bluuebunny/crossref_metadata_2025 huggingface dataset:
The relationship between contraction and intracellular sodium in rat and guinea‐pig ventricular myocytes.
Authors: Harrison S M , McCall E , Boyett M R
Published: December 2014

1. The contraction, measured optically, and the intracellular Na+ activity (aNai), measured with the Na(+)‐sensitive fluorescent dye SBFI, have been recorded simultaneously in rat and guinea‐pig ventricular myocytes. 2. In rat and guinea‐pig ventricular myocytes at rest, aNai was 7.8 +/‐ 0.3 mM (n = 4) and 5.1 +/‐ 0.3 mM (n = 16), respectively. 3. When both rat and guinea‐pig ventricular myocytes were stimulated at 1 Hz after a rest there was usually a gradual increase in twitch shortening (referred to as a 'staircase') over several minutes accompanied by an increase in aNai over a similar time course. Twitch shortening increased by 21 +/‐ 3% (n = 6) and 20 +/‐ 4% (n = 16) (of steady‐state twitch shortening during 1 Hz stimulation) per millimolar rise in aNai in rat and guinea‐pig ventricular myocytes, respectively. 4. When rat and guinea‐pig ventricular myocytes were exposed to strophanthidin to block the Na(+)‐K+ pump, there were increases in twitch shortening and aNai over similar time courses. Twitch shortening increased by 24 +/‐ 4% (n = 5) and 20 +/‐ 3% (n = 10) (of control twitch shortening) per millimolar rise in aNai in rat and guinea‐pig ventricular myocytes respectively. 5. The inotropic effect of cardiac glycosides, such as strophanthidin, is widely regarded to be principally the result of the rise in aNai. The similarity of the relation between twitch shortening and aNai during the staircase and on application of strophanthidin suggests that the progressive increase in the strength of contraction during the staircase was also linked to the rise in aNai. 6. In guinea‐pig, but not rat, ventricular myocytes there was hysteresis in the relation between twitch shortening and aNai on application and wash‐off of strophanthidin. This indicates that strophanthidin has another inotropic action in guinea‐pig ventricular myocytes. 7. A computer model of excitation‐contraction coupling has been developed to simulate the staircase and the action of cardiac glycoside and to account for the relation between contraction and intracellular Na+.
Changes in synovial PO2 and blood flow in the rabbit knee joint due to stimulation of the posterior articular nerve.
Authors: Ferrell W R , Najafipour H
Published: December 2014

1. Experiments were performed to measure the partial pressure of oxygen in the synovial fluid (Ps,O2) of the normal rabbit knee joint and assess the extent to which this varied with changes in knee joint blood flow. 2. With the hypodermic needle oxygen electrode sited just within the synovial cavity, Ps,O2 values ranged from 25 to 72 mmHg with a mean of 48.2 +/‐ 3.1 (S.E.M.; n = 18). 3. Ps,O2 was found to decrease with increasing depth of penetration of the oxygen electrode. Lowest values were observed close to the articular cartilage. 4. Electrical stimulation of the posterior articular nerve (PAN) of the knee resulted in vasoconstriction of knee joint blood vessels which was accompanied by a decrease in Ps,O2. The frequency‐response and voltage‐response profiles to electrical stimulation of PAN, although differing in magnitude, showed a high degree of correlation between blood flow and Ps,O2. 5. As judged by the conduction velocity, the vasoconstrictor response to nerve stimulation was mediated by unmyelinated nerve fibres, presumed to be sympathetic postganglionic fibres. 6. In view of the low Ps,O2 values occurring deep within the joint, avascular structures such as cartilage could be subject to injury if sustained reduction in synovial blood flow occurred. This could be a contributory factor in the pathogenesis of degenerative and inflammatory joint diseases.
GTP‐binding protein involvement in membrane currents evoked by carbachol and histamine in guinea‐pig ileal muscle.
Authors: Komori S , Kawai M , Takewaki T , Ohashi H
Published: December 2014

1. Single smooth muscle cells obtained by enzymic dispersion of the longitudinal muscle layer of guinea‐pig ileum were used for recording membrane currents under whole‐cell voltage clamp in response to carbachol (100 microM, unless otherwise stated) or histamine (100 microM) applied extracellularly. 2. At a holding potential of 0 mV, a transient outward current was evoked by carbachol and histamine. Responses to the two agonists were very similar in size and time course to the current response to caffeine (10 mM). The response to carbachol was virtually absent in the presence of histamine, and vice versa. Caffeine was without effect in the presence of either of these agonists. Inclusion of EGTA (10 or 20 mM) in the pipette abolished the responses to carbachol, histamine and caffeine. Thus, the outward current responses were considered to represent opening of Ca(2+)‐activated K+ channels in response to a massive release of Ca2+ from the same stores by these three agents. 3. An inward current was evoked by carbachol and histamine, but not by caffeine at a holding potential of ‐40 mV, which was considered to represent opening of cationic channels. The carbachol‐induced inward current was much longer in duration and larger in size than the histamine‐induced inward current. 4. Inclusion of GDP beta S (2 mM) in the pipette abolished the inward and outward current responses to histamine, but inhibited only part of those to carbachol. 5. When the holding potential was held at 0 mV with inclusion of GTP gamma S (0.1‐1 mM) in the pipette, spontaneous transient outward currents appeared immediately after break‐through but disappeared a few minutes later. Under these conditions, caffeine (10 mM) was almost without effect, suggesting that GTP gamma S had released Ca2+ stores. When the holding potential was held at ‐40 mV and GTP gamma S (0.1 or 0.2 mM) was present in the pipette, an inward current developed a few minutes after break‐through. During the GTP gamma S‐induced inward current, application of carbachol or histamine produced no further inward current. However, when 0.01 mM‐GTP gamma S was included in the pipette solution, carbachol‐ and histamine‐induced inward currents were potentiated. 6. Pretreated with 2‐5 micrograms/ml pertussis toxin (PTX) did not change noticeably the outward current responses to carbachol and histamine, but abolished or markedly reduced the inward current responses.(ABSTRACT TRUNCATED AT 400 WORDS)
Role of proton pump of mitochondria‐rich cells for active transport of chloride ions in toad skin epithelium.
Authors: Larsen E H , Willumsen N J , Christoffersen B C
Published: December 2014

