bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties of the transient outward current in rabbit atrial cells.
Authors: Clark R B , Giles W R , Imaizumi Y
Published: December 2014

1. Whole‐cell and patch clamp techniques have been used to study the steady‐state voltage dependence and the kinetics of a transient outward current, It, in single cells from rabbit atrium. 2. The steady‐state voltage dependence of both activation and inactivation of It are well described by Boltzmann functions. Inactivation is fully removed at potentials negative to ‐70 mV and it is complete near 0 mV. The threshold for activation of It is near ‐30 mV and it is fully activated at +30 mV. The region of overlap between the activation and inactivation curves indicates that a steady non‐inactivating current will be recorded over a membrane potential range from approximately ‐30 to 0 mV. 3. In general, the time course of inactivation at potentials in the range 0 to +50 mV is best described as a sum of two exponential functions. The kinetic parameters controlling these processes exhibit only very weak voltage dependence. 4. Comparison of the time course of the development of inactivation in response to long depolarizing voltage clamp steps with the development of inactivation in response to trains of brief depolarizing pulses indicates that inactivation develops very quickly and decays relatively slowly at potentials near the resting potential (e.g. ‐70 mV). Thus, in response to (i) a train of voltage‐clamp pulses or (ii) a series of action potentials, the magnitude of It decreases due to a progressive increase in the amount of inactivation. 5. A simple model of channel gating is presented: it can account for the major aspects of the voltage dependence and kinetics of It (cf. Aldrich, 1981). 6. Cell‐attached patch clamp recordings have been used to identify the single‐channel or unitary events underlying the current, It. In general, only one active channel is present per patch. The single‐channel conductance in normal Tyrode solution is approximately 14 pS and the current‐voltage relationship is approximately linear between +50 and +150 mV with respect to rest. This information, in combination with the fully activated current‐voltage characteristics from the whole‐cell data, can be used to estimate the number and density of It channels per cell: these are 1600 and one per 3‐4 micron 2, respectively. 7. Ensemble averages obtained from patch recordings are very similar in time course to the macroscopic or whole‐cell current itself: the ensemble current rises to a peak within approximately 5 ms and decays with a biexponential time course in response to depolarizations to approximately +50 mV.(ABSTRACT TRUNCATED AT 400 WORDS)
Membrane properties of primary sensory neurones of the cat after peripheral reinnervation.
Authors: Belmonte C , Gallego R , Morales A
Published: December 2014

1. The properties of sensory neurones in the petrosal ganglion of the cat were examined in vitro with intracellular electrodes 2‐30 days after cutting the carotid or the glossopharyngeal nerve or 30‐120 days after crushing the nerves and allowing time for peripheral reinnervation. Both glossopharyngeal and carotid neurones with conduction velocities faster than 2 m/s and humps on the falling phase of their action potentials were studied. 2. Two days after axotomy, the action potential of glossopharyngeal cells showed increased maximum rate of depolarization and decreased after‐hyperpolarization (AHP) amplitude. These cells also showed a decrease in time‐dependent inward rectification and conduction velocity. No significant changes were observed in carotid neurones at this stage. 3. Four days after axotomy, these changes were still present in glossopharyngeal cells and the duration of the AHP had also decreased. Among carotid neurones there was an increase in spike duration and a decrease in time‐dependent rectification and in the amplitude and duration of the AHP. 4. Eight days after axotomy glossopharyngeal cells additionally showed an increased spike duration and decreased rheobase, while carotid neurones had also decreased in conduction velocity. 5. All the changes described were still present 16 days after cutting the nerves. In addition, the input resistance increased in both types of cells. 6. The effects of axotomy persisted 30 days after cutting the nerve, except for the AHP amplitude in both types of cells and the spike duration in glossopharyngeal neurones which had recovered to near control values. 7. Thirty days after crushing the carotid nerve, some carotid neurones showed chemosensory activity, indicating that reinnervation of the carotid body had taken place. In these cells, the properties altered by axotomy had recovered except for the conduction velocity. 8. Four months after crushing the glossopharyngeal nerve, activity evoked by mechanical and chemical stimulation of the tongue could be recorded from the nerve, indicating that sensory reinnervation had taken place. The electrical properties of the glossopharyngeal cells were normal except for conduction velocity which was still slower than the control value. 9. It is concluded that the electrical membrane properties altered by axotomy revert to normal after peripheral sensory regeneration.
The ascending projection of interneurones activated by group I muscle afferent fibres of the cat hindlimb.
Authors: Fern R , Harrison P J , Riddell J S
Published: December 2014

1. The level of the ascending projection of lumbosacral interneurones with a monosynaptic input from group I muscle afferents of the cat hindlimb has been investigated. The study was concerned particularly with the interneurones mediating group I non‐reciprocal inhibition of motoneurones. 2. In chloralose‐anaesthetized cats, single‐unit microelectrode recordings were made, in the lateral funiculus at L6, from the ascending axons of lumbosacral interneurones. The axons studied were discharged by group I afferents primarily from extensor muscles of the knee and ankle. Some units were discharged in addition by cutaneous and/or joint afferents. 3. The extent of the ascending projection of the interneurones was assessed by antidromic activation of their axons in the lateral funiculus at different spinal levels. The great majority of axons tested (thirty‐four out of thirty‐six) were found to have an ascending projection to at least the L4 level and of these most (90%) did not project beyond the L4 or L3 segments of the cord. 4. The projection to at least L4, together with monosynaptic input from group I afferents and a pattern of peripheral input characteristic of interneurones in laminae V‐VI of lumbosacral segments, identified thirty‐four of the thirty‐six units as interneurones mediating group I non‐reciprocal inhibition. 5. These results suggest that the ascending group I inhibitory pathway, formed by these interneurones, is associated specifically with the group I relay of the dorsal spinocerebellar tract in Clarke's column, rather than being conterminous with group I afferents, which project throughout the rostral lumbar and lowest thoracic segments.
Plateau potentials in alpha‐motoneurones induced by intravenous injection of L‐dopa and clonidine in the spinal cat.
Authors: Conway B A , Hultborn H , Kiehn O , Mintz I
Published: December 2014

