bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of recombinant mouse acetylcholine receptors by acetylcholine, carbamylcholine and tetramethylammonium.
Authors: Zhang Y , Chen J , Auerbach A
Published: December 2014

1. The kinetic properties of cloned mouse embryonic nicotinic acetylcholine receptors (AChRs) expressed in HEK 293 cells or Xenopus oocytes were examined using high concentrations of acetylcholine (ACh), carbamylcholine (CCh), or tetramethylammonium (TMA). The rate constants of agonist binding and channel gating were estimated by fitting kinetic models to idealized open and closed intervals over a range of agonist concentrations. 2. Once doubly liganded, TMA‐activated receptors open at approximately 3000 s‐1. The equilibrium binding constants for TMA are 525 and 12,800 microM. Doubly liganded CCh‐activated receptors open at approximately 11,500 s‐1; the equilibrium binding constants for this agonist are 14 and 570 microM. If we assume that doubly liganded, ACh‐activated receptors open at 60,000 s‐1, then the equilibrium binding constants for ACh are 20 and > 650 microM, similar to those for CCh. For all three agonists the higher affinity site both binds and releases agonists more slowly than does the lower affinity site. 3. ACh and CCh bind to the two sites equally rapidly, at approximately 2 x 10(7) and 4 x 10(7) M‐1 s‐1 at the first and second binding sites, respectively. Compared with ACh, the TMA association rate is approximately 100 times slower at the first binding site, and approximately 30 times slower at the second binding site. These results indicate that at both binding sites the association rate of TMA is not limited by diffusional or steric factors. 4. All three agonists dissociate from the receptor binding sites at similar rates. The dissociation rate for all agonists was approximately 40 times slower at the first binding site than at the second. These results suggest that the interaction of the quarternary amine moiety with the receptor determines the rate of release of the agonist, and that the nature of this interaction is quite different at the two binding sites. 5. Although the channel opening rates for the three agonists varied approximately 20‐fold, the channel closing rates were not strongly agonist dependent, and varied less than 3‐fold. We speculate that the ester moiety of the agonist promotes both rapid binding and fast opening of the ligand receptors, and that interactions of the quarternary amine moiety of the agonist with the receptor determine the channel closing rate constant.
The effects of lyotropic anions on electric field‐induced guidance of cultured frog nerves.
Authors: Erskine L , McCaig C D
Published: December 2014

1. Dissociated Xenopus neurites turn cathodally in small applied electric fields. Increasing the external polycation concentration alters the direction and extent of field‐induced orientation. A decrease in membrane surface charge may underlie these effects. 2. Lyotropic anions increase membrane surface charge and we have examined the effect of perchlorate (ClO4‐), thiocyanate (SCN‐) and sulphate (SO4(2‐)) on galvanic nerve orientation. 3. Perchlorate and SCN‐ had no effect on field‐induced cathodal turning, whereas incubation with SO4(2‐) was inhibitory. In addition to its effects on surface charge, SO4(2‐) increases production of the second messengers diacylglycerol and inositol trisphosphate. Interestingly, lithium (Li+), a blocker of polyphosphoinositide metabolism, had a similar effect to SO4(2‐) on field‐induced neurite orientation. 4. We conclude that increasing surface charge with lyotropic anions neither enhances galvanotropic orientation nor underlies the inhibitory effects of SO4(2‐) and suggest that modulation of galvanotropism by SO4(2‐) occurs owing to changes in the inositolphospholipid second messenger system.
Imaging of calcium variations in living dendritic spines of cultured rat hippocampal neurons.
Authors: Segal M
Published: December 2014

1. Cultured rat hippocampal neurons were loaded with the Ca2+ indicator fura‐2 through micropipettes and visualized with an inverted microscope equipped with a high power objective and a cooled CCD camera. The responses of dendritic spines and their parent dendrites to stimuli which evoke a rise of [Ca2+]i were monitored. 2. NMDA caused a rapid and transient rise in [Ca2+]i, which was more evident in the spine than in the parent dendrite. The recovery in both compartments had the same time course, and was dependent on normal [Na+]o. 3. Application of alpha‐latrotoxin, which causes release of neurotransmitters from terminals, produced a rise of [Ca2+]i in the dendritic spines, more than in their parent dendrites. Prolonged exposure to the drug eliminated the spine/dendrite disparity. 4. The presence of voltage‐gated calcium channels in dendritic spines is indicated by the enhanced calcium rise in spines rather than dendrites of cells depolarized by either intracellular current injection or by raising [K+]o. This rise was attenuated by nifedipine or verapamil, both L‐type channel blockers. 5. It is suggested that the dendritic spine constitutes an independent calcium compartment that is closely linked to the parent dendrite.
Nitric oxide as a retrograde messenger in the nucleus tractus solitarii of rats during hypoxia.
Authors: Ogawa H , Mizusawa A , Kikuchi Y , Hida W , Miki H , Shirato K
Published: December 2014

