‐induced transient (n= 5 cells), an indication that fractional utilization of Sr2+ sequestered in the sarcoplasmic reticulum during CISR was low.

With increased Sr2+ loading, the amplitude of Ca2+ influx‐ and caffeine‐induced fluorescence transients increased, but fractional utilization of sarcoplasmic reticulum divalent cation stores was independent of the degree of Sr2+ loading. These data suggest that Ca2+ influx directly activated the release of divalent cations from the sarcoplasmic reticulum, but mechanisms promoting positive feedback of Sr2+ release were minimal during CISR.

By comparison, in Ca2+‐loaded myocytes, Ca2+ influx‐induced Ca2+ release (CICR) utilized a greater fraction of caffeine‐releasable stores than CISR. Fractional utilization of Ca2+ stores during CICR increased with the degree of Ca2+ loading.

Taken together, these results suggest that Ca2+‐dependent feedback mechanisms play a major role in determining the extent of sarcoplasmic reticulum Ca2+ release during cardiac excitation‐contraction coupling under a wide range of conditions.
Calcium dependence and distribution of calcium‐activated chloride channels in Xenopus oocytes
Authors: Gomez‐Hernandez Juan‐Manuel , Stühmer Walter , Parekh Anant B.
Published: September 2004

The Ca2+‐dependent Cl− current (ICl,Ca), expressed in the plasma membrane of Xenopus oocytes, was examined in excised inside‐out macropatches using a rapid perfusion system.

Application of Ca2+‐containing Ringer solution resulted in the activation of a current whose reversal potential shifted to the right by 51 + 5.2 mV when Cl− in the pipette solution was lowered from 119.3 to 10 mm. No currents were generated when Ca2+ was omitted from the solution. The current is therefore a Ca2+‐activated Cl− one.

Following exposure to Ca2+, the half‐time for activation of ICl,Ca was not voltage dependent, whereas deactivation was strongly so.

I
Cl,Ca was stable in the continuous presence of Ca2+ and showed no sign of inactivation or adaptation.

Comparison of the size of the currents (normalized to pipette resistance) from the animal and vegetal poles revealed that ICl,Ca had a highly polarized distribution. The current density was almost 10 times higher in the animal pole.

The results suggest that Cl− channels provide a continuous and reliable indication of submembranous Ca2+, at least in an excised patch, and the clustering of the Cl− channels renders it necessary to exert caution in interpreting results involving the kinetics of Ca2+ signalling, when ICl,Ca is used as the sole monitor of calcium.
Regulation of Na+–K+ pump activity in contracting rat muscle
Authors: Nielsen O. B. , Clausen T.
Published: September 2004

In rat soleus muscle, high frequency electrical stimulation produced a rapid increase in intra‐cellular Na+ (Na+i) content. This was considerably larger in muscles contracting without developing tension than in muscles contracting isometrically. During subsequent rest a net extrusion of Na+ took place at rates which, depending on the frequency and duration of stimulation, approached the maximum transport capacity of the Na+–K+ pumps present in the muscle.

In isometrically contracting muscles, the net extrusion of Na+ continued for up to 10 min after stimulation, reducing Na+i to values 30% below the resting level (P < 0.001). This undershoot in Na+i, seen in both soleus and extensor digitorum longus muscles, could be maintained for up to 30 min and was blocked by ouabain or cooling to 0 °C.

The undershoot in Na+i could be elicited by direct stimulation as well as by tubocurarine‐suppressible stimulation via the motor endplate. It could not be attributed to a decrease in Na+ influx, to effects of noradrenaline or calcitonin gene‐related peptide released from nerve endings, to an increase in extracellular K+ or the formation of nitric oxide.

The results indicate that excitation rapidly activates the Na+–K+ pump, partly via a change in its transport characteristics and partly via an increase in intracellular Na+ concentration. This activation allows an approximately 20‐fold increase in the rate of Na+ efflux to take place within 10 s.