1. Active Cl‐ currents were studied in short‐circuited toad skin epithelium in which the passive voltage‐activated Cl‐ current is zero. Under visual control double‐barrelled microelectrodes were used for impaling principal cells from the serosal side, or for measuring the pH profile in the solution bathing the apical border. 2. The net inward (active) 36Cl‐ flux of 27 +/‐ 8 pmol s‐1 cm‐2 (16) (mean +/‐ S.E.M (number of observation)) was abolished by 2 mM‐CN‐ (6.3 +/‐ 3.5 pmol s‐1 cm‐2 (8)). The active flux was maintained in the absence of active Na+ transport when the latter was eliminated by either 100 microM‐mucosal amiloride, replacement of mucosal Na+ with K+, or by 3 mM‐serosal ouabain. 3. In Ringer solution buffered by 24 mM‐HCO3‐ ‐5% CO2 mucosal amiloride reversed the short circuit current (ISC). The outward ISC was maintained when gluconate replaced mucosal Cl‐, and it was reversibly reduced in CO2‐free 5 mM‐Tris‐buffered Ringer solution (pH = 7.40) or by the proton pump inhibitor oligomycin. These observations indicate that the source of the outward ISC is an apical proton pump. 4. Amiloride caused principal cells to hyperpolarize from a basolateral membrane potential, Vb, of ‐73 +/‐ 3 (22) to ‐93 +/‐ 1 mV (26), and superfusion with CO2‐free Tris‐buffered Ringer solution induced a further hyperpolarization (Vb = ‐101 +/‐ 1 mV (26)) which could be blocked by Ba2+. The CO2‐sensitive current changes were null at Vb = EK (potassium reversal potential, ‐106 +/‐ 2 mV (55)) implying that they are carried by K+ channels in the basolateral membrane. Such a response cannot account for the inhibition of the outward ISC which by default seems to be located to mitochondria‐rich (MR) cells. 5. In the absence of mucosal Cl‐ a pH gradient was built up above MR cells with pH = 7.02 +/‐ 0.04 (42) and pH increasing to 7.37 +/‐ 0.02 (10) above principal cells (pH = 7.40 in bulk solution buffered by 0.1 mM‐Tris). This observation localizes a proton pump to the apical membrane of MR cells. Using the integrated diffusion equation it was shown that the measured external pH gradient would account within an order of magnitude for measured currents. 6. Standing gradients of protons were eliminated in the presence of mucosal Cl‐ suggesting that active uptake of Cl‐ is associated with the exit of base equivalents across the apical membrane of MR cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Ionic currents in cultured rat hypothalamic neurones.
Authors: Müller T H , Misgeld U , Swandulla D
Published: December 2014

1. Dissociated neurones from embryonic rat hypothalamus were grown for several weeks in culture where they formed complex networks. These synaptically coupled networks were capable of generating synchronized bursting activity. Voltage‐activated membrane currents were studied in these neurones using a patch clamp in the whole‐cell configuration. 2. Outward currents were carried by K+ ions and consisted of an inactivating and a non‐inactivating component. These components were similar to the transient K+ current (IA) and the delayed rectifier current (IK) reported in neurones from the postnatal rat hypothalamus. Application of Zn2+ (1 mM) blocked the transient component completely while reducing the non‐inactivating component by only approximately 20%. 3. Inward currents were carried by Na+ and Ca2+ ions. Rapidly activating transient Na+ currents were activated at approximately ‐25 mV. TTX entirely blocked these currents at low concentration (300 nM). Voltage sensitivity of the Na+ conductance was 5.8 mV per e‐fold change with half‐maximal activation occurring at ‐8 mV. Na+ current kinetics could be well described by the Hodgkin‐Huxley model (m3h). 4. With depolarizing pulses from a holding potential of ‐80 mV two Ca2+ current components with different ranges of activation were identified. Low voltage‐activated (LVA, T‐type) Ca2+ currents were activated at approximately ‐50 mV. High voltage‐activated (HVA; also called L‐ or N‐type) Ca2+ currents were observed at membrane potentials more positive to approximately ‐30 mV. LVA Ca2+ currents were observed in hypothalamic neurones that had developed a network of dendritic processes in the course of several weeks in culture. Activation and inactivation time constants of LVA Ca2+ currents were 15‐25 ms and 30‐100 ms (‐30 to ‐45 mV). In contrast to HVA Ca2+ currents, no LVA Ca2+ currents were seen in neuronal somata obtained from the network cultures by mechanical dissociation. This suggests that most of the LVA Ca2+ channels are located on the dendritic tree rather than on the soma membrane. 5. HVA Ca2+ currents were maximal between 0 and +10 mV (external [Ca2+] = 5 mM). The time‐to‐peak was in the range of 1.7‐5.4 ms (+30 to ‐10 mV). Tail currents following repolarization decayed monoexponentially with a time constant of approximately 210 microseconds. During 500 ms depolarizations, 90% of the current inactivated. The time course of inactivation showed two time constants of approximately 40 and approximately 700 ms.(ABSTRACT TRUNCATED AT 400 WORDS)
Co‐ordination of pumping in isolated bovine lymphatic vessels.
Authors: McHale N G , Meharg M K
Published: December 2014

