bluuebunny/crossref_metadata_2025 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.
Lambert‐Eaton syndrome antibodies inhibit acetylcholine release and P/Q‐type Ca2+ channels in electric ray nerve endings
Authors: Satoh Yasushi , Hirashima Naohide , Tokumaru Hiroshi , Takahashi Masanori P. , Ang Jin K , Viglione Michael P. , Kim Yong I. , Kirino Yutaka
Published: September 2004

The types of voltage‐dependent calcium channels (VDCCs) present in the cholinergic terminals isolated from the electric organ of the ray, Narke japonica, were characterized on the basis of their pharmacological sensitivity to specific antagonists. Inhibition of these channel types by autoantibodies from patients with the Lambert‐Eaton syndrome (LES) was then studied to determine the specificity of the pathogenic IgG.

In normal untreated synaptosomal preparations, maximal doses of N‐ and P and/or Q‐type Ca2+ channel antagonists, ω‐conotoxin GVIA and ω‐agatoxin IVA, inhibited depolarization‐evoked ACh release by 47 % and 43 %, respectively. Calciseptine, an L‐type VDCC antagonist, caused a 20 % reduction in the release. This indicates that the exocytotic release process is predominantly mediated by N‐ and P/Q‐type VDCCs.

LES IgG or sera caused an inhibition of ACh release by 39‐45 % in comparison with the control antibody‐treated preparations. The ionomycin‐induced ACh release, however, was not altered by the antibodies. Additionally, the same LES antibodies inhibited whole‐cell calcium currents (ICa) in bovine adrenal chromaffin cells. Thus, the pathogenic antibodies exert their action on VDCCs present in the synaptosomes.

The efficacy of three Ca2+ channel antagonists in blocking ACh release was determined in preparations pretreated with LES IgG. ω‐Agatoxin IVA produced only an additional 3‐5 % reduction in release beyond that obtained with LES antibodies. Despite the pretreatment with LES IgG, ω‐conotoxin GVIA and calciseptine inhibited the release to nearly their control levels.

These results indicate that LES antibodies mainly downregulate P/Q‐type Ca2+ channels which contribute to presynaptic transmitter release from the cholinergic nerve terminals of electric organ.

The present findings are consistent with the hypothesis that P/Q‐type VDCCs at the neuromuscular junction are the target of LES antibodies and that their inhibition by the antibodies produces the characteristic neuromuscular defect in this disease.
Chemosensitivity of rat medullary raphe neurones in primary tissue culture
Authors: Wang Wengang , Pizzonia John H. , Richerson George B.
Published: September 2004

The medullary raphe, within the ventromedial medulla (VMM), contains putative central respiratory chemoreceptors. To study the mechanisms of chemosensitivity in the raphe, rat VMM neurones were maintained in primary dissociated tissue culture, and studied using perforated patch‐clamp recordings. Baseline electrophysiological properties were similar to raphe neurones in brain slices and in vivo.

Neurones were exposed to changes in CO2 from 5 % to 3 or 9 % while maintaining a constant [NaHCO3]. Fifty‐one per cent of neurones (n= 210) did not change their firing rate by more than 20 % in response to hypercapnic acidosis. However, 22 % of neurones responded to 9 % CO2 with an increase in firing rate (‘stimulated’), and 27 % of neurones responded with a decrease in firing rate (‘inhibited’).

Chemosensitivity has often been considered an all‐or‐none property. Instead, a method was developed to quantify the degree of chemosensitivity. Stimulated neurones had a mean increase in firing rate to 298 ± 215 % of control when pH decreased from 7.40 to 7.19. Inhibited neurones had a mean increase in firing rate to 232 ± 265 % of control when pH increased from 7.38 to 7.57.

Neurones were also exposed to isocapnic acidosis. All CO2‐stimulated neurones tested (n= 15) were also stimulated by isocapnic acidosis, and all CO2‐inhibited neurones tested (n= 19) were inhibited by isocapnic acidosis. Neurones with no response to hypercapnic acidosis also had no response to isocapnic acidosis (n= 12). Thus, the effects of CO2 on these neurones were mediated in part via changes in pH.

