bluuebunny/crossref_metadata_2025 huggingface dataset:
Rate‐limiting energy‐dependent steps controlling oxidative metabolism‐contraction coupling in rabbit aorta.
Authors: Coburn R F , Moreland S , Moreland R S , Baron C B
Published: December 2014

1. We tested the hypotheses that coupling between oxidative metabolism and force in noradrenaline (NOR)‐activated rabbit aorta is controlled (a) by an energy‐dependent step or steps in receptor‐operated coupling mechanisms upstream to myosin light chain (MLC) kinase, or (b) by energy limitation of MLC kinase‐mediated phosphorylation of the MLC or actin‐activated myosin ATPase. 2. Oxidative energy production was rapidly inhibited by decreasing organ bath PO2 to less than 30 mmHg. There was no difference, comparing KCl‐ or NOR‐induced force, in the rates of decrease of [PCr] (phosphocreatine) or [ATP] following inhibition of oxidative energy production. (In this report we use the term [PCr] and [ATP] to indicate mean tissue values). Initial rates of decrease in [PCr] and [ATP] following inhibition of oxidative energy production were 0.05 mM/min and 0.06 mM/min, respectively. 3. Despite similar decreases in mean tissue [PCr] and [ATP], relaxations of KCl‐induced contractions following inhibition of oxidative energy production were markedly delayed and were blunted compared to relaxations seen during NOR‐induced contractions. The threshold mean tissue [PCr] and [ATP] for relaxation during KCl stimulation were 0.25 and 0.60 to 0.80 mM, respectively. During NOR stimulation, threshold values of [PCr] and [ATP] were 0.50 mM and 0.80 mM, respectively. Mean tissue [PCr] and [ATP] levels at 50% relaxation of KCl‐induced force were less than 0.1 mM and 0.1 mM, respectively. Fifty per cent relaxation of NOR‐induced force occurred at [PCr] and [ATP] values of 0.35 mM and 0.65 mM, respectively. 4. MLC phosphorylation levels decreased during relaxation of NOR force evoked by inhibition of oxidative energy production. There was no change in the level of MLC phosphorylation following inhibition of oxidative energy production in KCl‐contracted muscle even at mean tissue [PCr] and [ATP] lower than values associated with decreases in MLC phosphorylation during relaxations of NOR‐induced force. 5. Oxygen‐induced redevelopment of force during NOR exposure was not dependent on extracellular Ca2+. Mean tissue [PCr] increased prior to onset of O2‐evoked force redevelopment. Increases in MLC phosphorylation were seen at the time of onset of force redevelopment. 6. Oxidative metabolism‐contraction coupling during NOR‐stimulation seems not to be due to energy limitation of the MLC kinase reaction or actin‐activated myosin ATPase. Data suggest the rate‐limiting step is an energy‐dependent reaction in receptor‐operated coupling mechanisms upstream to MLC kinase.(ABSTRACT TRUNCATED AT 400 WORDS)
Respiratory muscle recruitment during selective central and peripheral chemoreceptor stimulation in awake dogs.
Authors: Saupe K W , Smith C A , Henderson K S , Dempsey J A
Published: December 2014

1. In four awake dogs we measured EMG activity of three inspiratory and four expiratory muscles during sustained central chemoreceptor stimulation (CO2 inhalation), and peripheral chemoreceptor stimulation (intravenous infusion of almitrine bismesylate (almitrine)). By using this selective pharmacological stimulation of the peripheral chemoreceptors and reversibly cold‐blocking pulmonary stretch receptors, we were able to determine the effects of each type of stimulation on respiratory muscle recruitment in the absence of such complicating influences as pulmonary stretch receptor feedback, cerebral hypoxia or hypocapnia, and differences in breathing pattern. 2. During 10 min of steady‐state hyperpnoea (minute ventilation VI, approximately twice eupnoea) caused by either hypercapnia or isocapnic stimulation of the carotid bodies with almitrine, all three inspiratory and all four expiratory muscles demonstrated significant and sustained elevations in EMG activity. 3. With both types of chemoreceptor stimulation, as tidal volume, VT, increased, so did the mean electrical activities of the crural diaphragm (r = 0.88), costal diaphragm (r = 0.93), parasternals (r = 0.82), triangularis sterni (r = 0.74), transversus abdominis (r = 0.77), external obliques (r = 0.68) and internal intercostals (r = 0.75). 4. In each dog, the response of ventilation and of the diaphragmatic EMG to a given level of central or peripheral chemoreceptor stimulation is highly reproducible from one test day to the next. On the other hand, accessory inspiratory and expiratory abdominal and rib cage muscles in two of the four dogs showed highly significant changes from day to day in the amount of their EMG activity at any given VT. 5. During steady‐state ventilatory stimulation, 2 min intervals were chosen during which the two types of chemoreceptor stimulation had caused hyperpnoeas with similar values for VT, total time per breath (TTOT) and inspiratory time divided by the total time (TI/TTOT). Comparison of EMG activities during these matched hyperpnoeas revealed that there were no differences in the activities of any of the muscles between the two forms of stimulation. We conclude that peripheral chemoreceptor stimulation causes significant and sustained recruitment of expiratory muscles even in the absence of pulmonary feedback and that both expiratory and inspiratory muscles are recruited to the same extent during peripheral chemoreceptor stimulation as they are during an identical hyperpnoea caused by central chemoreceptor stimulation.
Cyclic AMP‐and beta‐agonist‐activated chloride conductance of a toad skin epithelium.
Authors: Willumsen N J , Vestergaard L , Larsen E H
Published: December 2014

