bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative effects of inorganic phosphate and oxalate on uptake and release of Ca2+ by the sarcoplasmic reticulum in saponin skinned rat cardiac trabeculae.
Authors: Steele D S , McAinsh A M , Smith G L
Published: December 2014

1. Ventricular trabeculae from the right ventricle of rat heart were suspended in a 6 microliters bath and ‘skinned’ with saponin (50 mg ml‐1). Preparations were perfused with solutions mimicking the intracellular milieu and the [Ca2+] within the bath was monitored continuously using fura‐2. 2. Application of 20 mM caffeine released Ca2+ from the sarcoplasmic reticulum (SR), resulting in a transient increase in the fura‐2 fluorescence ratio. Caffeine‐induced Ca2+ transients were smaller in the presence of 30 or 60 mM inorganic phosphate (Pi). This depressive effect of Pi on SR function was reversed by 10 mM creatine phosphate (CP). Caffeine‐induced Ca2+ transients were also reduced in the presence of 10 mM oxalate, although this effect was not reversed by CP. 3. When perfusion was stopped in the presence of 30 or 60 mM Pi, the [Ca2+] within the bath remained constant. However, when the flow was stopped in the presence of 60 mM Pi and 10 mM CP, a prolonged decrease in [Ca2+] occurred, consistent with precipitation of calcium phosphate within the SR. A similar decrease in [Ca2+] was observed when perfusion was stopped in the presence of 2 or 20 mM oxalate, in the absence or presence of CP. 4. The SR was Ca2+ depleted by withdrawal of ATP and exposure to 20 mM caffeine. Perfusion was then stopped and ATP reapplied, resulting in a maintained decrease in [Ca2+] within the bath, due to SR Ca2+ uptake. Net Ca2+ uptake was markedly reduced in the presence of 30 mM Pi. In contrast, 20 mM oxalate increased Ca2+ uptake and the [Ca2+] within the bath continued to fall over 2‐3 min. 5. Introduction of Pi released Ca2+ from the SR. Ryanodine (100 microM) abolished caffeine‐induced Ca2+ release while Pi‐induced Ca2+ release was unaffected. Pi‐induced Ca2+ release was reduced in the constant presence of 20 mM caffeine or 10 mM CP and was abolished completely by disruption of the SR membrane with Triton X‐100. Pi‐induced Ca2+ release occurred after abolition of SR Ca2+ uptake by ATP withdrawal. 6. These results suggest that the Pi‐induced decrease in releasable Ca2+ does not result from precipitation of calcium phosphate within the SR lumen. Pi inhibits net SR Ca2+ uptake, but this appears to result from activation of a ryanodine‐insensitive Ca2+ efflux pathway rather then inhibition of Ca2+ uptake. Possible mechanisms are considered, including reversal of the SR Ca2+ pump.
A unique P‐region residue is required for slow voltage‐dependent gating of a G protein‐activated inward rectifier K+ channel expressed in Xenopus oocytes.
Authors: Kofuji P , Doupnik C A , Davidson N , Lester H A
Published: December 2014

1. The structural determinants of a G protein‐activated inwardly rectifying potassium channel, GIRK1 (KIR3.1), involved in voltage‐ and time‐dependent gating properties were investigated by heterologous expression of chimeric constructs and point mutants in Xenopus oocytes. 2. Chimeras between GIRK1 and the weakly rectifying potassium channel, ROMK1 (KIR1.1), indicate that residues in the putative transmembrane segments TM1 and TM2 affect the steep inward rectification of GIRK1, while residues in the main pore‐forming domain, the P‐region segment, are critical for the manifestation of GIRK1 time‐dependent activation. 3. Phenylalanine 137 in the P‐region of GIRK1 is unique; in ROMK1, as in other inward rectifiers, there is a serine residue at this position. Mutation of the phenylalanine 137 to serine leads to expression of currents with nearly time‐independent activation. 4. An acidic residue (aspartate) in TM2 partially controls the time‐ and voltage‐dependent gating in IRK1 (KIR2.1). Mutation of the equivalent aspartate 173 to glutamine in GIRK1 did not abolish the time‐dependent activation but did decrease the degree of inward rectification. 5. These results reveal an important role for the P‐region in controlling the time‐dependent gating of an inwardly rectifying potassium channel and suggest a close relationship between permeation and gating in this family of K+ channels.
Characterization of a beta‐adrenergically inhibited K+ current in rat cardiac ventricular cells.
Authors: Scamps F
Published: December 2014

