bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective depression of medium‐latency leg and foot muscle responses to stretch by an alpha 2‐agonist in humans.
Authors: Corna S , Grasso M , Nardone A , Schieppati M
Published: December 2014

1. In standing humans, toe‐up rotation of a platform induces a short‐latency (SLR) and a medium‐latency response (MLR) in both soleus (Sol) and flexor digitorum brevis (FDB) muscles. Toe‐down rotation evokes a MLR in the tibialis anterior (TA). The SLR is the counterpart of the monosynaptic stretch reflex, but the origin of the MLR is still debated. By means of tizanidine (an alpha 2‐adrenergic receptor agonist) we tested the hypothesis that the MLR is relayed by group II afferent fibres, since animal data indicate that tizanidine or stimulation of monoaminergic brainstem centres decrease the excitability of spinal interneurones supplied by those fibres. In addition, we compared the effect of the drug on these responses with that induced by stabilization of posture. 2. Eight subjects received tizanidine (150 micrograms kg‐1 orally) or placebo, in a single‐blind design. Platform rotations were delivered prior to administration and for 3 h afterwards. Both TA‐ and FDB‐MLRs decreased in size, starting from about 1 h after tizanidine administration. Sol‐SLR was unaffected. Response latencies were unchanged. Placebo induced no changes in any response. In each subject, the extent of TA‐MLR depression induced by holding onto a frame and by tizanidine was superimposable. 3. The selective effect of tizanidine on MLR supports the notion that it is relayed through group II afferent fibres. The similar effects of holding and tizanidine on the response suggests that it is modulated by monoaminergic centres.
Bi‐stable block by 4‐aminopyridine of a transient K+ channel (Kv1.4) cloned from ferret ventricle and expressed in Xenopus oocytes.
Authors: Rasmusson R L , Zhang Y , Campbell D L , Comer M B , Castellino R C , Liu S , Strauss H C
Published: December 2014

1. Using the two‐microelectrode, ‘cut open’ oocyte, and ‘torn off’ macropatch voltage clamp techniques, we studied the blocking effects of 4‐aminopyridine (4‐AP) on two cloned K+ channels expressed in Xenopus oocytes, an inactivating K+ channel isolated from ferret ventricle (FK1), and its NH2‐terminal deletion mutant (delta NCO) which lacks fast N‐type inactivation. 2. Experiments with a permanently charged, impermeant 4‐AP derivative, 4‐aminopyridine‐methyliodide, indicated that the cationic form of 4‐AP blocks at an intracellular site. 3. Block accumulated from pulse to pulse and was sensitive to the applied potential during hyperpolarizing deactivating pulses, indicating trapping of 4‐AP in deactivated channels. For long trains of depolarizing pulses (‐90 to +50 mV, 0.1 Hz), 4‐AP block increased with decreasing pulse duration. Block of FK1 was much more sensitive to pulse duration than was block of delta NCO, consistent with competition between N‐type inactivation and 4‐AP binding. 4. To elucidate these mechanisms further, in the absence of fast N‐type inactivation the following results were obtained on delta NCO channels: (1) application of 4‐AP caused the appearance of apparent inactivation; (2) 4‐AP, however, did not cause cross‐over of deactivating tail currents; (3) 4‐AP block developed with time for potentials positive to ‐40 mV; and (4) trapping of 4‐AP by delta NCO was insensitive to the degree of C‐type inactivation. 5. We conclude that the kinetics of 4‐AP block of FK1 and delta NCO channels cannot be accounted for by either a pure open channel or closed channel blocking scheme.
Potassium currents in rat prevertebral and paravertebral sympathetic neurones: control of firing properties.
Authors: Wang H S , McKinnon D
Published: December 2014

1. Intracellular recordings were made from rat sympathetic neurones in isolated superior cervical ganglia (SCG), coeliac ganglia (CG) and superior mesenteric ganglia (SMG). 2. Based on their response to a maintained depolarizing current stimulus, neurones were classified as ‘phasic’ or ‘tonic’. All neurones in the SCG were phasic, 85% of the neurones in the SMG and 58% of the neurones in the CG were tonic, and the remainder were phasic. 3. The voltage response of phasic and tonic neurones around threshold to a constant current step was markedly different. The response of phasic neurones was biphasic with an initial depolarizing response followed by significant repolarization of the membrane potential. In contrast, tonic neurones became more depolarized during a prolonged current step. 4. The underlying currents were studied using single‐electrode voltage‐clamp recording. The M‐current was present in all phasic neurones, but was very weak or absent in tonic neurones. 5. An A‐current was apparent in both phasic and tonic neurones. The voltage‐dependent activation, steady‐ state inactivation, and current density of the A‐current were all similar in phasic and tonic cells. 6. A low‐ threshold, slowly inactivating outward current (D2‐current) was observed exclusively in tonic neurones. The slow inactivation of this current appeared to underlie the slow depolarizing ramp seen in response to a maintained depolarizing current step. 7. Computer simulations, based on the voltage‐clamp data, suggested that the different firing properties of phasic and tonic neurones could be accounted for by differential expression of the M‐current.
Comparison of arterial potassium and ventilatory dynamics during sinusoidal work rate variation in man.
Authors: Casaburi R , Stringer W W , Singer E
Published: December 2014