1. We examined the role of nitric oxide (NO) in respiratory regulation in the nucleus tractus solitarii (NTS), where L‐glutamate release associated with peripheral chemoreceptor activation modulates the hypoxic ventilatory response. 2. Experiments were performed in unanaesthetized freely moving rats. First, the effects on the hypoxic ventilatory response of sodium nitroprusside (SNP, a NO donor) or NG‐monomethyl‐L‐arginine (L‐NMMA, a NO synthase inhibitor), microinjected into the NTS, were investigated. Second, using in vivo microdialysis, changes in extracellular L‐glutamate during hypoxia were examined in the presence of L‐NMMA. Third, the effect of L‐NMMA on ventilatory augmentation by exogenous L‐glutamate was examined. Furthermore, we measured extracellular L‐citrulline concentration changes during hypoxia in the NTS to assess NO formation indirectly and also examined the effect of MK‐801 (an NMDA receptor antagonist) on L‐citrulline levels during hypoxia. 3. SNP increased ventilation during both normoxia and hypoxia. L‐NMMA did not alter ventilation or L‐glutamate levels during normoxia but significantly attenuated the hypoxic ventilatory response and the increase in L‐glutamate during hypoxia. The inhibition by L‐NMMA was blocked by L‐arginine. The ventilatory augmentation by exogenous L‐glutamate was attenuated by L‐NMMA. L‐Citrulline increased during hypoxia, and this increase was inhibited by MK‐801. 4. We provide the first in vivo evidence that, in the NTS, NO works as a retrograde messenger in an L‐glutamate‐releasing positive feedback system contributing to the augmentation of ventilation during hypoxia.
Voltage‐gated calcium currents in the magnocellular neurosecretory cells of the rat supraoptic nucleus.
Authors: Fisher T E , Bourque C W
Published: December 2014

1. Whole‐cell patch‐clamp techniques were used to analyse voltage‐dependent calcium currents in acutely isolated somata of magnocellular neurosecretory cells (MNCs) from the supraoptic nucleus of the hypothalamus of adult rats. Currents were characterized on the basis of their rates of inactivation and their sensitivity to a series of calcium channel blocking agents. 2. Curve fitting analysis of series of long lasting depolarizing voltage steps from a holding potential of ‐80 mV revealed three current components with different voltage dependences and rates of inactivation (n = 36). These include a low threshold (‐60 mV), rapidly inactivating (tau = 42 +/‐ 3 ms at ‐10 mV) component, a high threshold (‐30 mV), slowly inactivating (tau = 1790 +/‐ 70 ms) component and a component with an intermediate threshold (‐50 mV) and rate of inactivation (tau = 187 +/‐ 15 ms). There is also a non‐inactivating portion of evoked calcium current with a threshold of ‐50 mV. 3. Based on its voltage dependence, rate of inactivation, greater sensitivity to the divalent cation nickel than to cadmium and insensitivity to omega‐conotoxin GVIA (omega‐CgTX), the low threshold current appears to be a T‐type calcium current. The rate of inactivation, voltage dependence, and sensitivity to omega‐CgTX of the slowly inactivating component suggests that it is an N‐type current. The characteristics of the intermediate component do not correspond to any identified calcium current type. 4. Portions of the non‐inactivating calcium current are sensitive to nifedipine (23 +/‐ 2% of the total non‐inactivating current at ‐10 mV; n = 10), suggesting the presence of L‐type currents, omega‐agatoxin‐IVA (omega‐Aga‐IVA; 20 +/‐ 6% of total; n = 11), suggesting the presence of P‐type channels, and omega‐CgTX (39 +/‐ 3% of total; n = 19), suggesting the presence of a non‐inactivating N‐type current. The non‐inactivating component at low potentials (> or = ‐50 mV) was selectively blocked by nifedipine, suggesting the presence of a novel, low threshold L‐type current. 5. We conclude that MNC soma express T‐, N‐, L‐, and P‐type calcium currents, as well as a novel low threshold nifedipine‐sensitive current and an unidentified inactivating component. This complement of currents is different from that seen in the terminals of these cells, suggesting a spatial and functional segregation of calcium current types in MNCs.
The multi‐ion nature of the cGMP‐gated channel from vertebrate rods.
Authors: Sesti F , Eismann E , Kaupp U B , Nizzari M , Torre V
Published: December 2014

1. Native cGMP‐gated channels were studied in rod outer segments of the larval tiger salamander, Ambystoma tigrinum. The alpha‐subunit of the cGMP‐gated channel, here referred to as the wild type (WT), and mutant channels were heterologously expressed in Xenopus laevis oocytes. These channels were studied in excised membrane patches in the inside‐out configuration and were activated by the addition of 100 or 500 microM cGMP. The current carried by monovalent cations was measured under voltage‐clamp conditions. 2. In the presence of 110 mM Na+ in the extracellular medium and different amounts of Na+ in the intracellular medium, the I‐V relations of the native channel could be described by a single‐site model with a profile of Gibbs free energy with two barriers and a well. A similar result was obtained in the presence of 110 mM Li+ in the extracellular medium and different amounts of Li+ in the intracellular medium. The well depth was 1.4RT (where R is the gas constant and T is the absolute temperature) for both Li+ and Na+. 3. The I‐V relations of the native channel in the presence of 110 mM Na+ on one side of the membrane and 110 mM Li+ on the other side could not be described by the same single‐site model with identical values of barriers and well obtained in the presence of Li+ or Na+ alone: the well for Li+ had to be at least 4RT. 4. In the presence of mixtures of 110 mM Li+ and Cs+ on the cytoplasmic side of the membrane, an anomalous mole fraction effect was observed both in the native and the WT channel. No anomalous behaviour was seen in the presence of Li(+)‐Na+ and Li(+)‐NH4+ mixtures. 5. The anomalous mole fraction effect with mixtures of Li+ and Cs+ was not observed in the channel where glutamate 363 was mutated to a glutamine (E363Q) or an asparagine (E363N). When glutamate 363 was mutated to an aspartate (E363D), the anomalous mole fraction effect with mixtures of Li+ and Cs+ was still observed, although significantly reduced. 6. When lysine 346, arginine 369, aspartate 370 and glutamate 372 were neutralized by mutation to glutamine, the ion permeation through the mutant channels and the WT channel had largely similar properties.(ABSTRACT TRUNCATED AT 400 WORDS)
Effects of angiotensin II in fetal sheep and modification of its actions by indomethacin.
Authors: Stevenson K M , Lumbers E R
Published: December 2014

