bluuebunny/crossref_metadata_2025 huggingface dataset:
Voltage‐dependent currents in isolated single Merkel cells of rats.
Authors: Yamashita Y , Akaike N , Wakamori M , Ikeda I , Ogawa H
Published: December 2014

1. Merkel cells were dissociated enzymatically from the footpad epidermis of 10‐ to 20‐day‐old rats pretreated with fluorescent dye, quinacrine, for purposes of staining. The fluorescent Merkel cells had an elongated or elliptic shape in situ, yet the dissociated ones were round (7‐12 microns in diameter). 2. Electrical recordings were performed in the whole‐cell configuration using a conventional patch‐clamp technique. The mean resting membrane potential of fluorescent Merkel cells was ‐54.0 mV, the value being greater than the ‐26.1 mV of non‐fluorescent epidermal cells. No voltage‐dependent channel was observed in non‐fluorescent cells. 3. The Merkel cells had no Na+ spike in an external standard solution, but tetrodotoxin‐resistant long‐lasting action potentials were evoked by depolarization with injection of constant currents in an external solution containing Ba2+. 4. In Merkel cells under voltage clamp, depolarizing step pulses (800 ms) from a holding potential (VH) of ‐80 mV elicited predominantly outward K+ currents composed of transient and sustained components: the former was selectively inhibited by 4‐aminopyridine (4‐AP), while the latter was inhibited by both tetraethylammonium (TEA) and quinacrine. Quinacrine was more effective and selective than TEA in blocking the sustained K+ current but had no effect on the current at the low concentration (10(‐7) or 3 x 10(‐6) M) used for staining the Merkel cells. 5. The sustained outward K+ current (IKD) was activated at potentials more positive than ‐20 or ‐10 mV at a VH of ‐50 mV, at which potential the transient outward K+ channel was completely inactivated. The potential for half‐inactivation in the steady‐state inactivation curve for IKD was ‐33 mV. 6. The transient outward K+ current (IA) was activated at potentials more positive than ‐50 mV at a VH of ‐80 mV. The potential for half‐inactivation in the steady‐state inactivation curve for IA was ‐64 mV. 7. When the outward K+ currents were blocked by adding both TEA and 4‐AP, only a sustained inward Ca2+ current was observed. In an external solution containing 10 mM‐Ca2+, ICa was evoked by potentials more positive than ‐20 mV at a VH of ‐80 mV, and the maximum inward current appeared around +10 mV. Increases in external Ca2+ concentration ([Ca2+]o) induced a hyperbolic increase in ICa and shifted the current‐voltage (I‐V) relationship along the voltage axis in a more positive direction. Saturation of ICa occurred at about 25 mM [Ca2+]o.(ABSTRACT TRUNCATED AT 400 WORDS)
All‐or‐nothing Ca2+ mobilization from the intracellular stores of single histamine‐stimulated HeLa cells.
Authors: Bootman M D , Berridge M J , Taylor C W
Published: December 2014

1. Histamine‐stimulated mobilization of intracellular Ca2+ stores was monitored in intact and permeabilized populations of HeLa cells using both the fluorescent Ca2+ indicator Fura‐2 and 45Ca2+ measurements. Digital video imaging of Fura‐2‐loaded cells was used to measure the intracellular calcium concentration ([Ca2+]i) of single cells. 2. In populations of HeLa cells, histamine caused a concentration‐dependent increase in cytoplasmic [Ca2+]. The initial transient increase was independent of extracellular Ca2+ (Ca2+o) and was followed by a sustained increase that was abolished by removal of Ca2+o. 3. In Ca(2+)‐free medium ([Ca2+]o < 1 microM), a maximal histamine concentration (25 microM) caused a transient increase in [Ca2+]i, and a subsequent challenge with histamine failed to evoke a further response indicating that the inositol 1,4,5‐trisphosphate (InsP3)‐sensitive Ca2+ stores had been completely emptied. Lower concentrations of histamine (0.5‐10 microM) caused smaller, concentration‐dependent increases in [Ca2+]i that were also transient. After exposure to these low histamine concentrations, where [Ca2+]i returned to baseline within 2 min, addition of a higher histamine concentration evoked a further increase in [Ca2+]i. The second increase in [Ca2+]i was inversely proportional to the increase caused by the first exposure to histamine, indicating that Ca2+ released in the initial response was not substantially resequestered into histamine‐sensitive stores. 4. Single HeLa cells challenged with low concentrations of histamine in Ca(2+)‐free medium responded with transient increases in [Ca2+]i, but individual cells differed in their sensitivity with 51% of cells responding to 1 microM, and 98% responding to 25 microM‐histamine. 5. When single cells in Ca(2+)‐free medium were challenged with stepwise increases in histamine concentration, they responded to each step with a transient [Ca2+]i increase after which [Ca2+]i returned to baseline within 1 min. Prolonging the interval between histamine additions by up to 25 min did not affect the [Ca2+]i increase evoked by a subsequent histamine addition. 6. Unidirectional 45Ca2+ efflux from saponin‐permeabilized HeLa cells showed that, under conditions that prevented Ca2+ resequestration, submaximal concentrations of InsP3 rapidly emptied only a fraction of the InsP3‐sensitive Ca2+ stores. The failure of low InsP3 concentrations to fully mobilize the InsP3‐sensitive Ca2+ stores was not a consequence of InsP3 degradation. 7. We conclude that within single HeLa cells, intracellular Ca2+ stores are heterogeneous in their sensitivity to InsP3, and the fraction of Ca2+ stores mobilized by InsP3 increases as the InsP3 concentration increases.
Na(+)‐HCO3‐ symport in the sheep cardiac Purkinje fibre.
Authors: Dart C , Vaughan‐Jones R D
Published: December 2014