1. The electrophysiological properties and beta‐adrenergic regulation of a non‐inactivating K+ current were studied using the whole‐cell patch‐clamp technique (22 +/‐ 2 degrees C) in adult rat ventricular cells. 2. In the presence of 4‐aminopyridine, an inhibitor of the rapidly inactivating current, the depolarization‐activated current consisted only of a slowly decaying outward current (IK). The presence of a non‐inactivating current (ISS) was revealed when analysing inactivation curves. 3. IK and ISS were both sensitive to 50 mM tetraethylammonium and 10 mM 4‐aminopyridine inhibition. IK was totally blocked by 100 microM clofilium, while ISS was not inhibited but rather enhanced by this class III anti‐arrhythmic agent. 4. Unlike IK, ISS was only slightly decreased by depolarizing prepulses and it did not show time‐dependent inactivation when measured during 500 ms depolarizations. 5. ISS was decreased by the beta‐adrenergic agonist isoprenaline (1 microM). Forskolin (10 microM) mimicked the effects of isoprenaline. The non‐specific beta‐adrenergic antagonist, propranolol (3 microM), and a specific beta 1‐adrenergic antagonist, CGP 20712A (0.3 microM), both prevented the effects of isoprenaline. Cell perfusion with 100 microM PKI6‐22, a peptide inhibitor of the cyclic AMP‐dependent protein kinase, reduced or abolished the effects of isoprenaline. 6. The dose‐response curve for the inhibition of ISS by isoprenaline was positioned to the left of that for the calcium current. The threshold dose and the dose giving 50% of the maximal effect were, respectively, 0.1 and 0.21 nM for ISS and 1 and 4.3 nM for ICa. 7. In view of the high sensitivity of ISS to isoprenaline, its possible physiological effect was evaluated on action potential duration during beta‐adrenergic stimulation. At 1 nM, a concentration that did not increase ICa, isoprenaline induced a significant prolongation of action potential duration as a consequence of ISS inhibition. With 1 microM isoprenaline, the action potential was further prolonged, due largely to an evoked increase in ICa. 8. In conclusion, a K+ current displaying a weak voltage‐dependent inactivation is present in rat ventricular cells. It is inhibited by stimulation of beta 1‐adrenergic receptors and is highly sensitive to phosphorylation by protein kinase A. This current may play an important role in the neuromodulation of excitation‐contraction coupling.
Effects of intracellular Mg2+ on channel gating and steady‐state responses of the NMDA receptor in cultured rat neurons.
Authors: Li-Smerin Y , Johnson J W
Published: December 2014

1. The effects of intracellular Mg2+ (Mgi2+) on the single N‐methyl‐D‐aspartate (NMDA)‐activated channel burst duration and frequency and on the mean NMDA‐activated patch current were studied in outside‐out patches from cultured rat cortical neurons. The inhibition by Mgi2+ of mean patch and whole‐cell currents were compared, and some possible explanations for the observed differences were investigated. 2. The burst duration at +60 mV did not depend on Mgi2+ concentration, suggesting that the channel can close when blocked by Mgi2+. The number of bursts per second increased significantly in the presence of Mgi2+, suggesting that the rate of channel opening is higher when Mg2+ from the intracellular solution occupies its binding site. 3. Mgi2+ caused a voltage‐ and concentration‐dependent inhibition of mean patch current. The inhibition is in quantitative agreement with the effects of Mgi2+ on the single‐channel current and on burst parameters. 4. Based on the effects of Mgi2+ on burst parameters and on single‐channel current, a four‐state model in which the NMDA‐activated channel can close while blocked by Mgi2+ is proposed. By fitting the model to the mean patch current data, we estimate that the rate of channel opening is increased by a factor of 1.4 when Mgi2+ occupies the channel. This estimation provides evidence that occupancy of the NMDA‐activated channel by Mgi2+ destabilizes the closed state. 5. Mgi2+ reduced NMDA‐activated whole‐cell currents in a voltage‐ and concentration‐dependent manner. However, normalized whole‐cell and mean patch currents at positive voltages differed in two significant respects. First, when currents were recorded in a 0 Mg2+ pipette solution, whole‐cell currents at positive voltages were smaller. Second, Mgi2+ appeared to inhibit whole‐cell current less effectively than it inhibited mean patch current. 6. Inclusion of the Mg2+ chelators EDTA and ATP in 0 Mg2+ pipette solutions did not increase the whole‐cell current measured at +60 mV. This observation suggests that the difference between normalized whole‐cell and mean patch currents with 0 Mg2+ pipette solution was not due to block of whole‐cell currents by residual Mgi2+. 7. When a pipette solution containing EGTA and Mg2+ was used to buffer Mgi2+, inhibition by Mgi2+ of the whole‐cell current was enhanced, suggesting that the free Mg2+ concentration inside a neuron can remain below the pipette Mg2+ concentration. However, we cannot exclude other explanations for the differences between the inhibition by Mg2+ of mean patch and whole‐cell currents.
Endogenous monoamines inhibit glutamate transmission in the spinal trigeminal nucleus of the guinea‐pig.
Authors: Travagli R A , Williams J T
Published: December 2014

1. With the use of whole‐cell patch clamp recordings in slices of guinea‐pig substantia gelatinosa (SG), we studied the serotonin (5‐HT)‐ and noradrenaline (NA)‐mediated inhibition of glutamate‐mediated EPSCs evoked from primary afferent stimulation. 2. The frequency of spontaneous EPSPs was reduced by 5‐HT and NA. 3. The inhibition of EPSCs caused by 5‐HT was mediated by the 5‐HT1D receptor subtype, since the 5‐HT1D agonist, sumatriptan (1 microM), was effective. 4. NA and the alpha 2‐agonist, 5‐bromo‐N‐(4,5‐dihydro‐1H‐imidazol‐2‐yl)‐6‐quinoxalinamine (UK 14304), decreased the EPSCs and this inhibition was blocked by the alpha 2‐antagonists, idazoxan (1 microM) and yohimbine (1 microM). 5. The 5‐HT‐releasing agent, fenfluramine (10 microM), and the Na‐releasing agent, amphetamine (1 microM), also depressed EPSCs. Pretreatment of slices with the 5‐HT‐depleting agent, p‐chloro‐amphetamine (10 microM), attenuated the inhibition of fenfluramine but failed to antagonize the effects of exogenously applied 5‐HT. 6. These in vitro results suggest that presynaptic inhibition of glutamate release from primary afferents can provide another mechanism to explain the antinociceptive effects of 5‐HT and NA obtained in vivo.
Inspiratory drive and phase duration during carotid chemoreceptor stimulation in the cat: medullary neurone correlations.
Authors: Morris K F , Arata A , Shannon R , Lindsey B G
Published: December 2014

