bluuebunny/crossref_metadata_2025 huggingface dataset:
Layer‐specific NO dependence of long‐term potentiation and biased NO release in layer V in the rat auditory cortex
Authors: Wakatsuki Hidemitsu , Gomi Hiroshi , Kudoh Masaharu , Kimura Shinji , Takahashi Kota , Takeda Masayuki , Shibuki Katsuei
Published: September 2004

We investigated the role of nitric oxide (NO) in the induction of long‐term potentiation (LTP) in slices prepared from the rat auditory cortex.

Tetanic stimulation of layer IV elicited LTP of field potentials in layer II–III (LTPII–III) and in layer V (LTPV). The magnitude of LTPII–III measured at 30 min after tetanic stimulation was 171 ± 9 % (n= 15, mean ± s.e.m.) of the control measured before tetanic stimulation, while that of LTPV was 138 ± 3 % (n= 17).

NO synthase (NOS) inhibitors had no apparent effect on LTPII–III, but LTPV was significantly suppressed (P < 0.001). This suppression of LTPV was significantly antagonized by a NO donor (P < 0.001) or a cGMP analogue (P < 0.001).

Small non‐pyramidal neurones in the auditory cortex were stained with an anti‐neuronal NOS antibody. More neurones were stained with the antibody in the deeper cortical layers.

We measured neocortical NO release with electrochemical NO probes. Layer IV stimulation elicited significantly more NO release in layer V than in layer II–III (P < 0.001). The amplitude of the increase in NO concentration elicited by stimulation at 20 Hz for 5 s was 380 ± 14 pM (n= 55) in layer V and 55 ± 8 pM (n= 5) in layer II–III.

NO release in layer V was partially but significantly suppressed by non‐NMDA (P < 0.002) or NMDA (P < 0.002) receptor antagonists. Simultaneous application of the antagonists of the two types blocked NO release almost completely.

These results clearly indicate the NO dependence of the induction of LTPV, and the greater NO release in the deeper layer of the rat auditory cortex.
Characterization of a hyperpolarization‐activated current in dedifferentiated adult rat ventricular cells in primary culture
Authors: Farès Nassim , Bois Patrick , Lenfant Jacques , Potreau Daniel
Published: September 2004

The presence of a hyperpolarization‐activated pacemaker (If)‐like current was tested in dedifferentiated adult rat ventricular myocytes up to 12 days in primary culture with the whole‐cell patch clamp technique.

An If‐like current was found and characterized on freshly isolated and cultured ventricular cells. Both activation and density of the current varied in relation to the stage of dedifferentiation. The current was activated from ‐92.0 ± 2.5 and ‐63.0 ± 1.0 mV at the beginning (4‐day‐cultured cells) and end of the dedifferentiation process (12 days), respectively. Its density measured at ‐170 mV progressively increased from ‐2.34 ± 0.36 to ‐6.12 ± 0.64 pA pF−1 between the two farthest stages of cellular remodelling. In freshly isolated cells the current was activated at ‐108.0 ± 1.5 mV and its current density measured at ‐170 mV was ‐1.97 ± 0.56 pA pF−1.

The current was blocked by extracellular CsCl (3 mM) in a voltage‐dependent manner. Modification of reversal potentials obtained at various values of [K+]o (5.4 or 25 mM) and [Na+]o (140 or 30 mM) suggests that the current was carried by both K+ and Na+ ions.

It is concluded that the hyperpolarization‐activated inward current, recorded in freshly isolated and in cultured ventricular cells has characteristics similar to those of If. In adult rat ventricular cells it is activated in a non‐physiological potential range, but can be elicited in a more physiological range when the cells are remodelled through a dedifferentiated way. It is suggested that such a current could be implicated in ventricular arrhythmias developed in pathological events.
Heterogeneous presynaptic Ca2+ channel types triggering GABA release onto medial preoptic neurons from rat
Authors: Haage David , Karlsson Urban , Johansson Staffan
Published: September 2004

Voltage‐dependent Ca2+ channels triggering GABA release onto neurons from the medial preoptic nucleus of rat were investigated. Acutely dissociated neurons with adherent functional synaptic terminals were investigated by tight‐seal whole‐cell recordings from the postsynaptic cells.

Spontaneous current events similar to miniature postsynaptic currents were recorded. They were blocked by bicuculline (100 μM), showed a roughly unimodal amplitude distribution and a reversal potential consistent with a Cl− current, and were therefore attributed to GABAA receptors activated by synaptically released GABA.

Application of 140 mM KCl, expected to depolarize presynaptic terminals, evoked currents that were ascribed to a more massive release of GABA. The KCl‐induced synaptic currents were abolished in Ca2+‐free solutions and showed a roughly hyperbolic relation to external Ca2+ concentration with half‐saturation at 0.15 mm. They further depended on the concentration of applied KCl in a way expected for high‐threshold Ca2+ channels.

The KCl‐evoked synaptic currents were completely blocked by 200 μm Cd2+, but only partially blocked by 200 μm Ni2+.

The KCl‐evoked synaptic currents were insensitive to the L‐type Ca2+ channel blocker nifedipine (10 μm). However, the synaptic currents were sensitive to either 1 μmω‐conotoxin GVIA, 25 nmω‐agatoxin IVA or 1 μmω‐conotoxin MVIIC.

It was concluded that, in many presynaptic terminals, the Ca2+ influx triggering GABA release onto medial preoptic neurons is mainly mediated by one predominant type of high‐ threshold Ca2+ channel that may be either of N‐, P‐ or Q‐type.

It was further concluded that terminals with similar predominant channel types often were clustered on the same postsynaptic cell.
Effect of chronic mitral valve damage on activity of pulmonary rapidly adapting receptors in the rabbit
Authors: Gunawardena S. , Bravo E. , Kappagoda C. T.
Published: September 2004

The effects of acute pulmonary venous congestion on the activity of rapidly adapting receptors (RARs) were determined in intact (control and sham‐operated) rabbits and in rabbits 6 and 12 weeks after surgical destruction of the mitral valve.

