bluuebunny/crossref_metadata_2025 huggingface dataset:
Noradrenaline induces IPSCs in rat medial septal/diagonal band neurons: involvement of septohippocampal GABAergic neurons.
Authors: Alreja M , Liu W
Published: December 2014

1. The physiological and pharmacological actions of noradrenaline (NA) on neurons of the medial septum and diagonal band of Broca (MSDB) were examined using extracellular, intracellular and whole‐cell patch‐clamp recordings in an in vitro rat brain slice preparation. 2. In current‐ and voltage‐clamp recordings with KCl‐ or potassium gluconate‐containing electrodes, bath‐applied NA increased the number of tetrodoxin‐ and bicuculline‐sensitive synaptic events in > 80% of cholinergic‐ and GABA‐type neurons tested. The NA‐induced synaptic activity originated from GABAergic neurons located within the MSDB itself, as a similar effect occurred in brain slices in which the MSDB had been surgically isolated from neighbouring structures. 3. In antidromic studies, NA dose‐dependently increased firing in a subpopulation of septohippocampal neurons with fast conducting fibres (mean conduction velocity, 1.78 +/‐ 0.10 m s‐1; presumably GABAergic). The NA excitation was mimicked by the alpha 1‐agonist phenylephrine (PE) and blocked by the alpha 1‐antagonists prazosin and WB‐4101, suggesting the presence of alpha 1‐receptors on septohippocampal GABAergic neurons. 4. Similarly, in whole‐cell recordings in both cholinergic‐ and non‐cholinergic‐type MSDB neurons, prazosin blocked the effects of NA and PE mimicked the effects of NA by inducing IPSCs with a similar amplitude distribution. 5. Consistent with the above findings, GABA‐type neurons that responded directly to NA and PE with a prazosin‐sensitive inward current were found within the MSDB. 6. In conclusion, NA, via alpha 1‐adrenoceptors, excites MSDB septohippocampal GABAergic neurons and influences both septal and septohippocampal circuitry.
The pattern of stimulation influences the amount of oscillatory work done by frog muscle.
Authors: Stevens E D
Published: December 2014

1. A doublet is the interjection of an additional action potential at the beginning of a regular motoneuron discharge pattern. The present experiments were designed to estimate the magnitude of the effect of doublets on the capacity of isolated frog sartorius muscle to do work during oscillatory length changes. 2. For the work loop method, the muscle was subjected to sinusoidal length changes at 4 Hz. Work was calculated from the loop formed when force was plotted against length. Work done was positive when the muscle was shortening and was negative when the muscle was lengthening; net work was the difference. 3. Adding pulses at the start of the stimulus pulse train increased isometric force and markedly increased net work per cycle. Adding one pulse increased the net work by about 52%, but increased isometric force by only 24%. 4. Eighty per cent of the maximum net work was achieved by adding only three pulses at the start of a low‐frequency pulse train, i.e. 80% of the work achieved with seventeen pulses could be achieved with only six pulses. 5. The maximum net work per stimulus pulse was achieved with a stimulus train that consisted of five pulses with two being additional initial pulses at 5 ms interpulse intervals, i.e. a triplet. 6. The results of the present study imply that doublet stimulation patterns may be important to reduce the fatigue that occurs during artificial neuromuscular stimulation of skeletal muscle.
Intracellular Ca2+ changes and Ca2+‐activated K+ channel activation induced by acetylcholine at the endplate of mouse skeletal muscle fibres.
Authors: Allard B , Bernengo J C , Rougier O , Jacquemond V
Published: December 2014

1. Enzymatically isolated skeletal muscle fibres were used to investigate the effects of applying acetylcholine (ACh) onto the endplate area on intracellular free calcium concentration ([Ca2+]i) measured using the indicator indo‐1 and single channel activity using the patch clamp technique. 2. Using a Tyrode solution containing 5 microM tetrodotoxin (TTX) as extracellular solution, ACh applications (at 0.1 or 1 mM) onto the endplate induced intracellular free calcium transients the mean maximal amplitude of which was 360 +/‐ 30 nM from a mean resting value of 72 +/‐ 7 nM (n = 13). In cells bathed with a K(+)‐rich solution (145 mM K+), applications of ACh (0.1 mM) induced transient rises in [Ca2+]i from a mean resting value of 53 +/‐ 7 nM to a maximum of 222 +/‐ 24 nM (n = 33). 3. In cell‐attached membrane patches at the endplate membrane of muscle fibres bathed in a K(+)‐rich external solution, using a pipette filled with Tyrode solution, external application of 0.1 mM ACh could induce a transient burst opening of channels carrying an outward current of an average amplitude of 4.6 +/‐ 0.2 pA at 0 mV (n = 8). 4. These channels were characterized as Ca2(+)‐activated K+ channels. At 0 mV, in inside‐out patches excised from the endplate membrane area, they displayed a conductance of 60 and 224 pS in the presence of Tyrode and K(+)‐rich solution in the pipette, respectively. Half‐maximum activation was found for a [Ca2+]i close to 4 microM. The channels showed a typical voltage dependence. In outside‐out patches these channels were shown to be blocked by 100 nM charybdotoxin (CTX). 5. In fibres bathed in a Tyrode solution containing TTX (5 microM), CTX had no clear effect on the change in membrane voltage, recorded near the endplate with a single intracellular microelectrode, in response to the application of ACh. 6. Although the physiological relevance of this ACh‐induced K+ channel activation remains unclear, results suggest that, in the presence of a physiological extracellular [Ca2+], Ca2+ entry through the endplate nicotinic receptors can produce a local increase in [Ca2+]i, sufficient to trigger the opening of Ca2+‐activated K+ channels in the adjacent surface membrane.
Downregulation of muscarinic M2 receptors linked to K+ current in cultured guinea‐pig atrial myocytes.
Authors: Bünemann M , Brandts B , Pott L
Published: December 2014

