bluuebunny/crossref_metadata_2025 huggingface dataset:
The tetravalent organic cation spermine causes the gating of the IRK1 channel expressed in murine fibroblast cells.
Authors: Ishihara K , Hiraoka M , Ochi R
Published: December 2014

1. The activation kinetics of the IRK1 channel stably expressed in L cells (a murine fibroblast cell line) were studied under the whole‐cell voltage clamp. Without polyamines or Mg2+ in the pipettes, inward currents showed an exponential activation on hyperpolarization. The steep inward rectification of the currents around the reversal potential (Erev) could be described by the open‐close transition of the channel with first‐order kinetics. 2. When the tetravalent organic cation spermine (Spm) was added in the pipettes, the activation kinetics changed; this was explicable by the increase in the closing rate constant. The activation of the currents observed without Spm or Mg2+ in the pipettes was ascribed to the unblocking of the ‘endogenous‐Spm block’. 3. In the presence of the divalent cation putrescine (Put) or of Mg2+ in the pipettes, a different non‐conductive state suppressed the outward currents on depolarization; the channels instantaneously changed to the open state on repolarization. As the depolarization was prolonged, this non‐conductive state was replaced by the non‐conductive state that shows an exponential activation on repolarization. This phenomenon was attributed to the redistribution of the channels from the Put‐ or Mg(2+)‐blocked state to the ‘endogenous Spm‐blocked state’ during depolarization. 4. In the presence of the trivalent cation spermidine (Spd) in the pipettes, two different non‐conductive states occurred, showing a faster and a slower activation on repolarization. The rectification around Erev was mainly due to the non‐conductive state showing a faster activation, which appeared to be the Spd‐blocked state. During depolarization, redistribution of the channels to the ‘endogenous Spm‐blocked state’ also occurred. 5. In the presence of Spd, Put or Mg2+ in the pipettes, the voltage dependence of the activation time constant reflecting the unblocking of the ‘endogenous Spm’ was shifted in the hyperpolarizing direction. 6. Our results suggest that the ‘intrinsic gating’ that shows the time‐dependent activation on repolarization, and that is responsible for the inward rectification around Erev, reflects the blocking kinetics of the tetravalent Spm.
KCl cotransport activation in human erythrocytes by high hydrostatic pressure.
Authors: Godart H , Ellory J C
Published: December 2014

1. Pressure induced a 4‐ to 5‐fold stimulation of the residual (i.e. oubain‐bumetanide insensitive) 86Rb+ influx across the human red cell membrane. This enhancement showed a broad pHo dependence with a maximum stimulation around pHo 7. 2. At atmospheric pressure, the protein kinase inhibitors staurosporine and chelerythrine stimulated a normally silent component of 86Rb+ influx in a dose‐dependent manner with a half‐maximum stimulatory concentration at about 550 nM and 140 microM, respectively. The component stimulated by staurosporine was entirely Cl‐ dependent, but part of the chelerythrine effect was Cl‐ independent. 3. Staurosporine (3 microM), chelerythrine (200 microM) and N‐ethylmaleimide (1 mM) stimulated further the increased residual 86Rb+ influx in cells at high pressure. 4. The serine/threonine protein phosphatase inhibitors okadaic acid, cantharidin and calyculin A inhibited the stimulatory pressure effect in a dose‐dependent manner with half‐maximum inhibitory concentrations of 70 nM, 2.5 microM and 3.3 nM, respectively. In contrast, deltamethrin, a specific protein phosphatase type 2B inhibitor, did not affect the stimulation by pressure, up to a concentration of 10 microM. 5. Decreasing the internal ionized magnesium concentration ([Mg2+]i) with A23187 and EDTA stimulated the increased residual 86Rb+ influx in cells at high pressure. On the other hand, increasing the [Mg2+]i nearly abolished the stimulatory pressure effect. 6. Decreasing the [Mg2+]i produced a marked change in the pHo dependence curve, with a linear increase of the 86Rb+ influx at higher pHo values. 7. We demonstrate that high pressure stimulates the normally silent component of 86Rb+ influx by modifying the phosphorylation/dephosphorylation ratio of the KCl cotransporter.
Acetylcholine activates two currents in guinea‐pig outer hair cells.
Authors: Evans M G
Published: December 2014

1. Whole‐cell voltage clamp recordings were made from outer hair cells (OHCs) isolated from the basal and middle regions of the cochlea. The current produced when acetylcholine (ACh) was applied to the basal pole was investigated. 2. Acetylcholine (50‐70 microM) activated an early inward current followed by a late outward current in most cells at ‐70 mV. The activation of the early current was very rapid, with no delay. 3. The late outward current was a K+ current and was the major component of the total ACh‐sensitive current at negative voltages. 4. The ACh‐sensitive current was voltage dependent. From tail current measurements, the current was maximal at ‐55 mV and declined steeply at more positive potentials. On average, 50% of the current was active at ‐22 mV and 10% was active at 0 mV. This decline was caused by a reduction in the K+ current with depolarization. Between +20 and +40 mV, the major component of the ACh‐sensitive current was the early current. 5. The steady‐state I‐V curve for the ACh‐sensitive current was N‐shaped, with a peak at ‐36 mV. The instantaneous I‐V curve for the ACh‐sensitive current taken from measurements just after a voltage step was outwardly rectifying and did not show this peak. This difference occurred because the voltage dependence was time dependent. 6. The reversal potential for the early current was estimated to be close to+13 mV, in accordance with it being a non‐specification current. 7. The K+ current was abolished by the removal of external Ca2+. The effect occurred with no measurable delay. When external Ca2+ was lowered to 0.4 mM, the peak of the steady‐state I‐V curve for the ACh‐sensitive current shifted by up to ‐20 mV and its amplitude was reduced. These results suggested that the K+ current was dependent on Ca2+ influx. 8. The inward current usually remained when the K+ current was abolished or greatly reduced by removing external Ca2+ or by dialysing the cells with BAPTA (5‐10 mM). 9. Both early and late currents were reversibly blocked by alpha‐bungarotoxin (0.2 microM) and curare (1 microM). 10. A simple scheme is proposed to account for the response. ACh, binding to a nicotinic receptor, directly gates the early cation current. Part of this current is carried by Ca2+, which once inside the cell leads to the activation of a Ca(2+)‐dependent K+ current.
Depolarization‐evoked increases in cytosolic calcium concentration in isolated smooth muscle cells of rat portal vein.
Authors: Kamishima T , McCarron J G
Published: December 2014

