bluuebunny/crossref_metadata_2025 huggingface dataset:
A relay for input from group II muscle afferents in sacral segments of the cat spinal cord.
Authors: Jankowska E , Riddell J S
Published: December 2014

1. A neuronal relay for input from group II afferents of hindlimb muscle nerves has been found in the previously little explored sacral segments of the cat spinal cord. 2. Electrical stimulation of group II muscle afferents of a number of nerves evoked negative potentials on the surface (cord dorsum potentials) and population postsynaptic potentials (field potentials) within the sacral segments. The largest potentials were evoked by stimulation of the posterior biceps‐semitendinosus and triceps surae nerves which evoke much smaller potentials in other segments. Group II afferents of other nerves, notably those which have their main relay within the middle lumbar segments, were much less effective. 3. The sites at which cord dorsum and field potentials evoked by group II muscle afferents were recorded varied in relation to the external topography of the L7‐S2 spinal segments but were consistent in their location relative to the pudendal motor nucleus (Onuf's nucleus). Potentials evoked by group II afferents of the posterior biceps and semitendinosus nerves peaked at a level corresponding to the rostral half of Onuf's nucleus and potentials evoked by afferents of the gastrocnemius nerves peaked just rostral to this nucleus. The largest field potentials (of 0.5‐1.0 mV) were recorded within the dorsal horn. Field potentials in the intermediate zone were much smaller (< 0.3 mV) and were seen less frequently. 4. Evidence was obtained that the dorsal horn field potentials are to a great extent evoked monosynaptically by the fast conducting fraction of group II muscle afferents: (i) they were evoked at short latencies (2.4‐2.7 ms from the stimulus; 1.3‐1.7 ms from group I components of afferent volleys and 0.5‐0.7 ms from group II components of these volleys), (ii) the conduction times of impulses in the fastest conducting fraction of group II afferents, between the sacral segments (where these impulses were induced by intraspinal stimuli) and the peripheral nerves, were only about 0.5 ms shorter than the latencies of field potentials recorded at the site of intraspinal stimulation and evoked by stimulation of the same peripheral nerves and, (iii) the field potentials followed repetitive stimuli without temporal facilitation. 5. Negative cord dorsum and field potentials were also evoked by small stretches of the semitendinosus and triceps surae muscles. Although they were smaller than potentials evoked by electrical stimulation of sensory fibres and appeared at longer latencies, their presence is consistent with a contribution of muscle spindle afferents to the actions of group II muscle afferents within the sacral segments.(ABSTRACT TRUNCATED AT 400 WORDS)
Histamine activates Cl‐ and K+ currents in guinea‐pig tracheal myocytes: convergence with muscarinic signalling pathway.
Authors: Janssen L J , Sims S M
Published: December 2014

1. We investigated the effects of histamine on membrane currents and contractile state of isolated guinea‐pig tracheal myocytes using perforated patch and whole‐cell recording techniques. The effects of histamine were compared to those of acetylcholine (ACh) and caffeine. 2. During voltage clamp (Vhold = ‐60 mV), histamine elicited contraction and an inward current (Ihist) which was often followed by current oscillations. Ihist had a reversal potential (Vrev) of ‐9 +/‐ 3 mV. 3. Ihist was dependent on the Cl‐ gradient and was antagonized by the Cl‐ channel blocker niflumic acid. Vrev was more positive (+2 +/‐ 1 mV) when K(+)‐selective currents were blocked by Cs+ and TEA. When all external Na+ was replaced with N‐methyl‐D‐glucamine, there was a small reduction in the amplitude of Ihist. 4. The histamine‐induced current was similar to that elicited by ACh and by caffeine with respect to time course, amplitude, and current‐voltage relationship. Responses to histamine and to ACh were non‐additive, consistent with a convergence of histaminergic and cholinergic signalling pathways. Ihist was antagonized by the H1 histaminergic receptor antagonist astemizole, but not by atropine. 5. When recorded using the perforated patch configuration, Ihist could be elicited repeatedly for more than 30 min. When cells were studied in the whole‐cell configuration using a pipette solution containing 0.025 mM EGTA, the amplitude of Ihist was initially the same as that obtained using perforated patch but then decreased; the time required for the responses to decrease to 50% (t1/2) was 8.2 +/‐ 1.0 min. When 1 mM EGTA was included in the pipette solution (whole‐cell configuration), the initial response to histamine was significantly decreased in size and t1/2 was reduced to 3.3 +/‐ 0.7 min. 6. The characteristics of the signalling pathway were examined in cells studied using the whole‐cell configuration with 0.025 mM EGTA in the recording pipette. Heparin significantly reduced t1/2 to 4.3 +/‐ 0.8 min. GTP gamma S elicited inward current and oscillations; both effects were enhanced by histamine. GTP gamma S also reduced t1/2 to 1.4 +/‐ 0.1 min. Pertussis toxin did not alter the amplitude or time course of Ihist. 7. We conclude that in guinea‐pig tracheal myocytes, binding of histamine to H1 receptors leads to release of Ca2+ from intracellular stores and subsequent activation of Cl‐ and K+ conductances as well as contraction. Furthermore, we demonstrate that ACh elicits similar physiological responses due to a convergence of the histaminergic and muscarinic signalling pathways.
Protein kinase C modulates hormone secretion regulated by extracellular polycations in bovine parathyroid cells.
Authors: Racke F K , Nemeth E F
Published: December 2014