1. This study addressed the hypothesis that there is a parallel processing of input from carotid chemoreceptors to brainstem neurones involved in inspiratory phase timing and control of inspiratory motor output amplitude. Data were from fifteen anaesthetized, bilaterally vagotomized, paralysed, artificially ventilated cats. Carotid chemoreceptors were stimulated by close arterial injection of 200 microliters of CO2‐saturated saline solution. 2. Planar arrays of tungsten microelectrodes were used to monitor simultaneously up to twenty‐two neurones in the nucleus tractus solitarii (NTS) and ventral respiratory group (VRG). Spike trains were analysed with two statistical tests of respiratory modulation, cycle‐triggered histograms, peristimulus‐time histograms, cumulative sum histograms and cross‐correlograms. 3. In NTS, 16 of 26 neurones with respiratory and 12 of 27 without respiratory modulation changed firing rate during carotid chemoreceptor stimulation. In the VRG 72 of 112 respiratory and 14 of 48 non‐respiratory neurones changed firing rate during stimulation. 4. The spike trains of 85 of 1276 pairs (6.7%) of cells exhibited short time scale correlations indicative of paucisynaptic interactions. Ten pairs of neurones were each composed of a rostral VRG phasic inspiratory neurone that responded to carotid chemoreceptor stimulation with a decline in firing rate and a caudal VRG phasic inspiratory neurone that increased its firing rate. Cross‐correlograms from two of the pairs had features consistent with excitation of the caudal neurones by the rostral cells. A decrease in the duration of activity of the rostral VRG neurones was paralleled by the decrease in inspiratory time of phrenic nerve activity. Caudal VRG inspiratory neurones increased their activity as phrenic amplitude increased. Spike‐triggered averages of all four neurones indicated post‐spike facilitation of phrenic motoneurones. 5. The results support the hypothesis that unilateral stimulation of carotid chemoreceptors results in parallel actions. (a) Inhibition of rostral VRG I‐Driver neurones decreases inspiratory duration. (b) Concurrent excitation of premotor VRG and dorsal respiratory group inspiratory neurones increases inspiratory drive to phrenic motoneurones. Other data suggest that responsive ipsilateral neurones act to regulate contralateral neurones.
Cholinergic inhibition of Ca2+ current in guinea‐pig gastric and tracheal smooth muscle cells.
Authors: Wade G R , Barbera J , Sims S M
Published: December 2014

1. Cholinergic regulation of L‐type Ca2+ channels was investigated in freshly dissociated guinea‐pig gastric and tracheal smooth muscle cells. Acetylcholine (ACh, 50 microM) decreased Ca2+ channel current (ICa) by 37 +/‐ 3% (mean +/‐ S.E.M., 46 cells). 2. ACh reduced ICa at all voltages, with no shift in the current‐voltage relationship. Effects of ACh were rapid (within 5 s) and repeatable, with multiple applications reproducibly inhibiting ICa in the continued presence of extracellular Ca2+ and in the presence of protein kinase C inhibitors. 3. The involvement of Ca2+ stores in this inhibition was investigated using Ca(2+)‐free solution or cyclopiazonic acid (CPA) to deplete the stores. ACh initially inhibited ICa in the Ca(2+)‐free solution (Na+ as charge carrier, 53 +/‐ 4% decrease, 18 cells) with subsequent responses significantly attenuated (n = 9). CPA (1 microM) reduced, then abolished, the effects of ACh on ICa (n = 5). 4. When studied in cell‐attached patches (Ba2+ as charge carrier), ACh reduced Ca2+ channel open probability in twenty‐two of thirty‐six cells, consistent with the involvement of a diffusible cytosolic messenger. 5. ACh also inhibited ICa in tracheal muscle cells (reduction of 38 +/‐ 6% in 1 mM Ca2+, 4 cells; 77 +/‐ 3% in Ca(2+)‐free solution, 7 cells). Furthermore, in cells where ACh elicited oscillating Ca(2+)‐activated Cl‐ current, oscillatory inhibition of ICa was also observed (3 cells). 6. In summary, ACh causes rapid and reversible inhibition of ICa in gastric and tracheal muscles. Ca2+ stores were required to initiate this effect, with the rapid onset and oscillatory inhibition consistent with Ca2+ inhibition of the channel. Suppression of ICa would reduce Ca2+ entry during cholinergic excitation.
The role of the renin‐angiotensin system in the renal response to moderate hypoxia in the rat.
Authors: Neylon M , Marshall J , Johns E J
Published: December 2014

1. In two groups of Saffan‐anaesthetized rats, we studied the role of the renin‐angiotensin system in mediating the antidiuresis and antinatriuresis induced by moderate systemic hypoxia. 2. In both groups, a first period of hypoxia (breathing 12% O2 for 20 min) induced a fall in arterial partial pressure of O2 (Pa,O2; to 42 mmHg), a fall in mean arterial pressure (MABP), no change in renal blood flow (RBF) due to an increase in renal vascular conductance (RVC = RBF/MABP) and falls in urine flow and absolute sodium excretion (UNaV). Concomitantly, plasma renin activity increased from 3.08 +/‐ 0.68 (mean +/‐ S.E.M.) to 8.36 +/‐ 1.8 ng ml‐1 hr‐1. 3. In group 1 (n = 11), Losartan (10 mg kg‐1, I.V.), the angiotensin (AII) AT1 receptor antagonist, induced a fall in MABP (115 +/‐ 3 to 90 +/‐ 3 mmHg), an increase in RVC such that RBF was unchanged, and falls in glomerular filtration rate (GFR), urine flow and UNaV. However, hypoxia induced qualitatively similar changes to those seen before Losartan treatment. 4. In group 2 (n = 9), we occluded the aorta distal to the renal artery to prevent basal MABP and renal perfusion pressure (RPP) from falling after addition of Losartan and to keep the hypoxia‐induced fall in MABP the same as before Losartan treatment. Nevertheless, Losartan induced an increase in basal RVC, RBF, urine flow and UNaV whilst hypoxia induced falls in urine flow and UNaV that were proportionately similar to those seen prior to addition of Losartan. 5. These results indicate that in the Saffan‐anaesthetized rat, AII exerts tonic, renal vasoconstrictor and consequent antidiuretic and antinatriuretic influences in normoxia, but does not contribute to the hypoxia‐induced antidiuresis and antinatriuresis. We propose that renin secretion is increased by the hypoxia‐induced fall in RPP rather than by an increase in renal sympathetic activity. Thus, the AII generated cannot produce antidiuresis and antinatriuresis by its known facilitatory influence on the actions of an increase in sympathetic activity on the renal tubules and is insufficient to produce these effects by direct actions. Rather, these results support the view that the antidiuresis and antinatriuresis of moderate hypoxia is predominantly due to the fall in RPP.
Selective facilitation of responses to cortical stimulation of proximal and distal arm muscles by precision tasks in man.
Authors: Schieppati M , Trompetto C , Abbruzzese G
Published: December 2014

