bluuebunny/crossref_metadata_2025_split huggingface dataset:
High‐affinity kainate‐type ion channels in rat cerebellar granule cells
Authors: Pemberton Karen E. , Belcher Scott M. , Ripellino James A. , Howe James R. 
Published: September 2004

Patch‐clamp recordings were made from rat cerebellar granule cells in primary culture. In cells pre‐exposed to concanavalin A (ConA) to remove kainate receptor desensitization, concentration‐response data for kainate showed two components. The EC50 value for the high‐affinity component (4 μM) was consistent with activation of kainate‐type channels. ConA enhanced the apparent potency of the kainate receptor ligand SYM 2081 by 100‐fold.
In ConA‐treated granule cells, currents evoked by 10 μM kainate were not significantly reduced by the AMPA receptor antagonist GYKI 53655, nor were these currents significantly reduced by the co‐application of 100 μM AMPA. Currents activated by low concentrations of kainate in the presence of AMPA were completely inhibited by 10 μM La3+.
Single‐cell reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis indicated that granule cells express both unedited (Q) and edited (R) versions of GluR5, with the majority of the GluR5 transcripts being unedited. In contrast, GluR6(R) was detected in seven cells and GluR6(Q) was detected in one granule cell.
Whole‐cell current‐voltage curves for kainate‐type currents in granule cells were measured and the ratio of the slope conductances at +40 mV and −40 mV was used as an index of rectification. The mean +40 mV/‐40 mV ratio determined from thirty‐six granule cells was 1.3 ± 0.1. Spectral density analysis of kainate‐evoked whole‐cell current noise gave values for the apparent single‐channel conductance, γnoise, that were on average about 1 pS.
To compare further the properties of recombinant kainate channels with the native kainate‐type channels in granule cells, we determined EC50 and γnoise values for SYM 2081 in stable cell lines expressing either GluR6(R) or GluR6(R) and KA2. Co‐expression of KA2 with GluR6(R) shifts the EC50 and γnoise values determined for SYM 2081 closer to the values typically found for native kainate‐type channels in granule cells.
The results demonstrate that cerebellar granule cells in culture express functional kainate‐type channels and that in most cells these channels show properties that are similar to those determined for heteromeric channels formed from GluR6(R) and KA2. However, the results also suggest that different granule cells express different repertoires of kainate‐type channels with different, and perhaps variable, subunit composition.
Cyclic ADP‐ribose and calcium‐induced calcium release regulate neurotransmitter release at a cholinergic synapse of Aplysia
Authors: Mothet J.‐P. , Fossier P. , Meunier F.‐M. , Stinnakre J. , Tauc L. , Baux G. 
Published: September 2004

Presynaptic injection of cyclic ADP‐ribose (cADPR), a modulator of the ryanodine receptor, increased the postsynaptic response evoked by a presynaptic spike at an identified cholinergic synapse in the buccal ganglion of Aplysia californica.
The statistical analysis of long duration postsynaptic responses evoked by square depolarizations of the voltage‐clamped presynaptic neurone showed that the number of evoked acetylcholine (ACh) quanta released was increased following cADPR injection.
Overloading the presynaptic neurone with cADPR led to a transient increase of ACh release followed by a depression.
cADPR injections did not modify the presynaptic Ca2+ current triggering ACh release.
Ca2+ imaging with the fluorescent dye rhod‐2 showed that cADPR injection rapidly increased the free intracellular Ca2+ concentration indicating that the effects of cADPR on ACh release might be related to Ca2+ release from intracellular stores.
Ryanodine and 8‐amino‐cADPR, a specific antagonist of cADPR, decreased ACh release.
ADP‐ribosyl cyclase, which cyclizes NAD+ into cADPR, was present in the presynaptic neurone as shown by reverse transcriptase‐polymerase chain reaction experiments.
Application of NAD+, the substrate of ADP‐ribosyl cyclase, increased ACh release and this effect was prevented by both ryanodine and 8‐amino‐cADPR.
These results support the view that Ca2+‐induced Ca2+ release might be involved in the build‐up of the Ca2+ concentration which triggers ACh release, and thus that cADPR might have a role in transmitter release modulation.
Non‐receptor‐mediated activation of IK(ATP) and inhibition of IK(ACh) by diadenosine polyphosphates in guinea‐pig atrial myocytes
Authors: Brandts Bodo , Brandts Annette , Wellner‐Kienitz Marie‐Cécile , Zidek Walter , Schlüter Hartmut , Pott Lutz 
Published: September 2004

