bluuebunny/crossref_metadata_2025 huggingface dataset:
Buffering of calcium influx by sarcoplasmic reticulum during the action potential in guinea‐pig ventricular myocytes.
Authors: Janczewski A M , Lakatta E G
Published: December 2014

1. Intracellular [Ca2+] ([Ca2+]i) transients, monitored by the fluorescent Ca2+ indicator, indo‐1, and twitch contractions elicited by action potentials, by voltage clamp pulses or by rapid, brief pulses of caffeine, were measured in guinea‐pig single ventricular myocytes. Experiments were designed to determine whether and to what extent the trans‐sarcolemmal Ca2+ influx is immediately sequestered by the sarcoplasmic reticulum (SR). 2. Rapid, brief (100‐200 ms) pulses of caffeine onto a rested myocyte elicited a [Ca2+]i transient and a contraction. Following exposure to specific SR inhibitors, ryanodine (100 nM) or thapsigargin (200 nM), the rapid application of caffeine onto a rested myocyte failed to elicit changes in [Ca2+]i or in cell length, indicating that caffeine increases [Ca2+]i by specifically discharging Ca2+ from the SR. In the absence of these inhibitors, a second pulse of caffeine, within 3 min following a prior pulse, failed to elicit a [Ca2+]i transient or contraction, indicating that a caffeine pulse depletes the SR releasable Ca2+ pool. 3. Following Ca2+ depletion of the SR by double caffeine pulses at rest, an electrical stimulation elicited a slow increase in [Ca2+]i, and, after a delay, a small, slow twitch contraction. The simultaneous application of caffeine and electrical stimulation of cells in which the SR was Ca2+ depleted elicited [Ca2+]i transients with an increased rate of rise and a larger amplitude (53 +/‐ 8 and 63 +/‐ 9% respectively; mean +/‐ S.E.M., n = 21) than those elicited by electrical stimulation alone. 4. Whether caffeine affected the L‐type calcium current (ICa) elicited by electrical stimulation was determined under whole‐cell voltage clamp. A caffeine pulse delivered at the onset of a depolarizing voltage clamp step also increased the rates of rise and the amplitudes of the [Ca2+]i transients and twitch contractions in cells in which the SR was depleted of Ca2+. However, Ca2+ influx via ICa decreased when caffeine was pulsed in conjunction with the voltage clamp, as the peak ICa was either unchanged or decreased while its inactivation was consistently accelerated. 5. Because the stimulation‐dependent trans‐sarcolemmal Ca2+ influx via ICa is not increased by a caffeine pulse, the augmentation of the rates of rise and the amplitudes of the electrically stimulated [Ca2+]i transients by caffeine pulsed in conjunction with the electrical stimulation in cells in which the SR had been depleted of Ca2+ indicates that a portion of Ca2+ influx during depolarization in the absence of caffeine is rapidly buffered by the SR.(ABSTRACT TRUNCATED AT 400 WORDS)
Effect of isoprenaline on Ca2+ channel current in single smooth muscle cells isolated from taenia of the guinea‐pig caecum.
Authors: Muraki K , Bolton T B , Imaizumi Y , Watanabe M
Published: December 2014

1. The effects of isoprenaline (Iso) on Ca2+ channel current in enzymatically isolated single cells of the guinea‐pig taenia caeci were examined using the standard whole‐cell voltage‐clamp method. 2. Iso potentiated the voltage‐dependent Ca2+ current; the threshold and maximally effective concentration of Iso to increase Ca2+ current were 3‐10 nM and 1‐3 microM, respectively. The average increase in Ca2+ current produced by 3 microM Iso was 42 +/‐ 6% (mean +/‐ S.E.M.) and the response could be obtained repeatedly in the same cell. The concentration‐response relationship could be fitted by a binding model with a Hill coefficient of 1 and a dissociation constant of 42 nM. 3. The effect of Iso on Ca2+ current was voltage dependent. Although potentiation of Ca2+ current by Iso was obvious between ‐30 and +10 mV, it was small or absent around +20 to +30 mV. Iso had little effect on the relationship between inactivation of the Ca2+ current and voltage obtained using a double‐pulse protocol. 4. External application of forskolin, an adenylyl cyclase activator, or internal perfusion of cAMP or dibutyryl cAMP from the recording pipette, did not increase Ca2+ current and potentiation of Ca2+ current by Iso was observed repeatedly and was unchanged. 5. Internal perfusion of GTP gamma S or GDP beta S increased or did not affect the Ca2+ current and potentiation of Ca2+ current by Iso was unchanged and could be recorded repeatedly for about 20 min after rupture of the cell membrane. In addition, treatment of cells with the potent protein kinase C inhibitor, chelerythrine, had no effect on Ca2+ current or on potentiation of Ca2+ current by Iso. 6. These results suggest that the Ca2+ current in guinea‐pig taenia caeci cells is potentiated by isoprenaline via mechanisms which do not involve either a cAMP pathway, a G‐protein pathway or a protein kinase C pathway. The receptor involved appeared to be an atypical adrenoreceptor not blocked by either alpha‐ or beta‐receptor blocking agents.
Ions required for the electrogenic transport of GABA by horizontal cells of the catfish retina.
Authors: Cammack J N , Schwartz E A
Published: December 2014