1. Ca2+ current through voltage‐dependent Ca2+ channels (ICa) and intracellular free Ca2+ concentration ([Ca2+]i) were measured simultaneously in rat portal vein smooth muscle cells using conventional whole‐cell voltage clamp technique and high temporal resolution microfluorimetry. 2. The relationship between depolarization‐evoked ICa and rise in [Ca2+]i was examined. The extracellular Ca2+ concentration dependence and the voltage dependence of the depolarization‐evoked increases in ICa and [Ca2+]i were similar. Both ICa and increased [Ca2+]i were blocked to a similar extent by nimodipine and cadmium and augmented by Bay K 8644. Furthermore, the time course of the measured increase in [Ca2+]i, closely followed the increase in [Ca2+]i expected from the time‐integrated ICa. These observations suggest that the depolarization‐evoked rise in [Ca2+]i was tightly coupled to ICa. 3. The cytosolic Ca2+ buffering capacity, determined as the ratio of the expected increase in [Ca2+]i (from ICa) divided by the measured increase in [Ca2+]i, was over 100. Therefore, less than 1 out of 100 Ca2+ ions entering the cell appears as a free Ca2+. 4. Ryanodine (30 microM), a blocker of the Ca(2+)‐induced Ca2+ release mechanism, had little effect on buffering capacity measured over the first 200 ms of the depolarizing voltage clamp pulse. Ryanodine also had little effect on the buffering capacity during 800‐1000 ms of the depolarizing voltage clamp pulse. Therefore, it was concluded that there is little Ca(2+)‐induced Ca2+ release from the stores in rat portal vein smooth muscle cells during depolarization‐evoked Ca2+ entry. 5. During brief depolarizations, the largest [Ca2+]i increase and ICa occurred at 0 mV. However, during steady‐state depolarization, the largest increase in [Ca2+]i occurred around ‐30 mV, and we estimate the peak steady‐state ICa to be about 0.6 pA.
Proton sensitivity of the GABA(A) receptor is associated with the receptor subunit composition.
Authors: Krishek B J , Amato A , Connolly C N , Moss S J , Smart T G
Published: December 2014

1. Modulation of GABA(A) receptors by external H(+) was examined in cultured rat sympathetic neurones, and in Xenopus laevis oocytes and human embryonic kidney (HEK) cells expressing recombinant GABA(A) receptors composed of combinations of alpha 1, beta 1, beta 2, gamma 2S and delta subunits. 2. Changing the external pH from 7.4 reduced GABA‐activated currents in sympathetic neurones. pH titration of the GABA‐induced current was fitted with a pH model which predicted that H(+) interact with two sites (PK(a) values of 6.4 and 7.2). 3. For alpha 1 beta 1 GABA(A) receptors, low external pH (< 7.4) enhanced responses to GABA. pH titration predicted the existence of two sites with PK(a) values of 6.6 and 7.5. The GABA concentration‐response curve was shifted to the left by low pH and non‐competitively inhibited at high pH (> 7.4). 4. alpha 1 beta 1 gamma 2S receptor constructs were not affected by external pH, whereas exchanging the beta 1 subunit for beta 2 conferred a sensitivity to pH, with predicted PK(a) values of 5.16 and 9.44. 5. Low pH enhanced the responses to GABA on alpha 1 beta 1 delta subunits, whilst high pH caused an inhibition (PK(a) values of 6.6 and 9.9). The GABA concentration‐response curves were enhanced (pH 5.4) or reduced (pH 9.4) with no changes in the GABA EC(50). 6. Immunoprecipitation with subunit and epitope‐specific antisera to alpha 1, beta 1 and delta subunits demonstrated that these subunits could co‐assemble in cell membranes. 7. Expression of alpha 1 beta 1 gamma 2S delta constructs resulted in a ‘bell‐shaped’ pH titration relationship. Increasing or decreasing external pH inhibited the responses to GABA. 8. The pH sensitivity of recombinant GABA(A) receptors expressed in HEK cells was generally in accordance with data accrued from Xenopus oocytes. However, rapid application of GABA to alpha 1 beta 1 constructs at high pH (> 7.4) caused an increased peak and reduced steady‐state current, with a correspondingly increased rate of desensitization. 9. Modulation of GABA(A) receptor function was apparently unaffected by the internal pH. Moreover, pH values between 5 and 9.5 did not significantly affect the charge distribution on the zwitterionic GABA molecules. 10. In conclusion, this study demonstrates that external pH can either enhance, have little effect, or reduce GABA‐activated responses, and this is apparently dependent on the receptor subunit composition. The potential importance of H(+) sensitivity of GABA(A) receptors is discussed.
Membrane potential difference and intracellular cation concentrations in human placental trophoblast cells in culture.
Authors: Greenwood S L , Clarson L H , Sides M K , Sibley C P
Published: December 2014