Destruction of the mitral valve increased the mean left atrial pressure (LAP) by approximately 2·6 and 3·8 mmHg, 6 and 12 weeks after surgery, respectively. These changes were accompanied by significant increases in left ventricular weight. The effect of acute increments in LAP on RAR activity was examined against this background of chronic pulmonary venous congestion.

In intact control and sham‐operated animals RAR activity increased from 48·8 ± 0·9 to 83·5 ± 3·6 and 121·1 ± 4·7 action potentials min−1 when the LAP was raised by 5 and 10 mmHg, respectively, above control values. Six weeks after surgery only 40 % of RARs were activated in this way.

In animals maintained for 12 weeks after surgery, RAR activity at LAPs of 6·6 ± 1·2 (control), 11·6 ± 1·2 and 16·6 ± 1·2 (mmHg) were 35·5 ± 2·3, 33·8 ± 14·4 and 34·0 ± 3·4 action potentials min−1, respectively. These changes were statistically not significant.

Slowly adapting receptors (SARs) in the lung showed a small but statistically significant increase in activity when the left atrial pressure was acutely elevated in both intact and mitral valve damaged animals .

It is concluded that chronic pulmonary venous congestion resulting from destruction of the mitral valve attenuates the ability of RARs to respond to acute moderate elevations of LAP.
Activation and co‐ordination of InsP3‐mediated elementary Ca2+ events during global Ca2+ signals in Xenopus oocytes
Authors: Callamaras Nick , Marchant Jonathan S. , Sun Xiao‐Ping , Parker Ian
Published: September 2004

The activation of elementary calcium release events (‘puffs’) and their co‐ordination to generate calcium waves was studied in Xenopus oocytes by confocal linescan imaging together with photorelease of inositol 1,4,5‐trisphosphate (InsP3) from a caged precursor.

Weak photolysis flashes evoked no responses or isolated calcium puffs, whereas flashes of increasing strength evoked more frequent puffs, often occurring in flurries as abortive waves, and then a near‐simultaneous calcium liberation originating at multiple sites. The numbers of sites activated increased initially as about the fourth power of photoreleased [InsP3].

Following repeated, identical photolysis flashes, puffs arose after stochastically varying latencies of a few hundred milliseconds to several seconds. The cumulative number of events initially increased as about the third power of time. No rise in free [Ca2+] was detected preceding the puffs, suggesting that this co‐operativity arises through binding of multiple InsP3 molecules, rather than through calcium feedback.

The mean latency to onset of calcium liberation shortened as about the square of the flash strength, and the dispersion in latencies between events reduced correspondingly.

Weak stimuli often evoked coupled puffs involving adjacent sites, and stronger flashes evoked saltatory calcium waves, propagating with non‐constant velocity. During waves, [Ca2+] rose slowly between puff sites, but more abruptly at active sites following an initial diffusive rise in calcium.

Initial rates of rise of local [Ca2+] at release sites were similar during puffs and release induced by much (> 10‐fold) greater [InsP3]. In contrast, macroscopic calcium measurements averaged over the scan line showed a graded dependence of rate of calcium liberation upon [InsP3], due to recruitment of additional sites and decreasing dispersion in activation latencies.

We conclude that the initiation of calcium liberation depends co‐operatively upon [InsP3] whereas the subsequent regenerative increase in calcium flux depends upon local calcium feedback and is largely independent of [InsP3]. Wave propagation is consistent with the diffusive spread of calcium evoking regenerative liberation at heterogeneous discrete sites, the sensitivity of which is primed by InsP3.
cGMP inhibits IP3‐induced Ca2+ release in intact rat megakaryocytes via cGMP‐ and cAMP‐dependent protein kinases
Authors: Tertyshnikova Svetlana , Yan Xiongwei , Fein Alan
Published: September 2004

Inhibition of inositol 1,4,5‐trisphosphate (IP3) receptor‐mediated Ca2+ release by cGMP was examined in intact rat megakaryocytes, by using a combination of single cell fluorescence microscopy to monitor intracellular free calcium ([Ca2+]i) and flash photolysis of caged second messengers.

Sodium nitroprusside (SNP), a nitric oxide (NO) donor, and the hydrolysis‐resistant cGMP analogue 8‐(4‐chlorophenylthio)guanosine 3′,5′‐cyclic monophosphate (pCPT‐cGMP) inhibited Ca2+ release induced by photolysis of caged IP3. Neither of them affected the rate of Ca2+ removal from the cytoplasm following photolysis of caged Ca2+.

Photolysis of the caged NO donor 3‐morpholinosydnonimine (SIN‐1) during agonist‐induced [Ca2+]i oscillations inhibited Ca2+ release without affecting the rate of Ca2+ uptake and/or extrusion.

We conclude that the inhibition of IP3‐induced Ca2+ release is the principal mechanism of NO‐cGMP‐dependent inhibition of [Ca2+]i mobilization.

IPG, a specific peptide inhibitor of cGMP‐dependent protein kinase (cGMP‐PK), blocked the inhibitory effect of pCPT‐cGMP, indicating that the inhibition of IP3‐induced Ca2+ release by pCPT‐cGMP is mediated by cGMP‐PK. However, the simultaneous application of both IPG and IP20, a specific peptide inhibitor of cAMP‐dependent protein kinase (cAMP‐PK), was required to block the inhibitory effect of SNP. These data strongly suggest that NO‐cGMP‐dependent inhibition of [Ca2+]i mobilization is mediated via the activation of both cGMP‐PK and cAMP‐PK.
Activation of Ca2+‐activated K+ channels by an increase in intracellular Ca2+ induced by depolarization of mouse skeletal muscle fibres
Authors: Jacquemond Vincent , Allard Bruno
Published: September 2004

Ionic currents were simultaneously recorded at macroscopic and unitary level using the whole‐cell and cell‐attached patch‐clamp procedures together on the same portion of isolated mouse skeletal muscle fibres.