1. Segments of bovine mesenteric lymphatic of varying diameter taken from different parts of the lymphatic tree were cut to 20 mm in length and set up so that measurements could be made of spontaneous isometric contractions. 2. There was considerable variability in frequency of spontaneous contractions but this was independent of resting tension. There was no significant correlation between lymphatic diameter and inherent frequency of contraction. 3. Isolated segments of bovine mesenteric lymphatic 70‐80 mm in length were cannulated and set up in a three‐compartment organ bath which allowed independent temperature control in each compartment. Pressure was recorded at inflow and outflow ends and experiments were video recorded. 4. Contractile activity was normally initiated at the end of the lymphatic maintained at the higher temperature and the contractile wave was propagated along the length of the vessel. 5. Propagation could occur either in the direction of valve orientation (orthograde) or retrogradely. The volume of fluid pumped was not significantly affected by the direction of propagation. 6. Perfusion of the central compartment with Krebs solution at 0‐2 degrees C disrupted normal propagation and allowed the two parts of the lymphatic to contract at different frequencies although the two parts maintained an approximately 2:1 ratio. 7. Perfusion of the central compartment with 10 mM‐heptanol also disrupted normal propagation but the rates on either side of the partition bore no harmonic relationship to one another. 8. These results suggest that relatively short segments of lymph duct have the ability to contract spontaneously and that their inherent frequencies are not determined by their position in the lymphatic tree. The results are consistent with the existence of electrical coupling along the lymphatic's length and they suggest that over distances of at least 80 mm independent pacemakers are capable of mutual entrainment at a frequency representing a compromise between the fastest and slowest components.
Independent control of human finger‐tip forces at individual digits during precision lifting.
Authors: Edin B B , Westling G , Johansson R S
Published: December 2014

1. Subjects lifted an object with two parallel vertical grip surfaces and a low centre of gravity using the precision grip between the tips of the thumb and index finger. The friction between the object and the digits was varied independently at each digit by changing the contact surfaces between lifts. 2. With equal frictional conditions at the two grip surfaces, the finger‐tip forces were about equal at the two digits, i.e. similar vertical lifting forces and grip forces were used. With different frictions, the digit touching the most slippery surface exerted less vertical lifting force than the digit in contact with the rougher surface. Thus, the safety margins against slips were similar at the two digits whether they made contact with surfaces of similar or different friction. 3. During digital nerve block, large and variable safety margins were employed, i.e. the finger‐tip forces did not reflect the surface conditions. Slips occurred more frequently than under normal conditions (14% of all trials with nerve block, <5% during normal conditions), and they only occasionally elicited compensatory adjustments of the finger‐tip forces and then at prolonged latencies. 4. The partitioning of the vertical lifting force between the digits was thus dependent on digital afferent inputs and resulted from active automatic regulation and not just from the mechanics of the task. 5. The safety margin employed at a particular digit was mainly determined by the frictional conditions encountered by the digit, and to a lesser degree by the surface condition at the same digit in the previous lift (anticipatory control), but was barely influenced by the surface condition at the other digit. 6. It was concluded that the finger‐tip forces were independently controlled for each digit according to a ‘non‐slip strategy’. The findings suggest that the force distribution among the digits represents a digit‐specific lower‐level neural control establishing a stable grasp. This control relies on digit‐specific afferent inputs and somatosensory memory information. It is apparently subordinated to a higher‐level control that is related to the total vertical lifting and normal forces required by the lifting task and the relevant physical properties of the manipulated object.
Presynaptic inhibitory action of enkephalin on excitatory transmission in superficial dorsal horn of rat spinal cord.
Authors: Hori Y , Endo K , Takahashi T
Published: December 2014

1. Tight‐seal whole‐cell recordings were made from marginal neurones visually identified in thin slices of 1‐ to 2‐week‐old rat lumbar spinal cord. Excitatory postsynaptic currents (EPSCs), either evoked by extracellular stimulation or those arising spontaneously in tetrodotoxin, i.e. miniature EPSCs (mEPSCs), were recorded after blocking inhibitory synaptic inputs with strychnine and bicuculline. 2. The EPSCs were abolished reversibly by kynurenic acid or 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) but not affected by (+)‐2‐amino‐5‐phosphonovalerate (APV), suggesting that they were mediated by non‐NMDA (N‐methyl‐D‐aspartate) glutamate receptors. Micromolar concentrations of methionine [Met5]enkephalin reversibly reduced the magnitude of evoked EPSCs and the frequency of mEPSCs. 3. The enkephalin action on the mEPSC frequency was blocked by naloxone. A specific agonist of mu‐opiate receptor, [D‐Ala2,N‐Me‐Phe4, Gly5]enkephalin‐ol (DAGO) suppressed the mEPSC frequency. In contrast, neither a delta‐opiate receptor agonist, [D‐Pen2, L‐Pen5]enkephalin (DPLPE) nor a kappa‐opiate receptor agonist, (5 alpha, 7 alpha, 8 beta)‐(‐)‐N‐methyl‐N‐[7‐(1‐pyrrolidinyl)‐1‐ oxaspiro(4,5)‐dec‐8‐yl]benzeneacetamide (U‐69,593) significantly affected the mEPSC frequency. 4. The amplitude of mEPSCs or of currents induced by exogenous L‐glutamate, was not affected by [Met5]enkephalin. It is suggested that [Met5]enkephalin presynaptically inhibits glutamatergic EPSCs by activating the mu‐opiate receptor. 5. The frequency of mEPSC was reduced by about 50% by replacement of external Ca2+ with Mg2+ or by addition of Cd2+. In Ca(2+)‐free‐Mg2+ solution, [Met5]enkephalin did not reduce the remaining mEPSCs' frequency any further. 6. It is concluded that the opiates may suppress presynaptic Ca2+ entry, thereby inhibiting synaptic transmission.
Characterization of membrane currents in single smooth muscle cells from the guinea‐pig gastric antrum.
Authors: Noack T , Deitmer P , Lammel E
Published: December 2014