1. The mechanisms underlying the exercise hyperpnoea have been difficult to define. Recently it has been suggested that exercise ventilation (VE) changes in proportion to changes in arterial potassium concentration ([K+]a). Similar VE and [K+]a time courses following work rate changes have been cited as supporting evidence. This study compared [K+]a and VE dynamics during moderate exercise in man. 2. We observed VE and gas exchange responses in five healthy men to sinusoidal work rate variation between 25 and approximately 105 W. Tests of approximately 30 min duration were performed at sinusoidal periods of 9, 6 and 3 min and in the steady state. In each test, during two or three sine periods, arterial blood was sampled (24 per test) and analysed for [K+] and blood gases. Response amplitude and phase (relative to work rate) were determined for each variable. 3. [K+]a fluctuated in response to sinusoidal work rate forcing with mean‐to‐peak amplitude averaging 0.15 mmol 1(‐1). However, among tests, VE amplitude and phase were not highly correlated with [K+]a (r = 0.36 and 0.67, respectively). Further, average [K+]a amplitude in the 9 and 6 min sinusoidal studies tended to exceed the steady‐state amplitude, while average VE amplitude fell progressively with increasing forcing frequency. The dissimilar dynamics of [K+]a and VE seem inconsistent with a major role for [K+]a as a proportional controller of ventilation during non‐steady state moderate exercise in man. 4. Among tests, VE and CO2 output (VCO2) amplitude and phase were closely correlated (r = 0.87 and 0.94, respectively). Further, arterial CO2 pressure (Pa,CO2) and arterial pH(pHa) did not fluctuate significantly in ten of twenty and thirteen of twenty studies, respectively. In tests where sinusoidal fluctuation was detected, amplitude averaged 1.1 mmHg and 0.008 units, respectively. Thus VE demonstrated a close dynamic coupling to CO2 output, with consequent tight regulation of Pa,CO2 and pHa.
Inactivation of the cardiac Na+ channels in guinea‐pig ventricular cells through the open state.
Authors: Mitsuiye T , Noma A
Published: December 2014

1. The inactivation kinetics of the Na+ current were investigated using the improved oil‐gap voltage clamp method in single ventricular cells of guinea‐pig hearts. 2. Activation of the Na+ current was observed on depolarization more positive than ‐50 mV from a holding potential of ‐100 mV, and inactivation was complete during these depolarizations. The time course of current decay was fitted by a double exponential at potentials between ‐40 and ‐15 mV, and virtually by a single exponential at more positive potentials. The decay time courses examined either by the double‐pulse protocol or the single‐pulse protocol were similar. 3. The double‐pulse protocol clearly revealed a sigmoidal onset of inactivation on depolarization. The initial delay of inactivation decreased with more positive potentials. The time course of double‐pulse inactivation was reconstructed by integrating the Na+ current recorded by a continuous depolarization. 4. These findings are consistent with the hypothesis that the cardiac Na+ channel inactivates exclusively through the open state.
Effects of divalent cations on muscarinic receptor cationic current in smooth muscle from guinea‐pig small intestine.
Authors: Zholos A V , Bolton T B
Published: December 2014