1. Desensitization of muscarinic K+ current (IK(ACh)) was studied in cultured atrial myocytes from guinea‐pig hearts using whole‐cell voltage clamp. 2. Three different types of desensitization could be identified. A fast component which upon rapid superfusion with ACh resulted in a partial relaxation of IK(ACh) within a few seconds to a plateau which was maintained in the presence of ACh. Recovery from this type of desensitization paralleled the decay of IK(ACh) after washout of the agonist. A second type of desensitization was observed within minutes. This was reversed around 10 min after washout of ACh. Both types were heterologous with regard to the A1 receptor and the novel phospholipid (Pl) receptor, both of which activate IK(ACh) via the same signalling pathway. 3. A third type of desensitization (downregulation) occurred upon exposure of the cultures for 24‐48 h to the muscarinic agonist carbachol (CCh). The level of downregulation depended on the concentration of CCh (0.1 microM < or = [CCh] < or = 10 microM). No recovery was observed within 5 h after washout of CCh. Thereafter sensitivity to ACh slowly returned (half‐time (t1/2), approximately 20 h). 4. Downregulation by CCh (0.1‐5 microM) was characterized by an increase in EC50 for ACh with no reduction in maximum IK(ACh). With 5 microM CCh, EC50 was increased from 0.1 to 3.7 microM. At 10 microM CCh EC50 was increased to 15 microM and maximal current that could be evoked by ACh was reduced to 15%. 5. Downregulation by CCh was homologous with regard to A1 and Pl receptors. Maximum IK(ACh), assayed by a saturating concentration of Pl, was not reduced in downregulated cells, suggesting a mechanism localized at the M2 receptor. 6. The changes in the concentration‐response curves can be accounted for by assuming an excess of M2 receptors relative to the subsequent component of the signalling pathway. 7. As the intact heart is under tonic vagal control, downregulation is likely to contribute to controlling the sensitivity of the heart to vagal activity in situ.
The vasodilator component of neurogenic inflammation is caused by a special subclass of heat‐sensitive nociceptors in the skin of the pig.
Authors: Lynn B , Schütterle S , Pierau F K
Published: December 2014

1. Skin blood flow has been imaged during stimulation of fine nerve filaments containing small numbers of identified C fibre units. Filaments were dissected from the saphenous nerve of anaesthetized pigs. 2. Stimulation of filaments containing C heat nociceptor units gave small areas of elevated blood flow (average increase 96%, n = 11) restricted to the afferent receptive field. The extent of the areas of raised blood flow was imaged completely for 8 units. The average extent of vasodilatation in the direction of greatest spread was 8 mm and the maximum spread in any unit was 13 mm. 3. Stimulation of C polymodal nociceptor units never caused increases in blood flow in or near their receptive fields. 4. Localized noxious stimuli (55 degrees C or intradermal injection of capsaicin) caused flare extending 7‐15 mm in the same skin region. 5. In agreement with the axon reflex model, spread of flare was restricted to the zone innervated by the terminals of single C fibre units. 6. It is concluded that the C heat nociceptor units are the major class of afferent involved in the flare reaction in the skin of the pig. C polymodal nociceptor units do not appear to be involved in flare in this species. The probable situation in human skin, which is also innervated by heat nociceptors, is discussed.
Oxidized glutathione mediates cation channel activation in calf vascular endothelial cells during oxidant stress.
Authors: Koliwad S K , Elliott S J , Kunze D L
Published: December 2014

1. The oxidant, tert‐butylhydroperoxide (tBuOOH) depolarizes calf pulmonary artery endothelial cells by activating a non‐selective cation channel. To identify the molecular mediator of channel activation during oxidant stress, the patch‐clamp technique was used to compare tBuOOH‐induced changes in membrane potential and channel activity with those induced by oxidized glutathione (GSSG), a cytosolic product of oxidant metabolism. 2. When recording pipettes contained GSSG (2 mM), whole‐cell zero‐current potential measured immediately following pipette break‐in was not different from control values (‐57 mV). However, within 20 min of break‐in, zero‐current potential was depolarized to ‐7 mV. The time course of depolarization was dependent on the concentration of GSSG and was accelerated by inhibition of GSSG metabolism. 3. In excised membrane patches, channels were activated by internal GSSG, but not by internal tBuOOH, reduced glutathione (GSH), or external GSSG. Channels were equal in size (28 pS) and in ionic selectivity to those activated by incubation of intact cells with tBuOOH. As little as 20 microM GSSG was sufficient to maximally activate channels. However, the time course of channel activation was concentration dependent between 20 microM and 2 mM GSSG. 4. Channel activation by GSSG was reversed by GSH and by increasing the [GSH]:[GSSG] ratio. Likewise, channel activation by pre‐incubation of intact cells with tBuOOH was reversed by GSH applied after patch excision. 5. These results strongly suggest that GSSG is an endogenous intracellular mediator of channel activation and depolarization during oxidant stress.
Contribution of oxygen‐sensitive neurons of the rostral ventrolateral medulla to hypoxic cerebral vasodilatation in the rat.
Authors: Golanov E V , Reis D J
Published: December 2014