1. Smooth muscle cells, enzymatically isolated from the antrum of the guinea‐pig stomach, were voltage clamped at room temperature using the whole‐cell patch clamp technique. In physiological salt solution (PSS), step depolarization from a holding potential of ‐90 mV elicited inward calcium current (ICa) followed and superimposed by outward potassium current. 2. Outward current was divided into components depending on the presence of extracellular Ca2+ and others which were not activated as a result of Ca2+ influx. Ca(2+)‐dependent components were (1) a fast transient component most likely representing Ca(2+)‐activated K+ current (IK(Ca)) immediately triggered by the initial peak of ICa and (2) spontaneous transient outward currents (STOCs) apparently reflecting synchronized opening of IK(Ca) channels by cyclic release of Ca2+ from intracellular stores. Ca2+ influx‐independent outward current could be divided into two main components: (1) a transient component (I(to)) showing voltage‐dependent activation and inactivation and (2) a non‐inactivating component (Ini). 3. I(to) activated with a threshold around ‐30 mV, was fully available at ‐90 mV and completely inactivated at ‐10 mV. The time course of both activation and inactivation of I(to) at different potentials could be described by single exponential functions. Time constants of activation decreased from 35 ms at ‐10 mV to 10 ms at +40 mV. The time constant of inactivation was about 2 s and only weakly voltage dependent. Time constants for exponentially developing recovery from inactivation of I(to) ranged from 0.1 s at ‐100 mV to 10 s at ‐30 mV. I(to) was insensitive to 4‐aminopyridine (4‐AP, 5 mmol/l), slightly sensitive to tetraethylammonium (TEA, 10 mmol/l), but substantially inhibited by caffeine (10 mmol/l) and Cd2+ (5 mmol/l). Estimates of the single‐channel conductance by current fluctuation analysis indicated a small value of about 2.5 pS. 4. The action of TEA on current elicited from a holding potential of ‐10 mV indicated a major contribution to Ini of a distinct component (Ini,K) that was completely blocked by this substance at a concentration of 10 mmol/l. Ini was almost unaffected by 4‐AP (5 mmol/l) and caffeine (10 mmol/l), but strongly suppressed by Cd2+ (5 mmol/l). Current fluctuation analysis of Ini,K gave a value for the single‐channel conductance of about 60 pS. 5. Ca2+ inward current was studied in PSS ([Ca2+]o = 2.5 mmol/l) using pipette solution in which K+ was replaced by Cs+ to suppress outward K+ currents.(ABSTRACT TRUNCATED AT 400 WORDS)
Ectopic activity in demyelinated spinal root axons of the rat.
Authors: Baker M , Bostock H
Published: December 2014

1. We have provoked ectopic discharges from demyelinated rat spinal roots by applying 1 mM‐4‐aminopyridine (4‐AP), and recorded membrane currents and action potentials extracellularly by spike‐triggered averaging. The demyelination was caused by intrathecal injection of diphtheria toxin, 6‐9 days previously. 2. Mapping the distribution of membrane currents in the vicinity of an ectopic site showed that in most cases (eight out of twelve recorded) the impulses arose from one end of a continuously conducting internode, and conducted in both directions. In the remaining cases the impulses also arose from a site of demyelination. 3. The 4‐AP‐induced activity resembled the activity occurring spontaneously in some preparations, and was often highly regular (5‐20 Hz). Recordings of membrane potential revealed a pacemaker potential, which was localized to the site of impulse initiation. One ectopic site was tested with applied currents and found to have a linear current‐frequency relation for steady currents. 4. The time course of the pacemaker potential resembled that of the small after‐hyperpolarization seen in normal fibres, due to a slow K+ conductance (GKs). Tetraethylammonium and barium ions, which block GKs, made spontaneously active fibres fire much more rapidly, or to fire bursts of action potentials. 5. Possible mechanisms for these ectopic discharges are discussed. GKs appears to contribute to the pacing of the activity, but not its generation. The increased excitability of the active fibres could not be attributed directly to the loss of myelin, nor to extracellular K+ accumulation. We suggest that they may have been depolarized by stretch‐activated or ligand‐gated channels in the demyelinated axon membrane.
Simultaneous measurements of cardiac noradrenaline spillover and sympathetic outflow to skeletal muscle in humans.
Authors: Wallin B G , Esler M , Dorward P , Eisenhofer G , Ferrier C , Westerman R , Jennings G
Published: December 2014

1. Muscle sympathetic nerve activity (MSA) was recorded in the peroneal nerve at the knee by microneurography in ten healthy subjects and determinations were made simultaneously of intra‐arterial blood pressure, and whole‐body and cardiac noradrenaline spillover to plasma. Measurements were made at rest, during isometric handgrip at 30% of maximum power and during stress induced by forced mental arithmetic. 2. At rest there were significant positive correlations between spontaneous MSA (expressed as number of sympathetic bursts min‐1) and both spillover of noradrenaline from the heart and concentration of noradrenaline in coronary sinus venous plasma. 3. Both isometric handgrip and mental arithmetic led to sustained increases of blood pressure, heart rate and MSA. Plasma concentrations of noradrenaline and spillover of noradrenaline (total body and cardiac) increased. In general the effects were more pronounced during handgrip than during stress. 4. When comparing effects during handgrip and stress the ratio between the fractional increases of MSA and cardiac noradrenaline spillover were significantly greater during handgrip. 5. The data suggest (a) that there are proportional interindividual differences in the strength of resting sympathetic activity to heart and skeletal muscle which are determined by a common mechanism and (b) that handgrip and mental stress are associated with differences in balance between sympathetic outflows to heart and skeletal muscle.
Mitochondrial and sarcolemmal Ca2+ transport reduce [Ca2+]i during caffeine contractures in rabbit cardiac myocytes.
Authors: Bassani R A , Bassani J W , Bers D M
Published: December 2014