1. Solitary horizontal cells were isolated from catfish retinas. Membrane currents activated by extracellular and intracellular GABA were characterized during a whole‐cell voltage clamp. 2. Extracellular GABA activated two currents: a GABAA current, and an ‘influx’ current mediated by a GABA transporter. The influx current was studied after the GABAA current was blocked with 0.5 mM picrotoxin. The influx current required extracellular Na+ and Cl‐. Extracellular Na+ could not be replaced by another alkali metal cation. 3. The influx current also depended upon the identity of ions in the intracellular solution. Either an intracellular alkali metal cation or Cl‐ was required to produce an influx current. 4. The influx current was inward at ‐75 mV and decreased as the membrane was depolarized towards +20 mV. When the membrane was depolarized beyond +25 mV, the polarity of the current depended upon the ion composition of the intracellular solution and could be inward, zero or outward. 5. The introduction of GABA into a cell during the course of an experiment produced an outward current. This ‘efflux’ current was small at ‐75 mV and increased with depolarization. The efflux current required intracellular Na+ and Cl‐. Intracellular Na+ could not be replaced by another alkali metal cation. 6. The efflux current also depended upon the identity of ions in the extracellular solution. An extracellular alkali metal cation was required to produce an efflux current. Removing extracellular Cl‐ did not affect the efflux current. 7. The outward movement of GABA produced a local accumulation in extracellular GABA concentration that could be detected by the activation of the GABAA current. GABA efflux only occurred during conditions that produced an efflux current. Electroneutral efflux did not occur. 8. In the absence of GABA, extracellular alkali metal cations produced a ‘leakage’ current. The leakage current was inward at ‐75 mV and decreased as the membrane was depolarized towards +20 mV. When the membrane was depolarized beyond +25 mV, the polarity of the leakage current depended, like the GABA influx current, upon the ion composition of the intracellular solution and could be inward, zero or outward. The addition of GABA to the intracellular solution produced an efflux current and suppressed the leakage current. 9. We conclude that the transporter mediates electrogenic influx, efflux and leakage. Each mode of operation depends upon ions on both sides of the membrane. Influx and efflux are not symmetrical.
Modulation by histamine of an inwardly rectifying potassium channel in human endothelial cells.
Authors: Nilius B , Schwarz G , Droogmans G
Published: December 2014

1. Whole‐cell and single‐channel currents were recorded together with intracellular Ca2+ in voltage clamped, single endothelial cells isolated from human umbilical vein. 2. The major current component under resting conditions in the whole‐cell configuration was a strongly inwardly rectifying potassium current. 3. This current is due to activation of a K+ channel with an inward conductance of 29 +/‐ 3 pS (n = 7) with symmetrical 140 mM K+ on both sides of the membrane. This channel could be measured both in the cell‐attached and in the inside‐out configuration. At potentials below ‐110 mV both whole‐cell and averaged single‐channel currents showed a fast inactivation. 4. During stimulation of endothelial cells with histamine, whole‐cell K+ currents initially increased but then substantially declined, despite the sustained increase in intracellular Ca2+ concentration ([Ca2+]i). 5. The blockade of the inwardly rectifying K+ channel by histamine could not be observed in cell‐attached patches if histamine was added to the bath. 6. It is concluded that endothelial cells possess K+ channels that are directly inhibited by agonists, such as histamine. Blocking these channels may depolarize the cell membrane and thereby reduce the driving force for Ca2+ influx.
Effect of positive and negative step changes in intrathoracic pressure on left ventricular function in conscious man.
Authors: De Cort S C , Innes J A , Guz A
Published: December 2014

1. Breathing affects left ventricular stroke volume (LVSV) in normal subjects. The observed relationship may result from interaction between the effects of changing lung volume and intrathoracic pressure (IP). 2. To investigate the effect of IP on LVSV with minimal changes in lung volume, beat‐by‐beat LVSV (pulsed Doppler ultrasound) and systemic blood pressure (Finapres) were measured during obstructed inspiratory and expiratory efforts causing step changes in IP of +/‐ 15 cmH2O for 10 s, in seven subjects. Changes in mouth pressure (MP) during airway occlusion were used to indicate changes in IP. Group‐averaged data for each second were compared to that in the second before the change in MP using Dunnet's multiple range test. 3. Step reductions in MP resulted in immediate and significant falls in LVSV (P < 0.05) and systolic blood pressure (P < 0.01) and increased heart rate, although this was not significant. These responses were transient, lasting only 3 s despite 10 s of reduced MP. 4. Step increases in MP caused biphasic cardiovascular responses. LVSV increased immediately, then fell significantly below control after 8 s (P < 0.01). Heart rate increased significantly between 5 and 9 s after the onset of the increase in MP (P < 0.05), suggesting activation of the baroreflexes by the accompanying progressive fall in systolic blood pressure. 5. The asymmetry in time course and magnitude between the responses to positive and negative pressure may reflect asymmetrical effects of MP on systemic venous return, right ventricular output, pulmonary venous return and left ventricular (LV) after‐load, with the baroreceptors limiting changes in arterial pressure.(ABSTRACT TRUNCATED AT 250 WORDS)
Neural and biomechanical specializations of human thumb muscles revealed by matching weights and grasping objects.
Authors: Kilbreath S L , Gandevia S C
Published: December 2014