1. Intracellular recordings were made from lumbar alpha‐motoneurones in unanaesthetized decerebrate acute spinal cats. The response of motoneurones to direct current pulse injection or synaptic excitation was investigated following intravenous injection of L‐beta‐3,4‐dihydroxyphenylalanine (L‐DOPA, 20‐120 mg/kg) alone, nialamide (10‐50 mg/kg) and L‐DOPA or clonidine (0.5‐1 mg/kg). 2. The response properties of motoneurones were tested with rectangular and triangular current waveforms. Before L‐DOPA treatment motoneuronal firing during a rectangular current pulse is characterized by an initial high firing frequency which rapidly decreases to a lower steady‐state firing which is maintained only for the duration of the pulse. Following administration of L‐DOPA an acceleration in firing frequency is apparent following the initial adaptation seen with rectangular current pulses. A transient after‐depolarization or an after‐discharge often followed the termination of the pulse. The frequency‐current relation in response to a triangular current injection changed from a clockwise to a counter‐clockwise hysteresis after L‐DOPA treatment (i.e. after L‐DOPA the firing frequency was higher for any given current during the descending phase than during the ascending phase of the triangular waveform). 3. Firing acceleration during and self‐sustained firing after rectangular current pulses and counter‐clockwise hysteresis of firing frequency with triangular current pulses are causally related to the presence of plateau potentials, which can be directly visualized after inactivation of the spikes. Plateau potentials in motoneurones could be generated by short‐lasting intracellular depolarizing current pulses or brief excitatory synaptic inputs and terminated by short‐lasting hyperpolarizing current pulses or brief inhibitory synaptic inputs. Plateau potentials were demonstrated in flexor and extensor motoneurones. 4. All bistable properties described in the preceding paragraphs following L‐DOPA administration could also be seen after administration of the alpha‐receptor agonist clonidine. 5. Slow rhythmic oscillations of the membrane potential (7.5‐10 Hz) were seen superimposed on plateau potentials in a few cells after administration of L‐DOPA and clonidine. The oscillations had an amplitude in the range 10‐20 mV and represent the expression of an intrinsic property of the motoneurone. 6. It is demonstrated that plateau potentials in the motoneurones contribute to the late long‐lasting reflexes observed in L‐DOPA‐treated spinal cats. 7. It is concluded that L‐DOPA (and clonidine) change the response properties of the motoneurones in an analogous way to 5‐hydroxy‐DL‐tryptophan (5‐HTP).(ABSTRACT TRUNCATED AT 400 WORDS)
Effects of urethane, ambient temperature and injection route on rat body temperature and metabolism due to endotoxins.
Authors: Bibby D C , Grimble R F
Published: December 2014

1. We have investigated the influence of environmental temperature, anaesthesia and route of administration on rectal temperature and other metabolic responses to two preparations of bacterial endotoxin in male adult Wistar rats. 2. Urethane anaesthesia, environmental temperatures of 20 and 28 degrees C, subcutaneous (S.C.) and intraperitoneal (I.P.) routes of administration and butanol and trichloroacetic acid (TCA) extracts of E. coli endotoxin (1.2 mg/kg) were used. 3. In addition to rectal temperature, serum zinc, albumin and urea concentrations and liver protein, RNA and zinc contents were measured. 4. Fevers were produced by injections of both endotoxins, by either route at 28 degrees C. Butanol‐extracted endotoxin produced a more rapid response than the TCA extract via the I.P. route whereas the TCA extract produced a higher temperature than the butanol extract when the S.C. route was used. 5. Fevers were inhibited at an environmental temperature of 20 degrees C and by anaesthesia, while the former had no effect on compositional changes the latter inhibited the fall in serum zinc in response to subcutaneous doses of either endotoxin and the increase in liver zinc content in response to the butanol extract of endotoxin. 6. At 20 degrees C a marked fall in rectal temperature occurred in conscious rats 2 h after receiving the TCA but not the butanol extract of endotoxin. Temperature depression was more severe when endotoxin was administered by the I.P. route. 7. Serum urea was elevated in conscious rats by the TCA extract of endotoxin via both routes but only by the I.P. route for the butanol extract of endotoxin. In anaesthetized animals only the TCA extract of endotoxin raised serum urea concentration when given intraperitoneally. 8. Serum albumin and liver protein and RNA were unaffected by endotoxin injections over the 7 h time course of the study. 9. Rectal temperature responses to endotoxins were influenced in direction and magnitude by all variables employed in the study, while compositional changes were unaffected by environmental temperature but influenced to varying degrees by urethane anaesthesia and the route of administration employed.
Whole‐cell recordings of ionic currents in bovine somatotrophs and their involvement in growth hormone secretion.
Authors: Mason W T , Rawlings S R
Published: December 2014