1. The control by intracellular cyclic AMP and beta‐adrenergic stimulation of chloride conductance was studied in toad skin epithelium mounted in a chamber on the stage of an upright microscope. Impalement of identified principal cells from the serosal side with single‐barrelled conventional or double‐barrelled Cl(‐)‐sensitive microelectrodes was performed at x500 magnification. For blocking the active sodium current 50 microM‐amiloride was present in the mucosal bath. 2. When clamped at transepithelial potential difference V = 0 mV, the preparations generated clamping currents of 0.9 +/‐ 1 microA/cm2 (mean +/‐ S.E.M.; number of observations n = 55). The intracellular potential of principal cells (Vb) was ‐96 +/‐ 2 mV with a fractional resistance of the basolateral membrane (fRb) of 0.016 +/‐ 0.003 (n = 54), and an intracellular Cl‐ activity of 40 +/‐ 2 mM (n = 24). 3. At V = 0 mV, serosal application of a cyclic AMP analogue, dibutyryl cyclic AMP (500 microM) or a beta‐adrenergic agonist, isoprenaline (5 microM) resulted in a sixfold increase in transepithelial Cl‐ conductance identified by standard 36Cl‐ tracer technique. 4. The clamping current at V = 0 mV was unaffected by cyclic AMP (short‐circuit current Isc = 0.1 +/‐ 0.3 microA/cm2, n = 16) indicating that subepidermal Cl(‐)‐secreting glands are not functioning in our preparations obtained by collagenase treatment. 5. Cyclic AMP‐ or isoprenaline‐induced chloride conductance (Gcl) activation (V = 0 mV) was not reflected in membrane potential and intracellular Cl‐ activity in principal cells. Intracellular chloride activity was constant at approximately 40 mM at membrane potentials between ‐90 and ‐100 mV. Therefore, it can be concluded that the principal cells are not contributing to activated Cl‐ currents. 6. At V = ‐100 mV where the voltage‐dependent chloride conductance of mitochondria‐rich (MR) cells was already fully activated, GCl was unaffected by cyclic AMP or isoprenaline. The major effect of these treatments was a rightward displacement of the MR cell‐generated GCl‐V relationship along the V axis. 7. Our results indicate that the beta‐adrenergically controlled cyclic AMP‐mediated chloride conductance is localized to the mitochondria‐rich cells.
Single C nociceptor responses and psychophysical parameters of evoked pain: effect of rate of rise of heat stimuli in humans.
Authors: Yarnitsky D , Simone D A , Dotson R M , Cline M A , Ochoa J L
Published: December 2014

1. Effects of rate of rise of temperature stimuli applied to skin on (i) unitary receptor threshold and frequency response often single C nociceptors, and (ii) on magnitude and reaction times of evoked pain were studied in fifteen healthy human volunteers. 2. Temperature ramps of 32 to 45 or 47 degrees C were applied at three consistent rates of rise to receptive fields of C nociceptors in dorsum of foot (n = 9) or hand (n = 1). For rates of rise of 0.3, 2.0 and 6.0 degrees C/s, mean receptor threshold for heat was remarkably uniform: 41.5 +/‐ 0.57, 41.5 +/‐ 0.61 and 41.9 +/‐ 0.71 degrees C respectively. 3. The mean discharge rate of the ten cutaneous C nociceptors increased with rate of rise of temperature stimuli: 1.22 +/‐ 0.13, 4.57 +/‐ 0.49 and 13.45 +/‐ 0.71 impulses/s, respectively, for stimulus temperature rates of 0.3, 2.0 and 6.0 degrees C/s. 4. Magnitude estimates of pain for thirteen subjects also increased with rate of rise of temperature stimuli. Mean normalized magnitude estimates of heat pain were: 11.8 +/‐ 1.55, 15.1 +/‐ 0.84 and 28.0 +/‐ 1.87 for stimulus rates of rise of 0.3, 2.0 and 6.0 degrees C/s, respectively. 5. Results of simultaneous recordings of reaction time for pain and of C nociceptor responses to heat ramps given at 2.0 degrees C/s, in three subjects, indicate that under those circumstances heat pain messages are exclusively mediated by C nociceptors.
The minimal inhibitory synaptic currents evoked in neonatal rat motoneurones.
Authors: Takahashi T
Published: December 2014

1. Tight‐seal whole‐cell recordings were made from lumbar motoneurones visually identified in thin slices of neonatal rat spinal cord. The inhibitory postsynaptic currents (IPSCs) were evoked by extracellular stimulation of a neighbouring internuncial neurone in the presence of glutamate receptor antagonists. 2. Glycinergic IPSCs were recorded in the presence of bicuculline. The IPSCs appeared in an all‐or‐none manner as the graded stimulus intensity exceeded a certain threshold. Their latencies showed a unimodal distribution with a mean of 0.81 ms at 37 degrees C. Thus, the observed IPSCs are suggested to be monosynaptically evoked unitary IPSCs. The mean conductance of unitary IPSCs was 2.9 +/‐ 1.2 nS (+/‐ S.D.). 3. When the external Ca2+ concentration ([Ca2+]o) was reduced, the number of failures in response to stimulation increased, thereby reducing the mean amplitude of IPSCs. The mean amplitude of IPSCs was linearly related to the [Ca2+]o (0.35‐1.4 mM) with a mean slope of 3.1 +/‐ 0.67 on double logarithmic co‐ordinates. 4. The amplitude of individual IPSCs decreased with decrease in [Ca2+]o. However, below 0.7 mM [Ca2+]o, the mean amplitude of IPSCs (excluding failures) reached a steady minimum level. The mean conductance of these IPSCs measured in 0.5 mM [Ca2+]o was 657 +/‐ 281 pS. 5. The minimal IPSCs had a coefficient of variation of 0.50 +/‐ 0.13. No clear correlation was observed between the rise time and the amplitude of minimal IPSCs evoked in individual motoneurones, indicating that the amplitude variability is not due to the different synaptic locations. 6. Spontaneous miniature IPSCs were recorded from motoneurones in the presence of tetrodotoxin. The miniature IPSCs had a mean conductance of 739 +/‐ 278 pS, being comparable to the minimal evoked IPSCs. 7. Under various internal and external Cl‐ concentration, the reversal potential of the IPSCs (EIPSC) approximately coincided with the Cl‐ equilibrium potential. A 730‐fold change in the potassium concentration gradient across the membrane did not affect the EIPSC. The permeability ratio of K+ to Cl‐ (Pk/PCl) was less than 0.05. 8. It is concluded that the IPSCs are carried almost exclusively by Cl‐ and that the minimal evoked IPSCs represent the quantal response of the transmitter.
Synergism in the autonomic regulation of parotid secretion of protein in sheep.
Authors: Edwards A V , Titchen D A
Published: December 2014