1. Human manual dexterity has been linked by some to biomechanical adaptations of the hand and by others to neural adaptations. To investigate neural adaptations, the present study using the performance of four muscles acting on the index and thumb, quantified weight matching and electromyography. 2. The accuracy (i.e. reproducibility) of weight matching was used to investigate whether thumb muscles (i.e. flexor pollicis longus (FPL) and adductor pollicis (AP)) perform differently from index muscles (i.e. flexor digitorum profundus (FDP) and first dorsal interosseous (FDI)), and whether intrinsic hand muscles (AP and FDI) perform differently from extrinsic ones (FPL and FDP). 3. Subjects lifted reference weights on the right which represented predetermined percentages of a force generated in a maximum voluntary contraction (MVC) ranging from 2.5% to 35% MVC (and to 50% MVC in two muscles) and matched them with a variable weight lifted in the same way on the left. 4. Analysis of the coefficients of variation (c.v., expressed as a percentage) and the standard deviations calculated for repeated estimates of perceived heaviness, revealed significant differences in the accuracy of weight matching between different muscles and between reference weights. Based on the c.v., subjects lifted more accurately with FPL and AP (the two thumb muscles) than with the two index muscles. The two intrinsic hand muscles (FDI and AP) were equally accurate, and significantly more accurate than FDP which was the least accurate muscle. The high accuracy for FPL remained when accuracy was expressed in terms of the torque produced by the muscles when lifting the reference weights, and also when the torques were converted to absolute intramuscular forces. 5. Accuracy (based on c.v.) decreased significantly with light weights and increased with heavy weights for all muscles except FPL, which was equally accurate over a very wide range of weights (< 2.5% to 50% MVC). When data from all muscles were pooled, the c.v. increased from 12.9 to 19.1 as the weights lifted decreased from 35% to 2.5% MVC. 6. To examine the functional implications of the weight‐matching study, electromyographic activity (EMG) was recorded with fine‐wire electrodes from the same four muscles while subjects lifted cylinders of different widths (17‐50 mm) and weights (15‐1000 g). For recordings in which integrated EMG was linearly related to force up to maximal levels, the amplitudes of the EMGs at ‘lift off’ and at the mid‐point of the ‘hold’ phase of the task were expressed relative to the maximal EMG during a MVC.(ABSTRACT TRUNCATED AT 400 WORDS)
Adrenaline‐regulated Cl‐ transport in cultured single rat epididymal cells measured by an entrapped Cl‐(‐)sensitive fluorophore.
Authors: Huang S J , Chan H C , Wong P Y
Published: December 2014

1. Isolated cells from primary cultures of rat epididymal epithelial cells were employed for the study of adrenaline‐stimulated Cl‐ transport using a Cl‐(‐)sensitive fluorophore 6‐methoxy‐N‐(3‐sulphopropyl) quinolinium (SPQ). SPQ was loaded into the cells by the hypotonic shock method. 2. The resting intracellular Cl‐ concentration, estimated in the presence of nigericin and tributyltin in high‐K+ solution, was 62.3 +/‐ 0.2 mM. This value was not altered in the presence of 1 microM adrenaline. When extracellular Cl‐ was replaced by NO3‐, an increase in fluorescence corresponding to a decrease in intracellular Cl‐ was observed. The initial outward Cl‐ movement was estimated to be 0.54 +/‐ 0.08 mM s‐1. This value was increased by incubating the cells with adrenaline. The stimulatory effect of adrenaline was reduced by 1 mM DPC. 3. Addition of Cl‐ to cells previously depleted of Cl‐ caused an instantaneous decrease in fluorescence due to the entry of Cl‐. The initial rate of Cl‐ entry was ‐0.62 +/‐ 0.13 mM s‐1. Adrenaline increased the rate of entry to ‐2.13 +/‐ 0.08 mM s‐1. The adrenaline‐stimulated rate of entry was reduced by DPC or frusemide (0.5 mM) and was completely blocked in the presence of both agents. 4. In Na(+)‐free solution, the adrenaline‐stimulated rise of rate of Cl‐ entry was reduced in the presence of DPC. Frusemide had no effect on the entry rate. 5. The stimulatory effect of adrenaline were abolished by propranolol (5 microM) but not by phentolamine (5 microM). Conversely, isoprenaline (1 microM) and forskolin (1 microM) mimicked the effects of adrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)
The intrinsic gating of inward rectifier K+ channels expressed from the murine IRK1 gene depends on voltage, K+ and Mg2+.
Authors: Stanfield P R , Davies N W , Shelton P A , Khan I A , Brammar W J , Standen N B , Conley E C
Published: December 2014

1. We describe the cloning of the inward rectifier K+ channel IRK1 from genomic DNA of mouse; the gene is intronless. 2. The IRK1 gene can be stably expressed in murine erythroleukaemia (MEL) cells. Such transfected cells show inward rectification under whole‐cell recording. 3. Channels encoded by the IRK1 gene have an intrinsic gating that depends on voltage and [K+]o. Rate constants are reduced e‐fold as the driving force on K+(V‐EK) is reduced by 24.1 mV. 4. Removal of intracellular Mg2+ permits brief outward currents under depolarization. The instantaneous current‐voltage relation may be fitted by an appropriate constant field expression. 5. Removal of intracellular Mg2+ speeds channel closure at positive voltages. In nominally zero [Mg2+]i, rate constants for the opening and closing of channels, processes which are first order, are similar to those of native channels.
Endogenous Na(+)‐K+ (or NH4+)‐2Cl‐ cotransport in Rana oocytes; anomalous effect of external NH4+ on pHi.
Authors: Keicher E , Meech R
Published: December 2014

1. In Rana oocytes, measurements with chloride‐sensitive microelectrodes show that the mean intracellular chloride activity (34.8 +/‐ 6.3 mM, n = 79) is three times higher than that expected for the passive distribution of chloride ions across the outer membrane (12.4 mM, mean membrane potential ‐43 +/‐ 8.8 mV, n = 79). 2. Reuptake of chloride into oocytes depleted by prolonged exposure to chloride‐free saline takes place against the electrochemical gradient. 3. Chloride reuptake does not take place in sodium‐free solution or in a sodium‐substituted potassium‐free solution. It is inhibited by bumetanide (10(‐5) M) in the bathing medium. 4. The overall stoichiometry of the transport mechanism deduced from simultaneous measurements of intracellular sodium and chloride using ion‐selective electrodes is 1Na+:1K+:2Cl‐. 5. Ammonium ions substitute for potassium on the cotransporter. 6. In oocytes smaller than 0.9 mm in diameter, exposure to external ammonium causes an alkaline shift in intracellular pH as the NH3 enters and takes up H+ to form NH4+. We propose that chloride‐dependent NH4+ transport contributes to the accumulation of NH4+ and causes the ‘postexposure’ acidification as the intracellular NH4+ releases H+ to form NH3 which is then lost from the cell. 7. In larger oocytes ammonium exposure produces a rapid reduction in pHi which may be explained in part by cotransport‐mediated uptake of NH4+. Evidence is also provided for a second chloride‐dependent NH4+ transport mechanism and a chloride‐independent process.
Regulation of intracellular pH in pyramidal neurones from the rat hippocampus by Na(+)‐dependent Cl(‐)‐HCO3‐ exchange.
Authors: Schwiening C J , Boron W F
Published: December 2014