1. The responses of the first dorsal interosseus (1DI), opponens pollicis (OP), extensor digitorum communis (EDC), brachioradialis (BR), biceps brachii (BB) and anterior deltoid (AD) muscles to magnetic stimulation of the motor cortex were recorded during different motor tasks. 2. Two precision and two power isometric tasks were investigated. The precision tasks were a pincer grip (‘grip’) and a thrust against a target with the wrist (‘push’). In the former, the prime movers were the intrinsic hand muscles, while the proximal muscles played a postural role. In the latter, the prime movers were the proximal muscles. In both tasks, force was controlled through visual feedback. The power tasks required encirclement of a cylinder with the fingers (‘grasp’), or sustaining a weight suspended at wrist level (‘load’). 3. Magnetic stimulation was applied in eight subjects by a coil placed over the vertex at 1.1‐1.2 times the motor threshold for the most excitable muscles. This produced in the prime mover muscles larger motor‐evoked responses (MEPs) during grip or push tasks than grasp or load tasks, in spite of similar background EMG levels. During grip tasks, only one of the two prime movers showed task‐dependent changes. In the postural muscle AD there was no significant difference between MEPs during grip and grasp tasks; however, BB responses were larger during grasp than grip tasks. 4. MEPs simultaneously recorded in the prime movers were plotted against each other. The slope of the regression line for AD versus BB was larger in push than load tasks, whilst the changes in MEPs of 1DI and OP were independent during both grip and grasp tasks. 5. In three subjects, MEPs were also elicited by electrical stimulation during grip and grasp tasks. MEP changes tended to parallel those obtained for magnetic stimulation, but the increase in size of the electrically evoked MEPs during the precision task was smaller. 6. In all subjects the median and ulnar nerves were stimulated during grip and grasp tasks, and an H reflex was evoked in the hand muscles of five subjects. In no case did the two tasks produce reflexes of different amplitude. 7. The motor response of both proximal and distal muscles can be task dependent, in spite of the differences in their principal functional role and cortical representation. The modulation is related to the degree of control requested by the task, and is likely to reflect selective changes in the excitability of corticospinal neurones.
Role in cation translocation of the N‐terminus of the alpha‐subunit of the Na(+)‐K+ pump of Bufo.
Authors: Wang X , Jaisser F , Horisberger J D
Published: December 2014

1. We have studied the effects on the physiological properties of the Na(+)‐K+ pump of both 31‐ and 40‐amino acid N‐terminal truncated forms of the alpha‐subunit of the Na(+)‐K(+)‐ATPase. 2. Na(+)‐K+ pumps that were moderately ouabain resistant (K1 = 50 microM) were expressed in the Xenopus oocyte by injection of wild‐type or truncated variants of the Bufo marinus Na(+)‐K(+)‐ATPase alpha‐subunit cRNA with Bufo beta‐subunit cRNA. The function of the Na(+)‐K+ pump was studied by electrophysiological methods after Na+ loading and inhibition of the endogenous Xenopus Na(+)‐K(+)‐ATPase by exposure to a low concentration (0.2 microM) of ouabain. 3. The voltage‐dependent potassium activation kinetics of the Na(+)‐K+ pump current and the ouabain‐sensitive proton‐dependent inward current were studied using the two‐electrode voltage‐clamp technique. A novel technique involving permeabilization of part of the oocyte membrane with digitonin was developed to enable study of the pre‐steady‐state current following fast voltage perturbation. 4. By comparison with the wild type, the 40‐amino acid N‐terminal truncation induced a lower level of Na(+)‐K+ pump current, a 2‐ to 3‐fold reduction in the apparent external K+ affinity when measured in the presence of extracellular Na+, a relative increase in the proton‐dependent inward current, and a reduction in the rate constant of the pre‐steady‐state current following a voltage step towards a positive membrane potential. The 31‐amino acid truncation induced changes that were qualitatively similar but of smaller magnitude. 5. We have analysed these results using a kinetic model of the Na(+)‐K+ pump cycle and have shown that all these effects can be explained by the change in a single rate constant in the cycle kinetics, namely a reduction in the rate of the main charge translocating part of the Na(+)‐K+ pump cycle, i.e. the forward E1 to E2 conformational change, the deocclusion and release of Na+ to the external side. 6. The highly charged N‐terminal segment seems to be directly involved in the mechanism that translocates Na+ ions across the membrane's electrical field.
Characterization, localization and axial distribution of Ca2+ signalling receptors in the rat submandibular salivary gland ducts.
Authors: Xu X , Diaz J , Zhao H , Muallem S
Published: December 2014