1. The role of protein kinase C (PKC) in the regulation of parathyroid hormone (PTH) secretion was examined in dissociated bovine parathyroid cells. 2. Increasing the concentration of extracellular Ca2+ from 0.5 to 2 mM inhibited PTH secretion by 60‐80%. Similar depressive effects on secretion were obtained by increasing the concentration of extracellular Mg2+ from 1 to 7 mM or by adding La3+ (to 40 microM). The PKC activator phorbol myristate acetate (PMA) depressed PTH secretion at the lower and potentiated secretion at the higher concentrations of extracellular Ca2+, Mg2+ or La3+. The inhibitory effect of PKC on secretion correlated positively with the magnitude of the inhibitory effect elicited by elevated extracellular Ca2+. 3. The stimulatory effects of PKC activators on PTH secretion were reversed completely and the inhibitory effects were reversed partially by the PKC inhibitor staurosporine. Staurosporine alone did not affect secretion at low (0.5 mM) or high (2 mM) concentrations of extracellular Ca2+ but it did depress secretion at intermediate concentrations (around 1 mM) of extracellular Ca2+. 4. The stimulatory effects of PKC activators on secretion were overcome by increases in the concentration of extracellular Ca2+ (to 5 or 10 mM) or La3+ (to 100 microM). In contrast, increasing the concentration of extracellular Mg2+ to 11 or 19 mM did not alleviate the potentiating effects of PKC activators. The different results obtained with Ca2+ and Mg2+ could not be explained by their different effects on cytosolic Ca2+ and suggests that different cations can have varying degrees of efficacy to activate functional responses linked to the Ca2+ receptor on bovine parathyroid cells. 5. PTH secretion stimulated by isoprenaline was not affected by PKC activators or staurosporine. Similarly, the inhibitory effects of extracellular ATP gamma S on secretion were unaffected by PKC activators. These results show that PKC activators affect specifically PTH secretion regulated by extracellular polycations. 6. The stimulatory effect of PKC activators on secretion parallels its inhibitory effects on [Ca2+]i and inositol trisphosphate formation, showing that PKC blunts the mechanisms associated with extracellular Ca(2+)‐induced inhibition of secretion. The specificity of these actions suggests that PKC acts at a very early step of stimulus‐secretion coupling in parathyroid cells, specific to that used by extracellular polycations and perhaps involving the Ca2+ receptor.
Non‐electrolyte solute permeabilities of human placental microvillous and basal membranes.
Authors: Jansson T , Powell T L , Illsley N P
Published: December 2014

1. Permeability to non‐electrolytes of isolated microvillous and basal membranes from human term placenta was measured using stopped‐flow light‐scattering techniques. The studied solutes were urea, ethylene glycol, glycerol, creatinine, erythritol, arabitol and mannitol. 2. At 37 degrees C, permeability of the microvillous membrane to mannitol and urea was 0.30 +/‐ 0.02 x 10(‐6) cm/s (mean +/‐ S.E.M.) and 3.2 +/‐ 0.2 x 10(‐6) cm/s, respectively. The corresponding permeabilities for the basal membrane were 1.2 +/‐ 0.1 x 10(‐6) cm/s (mannitol) and 4.4 +/‐ 0.3 x 10(‐6) cm/s (urea). The basal membrane was substantially more permeable to hydrophilic solutes than the microvillous membrane. This is probably due to differences in lipid composition, as illustrated by membrane cholesterol content, which was found to be approximately 50% lower in the basal as compared to the microvillous membrane. 3. Similarities between permeabilities in placental membranes and lipid bilayers and the linear relationship noted between solute hydrophobicity and placental permeability suggested that solutes permeate both human syncytiotrophoblast membranes by a solubility/diffusion mechanism. In the microvillous membrane this was supported by data obtained for activation energies (> 10 kcal/mol) and reflection coefficients (close to 1). In the basal membrane, low activation energies for glycerol and urea and a low reflection coefficient for urea indicated that these solutes may, in part, share a common pathway with water. 4. It was estimated that the placental permeability to molecules with a molecular weight under 200 observed in vivo can, to a great extent, be accounted for by transcellular permeation.
Sodium‐evoked, calcium‐independent vasopressin release from rat isolated neurohypophysial nerve endings.
Authors: Stuenkel E L , Nordmann J J
Published: December 2014

1. The effects of Na+ on vasopressin release and on redistribution of Ca2+, Na+ and H+ in isolated rat neurohypophysial nerve endings have been studied. 2. Substituting Na+ for a non‐permanent cation produced a pronounced and sustained release of vasopressin. This increase occurred in the absence of external Ca2+ and in nerve endings loaded with the Ca2+ chelator dimethyl‐BAPTA (1,2‐bis‐(O‐aminophenoxy)ethane‐N,N,N',N'‐tetraacetic acid). 3. The effect of Na+ was independent of a rise in intracellular Ca2+ as judged by the measurement of [Ca2+]i using the indicator fura‐2 and 45Ca2+ efflux studies. Although Na+ could release Ca2+ from internal reservoirs the small elevation in [Ca2+]i induced by Na+ could not explain the large and sustained increase in vasopressin secretion. 4. The channel blockers TTX (tetrodotoxin), D888 (desmethyoxyverapamil), N144 (5‐nitro‐2‐(phenylpropylamino)‐benzoic acid) or SITS (4‐acetamido‐4'‐isothiocyanatostilbene‐2,2'‐disulphonic acid) could not prevent the Na(+)‐dependent increase in vasopressin release. Similarly this increase was not affected by metabolic inhibitors (Ruthenium Red and KCN) nor by CCCP (carbonyl cyanide m‐chlorophenylhydrazone), an uncoupler of oxidative phosphorylation. 5. Selectivity among monovalent cations to promote secretion was found with the largest effect on the secretory response being produced by Na+. Similarly Cl‐ was found to be the most potent anion studied for inducing, in the presence of Na+, an increase in neurohormone release. 6. Measuring [Na+]i by means of the Na+ indicator SBFI showed that the extent of the secretory response was correlated with the intraterminal Na+ concentration. 7. The Na(+)‐induced, Ca(2+)‐independent release of vasopressin occurred by exocytosis as judged (i) by the linear relationship between the amount of vasopressin secreted and that of the co‐localized neurophysin and (ii) by the demonstration that the extracellular marker horseradish peroxidase was only found in endocytotic vacuoles and not in the cytoplasm of the stimulated nerve endings. 8. The Na(+)‐dependent secretory response found on addition of extracellular Na+ was not the result of the change in internal pH as measured with the indicator BCECF and as mimicked by addition of propionic acid. 9. Addition of Na+ to digitonin‐ or streptolysin‐O‐permeabilized nerve endings in the presence or absence of Ca2+ also gave rise to an increase in vasopressin secretion. 10. It is concluded that an increase in internal Na+ per se can promote, in the absence of a rise in intracellular Ca2+, an increase in neuropeptide secretion.
Ventilatory responses during arm and leg exercise at varying speeds and forces in untrained female humans.
Authors: Takano N
Published: December 2014