The excitation‐induced activation of the Na+–K+ pump may represent a feed‐forward mechanism that protects the Na+–K+ gradients and the membrane potential in working muscle. Contrary to previous assumptions, the Na+–K+ pump seems to play a dynamic role in maintenance of excitability during contractile activity.
Decay of calcium transients after electrical stimulation in rat fast‐ and slow‐twitch skeletal muscle fibres
Authors: Carroll Stefanie L. , Klein Michael G. , Schneider Martin F.
Published: September 2004

Calcium transients were calculated from fura‐2 fluorescence signals (corrected for kinetic delays in the Ca2+–fura‐2 reaction) from single rat skeletal muscle fibres, either fully dissociated from the fast‐twitch flexor digitorum brevis (FDB) muscle or in small bundles from the slow‐twitch soleus muscle. Fibres or bundles were embedded in agarose gel to inhibit movement and stimulated by single or trains of 1–2 ms electrical pulses (100 Hz, 2–400 ms train duration).

The rate constant of decay of [Ca2+] determined from single‐exponential fits to the final decay phase of [Ca2+] after a single action potential was considerably faster in FDB fibres than in soleus fibres. As the stimulation duration increased, the rate constant of [Ca2+] decay decreased for both the FDB and soleus fibres, but the effect was greater in FDB than in soleus fibres.

Using the magnitude of the decline in the rate constant of [Ca2+] decay with increasing stimulation duration as an index of relative contribution of the saturable Ca2+ binding sites on parvalbumin, subpopulations termed ‘high’, ‘medium’ and ‘low’, referring to estimated parvalbumin content, were determined within each group of FDB and soleus fibres. In fibres assigned to the ‘high’ and ‘medium’ groups, parvalbumin was the major contributor (50.73%) to the [Ca2+] decay rate constant after a single action potential. In fibres in the ‘low’ group, parvalbumin contributed only 0–28% to the rate constant of [Ca2+] decay.

Fluorescence recordings using mag‐fura‐2, a lower‐affinity Ca2+ indicator expected to be in equilibrium with myoplasmic Ca2+, gave similar values for both the [Ca2+] decay rate constant after a single action potential and the decrease in this rate constant with increased stimulation duration, as found for the fura‐2 [Ca2+] transients from FDB and soleus fibres. Thus, the observed differences in decay rate of Ca2+ were not introduced by kinetic correction of the fura‐2 recordings, but are attributed to differences in the Ca2+ binding and transport properties of fast‐ and slow‐twitch mammalian fibres.
Permeation and block of rat glur6 glutamate receptor channels by internal and external polyamines
Authors: Bähring Robert , Bowie Derek , Benveniste Morris , Mayer Mark L.
Published: September 2004

Polyamine block of rat GluR6(Q) glutamate receptor channels was studied in outside‐out patches from transiently transfected HEK 293 cells. With symmetrical 150 mm Na+ and 30 μm internal spermine there was biphasic voltage dependence with 95% block at +40 mV but only 20% block at +140mV. Dose–inhibition analysis for external spermine also revealed biphasic block; the Ka at +40 mV (54 μm) was lower than at +80 (167μm) and –80 mV (78 μm).

For internal polyamines relief from block was most pronounced for spermine, weaker for N‐(4‐hydroxyphenylpropanoyl)‐spermine (PPS), and virtually absent for philanthotoxin 343 (PhTX 343), suggesting that permeation of polyamines varies with cross‐sectional width (spermine, 0.44 nm; PPS, 0.70 nm; PhTX 343, 0.75 nm).

With putrescine, spermidine, or spermine as sole external cations, inward currents at –120 mV confirmed permeation of polyamines. For bi‐ionic conditions with 90 mm polyamine and 150 mm Na+i reversal potentials were –12.4 mV for putrescine (permeability ratio relative to Na+, PPut/PNa= 0.42) and –32.7 mV for spermidine (PSpd/PNa= 0.07). Currents carried by spermine were too small to analyse accurately in the majority of patches.