1. The whole‐cell mode of the patch‐clamp technique was used to record voltage‐activated cationic currents in immunocytochemically identified bovine somatotrophs. 2. In current‐clamp mode, cells had a resting membrane potential of ‐83.0 +/‐ 4.5 mV, and an input resistance of 8.4 +/‐ 2.1 G omega. Cells rarely fired action potentials spontaneously, but fired one to three action potentials in response to a suprathreshold current pulse. 3. Under voltage clamp, in Ca2+‐free media, the action potential was shown to be composed of a TTX‐sensitive inward Na+ current and an outward K+ current. 4. The isolated Na+ current had a threshold of approximately ‐50 mV, and rapidly activated and then inactivated to a small steady‐state current. Peak Na+ current amplitude with 140 mM‐external Na+ was 341.1 +/‐ 33.5 pA (n = 14) at a membrane potential of ‐32.1 +/‐ 2.4 mV (n = 14). 5. With Ca2+ or Ba2+ (5‐30 mM) as the only membrane‐permeable cation, voltage pulses to potentials more positive than ‐55 mV from a holding potential of ‐80 mV revealed a rapidly activating current component that was followed by a second, very slowly inactivating, current component, most clearly seen with Ba2+. Both components were maximally activated between 0 and +10 mV, were TTX insensitive, but were blocked by 4 mM‐Co2+. 6. Three components of the isolated K+ current were identified (IA, IK and IK(Ca] by their voltage sensitivity, Ca2+ dependence and their response to 4‐aminopyridine (4‐AP) and tetraethylammonium (TEA). 7. Growth hormone (GH)‐releasing hormone (GHRH) applied to cells under whole‐cell voltage clamp had no effect on either steady‐state or voltage‐activated ionic currents. This is probably due to dialysis of cytoplasmic compounds vital for GHRH activation of the cell. 8. Both basal and GHRH‐stimulated GH secretion were unaffected by TTX, implying that the Na+ action potential is not critical for such release. In contrast the Ca2+ channel blocker Co2+ attenuated GH release in both cases. The K+ channel blocker TEA stimulated GH release above basal and GHRH‐stimulated levels.
Inactivation of calcium release from the sarcoplasmic reticulum in frog skeletal muscle.
Authors: Schneider M F , Simon B J
Published: December 2014

1. The rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) in voltage clamped segments of frog skeletal muscle fibres was calculated from myoplasmic free calcium transients (delta[Ca2+]) measured with the calcium indicator dye Antipyrylazo III. 2. During a 100‐200 ms depolarizing pulse Rrel reached an early peak and then declined markedly. The time course and extent of decline of Rrel were independent of membrane potential over a range of potentials where release activation varied severalfold. 3. For test pulses applied shortly after relatively large or long conditioning pulses Rrel completely lacked the early peak. The peak gradually recovered as the interval between the conditioning and test pulses was increased to 1 s. 4. A latency was often observed before the start of recovery of the peak in Rrel. The latency appeared to be correlated with the time for delta[Ca2+] to fall below a certain level, indicating that recovery of the peak might represent reversal of a calcium‐dependent process. It was therefore proposed that the rapid decline in Rrel during a pulse was due to calcium‐dependent inactivation of the SR calcium release channels. 5. Inactivation continued to develop during the interval between a relatively large 20 ms conditioning pulse and a test pulse applied 20 ms later. This was as expected for calcium‐dependent inactivation of SR calcium release because of the elevated [Ca2+] between the conditioning and test pulses. It was not as expected for external membrane potential‐dependent inactivation. 6. Small steady elevations in [Ca2+] due to relatively small 200 ms conditioning pulses produced marked inactivation of Rrel, indicating an apparent dissociation constant for calcium‐dependent inactivation only slightly above resting [Ca2+]. 7. All observations could be well simulated by a two‐step model for inactivation in which myoplasmic free calcium equilibrates rapidly with a high‐affinity calcium receptor on the release channel and then the calcium‐receptor complex undergoes a slower conformational change to the inactivated state of the channel. 8. An alternative model in which calcium binds to a soluble receptor (e.g. free calmodulin) and then the calcium‐receptor complex binds to and directly inactivates the channel was shown to be formally identical to the preceding model. Either model could closely simulate all observations.
Sodium current kinetics in intact rat papillary muscle: measurements with the loose‐patch‐clamp technique.
Authors: Antoni H , Böcker D , Eickhorn R
Published: December 2014

1. Rapid inward sodium current (INa) was studied on intact rat papillary muscles and trabeculae excised from right or left ventricle using the loose‐patch‐clamp technique. All experiments were carried out at 25 degrees C. 2. Currents were recorded from patches with a large current density of mean 5.9 +/‐ 0.5 mA/cm2. 3. The current was reduced by tetrodotoxin (TTX) in a dose‐dependent manner. The concentration of TTX producing half‐maximal blockade of INa was 6.3 +/‐ 0.8 mumol/l. 4. Na+ current appeared upon depolarization at a threshold potential of about ‐55 mV and reached its maximum at about ‐20 mV. 5. Kinetic data were evaluated using the Hodgkin‐Huxley model. 6. Time constants of activation (tau m) were estimated using single‐pulse and tail‐current measurements. They had a maximum of about 0.4 ms near the threshold potential and declined at more positive and at more negative potentials to values near 0.1 ms. 7. Two time constants were necessary to describe inactivation. Both time constants had their maximal values of 135 +/‐ 8.1 and 29.1 +/‐ 5.9 ms at about ‐80 mV and decreased towards 4 and 0.5 ms at potentials positive to ‐20 mV.
Intracellular chloride regulation in amphibian dorsal root ganglion neurones studied with ion‐selective microelectrodes.
Authors: Alvarez‐Leefmans F J , Gamiño S M , Giraldez F , Noguerón I
Published: December 2014