1. The electrochemical gradients for Na+ and K+ were assessed in a cell culture model of trophoblast differentiation. 2. Membrane potential difference (Em), intracellular water and Na+ and K+ contents were measured in choriocarcinoma cells (JAr cell line; 96% of which are undifferentiated trophoblast cells) and in mononucleate and multinucleate (differentiated) cytotrophoblast cells isolated from the human placenta at term. 3. There was a significant fall in Em from ‐57 mV in JAr cells, to ‐48 and ‐40 mV in mono‐and multinucleate cytotrophoblast cells, respectively. Treatment with ouabain (1 mM for 15 min) depolarized the JAr cell membrane by 15 mV but did not affect cytotrophoblast cell membrane potential. 4. Intracellular K+ concentration was similar in JAr, mono‐ and multinucleate cytotrophoblast cells but Na+ concentration was higher in mononucleate cytotrophoblast cells compared with JAr cells. 5. Ouabain treatment (3 mM for 15 min) caused a small increase (4.5%) in cell water in mononucleate cytotrophoblast cells but lowered K+ (approximately 30%) and increased Na+ concentration (approximately 125%) in all the trophoblast cells studied. 6. The K+ equilibrium potential (EK) was more negative than Em in all cells and the difference between EK and Em was smaller in JAr cells (‐25 mV) than in mono‐ and multinucleate cytotrophoblast cells (‐33 and ‐43 mV, respectively). 7. The Na+ equilibrium potential (ENa) was positive in the trophoblast cells and the difference between ENa and Em was 122, 100 and 100 mV in JAr, mono‐ and multinucleate cytotrophoblast cells, respectively. 8. These results suggest that the electrochemical gradient for K+ is affected by the stage of trophoblast cell differentiation. In contrast, the electrochemical gradient for Na+ is similar in mono‐ and multinucleate cytotrophoblast cells.
Tensile strength and dilatational elasticity of giant sarcolemmal vesicles shed from rabbit muscle.
Authors: Nichol J A , Hutter O F
Published: December 2014

1. Mechanical properties of the surface membrane of skeletal muscle were determined on sarcolemmal vesicles (mean diameter, 71 microns) shed by rabbit psoas muscle swelling in 140 mM KC1 containing collagenase. 2. Vesicles were stressed by partial aspiration into parallel bore pipettes. The isotropic membrane tension so created caused an increase in membrane area which expresses itself in an elongation of the vesicle projection into the pipette. 3. For individual vesicles, a linear relationship between membrane tension and membrane area increase was found up to the point when the vesicle burst, i.e. sarcolemmal vesicles behaved as perfectly elastic structures. 4. The maximum tension sarcolemmal vesicles could sustain before bursting was 12.4 +/‐ 0.2 mN m‐1 (median +/‐ 95% confidence interval), and the corresponding fractional increase in membrane area was 0.026 +/‐ 0.005 (median +/‐ 95% confidence interval). The elastic modulus of area expansion was 490 +/‐ 88 mN m‐1 (mean +/‐ S.D.). 5. In conformity with cited comparable work on red blood cells and artificial lipid vesicles, the strength and area elasticity of the skeletal muscle membrane are considered properties of the fluid lipid matrix of the membrane and of the degree to which the bilayer is perturbed by lipid‐protein interaction.
High Ca2+ concentrations induce a low activity mode and reveal Ca2(+)‐independent long shut intervals in BK channels from rat muscle.
Authors: Rothberg B S , Bello R A , Song L , Magleby K L
Published: December 2014

1. Large‐conductance calcium‐activated K+ channels (BK channels) often display long closed intervals at higher levels of Ca2+. To gain further insight into possible mechanisms for these intervals, currents were recorded from single BK channels, using the patch clamp technique, from patches of membrane excised from primary cultures of rat skeletal muscle. 2. High intracellular calcium concentrations ([Ca2+]i; 10‐1000 microM) induced a low activity mode and revealed isolated long shut intervals. Neither of these phenomena were due to the Ba2+ that typically contaminates reagent grade salts. 3. The low activity mode was characterized by typically single brief open intervals with mean durations of 0.1 ms, separated by long shut intervals with mean durations of 100 ms. The very low open probability of about 0.001 during the low activity mode would make a sojourn to this mode functionally equivalent to a sojourn to an inactive state. The durations of sojourns in the low activity mode were exponentially distributed, with the mean durations ranging from about 1 s in 10 microM Ca(i)2+, to 4.5 s in 1000 microM Ca(i)2+. With increased filtering, the brief open intervals would escape detection so that a sojourn to the low activity mode would appear as a single shut interval. A typical channel spent less than 5% of its time in the low activity mode for [Ca2+]i < 10 microM. This increased to about 30% for [Ca2+]i > 100‐1000 microM. A kinetic model with three closed states and two open states could approximate the gating of the low activity mode. 4. The isolated long shut intervals were not from the low activity mode, suggesting a different underlying mechanism. Their frequency of occurrence of about 0.3 s‐1 did not increase with increasing [Ca2+]i, indicating that they did not arise from a slow Ca2+ block. Their durations were exponentially distributed, with a mean of 127 ms, which was independent of [Ca2+]i, suggesting that a single Ca(2+)‐independent closed state or block underlies the isolated long shut intervals. At higher [Ca2+]i, up to 60% of the shut time could be spent in the isolated long shut intervals. 5. These observations suggest that activation of BK channels by high [Ca2+]i can be limited by sojourns to a low activity mode and also by isolated long shut intervals, two additional phenomena that will have to be accounted for in the gating of BK channels.
Effects of extracellular pH on voltage‐gated Na+, K+ and Ca2+ currents in isolated rat CA1 neurons.
Authors: Tombaugh G C , Somjen G G
Published: December 2014