1. Angiotensin II (AII) was infused I.V. into seven chronically catheterized fetal sheep (gestational age, 120‐136 days). The effects of short‐term infusions of 6 and 12 micrograms kg‐1 h‐1 for 1.5 h were compared with the effects of infusing 6 micrograms kg‐1 h‐1 for 3 or 5 days (long‐term infusion). AII produced an immediate rise in fetal arterial blood pressure (P < 0.025). When infused for 3 or 5 days, 6 micrograms kg‐1 h‐1 AII caused a greater increase in arterial blood pressure (P < 0.05). 2. Infusions of 6 micrograms kg‐1 h‐1 AII for 1.5 h had no effect on fetal placental blood flow or on flow to the fetal membranes, but after AII infusion for 3 or 5 days both flows were reduced (P < 0.01 and P < 0.005, respectively). Fetal blood gas status and pH were maintained. The only change in fetal renal function observed with short‐term infusions of AII was a rise in sodium excretion when 12 micrograms kg‐1 h‐1 AII was given (P < 0.05). Infusion of 6 micrograms kg‐1 h‐1 for 3 or 5 days also caused a rise in sodium excretion (P < 0.025) because total and proximal fractional sodium reabsorptions were depressed (P < 0.01). Infusions of AII had no effects on the volume of lung liquid produced or on its composition. 3. Administration of indomethacin to the ewe (10 mg kg‐1) and to the fetus (12 mg kg‐1), during the infusion of AII, caused a rise in maternal arterial pressure (P < 0.01) but no change in fetal arterial pressure. 4. After indomethacin, umbilicoplacental blood flow rose (P < 0.05), as did fetal arterial PO2 (P < 0.05). Fetal arterial PCO2, pH and bicarbonate levels fell (P < 0.01). Glomerular filtration rate (GFR) rose (P < 0.01); there was a natriuresis (P < 0.01), chloriuresis (P < 0.01) and a kaliuresis (P < 0.05) but urine flow rate did not change. Lung liquid flow fell (P < 0.01). 5. It is concluded that in the fetus, long‐term infusions of AII at a constant dose rate cause a progressive rise in arterial pressure. In addition, effects of AII on placental blood flow and on renal function develop. Thus, short‐term infusions of AII cannot be used to predict the renal and cardiovascular effects of sustained high levels of this peptide in the fetus.(ABSTRACT TRUNCATED AT 400 WORDS)
Spatial profile of dendritic calcium transients evoked by action potentials in rat neocortical pyramidal neurones.
Authors: Schiller J , Helmchen F , Sakmann B
Published: December 2014

1. Simultaneous measurements of intracellular free calcium concentration ([Ca2+]i) and intrasomatic and intradendritic membrane potential (Vm) were performed using fura‐2 fluorimetry and whole‐cell recording in neocortical layer V pyramidal neurones in rat brain slices. 2. Back‐propagating action potentials (APs) evoked [Ca2+]i transients in the entire neurone including the soma, the axon initial segment, the apical dendrite up to the distal tuft branches, and the oblique and basal dendrites, indicating that following suprathreshold activation the entire dendritic tree is depolarized sufficiently to open voltage‐dependent calcium channels (VDCCs). 3. The [Ca2+]i transient peak evoked by APs showed large differences between various compartments of the neurone. Following a single AP, up to 6‐fold differences were measured, ranging from 43 +/‐ 14 nM in the soma to 267 +/‐ 109 nM in the basal dendrites. 4. Along the main apical dendrite, the [Ca2+]i transients evoked by single APs or trains of APs had the largest amplitude and the fastest decay in the proximal region; the [Ca2+]i transient peak and decay time constant following a single AP were 128 +/‐ 25 nM and 420 +/‐ 150 ms, respectively, and following a train of five APs (at 10‐12 Hz), 710 +/‐ 214 nM and 390 +/‐ 150 ms, respectively. The [Ca2+]i transients gradually decreased in amplitude and broadened in more distal portions of the apical dendrite up to the main bifurcation. 5. In the apical tuft branches, the profile of the [Ca2+]i transients was dependent on AP frequency.(ABSTRACT TRUNCATED AT 250 WORDS)
Interstitial space, electrical resistance and ion concentrations during hypotonia of rat hippocampal slices.
Authors: Chebabo S R , Hester M A , Jing J , Aitken P G , Somjen G G
Published: December 2014

1. The degree to which mammalian brain cells swell in hypotonic environments has not previously been determined. We exposed hippocampal tissue slices prepared from anaesthetized rats to artificial cerebrospinal fluid from which varying amounts of NaCl had been deleted. Interstitial volume (ISV) change was determined from the volume of dilution of the marker ions tetramethylammonium (TMA+) or tetraethylammonium (TEA+). Tissue electrical resistance was measured as the voltage generated by constant current pulses. 2. ISV decreased as a function of lowered extracellular osmolality (osmotic pressure, pi o), indicating cell swelling. After reaching a minimum, ISV recovered partially, suggesting regulatory volume decrease of cells. After restoring normal pi o the ISV expanded, indicating post‐hypotonic cell shrinkage. The electrical resistance of the tissue (Ro) increased when pi o was lowered, due to the reduced ionic strength, as well as restricted ISV. 3. To control for low NaCl concentration, reduced NaCl was replaced by mannitol or fructose. In isosmotic, NaCl‐deficient solution, ISV showed inconsistent change, and Ro corrected for ionic strength tended to decrease. 4. Extracellular K+ concentration decreased slightly in low pi o except when spreading depression caused it to increase. Extracellular Ca2+ concentration decreased substantially, consistently and reversibly. Administration of isosmotic low‐NaCl concentration solutions caused a similar decrease in extracellular Ca2+ concentrations. We propose that low Na+ concentration in extracellular fluid impaired the extrusion of Ca2+. 5. In severely hypotonic solution, ISV was reduced to 25% of its control volume, corresponding to a mean cell volume increase of at least 11%, probably more. From plotting relative changes in ISV against osmolarity we concluded that, within the range tested, hypotonic cell swelling was not opposed by the close approach of plasma membranes of neighbouring cells.
The relationship between light‐evoked synaptic excitation and spiking behaviour of salamander retinal ganglion cells.
Authors: Diamond J S , Copenhagen D R
Published: December 2014

