bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protein kinases modulate two glycine currents in salamander retinal ganglion cells
Authors: Han Yi , Slaughter Malcolm M. 
Published: September 2004

Protein kinase modulation of glycine‐activated currents was examined in acutely dissociated ganglion cells from tiger salamander retina using whole‐cell voltage‐clamp techniques.
Glycine (100 μM) induced an outward chloride current in cells clamped at 0 mV. Co‐application of 50 μM forskolin made the glycine‐induced current more transient. The combination of forskolin and glycine reduced the later portion of current response without changing the initial peak amplitude.
3‐Isobutyl‐1‐methylxanthine (IBMX) or 8‐bromoadenosine 3′,5′‐cyclic monophosphate (8‐Br‐cAMP) produced effects similar to those of forskolin. H‐89, a protein kinase A (PKA) inhibitor, blocked the effect of forskolin.
A protein kinase C (PKC) activator, OAG (1‐oleoyl‐2‐acetyl‐sn‐glycerol), also made the glycine response more transient. Unlike PKA analogues, OAG enhanced the glycine peak response without changing the glycine late response. OAG effects were blocked by 1 μM GF‐109203X, a PKC inhibitor.
Nanomolar concentrations of strychnine selectively blocked the fast phase of the glycine current and reversed the effect of OAG, but not that of forskolin. Conversely, forskolin occluded the effect of 5,7‐dichlorokynurenic acid, which selectively suppresses the late phase of the glycine current. The action of OAG was not blocked by 5,7‐dichlorokynurenic acid.
Thus, through a differential modulation, both protein kinase A and C shorten the decay time of the glycine current. PKA suppresses the slow component, while PKC potentiates the fast component.
Spontaneous activity regulates calcium‐dependent K+ current expression in developing ascidian muscle
Authors: Dallman Julia E. , Davis Anna K. , Moody William J. 
Published: September 2004

In embryonic ascidian muscle, outward K+ currents develop in two stages: the initial expression of a slowly activating, voltage‐gated K+ current (IKv) near the time of neurulation is followed about 6 h later by a rapidly activating calcium‐activated K+ current (IK(Ca)). During this 6 h interval, inward Ca2+ currents (ICa) appear and the inward rectifier (IK(IR)), the sole resting conductance, is transiently downregulated. These events predict a period of spontaneous activity. The following experiments were designed to test this prediction and to examine the relevance of spontaneous activity for muscle cell development.
By recording activity in cell‐attached patches, we have found that muscle cells generate spontaneous action potentials during this 6 h window of time when IK(IR) is downregulated and outward K+ currents are slow. Action potentials occur at a mean frequency of 13.9 min−1.
When activity is blocked by the transient application of the Ca2+ channel blocker Cd2+, IK(Ca) fails to develop. This disruption is specific for IK(Ca): IK(IR) and ICa develop normally in activity‐blocked cells. Application of Cd2+ either before or after the window of activity has no effect.
The reappearance of IK(IR) and the development of IK(Ca) and the mature form of ICa are all prevented by transcription blockers, with a sensitive period corresponding to the period of activity.
These data show that, although the expression of three channel types depends on transcription during the period of spontaneous activity, only the development of IK(Ca) depends on activity.
Presynaptic 5‐HT3 receptors evoke an excitatory response in dorsal vagal preganglionic neurones in anaesthetized rats
Authors: Wang Yun , Ramage Andrew G. , Jordan David 
Published: September 2004