1. We have measured intracellular pH (pHi) in freshly isolated pyramidal neurones from the CA1 region of the rat hippocampus using the fluorescent indicator 2',7'‐bis(carboxy‐ethyl)‐5‐(and‐6)‐carboxyfluorescein (BCECF). 2. The neurones selected by our isolation procedure, when studied in the nominal absence of CO2‐HCO3‐, had a mean steady‐state pHi of 6.81 +/‐ 0.02 (n = 163). The recovery of pHi from acid loads was very slow. The rate of recovery from acid loads was reduced by Na+ removal, but only very slightly inhibited by 1 mM amiloride. 3. The addition of 5% CO2‐25 mM HCO3‐ caused steady‐state pHi to increase from 6.74 +/‐ 0.05 to 7.03 +/‐ 0.03 (n = 28). In the presence of 5% CO2‐25 mM HCO3‐, the rate of pHi recovery from acid loads was much faster than in its absence. 4. The HCO(3‐)‐induced alkalinization was reversible, and did not occur in the absence of extracellular Na+ or in the presence of DIDS (4,4'‐diisothiocyanatostilbene‐ 2,2'‐disulphonic acid). 5. In the absence of external Cl‐, successive exposures to CO2‐HCO3‐ elicited alkalinizations that were progressively reduced in rate and amplitude. This effect, presumably due to gradual depletion of internal Cl‐, was rapidly reversed by returning Cl‐ to the external medium. 6. We conclude that the major acid‐extrusion mechanism in pyramidal CA1 neurones is the Na(+)‐dependent Cl(‐)‐HCO3‐ exchanger. The Na(+)‐dependent mechanism that operates in the nominal absence of HCO3‐ is far less active.
ACTH response induced in capsaicin‐desensitized rats by intravenous injection of interleukin‐1 or prostaglandin E.
Authors: Watanabe T , Morimoto A , Tan N , Makisumi T , Shimada S G , Nakamori T , Murakami N
Published: December 2014

1. We investigated whether afferent nerves are involved in the development of adrenocorticotrophic hormone (ACTH) responses induced either by systemic administration of interleukin‐1 beta (IL‐1 beta) and prostaglandin E2, or by psychological stress. The capsaicin desensitization method was used to impair afferent C fibres and we compared the ACTH responses between capsaicin desensitized and vehicle pretreated control rats. 2. The present results showed that the capsaicin desensitized rats had significantly smaller increases in plasma ACTH than the control rats in response to intravenous injection of IL‐1 beta or prostaglandin E2. 3. There were no significant differences between the capsaicin desensitized and control rats in the ACTH responses induced by cage switch stress. 4. The capsaicin desensitized rats responded to intravenous injection of corticotrophin releasing factor (CRF) with a greater increase in the plasma level of ACTH than the control rats, indicating that capsaicin pretreatment resulted in augmentation of pituitary gland sensitivity to CRF. 5. These results suggest that afferent neurons play an important role in the ACTH responses induced by systemic injection of IL‐1 beta or prostaglandin E2.
Intrarenal haemodynamic and glomerular responses to inhibition of nitric oxide formation in rabbits.
Authors: Denton K M , Anderson W P
Published: December 2014

1. The renal effects of inhibiting nitric oxide (NO) formation using N‐nitro‐L‐arginine (NOLA, 20 mg kg‐1) were examined using micropuncture techniques in pentobarbitone‐anaesthetized rabbits. 2. Renal vascular resistance doubled from 2.7 +/‐ 0.5 to 5.0 +/‐ 1.1 mmHg ml‐1 min‐1 after NOLA (P < 0.01), with similar percentage increases in both pre‐ (149 +/‐ 38%, P < 0.01) and postglomerular (158 +/‐ 42%, P < 0.01) resistance. 3. Glomerular capillary pressure rose from 33 +/‐ 1 to 40 +/‐ 1 mmHg after NOLA (P < 0.01) but despite this, glomerular filtration rate (GFR) and single nephron glomerular filtration rate did not significantly change. 4. Blood pressure increased 18 +/‐ 1 mmHg (P < 0.001) within 10 min of NOLA administration and remained near this level for the next 90 min. 5. The glomerular ultrafiltration coefficient (Kf) decreased significantly from 0.085 +/‐ 0.022 to 0.035 +/‐ 0.006 nl s‐1 mmHg‐1 (P < 0.05). 6. Urine flow and sodium excretion increased markedly (26 +/‐ 9 to 337 +/‐ 102 microliters min‐1 and 5 +/‐ 2 to 342 +/‐ 12 mumol min‐1 respectively, (P < 0.001)) and sodium fractional excretion rose from 1.0 +/‐ 0.3 to 8.0 +/‐ 2.2% (P < 0.01). 7. Thus, administration of NOLA to rabbits caused vasoconstriction of both pre‐ and postglomerular vessels, diuresis and natriuresis without significant change in GFR, and a reduction in Kf. The results suggest that NO may play an important role in the regulation of renal haemodynamics and glomerular function.
Differential effects of length on maximum force production and myofibrillar ATPase activity in rat skinned cardiac muscle.
Authors: Kentish J C , Stienen G J
Published: December 2014

