bluuebunny/crossref_metadata_2025 huggingface dataset:
Active ion transport pathways in the bovine retinal pigment epithelium.
Authors: Miller S S , Edelman J L
Published: December 2014

1. Radioactive tracer flux measurements demonstrate that active ion transport across the isolated bovine retinal pigment epithelium (RPE)‐choroid preparation can be maintained for hours after the eye is enucleated and the tissue removed from the eye. 2. It has been shown that 86Rb tracer fluxes can be used to monitor potassium (K+) transport across bull‐frog RPE. In bovine RPE, net 86Rb (K+) absorption is zero. Apical barium (Ba2+) elevated active K+ absorption from zero to approximately 0.3 mu equiv cm‐2 h‐1. This Ba2(+)‐induced increase in active K+ absorption was inhibited either by ouabain or bumetanide in the apical bath. 3. In control Ringer solution, buffered with bicarbonate and CO2, the RPE‐choroid actively absorbs chloride (Cl‐) at a rate of approximately 0.5 mu equiv cm‐2 h‐1. In contrast, sodium (Na+) is secreted at a rate of approximately 0.5 mu equiv cm‐2 h‐1. Chloride absorption was inhibited by apical bumetanide, and Na+ secretion was inhibited by apical ouabain. These drugs were only effective when placed in the solution bathing the apical or retinal side of the tissue. 4. Net Cl‐ absorption requires an exit mechanism at the basolateral membrane. DIDS (4,4'‐diisothiocyanostilbene‐2,2'‐disulphonic acid) in the basal bath completely inhibited net Cl‐ absorption in bicarbonate‐free Ringer solution. 5. These experiments show that the chloride transport pathway contains at least two components: (1) a bumetanide‐sensitive uptake mechanism at the apical membrane; and (2) an efflux mechanism at the basolateral membrane that is blocked by DIDS. 6. Three apical membrane mechanisms were identified that could help modulate [K+]o in the subretinal or extracellular space that separates the distal retina and the RPE apical membrane. They are: (1) an ouabain‐sensitive Na(+)‐K+ pump; (2) a bumetanide‐sensitive mechanism, the putative Na(+)‐K(+)‐Cl‐ co‐transporter; (3) a barium‐sensitive K+ channel that recycles, to the apical bath, most or all of the potassium that is actively taken up by the Na(+)‐K+ pump and the co‐transporter. 7. These data suggest that light‐induced alterations in subretinal potassium that occur in vivo can activate the chloride transport pathway and help modulate RPE intracellular Cl‐ during transitions between the light and dark.
The effect of dietary sodium and potassium intake on potassium secretion and kinetics in rat distal colon.
Authors: Edmonds C J , Willis C L
Published: December 2014

1. Potassium secretion by the distal colon was examined in relation to the secretion of chloride and absorption of sodium and to the epithelial turnover of 86Rb+ (as an analogue of potassium) in vivo in rats fed a standard, a potassium‐rich or a sodium‐depleted diet. 2. An acute intravenous potassium load stimulated potassium secretion two‐ to threefold. In rats fed the standard diet, sodium secretion was also increased but no significant change in the lumen‐to‐plasma sodium flux was detected. The potassium and sodium secretions were accompanied by increased chloride secretion which occurred even when the intravenous load contained no chloride. In rats fed the potassium‐rich diet, there was a small increase in sodium absorption and a less marked increased of chloride secretion. In the sodium‐depleted rats, however, about 70‐80% of the increased potassium secretion was balanced by increased sodium absorption. 3. The epithelial turnover rate of 86Rb+ in the rats fed the potassium‐rich (t1/2 12‐14 min) and the sodium‐depleted (t1/2 11‐13 min) diets was faster than that of those of the standard diet (t1/2 22‐27 min). 4. The epithelial potassium content was 304 +/‐ 9 nmol (mg dry weight)‐1 and was not significantly altered despite considerable variations in potassium secretion rate induced by dietary changes and acute potassium loading. 5. It is concluded that epithelial potassium turnover rate is increased during chronic states of increased potassium secretion and that the rise in potassium secretion is consistent with increased activity of the basolateral Na(+)‐K+ pump. Whether the increased potassium secretion is associated with chloride secretion or with increased sodium absorption appears to depend on the absence or presence of the amiloride‐sensitive sodium pathway in the apical membrane.
The effect of heart rate on the arrhythmogenic transient inward current in isolated sheep cardiac Purkinje fibres.
Authors: Henning B , Boyett M R
Published: December 2014

1. The effect of heart rate on the arrhythmogenic transient inward current (ITI) has been studied in sheep cardiac Purkinje fibres. 2. Transient inward current and force (active force, tonic force and the after‐contraction) have been measured in voltage‐clamped preparations. Intracellular Na+ activity (aiNa) was measured simultaneously using Na(+)‐sensitive microelectrodes. The effects of changes in voltage‐clamp pulse frequency on the ITI amplitude, active force, tonic force, the after‐contraction and aiNa have been investigated in different bathing solutions. 3. After a stepwise increase in pulse rate there was a biphasic response of ITI amplitude: a short‐lasting (50 s) phase during which ITI amplitude increased followed by a decline in ITI amplitude. The second phase could be fitted by an exponential with a time constant between 102 and 184 s. The after‐contraction changed in a similar biphasic manner whereas force and aiNa declined monophasically; active force first declined, increased and then declined again. These changes were greater at faster rates of pulsing. 4. Linear relationships between ITI amplitude, after‐contraction amplitude and tonic force were observed in all experiments. 5. In K(+)‐free solution the response of the ITI was markedly changed. After an increase in pulse rate the initial increase in ITI was enhanced, whereas the subsequent decline was strongly reduced or even abolished. Potassium‐free solution blocks the Na+ pump, and activation of the Na+ pump by addition of Cs+ or K+ restored the phase of decay of ITI amplitude during rapid stimulation. 6. A reduction of external Na+ to about 50% or elevation of external Ca2+ increased ITI but had little effect on the rate‐dependent changes in the current. 7. When stimulation was resumed at the basic pulse rate after a period of rapid pulsing the ITI amplitude initially continued to decline: this was followed by a slow recovery (over 10‐15 min) of ITI amplitude back to its steady‐state value prior to fast pulsing. 8. These results are consistent with the hypothesis that changes of ITI are the result of rate‐dependent changes in intracellular Ca2+, which in turn are in part dependent on changes of intracellular Na+.
Effects of intracellular pH on calcium‐activated potassium channels in rabbit tracheal smooth muscle.
Authors: Kume H , Takagi K , Satake T , Tokuno H , Tomita T
Published: December 2014