The effects of diadenosine polyphosphates (APnA, where n= 4–6) were studied on beating frequency of perfused guinea‐pig hearts and on muscarinic K+ current (IK(ACh)) and ATP‐regulated K+ current (IK(ATP)) in atrial myocytes from guinea‐pig hearts using whole‐cell voltage clamp.
Bradycardia induced by APnA in perfused hearts was completely inhibited by 8‐cyclopentyl‐ 1,3‐dipropylxanthine (CPX, 20 μm), a selective antagonist at A1 adenosine receptors, and was augmented by dipyridamole (Dipy), an inhibitor of cellular adenosine (Ado) uptake.
Whereas exposure of atrial myocytes to Ado (100 μm) within about 1 s induced a significant whole‐cell IK(ACh), APnA up to 1 mm applied for some tens of seconds failed to activate IK(ACh). If present for periods > 2 min, APnA caused inhibition of agonist‐evoked IK(ACh) and activation of a weakly inward rectifying K+ current, which was identified as IK(ATP) by its sensitivity to glibenclamide and its current‐voltage curve.
The actions of extracellular APnA on IK(ACh) and IK(ATP) were mimicked by intracellular loading of compounds via the patch clamp pipette and by intracellular loading of AMP.
The results from isolated myocytes exclude APnA acting as A1 agonists. It is suggested that myocytes can take up APnA, which are degraded to AMP. In the presence of ATP, AMP is converted to ADP, a physiological activator of ATP‐regulated K+ channels, by adenylate kinase. A similar mechanism resulting in a reduction of the [GTP]/[GDP] ratio might be responsible for inhibition of IK(ACh).
In the perfused heart and other multicellular cardiac preparations the actions of APnA are mediated by Ado via A1 receptors. It is suggested that APnA in multicellular cardiac tissue are hydrolysed by an ectohydrolase to yield AMP which is converted to Ado by ectonucleotidases.
Fluid secretion in interlobular ducts isolated from guinea‐pig pancreas
Authors: Ishiguro H. , Naruse S. , Steward M. C. , Kitagawa M. , Ko S. B. H. , Hayakawa T. , Case R. M. 
Published: September 2004

Pancreatic HCO3− and fluid secretion were studied by monitoring luminal pH (pHL) and luminal volume simultaneously in interlobular duct segments isolated from guinea‐pig pancreas. The secretory rate and HCO3− flux were estimated from fluorescence images obtained following microinjection of BCECF‐dextran (70 kDa, 20 μM) into the duct lumen.
Ducts filled initially with a Cl−‐rich solution swelled steadily (2.0 nl min−1 mm−2) when HCO3−/CO2 was introduced, and the luminal pH increased to 8.08. When Cl− was replaced by glucuronate, spontaneous fluid secretion was reduced by 75 %, and pHL did not rise above 7.3.
Cl−‐dependent spontaneous secretion was largely blocked by luminal H2DIDS (500 μM). We conclude that, in unstimulated ducts, HCO3− transport across the luminal membrane is probably mediated by Cl−‐HCO3− exchange.
Secretin (10 nM) and forskolin (1 μM) both stimulated HCO3− and fluid secretion. The final value of pHL (8.4) and the increase in secretory rate (1.5 nl min−1 mm−2) after secretin stimulation were unaffected by substitution of Cl−.
The Cl−‐independent component of secretin‐evoked secretion was not affected by luminal H2DIDS. This suggests that a Cl−‐independent mechanism provides the main pathway for luminal HCO3− transport in secretin‐stimulated ducts.
Ducts filled initially with a HCO3−‐rich fluid (125 mM HCO3−, 23 mM Cl−) secreted a Cl−‐rich fluid while unstimulated. This became HCO3−‐rich when secretin was applied.
Addition of H2DIDS and MIA (10 μM) to the bath reduced the secretory rate by 56 and 18 %, respectively. Applied together they completely blocked fluid secretion. We conclude that basolateral HCO3− transport is mediated mainly by Na+‐HCO3− cotransport rather than by Na+‐H+ exchange.
Relations between intracellular Ca2+ stores and store‐operated Ca2+ entry in primary cultured human glioblastoma cells
Authors: Hartmann Jana , Verkhratsky Alexej 
Published: September 2004

