bluuebunny/crossref_metadata_2025_split huggingface dataset:
α‐Adrenergic stimulation of cytosolic Ca2+ oscillations and exocytosis in identified rat corticotrophs
Authors: Tse Amy , Tse Frederick W. 
Published: September 2004

The patch clamp technique was used in conjunction with a fluorescent Ca2+ indicator (indo‐1, or indo‐1FF) to measure simultaneously cytosolic Ca2+ concentration ([Ca2+]i), ionic current and changes in membrane capacitance in single rat corticotrophs identified with the reverse haemolytic plaque assay.
Application of the adrenocorticotropin (ACTH) secretagogue noradrenaline (NA; norepinephrine), triggered [Ca2+]i oscillation in corticotrophs via α‐adrenergic receptors and the guanosine trisphosphate (GTP) binding protein‐coupled phosphoinositide pathway.
Simultaneous measurement of [Ca2+]i and capacitance shows that exocytosis was triggered during the first cycle of NA‐induced [Ca2+]i oscillation and the mean increase in cell membrane surface area was 1.4 ± 0.3 % (n= 6).
When Ca2+ was directly released from the inositol 1,4,5 trisphosphate (IP3)‐sensitive store via flash photolysis of caged IP3, the mean increase in cell surface area was 1.5 ± 0.5 % (n= 6). Thus, NA‐stimulated ACTH secretion in rat corticotrophs is closely coupled to intracellular Ca2+ release.
Large and rapid elevation of [Ca2+]i (>15 μm) via flash photolysis of caged Ca2+ triggered two phases of exocytosis: a rapid exocytic burst that was complete in ∼100 ms and a slow burst that continued for many seconds.
The rapid exocytic burst reflected the exhaustion of a pool of readily releasable granules and, on average, increased the cell surface by 2.8 ± 0.1 % (n= 14).
We suggest that the relatively weak exocytic response in corticotrophs during intracellular Ca2+ release may be partially attributed to a smaller pool of readily releasable granules.
Multiple modulatory effects of dopamine on calcium channel kinetics in adult rat sensory neurons
Authors: Formenti Alessandro , Martina Marzia , Plebani Alessio , Mancia Mauro 
Published: September 2004

The aim of this research was to study the modulatory effects induced on high‐voltage‐activated (HVA) calcium channels and pharmacologically isolated subtypes through dopamine receptor activation.
The experiments were carried out on acutely isolated adult rat sensory neurons, recorded by means of the whole‐cell patch‐clamp technique.
At saturating concentrations dopamine was effective in inducing: (a) a voltage‐dependent prolongation of activation kinetics, (b) a voltage‐independent scaling down of the currents without any changes in activation and inactivation kinetics, and (c) an acceleration of inactivation kinetics, not affected by a positive conditioning prepulse.
These three inhibitory effects were observed on N‐ and P/Q‐type currents, whereas only a voltage‐independent scaling up and/or scaling down was observed on L‐type current.
The inhibitory effects were sometimes observed in isolation in different neurons, but more frequently they were variously combined in the same cell. A correlation analysis of these effects shows no relationship between them, corroborating the conclusion that they are mechanistically distinct.
The existence of an inactivating effect accounts for the occurrence of a voltage‐dependent inhibitory effect in some cells without an apparent slowing down of activation kinetics, since the increased inactivation may mask the slow component of the activation.
The multiple modulatory effects on calcium channels, even on pharmacologically separated N‐, L‐ and P/Q‐currents, suggest that pharmacological and functional classifications do not necessarily match completely.
The multiple modulatory effects on HVA calcium currents may play a prominent role both in controlling the integrative properties of neurons and in regulating output at a presynaptic level.
The effect of extracellularly applied divalent cations on cytosolic Ca2+ in murine Leydig cells: evidence for a Ca2+‐sensing receptor
Authors: Adebanjo Olugbenga A. , Igietseme Joseph , Huang Christopher L.‐H. , Zaidi Mone 
Published: September 2004