In stimulated neurones, acidosis induced a small increase in the after‐hyperpolarization level of 1.38 ± 1.15 mV per −0.2 pH units, which was dependent on the level of tonic depolarizing current injection. In voltage clamp mode at a holding potential near resting potential, there were small and inconsistent changes in whole‐cell conductance and holding current in both stimulated and inhibited neurones. These results suggest that pH modulates a conductance in stimulated neurones that is activated during repetitive firing, with a reversal potential close to resting potential.

The two subtypes of chemosensitive VMM neurones could be distinguished by characteristics other than their response to acidosis. Stimulated neurones had a large multipolar soma, whereas inhibited neurones had a small fusiform soma. Stimulated neurones were more likely than inhibited neurones to fire with the highly regular pattern typical of serotonergic raphe neurones in vivo.

Within the medullary raphe, chemosensitivity is a specialization of two distinct neuronal phenotypes. The response of these neurones to physiologically relevant changes in pH is of the magnitude that suggests that this chemosensitivity plays a functional role. Elucidating their mechanisms in vitro may help to define the cellular mechanisms of central chemoreception in vivo.
T‐type Ca2+ current as a trigger for Ca2+ release from the sarcoplasmic reticulum in guinea‐pig ventricular myocytes
Authors: Sipido Karin R. , Carmeliet Edward , van de Werf F.
Published: September 2004

We have investigated whether Ca2+ entry through T‐type Ca2+ channels participates in triggering Ca2+ release from the sarcoplasmic reticulum (SR) in single guinea‐pig ventricular myocytes (whole‐cell voltage clamp, K5fura‐2 as [Ca2+]i indicator; all monovalent cations replaced by impermeant ions to record uncontaminated Ca2+ currents; T= 23 or 36 °C).

T‐type Ca2+ currents were elicited from a holding potential of ‐90 mV during steps to ‐50 to ‐20 mV. For steps to ‐50 mV, very small [Ca2+]i transients could be recorded with high loading of the SR (peak Δ[Ca2+]i, 67 ± 41 nM; n= 9).

For steps to ‐40, ‐30 and ‐20 mV, we compared the amplitude of Ca2+ release for a holding potential of ‐50 mV with L‐type Ca2+ current only to Ca2+ release for a holding potential of ‐90 mV with both T‐ and L‐type Ca2+ current. Significantly more Ca2+ release was observed with T‐type current present, and both the T‐type current and the additional Ca2+ release were suppressed by 50 μM NiCl2.

Ca2+ influx through T‐type Ca2+ channels triggered less Ca2+ release than a comparable Ca2+ influx through L‐type Ca2+ channels.

Rapid block of T‐type Ca2+ current during the action potential (50 μM NiCl2 during steady‐state stimulation at 1 or 2 Hz) did not immediately reduce Ca2+ release, although a small decrease was observed after longer application.

We conclude that T‐type Ca2+ current can trigger Ca2+ release from the SR albeit less efficiently than L‐type Ca2+ current. T‐type current is most likely to provide only a small contribution to the trigger for Ca2+ release in normal conditions. These results support the hypothesis that L‐type Ca2+ channels have a privileged role in excitation‐contraction coupling.
Absorptive apical amiloride‐sensitive Na+ conductance in human endometrial epithelium
Authors: Matthews C. Jane , McEwan Gordon T. A. , Redfern Christopher P. F. , Thomas Eric J. , Hirst Barry H.
Published: September 2004

Human endometrial epithelial cells cultured on porous tissue culture supports formed tight, polarized epithelial monolayers with features characteristic of tight epithelia. Endometrial epithelial layers generated significant transepithelial electrical resistance (750 Ω cm2) and potential difference (15.3 mV), with an inward short‐circuit current (Isc; 20.5 μA cm−2).