In primary cultured human glioblastoma cells extracellular application of ATP triggered elevation in cytoplasmic calcium concentration ([Ca2+]i) mediated entirely by generation of inositol 1,4,5‐trisphosphate (InsP3)‐dependent Ca2+ release from endoplasmic reticulum Ca2+ stores followed by the activation of store‐operated Ca2+ entry into the cells.
Sensitivity of P2Y purinoceptors to extracellular ATP was regulated by extracellular Ca2+: in Ca2+‐free extracellular solution the threshold concentration of ATP that induced an increase in [Ca2+]i was reduced by one order of magnitude.
Activation of Ca2+ release and store‐operated Ca2+ entry was dissociated: low concentrations of ATP induced substantial Ca2+ release without activation of Ca2+ entry; activation of the latter required higher ATP concentrations.
Mitochondria participated in buffering Ca2+ loads that resulted from store‐operated Ca2+ influx; in contrast Ca2+ released from intracellular stores was not accumulated by the mitochondrial depot.
We conclude that ATP‐induced Ca2+ responses are governed by several pathways with different sensitivities to the agonist. This enables cells to respond either with pure Ca2+ release from intracellular stores (at low ATP concentrations) or (at high ATP concentrations) the response is amplified by plasmalemmal Ca2+ influx. Store‐operated Ca2+ entry increases mitochondrial Ca2+ content providing a link between cellular activation and mitochondrial function.
The antagonist trinitrophenyl‐ATP reveals co‐existence of distinct P2X receptor channels in rat nodose neurones
Authors: Thomas Sarah , Virginio Caterina , North R. Alan , Surprenant Annmarie 
Published: September 2004

Whole‐cell recordings were made from rat nodose ganglion neurones in culture and from human embryonic kidney (HEK293) cells stably transfected to express P2X2, P2X3or both receptor subunits. We examined the blocking actions of 2′,3′‐O‐trinitrophenyl‐ATP (TNP‐ATP) on currents evoked by the agonists ATP and α,β‐methylene ATP.In cells expressing only P2X2or P2X3receptor subunits, the inhibition by TNP‐ATP was fitted by a single binding site model with half‐maximal concentrations of about 3 μM and 3 nM, respectively. In cells expressing both P2X2and P2X3receptor subunits, currents showed little or no desensitization, thus excluding contributions from homomeric P2X3receptors. When α,β‐methylene ATP was the agonist (activating heteromeric P2X2/3receptors), the inhibition by TNP‐ATP conformed to a single binding site (half‐maximal concentration about 3 nM). When ATP (30 μM) was the agonist, activating both heteromeric P2X2/3as well as homomeric P2X2receptors, the inhibition curve was biphasic (half‐maximal concentrations about 3 nM and 3 μM); the proportion of high affinity sites in all six cells tested was about 40 %.In nodose ganglion neurones, the inhibition by TNP‐ATP of currents evoked by ATP (30 μM) was also clearly biphasic. In this case, individual neurones showed more variability in the proportion of high and low affinity sites for TNP‐ATP.We conclude that more than one form of multimeric P2X receptor channels are functionally expressed on the cell bodies of individual nodose ganglion neurones. On the basis of sensitivity to TNP‐ATP, and other properties, one of these may correspond to the homomeric P2X2receptor and the other(s) to heteromeric P2X2/3receptors.
Mitochondrial Ca2+ uptake and release influence metabotropic and ionotropic cytosolic Ca2+ responses in rat oligodendrocyte progenitors
Authors: Simpson Peter B. , Russell Ames T. 
Published: September 2004

