bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of the cGMP‐gated ion channel in frog rods by calmodulin and an endogenous inhibitory factor.
Authors: Gordon S E , Downing-Park J , Zimmerman A L
Published: December 2014

1. Outer segment patches excised in the light were used to investigate the effects of exogenous calmodulin and an endogenous inhibitory factor on the cGMP‐gated channel of frog rods. 2. Calmodulin shifted to the right the dose‐response relation for activation of the channels by 8‐Br‐cGMP, but did not change the maximum current or the form of the relation. Reversal of this effect by removal of calmodulin was accelerated by brief exposure to saturating [8‐Br‐cGMP]. Inhibition by calmodulin required calcium and gave as much as a 5‐fold decrease in current for an [8‐Br‐cGMP] functionally comparable to the presumed physiological [cGMP]. 3. Exposure to low [Ca2+]i (tens of nanomolar) appeared to irreversibly remove or inactivate an endogenous channel inhibitory factor from the patches, increasing the current at low [8‐Br‐cGMP]. Like calmodulin, this factor slowed the voltage‐dependent channel‐gating kinetics and did not change the maximum current. However, unlike calmodulin, the endogenous factor remained stably associated with the patches at high [Ca2+]i (1 microM), even with exposure to saturating [8‐Br‐cGMP]. 4. After the low‐Ca2+ treatment increased the current, calmodulin reduced the current to about the same level as it had before the low‐Ca2+ treatment, giving a larger fractional suppression. Furthermore, patches with high initial sensitivity to 8‐Br‐cGMP had small low‐Ca2+ effects and large calmodulin effects, while the reverse was true for patches with low initial agonist sensitivity. 5. Application of trypsin to the intracellular surface of the patch prevented the responses to calmodulin and to low [Ca2+]i, suggesting involvement of a cytoplasmic portion of the channel. However, trypsin also reduced the total agonist‐induced patch current. 6. Our results are consistent with a model in which calmodulin and an endogenous calcium‐binding protein compete for the same site, inhibiting channel opening or cGMP binding. The tight association of the endogenous factor with the channel even at relatively low [Ca2+]i suggests that in the transducing rod it may inhibit the channels most of the time in darkness and in dim light, preventing any potential inhibitory effects of calmodulin. The endogenous factor would be expected to leave the channel only in bright or prolonged light, when the [Ca2+]i is thought to be very low.
Concentrations of Na+ and Cl‐ in transplacental ultrafiltrate in sheep.
Authors: Faber J J , Anderson D F
Published: December 2014

1. Maternal blood was made transiently hypertonic by rapid I.V. infusion of a concentrated mannitol solution into pregnant ewes bearing lambs with an indwelling flow sensor and vascular catheters. 2. The transplacental flows of water and of Na+ and Cl‐ were calculated from the umbilical arteriovenous differences in the concentrations of 125I‐labelled albumin and electrolytes, and the fetal placental blood flow. 3. The reflection coefficients of Na+ and Cl‐ were calculated by means of the Patlak equation and found to be 0.85 +/‐ 0.04 and 0.68 +/‐ 0.04 (means +/‐ S.E.M.). The filtration coefficient was 1.02 x 10(‐7) +/‐ 0.12 x 10(‐7) cm5 dyne‐1 s‐1. 4. The results fitted best to an equivalent pore radius in the placental barrier smaller than the currently accepted 0.44 nm but not less than 0.35 nm.
Nitric oxide evokes pain at nociceptors of the paravascular tissue and veins in humans.
Authors: Holthusen H , Arndt J O
Published: December 2014

1. Nitric oxide (NO) evokes pain on intracutaneous application, apparently by exciting cutaneous nociceptors. To look for similarities in the responsiveness and sensitivity of other nociceptive systems to NO we determined pain intensity‐concentration relations for NO applied to paravascular tissue and veins in humans. 2. NO solutions (0.4‐2.0 mM) were either injected paravascularly or perfused through a vascularly isolated hand vein segment. The subjects rated pain continuously with the help of an electronically controlled visual analog scale, which made it possible to determine both the time course (latency, duration) and the intensity of NO‐evoked pain. 3. Regardless of where it was applied, at concentrations above 0.7 mM NO always evoked pain of similar time course and concentration dependence. Pain increased proportionally to the concentration of applied NO, reaching subjects' tolerance maximum at four to five times the threshold concentration. 4. Pain intensity‐NO concentration relations were congruent, indicating that the respective nociceptive systems are equally sensitive to NO. 5. Our observations are consistent with the hypothesis that NO is a chemical link in peripheral nociception.
Receptor kinase‐dependent desensitization of the muscarinic K+ current in rat atrial cells.
Authors: Shui Z , Boyett M R , Zang W J , Haga T , Kameyama K
Published: December 2014

1. Activity of rat atrial muscarinic K+ channels has been measured in five configurations of the patch clamp technique. 2. In configurations in which the normal intracellular solution was lost, the slow phase of desensitization (a slow decline of channel activity during an exposure to ACh) was much reduced (or absent) and deactivation (on wash‐off of ACh) was slowed as compared with desensitization and deactivation in configurations in which normal intracellular solution was retained. This suggests that soluble intracellular regulators are involved in these processes. 3. When a G protein‐coupled receptor kinase (GRK2) was applied to the cytoplasmic surface of conventional outside‐out patches in the presence of ATP, the slow phase of desensitization was restored. In the absence of ATP, GRK2 failed to restore the slow phase. 4. It is concluded that (i) G protein‐coupled receptor kinase dependent phosphorylation of the muscarinic receptor is responsible for the slow phase of desensitization and (ii) a soluble factor (such as a GTPase activating protein or ‘GAP’) is responsible for normal rapid deactivation.
Concentration polarization of fluorescent dyes in rat descending colonic crypts: evidence of crypt fluid absorption.
Authors: Naftalin R J , Zammit P S , Pedley K C
Published: December 2014