1. Light‐evoked input‐output characteristics of ganglion cells in dark‐adapted tiger salamander retina were studied in the slice preparation using patch‐clamp techniques. Excitatory postsynaptic currents (EPSCs), isolated by blocking inhibitory inputs and evoked by a range of light stimulus intensities, were recorded under whole‐cell voltage clamp. Spike responses, evoked by the same light intensities, were recorded extracellularly from the same cells using the cell‐attached patch‐clamp technique. 2. When N‐methyl‐D‐aspartate (NMDA) receptor‐mediated input was blocked by the competitive NMDA antagonist DL‐2‐amino‐5‐phosphonoheptanoate (AP7), light‐evoked EPSC amplitude and peak firing rate were reduced at all light intensities. In both cases, the data obtained in the presence of AP7 scaled linearly to control data, indicating that NMDA and non‐NMDA receptors are activated in the same proportions across the entire 2 log unit stimulus response range of these ganglion cells. 3. The relationship between light‐evoked spike frequency and light‐evoked EPSC amplitude was linear. The slope of the light‐evoked synaptic current‐spike frequency relationship was close to the slope of the injected current‐spike frequency relationship, indicating that synaptic current and injected current drive spiking in a similar manner. The linearity of the synaptic current‐spike frequency relationship was not compromised when NMDA input was blocked by AP7. 4. Light‐evoked voltage responses, recorded under whole‐cell current clamp, revealed that the average membrane potential during a spike response was depolarized only slightly with increased firing rate. Once the membrane potential surpassed spike threshold, it was maintained by the voltage‐gated, spike‐generating conductances at a depolarized plateau upon which action potentials were fired. The potential of this plateau varied only slightly with spike frequency. We conclude that the voltage control exerted by the spike‐generating currents in ganglion cells prevents a substantial response‐dependent decrease in the electrical driving force of the excitatory currents, obviating the need for the voltage‐independent synaptic efficacy provided by the combination of NMDA and non‐NMDA inputs.
A maxi Cl‐ channel coupled to endothelin B receptors in the basolateral membrane of guinea‐pig parietal cells.
Authors: Kajita H , Kotera T , Shirakata Y , Ueda S , Okuma M , Oda-Ohmae K , Takimoto M , Urade Y , Okada Y
Published: December 2014

1. To study endothelin (ET) receptors in guinea‐pig stomach, ET‐binding assays and in vitro autoradiography were performed on fundic cell suspensions and on sections of the fundus, respectively. ETA and ETB receptor subtypes were found to coexist in the parietal cells. 2. Endothelin 1 (ET‐1) added to the (basolateral) bathing solution was found to activate noisy whole‐cell Cl‐ currents within about 1 min in both single, isolated parietal cells and those within gastric glands obtained from the fundus. 3. ET‐1‐induced Cl‐ currents were rapidly blocked by a Cl‐ channel blocker (NPPB) added to the (basolateral) bathing solution in a concentration‐dependent manner with a half‐maximum inhibition concentration of 33 microM. 4. The anion selectivity sequence of the ET‐1‐induced conductance was I‐ > Br‐ > Cl‐ > F‐, corresponding to Eisenman's sequence I. 5. Changes in extracellular pH between 5 and 8 did not affect the ET‐1‐induced activation of Cl‐ currents. 6. Similar activating effects were also observed with ET‐3 and a specific ETB receptor agonist (IRL1620). An ETB receptor antagonist (IRL1720) prevented the ET‐1 effect, whereas an ETA‐selective antagonist (FR139317 or BQ123) failed to antagonize the ET‐1 effect. 7. In the whole‐cell mode, unitary Cl‐ channel events could be observed in association with ET‐1‐activated macroscopic currents. The single‐channel conductances were around 200 and 350 pS at negative and positive membrane potentials, respectively. 8. It is concluded that gastric parietal cells of guinea‐pig possess pH‐insensitive ‘maxi’ Cl‐ channels coupled to ETB receptors in the basolateral membrane.
Perception of movement at the human ankle: effects of leg position.
Authors: Refshauge K M , Fitzpatrick R C
Published: December 2014

1. Recent studies show that subjects perceive smaller ankle movements when they are upright in the standing position than when they are seated. To examine this improvement, the ability to perceive ankle movements was tested in five positions of body, knee and ankle. Subjects reported the direction of slow ramp movements of the ankles. 2. The threshold for perceiving ankle movements was unchanged when only one ankle was moved rather than both together. When seated with the knees bent and ankles slightly plantarflexed, subjects perceived movements of 0.65 deg at 0.05 deg s‐1. However, when upright or when seated with their knees and ankles in the standing position, subjects perceived movements that were one‐third of this size. 3. These findings show that the knee and ankle positions, rather than being upright, explain the better performance in the standing position. During standing, knee extension and ankle dorsiflexion stretch the calf muscles. Thus, enhanced input from intramuscular stretch receptors appears responsible for the better performance.
A rapidly activating type of outward rectifier K+ current and A‐current in rat suprachiasmatic nucleus neurones.
Authors: Bouskila Y , Dudek F E
Published: December 2014