1. Intracellular pH (pHi) was recorded in isolated sheep cardiac Purkinje fibres using liquid sensor ion‐selective microelectrodes in conjunction with conventional (3 M‐KCl) microelectrodes (to record membrane potential). 2. In HEPES‐buffered solution (pH0 7.4), pHi recovery from an intracellular acid load (20 mM‐NH4Cl removal) was blocked by 1 mM‐amiloride, consistent with the inhibition of Na(+)‐H+ exchange. Replacement of the HEPES buffer with CO2‐HCO3‐ caused a transient acidosis followed by an amiloride‐resistant recovery of pHi to more alkaline levels (n = 43). This implies the presence of a HCO3(‐)‐dependent pHi regulatory mechanism. 3. Comparison of the membrane potential with the equilibrium potential for HCO3‐ ions (EHCO3) estimated during amiloride‐resistant pHi recovery, showed that for polarized fibres (membrane potential Em approximately ‐80 mV), there was a net outward electrochemical driving force for HCO3‐ ions. Hence the amiloride‐resistant pHi recovery cannot be explained in terms of passive HCO3‐ influx through membrane channels. 4. Removal of external Na+ (Na0+ replaced by N‐methyl‐D‐glucamine) inhibited HCO3(‐)‐dependent pHi recovery, whereas removal of external Cl‐ (leading to depletion of internal Cl‐; Cl0‐ replaced by glucuronate) or short‐term removal of extracellular K+ had no inhibitory effect. We suggest that a Na(+)‐HCO3‐ co‐influx causes the recovery. Replacement of external Na+ with Li+ greatly reduced HCO3(‐)‐dependent pHi recovery indicating that Li0+ cannot readily substitute for Na0+ on the co‐transport. 5. The stilbene drug DIDS (4,4‐diisothiocyano‐stilbene‐disulphonic acid, 500 microM) slowed HCO3(‐)‐dependent pHi recovery. 6. Depolarization of the membrane potential in high K0+ (44.5 mM) solution or with 5 mM‐BaCl2 had no effect upon the rate of HCO3(‐)‐sensitive pHi recovery. This observation, when coupled with the fact that activation of HCO3(‐)‐dependent pHi recovery was associated with no consistent change of membrane potential, suggests that the Na(+)‐HCO3‐ co‐influx is electroneutral and voltage insensitive. 7. HCO3(‐)‐dependent pHi recovery was unaffected by the Na(+)‐K(+)‐2Cl‐ co‐transport inhibitor, bumetanide (150 microM). 8. The contribution of Na(+)‐H+ exchange and Na(+)‐HCO3‐ co‐transport to net acid efflux was assessed. At a pHi of 6.6, we estimate that the co‐transport should account for 20% of total acid equivalent efflux.(ABSTRACT TRUNCATED AT 400 WORDS)
Glutamate receptor channels in isolated patches from CA1 and CA3 pyramidal cells of rat hippocampal slices.
Authors: Jonas P , Sakmann B
Published: December 2014

1. Currents activated by glutamate receptor (GluR) agonists were recorded from outside‐out patches isolated from the soma of visually identified pyramidal neurones of the CA3 and CA1 region of rat hippocampal slices. alpha‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA), L‐glutamate (L‐Glu), and kainate (KA) were delivered either by bath application through perfusion of the recording chamber or by rapid application via a piezo‐driven two‐barrelled fast application system. 2. Bath application of each of the three agonists activated inward currents in all patches (n = 134) at holding potentials of ‐50 or ‐60 mV. The current amplitude increased in size between 3 to 30 microM‐AMPA and 100 microM to 1 mM‐KA. With this slow mode of bath application, the responses showed no apparent desensitization even at saturating concentrations of AMPA (30 microM) and KA (1 mM). 3. The ratio of currents activated by 30 microM‐AMPA and 300 microM‐KA showed a characteristic difference between CA3 and CA1 neurones. The ratio was 0.242 +/‐ 0.028 (mean +/‐ S.E.M., n = 16) for CA3 cell patches and 0.097 +/‐ 0.012 (n = 8) for CA1 cell patches indicating that GluRs in the two cell populations are different. 4. The steady‐state current‐voltage relations (I‐Vs) for AMPA‐ and KA‐activated currents showed pronounced outward rectification for both cell types (when the main cations are Na+ in the bath and Cs+ in the pipette solution). The current reversed close to 0 mV and the ratio of chord conductances 80 mV on either side of the reversal potential was 2.66 for KA‐activated currents in CA3 cell patches and 2.60 in CA1 cell patches. AMPA‐activated currents showed a time‐dependent increase after steps to positive membrane potentials and a decrease after steps to negative voltages, indicating that a gating process is responsible for outward rectification of the steady‐state I‐V. 5. The permeability (P) of GluR channels was high for Na+ as compared to Cs+ for both cell types (PNa/PCs = 0.88 and 0.84). The permeability was low for N‐methyl‐D‐glucamine+ (PNMG/PCs < or = 0.03) and Ca2+ (PCa/PCs < or = 0.05). 6. The current noise level increased during application of AMPA or KA. Apparent single‐channel conductances obtained from fluctuation analysis were higher for AMPA than for KA, but similar for both cell types. In CA3 cell patches, AMPA activated channels with an apparent chord conductance of 7.2 pS, KA of 3.0 pS conductance.(ABSTRACT TRUNCATED AT 400 WORDS)
Ca(2+)‐dependent and ‐independent components of transmitter release at the frog neuromuscular junction.
Authors: Tanabe N , Kijima H
Published: December 2014