1. The effects of intracellular pH (pHi) on calcium‐activated potassium channels (Ca2(+)‐activated K+ channels) were studied in membrane patches of smooth muscle freshly dispersed from the rabbit trachea. Single‐channel currents were recorded with an ‘inside‐out’ patch clamp technique, mainly at 0 mV, with the external (electrode) medium containing 130 mM‐K+ and the internal (bath) medium 6 mM‐K+. 2. With an internal Ca2+ concentration ([Ca2+]i) of 1 microM, the fraction of time during which the channel was in an open state (the open probability, Po) was more than 0.8 at pHi 7.4. The channel activity nearly disappeared at pHi 7.0. The [Ca2+]i‐Po relationship was shifted to higher [Ca2+]i by acidosis, the shift being approximately an 8‐fold increase for a fall in pHi of 0.5 units. 3. The membrane potential and current intensity (V‐I) relationship of single channels between +30 and ‐50 mV was shifted in a hyperpolarizing direction by intracellular acidosis. The shift was roughly 10 mV for 1 pH unit at 1 microM [Ca2+]i. At pHi 7.4 [Ca2+]i 1 microM, the V‐Po relationship was shifted in a depolarizing direction by acidification. When [Ca2+]i was increased to 10 microM, V‐Po relationship became less sensitive to V as well as pHi changes. 4. When Po was high, the probability density function of open and closed time distributions could be fitted by two exponentials. When Po was decreased to less than 0.3, either by reducing [Ca2+]i or by lowering pHi, another component having long closed times appeared. At similar Po values, the time constant of open time distribution was smaller with lower pHi. 5. It is concluded that the main effect of an increase in intracellular hydrogen ions is to decrease the open probability of the Ca2(+)‐activated K+ channel, by reducing the sensitivity to Ca2+ and also shortening the open state.
Paired‐pulse depression of monosynaptic GABA‐mediated inhibitory postsynaptic responses in rat hippocampus.
Authors: Davies C H , Davies S N , Collingridge G L
Published: December 2014

1. Intracellular recording techniques were used to characterize monosynaptic inhibitory postsynaptic potentials (IPSPs) and currents (IPSCs) in rat hippocampal slices and to study the mechanism of paired‐pulse depression of these synaptic responses. This was achieved by stimulation in stratum radiatum close (less than 0.5 mm) to an intracellularly recorded CA1 neurone after pharmacological blockade of all excitatory synaptic transmission. 2. Under these conditions, low‐frequency stimulation (0.033 Hz) evoked a pure biphasic IPSP, which had a short and constant latency to onset. This IPSP was blocked by tetrodotoxin (1 microM) suggesting that it resulted from the electrical stimulation of the axons and/or cell bodies of a monosynaptic inhibitory pathway. 3. Picrotoxin (100 microM) abolished the early component of the biphasic IPSP/C. It left an intact, pure late IPSP/C (IPSP/CB) which had a latency to onset of 29 +/‐ 2 ms, latency to peak of 139 +/‐ 4 ms, a duration of 723 +/‐ 135 (range 390‐1730) ms and a reversal potential of ‐93 +/‐ 2 mV. The duration was highly dependent on the stimulus intensity whereas the latency to onset was largely independent of the stimulus intensity. The IPSP/CB was reduced or abolished by 1 mM‐phaclofen. 4. Phaclofen (1 mM) and 2‐hydroxy‐saclofen (0.1‐1.0 mM) reversibly depressed (60‐100%) the late component of the biphasic IPSP/C and, where maximally effective, left a pure, early IPSP/C (IPSP/CA). The IPSP/CA had a latency to onset of 3 ms or less, a latency to peak of 17 +/‐ 1 ms, a duration of 225 +/‐ 3 ms and a reversal potential of ‐75 +/‐ 2 mV. 5. Two shocks of identical strength were applied in close succession to characterize, and to study the mechanisms underlying, frequency‐dependent depression of inhibitory synaptic responses. Paired‐pulse depression was seen for both phases of the biphasic IPSP/C and of the pure IPSP/CB, recorded in the presence of picrotoxin. Paired‐pulse depression was not accompanied by changes in the reversal potential of either component, indicating that it was caused by a reduction in the two synaptic conductances. Paired‐pulse depression was greater when high stimulus intensities were employed. 6. Paired stimuli were applied at separation intervals of between 5 ms and 10 s to determine the temporal profile of frequency‐dependent depression. Paired‐pulse depression of both IPSCA and IPSCB was most pronounced at an interstimulus interval of 100‐125 ms and ceased to occur at intervals greater than 5 10s.(ABSTRACT TRUNCATED AT 400 WORDS)
Effects of vagal stimulation and applied acetylcholine on the arrested sinus venosus of the toad.
Authors: Bywater R A , Campbell G D , Edwards F R , Hirst G D
Published: December 2014