1. Contraction and intracellular Ca2+ (Ca2+i) transients were measured in isolated rabbit ventricular myocytes during twitches and contractures induced by rapid application of 10 mM‐caffeine. 2. The amplitude of caffeine‐induced contractures and the accompanying Ca2+i transients were larger than during normal twitches and also declined more slowly. This may be because only a fraction of sarcoplasmic reticulum (SR) Ca2+ is released during a normal twitch, or because of a temporal overlap of SR Ca2+ release and uptake during the twitch. 3. When a caffeine contracture was initiated in Na(+)‐free, Ca(2+)‐free medium (to prevent sarcolemmal Na(+)‐Ca2+ exchange) the contracture and Ca2+i transient were larger and decreased much more slowly. Thus, Ca2+ extrusion via Na(+)‐Ca2+ exchange may limit the amplitude of caffeine‐induced contractures. 4. Relaxation half‐time (t1/2) for the twitch (0.17 +/‐ 0.03 s) was increased to 0.54 +/‐ 0.07 s for caffeine contractures in control solution and 8.8 +/‐ 1 s for caffeine‐induced contractures in Na(+)‐free, Ca(2+)‐free solution. These results confirm that the SR Ca2+ pump and Na(+)‐Ca2+ exchange are the predominant mechanisms for cytoplasmic Ca2+ removal during relaxation. However slower mechanisms can still reduce intracellular [Ca2+]. 5. Relaxation of caffeine contractures in Na(+)‐free solution was further slowed when (a) mitochondrial Ca2+ uptake was inhibited with the oxidative phosphorylation uncoupler, FCCP (t1/2 = 19.7 +/‐ 3.2 s), or (b) the sarcolemmal Ca(2+)‐ATPase pumping ability was depressed by a large transmembrane [Ca2+] gradient (t1/2 = 27.5 +/‐ 6.9 s). 6. When the four Ca2+ transport systems were simultaneously inhibited (i.e. SR Ca2+ pump, Na(+)‐Ca2+ exchange, mitochondrial Ca2+ uptake and sarcolemmal Ca2+ pump), relaxation was practically abolished, but the cell could recover quickly when Na+ was reintroduced and caffeine removed. 7. We conclude that, under our experimental conditions, the sarcolemmal Ca2+ pump and mitochondria are approximately 37‐ and 50‐fold slower than the Na(+)‐Ca2+ exchange at removing Ca2+ from the cytoplasm. Additionally, the SR Ca2+ pump is about 3‐4 times faster than Na(+)‐Ca2+ exchange.
A patch‐clamp study on the muscarine‐sensitive potassium channel in bullfrog sympathetic ganglion cells.
Authors: Koyano K , Tanaka K , Kuba K
Published: December 2014

1. A voltage‐independent K+ channel was characterized and effects of muscarine were studied in cultured bullfrog sympathetic ganglion cells using the cell‐attached patch‐clamp configuration. 2. Three types of single‐channel current were recorded from 2‐ to 10‐day‐old cultured cells in the presence of tetraethylammonium (2‐20 mM), tetrodotoxin (1‐2 microM), Cd2+ (0.1 mM) and apamin (20 nM). 3. The most frequently observed channel was a voltage‐independent K+ channel which was open at the resting membrane potential and had a conductance of 52.6, 78.9 and 114.9 pS at a [K+]o of 2, 40 and 100 mM, respectively. This channel was designated background K+ channel. 4. Two other channel types were observed less frequently. One had a conductance of 26 pS (external K+, 118 mM) and a long open time of several seconds at the resting membrane potential. The second channel had a smaller conductance (20 pS) and displayed a voltage‐dependent activation. 5. The open probability of the background K+ channel varied between patches, ranging from 0.0005 to 0.486. The open time distribution was fitted by a single exponential with a time constant of 0.51 ms. Both of these parameters were independent of the membrane potential. The closed time distribution consisted of at least four exponentials having time constants of 0.17, 3.7, 120 ms and several seconds. 6. Muscarine (10‐20 microM) applied to the membrane outside the patch pipette reversibly enhanced the activity of the background K+ channel. This effect was associated with an increase in the open probability, which resulted from an increase in the mean open time concomitant with a decrease in the mean closed time. Muscarine did not change the single‐channel conductance of this channel. 7. The effects of muscarine were blocked by atropine (1 microM). 8. It is concluded that there exists a muscarine‐sensitive, voltage‐independent K+ channel in cultured bullfrog ganglion cells. This K+ channel appears to contribute to the generation of the resting membrane potential and underlie the slow inhibitory postsynaptic potential of these neurones in situ.
Mechanisms of extracellular divalent and trivalent cation block of the sodium current in canine cardiac Purkinje cells.
Authors: Sheets M F , Hanck D A
Published: December 2014