1. Using confocal microscopy, the rate of fluid absorption into isolated perifused descending rat colonic crypt lumens is estimated from the concentration polarization and distribution of fluorescein sulphonate (FS) and fluorescein isothiocyanate‐dextran (FITC‐dextran; molecular weight, 10,000) within the crypt lumens and pericryptal fluid. 2. The probe dyes enter the crypt via the luminal opening, are concentrated in the lumen, then escape into the pericryptal space via the paracellular spaces spanning the crypt wall. 3. FITC‐dextran is maximally accumulated at a luminal depth of 60 microns to 5 times the concentration at the crypt opening (p < 0.001) and penetrates 150‐200 microns along the lumen. FS is maximally accumulated within crypt lumen close to the opening. At crypt luminal depths 10‐60 microns from the opening FS is accumulated by a factor of 1.5‐2.0 above that found in HgCl2‐treated tissue (p < 0.001). 4. Dye enters the crypt lumen slowly from the basal side, but from this side does not accumulate above the bathing solution concentration. 5. HgCl2 (20 microM) or theophylline (10 mM) completely inhibit concentrative accumulation of FITC‐dextran and FS within the crypts and pericryptal space (p < 0.001). 6. Computer simulation of the dye uptake indicates that the rate of water flow into the crypt luminal opening is 1 x 10(‐3) cm s‐1 which is equivalent to 15 microliters (cm mucosa)‐2 h‐1. Approximately 75% of the fluid entering the crypt is abosrbed across the proximal 50 microns of crypt wall as a consequence of the large osmotic pressure gradient between the pericryptal and crypt luminal solutions. A pericryptal diffusion barrier with lower permeability than that across the crypt wall is required to simulate dye accumulation in the pericryptal space. Differences between FITC‐dextran and FS accumulation are explained by the lower diffusion coefficient within the crypt lumen, and lower crypt wall permeability of FITC‐dextran.
The effect of voluntary contraction on cortico‐cortical inhibition in human motor cortex.
Authors: Ridding M C , Taylor J L , Rothwell J C
Published: December 2014

1. It has been previously shown that in a relaxed target muscle, at short interstimulus intervals (ISIs) (up to 6 ms) a conditioning subthreshold transcranial magnetic stimulus can cause suppression of the EMG response evoked by a magnetic test stimulus. At longer ISIs (7‐15 ms) facilitation of the test response is seen. This type of inhibition has been termed ipsilateral cortico‐cortical inhibition. 2. The effect of a minimal tonic contraction on ipsilateral cortico‐cortical inhibition has been investigated in the first dorsal interrosseous (FDI). 3. At short ISIs there was significantly less inhibition of the test response during the maintenance of minimal voluntary tonic contraction of the target muscle (FDI). 4. At longer ISIs (7‐15 ms) there was significantly less facilitation of the test response during a tonic contraction than during relaxation. 5. Minimal activation of an ipsilateral proximal muscle (biceps) had no significant effect on the degree of inhibition seen in the relaxed target muscle (FDI). 6. We suggest that voluntary drive reduces the excitability of inhibitory circuits in cortical areas that project to the active muscle.
Mechanics of pulsatile transpyloric flow in the pig.
Authors: Anvari M , Dent J , Malbert C , Jamieson G G
Published: December 2014

1. In eight conscious pigs equipped with gastric and duodenal cannulae, the relationship of transpyloric flow to gastro‐duodenal motor events was evaluated during gastric emptying of 1000 ml of saline. Rates of liquid gastric emptying were correlated with pressures at the antrum, pylorus and duodenum, recorded by a sleeve sensor and multiple perfused side‐holes. Transpyloric flow was recorded concurrently by continuous collection and weighing of the duodenal effluent. 2. In three pigs the above measurements were repeated during concurrent videofluoroscopy of gastric emptying after adding 100 ml of liquid barium to the gastric instillate. 3. The mean volume of saline emptied in 30 min was 627 +/‐ 51.2 ml. Pulsatile flow accounted for 71% of total emptying. Pulses had a mean flow rate of 3.9 +/‐ 0.44 ml s‐1. Most flow pulses (59%) occurred during the first 5 min of emptying. 4. Distinctive, low‐amplitude (4.8 +/‐ 0.33 mmHg), relatively long‐lasting (15.8 +/‐ 0.46 s) antral pressure waves were associated with 58% of flow pulses. In all antral pressure recording points, the first and longest duration component of these pressure waves had an identical timing, amplitude and waveform consistent with pressurization of the entire antrum‐gastric cavity. 5. Videofluoroscopy and concurrent manometry showed that these antral common cavity pressure waves were associated with non‐lumen‐occlusive contractions of the gastric wall, initially observed at the corpus which propagated down to the pylorus; 93% of these contractions became lumen occlusive in the terminal antrum and pylorus when pressure waves of a unique pattern for each recording point were recorded at this level. 6. The onset of 68% of the flow pulses which accounted for 62% of pulsatile emptying occurred in the interval (mean 7.9 +/‐ 0.65 s) between the onset of the common cavity wave and the onset of localized, lumen‐occlusive distal antral‐pyloric pressure waves. 7. These findings indicate that in the pig, pulsatile emptying of non‐nutrient liquids into the duodenum occurs predominantly during the non‐lumen‐occlusive stage of a propagated gastric contraction, which is recognisable as a common cavity pressure wave. This is a previously inadequately recognized pattern of gastric pumping.
Role of nitric oxide in exercise hyperaemia during prolonged rhythmic handgripping in humans.
Authors: Dyke C K , Proctor D N , Dietz N M , Joyner M J
Published: December 2014