The Isc was linearly proportional to the external Na+ concentration and was abolished in the absence of Na+. The Isc was sensitive to apical amiloride. Net 22Na+ flux was in the absorptive apical to basolateral direction and fully accounted for the inward Isc. In addition, apical to basolateral and net 22Na+ transport were reduced in the presence of amiloride.

The Isc was also sensitive to addition of ouabain and Ba2+ to the basal solution, consistent with a role for basolateral Na+−K+‐ATPase and K+ channels in generation of the current.

These data demonstrate that human endometrial epithelial cells in primary culture produce tight, functional monolayers on permeable supports. We provide the first evidence that human endometrial epithelial cells have an inward Isc accounted for by an amiloride‐sensitive Na+ conductance. The Na+‐absorptive function of the endometrium may provide an appropriate environment for sperm function and embryo growth.
Sodium pump evokes high density pump currents in rat midbrain dopamine neurons
Authors: Shen Ke‐Zhong , Johnson Steven W.
Published: September 2004

Patch pipettes contained various concentrations of Na+ ([Na+]pip) in order to record strophanthidin‐sensitive currents under voltage clamp in dopamine neurons in slices of rat substantia nigra and ventral tegmental area.

When [Na+]pip was 40 mm and the external K+ concentration ([K+]o) was 2.5 mm, strophanthidin (10 μm) evoked 461 ± 121 pA of inward current. This effect was concentration dependent, with an EC50 of 7.1 ± 2.6 μm. At potentials of −60 to −120 mV, strophanthidin‐induced currents were not associated with significant changes in chord conductance.

Strophanthidin (10 μm) evoked 234 ± 43 pA of inward current when [Na+]pip was 0.6 mm, and 513 ± 77 pA when [Na+]pip was 80 mm. Despite higher pump currents with greater [Na+]pip, the strophanthidin EC50 was not significantly different for any of six different [Na+]pip.

Sodium pump currents were half‐maximal when the [Na+]pip was about 1.3 mm. Maximum pump current was estimated at 830 pA (29 μA cm−2) at concentrations of intracellular Na+ that were assumed to be saturating (50–100 mm).

Strophanthidin currents were smaller in a reduced [K+]o (EC50= 0.2 mm).

These data show that intracellular Na+ loading evokes relatively large pump currents. Our results are consistent with the physiological role of the sodium pump in burst firing in midbrain dopamine neurons
Prejunctional effects of the nicotinic ACh receptor agonist dimethylphenylpiperazinium at the rat neuromuscular junction
Authors: Singh Shila , Prior Chris
Published: September 2004

We have studied the effects of the nicotinic acetylcholine (ACh) receptor agonist dimethylphenylpiperazinium (DMPP) on the evoked release of ACh from motor terminals in the rat isolated hemidiaphragm using an electrophysiological approach.

DMPP (1–4 μM) had no effect on the rate of spontaneous quantal ACh release but increased the number of quanta of ACh released per impulse during 50 Hz stimulation. The DMPP‐induced increase in evoked ACh release was dependent on the frequency of stimulation, being absent when it was reduced to 0.5 Hz, but was not Ca2+ dependent, being unaffected at 50 Hz by a 4‐fold decrease in the extracellular Ca2+ concentration.

The facilitation of evoked ACh release at 50 Hz by 2 μM DMPP was abolished by 10 μM of the calmodulin antagonist W7 (N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulphonamide hydrochloride) and, in the presence of W7, 2 μM DMPP depressed evoked ACh release at 0.5 Hz. The ability of the nicotinic ACh receptor antagonist vecuronium (1 μM) to depress evoked ACh release at 50 Hz was also abolished by 10 μM W7.