1. Intracellular Cl‐ activity (aiCl) and membrane potential (Em) were measured in frog dorsal root ganglion neurones (DRG neurones) using double‐barrelled Cl‐ ‐selective microelectrodes. In standard Ringer solution buffered with HEPES (5 mM), equilibrated with air or 100% O2, the resting membrane potential was ‐57.7 +/‐ 1.0 mV and aiCl was 23.6 +/‐ 1.0 mM (n = 53). The value of aiCl was 2.6 times the activity expected for an equilibrium distribution and the difference between Em and ECl was 25 mV. 2. Removal of external Cl‐ led to a reversible fall in aiCl. Initial rates of decay and recovery of aiCl were 4.1 and 3.3 mM min‐1, respectively. During the recovery of aiCl following return to standard Ringer solution, most of the movement of Cl‐ occurred against the driving force for a passive distribution. Changes in aiCl were not associated with changes in Em. Chloride fluxes estimated from initial rates of change in aiCl when external Cl‐ was removed were too high to be accounted for by electrodiffusion. 3. The intracellular accumulation of Cl‐ was dependent on the extracellular Cl‐ activity (aoCl). The relationship between aiCl and aoCl had a sigmoidal shape with a half‐maximal activation of about 50 mM‐external Cl‐. 4. The steady‐state aiCl depended on the simultaneous presence of extracellular Na+ and K+. Similarly, the active reaccumulation of Cl‐ after intracellular Cl‐ depletion was abolished in the absence of either Na+ or K+ in the bathing solution. 5. The reaccumulation of Cl‐ was inhibited by furosemide (0.5‐1 x 10(‐3) M) or bumetanide (10(‐5) M). The decrease in aiCl observed in Cl‐ ‐free solutions was also inhibited by bumetanide. 6. Cell volume changes were calculated from the observed changes in aiCl. Cells were estimated to shrink in Cl‐ ‐free solutions to about 75% their initial volume, at an initial rate of 6% min‐1. 7. The present results provide direct evidence for the active accumulation of Cl‐ in DRG neurones. The mechanism of Cl‐ transport is electrically silent, dependent on the simultaneous presence of external Cl‐, Na+ and K+ and inhibited by loop diuretics. It is suggested that a Na+:K+:Cl‐ co‐transport system mediates the active transport of Cl‐ across the cell membrane of DRG neurones.
The role of periodontal receptors in the jaw‐opening reflex in the cat.
Authors: Dessem D , Iyadurai O D , Taylor A
Published: December 2014

1. In anaesthetized cats, graded electrical stimulation of the inferior alveolar nerve at just above threshold for the largest afferent fibres caused inhibition of jaw‐closer motoneurones. Stimulus strength had to be increased to 1.5 times threshold with double shocks to cause reflex contraction of the digastric muscle. 2. Inhibition of jaw‐closer muscles and excitation of digastric muscle resulted from transients of force applied to the upper canine tooth. However, the threshold for the digastric response was approximately 11 times higher than that of the periodontal afferent units recorded in the mesencephalic nucleus of the fifth nerve (MesV). Vibration of the upper canine at 50 Hz, with amplitude adequate to excite periodontal afferents, caused no digastric contraction. 3. Stimulation in the caudal part of the MesV so as to excite periodontal afferents caused no digastric reflex, provided that the stimulus did not spread to other parts of the fifth nerve nuclei. 4. It is concluded that under these conditions the low‐threshold periodontal mechanoreceptors cause inhibition of jaw‐closer muscles, but no significant excitation of jaw‐opener muscles. 5. These findings are discussed from the point of view of the control which periodontal mechanoreceptors may exert over the biting force during mastication.
Electrical transients produced by the toad urinary bladder in response to altered medium osmolality.
Authors: Gordon L G
Published: December 2014

1. The effects of changes in media osmolality on the transepithelial current through the toad urinary bladder under voltage‐clamp conditions have been studied. Over the limited range (+/‐ 24 mosmol/kg H2O) used in these investigations, changes in the osmolality of the mucosal bathing fluid produced no changes in transepithelial current. 2. Changes in osmolality of the serosal fluid greatly affected the transepithelial current with a decrease (increase) in osmolality producing a sustained increase (decrease) in current. 3. The changes in steady‐state current were approximately proportional to the magnitude of the osmotic steps and were reproducible and reversible if the osmolalities of the solutions were confined to a domain of 220‐260 mosmol/kg H2O. 4. Amiloride, which was used to block all active current, also eliminated the electrical responses to an osmotic pulse, indicating that the responses were of cellular origin. 5. The effects of substituting gluconate for medium chloride were examined. Similar responses were observed, indicating that they were not due to changes in a plasma membrane chloride conductance. 6. The transient currents observed during the changes from one steady state to the other often contained an oscillatory component, the amplitude and the degree of damping of which varied between bladders. The amplitude of the oscillations, but not their frequency, could be varied by altering the magnitude of the osmotic pulse and by changing the imposed transepithelial voltage. Decreasing the electrical potential of the mucosal solution with respect to that of the serosal solution decreased the amplitude of the oscillations, as did increased serosal potassium or substitution of gluconate for serosal chloride. The period of the oscillations always remained within the range of 9‐12 min. 7. The results suggest that two major processes are initiated by an osmotic step in the serosal bathing medium. The first involves the establishment of new ion gradients and the second, alterations in sodium pump activity. In addition, there is evidence for a voltage‐dependent sodium conductance in the apical membrane.
Effects of acetylcholine, isoprenaline and forskolin on electrolyte and protein composition of rabbit mandibular saliva.
Authors: Case R M , Howorth A J , Padfield P J
Published: December 2014