1. We sought to determine whether hypoxic stimulation of neurons of the rostral ventrolateral reticular nucleus (RVL) would elevate regional cerebral blood flow (rCBF) in anaesthetized paralysed rats. 2. Microinjection of sodium cyanide (NaCN; 150‐450 pmol) into the RVL rapidly (within 1‐2 s), transiently, dose‐dependently and site‐specifically elevated rCBF1 measured by laser Doppler flowmetry, by 61.3 +/‐ 22.1% (P < 0.01), increased arterial pressure (AP; +30 +/‐ 8 mmHg; P < 0.01)1 and triggered a synchronized 6 Hz rhythm of EEG activity. 3. Following cervical spinal cord transection, NaCN and also dinitrophenol (DNP) significantly (P < 0.05) elevated rCBF and synchronized the EEG but did not elevate AP; the response to NaCN was attenuated by hyperoxia and deepening of anaesthesia. 4. Electrical stimulation of NaCN‐sensitive sites in the RVL in spinalized rats increased rCBF measured autoradiographically with 14C iodoantipyrine (Kety method) in the mid‐line thalamus (by 182.3 +/‐ 17.2%; P < 0.05) and cerebral cortex (by 172.6 +/‐ 15.6%; P < 0.05) regions, respectively, directly or indirectly innervated by RVL neurons, and in the remainder of the brain. In contrast regional cerebral glucose utilization (rCGU), measured autoradiographically with 14C‐2‐deoxyglucose (Sokoloff method), was increased in proportion to rCBF in the mid‐line thalamus (165.6 +/‐ 17.8%, P < 0.05) but was unchanged in the cortex. 5. Bilateral electrolytic lesions of NaCN sensitive sites of RVL, while not altering resting rCBF or the elevation elicited by hypercarbia (arterial CO2 pressure, Pa,CO2, approximately 69 mmHg), reduced the vasodilatation elicited by normocapnic hypoxaemia (arterial O2 pressure, Pa,O2, approximately 27 mmHg) by 67% (P < 0.01) and flattened the slope of the Pa,O2‐rCBF response curve. 6. We conclude that the elevation of rCBF produced in the cerebral cortex by hypoxaemia is in large measure neurogenic, mediated trans‐synaptically over intrinsic neuronal pathways, and initiated by excitation of oxygen sensitive neurons in the RVL.
Modulation of glycogen synthesis in rat skeletal muscle by changes in cell volume.
Authors: Low S Y , Rennie M J , Taylor P M
Published: December 2014

1. The hypothesis that cellular hydration state modulates muscle glycogen synthesis was tested by measuring the incorporation of [14C]glucose into glycogen (glycogen synthesis) in primary rat myotubes after experimentally induced volume changes. 2. Glycogen synthesis in myotubes increased (by 75%, P < 0.01) after swelling induced by 60 min exposure to hyposmotic media (170 mosmol kg‐1) relative to isosmotic control (300 mosmol kg‐1) values, it decreased (by 31%, P < 0.05) after shrinkage induced by 60 min exposure to hyperosmotic (430 mosmol kg‐1) media. Myotube 2‐deoxy‐D‐glucose (0.05 mM) uptake was unaffected by changes in external osmolality. 3. Wortmannin (100 nM; 60 min), a phosphatidylinositol 3‐kinase inhibitor, decreased basal glycogen synthesis by 28% whereas rapamycin (100 nM; 60 min), which blocks the activation of p70 S6 kinase, had no effect. Both wortmannin (100 nM; 60 min) and rapamycin (100 nM; 60 min) blocked the changes in glycogen synthesis resulting from hypo‐ and hyperosmotic exposure. 4. Myotube glycogen synthesis is modulated by volume changes independently of changes in glucose uptake. The phenomenon may be physiologically important in promoting glycogen storage during circumstances of myofibrillar swelling, e.g. after feeding or exercise.
Activation of MAP kinases and phosphorylation of caldesmon in canine colonic smooth muscle.
Authors: Gerthoffer W T , Yamboliev I A , Shearer M , Pohl J , Haynes R , Dang S , Sato K , Sellers J R
Published: December 2014

1. Phosphorylation of caldesmon was assayed in canine colonic circular smooth muscle strips labelled with 32P and stimulated with 10 microM acetylcholine. Caldesmon was isolated by two‐dimensional non‐equilibrium pH gel electrophoresis. Stimulation with acetylcholine increased caldesmon phosphorylation significantly from a basal level of 0.6 +/‐ 0.07 to 1.1 +/‐ 0.15 mol P1 (mol caldesmon)‐1 after 2 min. 2. MAP kinase activities were measured in SDS extracts of muscle by a gel reconstitution method using myelin basic protein. Myelin basic protein kinase activities were observed at 38, 44, 50 and 57 kDa by the gel reconstitution method. Endogenous caldesmon kinase activities were also identified by the gel reconstitution method at 38, 44 and 50 kDa. The 38 and 44 kDa kinases comigrated with proteins labelled by anti‐ERK1 MAP kinase antibodies on Western blots. Both 38 and 44 kDa MBP kinase activities increased significantly during contractions induced by 10 microM acetylcholine, 0.1 microM neurokinin A and 70 mM potassium. 3. Phorbol dibutyrate (0.1 microM) potentiated activation of MAP kinases and contraction of depolarized muscles while producing a decrease in fura‐2 fluorescence ratio. This suggests that protein kinase C activation is coupled to MAP kinase activity in colonic smooth muscle. 4. MAP kinases isolated form muscle homogenates by Mono Q chromatography were assayed using the specific MAP kinase substrate peptide APRTPGGRR. Stimulation of muscles for 2 min with 10 microM acetylcholine activated both ERK1 and ERK2 MAP kinase activities 2‐fold. 5. To determine the effects of caldesmon phosphorylation by MAP kinase on the cross‐bridge cycle, actin sliding velocity was measured with an in vitro motility assay. Unphosphorylated turkey gizzard caldesmon (3 microM) significantly reduced mean sliding velocity. Phosphorylation of caldesmon with sea star ERK1 MAP kinase reversed the inhibitory effect of caldesmon on sliding velocity. The results are consistent with a protein kinase cascade being activated by contractile agonists in gastrointestinal smooth muscle which activates ERK MAP kinases leading to phosphorylation of caldesmon. Phosphorylation of caldesmon in vivo may reverse inhibitory influences of caldesmon on cross‐bridge cycling.
Distinct voltage‐dependent gating behaviours of a swelling‐activated chloride current in human epithelial cells.
Authors: Braun A P , Schulman H
Published: December 2014