Many physiologically important activities of oligodendrocyte progenitor cells (O‐2A cells), including proliferation, migration and differentiation, are regulated by cytosolic Ca2+ signals. However, little is known concerning the mechanisms of Ca2+ signalling in this cell type. We have studied the interactions between Ca2+ entry, Ca2+ release from endoplasmic reticulum and Ca2+ regulation by mitochondria in influencing cytosolic Ca2+ responses in O‐2A cells.
Methacholine (MCh; 100 μM) activated Ca2+ waves that propagated from several initiation sites along O‐2A processes.
During a Ca2+ wave evoked by MCh, mitochondrial membrane potential was often either depolarized (21 % of mitochondria) or hyperpolarized (20 % of mitochondria), as measured by changes in the fluorescence of 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazole carbocyanine iodide (JC‐1).
Stimulation with kainate (100 μM) evoked a slowly rising, sustained cytosolic Ca2+ elevation in O‐2A cells. This also, in some cases, resulted in either a depolarization (15 % of mitochondria) or hyperpolarization (12 % of mitochondria) of mitochondrial membrane potential.
Simultaneous measurement of cytosolic (fluo‐3 AM) and mitochondrial (rhod‐2 AM) Ca2+ responses revealed that Ca2+ elevations in the cytosol evoked by either MCh or kainate were translated into long‐lasting Ca2+ elevations in subpopulations of mitochondria. In some mitochondria, Ca2+ signals appeared to activate Ca2+ release into the cytosol.
Inhibition of the mitochondrial Na+‐Ca2+ exchanger by CGP‐37157 (25 μM) decreased kainate Ca2+ response amplitude and increased the rate of return of the response to basal Ca2+ levels.
Thus, both ionotropic and metabotropic stimulation evoke changes in mitochondrial membrane potential and Ca2+ levels in O‐2A cells. Ca2+ uptake into some mitochondria is activated by Ca2+ entry into cells or release from stores. Mitochondrial Ca2+ release appears to play a key role in shaping kainate‐evoked Ca2+ responses.
Developmental changes in calcium channel types mediating synaptic transmission in rat auditory brainstem
Authors: Iwasaki Shinichi , Takahashi Tomoyuki 
Published: September 2004

Calcium channel blockers were tested on excitatory postsynaptic currents (EPSCs) at the synapse formed by the calyx of Held on the principal cells in the medial nucleus of trapezoid body (MNTB) in brainstem slices of 4‐ to 14‐day‐old rats.
At postnatal day 4‐9 (P4‐9), EPSCs were irreversibly suppressed by the P/Q‐type Ca2+ channel blocker ω‐agatoxin‐IVA (ω‐Aga‐IVA, 200 nM) and also by the N‐type Ca2+ channel blocker ω‐conotoxin GVIA (ω‐CgTx, 2 μM). A small fraction of EPSCs was resistant to both toxins but abolished by Cd2+ (100 μM).
After P7, the ω‐CgTx‐sensitive EPSC fraction diminished and eventually disappeared after P10. Concomitantly the fraction insensitive to both toxins decreased and became undetectable after P10.
In contrast, the ω‐Aga‐IVA‐sensitive EPSC fraction increased with development and became predominant after P10. All through the developmental period examined, the L‐type Ca2+ channel blocker nicardipine (10 μM) had no effect.
We conclude that presynaptic Ca2+ channel types triggering transmitter release undergo developmental switching during the early postnatal period.
Anoxia differentially modulates multiple K+ currents and depolarizes neonatal rat adrenal chromaffin cells
Authors: Thompson Roger J. , Nurse Colin A. 
Published: September 2004