1. Effects of Mg2+ and Ca2+ on muscarinic receptor cationic current (Icat) in guinea‐pig ileal smooth muscle cells have been studied using patch‐clamp techniques (whole‐cell recording). Icat was activated either by externally applied carbachol or, to bypass receptors, by intracellular GTP‐gamma‐S. 2. Independently of the main permeant cation the current‐voltage (I‐V) relation for Icat was U‐shaped between the reversal potential (usually 0 mV) and very negative potentials such as ‐120 mV where current could be virtually lost. Adding Ca2+ to Ca(2+)‐ and Mg(2+)‐free external solution reduced inward current and made it less U‐shaped whereas adding Mg2+ reduced inward current and shifted more positively the potential at which maximum inward current occurred. 3. Activation of the conductance underlying Icat could be described by the Boltzmann relation which was shifted positively by adding Ca2+ or Mg2+. Extracellular Ca2+ also distorted the relation by increasing the slope factor; maximal conductance was reduced in all cases. Icat relaxation at negative potentials was accelerated by increasing Mg2+ and slowed down by Ca2+. 4. These data suggest the presence of fixed negative surface charges on or near the muscarinic receptor cationic channel, which allow its modulation through alteration of surface potential. Additional more direct ion binding to and blocking of the channel cannot be ruled out. Some additional effects of Ca2+ (if compared with Mg2+) could be explained on the assumption that the Ca(2+)‐binding activation site known to be present on the internal side of the channel can be accessible to Ca2+ entering through the open channel during muscarinic receptor stimulation, as Ca2+ ions contribute to a limited extent to Icat. 5. We conclude that voltage‐dependent gating of muscarinic receptor cationic channels is an intrinsic channel property and that Ca2+ and Mg2+ have strong modulatory effects.
The electrical activity of mouse pancreatic beta‐cells recorded in vivo shows glucose‐dependent oscillations.
Authors: Sánchez-Andrés J V , Gomis A , Valdeolmillos M
Published: December 2014

1. The characteristics of the electrical activity of beta‐cells from islets of Langerhans recorded in vivo are described. For blood glucose concentrations from 4 to 11 mM, the electrical activity of pancreatic beta‐cells is oscillatory, with alternating depolarized and hyperpolarized phases. During the depolarized phases, action potentials are triggered. 2. The main effect of increasing glucose concentration consists of an increase in the duration of the depolarized phase. The relationship between blood glucose concentration and the percentage of time in the depolarized phase can be described by a sigmoidal function with half‐activation at 6.8 mM glucose. The equivalent value obtained in parallel experiments in vitro is 13.3 mM, a significant rightward shift in the activation curve that suggests a role for other neural or humoral factors in determining the islet sensitivity to glucose. 3. The injection of glucose into the bloodstream produces a transitory phase of continuous electrical activity that is recorded within seconds after the change and that leads to a decrease of the glycaemia to the prestimulatory value. 4. The results demonstrate that under physiological conditions the electrical response of beta‐cells to glucose consists of membrane potential oscillations, validating previous data obtained with isolated preparations. Furthermore, the electrical response occurs at lower levels of glycaemia than those predicted from recordings in isolated preparations and is maximal within the physiological range of blood glucose.
Analysis of bursting responses of oxytocin neurones in the rat in late pregnancy, lactation and after weaning.
Authors: Jiang Q B , Wakerley J B
Published: December 2014

1. Electrophysiological recordings were undertaken to compare bursting characteristics of oxytocin (OT) neurones at four reproductive stages: day 20 pregnancy, day 22 of pregnancy (expected day of parturition), day 7‐11 of lactation, and day 5‐6 after weaning. 2. Each OT neurone was recorded for 1 h of suckling, combined with cervico‐vaginal probing at 5 min intervals as an additional stimulus for bursting. Intracerebroventricular (I.C.V.) oxytocin (2.2 ng) was given after 30 min to facilitate bursting responses. Bursts observed during suckling were classified as ‘spontaneous’ or ‘probe‐evoked’. 3. The percentage of cells displaying spontaneous and/or probe‐evoked bursts during the recording was low in day 20 pregnant animals, high in lactators and intermediate in day 20 pregnant and weaner groups. These differences may relate to variation in the proportion of animals with a responsive milk‐ejection reflex, as well as the relative size of the population of bursting OT neurones. 4. In the period before I.C.V. OT, overall burst frequency (including both spontaneous and probe‐evoked bursts) was similar across groups. After I.C.V. OT, overall burst frequency was much higher in lactators compared with other groups. Similar results were obtained when spontaneous bursts were analysed separately. 5. Burst amplitude (action potentials per burst, including both spontaneous and probe‐evoked bursts) prior to I.C.V. OT was similar between the day 20 pregnant, day 22 pregnant and lactating groups, but was lower in weaners. All groups showed an increase in burst amplitude after I.C.V. OT, but values in weaners remained lower than in other groups. In a separate analysis of spontaneous bursts, burst amplitude after I.C.V. OT was higher in lactators, and lower in weaners, than in pregnant animals. 6. Background firing rates of OT cells were higher in the day 20 and day 22 pregnant groups compared with lactators, and lower in weaners. Only OT cells in lactators showed a significant increase in background firing rates following I.C.V. OT. 7. It is concluded that the bursting characteristics of OT cells change markedly between late pregnancy, mid‐lactation and weaning. The factors underlying these changes, which are only loosely correlated with the sequence of morphological adaptations in OT cells surrounding lactation, remain to be established.
Control of Ca2+ entry into rat lactotrophs by thyrotrophin‐releasing hormone.
Authors: Carew M A , Mason W T
Published: December 2014