In the presence of Tyrode solution in the patch pipette and Tyrode‐TTX solution in the bath, macroscopic and unitary currents through delayed rectifier K+ channels were simultaneously recorded in response to depolarizing pulses of 1 s duration.

In five fibres, successive long‐lasting incremental depolarizing levels induced, at ‐40 mV or ‐30 mV, the opening of a high conductance channel carrying an outward current superimposed on delayed rectifier K+ channel activity. Opening of this high conductance channel was not observed when the depolarization steps were applied in the patch pipette.

Using the same depolarizing protocol, activation of a high conductance channel was also observed in two fibres in the presence of a K+‐rich solution in the pipette (145 mm K+).

With either Tyrode or K+‐rich solution in the pipette, unitary current amplitudes of the high conductance channel matched well with the values obtained for Ca2+‐activated K+ (KCa) channels in inside‐out patches under similar ionic conditions.

Indo‐1 fluorescence measurements showed that the stimulation protocol that led to KCa channel opening induced stepwise increases in intracellular [Ca2+] in the submicromolar range.

Our results provide evidence that activation of sarcolemmal KCa channels can be induced by a rise in intracellular [Ca2+] following voltage‐activated sarcoplasmic reticulum Ca2+ release.
Differential inhibition of N and P/Q Ca2+ currents by 5‐HT1A and 5‐HT1D receptors in spinal neurons of Xenopus larvae
Authors: Sun Qian‐Quan , Dale Nicholas
Published: September 2004

In whole‐cell patch clamp recordings made from non‐sensory neurons acutely isolated from the spinal cord of Xenopus (stage 40–42) larvae, two forms of inhibition of the high voltage‐activated (HVA) Ca2+ currents were produced by 5‐HT. One was voltage dependent and associated with both slowing of the activation kinetics and shifting of the voltage dependence of the HVA currents. This inhibition was relieved by strong depolarizing prepulses. A second form of inhibition was neither associated with slowing of the activation kinetics nor relieved by depolarizing prepulses and was thus voltage independent.

In all neurons examined, 5‐HT (1 μM) reversibly reduced 34 ± 1.6 % (n= 102) of the HVA Ca2+ currents. In about 40 % of neurons, the inhibition was totally voltage independent. In another 5 %, the inhibition was totally voltage dependent. In the remaining neurons, inhibition was only partially (by around 40 %) relieved by a large depolarizing prepulse, suggesting that in these, the inhibition consisted of both voltage‐dependent and ‐independent components.

By using selective channel blockers, we found that 5‐HT acted on both N‐ and P/Q‐type channels. However, whereas the inhibition of P/Q‐type currents was only voltage independent, the inhibition of N‐type currents had both voltage‐dependent and ‐independent components.

The effects of 5‐HT on HVA Ca2+ currents were mediated by 5‐HT1A and 5‐HT1D receptors. The 5‐HT1A receptors not only preferentially caused voltage‐independent inhibition, but did so by acting mainly on the ω‐agatoxin‐IVA‐sensitive Ca2+ channels. In contrast, the 5‐HT1D receptor produced both voltage‐dependent and ‐independent inhibition and was preferentially coupled to ω‐conotoxin‐GVIA sensitive channels. This complexity of modulation may allow fine tuning of transmitter release and calcium signalling in the spinal circuitry of Xenopus larvae.
Anemone toxin (ATX II)‐induced increase in persistent sodium current: effects on the firing properties of rat neocortical pyramidal neurones
Authors: Mantegazza Massimo , Franceschetti Silvana , Avanzini Giuliano
Published: September 2004

The experiments were performed on sensorimotor cortex using current‐clamp intracellular recordings in layer V pyramidal neurones and whole‐cell voltage‐clamp recordings in dissociated pyramidal neurones. The intracellularly recorded neurones were classified on the basis of their firing characteristics as intrinsically bursting (IB) and regular spiking (RS). The RS neurones were further subdivided into adapting (RSAD) or non‐adapting (RSNA), depending on the presence or absence of spike frequency adaptation. Since burst firing in neocortical pyramidal neurones has previously been suggested to depend on the persistent fraction of Na+ current (INa,p), pharmacological manipulations with drugs affecting INa inactivation have been employed.

ATX II, a toxin derived from Anemonia sulcata, selectively inhibited INa fast inactivation in dissociated neurones. In current‐clamp experiments on neocortical slices, ATX II enhanced the naturally occurring burst firing in IB neurones and revealed the ability of RSNA neurones to discharge in bursts, whereas in RSAD neurones it increased firing frequency, without inducing burst discharges. During the ATX II effect, in all the three neuronal subclasses, episodes of a metastable condition occurred, characterized by long‐lasting depolarizing shifts, triggered by action potentials, which were attributed to a peak in the toxin‐induced inhibition of INa inactivation. The ATX II effect on IB and RSNA neurones was compared with that induced by veratridine and iodoacetamide. Veratridine induced a small increase in the INa and a large shift to the left in the voltage dependence of INa activation. Accordingly, its major effect on firing characteristics was the induction of prolonged tonic discharges, associated with burst facilitation less pronounced than that induced by ATX II. The alkylating agent iodoacetamide was able to induce a selective small increase in the INa,p, with a similar but less pronounced effect than ATX II on firing behaviour.