Recordings were made from a total of sixty‐four vagal preganglionic neurones in the dorsal vagal motor nucleus (DVMN) of pentobarbitone sodium anaesthetized rats. The effects of ionophoretic administration of Mg2+ and Cd2+, inhibitors of neurotransmitter release, and the selective NMDA and non‐NMDA receptor antagonists (±)‐2‐amino‐5‐phosphono‐pentanoic acid (AP5) and 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX) on the excitatory actions of the 5‐HT3 receptor agonist 1‐phenylbiguanide (PBG) were studied.
In extracellular recording experiments, PBG (0‐40 nA) increased the firing rate of thirty‐five of the thirty‐nine neurones tested. The PBG‐evoked excitation was attenuated by application of Mg2+ (1‐10 nA) in sixteen of seventeen neurones or Cd2+ (2‐10 nA) in seven of eight neurones tested. At these low ejection currents neither Mg2+ nor Cd2+ altered baseline firing rates and Mg2+ had no effect on the excitations evoked by DL‐homocysteic acid (n= 4), NMDA (n= 4) or (AMPA; n= 2).
Ionophoresis of AP5 (2‐10 nA), at currents which selectively inhibited NMDA‐evoked excitations, attenuated PBG‐evoked excitations in all eight neurones tested. DNQX (5‐20 nA), at currents which selectively inhibited AMPA‐evoked excitations, also attenuated PBG‐evoked excitations (n= 3).
Intracellular activity was recorded in nine DVMN neurones. In six neurones ionophoretic application of PBG (10‐200 nA) depolarized the membrane and increased firing rate whilst in the other three neurones, PBG had no effect on membrane potential though it increased synaptic noise (n= 3) and firing rate (n= 2). In all six neurones tested, ionophoresis of Mg2+ (10‐120 nA) attenuated the PBG‐evoked increases in synaptic noise and firing rate.
In conclusion, the data are consistent with the hypothesis that 5‐HT3 receptor agonists activate DVMN neurones partly by acting on receptors located at sites presynaptic to the neurones. Activation of these receptors appears to facilitate release of glutamate, which, in turn, acts on postsynaptic NMDA and non‐NMDA receptors to activate the neurones.
Human sodium channel gating defects caused by missense mutations in S6 segments associated with myotonia: S804F and V1293I
Authors: Green Donnella S. , George Alfred L. , Cannon Stephen C. 
Published: September 2004

Missense mutations in the α‐subunit of the human skeletal muscle sodium channel (hSkM1) have been detected in some heritable forms of myotonia. By recording Na+ currents from cells transfected with cDNA encoding either wild‐type or mutant hSkM1, we characterized the functional consequences of two myotonia‐associated mutations that lie at the cytoplasmic end of the sixth transmembrane segment in domain II (S804F) or domain III (V1293I).
Both mutations caused modest, but unequivocal, alterations in the voltage‐dependent gating behaviour of hSkM1. For S804F, the abnormalities were limited to fast inactivation: the persistent Na+ current at the end of a 50 ms depolarization was increased 3‐fold, the rate of inactivation from the open state was slowed 2‐fold, and the voltage dependence of fast inactivation (h∞) was shifted by +3 mV. V1293I also disrupted fast inactivation, as evidenced by a 3‐fold faster rate of recovery at hyperpolarized potentials (<= ‐70 mV). Activation was altered as well for V1293I: the voltage dependence was shifted by ‐6 mV (hyperpolarized).
Slow inactivation was not altered by S804F or V1293I.
We conclude that S804F and V1293I are not benign polymorphisms. Either mutation causes detectable alterations in channel gating and, in model simulations, the magnitude of the defects is sufficient to produce runs of myotonic discharges.
Calcitonin gene‐related peptide acts presynaptically to increase quantal size and output at frog neuromuscular junctions
Authors: Van der Kloot William , Benjamin William B. , Balezina Olga P. 
Published: September 2004