1. Lactotrophs are adenohypophysial cells that synthesize and secrete prolactin (PRL), a hormone principally involved in mammalian milk production. An increase in the intracellular Ca2+ concentration ([Ca2+]i) is an important signal for PRL secretion. Thyrotrophin‐releasing hormone (TRH) generates Ca2+ signals derived from both the release of Ca2+ from intracellular stores and the entry of extracellular Ca2+, the latter being particularly important for PRL secretion. The identity of this TRH‐sensitive Ca2+ entry pathway is unknown and therefore the subject of the present study. 2. [Ca2+]i was measured by video imaging of fura‐2 loaded into single rat anterior pituitary cells. Ca2+ influx was detected by quenching of fura‐2 fluorescence by external Mn2+. All data are from lactotrophs isolated from lactating female rats. Individual lactotrophs were identified by postexperimental immunofluorescent detection of PRL in fixed cells. 3. TRH induced the release of Ca2+ from intracellular stores and also stimulated Mn(2+)‐permeable Ca2+ influx. U73122 (1 microM), a phospholipase C inhibitor, prevented the Ca(2+)‐mobilizing actions of TRH. The chemically similar but inactive analogue, U73343 (1 microM), had no effect on TRH responses. U73122 did not act as a global G protein inhibitor because the reduction of basal [Ca2+]i by dopamine (1 microM, a G protein‐mediated event) was not affected. 4. TRH‐stimulated Mn2+ influx occurred either immediately after addition of TRH (early entry) or after a delay of about 130 s (late entry). There were no statistically significant differences in the magnitude or temporal characteristics of the Ca2+ signals evoked from cells showing early or late Mn2+ entry. 5. The identity of Ca2+ channels permeable to Mn2+ was investigated. Cell depolarization with 50 mM KCl stimulated Ca2+/Mn2+ influx and was prevented by nifedipine (1 microM). Bay K 8644 (1 microM) also stimulated Mn2+ influx. Thus, the presence of Mn(2+)‐permeable L‐type voltage‐operated Ca2+ channels is likely. A second Mn(2+)‐permeable pathway was present in lactotrophs. Depletion of Ca2+ stores by thapsigargin (1 microM) stimulated a Ca2+ signal and Mn2+ influx. This ‘capacitative entry pathway’ was insensitive to nifedipine (1 microM), indicating that putative L‐type Ca2+ channels were not activated. 6. TRH‐stimulated Mn2+ influx was not prevented by nifedipine (1 microM). TRH added during KCl‐induced Mn2+ influx reduced the quench rate within the time frame of the TRH‐induced Ca2+ spike. TRH may therefore inhibit putative L‐type Ca2+ channels. 7. Addition of thapsigargin in Ca(2+)‐free medium transiently increased [Ca2+]i and prevented subsequent Ca2+ responses to TRH.(ABSTRACT TRUNCATED AT 400 WORDS)
Ryanodine receptor‐mediated intracellular calcium release in rat cerebellar Purkinje neurones.
Authors: Kano M , Garaschuk O , Verkhratsky A , Konnerth A
Published: December 2014

1. Ryanodine receptor‐mediated Ca2+ release was investigated in Purkinje neurones of rat cerebellar slices by using whole‐cell patch‐clamp recordings combined with fluorometric digital imaging of cytoplasmic Ca2+ concentration ([Ca2+]i). 2. Caffeine caused a transient increase in [Ca2+]i in the somata and dendrites of Purkinje neurones. Caffeine‐induced Ca2+ transients were not associated with a membrane inward current and persisted in Ca(2+)‐free external solutions, indicating that they are caused by Ca2+ released from intracellular stores. The amplitudes of the caffeine‐mediated elevations in [Ca2+]i were strongly dependent on the baseline level of [Ca2+]i. 3. Intracellular application of Ruthenium Red through the patch pipette blocked caffeine‐induced Ca2+ transients in Purkinje neurones. Ryanodine when applied either intra‐ or extracellularly caused a use‐dependent block of caffeine‐induced Ca2+ release. 4. Depolarization‐induced Ca2+ transients were strongly prolonged by caffeine. Several lines of evidence suggest that these prolongations reflect Ca(2+)‐induced Ca2+ release. 5. Despite the presence of skeletal muscle type ryanodine receptors in Purkinje neurones, depolarizing pulses failed to induce any changes in [Ca2+]i when the influx of Ca2+ through voltage‐gated channels was prevented by using Ca(2+)‐free solution, or when applying blockers of voltage‐gated Ca2+ channels. 6. Dendritic Ca2+ transients produced by stimulation of the excitatory climbing fibre synaptic input were also prolonged by caffeine, indicating that ryanodine receptor‐mediated release of Ca2+ may be involved in synaptic signalling in cerebellar Purkinje neurones. 7. Ryanodine receptor‐mediated release of Ca2+ in cerebellar Purkinje neurones can be explained by a model in which release of Ca2+ is strongly facilitated by the co‐operative action of Ca2+, caffeine and/or ryanodine. Our results suggest that Ca2+ release in these central neurones becomes prominent only during episodes of intensive electrical activity associated with increased Ca2+ entry.
Characterization of single voltage‐gated Na+ and Ca2+ channels in apical dendrites of rat CA1 pyramidal neurons.
Authors: Magee J C , Johnston D
Published: December 2014

