bluuebunny/crossref_metadata_2025_split huggingface dataset:
Oxygen deprivation inhibits Na+ current in rat hippocampal neurones via protein kinase C
Authors: O'Reilly John P. , Cummins Theodore R. , Haddad Gabriel G. 
Published: September 2004

Hippocampal neurones respond to acute oxygen deprivation (hypoxia) with an inhibition of whole‐cell Na+ current (INa), although the mechanism of the inhibition is unknown. Kinases can modulate INa and kinases are activated during hypoxia. We hypothesized that kinase activation may play a role in the hypoxia‐induced inhibition of INa.
Single electrode patch clamp techniques were used in dissociated hippocampal CA1 neurones from the rat. INa was recorded at baseline, during exposure to kinase activators (with and without kinase inhibitors), and during acute hypoxia (with and without kinase inhibitors).
Hypoxia (3min) reduced INa to 38.1 ± 4.5% of initial values, and shifted steady‐state inactivation in the negative direction. Hypoxia produced no effect on activation or fast inactivation.
Protein kinase A (PKA) activation with 2.5 mm adenosine 3′,5′‐cyclic adenosine monophosphate, N6O2‐dibutyryl, sodium salt (db‐cAMP) resulted in reduction of INa to 62.8 ± 5.5% without an effect on activation or steady‐state inactivation. INa was also reduced by activation of protein kinase C (PKC) with 5 nm phorbol 12‐myristate 13‐acetate (PMA; to 40.0 ± 3.7%) or 50 μm 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG; to 46.1 ± 2.8%). In addition, steady‐state inactivation was shifted in the negative direction by PKC activation. Neither the activation curve nor the kinetics of fast inactivation was altered by PKC activation.
The response to PKA activation was blocked by the PKA inhibitor N‐[2‐(p‐bromocinnamyl‐amino) ethyl]‐5‐isoquinolinesulphonamide (H‐89; 30 μm) and by 30 μm of PKA inhibitory peptide PKA5–24 (PKAi). PKC activation was blocked by the kinase inhibitor 1‐(5‐iso‐quinolinesulphonyl)‐2‐methylpiperazine (H‐7; 100 μm), by the PKC inhibitor calphostin C (10 μm), and by 20 μm of the inhibitory peptide PKC19–31 (PKCi).
The hypoxia‐induced inhibition of INa and shift in steady‐state inactivation were greatly attenuated with H‐7, calphostin C, or PKCi, but not with H‐89 or PKAi.
We conclude that hypoxia activates PKC in rat CA1 neurones, and that PKC activation leads to the hypoxia‐induced inhibition of INa.
Thrombin‐dependent calcium signalling in single human erythroleukaemia cells
Authors: Somasundaram Baggi , Mason Michael J. , Mahaut‐Smith Martyn P. 
Published: September 2004

A combination of single cell fluorescence and patch clamp techniques were used to study the mechanisms underlying thrombin‐evoked Ca2+ signals in human erythroleukaemia (HEL) cells, a leukaemic cell line of platelet–megakaryocyte lineage.
Thrombin caused a transient increase in intracellular Ca2+ ([Ca2+]j), consisting of both release of Ca2+ from intracellular stores and influx of extracellular Ca2+. Mn2+ quench studies indicated that the thrombin‐evoked divalent cation‐permeable pathway was activated during, but not prior to, release from internal stores.
Thapsigargin (1 μm) irreversibly released internal Ca2+ from the same store as that released by thrombin and continuously activated a Ca2+‐influx mechanism. The amplitude of the thrombin‐ and thapsigargin‐induced Ca2+ influx displayed a marked single cell heterogeneity which showed no correlation with the size of the store Ca2+ transient.
In whole‐cell patch clamp recordings, both thrombin and thapsigargin evoked an inwardly rectifying Ca2+ current which developed with little or no increase in current noise, showed no reversal in the voltage range −110 to +60 mV and was blocked by 1 mm Zn2+. The apparent divalent cation permeability sequence of this pathway was Ca2+≫ Ba2+ > Mn2+, Mg2+. The thapsigargin‐evoked current density at −100 mV varied between 0.42 and 2.1 pA pF−1 in different cells. Thrombin failed to activate additional Ca2+ current if it was added after the thapsigargin‐induced inward current had fully developed.
These studies indicate that thrombin activates Ca2+ influx in HEL cells entirely via a Ca2+‐store‐release‐activated Ca2+ current (ierac) rather than via receptor‐operated or second messenger‐dependent Ca2+ channels. The level of expression of Ierac appears to be a major factor in determining the duration of the thrombin‐evoked [Ca2+]i response and therefore represents a means by which cells can exert control over [Ca2+]i‐dependent events.
Dissociation between lactate and proton exchange in muscle during intense exercise in man
Authors: Bangsbo J. , Juel C. , Hellsten Y. , Saltin B. 
Published: September 2004