1. When a Ca2+ chelator, bis (O‐aminophenoxy)ethane‐N,N,N',N'‐tetraacetic acid (BAPTA), was loaded into the presynaptic nerve terminal of the frog neuromuscular junction (NMJ), facilitation, measured as an increase in endplate potential (EPP) amplitudes during a train of ten stimulations at 100 Hz, was greatly decreased within 20 min of BAPTA‐AM (the acetoxymethyl ester of BAPTA) perfusion, and remained at a constant low level thereafter, suggesting that [Ca2+]i at the presynaptic nerve terminal was buffered by BAPTA. 2. Detailed examination of the two components of facilitation of EPP amplitude in the BAPTA‐loaded NMJs showed that the fast component was lost almost completely, while the slow component was unaffected by loaded BAPTA. Augmentation and potentiation were also unaffected by BAPTA. 3. Under external Ca(2+)‐free conditions (with 1 mM‐EGTA), both augmentation and potentiation of miniature endplate potential (MEPP) frequency were clearly observed after tetanic stimulation in the normal NMJ, and were also unaffected by loaded BAPTA. 4. The above findings strongly support the residual Ca2+ hypothesis for the fast component of facilitation, and suggest that the three slower processes (the slow component of facilitation, augmentation and potentiation) occur independently of [Ca2+]i. This Ca2+ independence was supported by the fact that facilitation and potentiation have multiplicative effects on the amount of release. 5. The quantal content of the first EPP in the train remained unchanged throughout the time course of BAPTA loading for most NMJs. This suggests that [Ca2+]i immediately adjacent to Ca2+ channels at the active zone triggers transmitter release and is little affected by loaded BAPTA. 6. MEPP frequency was almost unchanged during BAPTA loading, suggesting that the basal [Ca2+]i remained unchanged close to the dissociation constant of BAPTA for Ca2+ (108 nM). 7. The slow component of facilitation had a multiplicative relationship with augmentation and potentiation, suggesting that the underlying mechanism for the slow component of facilitation differs from that for augmentation and potentiation.
Estimation of the carnosine content of different fibre types in the middle gluteal muscle of the thoroughbred horse.
Authors: Sewell D A , Harris R C , Marlin D J , Dunnett M
Published: December 2014

1. Skeletal muscle samples were obtained by needle biopsy from one of two depths of the m. gluteus medius in a group of twenty race‐trained thoroughbred horses. 2. The content of carnosine was determined in each muscle sample, part of which was used for histochemical analysis. Fibres were classified as type I, type IIA or type IIB on the basis of the pH dependent lability of the myosin ATPase reaction. 3. Muscle samples with a higher type II fibre section area (FSA) have a higher carnosine content than those with a higher type I FSA. 4. Multiple linear regression analysis was used to estimate the mean carnosine content of individual fibre types. The results estimated a mean carnosine content in type I fibres of 54 mmol (kg dry muscle (DM))‐1, in type IIA fibres 85 mmol (kg DM)‐1 and in type IIB fibres 180 mmol (kg DM)‐1. 5. Based on the estimated values of single fibre carnosine content, there was close concordance between the estimated and the measured carnosine content of mixed fibre samples. 6. It would appear from this and other studies that carnosine has an important role as a physico‐chemical buffer in equine middle gluteal muscle and that this is greatest in type IIB fibres, where it may account for up to 50% of physico‐chemical buffering of H+ produced by muscle in the pH range 7.1‐6.5.
Regulation of perfusive O2 transport during exercise in humans: effects of changes in haemoglobin concentration.
Authors: Ferretti G , Kayser B , Schena F , Turner D L , Hoppeler H
Published: December 2014

1. Recently it was suggested that submaximal cardiac output (Q) could vary in response to changes in arterial O2 concentration (Ca,O2), so that arterial O2 delivery (Qa,O2 = Q x Ca,O2, in ml min‐1) is kept constant. 2. This hypothesis was tested on eight healthy male subjects, at rest and during exercise (50, 100 and 150 W) in three conditions: normaemia (N), after 6 weeks of endurance training (T), and 2 days after subsequent autologous blood reinfusion (P). 3. Measured variables were oxygen consumption (VO2), by open circuit method, Q, by a CO2 rebreathing method, and haemoglobin concentration ([Hb]), by a photometric method. Ca,O2 was calculated as the product of [Hb], arterial O2 saturation (0.97), and the O2 binding coefficient. 4. [Hb] and thus Ca,O2 increased by 2.6% (T vs. N) and subsequently by further 5.8% (P vs. T). VO2 and Qa,O2 were linear functions of power (w), both relationships being unaffected by changes in Ca,O2. As a consequence, the linear Q vs. VO2 relationships were shifted downward as Ca,O2 increased. 5. The VO2 vs. w and the Qa,O2 vs. w relationships had the same slope. Therefore, the difference between Qa,O2 (w) and VO2 (w), equal to O2 flow in mixed venous blood (Qv,O2), was constant. 6. In conclusion, the tested hypothesis was supported by the present results. The observed constancy of Qv,O2 suggested that Qv,O2 may play a key role in regulating the cardiovascular response to exercise.
Corticospinal volleys evoked by electrical stimulation of human motor cortex after withdrawal of volatile anaesthetics.
Authors: Hicks R , Burke D , Stephen J , Woodforth I , Crawford M
Published: December 2014