1. We have used dendrite‐attached patch‐clamp techniques to record single Na+ and Ca2+ channel activity from the apical dendrites (up to 350 microns away from soma) of CA1 pyramidal neurons in rat hippocampal slices (ages: 2‐8 weeks). 2. Na+ channels were found in every patch examined (range: 2 to > 20 channels per patch). Channel openings, which had a slope conductance of 15 +/‐ 0.3 pS (mean +/‐ S.E.M.), began with test commands to around ‐50 mV and consisted of both early transient channel activity and also later occurring prolonged openings of 5‐15 ms. All Na+ channel activity was suppressed by inclusion of TTX (1 microM) in the recording pipette. 3. Ca2+ channel activity was recorded in about 80% of the patches examined (range: 1 to > 10 channels per patch). Several types of channel behaviour were observed in these patches. Single channel recordings in 110 mM BaCl2, revealed an approximately 10 pS channel of small unitary current amplitude (‐0.5 pA at ‐20 mV). These channels began activating at relatively hyperpolarized potentials (‐50 mV) and ensemble averages of this low voltage‐activated (LVA) channel activity showed rapid inactivation. 4. A somewhat heterogeneous population of high voltage‐activated, moderate conductance (HVAm; approximately 17 pS), Ca2+ channel activity was also encountered. These channels exhibited a relatively large unitary amplitude (‐0.8 pA at 0 mV) and ensemble averages demonstrated moderate inactivation. The HVAm population of channels could be tentatively subdivided into two separate groups based upon mean channel open times. 5. Less frequently, HVA, large conductance (27 pS) Ca2+ channel activity (HVA1) was also observed. This large unitary amplitude (‐1.5 pA at 0 mV) channel activity began with steps to approximately 0 mV and ensemble averages did not show any time‐dependent inactivation. The dihydropyridine Ca2+ channel agonist Bay K 8644 (0.5 or 1 microM) was found to characteristically prolong these channel openings. 6. omega‐Conotoxin MVIIC (10 microM), did not significantly reduce the amount of channel activity recorded from the LVA, HVAm or HVA1 channel types in dendritic patches. In patches from somata, omega‐conotoxin MVIIC was effective in eliminating a significant amount of HVAm Ca2+ channel activity. Inclusion of 50 or 100 microM NiCl2 to the recording solution significantly reduced the amount of channel activity recorded from LVA and HVAm channel types in dendritic patches. A subpopulation of HVAm channels was, however, found to be Ni2+ insensitive. Dendritic HVA, channel activity was unaffected by these low concentrations of Ni2+.(ABSTRACT TRUNCATED AT 400 WORDS)
Phosphorylation of caldesmon by mitogen‐activated protein kinase with no effect on Ca2+ sensitivity in rabbit smooth muscle.
Authors: Nixon G F , Iizuka K , Haystead C M , Haystead T A , Somlyo A P , Somlyo A V
Published: December 2014

1. Recombinant, activated mitogen‐activated protein kinase (3.3 microM; p42mapk) phosphorylated caldesmon in phasic (rabbit portal vein) and tonic (rabbit femoral artery) smooth muscle strips permeabilized with Triton X‐100. 2. Phosphorylation of caldesmon by p42mapk neither induced contraction of relaxed smooth muscle nor affected the Ca2+ sensitivity of submaximally contracted permeabilized phasic or tonic smooth muscle.
Fast activation and inactivation of inositol trisphosphate‐evoked Ca2+ release in rat cerebellar Purkinje neurones.
Authors: Khodakhah K , Ogden D
Published: December 2014

