bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the hyperpolarization‐activated chloride current in dissociated rat sympathetic neurons
Authors: Clark Samantha , Jordt Sven‐Eric , Jentsch Thomas J. , Mathie Alistair
Published: September 2004

Dissociated rat superior cervical ganglion (SCG) neurons have been shown to possess a hyperpolarization‐activated inwardly rectifying chloride current. The current was not altered by changes in external potassium concentration, replacing external cations with NMDG (N‐methyl‐D‐glucamine) or by addition of 10 mM caesium or barium ions.

The reversal potential of the current was altered by changing external anions. The anion selectivity of the current was Cl− > Br− > I− > cyclamate. All substituted permeant anions also blocked the current.

The current was blocked by DIDS (4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid), 9AC (anthracene‐9‐carboxylic acid) and NPPB (5‐nitro‐2‐(3‐phenylpropylamino)benzoic acid) but was unaffected by SITS (4‐acetamido‐4′‐isothiocyanatostilbene‐2,2′‐disulphonic acid) and niflumic acid. The effective blockers were voltage dependent; DIDS and NPPB were more effective at depolarized potentials while 9AC was more effective at hyperpolarized potentials.

The current was enhanced by extracellular acidification and reduced by extracellular alkalinization. Reducing external osmolarity was without effect in conventional whole‐cell recording but enhanced current amplitude in those perforated‐patch recordings where little current was evident in control external solution.

The current in SCG neurons was blocked by external cadmium and zinc. ClC‐2 chloride currents expressed in Xenopus oocytes were also sensitive to block by these divalent ions and by DIDS but the sensitivity of ClC‐2 to block by cadmium ions was lower than that of the current in SCG neurons.

Reverse transcriptase‐polymerase chain reaction (RT‐PCR) experiments showed the presence of mRNA for ClC‐2 in SCG neurons but not in rat cerebellar granule cells which do not possess a hyperpolarization‐activated Cl− current.

The data suggest that ClC‐2 may be functionally expressed in rat SCG neurons. This current may play a role in regulating the internal chloride concentration in these neurons and hence their response to activation of GABAA receptors.1. Dissociated rat superior cervical ganglion (SCG) neurons have been shown to possess a hyperpolarization‐activated inwardly rectifying chloride current. The current was not altered by changes in external potassium concentration, replacing external cations with NMDG (N‐methyl‐D‐glucamine) or by addition of 10 mM caesium or barium ions.

The reversal potential of the current was altered by changing external anions. The anion selectivity of the current was Cl− > Br− > I− > cyclamate. All substituted permeant anions also blocked the current.
Functional contribution of the α5 subunit to neuronal nicotinic channels expressed by chick sympathetic ganglion neurones
Authors: Yu C. Ron , Role Lorna W.
Published: September 2004

Heterologous expression studies of the α5 subunit of the neuronal acetylcholine receptor (nAChR) gene family have demonstrated that it can participate in the function of ACh‐gated channels if co‐expressed with another α‐ and a β‐subunit. Previous studies also indicate prominent expression of α5 in both central and peripheral nervous systems. The participation of α5 in native nAChRs and its functional role in these channels is, however, unknown.

In this study, we present evidence that α5 has a role in at least two distinct subtypes of nAChR complexes expressed by embryonic chick sympathetic neurones.

α5 contributes not only to agonist but also to antagonist sensitivity of natively expressed nAChR channels. Functional deletion of the α5 subunit by antisense oligonucleotide treatment removes the nAChRs with relatively low affinity to ACh and cytisine. Deletion of α5 also eliminates channels that are blocked by the α7‐specific antagonist methyllycaconitine (MLA) while increasing the percentage of current carried by nAChRs that are sensitive to α‐bungarotoxin (α‐BgTx).

Single channel analyses indicate that functional deletion of α5 results in the deletion of both the ‘brief’ and ‘long’ open duration, 50 pS subtypes of nAChR channels while increasing the expression of the 18 pS, α‐BgTx‐sensitive native nAChRs normally detected in sympathetic neurones at later developmental stages.

The biophysical and pharmacological profiles of native nAChRs revealed by this study and previous work are discussed in the context of a proposed model of the nAChR channels expressed by chick sympathetic neurones throughout development.
Intercellular Ca2+ waves in rat heart muscle
Authors: Lamont Christine , Luther Paul W. , Balke C. William , Wier W. Gil
Published: September 2004

Confocal laser scanning microscopy was used to visualize intercellular transmission of Ca2+ waves in intact rat ventricular trabeculae micro‐injected with the calcium indicator fluo‐3.

Ca2+ waves usually failed to be transmitted from cell to cell. At identified individual end‐to‐end cell contacts, successful transmission interspersed with failure, which sometimes occurred despite an apparent small spritz of Ca2+ between cells. The probability of cell to cell transmission (Ptran) was 0.13.

Ca2+ waves arose preferentially near junctions of connected cells, where connexin‐43 was found, but randomly in enzymatically disconnected heart cells.

β‐Adrenergic stimulation significantly increased Ptran (to 0.22) and heptanol, an uncoupler of gap junction channels, significantly decreased it (to 0.045).

In regions of high [Ca2+]i due to damage, wave frequency decreased markedly with each cell‐cell junction passed.

The Ca2+ permeability of cardiac gap junctions may be regulated, and the low ability of cardiac gap junctions to transmit Ca2+ may help control the spread of Ca2+ from damaged regions.
Cloning of a mammalian elk potassium channel gene and EAG mRNA distribution in rat sympathetic ganglia
Authors: Shi Wenmei , Wang Hong‐Sheng , Pan Zongming , Wymore Randy S. , Cohen Ira S. , McKinnon David , Dixon Jane E.
Published: September 2004

Three new members of the EAG potassium channel gene family were identified in rat and the complete coding sequence of one of these genes (elk1) was determined by cDNA cloning.

The elk1 gene, when expressed in Xenopus oocytes, encodes a slowly activating and slowly deactivating potassium channel.