1. In twenty‐two neurologically normal patients undergoing surgery for scoliosis, corticospinal volleys to transcranial electrical stimulation of the motor cortex were recorded from the spinal cord using epidural electrodes. While anaesthesia was maintained by nitrous oxide and narcotics, volatile anaesthetics were withdrawn to determine whether such agents had a depressant effect on the evoked corticospinal volley. 2. Profound changes were documented in liminal D waves, there being an increase in amplitude averaging 392% following withdrawal of the volatile anaesthetic. There was a proportionately smaller increase (averaging 26%) in supraliminal D waves; these had a complex bifid or trifid shape indicating that some corticofugal axons were being activated deep to cortex. In general the effect on the D wave of withdrawing the anaesthetic agent was similar to that of increasing stimulus intensity. 3. Withdrawal of isoflurane dramatically increased the number of I waves and their mean amplitude. In the absence of isoflurane, I3 (mean latency 3.5 ms after the D wave) became the dominant I wave. The amplitude of I2 (mean latency 2.2 ms) became slightly smaller. The change in I waves could not be likened to an increase in stimulus intensity, because I waves invariably increase in, or remain of the same, amplitude as stimulus intensity is increased. 4. These findings indicate that changes in motor cortex excitability can result in major changes in the corticospinal volley produced by transcranial electrical stimulation, affecting both the D wave and I waves. They caution against identifying a cortical action solely on the basis of a change in the responses to magnetic stimulation of motor cortex but no such change to electrical stimulation.
Histochemical arguments for muscular non‐shivering thermogenesis in muscovy ducklings.
Authors: Duchamp C , Cohen‐Adad F , Rouanet J L , Barré H
Published: December 2014

1. The histochemical characteristics of gastrocnemius muscle were investigated in 6‐week‐old cold‐acclimated (5 weeks, 4 degrees C) and glucagon‐treated (5 weeks, 25 degrees C, 103 nmol/kg I.P. twice daily) muscovy ducklings, two groups able to develop non‐shivering thermogenesis in vivo. A comparison was made with thermoneutral controls (25 degrees C) of the same age. All animals were fed ad libitum. Fibre type, fibre area and capillary supply have been studied. Further, a quantitative histochemical method for mitochondrial Mg(2+)‐ATPase activity was developed to characterize the mitochondrial coupling state in situ. 2. White gastrocnemius was composed of fast glycolytic (FG) and fast oxidative glycolytic (FOG) fibres, while red gastrocnemius contained FOG and slow oxidative (SO) fibres. In white gastrocnemius, the proportion of FG fibres was higher in glucagon‐treated than in control or cold‐acclimated ducklings. In red gastrocnemius, the proportion of SO fibres was higher in both cold‐acclimated and glucagon‐treated ducklings than in controls. The area of all fibres was generally lower in glucagon‐treated than in other ducklings. 3. The capillary density was higher in both red and white components of the gastrocnemius muscle in cold‐acclimated and glucagon‐treated than in control ducklings, as a result of an increased number of capillaries around each fibre. 4. In all fibres, except the FG type in cold‐acclimated ducklings, the staining intensity of the Mg(2+)‐ATPase reaction was higher in cold‐acclimated and glucagon‐treated than in control ducklings whereas the staining intensity with maximal decoupling of oxidative phosphorylation by dinitrophenol was unchanged. This indicated a more loose‐coupled state of mitochondria in situ in all fibres of cold‐acclimated ducklings, and in FOG fibres of white gastrocnemius and SO fibres of red gastrocnemius in glucagon‐treated ducklings. 5. These results indicated a higher oxidative metabolism of skeletal muscle in both cold‐acclimated and glucagon‐treated than in control ducklings, and for most of the parameters studied, a similarity between cold acclimation and glucagon treatment. Because of the higher loose‐coupled state of muscle mitochondria in cold‐acclimated and glucagon‐treated than in control ducklings, the higher oxidative capacity of skeletal muscle in these ducklings could be used for heat production rather than ATP synthesis and account for muscular non‐shivering thermogenesis.
The effects of ventricular end‐diastolic and systolic pressures on action potential and duration in anaesthetized dogs.
Authors: Coulshed D S , Cowan J C , Drinkhill M J , Hainsworth R
Published: December 2014

1. Although it is known that mechanical events in the heart influence the duration of the cardiac action potential, there is no quantitative information on the effects of independent changes in ventricular end‐diastolic and systolic pressures. 2. Experiments were carried out on open‐chest anaesthetized dogs in which the autonomic nervous influences on the heart were prevented and monophasic action potentials were recorded form the epicardial surface of the left ventricle. The duration of these action potentials was taken as the interval from the upstroke to the point of 90% repolarization. 3. Elevation of left ventricular peak systolic pressure, at constant end‐diastolic pressure, significantly shortened the monophasic action potential. 4. Elevation of end‐diastolic pressure at constant peak systolic pressure significantly lengthened the monophasic action potential. 5. Responses were not dependent on release of noradrenaline from sympathetic nerve terminals because they persisted after administration of bretylium tosylate. They were also not due to myocardial ischaemia because they persisted when coronary perfusion pressure was maintained at a constant high level. 6. Simultaneous recordings of changes in myocardial segment length showed the expected responses to changes in ventricular pressures: increases in shortening in response to increases in diastolic pressure and no consistent effect from changes in systolic pressure. 7. These investigations demonstrate the independent effects of changes in systolic and end‐diastolic pressures on cardiac action potential duration. This effect is likely to be an effect of the mechanical events, i.e. contraction‐excitation feedback. This response may be mediated through changes in myocardial fibre tension, the consequent changes in fibre shortening, or both.
Effects of tetrodotoxin, Ca2+ absence, d‐tubocurarine and vesamicol on spontaneous acetylcholine release from rat muscle.
Authors: Dolezal V , Tucek S
Published: December 2014

1. Rat hemidiaphragms were incubated in a physiological low‐K+ medium without stimulation and the amount of acetylcholine (ACh) released was measured radioenzymatically. Cholinesterases were inhibited by paraoxon. 2. In the presence of 1 microM tetrodotoxin (TTX), the amount of ACh released during a 2 h incubation was lowered by 40%. A similar decrease was observed in the absence of Ca2+ and in the presence of 10 microM‐d‐tubocurarine (dTC). The effects of TTX combined with Ca2+ removal, and of TTX combined with dTC were no greater than those of TTX, dTC or Ca2+ removal alone. TTX and dTC had no effect on the release of ACh from diaphragms 4 days after denervation. 3. The reduction of spontaneous ACh release observed in the presence of TTX or dTC or in the absence of Ca2+ is best interpreted on the assumption that about 40% of the ACh release was due to the impulse activity known to be generated in intramuscular motor nerve branches by the ACh which accumulates after the inhibition of cholinesterases. 4. In the presence of 1 and 10 microM vesamicol (AH5183, 2‐(4‐phenylpiperidino)‐cyclohexanol), the release of ACh was also diminished by approximately 40%. Vesamicol did not augment the inhibition of release produced by TTX or by the omission of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)
Energy metabolism in single human muscle fibres during intermittent contraction with occluded circulation.
Authors: Greenhaff P L , Söderlund K , Ren J M , Hultman E
Published: December 2014