1. Calcium release from stores via inositol trisphosphate (InsP3) activation of intracellular Ca2+ receptor‐channels is thought to have a role in regulating the excitability of cerebellar Purkinje neurones. The kinetic characteristics of InsP3 receptor activation in Purkinje neurones are reported here. 2. InsP3 was applied by flash photolysis of caged InsP3 during whole‐cell patch clamp. Ca2+ flux into the cytosol was measured with a low‐affinity fluorescent Ca2+ indicator and by activation of Ca(2+)‐dependent membrane conductance. 3. InsP3 produced Ca2+ release from stores with an initial well‐defined delay (mean, 85 ms at 10 microM InsP3), which decreased to less than 20 ms at high InsP3 concentrations. 4. The rate of rise of free [Ca2+], which provides a measure of Ca2+ efflux and InsP3 receptor activation, increased with increasing InsP3 concentration in each cell and had a high absolute value of up to 1400 microM s‐1 at 40 microM InsP3. The period of fast efflux was brief, inactivating in 25 ms at low and in 9 ms at high InsP3 concentration. 5. Peak free [Ca2+] was high (mean, 23 microM with a pulse of 40 microM InsP3) and increased with InsP3 concentration up to 80 microM InsP3 tested here. 6. Experiments with a flash‐released, stable 5‐thio‐InsP3 confirm that the low InsP3 sensitivity of Purkinje neurones does not result from metabolism of InsP3. 7. The low functional affinity and fast activation by InsP3 suggest a difference in InsP3 receptor properties from non‐neuronal cells tested in the same way. The large Ca2+ efflux and high peak [Ca2] probably result from high InsP3 receptor‐channel density. 8. Elevated cytosolic [Ca2+] produced by Ca2+ influx through plasmalemmal Ca2+ channels strongly suppressed InsP3‐evoked Ca2+ release from stores. Rapid termination of InsP3‐evoked efflux results mainly from inhibition by high [Ca2+]. 9. The fast InsP3 activation kinetics and rapid, strong inactivation by Ca2+ influx suggest that interactions between InsP3‐mediated and membrane Ca2+ signalling could occur on a time scale compatible with neuronal excitation.
Patch‐clamp analysis of glycine‐induced currents in chick ciliary ganglion neurons.
Authors: Zhang Z W , Berg D K
Published: December 2014

1. The whole‐cell configuration of the patch‐clamp technique was used to analyse currents induced by glycine in chick ciliary ganglion neurons freshly dissociated from 14‐ to 15‐day‐old embryos. 2. Application of glycine to cells voltage‐clamped at ‐60 mV induced inward currents in all neurons tested. Dose‐response curves yielded an EC50 of about 50 microM. Similar responses were elicited by beta‐alanine and taurine though higher concentrations were required. 3. Strychnine reversibly inhibited the glycine‐induced responses. The effect was dose dependent with a half‐maximal effect being obtained with 20 nM strychnine. 4. Glycine‐induced currents were inhibited by 100 microM Zn2+. The inhibition had slow rates of onset and recovery, in contrast to Zn2+ inhibition of GABA responses. Both bicuculline and d‐tubocurarine inhibited glycine responses in a dose‐dependent manner. 5. The steady‐state I‐V curve for glycine‐induced currents was linear over the range ‐60 to +60 mV, but showed an outward rectification at very hyperpolarized membrane potentials. The reversal potential of glycine‐induced currents shifted with changes in intracellular chloride concentration in a manner expected for chloride‐selective channels. 6. Expression of functional glycine receptors during development was examined at embryonic day 8 (E8), 11, 14, and 18. The mean peak current was about 60‐fold larger at E14 than at E8 and vastly exceeded changes in cell size. During the same period, responses to GABA increased only 2‐fold in amplitude and correlated with changes in cell size.(ABSTRACT TRUNCATED AT 250 WORDS)
A study on rats of the effects of chronic hypoxia from birth on respiratory and cardiovascular responses evoked by acute hypoxia.
Authors: Thomas T , Marshall J M
Published: December 2014