1. The properties of calcium‐independent (i.e. persisting in the absence of external calcium) depolarization‐activated potassium currents in suprachiasmatic nucleus (SCN) neurones (n = 75) were studied under voltage‐clamp conditions with whole‐cell patch‐clamp recordings in rat hypothalamic slices (150‐175 microns). 2. Two distinct types of potassium currents were found. One was a rapidly activating and slowly inactivating type of outward rectifier (named IK(FR) for fast rectifier potassium current), similar to a potassium current described in cardiac muscle, and the other was a transient A‐current (IA). 3. The rates of activation and deactivation of IK(FR) were voltage dependent. Time constants of activation fitted to n4 kinetics and declined from 3.5 ms (at ‐20 mV) to 1.1 ms (at 60 mV). Inactivation had a biexponential time course with voltage‐independent time constants of 0.3 s (minor component) and 3.0 s (major component) between 10 and 50 mV. IK(FR) was activated above ‐40 mV with a V1/2 (membrane potential at half‐maximal activation) of 14 +/‐ 2 mV and slope factor of ‐17 +/‐ 1 mV reaching a conductance (not maximal) of 10.8 +/‐ 1.7 nS at 60 mV. Steady‐state inactivation had a slope factor of 11 +/‐ 1 mV. 4. IK(FR) was highly selective for K+ (PNa/PK = 0.002). Tetraethylammonium (TEA) reduced IK(FR) reversibly (IC50 = 20 mM), while 4‐aminopyridine (4‐AP) at 10 mM had little effect. The remaining current in 30 mM TEA was similar to that in control conditions, indicating that TEA reduced IK(FR) rather than revealed an additional TEA‐resistant current. 5. The rate of IA activation was voltage dependent with time to peak declining from 8.5 ms (at ‐40 mV) to 3.6 ms (at 60 mV). Inactivation had a biexponential time course with voltage‐dependent and voltage‐independent time constants. The two components were similar in amplitude. IA was activated above ‐60 mV, reaching a maximal conductance of 3.6 +/‐ 0.4 nS at above 20 mV. Steady‐state inactivation was complete above ‐10 mV. Rates of onset of inactivation (at ‐40 mV) and recovery from inactivation (at ‐100 mV) were similar with time constants of 39 +/‐ 5 and 41 +/‐ 9 ms, respectively. 6. IK(FR) and IA were found in every neurone tested in the SCN and in all locations throughout the nucleus. The possible function of these currents is discussed, particularly in relation to the circadian rhythm of firing rate in the SCN.
Transcellular gaps in microvascular walls of frog and rat when permeability is increased by perfusion with the ionophore A23187.
Authors: Neal C R , Michel C C
Published: December 2014

1. The experiments described in this paper aimed to determine whether the gaps which develop in microvascular endothelium in association with increases in permeability are located in the intercellular clefts or are openings passing through the endothelial cells. 2. Hydraulic permeability (Lp) was estimated in frog mesenteric capillaries and single rat venules using a microperfusion‐micro‐occlusion technique before and during perfusion with solutions containing the ionophore A23187 at a concentration of 10 microM. When Lp was seen to have increased, the tissues were fixed in situ with 2.5% glutaraldehyde. 3. The increases in Lp varied considerably from vessel to vessel. In six frog vessels Lp increased from 2.6 +/‐ 0.9 x 10(‐7) to 266 +/‐ 159 x 10(‐7) cm s‐1 cmH2O‐1 and in three rat venules Lp rose from 0.94 +/‐ 0.09 x 10(‐7) to 16.4 +/‐ 4.9 x 10(‐7) cm s‐1 cmH2O‐1 (means +/‐ S.E.M.). 4. Forty openings or gaps were completely reconstructed from electron micrographs of serial ultrathin sections of the six frog vessels. Thirty‐nine of these gaps passed through the endothelial cells and did not communicate with the intercellular clefts; one was intercellular. Similarly, fifteen out of sixteen gaps reconstructed from electron micrographs of the rat venules were transcellular and clearly separated from the intercellular clefts. 5. The increased Lp and associated ultrastructural changes induced by A23187 were reversed by perfusion with ionophore‐free solutions.
Developmental changes in blood‐brain barrier potassium permeability in the rat: relation to brain growth.
Authors: Keep R F , Ennis S R , Beer M E , Betz A L
Published: December 2014

1. The potassium permeability of the blood‐brain barrier (BBB) was determined in anaesthetized rats aged between 21 days gestation and adult using 86Rb+ as a marker for potassium. 2. The brain influx rate constant for 86Rb+ was high in fetal cortex at 21 days gestation (42.5 +/‐ 4.3 microliters g‐1 min‐1) but had decreased markedly by just after birth (12.2 +/‐ 0.6 microliters g‐1 min‐1). There was a further, gradual, postnatal decline to 7.0 +/‐ 0.3 microliters g‐1 min‐1 by 50 days after birth. 3. Developmental changes in passive BBB permeability were examined over the same age range using 14[C]urea. These studies showed similar developmental changes in influx rate to those found for 86Rb+. Specifically, a marked perinatal decline followed by a more gradual postnatal fall. Thus, the changes in potassium permeability probably reflect a decrease in the BBB paracellular leak during development. 4. The changes in BBB permeability coincide with changes in the rate of brain growth and the associated rate of brain potassium accumulation. As the potassium permeability properties of the adult BBB would provide insufficient potassium influx to meet the requirement associated with fetal brain growth, it is suggested that need for potassium may be the reason for the greater BBB permeability early in development.
Specific motifs in the external loops of connexin proteins can determine gap junction formation between chick heart myocytes.
Authors: Warner A , Clements D K , Parikh S , Evans W H , DeHaan R L
Published: December 2014