1. The major purpose of this study was to investigate cellular regulation of the ductal transport processes in salivary glands which act to modify the electrolyte composition of primary saliva and cause it to become hypotonic. This was achieved using an isolated mandibular gland preparation by observing the effect of different stimuli on the electrolyte composition of saliva secreted at the same flow rate, on the assumption that these stimuli do not influence primary saliva composition. The effects of the same stimuli on the volume of primary fluid secretion and on protein secretion were also observed. Proteins were measured in total and as individual components after their separation by high‐performance liquid chromatography. 2. Acetylcholine was used as a ‘Ca2+‐mobilizing’ agonist (i.e. one which both elevates intracellular Ca2+ concentration and activates protein kinase C). Isoprenaline was initially used to elevate intracellular cyclic AMP concentration but was subsequently abandoned in favour of forskolin. 3. Acetylcholine was a very potent stimulus of primary fluid secretion. By contrast, isoprenaline and forskolin were essentially without effect, even when superimposed on acetylcholine stimulation. 4. As judged by saliva electrolyte composition, increasing the concentration of acetylcholine enhanced ductal absorption of Na+ and Cl‐ and secretion of K+ (and presumably HCO3‐). Forskolin had the opposite effect: when superimposed on submaximal acetylcholine stimulation it caused saliva concentrations of Na+ and Cl‐ to remain high and K+ low (i.e. it inhibited ductal transport processes). The inhibitory effect of forskolin on ductal transport could be overcome by increasing the concentration of acetylcholine, and vice versa. 5. Acetylcholine, isoprenaline and forskolin each increased salivary protein secretion, although the kinetics of secretion differed. The spectrum of proteins secreted in response to the three stimuli was the same. The relative proportions of the individual proteins was influenced by the strength of stimulation (i.e. the proportions at high total protein output differed from those at low total protein output) but not apparently by the nature of the stimulus. 6. Thus, the three major secretory processes in the rabbit mandibular salivary gland respond differently to the two major signal transduction mechanisms. For primary fluid secretion, Ca2+ is stimulatory and cyclic AMP almost without effect; for ductal transport, Ca2+ is stimulatory and cyclic AMP inhibitory; and for protein secretion both Ca2+ and cyclic AMP are stimulatory.
Effect of tachycardia and constriction of left circumflex artery on coronary flow and pressure in anaesthetized dogs.
Authors: Di Lavore P , Gattullo D , Guiot C , Losano G , Mary D A , Vacca G , Vono P
Published: December 2014

1. The effect of graded changes in heart rate between 100 and 160 beats/min and constriction of the left circumflex coronary artery which reduced coronary blood flow was examined in seven anaesthetized and artificially ventilated dogs in the absence of significant changes in aortic blood pressure. Mean diastolic coronary blood flow, and the difference between the mean diastolic pressures in the coronary artery and the left ventricle were related to the increase in heart rate. 2. In all seven dogs diastolic coronary blood flow showed linear increases with heart rate increments with and without coronary narrowing which averaged 70 and 82% respectively. 3. A significant shift to the right in the relation between heart rate and mean diastolic coronary blood flow occurred with each grade of coronary constriction. Coronary blood flow became lower at any given heart rate. 4. The shift to the right in the relation between heart rate and coronary blood flow was associated with decreases in the difference between the mean diastolic pressures in the coronary artery and the left ventricle which accompanied the increase in heart rate. 5. The results suggest that increases in heart rate can enhance diastolic coronary blood flow despite coronary narrowing which reduced flow, possibly through dilatation in myocardial blood vessels.
Guanosine 5'‐triphosphate analogue activates potassium current modulated by neurotransmitters in Aplysia neurones.
Authors: Brezina V
Published: December 2014