1. We sought to determine whether the vasodilating molecule nitric oxide (NO) contributes to the forearm hyperaemia observed during prolonged rhythmic handgripping in humans. 2. Two bouts of exercise were performed during experimental protocols conducted on separate days. During each protocol the subject performed a 10 min and a 20 min bout of rhythmic (30 min‐1) handgripping at 15% of maximum. Two exercise bouts were required to facilitate pharmacological interventions during the second protocol. Blood flow in the exercising forearm was measured every minute with plethysmography during brief pauses in the contractions. During both exercise bouts in the first protocol, forearm blood flow increased 2‐ to 3‐fold above rest after 1 min of handgripping and remained constant at that level throughout the exercise. 3. During the 10 min bout of exercise in the second protocol, acetylcholine was given via a brachial artery catheter at 16 micrograms min‐1 for 3 min to evoke NO release from the vascular endothelium. This caused forearm blood flow to increase above the values observed during exercise alone. 4. During the 20 min trial of handgripping in the second protocol, the NO synthase blocker NG‐monomethyl‐L‐arginine (L‐NMMA) was infused in the exercising forearm via the brachial catheter after 5 min of handgripping. The L‐NMMA was infused at 4 mg min‐1 for 10 min. 5. L‐NMMA during exercise caused forearm blood flow to fall to values approximately 20‐30% lower than those observed during exercise alone. When ACh was given during exercise after L‐NMMA administration the rise in blood flow was also blunted, indicating blockade of NO synthase. These data suggest NO plays a role in exercise hyperaemia in humans.
Temperature effects on membrane potential and input resistance in rat hypothalamic neurones.
Authors: Griffin J D , Boulant J A
Published: December 2014

1. Whole‐cell recordings were conducted in rat hypothalamic tissue slices to test the hypothesis that thermal changes in membrane potential contribute to neuronal thermosensitivity. Intracellular recordings of membrane potential and input resistance were made in eighty‐two neurones, including twenty‐four silent neurones and fifty‐eight spontaneously firing neurones (22 warm‐sensitive neurones and 36 temperature‐insensitive neurones). Fifty‐seven of the neurones were recorded in the preoptic and anterior hypothalamus. 2. Warm‐sensitive neurones increased their firing rates during increases in temperature (1.07 +/‐ 0.06 impulses s‐1 degree C‐1), but their resting membrane potentials were not affected by temperature (0.06 +/‐ 0.06 mV degree C‐1). Similarly, temperature did not affect the membrane potentials of temperature‐insensitive neurones or silent neurones. 3. Silent neurones had significantly lower input resistances (256.9 +/‐ 20.0 M omega), compared with temperature‐insensitive (362.6 +/‐ 57.2 M omega) and warm‐sensitive neurones (392.2 +/‐ 50.0 M omega). Temperature had the same effect on all three types of neurones, such that resistance increased during cooling and decreased during warming. 4. If hyperpolarizing or depolarizing holding currents were applied to neurones, temperature caused changes in the membrane potentials. This spurious effect can be explained by thermally induced changes in the input resistance. 5. Measurements of electrode tip potentials indicated that artificial changes in membrane potential may also be recorded if grounding electrodes are not isolated from the changes in temperature. 6. These results suggest that physiological changes in resting membrane potentials do not determine neuronal warm sensitivity, and thermal changes in input resistance do not determine the primary differences between warm‐sensitive and temperature‐insensitive hypothalamic neurones.
A voltage‐gated chloride channel in ascidian embryos modulated by both the cell cycle clock and cell volume.
Authors: Villaz M , Cinniger J C , Moody W J
Published: December 2014

1. Eggs of the ascidian Boltenia villosa have an inwardly rectifying Cl‐ current whose amplitude varies by more than 10‐fold during each cell cycle, the largest amplitude being at exit from M‐phase. We examined whether this current was also sensitive to changes in cell volume. 2. Cell swelling, produced by direct inflation through a whole‐cell recording pipette, greatly increased the amplitude of the Cl‐ current at all stages of the cell cycle in activated eggs. Swelling was much less effective in unfertilized eggs. 3. The increase in Cl‐ current amplitude continued for 10‐20 min after an increase in diameter that was complete in 10 s, suggesting the involvement of a second messenger system in the response. 4. Treatment of unfertilized eggs with 6‐dimethylaminopurine (DMAP), an inhibitor of cell cycle‐dependent protein kinases, increased the amplitude of the Cl‐ current and its sensitivity to swelling to levels characteristic of fertilized eggs. 5. Osmotically produced swelling also increased Cl‐ current amplitude in unfertilized eggs. 6. We propose that dephosphorylation renders the Cl‐ channel functional, and that swelling or activation of the egg increases the sensitivity of the channel to dephosphorylation, perhaps by disrupting its links to the cytoskeleton.
Diabetes‐induced activation of system y+ and nitric oxide synthase in human endothelial cells: association with membrane hyperpolarization.
Authors: Sobrevia L , Cesare P , Yudilevich D L , Mann G E
Published: December 2014