Using perforated‐patch, whole cell recording, we investigated the membrane mechanisms underlying O2 chemosensitivity in neonatal rat adrenomedullary chromaffin cells (AMC) bathed in extracellular solution containing tetrodotoxin (TTX; 0.5–1 μm), with or without blockers of calcium entry.
Under voltage clamp, low PO2 (0–15 mmHg) caused a graded and reversible suppression in macroscopic outward K+ current. The suppression during anoxia (PO2= 0 mmHg) was ∼35 % (voltage step from −60 to +30 mV) and was due to a combination of several factors: (i) suppression of a cadmium‐sensitive, Ca2+‐dependent K+ current, IK(CaO2); (ii) suppression of a Ca2+‐insensitive, delayed rectifier type K+ current, IK(VO2); (iii) activation of a glibenclamide‐ (and Ca2+)‐sensitive current, IK(ATP).
During normoxia (PO2= 150 mmHg), application of pinacidil (100 μm), an ATP‐sensitive potassium channel (KATP) activator, increased outward current density by 45.0 ± 7.0 pA pF−1 (step from −60 to + 30 mV), whereas the KATP blocker glibenclamide (50 μm) caused only a small suppression by 6.3 ± 4.0 pA pF−1. In contrast, during anoxia the presence of glibenclamide resulted in a substantial reduction in outward current density by 24.9 ± 7.9 pA pF−1, which far exceeded that seen in its absence. Thus, activation of IK(ATP) by anoxia appears to reduce the overall K+ current suppression attributable to the combined effects of IK(CaO2) and IK(VO2).
Pharmacological tests revealed that IK(CaO2) was carried predominantly by maxi‐K+ or BK potassium channels, sensitive to 50–100 nm iberiotoxin; this current also accounted for the major portion (∼60 %) of the anoxic suppression of outward current. Tetraethylammonium (TEA; 10–20 mm) blocked all of the anoxia‐sensitive K+ currents recorded under voltage clamp, i.e. IK(CaO2), IK(VO2) and IK(ATP).
Under current clamp, anoxia depolarized neonatal AMC by 10–15 mV from a resting potential of ∼‐55 mV. At least part of this depolarization persisted in the presence of either TEA, Cd2+, 4‐aminopyridine or charybdotoxin, suggesting the presence of anoxia‐sensitive mechanisms additionalto those revealed under voltage clamp. In Na+/Ca2+‐free solutions, the membrane hyperpolarized, though at least a portion of the anoxia‐induced depolarization persisted.
In the presence of glibenclamide, the anoxia‐induced depolarization increased significantly to ∼25 mV, suggesting that activation of KATP channels may function to attenuate the anoxia‐induced depolarization or receptor potential.
Muscarinic IPSPs in rat striatal cholinergic interneurones
Authors: Calabresi Paolo , Centonze Diego , Pisani Antonio , Sancesario Giuseppe , North R. Alan , Bernardi Giorgio 
Published: September 2004

Intracellular recordings were made from neurones in slices of rat striatum in vitro.
The forty‐nine neurones studied were immunoreactive for choline acetyltransferase and had the electrophysiological characteristics typical of large aspiny interneurones.
Focal stimulation of the slice elicited a hyperpolarizing inhibitory postsynaptic potential in thirty‐five neurones. This IPSP lasted 0.5–1 s and reversed polarity at a membrane potential which was dependent on the logarithm of the extracellular potassium concentration.
The IPSP was reversibly blocked by scopolamine and methoctramine, which has some selectivity for the M2 subtype of muscarinic receptor. It was unaffected by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (10 μM), DL‐2‐amino‐phosphonovaleric acid (30 μM) and bicuculline (30 μM).
Exogenous acetylcholine and muscarine also hyperpolarized the neurones, and this was blocked by methoctramine but not by pirenzepine, which is an M1 receptor‐selective antagonist.
The findings demonstrate that muscarinic IPSPs occur in the central nervous system. The IPSP may mediate an ‘autoinhibition’ of striatal cholinergic neurone activity.
Density of transient K+ current influences excitability in acutely isolated vasopressin and oxytocin neurones of rat hypothalamus
Authors: Fisher Thomas E. , Voisin Daniel L. , Bourque Charles W. 
Published: September 2004