The present findings demonstrate, using an electrophysiological technique, that DMPP can produce changes in the evoked ACh release from rat motor nerve terminals that are consistent with the existence of facilitatory nicotinic ACh receptors on the motor nerve endings. Further, they indicate a role for calmodulin‐dependent systems in this facilitatory effect of the compound.
Characterization of cell volume‐sensitive chloride currents in freshly prepared and cultured pancreatic acinar cells from early postnatal rats
Authors: Schmid Andreas , Blum Robert , Krause Elmar
Published: September 2004

In freshly prepared and cultured exocrine pancreatic acinar cells from 5‐ to 7‐day‐old rats a chloride‐selective membrane conductance could be activated by intracellular application of GTPγS (40–100 μM), by application of positive pressure (5 cmH2O) to the pipette interior or by challenging the cells with a hyposmolar bath solution. Hyperosmolar bath solutions inhibited the cell volume‐sensitive chloride currents.

The anion permeability sequence of the cell volume‐sensitive chloride conductance was I− > Cl−≈ Br− > F− > methanesulphonate− > glutamate−. I− had a higher permeability but lower conductance than Cl−. The permeability ratio for Pglutamate/PCl was 0.12.

The cell volume‐sensitive chloride conductance showed outward rectification. Membrane depolarization to high positive voltages (≥+60 mV) caused a time‐dependent decay in outward currents.

DIDS (4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid) and SITS (4‐acetamido‐4′‐isothiocyanatostilbene‐2,2′‐disulphonic acid) reversibly inhibited the cell volume‐sensitive chloride current in a voltage‐dependent manner. NPPB (5‐nitro‐2‐(3‐phenylpropylamino)‐benzoic acid), quinidine, quinine and tamoxifen caused voltage‐independent current inhibition.

Combined fura‐2 and whole‐cell current measurements showed that activation of the cell volume‐sensitive chloride current does not involve cytosolic Ca2+ signals. Furthermore, there is no evidence that Ca2+‐activated chloride currents play a significant role in cultured pancreatic acinar cells from 5‐ to 7‐day‐old rats.

Polymerase chain reaction followed by DNA sequence analysis indicated the presence of mRNA homologous to the ClC‐3 chloride channel in pancreatic tissue from 5‐day‐old rats.
Interaction of the Na+‐K+ pump and Na+‐Ca2+ exchange via [Na+]i in a restricted space of guinea‐pig ventricular cells
Authors: Fujioka Yasutada , Matsuoka Satoshi , Ban Toshihiko , Noma Akinori
Published: September 2004

The whole‐cell Na+‐K+ pump current (INa‐K) and Na+‐Ca2+ exchange current (INa‐Ca) were recorded in guinea‐pig ventricular myocytes to study the interaction between the two Na+ transport mechanisms.

I
Na‐K was isolated as an external K+‐induced current, and INa‐Ca as an external Ca2+‐ induced or Ni2+‐sensitive current. The experimental protocol used for one ion carrier did not affect the other.

The amplitude of INa‐K decreased to 54 ± 17 % of the initial peak during continuous application of K+ with 20 mM Na+ in the pipette. The outward INa‐Ca, which was intermittently activated by brief applications of Ca2+, decreased during activation of INa‐K, and recovered after cessation of INa‐K activation. These findings revealed a dynamic interaction between INa‐K and INa‐Ca via a depletion of Na+ under the sarcolemma.

To estimate changes in Na+ concentration ([Na+]i) under the sarcolemma, the reversal potential (Vrev) of INa‐Ca was measured. Unexpectedly, Vrev hardly changed during activation of INa‐K. However, when INa‐Ca was blocked by Ni2+ at the same time that INa‐K was activated, Vrev changed markedly, maximally by +100 mV, immediately after the removal of Ni2+ and K+.

Subsarcolemmal [Na+]i was calculated from the Vrev of INa‐Ca on the assumption that the subsarcolemmal Ca2+ concentration ([Ca2+]i) was fixed with EGTA, and mean [Na+]i was calculated from both the time integral of INa‐K and the cell volume. The subsarcolemmal [Na+]i was about seven times greater than the mean [Na+]i.