1. The activity of the human endothelial cell L‐arginine transporter (system y+) has been correlated with cGMP production (index of nitric oxide) and prostacyclin (PGI2) release in umbilical vein endothelial cells cultured from normal or gestational diabetic pregnancies. 2. In non‐diabetic and diabetic cells, transport of L‐arginine was Na+ and pH independent, inhibited by other cationic L‐arginine analogues and unaffected by neutral amino acids. 3. Diabetes was associated with an increased Vmax for saturable L‐arginine transport (4.6 +/‐ 0.13 vs. 9.9 +/‐ 0.5 pmol (microgram protein)‐1 min‐1, P < 0.01), but had no effect on initial rates of transport for L‐serine, L‐citrulline, L‐leucine or 2‐deoxyglucose. 4. In non‐diabetic and diabetic cells, elevated K+ resulted in a concentration‐dependent inhibition in the initial rates of transport for L‐arginine and the membrane potential‐sensitive probe tetra[3H]phenylphosphonium (TPP+). 5. When resting membrane potential was measured using the whole‐cell patch voltage clamp technique, diabetic cells were hyperpolarized (‐78 +/‐ 0.3 mV) compared with non‐diabetic cells (‐70 +/‐ 0.04 mV, P < 0.04). Accumulation of [3H]TPP+ was also increased in diabetic compared with non‐diabetic cells. 6. Basal intracellular cGMP levels were elevated 2.5‐fold in diabetic cells, and L‐NAME (100 microM), an inhibitor of nitric oxide synthase, abolished basal cGMP accumulation in non‐diabetic and diabetic cells. 7. Histamine (10 microM) had no effect on L‐arginine transport but evoked significant increases in cGMP in non‐diabetic and diabetic cells, which were completely inhibited by L‐NAME but unaffected by superoxide dismutase. 8. Basal and histamine‐stimulated PGI2 release was decreased markedly in diabetic cells. 9. Our findings demonstrate that gestational diabetes is associated with phenotypic changes in fetal endothelial cells, which result in a membrane hyperpolarization, activation of the human endothelial cell L‐arginine transporter (system y+), elevation of basal nitric oxide synthesis and decreased PGI2 production.
The role of adenosine in mediating vasodilatation in mesenteric circulation of the rat in acute and chronic hypoxia.
Authors: Mian R , Marshall J M
Published: December 2014

1. We have compared the roles of adenosine in mediating dilator responses to acute hypoxia in mesenteric microcirculation of control, normoxic (N) rats and in chronically hypoxic (CH) rats kept in an hypoxic chamber at 10% O2 for 3‐4 weeks. 2. In fifteen N rats, acute hypoxia (breathing 6% O2 for 3 min) induced mean increases in the diameter of arterial vessels of mesentery (whose internal diameter was 10‐350 microns) of 8.0 +/‐ 1.9% (mean +/‐ S.E.M.) and of venous vessels (whose internal diameter was 12‐360 microns) of 10.4 +/‐ 2.6%. These diameter changes were reduced by approximately 30% when the adenosine receptor antagonist 8‐sulpho‐phenyltheophylline (8‐SPT, 10(‐3) M) was applied topically to the mesentery. 3. In a further six N rats, topical application of graded concentrations of adenosine (10(‐7)‐10(‐3) M) to the mesentery evoked graded increases in the diameter of all arterial and venous vessels, maximum increases with 10(‐3) M being 12.5 +/‐ 3.3 and 8.4 +/‐ 4.3%, respectively; these responses were abolished by 8‐SPT. 4. By contrast, in fourteen CH rats, the smaller change in inspirate from 10 to 8% O2 induced increases in diameter of arterial and venous vessels which had control diameters that were comparable to those of N rats, of 14.1 +/‐ 2.4 and 12.9 +/‐ 2.7%, respectively, and which were virtually equivalent to the responses induced by topical application of 10(‐3) M adenosine (13.3 +/‐ 1.3 and 16.3 +/‐ 2.0% in arterial and venous vessels, respectively). The changes induced by acute hypoxia were abolished by 8‐SPT, as were those induced by adenosine. 5. These results suggest that in the intestinal mesentery, where the blood vessels have negligible tissue parenchyma around them, locally released or synthesized adenosine makes a substantial contribution to the dilatation that is evoked in arteriolar vessels by acute hypoxia and to the active dilatation, or passive distension of the venous vessels. The results also suggest that this contribution is accentuated in chronic hypoxia either by greater release of adenosine or greater vascular sensitivity to it.
Voltage‐activated proton currents in membrane patches of rat alveolar epithelial cells.
Authors: DeCoursey T E , Cherny V V
Published: December 2014

1. Voltage‐activated H(+)‐selective currents were studied in cell‐attached and excised, inside‐out patches of membrane from rat alveolar epithelial cells in primary culture. The pH was varied from 5.5 to 7.5 in the pipette (pHo) and in the bath (pHi). 2. H+ currents in cell‐attached patches exhibited activation kinetics and voltage dependence intermediate between their behaviour after excision into pH 6.5 and pH 7.5 solutions, consistent with reported pHi values for alveolar epithelial cells. 3. In inside‐out patches, increasing pHo shifted the threshold voltage for activating H+ currents and the relationship between the time constant of activation of H+ currents (tau act) and voltage (V) negatively along the voltage axis by 40‐50 mV (unit pH)‐1. 4. Decreasing pHi shifted the activation threshold and the tau act‐V relationship negatively along the voltage axis by 40‐50 mV (unit pH)‐1. In addition, at lower pHi the activation of H+ currents upon patch depolarization was markedly faster, with tau act increasing approximately 4‐ to 5‐fold per unit pHi. 5. The limiting slope H+ conductance increased only by a factor of 1.7 per unit pH when pHi was lowered in the range 7.5‐5.5 (at constant pHo 7.5). This result suggests that H+ permeation through voltage‐activated H+ ‘channels’ occurs by a mechanism distinct from H+ permeation through water‐filled ion channels, in which the conductance might be expected to increase in direct proportion to [H+].
Hyperpnoea during and immediately after exercise in man: evidence of motor cortical involvement.
Authors: Fink G R , Adams L , Watson J D , Innes J A , Wuyam B , Kobayashi I , Corfield D R , Murphy K , Jones T , Frackowiak R S
Published: December 2014