1. To characterize [Ca2+]i signalling in salivary duct cells a procedure was developed for the rapid preparation and isolation of intralobular ducts, some of which had attached intercalated ducts. The isolated ducts retained agonist‐induced Ca2+ signalling after permeabilization with streptolysin O (SLO). 2. The improved cell preparation technique was reflected in the repertoire and intensity of agonist responsiveness of the cells. Measurements of [Ca2+]i in intact cells showed that all agonists previously reported to affect electrolyte transport by the submandibular salivary gland (adrenaline, carbachol, isoprenaline and forskolin) mobilized Ca2+ from internal stores and increased Ca2+ influx across the plasma membrane. 3. The use of the SLO‐permeabilized ducts showed that all agonists, including isoprenaline and forskolin, mobilized Ca2+ exclusively from the inositol 1,4,5 trisphosphate (IP3)‐sensitive pool. However, in granular ducts only adrenaline mobilized the entire IP3‐sensitive pool whereas all other agonists mobilized only part of the pool. 4. All regions of the duct responded to substance P and the luminally secreted agonist ATP. Interestingly, the intercalated duct was most responsive to ATP and demonstrated only a minimal response to all other agonists. The granular region of the same duct and the extralobular duct always responded best to stimulation by adrenaline. 5. The perfused extralobular duct was used to show that adrenaline and carbachol stimulated the duct through the basolateral membrane whereas the receptors for ATP were localized in the luminal membrane of the duct. This suggests the presence of an ATP‐dependent positive feedback loop in salivary duct with decreased activity along the ductal tree.
Effect of intracellular calcium on ATP‐activated, GTP‐dependent calcium channels in rat macrophages.
Authors: Mamin A G , Kiselyov K I , Mozhayeva G N
Published: December 2014

1. In order to study the effect of intracellular free Ca2+ concentration ([Ca2+]i) on the activity of ATP‐activated, GTP‐dependent Ca2+ channels in rat macrophages, experiments were performed using the inside‐out configuration of the patch‐clamp technique. 2. Channel activity was observed in the cell‐attached mode when 100 microM ATP was added to the pipette solution containing 105 mM Ba2+, but it disappeared rapidly after patch excision. The activity could be restored by the application of 100 microM GTP or GTP gamma S onto the internal surface of the plasma membrane. 3. The properties of the GTP gamma S‐evoked channels are identical to those of channels activated by extracellular application of ATP. The channels exhibited four current sublevels with conductances of about 3.5, 7, 10 and 15 pS when 105 mM Ba2+ was the only permeant cation. The extrapolated reversal potentials were similar for all the sublevels and averaged about +40 mV. 4. Elevation of [Ca2+]i within the range 0.01‐1 microM resulted in a decrease in mean inward current. The half‐maximal value of the mean current was about 0.08 microM. 5. This decreases in mean current resulted from a redistribution of sublevel occupancies: the 1st sublevel tended to be come more abundant with elevation of [Ca2+]i, while the relative weights of the high‐conductance 3rd and 4th sublevels decreased. 6. The open‐channel current fell with an increase in [Ca2+]i as quickly as the mean current did, indicating that the sublevel redistribution alone is sufficient to produce the revealed decrease in net inward current. 7. It is concluded that [Ca2+]i elevation does not fix the channel in a closed state but rather decreases the ability of the channel to operate in high‐conductance states.
Submicroscopic calcium signals as fundamental events of excitation‐‐contraction coupling in guinea‐pig cardiac myocytes.
Authors: Lipp P , Niggli E
Published: December 2014

1. Subcellularly localized Ca2+ signals have been proposed to represent elementary events of cardiac Ca2+ signalling (Ca2+ sparks), whereby an individual sarcolemmal L‐type Ca2+ channel locally controls opening of a single (or a few) Ca2+ release channels in the sarcoplasmic reticulum (SR). 2. To investigate directly the elementary nature of this Ca(2+)‐induced Ca2+ release mechanism we used flash photolysis of caged Ca2+ while simultaneously measuring the intracellular Ca2+ concentration ([Ca2+]i) with a laser‐scanning confocal microscope. 3. Power spectral analysis of the confocal images performed in the spatial domain revealed that only Ca2+ signalling events involving the L‐type Ca2+ channel pathway gave rise to Ca2+ sparks. In contrast, SR Ca2+ release triggered by photolytic [Ca2+]i jumps resulted in Ca2+ transients that were always spatially homogeneous. 4. From these findings we conclude that the fundamental event of Ca2+ signalling in cardiac muscle may be smaller in size or amplitude than a Ca2+ spark. 5. We term this event a ‘Ca2+ quark’ possibly resulting from gating of a single SR Ca2+ release channel. It is proposed that concerted activation of several ‘Ca2+ quarks’ may be required for a Ca2+ spark. The ‘Ca2+ quark’ could also be the fundamental event in other cell types implementing a hierarchical Ca2+ signalling concept.
Dual effect of external Ca2+ on noradrenaline‐activated cation current in rabbit portal vein smooth muscle cells.
Authors: Helliwell R M , Large W A
Published: December 2014