1. The effects of vagal stimulation and applied acetylcholine were compared on sinus venosus preparations of the toad, Bufo marinus, in which beating had been inhibited by adding the organic calcium antagonist nifedipine. 2. Bath‐applied acetylcholine and vagal stimulation each caused membrane hyperpolarizations which were abolished by hyoscine. 3. Whereas the hyperpolarization that accompanied vagal stimulation was largely unaffected by barium ions, that produced by bath‐applied acetylcholine was almost abolished. 4. Caesium ions also prevented the hyperpolarization produced by bath‐applied acetylcholine but potentiated the responses to vagal stimulation. 5. The membrane resistance of arrested sinus venosus cells was found to be higher during vagal stimulation than in the absence of stimulation. In contrast when a similar hyperpolarization was produced by adding acetylcholine, the membrane resistance was found to be lower than in control solution. 6. The results are discussed in relation to the idea that neuronally released acetylcholine causes membrane hyperpolarization by suppressing inward current flow and applied acetylcholine acts to increase outward current flow.
The role of [Ca2+]i and [Ca2+] sensitization in the caffeine contracture of rat myocytes: measurement of [Ca2+]i and [caffeine]i.
Authors: O'Neill S C , Donoso P , Eisner D A
Published: December 2014

1. Fluorescence measurements have been made in single, isolated rat ventricular myocytes using the Ca2(+)‐sensitive indicators Fura‐2 and Indo‐1. In Fura‐2‐loaded cells, the application of caffeine (2‐20 mM) produced a change of fluorescence indicating an increase of [Ca2+]i which then spontaneously decayed to control levels. These changes of [Ca2+]i were accompanied by a contracture. 2. In contrast, in Indo‐1‐loaded cells, in addition to the changes of fluorescence expected for the transient increase of [Ca2+]i produced by caffeine, there was a maintained decrease of fluorescence. 3. Measurements in vitro showed that caffeine quenches the fluorescence of Indo‐1 (but not of Fura‐2) in a [Ca2+]‐and wavelength‐independent manner. Caffeine therefore had no effect on the ratio of Indo‐1 fluorescence measured at two wavelengths. This inhibition by caffeine could be described by an apparent Ki of 4 mM. In the cell the Ki was considerably larger (18 mM). 4. We have separated the Indo‐1 fluorescence changes into caffeine‐ and [Ca2+]i‐dependent components. The time course of change of intracellular caffeine was calculated. When [caffeine]o was rapidly increased, [caffeine]i changed with a rate constant of 8 s‐1 giving an apparent permeability to caffeine of 2 x 10(‐3) cm s‐1. 5. This method was used to measure [caffeine]i and [Ca2+]i simultaneously during caffeine‐induced contractures. The shape of the caffeine contracture was found to depend on both the speed of application of caffeine and the concentration applied. If caffeine was applied quickly then the contracture developed within 1 s to a maximum level and then relaxed to a lower maintained level. With slower application, there was a more complete relaxation of the initial contraction followed by a slower redevelopment of contraction. 6. Despite the difference in contraction time course, irrespective of the flow rate, [Ca2+]i decayed monotonically. The slow secondary development of contraction has the same time course as the increase of [caffeine]i. The caffeine contracture can be reproduced by a model in which both [Ca2+]i and [caffeine]i affect contraction. 7. The increase of [Ca2+]i is not greatly affected by altering the caffeine concentration from 2.5 to 50 mM. In contrast the maintained level of contraction increases over this range showing that the Ca2(+)‐independent effects of caffeine on the myofilaments have a low affinity for caffeine.
Ionic control of intracellular pH in rat cerebellar Purkinje cells maintained in culture.
Authors: Gaillard S , Dupont J L
Published: December 2014

1. Intracellular pH (pHi) was measured in single rat cerebellar Purkinje cells maintained in primary culture using microspectrofluorescence analysis of the intracellularly trapped pH‐sensitive dye 2',7'‐bis‐(2‐carboxyethyl)‐5 (and ‐6)‐carboxyfluorescein (BCECF). 2. The ratio of the fluorescence signals measured at 530 nm in response to an alternating excitation at 450 and 490 nm was calibrated using the K(+)‐H+ ionophore nigericin. This calibration gave a steady‐state pHi of 7.06 +/‐ 0.02 (S.E.M., n = 17) when cells were perfused by a 5% CO2‐25 mM‐HCO3(‐)‐buffered solution at an external pH of 7.40 at 37 degrees C. 3. Replacement of external chloride with gluconate in the presence of bicarbonate induced a cytoplasmic alkalinization of about 0.3 pH unit. This alkalinization was independent of external sodium and was greatly reduced by 0.5 mM‐DIDS, indicating the presence of a chloride‐bicarbonate exchange. 4. In bicarbonate‐free (HEPES‐buffered) solution the steady‐state pHi was 7.37 +/‐ 0.02 (n = 19), significantly higher than in bicarbonate‐buffered solution. Recovery from an intracellular acid load brought about by the ammonium chloride pre‐pulse technique was blocked by the removal of external sodium or the addition of 1.5 mM‐amiloride, indicating the presence of a sodium‐hydrogen exchange. 5. In bicarbonate‐buffered solution pHi recovery after an acid load was also completely blocked by addition of 1.5 mM‐amiloride indicating the absence of a bicarbonate‐dependent acid extrusion mechanism. 6. Addition of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA, 100 nM) induced an amiloride‐sensitive alkalinization of about 0.3 pH unit in bicarbonate‐buffered solution but had no effect in HEPES‐buffered solution. This observation suggests that in cultured Purkinje cells the sodium‐hydrogen exchanger could be activated through a protein kinase C pathway only when pHi is maintained at a low physiological value by the activity of the chloride‐bicarbonate exchange.
Capsaicin‐sensitive afferents activate a sympathetic intestinointestinal inhibitory reflex in dogs.
Authors: Mizutani M , Neya T , Nakayama S
Published: December 2014