The effect of extracellularly applied divalent cations upon cytosolic Ca2+ levels ([Ca2+]) was investigated in fura‐2‐loaded mouse Leydig (TM3) cells.
The extracellular application of Ca2+ (2.5–15 mM) or Ni2+ (0.5–5 mM) elicited concentration‐dependent elevations in cytosolic [Ca2+] that were followed by decays to baseline levels. Extracellular Mg2+ (0.8–15 mM) failed to influence cytosolic [Ca2+].
Conditioning applications of Ca2+ (2.5–10 mM), Mg2+ (2.5–15 mM) or Ni2+ (0.5–5 mM) all attenuated the cytosolic Ca2+ response to a subsequent test application of 5 mM [Ni2+].
The amplitude of Ni2+‐induced cytosolic Ca2+ signals remained constant in low‐Ca2+ solutions. Such findings suggest a participation of Ca2+ release from intracellular stores. In parallel, depletion of Ca2+ stores by either ionomycin (5 μM, in low‐Ca2+ solutions) or thapsigargin (4 μM) abolished or attenuated Ni2+‐induced Ca2+ transients.
Ionomycin (5 μM) elevated cytosolic [Ca2+] in Ca2+‐free solutions even after prior Ni2+ application, indicating the presence of Ni2+‐insensitive stores.
Caffeine (250 and 500 μM) elevated cytosolic [Ca2+] and attenuated Ni2+‐induced Ca2+ release. Furthermore, TM3 cells stained intensely with a specific anti‐ryanodine receptor antiserum, Ab34. These findings suggest that Ca2+ release is regulated by ryanodine receptors.
Both membrane depolarization and hyperpolarization, brought about by changes in extracellular [K+] ([K+]e) in the presence of valinomycin (5 μM), altered the waveform of the Ni2+‐induced cytosolic Ca2+ signal. Hyperpolarization, in addition, diminished the response magnitude. Such voltage‐induced response modulation localizes the regulatory events to the Leydig cell plasma membrane.
We propose the existence of a cell surface divalent cation (Ca2+) receptor in Leydig cells, the activation of which triggers Ca2+ fluxes through ryanodine receptors.
The effect of polyamines on KATP channels in guinea‐pig ventricular myocytes
Authors: Niu X. W. , Meech R. W. 
Published: September 2004

The effect of natural polyamines on KATP channels was studied using inside‐out patches from guinea‐pig ventricular myocytes.
At a holding potential of +40 mV, spermine at the intracellular membrane surface reduced the KATP channel open probability (Popen) in a dose‐dependent manner. Half‐maximal inhibition occurred at 29 μM with a Hill coefficient of 1.2.
The effect of spermine on Popen was not greatly influenced by the membrane potential but there appeared to be a small reduction in unitary current amplitude during strong depolarizations.
Analysis of KATP single channel kinetics showed that spermine inhibited the channel by decreasing the mean open time and introducing transitions to a long closed state.
Spermidine (0.1 mM) was found to have a similar effect to spermine. Putrescine (10 mM) was found to block more effectively at positive membrane potentials. Up to 20 mM arginine had no significant effect on KATP channels.
Our results indicate that natural polyamines influence native KATP channel gating in cardiac myocytes.
Presynaptic calcium channels mediating synaptic transmission in submucosal neurones of the guinea‐pig caecum
Authors: Cunningham Shauna M. C. , Mihara Satoshi , Higashi Hideho 
Published: September 2004