1. The fall of maximum Ca(2+)‐activated force of cardiac myofibrils at short muscle lengths could be due to a reduction of cross‐bridge cycling or to development of an opposing (restoring) force. To try to distinguish between these possibilities, we measured simultaneously myofibrillar force development and MgATPase activity (a measure of cross‐bridge cycling) in rat skinned trabeculae at different muscle lengths. ATPase activity was measured photometrically from the utilization of NADH in a coupled enzyme assay. Muscle length was varied to give estimated 0.2 micron changes in sarcomere length (SL) over the range 1.4‐2.4 microns. 2. Both Ca(2+)‐activated force development and ATPase activity were optimal at a muscle length (Lo) where the resting SL was 2.2 microns. At Lo the maximum ATPase activity at 21 degrees C was 0.56 +/‐ 0.05 mM s‐1 (mean +/‐ S.E.M., n = 6), which was equivalent to an ATP turnover per myosin S1 head of 3.3 s‐1. 3. The relationship between ATPase activity and SL was curved, with rather little change in ATPase activity over the SL range 2.0‐2.4 microns, but significant falls at 1.8 microns and below. At 65% of Lo (corresponding to a mean active SL of approximately 1.4 microns), the ATPase activity was only 50% of its value at 2.2 microns SL. 4. Force development decreased linearly as SL was reduced below 2.2 microns. Force fell by more than ATPase activity, particularly at SL 1.6 and 1.8 microns. 5. The fall of ATPase activity indicates that some of the decline of force production at short SL results from a fall in the net rate of cross‐bridge cycling. This is probably the result of double overlap of thin filaments. However, the differential effect on force and ATPase reveals that, in the intermediate range of SL, decreased cross‐bridge cycling can account for only part of the fall of force; the remainder is probably due to an increase in a restoring force, which may arise from deformation of the connective tissue in the muscle preparations used.
Effects of heterotopic noxious stimuli on activity of neurones in subnucleus reticularis dorsalis in the rat medulla.
Authors: Villanueva L , Bing Z , Le Bars D
Published: December 2014

1. In anaesthetized rats, recordings were made in the medullary subnucleus reticularis dorsalis. Neurones with total nociceptive convergence (TNC) responded to percutaneous electrical stimuli with early and late peaks due to the activation of A delta and C fibres respectively, no matter which part of the body was stimulated. Neurones with partial nociceptive convergence (PNC) responded with an A delta peak regardless of which part of the body was stimulated, and with a C peak of activation from some, mainly contralateral, parts of the body. 2. All TNC neurones responded to noxious thermal stimulation of the limbs with a phasic discharge followed by tonic activity that lasted throughout the stimulation. Discharges elicited by applying stimuli simultaneously to both forepaws or to a hindpaw and a forepaw were lower than the individual responses to stimulation of a single limb. Similar negative interactions were observed in the responses of PNC neurones following noxious thermal stimulation of two paws. 3. In both neuronal populations, the simultaneous application of noxious thermal stimuli and microelectrophoretic application of D,L‐homocysteic acid (DLH) induced responses of greater magnitude than those evoked by each stimulus alone. 4. TNC neurones responded to electrical stimulation of the contralateral hindpaw with A delta and C fibre responses. Noxious thermal stimuli applied to different areas of the body induced an excitatory response during the period that preceded the electrical stimulation, but an inhibition of both A delta and C fibre responses. By contrast, using a similar protocol, application of DLH induced a steady discharge in the period preceding the electrical stimulation and also in between the A delta and C fibre responses, which were not themselves inhibited. 5. The negative heterotopic influences in normal rats during simultaneous immersion of the ipsi‐ and contralateral hindpaws was strongly reduced in rats with bilateral lesions of the dorsolateral funiculi at the cervical level (C3‐C4). 6. It is concluded that strong negative interactions elicited by heterotopic noxious stimuli are observed in the spinoreticular system. Such interactions do not occur at the subnucleus reticularis dorsalis level but are mediated by supraspinal structures which control, through the dorsolateral funiculi, the spinal transmission of nociceptive information.
The intracellular Ca2+ transient and tension in frog skeletal muscle fibres measured with high temporal resolution.
Authors: Claflin D R , Morgan D L , Stephenson D G , Julian F J
Published: December 2014

1. The purpose of this study was to determine, with high temporal resolution, the relationship between the intracellular Ca2+ transient (ICT) and the mechanical responses of intact, single skeletal muscle fibres of frogs following stimulation by a single, brief depolarization. 2. The time course of the ICT was monitored using the Ca(2+)‐sensitive fluorescent dyes mag‐fura‐2 (furaptra) and mag‐fura‐5. The mag‐fura dyes have a low affinity for Ca2+ and have been shown to track the ICT with no appreciable kinetic delay. Continuous records of mag‐fura fluorescence, tension and stiffness responses were obtained simultaneously at high time resolution at a sarcomere length of 2.9 microns. Experimental temperature was 3 degrees C. 3. When a delay of 0.4 ms due to the low‐pass filter associated with the photodetector was included, the onset of the fluorescence response preceded the onset of latency relaxation (the small fall in tension that precedes positive tension generation) by 3.1 +/‐ 0.2 ms (mean +/‐ S.E.M., n = 8). After its onset, the mag‐fura fluorescence signal continued to change rapidly (indicating increasing intracellular [Ca2+]) to an extreme level that occurred 1.5 +/‐ 0.5 ms (mean +/‐ S.E.M., n = 7) before tension had recovered to its resting level following latency relaxation. The time delay from the extreme of the fluorescence signal to the peak of the tension signal was 239 +/‐ 27 ms (mean +/‐ S.E.M., n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)
Modulation of AMPA/kainate receptors in cultured murine hippocampal neurones by protein kinase C.
Authors: Wang L Y , Dudek E M , Browning M D , MacDonald J F
Published: December 2014