1. The swelling‐activated chloride current is critical in the homeostatic regulatory volume decrease (RVD) of both excitable and non‐excitable cells. Although not activated by voltage, it displays kinetic behaviour similar to voltage‐gated Shaker‐type potassium currents. We have studied the voltage‐dependent properties of this current in single T84 human cell line epithelial cells using whole‐cell patch clamp methodology. 2. An external anion permeability sequence of I‐ > Cl‐ > methanesulphonate (MeSO3‐) was observed for the swelling‐activated current. Extracellular application of the chloride channel blocker DIDS (100 microM) resulted in approximately 50% block of the current in a voltage‐dependent manner. 3. At positive membrane potentials, the swelling‐activated chloride current undergoes time‐dependent inactivation. Following such inactivation, recovery of both the inward and outward components of the macroscopic current was found to be voltage dependent. The time constants describing these two individual recovery processes were identical over a range of membrane potentials. In addition, the magnitude of current recovery was directly dependent upon the degree prior inactivation of current at positive voltage. 4. We further observed that the swelling‐activated current undergoes a form of steady‐state, voltage‐dependent inactivation that appears to differ from the inactivation observed at positive potentials. This steady‐state inactivation occurred over the physiological voltage range, with a membrane potential at half‐maximal inactivation (V1/2) of ‐72 mV, and differed from the time‐dependent inactivation observed at positive membrane potentials, which occurred with a V1/2 of 40 mV. These observations demonstrate two distinct forms of voltage‐dependent inactivation, probably reflecting two separate gating processes at the level of the channel. 5. These latter properties are thus anticipated to regulate voltage‐dependent chloride efflux under cell swelling conditions and further influence RVD and membrane excitability in cells generating action potentials.
Ventilatory chemoreflexes at rest following a brief period of heavy exercise in man.
Authors: Clement I D , Pandit J J , Bascom D A , Robbins P A
Published: December 2014

1. Ventilatory chemoreflex responses have been studied at rest during the recovery from a brief period of heavy exercise. 2. Six young, healthy male subjects each undertook four experimental studies. In each study measurements were made at rest during recovery from an exhaustive 1‐2 min sprint on a bicycle ergometer with a workload of 400 W. Three levels of end‐tidal O2 pressure (Po2) were employed. Continuous ventilatory measurements were made during euoxia (end‐tidal Po2, 100 Torr), hypoxia (end‐tidal Po2, 50 Torr) and hyperoxia (end‐tidal Po2, 300 Torr). Arterialized venous blood samples were drawn during each of the measurement periods for the estimation of arterial pH. In two of the studies, end‐tidal CO2 pressure (Pco2) was maintained throughout at 1‐2 Torr above the eucapnic level that existed prior to exercise (isocapnic post‐exercise protocol, IPE). In the other two studies, end‐tidal Pco2 was allowed to vary (poikilocapnic post‐exercise protocol, PPE). Data from a previously published study on the same subjects involving an infusion of hydrochloric acid were used to provide control data with a varying level of metabolic acidosis, but with no prior exercise. 3. Ventilation‐pH slopes in the IPE protocol were no different from control. Ventilation‐pH slopes in the PPE protocol were significantly lower than in the IPE and control protocols (P < 0.05, ANOVA). This difference may be due to the progressive change in end‐tidal Pco2 in the PPE protocol compared with the constant end‐tidal Pco2 in the IPE and control protocols. 4. An arterial pH value of 7.35 was attained 30.4 +/‐ 2.7 min (mean +/‐ S.E.M.) after the end of exercise in the IPE protocol and 17.1 +/‐ 1.4 min after the end of exercise in the PPE protocol. 5. Hypoxic sensitivities at an arterial pH of 7.35 were not significantly different between the IPE, PPE and control protocols (ANOVA). 6. Euoxic ventilation at an arterial pH 7.35 was significantly greater than control for the IPE protocol (P < 0.001, Student's paired t test) and no different from control for the PPE protocol. 7. The results suggest that 30 min after heavy exercise, ventilation remains stimulated by processes other than the post‐exercise metabolic acidosis, and that changes in peripheral chemoreflex sensitivity to hypoxia and acid are not implicated in this.
Single L‐type calcium channel conductance with physiological levels of calcium in chick ciliary ganglion neurons.
Authors: Church P J , Stanley E F
Published: December 2014