1. Stimulation of the cervical sympathetic nerve continuously at 2 Hz, or in bursts of 1 s at 20 Hz every 10 s, for 5 min doubled the resting flow of parotid secretion in anaesthetized sheep and increased its concentration of protein. It also caused a significant increase in the concentration of potassium and decreased that of sodium: these latter effects were linearly inversely related. 2. Stimulation of the parotid nerves (the parasympathetic innervation) continuously at 2 Hz increased the flow of parotid secretion over 15‐fold which was significantly more than that which occurred in response to stimulation in bursts at 20 Hz. However, the latter pattern of stimulation significantly increased the concentration of protein in the saliva, which was not altered by stimulation at 2 Hz continuously. 3. Simultaneous stimulation of both the parotid and sympathetic nerves at 20 Hz in bursts substantially increased the output of protein from the gland, above that recorded in response to stimulation of either alone, without potentiating either the volume of secretion produced or the outputs of sodium or potassium. 4. The output of protein was also potentiated when both nerves were stimulated simultaneously at 2 Hz continuously but this pattern of stimulation elicited the secretion of a significantly smaller amount of protein. 5. All parotid responses to stimulation of the parotid nerves, either continuously at 2 Hz or in bursts at 20 Hz, were abolished by atropine (greater than or equal to 0.5 mg kg‐1 I.V.), as was potentiation of protein secretion during combined stimulation with the sympathetic innervation. 6. Stimulation of the parotid nerves in bursts at a higher frequency (60 Hz for 1 s at 20 s intervals) elicited a sparse flow of parotid saliva in atropinized animals without significant increase in protein output, with or without simultaneous stimulation of the sympathetic innervation. 7. The results of these experiments show that secretion of protein by the parotid gland, in response to stimulation of the parasympathetic innervation, depends on the pattern of stimulation and is potentiated by concomitant stimulation of the sympathetic innervation in anaesthetized sheep.
Alpha‐melanocyte‐stimulating hormone suppresses fever and increases in plasma levels of prostaglandin E2 in the rabbit.
Authors: Davidson J , Milton A S , Rotondo D
Published: December 2014

1. The effect of alpha‐melanocyte‐stimulating hormone (alpha‐MSH) on changes in body temperature and plasma levels of prostaglandin E2 (PGE2) were measured in the rabbit following intravenous injection of bacterial lipopolysaccharide (LPS), rabbit endogenous pyrogen (EP), human recombinant tumour necrosis factor‐alpha (TNF‐alpha), human recombinant interleukin‐1 beta (IL‐1 beta) and intracerebroventricular injection of PGE2. 2. LPS (25 ng kg‐1), EP (25 microliters kg‐1), TNF‐alpha (11 micrograms kg‐1) and IL‐1 beta (5 ng kg‐1) produced increases in body temperature simultaneously with increases in plasma PGE2 levels. alpha‐MSH (5 or 10 micrograms kg‐1) attenuated both the increase in body temperature and increases in plasma levels of PGE2. 3. Intracerebroventricular injection of PGE2 (500 ng) produced a monophasic increase in body temperature. alpha‐MSH (5 micrograms kg‐1) administered 20 min after PGE2 had no effect on the hyperthermic response. 4. alpha‐MSH (10 micrograms kg‐1) had no effect on either body temperature or plasma levels of PGE2 in response to I.V. injection of sterile saline. 5. These data demonstrate that alpha‐MSH inhibits both the pyrogenic actions of LPS, EP, TNF‐alpha and IL‐1 beta and their ability to increase PGE2 release without affecting the direct actions of PGE2, suggesting the possibility that alpha‐MSH may prevent the synthesis of PGE2 either by preventing the actions or release of mediators such as TNF‐alpha and IL‐1 in response to LPS.
Properties of spontaneous inward currents recorded in smooth muscle cells isolated from the rabbit portal vein.
Authors: Wang Q , Hogg R C , Large W A
Published: December 2014