Increasing [Na+]i from 44 to 330 mm had no effect on the potential for 50% block (V½) by 30 μm internal spermine; however, relief from block at positive membrane potentials increased with [Na+]i. In contrast, raising [Na+]o from 44 to 330 mm resulted in a depolarizing shift in V½, indicating a strong interaction between internal polyamines and external per meant ions.

The Woodhull infinite barrier model of ion channel block adequately described the action of spermine at membrane potentials insufficient to produce relief from block. For 30 μm internal spermine such analysis gave Kd(0)= 2.5 μm, z θ= 1.97; block by 30 μm external spermine was weaker and less voltage dependent (Kd(0)= 37.8 μm and zδ= 0.55); δ and θ are electrical distances measured from the outside and inside, respectively.

Fits of the Woodhull equation for a permeable blocker adequately described both onset and relief from block by spermine over a wide range of membrane potentials. However, the rate constants and zδ values estimated for block by internal spermine predicted much stronger external block than was measured experimentally, and vice versa.

An Eyring rate theory model with two energy wells and three barriers explained qualitatively many characteristic features of the action of polyamines on GluPvs, including biphasic I–V relationships, weaker block by external than internal spermine and low permeability.
Calcium homeostatic mechanisms operating in cultured postnatal rat hippocampal neurones following flash photolysis of nitrophenyl‐EGTA
Authors: Sidky Adam O. , Baimbridge Kenneth G.
Published: September 2004

We examined Ca2+ homeostatic mechanisms in cultured postnatal rat hippocampal neurones by monitoring the recovery of background‐subtracted fluo‐3 fluorescence levels at 20–22°C immediately following a rapid increase in Ca2+ levels induced by flash photolysis of the caged Ca2+ compound nitrophenyl‐EGTA (NP‐EGTA).

A variety of methods or drugs were used in an attempt to block specifically efflux of Ca2+ by the plasmalemmal Na+–Ca2+ exchanger or uptake of Ca2+ into mitochondria.

Many of the experimental manipulations produced a decrease in intracellular pH (pHi) measured in sister cultures using the pH‐sensitive dye 2′,7′‐bis‐(2‐carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein (BCECF). Accordingly, in each case, we determined the appropriate amount of the weak base trimethylamine (TMA) required to restore baseline pHi prior to flash photolysis.

Blockade of the plasmalemmal Na+‐Ca2+ exchanger by replacement of external Na+ with either Li+ or N‐methyl‐d‐glucamine (NMDG) markedly reduced pHi, but did not affect the rate of recovery of fluo‐3 fluorescence intensities once pHi was restored.

Inhibition of mitochondrial Ca2+ uptake, using the protonophore carbonyl cyanide m‐chlorophenylhydrazone (CCCP), resulted in a reduction in pHi, which could be restored by the addition of 2 mm TMA. Under these conditions the rate of recovery of Ca2+ levels was significantly slower than in the controls. Similar results were found using the respiratory chain inhibitor rotenone.

We conclude that, when the potential effects of changes in pHi are taken into account, mitochondria appear to sequester significant amounts of Ca2+ in the neuronal preparations used.
H+ transporters in the main excretory duct of the mouse mandibular salivary gland
Authors: Chaturapanich G. , Ishibashi H. , Dinudom A. , Young J. A. , Cook D. I.
Published: September 2004

We used microspectrofluorimetry with the pH‐sensitive fluoroprobe 2′,7′‐bis(2‐carboxy‐ethyl)‐5(and‐6)‐carboxyfluorescein (BCECF) to study the regulation of cytosolic pH (pHi) in the isolated, perfused main excretory duct of the mouse mandibular gland.

In nominally HCO3−free solutions, removal of Na+ from the lumen alone caused pHi to decline whereas removing it from the bath alone did not.