1. Comparative studies were performed on eighteen rats 54 days old made chronically hypoxic from birth in an hypoxic chamber at 12% O2 (CHB), and in eight weight‐matched control rats (NB, 42 days old); both CHB and NB rats were anaesthetized with Saffan. 2. In NB rats, breathing 12 or 8% O2 for 5 min induced a pattern of response comparable to that described in older rats (10‐11 weeks old): an initial increase and secondary fall in minute volume (VE), a fall in arterial pressure (ABP), an increase in muscle vascular conductance, while cerebral blood flow (CBF) increased at the 1st minute in six animals and fell by the 5th minute in all animals. The adenosine receptor antagonist 8‐phenyl‐theophylline (8‐PT, 10 mg kg‐1) reduced the secondary fall in VE, the fall in ABP and muscle vasodilatation, indicating they were partly mediated by adenosine. 3. In CHB rats breathing 12% O2, VE was higher (277 +/‐ 12 vs. 204 +/‐ 18 ml min‐1), arterial partial pressures of O2 (45 +/‐ 2 vs. 88 +/‐ 3 mmHg), CO2 (32 +/‐ 1 vs. 44 +/‐ 1 mmHg) and ABP (105 +/‐ 5 vs. 131 +/‐ 5 mmHg) were lower, while muscle vascular conductance was higher (0.08 +/‐ 0.01 vs. 0.03 +/‐ 0.01 ml min‐1 mmHg‐1) than in NB rats breathing air; these differences were reduced, but not abolished, when CHB rats acutely breathed air for 5 min. 4. In CHB rats, the smaller change from 12 to 8% O2 for 5 min evoked a similar pattern of response to that evoked by 8% O2 in NB rats, except that heart rate (HR) and CBF decreased progressively. However, 8‐PT increased baseline VE and reduced ABP in 12% O2 and reduced the secondary decrease in VE and HR evoked by 8% O2, but had no effect on the fall in ABP, or change in muscle vascular conductance. 5. We propose that in CHB rats (i) there is accentuation of the components of the response to acute hypoxia (the fall in ABP, HR and CBF) that form a positive feedback loop which promotes central ventilatory depression and (ii) that adenosine exerts a tonic inhibitory influence on VE and vasodilator influence in muscle and mediates the secondary fall in VE, but not the muscle vasodilation induced by acute hypoxia.
Control of the amiloride‐sensitive Na+ current in mouse salivary ducts by intracellular anions is mediated by a G protein.
Authors: Dinudom A , Komwatana P , Young J A , Cook D I
Published: December 2014

1. We have previously reported that the Na+ conductance in mouse intralobular salivary duct cells is controlled by cytosolic anions, being inhibited by high cytosolic concentrations of Cl‐ and NO3‐ but not of glutamate. In the present paper, we use whole‐cell patch‐clamp methods to investigate whether this anion effect is mediated by a G protein. 2. Inclusion of 100 mumol l‐1 GTP‐gamma‐S, a non‐hydrolysable GTP analogue, in the glutamate‐containing pipette solution, i.e. when the Na+ conductance is active, reduced the size of the Na+ conductance whereas inclusion of 100 mumol l‐1 GDP‐beta‐S, a non‐hydrolysable GDP analogue, had no effect. 3. Inclusion of 100 mumol l‐1 GDP‐beta‐S in the NO3(‐)‐containing pipette solution, i.e. when the Na+ conductance is inhibited, reactivated the conductance. Inclusion of 500 ng ml‐1 activated pertussis toxin in the NO3(‐)‐containing pipette solution had a similar effect on the Na+ conductance. 4. We conclude that the inhibitory effect of intracellular anions such as NO3‐ and Cl‐ on the amiloride‐sensitive Na+ conductance in mouse mandibular intralobular duct cells is mediated by a G protein sensitive to pertussis toxin.
Modulation of cardiac ryanodine receptors of swine and rabbit by a phosphorylation‐dephosphorylation mechanism.
Authors: Lokuta A J , Rogers T B , Lederer W J , Valdivia H H
Published: December 2014