1. The effects of extracellular H+ (pHo) in the pathophysiological range (pH 6‐8) on voltage‐gated sodium, potassium, and calcium currents were examined in acutely dissociated rat hippocampal CA1 neurons using the whole‐cell patch clamp technique. All experiments were conducted in Hepes‐buffered solutions and were performed at room temperature (21‐23 degrees C). 2. TTX‐sensitive sodium currents, evoked by both step and ramp depolarization, were reversibly depressed by moderate acidosis and enhanced slightly by alkaline exposure. Changes in current amplitude were coincident with small reversible shifts (+/‐ 3 mV) in the voltage dependence of activation. In contrast, sodium current activation and decay kinetics as well as steady‐state inactivation were unaffected by acidosis. 3. Outward potassium currents could be separated into a transient, rapidly inactivating current (IA) and a sustained, slowly inactivating component (IK). Steady‐state activation of both currents was unaffected by an increase or decrease in pHo. Similarly, IK activation and IA decay kinetics remained stable during pHo exchange. In contrast, the steady‐state inactivation (h infinity) of both potassium currents was reversibly shifted by approximately +10 mV during acid exposure, but remained unchanged during alkaline treatment. 4. Calcium currents were found to be predominantly of the high‐voltage‐activated (HVA) type, which could be carried by Ba2+ and inhibited completely by cadmium. Moderate acidosis (pH 6.9‐6.0) reversibly depressed HVA Ca2+ current amplitude and caused a positive shift in its voltage dependence. For both of these parameters, alkaline treatment (pH 8.0) had the opposite effect. The depression of HVA Ca2+ currents by low pHo was unaffected by raising the internal Hepes concentration from 10 to 50 mM in the patch pipette. A Hill plot of the effect of pH on Ca2+ current amplitude revealed a pK value (defined as the mid‐point of the titration curve) of 7.1 and a slope of 0.6. 5. The rate of Ca2+ current activation was unaffected by pHo at positive potentials, but below 0 mV the activation rate increased at low pH and decreased at high pH, becoming significant at ‐20 mV. At this membrane voltage, a second HVA current was revealed during acid exposure as the whole‐cell HVA current was depressed. Ca2+ current decay was described by two time constants, both of which were significantly reduced at pH 6.4 and slightly enhanced at pH 8.0. Steady‐state Ca2+ current inactivation reached 50% near ‐50 mV and was not affected at either pH extreme. 6. These results demonstrate that extracellular pH shifts within the pathophysiological range are capable of modulating both the conductance and gating properties of voltage‐gated ion channels in hippocampal CA1 neurons. The effects we describe are consistent with the wellknown effects of pHo on neuronal excitability and strengthen the idea that endogenous pHo shifts may help regulate cell activity in situ.
External pore residue mediates slow inactivation in mu 1 rat skeletal muscle sodium channels.
Authors: Balser J R , Nuss H B , Chiamvimonvat N , Pérez-García M T , Marban E , Tomaselli G F
Published: December 2014

1. Upon depolarization, voltage‐gated sodium channels assume non‐conducting inactivated states which may be characterized as ‘fast’ or ‘slow’ depending on the length of the repolarization period needed for recovery. Skeletal muscle Na+ channel alpha‐subunits expressed in Xenopus laevis oocytes display anomalous gating behaviour, with substantial slow inactivation after brief depolarizations. We exploited this kinetic behaviour to examine the structural basis for slow inactivation. 2. While fast inactivation in Na+ channels is mediated by cytoplasmic occlusion of the pore by III‐IV linker residues, the structural features of slow inactivation are unknown. Since external pore‐lining residues modulate C‐type inactivation in potassium channels, we performed serial cysteine mutagenesis in the permeation loop (P‐loop) of the rat skeletal muscle Na+ channel (mu 1) to determine whether similarly placed residues are involved in Na+ channel slow inactivation. 3. Wild‐type and mutant alpha‐subunits were heterologously expressed in Xenopus oocytes, and Na+ currents were recorded using a two‐electrode voltage clamp. Slow inactivation after brief depolarizations was eliminated by the W402C mutation in domain I. Cysteine substitution of the homologous tryptophan residues in domains II, III and IV did not alter slow inactivation. 4. Analogous to the W402C mutation, coexpression of the wild‐type alpha‐subunit with rat brain Na+ channel beta 1‐subunit attenuated slow inactivation. However, the W402C mutation imposed a delay on recovery from fast inactivation, while beta 1‐subunit coexpression did not. We propose that the W402C mutation and the beta 1‐subunit modulate gating through distinct mechanisms. 5. Removal of fast inactivation in wild‐type alpha‐subunits with the III‐IV linker mutation I1303Q; F1304Q; M1305Q markedly slowed the development of slow inactivation. We propose that slow inactivation in Na+ channels involves conformational changes in the external pore. Mutations that affect fast and slow inactivation appear to interact despite their remote positions in the channel.
Eccentric exercise decreases maximal insulin action in humans: muscle and systemic effects.
Authors: Asp S , Daugaard J R , Kristiansen S , Kiens B , Richter E A
Published: December 2014

1. Unaccustomed eccentric exercise decreases whole‐body insulin action in humans. To study the effects of one‐legged eccentric exercise on insulin action in muscle and systemically, the euglycaemic clamp technique combined with arterial and bilateral femoral venous catheterization was used. Seven subjects participated in two euglycaemic clamps, performed in random order. One clamp was preceded 2 days earlier by one‐legged eccentric exercise (post‐eccentric exercise clamp (PEC)) and one was without the prior exercise (control clamp (CC)). 2. During PEC the maximal insulin‐stimulated glucose uptake over the eccentric thigh was marginally lower when compared with the control thigh, (11.9%, 64.6 +/‐ 10.3 vs. 73.3 +/‐ 10.2 mumol kg‐1 min‐1, P = 0.08), whereas no inter‐thigh difference was observed at a submaximal insulin concentration. The glycogen concentration was lower in the eccentric thigh for all three clamp steps used (P < 0.05). The glucose transporter GLUT4 protein content was on average 39% lower (P < 0.05) in the eccentric thigh in the basal state, whereas the maximal activity of glycogen synthase was identical in the two thighs for all clamp steps. 3. The glucose infusion rate (GIR) necessary to maintain euglycaemia during maximal insulin stimulation was lower during PEC compared with CC (15.7%, 81.3 +/‐ 3.2 vs. 96.4 +/‐ 8.8 mumol kg‐1 min‐1, P < 0.05). 4. Our data show that 2 days after unaccustomed eccentric exercise, muscle and whole‐body insulin action is impaired at maximal but not submaximal concentrations. The local effect cannot account for the whole‐body effect, suggesting the release of a factor which decreases insulin responsiveness systemically.
Kinetics of desensitization induced by saturating flashes in toad and salamander rods.
Authors: Murnick J G , Lamb T D
Published: December 2014