1. Single canine cardiac Purkinje cells were internally perfused and voltage clamped with a large‐bore perfusion pipette for measurement of sodium ionic current (INa) in the absence and presence of extracellular group IIA divalent cations (Mg2+, Ba2+ and Ca2+), transition divalent cations (CO2+, Mn2+ and Ni2+), group IIB divalent cations (Cd2+ and Zn2+), and the trivalent cation La3+. 2. Open channel block of cardiac INa by external Ca2+, assessed from instantaneous INa‐voltage (I‐V) relationships, has been well described by a two‐barrier, one‐well model with a dissociation constant at 0 mV, KB(0), of 37 mM and an electrical distance, z' = delta, of 0.34. At the most negative test potentials there was less block of INa than predicted by the model, but correction of INa for the contribution of Na+ channel gating current (Ig) to the peak current improved the fit by the model. 3. The divalent cations Ba2+, Mg2+, CO2+ and Mn2+ produced voltage‐dependent, open channel block of INa, which by the two‐barrier, one‐well model predicted a similar z' about one‐third into the membrane field. The relative efficacy for voltage‐dependent block was CO2+ > Mn2+ > Ca2+ > Mg2+ > Ba2+ with respective KB(0)s of 11, 13, 37, 43 and 61 mM. 4. Cd2+, Zn2+ and La3+ produced block of INa at low concentrations that was nearly voltage independent with z' < or = 0.13. Fits of single‐site binding curves to peak INa in response to step depolarizations at positive test potentials gave the following apparent KD values: Zn2+ 0.14 mM, Cd2+ 0.27 mM and La3+ 0.50 mM. 5. In the presence of Cd2+, INa tail current relaxations were much faster than could be accounted for by Cd2+ binding to and/or screening of extracellular surface charges. Fits of the data to a model that assumed voltage‐dependent open channel block during the tail current relaxations estimated the KB(0) for Cd2+ to be 0.80 mM. 6. Both z' and KB(0) for Ni2+ from fits of the two‐barrier, one‐well model to instantaneous I‐V relationships varied as a function of [Ni2+], consistent with the hypothesis that Ni2+ blocked with similar affinity at a voltage‐dependent and a voltage‐independent site. At [Ni2+] > or = 5 mM, KB(0) was 7.6 mM and z' was 0.21.(ABSTRACT TRUNCATED AT 400 WORDS)
Anoxia induces time‐independent K+ current through KATP channels in isolated heart cells of the guinea‐pig.
Authors: Benndorf K , Bollmann G , Friedrich M , Hirche H
Published: December 2014

1. Isolated ventricular heart cells of the guinea‐pig were exposed to anoxia (PO2 < 0.1 Torr) which induced a time‐independent K+ current. This current was studied with the patch clamp technique in the whole‐cell and cell‐attached configuration. 2. The latency until anoxia‐induced changes of whole‐cell current developed was distributed exponentially (mean 10.5 min; n = 41). The current was abolished within 2‐4 s of reoxygenation. 3. The reversal potential of the anoxia‐induced change of whole‐cell current at 5.4 and 15 mM [K+]o was ‐82 and ‐61 mV, respectively. 4. Analysis of current noise in whole‐cell current during the phase of the slow development of the anoxia‐induced current yielded a slope conductance of unitary currents of 8.1 pS (5.4 mM [K+]o) which is far below the 30 pS of KATP channels in inside‐out patches with no Na+ and Mg2+ in the bath. 5. Reduced unitary current induced by anoxia was recorded in single‐channel measurements with 10.4 mM‐K+ in the pipette. 6. Using 150 mM‐K+ in the pipette, anoxia caused unitary inward currents with a slope conductance of 83 pS. The open probability of the channels (P(o)) reached maximum values between 0.6 and 0.95. The channels closed within 1‐3 s of reoxygenation. 7. At voltages between ‐85 and ‐45 mV and maximum P(o), open time histograms were dominated by a fast exponential (tau 01 = 0.55 +/‐ 0.21 ms, mean +/‐ S.D.) and one or two slow exponentials. 8. Voltage ramp experiments showed that single‐channel currents were slightly rectifying in the inward direction. 9. Glibenclamide (1 microM) reversibly blocked the anoxia‐induced whole‐cell and single‐channel currents. 10. It is concluded that during anoxia it is only KATP channels which open by a sufficient decrease of submembrane ATP levels.
Crossed actions on group II‐activated interneurones in the midlumbar segments of the cat spinal cord.
Authors: Bajwa S , Edgley S A , Harrison P J
Published: December 2014

1. Evidence has been sought for crossed actions on midlumbar propriospinal neurones activated by ipsilateral group II muscle afferents, with particular emphasis on those neurones with projections to the ipsilateral hindlimb motor nuclei. 2. A large majority of group II‐activated midlumbar neurones were influenced by stimulation of contralateral group II afferents. The most frequent and most powerful actions were from those nerves which most effectively influenced ipsilateral midlumbar neurones. Crossed actions from group I afferents were rare. 3. In the great majority of neurones the pattern of actions was similar from both limbs, the neurones being bilaterally excited, bilaterally inhibited or had both EPSPs and IPSPs from both sides. 4. The latencies of crossed actions suggest that the earliest crossed EPSPs from group II afferents were evoked disynaptically (i.e. via a single commissural neurone) and that the crossed IPSPs were evoked trisynaptically. 5. The pattern of crossed actions suggests a strong bilateral interaction between midlumbar neurones. The possible role of these neurones in postural control and the production of co‐ordinated movements of the hindlimbs is discussed.
Upstroke component of electrical slow waves in canine colonic smooth muscle due to nifedipine‐resistant calcium current.
Authors: Ward S M , Sanders K M
Published: December 2014