1. Involvement of neural stimuli, central and/or peripheral in origin, in exercise ventilatory control was ascertained by examining the ventilatory responses to varying mechanical conditions of arm and leg cycle ergometries. Twelve untrained women underwent each of two modes of exercise at three levels of loading (0, 5 and 10 N), each at three levels of speed (30, 50 and 72 r.p.m.), during which steady‐state values of minute ventilation (VE), tidal volume (VT), respiratory frequency (f) and CO2 excretion (VCO2) were measured. 2. Using the data obtained at the aerobic work intensities, the relationship of ventilatory responses (VE, VT and f) to the metabolic (VCO2) and mechanical (speed and load) variables were studied by multiple linear regression analysis. Coefficient of determination (r2) of the regression model was lowest (0.84) for f in the arm exercise and highest (0.99) for VE in the leg exercise. 3. Standardized partial regression coefficients of the model indicated that VE response is related to VCO2 at the rate of 94 +/‐ 3% (mean +/‐ S.E.M.) and to the pedal rate at 8 +/‐ 3% during the leg exercise, while it is closely related to VCO2 in the arm exercise. For f response, influence of the rate of limb movement was seen in the leg exercise but not in the arm exercise. The different effects of the rate of limb movement between the two exercise modes may be related to familiarity with the exercise modes, suggesting that a familiarity‐related mechanism is involved in exercise ventilatory control. 4. A heavier load imposed on the limb muscles elicited a greater VT both in the arm and leg exercise and a lower f in the arm exercise. Postural control in the upper torso during increased limb muscle tension seems to affect VT and f.
The neuropeptide CRF is involved in the modulation of the baroreflex during hindlimb ischaemia in the anaesthetized rat.
Authors: Turnbull A V , Kirkman E , Rothwell N J , Little R A
Published: December 2014

1. The effects of bilateral hindlimb ischaemia (BHLI), and of administration of the neuropeptide corticotrophin‐releasing factor (CRF), were tested on cardiovascular variables in propofol‐anaesthetized Sprague‐Dawley rats. In addition, the effects of antagonizing endogenous CRF on the cardiovascular response to BHLI was investigated. 2. BHLI produced significant increases in mean arterial pressure and heart rate. Baroreflex sensitivity was reduced in all animals tested both 10 and 30 min after the induction of ischemia and this was accompanied by changes which are consistent with an underlying baroreflex resetting. 3. Intracerebroventricular (I.C.V.) administration of CRF (1.5 nmol) produced significant increases in heart rate but not mean arterial pressure. CRF also produced significant reductions in baroreflex sensitivity 10 and 30 min after administration, but there was no consistent evidence of baroreflex resetting. These effects of CRF could be prevented by pretreatment with the synthetic CRF antagonist, alpha‐helical CRF(9‐41) (alpha hCRF, 6.5 nmol, I.C.V.), which itself had no effect on these cardiovascular variables. 4. Pretreatment with alpha hCRF (6.5 nmol, I.C.V.) prevented the reduction in baroreflex sensitivity observed 10 min after the induction of BHLI, but had no consistent effect on the pressor or tachycardiac responses to BHLI and allowed a clear expression of BHLI‐induced baroreflex resetting. alpha‐Helical CRF(9‐41) had no effect on the reduction in baroreflex sensitivity after 30 min of BHLI. 5. We conclude that corticotrophin‐releasing factor mediates, at least in part, the early reduction in baroreflex sensitivity observed during hindlimb ischaemia in the rat.
Changes in crossbridge and non‐crossbridge energetics during moderate fatigue of frog muscle fibres.
Authors: Barclay C J , Curtin N A , Woledge R C
Published: December 2014

1. The effect of sarcomere length (SL) during a fatiguing series of isometric tetani of frog muscle fibres was investigated. Tetani at 2.3 microns SL were more fatiguing than tetani at 3.2 microns SL, in that force declined twice as much as relaxation became much slower. 2. In a second set of experiments the force and heat production were measured during a series of fatiguing tetani. Heat was separated into two components: (a) crossbridge heat which is dependent on filament overlap and interaction, and (b) non‐crossbridge heat which is independent of filament overlap and due to Ca2+ turnover. 3. In a series of fifty tetani, force, crossbridge heat and non‐crossbridge heat each declined by 25‐30% of its initial value. 4. The 25% reduction in non‐crossbridge heat occurred completely during the first few tetani of the fatiguing series while force declined by less than 3%. This may be due to a reduction in Ca2+ binding to parvalbumin and to Ca2+ remaining bound during the remainder of the fatigue series. 5. After the first few tetani of the fatigue series the non‐crossbridge heat hardly changed as force declined by a further 25% of its initial value. Continuing reduction of force with constant Ca2+ turnover indicates a reduction in the Ca2+ sensitivity of the filaments, and/or a reduction in the average force per attached crossbridge. 6. At the start of the fatiguing series, as force declines by about 7.5% there is a much larger decline of crossbridge heat (17%). The reason for this is unknown. Later in the series, force declined more rapidly than heat. This is probably due to a progressive accumulation of inorganic phosphate which acts by depressing force more than it depresses ATP breakdown.
Enhancement by atropine of the pancreatic exocrine secretions evoked by vagal stimulation in the pithed rat.
Authors: Barrett J , McDougall J J , Morrison J D
Published: December 2014