The present results show that pharmacological manipulations capable of slowing down INa inactivation significantly enhance burst behaviour in IB neurones and promote burst firing in otherwise non‐bursting RSNA neurones. We suggest that IB and, to a lesser extent, RSNA neurones are endowed with a relatively large fraction of INa,p which, in physiological conditions, is sufficient to sustain bursting in IB but not in RSNA neurones.
ATP‐sensitive K+ channel activation by calcitonin gene‐related peptide and protein kinase A in pig coronary arterial smooth muscle
Authors: Wellman G. C. , Quayle J. M. , Standen N. B.
Published: September 2004

We used patch clamp to study whole‐cell K+ currents activated by calcitonin gene‐related peptide (CGRP) in smooth muscle cells freshly dissociated from pig coronary arteries.

CGRP (50 nm) activated an inward current at −60 mV in symmetrical 140 mm K+ that was blocked by glibenclamide (10 μm), an inhibitor of ATP‐sensitive potassium (KATP) channels. CGRP‐induced currents were larger in cells dialysed with 0.1 mm ATP than with 3.0 mm ATP.

Forskolin (10 μm) activated a glibenclamide‐sensitive current, as did intracellular dialysis with cAMP (100 μm). The catalytic subunit of cAMP‐dependent protein kinase (protein kinase A, PKA), added to the pipette solution, activated equivalent currents in five out of twelve cells.

CGRP‐induced currents were reduced by the PKA inhibitors adenosine 3′,5′‐cyclic monophosphorothioate, RP‐isomer, triethylammonium salt (Rp‐cAMPS; 100 μm) and N‐[2‐((p‐bromocinnamyl)amino)ethyl]‐5‐isoquinolinesulphonamide dihydrochloride (H‐89; 1 μm), and abolished by inclusion of a PKA inhibitor peptide in the pipette solution.

The β‐adrenergic agonist isoprenaline (10 μm) also activated a glibenclamide‐sensitive K+ current.

CGRP‐induced currents were unaffected by the inhibitor of cGMP‐dependent protein kinase (PKG) KT5823 (1 μm). Sodium nitroprusside (10 μm) did not activate a glibenclamide‐sensitive current in cells held at −60 mV, but did activate an outward current at +60 mV that was abolished by KT5823, or by 100 nm iberiotoxin (an inhibitor of BKCa channels).

Our findings suggest that CGRP activates coronary KATP channels through a pathway that involves adenylyl cyclase and PKA, but not PKG.
Role of Na+ conductance, Na+‐H+ exchange, and Na+‐K+‐2Cl− symport in the regulatory volume increase of rat hepatocytes
Authors: Wehner Frank , Tinel Hanna
Published: September 2004

In rat hepatocytes under hypertonic stress, the entry of Na+ (which is thereafter exchanged for K+ via Na+‐K+‐ATPase) plays the key role in regulatory volume increase (RVI).

In the present study, the contributions of Na+ conductance, Na+‐H+ exchange and Na+‐K+‐2Cl− symport to this process were quantified in confluent primary cultures by means of intracellular microelectrodes and cable analysis, microfluorometric determinations of cell pH and buffer capacity, and measurements of frusemide (furosemide)/bumetanide‐sensitive 86Rb+ uptake, respectively. Osmolarity was increased from 300 to 400 mosmol l−1 by addition of sucrose.

The experiments indicate a relative contribution of approximately 4:1:1 to hypertonicity‐induced Na+ entry for the above‐mentioned transporters and the overall Na+ yield equalled 51 mmol l−1 (10 min)−1.

This Na+ gain is in good agreement with the stimulation of Na+ extrusion via Na+‐K+‐ATPase plus the actual increase in cell Na+, namely 55 mmol l−1 (10 min)−1, as was determined on the basis of ouabain‐sensitive 86Rb+ uptake and by means of Na+‐sensitive microelectrodes, respectively.

The overall increase in Na+ and K+ activity plus the expected concomitant increase in cell Cl− equalled 68 mmol l−1, which fits well with the increase in osmotic activity expected to occur from an initial cell shrinkage to 87.5 % and a RVI to 92.6 % of control, namely 53 mosmol l−1.

The prominent role of Na+ conductance in the RVI of rat hepatocytes could be confirmed on the basis of the pharmacological profile of this process, which was characterized by means of confocal laser‐scanning microscopy.
The development of conduction block in single human axons following a focal nerve injury
Authors: Inglis J. Timothy , Leeper James B. , Wilson Linda R. , Gandevia Simon C. , Burke David
Published: September 2004

Using microneurography with a conventional monopolar electrode, the action potentials of ten myelinated axons in the peripheral nerves of human subjects were followed while they developed conduction block.

The action potentials had initially (n= 6) or developed (n= 4) a positive double‐peaked morphology. The time interval between the two positive peaks represents the conduction time across the impaled internode.

When the interpeak interval was < 500 μs, conduction across the site of impalement was secure, even if the conduction time was markedly prolonged. When the interval was > 600 μs, intermittent conduction failure occurred. For all units the longest interpeak interval recorded just prior to complete conduction failure was, on average, 1.12 ms (range, 0.8–1.4 ms).

For five axons, there was evidence that natural activity triggered the conduction failure.

Impalement of the nerve fibre by the microelectrode impairs the ability of the axon to conduct impulses across the site of injury, but impulse transmission can be secure even when the conduction time across individual internodes is prolonged to 500 μs. These findings are therefore relevant to the conduction deficits that occur in focal injuries of human axons.
A mutation in the pore region of HERG K+ channels expressed in Xenopus oocytes reduces rectification by shifting the voltage dependence of inactivation
Authors: Zou Anruo , Xu Qing P. , Sanguinetti Michael C.
Published: September 2004

The effects of a mutation in the human ether‐a‐go‐go‐related gene (HERG) (Ser631 to Ala, S631A) on the voltage‐ and extracellular [K+] dependence of inactivation were studied in Xenopus oocytes using two microelectrode and single channel voltage‐clamp techniques.