1. The patch clamp technique, together with intracellular perfusion of the catalytic fragment of protein kinase C (PKCM), was employed to investigate the role of this enzyme in the intracellular regulation of alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA)/kainate receptors in cultured hippocampal neurones. 2. The responses evoked by near‐maximal concentrations of kainate (250 microM) and AMPA (100 microM) were potentiated by the introduction of PKCM, whilst co‐application of the inhibitory peptide fragment PKCI(19‐36) prevented this action. 3. Modulation of kainate responses by PKCM was dependent upon the concentration of agonist applied. Currents evoked by kainate were potentiated at concentrations above those which caused 50% of the maximal response (EC50) and depressed at lower concentrations. Furthermore, okadaic acid, a specific inhibitor of phosphatases 1 and 2A, had a similar effect upon concentration‐response relationships when currents activated by kainate were recorded using the perforated patch technique. 4. In addition, the mean amplitude and/or time constant of decay of miniature excitatory synaptic currents (mediated by AMPA/kainate receptors) was increased by the intracellular injection of PKCM. 5. These observations suggest that the function of postsynaptic excitatory amino acid receptors can be modulated by the activity of PKC as well as by endogenous phosphatases. This regulation may contribute to some forms of synaptic plasticity within the central nervous system.
Relaxation in rabbit and rat cardiac cells: species‐dependent differences in cellular mechanisms.
Authors: Bassani J W , Bassani R A , Bers D M
Published: December 2014

The roles of the sarcoplasmic reticulum (SR) Ca(2+)‐ATPase and Na(+)‐Ca2+ exchange in Ca2+ removal from cytosol were compared in isolated rabbit and rat ventricular myocytes during caffeine contractures and electrically stimulated twitches. Cell shortening and intracellular calcium concentration ([Ca2+]i) were measured in indo‐1‐loaded cells. Na(+)‐Ca2+ exchange was inhibited by replacement of external Na+ by Li+. To avoid net changes in cell or SR Ca2+ load during a twitch in 0 Na+ solution, intracellular Na+ (Na+i) was depleted using a long pre‐perfusion with 0 Na+, 0 Ca2+ solution. SR Ca2+ accumulation was inhibited by caffeine or thapsigargin (TG). Relaxation of steady‐state twitches was 2‐fold faster in rat than in rabbit (before and after Na+i depletion). In contrast, caffeine contractures (where SR Ca2+ accumulation is inhibited), relaxed faster in rabbit cells. Removal of external Na+ increased the half‐time for relaxation of caffeine contractures 15‐ and 5‐fold in rabbit and rat myocytes respectively (and increased contracture amplitude in rabbit cells only). The time course of relaxation in 0 Na+, 0 Ca2+ solution was similar in the two species. Inhibition of the Na(+)‐Ca2+ exchange during a twitch increased the [Ca2+]i transient amplitude (delta[Ca2+]i) by 50% and the time constant of [Ca2+]i decline (tau) by 45% in rabbit myocytes. A smaller increase in tau (20%) and no change in delta[Ca2+]i were observed in rat cells in 0 Na+ solution. [Ca2+]i transients remained more rapid in rat cells. Inhibition of the SR Ca(2+)‐ATPase during a twitch enhanced delta[Ca2+]i by 25% in both species. The increase in tau after TG exposure was greater in rat (9‐fold) than in rabbit myocytes (2‐fold), which caused [Ca2+]i decline to be 70% slower in rat compared with rabbit cells. The time course of [Ca2+]i decline during twitch in TG‐treated cells was similar to that during caffeine application in control cells. Combined inhibition of these Ca2+ transport systems markedly slowed the time course of [Ca2+]i decline, so that tau was virtually the same in both species and comparable to that during caffeine application in 0 Na+, 0 Ca2+ solution. Thus, the combined participation of slow Ca2+ transport mechanisms (mitochondrial Ca2+ uptake and sarcolemmal Ca(2+)‐ATPase) is similar in these species. We conclude that during the decline of the [Ca2+]i transient, the Na(+)‐Ca2+ exchange is about 2‐ to 3‐fold faster in rabbit than in rat, whereas the SR Ca(2+)‐ATPase is 2‐ to 3‐fold faster in the rat.(ABSTRACT TRUNCATED AT 400 WORDS)
Relaxation in ferret ventricular myocytes: unusual interplay among calcium transport systems.
Authors: Bassani R A , Bassani J W , Bers D M
Published: December 2014

Transport systems responsible for removing Ca2+ from the myoplasm during relaxation in isolated ferret ventricular myocytes were studied using caffeine‐induced contractures. Internal calcium concentration ([Ca2+]i) was measured with the fluorescent calcium indicator indo‐1, and the results were compared with our recent detailed characterizations in rabbit and rat myocytes. Relaxation and [Ca2+]i decline during a twitch in ferret myocytes were fast and similar to that in rat myocytes (i.e. half‐time, t 1/2 approximately 100‐160 ms). During a caffeine‐induced contracture (SR Ca2+ accumulation prevented), relaxation was still relatively fast (t 1/2 = 0.57 s) and similar to relaxation in rabbit supported mainly by a strong Na(+)‐Ca2+ exchange. When both the SR Ca2+ uptake and Na(+)‐Ca2+ exchange are blocked (by caffeine and 0 Na+, 0 Ca2+ solution) relaxation in the ferret myocyte is remarkably fast (approximately 5‐fold) compared with rabbit and rat myocytes. The decline of the Cai2+ transient was also fast under these conditions. These values were similar to those in rat under conditions where relaxation is due primarily to Na(+)‐Ca2+ exchange. Additional inhibition of either the sarcolemmal Ca(2+)‐ATPase or mitochondrial Ca2+ uptake caused only modest slowing of the relaxation of caffeine‐induced contracture in 0 Na+, 0 Ca2+ (t 1/2 increased to approximately 3 s). In rabbit myocytes the relaxation t 1/2 is slowed to 20‐30 s by these procedures. Even when the systems responsible for slow relaxation in rabbit ventricular myocytes are inhibited (i.e. sarcolemmal Ca(2+)‐ATPase and mitochondrial Ca2+ uptake) along with the SR Ca(2+)‐ATPase and Na(+)‐Ca2+ exchange, relaxation and [Ca2+]i decline in ferret myocytes remain rapid compared with rabbit myocytes. Ca2+ taken up by mitochondria in rabbit myocytes during a caffeine contracture in 0 Na+, 0 Ca2+ solution gradually returns to the SR after caffeine removal, but this component appears to be much smaller in ferret myocytes under the same conditions. We tested for possible residual Ca2+ transport by each of the four systems which suffice to explain Ca2+ removal from the cytoplasm in rabbit (SR Ca(2+)‐ATPase, Na(+)‐Ca2+ exchange, sarcolemmal Ca(2+)‐ATPase and mitochondrial Ca2+ uptake). We conclude that there is an additional calcium transport system at work in ferret myocytes. For this additional system, our results are most compatible with a trans‐sarcolemmal Ca2+ transport, but neither a cation exchanger nor a Ca(2+)‐ATPase with characteristics like that in other cardiac cells. This additional system appears able to transport Ca2+ nearly as fast as the Na(+)‐Ca2+ exchange in rat ventricular myocytes.
Effect of raised potassium on ventilation in euoxia, hypoxia and hyperoxia at rest and during light exercise in man.
Authors: Qayyum M S , Barlow C W , O'Connor D F , Paterson D J , Robbins P A
Published: December 2014