Readmission of Na+ to the lumen of ducts studied under zero‐Na+ conditions caused pHi to recover fully. This recovery was blocked by 5‐(N‐ethyl‐N‐isopropyl)‐amiloride (EIPA) with a half‐maximum concentration of 0.5 μmol l−1, indicating the presence of an apical Na+–H+ exchanger.

Readmission of Na+ to the bath of ducts studied under zero‐Na+ conditions also caused pHi to recover. This recovery was blocked by 100 μmol l−1 EIPA, indicating the presence of a basolateral Na+‐H+ exchanger.

Measurements of H+ fluxes indicated that the apical Na+–H+ exchanger was approximately four times more active than the basolateral Na+‐H+ exchanger.

In three sets of experiments (in the absence of Na+, in the presence of Na+, and in the presence of Na+ plus 100 μmol l−1 EIPA), the effects of changing luminal K+ concentration on pHi were examined. We found no evidence for the presence of K+–H+ exchange or Na+‐coupled K+–H+ exchange in the apical membranes of duct cells.

pHi recovery under nominally HCO3−‐free conditions following acidification with an NH4Cl pulse was abolished by removal of Na+ from the bath and luminal solutions, indicating that no Na+‐independent systems such as H+‐ATPases were present.

A repeat of the above experiments in the presence of 25 mmol l−1 HCO3− plus 5% CO2 did not reveal any additional H+ transport systems. The removal of luminal Cl−, however, caused a small rise in pHi. This latter effect was blocked by 500 μmoll−1 4,4′‐diisothio‐cyanatodihydrostilbene‐2,2′‐disulphonic acid (H2‐DIDS), suggesting that a Cl−–HCO3− exchanger in the apical membrane might contribute in a minor way to pHi regulation.

We conclude that the predominant H+ transport systems in the mouse mandibular main excretory duct are Na+‐H+ exchangers in the apical and the basolateral membranes. The model we postulate to account for electrolyte transport across the main duct in the mouse mandibular gland is quite different from that previously developed for the rat duct but is similar to that developed for the rabbit duct. The difference is in concordance with the known ability of the mandibular gland of the rat, but not the rabbit or the mouse, to secrete a HCO3−‐rich final saliva.
Localized detection of action potential‐induced presynaptic calcium transients at a Xenopus neuromuscular junction
Authors: DiGregorio David A. , Vergara Julio L.
Published: September 2004

Action potential (AP)‐induced fluorescence transients were measured, using Ca2+ indicators and a spot‐detection method, at single nerve terminals of a cultured Xenopus neuromuscular junction preparation with simultaneous measurement of neurotransmitter release.

Transients obtained using the low affinity Ca2+ indicator Oregon Green™ 488 BAPTA‐5N (OGB‐5N) exhibited rapid rising (t1/2(time at which one‐half of the peak fluorescence was attained) = 0.54 ms) and decaying (Tfast= 1.9 ms) phases. The higher affinity indicator Oregon Green™ 488 BAPTA‐2 (OGB‐2) produced transients with significantly slower kinetics (t1/2= 2 ms; Tslow= 73 ms).

Tetanic stimulation elicited distinct increases in fluorescence in response to each AP. Each OGB‐5N fluorescence increase was more rapid than those observed using OGB‐2. Furthermore, a smaller proportion of residual fluorescence at the end of the train was observed using OGB‐5N.

When OGB‐5N was used, a significant [Ca2+] increase was observed prior to the release of neurotransmitter. This was not observed when OGB‐2 was used.

We conclude that the use of localized optical detection coupled with low affinity Ca2+ indicators can help elucidate rapid changes in presynaptic [Ca2+] dynamics underlying evoked neurotransmitter release.
Spatiotemporal patterns of γ frequency oscillations tetanically induced in the rat hippocampal slice
Authors: Whittington Miles A. , Stanford Ian M. , Colling Simon B. , Jefferys John G. R. , Traub Roger D.
Published: September 2004

We used transverse and longitudinal rat hippocampal slices to study the synchronization of γ frequency (> 20 Hz) oscillations, across distances of up to 4.5 mm. γ oscillations were evoked in the CA1 region by tetanic stimulation at one or two sites simultaneously, and were associated with population spikes. Tetanic stimuli that were strong enough to induce oscillations were associated with depolarization of both pyramidal cells and interneurones, largely produced by activation of metabotropic glutamate receptors.