Calcitonin gene‐related peptide (CGRP) is found in dense‐cored vesicles in the motor nerve terminal.
Exogenous CGRP increased the size of the quanta. The increase in size reached a maximum after about 40 min. The lowest effective concentration of human CGRP (hCGRP) was 0.8 nM. The action of hCGRP was antagonized by (−)‐vesamicol, a drug that blocks active acetylcholine (ACh) uptake into synaptic vesicles, so it appears that hCGRP increases size by adding more ACh to the quanta. The action of hCGRP was antagonized by drugs that block the activation of protein kinase A (PKA). (In other preparations CGRP also activates PKA.)
The hCGRP effect was not blocked by fragment 8–37, an antagonist of one class of CGRP receptor.
hCGRP increases evoked quantal output and miniature endplate potential (MEPP) frequency, again by activating PKA.
CGRP release was measured by radioimmunoassay. Release was increased by depolarization with elevated K+, but the amounts released appear to be below those needed to affect quantal size or output. Moreover, although elevated K+ can increase quantal size it acts by a pathway that does not involve PKA. We suggest that the most likely target of endogenously released CGRP is the regulation of circulation of the muscle.
NH4+ as a substrate for apical and basolateral Na+‐H+ exchangers of thick ascending limbs of rat kidney: evidence from isolated membranes
Authors: Blanchard Anne , Eladari Dominique , Leviel Françoise , Tsimaratos Michel , Paillard Michel , Podevin René‐Alexandre 
Published: September 2004

We have used highly purified right‐side‐out luminal and basolateral membrane vesicles (LMVs and BLMVs) isolated from rat medullary thick ascending limb (MTAL) to study directly the possible roles of the LMV and BLMV Na+‐H+ exchangers in the transport of NH4+.
Extravesicular NH4+ ((NH4+)o) inhibited outward H+ gradient‐stimulated 22Na+ uptake in both types of vesicles. This inhibition could not be accounted for by alteration of intravesicular pH (pHi).
Conversely, in both plasma membrane preparations, the imposition of outward NH4+ gradients stimulated 22Na+ uptake at the acidic pHi (6.60) of MTAL cells, under conditions in which possible alterations in pHi were prevented. All NH4+ gradient‐stimulated Na+ uptake was sensitive to 0.5 mM 5‐(N,N‐dimethyl)‐amiloride.
The BLMV and LMV Na+‐H+ exchangers had a similar apparent affinity for internal H+ (H+i), with pK (‐log of dissociation constant) values of 6.58 and 6.52, respectively.
These findings indicate that NH4+ interacts with the external and internal transport sites of the LMV and BLMV Na+‐H+ antiporters, and that both of these exchangers can mediate the exchange of internal NH4+ ((NH4+)i) for external Na+ (Na+o) at the prevailing pHi of MTAL cells.
We conclude that operation of the BLMV Na+‐H+ exchanger on the NH4+‐Na+ mode may represent an important pathway for mediating the final step of NH4+ absorption, whereas transport of NH4+ on the apical antiporter may provide negative feedback regulation of NH4+ absorption.
ATP and glutamate are released from separate neurones in the rat medial habenula nucleus: frequency dependence and adenosine‐mediated inhibition of release
Authors: Robertson Susan J. , Edwards Frances A. 
Published: September 2004

ATP and glutamatergic synaptic currents were compared in slices of rat medial habenula nucleus using whole‐cell patch‐clamp techniques.
In most cells low voltage stimulation resulted in glutamatergic responses and not purinergic responses. In five cells where ATP currents could be stimulated with low voltages, wash out of glutamate antagonists did not reveal evoked glutamate currents. Spontaneous glutamate currents confirmed washout of antagonist.
Modulation of release probability of glutamate and ATP, assessed by changes in failure rate of synaptic currents, was compared under conditions of different stimulation frequencies and in the presence of adenosine agonists and antagonists.
ATP release, but not glutamate release, was shown to be modulated by increased stimulation frequency which resulted in inhibition of ATP release via A2‐like adenosine receptors. A1 receptors caused inhibition of both ATP and glutamate release.
Endogenous adenosine inhibited glutamate release via A1 receptors but only inhibited ATP release via A2‐like receptors.
Attempts to inhibit the degradation of ATP to adenosine did not alter the frequency dependence of the failure rate.
We conclude, from the direct demonstration and from the differences in pharmacology and frequency dependence of the modulation of release, that ATP and glutamate responses are due to release from separate neurones.
Insulin occludes leptin activation of ATP‐sensitive K+ channels in rat CRI‐G1 insulin secreting cells
Authors: Harvey J. , Ashford M. L. J. 
Published: September 2004