1. Pancreatic secretions were collected in response to 15 min periods of bilateral stimulation of the cervical vagus nerves in the pithed rat. 2. The weight of juice, total HCO3‐ and total protein evoked by a second period of vagal stimulation were essentially similar to those of the first period of vagal stimulation. 3. When the second period of vagal stimulation was preceded by an intravenous bolus injection of atropine sufficient to block the vagally induced bradycardia, the weight of secretion and total protein were greatly potentiated over an extended time course far exceeding that of the period of vagal stimulation. Total HCO3‐ was unchanged. 4. By contrast, atropine was effective in antagonizing the stimulatory effects of the muscarinic agonist methacholine injected intravenously. 5. The putative VIP (vasoactive intestinal polypeptide) antagonist [D‐p‐chloro‐Phe6, Leu17]‐VIP injected intravenously also increased the vagally evoked weight of juice, with total HCO3‐ and total protein unchanged. This was explicable by a partial agonist effect which was additive to the stimulatory action of vagal stimulation. 6. To explain these results, it is proposed that endogenously released acetylcholine exerts a negative feedback effect on the postganglionic varicosities which release both acetylcholine and another cotransmitter which was not excluded as being VIP. In the presence of atropine, the cotransmitter is proposed to be released from the inhibitory feedback, thus enhancing the response to vagal stimulation.
Electrical coupling of circular muscle to longitudinal muscle and interstitial cells of Cajal in canine colon.
Authors: Liu L W , Huizinga J D
Published: December 2014

1. Electrical communication between circular muscle, longitudinal muscle and interstitial cells of Cajal (ICC) was investigated; the hypothesis was tested that the resting membrane potential (RMP) gradient in the circular muscle of canine colon is caused by electrical coupling to neighbouring cells. 2. Isolated longitudinal muscle exhibited spike‐like action potentials at a RMP of ‐45 mV with a frequency and amplitude of 20 cycles/min and 12 mV, respectively. 3. The circular muscle (CM), devoid of longitudinal muscle, myenteric plexus and submuscular ICC‐smooth‐muscle network, was electrically quiescent at a uniform RMP of ‐62 mV across the entire circular muscle layer. 4. Preparations consisting of only the submuscular ICC network and a few adjacent layers of circular muscle cells exhibited slow wave‐type action potentials at a RMP of about ‐80 mV. 5. In ICC‐CM preparations, consisting of the submuscular ICC network and circular muscle, a RMP gradient of 10 mV was observed near the submucosal border, whereas the RMP was constant at ‐62 mV in the myenteric half of the circular muscle. 6. In full thickness (FT) preparations, a RMP gradient of 23 mV was observed. The RMP decreased gradually from ‐71 mV at the submucosal border to ‐48 mV at the myenteric border of the circular muscle. 7. Coupling of longitudinal muscle to circular muscle caused circular muscle cells at the myenteric surface to depolarize by 14 mV and longitudinal muscle cells to hyperpolarize by 3 mV. 8. In the ICC‐CM preparations, the slow wave amplitudes did not decay exponentially away from the ICC network indicating that slow waves propagated actively into the circular muscle; in the FT preparations there was an apparent exponential decay but this was due to the RMP gradient. 9. Spike‐like action potentials (SLAPs) superimposed on the plateau phase of slow waves did not decay exponentially away from the myenteric border suggesting that SLAPs were generated within the circular muscle layer. 10. In summary, circular muscle cells possess a uniform intrinsic RMP of ‐62 mV. The RMP gradient in situ is caused by electrical coupling of circular muscle cells to longitudinal muscle cells and the submuscular network of ICC. In situ, slow wave‐type action potentials propagate actively into the circular muscle layer, and, dependent on the level of excitation, circular muscle cells actively generate spikes.
Modulation of basal L‐type Ca2+ current by adenosine in ferret isolated right ventricular myocytes.
Authors: Qu Y , Campbell D L , Whorton A R , Strauss H C
Published: December 2014

1. The whole‐cell configuration of the gigaohm seal voltage clamp and an internal perfusion technique were used to study the effects of adenosine on the basal L‐type Ca2+ current (ICa) in enzymatically isolated right ventricular myocytes of ferrets. Basal L‐type ICa was isolated by using a Na(+)‐ and K(+)‐free saline (replacement by N‐methyl‐D‐glucamine+, Cs+ and TEA+, respectively). All experiments were conducted at room temperature (22‐24 degrees C). 2. Basal ICa was markedly reduced during exposure to adenosine in a concentration‐dependent manner with a half‐inhibitory concentration (IC50) of 0.3 microM and maximum inhibition of 35%. This effect was completely abolished by 50 nM 8‐cyclopentyl‐1,3‐dipropylxanthine (CPDPX), a specific A1 adenosine receptor antagonist with an inhibition constant, Ki = 0.48 nM. Inhibition was also observed in the presence of 1 microM atropine. 3. Adenosine decreased basal ICa by decreasing the peak amplitude of ICa without significantly altering (i) the voltage dependence of the current‐voltage relationship, (ii) the apparent reversal potential, (iii) the voltage dependence of steady‐state activation and inactivation, (iv) the kinetics of inactivation at 0 mV, and (v) the kinetics of recovery from inactivation at ‐70 mV. 4. Pretreatment of cells with 0.4 microns/ml pertussis toxin (PTX) for 4 h at 37 degrees C produced greater than 90% ADP ribosylation of PTX‐sensitive G proteins. PTX pretreatment significantly attenuated the adenosine‐mediated decrease in ICa (35% in control; 4.6% after PTX pretreatment). 5. The peptide inhibitor (PKI) of cyclic AMP‐dependent protein kinase A at a concentration of 2 microM neither inhibited basal ICa nor attenuated the effects of adenosine on basal ICa. However, PKI decreased the stimulatory effects of 100 microM cAMP on ICa. 6. Increasing intracellular cAMP to a supra‐saturable level by using 10 mM cAMP and 100 microM papaverine did not prevent adenosine from inhibiting ICa. 7. Consistent with the reduction of basal ICa, adenosine produced an inhibitory effect on the action potential under basal conditions, i.e. hyperpolarization of the plateau phase and marked shortening of action potential duration. These effects were concentration dependent. 8. These results demonstrate a reduction of the basal L‐type ICa by adenosine in ferret ventricular myocardium. This reduction is not mediated by modification of voltage‐dependent properties of macroscopic ICa. The shortening of action potentials may be explained in part by the reduction in ICa.(ABSTRACT TRUNCATED AT 400 WORDS)
The anion transport inhibitor DIDS increases rat hepatocyte K+ conductance via uptake through the bilirubin pathway.
Authors: Wehner F , Rosin‐Steiner S , Beetz G , Sauer H
Published: December 2014