Computer simulations of γ oscillations were also performed in a model with pyramidal cells and interneurones, arranged in a chain of five cell groups. This model had suggested previously that interneurone networks alone could generate synchronous γ oscillations locally, but that pyramidal cell firing, by inducing spike doublets in interneurones, was necessary for the occurrence of highly correlated oscillations with small phase lag (< 2.5 ms), in a distributed network possessing long axon conduction delays.

In both experiment and model, pyramidal cell spikes occurred in phase with local population spikes, as did the first spike of the interneurone doublet.

The conductance of the interneurone α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) receptor‐mediated conductance was manipulated in the model, while the relation between oscillations at opposite ends of the chain was examined. When the conductance was large enough for doublet firing to be synaptically induced in interneurones, oscillation phase lags were < 2.25 ms across the chain. As predicted, experimental blockade of AMPA receptors resulted in increased phase lags between two sites oscillating simultaneously, compared with control conditions.

Both in model and in experiment, when stimuli to the two ends of the network were slightly different, cross‐network synchronization occurred with a shorter phase lag at high frequencies than at lower frequencies.

These data suggest that, while interneurone networks alone can generate locally synchronized γ oscillations, firing of pyramidal cells, and the synaptically induced doublet firing in interneurones, contribute to the stability and tight synchrony of the oscillations in distributed networks.
Functional differences in Na+ channel gating between fast‐spiking interneurones and principal neurones of rat hippocampus
Authors: Martina Marco , Jonas Peter
Published: September 2004

GABAergic interneurones differ from glutamatergic principal neurones in their ability to discharge high‐frequency trains of action potentials without adaptation. To examine whether Na+ channel gating contributed to these differences, Na+ currents were recorded in nucleated patches from interneurones (dentate gyrus basket cells, BCs) and principal neurones (CA1 pyramidal cells, PCs) of rat hippocampal slices.

The voltage dependence of Na+ channel activation in BCs and PCs was similar. The slope factors of the activation curves, fitted with Boltzmann functions raised to the third power, were 11.5 and 11.8 mV, and the mid‐point potentials were −25.1 and −23.9 mV, respectively.

Whereas the time course of Na+ channel activation (−30 to +40 mV) was similar, the deactivation kinetics (−100 to −40 mV) were faster in BCs than in PCs (tail current decay time constants, 0.13 and 0.20 ms, respectively, at −40 mV).

Na+ channels in BCs and PCs differed in the voltage dependence of inactivation. The slope factors of the steady‐state inactivation curves fitted with Boltzmann functions were 6.7 and 10.7 mV, and the mid‐point potentials were −58.3 and −62.9 mV, respectively.

The onset of Na+ channel inactivation at −55 mV was slower in BCs than in PCs; the inactivation time constants were 18.6 and 9.3 ms, respectively. At more positive potentials the differences in inactivation onset were smaller.

The time course of recovery of Na+ channels from inactivation induced by a 30 ms pulse was fast and mono‐exponential (τ= 2.0 ms at −120 mV) in BCs, whereas it was slower and bi‐exponential in PCs (τ1= 2.0 ms and τ2= 133 ms; amplitude contribution of the slow component, 15%).

We conclude that Na+ channels of BCs and PCs differ in gating properties that contribute to the characteristic action potential patterns of the two types of neurones.
Agonist action of taurine on glycine receptors in rat supraoptic magnocellular neurones: possible role in osmoregulation
Authors: Hussy Nicolas , Deleuze Charlotte , Pantaloni Antoine , Desarménien Michel G. , Moos Françoise
Published: September 2004

To evaluate the implication of taurine in the physiology of supraoptic neurones, we (i) investigated the agonist properties of taurine on glycine and GABAA receptors of supraoptic magnocellular neurones acutely dissociated from adult rats, using whole‐cell voltage clamp, (ii) studied the effects of taurine and strychnine in vivo by extracellular recordings of supraoptic vasopressin neurones in anaesthetized rats, and (iii) measured the osmolarity‐dependent release of endogenous taurine from isolated supraoptic nuclei by HPLC.