1. Glycogenolysis in type I and II muscle fibres was investigated in five healthy volunteers during electrical stimulation of the quadriceps muscle group with blood flow occluded. 2. The quadriceps femoris muscles were stimulated intermittently (1.6 s stimulation, 1.6 s rest) at a frequency of 50 Hz for 64 s and isometric contraction force was recorded. Muscle biopsies were obtained at rest prior to and immediately after stimulation. Single muscle fibres were dissected free and were identified as type I and II fibres. ATP, phosphocreatine (PCr) and glycogen contents were measured luminometrically and enzymatically in single fibres and mixed fibre muscle. 3. Electrical stimulation resulted in a marked decline in contraction force and near total depletion of PCr in both fibre types. The ATP turnover rate (P < 0.05) and the magnitude of the decline in ATP (P < 0.05) were greater in type II fibres. Prior to stimulation the muscle glycogen content was 32% higher in type II fibres compared with type I fibres (P < 0.01). During stimulation the rate of glycogenolysis in type II fibres (4.32 +/‐ 0.54 mmol (kg dry matter (DM)‐1 s‐1 was twofold greater than the rate in type I fibres (2.05 +/‐ 0.70 mmol (kg DM)‐1 s‐1, P < 0.05). 4. The data suggest that the relatively higher rate of glycogenolysis observed in type I fibres during intermittent electrical stimulation with occluded circulation (2.05 +/‐ 0.70 mmol (kg DM)‐1 s‐1), when compared with the corresponding rate recorded during intense contraction with circulation intact (0.18 +/‐ 0.14 mmol (kg DM)‐1 s‐1, P < 0.05), may result from an accelerated ATP turnover rate in this fibre type increasing the cellular concentrations of free AMP and inosine 5'‐monophosphate (IMP), which are known activators of glycogen phosphorylase. 5. The similarity in the rate of type II fibre glycogenolysis during contraction with circulatory occlusion (4.32 +/‐ 0.54 mmol (kg DM)‐1 s‐1), when compared with the corresponding rate recorded during non‐occluded circulation (3.54 +/‐ 0.53 mmol (kg DM)‐1 s‐1, P > 0.05), is in agreement with the suggestion that glycogenolysis in this fibre type is already occurring at a near‐maximal rate with circulation intact.
Dopamine and baclofen inhibit the hyperpolarization‐activated cation current in rat ventral tegmental neurones.
Authors: Jiang Z G , Pessia M , North R A
Published: December 2014

1. Whole‐cell patch‐clamp recordings were made from dopamine‐containing ventral tegmental area neurones in slices of rat midbrain. An inward current (Ih) was activated by hyperpolarization from ‐60 mV. 2. Dopamine (30 microM) reduced the amplitude of Ih by 10‐30% at potentials from ‐70 to ‐120 mV. The effect was concentration dependent, mimicked by the D2 agonist quinpirole, and prevented by the D2 antagonist (‐)‐sulpiride. Baclofen (0.3‐3 microM) also inhibited Ih; this action was antagonized by 2‐hydroxysaclofen but not by (‐)‐sulpiride. The decrease in Ih resulted from a reduction in the maximal current with no change in the voltage dependence. 3. The action of dopamine was unaffected by cadmium (200 microM), forskolin (10 microM), the adenylyl cyclase inhibitor 2',3'‐dideoxyadenosine (100 microM), or by intracellular solution containing cyclic AMP (2 mM). 4. Ih was progressively reduced during the first 5‐10 min of recording with electrodes containing guanosine 5'‐O‐(3‐thiotriphosphate); after this time, dopamine had no further effect. 5. It is concluded that agonists acting at D2 receptors and GABAB receptors reduce Ih in ventral tegmental neurones.
Stimulation of the KATP channel by ADP and diazoxide requires nucleotide hydrolysis in mouse pancreatic beta‐cells.
Authors: Larsson O , Ammälä C , Bokvist K , Fredholm B , Rorsman P
Published: December 2014