1. In confluent primary cultures of rat hepatocytes, membrane effects of the anion transport inhibitor 4,4'‐diisothiocyanatostilbene‐2,2'‐disulphonic acid (DIDS) were recorded with conventional microelectrodes. In addition, cell pH and cell Ca2+ were monitored by use of the fluorescent dyes BCECF and fluo‐3, respectively. Uptake of DIDS was determined by measuring intracellular DIDS fluorescence between 470 and 520 nm (excitation wavelength 390 nm). 2. In the presence of 0.2 mM DIDS, membrane voltages hyperpolarized from ‐44.0 +/‐ 1.8 to ‐73.1 +/‐ 1.9 mV (n = 16). This change was monophasic and occurred with a time constant of 170 +/‐ 25 s. The effect was only partly reversible. 3. Cable analysis revealed a concomitant decrease in the specific cell membrane resistance from 3.2 to 1.5 k omega cm2. 4. In ion substitution experiments, a 10‐fold elevation of external K+ (from 2.5 to 25 mM) depolarized cell membranes by 6.2 +/‐ 1.5 mV (n = 5). In the presence of 0.2 mM DIDS, this membrane response was increased 5‐fold to 32.2 +/‐ 4.1 mV. 5. Replacement of Cl‐ by 99% with gluconate depolarized the cells by 9.3 +/‐ 1.9 mV. In contrast, with 0.2 mM DIDS present, Cl‐ removal led to a membrane hyperpolarization of 5.9 +/‐ 0.9 mV (n = 4). 6. DIDS had no effect on cytosolic pH or Ca2+. 7. To determine the sidedness of the DIDS effect, i.e. to analyse if the increase in K+ conductance is mediated by uptake of the compound, DIDS was added in the presence of different substrates of hepatocellular anion transport. Taurocholate (50 microM) and frusemide (0.5 mM), which are both taken up via the sinusoidal multi‐specific bile acid transporter, did not change DIDS‐induced membrane hyperpolarization. 8. In contrast, 0.5 mM bromosulphthalein (BSP), a substrate of the bilirubin transporter, competitively inhibited the membrane hyperpolarization elicited by various concentrations of DIDS (0.1‐1.0 mM). 9. Hepatocellular uptake of BSP is known to be, in part, Cl‐ dependent and to be competitively inhibited by Indocyanine Green. When 0.2 mM DIDS was added to a superfusate, in which 99% of Cl‐ had been exchanged by gluconate, the velocity of membrane hyperpolarization was decreased by 45%. In the presence of Indocyanine Green (0.1 mM) DIDS‐induced membrane hyperpolarization was reduced to approximately 20%. 10. Addition of 0.2 mM DIDS to hepatocyte monolayers led to a time‐dependent increase in cell fluorescence which was absent at 4 degrees C and which was completely blocked by 0.5 mM BSP.(ABSTRACT TRUNCATED AT 400 WORDS)
Opposite effects of phosphatase inhibitors on L‐type calcium and delayed rectifier currents in frog cardiac myocytes.
Authors: Frace A M , Hartzell H C
Published: December 2014

1. Application of the phosphatase inhibitors okadaic acid (OA) and microcystin (MC) to frog cardiomyocytes caused large increases in L‐type calcium current (ICa) in the absence of beta‐adrenergic agonists. The increase occurred without effects on the peak current‐voltage relation or voltage‐dependent inactivation. OA and MC caused a decrease in amplitude of delayed rectifier current (IK), which is opposite to the increase produced by cAMP‐dependent phosphorylation. The decrease occurred without effects on voltage‐dependent activation or reversal potential. 2. Analysis of the dose‐response relations for OA and MC on ventricular cell ICa were best fitted with a single‐site relationship with a K1/2 of 1.58 microM and 0.81 microM, respectively. These data suggest the predominant form of phosphatase active on ICa in this cell type is produced by protein phosphatase 1. Inhibition of phosphatase 2B (calcineurin) was without appreciable effect. 3. Reducing intracellular ATP levels was without effect on basal ICa suggesting that calcium channels may not need to be phosphorylated to open. ATP depletion was able to block completely the ICa increase induced by OA or MC. This demonstrates that the effects of OA and MC on ICa are mediated by a phosphorylation reaction. In contrast, ATP depletion totally abolished IK, suggesting either a requirement for ATP or phosphorylation for basal function of the delayed rectifier channel. 4. Internal perfusion of a peptide inhibitor (PKI(5‐22)) of protein kinase A (PK‐A) was without effect on basal current levels of ICa or IK, suggesting that this kinase is not phosphorylating these channels under basal conditions. Furthermore, although PKI is capable of completely blocking the response of ICa to isoprenaline or forskolin, PKI does not affect the increase in ICa induced by MC or OA. Inhibition of adenylate cyclase with acetylcholine or inhibition of PK‐A with adenosine cyclic 3',5'‐(Rp)‐phosphothioate (Rp‐cAMPS) also had no effect on the response to OA or MC. 5. Application of beta‐adrenergic agonist, forskolin or cAMP all produced additional increases in the presence of saturating doses of MC or OA. This supports the hypothesis that PK‐A is not mediating the OA response and that phosphatase inhibition does not result in complete phosphorylation of PK‐A sites. 6. To attempt to identify the protein kinase activity responsible for OA effects on ICa and IK, several types of protein kinase inhibitors were internally perfused.(ABSTRACT TRUNCATED AT 400 WORDS)
Pyloric motility and liquid gastric emptying during barostatic control of gastric pressure in pigs.
Authors: Treacy P J , Jamieson G G , Dent J
Published: December 2014