The voltage required for half‐inactivation of S631A HERG was 102 mV more positive than for wild‐type (WT)‐HERG, resulting in reduced rectification of the steady‐state current‐voltage relationship. In contrast, the voltage dependence of channel activation was not altered by the S631A mutation. These findings indicate that inactivation of HERG channels is not linked to activation.

Rectification of whole‐cell S631A HERG current was caused by a voltage‐dependent reduction in open probability, and inward rectification of the current‐voltage relationship of single channels.

Elevation of extracellular [K+] from 2 to 20 mm shifted the half‐point for inactivation by +20 mV for WT‐HERG, and +25 mV for S631A HERG. Thus, elevated [K+]o and the S631A mutation affect HERG inactivation by different mechanisms.

The S631A mutation altered the ion translocation rate of HERG channels. The single channel conductance (γ) of S631A HERG was 20 pS between ‐40 and‐100 mV, and 6.0 pS between +40 and +100 mV (120 mm extracellular K+). This compares to a γ of 12.1 and 5.1 pS for WT‐HERG channels under the same conditions.
Contribution of sarcoplasmic reticular calcium to smooth muscle contractile activation: gestational dependence in isolated rat uterus
Authors: Taggart Michael J. , Wray Susan
Published: September 2004

The contribution of Ca2+ released from the sarcoplasmic reticulum (SR) to smooth muscle contractile activation remains poorly understood. By simultaneously monitoring cytosolic [Ca2+] ([Ca2+]i) and force in isolated rat uterine smooth muscle, we report the influence of SR Ca2+ release on contractility during conditions (a) of altered SR Ca2+ homeostasis and (b) where the only source of activating Ca2+ was derived from the SR.

In myometria of non‐pregnant rats, ryanodine (1‐50 μM), a modulator of calcium‐induced calcium release (CICR), had no effect on the spontaneous [Ca2+]i or force transients. However, depletion of SR Ca2+ by inhibiting the SR Ca2+‐ATPase (with cyclopiazonic acid (CPA), 20 μM) resulted in an enhancement of spontaneous [Ca2+]i and force transients.

In myometria of pregnant rats, although ryanodine had no effect in 40 % of tissues studied it produced a small but significant enhancement of the integrated spontaneous [Ca2+]i and force transient in 60 % of cases. The potentiating effects of CPA were enhanced in myometria of pregnant rats compared with non‐pregnant rats, often resulting in maintained [Ca2+]i increases and contraction.

In zero external Ca2+, agonist‐induced SR Ca2+ release resulted in transient increases in [Ca2+]i and force. The magnitude of these agonist‐induced [Ca2+]i and force changes were significantly enhanced in myometria of pregnant rats. No evidence for agonist‐induced Ca2+‐independent force production was observed.

These results indicate that CICR plays little role in SR Ca2+ release from the myometrium, and that there are gestational‐dependent alterations in the ability of SR Ca2+ mobilization to contribute to contractile activation. The implications of these findings for the co‐ordination of myometrial [Ca2+]i signalling and contractility are discussed.
Transmitter release modulation in nerve terminals of rat neocortical pyramidal cells by intracellular calcium buffers
Authors: Ohana Ora , Sakmann Bert
Published: September 2004

Dual whole‐cell voltage recordings were made from synaptically connected layer 5 (L5) pyramidal neurones in slices of the young (P14‐P16) rat neocortex. The Ca2+ buffers BAPTA or EGTA were loaded into the presynaptic neurone via the pipette recording from the presynaptic neurone to examine their effect on the mean and the coefficient of variation (c.v.) of single fibre EPSP amplitudes, referred to as unitary EPSPs.

The fast Ca2+ buffer BAPTA reduced unitary EPSP amplitudes in a concentration dependent way. With 0.1 mm BAPTA in the pipette, the mean EPSP amplitude was reduced by 14 ± 2.8 % (mean ±s.e.m., n= 7) compared with control pipette solution, whereas with 1.5 mm BAPTA, the mean EPSP amplitude was reduced by 72 ± 1.5 % (n= 5). The concentration of BAPTA that reduced mean EPSP amplitudes to one‐half of control was close to 0.7 mm.

Saturation of BAPTA during evoked release was tested by comparing the effect of loading the presynaptic neurone with 0.1 mm BAPTA at 2 and 1 mm[Ca2+]o. Reducing [Ca2+]o from 2 to 1 mm, thereby reducing Ca2+ influx into the terminals, decreased the mean EPSP amplitude by 60 ± 2.2 % with control pipette solution and by 62 ± 1.9 % after loading with 0.1 mm BAPTA (n= 7).

The slow Ca2+ buffer EGTA at 1 mm reduced mean EPSP amplitudes by 15 ± 2.5 % (n= 5). With 10 mm EGTA mean EPSP amplitudes were reduced by 56 ± 2.3 % (n= 4).

With both Ca2+ buffers, the reduction in mean EPSP amplitudes was associated with an increase in the c.v. of peak EPSP amplitudes, consistent with a reduction of the transmitter release probability as the major mechanism underlying the reduction of the EPSP amplitude.

The results suggest that in nerve terminals of thick tufted L5 pyramidal cells the endogenous mobile Ca2+ buffer is equivalent to less than 0.1 mm BAPTA and that at many release sites of pyramidal cell terminals the Ca2+ channel domains overlap, a situation comparable with that at large calyx‐type terminals in the brainstem.
Electrophysiological characterization of Na+ currents in acutely isolated human hippocampal dentate granule cells
Authors: Reckziegel G. , Beck H. , Schramm J. , Elger C. E. , Urban B. W.
Published: September 2004

Properties of voltage‐dependent Na+ currents were investigated in forty‐two dentate granule cells (DGCs) acutely isolated from the resected hippocampus of twenty patients with therapy‐refractory temporal lobe epilepsy (TLE) using the whole‐cell patch‐clamp technique.