1. In urethane‐anaesthetized dogs, an intra‐arterial infusion of capsaicin (0.7‐14 nmol min‐1) into a separated jejunal segment inhibited a vagally evoked cholinergic contraction of the other non‐infused segments. The mechanism of this reflex was investigated. 2. The inhibition by capsaicin was abolished after bilateral splanchnic nerve section or cervical spinal cord transection (C5 or C6), but was unaffected by bilateral vagotomy. Decerebration partially reduced the inhibition. 3. The inhibition by capsaicin was abolished by pre‐treatment with phentolamine or yohimbine, but was unaffected by prazosin or propranolol. 4. Sympathetic efferent discharge of the mesenteric nerve increased with capsaicin application, during which time vagally evoked contractions were inhibited. 5. Single‐unit discharges of the major splanchnic and mesenteric afferents increased with capsaicin infusion to the loop which was innervated by the units. 6. Together the results implied that capsaicin stimulated canine intestinal primary afferents, resulting in the sympathetic intestinointestinal inhibitory reflex supraspinally. The inhibition of vagally evoked contractions may be due to a presynaptic inhibition via alpha 2‐adrenoceptors, which are activated by the reflex.
Sodium currents in Schwann cells from myelinated and non‐myelinated nerves of neonatal and adult rabbits.
Authors: Howe J R , Ritchie J M
Published: December 2014

1. Patch‐clamp methods were used to study sodium channels in Schwann cells obtained from four different tissue sources. Primary cultures of Schwann cells were prepared from the sciatic nerve and from the vagus nerve of neonatal and of adult rabbits. In the adult, the sciatic is predominantly myelinated whereas the vagus is predominantly non‐myelinated. Whole‐cell currents, and single‐channel currents in outside‐out membrane patches, were analysed. 2. No substantial differences were noted in the passive electrical properties (input resistance, cell capacitance, resting membrane potential) of the four groups of cells. Similarly, no substantial differences were found in the average properties of sodium currents (maximum current, maximum conductance, time‐to‐peak current, current‐voltage relation, h infinity relation) recorded from each type of cell in cultures less than 8 days old. At 10‐17 days a fall in the size of the sodium currents recorded from cells in the vagal cultures was found. 3. Exposure of the cells to proteolytic enzymes or collagenase, under conditions similar to those used when the cells were put in culture initially, substantially reduced the size of the peak sodium currents recorded from the cells 24 h later. 4. The results of experiments on Schwann cells with retracted processes indicated that sodium channels are present in the processes extending from each pole of the cell soma and that the plasmalemmal density of these channels in the processes is about the same as it is at the soma. 5. Recordings from outside‐out patches revealed no apparent differences in the properties of single‐channel sodium currents in patches from cells obtained from the four different sources. The single‐channel conductance was about 20 pS for each of the four groups. Ensemble currents from single‐channel records were similar in time course to those of whole‐cell currents. 6. Saxitoxin reduced the maximum sodium conductance in Schwann cells and bound to the cells with equally high affinity. The equilibrium dissociation constant was about 2 nM at 20‐22 degrees C. 7. It is argued that the expression of sodium channels in myelinating Schwann cells does not differ substantially from that of non‐myelinating Schwann cells.
Development of the arterial chemoreflex and turnover of carotid body catecholamines in the newborn rat.
Authors: Hertzberg T , Hellström S , Lagercrantz H , Pequignot J M
Published: December 2014

1. The peripheral, arterial chemoreceptors in the carotid body are active and responsive in the fetus. At birth, when oxygenation increases, the chemoreceptors are silenced. Over the next few days the sensitivity is reset toward the adult level and the chemoreceptors influence breathing during normal conditions. In order to investigate the underlying mechanisms of this resetting we examined the strength of the chemoreflex in newborn rats and correlated this to the contents of dopamine and noradrenaline in the carotid bodies of the newborn pups and near‐term fetuses. Furthermore, turnover rates of dopamine and noradrenaline were determined in newborn rats up to 1 week of age by analysis of catecholamine decreases after inhibition of synthesis with alpha‐methyl‐p‐tyrosine. 2. Chemoreceptor influence was assessed by the method of ‘physiological chemodenervation’ with hyperoxia of 15‐20 s duration in unanaesthetized rat pups. Relative changes in ventilation elicited by hyperoxia were determined by body plethysmography. We found no change in ventilation on the day of birth either in vaginally born rats or in near‐term pups delivered by Caesarean section. After 1 day there was a significant decrease in ventilation of ‐19.4 +/‐ 2.3% (mean +/‐ S.E.M.) and at 7 days of age the decrease was ‐28.8 +/‐ 2.2%, suggesting an increasing influence from the peripheral chemoreceptors. 3. The contents of dopamine and noradrenaline were measured by high‐performance liquid chromatography. Dopamine increased from 3.7 +/‐ 0.4 pmol (pair of carotid bodies)‐1 in the fetus to a peak of 15.9 +/‐ 2.6, 6‐12 h after birth followed by a decline to 7.1 +/‐ 0.7 at 7 days of age. Noradrenaline levels increased from 1.3 +/‐ 0.3 in the fetus to 9.6 +/‐ 1.1 pmol (pair of carotid bodies)‐1 after 4 days. The turnover rate of dopamine decreased from 4.4 pmol (pair of carotid bodies)‐1 h‐1 0‐6 h after birth to 1.0 at 6‐12 h of age. The turnover rate of noradrenaline also decreased over the first hours following delivery. 4. Since dopamine is an inhibitory neuromodulator in this system, we suggest that the increase in sensitivity seen after the first day of life is, at least in part, due to a decrease in the release of dopamine and thus a removal of an inhibitory mechanism.
Changes in kinetics of acetylcholine receptor channels after initial expression in Xenopus myocyte culture.
Authors: Rohrbough J , Kidokoro Y
Published: December 2014