The elk1 gene is expressed in sympathetic ganglia and is also expressed in sciatic nerve.

Six of the seven known EAG genes were found to be expressed in rat sympathetic ganglia, suggesting an important functional role for these channels in the sympathetic nervous system.
Oxygen‐dependent K+ fluxes in sheep red cells
Authors: Campbell E. H. , Gibson J. S.
Published: September 2004

This study was designed to investigate the O2 dependence of K+ influx in sheep red cells. Influx was determined using 86Rb+ as a tracer for K+; glass tonometers coupled to a gas mixing pump were used to equilibrate cell samples to the requisite oxygen tension (PO2).

Both volume‐ and H+‐stimulated K+ influxes in low potassium‐containing (LK) sheep red cells were approximately doubled on equilibration with O2 relative to influxes measured in N2. O2‐dependent influxes were abolished when Cl− was replaced with NO3−, consistent with mediation by the KCl cotransporter. At pH 7, PO2 required for half‐maximal stimulation was 56 ± 1 mmHg (mean ± s.e.m., 3 sheep) for the O2‐dependent component of K+ influx: thus PO2 values over the physiological range affected K+ influx.

K+ influx in fully deoxygenated sheep red cells showed substantial volume and H+ sensitivity. These residual components in N2 were also Cl− dependent, indicating that the KCl cotransporter of LK sheep red cells was active in the absence of O2.

Volume‐sensitive K+ influxes in high potassium‐containing (HK) sheep red cells responded in a similar way to those in cells from LK sheep, although much smaller in magnitude, showing that intracellular [K+] had no significant effect on the O2 dependence of the cotransporter.

Intracellular [Mg2+] ([Mg2+]i) was altered by incubating sheep red cells with A23187 (20 μM) and different values of extracellular [Mg2+] ([Mg2+]o). Total [Mg2+]i was determined by atomic absorption spectroscopy and free [Mg2+]i from [Mg2+]o and the Donnan ratio. Total [Mg2+]i was 1.29 ± 0.08 mM (mean ± s.e.m., n= 5), similar to that reported in the literature. Estimates of free [Mg2+]i showed an increase from 0.39 ± 0.05 in oxygenated cells to 0.52 ± 0.04 mM (mean ± s.e.m., n= 5; P < 0.05) in deoxygenated ones.

Finally, although K+ influxes were altered by pharmacological loading or depletion of cells with Mg2+, the free [Mg2+]i required to affect influxes significantly was outside the physiological range. Results are difficult to reconcile with PO2 modulating KCl cotransport activity directly via changes in free [Mg2+]i or [Mg2+‐ATP]i.
Non‐additive interaction between nicotinic cholinergic and P2X purine receptors in guinea‐pig enteric neurons in culture
Authors: Zhou Xiaoping , Galligan James J.
Published: September 2004

Acetylcholine (ACh)‐activated currents and their interaction with ATP‐activated currents were studied in primary cultures of myenteric neurons from guinea‐pig small intestine using patch clamp techniques. Peak currents caused by co‐application of ACh (1 mm) and ATP (300 μm) were 78 ± 2 % of the sum of currents activated by each agonist alone (P < 0.05, n= 29). Reversal potentials measured during co‐application of ACh and ATP did not differ from those measured during application of ACh or ATP alone. Addition of BAPTA (10 mm) to the pipette solution or replacement of extracellular Ca2+ with Na+ did not prevent occlusion.

Responses caused by co‐application of 5‐HT (300 μm), acting at 5‐HT3 receptors, and ACh (3 mm) or ATP (1 mm) were additive (94 ± 3 or 96 ± 4 %, respectively, of the sum of currents activated by 5‐HT and ACh or ATP alone; P > 0.05). Currents caused by GABA (1 mm), acting at GABAA receptors, and ACh (3 mm) or ATP (1 mm) were also additive (105 ± 4 or 100 ± 3 %, respectively, of the sum of currents activated by GABA and ACh or GABA and ATP applied separately; P > 0.05).

Single channel currents caused by ACh and ATP in the same outside‐out patches were less than additive (85 ± 10 % of the predicted sum, P < 0.05).

P2X receptors and nicotinic cholinergic receptors (nAChRs) are linked in a mutually inhibitory manner in guinea‐pig myenteric neurons. The functional interaction does not involve ligand binding sites, Ca2+‐dependent mechanisms, a change in the driving force for Na+ or cytoplasmic signalling mechanisms.
Mechanistic link between lidocaine block and inactivation probed by outer pore mutations in the rat μ1 skeletal muscle sodium channel
Authors: Kambouris Nicholas G. , Hastings Laura A. , Stepanovic Svetlana , Marban Eduardo , Tomaselli Gordon F. , Balser Jeffrey R.
Published: September 2004

Mutations that disrupt Na+ channel fast inactivation attenuate lidocaine (lignocaine)‐induced use dependence; however, the pharmacological role of slower inactivation processes remains unclear. In Xenopus oocytes, tryptophan substitution in the outer pore of the rat skeletal muscle channel (μ1‐W402) alters partitioning among fast‐ and slow‐inactivated states. We therefore examined the effects of W402 mutations on lidocaine block.

Recovery from inactivation exhibited three kinetic components (IF, fast; IM, intermediate; IS, slow). The effects of W402A and W402S on IF and IS differed, but both mutants (with or without β1 subunit coexpression) decreased the amplitude of IM. In wild‐type channels, lidocaine imposed a delayed recovery component with intermediate kinetics, and use‐dependent block was attenuated in both W402A and W402S.

To examine the pharmacological role of IS relative to IM, drug‐exposed β1‐coexpressed channels were subjected to 2 min depolarizations. Lidocaine had no effect on sodium current (INa) after a 1 s hyperpolarization interval that allowed recovery from IM but not IS, suggesting that lidocaine affinity for IS is low.