1. Identified neurones in the abdominal ganglion of Aplysia californica were voltage clamped in order to investigate how guanosine 5'‐O‐(3‐thiotriphosphate) (GTP‐gamma‐S), a GTP analogue that irreversibly activates guanine nucleotide‐binding (G) proteins, modifies activation by the neuropeptide FMRFamide (Phe‐Met‐Arg‐Phe‐NH2) of a slow K+ current resembling the serotonin‐ and adenosine 3',5'‐cyclic monophosphate (cyclic AMP)‐sensitive ‘S’ current, and a similar response to acetylcholine. 2. Ionophoretic or pressure injection of GTP‐gamma‐S into the cell triggered the slow and irreversible development of a large K+ current, rendered the K+ current responses to FMRFamide and acetylcholine irreversible, and finally, once the GTP‐gamma‐S‐induced current had fully developed, occluded the neurotransmitter responses altogether. 3. The K+ currents activated by GTP‐gamma‐S and acetylcholine had properties identical to those previously found for the FMRFamide‐induced ‘S'‐like K+ current: they were Ca2+ and voltage independent, relatively insensitive to block by extracellular tetraethylammonium (TEA) and 4‐aminopyridine (when high concentrations of acetylcholine were used to overcome an additional block by these agents of the receptor), and suppressed in Ba2+‐containing solution, by injection of TEA+ or Cs+ into the cell, and by serotonin and elevation of the intracellular concentration of cyclic AMP. 4. The K+ current responses to FMRFamide and acetylcholine were not additive when the agonist concentrations used were high enough to activate most of the available current. 5. Desensitization of either response did not affect the other, and the effect of acetylcholine, but not that of FMRFamide, could be blocked by the known acetylcholine‐receptor blockers phenyltrimethylammonium and TEA. 6. These results suggest that FMRFamide and acetylcholine, acting through different receptors, activate the same ‘S’‐like K+ current by a mechanism involving a G protein. 7. In addition to activating the slow K+ current, FMRFamide and acetylcholine each activate a faster current in these cells, carried by Na+ in the case of FMRFamide, and by Cl‐ in the case of acetylcholine. Neither fast response was affected by GTP‐gamma‐S.
Rapid regulation of the 'second inward current' by intracellular calcium in isolated rat and ferret ventricular myocytes.
Authors: Boyett M R , Kirby M S , Orchard C H
Published: December 2014

1. Single cells were isolated from the ventricles of ferret and rat hearts. Cells were voltage clamped using a single conventional microelectrode. Membrane voltage, membrane currents and cell length were monitored. 2. The current elicited by decreasing the membrane potential from a holding potential of ‐40 or ‐45 mV to potentials more positive than ‐20 mV was abolished by D600, by Cd2+ and by removal of Ca2+ from the cell superfusate. This current activated within 20 ms and inactivated over several hundred milliseconds; it had a bell‐shaped current‐voltage relation, and was maximal at about +10 mV. It is concluded that this is the fast Ca2+ current ICa. 3. Increasing bathing [Ca2+] (Ca2+o) led to the appearance of transient inward currents (Iti). If ICa was triggered during Iti, it was reduced in magnitude, and inactivated more slowly. 4. The sarcoplasmic reticulum inhibitor ryanodine (1 mumol/l) abolished Iti, and reduced twitch contraction, but had no direct effect on the magnitude of ICa, although its rate of inactivation was slowed. 5. Iti produced by depolarization of the holding potential, or by lowering bathing [K+] or [Na+], led to similar changes to those described in paragraph 3. 6. Gradually increasing diastolic cytoplasmic [Ca2+] (Ca2+i) by rapid stimulation in the presence of ryanodine, by lowering bathing [K+], or lowering bathing [Na+], led to a parallel decrease of ICa. 7. The effects of lowering bathing [Na+] could be abolished by using an electrode‐filling solution containing EGTA. 8. In some ferret cells a slow component of the second inward current was observed. The size of this current was directly related to the size of the twitch: changes in the size of the twitch produced by changing the pattern of stimulation or application of ryanodine were paralleled by changes in the size of this current, but had no effect on the size of ICa. 9. It is concluded that the magnitude of ICa can be decreased by an increase of either resting Ca2+i, or the spontaneous increase of Ca2+ which underlies Iti, but it is not affected by the size of the stimulated calcium transient (although the time course of inactivation is dependent on the calcium transient). The size of the slow component of the second inward current, however, is directly related to the size of the twitch and may, therefore, be activated by Ca2+.
Stiffness changes during enhancement and deficit of isometric force by slow length changes in frog skeletal muscle fibres.
Authors: Sugi H , Tsuchiya T
Published: December 2014

1. The mechanism of the enhancement and the deficit of isometric force by slow length changes in frog fast muscle fibres was studied by recording muscle fibre stiffness changes as measured with sinusoidal vibrations (0.5‐1.9 kHz, peak‐to‐peak amplitude 0.1% of slack length, L0). 2. When a tetanized fibre was slowly stretched by 5‐9% from sarcomere lengths 2.4‐2.6 microns, the force rose to a peak during the stretch and then decreased towards a steady level higher than that during the ordinary isometric tetanus at the same sarcomere length. 3. The stiffness of the fibre first rose abruptly in response to stretch and then started to decrease linearly while the stretch went on; after the completion of stretch the stiffness decreased towards a steady value which was equal to that during the isometric tetanus at the same sarcomere length, indicating that the enhancement of isometric force is associated with decreased stiffness. 4. If a tetanized fibre was slowly released by 4‐12% from sarcomere lengths 2.55‐2.7 microns, the steady force attained after the completion of release was lower than that during an isometric tetanus at the same sarcomere length. 5. The stiffness of the fibre changed in parallel with the force both during and after the applied release. 6. Recordings of the segmental length changes along the fibre with a high‐speed video system (200 frames/s) indicated that all segments lengthened in response to the applied stretch. 7. The segmental length changes in response to the applied release were markedly non‐uniform; the length of a segment located at the centre of the fibre did not change appreciably both during and after the release. 8. These results are discussed in terms of cross‐bridge performance and structure of the myofilament lattice.
A study of stretch‐activated channels in the membrane of frog oocytes: interactions with Ca2+ ions.
Authors: Taglietti V , Toselli M
Published: December 2014