1. Single L‐type calcium channels in chick ciliary ganglion neurons were studied at high current resolution in cell‐attached patch recordings using quartz‐glass micropipettes. 2. A single open‐channel current amplitude was observed when Ba2+ was the charge carrier with a conductance of 26 pS at 110 mM barium. However, with 110 mM calcium two current fluctuation amplitudes were observed. These were termed low and high fluctuation amplitudes, CaL and CaH, and had conductances of 8.8 and 12 pS, respectively. These two levels probably reflect two different channel species. CaL was identified as an L‐type calcium channel on the basis of resistance to inactivation, conductance, and dihydropyridine sensitivity. 3. Single‐channel current fluctuations could be detected with calcium concentrations as low as 1.0 mM. Although the unitary conductance (gamma) was much greater with barium than calcium at every concentration tested, the concentration dependence of conductance was similar for gamma Ba, gamma CaH and gamma CaL. Fitting the concentration dependencies of these conductances with a Langmuir isotherm gave KD estimates of 4.7, 5.6 and 5.0 mM for barium, CaL and CaH, respectively 4. The single‐channel conductance of the L‐type channel (gamma L) can be described by the relation: conductance (in pS) = 9.2/(1 + 5.6/[Ca]) where [Ca] is the external calcium concentration in the 1.0‐110 mM range. Thus, at a physiological external calcium concentration of 2 mM the conductance is 2.4 pS. 5. Ca2+ transport through the L‐type calcium channel is particularly sensitive to changes in external calcium concentration in the physiological range but approaches saturation at about 10 mM. this characteristic may optimize the responsiveness of the cell to small changes in ambient calcium concentrations while limiting excess entry in the presence of abnormally high calcium levels.
Salbutamol and chronic low‐frequency stimulation of canine skeletal muscle.
Authors: Hu P , Zhang K M , Feher J J , Wang S W , Wright L D , Wechsler A S , Spratt J A , Briggs F N
Published: December 2014

1. The effect of simultaneous application of chronic muscle stimulation and salbutamol on the expression of mRNAs and proteins normally expressed by fast‐ or slow‐twitch fibres was followed and the effects of changes in protein expression on mechanical performance were evaluated. Chronic low‐frequency stimulation increased the myosin heavy chain (HC)‐I level in the canine latissimus dorsi muscle and simultaneous administration of salbutamol partially blocked this change. Associated with the increase in HC‐I level was a decrease in the velocity of shortening at zero load, VMAX. The change in VMAX was partially blocked by salbutamol. 2. Chronic low‐frequency stimulation increased the levels of slow‐twitch cardiac isoform sarco‐/endoplasmic reticulum Ca(2+)‐ATPase (SERCA2a) and phospholamban mRNA, and SERCA2a and phospholamban protein expression. These changes were associated with an increase in time‐to‐peak tension and a decrease in fusion frequency. Simultaneous administration of salbutamol blocked these changes in protein expression and muscle mechanics. Chronic stimulation of latissimus dorsi decreased the levels of the fast‐twitch isoform of sarco‐/endoplasmic reticulum Ca(2+)‐ATPase (SERCA1a) and increased SERCA2a protein expression and decreased calcium uptake rate by muscle homogenates. These changes were blocked by salbutamol. 3. The loss of latissimus dorsi muscle weight by chronic stimulation was partially blocked by salbutamol.
Dopamine modulates unitary conductance of single PL‐type calcium channels in Roccus chrysops retinal horizontal cells.
Authors: Pfeiffer-Linn C L , Lasater E M
Published: December 2014

1. Dopamine modulation of the PL‐type calcium channel of white bass retinal horizontal cells was studied in isolated, cultured neurons. Single‐channel recordings were made of calcium channels in outside‐out patches, under conditions which favoured the expression of calcium channel activity. 2. Analysis of single‐channel properties revealed that dopamine potentiated the activity of the sustained calcium channel in three ways. First, it increased unitary conductance through individual channels. Under the influence of dopamine, single‐channel conductance doubled. 3. Dopamine also increased the probability of channel opening and increased channel mean open time. The probability of opening increased 4‐fold while mean open time doubled. 4. The mean closed time was also affected. The time between individual openings was not affected but the closed time between bursts of openings was shortened by over 50%. 5. The effects of dopamine were mediated via the activation of a D1‐type receptor and the resulting activation of a cAMP‐mediated second messenger system. 6. The combination of the effects of dopamine significantly increased the net calcium influx into the cell.
Purinergic stimulation of rabbit ciliated airway epithelia: control by multiple calcium sources.
Authors: Korngreen A , Priel Z
Published: December 2014

1. Simultaneous measurements of average intracellular calcium concentration ([Ca2+]i) and ciliary beat frequency (CBF) were carried out on ciliated rabbit tracheal cells in order to determine quantitatively the role of calcium in the regulation of mucus‐transporting cilia. 2. Extracellular ATP caused a rapid increase in both [Ca2+]i and CBF in the 0.1‐1000 microM concentration range. The rise in [Ca2+]i levelled off to an elevated [Ca2+]i plateau while the cilia remained in a high activation state. The magnitude of the rise in [Ca2+]i and CBF as well as the value of the elevated [Ca2+]i plateau and the value of the sustained CBF were dependent on the concentration of ATP in the solution. 3. No correlation was found between the mean values of [Ca2+]i and CBF at rest but a sigmoidal relationship was found to exist between the maximal rises of these parameters following excitation with extracellular ATP. This sigmoidal correlation incorporated the experiments where [Ca2+]i rise was induced by depletion of internal calcium stores with thapsigargin or by entry of calcium induced by ionomycin. 4. Extracellular ATP caused both the release of calcium from internal stores and calcium influx from the extracellular solution. The release of calcium was identified as originating from a thapsigargin‐sensitive and a thapsigargin‐insensitive calcium store. It is suggested that the release of calcium from these stores induces the initial rise in CBF. 5. The sustained activation of the cilia and elevated calcium plateau were found to be the result of the extracellular ATP‐induced calcium influx. This calcium influx was insensitive to the voltage‐gated calcium channel inhibitors verapamil and diltiazem, but was completely eliminated by lowering the extracellular calcium concentration to 0.1 microM. 6. We propose that the initial jump in the CBF is mediated by the calcium released from a thapsigargin‐insensitive calcium store adjacent to the cilia, while the later, and longer, rise in CBF is the result of the calcium emanating from the thapsigargin‐sensitive store which is positioned further away from the cilia within the cell cytoplasm. The calcium influx that follows is responsible for sustaining the cilia at a high level of excitation.
Repriming and activation alter the frequency of stereotyped discrete Ca2+ release events in frog skeletal muscle.
Authors: Lacampagne A , Lederer W J , Schneider M F , Klein M G
Published: December 2014