Both W402 mutations reduced occupancy of IM in drug‐free conditions, and also induced resistance to use‐dependent block. We propose that lidocaine‐induced use dependence may involve an allosteric conformational change in the outer pore.
Effect of depletion of interstitial hyaluronan on hydraulic conductance in rabbit knee synovium
Authors: Coleman P. J. , Scott D. , Abiona A. , Ashhurst D. E. , Mason R. M. , Levick J. R.
Published: September 2004

The hydraulic resistance of the synovial lining to fluid outflow from a joint cavity (Q̇s) is important for the retention of intra‐articular lubricant. The resistance has been attributed in part to extracellular glycosaminoglycans, including hyaluronan and chondroitin sulphates. Increased permeability in joints infused with testicular hyaluronidase, which digests both chondroitin sulphates and hyaluronan, supports this view. In this study the importance of interstitial hyaluronanper sewas assessed using leech andStreptomyceshyaluronidases, which degrade only hyaluronan.

Ringer solution was infused into the knee joint cavity of anaesthetized rabbits for 30 min, with or without hyaluronidase, after which intra‐articular pressure (Pj) was raised and the relation between pressure and outflow determined.

Treatment withStreptomyces, leech or testicular hyaluronidases increased the fluid escape rates by similar factors, namely 4‐ to 6‐fold. AfterStreptomyceshyaluronidase treatment the slope dQ̇s/dPj, which at low pressures represents synovial hydraulic conductance, increased from a control of 0.90 ± 0.20 μl min−1cmH2O−1(mean ± s.e.m.,n= 6) to 4.52 ± 0.70 μl min−1cmH2O−1. The slope dQ̇s/dPjincreased to a similar level after testicular hyaluronidase, namely to 4.14 ± 1.06 μl min−1cmH2O−1(control, 0.54 ± 0.24 μl min−1cmH2O−1).Streptomycesand leech hyaluronidases were as effective as testicular hyaluronidase (no statistically significant differences) despite differences in substrate specificity.

It was shown using histochemical and immunohistochemical techniques that hyaluronan was removed from the synovium by leech,Streptomycesand testicular hyaluronidases. The binding of antibodies 2‐B‐6 and 3‐B‐3 showed that the core proteins of the chondroitin sulphate proteoglycans remained intact after treatment with hyaluronidases, and the binding of 5‐D‐4 showed that keratan sulphate was unaffected. An azocasein digestion assay confirmed that the hyaluronidase preparations had no significant proteolytic activity.

The effect of the hyaluronidases was four times greater than predicted from the low concentration of interstitial hyaluronan and its resistivity. Factors that might amplify the effect of hyaluronan depletion include the matrix‐organizing role of hyaluronan, and/or non‐uniformity of hyaluronan distribution. It is concluded that interstitial hyaluronan makes a major contribution to synovial hydraulic resistance, but the mechanisms are as yet poorly understood.
Activation of multiple intracellular transduction signals by vasopressin in vasopressin‐sensitive neurones of the rat supraoptic nucleus
Authors: Sabatier Nancy , Richard Philippe , Dayanithi Govindan
Published: September 2004

The intracellular mechanisms activated by the binding of vasopressin to its receptor(s) and which result in the increase of [Ca2+]i were investigated in freshly dissociated supraoptic nucleus neurones. Various pharmacological agents were used to investigate the possible involvement of phospholipase C (PLC) and adenylate cyclase (AC) intracellular pathways in the transduction of the vasopressin action.

Both the PLC inhibitor U‐73122 and the protein kinase C (PKC) inhibitor calphostin C, reduced the [Ca2+]i rise elicited by vasopressin. The cAMP analogue, 8‐Br‐cAMP produced an increase in [Ca2+]i and IBMX, a phosphodiesterase inhibitor, potentiated the response to vasopressin.

After pre‐incubation with the AC inhibitor SQ‐22536, 7 out of 18 vasopressin‐sensitive neurones showed no inhibition of the vasopressin response, while the response to vasopressin was reduced by greater than 35 % in each of the other 11 neurones.

The activation of protein kinase A (PKA) with Sp‐cAMPS caused an increase in [Ca2+]i which was additive to the vasopressin‐elicited [Ca2+]i increase. After incubation with the PKA inhibitors Rp‐cAMPS or H‐89, the [Ca2+]i responses triggered by Sp‐cAMPS and vasopressin were, respectively, abolished and greatly reduced.

A combined administration of SQ‐22536 (AC inhibitor) followed by U‐73122 (PLC inhibitor), or U‐73122 followed by H‐89 (PKA inhibitor), virtually abolished the response to vasopressin.

In vasopressin‐responsive neurones, the pituitary adenylate cyclase‐activating polypeptide (PACAP) induced a [Ca2+]i increase similar to the response to vasopressin and in both cases the increase was inhibited to the same extent by a combination of U‐73122 and Rp‐cAMPS.

In conclusion, we suggest that the autoregulation exerted specifically by vasopressin on vasopressin‐sensitive neurones involves the activation of both PLC‐ and AC‐linked pathways.
The influence of perchlorate ions on complex charging transients in amphibian striated muscle
Authors: Huang Christopher L.‐H.
Published: September 2004

The effects of perchlorate ions on intramembrane charge movements were examined under different conditions of ryanodine receptor (RyR) modification in intact voltage‐clamped amphibian skeletal muscle fibres studied in the gluconate‐containing solutions previously reported to emphasize the features of qγ at the expense of those of the qβ charge.

The introduction of graded increases in perchlorate concentration to the experimental solutions selectively shifted the threshold of appearance of the qγ‘hump’ currents to more negative test potentials at which they actually appeared in the absence of prior qβ transients at perchlorate concentrations of 4.0‐8.0 mM. Such findings suggested that the delayed (qγ) transitions can take place independently of any previous exponential (qβ) decay.