GABA, glycine and taurine evoked rapidly activating currents that all reversed close to the equilibrium potential for Cl−, indicating activation of Cl− selective channels. Glycine‐activated currents were reversibly blocked by strychnine (IC50 of 35 nM with 100μm glycine), but were unaffected by the GABAA antagonist gabazine (1–3 μm). GABA‐activated currents were reversibly antagonized by 3 μm gabazine, but not by strychnine (up to 1 μm).

Responses to 1 mm taurine were blocked by strychnine but not by gabazine and showed no additivity with glycine‐induced currents, indicating selective activation of glycine receptors. Responses to 10 mm taurine were partially antagonized by gabazine, the residual current being blocked by strychnine. Thus, taurine is also a weak agonist of GABAA receptors.

In the presence of gabazine, taurine activated glycine receptors with an EC50 of 406 μm. Taurine activated at most 70% of maximal glycine currents, suggesting that it is a partial agonist of glycine receptors.

In vivo, locally applied strychnine (300 mm) increased and taurine (1 mm) decreased the basal electrical activity of vasopressin neurones in normally hydrated rats. The effect of strychnine was markedly more pronounced in water‐loaded rats.

Taurine, which is concentrated in supraoptic glial cells, could be released from isolated supraoptic nuclei upon hyposmotic stimulation. Decreases in osmolarity of 15 and 30% specifically enhanced basal release of taurine by 42 and 124%, respectively.

We conclude that supraoptic neurones express high amounts of glycine receptors, of which taurine may be regarded as a major natural agonist. We postulate that taurine, which can be released in hyposmotic situations, acts on glycine receptors to exert an inhibitory control on magnocellular neurones during alterations of body fluid homeostasis, implicating an active participation of glial cells in this neuroendocrine regulatory loop.
Interchangeable discharge patterns of neurons in caudal nucleus tractus solitarii in rat slices: role of GABA and NMDA
Authors: Yen J. C. , Chan Samuel H. H.
Published: September 2004

We characterized in rat brain slices the discharge patterns of spontaneously active neurons in the caudal region of the nucleus tractus solitarii (cNTS) and the neuromodulatory role of GABA and glutamate, via GABAA and NMDA receptors.

Spontaneous action potentials recorded intracellularly from cNTS neurons manifested either a regular or an irregular discharge pattern, alongside characteristic waveforms of the action potentials. These discharge patterns were interchangeable, and were highly sensitive to fluctuations in membrane potentials. In addition, the repolarizing rate of the after‐hyper‐polarization (AHP) in cNTS neurons that exhibited a regular discharge pattern was significantly higher than that of neurons that displayed irregular discharges.

cNTS neurons that manifested a regular discharge pattern were converted to irregular discharges upon superfusion with GABA (200 μm). This was accompanied by a reduction in the repolarizing rate of the AHP of both spontaneous and evoked action potentials. Conversion of discharge patterns in the opposite direction was elicited by superfusion with NMDA(6.8/μm).

The irregular discharges of spontaneous or evoked cNTS neurons were converted to a regular discharge pattern by bicuculline (200 μm). Subsequent application of d(–)‐2‐amino‐5‐phosphonopentanoic acid (250 μm) essentially led the neuronal discharges to revert to an irregular pattern.

Our results support the presence of two interchangeable modes of electrophysiological manifestations from the same cNTS neuronal population. They also showed that GABA and glutamate, via GABAA and NMDA receptors, may provide a novel form of neuromodulation at the cNTS by switching the patterns of neuronal discharges.