Intracellular recording techniques were used to examine the voltage‐activated calcium channels mediating neurotransmitter release from nerve terminals of extrinsic, sympathetic origin and intrinsic (enteric) origin innervating submucosal neurones of the guinea‐pig caecum.
The noradrenergic slow inhibitory postsynaptic potential (IPSP) was abolished by superfusion of ω‐conotoxin (ω‐CTX) GVIA (3‐300 nM), with an apparent IC50 of 8.6 nM. Superfusion of ω‐CTX MVIIC (500 nM) also suppressed the amplitude of slow IPSPs, but both ω‐agatoxin IVA (100 nM) and nicardipine (1‐10 μM) were ineffective. The hyperpolarization induced by exogenous noradrenaline was not affected by ω‐CTX GVIA (100 nM).
In contrast to the slow IPSP, the amplitude of the cholinergic fast excitatory postsynaptic potential (EPSP) was partially inhibited, but not abolished, by ω‐CTX GVIA (0.1‐1 μM). Furthermore, ω‐agatoxin IVA (0.1‐1 μM) or ω‐CTX MVIIC (0.1‐1 μM) also affected the fast EPSP, but nicardipine (1–10 μM) was ineffective. In combination, ω‐CTX GVIA (100 nM) and ω‐agatoxin IVA (100 nM) inhibited the fast EPSP by 74 ± 6 %; the residual fast EPSP was not affected by ω‐CTX MVIIC (100 nM). The fast EPSP was completely abolished by low Ca2+, high Mg2+ Krebs solution or Krebs solution containing Co2+ (2 mM) and Cd2+ (400 μM). The depolarization induced by exogenous acetylcholine was not affected by either ω‐CTX GVIA (100 nM), ω‐agatoxin IVA (100 nM) or ω‐CTX MVIIC (100 nM).
Taken together, these results suggest that, in the submucosal plexus of the guinea‐pig caecum, release of noradrenaline from extrinsic nerve terminals is regulated by N‐type calcium channels, whereas release of acetylcholine from intrinsic nerve terminals involves several types of calcium channel.
Gap junctional communication and pharmacological heterogeneity in astrocytes cultured from the rat striatum
Authors: Venance Laurent , Prémont Joël , Glowinski Jacques , Giaume Christian 
Published: September 2004

Indo‐1 and fluo‐3 imaging techniques were used to investigate the role of gap junctions in the changes in cytosolic calcium concentrations ([Ca2+]i) induced by several receptor agonists. Subpopulations of confluent cultured astrocytes from the rat striatum were superfused with submaximal concentrations of endothelin‐1 (Et1) and the α1‐adrenergic and muscarinic receptor agonists, methoxamine and carbachol, respectively.
Combined binding and autoradiographic studies indicated that all striatal astrocytes possess binding sites for Et1. In contrast, α1‐adrenergic and muscarinic binding sites were found to be heterogeneously distributed. In agreement with these findings, Et1 induced fast calcium responses in all cells while only subsets of striatal astrocytes responded to the application of either methoxamine or carbachol.
Halothane, heptanol and octanol, which are commonly used as gap junction inhibitors, drastically reduced the amplitude of Et1‐induced calcium responses. In contrast, 18‐α‐glycyrrhetinic acid (αGA) used at a concentration known to block gap junction permeability in astrocytes had no significant effect on the amplitude of these calcium responses.
As demonstrated by quantitative and topological analysis, Et1 application similarly increased [Ca2+]i levels in all astrocytes in both the absence and presence of αGA.
In control conditions, subpopulations of cells responding to methoxamine or carbachol exhibited two main types of calcium responses which differed in their shape and kinetic characteristics. In the presence of αGA the number of cells responding to these receptor agonists was significantly reduced. Indeed, responses characterized by their long latency, slow rise time and weak amplitude disappeared in the presence of αGA while responses with short latency and fast rise time were preserved.
These results indicate that permeable gap junction channels tend to attenuate the pharmacological and functional heterogeneity of populations of astrocytes, while their inhibition restricts calcium responses in astrocytes expressing high densities of transmitter receptors coupled to phospholipase C.
Non‐specificity of chloride channel blockers in rat cerebral arteries: block of the L‐type calcium channel
Authors: Doughty Joanne M. , Miller Alastair L. , Langton Philip D. 
Published: September 2004