These kinetic effects were accompanied by hyperpolarizing shifts in the transition potentials (V*) of the steady‐state voltage dependences of either the overall or the isolated qγ charge. These shifts were graded with concentration and reached their maximum effects at 4.0‐8.0 mM perchlorate. However, both the total charge (Qmax) and the steepness factor (k) remained conserved at values consistent with a system that included significant contributions from the steeply voltage‐sensitive qγ component (overall charge: Qmax≈ 19‐21 nC μF−1, k≈ 7‐9 mV; qγ component alone: Qmax≈ 10‐12 nC μF−1, k≈ 4‐6 mV). This contrasts with earlier reports on the effects of perchlorate in fibres that were studied in sulphate‐ or methanesulphonate‐containing extracellular solutions.

Perchlorate (8.0 mM) restored the ‘hump’ waveform associated with qγ charge movements that had previously been obliterated by the prior application of fully effective (0.1 mM) concentrations of either ryanodine or daunorubicin.

Perchlorate similarly reversed the positive shift in the transition potential of the qγ component that was brought about by such RyR modification (from V*≈ ‐40 mV back to V*≈ ‐60 mV). In contrast, the values of either Qmax (overall charge, 19‐21 nC μF−1; qγ component, 10‐13 nC μF−1) or k (overall charge, 7‐9 mV; qγ component, 4‐6 mV) remained conserved through all these experimental manoeuvres.

The inclusion of perchlorate also reversed the action of 2 mM tetracaine and restored delayed qγ transients to an extent that was graded with concentration (0.5‐8.0 mM perchlorate). There was an accompanying recovery of the steeply voltage‐dependent steady‐state (qγ) component consistent with a competitive interaction between these agents upon the qγ intramembrane charge.

The present findings suggest that perchlorate exerts a specific action upon the qγ charge in independent transitions that are driven by the tubular membrane field. Its interactions with the known RyR inhibitors that nevertheless conserve both the charge and its voltage sensitivity suggest a primary action upon the RyR that in turn exerts reciprocal actions upon the voltage sensor.
The effects of flow on the transport of potassium ions through the walls of single perfused frog mesenteric capillaries
Authors: Kajimura M. , Head S. D. , Michel C. C.
Published: September 2004

We have investigated the effects of varying flow velocity upon permeability to potassium ions (PK) of single perfused mesenteric microvessels in pithed frogs.

P
K was estimated using a development of the single bolus microperfusion technique at chosen flow velocities (U) in the range of 150–7000 μm s−1.

In thirty‐seven out of forty‐three vessels, there was a strong positive correlation between PK and U. Average values (median) for PK (μm s−1) were related to U (μm s−1) by the expression: PK= 0.0043U+ 4.05 (n= 43).

The correlation between PK and U was independent of microvascular pressure (and hence fluid filtration) over the range of 5–70 cmH2O.

The correlation between PK and U was independent of the potassium concentration in the bolus over the range of 2–40 mmol l−1 and of the direction of the potassium flux through the capillary walls.
GABAA receptor‐mediated currents in interneurons and pyramidal cells of rat visual cortex
Authors: Xiang Zixiu , Huguenard John R. , Prince David A.
Published: September 2004

We compared γ‐aminobutyric acid (GABA)‐mediated responses of identified pyramidal cells and fast spiking interneurons in layer V of visual cortical slices from young rats (P11‐14).

The frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) was similar in pyramidal cells and interneurons (1.7 vs. 1.9 Hz). For events with 10‐90 % rise times less than 0.9 ms, no significant differences were found in mean amplitude (61 vs. 65 pA), mean rise time (0.58 vs. 0.61 ms), or the first time constant of decay (τ1, 6.4 vs. 6.5 ms) between pyramidal cells and interneurons. The second decay time constant (τ2) was significantly longer in interneurons than in pyramidal cells (49 vs. 22 ms). The difference in sIPSC decay kinetics between two cell types also existed in adult rats (P36‐42), suggesting the kinetic difference is not due to differential development of GABAA receptors in these cell types.

The decay kinetics of monosynaptic evoked IPSCs were also longer in interneurons. As in the case of sIPSCs, the difference was accounted for by the second decay time constant. τ1 and τ2 were, respectively, 13 and 64 ms for interneurons and 12 and 47 ms for pyramidal cells.

Cell‐attached patch recordings revealed that the mean open time for single Cl− channels in response to 2 μM GABA was significantly longer in interneurons than pyramidal cells (5.0 vs. 2.8 ms). The chord conductance of these channels in interneurons (12 pS) was significantly smaller than in pyramidal cells (15 pS). Single channel currents reversed polarity when the pipette potential was approximately ‐10 mV for both cell types.

These results show that there is a functional diversity of GABAA receptors in electrophysiologically and morphologically identified cortical pyramidal cells and interneurons. This diversity might derive from the different molecular composition of the receptors in these two cell types.
Sensitivity to 5‐hydroxytryptamine in different afferent subpopulations within mesenteric nerves supplying the rat jejunum
Authors: Hillsley K. , Grundy D.
Published: September 2004

This study was performed to elucidate the type of afferents that mediate the multiple actions of 5‐hydroxytryptamine (5‐HT) on mesenteric nerve discharge. Electrophysiological recordings were made from mesenteric afferents innervating the mid‐jejunum of the urethane‐anaesthetized rat. The discharge of single nerves within the whole nerve recording was monitored using waveform discrimination software.

Afferents responded to 5‐HT in one of two ways: a short latency, transient excitation mediated by 5‐HT3 receptors, or a delayed onset, more prolonged effect that was 5‐HT2A receptor mediated. Afferents showing the 5‐HT3‐mediated response did not respond to luminal distension but were sensitive to intraluminal hydrochloric acid (150 mM) in twenty‐eight of twenty‐nine experiments. In eight experiments, the 5‐HT3‐mediated response was reversibly abolished by a 2 min exposure to intraluminal application of local anaesthetic (2 % Xylocaine).

Mechanosensitive afferents which responded to distension (< 10 cmH2O) did not show a 5‐HT3‐mediated response (P= 0.92, n= 14), and maintained this mechanosensitivity after luminal anaesthesia. Mechanosensitive afferents did show a secondary response to 5‐HT that was significantly attenuated by atropine (100‐200 μg kg−1), whereas hexamethonium (8 mg kg−1) had no effect.