1. The mechanisms by which ADP and the hyperglycaemic compound diazoxide stimulate the activity of the ATP‐regulated K+ channel (KATP channel) were studied using inside‐out patches isolated from mouse pancreatic beta‐cells maintained in tissue culture. 2. The ability of diazoxide and ADP to increase KATP channel activity declined with time following patch excision and no stimulation was observed after 15‐40 min. 3. Activation of KATP channels by ADP required the presence of intracellular Mg2+. The stimulatory effect of ADP was mimicked by AMP but only in the presence of ATP. Replacement of ATP with the non‐hydrolysable analogue beta, gamma‐methylene ATP did not interfere with the ability of ADP to stimulate KATP channel activity. By contrast, enhancement of KATP channel activity was critically dependent on hydrolysable ADP and no stimulation was observed after substitution of alpha,beta‐methylene ADP for standard ADP. 4. The ability of diazoxide to enhance KATP channel activity was dependent on the presence of both internal Mg2+ and ATP. Diazoxide stimulation of KATP channel activity was not observed after substitution of beta,gamma‐methylene ATP for ATP. However, in the presence of ADP, at a concentration which in itself had no stimulatory action (10 microM), diazoxide was stimulatory also in the presence of the stable ATP analogue. 5. The stimulatory action of diazoxide on KATP channel activity in the presence of ATP was markedly enhanced by intracellular ADP. This potentiating effect of ADP was not reproduced by the stable analogue alpha,beta‐methylene ADP and was conditional on the presence of intracellular Mg2+. A similar enhancement of channel activity was also observed with AMP (0.1 mM). In the absence of ATP, diazoxide was still capable of stimulating channel activity provided ADP was present. This effect was not reproduced by AMP. 6. In both nucleotide‐free solution and in the presence of 0.1 mM ATP, the distribution of the KATP channel open times were described by a single exponential with a time constant of approximately 20 ms. Addition of ADP or diazoxide resulted in the appearance of a second component with a time constant of > 100 ms which comprised 40‐70% of the total number of events. Under the latter experimental conditions, the open probability of the channel increased more than fivefold relative to that observed in the presence of ATP alone.(ABSTRACT TRUNCATED AT 400 WORDS)
Effects of distension of the urinary bladder on the cardiovascular reflexes from the carotid baroreceptors in the dog.
Authors: de Burgh Daly M , Ward J , Wood L M
Published: December 2014

1. The hindlimb vasoconstrictor effects of distension of the urinary bladder were studied at different levels of input from the carotid sinus baroreceptors in the dog anaesthetized with a mixture of chloralose and urethane. 2. The vascularly isolated hindlimb was perfused at constant blood flow through its femoral artery, so that a change in pressure gradient (mean femoral arterial perfusion pressure minus mean inferior vena caval pressure) indicated a similar directional change in vascular resistance. The vascularly isolated carotid sinus regions were perfused with blood at a constant pulsatile flow. 3. Raising the carotid sinus mean perfusion pressure in randomly selected steps of 30 mmHg from 60 to 210 mmHg had little effect on heart rate unless the blood pressure was controlled, when a progressive bradycardia occurred, but caused a progressive reduction in arterial blood pressure and vasodilatation in the perfused hindlimb. Distension of the bladder at each level of carotid sinus pressure resulted in tachycardia, hypertension and hindlimb vasoconstriction. 4. The cardiac responses to bladder distension were the same at all carotid sinus pressures. When the blood pressure was controlled, however, the response was reduced at high and low sinus pressures. 5. The relationship between the carotid sinus perfusion pressure and hindlimb perfusion pressure (i.e. vascular resistance) was affected by distension of the bladder in two ways. In the one, hindlimb perfusion pressure increased by approximately the same amount at all levels of carotid sinus pressure indicating resetting of the carotid sinus baroreceptor reflex control of hindlimb vascular resistance towards vasoconstriction without change in gain of the reflex. In the other, the pressure increases were diminished at the higher levels of carotid sinus pressure indicating both resetting and an increase in gain of the reflex. 6. Both types of response occurred in the spontaneously breathing animal, in animals artificially ventilated, while pacing the heart, with the arterial blood pressure maintained constant at about 100 mmHg, and after division of the cervical vagosympathetic nerves. The frequency of occurrence of each type of response, however, varied under the different conditions. 7. The possible reasons for the two types of vascular response are discussed.
A reassessment of mercury in silastic strain gauge plethysmography for microvascular permeability assessment in man.
Authors: Gamble J , Gartside I B , Christ F
Published: December 2014

1. We have used non‐invasive mercury in a silastic strain gauge system to assess the effect of pressure step size, on the time course of the rapid volume response (RVR) to occlusion pressure. We also obtained values for hydraulic conductance (Kf), isovolumetric venous pressure (Pvi) and venous pressure (Pv) in thirty‐five studies on the legs of twenty‐three supine control subjects. 2. The initial rapid volume response to small (9.53 +/‐ 0.45 mmHg, mean +/‐ S.E.M.) stepped increases in venous pressure, the rapid volume response, could be described by a single exponential of time constant 15.54 +/‐ 1.14 s. 3. Increasing the size of the pressure step, to 49.8 +/‐ 1.1 mmHg, gave a larger value for the RVR time constant (mean 77.3 +/‐ 11.6 s). 4. We propose that the pressure‐dependent difference in the duration of the rapid volume response, in these two situations, might be due to a vascular smooth muscle‐based mechanism, e.g. the veni‐arteriolar reflex. 5. The mean (+/‐ S.E.M.) values for Kf, Pvi and Pv were 4.27 +/‐ 0.18 (units, ml min‐1 (100 g)‐1 mmHg‐1 x 10(‐3), 21.50 +/‐ 0.81 (units, mmHg) and 9.11 +/‐ 0.94 (units, mmHg), respectively. 6. During simultaneous assessment of these parameters in arms and legs, it was found that they did not differ significantly from one another. 7. We propose that the mercury strain gauge system offers a useful, non‐invasive means of studying the mechanisms governing fluid filtration in human limbs.
Effects of deoxygenation on active and passive Ca2+ transport and cytoplasmic Ca2+ buffering in normal human red cells.
Authors: Tiffert T , Etzion Z , Bookchin R M , Lew V L
Published: December 2014