Transport of lactate, H+ and fluid across muscle sarcolemma was studied in contracting muscles under varying blood acid–base conditions.
Subjects performed two‐legged submaximal knee‐extensor exercise for 29–35 min consisting of warming up for 5 min followed by 10 min of leg exercise (L1), leg and arm exercise for 6–10 min (L2+A) and leg exercise for 10 min (L3). The experimental protocol was performed on two occasions; inspiring air (normoxia, N) or breathing 14% O2 in N2 (hypoxia, H). Leg blood flow was measured and femoral arterial and venous blood was sampled before and during each phase of exercise.
Arterial blood lactate concentration increased progressively during exercise to 5.9 ± 0.8 (N) and 8.2 ± 0.8 mmol l−1 (H) (P≤ 0.05) after 5.5 min of L2+A. Arterial blood pH was higher (P≤ 0.05) in H than in N, whereas arterial blood HCO3‐ concentrations were the same. Leg lactate release was higher in H than in N (3.1 ± 0.7 vs. 2.0 mmol l−1 (P≤ 0.05) during L1. In L2+A a net uptake of lactate was observed in both N and H. The concentration of lactate in the red blood cells increased during exercise to 2.3 ± 0.4 (N) and 4.3 ± 0.7 mmol l−1 (H) (P≤ 0.05) after 5.5 min of L2+A, but no red blood cell femoral arterial–venous lactate difference was observed.
Net proton release, estimated from actual base excess (ABE) adjusted for changes in reduced haemoglobin, was significant (P≤ 0.05) both at rest and during each phase of exercise. Furthermore, the difference between net proton and lactate release was positive throughout exercise and of similar magnitude in N and H.
The present data suggest that (1) H+ exchange in muscle during submaximal exercise can to a large extent occur through mechanisms other than via coupling to lactate; (2) muscle transport of H+ is insensitive to changes in blood pH in the range of 0.02–0.08 pH units; (3) transport of lactate across the membrane of red blood cells appears to be of minor importance for lactate release from active muscles.
Swelling‐ and cAMP‐Activated Cl− Currents in Isolated Rat Carotid Body Type I Cells
Authors: Carpenter E. , Peers C. 
Published: September 2004

In the whole‐cell configuration of the patch clamp technique, isolated rat carotid body type I cells exhibited reversible activation of Cl− currents during cell swelling effected by hypotonic extracellular solutions.
Hypotonic solutions evoked outwardly rectifying, non‐inactivating currents which showed time‐independent activation. The reversal potential (Erev) for the hypotonically evoked current was 1.6 ± 0.6 mV (n= 26). Reduction of extracellular Cl− from 133 to 65.5 mm caused a shift in Erev of ± 14.7 ± 0.4 mV (n= 5).
The swelling‐activated Cl− current could not activate when ATP was omitted from the patch pipette or when substituted for the non‐hydrolysable ATP analogues 5′‐adenylylimido‐diphosphate, AMP‐PNP (2 mm) or β,γ–methylene‐adenosine 5′‐triphosphate, AMP‐PCP (2 mm). The current also failed to activate in the absence of free intracellular Ca2+.
The swelling‐activated Cl− current was sensitive to blockade by the Cl− channel blockers niflumic acid (300 μm and 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid (DIDS; 200 μm), although the blockade by DIDS was voltage dependent.
A similar, non‐inactivating, outwardly rectifying Cl− current was evoked by the inclusion of cAMP (200 μm) in the patch pipette. This current could be inhibited by niflumic acid (300 μm) DIDS (200 μm) and hypertonic solutions, and was virtually abolished in the absence of intracellular ATP.
In conclusion, carotid body type I cells possess Cl− currents activated by cell swelling and rises in intracellular cAMP concentration. These currents may be involved in cell volume regulation, blood volume and osmolarity regulation and the response of the type I cell to chemostimuli.
Activation of small conductance Ca2+‐dependent K+ channels by purinergic agonists in smooth muscle cells of the mouse ileum
Authors: Vogalis Fivos , Goyal Raj K. 
Published: September 2004