1. Brief localized elevations in myoplasmic [Ca2+] (Ca2+ sparks) in individual sarcomeres of voltage‐clamped frog skeletal muscle fibres were examined by laser scanning confocal microscopy. 2. Fibres held at 0 mV were briefly repolarized to ‐90 mV (repriming pulse) to restore only a small fraction of sarcoplasmic reticulum (SR) calcium release. Subsequent depolarization to 0 mV (test pulse) caused the appearance of small numbers of Ca2+ sparks at different sarcomeres from pulse to pulse. Increasing the repriming time resulted in an increase in the frequency of occurrence of the Ca2+ sparks. 3. The amplitude and spatio‐temporal extent of the Ca2+ sparks were independent of the repriming time and test pulse voltage. Ca2+ sparks recorded during small depolarizations of fibres held at ‐90 mV had a similar amplitude and spatio‐temporal extent as those recorded after brief repriming of the same fibre held at 0 mV. 4. We conclude that stereotyped Ca2+ sparks underlie calcium release at all voltages and all extents of repriming. The amplitude of Ca2+ release is thus graded by the frequency but not by the amplitude or spatio‐temporal extent of the individual SR Ca2+ release events.
Subcellular properties of triggered Ca2+ waves in isolated citrate‐loaded guinea‐pig atrial myocytes characterized by ratiometric confocal microscopy.
Authors: Lipp P , Hüser J , Pott L , Niggli E
Published: December 2014

1. Spatiotemporal aspects of subcellular Ca2+ signalling were studied in cultured adult guinea‐pig atrial myocytes. A mixture of the Ca2+ indicators fluo‐3 and Fura Red in combination with laser‐scanning confocal microscopy was used for [Ca2+]i measurements while membrane currents were recorded simultaneously. 2. In citrate‐loaded atrial myocytes not every Ca2+ current (ICa) could trigger Ca2+ release from the sarcoplasmic reticulum (SR). Two types of Ca2+ signals could be observed: Ca2+ transients resulting from (i) Ca2+ influx alone and (ii) additional Ca2+ release. 3. Ca2+ release elicited by voltage steps of 100‐150 ms duration was either apparently homogeneous or propagated as Ca2+ waves through the entire cell. With brief ICa (50‐75 ms), Ca2+ waves with limited subcellular propagation were observed frequently. These waves always originated from either end of the myocyte. 4. The time course of changes in Na(+)‐Ca2+ exchange current (INaCa) depended on the subcellular properties of the underlying Ca2+ transient and on the particular cell geometry. Apparently homogeneous Ca2+ release was accompanied by an inward change of INaCa the onset phase of which was fused with ICa. Changes in INaCa caused by a Ca2+ wave propagating through the entire cell showed a W shape, which could be attributed to differences of the fractional surface‐to‐volume ratio in different cell segments during propagation of the Ca2+ wavefront. Those waves with limited spreading only activated a small component of INaCa. 5. The different subcellular patterns of Ca2+ release signals can be explained by spatial inhomogeneities in the positive feedback of the SR. This depends on the local SR Ca2+ loading state under the control of the local Ca2+ influx during activation of ICa. Due to the higher surface‐to‐volume ratio at the two ends of the myocyte, SR loading and therefore the positive feedback in Ca(2+)‐induced Ca2+ release may be higher at the ends, locations where Ca2+ waves are preferentially triggered. 6. We conclude that the individual cell geometry may be an important determinant of subcellular Ca2+ signalling not only in cardiac muscle cells but presumably also in other types of cells that depend on Ca2+ signalling. In addition, the cell geometry in combination with varying subcellular Ca2+ release patterns can greatly affect the time course of Ca(2+)‐activated membrane currents.
The effects of repeated active stretches on tension generation and myoplasmic calcium in frog single muscle fibres.
Authors: Morgan D L , Claflin D R , Julian F J
Published: December 2014

1. A series of contractions with stretches (eccentric contractions) beyond the optimal length for tension generation (optimum) were shown to induce a shift in that optimum in single muscle fibres of frog, as has been previously reported for whole muscles. Shifts averaging 0.129 micron (sarcomere)‐1 or 6% were found, without apparent damage to the fibre. 2. The stiffness of fibres was found to fall during a stretch, even though tension was rising. In addition, the isometric stiffness fell as a result of a series of eccentric contractions. 3. Calcium‐sensitive fluorescent dyes indicated that such contractions did not reduce the amplitude of the intracellular calcium transient, but did increase its duration. A rise in resting [Ca2+] was found to accompany damage, but not necessarily the shift in optimum. 4. The twitch potentiator nitrate was shown to increase myoplasmic [Ca2+] during twitch and tetani, but not to reverse the shift in optimum length due to eccentric contractions. Both eccentric contractions and twitch potentiation reduced the maximum stimulation rate to which a fibre could respond with propagated action potentials. 5. These results exclude reduced myoplasmic [Ca2+] as the cause of the shift in optimum length in this preparation.
Invariance of the pattern electroretinogram evoked by psychophysically equivalent stimuli in human ageing.
Authors: Muir J A , Barlow H L , Morrison J D
Published: December 2014