The purpose of this study was to determine whether changes in arterial plasma potassium concentration [K+]a affect expired ventilation (VE) in euoxia, hypoxia and hyperoxia during rest and light exercise in humans. Three periods of ventilatory measurements were undertaken in eight healthy subjects at rest and in seven other subjects during cycle ergometry (70 W). The first period of measurement was before the ingestion of 64 mmol of potassium chloride (KCl), the second 20 min after ingestion of KCl when [K+]a levels were elevated, and the third 3 h after the ingestion of KCl when [K+]a had returned substantially to normal. During each period, end‐tidal PO2 was cycled between euoxia, hypoxia and hyperoxia, whilst the end‐tidal PCO2 was maintained constant. The acute ventilatory response to hypoxia (AHVR) was calculated as the difference in VE during hypoxia and hyperoxia within each period of measurement. Oral KCl produced a 1.3 +/‐ 0.2 mM (mean +/‐ S.E.M.) increase in [K+]a at rest and a 0.8 +/‐ 0.2 mM increase during exercise. There was no significant difference in ventilation during euoxia between the three periods of measurement at rest or during exercise. There was a significant increase in AHVR with the rise in [K+]a of 21 min‐1 mM‐1 at rest (arterial PO2 during hypoxia ca 57 Torr) and 10 l min‐1 mM‐1 during exercise (arterial PO2 during hypoxia ca 52 Torr). There was a significant difference in the absolute increase in AHVR with [K+]a between rest and exercise, but this difference was not significant if the increase in AHVR with [K+]a was expressed as a percentage of the initial AHVR. We conclude that changes in [K+]a of the order of 1 mM have little effect on euoxic ventilation at rest or during light exercise in humans. We also conclude that [K+]a changes of this order increase AHVR at rest and during light exercise and that increases in [K+]a contribute to the increase in AHVR with exercise in humans.
Mechanism of force inhibition by 2,3‐butanedione monoxime in rat cardiac muscle: roles of [Ca2+]i and cross‐bridge kinetics.
Authors: Backx P H , Gao W D , Azan‐Backx M D , Marban E
Published: December 2014

1. We investigated the mechanism of force inhibition by 2,3‐butanedione monoxime (BDM) on rat cardiac trabeculae. [Ca2+]i was measured by iontophoretic injection of fura‐2 salt. Isometric force was recorded at an end‐systolic sarcomere length of 2.1‐2.2 microns. 2. With an external [Ca2+] of 1 mM, peak twitch force was monotonically reduced with increasing [BMD]; at 5 and 20 mM [BDM], force was 35 and 1% of the control force. In contrast, the mean peak [Ca2+]i during transients was only reduced at [BDM] > or = 10 mM. 3. The duration of the twitch was dramatically reduced by BDM in a dose‐dependent fashion with no significant change in the time course of the underlying Ca2+ transients. The abbreviation of twitch force duration was much greater than expected for the observed reduction in peak force by this agent. 4. The mechanism of the inhibition of force by BDM was explored by examining the relationship between twitch force and Ca2+ transients at various values of external [Ca2+]. In the presence of BDM, the steepness of the relationship between peak force and peak [Ca2+]i was reduced compared to control conditions. As a result, significant elevation in the [Ca2+]i transient was unable to reverse the reduction in force observed in the presence of BDM. 5. The direct inhibitory effects of BDM on the contractile system were examined using ryanodine tetani in intact trabeculae to measure the steady‐state force‐[Ca2+]i relationship. In contrast to the effects on twitch force at 5 mM BDM, maximal force was only reduced to 71% of control. Furthermore, the [Ca2+]i required for half‐maximal activation (Ca50) was increased while the Hill coefficient was reduced slightly by BDM. 6. BDM dramatically slowed the rate of rise of tetanic force. At maximal activation, the time required to reach 90% maximal force was prolonged by a factor of 3‐8 in the presence of 5 mM BDM. This suggests that the observed reduction in twitch force and steady‐state force may result from slowed kinetics of cross‐bridge attachment, consistent with recent biochemical studies. 7. The contribution of altered cross‐bridge kinetics to the effects of BDM was investigated using a co‐operative cross‐bridge model of the contractile system. Changing the rate constants for cross‐bridge attachment in the model to mimic the reported biochemical effects of BDM reproduced the observed effects of BDM.(ABSTRACT TRUNCATED AT 400 WORDS)
Epidermal growth factor as a diuretic in sheep.
Authors: Gow C B , Phillips P A
Published: December 2014