The effects of chloride channel blockers on pressure‐induced constriction, K+‐induced force, and whole‐cell calcium channel currents were tested in rat cerebral arteries using isobaric and isometric myography, and patch clamp.
Under isobaric conditions at 75 mmHg, NPPB), a chloride channel blocker, reversibly depressed the myogenic constriction with an IC50 of 32.8 ± 0.52 μm (mean ± s.e.m., n= 5). Blockers of Ca2+‐activated chloride channels, flufenamic acid (100 μm) and 9‐AC; 1 mm), and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel blocker, glibenclamide (100 μm), were without effect in this tissue (n= 3).
Under isobaric conditions at 20 mmHg, 37 °C, raising [K+]o to 45 mm induced a constriction which was unaffected by 100 μm NPPB (n= 4). In contrast, at 75 mmHg and 18‐21 °C, 100 μm NPPB completely and reversibly blocked a 45 mm K+‐induced constriction (n= 3).
Under isometric conditions, NPPB reversibly depressed a 45 mm K+‐induced force with an IC50 of 10.0 ± 0.76 μm (mean ± s.e.m., n= 5). IAA‐94), another chloride channel blocker, depressed the K+‐induced force with an IC50 of 17.0 ± 1.2 μm (mean ± s.e.m., n= 4).
Using whole‐cell patch clamp, 100 μm NPPB or 200 μm IAA‐94 blocked calcium channel currents carried by 10 mm Ba2+ by 79.1 ± 1.7 and 39.8 ± 7.0 %, respectively (mean ± s.e.m., n= 6).
In summary, chloride channel blockers depress calcium channel currents in rat cerebral arteries, which could contribute to a reduction in myogenic contraction.
Involvement of PKC‐α in regulatory volume decrease responses and activation of volume‐sensitive chloride channels in human cervical cancer HT‐3 cells
Authors: Chou Cheng‐Yang , Shen Meng‐Ru , Hsu Kuei‐Sen , Huang Hsueh‐Yin , Lin Hui‐Chuan 
Published: September 2004

The present study was carried out to identify the specific protein kinase C (PKC) isoform involved in regulatory volume decrease (RVD) responses, and to investigate the signal transduction pathways underlying the activation of volume‐sensitive chloride channels in human cervical cancer HT‐3 cells. The role of Ca2+ in RVD and in the activation of chloride currents was also studied.
The time course of RVDs was prolonged by microinjection of PKC‐α antibody but not by PKC‐β or PKC‐γ antibody, and also by exposure to Ca2+‐free medium, in particular when combined with microinjection of EDTA. Immunofluorescence staining showed that hypotonic superfusion evoked the translocation of PKC‐α to the cell membrane, whereas PKC‐β or PKC‐γ remained unaffected. The translocation of PKC‐α was observed a few minutes after hypotonic stress, reaching peak intensity at 30 min, and returned to the cytoplasm 60 min after hypotonic exposure. Western blot analyses showed an increased PKC‐α level in terms of intensity and phosphorylation in the cell membrane, while neither PKC‐β nor PKC‐γ was activated upon hyposmotic challenge.
Whole‐cell patch‐clamp studies demonstrated that neomycin and PKC blockers such as staurosporine and H7 inhibited volume‐sensitive chloride currents. The inhibitory effect of neomycin on chloride currents can be reversed by the PKC activator phorbol 12‐myristate, 13‐acetate (PMA). Moreover, the PKC inhibitor and PKC‐α antibody, but not PKC‐β or PKC‐γ antibody, significantly attenuated the chloride currents. The activation of volume‐sensitive chloride currents were insensitive to the changes of intracellular Ca2+ but required the presence of extracellular Ca2+.
Our results suggest the involvement of PKC‐α and extracellular Ca2+ in RVD responses and the activation of volume‐sensitive chloride channels in HT‐3 cells.
Dissociation between electrical and mechanical responses to nitrergic stimulation in the canine gastric fundus
Authors: Bayguinov Orline , Sanders Kenton M. 
Published: September 2004

