bluuebunny/crossref_metadata_2025_split huggingface dataset:
A defect in skeletal muscle sodium channel deactivation exacerbates hyperexcitability in human paramyotonia congenita
Authors: Featherstone David E. , Fujimoto Esther , Ruben Peter C. 
Published: September 2004

Paramyotonia congenita (PC) is a human hereditary disorder wherein missense mutations in the skeletal muscle sodium channel lead to cold‐exacerbated muscle hyperexcitability. The most common site for PC mutations is the outermost arginine of domain IV segment 4 (human R1448, rat R1441).
We examined the rat homologues of two PC mutants with changes at this site: R1441P and R1441C. The R→P mutation leads to the most clinically severe form of the disease. Since PC has so far been attributed to defects in fast inactivation, we expected the R→P substitution to have a more dramatic effect on fast inactivation than R→C. Both mutants (R1441P and R1441C), however, had identical rates and voltage dependence of fast inactivation and activation.
R1441P and R1441C also had slowed deactivation, compared with wild‐type, raising the possibility that slowed deactivation, in combination with defective fast inactivation, might be a contributing cause of paramyotonia congenita. Furthermore, deactivation was slower in R1441P than in R1441C, suggesting that the worse phenotype of the human R→P mutation is due to a greater effect on deactivation, and supporting our hypothesis that slowed sodium channel deactivation contributes to paramyotonia congenita.
We show that the downstroke of the muscle action potential produced a sodium tail current, and thus slowed deactivation opposes repolarization and therefore leads to hyperexcitability. Hyperexcitability due to slowed deactivation, which has previously been overlooked, also predicts the temperature sensitivity of PC, which has otherwise not been adequately explained.
Regulation of distal nephron K+ channels (ROMK) mRNA expression by aldosterone in rat kidney
Authors: Beesley A. H. , Hornby D. , White S. J. 
Published: September 2004

The expression of ROMK mRNA isoforms in rat kidney was measured using competitive polymerase chain reaction. Under basal conditions the expression of ROMK2 and 3 mRNA was significantly higher than that of ROMK1 or 6.
Administration of aldosterone for a period of 1 week significantly increased the mRNA expression not only of the α1‐subunit of the Na+‐K+‐ATPase, but also of ROMK2, 3 and 6.
These data not only provide evidence that ROMK K+ channels may be involved with mineralocorticoid‐sensitive K+ secretion in the distal nephron, but also demonstrate for the first time that ROMK6 may be involved in this process.
Modulation of guinea‐pig cardiac L‐type calcium current by nitric oxide synthase inhibitors
Authors: Gallo Maria Pia , Ghigo Dario , Bosia Amalia , Alloatti Giuseppe , Costamagna Costanzo , Penna Claudia , Levi Renzo C. 
Published: September 2004

Electrophysiological (whole‐cell clamp) techniques were used to study the effect of NO synthase (NOS) inhibitors on guinea‐pig ventricular calcium current (ICa), and biochemical measurements (Western blot and citrulline synthesis) were made to investigate the possible mechanisms of action.
The two NOS inhibitors, NG‐monomethyl‐L‐arginine (L‐NMMA, 1 mM) and NG‐nitro‐L‐arginine (L‐NNA, 1 mM), induced a rapid increase in ICa when applied to the external solution. D‐NMMA (1 mM), the stereoisomer of L‐NMMA, which has no effect on NOS, did not enhance ICa.
Western blot experiments gave no indication of the presence of inducible NOS protein (iNOS) in our cell preparation, neither immediately after dissociation nor after more than 24 h. Statistically, there was no significant difference between electrophysiological experiments performed on freshly dissociated cells and experiments performed the next day. Moreover cells prepared and kept in the presence of dexamethasone (3 μM), to inhibit the expression of iNOS, gave the same response to L‐NMMA as control cells.
The stimulatory effect of L‐NMMA (1 mM) on basal ICa was reversed by competition with higher doses (5 mM) of externally applied L‐arginine, the natural substrate of NOS. The effect of L‐NMMA was also eliminated by L‐arginine in the patch pipette solution.
Intracellular perfusion with GDPβS (0.5 mM), which stabilizes the G‐proteins in the inactive state, did not affect the L‐NMMA‐induced stimulation of ICa.
Carbachol (1 μM) reduced the ICa previously stimulated by L‐NMMA, and intracellular cGMP (10 μM) prevented L‐NMMA enhancement.
Simultaneous treatment with L‐NMMA and isoprenaline (1 μM) induced a non‐cumulative enhancement of ICa that could not be reversed by carbachol (1 μM).
NO synthesis, measured by the formation of [3H]citrulline from L‐[3H]arginine during a 15 min incubation, showed a relatively high basal NO production, which was inhibited by L‐NMMA but not affected by carbachol.
These results suggest that inhibitors of NOS are able to modulate the basal ventricular ICa in the absence of a receptor‐mediated pathway, and that NO might be required for the muscarinic reduction of ICa under isoprenaline stimulation, even if NO production is not directly controlled by the muscarinic pathway.
Laminar difference in GABA uptake and GAT‐1 expression in rat CA1
Authors: Engel Dominique , Schmitz Dietmar , Gloveli Tengis , Frahm Christiane , Heinemann Uwe , Draguhn Andreas 
Published: September 2004