1. Epidermal growth factor (EGF) has recently been shown to stimulate both polydipsia and polyuria and the aim of this study was to determine which was the primary response. Ewes received a continuous intravenous saline infusion (100 ml day‐1) for 12 days (days 1‐12) and EGF at doses of 0 (n = 6) or 10 micrograms h‐1 (n = 6) over days 5‐8. The supply of water was ad libitum during days 1‐4 and 9‐12, but was fixed at the pretreatment mean of days 1‐4 for each ewe during days 5‐8. 2. During the period of fixed water intake, the EGF‐treated ewes experienced mild dehydration with elevated plasma osmolality, sodium, renin and arginine vasopressin (AVP) concentrations and slightly reduced plasma atrial natriuretic peptide (ANP) concentrations. 3. When the supply of water returned to ad libitum, the EGF‐treated ewes increased their water intake by 105% (5.25 +/‐ 0.28 vs. 2.55 +/‐ 0.19 l day‐1) and subsequently fluid balance was restored; plasma electrolyte and hormone responses also returned to normal. 4. This experiment demonstrates that EGF infused at a dose rate of 10 micrograms h‐1 I.V. into sheep has a direct renal diuretic effect.
Synchronization of inhibitory neurones in the guinea‐pig hippocampus in vitro.
Authors: Michelson H B , Wong R K
Published: December 2014

1. Intracellular recordings were obtained from pyramidal, granule and hilar cells in transverse slices of guinea‐pig hippocampus to examine synaptic interactions between GABAergic neurones. 2. In the presence of the convulsant compound 4‐aminopyridine (4‐AP), after fast excitatory amino acid (EAA) neurotransmission was blocked pharmacologically, large amplitude inhibitory postsynaptic potentials (IPSPs) occurred rhythmically (every 4‐8 s) and synchronously in all principal cell populations (triphasic synchronized IPSPs). In the presence of the GABAA receptor blocker picrotoxin (PTX), a large amplitude IPSP continued to occur spontaneously in all principal cells simultaneously (monophasic synchronized IPSP). 3. Burst firing occurred simultaneously in a group of hilar neurones (synchronized bursting neurones) coincident with triphasic synchronized IPSPs in principal cells. After PTX was added, the bursts and the underlying depolarizing synaptic potentials were completely suppressed in some of the synchronized bursting neurones (type I hilar neurones), while others (type II hilar neurones) continued to fire in bursts coincident with monophasic synchronized IPSPs in principal cells. Intense hyperpolarization blocked burst firing and revealed underlying attenuated spikes of less than 10 mV, but did not uncover any underlying depolarizing synaptic potentials. 4. In type II hilar neurones, during sufficient hyperpolarization, spontaneous activity consisted of attenuated spikes. With depolarization, the small spikes began to trigger full size action potentials. These data suggest the presence of electrotonically remote spike initiation sites. 5. The morphology of synchronized bursting neurones was revealed by intracellular injection of the fluorescent dye Lucifer Yellow. Attempts to inject dye into one type II hilar neurone often resulted in the labelling of two to four cells (dye coupling). Dye coupling was not observed in type I hilar neurones. 6. These findings indicate that excitatory interactions synchronizing the firing of GABAergic neurones can occur in the absence of fast EAA neurotransmission. GABAergic neurones can become synchronized via their recurrent collaterals through the depolarizing action of synaptically activated GABAA receptors. In addition, a subpopulation of GABAergic neurones can become synchronized by a mechanism probably involving electrotonic coupling.
Role of wall tension in the vasoconstrictor response of cannulated rat mesenteric small arteries.
Authors: VanBavel E , Mulvany M J
Published: December 2014

1. We have studied the influence of mechanical loading conditions on the responses of cannulated rat mesenteric small arteries to noradrenaline, vasopressin and potassium. 2. The cross‐sectional area (CSA) of vessels was continuously monitored. Isometric loading (CSA‐controlled conditions) or isobaric loading (pressure‐controlled conditions) was achieved by feedback adjustment of the distending pressure. 3. Noradrenaline (0.3 microM) and vasopressin (0.05 u l‐1) induced myogenic responsiveness, resulting in a constant or declining CSA with increasing pressure. Potassium (32 mM) induced weak myogenic responsiveness. 4. At a constant pressure of 60 cmH2O, noradrenaline and vasopressin concentration‐response curves were graded, the concentration‐response curves of individual vessels being extended over two to three decades. Sensitivity to the vasoconstrictors, expressed as pD2 values (‐log10 EC50), averaged 6.45 +/‐ 0.18 log M and 1.27 +/‐ 0.20 log u l‐1 for the noradrenaline and vasopressin concentration‐response curves respectively. The isobaric pD2 for K+ was 1.54 +/‐ 0.07 log M. 5. During CSA‐controlled conditions, noradrenaline and vasopressin induced all‐or‐none responses to stretch. Potassium induced graded responses to stretch. 6. During CSA‐controlled conditions, noradrenaline and vasopressin concentration‐response curves also showed all‐or‐none behaviour. Almost the full response occurred through only a doubling of the concentration. pD2 values were 6.88 +/‐ 0.38 log M (noradrenaline) and 1.87 +/‐ 0.43 log u l‐1 (vasopressin). Isometric vessels were significantly more sensitive to noradrenaline and vasopressin than isobaric vessels. Isometric K+ curves were gradual. pD2 was 1.54 +/‐ 0.07 log M, a value not different from the isobaric value. 7. These findings can be explained by assuming that agonist sensitivity is wall tension dependent, such that sensitivity increases with increasing wall tension. This concept accounts for partial regulation of wall tension during pressure‐controlled conditions, as well as instability due to a positive feedback loop of active tension development and tension‐induced sensitization during CSA‐controlled conditions.