1. Characteristics of spontaneous transient inward currents (STICs) which produced membrane depolarization were analysed with the perforated patch technique in single smooth muscle cells isolated from the rabbit portal vein. 2. In K(+)‐free solutions the amplitude of STICs was linearly related to membrane potential and the reversal potential (Er) was ‐3.0 +/‐ 0.9 mV. Replacement of external NaCl with NaI shifted Er to ‐40.0 +/‐ 1.0 mV. Substitution of external NaCl by NaSCN also moved Er to negative values but replacement of sodium with Tris and choline did not change Er. It is concluded that STICs are generated by an increase in chloride conductance. 3. STICs were abolished or reduced by the chloride channel antagonists anthracene‐9‐carboxylic acid (1 mM) and 4‐acetamido‐4'‐isothiocyanato‐2,2'‐ stilbene‐disulphonic acid (2 mM). 4. STICs were blocked by noradrenaline and caffeine which deplete intracellular calcium stores. This effect was reversible and this result indicates that the primary trigger for STICs is calcium released from intracellular stores and therefore STICs are calcium‐activated chloride currents (ICl(Ca)). 5. Removal of calcium from the bathing solution abolished STICs in six out of seven cells but STICs persisted in Ca(2+)‐free solution in one cell. When STICs were abolished in Ca(2+)‐free external solution the size of the internal calcium store, as estimated from the noradrenaline‐induced ICl(Ca), was not altered. It appears that an influx of calcium is usually necessary for STICs to be observed. 6. The frequency and amplitude of STICs were not altered by the voltage‐dependent calcium channel antagonist cadmium (1 mM). However, in some quiescent cells influx of calcium through voltage‐dependent channels did activate STICs. 7. It was concluded that in isolated portal vein cells STICs represent a Ca(2+)‐activated chloride current which leads to spontaneous depolarization of the membrane and may play an important physiological or pathophysiological role to produce smooth muscle contraction.
Membrane recapture and early triggered secretion from the newly formed endocytotic compartment in bovine chromaffin cells.
Authors: von Grafenstein H , Knight D E
Published: December 2014

1. Recycling of secretory vesicles in cultured bovine adrenal medullary cells was investigated. 2. Extracellular horseradish peroxidase (HRP), a fluid phase marker, was taken up into cultured adrenal medullary cells following carbamylcholine‐induced secretion of catecholamine. 3. The endocytosed HRP remained compartmentalized within the cell, migrating to a low density band on a Percoll density gradient. The endocytotic compartment was distinct from the major pool of catecholamine‐containing chromaffin granules, which were found at much higher densities on the Percoll gradient. 4. The chromaffin granule membrane marker dopamine beta‐hydroxylase was associated with the endocytosed HRP compartment as well as with the heavier chromaffin granules. 5. A subsequent challenge of the cells with carbamylcholine triggered the release of up to forty per cent of the endocytosed HRP. 6. The time course for secretion of the fluid phase marker was similar to that for catecholamine secretion. 7. Triggered release of HRP was dependent on extracellular calcium. The dependence on the extracellular calcium concentration was similar to that of catecholamine release. 8. Release of HRP could be triggered from electropermeabilized cells by raising the intracellular Ca2+ into the micromolar range. The intracellular Ca2+ dependence of triggered HRP release was similar to that for catecholamine release. 9. HRP could be secreted as early as 5 min, and as late as 2 h after endocytosis. 10. These data provide evidence that endocytotic vesicles can rapidly re‐enter the secretory cycle. Endocytosed vesicles may therefore not have to recycle via the trans‐Golgi reticulum to form high‐density chromaffin granules in order to re‐enter the regulated secretory pathway.
Uncertainty effects in orientation discrimination of foveally seen lines in human observers.
Authors: Lindblom B , Westheimer G
Published: December 2014

1. The effect of spatial uncertainty on line orientation thresholds was studied in normal observers. Vertical lines, 5 min of arc long, built up a matrix in which one of the line elements could be tilted to the left or right. The orientation thresholds depended strongly on the number of alternative test positions. There was a linear relation between log (threshold) and log (P), where P is the probability that a particular line element was the one being tested. 2. The uncertainty effect was shown to be time dependent. The effect was more marked for the shortest stimulus duration (1 s). However, even with a 6 s stimulus duration, allowing several re‐fixations, the thresholds were significantly higher in the presence of uncertainty, compared to the situation in which the test position was fixed and known to the observer. 3. When the measurements were restricted to the centre line in a matrix, thresholds were more than twice as high when the test line could be in any of the centre 3 x 3 positions, compared to the case in which there was no uncertainty as to the test position. Foreknowledge of location of the test line within the matrix improved the threshold further, even if the whole matrix was displaced to different retinal positions. 4. It is concluded that the physiological mechanism mediating threshold improvement probably operates on a cortical processing apparatus more central than V1.
Markov modelling of ensemble current relaxations: bovine adrenal nicotinic receptor currents analysed.
Authors: Maconochie D J , Knight D E
Published: December 2014

1. A general approach to the analysis of ensemble currents of ligand‐gated channels is presented, with a variety of examples that include single and multiple agonist binding steps, desensitization and several blocking pathways. 2. The use of matrix methods to describe model reaction schemes leads to a simplification if the reaction scheme is irreversible: the product of the exponential relaxation rate constants is exactly equal to the product of the forward reaction steps. 3. This method of analysis applied to the bovine adrenal nicotinic receptor suggests that in the range of acetylcholine concentrations from 1 microM to 2 mM, a model with a single kinetically relevant agonist binding step is appropriate. 4. Complex models, to explain the presence of two desensitizing components in currents recorded from whole cells, may be discounted in favour of two distinct receptor types. 5. A simple model of open channel block is discounted, and desensitization of the blocked state proposed.
Metabolic changes during ischaemia and their role in contractile failure in isolated ferret hearts.
Authors: Elliott A C , Smith G L , Eisner D A , Allen D G
Published: December 2014