1. The neurophysiological basis for the increase in breathing associated with exercise remains obscure. The present study uses positron emission tomography (PET) to measure relative regional cerebral blood flow (rCBF) in order to identify sites of increased neuronal activation during and immediately following exercise. 2. Male volunteers underwent H2(15)O PET scanning during two complementary studies. Firstly, six subjects performed right leg exercise, adequate to increase oxygen uptake 2.5‐fold. Secondly, five different subjects were scanned immediately following bicycle exercise (adequate to increase oxygen uptake 5‐fold) while breathing was still increased. In each study, as a control, scanning was also performed during matched passive isocapnic positive pressure ventilation; additionally, in the first study, passive right leg movement was performed. 3. Increases in relative rCBF were obtained in each individual and co‐registered with their magnetic resonance image of the brain defining individual gyral morphology. 4. During exercise, individual and group analysis revealed significant relative rCBF increases in the left and right superomedial primary motor cortex (the motor cortical ‘leg’ areas) and also in the left and right superolateral primary motor cortex in areas previously shown to be associated with volitional breathing. After exercise, there was no significant increase in relative rCBF in the superomedial areas but such increases were still present bilaterally in the superolateral areas which had been activated during the exercise. Other relative rCBF increases were also found, both during and after exercise, in cortical and subcortical areas known to be involved in motor control. 5. The results from PET scans during and after exercise, taken together, provide evidence for motor cortical involvement in the exercise‐related hyperpnoea in man.
Activation of muscarinic K+ current in guinea‐pig atrial myocytes by sphingosine‐1‐phosphate.
Authors: Bünemann M , Brandts B , zu Heringdorf D M , van Koppen C J , Jakobs K H , Pott L
Published: December 2014

1. Activation of muscarinic K+ current (IK(ACh)) by sphingosine‐1‐phosphate (Sph‐1‐P) was studied in isolated cultured guinea‐pig atrial myocytes using whole‐cell voltage clamp. 2. Sph‐1‐P caused activation of IK(ACh) with an EC50 of 1.2 nM. The maximal current that could be activated by Sph‐1‐P amounted to about 90% of the IK(ACh) caused by a saturating concentration of acetylcholine (ACh, 10 microM). Sphingosine (1 microM), which can mimic the signalling effects of Sph‐1‐P in other cells, failed to cause measurable activation of IK(ACh). 3. IK(ACh) activation by Sph‐1‐P was completely suppressed in cells treated with pertussis toxin. 4. Desensitization of muscarinic receptors by pre‐incubation of the cells with carbachol did not affect the response to Sph‐1‐P; likewise, pre‐incubation of the cells with Sph‐1‐P resulted in a reduced sensitivity to the phospholipid but not to ACh. In contrast, pre‐incubation with either Sph‐1‐P or a serum phospholipid previously described as activating atrial IK(ACh) resulted in reduced sensitivity to both phospholipids. 5. It is concluded that activation of IK(ACh) by Sph‐1‐P in atrial myocytes is induced by binding to a novel G protein‐coupled phospholipid receptor.
Activation of nicotinic acetylcholine receptors increases the rate of fusion of cultured human myoblasts.
Authors: Krause R M , Hamann M , Bader C R , Liu J H , Baroffio A , Bernheim L
Published: December 2014

1. Fusion of myogenic cells is important for muscle growth and repair. The aim of this study was to examine the possible involvement of nicotinic acetylcholine receptors (nAChR) in the fusion process of myoblasts derived from postnatal human satellite cells. 2. Acetylcholine‐activated currents (ACh currents) were characterized in pure preparations of freshly isolated satellite cells, proliferating myoblasts, myoblasts triggered to fuse and myotubes, using whole‐cell and single‐channel voltage clamp recordings. Also, the effect of cholinergic agonists on myoblast fusion was tested. 3. No nAChR were observed in freshly isolated satellite cells. nAChR were first observed in proliferating myoblasts, but ACh current densities increased markedly only just before fusion. At that time most mononucleated myoblasts had ACh current densities similar to those of myotubes. ACh channels had similar properties at all stages of myoblast maturation. 4. The fraction of myoblasts that did not fuse under fusion‐promoting conditions had no ACh current and thus resembled freshly isolated satellite cells. 5. The rate of myoblast fusion was increased by carbachol, an effect antagonized by alpha‐bungarotoxin, curare and decamethonium, but not by atropine, indicating that nAChR were involved. Even though a prolonged exposure to carbachol led to desensitization, a residual ACh current persisted after several days of exposure to the nicotinic agonist. 6. Our observations suggest that nAChR play a role in myoblast fusion and that part of this role is mediated by the flow of ions through open ACh channels.
Effect of chronic vitamin E deficiency on sympathetic and sensorimotor function in rat mesenteric arteries.
Authors: Ralevic V , Hoyle C H , Goss-Sampson M A , Milla P J , Burnstock G
Published: December 2014

1. Mesenteric arterial beds from male rats deprived of vitamin E for 12 months postweaning were isolated and perfused at 5 ml min‐1. 2. The basal perfusion pressure of vitamin E‐deficient preparations was significantly higher (34.0 +/‐ 1.9 mmHg, n = 15) than in age‐matched controls (26.1 +/‐ 2 mmHg, n = 14; P < 0.01). 3. At basal tone, vasoconstrictor responses to electrical field stimulation (EFS) were not attenuated by vitamin E deficiency; at high stimulation frequencies, responses were enhanced. According to dose‐response curves, exogenous noradrenaline was significantly more efficacious in preparations from vitamin E‐deficient rats (P < 0.05). 4. In preparations with tone raised by methoxamine (6‐20 microM) and in the presence of guanethidine (5 microM), EFS of perivascular sensorimotor nerves elicited frequency‐dependent vasodilatation which was significantly attenuated by vitamin E deficiency. There was no difference in relaxation to calcitonin gene‐related peptide (CGRP; 1.5 x 10(‐11) mol), or to the sensory neurotoxin capsaicin (5 x 10(‐11) mol). 5. Immunohistochemical analysis of CGRP‐containing nerves in the superior mesenteric artery showed no differences in density of innervation. 6. In conclusion, chronic vitamin E deficiency impairs sensorimotor vasodilatation in rat mesenteric arteries; this does not appear to be due to changes in postjunctional receptors, or to a depletion of transmitter (CGRP) content of the superior mesenteric artery. Sensorimotor nerves appear to be more vulnerable than sympathetic nerves to chronic vitamin E deficiency.
Regulation of renal Na+,K(+)‐ATPase in rat thick ascending limb during K+ depletion: evidence for modulation of Na+ affinity.
Authors: Buffin‐Meyer B , Marsy S , Barlet-Bas C , Cheval L , Younes-Ibrahim M , Rajerison R , Doucet A
Published: December 2014