1. The influence of divalent cations was investigated on the noradrenaline‐activated non‐selective cation current (Icat) in freshly dispersed smooth muscle cells of rabbit portal vein. 2. Reduction of external calcium concentration ([Ca2+]o) from 1.5 mM to ‘test’ levels of 100‐500 microM during stimulation of Icat produced a sustained potentiation of Icat, which indicates an inhibitory action of Cao2+. With ‘test’ concentrations lower than 100 microM Cao2+ the amplitude of Icat was initially potentiated and then the current subsequently declined. Under these conditions re‐addition of 1.5 mM Cao2+ transiently activated Icat which demonstrates an additional facilitatory action of [Ca2+]o. The half‐maximal [Ca2+]o for the facilitatory and inhibitory action on Icat was 6 and 400 microM, respectively. 3. When [Ca2+]o was decreased to very low levels (< 10 nM) by inclusion of 1 mM BAPTA in nominally Ca(2+)‐free external solution, Icat could still be evoked by noradrenaline. This suggested that Ca2+ is not obligatory for activation of Icat but merely modulatory. 4. In 1.5 mM Cao2+ the current‐voltage (I‐V) relationship of Icat was S‐shaped with a reversal potential (Er) of about +9 mV. The I‐V characteristics and Er of the potentiated current in 200 microM Cao2+ were not markedly different from those in 1.5 mM Cao2+. 5. During the noradrenaline‐induced Icat, removal of external Mg2+ caused a small increase in the amplitude of Icat which was not as great as that seen on reduction of [Ca2+]o and there was no facilitatory effect of Mgo2+. 6. These results indicate that external Ca2+ has a dual action on Icat, both facilitating and inhibiting the current. This dual action is not shared with external Mg2+.
Effects of troponin C isoforms on pH sensitivity of contraction in mammalian fast and slow skeletal muscle fibres.
Authors: Metzger J M
Published: December 2014

1. The effects of troponin C (TnC) isoforms on the acidic pH‐induced rightward shift in the tension‐pCa (‐log[Ca2+]) relationship were examined in slow soleus and fast psoas skeletal muscle fibers. Endogenous TnC was partially extracted from skinned single fibres and the extracted fibres were subsequently reconstituted with purified TnC. The pCa producing one‐half maximal tension (pCa50) was determined at pH 7.00 and 6.20 in each fibre and then the pH‐induced shift in pCa50 (delta pCa50) was calculated. 2. In control fast fibres which express fast skeletal TnC (sTnC), the delta pCa50 was 0.64 +/‐ 0.02 pCa units (n = 10), and this increased significantly to 0.78 +/‐ 0.04 pCa units (n = 8) following extraction and reconstitution with cardiac TnC (cTnC). In each fibre, the reconstituted delta pCa50 was subtracted from the control delta pCa50 which yielded a significant shift of ‐0.13 +/‐ 0.05 pCa units (n = 8; P < 0.05). Thus, the pH sensitivity of contraction was increased in the cTnC‐reconstituted psoas fibres. 3. In extracted psoas fibres that were reconstituted with fast sTnC the pH sensitivity of contraction was unchanged, indicating that the above effects were related to the TnC isoform and not a non‐specific effect of the extraction procedure. 4. In a second series of experiments cTnC was specifically extracted from slow soleus fibres which were subsequently reconstituted with purified fast sTnC. Skeletal TnC reconstituted soleus fibres demonstrated a significant decrease in pH sensitivity. In each fibre, the reconstituted delta pCa50 (mean, 0.58 +/‐ 0.02 pCa units) was subtracted from the control delta pCa50 (mean, 0.63 +/‐ 0.02 pCa units) which yielded a significant shift of 0.05 +/‐ 0.01 pCa units (n = 4; P < 0.05). The pH sensitivity was not altered in cTnC‐reconstituted soleus fibres (‐0.01 +/‐ 0.01 pCa units, n = 4). 5. These findings indicate that TnC isoforms alter the pH sensitivities of contraction in slow and fast skeletal muscle fibres. However, the magnitude of the change in pH sensitivity is muscle lineage dependent, indicating that differential expression of other myofilament protein isoforms, together with TnC, is necessary to confer full pH sensitivity of contraction in striated muscles.
Rib motion modulates inspiratory intercostal activity in dogs.
Authors: De Troyer A
Published: December 2014

1. A test was performed of the hypothesis that the motion of the ribs during inspiration modulates, via changes in spindle afferent activity, the activation of the inspiratory intercostal muscles. The electrical activity of the parasternal intercostal, external intercostal, and levator costae muscles in anaesthetized spontaneously breathing dogs was thus recorded during manipulation of the inspiratory displacement of the ribs over a wide range of rib motion. 2. In agreement with the hypothesis, the external intercostal and levator costae muscles lengthened and showed increased inspiratory activities when the normal inspiratory cranial motion of the lower rib was reduced or reversed into an inspiratory caudal motion. Conversely, the inspiratory activities decreased when the inspiratory cranial motion of the rib and the inspiratory shortening of the muscles was augmented. The inspiratory activity of the parasternal intercostal remained unchanged throughout. 3. However, when the two ribs making up the interspace were linked together so that the external intercostal muscle was constant in length, the relationship of muscle activity to rib motion was maintained. 4. In addition, when the upper rather than the lower rib of the interspace was manipulated, the relationship between the change in muscle length and inspiratory activity was reversed, so that activity decreased when the muscle was lengthened and increased when the muscle was shortened. The relationship of muscle activity to lower rib motion, however, was still maintained. 5. These observations thus indicate that rib motion triggers proprioceptive reflexes which, regardless of the changes in length of the individual muscles, make the external intercostal inspiratory activity exquisitely sensitive to the direction of rib displacement.
Responses of glutamine transport in cultured rat skeletal muscle to osmotically induced changes in cell volume.
Authors: Low S Y , Taylor P M , Rennie M J
Published: December 2014