1. The effects of global ischaemia on phosphorus metabolites, intracellular pH (pHi) and developed pressure were measured in isolated whole ferret hearts using 31P nuclear magnetic resonance (NMR) spectroscopy. 2. Brief (10 min) periods of global ischaemia reduced left ventricular developed pressure (LVDP) to undetectable levels. This fall in LVDP was accompanied by a fall in the intracellular concentration of phosphocreatine (PCr) and increases in the concentrations of inorganic phosphate (Pi) and phosphomonoesters. There was no change in the intracellular ATP concentration ([ATP]i). pHi fell approximately linearly at a rate of 0.04 pH units min‐1. 3. When ferret hearts were exposed to cyanide (CN‐) in the presence of alpha‐cyano‐4‐hydroxycinnamate (CHC), a blocker of lactate efflux, the changes in pHi and [Pi]i which occurred were similar to those observed during global ischaemia. However, developed pressure only fell to around 15% of the control value. 4. Removing the intracellular acidosis (by reducing the CO2 level of the gas with which the perfusate was equilibrated) during exposure to CN‐ and CHC caused an increase in developed pressure, consistent with the fall in pHi being responsible for a substantial fraction of the fall in developed pressure. 5. Taken together, these results suggest that most, but not all, of the fall in developed pressure during ischaemia can be explained by the effects of the changes in pHi and [Pi]i on the contractile apparatus. 6. Action potential recordings made with a suction electrode during short periods of global ischaemia showed that there was no decrease in action potential duration over the period when developed pressure was falling, eliminating action potential shortening as a possible cause of the fall in developed pressure. 7. In hearts in which the rate of glycolysis had been reduced by glycogen depletion, global ischaemia led to a marked shortening of the action potential. NMR experiments showed that under these conditions [ATP]i decreased by around 50% over the first 10 jin of ischaemia, while the intracellular acidosis which occurred was smaller than that in a control ischaemic period. 8. The time course of the decline of [ATP]i was examined in several hearts during long (45 min and over) ischaemic periods without prior glycogen depletion. After 45 min of ischaemia [ATP]i fell to around two‐thirds of the control value, while pHi declined to approximately 6.1. Resting pressure did not increase. On reperfusion pHi recovered rapidly to control levels. [ATP]i, however, did not recover. 9. If ischaemia was prolonged further, [ATP]i eventually became undetectable after 70‐90 min.(ABSTRACT TRUNCATED AT 400 WORDS)
Central and peripheral fatigue of human diaphragm and limb muscles assessed by twitch interpolation.
Authors: McKenzie D K , Bigland‐Ritchie B , Gorman R B , Gandevia S C
Published: December 2014

1. This study used a sensitive modification of the twitch interpolation technique to compare the extent of voluntary neural drive to the diaphragm and the elbow flexors during fatigue. For the diaphragm both inspiratory and expulsive efforts were tested, and fatigue was induced by expulsive efforts which were either maximal voluntary contractions (MVCs, 10 s duration, 50% duty cycle) or submaximal contractions (50% MVC, 3 s duration, 60% duty cycle). 2. Over the series of thirty MVCs peak elbow torque declined to 57.9 +/‐ 3.0% (mean +/‐ S.E.M.) of the initial value while maximal inspiratory pressure declined to 78.7 +/‐ 7.3% (P < 0.05). For the diaphragm the relative decline in voluntary peak inspiratory (and expulsive) force was similar to the decline in twitch responses to single and twin (10 ms interval) stimuli. However, for the elbow flexors the decline in twitch force was disproportionately greater than the decline in maximal voluntary force. The decline in twitch force for the diaphragm could not be attributed to failure at the neuromuscular junction. 3. At the start of the exercise, twitch potentiation (following three brief MVCs) was significantly less for the diaphragm than for the elbow flexors (20% versus 61%, P < 0.01). 4. In the unfatigued state maximal voluntary efforts by subjects activated 98.4 +/‐ 0.4% of the stimulated elbow flexors compared with 95.0 +/‐ 1.5% of the diaphragm (P < 0.05). During the exercise period there was a progressive failure in the ability to activate the limb muscle ('central fatigue'; voluntary drive declined from 98.4 +/‐ 0.4 to 86.8 +/‐ 2.2%, P < 0.01) whereas the decline in voluntary activation during inspiratory contractions was not significant (from 95.0 +/‐ 15 to 91.5 +/‐ 2.5%). 5. Voluntary activation during attempted maximal efforts was less complete for both muscles when stimuli were delivered without warning. The index of voluntary activation for unwarned stimuli was lower for the diaphragm (performing expulsive efforts, 81.0 +/‐ 2.8%) than for the limb muscle (89.9 +/‐ 1.5%, P < 0.01). 6. During repeated submaximal expulsive efforts we confirmed that subjects develop a marked inability to contract the diaphragm voluntarily, but when the diaphragm performed inspiratory manoeuvres at the same level of contractile fatigue, the index of voluntary drive was greater than 94%. 7. In conclusion, when tested with inspiratory efforts the diaphragm developed less central fatigue than the limb muscle over the same exercise period.(ABSTRACT TRUNCATED AT 400 WORDS)
Pipette GTP is essential for receptor‐mediated regulation of Cl‐ current in dialysed myocytes from guinea‐pig ventricle.
Authors: Horie M , Hwang T C , Gadsby D C
Published: December 2014