1. The suction pipette technique was used to examine the effect of a conditioning pre‐flash on the saturation time (tsat) of a bright test flash (intensity 10,000‐250,000 isomerizations) delivered to intact salamander or toad rod outer segments. The conditioning flash was delivered 0‐60 s before the test flash; its intensity was typically between six and sixty times dimmer than the test flash, and it was sufficient by itself to fully saturate the photocurrent. 2. A saturating pre‐flash delivered before a saturating test flash reduced the tsat of the test flash. This was equivalent to a reduction in phototransduction gain (psi). 3. The pre‐flash had little effect on tau zero the time constant of decay of the rate‐limiting species in photoresponse inactivation (activated rhodopsin or the activated G‐protein‐phosphodiesterase complex). 4. The tsat declined exponentially as the separation time between a fixed intensity pre‐flash and test flash was increased. The time constant (tau p) of decline in tsat was approximately 2.4s. The maximum reduction in tsat corresponded to a reduction in the apparent gain of phototransduction to approximately 0.10 of its original level. This exponential decline is consistent with a [Ca2+]i‐mediated effect. 5. We conclude that the rate‐limiting step in response inactivation and the step responsible for light‐induced gain reduction constitute separate and distinct steps of the phototransduction cascade.
Contrasting effects of hypoxia on cytosolic Ca2+ spikes in conduit and resistance myocytes of the rabbit pulmonary artery.
Authors: Ureña J , Franco-Obregón A , López-Barneo J
Published: December 2014

1. The effects of hypoxia on cytosolic Ca2+ ¿[Ca2+]i) and spontaneous cytosolic Ca2+ spikes were examined in fura 2‐loaded myocytes isolated from conduit and resistance branches of the rabbit pulmonary artery. In all myocyte classes, generation of the Ca2+ spikes was modulated by basal [Ca2+]i which, in turn, was influenced by the influx of Ca2+ through L‐type Ca2+ channels of the plasmalemma. 2. Conduit and resistance myocytes responded distinctly to hypoxia. In most conduit myocytes (approximately 82% of total; n = 23) exposure to hypoxia reduced basal [Ca2+]i. This effect was often associated with the abolition of the Ca2+ spikes. Hypoxia gave rise to two main responses in resistance myocytes. In a subset of resistance myocytes (41 % of total; n = 34) hypoxia incremented basal [Ca2+]i but reduced Ca2+ spike amplitude. This response mimicked the effect of membrane depolarization with K+ and was reverted by nifedipine or the removal of extracellular Ca2+. In a second subset of resistance myocytes (59% of total; n = 34) hypoxia decreased basal [Ca2+]i and, in most cases, increased spike amplitude; a response counteracted by depolarization with K+. 3. These results indicate that hypoxia can differentially modulate [Ca2+]i in smooth muscle cells from large and small diameter pulmonary vessels through a dual effect on transmembrane Ca2+ influx. Our observations further demonstrate the longitudinal heterogeneity of myocytes along the pulmonary arterial tree and help to explain the hypoxic vasomotor responses in the pulmonary circulation.
The relationship between mitochondrial state, ATP hydrolysis, [Mg2+]i and [Ca2+]i studied in isolated rat cardiomyocytes.
Authors: Leyssens A , Nowicky A V , Patterson L , Crompton M , Duchen M R
Published: December 2014

1. As ATP has a higher affinity for Mg2+ than ADP, the cytosolic magnesium concentration rises upon ATP hydrolysis. We have therefore used the Mg(2+)‐sensitive fluorescent indicator Magnesium Green (MgG) to provide an index of changing ATP concentration in single rat cardiomyocytes in response to altered mitochondrial state. 2. In response to FCCP, [Mg2+]i rose towards a plateau coincident with the progression to rigor, which signals ATP depletion. Contamination of the MgG signal by changes in intracellular free Ca2+ concentration (the KD of MgG for Ca2+ is 4.7 microM) was excluded by simultaneous measurement of [Ca2+]i and [Mg2+]i in cells dual loaded with fura‐2 and MgG. The response to FCCP was independent of external Mg2+, confirming an intracellular source for the rise in [Mg2+]i. 3. Simultaneous measurements of mitochondrial NAD(P)H autofluorescence and mitochondrial potential (delta psi m; .‐1 fluorescence) and of autofluorescence and MgG allowed closer study of the relationship between [Mg2+]i and mitochondrial state. Oligomycin abolished the FCCP‐induced rise in [Mg2+]i without altering the change in autofluorescence. Thus, the rise in [Mg2+]i in response to FCCP is consistent with the release of intracellular Mg2+ following ATP hydrolysis by the mitochondrial F1F0‐ATPase. 4. The rise in [Mg2+]i was correlated with cell‐attached recordings of ATP‐sensitive K+ channel (KATP) activity. In response to FCCP, an increase in KATP channel activity was seen only as [Mg2+]i reached a plateau. In response to blockade of mitochondrial respiration and glycolysis with cyanide (CN‐) and 2‐deoxyglucose (DOG), [Mg2+]i rose more slowly but again KATP channel opening increased only when [Mg2+]i reached a plateau and the cells shortened. 5. Oligomycin decreased the rate of rise of [Mg2+]i delayed the onset of rigor and increased the rate of mitochondrial depolarization in response to CN‐_DOG. Thus, with blockade of mitochondrial respiration delta psi m is maintained by the mitochondrial F1F0‐ATPase at the expense of ATP reserves. 6. In response to CN‐_DOG, the initial rise in [Mg2+]i was accompanied by a small rise in [Ca2+]i. After [Mg2+]i reached a plateau and rigor developed, [Ca2+]i rose progressively. On reperfusion, in hypercontracted cells, [Ca2+]i recovered before [Mg2+]i and [ca2+]i oscillations were sustained while [Mg2+]i decreased. Thus on reperfusion, full recovery of [ATP]i is slow, but the activation of contractile elements and the restoration of [Ca2+]i does not require the re‐establishment of millimolar concentrations of ATP.
Bidirectional plasticity expressed by GABAergic synapses in the neonatal rat hippocampus.
Authors: McLean H A , Caillard O , Ben-Ari Y , Gaiarsa J L
Published: December 2014