1. Developmental changes in single acetylcholine (ACh) receptor channel properties were analysed in cultured Xenopus myocytes at low temperatures (11‐16 degrees C) using the cell‐attached patch clamp technique. Single‐channel recordings were done at fourteen stages of development ranging from several hours (stage 21) to 5 days (stage 47) after ACh receptors first appear in the muscle membrane. 2. Two types of channels, low conductance and high conductance, which have been described previously, were observed at all stages. At low concentrations of ACh, channel events often occurred as bursts of openings separated by closures briefer than 1 ms. Such bursts were treated as one event. Many brief, isolated channel openings, which were described in the preceding paper, were also observed. Developmental changes in burst duration, brief openings and brief closures were assessed for the period studied in culture. 3. Throughout development, most of the burst duration histograms for the low‐conductance channel were not well fitted by a single exponential, having an excess of brief openings. The brief component could be largely accounted for by singly liganded openings, as described in the preceding paper. The burst durations reported here represent the main component of the distribution. At 150 nM‐ACh, high‐conductance channel burst duration histograms were well fitted by single exponentials. 4. There was a developmental increase in the percentage of single‐channel events belonging to the high‐conductance class. The percentage of high‐conductance events remained low (less than 10%) for the first day after ACh receptors appeared (stages 20‐34), and increased to 60% at stage 47. 5. In addition to a shift in channel population, there was a decrease in both low‐ and high‐conductance channel burst durations during early stages of development (mostly within the first day in culture). The effect was more dramatic for the low‐conductance channel: overall, low‐conductance channel burst duration at 50 mV hyperpolarization over the resting potential decreased from 24 ms at stage 24/25 to 6 ms at stage 47, a 4‐fold decrease. During the same period, high‐conductance burst duration decreased from 10.6 to 4 ms. a 2.5‐fold decrease. In contrast to the burst duration, the mean open time of 60 microseconds for the brief isolated class of opening in the low‐conductance channel did not change with development. 6. Neither the single‐channel conductance nor the voltage dependence of burst duration changed with development.(ABSTRACT TRUNCATED AT 400 WORDS)
Cell swelling, co‐transport activation and potassium conductance in isolated perfused rabbit kidney proximal tubules.
Authors: Beck J S , Potts D J
Published: December 2014

1. Isolated, perfused rabbit proximal tubules were used to study the effects of activation of the apical membrane sodium co‐transporters, and of the effects of osmotically induced cell swelling, upon cell volume, basolateral membrane potential and apparent partial conductance of potassium. 2. Activation of electrogenic apical sodium co‐transport caused a depolarization of the basolateral membrane and a reduction of the basolateral apparent potassium transference number. This was followed by a spontaneous partial recovery of potential and increase in apparent potassium transference number. 3. Stimulation of apical sodium co‐transport led to a sustained increase in cell volume. 4. A sustained increase in cell volume (of similar magnitude to that seen after activation of apical membrane sodium co‐transporters) was also caused by reduction of bath and perfusate osmolality by removal of 89 mmol l‐1 mannitol from both lumen and bath solutions. 5. This reduction in bath and perfusate osmolality also led to a basolateral membrane hyperpolarization and an increase in basolateral apparent potassium transference number. 6. These observations support the possibility that some of the partial recovery of basolateral membrane potential (Vb1) during apical sodium co‐transport stimulation is due to a cell volume sensitive change in basolateral potassium conductance.
Contribution of two types of calcium channels to membrane conductance of single myocytes from guinea‐pig coronary artery.
Authors: Ganitkevich VYa  , Isenberg G
Published: December 2014

1. Whole‐cell and single‐channel current recordings were used to study calcium channels in single smooth muscle cells isolated from guinea‐pig coronary artery. Potassium currents were blocked by intracellular Cs+ ions. 2. Whole‐cell currents were recorded with 10 mM‐barium in the bath. Step pulses of 200 ms from a holding potential of ‐90 mV activated calcium channel current when the depolarization reached ‐55 to ‐50 mV. All cells showed a current component which inactivated slowly and incompletely. About half of the cells showed an additional current component with a rapid inactivation time course. Both components were abolished by Cd2+ ions (1 mM) and were reduced by changing the holding membrane potential to ‐40 mV or by addition of 0.1 mM‐Ni2+. 3. Single calcium channel currents were measured in cell‐attached patches with 110 or 10 mM‐Ba2+ as a current carrier. Two different types of single calcium channel activity were observed. 4. A high‐conductance calcium channel was activated near ‐30 mV with 110 mM‐Ba2+ and this threshold was changed to about ‐60 mV with 10 mM‐Ba2+ in the patch pipette. The conductance was 28.0 +/‐ 1.5 pS (mean +/‐ S.D.) in 110 mM‐Ba2+ and 16.0 +/‐ 1.0 pS in 10 mM‐Ba2+. Dependence of the conductance on the concentration of Ba2+ in the patch pipette followed a Langmuir curve: the apparent dissociation constant of Ba2+ was 8 mM. It was concluded that this channel type corresponds to L‐type calcium channels. 5. Another calcium channel was found in these experiments. It had a low conductance and was activated at around ‐50 mV with 110 mM‐Ba2+, and this threshold was shifted to about ‐70 mV when 10 mM‐Ba2+ was the charge carrier. The conductance of this calcium channel was 7.5 +/‐ 0.6 pS in 110 mM‐Ba2+ and 5.5 +/‐ 1.0 pS in 10 mM‐Ba2+. With 10 mM‐Ba2+, inactivation of the mean current was slow at potentials ‐70 to ‐50 mV, but fast and complete (within 100 ms) at more positive potentials. It was concluded that this type of calcium channel corresponds to T‐type calcium channels. 6. With the membrane potential continuously held at ‐50 to ‐40 mV (with 10 mM‐Ba2+ in the patch pipette), i.e. close to the usual resting potential of these cells, T‐type calcium channels were completely inactivated whereas rare openings of L‐type calcium channels could be detected.(ABSTRACT TRUNCATED AT 400 WORDS)
Synergistic activation by serotonin and GTP analogue and inhibition by phorbol ester of cyclic Ca2+ rises in hamster eggs.
Authors: Miyazaki S , Katayama Y , Swann K
Published: December 2014