1. Wide‐tipped, low‐resistance (approximately 1 M omega) pipettes were used to record the whole‐cell Cl‐ current activated by cAMP‐dependent protein kinase (PKA) in guinea‐pig ventricular myocytes internally dialysed with or without GTP. Without GTP in the pipette, the response to 1 microM‐isoprenaline declined with time and eventually disappeared, usually within approximately 20 min of rupturing the membrane and beginning cell dialysis. 2. This rundown of the isoprenaline response occurred more quickly with wider, lower‐resistance pipette tips. 3. After complete rundown of the isoprenaline response, histamine (10 microM), another agonist known to elicit the Cl‐ current, also had no effect, but extracellular forskolin (1 microM) or intrapipette cAMP (1 mM) could still readily elicit the Cl‐ current. 4. In contrast, with 100 microM‐GTP in the pipette, the response to 1 microM‐isoprenaline was well maintained for periods greater than 20 min. But, if GTP was then withdrawn from the pipette, a rundown of the isoprenaline response was seen comparable to that in the experiments begun with GTP‐free pipette solution. Moreover, in experiments begun without pipette GTP, the addition of 100 microM‐GTP to the pipette solution, after the response to isoprenaline had disappeared, was able to restore that Cl‐ current response. 5. With GTP in the pipette, the forskolin‐induced Cl‐ current could be suppressed by concurrent exposure to carbachol (10 microM). That inhibition was not seen in myocytes pretreated with pertussis toxin. In untreated myocytes dialysed with GTP‐free pipette solution, after disappearance of the isoprenaline response, the muscarinic receptor‐mediated inhibition was itself abolished. 6. We confirm that both beta‐adrenoceptor‐mediated activation of the Cl‐ current by isoprenaline, and muscarinic receptor‐mediated inhibition of the forskolin‐induced Cl‐ current, are mediated by G proteins, and conclude that the disappearance of both receptor‐mediated responses during whole‐cell recording with GTP‐free pipette solution reflects the fall of cellular [GTP] below the level required to maintain G protein‐dependent signal transduction.
Intracellular pH and its relationship to regulation of ion transport in normal and cystic fibrosis human nasal epithelia.
Authors: Willumsen N J , Boucher R C
Published: December 2014

1. Intracellular pH (pHi) of cultured human airway epithelial cells from normal and cystic fibrosis (CF) subjects were measured with double‐barrelled pH‐sensitive liquid exchanger microelectrodes. The cells, which were grown to confluence on a permeable collagen matrix support, were mounted in a modified miniature Ussing chamber. All studies were conducted under open circuit conditions. Values are given as means +/‐ S.E.M. and n refers to the number of preparations. 2. Normal preparations (n = 15) were characterized by a transepithelial potential difference (Vt) of ‐18 +/‐ 2 mV, an apical membrane potential (Va) of ‐19 +/‐ 2 mV, a basolateral membrane potential (Vb) of ‐37 +/‐ 2 mV, a transepithelial resistance (Rt) of 253 +/‐ 15 omega cm2, a fractional apical membrane resistance (fRa) of 0.40 +/‐ 0.04 and an equivalent short circuit current (Ieq) of ‐73 +/‐ 7 microA cm‐2. 3. CF preparations (n = 13) were characterized by a Vt of ‐46 +/‐ 7 mV, a Va of 3 +/‐ 5 mV, a Vb of ‐43 +/‐ 3 mV, Rt of 373 +/‐ 47 omega cm2, fRa of 0.44 +/‐ 0.04 and an Ieq of ‐130 +/‐ 16 microA cm‐2. All parameters except Vb and fRa were significantly different (P < 0.025) from those of normal preparations. 4. Despite large differences in electrochemical driving force for proton flow across the apical cell membranes between normal and CF preparations (‐4 +/‐ 3 mV and 20 +/‐ 7 mV, respectively), pHi was similar (7.15 +/‐ 0.02 and 7.11 +/‐ 0.05, respectively). The driving force across the basolateral membrane was similar in normal and CF preparations (22 +/‐ 3 and 26 +/‐ 3 mV, respectively). 5. Intracellular alkalinization achieved by removal of CO2 from the luminal Ringer solution or by luminal ammonium prepulse led to stimulation of Ieq in both normal (from ‐58 to ‐70 microA cm‐2, n = 4; P < 0.05) and CF (from ‐144 to ‐163 microA cm‐2, n = 4; P < 0.005) preparations. The increase in Ieq was associated with a reduction of Rt, increase in fRa, and hyperpolarization of Vb. All changes in bioelectric properties in response to intracellular alkalinization were fully reversible. 6. Intracellular acidification achieved by serosal ammonium prepulse led to marked reductions of Ieq in both normal (from ‐95 to ‐31 microA cm‐2, n = 6; P < 0.05) and CF (from ‐111 to ‐67 microA cm‐2, n = 7; P < 0.005) preparations.(ABSTRACT TRUNCATED AT 400 WORDS)
Activation of N‐methyl‐D‐aspartate receptors by L‐glutamate in cells dissociated from adult rat hippocampus.
Authors: Gibb A J , Colquhoun D
Published: December 2014

1. Single channel recording techniques were used to study the ion channel openings resulting from activation of N‐methyl‐D‐aspartate (NMDA) receptors by the agonist glutamate. Patches were from cells acutely dissociated from adult rat hippocampus (CA1). Channel activity was studied at low glutamate concentrations (20‐100 nM) with 1 microM‐glycine, in the absence of extracellular divalent cations. 2. Channel openings were to two main conductance levels corresponding to 50 pS and 40 pS openings in extracellular solution with 1 mM‐Ca2+. Around 80% of openings were to the large conductance level. The single channel conductances increased as extracellular Ca2+ was reduced. 3. Distributions of channel open times were described by three exponential components of 87 microseconds, 0.91 ms and 4.72 ms (relative areas of 51, 31 and 18%). Most long openings were to the large conductance level. 4. The channel closed time distribution was complex, requiring five exponential components to describe it adequately. Of these five components, at least three, with time constants of 68 microseconds, 0.72 ms and 7.6 ms (relative areas of 38, 12 and 17%) represent gaps within single activations of the receptor. The presence of a component with a mean of 7.6 ms is notable because gaps of this length have not previously been identified as being within single NMDA receptor channel activations. 5. Channel activations were identified as including gaps underlying at least the first three closed time components. Activations consisted of clusters of channel openings. Distributions of the length of these clusters had mean time constants of 88 microseconds, 3.4 ms and 32 ms (relative areas of 45, 25 and 30%). Long clusters contained short, intermediate and long duration openings as well as subconductance openings. The open probability within clusters averaged 0.62. Three components were evident in distributions of the number of openings per cluster. These had mean values of 1.22, 3.2 and 11 openings per cluster. 6. An inverse correlation was evident between the length of adjacent open and closed times. When open intervals were separated into groups based on the length of adjacent gaps, the time constants of the exponential components in these conditional open time distributions were independent of the length of the adjacent gap. This supports the idea that the NMDA receptor channel gating has the properties of a discrete Markov process. 7. The long duration of NMDA receptor channel clusters suggests that they contribute to the slow time course of the NMDA receptor‐mediated synaptic current.
Macroscopic and unitary properties of physiological ion flux through T‐type Ca2+ channels in guinea‐pig heart cells.
Authors: Balke C W , Rose W C , Marban E , Wier W G
Published: December 2014