1. Electrical slow waves of gastrointestinal smooth muscles are not abolished by organic Ca2+ channel blocking drugs, such as nifedipine or D600. These compounds reduce the amplitude and duration of the plateau phase, but the upstroke phase of slow waves persists. 2. Voltage clamp experiments were performed on isolated circular muscle cells from the canine proximal colon to characterize the dihydropyridine‐resistant component of inward current. Inward currents were measured at 25 and 35 degrees C. The higher temperature increased the amplitudes of the transient and sustained phases of the inward current. The voltage dependence of activation and inactivation of the inward current was not significantly changed at 35 vs. 25 degrees C. 3. At 35 degrees C the transient phase of the inward current was reduced but not blocked by nifedipine (10(‐6) M). The sustained phase was blocked by nifedipine. 4. The block by nifedipine was voltage dependent, increasing with depolarization. At voltages reached during the upstroke depolarization about 35% of the inward current persisted in the presence of nifedipine (10(‐6) M). This may be sufficient inward current to sustain the upstroke depolarization in intact muscles. 5. Nifedipine caused a 20 mV negative shift in the voltage dependence of inactivation suggesting that dihydropyridines may preferentially bind to Ca2+ channels in an inactivated state. 6. Ni2+ (< 100 microM) significantly decreased the transient phase of inward current. A combination of Ni2+ (40 microM) and nifedipine (10(‐6) M) blocked all of the inward current at 35 degrees C. Combination of nifedipine (10(‐6) M) and Ni2+ (40 microM) blocked slow waves in intact muscles. 7. Bay K 8644 (10(‐6) M) increased the amplitude of the transient and sustained components of inward current. On a percentage basis the increase in the sustained component was greater than the increase in the transient component with test potentials in the range of ‐50 to ‐20 mV. This may explain why Bay K 8644 preferentially increases the plateau component of slow waves vs. the upstroke component. 8. The findings of this study suggest that the nifedipine resistance of the upstroke depolarization could be due to the voltage dependence of the block of Ca2+ channels by dihydropyridines. Thus a single class of voltage‐dependent Ca2+ channels could be responsible for the upstroke and plateau phases of slow waves.
Muscarine increases cation conductance and decreases potassium conductance in rat locus coeruleus neurones.
Authors: Shen K Z , North R A
Published: December 2014

1. Whole‐cell patch‐clamp recordings were made from rat locus coeruleus neurones in slices of brain tissue in vitro. Muscarine (30 microM) caused an inward current of about 100 pA in neurones voltage clamped at ‐60 mV. 2. In about 75% of cells, the current elicited by muscarine was independent of potential in the range ‐60 to ‐120 mV and had no associated conductance change. 3. In about 25% of cells, the current became smaller with hyperpolarization, was associated with a decreased conductance, and reversed polarity between ‐100 and ‐140 mV. The reversal potential changed with the logarithm of the extracellular potassium concentration. Barium and caesium blocked inward rectification and also prevented reversal of the muscarine current. 4. When potassium ions of the intracellular and extracellular solutions were replaced by caesium, the current evoked by muscarine became smaller with depolarization at reversed polarity at +9 mV. This current was associated with an increase in conductance, and was greatly reduced when the extracellular sodium concentration was reduced to 20 mM. 5. The results could be quantitatively accounted for by a model in which muscarine both increases a voltage‐independent cation conductance and decreases the inward rectifier potassium conductance.
Phenylephrine‐induced translocation of protein kinase C and shortening of two types of vascular cells of the ferret.
Authors: Khalil R A , Morgan K G
Published: December 2014

1. The relationship between phenylephrine‐induced smooth muscle contraction and the subcellular distribution of protein kinase C (PKC) was investigated. 2. Cell shortening induced by phenylephrine (10(‐5) M) was measured in single vascular cells freshly isolated from ferret portal vein and aorta. 3. At various time points during phenylephrine activation, single cells were fixed with paraformaldehyde and the distribution of PKC was imaged in cells labelled with the fluorescent PKC probe 12‐(1,3,5,7‐tetramethylBODIPY‐2‐propionyl)phorbol‐13‐acetate. 4. The PKC probe located to a perinuclear region, the cytosol and surface membrane. The amplitude and time course of the phenylephrine‐induced changes in the surface membrane/cytosol fluorescence ratio were measured and compared with the amplitude and time course of phenylephrine‐induced cell shortening. 5. In portal vein cells incubated in 1 mM‐external Ca2+, phenylephrine caused significant shortening and time‐dependent translocation of the PKC probe to the surface membrane, but cell shortening preceded PKC translocation. In Ca2+free solution both cell shortening and translocation of the probe were completely inhibited. 6. Verapamil (3 x 10(‐7) M) partially, but significantly, inhibited the magnitude of cell shortening and delayed the onset and time to peak shortening. Translocation of PKC in verapamil preceded or coincided with cell shortening. 7. In aorta cells incubated in 1 mM‐extracellular Ca2+, phenylephrine induced significant shortening and time‐dependent translocation of the PKC probe. Cell shortening preceded PKC translocation. In Ca(2+)‐free solution, shortening was only partially, but significantly, inhibited and PKC translocation preceded the fraction of the shortening response that remained. 8. These data are consistent with a role for PKC in the maintenance of the phenylephrine‐induced contraction in both portal vein and aorta. The data also suggest that phenylephrine‐induced contraction may involve activation of a Ca(2+)‐dependent PKC isoform in ferret portal vein but a Ca(2+)‐independent isoform in ferret aorta.
A contraction‐related component of slow inward current in dog ventricular muscle and its relation to Na(+)‐Ca2+ exchange.
Authors: Simurda J , Simurdová M , Bravený P , Sumbera J
Published: December 2014