1. NaCl reabsorption along the loop of Henle is reduced in K(+)‐depleted rats. Because Na+,K(+)‐ATPase energizes this transport and because K+ depletion is known to induce an upregulation of Na+,K(+)‐ATPase in most tissues, the regulation of this enzyme was investigated at the level of single thick ascending limbs of the loop of Henle freshly microdissected from rats fed either a normal (control rats) or a low‐K+ diet (LK rats). 2. Within 2 weeks of K+ depletion, Na+,K(+)‐ATPase activity and [3H]ouabain binding were increased by 30‐50% in the medullary portion of the thick ascending limb (MTAL). 3. Despite this increase in the number of Na+,K(+)‐ATPase units, the transport capacity of the Na+,K+ pump, determined by ouabain‐sensitive Rb+ uptake in the presence of an extracellular concentration of Rb+ mimicking the kalaemia determined in control (4.0 mM Rb+) and LK rats (2.3 mM Rb+), was reduced in MTAL from LK rats. 4. Inhibition of the Na+,K+ pump was not accounted for by changes in either extracellular K+ or intracellular Na+ concentrations, but by a decrease in the pump affinity for Na+. 5. Because this change in the apparent affinity of the Na+,K+ pump for Na+ was detectable in intact but not in permeabilized MTAL cells, it is probably induced by a rapidly reversible cytosolic factor.
Properties of presympathetic neurones in the rostral ventrolateral medulla in the rat: an intracellular study "in vivo'.
Authors: Lipski J , Kanjhan R , Kruszewska B , Rong W
Published: December 2014

1. Intracellular recordings were made in pentobarbitone‐anaesthetized rats from sixty‐eight neurones located in the rostral ventrolateral medulla (RVLM), which responded with inhibition (latency, 33.6 +/‐ 9.3 ms) after stimulation of the aortic depressor nerve with short bursts of pulses. This inhibition was due to chloride‐ and voltage‐dependent IPSPs. 2. Seventeen neurones could be excited antidromically after stimulation in the T2 spinal segment (conduction velocity 1.9‐8.5 m.s‐1) and were classified as RVLM presympathetic vasomotor neurones. 3. ‘Spontaneously’ active neurones (n = 29) displayed a largely irregular pattern of firing, with no clear relationship between the level of the membrane potential and cycles of phrenic nerve activity at end‐tidal CO2 < 5.0%. Cardiac cycle‐related shifts of the membrane potential were not considered indicative of baroreceptor input as they could be due to movement artifacts. 4. All neurones displayed large synaptic activity (EPSPs and IPSPs, peak‐to‐peak amplitude > 5.0 mV). The depolarizing IPSPs observed during injection of chloride and/or negative current consisted of a phasic and a tonic component. 5. The on‐going activity of these neurones resulted from synaptic inputs, with individual action potentials usually preceded by identifiable fast EPSPs. 6. No evidence was found for the presence of gradual depolarizations (autodepolarizations) between individual action potentials, and therefore under these experimental conditions the activity of RVLM presympathetic neurones did not depend on intrinsic pacemaker properties. 7. These results are consistent with the ‘network’ hypothesis for the generation of sympathetic vasomotor tone.
Oxidant stress activates a non‐selective cation channel responsible for membrane depolarization in calf vascular endothelial cells.
Authors: Koliwad S K , Kunze D L , Elliott S J
Published: December 2014

1. In vascular endothelial cells, oxidant stress increases cell Na+ content and inhibits the agonist‐stimulated influx of external Ca2+. Further, oxidant stress increases uptake of Ca2+ into otherwise quiescent endothelial cells. To determine the mechanism responsible for altered Na+ and Ca2+ homeostasis, the present study examined the effect of oxidant stress on ionic current and channel activity in calf pulmonary artery endothelial cells. 2. Voltage‐clamped control cells had a zero‐current potential of ‐60 mV. Incubation of cells with the oxidant tert‐butylhydroperoxide (tBuOOH; 0.4 mM, 1 h) caused depolarization to ‐4 mV and activation of ionic current equally selective for Na+ and K+. 3. Cell‐attached membrane patches made on tBuOOH‐treated cells contained ion channels that had a bidirectional conductance of 30 pS and that were not present in patches from control cells. Inside‐out patches excised from oxidant‐treated cells showed the channel to be equally selective for Na+ and K+ and to allow inward Ca2+ current. 4. Oxidant‐activated channels were observed to display two gating modalities that were further evident during analysis of single‐channel open probability. Neither modality was significantly affected by altering internal [Ca2+] (1 microM‐10 nM). 5. Activation of non‐selective channels provides a possible mechanism by which oxidants may increase endothelial cell Na+ content. Channel permeability to Ca2+ may account in part for the elevation of cytosolic free [Ca2+] that occurs in oxidant‐treated cells. 6. Channel activation is associated with membrane depolarization, a mechanism that may contribute to oxidant inhibition of the agonist‐stimulated Ca2+ influx pathway.
Paired‐pulse facilitation and depression at unitary synapses in rat hippocampus: quantal fluctuation affects subsequent release.
Authors: Debanne D , Guérineau N C , Gähwiler B H , Thompson S M
Published: December 2014