Using whole‐cell and cell‐attached recording configurations, the effects of insulin on leptin activation of ATP‐sensitive K+ (KATP) channels were examined in the CRI‐G1 insulinoma cell line.
Whole‐cell recordings demonstrated that the leptin‐induced hyperpolarization and increased potassium conductance are completely occluded by prior exposure to insulin (1‐50 nM). In cell‐attached recordings, insulin prevented leptin activation of tolbutamide‐sensitive KATP channels. Furthermore, insulin (50 nM) slowly and completely reversed the effects of leptin (10 nM), an action not attributable to direct inhibition of KATP channels per se.
Low concentrations of insulin‐like growth factor‐1 (IGF‐1; 10–100 nM) failed to prevent leptin activation of KATP channels, although higher concentrations (1 μm) did inhibit leptin actions.
The action of insulin was specific for leptin, as the hyperglycaemic agent diazoxide activated KATP channels following prior exposure to insulin.
Wortmannin (1‐10 nM) and LY 294002 (10 μm) prevented leptin activation of KATP channels, indicating an involvement of phosphoinositide 3‐kinase (PI 3‐kinase).
In conclusion, leptin activation of KATP channels is counter‐regulated by insulin in the CRI‐G1 insulinoma cell line. This feedback mechanism may be important in the local integration of hormonal signals which regulate insulin secretion and in alterations of metabolic homeostasis associated with obesity and non‐insulin dependent diabetes mellitus (NIDDM).
Substrate upregulation of the human small intestinal peptide transporter, hPepT1
Authors: Walker Dianne , Thwaites David T. , Simmons Nicholas L. , Gilbert Harry J. , Hirst Barry H. 
Published: September 2004

Molecular mechanisms underlying physiological adaptation to increased levels of dietary peptides have been elucidated by studying the response to the substrate glycyl‐L‐glutamine (Gly‐Gln) of the proton‐coupled peptide transporter, hPepT1, in the Caco‐2 human intestinal cell line. Vmax for apical uptake of [14C]glycyl‐[14C]sarcosine was increased 1.64 (± 0.34)‐fold after incubation of Caco‐2 cells for 3 days in a peptide‐rich medium (4 mM Gly‐Gln replacing 4 mM Gln).
A full‐length Caco‐2 hPepT1 cDNA clone was identical to human small intestinal hPepT1 with the exception of a single amino acid substitution Ile‐662 to Val. Transcript sizes, on Northern blots of Caco‐2 poly(A)+ RNA probed with a 630 bp 5′ hPepT1 cDNA probe, correspond to the reported band pattern seen with human small intestinal RNA. The dipeptide‐induced increase in substrate transport was accompanied by a parallel increase of 1.92 (± 0.30)‐fold (n= 9) in hPepT1 mRNA. This was in part due to an increase in hPepT1 mRNA half‐life from 8.9 ± 1.1 to 12.5 ± 1.6 h (n= 3), but the increase in half‐life does not account fully for the observed increase in mRNA levels, suggesting that there was also a dipeptide‐mediated increase in hPepT1 transcription.
Anti‐hPepT1‐specific antipeptide antibodies localized hPepT1 exclusively to the apical membrane of human small intestinal enterocytes and Caco‐2 cells. Gly‐Gln supplementation of media resulted in a 1.72 (± 0.26)‐fold (n= 5) increase in staining intensity of Caco‐2 cells.
We conclude that Caco‐2 cells provide an appropriate model for the study of adaptation of intestinal hPepT1, at the molecular level, to increased levels of dietary peptide. The magnitude of functional increase in apical peptide transport activity in response to Gly‐Gln can be fully accounted for by the increased levels of hPepT1 protein and mRNA, the latter mediated by both enhanced hPepT1 mRNA stability and increased transcription. The signalling pathway between increased dietary peptide and hPepT1 upregulation, therefore, involves direct action on the enterocyte, independent of hormonal and/or neural control.
Extracellular ATP directly gates a cation‐selective channel in rabbit airway ciliated epithelial cells
Authors: Korngreen Alon , Ma Weiyuan , Priel Zvi , Silberberg Shai D. 
Published: September 2004