We examined the relationships between membrane potential, intracellular [Ca2+] ([Ca2+]i), and tension in muscles of the canine gastric fundus in response to nitrergic stimulation by NO donors and electrical field stimulation (EFS) of intrinsic enteric inhibitory neurons when adrenergic and cholinergic responses were blocked.
NO donors reduced [Ca2+]i and tension in a concentration‐dependent manner. A close relationship was noted between these parameters.
In terms of the [Ca2+]vs. force relationship, relaxation responses to EFS differed from responses to NO donors. EFS resulted in smaller decreases in [Ca2+]i to produce a given relaxation compared with responses to NO donors. Thus, muscles stimulated with EFS were less sensitive to [Ca2+]i than muscles stimulated with exogenous NO.
When membrane potential, [Ca2+]i and tension were monitored simultaneously in the same muscles, a temporal dissociation was noted between the electrical responses and changes in [Ca2+]i and tension. Brief electrical responses were associated with more sustained changes in [Ca2+]i and tension.
Further dissociation between electrical and mechanical effects was noted. Changes in [Ca2+]i and tension caused by sodium nitroprusside and EFS were blocked by arginine analogues and by oxyhaemoglobin, but electrical responses were unaffected. 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ), an inhibitor of soluble guanylyl cyclase, also blocked the effects of nitrergic stimulation on [Ca2+]i and tension, without affecting hyperpolarization. Thus, in the presence of continued hyperpolarization, the reductions in [Ca2+]i and tension caused by nitrergic stimulation were blocked.
Block of hyperpolarization in response to nitrergic stimulation with tetrapentylammonium chloride (TPEA) had relatively little effect on the [Ca2+]i and tension responses. Thus, hyperpolarization is not required for nitrergic effects on [Ca2+]i and tension.
In summary, reduction in [Ca2+]i and tension in response to nitrergic stimulation of the canine gastric fundus does not depend upon electrical hyperpolarization. Non‐electrical mechanisms such as enhanced uptake of Ca2+ by the sarcoplasmic reticulum or reduction in the Ca2+ sensitivity of the contractile apparatus may be the primary mechanisms mediating nitrergic responses in these muscles.
Identification of an ATP‐sensitive potassium channel current in rat striatal cholinergic interneurones
Authors: Lee K. , Dixon A. K. , Freeman T. C. , Richardson P. J. 
Published: September 2004

Whole‐cell patch‐clamp recordings were made from rat striatal cholinergic interneurones in slices of brain tissue in vitro. In the absence of ATP in the electrode solution, these neurones were found to gradually hyperpolarize through the induction of an outward current at −60 mV. This outward current and the resultant hyperpolarization were blocked by the sulphonylureas tolbutamide and glibenclamide and by the photorelease of caged ATP within neurones.
This ATP‐sensitive outward current was not observed when 2 mM ATP was present in the electrode solution. Under these conditions, 500 μM diazoxide was found to induce an outward current that was blocked by tolbutamide.
Using permeabilized patch recordings, neurones were shown to hyperpolarize in response to glucose deprivation or metabolic poisoning with sodium azide (NaN3). The resultant hyperpolarization was blocked by tolbutamide.
In cell‐attached recordings, metabolic inhibition with 1 mM NaN3 revealed the presence of a tolbutamide‐sensitive channel exhibiting a unitary conductance of 44.1 pS.
Reverse transcription followed by the polymerase chain reaction using cytoplasm from single cholinergic interneurones demonstrated the expression of the ATP‐sensitive potassium (KATP) channel subunits Kir6.1 and SUR1 but not Kir6.2 or SUR2.
It is concluded that cholinergic interneurones within the rat striatum exhibit a KATP channel current and that this channel is formed from Kir6.1 and SUR1 subunits.
Intracellular recordings in thalamic neurones during spontaneous spike and wave discharges in rats with absence epilepsy
Authors: Pinault Didier , Leresche Nathalie , Charpier Stéphane , Deniau Jean‐Michel , Marescaux Christian , Vergnes Marguerite , Crunelli Vincenzo 
Published: September 2004