1. In conscious pigs, intragastric pressure was controlled by a water barostat such that the gastroduodenal pressure gradient was always positive with the barostat set above 15 cm. In six pigs pyloric motor function was removed by prior pylorectomy. 2. In pylorus‐intact animals, isolated pyloric pressure waves (IPPWs) occurred at a median of 2.9 min‐1 during duodenal dextrose infusion with the barostat set at or above a threshold of 15 cm. Increases of intragastric pressure above this threshold had no additional effect upon rates of IPPWs. Below this threshold, with identical duodenal dextrose infusion, IPPWs occurred infrequently (0.5 min‐1). In pylorus‐excised animals virtually no IPPWs were seen. 3. In both pylorus‐intact and ‐excised animals, the rate of antral pressure waves (APWs) was dependent upon intragastric pressure during duodenal infusion of both saline and dextrose. No threshold for stimulation was seen. Duodenal dextrose infusion inhibited APWs in pylorus‐intact animals only. 4. With a positive gastroduodenal pressure gradient, gastric emptying was more rapid in pylorus‐excised animals than pylorus‐intact animals during duodenal dextrose infusion. In contrast, gastric emptying rates were not different between pylorus‐intact and ‐excised animals during saline infusion. 5. These findings suggest an all‐or‐none‐type stimulation of localized pyloric contractions by distension of the stomach beyond a threshold, in synergism with stimulation by nutrients within the upper small bowel. In contrast, stimulation of antral motility is in proportion to distension of the stomach. The pylorus, by way of localized pyloric contractions, is an effective resistor to transpyloric flow in the face of a positive gastroduodenal pressure gradient.
Regulation of A‐currents by cell‐cell interactions and neurotrophic factors in developing chick parasympathetic neurones.
Authors: Dourado M M , Dryer S E
Published: December 2014

1. The developmental regulation of ion channel expression was studied in parasympathetic neurones isolated from the chick ciliary ganglion. Whole‐cell patch clamp recordings were made from ciliary ganglion neurones that were removed from the embryo on the ninth embryonic day (E9) and maintained in dissociated cell culture for an additional 4 days. Previous studies have shown that the expression of a transient voltage‐activated K+ current (IA) is regulated by unidentified environmental stimuli during these developmental stages. 2. The effect of interactions between neurones and target tissue on the expression of IA was tested by co‐culturing ciliary ganglion neurones with chick striated muscle cells. Neurones from the nerve‐muscle co‐cultures expressed normal amplitudes of IA, but the neurones did not express normal levels of IA when they were plated onto lysed muscle fibres. 3. The effect of interactions between ganglionic neurones and non‐neuronal ganglionic cells was tested by culturing ganglia as explants rather than as dissociated cells. Neurones isolated from the explant cultures did not express normal levels of IA. Similarly, when dissociated ganglionic neurones were co‐cultured with fibroblasts isolated from embryonic chick skin, they did not express normal amplitudes of IA. 4. Chronic depolarization caused by growing ciliary ganglion neurones in the presence of elevated K+ concentrations did not allow for the normal expression of IA, although it did promote the survival of these neurones in vitro. 5. Addition of 40 ng ml‐1 of recombinant human ciliary neurotrophic factor (CNTF) or basic fibroblast growth factor (bFGF) to the cell culture medium had no effect on IA expression in developing chick ciliary ganglion neurones. However, 40 ng ml‐1 of acidic fibroblast growth factor (aFGF) stimulated the expression of IA. All trophic factors promoted the growth and survival of ciliary ganglion neurones in vitro. 6. Dissociated ciliary ganglion neurones were maintained in a culture medium containing an aqueous extract of the whole brain. Neurones developing under these conditions expressed normal levels of IA. The stimulatory activity of the brain extract was destroyed by heating. 7. The expression of IA in chick ciliary ganglion neurones developing in vitro can be regulated by soluble growth factors and by interactions with certain other cell types. Similar interactions may regulate the expression of IA in ciliary ganglion neurones developing in situ.
Processes that remove calcium from the cytoplasm during excitation‐contraction coupling in intact rat heart cells.
Authors: Balke C W , Egan T M , Wier W G
Published: December 2014