1. Activity‐dependent plasticity of GABAergic synaptic transmission was investigated in neonatal rat hippocampal slices obtained between postnatal day (P) 2‐10 using intracellular recording techniques. In all experiments, AMPA receptors were blocked by continual application of CNQX (10 microM). 2. Between P2 and P4, tetanic stimulation (TS) evoked NMDA receptor‐dependent long‐term depression of monosynaptic GABAA EPSPS (LTDGABAA). In contrast, when NMDA receptors were blocked by D‐AP5 (50 microM), the same TS evoke long‐term potentiation of GABAA EPSPS (LTPGABAA). 3. Between P6 and P10, TS failed to produce either LTP or LTD or hyperpolarizing monosynaptic GABAA IPSPS under the same recording conditions. However, when GABAergic potentials were rendered depolarizing (KCl‐filled electrode) Ts induced either LTPGABAA or LTDGABAA in the presence or absence of D‐AP5, respectively. 4. Both LTPGABAA and LTDGABAA were specific to the conditioned pathway and could be sequentially expressed at the same synapses. Potentiation of GABAergic synaptic efficacy was induced more easily following previous induction of LTDGABAA than in naive slices. 5. In conclusion, early in development, bidirectional synaptic plasticity is expressed by GABAA receptors and the activation (or not) of NMDA receptors determines the induction of either LTPGABAA or LTDGABAA.
Evidence for the differential release of the cotransmitters ATP and noradrenaline from sympathetic nerves of the guinea‐pig vas deferens.
Authors: Todorov L D , Mihaylova-Todorova S , Craviso G L , Bjur R A , Westfall D P
Published: December 2014

1. Experiments were carried out to quantify the stimulation‐evoked overflow of catecholamines and purines (ATP, ADP, AMP and adenosine) from an in vitro sympathetic nerve‐smooth muscle preparation of the guinea‐pig vas deferens and from isolated bovine adrenal chromaffin cells. The superfused preparations were stimulated for 60 s with electrical field stimulation (EFS; vas deferens), dimethylphenylpiperazinium (chromaffin cells) or KCl (both preparations). 2. Samples of superfusate were taken at 10 s intervals during the 60 s stimulation period for analysis of purines by HPLC‐fluorescence detection and catecholamines by HPLC‐electrochemical detection. 3. The evoked overflow of catecholamines and purines from chromaffin cells occurred with the same time course and in a constant ratio of approximately 4:1 (catecholamine to purine). These findings are compatible with the release of catecholamines and purines from a homogeneous population of exocytotic vesicles in the chromaffin cells. 4. The evoked overflow of purines and noradrenaline (NA) from the vas deferens preparation differed from the pattern of overflow from chromaffin cells and there was also some temporal disparity in the overflow of the two cotransmitters. The evoked overflow of ATP exceeded that of NA. In addition, the overflow of NA was tonic while the overflow of ATP and the other purines was phasic. 5. The EFS‐evoked overflow of NA and the purines from the guniea‐pig vas deferens preparation was examined after treatment with the neuronal amine‐uptake inhibitors desipramine and cocaine, the alpha 1‐adrenoceptor agonist methoxamine, the alpha 1‐adrenoceptor antagonist prazosin, the alpha 2‐adrenoceptor antagonists idazoxan and yohimbine, the noradrenaline‐depleting drug reserpine and the adrenergic neuron‐blocking agent guanethidine. The results of these studies, together with an analysis of the metabolic degradation of extracellular ATP, indicated that the temporal disparity in the overflow of NA and ATP is unlikely to be due to differences in the clearance of the cotransmitters or to the release of purines from non‐neuronal sites. These results indicate that evoked overflow of the cotransmitters accurately reflects release from nerves. This pattern of release from nerves suggests that the two cotransmitters are released from two separate populations of exocytotic vesicles. 6. Superfusion of the vas deferens with exogenous epsilon‐ATP, a fluorescent derivative of ATP, revealed that there was essentially no metabolism of the nucleotide over 60 s unless the tissue was subjected to EFS. Upon EFS, there was a rapid and nearly complete degradation of ATP with a corresponding increase in ADP, AMP and adenosine. This indicates the presence of a nerve stimulation‐dependent metabolism of ATP.
GABA and glutamate mediate rapid neurotransmission from suprachiasmatic nucleus to hypothalamic paraventricular nucleus in rat.
Authors: Hermes M L , Coderre E M , Buijs R M , Renaud L P
Published: December 2014