Whole‐cell and single‐channel K+ currents were recorded at room temperature (22–24 °C), from smooth muscle cells enzymatically dispersed from the mouse ileum, using variations of the patch‐clamp technique.
Net outward K+ currents recorded through amphotericin‐B‐perforated patches in response to step depolarizations positive to −50 mV from a holding potential of −80 mV were decreased by up to 70% by external apamin (0.5/μM). Apamin‐sensitive whole‐cell currents were also recorded from cells perfused internally with 150 nm Ca2+ but not from cells perfused internally with 85nM Ca2+.
Three types of non‐inactivating Ca2+‐sensitive K+ channels were identified in cell‐attached and excised patches under an asymmetrical K+ gradient: (i) large conductance (BKCa; ∼200 pS) channels blocked by 2 mm external TEA; (ii) intermediate conductance (IKCa; ∼39 pS) channels blocked by 2mm external TEA and inhibited by external apamin (0.5 μm); and (iii) small conductance (SKCa; ∼10pS) channels that were not blocked by 5mm external TEA but were sensitive to extracellular apamin (0.5 μm).
The TEA‐resistant SKCa channels were activated by an increase in [Ca2+]i with an EC50 of 1.5 μm and a Hill coefficient of 1.3.
P2 purinoceptor agonists 2‐methylthioATP (2‐MeSATP), 2‐chloroATP and ATP (10–50μm) increased an apamin‐sensitive whole‐cell outward K+ current. Extrapatch application of 2‐MeSATP (20–100μm) stimulated the apamin‐sensitive IKCa and SKCa channels and activated an apamin‐sensitive steady outward current at 0mV.
Smooth muscle cells from the mouse ileum possess two apamin‐sensitive K+ channels (IKCa and SKCa); of these, the IKCa channels are TEA sensitive while the SKCa channels are TEA resistant. These channels, along with an apamin‐sensitive but TEA‐resistant steady outward current, may mediate membrane hyperpolarization elicited by purinergic agonists.
Regulation of the cytosolic Ca2+ concentration by Ca2+ stores in single smooth muscle cells from rat cerebral arteries
Authors: Kamishima T. , McCarron J. G. 
Published: September 2004