1. We sought to distinguish two types of Ca2+ channel in guinea‐pig ventricular cells (T‐type and L‐type) and to characterize their respective gating and permeation properties when Ca2+ (1‐10 mM) is the charge carrier, as is the case physiologically. 2. Na+ was removed from both the external and internal solutions to eliminate currents through Na+ channels and Na(+)‐Ca2+ exchange. Major differences in the voltage dependence of steady‐state inactivation were exploited to separate the two Ca2+ current components. 3. From a holding potential of ‐50 mV, only L‐type channels were available to open with depolarization. When holding at ‐90 mV, T‐type channels contributed an additional rapidly inactivating component superimposed upon the L‐type current. Only the L‐type channels thus identified were sensitive to the dihydropyridine Ca2+ channel blocker nitrendipine. 4. T‐type currents, measured by taking the difference between the currents elicited from a holding potential of ‐90 mV and those elicited from ‐50 mV, peaked within 10 ms and decayed completely within 50‐100 ms. 5. Macroscopic T‐type currents were largest during depolarizing pulses between ‐40 and ‐30 mV (peak current density of 0.62 +/‐ 0.21 nA nF‐1) and decreased at more positive potentials, becoming unmeasurably small above 0 mV. 6. Unitary currents recorded with similar ionic conditions and voltage protocols exhibited a single‐channel conductance of 4‐5 pS in 10 mM Ca2+. Ensemble average currents through a single channel reproduced accurately the time course of whole‐cell T‐type current. Permeation properties could not explain the absence of macroscopic T‐type currents at positive test potentials, which must therefore be attributable to gating. 7. Convolution analysis was employed to clarify the single‐channel basis of the rapidly decaying current waveform of T‐type channels. The latencies to first opening and reopening, which reflect activation and deactivation, influenced the waveform most strikingly. Open times were sufficiently brief that they contributed little to shaping the average current. Thus, macroscopic inactivation largely reflects rate‐limiting activation events. 8. The unitary current amplitudes and peak open probabilities measured for single T‐type channels, when compared to the average macroscopic T‐type current density, predict 10.6 functional channels per picofarad, or approximately 1700 T‐type channels per typical ventricular myocyte.
Hyperosmotic activation of the Na(+)‐H+ exchanger in a rat bone cell line: temperature dependence and activation pathways.
Authors: Dascalu A , Nevo Z , Korenstein R
Published: December 2014

1. The hyperosmotic activation of the Na(+)‐H+ exchanger was studied in an osteoblast‐like rat cell line (RCJ 1.20). The activation was monitored by recording the intracellular pH (pHi) changes employing double excitation of the pH‐sensitive fluorescent dye 2'7'‐bis(carboxyethyl)‐5(6)‐carboxyfluorescein acetoxymethyl ester (BCECF‐AM). 2. Exposure of the cells to a hyperosmotic HCO(3‐)‐free medium at 37 degrees C produced an initial cytosolic acidification of 0.05 pH units followed by a lag period and an alkalinization overshoot of about 0.2 pH units, without a concomitant change of the free cytosolic calcium [Ca2+]i by the use of Fura‐2 calcium‐sensitive probes. This response was completely inhibited by amiloride (0.33 mM) or by Na+ depletion from the external medium and insensitive to the extracellular Cl‐ replacement, indicating the involvement of a Na(+)‐H+ exchanger in the hyperosmotic response. 3. Hyperosmotic stimuli (200 moSM sucrose) applied in the temperature range of 17‐37 degrees C demonstrated a shortening of the lag period preceding alkalinization and an increased rate of proton extrusion upon temperature elevation. The biochemical reaction underlying the lag period and the proton extrusion resulted in apparent activation energies of 19 and 29 kcal mol‐1, respectively, as calculated from the appropriate Arrhenius plots. 4. Stimulation of the exchanger under isosmotic conditions by 25 nM 4 beta‐phorbol 12‐myristate 13‐acetate (PMA) and 0.1 mM vanadate resulted in an amiloride‐sensitive pHi increase of about 0.08 pH units. The hyperosmotic stress was additive to the stimulatory effects of these agents, suggesting an independent hyperosmotic activation pathway. 5. The hyperosmotic activation of the Na(+)‐H+ exchanger was independent of cAMP, cGMP, cytosolic Ca2+ and protein kinase C. Thus, none of the classical transduction mechanisms seem to be involved directly in the hyperosmotic activation of the antiporter. 6. The pHi response induced by the hyperosmotic stress was abolished by two calmodulin inhibitors, W‐7 and chlorpromazine (50% inhibition, Ki at 28 and 20 microM, respectively), 20 microM cytochalasin B, but not by 10 microM colchicine. The results suggest the involvement of actin and calmodulin‐like structural elements of the cytoskeleton in the transduction process leading to the activation of the Na(+)‐H+ exchanger.
Influence of apical Na+ entry on Na(+)‐K(+)‐ATPase in amphibian distal nephron cells in culture.
Authors: Lyoussi B , Crabbé J
Published: December 2014