In animals whose vagal afferent contribution to their mesenteric nerves had been eliminated by chronic truncal vagotomy, the 5‐HT3‐mediated response was absent in thirty‐six of thirty‐six nerve bundles. In contrast, mechanosensitivity to distension and the secondary response to 5‐HT could still be evoked.

These results suggest that 5‐HT stimulates mesenteric afferents by a direct action on 5‐HT3 receptors that are present on vagal mucosal afferent terminals. The mucosal afferent response to luminal acid, however, was unaffected by treatment with granisetron (0.5 mg kg−1) indicating that endogenous 5‐HT from enterochromaffin cells is not essential for transduction of this luminal signal. In contrast, mechanosensitivity in non‐vagal afferents was modulated by 5‐HT following an intestinal motor response which was influenced by cholinergic tone.
Cyclic GMP‐dependent but G‐kinase‐independent inhibition of Ca2+‐dependent Cl− currents by NO donors in cat tracheal smooth muscle
Authors: Waniishi Yoshiki , Inoue Ryuji , Morita Hiromitsu , Teramoto Noriyoshi , Abe Kihachiro , Ito Yushi
Published: September 2004

The effects of NO donors on Ca2+‐dependent Cl− currents (ICl(Ca)) were investigated in freshly isolated cat tracheal myocytes using the whole‐cell patch clamp technique.

With nystatin‐perforated whole‐cell recording, carbachol (CCh, ≥ 1 μm) induced a transient inward current (ICCh) with a reversal potential of about ‐20 mV. Activation of ICCh probably occurred through the M3 muscarinic receptor, since nanomolar concentrations of 4‐diphenylacetoxy‐N‐methylpiperidine methobromide (4‐DAMP) greatly inhibited this current, while 11‐(2‐(diethylamino)methyl)‐1‐piperidinylacetyl)‐5,11‐dihydro‐6H‐pyrido (2,3β) (1,4)benzodiazepine‐6‐one (AF‐DX 116) or pirenzepine at concentrations of up to 1 μm were almost ineffective.

Chloride channel/transporter blockers such as DIDS (100 μm), anthracene‐9‐carboxylic acid (9‐AC, 100 μm) and niflumic acid (100 μm) greatly inhibited ICCh, but cation channel blockers, such as nifedipine (10 μm), Zn2+ (500 μm) or Gd3+ (500 μm), were without effect.

Activation of ICCh was strongly attenuated by pretreatment with ryanodine (4 μm) plus caffeine (10 mM). Addition of neomycin (1 mM) into the bath or inclusion of heparin (3 mg ml−1) in the pipette abolished a substantial part of ICCh. These results suggest that ICCh is ICl(Ca), which is activated by inositol 1,4,5‐trisphosphate (IP3)‐mediated Ca2+ release.

The nitric oxide donor S‐nitroso‐N‐acetyl penicillamine (SNAP) reduced the amplitude of ICCh dose dependently (IC50, ≈10 μm). Similar inhibition was also exerted by other types of NO donor such as glyceryl trinitrate (GTN) and (±)‐E‐methyl‐2‐(E‐hydroxyimitol)‐5‐nitro‐6‐methoxy‐3‐hexeneamide (NO‐R).

SNAP‐induced ICCh inhibition was effectively antagonized by Methylene Blue (1‐100 nM), and mimicked by dibutyryl cGMP (db‐cGMP) (0.5‐1 mM), whereas two structurally distinct types of cGMP‐dependent (G)‐kinase inhibitor, N‐(2‐aminoethyl)‐5‐isoquinilinesulphonamide (H‐8, 2.5 μm) and KT5823 (1 μm), failed to counteract the inhibitory effects of SNAP or db‐cGMP. Another G‐kinase‐specific inhibitor Rp‐8‐(para‐chlorophenylthio)guanosine‐3′,5′‐cyclic monophosphorothioate (Rp‐8‐pCPT‐cGMPS; 1 μm) itself caused a marked reduction in ICCh.

SNAP (100 μm) or db‐cGMP (100 μm) exhibited no inhibitory actions, when caffeine (10 mM) or photolytically released IP3 were used instead of CCh to activate the inward current.

These results suggest that inhibition of ICCh by NO donors involves a cGMP‐dependent but G‐kinase‐independent mechanism, which may operate at a site(s) between the muscarinic (M3) and IP3 receptors.
Ca2+‐dependent inactivation of large conductance Ca2+‐activated K+ (BK) channels in rat hippocampal neurones produced by pore block from an associated particle
Authors: Hicks Gareth A. , Marrion Neil V.
Published: September 2004

Recordings of the activity of the large conductance Ca2+‐activated K+ (BK) channel from over 90 % of inside‐out patches excised from acutely dissociated hippocampal CA1 neurones revealed an inactivation process dependent upon the presence of at least 1 μM intracellular Ca2+. Inactivation was characterized by a sudden switch from sustained high open probability (Po) long open time behaviour to extremely low Po, short open time channel activity. The low Po state (mean Po, 0.001) consisted of very short openings (time constant (τ), ≈0.14 ms) and rare longer duration openings (τ, ≈3.0 ms).

Channel inactivation occurred with a highly variable time course being observed either prior to or immediately upon patch excision, or after up to 2 min of inside‐out recording. Inactivation persisted whilst recording conditions were constant.

Inactivation was reversed by membrane hyperpolarization, the rate of recovery increasing with further hyperpolarization and higher extracellular K+. Inactivation was also reversed when the intracellular Ca2+ concentration was lowered to 100 nM and was permanently removed by application of trypsin to the inner patch surface. In addition, inactivation was perturbed by application of either tetraethylammonium ions or the Shaker (Sh)B peptide to the inner membrane face.

During inactivation, channel Po was greater at hyperpolarized rather than depolarized potentials, which was partly the result of a greater number of longer duration openings. Depolarizing voltage steps (‐40 to +40 mV) applied during longer duration openings produced only short duration events at the depolarized potential, yielding a transient ensemble average current with a rapid decay (τ, ≈3.8 ms).