1. Synergistic activation of a GTP‐binding protein (G protein) by external serotonin (5‐hydroxytryptamine, 5‐HT) and internally applied guanosine‐5'‐O‐(3‐thiotriphosphate (GTP gamma S) in hamster eggs was demonstrated by the facilitation of repetitive increases in cytoplasmic Ca2+ as measured by their associated hyperpolarizing responses (HRs) and by aequorin luminescence. 2. Rapid application of 70 nM‐5‐HT caused a single HR of 10‐12 s duration and with a delay of 80 s. The critical concentration of 5‐HT to cause an HR was 50 nM. 3. With 10 microM‐5‐HT four to six HRs were often elicited with a delay to the first HR of 8‐30 s. HRs disappeared after prolonged or repeated application of 5‐HT, indicating an apparent desensitization. 4. 5‐HT‐induced HRs were completely inhibited by the protein kinase C (PKC) activator phorbol 12‐myristate 13‐acetate (TPA) (100 nM). Conversely, the PKC inhibitor sphingosine (2 microM) enhanced the series of HRs by shortening the delay to the first HR (3‐9 s) and by causing more HRs. 5. Ionophoretic injection of GTP gamma S into the egg usually produced a large HR with a delay of 120‐240 s followed by a series of much smaller HRs. When 5‐HT was applied within 1 min of injection of GTP gamma S. 70 nM‐5‐HT induced a number of large HRs and even 1 nM‐5‐HT could induce HR(s). In contrast, when 5‐HT was applied after the size of GTP gamma S‐induced HRs had declined, as much as 10 microM‐5‐HT could only elicit a single large HR. Thus, GTP gamma S apparently caused a sensitization and then a desensitization of the action of 5‐HT. 6. GTP gamma S‐induced Ca2+ transients were facilitated when injected in the presence of 5‐HT concentrations as low as 0.1 nM. The time delay to the first HR was 65 s in 0.1 nM‐5‐HT or 4 s in 100 nM‐5‐HT whereas it was 170 s without 5‐HT (mean values). The magnitude as well as frequency of HRs succeeding the first HR was enhanced by 5‐HT at concentrations above 0.01 nM. 7. TPA (100 nM) blocked the GTP gamma S‐plus‐5‐HT‐potentiated HRs after the first four to five HRs. Sphingosine (2 microM) augmented the series of HRs.(ABSTRACT TRUNCATED AT 400 WORDS)
ATP regulates muscarine‐sensitive potassium current in dissociated bull‐frog primary afferent neurones.
Authors: Tokimasa T , Akasu T
Published: December 2014

1. Bull‐frog dorsal root ganglion cells in primary culture were voltage clamped in the whole‐cell configuration. The pipette solution contained ATP (5 mM). 2. Step depolarizations (5‐70 mV, 0.1‐1 s) from a holding potential close to the resting potential (range, ‐64 to ‐79 mV) evoked a non‐inactivating potassium current with properties indistinguishable from those which have been reported for the M‐current of bull‐frog sympathetic neurones. 3. An unhydrolysable ATP analogue APP(NH)P (5 mM), substitute with ATP in the pipette solution, did not support the M‐current activation. 4. Bath application of ATP (30 nM‐30 microM) reduced the amplitude of the M‐current in a concentration‐dependent manner, congruent to 50% inhibition of the current occurring with 1 microM‐ATP. The main effect of ATP was to reduce the maximum M‐conductance without changing the activation and deactivation kinetics of the M‐current. 5. Essentially the same results were obtained with ADP (0.1‐30 microM) and alpha, beta‐methylene‐ATP (10‐30 microM). AMP (10‐100 microM) and adenosine (10‐30 microM) were without effect on the M‐current. 6. The ATP‐induced inhibition of the M‐current was irreversible when an unhydrolysable GTP analogue GTP‐gamma‐S (10‐30 microM) was present in the pipette solution. ATP (3 microM) reduced the amplitude of the M‐current only by about 10% when GDP‐beta‐S (100 microM) was present in the pipette solution. Pre‐treatment of the cells with pertussis toxin (IAP; 500 ng ml‐1) for 24 h at 24 degrees C did not prevent the ATP‐induced M‐current inhibition. 7. Phorbol 12‐myristate 13‐acetate (PMA; 1‐3 microM) reduced the amplitude of the M‐current to about 50%. A reduction in the M‐current amplitude by PMA (3 microM) and ATP (10 microM) was attenuated when staurosporine (200 nM) was present in the pipette solution. Forskolin (10 microM) was without effect on the M‐current. 8. It is concluded that ATP acting at P2 receptors, associated with an IAP‐insensitive GTP‐binding protein, inhibits the M‐current in amphibian primary afferent neurones.
Developmental changes in the half‐life of acetylcholine receptors in the myotomal muscle of Xenopus laevis.
Authors: Cohen M W , Frair P F , Cantin C , Hébert G
Published: December 2014