1. Excitatory synaptic transmission between pairs of monosynaptically coupled pyramidal cells was examined in rat hippocampal slice cultures. Action potentials were elicited in single CA3 pyramidal cells impaled with microelectrodes and unitary excitatory postsynaptic currents (EPSCs) were recorded in whole‐cell voltage‐clamped CA1 or CA3 cells. 2. The amplitude of successive unitary EPSCs in response to single action potentials varied. The amplitude of EPSCs was altered by adenosine or changes in the [Mg2+]/[CA2+] ratio. We conclude that single action potentials triggered the release of multiple quanta of glutamate. 3. When two action potentials were elicited in the presynaptic cell, the amplitude of the second EPSC was inversely related to the amplitude of the first. Paired‐pulse facilitation (PPF) was observed when the first EPSC was small, i.e. the second EPSC was larger than the first, whereas paired‐pulse depression (PPD) was observed when the first EPSC was large. 4. The number of trials displaying PPD was greater when release probability was increased, and smaller when release probability was decreased. 5. PPD was not postsynaptically mediated because it was unaffected by decreasing ionic flux with 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) or receptor desensitization with aniracetam. 6. PPF was maximal at an interstimulus interval of 70 ms and recovered within 500 ms. Recovery from PPD occurred within 5 s. 7. We propose that multiple release sites are formed by the axon of a CA3 pyramidal cell and a single postsynaptic CA1 or CA3 cell. PPF is observed if the first action potential fails to release transmitter at most release sites. PPD is observed if the first action potential successfully triggers release at most release sites. 8. Our observations of PPF are consistent with the residual calcium hypothesis. We conclude that PPD results from a decrease in quantal content, perhaps due to short‐term depletion of readily releasable vesicles.
Modulation by opioid peptides of mechanosensory pathways supplying the guinea‐pig inferior mesenteric ganglion.
Authors: Ma R C , Szurszewski J H
Published: December 2014

1. Radioimmunological techniques were used in isolated guinea‐pig inferior mesenteric ganglion (IMG)‐colon preparations to determine whether opioid peptides and neurotensin8‐13 (NT8‐13), the C‐terminal region of NT1‐13 recognized by neurotensin receptors, modulate distension‐induced release of substance P (SP)‐ and vasoactive intestinal polypeptide (VIP)‐like immunoreactive (LI) material. 2. Colonic distension significantly increased the amount of SP‐ and VIP‐LI material released in the ganglionic superfusate. A low‐Ca2+ (0.1 mM), high‐Mg2+ (15 mM) solution blocked their release. 3. In vivo capsaicin pretreatment abolished release of SP‐LI material during colonic distension but had no significant effect on distension‐induced release of VIP‐LI material. 4. The addition of [Leu5]enkephalin, [Met5]enkephalin, PL017 (a mu‐receptor agonist) and DPDPE (a delta‐receptor agonist) to the ganglion side of a two‐compartment chamber blocked distension‐induced release of SP‐LI material. The addition of naloxone and ICI‐174,864 (a delta‐receptor antagonist) to the ganglion compartment reversed the inhibitory effect of the mu‐ and delta‐receptor agonists. 5. Addition of [Leu5]enkephalin and [Met5]enkephalin to the ganglion compartment had no significant effect on release of VIP‐LI material during colonic distension. 6. Addition of NT8‐13 to the ganglion compartment significantly increased in the amount of SP‐LI material released during colonic distension but had no affect on distension‐induced release of VIP‐LI material. 7. The results suggest the hypothesis that under in vivo conditions, enkephalinergic nerves decrease and neurotensinergic nerves increase the release of SP from peripheral branches of primary afferent sensory nerves.
Relationship between intracellular calcium and its muffling measured by calcium iontophoresis in snail neurones.
Authors: Schwiening C J , Thomas R C
Published: December 2014

1. We have measured intracellular free calcium ion concentration ([Ca2+]i) with fura‐2, and intracellular chloride with chloride‐sensitive microelectrodes, in voltage‐clamped snail neurones. By making iontophoretic injections of CaCl2 we have investigated calcium muffling, the sum of the processes which minimize the calcium transient, at different values of [Ca2+]i. 2. By injection of calcium into cell‐sized droplets of buffer we measured the calcium transport index. It was stable over the range pCa 6‐7.4 (0.48 +/‐ 0.06 measured at pCa 6.70 +/‐ 0.12, n = 5). 3. Measurement of intracellular chloride activity during a series of fura‐2‐KCl pressure injections revealed a nearly linear relationship between fura‐2 Ca(2+)‐insensitive fluorescence and the sum of the increments in intracellular chloride. This allowed us to calculate the intracellular fura‐2 concentration ([fura‐2]i). 4. The rate of recovery of [Ca2+]i following a depolarization‐induced load was increased by low [fura‐2]i (10‐20 microM) but decreased by higher [fura‐2]i (40‐80 microM). These effects are consistent with the addition of a mobile buffer to the cytoplasm. 5. Iontophoresis of Ca2+ at various membrane potentials allowed us to calculate the intracellular calcium muffling power (the amount of calcium required to cause a transient tenfold increase in [Ca2+]i per unit volume) and calcium muffling ratio (number of Ca2+ ions injected divided by the maximum increase in [Ca2+]i per unit volume) at different values of [Ca2+]i. 6. Calcium muffling power at resting [Ca2+]i was approximately 40 microM Ca2+ (pCa unit)‐1, (about 250 times less than for hydrogen ions). It increased linearly about fivefold with [Ca2+]i over the range 20‐120 nM (10 cells, 153 measurements) and therefore exponentially with decreasing pCa. 7. The calcium muffling ratio appeared to be constant (361 +/‐ 14, n = 10 cells, 130 measurements) over the range 20‐120 nM Ca2+. 8. In three experiments we modelled the additional calcium buffering power produced by multiple pressure injections of fura‐2 into voltage‐clamped snail neurones. Back‐extrapolation of the increases in calcium buffering power allowed us to calculate the calcium muffling power of the neurones. 9. Small increases in [fura‐2]i (approximately 10 microM) significantly increased intracellular calcium muffling power in individual experiments. However, the variability among neurones in intracellular calcium muffling power was large enough to obscure the additional buffering produced by fura‐2 in pooled experiments.
ATP activates non‐selective cation channels and calcium release in inner hair cells of the guinea‐pig cochlea.
Authors: Sugasawa M , Erostegui C , Blanchet C , Dulon D
Published: December 2014