These data suggest that hippocampal BK channels exhibit a Ca2+‐dependent inactivation that is proposed to result from block of the channel by an associated particle. The findings that inactivation was removed by trypsin and prolonged by decreasing extracellular potassium suggest that the blocking particle may act at the intracellular side of the channel.
Role of 5‐HT3 receptors in activation of abdominal sympathetic C fibre afferents during ischaemia in cats
Authors: Fu Liang‐Wu , Longhurst John C.
Published: September 2004

Activation of abdominal sympathetic afferents during ischaemia reflexly excites the cardiovascular system. We have shown previously that exogenous 5‐hydroxytryptamine (5‐HT, i.e. serotonin) stimulates abdominal sympathetic afferent nerve endings, and recently have documented increased concentrations of 5‐HT in intestinal lymph and portal venous plasma during brief abdominal ischaemia. The present investigation evaluated the role of endogenously produced 5‐HT in activation of ischaemically sensitive abdominal sympathetic afferents.

Nerve activity of single‐unit C fibre afferents innervating duodenum, mesentery, pancreas, portal hepatis, bile duct, gall bladder and jejunum was recorded from the right thoracic sympathetic chain of anaesthetized cats. Ischaemically sensitive C fibre afferents were identified according to their response to 5‐10 min of abdominal ischaemia.

Intra‐arterial injection of 5‐HT (20 μg kg−1) increased discharge activity of twelve afferents from 0.23 ± 0.05 to 0.96 ± 0.09 impulses s−1 after an onset latency of 5.7 ± 1.4 s. Also, 2‐methylserotonin (100 μg kg−1, i.a.), a 5‐HT3 receptor agonist, stimulated eleven of twelve afferents to significantly increase their discharge activity from 0.25 ± 0.05 to 0.90 ± 0.10 impulses s−1 after a latency of 3.3 ± 0.4 s. Furthermore, intravenous injection of tropisetron (200 μg kg−1), a 5‐HT3 receptor antagonist, significantly attenuated the increase in activity of twelve other C fibre afferents during 10 min of abdominal ischaemia from 1.62 ± 0.18 to 0.94 ± 0.22 impulses s−1, and eliminated the response of eleven other afferents to 5‐HT.

Both the 5‐HT2 receptor agonist, α‐methylserotonin (100 μg kg−1, i.a.), and the 5‐HT1 receptor agonist, 5‐carboxamidotryptamine (100 μg kg−1, i.a.), did not alter the impulse activity of these twelve afferents (0.29 ± 0.05 to 0.31 ± 0.06, and 0.26 ± 0.06 to 0.29 ± 0.06 impulses s−1, respectively).

Treatment with indomethacin (5 mg kg−1, i.v.) in eight different cats did not alter the response of nine C fibre afferents to exogenous 5‐HT (0.91 ± 0.17 vs. 1.19 ± 0.25 impulses s−1, P > 0.05).

The results suggest that, during mesenteric ischaemia, endogenous 5‐HT contributes to the activation of abdominal sympathetic afferents, mainly through direct stimulation of 5‐HT3 receptors and that the action of 5‐HT on these afferents appears to be independent of the cyclo‐oxygenase pathway.
Mode switching characterizes the activity of large conductance potassium channels recorded from rat cortical fused nerve terminals
Authors: Smith M. A. , Ashford M. L. J.
Published: September 2004

Inside‐out recordings from rat cortical fused nerve terminals indicate that the most common channel observed was a large conductance K+ (BK) channel with characteristics dissimilar to conventional cell body calcium‐activated BK (BKCa) channels.

BK channels exhibit mode switching between low (mode 1) and high (mode 2) activity, an effect not influenced by membrane voltage. Increasing internal Ca2+ concentration increased time spent in mode 2 as did application of protein kinase A, an effect not mimicked by protein kinase C or protein kinase G.

Mode 1 activity was voltage independent although depolarization increased mode 2 channel activity. Global average channel activity was voltage and Ca2+ dependent.

Alkaline phosphatase treatment induced channel activity to reside permanently in mode 2, where activity was voltage and Ca2+ dependent but unaffected by protein kinases A, G or C.

Internal application of tetraethylammonium blocked BK channel activity in a manner identical to that reported for BKCa channels.

These results indicate that nerve terminal membranes have large conductance K+ channels with significant differences in gating kinetics and regulation of activity compared with BKCa channels of other neuronal preparations. The BK channel subtype may play a unique physiological role specific to the nerve terminal.
Characteristics of nitric oxide‐mediated cholinergic modulation of calcium current in rabbit sino‐atrial node
Authors: Han X. , Kobzik L. , Severson D. , Shimoni Y.
Published: September 2004

We have previously shown that nitric oxide (NO) production is essential for cholinergic inhibition of the β‐adrenergic stimulated L‐type calcium current (ICa‐L) in rabbit pacemaker (sino‐atrial node (SAN)) cells. The present experiments demonstrate the presence of constitutive nitric oxide synthase (cNOS) in SAN cells, and characterize the NO‐mediated cholinergic response.

Immunohistochemical staining, using an antibody prepared against endothelial cNOS, demonstrated that this enzyme was present in single myocytes obtained from the SAN.

The activation of cNOS is known to be Ca2+ and calmodulin dependent. Strongly buffering intracellular Ca2+ with the membrane‐permeable chelator BAPTA‐AM (10 μM) significantly reduced (and in some cases abolished) the attenuation of ICa‐L by the muscarinic agonist carbamylcholine (CCh). In contrast, the CCh‐induced activation of an outward K+ current, IK,ACh, was unaffected by buffering of [Ca2+]i. The calmodulin inhibitor 48/80 (20 μM) also abolished the attenuation of ICa‐L by CCh, with no change in the activation of IK,ACh.

Neither thapsigargin nor ryanodine (5‐10 μM), agents which deplete intracellular Ca2+ stores, significantly changed the attenuation of ICa‐L by CCh.