1. Tail preparations, containing myotomal muscle and associated spinal cord, were isolated from embryos and tadpoles of Xenopus laevis between stages 25 and 49 (1.15‐12 days) and were pulse‐labelled with 125I‐alpha‐bungarotoxin (125I alpha BT) so that the half‐life (T1/2) of their acetylcholine receptors (AChRs) could be estimated in organ culture. 2. For the entire population of AChRs, estimates of T1/2 based on a single exponential decline in radioactivity (but see item 4 below) increased from 53‐55 h at stages 25‐31 (1.15‐1.56 days) to approximately 135 h at stage 47 (5.5 days). Beyond stage 47 T1/2 increased only slightly. 3. Radioautographic estimates of the T1/2 of extrajunctional AChRs at stages 47‐48 (5.5‐7.5 days) were 41‐50 h. It follows that the developmental change in the T1/2 of the entire population of AChRs was due to the junctional AChRs. 4. At stages 47‐49 (5.5‐12 days) the decline in radioactivity for the entire population of AChRs was fitted well by a double exponential. Assuming a T1/2 of 50 h for the extrajunctional AChRs and 210 h for the junctional AChRs, the correlation coefficient (r) was 0.9947 +/‐ 0.0014 (mean +/‐ S.E.M.; n = 14) and junctional AChRs were estimated to comprise 80 +/‐ 3% of the entire population. Similar analysis, as well as experiments in which the degradation of junctional AChRs was assessed by pulse‐labelling with fluorescent alpha‐bungarotoxin, suggested that at earlier stages of development the junctional AChRs have a shorter T1/2 and comprise a smaller fraction of the entire population. 5. The developmental increase in T1/2 occurred even when animals were raised in the anaesthetic tricaine or in tetrodotoxin, conditions which abolished all motor activity. 6. Developmental increases in T1/2 also occurred in culture but were smaller than those in vivo. The increases in culture did not occur amongst those AChRs which were pre‐labelled with 125I alpha BT. 7. It is concluded that in Xenopus myotomal muscle the T1/2 of junctional AChRs begins to increase within a day after the onset of innervation and that the increase does not require nerve or muscle impulse activity. We suggest, among other possibilities, that it may depend upon incorporation of a different molecular species of AChR into the postsynaptic membrane.
Rod phototransduction modulated by bicarbonate in the frog retina: roles of carbonic anhydrase and bicarbonate exchange.
Authors: Donner K , Hemilä S , Kalamkarov G , Koskelainen A , Shevchenko T
Published: December 2014

1. Effects on rod phototransduction following manipulation of retinal CO2‐HCO3‐ and H+ fluxes were studied in dark‐adapted retinas of the frog and the tiger salamander. 2. Rod photoresponses to brief flashes of light were recorded from the isolated sensory retina as electroretinogram mass receptor potentials and from isolated rods by the suction‐pipette technique. The experimental treatments were: (1) varying [CO2] + [HCO3‐] in the perfusion fluid: (2) applying acetazolamide (AAA), which inhibits the enzyme carbonic anhydrase (CA); and (3) applying 4,4'‐diisothiocyanatostilbene‐2,2'‐disulphonic acid (DIDS) which blocks exchange mechanisms transporting HCO3‐ across cell membranes. 3. The concentration of the internal transmitter of the rods, cyclic GMP, was biochemically determined from the rod outer segment layer of retinas that had been incubated in the same solutions as were used for perfusion in the electrophysiological experiments. 4. The introduction of 6 mM‐sodium bicarbonate to replace half the buffer of a nominally CO2‐HCO3(‐)‐free (12 mM‐phosphate or HEPES, [Na+] constant) Ringer solution doubled the cyclic GMP concentration in the rod outer segment layer and increased the saturating response amplitude and the relative sensitivity of rods in the intact retina. 5. The introduction of 0.5 mM‐AAA into bicarbonate‐containing Ringer solution accelerated the growth of saturated responses and sensitivity. Incubation of the retina in AAA‐bicarbonate Ringer solution elevated the concentration of cyclic GMP ninefold compared with the phosphate control. 6. No effects of switching to bicarbonate‐AAA Ringer solution were observed in the photocurrent of isolated rods drawn into suction pipettes with only the outer segment protruding into the perfusion fluid. The target of AAA is probably the CA‐containing Müller cell. 7. The introduction of DIDS into the perfusate (at normal pH 7.5) set off a continuous decay of photoresponses which finally abolished light sensitivity completely. The decay proceeded regardless of whether bicarbonate and AAA were present or not. 8. Rods that had lost their photosensitivity in DIDS recovered almost fully when the pH of the DIDS perfusate was raised to 8.5. They also recovered when DIDS was washed out with bicarbonate Ringer solution at constant pH (7.5). 9. It is proposed that all our treatments ultimately modulate the intracellular pH of the rods which is determined by the relative rates of H+ leakage and HCO3‐ transport into the cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Effects of systemic hypoxia on the distribution of cardiac output in the rat.
Authors: Marshall J M , Metcalfe J D
Published: December 2014

1. Studies were made in unanaesthetized rats of cardiovascular responses induced during 3 min periods of systemic hypoxia (inspirate 8 or 6% O2). Arterial pressure and heart rate were recorded continuously; cardiac index and regional blood flows were measured in normoxia and at the 2nd min of hypoxia by injection of radiolabelled microspheres. Comparisons are made with changes recorded in Saffan‐anaesthetized rats during 8% O2 using microspheres and in previous studies using electromagnetic transducers on renal, mesenteric and femoral arteries (Marshall & Metcalfe, 1988a). 2. In unanaesthetized rats, the initial 1‐1.5 min of hypoxia evoked behavioural arousal associated with a short‐lasting rise in arterial pressure and heart rate. This agrees with our previous proposal that hypoxia activates the brain stem defence areas by stimulating peripheral chemoreceptors. 3. In unanaesthetized rats, these changes were superimposed upon a gradual fall in arterial pressure and tachycardia, the responses being greater during 6 than 8% O2 (cf. Saffan‐anaesthetized rats). Further, in all rats at the 2nd min of hypoxia, cardiac index and vascular conductance of most body tissues was increased. It is concluded that the fall in arterial pressure is due to peripheral vasodilatation. 4. In the unanaesthetized rats, the tendency for vascular conductance in kidney, intestine and gastrocnemius muscle to increase (more during 6 than 8% O2) allowed increases in blood flow in the last two regions. These changes accord with those recorded under Saffan anaesthesia. 5. In both unanaesthetized and anaesthetized rats, hypoxia induced pronounced increases in vascular conductance of diaphragm, adrenal gland, cerebral hemispheres, cerebellum and brain stem, the resultant increases in blood flow being larger in the unanaesthetized rats. 6. It is proposed that in unanaesthetized, as in anaesthetized, rats the regional dilator responses predominantly reflect the local dilator effects of tissue hypoxia. Possible dilator factors are considered.
Human placental L‐tyrosine transport: a comparison of brush‐border and basal membrane vesicles.
Authors: Kudo Y , Boyd C A
Published: December 2014