There is no general agreement on the presence or role of Ca2+‐induced Ca2+ release in smooth muscle. In this paper, Ca2+‐induced Ca2+ release has been investigated in rat resistance‐sized superior cerebral arteries to determine its role in regulating the cytosolic Ca2+ concentration ([Ca2+]i.
Pressurized superior cerebral arteries developed spontaneous oscillations in diameter. These oscillations were abolished by ryanodine (an inhibitor of Ca2+‐induced Ca2+ release) and removal of extracellular Ca2+. This suggests, indirectly, that Ca2+‐induced Ca2+ release may regulate [Ca2+]i in the resistance arteries.
To determine if Ca2+‐induced Ca2+ release could regulate [Ca2+]i, single smooth muscle cells were isolated from the superior cerebral artery, voltage clamped in the whole cell configuration and high temporal resolution [Ca2+]i measurements made. The relationship between the Ca2+ current (ICa) and rise in [Ca2+]i was examined.
Depolarization triggered ICa and increased [Ca2+]i. The time course of the measured increase in [Ca2+]i closely followed the increase in [Ca2+]i expected from the time‐integrated ICa, although about 140‐fold more Ca2+ entered the cytosol than appeared as free Ca2+. When the cells were dialysed with ryanodine (30 μm), the Ca2+ transient evoked by the ICa was substantially reduced indicating that Ca2+ influx triggered Ca2+ release from an internal store.
Voltage pulses to negative membrane potentials were more effective in triggering Ca2+ release than pulses to positive potentials suggesting that the Ca2+‐induced Ca2+ release was voltage dependent. However, the release of Ca2+ from the internal store triggered by caffeine was voltage independent. These results suggest that the voltage dependence of Ca2+ release is indirect and possibly related to the plasmalemma unitary Ca2+ current magnitude.
The results establish that Ca2+‐induced Ca2+ release contributes to depolarization‐evoked increases in [Ca2+]i in rat resistance‐sized superior cerebral arteries over the physiological [Ca2+]i range (100‐200 nm). Compared with more positive membrane potentials the efficacy of Ca2+ in triggering release is high at physiological membrane potentials.
Spatial heterogeneity of intracellular Ca2+ signals in axons of basket cells from rat cerebellar slices
Authors: Llano Isabel , Tan Yusuf P. , Caputo Carlo 
Published: September 2004

Using tight‐seal whole‐cell recording and digital fluorescence imaging, we studied intracellular calcium (Cai2+) dynamics in cerebellar basket cells, whose dendrites, axon and presynaptic terminals are coplanar, an optimal configuration for simultaneous optical measurements of all functional domains.
In Cs+‐loaded neurones, depolarizing pulses induced large Cai2+ transients in single axonal varicosities and synaptic terminals, contrasting with much weaker signals between varicosities or in the somato‐dendritic domain.
Axonal branch points consistently displayed [Cai2+] rises of similar magnitude and time course to those in axonal terminals and varicosities.
In biocytin‐filled basket cells, varicosity‐like swellings were present along the axon including its branch points. Thus, axonal enlargements are not due to fluorescence‐induced cell damage.
The spatial heterogeneity of Cai2+ signals was also observed in K+‐loaded cells upon depolarizing trains, suggesting that this behaviour is an intrinsic property of Cai2+ homeostasis in basket cells.
We conclude that depolarization of basket cell axons evokes high local Cai2+ signals in synaptic terminals, en passant varicosities and branch points. While high [Ca2+]1 in presynaptic structures presumably triggers transmitter release, Cai2+ transients at branch points may control signal transmission in the axonal arborization.
Evidence of two mechanisms for the activation of the glucose transporter GLUT1 by anisomycin: p38(MAP kinase) activation and protein synthesis inhibition in mammalian cells
Authors: Barros Luis Felipe , Young Michelle , Saklatvala Jeremy , Baldwin Stephen A. 
Published: September 2004

Inhibitors of protein synthesis stimulate sugar transport in mammalian cells through activation of plasma membrane GLUT1, the housekeeping isoform of the glucose transporter. However, it has been reported that some of these compounds, in addition to their effect on protein synthesis, also activate protein kinases.
In the present study we have explored the role of these two effects on GLUT1 activation. In 3T3‐L1 adipocytes and Clone 9 cells, stimulation of sugar transport by puromycin, a translational inhibitor that does not activate kinases, was not detectable until 90 min after exposure. In contrast, stimulation by anisomycin, a potent Jun‐NH2‐terminal kinase (JNK) agonist, exhibited no lag phase. An intermediate response was observed to emetine and cycloheximide, weak activators of JNK.
The potency of anisomycin to stimulate transport acutely (30 min of exposure) was 5‐ to 10‐fold greater than for its chronic stimulation of transport, measured after 4 h of exposure. The stimulation of transport by a low concentration of anisomycin (0.3 μm) was transient, peaked at 30–60 min and it was inhibited (IC50 < 1 μm) by SB203580, which indicates that its mediator is not JNK, but the homologous p38(MAP kinase) (p38(MAPK)). In contrast, the responses to 4 h exposure to 300 μm anisomycin or puromycin were refractory to SB203580.
Exposure to anisomycin resulted in rapid activation of p38(MAPK). Activation of both p38(MAPK) and GLUT1 by 0.3 μm anisomycin was cancelled by puromycin.
We conclude that the activation of GLUT1 in response to anisomycin includes two components: a delayed component involving translational inhibition and a fast, puromycin‐inhibitable component that is secondary to activation of p38(MAPK).
Leptin activates ATP‐sensitive potassium channels in the rat insulin‐secreting cell line, CRI‐G1
Authors: Harvey J. , McKenna F. , Herson P. S. , Spanswick D. , Ashford M. L. J. 
Published: September 2004