1. The processes that remove Ca2+ rapidly from the cytoplasm were studied in isolated rat ventricular myocytes subjected to whole‐cell voltage clamp and internal perfusion with the Ca2+ indicator, indo‐1. Na(+)‐Ca2+ exchange was eliminated in most experiments by removing Na+ both internally and externally. 2. When the Ca(2+)‐pumping ATPase of the sarcoplasmic reticulum (SR) was inhibited with cyclopiazonic acid and ryanodine interfered with the release of Ca2+ from the SR, [Ca2+]i transients rose slowly and declined extremely slowly. We concluded that transport of Ca2+ by mitochondria and the surface membrane Ca(2+)‐pumping ATPase would be negligible over the time course of a single [Ca2+]i transient. 3. The influence of cytoplasmic Ca2+ ligands was characterized by internal perfusion with high concentrations of diffusible Ca2+ ligands (indo‐1) or by superfusion with the membrane‐permeant Ca2+ ligand, BAPTA AM. As the concentration of indo‐1 in the cell increased from < 0.1 mM to at least 0.5 mM, the time constant of the decline of [Ca2+]i increased from about 0.15 s to nearly 3 s. 4. Calcium bound to endogenous Ca2+ ligands during depolarizing clamp pulses was characterized quantitatively as the difference between the total Ca2+ entering the cell via L‐type Ca2+ channels and [Ca2+]i, in experiments in which SR function had been abolished. As total calcium increased during the entry of Ca2+, total calcium was found to agree reasonably well with that predicted by assuming that Ca2+ could bind to endogenous intracellular Ca2+ ligands and to indo‐1. 5. The results indicate that, in the absence of Na+, the major factors determining the removal of cytoplasmic free Ca2+ are the Ca(2+)‐pumping ATPase of the SR and the binding of Ca2+ to endogenous and exogenous Ca2+ ligands. 6. Several hypothetical ‘Ca2+ removal functions’ were fitted to the declining phase of [Ca2+]i transients. The best fit was one in which the flux of Ca2+ through the SR Ca(2+)‐pumping ATPase was described by a Michaelis‐Menten‐type equation. The decline of the [Ca2+]i transient was thus described by a linear, first‐order differential equation having terms giving the rate of Ca2+ transport by the SR Ca(2+)‐pumping ATPase (Vmax and KM), the rates of complexation of Ca2+ with the various Ca2+ ligands (L), and a leak of Ca2+ into the cytoplasm from the SR (FSR,leak).(ABSTRACT TRUNCATED AT 400 WORDS)
Quantal and non‐quantal ACh release at developing Xenopus neuromuscular junctions in culture.
Authors: Young S H , Grinnell A D
Published: December 2014

1. Single acetylcholine receptor (AChR) channel openings, detected by the whole‐cell patch clamp technique, were used to monitor quantal and non‐quantal ACh release at synapses in 1‐ and 2‐day‐old co‐cultures of Xenopus embryonic motoneurons and muscle cells. motoneuron growth cones in ways that presumably reflect muscle‐nerve inductive influences and the development of neurotransmitter release mechanisms. 2. Miniature endplate currents (MEPCs) occurred at a mean frequency of approximately 0.6 s‐1 with a skewed distribution and mean amplitude of about twenty channel openings. In addition, occasional brief episodes of rapid deviations in the baseline were observed in some cells, with mean amplitudes of 4‐8 pA and durations of a few hundred milliseconds. However, these episodes did not closely resemble summated openings of AChR channels. Moreover, where tested, these episodes were not blocked by curare; and comparable episodes were seen in an uninnervated myocyte. Thus they appear not to reflect ACh release from the nerve terminal. 3. Single‐channel openings that might have been responses to non‐quantal release of ACh were observed at rates of 0.9‐12.3 min‐1 (mean 3.0 min‐1), only 1‐5 times the rate of spontaneous AChR channel openings in uninnervated myocytes (mean 1.4 min‐1). 4. We conclude that there is no significant non‐quantal ACh leak from the presynaptic contacts in these immature synapses under these culture conditions. This is in disagreement with other, less direct, experimental reports, but consistent with findings in mature frog motor nerve terminals.
Sodium and potassium currents in freshly isolated and in proliferating human muscle satellite cells.
Authors: Hamann M , Widmer H , Baroffio A , Aubry J P , Krause R M , Kaelin A , Bader C R
Published: December 2014

1. Human muscle satellite cells (SC) were studied either immediately after dissociation of muscle biopsies or later, as they proliferated in culture. A purification procedure combined with clonal cultures ensured that electrophysiological recordings were done in myogenic cells. Hoechst staining for the DNA attested that cells were mononucleated. 2. The goals of this study were to examine (i) whether the electrophysiological properties of freshly isolated SC resembled those of SC that proliferated in culture for several weeks, (ii) whether freezing and thawing affected these properties, and (iii) whether SC constituted a homogeneous population. 3. We found that there were only subtle differences between the electrophysiological results obtained in freshly isolated SC and in proliferating SC with or without previous freezing and thawing. Most SC expressed two voltage‐gated currents, a TTX‐resistant Na+ current and a calcium‐activated potassium current (IK, Ca). 4. The level of expression of the Na+ current and of IK, Ca was affected in a different way by cellular proliferation; the normalized Na+ conductance (pS pF‐1) of proliferating cells resembled that of freshly isolated SC, whereas the IK, Ca conductance increased 10 times. The analysis of the amplitude distributions of the Na+ current and of IK, Ca in the various SC preparations suggested that there was only one class of SC.
Force‐velocity relation for frog muscle fibres: effects of moderate fatigue and of intracellular acidification.
Authors: Curtin N A , Edman K A
Published: December 2014

1. Intact frog single fibres were investigated under control conditions (1 s tetanus every 2, 3 or 5 min) and during moderate fatigue (interval between tetani 15 or 30 s). 2. Fatigue reduced isometric force (P0) by 25.8 +/‐ 1.6% (S.E.M.; n = 13) and depressed the maximum velocity of shortening (Vmax) by 10.2 +/‐ 2.2% (n = 13). The force‐velocity relation became less curved, a/P0* (see Methods) being increased by 29.5 +/‐ 8.8% (n = 13). Thus, power was less affected than isometric force or Vmax. 3. The velocity of unloaded shortening (V0), from slack test measurements, was reduced proportionally more than Vmax during fatigue. Under control conditions V0 was larger than Vmax, but during fatigue their values were not significantly different. 4. Stiffness during shortening was reduced during fatigue indicating fewer attached cross‐bridges in fatigue. Force was reduced more than stiffness indicating that, on average, there is less force per attached cross‐bridge. 5. The force‐lengthening velocity relation showed that the ability to resist forces greater than isometric was well preserved in fatigue. 6. Compared with fatigue, intracellular acidification with CO2 produced a smaller reduction in isometric force. However, reduction in Vmax was not significantly different from that in fatigue. These results are consistent with both inorganic phosphate and H+ increasing in fatigue, but only H+ increasing during acidification, and isometric force being reduced by both, Vmax being sensitive only to H+.
Excitation‐contraction coupling in single guinea‐pig ventricular myocytes exposed to hydrogen peroxide.
Authors: Goldhaber J I , Liu E
Published: December 2014