1. The effects of deoxygenation on cytoplasmic Ca2+ buffering, saturated Ca2+ extrusion rate through the Ca2+ pump (Vmax), passive Ca2+ influx and physiological [Ca2+]i level were investigated in human red cells to assess whether or not their Ca2+ metabolism might be altered by deoxygenation in capillaries and venous circulation. 2. The study was performed in fresh human red cells maintained in a tonometer either fully oxygenated or deoxygenated. Cytoplasmic Ca2+ buffering was estimated from the equilibrium distribution of 45Ca2+ induced by the divalent cation ionophore A23187 and the Vmax of the Ca2+ pump was measured either by the Co(2+)‐exposure method or following ionophore wash‐out. The passive Ca2+ influx and physiological [Ca2+]i were determined in cells preloaded with the Ca2+ chelator benz‐2 and resuspended in autologous plasma. 3. Deoxygenation increased the fraction of ionized Ca2+ in cell water by 34‐74% and reduced the Vmax of the Ca2+ pump by 18‐32%. 4. To elucidate whether or not these effects were secondary to deoxygenation‐induced pH shifts, the effects of deoxygenation on cell and medium pH, and of pH on cytoplasmic Ca2+ binding and Ca2+ pump Vmax in oxygenated cells were examined in detail. 5. Deoxygenation generated large alkaline pH shifts that could be explained if the apparent isoelectric point (pI) of haemoglobin increased by 0.2‐0.4 pH units in intact cells, consistently higher than the value of 0.15 reported for pure haemoglobin solutions. 6. In oxygenated cells, the fraction of ionized cell calcium, alpha, was little affected by pH within the 7.0‐7.7 range. Ca2+ pump Vmax was maximal at a medium pH of about 7.55. Comparison between pH effects elicited by HCl‐NaOH additions and by replacing Cl‐ with gluconate suggested that Vmax was inhibited by both internal acidification and external alkalinization. Since deoxygenation alkalinized cells and medium within a range stimulatory for Vmax, the inhibition observed was not due to pH. 7. There was no significant effect of deoxygenation on passive Ca2+ uptake, or steady‐state physiological [Ca2+]i level. 8. The deoxygenation‐induced reduction in Ca2+ binding capacity may result from the increased protonation of haemoglobin on deoxygenation and from binding of 2,3‐diphosphoglyceric acid (2,3‐DPG) and ATP to deoxyhaemoglobin; inhibition of the Ca2+ pump may result from shifts in the [Mg2+]i/[ATP]i ratio away from a near optimal stimulatory value in the oxygenated state.
Postsynaptic activation at the squid giant synapse by photolytic release of L‐glutamate from a ‘caged’ L‐glutamate.
Authors: Corrie J E , DeSantis A , Katayama Y , Khodakhah K , Messenger J B , Ogden D C , Trentham D R
Published: December 2014

1. Pharmacological evidence suggests L‐glutamate is a strong candidate as a transmitter at the giant synapse of the squid. Postsynaptic activation at the giant synapse cannot be effected by conventional application of putative neurotransmitters by iontophoresis or perfusion, apparently because the complex structure of the synapse prevents a sufficiently rapid change in concentration at the postsynaptic membrane. Flash photolytic release of L‐glutamate from a pharmacologically inert ‘caged’ L‐glutamate pre‐equilibrated in the stellate ganglion of Alloteuthis or Loligo was used to determine whether L‐glutamate can produce postsynaptic activation when released rapidly in the synaptic clefts. 2. The preparation, reaction mechanism and properties of the caged L‐glutamate, N‐1‐(2‐nitrophenyl)ethoxycarbonyl‐L‐glutamate, are described. The product quantum yield on photolysis was 0.65 (+/‐ 0.05). On flash photolysis glutamate release followed a single exponential time‐course in the pH range 5.5‐7.8. The rate constant was proportional to [H+] and was 93 s‐1 at pH 5.5 and 16 degrees C in artificial sea water (ionic strength, I = 0.68 M). 3. At pH 7.8 flash photolysis of caged glutamate pre‐equilibrated in the synapse caused only a slow depolarization. A second photolytic release of L‐glutamate or transsynaptic activation produced no further depolarization, suggesting desensitization and inactivation of postsynaptic mechanisms by the initial pulse of L‐glutamate. 4. Synaptic transmission in the giant synapse was normal at pH 5.5. Flash photolysis at pH 5.5 caused rapid production of L‐glutamate within the synaptic cleft and a fast postsynaptic depolarization which generated postsynaptic action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)
Calcium release‐activated calcium current in rat mast cells.
Authors: Hoth M , Penner R
Published: December 2014

1. Whole‐cell patch clamp recordings of membrane currents and fura‐2 measurements of free intracellular calcium concentration ([Ca2+]i) were used to study the biophysical properties of a calcium current activated by depletion of intracellular calcium stores in rat peritoneal mast cells. 2. Calcium influx through an inward calcium release‐activated calcium current (ICRAC) was induced by three independent mechanisms that result in store depletion: intracellular infusion of inositol 1,4,5‐trisphosphate (InsP3) or extracellular application of ionomycin (active depletion), and intracellular infusion of calcium chelators (ethylene glycol bis‐N,N,N',N'‐tetraacetic acid (EGTA) or 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N',N'‐tetraacetic acid (BAPTA)) to prevent reuptake of leaked‐out calcium into the stores (passive depletion). 3. The activation of ICRAC induced by active store depletion has a short delay (4‐14 s) following intracellular infusion of InsP3 or extracellular application of ionomycin. It has a monoexponential time course with a time constant of 20‐30 s and, depending on the complementary Ca2+ buffer, a mean normalized amplitude (at 0 mV) of 0.6 pA pF‐1 (with EGTA) and 1.1 pA pF‐1 (with BAPTA). 4. After full activation of ICRAC by InsP3 in the presence of EGTA (10 mM), hyperpolarizing pulses to ‐100 mV induced an instantaneous inward current that decayed by 64% within 50 ms. This inactivation is probably mediated by [Ca2+]i, since the decrease of inward current in the presence of the fast Ca2+ buffer BAPTA (10 mM) was only 30%. 5. The amplitude of ICRAC was dependent on the extracellular Ca2+ concentration with an apparent dissociation constant (KD) of 3.3 mM. Inward currents were nonsaturating up to ‐200 mV. 6. The selectivity of ICRAC for Ca2+ was assessed by using fura‐2 as the dominant intracellular buffer (at a concentration of 2 mM) and relating the absolute changes in the calcium‐sensitive fluorescence (390 nm excitation) with the calcium current integral. This relationship was almost identical to the one determined for Ca2+ influx through voltage‐activated calcium currents in chromaffin cells, suggesting a similar selectivity. Replacing Na+ and K+ by N‐methyl‐D‐glucamine (with Ca2+ ions as exclusive charge carriers) reduced the amplitude of ICRAC by only 9% further suggesting a high specificity for Ca2+ ions. 7. The current amplitude was not greatly affected by variations of external Mg2+ in the range of 0‐12 mM. Even at 12 mM Mg2+ the current amplitude was reduced by only 23%. 8. ICRAC was dose‐dependently inhibited by Cd2+.(ABSTRACT TRUNCATED AT 250 WORDS)
Interaction between central pattern generators for breathing and swallowing in the cat.
Authors: Dick T E , Oku Y , Romaniuk J R , Cherniack N S
Published: December 2014