1. Transepithelial Na+ transport, Na(+)‐K(+)‐ATPase activity and ouabain binding were measured in cells originating from the distal part of amphibian nephron (A6) which form ‘tight’ monolayers in culture, under standard (control) incubation conditions and after various manoeuvres designed to reversibly interfere with Na+ transport. 2. At spontaneous transport rate, the short‐circuit current (which reflects transepithelial Na+ transport) and the Na(+)‐K(+)‐ATPase activity averaged 7.0 microA/cm2 and 5.9 mumol Pi/(mg protein.h), respectively (n = 53). Short‐circuit current and Na(+)‐K(+)‐ATPase activity appeared to be directly related over a wide range. 3. Suppression of Na+ transport led to a progressive decrease in Na(+)‐K(+)‐ATPase activity over several hours, with an apparent half‐life of approximately 6 h after subtraction of baseline enzyme activity. 4. When A6 cells were allowed to resume sodium transport, the short‐circuit current and Na(+)‐K(+)‐ATPase activity returned to control levels within 12‐24 h, the former recovering somewhat faster. 5. When apical sodium concentration was reduced, a decrease in enzyme level occurred inasmuch as short‐circuit current decreased. 6. There was good agreement between the measured enzyme activity and ouabain binding onto dispersed A6 cells, which suggests that it is unlikely that the changes observed result from internalization vs. insertion in the plasma membrane of sodium pumps.
Modulation of beta‐adrenergic responses of chloride and calcium currents by external cations in guinea‐pig ventricular cells.
Authors: Tareen F M , Yoshida A , Ono K
Published: December 2014

1. The catecholamine‐induced Cl‐ current and the Ca2+ current were recorded in the single ventricular cells of guinea‐pig hearts, using the whole‐cell patch clamp technique combined with internal perfusion. Dependence of the beta‐adrenergic responses on external monovalent cations was investigated. The Cl‐ current was recognized by measuring the reversal potential of the agonist‐induced current. 2. The amplitude of the Cl‐ current, activated by 1 microM adrenaline or 0.01‐0.1 microM isoprenaline, was decreased when the external Na+ concentration ([Na+]o) was reduced by replacement with Tris+. The conductance of the catecholamine‐induced Cl‐ current was proportional to the logarithm of the [Na+]o over a range of 15‐140 mM. When the conductance was plotted against the concentration of Tris+, a dose‐dependent inhibition of the Cl‐ response by Tris+ was suggested with a half‐maximum concentration of 95 mM. 3. The inhibitory effect of the Na+ substitute TEA+ on the Cl‐ current was not affected by either increasing the buffer for the internal Ca2+ (10 mM BAPTA) or for the pH (50 mM HEPES). 4. In the relationship between agonist concentration and the Cl‐ conductance, the half‐maximum concentration (K1/2) of isoprenaline was 0.013 microM in the control Na+ solution, and was shifted to 0.07, 0.08, 0.1 and 0.3 microM in the Li+, Cs+, TEA+ and Tris+ external solutions, respectively. The maximum slope conductance was not significantly affected, except for a slight depression on the Tris+ solution. When the current was induced by adrenaline, qualitatively the same finding was obtained; K1/2 was 0.15 and 3.2 microM in the Na+ and Tris+ solutions, respectively. 5. As a substitute for the external Na+, sucrose seemed to be inert. The activation of the inward Cl‐ current was conserved in the 300 mM sucrose solution ([Cl‐]o = 8 mM) with a K1/2 value of 0.015 microM isoprenaline. 6. The Cl‐ current, when activated by either an external application of forskolin (0.2‐10 microM) or an internal perfusion of cyclic AMP (100‐500 microM), was not affected by replacing external Na+ with other cations. Activation of the Cl‐ current by 0.2‐5 microM histamine was also insensitive to a substitution of Na+. These findings indicate that the inhibition by the Na+ substitute is at a point before the activation of GTP‐binding protein. 7. The effects of Na+ substitution were not affected by varying the Na+ concentration (0‐115 mM) in the internal solution, excluding an involvement of a change in the [Na+]i.(ABSTRACT TRUNCATED AT 400 WORDS)
Activation of protein kinase C suppresses responses to NMDA in rat CA1 hippocampal neurones.
Authors: Markram H , Segal M
Published: December 2014

1. The effects of 1‐oleoyl‐2‐acetylglycerol (OAG), an activator of protein kinase C (PKC), on NMDA receptor‐mediated responses were investigated in CA1 neurones of hippocampal slices using current‐ and voltage‐clamp techniques. 2. Topical application of OAG caused a suppression of the slow, voltage‐sensitive, NMDA receptor‐mediated component of excitatory postsynaptic potentials (EPSPs) evoked by stimulating the schaffer‐collateral commissural afferents and had no effect on the fast, voltage‐insensitive, quisqualate/kainate component. 3. OAG suppressed the amplitude of inward current responses to NMDA down to about one‐third of control responses. OAG could also increase the duration of the responses to NMDA by up to twofold. The effect of OAG on the duration but not on the amplitude of the response to NMDA was blocked by pre‐loading cells with the K+ channel blocker, Cs+. Topical application of OAG had no significant effect on current responses to quisqualate. 4. An OAG isomer, which does not activate PKC, had no effect on responses to NMDA. Intracellular application of the kinase inhibitor, H‐7, completely blocked the effect of OAG on the amplitude and duration of responses to NMDA, as well as on the slow EPSP. Finally, topical application of another activator of PKC, phorbol 12‐myristate 13‐acetate (PMA), also suppressed responses to NMDA. PMA reduced the slow component of synaptic responses in about half of the cells tested. 5. We propose that activation of PKC in CA1 hippocampal neurones suppresses NMDA receptor‐mediated responses.