1. Gap junction formation was compared in the absence and presence of small peptides containing extracellular loop sequences of gap junction (connexin) proteins by measuring the time taken for pairs of spontaneously beating embryonic chick heart myoballs to synchronize beat rates. Test peptides were derived from connexin 32. Non‐homologous peptides were used as controls. Control pairs took 42 +/‐ 0.5 min (mean +/‐ S.E.M.; n = 1088) to synchronize. 2. Connexins 32 and 43, but not 26, were detected in gap junction plaques. The density and distribution of connexin immunolabelling varied between myoballs. 3. Peptides containing conserved motifs from extracellular loops 1 and 2 delayed gap junction formation. The steep portion of the dose‐response relation lay between 30 and 300 microM peptide. 4. In loop 1, the conserved motifs QPG and SHVR were identified as being involved in junction formation. In loop 2, the conserved SRPTEK motif was important. The ability of peptides containing the SRPTEK motif to interfere with the formation of gap junctions was enhanced by amino acids from the putative membrane‐spanning region. 5. Peptides from loop 1 and loop 2 were equivalently effective; there was no synergism between them. 6. The inclusion of conserved cysteines in test peptides did not make them more effective in the competition assay.
Effects of Ca2+ and K+ channel blockers on nerve impulses recorded from guinea‐pig postganglionic sympathetic nerve terminals.
Authors: Brock J A , Cunnane T C
Published: December 2014

1. A focal extracellular suction electrode was used to investigate the contributions of K+ and Ca2+ currents to the nerve impulse recorded from sympathetic nerve terminals innervating the guinea‐pig vas deferens in vitro. 2. Perfusing the electrode with Cd2+ (0.1‐0.5 mM) had little effect on the configuration of the nerve impulse. 3. Perfusing the electrode with Ba2+ (1‐3 mM) caused the appearance of a second negative‐going component of the nerve impulse. Local application of Cd2+ (0.1 mM) had little affect on this component of the nerve impulse. 4. Perfusing the electrode with 4‐aminopyridine (4‐AP) and/or tetraethylammonium (TEA) caused the appearance of a second negative‐going component of the nerve impulse. This component has been termed the late negative‐going component (LNC). 5. The LNC produced by local application of 1 mM 4‐AP and 10 mM TEA was not changed when the solution perfusing the electrode contained no added Ca2+, 10 mM Ca2+ or omega‐conotoxin GVIA (0.1 microM). Perfusion of the electrode with Cd2+ (0.1 mM) reduced the amplitude and slowed the time course of the LNC. 6. The LNC was markedly inhibited when the organ bath was perfused with TEA (10 mM) or 4‐AP and TEA (1 and 10 mM, respectively). In some experiments the LNC was completely abolished. 7. The LNC was reduced in amplitude and slowed in time course when the solution perfusing the organ bath contained no added Ca2+. A similar effect on the LNC was observed when the solution perfusing the organ bath contained omega‐conotoxin GVIA (0.1 microM), charybdotoxin (0.05 microM) or low concentrations of TEA (0.3‐1 mM) or Ba2+ (10‐500 microM). 8. Bath application of the alpha 2‐adrenoceptor agonist clonidine (0.1‐0.3 microM) did not detectably change the LNC. 9. The results demonstrate that the LNC produced by the local application of K+ blockers is due primarily to K+ efflux from sites outside the recording electrode and that a part of the change in conductance that underlies this component is due to opening of Ca(2+)‐activated K+ channels. The failure to detect an effect of clonidine on the LNC suggests that activation of presynaptic alpha 2‐adrenoceptors does not change either the K+ or the Ca2+ conductance of the nerve terminals.
Expression and regulation of a vesicular monoamine transporter in rat stomach: a putative histamine transporter.
Authors: Dimaline R , Struthers J
Published: December 2014

1. Vesicular monoamine transporters (VMATs) translocate monoamines from the cytoplasm into secretory vesicles of endocrine cells and neurones, but they have limited affinity for histamine, and the identity of the vesicular transporter for this monoamine is uncertain. The aims of the present study were to characterize VMAT representatives in rat gastric corpus, and to determine if their expression was regulated by factors that modulate histamine biosynthesis. 2. Polymerase chain reaction (PCR) cloning using oligonucleotide primers to DNA sequences conserved within the VMAT family provided evidence for VMAT2, but not VMAT1 in rat gastric corpus. Northern analysis using a VMAT2 complementary RNA probe revealed a single 4 kb mRNA species in corpus endocrine cells. 3. In rats treated for up to 5 days with the H(+)‐K(+)‐ATPase inhibitor omeprazole, VMAT2, histidine decarboxylase and chromogranin A mRNA abundance in gastric corpus, and plasma gastrin concentrations increased progressively. Omeprazole also elevated VMAT2 expression in rats fasted for 48 h, but fasting alone, or refeeding fasted animals had no effect. 4. The results are consistent with a role for VMAT2 in the transport of histamine into enterochromaffin‐like cell secretory vesicles, and with upregulation of the transporter to accommodate the increased histamine biosynthesis and secretion that accompanies achlorhydria.
Sodium‐dependent GABA‐induced currents in GAT1‐transfected HeLa cells.
Authors: Risso S , DeFelice L J , Blakely R D
Published: December 2014