1. The slow inward current component related to contraction (Isic) was studied in voltage clamp experiments on canine ventricular trabeculae at 30 degrees C with the aims of (a) estimating its relation to electrogenic Na(+)‐Ca2+ exchange and (b) comparing it with similar currents as reported in cardiac myocytes. 2. Isic may be recorded under conditions of augmented contractility in response to depolarizing pulses below the threshold of the classic slow inward current (presumably mediated by L‐type Ca2+ channels). In responses to identical depolarizing clamp pulses the peak value of Isic is directly related to the amplitude of contraction (Fmax). Isic peaks about 60 ms after the onset of depolarization and declines with a half‐time of about 110 ms. 3. The voltage threshold of Isic activation is the same as the threshold of contraction. The positive inotropic clamp preconditions shift both thresholds to more negative values of membrane voltage, i.e. below the threshold of the classic slow inward current. 4. Isic may also be recorded as a slowly decaying inwardly directed current ‘tail’ after depolarizing pulses. In this representation the peak value of Isic changes with duration of the depolarizing pulses, again in parallel with Fmax. In response to pulses shorter than 100 ms both variables increase with depolarization time. If initial conditions remain constant, further prolongation of the pulse does not significantly influence either one (tail currents follow a common envelope). 5. Isic differs from classic slow inward current by: (a) its direct relation to contraction, (b) the slower decay of the current tail on repolarization, (c) slower restitution corresponding to the mechanical restitution, (d) its relative insensitivity to Ca(2+)‐blocking agents (the decrease of Isic is secondary to the negative inotropic of Ca(2+)‐blocking agents (the decrease of Isic is secondary to the negative inotropic effect) and (e) its disappearance after Sr2+ substitution for Ca2+. 6. The manifestations of Isic in multicellular preparations do not differ significantly from those reported in isolated myocytes (in contrast to calcium current). 7. The analysis of the correlation between Isic and Fmax transients during trains of identical test depolarizing pulses at variable extra‐ and intracellular ionic concentrations (changes of [Ca2+]o, 50% Li+ substitution for Na+, strophanthidin) indicate that the observed effects conform to the predictions based on a quantitative model of Na(+)‐Ca2+ exchange. 8. It is concluded that Isic is activated by a transient increase of [Ca2+]i, in consequence of the release from the reticular stores.(ABSTRACT TRUNCATED AT 400 WORDS)
Resting membrane potential and potassium currents in cultured parasympathetic neurones from rat intracardiac ganglia.
Authors: Xu Z J , Adams D J
Published: December 2014

1. Whole‐cell K+ currents contributing to the resting membrane potential and repolarization of the action potential were studied in voltage‐clamped parasympathetic neurones dissociated from neonatal rat intracardiac ganglia and maintained in tissue culture. 2. Rat intracardiac neurones had a mean resting membrane potential of ‐52 mV and mean input resistance of 850 M omega. The current‐voltage relationship recorded during slow voltage ramps indicated the presence of both leakage and voltage‐dependent currents. The contribution of Na+, K+ and Cl‐ to the resting membrane potential was examined and relative ionic permeabilities PNa/PK = 0.12 and PCl/PK < 0.001 were calculated using the Goldman‐Hodgkin‐Katz voltage equation. Bath application of the potassium channel blockers, tetraethylammonium ions (TEA; 1 mM) or Ba2+ (1 mM) depolarized the neurone by approximately 10 mV. Inhibition of the Na(+)‐K+ pump by exposure to K(+)‐free medium or by the addition of 0.1 mM ouabain to the bath solution depolarized the neurone by 3‐5 mV. 3. In most neurones, depolarizing current pulses (0.5‐1 s duration) elicited a single action potential of 85‐100 mV, followed by an after‐hyperpolarization of 200‐500 ms. In 10‐15% of the neurones, sustained current injection produced repetitive firing at maximal frequency of 5‐8 Hz. 4. Tetrodotoxin (TTX; 300 nM) reduced, but failed to abolish, the action potential. The magnitude and duration of the TTX‐insensitive action potential increased with the extracellular Ca2+ concentration, and was inhibited by bath application of 0.1 mM Cd2+. The repolarization rate of the TTX‐insensitive action potential was reduced, and after‐hyperpolarization was replaced by after‐depolarization upon substitution of internal K+ by Cs+. The after‐hyperpolarization of the action potential was reduced by bath application of Cd2+ (0.1 mM) and abolished by the addition of Cd2+ and TEA (10 mM). 5. Depolarization‐activated outward K+ currents were isolated by adding 300 nM TTX and 0.1 mM Cd2+ to the external solution. The outward currents evoked by step depolarizations increased to a steady‐state plateau which was maintained for > 5 s. The instantaneous current‐voltage relationship, examined under varying external K+ concentrations, was linear, and the reversal (zero current) potential shifted in accordance with that predicted by the Nernst equation for a K(+)‐selective electrode. The shift in reversal potential of the tail currents as a function of the extracellular K+ concentration gave a relative permeability, PNa/PK = 0.02 for the delayed outward K+ channel(s).(ABSTRACT TRUNCATED AT 400 WORDS)
A Cl‐ conductance sensitive to external pH in the apical membrane of rat duodenal enterocytes.
Authors: Brown C D , McNicholas C M , Turnberg L A
Published: December 2014

1. The pH dependence of a chloride conductance in the apical membrane of rat duodenal enterocytes was examined. 2. A stepwise reduction of both internal and external pH from 7.4 to 6.8 resulted in a significant stimulation of 36Cl flux driven by an inside‐positive membrane potential. 3. A stepwise reduction in pH had no significant effect upon other parameters such as the initial rate of D‐[3H]glucose or voltage‐independent 36Cl uptake, suggesting a specific effect upon the chloride conductance. 4. The pH‐dependent stimulation of 36Cl uptake exhibited saturation kinetics, with an apparent Vmax (maximum velocity) of 5.5 nmol (mg protein)‐1 (4 s)‐1 and an apparent Km (Michaelis‐Menten constant) of 88 nM H+ ions. 5. To determine the site of action of protons upon the conductance the effect of asymmetrically reducing either the internal or external pH was examined. 6. A step reduction of extracellular pH from 7.8 to 6.8 significantly stimulated the rate of 36Cl uptake. In contrast, a step reduction of internal pH from 7.8 to 6.8 was without effect upon the rate of 36Cl uptake. 7. These results suggest that the chloride conductance on the apical membrane of rat duodenal enterocytes is allosterically regulated by protons at an external site.