1. The effects of hydrogen peroxide (H2O2), an in vitro free radical generating system, on excitation‐contraction (E‐C) coupling were studied in isolated adult guinea‐pig ventricular myocytes using Ca(2+)‐sensitive dyes and the patch‐clamp technique. 2. In paced myocytes loaded with indo‐1 AM, 1 mM H2O2 briefly increased, then decreased the amplitude of intracellular Ca2+ ([Ca2+]i) transients and cell contractions. Diastolic [Ca2+]i increased in association with cell shortening. Automaticity also developed, followed shortly by inexcitability. In contrast, paced myocytes exposed to the metabolic inhibitors carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP) and 2‐deoxyglucose (DG), rapidly became inexcitable and exhibited marked diastolic shortening prior to increases in diastolic [Ca2+]i. 3. In patch‐clamped myocytes loaded with fura‐2, H2O2 reduced the amplitude of the Ca2+ current (ICa), the [Ca2+]i transient, and active cell shortening. H2O2 prolonged the relaxation phase of the [Ca2+]i transient, and activated an outward membrane current consistent with the ATP‐sensitive K+ current (IK,ATP), but did not change the voltage dependence of ICa, the peak [Ca2+]i transient or active cell shortening. These responses were qualitatively similar to patch‐clamped myocytes exposed to FCCP and DG. 4. Following exposure to H2O2, ICa elicited smaller [Ca2+]i transients than under control conditions. This was consistent with the observation that H2O2 reduced sarcoplasmic reticulum (SR) stores of Ca2+ by 42%, when assessed by observing the [Ca2+]i transients elicited by rapid extracellular application of 5 mM caffeine. In contrast FCCP‐DG tended to increase SR Ca2+ stores. 5. Despite the decrease in the caffeine‐induced Ca2+i release after H2O2, there was an increase in the Na(+)‐Ca2+ exchange current associated with the caffeine‐induced [Ca2+]i transient. 6. We conclude, therefore, that as with metabolic inhibitors, H2O2 interferes with E‐C coupling in guinea‐pig myocytes by impairing ICa and activating IK,ATP. However, unlike metabolic inhibitors, H2O2 stimulates Na(+)‐Ca2+ exchange and depletes SR Ca2+ stores. Furthermore, diastolic [Ca2+]i becomes elevated while the myocyte is still excitable. These observations suggest that free radicals have primary effects on cardiac E‐C coupling independent of their depressant effects on metabolism.
Regulation of the frequency‐dependent facilitation of L‐type Ca2+ currents in rat ventricular myocytes.
Authors: Tiaho F , Piot C , Nargeot J , Richard S
Published: December 2014

1. An increase in the rate of stimulation induces an augmentation of L‐type Ca2+ currents (ICa) and concomitant slowing of current decay in rat ventricular cells. This facilitation is quasi immediate (1‐3 s), graded with the rate of stimulation, and occurs only from negative holding potentials. We investigated this effect using trains of stimulation at 1 Hz and the whole‐cell patch‐clamp technique (18‐22 degrees C). 2. The decay of ICa is normally bi‐exponential and comprises fast and slow current components (ICa,fc and ICa,sc, respectively). Facilitation of ICa was observed only when ICa,fc was predominant. 3. Facilitation developed during the run‐up of ICa with the interconversion of ICa,sc into ICa,fc, and vanished during the run‐down of ICa with the loss of ICa,fc.Ni2+ (300 microM) and nifedipine (1 microM) suppressed facilitation owing to the preferential inhibition of ICa,fc. 4. Facilitation of ICa was not altered (when present) or favoured (when absent) by the cAMP‐dependent phosphorylation of Ca2+ channels promoted by isoprenaline or by intracellular application of cAMP or of the catalytic subunit of protein kinase A (C‐sub). A similar effect was observed when the dihydropyridine agonist Bay K 8644 was applied. In both cases, facilitation was linked to a preferential increase of ICa,fc. 5. Following intracellular application of inhibitors of protein kinase A in combination with a non‐hydrolysable ATP analogue, ICa consisted predominantly of ICa,sc and no facilitation was observed. The calmodulin antagonist naphthalenesulphonamide had no effect on facilitation. 6. When Bay K 8644 was applied in combination with isoprenaline, cAMP or C‐sub, the decay of ICa was slowed with the predominant development of ICa,sc, and facilitation of ICa was nearly abolished. Facilitation also depended on extracellular Ca2+, and was suppressed when Ba2+ replaced Ca2+ as the permeating ion. 7. When no EGTA was included in the patch pipette, facilitation was not further enhanced but a use‐dependent decrease of ICa frequently occurred. When BAPTA was used in place of EGTA, the rate of inactivation of ICa was reduced and facilitation was abolished. 8. In conclusion, the facilitation of ICa that reflects a voltage‐driven interconversion of ICa,fc into ICa,sc is also regulated by Ca2+ and by cAMP‐dependent phosphorylation. The presence of the gating pattern typified by ICa,fc is required. Ca2+ may exert its effect near the inner pore of the Ca2+ channel protein and control the distribution between the closed states of the two gating pathways.