1. HeLa cells were infected with recombinant vaccinia virus containing the T7 RNA polymerase gene and transfected with the cDNA for a rat GABA transporter, GAT1, cloned downstream of a T7 RNA polymerase promoter. Six to sixteen hours after transfection, whole‐cell recording with a voltage ramp in the range ‐90 to 50 mV revealed GABA‐induced currents (approximately ‐100 pA at ‐60 mV in 100 microM GABA, 16 h after transfection at room temperature). No GABA‐induced currents were observed in parental HeLa cells or in mock‐transfected cells. 2. GABA‐induced currents were suppressed by extracellular perfusion with GABA‐free solutions or addition of GAT1 inhibitors SKF89976‐A or SKF100330‐A. At fixed voltage the GABA dependence of the inward current fitted the Michaelis‐Menten equation with a Hill coefficient, n, near unity and an equilibrium constant, K(m), near 3 microM. The Na+ dependence of the inward currents fitted the Michaelis‐Menten equation with n approximately equal to 2 and K(m) approximately equal to 10 mM. The constants n and K(m) for GABA and Na+ were independent of voltage in the range ‐90 to ‐30 mV. 3. GABA‐induced currents reverse direction in the range 5‐10 mV. The implication of this result is that GAT1 can mediate electrogenic (electrophoretic) influx or efflux of GABA depending on the membrane voltage. The presence of an outward current in our experiments is consistent with radioactive‐labelled flux data from resealed vesicle studies. However, it is inconsistent with frog oocyte expression experiments using the sample clone. In oocytes, GAT1 generates no outward current in a similar voltage range. Smaller intracellular volume or higher turnover rates in the mammalian expression system may explain the outward currents. 4. External GABA induces inward current, and internal GABA induces outward current. However, in cells initially devoid of internal GABA, external GABA can also facilitate an outward current. This GAT1‐mediated outward current occurs only after applying negative potentials to the cell. These data are consistent with the concept that negative potentials drive GABA and Na+ into the cell, which then leads to electrogenic efflux through GAT1 at positive voltages. 5. Assuming coupled transport, we estimate the number of transporters, N, times the turnover rate, r, to be Nr approximately 10(9) s‐1 under nominal conditions (V = ‐60 mV, 30 microM GABA, 130 mM Na+ and room temperature). This indicates either very high levels of expression (approximately 10(4) microns‐2), assuming published turnover rates (approximately 10 s‐1), or turnover rates that are significantly greater than previously reported. As an alternative, a channel may exist in the GAT1 protein that is gated by GABA and Na+ and blocked by GAT1 antagonists. The channel mode of conduction would exist in addition to the coupled, fixed‐stoichiometry transporter mode of conduction.
Behaviour of raphe cells projecting to the dorsomedial medulla during carbachol‐induced atonia in the cat.
Authors: Woch G , Davies R O , Pack A I , Kubin L
Published: December 2014

1. The activity of most brainstem serotonergic cells is suppressed during sleep, particularly the rapid eye movement (REM) phase. Thus, they may play a major role in state‐dependent changes in CNS functioning. Our main goal was to search for medullary raphe cells having axonal branches in the region of the hypoglossal (XII) motor nucleus and assess their behaviour during the atonia produced by microinjections of a cholinergic agonist, carbachol, into the dorsal pontine tegmentum. In chronic animals, such microinjections evoke a desynchronized sleep‐like state similar to natural REM sleep; in decerebrate animals, they produce eye movements and a motor suppression similar to the postural atonia of REM sleep. 2. In decerebrate, paralysed, vagotomized and artificially ventilated cats, we recorded extracellularly from medullary raphe cells antidromically activated from the XII nucleus region. Forty‐five cells recorded in the raphe obscurus and pallidus nuclei were antidromically activated with latencies characteristic of non‐myelinated fibres (4.4‐42.0 ms). For thirty‐three of the forty‐five cells, we found one or more axonal branches within or just below the XII nucleus. The remaining twelve cells, in addition to the XII nucleus, had axonal ramifications in the medial nucleus of the solitary tract (NTS) and/or the dorsal motor nucleus of the vagus (DMV). 3. A subset of fourteen spontaneously active cells with identified axonal projections were held long enough to be recorded during the carbachol‐induced atonia, and eight of these also during the subsequent recovery and a systemic administration of the serotonergic 1A receptor agonist (+/‐)8‐hydroxy‐2‐(di‐N‐propylamino)tetrealin hydrobromide (8‐OH‐DPAT). All but one were suppressed during the atonia in parallel to the suppression of XII, phrenic and postural nerve activities (firing rate, 1.3 +/‐ 0.7 Hz before and 0.1 +/‐ 0.2 Hz after carbachol (means +/‐ S.D.)). Following the recovery from the atonia, the firing rates of the eight cells increased to the pre‐carbachol level (1.6 +/‐ 1.0 Hz). Subsequently, all were silenced by 8‐OH‐DPAT. 4. These cells fulfil most physiological criteria for serotonergic cells and have the potential to modulate, in a state‐dependent manner, activities in the motor XII nucleus, visceral sensory NTS, and DMV. The decrements in serotonergic neuronal activity that occur during the carbachol‐induced atonia suggest that a similar withdrawal of serotonergic input may occur during REM sleep and contribute to the characteristic reductions in upper airway motor tone.
A rate theory model for Mg2+ block of ATP‐dependent potassium channels of rat skeletal muscle.
Authors: Davies N W , McKillen H C , Stanfield P R , Standen N B
Published: December 2014