Whole‐cell current‐clamp recordings demonstrate that leptin (0.3–10 nm) hyperpolarizes CRI‐G1 insulin‐secreting cells. This effect is slow on onset and is not reversed on washout of the leptin.
Voltage‐clamp recordings indicate that leptin activates a potassium conductance in the presence of intracellular ATP (5 mm), but has no effect in its absence. Following activation of ATP‐sensitive K+ (KATP) current by diazoxide (0.2 mm), addition of leptin did not alter cell membrane potential or potassium current further.
The leptin‐induced hyperpolarization and increased potassium conductance are completely inhibited by the application of the sulphonylureas tolbutamide (100 μm) and glibenclamide (0.5 μm).
Cell‐attached and inside‐out single‐channel recordings indicate that leptin activates tolbutamide‐sensitive KATP channels in CRI‐G1 insulin‐secreting cells.
Effects of acetylcholine on the Na+–K+ pump current in guinea‐pig ventricular myocytes
Authors: Gao J. , Mathias R. T. , Cohen I. S. , Baldo G. J. 
Published: September 2004

The whole‐cell patch clamp technique was used to study the effects of acetylcholine (ACh) on Na+‐K+ pump current (Ip) in acutely isolated guinea‐pig ventricular myocytes. Studies were performed in the absence and presence of the β‐agonist isoprenaline (Iso).
ACh had no effect on Ip at low or high [Ca2+]i at any voltage in the absence of Iso. Iso alone inhibited Ip at low [Ca2+]i and shifted the Ip–V relationship at high [Ca2+]i in a negative direction. Addition of 1 μm ACh reversed these effects of Iso. K0.5 for the effects of ACh was about 16 nm, regardless of [Ca2+]i.
The actions of ACh on the heart are usually mediated via muscarinic receptors. Atropine, a muscarinic antagonist, blocked the effects of ACh on Ip in the presence of Iso, suggesting that these effects are also mediated by muscarinic receptors.
Muscarinic receptors are usually coupled to a Gi protein, leading to inhibition of adenylyl cyclase and a reduction of cAMP levels. We have shown previously that basal levels of cAMP are very low in guinea‐pig ventricular myocytes, and that a membrane‐permeant cAMP analogue, chlorophenylthio‐cAMP (CPTcAMP), mimics the effects of Iso. ACh did not reverse the effects of CPTcAMP, supporting the hypothesis that the effects of ACh on Ip are also mediated via inhibition of adenylyl cyclase.
The present results suggest that a high level of parasympathetic tone alone does not affect the activity of ventricular Na+–K+ pumps. However, if sympathetic tone is high, then muscarinic stimulation can reciprocally modulate Na+–K+ pump activity.
How the thyroid controls metabolism in the rat: different roles for triiodothyronine and diiodothyronines
Authors: Moreno Maria , Lanni Antonia , Lombardi Assunta , Goglia Fernando 
Published: September 2004