Depolarizing voltage commands elicited large, rapidly activating and inactivating Na+ currents (140 pS μm−2; 163 mm extracellular Na+) that were reduced in amplitude by lowering the Na+ gradient (43 mm extracellular Na+). At low temperatures (8‐12 °C), the time course of Na+ currents slowed and could be well described by the model of Hodgkin & Huxley.

Na+ currents were reversibly blocked by tetrodotoxin (TTX) and saxitoxin (STX) with a half‐maximal block of 4.7 and 2.6 nm, respectively. In order to reduce series resistance errors, the Na+ current was partially blocked by low toxin concentrations (10‐15 nm) in the experiments described below. Under these conditions, Na+ currents showed a threshold of activation of about ‐50 mV, and the voltages of half‐maximal activation and inactivation were ‐29 and ‐55 mV, respectively.

The time course of recovery from inactivation could be described with a double‐exponential function (time constants, 3‐20 and 60‐200 ms). The rapid and slow time constants showed a distinct voltage dependence with maximal values around ‐55 and ‐80 mV, respectively. These properties contributed to a reduction of the Na+ currents during repetitive stimulation that was more pronounced with higher stimulation frequencies and also showed a dependence on the holding potential.

In summary, the most striking features of DGC Na+ currents were the large current density and the presence of a current component showing a slow recovery from inactivation. Our data provide a basis for comparison with properties of Na+ currents in animal models of epilepsy, and for the study of drug actions in therapy‐refractory epilepsy.
Adrenergic regulation of calcium‐activated potassium current in cultured rabbit pigmented ciliary epithelial cells
Authors: Ryan Jennifer S. , Tao Qian‐Ping , Kelly Melanie E. M.
Published: September 2004

The effects of adrenergic agonists on K+ currents were studied in cultured rabbit pigmented ciliary epithelial (PCE) cells.

Outward K+ current (IK) was reduced by tetraethylammonium chloride, the Ca2+‐activated K+ (K(Ca)) channel blocker iberiotoxin (IbTX), or Ca2+‐free external Ringer solution. The calcium ionophore ionomycin increased an IbTX‐sensitive IK in PCE cells.

The adrenergic agonists adrenaline and phenylephrine increased IK in PCE cells. The induced current was blocked by IbTX and the α1‐antagonist prazosin, suggesting that adrenergic agonists activate IK(Ca) via α1‐adrenoreceptors.

Internal dialysis of D‐myo‐inositol 1,4,5‐trisphosphate (IP3) increased IK, whilst pre‐incubation of PCE cells with thapsigargin or the phospholipase C (PLC) inhibitor U‐73122 reduced phenylephrine‐induced increases in IK(Ca). Adrenergic increases in IK(Ca) were mediated by a pertussis toxin‐insensitive G protein.

These results demonstrate that IK(Ca) channels in rabbit PCE cells are coupled to α1‐adrenergic receptors and a PLC/IP3 signalling pathway. Activation of these channels may modulate fluid secretion by the ciliary epithelium.
The role of Ca2+ in volume regulation induced by Na+‐coupled alanine uptake in single proximal tubule cells isolated from frog kidney
Authors: Mounfield P. R. , Robson L.
Published: September 2004

It has been suggested that epithelial cells maintain cell volume and function, in the face of changes in the rate of transepithelial transport, by activation of volume‐regulatory pathways.

The aim of the following study was to examine directly the effect of an alteration in Na+‐coupled alanine transport on cell length in single proximal tubule cells isolated from frog kidney.

An optical technique was used to examine the change in cell length induced by 5 mM L‐alanine.

On addition of L‐alanine to the bath there was an initial increase in cell length to a peak value. This was followed by two types of response. In eighteen out of thirty‐one cells a typical volume‐regulatory response was observed. The remaining cells showed no volume regulation.

Volume regulation was not affected by the removal of extracellular Ca2+. The mean degrees of recovery were 159 ± 21 % (n= 18) and 144 ± 18 % (n= 8) in the presence and absence of Ca2+, respectively.

Volume regulation was inhibited by depletion of intracellular Ca2+ stores, or in the presence of either Gd3+ or DIDS. The mean degrees of regulation were 55.4 ± 9.2 % (n= 7), 68.2 ± 18.8 % (n= 7) and 69.1 ± 14.3 % (n= 7), respectively.

The alanine‐induced increases in cell length were both stereospecific and Na+ dependent.

The evidence suggests that volume regulation induced by Na+‐coupled alanine uptake may be dependent on the release of Ca2+ from intracellular stores. This is in contrast to volume regulation induced by hypotonic shock, which appears to require extracellular Ca2+. Results obtained using a hypotonic shock should, therefore, be viewed with caution.
Facilitation of presynaptic calcium currents in the rat brainstem
Authors: Borst J. G. G. , Sakmann B.
Published: September 2004

To study use‐dependent changes in the presynaptic Ca2+ influx and their contribution to transmitter release, we made simultaneous voltage clamp recordings from presynaptic terminals (the calyces of Held) and postsynaptic cells (the principal cells of the medial nucleus of the trapezoid body) in slices of the rat auditory brainstem.

Following a short (2 ms) prepulse to 0 mV, calcium channels opened faster during steps to negative test potentials. During trains of action potential waveforms the Ca2+ influx per action potential increased. At the same time, however, the amplitude of the EPSCs decreased.

The facilitation of the calcium currents appeared to depend on a build‐up of intracellular Ca2+, since its magnitude was proportional to the Ca2+ influx and it was reduced in the presence of 10 mm BAPTA.