1. The aim of this study was to assess the contribution of retinal ganglion cells to the decline in contrast sensitivity during human ageing. 2. After determination of the appropriate refraction for each subject, younger subjects were arranged to be exposed to a display luminance which was suprathreshold by the same amount as in older subjects wearing a 4.0 mm diameter artificial pupil with a neutral density filter. 3. In fifty‐four subjects, aged 20‐99 years, contrast sensitivities measured in response to phase‐reversed grating patterns of 2, 5 and 8 cycles per degree declined significantly with increasing age at each spatial frequency studied. 4. Subjects were made psychophysically equivalent by setting the display contrast at x5 and x10 contrast threshold for each subject. The pattern electroretinogram (PERG) was recorded with a sterile silver thread (DLT) electrode placed in the lower canthus of one eye, with the indifferent electrode on the temple and the earth on the forehead. 5. For each contrast multiple at each spatial frequency, the PERG implicit time showed no significant change with age, indicating equivalence of the response across the age range. 6. Control experiments with two young and two elderly subjects established that the PERG implicit time decreased appreciably with increasing contrast, over a range of x2 to x20 contrast threshold. 7. Since the psychophysically equivalent stimulus displays had generated equivalent PERGs in terms of implicit time in young and elderly subjects, this was consistent with the equivalence of retinal ganglion cell function under these conditions. 8. Adverse changes within the retina were therefore inferred to play a major role in the decline in contrast sensitivity with age.
Regulation of intracellular pH in salamander retinal rods.
Authors: Saarikoski J , Ruusuvuori E , Koskelainen A , Donner K
Published: December 2014

1. We measured intracellular pH (pHi) in rods isolated from the retina of the axolotl salamander, Ambystoma mexicanum, using the fluorescent indicator 2',7'‐bis(carboxyethyl)‐5(and ‐6)‐carboxyfluorescein (BCECF). 2. The light exposures associated with data acquisition had no marked effect on pHi. There was no sharp change between the value obtained from the first exposure of dark‐adapted rods and subsequent readings. Increasing the acquisition frequency from 1 to 10 min‐1 either had no effect, or brought about a slow acidification, which was stopped or reversed when the low frequency was restored. 3. In nominally HCO3(‐)‐free solution at pH 7.5, the rods had a steady‐state pHi of 7.09 +/‐ 0.02 (n = 46) and a buffering power (beta i) of 24 +/‐ 1 mM (pH unit)‐1 (n = 48). The buffering power was virtually constant in the pH range 6.6‐8.0. In the same range, pHi dependent linearly on perfusion pH (pHo) with regression coefficients of 0.4‐0.5. 4. There were no significant differences between the inner and outer segment of intact rods as regards steady‐state pHi or responses to experimental treatments. 5. Recovery from an intracellular acid load imposed by sodium propionate or an NH4Cl prepulse in nominally bicarbonate‐free perfusate was completely blocked by decreasing the extracellular Na+ concentration to 7 mM, and slowed by 86% by applying 1 mM amiloride. 6. Introduction of 2% CO2‐13 mM HCO3‐ caused an alkalinization that was often preceded by a transient acidification. Steady‐state pHi was on average 0.1 pH units higher than in nominally bicarbonate‐free solution. The mean acid extrusion rate, calculated on the assumption that CO2‐HCO3‐ behaves as an open system, was 19% higher (31 +/‐ 2 mM h‐1) than in a solution buffered only by Hepes (26 +/‐ 2 mM h‐1). 7. In the presence of CO2‐HCO3‐, 100 microM 4,4'‐diisothiocyanatostilbene‐2,2'‐disulphonic acid (DIDS) decreased the acid extrusion rate by 20% on average. Lowering the extracellular Cl‐concentration to 7 mM raised pHi, but did not significantly affect the acid extrusion rate. 8. We conclude that retinal rods regulate pHi by both Na(+)‐H+ exchange and mechanism(s) involving HCO3(‐)‐Cl‐ exchange. In the present conditions, the Na(+)‐H+ exchanger appears as the dominant mechanism for acid extrusion.
Solvent‐dependent rate‐limiting steps in the conformational change of sodium channel gating in squid giant axon.
Authors: Kukita F
Published: December 2014

1. The time course of sodium currents (INa) in squid giant axon was analysed using viscous non‐electrolyte solutions on both sides of the axolemma. It slowed reversibly as the non‐electrolyte concentration increased. The activation, deactivation (closing) and inactivation processes were slowed in a similar manner. The gating current of the sodium channel was also slowed to the same extent as the activation time constant. 2. The voltage dependence observed in a time constant vs. voltage relationship and a chord conductance vs. voltage relationship (activation curve), did not change significantly. 3. The gating kinetics have a similar temperature dependence in non‐electrolyte solutions, showing that the basic gating mechanism did not change in these solutions and only a slight increase in the activation free energy was one of the main causes of slowing. 4. Eight non‐electrolytes, formamide, ethylene glycol, glycerol, erythritol, glucose, sorbitol, sucrose and polyethylene glycol (mean molecular weight 600) were used. The amount of slowing was correlated with the gram concentration (g l‐1) of non‐electrolytes, but not with molar concentration (M) and solution osmolarity (osmol l‐1). 5. The percentage changes of the time constant were expressed as a function of the relative change in solution viscosity, eta/eta0. The proportionality constants alpha in the relationship alpha (eta/eta0), and gamma in the relationship 100 (eta/eta0)gamma, obtained using different non‐electrolytes, were close to 100% and 1, respectively. The simplest model to explain the results assumes that a slowing of a global conformational change is a consequence of sequential viscosity‐dependent movements of local structures (viscosity model). 6. Values of alpha and gamma deviated frequently from those in an ideal case, i.e. 100% for alpha and 1 for gamma, and they scattered, having a tendency to decrease as a function of molecular weight. 7. The slowing was also expressed as an exponential function of the solution osmolarity. A predicted solute‐inaccessible volume Va ranged (in nm3 per molecule) between 0.09 and 1.45. The value of Va increased as a logarithmic function of the molecular weight of the non‐electrolyte. 8. This solute‐inaccessible volume should be distributed in all hydrophilic parts of the sodium channel protein, but is not located in the channel conducting pore itself. The slowing of gating could be explained by a model in which a rate‐limiting step is a hydration process that occurs after local small structural changes have exposed new, unhydrated faces (transient hydrated‐states model). 9. Considering the opposite dependencies of parameters alpha (or gamma) and beta on the molecular weight, sodium channel gating is likely to reflect a combination of these two models, which are coupled in microscopic segment movements. We emphasize with this combination of models that fluctuating hydrophilic structures play an important role in determining time constants in the gating process.
Pacemaker activity in a sensory ending with multiple encoding sites: the cat muscle spindle primary ending.
Authors: Banks R W , Hulliger M , Scheepstra K A , Otten E
Published: December 2014