The interaction between the Na+‐K+ pump and Na+‐Ca2+ exchange was well simulated by a diffusion model, in which Na+ diffusion was restricted to one‐seventh (14 %) of the total cell volume.
Acid/base transport across the leech giant glial cell membrane at low external bicarbonate concentration
Authors: Deitmer Joachim W. , Schneider Hans‐Peter
Published: September 2004

We have studied acid/base transport across the cell membrane of the giant neuropile glial cell in the leech (Hirudo medicinalis) central nervous system induced by changing the external pH (pHo), using double‐barrelled, pH‐sensitive microelectrodes. In the presence of 5 % CO2 and 24 mm HCO3−, the intracellular pH (pHi) rapidly changes due to a potent, reversible Na+‐HCO3− cotransport across the glial membrane. We have now investigated the transport mechanism which leads to pHi changes in the nominal absence of CO2/HCO3−, where the HCO3− concentration is expected to be below 1 mm.

The intracellular pH increased and then decreased when pHo was altered from 7.4 to 7.8 and then 7.0 with a rate of increase of +0.026 ± 0.008 and a rate of decrease of −0.028 ± 0.009 pH units min−1 (± s.d., n= 49), indicating an acid/base flux rate of 0.64 and 0.71 mm min−1 across the glial membrane, respectively.

In the absence of external sodium (Na+replaced by N‐methyl‐D‐glucamine), pHi slowly decreased, and the rate of alkali and acid loading was reduced to 19 and 28 %, respectively, (n= 12). Amiloride (2 mm), which inhibits Na+‐H+ exchange, had no effect on the alkali/acid loading (n= 6).

The alkali and acid loading were not impaired after the removal of external chloride (Cl−o, replaced by gluconate; n= 11), but were significantly reduced by the anion transport inhibitor 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid (DIDS, 0.5 mm) to 23 and 16 %, respectively, of the control (P < 0.001; n= 5).

Alkali and acid loading were affected differently by manipulating the availability of residual HCO3−. After adding the membrane‐permeable carbonic anhydrase inhibitor ethoxyzolamide (EZA, 2 μm) to the saline, the acid loading, but not the alkali loading, was significantly reduced (by 25 %, P < 0.01), while lowering the residual CO2/HCO3− concentration in the saline by O2 bubbling significantly reduced the alkali loading (by 59 %, P < 0.02), but not the acid loading.

Changing the membrane holding potential in voltage‐clamped glial cells or raising the external K+ concentration to 30 mm had no significant effect on acid/base loading.

It is concluded that a residual HCO3− concentration of less than 1 mm in nominally CO2/HCO3−‐free salines and HCO3− produced endogenously in the glial cells support alkali and acid loading across the glial cell membrane, presumably by activation of the reversible Na+‐HCO3− cotransporter. The results suggest a very high selectivity and affinity of this cotransporter for HCO3−; they imply that HCO3−‐dependent processes may not be negligible even in the nominal absence of CO2/HCO3−, when the HCO3− concentration is expected to be in the submillimolar range.
Analysis of the periodicity of synaptic events in neurones in the superior cervical ganglion of anaesthetized rats
Authors: McLachlan Elspeth M. , Häbler Heinz‐Joachim , Jamieson John , Davies Philip J.
Published: September 2004

The patterns of on‐going synaptic events recorded intracellularly in neurones of superior cervical ganglia (SCG)of anaesthetized female rats were analysed by constructing inter‐event interval histograms, autocorrelograms, ln‐survivor curves and histograms triggered by the arterial pulse wave and by the intercostal EMG.

In 11/12 cells with on‐going frequencies > 0.5 Hz, one or two inputs were strong (i.e. always suprathreshold). In five cells, action potentials also arose from synaptic potentials with amplitudes close to threshold.

Synaptic events in 5/11 neurones tested were phase‐related to the arterial pressure wave (i.e. had cardiac rhythmicity, CR).

Synaptic events in 9/10 neurones tested (including all with CR) were phase‐related to the intercostal EMG and/or their autocorrelograms showed peaks at multiples of the respiratory interval (i.e. had respiratory rhythmicity, RR).