1. We examined the interaction between central pattern generators for respiration and deglutition in decerebrate, vagotomized, paralysed and ventilated cats (n = 10), by recording activity from the following nerves: hypoglossal, phrenic, thyroarytenoid and triangularis sterni. Fictive breathing was spontaneous with carbon dioxide above the apnoeic threshold (end‐tidal PCO2, 32 +/‐ 4 mmHg) and fictive swallowing was induced by stimulating the internal branch of the left superior laryngeal nerve (SLN) continuously (0.2 ms pulse duration, 10 Hz). 2. In all ten animals, SLN stimulation evoked short bursts of thyroarytenoid and hypoglossal nerve activity indicative of fictive swallowing. In two of ten animals, respiration was inhibited completely during deglutition. In the other eight animals, fictive breathing and swallowing occurred simultaneously. 3. With SLN stimulation below threshold for eliciting swallowing, the respiratory rhythm decreased, the duration of inspiration did not change but the duration of expiration, especially stage II, increased. Integrated nerve activities indicated that the rate of rise and peak of phrenic nerve activity decreased, stage I expiratory activity of the thyroarytenoid and especially that of the hypoglossal nerve increased and stage II expiratory activity of the triangularis sterni nerve was suppressed completely. However, if inspired carbon dioxide was increased, i.e. hypercapnic ventilation, stage II expiratory activity remained partially during continuous SLN stimulation. 4. Fictive‐swallowing bursts occurred only at respiratory phase transitions. At the minimal stimulus intensity that evoked repetitive swallowing bursts, the pattern of interaction between breathing and swallowing central pattern generators was consistent for each animal (n = 7) but was different across animals. In four animals, fictive swallows occurred at the phase transition between stage II expiration and inspiration, at the transition between inspiration and stage I expiration in one animal; and in two other animals, at the transition between stage I and II of expiration. 5. The response to SLN stimulation accommodated during the stimulus train. Accommodation was evident in both the interswallow interval (ISI) which lengthened, and the interaction pattern which had fewer swallows per breath as the stimulus period progressed. In contrast to the ISI, characteristics of the fictive swallow did not accommodate. For example, duration of the swallow was constant, distributed over a narrow range throughout the stimulus train. 6. We conclude that the central pattern generators for swallowing and breathing interact. The pattern of interaction supports the three‐phase theory of respiratory pattern generation.(ABSTRACT TRUNCATED AT 400 WORDS)
Calcium‐activated hyperpolarizations in rat locus coeruleus neurons in vitro.
Authors: Osmanović S S , Shefner S A
Published: December 2014

1. Intracellular recordings were made from rat locus coeruleus (LC) neurons in completely submerged brain slices. Trains of action potentials in LC neurons were followed by a prolonged post‐stimulus hyperpolarization (PSH). If trains were elicited with depolarizing current pulses of sufficient intensity, PSH was composed of a fast, early component (PSHE) and a slow, late component (PSHL). PSH which followed trains elicited with lower intensity depolarizing current pulses consisted only of PSHL. 2. Both PSHE and PSHL were augmented by increasing the number of action potentials in the train and both were associated with an increase in membrane conductance. The reversal potential for PSHE was ‐108 mV and for PSHL it was ‐114 mV. 3. When a hybrid voltage clamp protocol was used, the current underlying PSH (IPSH) was observed to consist of an early, rapidly decaying component, IE, followed by a late, slower decaying component, IL. The time course of decay of IPSH was biexponential with the time constant of decay of IL more than one order of magnitude larger than the time constant of decay of IE. An increase in the concentration of external K+ shifted the reversal potentials for IE and IL in the depolarizing direction; the mean value of shift per tenfold increase in external K+ concentration was 57.1 mV for IE and 57.6 mV for IL. 4. Both PSHE and PSHL were inhibited by lowering the external Ca2+ concentration or by application of the Ca2+ channel blockers Cd2+ (200‐500 microM) or nifedipine (100 microM). Intracellular injection of EGTA abolished both components of PSH. Increasing the external Ca2+ concentration augmented both PSH components. 5. Superfusion of dantrolene (25 microM) or ryanodine (20 microM) decreased the amplitude and duration of PSHL with much less effect on PSHE. 6. d‐Tubocurarine (20‐200 microM) selectively blocked PSHE with no effect on PSHL; this effect is the same as that of apamin which we have previously described. Superfusion with charybdotoxin (40 nM) or TEA (400 microM‐1 mM) did not reduce PSHE or PSHL. 7. Inhibition of IA by 4‐aminopyridine or 2,4‐diaminopyridine also did not reduce either component of PSH. In fact, these agents slightly augmented both components of PSH; this effect was probably secondary to the prolongation of action potential duration. Superfusion of TEA in concentrations of 2‐10 mM increased the size and duration of PSHL and increased the duration but decreased the size of PSHE.(ABSTRACT TRUNCATED AT 400 WORDS)