1. A combined physiological, histological and computer modelling study was carried out on muscle spindles of the cat tenuissimus muscle to examine whether there was any correlation between the functional interaction of putative encoding sites, operated separately by static and dynamic fusimotor neurones, and the topological structure of the preterminal branches of the primary sensory ending. 2. Spindles, whose I a responses to stretch and separate and combined static and dynamic fusimotor stimulation were recorded in physiological experiments, were located in situ. Subsequently the ramifications of the sensory ending were reconstructed histologically, and the topology of the branch tree was used in computer simulations of I a responses to examine the effect of the electronic separation of encoding sites on the static‐dynamic interaction pattern. 3. Interactions between separate static and dynamic inputs, manifest in responses to combineed stimulation, were quantified by a coefficient of interaction (Ci) which, by definition, was 1 for strictly linear summation of separate inputs and zero for maximum occlusion between inputs. 4. For the majority of spindles static‐dynamic interactions were characterized by pronounced occlusion (C1 < 0.35). In these spindles putative encoding sites (the peripheral heminodes of the branches supplying the intrafusal fibres activated by individual fusimotor efferents) were separated by a minimum conduction path of between three and ten myelinated segments (2‐9 nodes of Ranvier). In contrast, significant summation (C1, approximately 0.7) was found in only one spindle. In this case putative encoding sites were separated by a single node. 5. Occlusion was not due to encoder saturation and it could not be accounted for by any other known physiological mechanisms (intrafusal fatigue or unloading). It is therefore attributed to competitive pacemaker interaction between encoding sites which are largely selectively operated by static and dynamic fusimotor efferents. 6. Model simulations of real preterminal‐branch tree structures confirmed that short conduction paths between encoding sites were associated with manifest summation, whereas longer minimum conduction paths favoured pronounced occlusion. 7. In the extreme, occlusion could be so pronounced as to give rise to negative values of C1 during critical segments of response cycles. This was associated with lower discharge rates during combined static and dynamic stimulation than the higher of the individual stimulation effects. This phenomenon is referred to as hyperocclusion. Computer simulations demonstrated that hyperocclusion could be accounted for by a slow ionic adaptation process. e.g. by a very slowly activating K+ conductance.
The shape of indicator dilution curves used for cardiac output measurement in man.
Authors: Band D M , Linton R A , O'Brien T K , Jonas M M , Linton N W
Published: December 2014

1. In six patients arterial plasma lithium concentration‐time curves were recorded following injection of lithium chloride into the right or left atrium. 2. Lognormal curve fitting was used to derive the areas under the first pass dilution curves. 3. Subjecting the curves produced by left atrial injection to a delay and sequential filtering produced curves that closely approximated those produced by right atrial injection. 4. We conclude that the transfer function of the right heart and lungs is equivalent to a delay and sequential filtering, that the primary indicator dilution curve is closely approximated by a lognormal curve and that loss of lithium in the lungs following right atrial injection is clinically insignificant.
Role of caloric content on gastric emptying in humans.
Authors: Calbet J A , MacLean D A
Published: December 2014

1. This study examined the effects of caloric content (caloric density and the nature of calories) on the rate of gastric emptying using the double‐sampling gastric aspiration technique. Four test meals of 600 ml (glucose, 0.1 kcal ml‐1; pea and whey peptide hydrolysates, both 0.2 kcal ml‐1; milk protein, 0.7 kcal ml‐1) were tested in six healthy subjects in random order on four separate occasions. 2. The glucose solution was emptied the fastest with a half‐time of 9.4 +/‐ 1.2 min (P < 0.05) and the milk protein the slowest with a half‐time of 26.4 +/‐ 10.0 min (P < 0.05); the pea peptide hydrolysate and whey peptide hydrolysate solutions had half‐times of emptying of 16.3 +/‐ 5.4 and 17.2 +/‐ 6.1 min, respectively. The rates of gastric emptying for the peptide hydrolysate solutions derived from different protein sources were not different. 3. Despite the lower rate of gastric emptying for the milk protein solution, the rate of caloric delivery to the duodenum during the early phase of the gastric emptying process was higher than that for the other three solutions (46.3 +/‐ 6, 63.5 +/‐ 22, 62.5 +/‐ 19 and 113.8 +/‐ 25 cal min‐1 kg‐1 for the glucose, pea peptide hydrolysate, whey peptide hydrolysate and milk protein meals, respectively; P < 0.05). The caloric density of the test solutions was linearly related to the half‐time of gastric emptying (r = 0.96, P < 0.05) as well as to the rate at which calories were delivered to the duodenum (r = 0.99, P < 0.001). 4. This study demonstrates that the rate of gastric emptying is a function of the caloric density of the ingested meal and that a linear relationship exists between these variables. Furthermore, the nature of the calories seems to play a minor role in determining the rate of gastric emptying in humans.