Although the first evidence of a relationship between the thyroid and metabolism was reported in 1895, the mechanism by which thyroid hormones influence resting metabolic rate in whole animals is still poorly understood. This paper reports an attempt to test whether diiodothyronines (T2s) and triiodothyronine (T3) have different roles in the control of resting metabolism (RM).
Changes in resting metabolic rate were measured in hypothyroid rats treated acutely (25μg (100 g body weight)−1) either with one of the T2s or with T3. Injection of T3 induced an increase of about 35% in RM that started 25–30 h after the injection and lasted until 5–6 days after the injection, the maximal value being observed at 50–75 h. The injection of T2s evoked a temporally different pattern of response. The increases in RM started 6–12 h after the injection, had almost disappeared after 48 h, and the maximal stimulation was observed at 28–30 h.
When actinomycin D (an inhibitor of protein synthesis) and T3 were given together, the stimulation of RM was almost completely abolished. The simultaneous injection of actinomycin D and either of the T2s, on the other hand, did not cause any attenuation of the stimulation seen with the T2s alone.
Following chronic treatment (3 weeks) with either T3 or T2s there was a stimulation of organ growth only after the administration of T3.
Chronic administration of either T2s or T3 to hypothyroid rats significantly enhanced the oxidative capacity of each of the tissues considered. In the case of T2s the stimulation was almost the same whether it was expressed as an increase in specific activity or total tissue activity. In the case of T3 the increases were, in the main, secondary to the hypertrophic or hyper plastic effect.
These results indicate that T2s and T3 exert different effects on RM. The effects of T2s are rapid and possibly mediated by their direct interaction with mitochondria. Those of T3 are slower and more prolonged, and at least partly attributable to a modulation of the cellularity of tissues that are metabolically very active.
Histamine H1 receptor activation blocks two classes of potassium current, IK(rest) and IAHP to excite ferret vagal afferents
Authors: Jafri M. Samir , Moore Kimberly A. , Taylor Glen E. , Weinreich Daniel 
Published: September 2004

Intracellular recordings were made in intact and acutely dissociated vagal afferent neurones (nodose ganglion cells) of the ferret to investigate the membrane effects of histamine.
In current‐clamp or voltage‐clamp recordings, histamine (10 μm) depolarized the membrane potential (10 ± 0.8 mV; mean ±s.e.m.; n= 27) or produced an inward current of 1.6 ± 0.35 nA (n= 27) in ∼80% of the neurones.
Histamine (10 μm) also blocked the post‐spike slow after‐hyperpolarization (AHPslow) present in 80% of these neurones (95 ± 3.2%; n= 5). All neurones possessing AHPslow in ferret nodose were C fibre neurones; all AHPslow neurones had conduction velocities ≤ 1 m s−1(n= 7).
Both the histamine‐induced inward current and the block of AHPslow were concentration dependent and each had an estimated EC50 value of 2 μm. These histamine‐induced effects were mimicked by the histamine H1 receptor agonist 2‐(2‐aminoethyl)thiazole dihydrochloride (10 μm) and blocked by the H1 antagonists pyrilamine (100 nm) or diphenhydramine (100 nm). Schild plot analysis of the effect of pyrilamine on the histamine‐induced inward current revealed a pA2 value of 9.7, consistent with that expected for an H1 receptor. Neither impromidine (10 μm) nor R(−)‐α‐methylhistamine (10 μm), selective H2 or H3 agonists, respectively, significantly affected the membrane potential, input resistance or AHPslow.
The reversal potential (Vrev) for the histamine‐induced inward current was −84 ± 2.1 mV (n= 4). The Vrev for the histamine response shifted in a Nernstian manner with changes in the extracellular potassium concentration. Alterations in the extracellular chloride concentration had no significant effect on the Vrev of the histamine response (n= 3). The Vrev for the AHPslow was –85 ± 1.7 mV (n= 4).
These results indicate that histamine increases the excitability of ferret vagal afferent somata by interfering with two classes of potassium current: the resting or ‘leak’ potassium current (IK(rest)) and the potassium current underlying a post‐spike slow after‐hyper‐polarization (IAHP). Both these effects can occur in the same neurone and involve activation of the same histamine receptor subtype, the histamine H1 receptor.