1. ATP‐evoked currents and Ca2+ signals were simultaneously recorded in isolated inner hair cells (IHC) of guinea‐pig cochlea by combining conventional whole‐cell or perforated patch clamp recording with indo‐1 dual emission microfluorometry. 2. In most IHCs, voltage clamped near resting membrane potential (‐40 mV), extracellular ATP evoked a rapid inward current (time constant, 150 ms). This current was concomitant with a slow rise in [Ca2+]i (time constant, 5 s). The ATP‐evoked inward currents could be repeated several times with only a small run‐down in amplitude (< 10%), while the ATP‐evoked Ca2+ responses showed a rapid run‐down (> 80% at the third ATP application). 3. The current‐voltage relationship of ATP‐evoked currents showed a reversal potential at ‐11 +/‐ 6 mV (n = 8), suggesting that ATP essentially activated a non‐specific cationic conductance. On the contrary, the amplitude of the ATP‐evoked Ca2+ responses did not show significant dependence on holding membrane potential. 4. The Ca2+ response showed an apparent Kd for ATP (EC50, 1.8 +/‐ 0.3 microM; Hill coefficient, 1.0 +/‐ 0.1) eightfold smaller than for the evoked currents (EC50, 13.7 +/‐ 3.0 microM; Hill coefficient, 2.0 +/‐ 0.7). 5. Perfusion with high extracellular Ca2+ solution (10 mM CaCl2) reduced the amplitude of the ATP‐evoked currents by 90%, while perfusion with zero Ca2+ solution increased it by more than 100%. However, similar variations in external Ca2+ concentration did not change the amplitude of the ATP‐evoked Ca2+ responses. Furthermore, intracellular heparin (1 mg mL‐1), a potent inhibitor of InsP3 receptors, did not significantly change the amplitude of ATP‐evoked currents but reduced the ATP‐evoked Ca2+ response, suggesting again that the latter is related to Ca2+ release from intracellular stores. 6. The results suggested that two types of P2‐purinergic receptor are expressed in IHCs: ATP‐gated ion channels and ATP‐activated metabotropic receptors. At submicromolar ATP concentrations, the metabotropic receptors raising intracellular [Ca2+] would hyperpolarize IHCs via Ca(2+)‐sensitive K+ channels. The ATP‐gated ion channels activated at higher ATP concentrations would mainly have a depolarizing effect on IHCs.
P‐glycoprotein regulates a volume‐activated chloride current in bovine non‐pigmented ciliary epithelial cells.
Authors: Wu J , Zhang J J , Koppel H , Jacob T J
Published: December 2014

1. The whole‐cell patch clamp technique was used to investigate the swelling‐activated currents in bovine non‐pigmented ciliary epithelial (NPCE) cells. 2. Exposure to hypotonic solution activated a current that was blocked by 5‐nitro‐2‐(3‐phenylpropylamino)benzoic acid (NPPB). The I‐V relationship was shifted in the direction expected for a Cl‐ current when the external Cl‐ was replaced by gluconate (permeability ratio P(gluconate)/PCl = 0.17). The inhibition of the current evoked by voltage clamp steps of +80 mV yielded an IC50 for NPPB of 13.4 microM. 3. The current was found to be dependent on ATP. With ATP in the patch pipette the current could be repeatedly activated by exposure to hypotonic solution but when ATP was omitted the current ran down with time. 4. The development of this current was associated with visible cell swelling and inhibitors of regulatory volume decrease in these cells, e.g. tamoxifen, 4‐acetamido‐4'‐isothiocyanatostilbene‐2,2'‐disulphonic acid (SITS) and 4,4'‐diisothiocyanostilbene‐2,2'‐disulphonic acid (DIDS), also inhibited this current. 5. The volume‐activated current was additionally blocked by NPPB, verapamil, quinidine and dideoxyforskolin. 6. The current was independent of external calcium and exhibited slight outward rectification and time‐dependent inactivation at strong depolarizing potentials. 7. Disrupting the cytoskeleton and microtubules with cytochalasin B and colchicine had no effect on the activation of the Cl‐ current. 8. An antibody (C219) to the MDR1 gene product, P‐glycoprotein, caused a functional block of the swelling‐activated Cl‐ current when added to the patch pipette. 9. Immunofluorescence studies using the monoclonal antibodies C219 and JSB‐1 demonstrated the presence of P‐glycoprotein in the ciliary epithelial cells. The immunofluorescence was stronger on the non‐pigmented than on the pigmented cells. 10. It is concluded that swelling in NPCE cells activates a Ca(2+)‐independent, ATP‐dependent Cl‐ current and that the activity of this current is associated with P‐glycoprotein. 11. It is suggested that this Cl‐ current contributes to regulatory volume decrease and may participate in the secretory activity of these cells.