1. The mechanisms responsible for L‐tyrosine transport at both the maternal‐facing and fetal‐facing surfaces of the human full‐term placenta have been studied using isolated brush‐border and basal membrane vesicles under conditions where a direct comparison of the transport properties of the two membranes can be made. 2. Brush‐border vesicle uptake of L‐tyrosine was substantially into an osmotically active space. Transport was Na(+)‐independent, N‐ethylmaleimide‐sensitive (half‐maximal inhibition, Ki = 1.1 mM), and insensitive to pH over the range 5.5‐8.5. The initial rate of brush‐border L‐tyrosine uptake as a function of concentration showed saturation and obeyed Michaelis‐Menten kinetics with Michaelis constant (Km) and maximum velocity (Vmax) values of 54.2 microM and 1.28 pmol (mg protein)‐1 s‐1, respectively. Influx of L‐tyrosine was stereospecific and was virtually completely abolished by L‐phenylalanine, L‐tryptophan, L‐leucine or by 2‐aminobicycloheptane‐2‐carboxylic acid. These properties suggest that system L is responsible for brush‐border L‐tyrosine transport. 3. Basal membrane transport of L‐tyrosine was more complex and uptake was slower than that found in the brush border. Although, as in the brush‐border membranes, uptake was completely Na(+)‐independent, N‐ethylmaleimide was a less effective inhibitor, there was stimulation of transport at more alkaline pH and uptake did not show marked stereospecificity. An apparent Km of 168.9 microM and a Vmax of 0.31 pmol (mg protein)‐1 s‐1 were calculated for basal L‐tyrosine transport. There was clear inhibition by L‐ and D‐tyrosine, L‐phenylalanine and L‐tryptophan. 2‐Aminobicycloheptane‐2‐carboxylic acid was not as effective. 4. These findings suggest the existence of non‐identical carrier‐mediated transport systems for L‐tyrosine in brush‐border and basal membranes. Brush‐border transport resembles that by system L; L‐tyrosine transport at the basal membrane may be via system t.
Synchronization of motor units in human masseter during a prolonged isometric contraction.
Authors: Nordstrom M A , Miles T S , Türker K S
Published: December 2014

1. The action potentials of concurrently active motor units in human masseter were recorded on several fine‐wire intramuscular electrodes and synchronization was assessed by cross‐correlation of their firing times. 2. Long recording sessions and a test of significance for peaks in histograms were used to enhance detection of synchrony. 3. Synchrony in masseter units was found to be very weak, and was observed consistently only in histograms with a large number of counts. However, synchrony was widespread within the motoneurone pool; of the unit pairs whose cross‐correlation histograms contained more than 100 counts per 1 ms bin, 89% were significantly synchronized. 4. The strength of synchrony between pairs of units did not change consistently during 15 min of continuous activity. 5. It was concluded that most, if not all, low‐threshold masseter motoneurones have some common synaptic inputs, although the origin of those inputs remains unclear.
Substance P mediates synaptic transmission between rat myenteric neurones in cell culture.
Authors: Willard A L
Published: December 2014

1. Whole‐cell patch‐clamp recordings were made from pairs of neurones in cell cultures of rat myenteric neurones. In some pairs, action potentials evoked in the first neurone evoked a slow excitatory postsynaptic potential (EPSP) in the second neurone. 2. Action potentials at a frequency of at least 5 Hz were required to evoked slow EPSPs. In one group of cells, the slow EPSP followed a series of nicotinic fast EPSPs; in another group, fast EPSPs did not precede the slow EPSP. 3. The slow EPSPs were 2‐16 mV in amplitude and were accompanied by decreased resting potassium conductance. 4. Most (17/28) neurones in which action potentials evoked only slow EPSPs in a follower cell contained substance P (SP)‐like immunoreactivity; they were not immunoreactive for 5‐hydroxytryptamine (0/15) or vasoactive intestinal peptide (0/22). 5. Postsynaptic responses to SP, neurokinin A and a synthetic tachykinin [( pGlu6, Pro9]SP6‐11) mimicked the slow EPSPs. The non‐tachykinin peptide vasoactive intestinal polypeptide (VIP), which was not found in neurones that evoked only slow EPSPs, also mimicked the slow EPSPs. Responsiveness to SP decreased significantly during slow EPSPs. 6. Desensitization to either SP or VIP reduced or prevented the slow EPSPs and also responses to each other. Two proposed antagonists of SP receptors, [D‐Arg1, D‐Pro2,D‐Trp7,9,Leu11]substance P and [D‐Arg1,D‐Trp7,9,Leu11]substance P, did not affect the slow EPSPs significantly. 7. Antisera against SP reversibly blocked or reduced slow EPSPs evoked by eight of thirteen presynaptic neurones that evoked slow EPSPs without evoking fast EPSPs. All eight of the presynaptic neurones that evoked anti‐SP‐sensitive slow EPSPs contained SP‐like immunoreactivity. None of the presynaptic neurones that evoked anti‐SP‐insensitive slow EPSPs contained detectable SP‐like immunoreactivity. Normal sera and anti‐VIP antisera did not alter the slow EPSPs detectably. 8. It is concluded that subsets of myenteric neurones release an SP‐like transmitter to evoke slow EPSPs. These neurones appear to lack a ‘classical’ neurotransmitter that evokes fast EPSPs.