1. We have carried out patch‐clamp measurements on a cationic channel in the plasma membrane of the frog oocyte, which can be specifically activated by membrane stretch. The kinetics of this channel also display a distinct dependence upon membrane potential, the probability of the channel being open increasing with membrane depolarization. 2. When the patch‐clamp pipette filling solution was standard Ringer solution, the single‐channel current‐voltage (I‐V) relationship was linear, the elementary conductance being 38 pS and the reversal potential +7 mV, suggesting very poor selectivity of the channel for the various cations. 3. The I‐V relationship was highly non‐linear having a strong inward‐going rectification when Ca2+‐free solutions were used to fill the patch pipette. These solutions also resulted in a selective, inward cationic permeability through the membrane, with K+ being more permeable than Na+ greater than Li+ greater than Ba2+ greater than Ca2+. 4. Though permeant through the stretch‐activated channel, Ca2+ inhibited in a concentration‐dependent manner the currents carried by other cations. La3+ (0.1 mM) was also an effective channel blocker. 5. The inward current carried by individual cations at a given membrane potential increased with increasing external cation concentration up to a saturating level, this level being maximal for K+ and minimal for Ca2+. Also the half‐saturating concentration was maximal for K+ and minimal for Ca2+ at all membrane potentials. 6. In the presence of a constant Ca2+ concentration (50 microM) increasing [K+] did not change the absolute level at which the current saturated; however the half‐saturating K+ concentration was greatly increased, indicating competitive inhibition between Ca2+ and K+ for the same site. 7. The data are consistent with a model based on Eyring rate theory for current conduction through ionic channels, in which we assume that the ions capable of entering the channel compete for a binding site that they must first occupy before proceeding on. The possible energy profile of the stretch‐activated channel was defined by optimizing the model parameters to obtain the best fit of the experimental data. Ca2+ was found to have a smaller dissociation constant and much longer occupancy time than Na+ or K+, thus accounting for its lower permeability and inhibitory effect on current conduction by other cations through the stretch‐activatable channel.
Two‐suction‐electrode voltage‐clamp analysis of the sustained calcium current in cat sensory neurones.
Authors: Taylor W R
Published: December 2014

1. The kinetics of the sustained calcium current were examined in cat dorsal root ganglion (DRG) neurones, using a two‐suction‐electrode voltage clamp. It was shown that this current could be examined with minimal contamination from other ionic currents. Experiments were performed at 20 degrees C, with a concentration of 10 mM‐calcium externally. 2. The transient calcium current was eliminated by using a holding potential of ‐50 mV. The sustained calcium current showed no evidence of steady‐state inactivation at potentials between ‐90 and ‐30 mV. 3. The activation and deactivation time course of the calcium current was described by a double‐exponential function. The activation process was examined without interference from significant inactivation under the conditions used. 4. The steady‐state activation of the calcium channels was approximated by a two‐step activation process. Both reactions were voltage sensitive, the first having an equivalent valency of 3.4 +/‐ 0.1 electronic charges (e‐), and the second with an equivalent valency of 1.0 +/‐ 0.2 e‐. On average, half‐maximal channel activation occurred at +10 mV. 5. The fast and the slow time constants of the exponential relaxations differed by a factor of 4‐10 and both showed significant voltage dependence. Both the fast and slow time constants were greatest at potentials where approximately half the available channels were activated in the steady state. The slow time constant measured from activation and deactivation appeared to be independent of the starting potential. 6. The fractional amplitude of the slow exponential component of the tail currents was 0.09 +/‐ 0.01 at ‐70 mV and increased steadily at more positive potentials passing through a clear maximum of 0.59 +/‐ 0.03 at ‐10 mV. 7. Reducing the temperature decreased the magnitude of the peak inward current, with an apparent activation energy (Ea) of 67 kJ/mol. The slow time constants measured from activation and deactivation were also reduced at lower temperatures. The slow activation time constant had a higher temperature sensitivity (Ea = 85 kJ/mol) than the slow tail current time constant (Ea = 29 kJ/mol). The fast tail current time constant was reduced at lower temperatures with an apparent Ea of 79 kJ/mol. 8. Application of 10 microM‐(+/‐)Bay K 8644 prolonged the tail currents and shortened the activation time constant. The calcium current was activated at more negative potentials and the slope of the steady‐state activation curve was reduced.(ABSTRACT TRUNCATED AT 400 WORDS)
Detection and separation of human red cells with different calcium contents following uniform calcium permeabilization.
Authors: García‐Sancho J , Lew V L
Published: December 2014

1. The human red cell, permeabilized to calcium with the ionophore A23187, is extensively used to study Ca2+ transport and the effects of intracellular Ca2+ on transport and metabolism. The interpretation of results with calcium‐permeabilized cells, in general, has depended on the implicit assumption that the ionophore‐induced calcium distribution among the cells is uniform. 2. To establish whether or not calcium permeabilization with the ionophore A23187 generated a uniform calcium distribution in normal‐ATP red cells, a method was developed to detect and separate calcium‐permeabilized red cells with different calcium contents. For the method to uncover pre‐existing heterogeneity without itself inducing it, it was essential to preserve the calcium distribution which existed at the time of sampling. The method was based (i) on the ability of cytoplasmic Ca2+ to activate K+‐selective channels in the membrane, and (ii) on the demonstration here that thiocyanate (SCN‐) is a non‐limiting co‐ion for rapid net KSCN efflux and cell shrinkage in the cold. 3. Calcium‐permeabilized cells in pump‐leak steady state were washed free of ionophore using ice‐cold, albumin‐containing media. Subsequent incubation at 0 degrees C in low‐K+ media with 45‐75 mM‐SCN‐ generated dense‐cell fractions (H cells) in less than 10 min. These could be separated from the remaining light cells (L cells) by either centrifugation over phthalate oils, or differential osmotic haemolysis, with conservation of the mean total cell calcium. 4. Analysis of the calcium content of H and L cell fractions revealed striking differences in their calcium content, with 70‐99% of the mean total cell calcium in the H cell fraction. 5. The ionophore content of density‐separated cells, processed with omission of the ionophore removal step, was similar for cells with high‐ and low‐calcium. Magnesium loss from ionophore‐treated red cells suspended in magnesium‐free media followed single exponentials. Thus ionophore distribution and induced permeability were uniform, and the unequal cell calcium content must be due to factors affecting active calcium extrusion.
Calcium‐induced conversion of adenine nucleotides to inosine monophosphate in human red cells.
Authors: Almaraz L , García‐Sancho J , Lew V L
Published: December 2014