Facilitation of the presynaptic calcium currents may contribute to short‐term facilitation of transmitter release, observed when quantal output is low. Alternatively, it may counteract processes that contribute to synaptic depression.
Two types of parasympathetic preganglionic neurones in the superior salivatory nucleus characterized electrophysiologically in slice preparations of neonatal rats
Authors: Matsuo Ryuji , Kang Youngnam
Published: September 2004

The electrophysiological properties of parasympathetic preganglionic neurones in the superior salivatory nucleus were studied in thin‐ and thick‐slice preparations of rats aged 1 and 2 weeks using the whole‐cell patch‐clamp technique.

The superior salivatory neurones were identified by a retrograde tracing method with dextran‐tetramethylrhodamine‐lysine. The injection of the tracer into the chorda‐lingual nerve labelled the neurones innervating the submandibular ganglia and those innervating the intra‐lingual ganglia, while the injection into the tip of the tongue labelled the latter group of neurones.

Firing characteristics were investigated mainly in the neurones of 6–8 days postnatal rats. In response to an injection of long depolarizing current pulses at hyperpolarized membrane potentials (< −80 mV) under a current clamp, the neurones labelled from the nerve displayed a train of action potentials with either a long silent period preceding the first spike (late spiking pattern) or a long silent period interposed between the first and second spikes (interrupted spiking pattern). The neurones labelled from the tongue invariably displayed the interrupted spiking pattern.

Under a voltage clamp, among the neurones from 6–8 days postnatal rats, those labelled from the nerve expressed either a fast or a slow transient outward current (A‐current), while those labelled from the tongue invariably showed a slow transient outward current. Both the fast and slow A‐currents were largely depressed by 1 mm 4‐aminopyridine.

Similar fast and slow A‐currents were observed in the neurones of rats aged 14–15 days. Both the time to peak and decay time constant of these A‐currents were accelerated, suggesting a developmental trend of maturation in the activation and inactivation kinetics between 6 and 15 days postnatal.

Based on the differences in the firing pattern and outward current, the superior salivatory neurones can be separated into two distinct types. We discuss the functional aspects of these two types of neurones with reference to their target organs.
Functions of large conductance Ca2+‐activated (BKCa), delayed rectifier (KV) and background K+ channels in the control of membrane potential in rabbit renal arcuate artery
Authors: Prior H. M. , Yates M. S. , Beech D. J.
Published: September 2004

The types of K+ channel which determine the membrane potential of arcuate artery smooth muscle cells were investigated by patch‐clamp recording from isolated cells and lumenal diameter measurements from intact pressurized renal arcuate arteries.

Single cells had a mean resting potential of ‐38 mV and were depolarized by 130 mM K+ but not by the Cl− channel blocker 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid (DIDS).

Iberiotoxin did not affect the resting potential but inhibited spontaneous transient hyperpolarizations. Iberiotoxin or 1 mM tetraethylammonium (TEA+) constricted intact arteries. 3,4‐Diaminopyridine (3,4‐DAP)‐sensitive delayed rectifier K+ (KV) channel current was elicited by depolarization but 3,4‐DAP did not affect the resting potential or induce constriction in the intact artery.

A voltage‐independent K+ current was inhibited by ≥ 0·1 mM barium (Ba2+) and unaffected by iberiotoxin, glibenclamide, apamin, 3,4‐DAP and ouabain. In six out of ten cells, 1 mM Ba2+ depolarized the resting potential, while in the other cells the potential was resistant to all of the K+ channel blockers and ouabain. Ba2+ (0·1‐1 mM) constricted the intact artery, but 10 μM Ba2+, 1 μM glibenclamide or 100 nM apamin had no effect.

The data suggest that resting potential is determined by background K+ channels, one type being Ba2+ sensitive and voltage independent, and another type being poorly defined due to its resistance to any inhibitor. Large conductance Ca2+‐activated K+ (BKCa) and KV channels do not determine the resting potential but have separate functions to underlie transient Ca2+‐induced hyperpolarizations and to protect against depolarization past about ‐30 mV.
Calcium permeation of the turtle hair cell mechanotransducer channel and its relation to the composition of endolymph
Authors: Ricci A. J. , Fettiplace R.
Published: September 2004

Recordings of mechanoelectrical transducer currents were combined with calcium imaging of hair bundles in turtle auditory hair cells located near the high‐frequency end of the cochlea. The external face of the hair bundles was perfused with a range of Ca2+ concentrations to study the quantitative relationship between Ca2+ influx and transducer adaptation.

With Na+ as the monovalent ion, the peak amplitude of the transducer current decreased monotonically as the external [Ca2+] was raised from 25 μM to 20 mM. When Na+ was replaced with the impermeant Tris the transducer current increased with external [Ca2+]. These results indicate that Ca2+ can both permeate and block the transducer channels. The Ca2+ concentration for half‐block of the monovalent current was 1 mM.

To quantify the Ca2+ influx, the fraction of transducer current carried by Ca2+ was measured using the change in bundle fluorescence in cells loaded with 1 mM Calcium Green‐1. The fluorescence change was calibrated by substituting an impermeable monovalent ion to render Ca2+ the sole charge carrier.

In the presence of Na+, the fractional Ca2+ current was ≈10 % in 50 μM Ca2+, a concentration similar to that in endolymph, which bathes the hair bundles in vivo. The amount of Ca2+ entering was dependent on the identity of the monovalent ion, and was larger with K+, suggesting that the transducer channel is a multi‐ion pore.

Over a range of ionic conditions, the rate of transducer adaptation was proportional to Ca2+ influx indicating that adaptation is driven by a rise in intracellular [Ca2+].

Shifts in the current‐displacement function along the displacement axis in different external Ca2+ concentrations were predictable from variation in the resting Ca2+ influx. We suggest that changes in the resting open probability of the transducer channels adjust the entry of Ca2+ to keep its concentration constant at an internal site.