1. We have studied the block by intracellular Mg2+ (0.08‐4mM) of ATP‐dependent potassium channels (KATP channels) from rat skeletal muscle using inside‐out excised sarcolemmal patches. The block is voltage dependent, is relieved by extracellular potassium and has rapid kinetics, allowing the use of amplitude distribution analysis to estimate on and off rates. 2. To gain insight into the pore properties necessary to produce such a block, we have used an energy barrier model based on Eyring rate theory. The model has two energy wells and three barriers for K+ within the pore, while intracellular Mg2+ has access only to the inner well. We fitted the model to unitary current‐voltage relations in different [Mg2+], to on and off rates, and to dissociation constants for Mg2+ block. 3. The voltage dependence of block was almost entirely due to the rate constant for unblocking. This implies that the inner energy barrier is asymmetrical, so that Mg2+ entry senses little of the voltage field, but Mg2+ exit senses about 20% of the voltage field. Best fits were obtained by placing the barrier and binding site 0.01 and 0.22, respectively, of the electrical distance through the pore from the inside. 4. The relief of block by [K+]o resulted from an increase in the unblocking rate for Mg2+, implying ionic repulsion between ions in the pore.
Control of ion flux and selectivity by negatively charged residues in the outer mouth of rat sodium channels.
Authors: Chiamvimonvat N , Pérez-García M T , Tomaselli G F , Marban E
Published: December 2014

1. The sodium channel has a ring of negatively charged amino acids on its external face. This common structural feature of cation‐selective channels has been proposed to optimize conduction by electrostatic attraction of permeant cations into the channel mouth. We tested this idea by mutagenesis of mu1 rat skeletal sodium channels expressed in Xenopus oocytes. 2. Replacement of the external glutamate residue in domain II by cysteine reduces sodium current by decreasing single‐channel conductance. While this effect can be reversed by the negatively charged sulfhydryl modifying reagent methanethiosulphonate ethylsulphonate (MTSES), the flux saturation behaviour cannot be rationalized simply by changes in the surface charge. 3. The analogous mutations in domains I, III and IV affect not only conductance but also selectivity. These changes in selectivity are only partially reversed by exposure to MTSES. 4. Our findings necessitate revision of prevailing concepts regarding the role of superficial negatively charged residues in the process of ion permeation. These residues do not act solely by electrostatic attraction of permeant ions, but instead may help to form ion‐specific binding sites within the pore.
Intracellular sodium homeostasis in rat hippocampal astrocytes.
Authors: Rose C R , Ransom B R
Published: December 2014

1. We determined the intracellular Na+ concentration ([Na+]i) and mechanisms of its regulation in cultured rat hippocampal astrocytes using fluorescence ratio imaging of the Na+ indicator SBFI‐AM (acetoxymethylester of sodium‐binding benzofuran isophthalate, 10 microM). Dye signal calibration within the astrocytes showed that the ratiometric dye signal changed monotonically with changes in [Na+]i from 0 to 140 nM. The K+ sensitivity of the dye was negligible; intracellular pH changes, however, slightly affected the ‘Na+’ signal. 2. Baseline [Na+]i was 14.6 +/‐ 4.9 mM (mean +/‐ S.D.) in CO2/HCO3(‐)‐containing saline with 3 mM K+. Removal of extracellular Na+ decreased [Na+]i in two phases: a rapid phase of [Na+]i reduction (0.58 +/‐ 0.32 mM min‐1) followed by a slower phase (0.15 +/‐ 0.09 mM min‐1). 3. Changing from CO2/HCO3(‐)‐free to CO2/HCO3(‐)‐buffered saline resulted in a transient increase in [Na+]i of approximately 5 mM, suggesting activation of inward Na(+)‐HCO3‐ cotransport by CO2/HCO3‐. During furosemide (frusemide, 1 mM) or bumetanide (50 microM) application, a slow decrease in [Na+]i of approximately 2 mM was observed, indicating a steady inward transport of Na+ via Na(+)‐K(+)‐2Cl‐ cotransport under control conditions. Tetrodotoxin (100 microM) did not influence [Na+]i in the majority of cells (85%), suggesting that influx of Na+ through voltage‐gated Na+ channels contributed to baseline [Na+]i in only a small subpopulation of hippocampal astrocytes. 4. Blocking Na+, K(+)‐ATPase activity with cardiac glycosides (ouabain or strophanthidin, 1 mM) or removal of extracellular K+ led to an increase in [Na+]i of about 2 and 4 mM min‐1, respectively. This indicated that Na+, K(+)‐ATPase activity was critical in maintaining low [Na+]i in the face of a steep electrochemical gradient, which would favour a much higher [Na+]i. 5. Elevation of extracellular K+ concentration ([K+]o) by as little as 1 mM (from 3 to 4 mM) resulted in a rapid and reversible decrease in [Na+]i. Both the slope and the amplitude of the [K+]o‐induced reductions in [Na+]i were sensitive to bumetanide. A reduction of [K+]o by 1 mM increased [Na+]i by 3.0 +/‐ 2.3 mM. In contrast, changing extracellular Na+ concentration by 20 mM resulted in changes in [Na+]i of less than 3 mM. 6. These results implied that in hippocampal astrocytes low baseline [Na+]i is determined by the action of Na(+)‐HCO3‐ cotransport, Na(+)‐K(+)‐2Cl‐ cotransport and Na+, K(+)‐ATPase, and that both Na+, K(+)‐ATPase and inward Na(+)‐K(+)‐2Cl cotransport are activated by small, physiologically relevant increases in [K+]o. These mechanisms are well suited to help buffer increases in [K+]o associated with neural activity.