1. In order to investigate the relationship between cellular hydration state and the rate of glutamine transport, tracer glutamine uptake into primary rat myotubes was studied at external osmolalities of 170, 320 or 430 mosmol kg‐1. 2. Incubation of myotubes with glutamine (2 mM; 30 min) at 320 mosmol kg‐1 increased cell volume and glutamine transport (by 35 and 36%, respectively); insulin (66 nM; 30 min) also increased cell volume and glutamine transport (by 22 and 40%, respectively) and the effects of insulin and glutamine combined were additive. The increase in glutamine uptake following glutamine pre‐incubation represented an increase in Vmax of Na(+)‐dependent glutamine transport. 3. There was an inverse relationship between myotube glutamine transport and external osmolality after 30 min exposure. 4. During hyposmotic (170 mosmol kg‐1) exposure there were large, rapid increases of cell volume and glutamine transport; the latter increased transiently (during the cell swelling phase) by a maximum of approximately 80% at 2 min, (due to an increased Vmax for Na(+)‐dependent glutamine transport) then decayed to a new elevated steady state after 30 min exposure. 5. During hyperosmotic (430 mosmol kg‐1) exposure there were rapid decreases in glutamine transport and myotube cell volume (both by approximately 30%) to values which were maintained for at least 15 min. 6. The volume‐sensitive glutamine transport process features characteristics of the insulin‐sensitive system Nm transporter. 7. Modulation of Na(+)‐dependent glutamine transport by insulin and cell volume changes may contribute towards regulation of muscle metabolism.
Force responses following stepwise length changes of rat skeletal muscle fibre types.
Authors: Galler S , Hilber K , Pette D
Published: December 2014

1. Force responses following stepwise length changes of Ca(2+)‐activated skinned leg muscle fibres (6 degrees C) of the rat were correlated with their myosin heavy chain (HC) isoforms (myosin HC I, fibre type I; myosin HC IIA, type IIA; myosin HC IID (HC IIX), type IID (type IIX); myosin HC IIB, type IIB) in order to study the mechanical properties of these molecules. 2. Marked differences in the time behaviour of force transients following quick releases of fibre length existed between various muscle fibres, and a conspicuous correlation with their myosin HC complement was noticed (order of velocity: IIB > IID > IIA > > I). No differences were found in the relationship between the applied length step and the resulting force (T1, T2 curves). 3. Our results suggest that the heads of various myosin heavy chain isoforms exhibit different kinetic properties. The differences concern the kinetics of the myosin head movements and the duration of cyclic interactions between myosin heads and thin filaments. The extent of force‐generating movements and the mean elongation of attached heads in the isometric state seem to be independent of the isoform.
Caffeine enhances intramembranous charge movement in frog skeletal muscle by increasing cytoplasmic Ca2+ concentration.
Authors: Shirokova N , Ríos E
Published: December 2014

1. Currents of intramembranous charge movement were recorded, together with intracellular [Ca2+], in single muscle fibres subjected to voltage‐clamp depolarization and ‘pulses’ of extracellular solution with a Ca2+ release‐inducing concentration of caffeine (10 mM). 2. When caffeine was present prior to and during the voltage pulses, the charge transferred by pulses to between ‐60 and ‐40 mV increased by about 40%. 3. In fibres depleted of Ca2+ in the sarcoplasmic reticulum (SR), caffeine had no effect on charge transfer or kinetics. 4. Whenever the prior exposure to caffeine resulted in a large elevation in [Ca2+]i at the start of the depolarizing pulse, there was an increase in I beta, the monotonically decaying component of charge movement. When the presence of caffeine enhanced Ca2+ release induced by the pulse, there was increase in I gamma, the hump‐like component. 5. The charge transferred during a pulse to ‐50 mV increased with time of exposure to caffeine. Ca2+ release induced by the voltage pulse grew during the first second of caffeine exposure, then decreased with longer exposure time. The enhancement of charge transfer by caffeine was therefore not due to the increase in Ca2+ release caused by the drug. 6. The increase in charge transfer was a uniform, monotonically increasing function of the [Ca2+]i attained at the end of the voltage pulse. 7. Charge transfer, as a function of [Ca2+]i, pulse voltage and time, was simulated with a model, used previously, in which Ca2+ binds to intracellular sites and increases the electrical potential near the voltage sensors. Two sites were needed to fit the observations, with dissociation constants of 60 nM and 2 to 10 microM. 8. In the presence of caffeine, the voltage‐driven movement of a given amount of intra‐membranous charge resulted in greater activation of release permeability. 9. All effects of caffeine observed in this and the preceding paper could be explained assuming a single action: caffeine increases the tendency of the release channels to open. This results in opening of closed channels and an increase in their susceptibility to activation by the voltage sensors.
Properties of single motor units in medial gastrocnemius muscles of adult and old rats.
Authors: Kadhiresan V A , Hassett C A , Faulkner J A
Published: December 2014

1. The purpose of this study was to determine the role of motor unit remodelling in the deficit that develops in the maximum isometric tetanic force (Fo) of whole medial gastrocnemius (MGN) muscles in old compared with adult rats. The Fo values and morphological data were determined for MGN muscles and eighty‐two single motor units in muscles of adult (10‐12 months) and sixty‐two units in those of old (24‐26 months) F344 rats. During an unfused tetanus, fast and slow (S) motor units were identified by the presence and absence of sag, respectively. Fast‐fatigable (FF) and fast‐fatigue‐resistant (FR) units were classified by fatigue indices less than or greater than 0.50, respectively. 2. For old rats, whole MGN muscle Fo was 29% less than the value of 11.2 N measured for adult rats. The deficit in whole muscle Fo of old rats resulted from equivalent decreases in the number of motor units, 16% smaller than the adult value of ninety‐seven, and in the mean motor unit Fo value, 14% less than the adult value of 117 mN. 3. With ageing, little motor unit remodelling occurred in FR units, whereas the S and FF motor units demonstrated dramatic, but opposing, changes. For S units, the number of units remained constant, but the number of fibres per motor unit increased 3‐fold from 57 to 165. In contrast, the number of FF units decreased by 34% and the number of fibres per motor unit of the remaining units decreased to 86% of the adult value of 333. The age‐related remodelling of motor units appeared to involve denervation of fast muscle fibres with reinnervation of denervated fibres by axonal sprouting from slow fibres.
Effects of arachidonic acid upon the volume‐sensitive chloride current in rat osteoblast‐like (ROS 17/2.8) cells.
Authors: Gosling M , Poyner D R , Smith J W
Published: December 2014