A membrane conductance activated by extracellular ATP was identified and characterized in freshly dissociated rabbit airway ciliated cells using the whole‐cell and outside‐out patch configurations of the patch‐clamp technique.
In solutions designed to maximize currents through voltage‐gated calcium channels, there were no indications of voltage‐gated Ba2+ currents.
Extracellular ATP (but not UTP or ADP) activated a membrane conductance which remained activated for several minutes in the presence of ATP. The conductance was permeable to monovalent and divalent cations with approximate relative permeabilities (P) for PBa:PCs:PTEA of 4:1:0.1. Permeability to Cl− was negligible.
Including GDP‐β‐S in the intracellular solution did not inhibit the effects of ATP, nor did GTP‐γ‐S irreversibly activate the conductance.
In outside‐out membrane patches, with GDP‐β‐S in the pipette solution, ATP activated ion channels which had a chord conductance of approximately 6 pS in symmetrical 150 mM CsCl solutions at ‐120 mV.
Suramin (100 μM) inhibited the whole‐cell currents activated by ATP (200 μM) by 93 ± 3 %. Similar effects of suramin were observed on ATP‐activated channels in outside‐out membrane patches.
Extracellular ATP had a priming action on the response to subsequent exposure to ATP. At ‐40 mV, the time to half‐maximal current activation (t½) was 46 ± 9 s during the first exposure to 200 μM ATP and decreased to 5 ± 3 s during a second exposure to the same concentration of ATP. The priming action of ATP was not inhibited by including GDP‐β‐S in the intracellular solution.
The initial rate of activation increased with the concentration of ATP, and was voltage sensitive. During the first exposure to 200 μM ATP, t½ at +40 mV was 4‐fold longer than t½ at ‐40 mV.
Half‐maximal activation of the conductance shifted from 210 ± 30 to 14 ± 4 μM added ATP when CaCl2 in the extracellular solution was reduced from 1.58 to 0.01 mM. The Hill coefficient for ATP was 1.2 in both solutions.
These observations suggest that a form of ATP uncomplexed with divalent cations directly gates an ion channel (P2X receptor) in rabbit airway ciliated cells, which serves as a pathway for Ca2+ influx. This purinoceptor may contribute to sustained ciliary activation during prolonged exposures to ATP.
Ca2+ images and K+ current during depolarization in smooth muscle cells of the guinea‐pig vas deferens and urinary bladder
Authors: Imaizumi Yuji , Torii Yuichi , Ohi Yoshiaki , Nagano Norihiro , Atsuki Kaoru , Yamamura Hisao , Muraki Katsuhiko , Watanabe Minoru , Bolton Thomas B. 
Published: September 2004