1. When inosine‐fed human red cells are permeabilized to calcium by exposure to the ionophore A23187, progressively larger proportions of the cell population become irreversibly depleted of ATP as calcium influx is increased (Brown & Lew, 1983; García‐Sancho & Lew, 1988b). When calcium influx is over 30 mmol/(l cells.h), all cells become ATP depleted and calcium equilibrated (E cells) (García‐Sancho & Lew, 1988b). When calcium influx is lower, E cells co‐exist with cells able to maintain normal ATP and low calcium contents in vigorous pump‐leak balance (B cells). The experiments reported here investigate why calcium‐induced ATP depletion of E cells is irreversible. 2. The inosine monophosphate (IMP) content of cells after 30 min of calcium permeabilization increased with the magnitude of the calcium load, roughly in inverse proportion to the fall in ATP. The calcium‐induced increase in IMP was confined to the fraction of cells which became osmotically resistant after SCN‐ treatment (H cells), and which contained the E cells. 3. Cell nucleotides were measured after calcium permeabilization [( A23187]c = 100 mumol/l cells) in substrate‐free media with different [Ca2+]o (0‐0.5 mM). Calcium entry caused rapid ATP fall, AMP and IMP accumulation, and delayed ADP fall at all [Ca2+]o concentrations. Initial IMP formation increased with [Ca2+]o along a sigmoid saturation‐like curve whereas AMP accumulation and ATP fall were maximal at [Ca2+]o = 20 microM and declined at the higher [Ca2+]o. The rate of IMP formation correlated positively with cell ATP and negatively with cell AMP at all [Ca2+]o values. 4. The AMP deaminase activity of red cell lysates was reversibly increased over tenfold by calcium. Half‐maximal stimulation was observed at a Ca2+ concentration of about 50 microM. 5. These results suggest that the irreversibility of calcium‐induced ATP depletion results from irreversible trapping of the adenine nucleotide as IMP, and help explain the mechanism of E cell formation.
Spontaneous electrical and contractile activity correlated to 86Rb+ efflux in smooth muscle of guinea‐pig mesotubarium.
Authors: Hellstrand P , Lydrup M L
Published: December 2014

1. The spontaneous mechanical activity of guinea‐pig mesotubarium consists of fused tetanic contractions lasting about 6 min, with a frequency of about four per hour. The muscle is completely relaxed between the contractions. Stretching the relaxed muscle elicits a contraction of the same appearance as the spontaneous ones. Comparison of preparations from oestrus (day 1 of the hormonal cycle), dioestrus (days 9‐11) and prooestrus (days 14‐15) showed no variation in the pattern of mechanical activity. 2. The resting membrane potential, measured by intracellular microelectrodes, did not differ with hormonal phase (prooestrus: ‐63.5 +/‐ 0.84 (n = 16); oestrus: ‐63.7 +/‐ 1.6 (n = 5); dioestrus: ‐61.6 +/‐ 0.77 (n = 17]. In most recordings a depolarization of a few millivolts occurred during the relaxation period (5‐10 min), but in a few cells a more pronounced spontaneous depolarization of 10‐15 mV was found. Ouabain (1 microM) caused depolarization by about 9 mV, both in pro‐ and dioestrus, leading to the initiation of maintained repetitive spiking. 3. Contraction is preceded by a depolarization lasting 10‐30 s, and when a threshold is reached a train of slow waves and spikes is elicited. The frequency of slow waves and the number of spikes on each slow wave progressively decrease during the contraction, until spiking eventually ceases. 4. The spikes are resistant to tetrodotoxin (0.5 microM) and disappear in Ca2+‐free medium, which also causes membrane depolarization. The duration of contractions increased with Ca2+ concentration in the range 1‐5 mM. 5. The rate of 86Rb+ efflux, expressed as fractional release in 2 min intervals, showed a consistent variation during the contractile cycle in preparations with regular spontaneous activity. Relative to the value at the end of the relaxed period the efflux rate increased by about twofold during the contraction. From the beginning of the relaxed period after the contraction the efflux rate decreased by about 25% until the beginning of the next contraction. 6. It is concluded that the contractile activity in the mesotubarium, as opposed to that of the fallopian tube (Lydrup & Hellstrand, 1986a), is independent of the hormonal phase, including the period around ovulation. The mechanism for initiation of the trains of action potentials associated with spontaneous contractions may involve a gradual decrease of permeability of K+ channels or activity of the Na+‐K+ pump during the relaxed period.