1. The effects of arachidonic acid upon the volume‐sensitive Cl‐ current present in cultured osteoblastic cells (ROS 17/2.8) was studied using the whole‐cell patch‐clamp technique. 2. Arachidonate produced two distinct phases of inhibition, a rapid phase occurring within 10‐15 s of application, preceding a slower phase that occurred 2 min after onset of arachidonate superfusion. Accompanying the slower inhibitory phase was an acceleration of the time‐dependent inactivation exhibited by the current at strongly depolarized potentials (> + 50 mV). The half‐maximal inhibitory concentrations (IC50) were 177 +/‐ 31 and 10 +/‐ 4 microM for the two phases, respectively. 3. Arachidonate was still effective in the presence of inhibitors of cyclo‐oxygenase (indomethacin, 10 microM), lipoxygenase (nordihydroguaretic acid, 10‐100 microM) and cytochrome P450 (SKF525A, 100 microM; ethoxyresorufin, 10 microM; metyrapone, 500 microM; piperonyl butoxide, 500 microM; cimetidine, 1 mM). The effects of arachidonate could not be produced by another cis unsaturated fatty acid, oleic acid. 4. Measurements of cell volume showed that arachidonate effectively inhibited the regulatory volume decrease elicited by ROS 17/2.8 cells in response to a reduction in extracellular osmolarity. 5. It is concluded that the volume‐sensitive Cl‐ conductance in ROS 17/2.8 cells is directly modulated by arachidonate and may represent a physiological mechanism by which volume regulation can be controlled in these cells.
Activation of protein kinase C inhibits calcium‐activated potassium channels in rat pituitary tumour cells.
Authors: Shipston M J , Armstrong D L
Published: December 2014

1. The regulation of large‐conductance, calcium‐ and voltage‐dependent potassium (BK) channels by protein kinase C (PKC) was investigated in clonal rat anterior pituitary cells (GH4C1), which were voltage clamped at ‐40 mV in a physiological potassium gradient through amphotericin‐perforated patches. 2. Maximal activation of PKC by 100 nM phorbol 12, 13‐dibutyrate (PdBu) almost completely inhibited the voltage‐activated outward current through BK channels. In contrast PdBu had no significant effect on the residual outward current after block of BK channels with 2 mM TEA or 30 nM charybdotoxin. In single‐channel recordings from cell‐attached patches, PdBu reduced the open probability of BK channels more than eightfold with no significant effect on mean open lifetime or unitary conductance. 3. The effects of PdBu on BK channels were not mimicked by the 4 alpha‐isomer, which does not activate PKC, and were blocked almost completely by 25 microM chelerythrine, a specific, noncompetitive PKC inhibitor. 4. PdBu had no significant effect on the amplitude of the pharmacologically isolated, high voltage‐activated calcium current. 5. Inhibition of BK channel activity by PKC provides the first molecular mechanism linking hormonal activation of phospholipase C to sustained excitability in pituitary cells.
The ventral medullary respiratory network of the mature mouse studied in a working heart‐brainstem preparation.
Authors: Paton J F
Published: December 2014

1. This report provides the first description of respiratory network activity within the ventrolateral medulla of the mature mouse obtained from a unique working heart‐brainstem preparation (WHBP). 2. In the WHBP three distinct respiratory phases were evident in recordings of both phrenic and vagal efferent nerves. These included a ramp inspiratory (I) discharge, post‐inspiratory (PI) activity and a silent or expiratory interval (E2). 3. Extracellular recordings were made from different types of respiratory neurones located within, or in close proximity to, the nucleus ambiguus. Based on firing patterns and phase relative to phrenic nerve discharge, respiratory neurone types, including pre‐inspiratory (PreI), early‐inspiratory, throughout inspiratory (I), late‐inspiratory, post‐inspiratory (PI) and stage II expiratory or E2 neurones were characterized. 4. Intracellular recordings were made from four types of respiratory neurones (PreI, I, PI and E2 neurones). PreI neurones were depolarized maximally during the E2‐inspiratory transition. I neurones exhibited a ramp depolarization which started either before or at the onset of phrenic discharge. Based on the kinetics of the inspiratory‐related hyperpolarizations and duration of discharge, two types of PI neurones were found (rapidly adapting and slowly adapting). E2 neurones were hyperpolarized during both the inspiratory and post‐inspiratory phases. 5. Phase‐dependent chloride‐mediated inhibition was studied in PreI, PI and E2 neurones and included: late inspiratory inhibition of PreI neurones; inspiratory‐related inhibition of PI and E2 neurones; and post‐inspiratory inhibition of PreI and E2 neurones. In addition, pre‐inspiratory inhibition of PI neurones was also demonstrated. 6. The WHBP appears to be viable for analysing reflex, synaptic and cellular mechanisms regulating respiratory activity in an in vitro milieu. The synaptic organization of the respiratory network of the mouse appears comparable to that of the rat and cat. The possibility of a mutual inhibitory interaction between PreI and PI neurones is discussed in terms of the functional organization of the respiratory network in the mouse.