The transient outward K+ current (ITO) was studied using whole‐cell recording in immunocytochemically identified oxytocin (OT; n= 23) and vasopressin (VP; n= 67) magnocellular neurosecretory cells (MNCs) acutely isolated from the supraoptic nucleus of adult rats.
The peak density of ITO during steps to −10 mV was 26 % smaller in OT‐MNCs (355 ± 23 pA pF−1; mean ± s.e.m.; n= 18) than in VP‐MNCs (478 ± 17 pA pF−1; n= 52). No differences were observed in the voltage dependence of activation or inactivation.
Kinetic analysis revealed two components of ITO inactivation in both OT‐MNCs (τ1= 9.2 ± 0.4 ms and τ2= 41.2 ± 1.6 ms; n= 18) and VP‐MNCs (τ1= 12.4 ± 0.4 ms and τ2= 37.1 ± 1.2 ms; n= 52). Although the density of the rapid component (τ1) was not different (275 ± 13 versus 265 ± 16 pA pF−1, respectively), the slow component (τ2) was markedly smaller in OT‐MNCs (183 ± 19 versus 331 ± 16 pA pF−1 in VP‐MNCs).
In unidentified MNCs, 0.5 mM 4‐aminopyridine reduced ITO amplitude by 29 % and decreased the latency to spike discharge by about 70 % during depolarization from −70 mV. Latency to discharge from potentials less negative than −60 mV, where ITO is inactivated, was unaffected.
Comparison of latency to spike discharge in identified cells showed that OT‐MNCs achieve spike threshold twice as fast as VP‐MNCs when depolarized from −70 mV. The lower density of ITO in OT‐MNCs, therefore, accelerates the rate at which excitation can occur in response to depolarizing stimuli and may facilitate the occurrence of higher frequency discharges in OT‐MNCs during physiological activation.
Control of L‐type calcium current during the action potential of guinea‐pig ventricular myocytes
Authors: Linz Klaus W. , Meyer Rainer 
Published: September 2004

During an action potential the L‐type Ca2+ current (ICa,L) activates rapidly, then partially declines leading to a sustained inward current during the plateau phase. The reason for the sustained part of ICa,L has been investigated here.
In the present study the mechanisms controlling the ICa,L during an action potential were investigated quantitatively in isolated guinea‐pig ventricular myocytes by whole‐cell patch clamp. To measure the actual time courses of ICa,L and the corresponding L‐type channel inactivation (fAP) during an action potential, action potential‐clamp protocols combined with square pulses were applied.
Within the first 10 ms of the action potential the ICa,L rapidly inactivated by about 50 %; during the plateau phase inactivation proceeded to 95 %. Later, during repolarization, the L‐type channels recovered up to 25 %.
The voltage‐dependent component of inactivation during an action potential was determined from measurements of L‐type current carried by monovalent cations. This component of inactivation proceeded rather slowly and contributed only a little to fAP. ICa,L during an action potential is thus mainly controlled by Ca2+‐dependent inactivation.
In order to investigate the source of the Ca2+ controlling fAP, internal Ca2+ homeostasis was manipulated by the use of Ca2+ buffers (EGTA, BAPTA), by blocking Na+−Ca2+ exchange, or by blocking Ca2+ release from the sarcoplasmic reticulum (SR). Internal BAPTA markedly reduced the L‐type channel inactivation during the entire action potential, whereas EGTA affected fAP only during the middle and late plateau phases. Inhibition of Na+−Ca2+ exchange markedly increased inactivation of L‐type channels. Although blocking SR Ca2+ release decreased the fura‐2‐measured cytoplasmic Ca2+ concentration ([Ca2+]i) transient by about 90 %, it reduced L‐type channel inactivation only during the initial 50 ms of the action potential. Thus, it is Ca2+ entering the cell through the L‐type channels that controls the inactivation process for the majority of the action potential. Nevertheless, SR Ca2+‐release contributes 40–50 % to L‐type channel inactivation during the initial period of the action potential. However, the maximum extent of inactivation reached during the plateau is independent of Ca2+ released from the SR.
For the first time, the actual time course of L‐type channel inactivation has been directly determined during an action potential under various defined [Ca2+]i conditions. Thereby, the relative contribution to ICa,L inactivation of voltage, Ca2+ entering through L‐type channels, and Ca2+ being released from the SR could be directly demonstrated.