The axonal plexus of most hippocampal interneurons is restricted to certain strata within the target region. This lamination suggests a possible functional heterogeneity of inhibitory synapses between different interneurons and CA1 pyramidal cells.
We therefore compared inhibitory postsynaptic potentials (IPSPs) and currents (IPSCs) in CA1 pyramidal cells, which were evoked from two stimulation sites (stratum oriens and stratum radiatum). Stimulation in stratum oriens yielded faster decaying IPSPs and IPSCs than stimulation in stratum radiatum.
IPSP and IPSC kinetics were regulated by GABA uptake in both layers as indicated by the prolongation of the signals under tiagabine, a GAT‐1 (neuronal GABA plasma membrane transporter)‐specific GABA‐uptake blocker. However, the effect of tiagabine was significantly more pronounced following stimulation in stratum radiatum than in stratum oriens (prolongation of IPSC half‐decay time by 167 vs. 115 %, respectively).
In situ hybridization with antisense mRNA for the GABA‐synthesizing enzyme glutamate decarboxylase (GAD65/67) and the GABA transporter GAT‐1 showed that the proportion of interneurons expressing GAT‐1 was lower in stratum oriens than in stratum radiatum/lacunosum‐moleculare.
From these functional and molecular data we conclude that the regulation of IPSP and IPSC kinetics in CA1 pyramidal cells by neuronal GABA uptake differs between layers. Our findings suggest that this laminar difference is caused by a lower expression of GAT‐1 in interneurons in stratum oriens than in stratum radiatum/lacunosum‐moleculare.
The role of bradykinin in the regulation of blood flow to hindlimb muscle groups of the anaesthetized cat
Authors: Poucher S. M. , Garcia S. , Brooks R. 
Published: September 2004

Male cats were anaesthetized with alphaxalone‐alphadolone and breathed spontaneously following tracheotomy. Using coloured microspheres, muscle blood flow was measured at rest and during two periods of contraction elicited by simultaneous stimulation of the left sciatic and femoral nerves at 3 Hz for 10 min. In one group, the hindlimb blood flow was allowed to increase during muscle contraction and in another group the perfusion of the hindlimb was limited by a stenosis on the left external iliac artery.
In the absence of flow restriction, soleus muscle blood flow increased from 18.9 ± 3.8 to 30.4 ± 3.3 ml min−1 (100 g tissue)−1 (n= 6; P < 0.02) and gastrocnemius muscle blood flow increased from 24.8 ± 5.9 to 61.6 ± 12.8 ml min−1 (100 g tissue)−1 (n= 6; P < 0.01) during contraction. The bradykinin (BK) antagonist HOE 140 (1 mg kg−1, i.v.) did not affect the response in either the soleus (37.7 ± 7.3 ml min−1 (100 g tissue)−1; n= 6; n.s.) or the gastrocnemius (62.9 ± 7.9 ml min−1 (100 g tissue)−1; n= 6; n.s.) muscles.
In the stenosis group, soleus muscle blood flow increased from 9.8 ± 2.3 to 22.9 ± 4.9 ml min−1 (100 g tissue)−1 (n= 6; P < 0.01) and gastrocnemius muscle blood flow increased from 15.8 ± 3.4 to 36.4 ± 5.5 ml min−1 (100 g tissue)−1 (n= 6; P < 0.01) during contraction. Following administration of HOE 140, functional hyperaemia in the soleus muscle was unaffected (blood flow, 17.8 ± 2.2 ml min−1 (100 g tissue)−1; n= 6; n.s.) whilst blood flow in the gastrocnemius muscle was reduced to 21.8 ± 6.0 ml min−1 (100 g tissue)−1 (n= 6; P < 0.05).
The results show that BK does not contribute to functional hyperaemia associated with twitch contraction at 3 Hz when blood flow is unrestricted, but may contribute up to 40 % of the vasodilatation of predominantly glycolytic muscle groups such as the gastrocnemius when flow is restricted. BK plays no role in hyperaemia associated with twitch contraction of oxidative muscle groups such as the soleus.
Functional contribution of the α7 subunit to multiple subtypes of nicotinic receptors in embryonic chick sympathetic neurones
Authors: Yu C. Ron , Role Lorna W. 
Published: September 2004

Many studies of the α7 subunit of the neuronal nicotinic acetylcholine receptor (nAChR) family have demonstrated that this α‐bungarotoxin (α‐BgTx)‐binding neuronal receptor can participate in ACh‐gated channels. Heterologous expression studies reveal that α7 subunits form homomeric channels of unusually high Ca2+ permeability. However, the physiological role of the α7 subunit in native neuronal nAChR channels is less clear.
We present evidence that the α7 subunit contributes to the function of at least three subtypes of native nAChR expressed by embryonic chick sympathetic neurones. These subtypes are functionally distinct from heterologously expressed homomeric α7 nAChRs as well as homomeric‐like currents described in studies of hippocampal and parasympathetic neurones.
The proposed nAChRs differ from one another and from homomeric α7 nAChRs in their sensitivity to block by α7 subunit‐specific antagonists: α‐BgTx and methyllycaconitine (MLA). While MLA blocks 60 % of the macroscopic ACh response, α‐BgTx inhibits a small component of the macroscopic current described by slow‐on and slow‐off kinetics.
Functional deletion of the α7 subunit by antisense oligonucleotide treatment eliminates the susceptibility of the nAChRs to block by both MLA and α‐BgTx.
Single channel recordings combined with pharmacological and antisense‐mediated ‘deletion’ techniques reveal that α‐BgTx‐sensitive α7‐containing nAChRs have a small unitary conductance (18 pS), brief open time kinetics and relatively low open probability (Po). MLA‐sensitive α7 nAChRs are characterized by a conductance of ≈35 pS, intermediate burst duration, and a relatively high Po.
The third nAChR subtype deleted by α7 antisense treatment is characterized by a unitary conductance of 50 pS and prolonged opening duration.
We propose that these three populations of native α7‐containing nAChRs are distinct heteromeric complexes that include other α and/or β nAChR subunits.
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.