The results demonstrate that endolymph containing high K+, 50 μM Ca2+ and low Mg2+ concentrations, maximizes the transducer current while still allowing sufficient Ca2+ entry to drive adaptation. The hair cell mechanotransducer channel, in its permeation and block by Ca2+, shows behaviour similar to the voltage‐gated Ca2+ channel and the cyclic nucleotide‐gated channel.
Nitric oxide lowers the calcium sensitivity of tension in the rat tail artery
Authors: Tran Nguyen N. P. , Spitzbarth Esther , Robert Alain , Giummelly Philippe , Atkinson Jeffrey , Capdeville‐Atkinson Christine
Published: September 2004

Controversy exists as to whether a fall in the intracellular Ca2+ concentration ([Ca2+]i) is a requisite element of the vasodilatory response to nitric oxide (NO).

We studied the effect of NO on the coupling between [Ca2+]i and vasoconstriction in arterial segments loaded with the [Ca2+]i‐sensitive, intracellular dye fura‐2. As data interpretation is equivocal when fura‐2 is loaded into both endothelial and smooth muscle cells, we compared results from in vitro experiments on segments of the rat tail artery in which fura‐2 and noradrenaline were applied on the luminal or adventitial side, and endothelium was removed ‘physically’ (rubbing or air) or ‘functionally’ (Nω‐nitro‐l‐arginine methyl ester). The use of air perfusion to remove endothelium is of considerable benefit since it allows paired observations in a single tissue.

Fura‐2 loaded into endothelial cells but endothelial ‘contamination’ of the smooth muscle cell [Ca2+]i signal was minimal.

Endogenous NO decreased vasoconstrictor responses to noradrenaline but had no effect on [Ca2+]i.

Nitroglycerine decreased vasoconstrictor responses in a concentration‐dependent fashion but had no effect on [Ca2+]i.

In conclusion, NO causes vasodilatation via a mechanism which is downstream of [Ca2+]i mobilization.
Evidence for enhancement of gap junctional coupling between rat island of Calleja granule cells in vitro by the activation of dopamine D3 receptors
Authors: Halliwell J. V. , Horne A. L.
Published: September 2004

Using patch‐clamp techniques, we have studied actions of dopamine and related compounds on granule neurones within the islands of Calleja in vitro, in slices of ≈200 μm thickness or as groups of varying cell number following enzymic digestion.

Prior to agonist application, island of Calleja granule cells displayed spontaneous stepwise shifts in whole‐cell conductance ranging from 104 to 632 pS. The reversal potentials of these conductance changes ranged widely and matched the distribution of the cells' membrane potentials. Reversal potentials and membrane potentials shifted equally when cells were uniformly depolarized in 24 mM external K+.

Bath‐applied dopamine elicited, after a delay of 4‐9 min, an exaggerated form of the spontaneous behaviour that frequently gave way to a sudden large (up to thirtyfold) conductance change. At concentrations of 100‐300 nM, a range of agonists with increasing affinity for the D3 receptor (apomorphine, quinpirole, 7‐OH DPAT and PD 128907) triggered the response. The actions were neither mimicked by SKF‐38393 nor antagonized by SCH‐23390 (a selective D1 agonist and antagonist, respectively). Haloperidol reversibly blocked responses elicited by the D3/D2 agonist quinpirole. The action of effective agonists was maintained when transmitter release was abolished. Given the reported lack of D2 receptors in the islands of Calleja, these findings indicate a direct action of dopamine at the D3 receptor.

The dopaminergic effects were not affected by Gd3+ or substantial replacement of external Na+ with TEA, Tris or choline, eliminating stretch‐activated channels but suggesting that if transmembrane channels were to be involved in this dopaminergic action they posseses a non‐selective permeability to large cations. The reported presence of gap junctions in the islands of Calleja offers the explanation that these effects derive from enhanced activity of such channels or their hemi‐constituents.

In testing the possible involvement of gap junctional coupling the following experimental observations were made: (i) alkalinization of slices mimicked the effect of D3 agonists; (ii) in cell groups, recording from pairs provided evidence of intercellular coupling, and mechanical separation of recorded neurones from neighbouring cells during the agonist‐evoked response caused shutdown of the additional conductance; (iii) when applied to slices, the gap junctional blocker, 18α‐glycyrrhetinic acid, whilst not preventing the full‐blown dopamine response, significantly reduced both the variance of recorded granule cell input conductance and the cells’ apparent capacitance.

Taken together the results indicate a D3 action in granule cells, which is best explained by a dopaminergic promotion of intercellular coupling. The physiological relevance of such a mechanism is discussed.
Single channel currents at six microsecond resolution elicited by acetylcholine in mouse myoballs
Authors: Parzefall Franz , Wilhelm Robert , Heckmann Manfred , Dudel Josef
Published: September 2004

A patch‐clamp set‐up was optimized for low noise and high time resolution. An Axoclamp 200B amplifier was modified to incorporate a Teflon connector to the electrode. An electrode puller was equipped with a hydrogen‐oxygen burner to produce quartz‐glass pipettes with optimally 0.2 μm openings and 20 MΩ resistance.

The r.m.s. (root mean square) noise of sealed pipettes in the bath ranged from 3.6 fA with 100 Hz filter cut‐off to 1.5 pA with 61 kHz filter cut‐off. At these extremes currents of 17 fA and more than 3 ms, or 9 pA and more than 6 μs could be resolved with a negligible error rate.

The system was tested on mouse myoballs, recording 9–10 pA single channel currents on‐cell at −200 mV polarization which were elicited by 0.1–5000 μm acetylcholine (ACh).

Distributions of open and closed times and of correlations of open times to the preceding closed time defined several open states: single openings with mean durations of 1.2 and 25 μs, from single‐liganded receptors, and bursts of 10 ms mean duration containing on average 800 μs openings and 16 μs closings, from double liganded receptors. Above 0.1 mm ACh these openings are interrupted increasingly by on average 18 μs and 72 μs channel blocks by ACh.