1. Intracellular sharp electrode and whole‐cell patch‐clamp recording from characterized paraventricular nucleus (PVN) neurones in rat hypothalamic slices were used to study the synaptic mechanism and associated neurotransmitters that mediate their response to suprachiasmatic nucleus (SCN) stimulation. 2. Electrical stimulation restricted to SCN evoked short‐latency inhibitory postsynaptic potentials (IPSPs) or combinations of IPSPs and excitatory postsynaptic potentials (EPSPs) in all (n = 59) PVN neurones tested. Type I neurones (n = 18) were magnocellular and a majority (13/18) demonstrated monosynaptic IPSPs that reversed polarity at the chloride equilibrium potential and were sensitive to bicuculline. 3. Type II (n = 10) and III parvocellular (n = 13), and unclassifiable neurones (n = 18) displayed combinations of IPSPs and EPSPs following similar stimuli applied to SCN. IPSP blockade with bicuculline uncovered SCN‐evoked monosynaptic dual‐component EPSPs that were sensitive to N‐methyl‐D‐aspartate (NMDA) and non‐NMDA receptor antagonists. In addition, chemical microstimulation within SCN was associated with transient increases in spontaneous EPSPs recorded from these PVN neurones. 4. These data imply that the amino acids GABA and glutamate are important mediators of fast monosynaptic transmission from SCN to defined neurones in PVN, and are candidates for conveying circadian rhythmicity to PVN regulation of neuroendocrine and autonomic processes.
Localization of endothelin ETA and ETB receptor‐mediated constriction in the renal microcirculation of rats.
Authors: Endlich K , Hoffend J , Steinhausen M
Published: December 2014

1. The aim of the study was to visualize endothelin‐1 (ET‐1)‐mediated constriction in renal vessels of cortical and juxtamedullary glomeruli in the split hydronephrotic rat kidney in vivo and to functionally characterize the ET receptor subtypes involved. 2. ET‐1 (10(‐9) M) constricted preglomerular vessels (by 6‐18%) and efferent arterioles (by 11‐13%), and decreased glomerular blood flow (GBF, by 55%) of cortical and juxtamedullary glomeruli. 3. The ETA antagonist BQ‐123 (10(‐6) M), as well as the ETB antagonist BQ‐788 (2 x 10(‐7) M) and IRL 1038 (10(‐6) M), shifted the concentration‐response curve of GBF for ET‐1 to the right by one order of magnitude. While BQ‐123 antagonized ET‐1 constriction only in preglomerular vessels, BQ‐788 and IRL 1038 were effective both in preglomerular vessels and efferent arterioles. 4. The ETB agonist IRL 1620 (10(‐8) M) reduced GBF by 50% and constricted efferent arterioles (by 20‐33%) about two times more than preglomerular vessels (by 6‐14%). 5. Our results suggest that in renal cortical and juxtamedullary vessels of rats, ET‐1‐induced preglomerular vasoconstriction is mediated by ETA and ETB receptors, while efferent vasoconstriction is predominantly mediated by ETB receptors, which might have important consequences for the regulation of glomerular filtration pressure by ET.
The action of angiotensin II on the intracellular sodium content of suspensions of rat proximal tubules.
Authors: Wong P S , Johns E J
Published: December 2014

1. Intracellular sodium levels in isolated suspensions of rat proximal tubles were measured by 23Na NMR spectroscopy and the effect of angiotensin II (AII) on these levels was recorded. 2. AII at 10(‐11) M produced an increase in intracellular sodium content of approximately 20% (P < 0.001) from the steady‐state level 5 min after the addition of the drug; intracellular sodium content then gradually returned to baseline levels over the subsequent 25 min. 3. Addition of AII at 10(‐5) M resulted in a significant 20% decrease (P < 0.01) in the steady‐state intracellular sodium level within 5 min. Again the effect was transient and steady‐state intracellular sodium levels were re‐established after 25 min. 4. Amiloride at 10(‐4) M significantly attenuated the action of AII at 10(‐11) M (P < 0.0001) and inhibited the transient response to AII at 10(‐5) M (P < 0.01). When amiloride alone was added to the tubular suspension, intracellular sodium content decreased significantly by 18‐22% (P < 0.001), and addition of both the high and low doses of AII did not have any further effect on intracellular sodium level. 5. The actions of both concentrations of AII were unaffected by an inhibitor of endopeptidase‐24.11, phosphoramidon at 10(‐6) M, which suggests that the transient action of AII was not due to the breakdown of AII by endopeptidase‐24.11. 6. It is well known that AII at high doses inhibits and at low doses stimulates sodium transport across proximal tubular epithelial cells. From the present data it is proposed that AII has a transient biphasic action on intracellular sodium content which may reflect the stimulation of the Na(+)‐H+ exchanger.
Coupling between charge movement and pore opening in vertebrate neuronal alpha 1E calcium channels.
Authors: Olcese R , Neely A , Qin N , Wei X , Birnbaumer L , Stefani E
Published: December 2014

1. Neuronal alpha 1E Ca2+ channels were expressed alone and in combination with the beta 2a subunit in Xenopus laevis oocytes. 2. The properties of ionic and gating currents of alpha 1E were investigated: ionic currents were measured in 10 mM external Ba2+; gating currents were isolated in 2 mM external Co2+. 3. Charge movement preceded channel opening. The charge movement voltage curve (Q(V)) preceded the ionic conductance voltage dependence (G(V)) by approximately 20 mV. 4. Coexpression of alpha 1E with the beta 2a subunit did not modify the voltage dependence of charge movement but shifted the G(V) curve to more negative potentials. The voltage gap between Q(V) and G(V) curves was reduced by the beta 2a subunit and both curves overlapped at potentials near 0 mV. 5. The coupling efficiency between the charge movement and pore opening was estimated by the ration between limiting conductance and maximum charge movement (Gmax/Qmax). Coexpression of the beta 2a subunit increased the Gmax/Qmax ratio from 9.2 x 10(5) +/‐ 1.4 x 10(5) to 21.9 x 10(5) +/‐ 2.8 X 10(5) S C‐1 for alpha 1E and alpha 1E + beta 2a, respectively. 6. We conclude that in the neuronal alpha 1E the charge movement is tightly coupled with the pore opening and that the beta 2a subunit coexpression further improves this coupling.
Activity and expression of Na(+)‐K(+)‐ATPase in human placental cytotrophoblast cells in culture.
Authors: Clarson L H , Glazier J D , Greenwood S L , Jones C J , Sides M K , Sibley C P
Published: December 2014