Electrical events and intracellular calcium concentration ([Ca2+]) imaged using fluo‐3 and laser scanning confocal microscopy were simultaneously monitored in single smooth muscle cells freshly isolated from guinea‐pig vas deferens or urinary bladder.
Images obtained every 8 ms, during stepping from ‐60 to 0 or +10 mV for 50 ms under voltage clamp, showed that a rise in [Ca2+] could be detected within 20 ms of depolarization in five to twenty small (< 2 μm diameter) ‘hot spots’, over 95 % of which were located within 1.5 μm of the cell membrane. Depolarization at 30 s intervals activated hot spots at the same places.
Cd2+ or verapamil abolished both hot spots and Ca2+‐activated K+ current (IK,Ca). Caffeine almost abolished hot spots and markedly reduced IK,Ca. Cyclopiazonic acid, which raised basal global [Ca2+], decreased the rise in hot spot [Ca2+] and IK,Ca amplitude during depolarization. These results suggest that Ca2+ entry caused Ca2+‐induced Ca2+ release (CICR).
Under voltage clamp, hot spot [Ca2+] closely paralleled the rise in IK,Ca and reached a peak within 20 ms of the start of depolarization, but the rise in global [Ca2+] over the whole cell area was much slower. Step depolarization to potentials positive to ‐20 mV caused hot spots to grow in size and coalesce, leading to a rise in global [Ca2+] and contraction. Ca2+ hot spots also occurred during the up‐stroke of an evoked action potential under current clamp.
It is concluded that the entry of Ca2+ in the early stages of an action potential evokes CICR from discrete subplasmalemma Ca2+ storage sites and generates hot spots that spread to initiate a contraction. The activation of Ca2+‐dependent K+ channels in the plasmalemma over hot spots initiates IK,Ca and action potential repolarization.
The influence of caffeine on intramembrane charge movements in intact frog striated muscle
Authors: L.‐H. Huang Christopher 
Published: September 2004

The influence of caffeine, applied over a 25‐fold range of concentrations, on intramembrane charge movements was examined in intact voltage‐clamped amphibian muscle fibres studied in the hypertonic gluconate‐containing solutions that were hitherto reported to emphasize the features of qγ at the expense of those of qβ charge.
The total charge, Qmax, the transition voltage, V*, and the steepness factor, k, of the steady‐state charge‐voltage relationships, Q(V), were all conserved to values expected with significant contributions from the steeply voltage‐dependent qγ species (Qmax≈ 20 nC μF−1, V*≈−50 mV, k≈ 8 mV) through all the applications of caffeine concentrations between 0.2 and 5.0 mm. This differs from recent reports from studies in cut as opposed to intact fibres.
The delayed transients that have been attributed to transitions within the qγ charge persisted at low (0.2 mm) and intermediate (1.0 mm) caffeine concentrations.
In contrast, the time courses of such qγ currents became more rapid and their waveforms consequently merged with the earlier qβ decays at higher (5.0 mm) reagent concentrations. The charging records became single monotonic decays from which individual contributions could not be distinguished. This suggests that caffeine modified the kinetic properties of the qγ system but preserved its steady‐state properties. These findings thus differ from earlier reports that high caffeine concentrations enhanced the prominence of delayed transient components in cut fibres.
Caffeine (5.0 mm) and ryanodine (0.1 mm) exerted antagonistic actions upon qγ charge movements. The addition of caffeine restored the delayed time courses that were lost in ryanodine‐containing solutions, reversed the shift these produced in the steady‐state charge‐voltage relationship but preserved both the maximum charge, Qmax, and the steepness, k, of the steady‐state Q(V) relationships.
Caffeine also antagonized the actions of tetracaine on the total available qγ charge, but did so only at the low and not at the high applied concentrations. Thus, 0.2 mm caffeine restored the steady‐state qγ charge, the steepness of the overall Q(V) function and the appearance of delayed qγ charge movements that had been previously abolished by the addition of 2.0 mm tetracaine.
In contrast, the higher applied (1.0 and 5.0 mm) caffeine concentrations paradoxically did not modify these actions of tetracaine. The total charge and voltage dependence of the Q(V) curves, and the amplitude and time course of charge movements remained at the reduced values expected for the tetracaine‐resistant qβ charge.
These results permit a scheme in which caffeine acts directly upon ryanodine receptor (RyR)‐Ca2+ release channels whose consequent activation then dissociates them from the tubular dihydropyridine receptor (DHPR) voltage sensors that produce qγ charge movement, with which they normally are coupled in reciprocal allosteric contact.