Pertussis toxin (PTX) completely abolished both the inhibitory action of CCh on ICa‐L and the activation of IK,ACh. This establishes that a PTX‐sensitive GTP‐binding protein links the muscarinic receptor to NO synthase activation in SAN cells.

Our hypothesis is that NO leads to activation of a cyclic GMP (cGMP)‐activated phosphodiesterase (PDE II) as a mechanism for enhanced cyclic AMP breakdown and ICa‐L attenuation. This was supported by showing that a specific inhibitor of PDE II, erythro‐9‐(2‐hydroxy‐3‐nonyl) adenine (EHNA), blocks the effect of CCh on ICa‐L, but not on IK,ACh. Using reverse transcriptase‐polymerase chain reaction techniques, we have established that PDE II is the dominant cyclic nucleotide phosphodiesterase isoform in SAN cells.
Inhibition of Ca2+‐dependent K+ channels in rat carotid body type I cells by protein kinase C
Authors: Peers C. , Carpenter E.
Published: September 2004

Whole‐cell patch clamp recordings were used to investigate the effects of protein kinase C (PKC) activation on K+ and Ca2+ currents in type I cells isolated from the rat carotid body.

Pretreatment of cells for 10 min at 37 °C with 4α‐phorbol 12,13‐didecanoate (4α‐PDD, 200 nm), a phorbol ester which does not activate PKC, did not affect K+ current density as compared with cells pretreated with vehicle alone. By contrast, identical pretreatment with 200 nm 12‐O‐teradecanoylphorbol‐13‐acetate (TPA, a PKC activator) dramatically reduced K+ current density in type I cells. This effect was prevented by co‐incubation of cells with the PKC inhibitor bisindolylmaleimide (BIM, 3 μm).

The sensitivity of K+ currents to inhibition by 200 μm Cd2+ (indicative of the presence of Ca2+‐dependent K+ channels) was markedly reduced in TPA‐treated cells as compared with sham‐treated cells, cells treated with 4α‐PDD, and cells treated with both TPA and BIM. Cd2+‐resistant K+ current densities were of similar magnitude in all four groups of cells, as were the input resistances determined over the voltage range −100 mV to −50 mV.

Ca2+ channel current density was not significantly different in type I cells pretreated with 200 nm 4α‐PDD as compared with cells treated with the same concentration of TPA.

The degree of inhibition of K+ currents caused by hypoxia (Po2 15–20 mmHg) was unaltered by pretreatment of cells with 3 μm BIM.

The resting membrane potential of cells pretreated with TPA was depolarized as compared with controls, and the Ca2+‐dependent K+ channel inhibitor iberiotoxin (20 nm) failed to depolarize these cells further.

Our results suggest that activation of PKC causes a marked, selective inhibition of Ca2+‐dependent K+ currents in type I carotid body cells, but that PKC activation is unlikely to account for inhibition of these channels by acute hypoxia.
Na+‐activated K+ channels in small dorsal root ganglion neurones of rat
Authors: Bischoff Ulrike , Vogel Werner , Safronov Boris V.
Published: September 2004

Whole‐cell Na+‐activated K+ (KNa) channel currents and single KNa channels were studied with the patch‐clamp method in small (20‐25 μm) dorsal root ganglion (DRG) neurones in slices of rat dorsal root ganglia.

The whole‐cell KNa channel current was identified as an additional K+‐selective leakage current which appeared after cell perfusion with internal solutions containing different [Na+]. The concentration for half‐maximal activation of KNa channel current was 39 mM and the Hill coefficient was 3.5. At [Na+]i above 12 mM, KNa channel current dominated the unspecific leakage current. The ratio of maximum KNa channel current to unspecific leakage current was 45.

KNa channel current was not activated by internal Li+. It was suppressed by external 20 mM Cs+ but not by 10 mM tetraethylammonium.

Single KNa channels with a conductance of 142 pS in 155 mM external K+ (K+o)‐85 mM internal K+ (K+i) solutions were observed at a high density of about 2 channels μm−2.

In two‐electrode experiments, a direct correlation was seen between development of whole‐cell KNa channel current and activation of single KNa channels during perfusion of the neurone with Na+‐containing internal solution.

Under current‐clamp conditions, KNa channels did not contribute to the action potential. However, internal perfusion of the neurone with Na+ shifted the resting potential towards the equilibrium potential for K+ (EK). Varying external [K+] indicated that in neurones perfused with Na+‐containing internal solution the resting potential followed the EK values predicted by the Nernst equation over a broader voltage range than in neurones perfused with Na+‐free solution.

It is concluded that the function of KNa channels has no links to firing behaviour but that the channels could be involved in setting or stabilizing the resting potential in small DRG neurones.
A characterization of muscarinic receptor‐mediated intracellular Ca2+ mobilization in cultured rat hippocampal neurones
Authors: Irving Andrew J. , Collingridge Graham L.
Published: September 2004

The properties of muscarinic receptor‐mediated Ca2+ mobilization were investigated in hippocampal cultures using fluorescent imaging techniques.

Somatic responses to carbachol (1‐10 μm) were observed in 21 % of neurones under control conditions (5.4 mM K+, 1.8 mM Ca2+, 0.5‐1 μm tetrodotoxin). Smaller responses were observed in Ca2+‐free medium.

In cells where responses to carbachol were absent under control conditions, responses were often observed following depolarization with high extracellular K+ (16.2‐25 mM). These responses decreased in magnitude with time after the depolarizing episode. Mobilization of Ca2+ from stores using caffeine (50 mM) exhibited similar properties.

Carbachol responses were greatly facilitated in the presence of moderate elevations in extracellular K+ or Ca2+ levels (2‐ or 3‐fold, respectively). These conditions were usually, but not always, associated with a small increase in cytosolic Ca2+ levels (< 50 nM).

Muscarinic responses in 10.8 mM K+ were inhibited by 80–95 % in the presence of the L‐type voltage‐gated Ca2+ channel antagonists nitrendipine (2‐5 μm) or nifedipine (10 μm). Depletion of intracellular Ca2+ stores with thapsigargin (2‐10 μm) blocked responses.