1. To determine whether there is a change during differentiation, the activity and expression of Na(+)‐K(+)‐ATPase were studied in mononucleate cytotrophoblast cells (18 h culture) and syncytiotrophoblast‐like cells (66 h culture). A choriocarcinoma‐derived cell line (JAr) which, unlike the cytotrophoblast cells, divides in culture, was also studied for comparison. 2. Na(+)‐K(+)‐ATPase activity was assessed by measurement of ouabain‐sensitive 86Rb+ uptake. Na(+)‐K(+)‐ATPase expression was determined by (i) measurement of [3H]ouabain binding and (ii) Northern hybridization to measure expression of alpha‐1 and beta 1‐subunit mRNA. 3. There was no significant difference in either activity or expression of Na(+)‐K(+)‐ATPase during differentiation of cytotrophoblast cells. However, expression of alpha 1‐ and beta 1‐subunit mRNA was significantly lower in 66 vs. 18 h cultured cytotrophoblast cells. 4. Both Na(+)‐K(+)‐ATPase activity and [3H]ouabain binding was significantly greater in JAr cells than either cytotrophoblast cell groups, although expression of alpha 1‐ and beta 1‐subunit mRNA was the same as cytotrophoblast cells cultured for 18 h. 5. It is concluded that N(+)‐K(+)‐ATPase activity and protein expression does not change during differentiation of cytotrophoblast cells but that there are changes in expression at the transcriptional or post‐transcriptional level.
Properties of cloned ATP‐sensitive K+ currents expressed in Xenopus oocytes.
Authors: Gribble F M , Ashfield R , Ammälä C , Ashcroft F M
Published: December 2014

1. We have studied the electrophysiological properties of cloned ATP‐sensitive K+ channels (KATP channels) heterologously expressed in Xenopus oocytes. This channel comprises a sulphonylurea receptor subunit (SUR) and an inwardly rectifying K+ channel subunit (Kir). 2. Oocytes injected with SUR1 and either Kir6.2 or Kir6.1 exhibited large inwardly rectifying K+ currents when cytosolic ATP levels were lowered by the metabolic inhibitors azide or FCCP. No currents were observed in response to azide in oocytes injected with Kir6.2, Kir6.1 or SUR1 alone, indicating that both the sulphonylurea receptor (SUR1) and an inward rectifier (Kir6.1 or Kir6.2) are needed for functional channel activity. 3. The pharmacological properties of Kir6.2‐SUR1 currents resembled those of native beta‐cell ATP‐sensitive K+ channel currents (KATP currents): the currents were > 90% blocked by tolbutamide (500 microM), meglitinide (10 microM) or glibenclamide (100 nM), and activated 1.8‐fold by diazoxide (340 microM), 1.4‐fold by pinacidil (1 mM) and unaffected by cromakalim (0.5 mM). 4. Macroscopic Kir6.2‐SUR1 currents in inside‐out patches were inhibited by ATP with a Ki of 28 microM. Kir6.1‐SUR1 currents ran down within seconds of patch excision preventing analysis of ATP sensitivity. 5. No sensitivity to tolbutamide or metabolic inhibition was observed when SUR1 was coexpressed with either Kir1.1a or Kir2.1, suggesting that these proteins do not couple in Xenopus ocytes. 6. Our data demonstrate that the Xenopus oocyte constitutes a good expression system for cloned KATP channels and that expression may be assayed by azide‐induced metabolic inhibition.
The decrease of maximal oxygen consumption during hypoxia in man: a mirror image of the oxygen equilibrium curve.
Authors: Ferretti G , Moia C , Thomet J M , Kayser B
Published: December 2014

1. Endurance athletes (E) undergo a marked reduction of arterial O2 saturation (Sa,O2) at maximal exercise in normoxia, which disappears when they breathe hyperoxic mixtures. In addition, at a given level of hypoxia, the drop in maximal O2 consumption (VO2,max) is positively related to the individual normoxic VO2,max. 2. These data suggest that the curve relating VO2,max to PI,O2 may be steeper and perhaps less curved in E than in sedentary subjects (S) with low VO2,max values because of the greater hypoxaemia in the latter, whence the hypotheses that (i) the relationship between VO2,max and PI,O2 may be set by the shape of the oxygen equilibrium curve; and (ii) the differences between E and S may be due to the different position on the oxygen equilibrium curve on which these subjects operate. These hypotheses have been tested by performing a systematic comparison of the VO2,max or Sa,O2 vs. PI,O2 relationships in E and S. 3. On ten subjects (five S and five E), VO2,max was measured by standard procedure during cycloergometric exercise. Sa,O2 was measured by finger‐tip infrared oximetry. Arterialized blood PO2 (Pa,O2) and PCO2 (Pa,CO2) were determined in 80 microliters blood samples from an ear lobe. The subjects breathed ambient air or a N2‐O2 mixture with an inspired O2 fraction (FI,O2) of 0.30, 0.18, 0.16, 0.13 and 0.11, respectively, VO2,max was normalized with respect to that obtained at the highest FI,O2. 4. The relationships between Sa,O2 or normalized VO2,max and FI,O2 (or PI,O2) had similar shapes, the data for E being systematically below and significantly different from those for S. Linear relationships between Sa,O2 and normalized VO2,max, statistically equal between E and S, were found. 5. We conclude that the relationships between either VO2,max or Sa,O2 and FI,O2 (or Pa,O2) may indeed be the mirror images of one another, implying a strict link between the decrease of VO2,max in hypoxia and the shape of the oxygen equilibrium curve, as hypothesized.