The intervals between all synaptic events were exponentially distributed in 8/12 neurones although intervals between single strong events showed peaks related to the respiratory cycle. Bursts occurred only by chance.

Event patterns could be simulated by combining events from several respiration‐modulated inputs with their timing distributed over nearly half the cycle. From the simulations, the mean number of active preganglionic inputs was estimated to be ≈6 with mean discharge frequency ≈0.4 Hz.

We conclude that, in the spontaneously breathing anaesthetized rat, most preganglionic neurones to the SCG fire with relatively low probability in relation to the respiratory cycle. Rhythms in a postganglionic neurone reflect the activity of its suprathreshold preganglionic inputs.
Properties and glial origin of osmotic‐dependent release of taurine from the rat supraoptic nucleus
Authors: Deleuze Charlotte , Duvoid Anne , Hussy Nicolas
Published: September 2004

Taurine, prominently concentrated in glial cells in the supraoptic nucleus (SON), is probably involved in the inhibition of SON vasopressin neurones by peripheral hypotonic stimulus, via activation of neuronal glycine receptors. We report here the properties and origin of the osmolarity‐dependent release of preloaded [3H]taurine from isolated whole SO nuclei.

Hyposmotic medium induced a rapid, reversible and dose‐dependent increase in taurine release. Release showed a high sensitivity to osmotic change, with a significant enhancement with less than a 5 % decrease in osmolarity. Hyperosmotic stimulus decreased basal release.

Evoked release was independent of extracellular Ca2+ and Na+, and was blocked by the Cl− channel blockers DIDS (4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid) and DPC (N‐phenylanthranilic acid), suggesting a diffusion process through volume‐sensitive Cl− channels.

Evoked release was transient for large osmotic reductions (≥ 15 %), probably reflecting regulatory volume decrease (RVD). However, it was sustained for smaller changes, suggesting that taurine release induced by physiological variations in osmolarity is not linked to RVD.

Basal and evoked release were strongly inhibited by an incubation of the tissue with the glia‐specific toxin fluorocitrate, but were unaffected by a neurotoxic treatment with NMDA, demonstrating the glial origin of the release of taurine in the SON.

The high osmosensitivity of taurine release suggests an important role in the osmoregulation of the SON function. These results strengthen the notion of an implication of taurine and glial cells in the regulation of the whole‐body fluid balance through the modulation of vasopressin release.
Role of protein kinase C in the induction of homosynaptic long‐term depression by brief low frequency stimulation in the dentate gyrus of the rat hippocampus in vitro
Authors: Wang Y. , Wu J. , Rowan M. J. , Anwyl R.
Published: September 2004

Enhancement of the induction of long‐term depression (LTD) of excitatory postsynaptic currents (EPSCs) by a priming stimulus was investigated in the medial perforant pathway of the dentate gyrus of the hippocampus in vitro.

In control, LTD could be induced by a conditioning low frequency stimulation (LFS) consisting of sixty, although not thirty or fewer, stimuli at 1 Hz applied at a holding potential of −40 mV.

A conditioning LFS of just five stimuli at 1 Hz was found to induce LTD if preceded 1–5 min, but not 15 min, by a priming LFS of five stimuli at 1 Hz, −40 mV, which did not by itself induce LTD.

A low concentration of the protein kinase C (PKC) activator (−)‐indolactam V, which did not itself induce LTD, reduced the threshold for the number of stimuli inducing LTD following the priming stimulus, while a high concentration of (−)‐indolactam V directly induced a depression of the test excitatory postsynaptic current (EPSC), which occluded LFS‐induced LTD. This suggests that the priming of LTD and also the direct induction of LTD involves the activation of PKC.

The pseudosubstrate peptide inhibitor PKC19–36 inhibited the induction of LTD by the priming protocol and by the control induction conditioning protocol.

These experiments demonstrate that a covert synaptic change involving generation of PKC is very effective in producing conditions whereby LTD is induced by very brief synaptic stimulation.