In vivo extracellular and intracellular recordings were performed from thalamocortical (TC) neurones in a genetic model of absence epilepsy (genetic absence epilepsy rats from Strasbourg) during spontaneous spike and wave discharges (SWDs).
Extracellularly recorded single units (n= 14) fired either a single action potential or a high frequency burst of up to three action potentials, concomitantly with the spike component of the spike‐wave complex.
Three main events characterized the intracellular activity of twenty‐six out of twenty‐eight TC neurones during SWDs: a small amplitude tonic hyperpolarization that was present throughout the SWD, rhythmic sequences of EPSP/IPSPs occurring concomitantly with the spike‐wave complexes, and a small tonic depolarization at the end of the SWD. The rhythmic IPSPs, but not the tonic hyperpolarization, were mediated by activation of GABAA receptors since they reversed in polarity at ‐68 mV and appeared as depolarizing events when recording with KCl‐filled electrodes.
The intracellular activity of the remaining two TC neurones consisted of rhythmic low threshold Ca2+ potentials, with a few EPSP/IPSP sequences present at the start of the SWD.
These results obtained in a well‐established genetic model of absence epilepsy do not support the hypothesis that the intracellular activity of TC neurones during SWDs involves rhythmic sequences of GABAB IPSPs and low threshold Ca2+ potentials.
Ca2+‐permeable non‐NMDA glutamate receptors in rat magnocellular basal forebrain neurones
Authors: Waters D. Jack , Allen Timothy G. J. 
Published: September 2004

Ionotropic glutamate receptor‐mediated responses were recorded from rat magnocellular basal forebrain neurones under voltage clamp from a somatically located patch‐clamp pipette. Currents were recorded from both acutely dissociated neurones and neurones maintained in culture for up to 6 weeks.
Non‐NMDA and NMDA receptor‐mediated events could be distinguished pharmacologically using the selective agonists (S)‐α‐amino‐3‐hydroxy‐5‐methyl‐isoxazolepropionic acid (AMPA), kainate and N‐methyl‐D‐aspartate (NMDA), and antagonists 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and D(‐)‐2‐amino‐5‐phosphonopentanoic acid (AP5).
Responses to rapid application of AMPA displayed pronounced and rapid desensitization. Responses to kainate showed no desensitization. Steady‐state EC50 values for AMPA and kainate were 2.7 ± 0.4 μM (n= 5) and 138 ± 25 μM (n= 10), respectively. Cyclothiazide markedly increased current amplitude of responses to both agonists, whereas concanavalin A had no clear effect on either response. The selective AMPA receptor antagonist GYKI 53655 inhibited responses to kainate with an IC50 of 1.2 ± 0.08 μM (n= 5) at ‐70 mV. These data strongly suggest that AMPA receptors are the predominant non‐NMDA receptors expressed by basal forebrain neurones.
At ‐70 mV, approximately 6 % of control current amplitude remained, at a maximally effective concentration of GYKI 53655. This residual response displayed desensitization, was insensitive to cyclothiazide and was potentiated by concanavalin A, suggesting that it was mediated by a kainate receptor.
Current‐voltage relationships for non‐NMDA receptor‐mediated currents were obtained from both nucleated patches pulled from neurones in culture and from acutely dissociated neurones. With 30 μM spermine in the recording pipette, currents frequently displayed double‐rectification characteristic of non‐NMDA receptors with high Ca2+ permeabilities. Ca2+ permeability, relative to Na+ and Cs+, was investigated using constant field theory. The measured Ca2+ to Na+ permeability coefficient ratio was 0.26‐3.6; median, 1.27 (n= 15).
Current flow through non‐NMDA receptors was inhibited by Ca2+, Cd2+ and Co2+ ions. At a holding potential of ‐70 mV, a maximally effective concentration of Cd2+ (> 30 mM) reduced current amplitude by approximately 90 %, with an IC50 of 44 μM. In six out of seven cells tested, block by Cd2+ was voltage sensitive.
Ca2+ permeability of many of the non‐NMDA receptors expressed by magnocellular basal forebrain neurones may underlie the unusual sensitivity of cholinergic basal forebrain neurones to non‐NMDA receptor‐mediated excitotoxicity.