Oscillatory Ca2+ mobilizing responses were observed in some cells. Their expression was facilitated by moderate cytosolic Ca2+ elevations and by increasing the duration of carbachol exposure.

Ca2+ mobilizing responses were also observed in dendritic regions. These were smaller than somatic responses, but had faster decay kinetics.

In conclusion, muscarinic receptor‐mediated Ca2+ mobilization in cultured hippocampal neurones shows a strong Ca2+ dependence. Moderate intracellular Ca2+ rises greatly facilitate muscarinic responses and uncover, in some cells, oscillatory Ca2+ mobilization. These effects appear to reflect the loading state of intracellular Ca2+ stores.
Dorsal medullary 5‐HT3 receptors and sympathetic premotor neurones in the rat
Authors: Sévoz‐Couche Caroline , Nosjean Anne , Franc Bernard , Hamon Michel , Laguzzi Raul
Published: September 2004

Our aim was to determine whether the cardiovascular neurones in the rostro‐ventrolateral medulla (CV‐RVLM neurones) were involved in the sympathoexcitation induced by stimulation of 5‐HT3 receptors in the region of the nucleus tractus solitarii (NTS). Experiments were performed in pentobarbitone‐anaesthetized rats, artificially ventilated and paralysed with pancuronium bromide.

Using extracellular recordings, different types of RVLM neurones were characterized: cardiovascular (CV), ventilation‐related and baroreflex‐insensitive (unidentified) neurones. The CV‐RVLM cells were further subdivided into three populations according to their axonal conduction velocities: A (1.2 ± 0.1 m s−1), B (2.5 ± 0.2 m s−1) and C (6.8 ± 1.1 m s−1).

Only the CV‐RVLM neurones of the A and B categories were partially inhibited (‐30 %) by a hypotensive dose (2.5 μg kg−1 i.v.) of clonidine.

Microinjections into the region of the commissural NTS of 1‐(m‐chlorophenyl)‐biguanide (CPBG, 2 nmol), a selective 5‐HT3 receptor agonist, elicited an increase in both lumbar sympathetic nerve discharge (SND) and arterial pressure. In addition, this treatment produced a marked excitation of CV‐RVLM neurones of the A and B categories, without affecting those of the C type, as well as ventilation‐related and unidentified RVLM cells.

The activity of the CV neurones in the caudo‐ventrolateral part of the medulla oblongata (CV‐CVLM) was not modified by 5‐HT3 receptor stimulation in the NTS.

Prior intra‐NTS microinjections of ondansetron (300 pmol, a selective 5‐HT3 receptor antagonist) into the region of the commissural NTS prevented the excitation of A and B CV‐RVLM neurones induced by CPBG.

Intracarotid administration of saline saturated with CO2 (chemoreceptor activation) elicited both an increase in the SND and an excitation of the clonidine‐insensitive CV‐RVLM neurones of the C type, without affecting A and B neurones.

In conclusion, the sympathoexcitation elicited following 5‐HT3 receptor stimulation in the region of the commissural NTS of pentobarbitone‐anaesthetized rats seems to result from the excitation of two different pools of clonidine‐sensitive CV‐RVLM neurones. These neurones are apparently not involved in the sympathetic component of the chemoreceptor reflex.
A repolarization‐induced transient increase in the outward current of the inward rectifier K+ channel in guinea‐pig cardiac myocytes
Authors: Ishihara Keiko , Ehara Tsuguhisa
Published: September 2004

Outward currents of the inwardly rectifying K+ current (IKir) in guinea‐pig ventricular myocytes were studied in the presence of 1 mM intracellular free Mg2+ using the whole‐cell patch‐clamp technique.

During repolarizing voltage steps following a large depolarizing pulse (> 0 mV), outward IKir increased transiently at voltages positive to the K+ equilibrium potential (EK, ‐84 mV for 5.4 mM extracellular [K+]). The rising phase was almost instantaneous, while the decay was exponential. The decay rate was faster at voltages closer to EK (time constants, 33.9 ± 9.8 and 4.8 ± 1.4 ms at ‐30 and ‐50 mV, respectively).

The transient outward IKir was absent when the preceding depolarization was applied from ‐40 mV. Larger transient currents developed as the voltage before the depolarization was shifted to more hyperpolarized levels.

Shift of the depolarizing voltage from > 0 mV to more negative ranges diminished the amplitudes of transient outward IKir and instantaneous inward IKir during the subsequent repolarizing steps positive and negative to EK, respectively. Since blockage of IKir by internal Mg2+ occurs upon large depolarization, and the block is instantaneously relieved at voltages negative to EK, the rising phase of the transient outward IKir was attributed to the relief of Mg2+ block at voltages positive to EK. Transient outward IKir was absent when intracellular [Mg2+] was reduced to 10 μM or lower.

Prolongation of the repolarizing voltage step increased the amplitude of time‐dependent inward IKir during the subsequent hyperpolarization, indicating the progress of a gating process (presumably the channel block by intracellular polyamine) during the decaying phase of outward IKir.

Progressive prolongation of the depolarizing pulse (> 0 mV) from 100 to 460 ms decreased the transient outward IKir amplitude during the subsequent repolarizing step due to slow progress of the gating (polyamine block) at > 0 mV.

Current‐voltage relations measured using repolarizing ramp pulses (‐3.4 mV ms−1) showed an outward hump at around ‐50 mV, the magnitude of which increased as the voltage before the conditioning depolarization (10 mV) was shifted to more negative levels. With slower ramp speeds (‐1.5 and ‐0.6 mV ms−1), the hump was depressed at voltages near EK.

Our study suggests that the relief of Mg2+ block may increase outward IKir during repolarization of cardiac action potentials, and that the resting potential, the level/duration of action potential plateau and the speed of repolarization influence the outward IKir amplitude.

A kinetic model incorporating a competition between polyamine block and Mg2+ block was able to account for the time dependence of outward IKir.