1. The regulation of the cardiac Ca2+ release channel‐ryanodine receptor (RyR) by exogenous acid phosphatase (AcPh) and purified Ca(2+)‐calmodulin‐dependent protein kinase II (CaMKII) was studied in swine and rabbit sarcoplasmic reticulum (SR) vesicles using [3H]ryanodine binding and planar bilayer reconstitution experiments. 2. Addition of AcPh (1‐20 U ml‐1) to a standard incubation medium increased [3H]ryanodine binding in a Ca(2+)‐dependent manner. Stimulation was only readily apparent in media containing micromolar Ca2+ concentrations. 3. Scatchard analysis of [3H]ryanodine binding curves revealed that AcPh enhanced binding by increasing the affinity of the receptor for [3H]ryanodine without recruiting additional receptor sites (Kd, 9.8 +/‐ 0.85 and 3.9 +/‐ 0.65 nM; Bmax (the maximal receptor density), 1.45 +/‐ 0.14 and 1.47 +/‐ 0.12 pmol mg‐1 for control and AcPh, respectively). The failure of AcPh to increase Bmax suggested that the number of receptors that were ‘dormant’ due to phosphorylation in the SR preparation was very small. 4. At the single channel level, AcPh increased the open probability (Po) of RyR channels by increasing the opening rate and inducing the appearance of a longer open state while having no effect on single channel conductance. Thus AcPh acted directly on RyR channels or a closely associated regulatory protein. 5. CaMKII decreased both [3H]ryanodine binding and Po of RyRs when added to medium supplemented with micromolar levels of Ca2+ and calmodulin (CaM). Addition of a synthetic peptide inhibitor of CaMKII, or replacement of ATP with the non‐hydrolysable ATP analogue adenylyl[beta, gamma‐methylene]‐diphosphate (AMP‐PCP), prevented CaMKII inhibition of RyRs, suggesting that CaMKII acted specifically through a phosphorylation mechanism. 6. The inhibition of RyR channel activity by CaMKII was reversed by the addition of AcPh. Thus we showed that an in vitro phosphorylation‐dephosphorylation mechanism effectively regulates RyRs. 7. The results suggest that intracellular signalling pathways that lead to activation of CaMKII may reduce efflux of Ca2+ from the SR by inhibition of RyR channel activity. The Ca2+ dependence of CaMKII inhibition suggests that the role of the phosphorylation mechanism is to modulate the RyR response to Ca2+.
Adenosine‐induced apoptosis in chick embryonic sympathetic neurons: a new physiological role for adenosine.
Authors: Wakade T D , Palmer K C , McCauley R , Przywara D A , Wakade A R
Published: December 2014

1. A newly found action of adenosine in neurons, which may have an important physiological function in the growth and development of the sympathetic nervous system, is described. Adenosine (1‐100 microM) inhibited neurite outgrowth within the first 24 h and killed about 80% of sympathetic neurons supported by nerve growth factor over the next 2 days in culture. Neurons supported by excess KCl, forskolin or phorbol 12,13‐dibutyrate were equally susceptible to the toxic actions of adenosine. Inosine, guanosine or hypoxanthine (all 100‐300 microM) were without effect on neuronal growth and survival. 2. Specific agonists of adenosine A1 and A2 receptors were not neurotoxic, and toxic effects of adenosine were not antagonized by aminophylline. These results rule out involvement of adenosine receptors and the adenylyl cyclase‐cAMP signalling system in neurotoxic actions of adenosine. 3. Adenosine toxicity was prevented by inhibitors of the adenosine membrane transporter, suggesting an intracellular site of action of adenosine. 4. Inhibitors of adenosine deaminase dramatically facilitated the toxic action so that physiologically relevant concentrations of adenosine were neurotoxic. 5. Adenosine kinase activity of sympathetic neurons was dose‐dependently inhibited by 5'‐iodotubercidin (3‐100 nM). 5'‐Iodotubercidin (100 nM) completely protected neurons against toxicity of adenosine plus adenosine deaminase inhibitors. These results provide convincing evidence that phosphorylation of the nucleoside is an essential requirement for initiation of adenosine toxicity. 6. Sympathetic neurons were successfully rescued from the lethal effects of adenosine deaminase inhibitor plus adenosine by uridine or 2‐deoxycytidine, but not by nicotinamide or 2‐deoxyguanosine, suggesting that depletion of pyrimidine nucleotides by phosphorylated adenosine compounds and consequent inhibition of DNA synthesis produces neuronal death. 7. DNA fragmentation, assessed by the fluorescent dye bisbenzimide and by the TUNEL (terminal deoxynucleotidyl transferase‐mediated nick end labelling) method, indicated that neuronal death induced by adenosine was apoptotic. 8. We conclude that adenosine deaminase and adenosine kinase play an important role in the metabolism of intracellular concentrations of adenosine and thereby regulate the growth and development of sympathetic neurons. Our study highlights, for the first time, the importance of adenosine as a mediator of programmed cell death of neurons supported by nerve growth factor.
Control of sarcoplasmic reticulum calcium release during calcium loading in isolated rat ventricular myocytes.
Authors: Spencer C I , Berlin J R
Published: December 2014

1. Isolated rat ventricular myocytes were whole‐cell voltage clamped using electrodes containing fluorescent Ca2+ indicators. Cytosolic [Ca2+] ([Ca2+]i) was estimated with calcium green‐2 in combination with carboxy SNARF‐1 to remove movement artifacts, or with indo‐1. 2. Sarcoplasmic reticulum (SR) Ca2+ was depleted using 20 mM caffeine in Na(+)‐containing superfusion solution, and cells were Ca2+ loaded by voltage clamp depolarizations applied during superfusion with Na(+)‐free 2 mM Ca2+ solution. Ca2+ currents (ICa) and fluorescence transients elicited by these depolarizations were measured, and the releasable Ca2+ content of the Sr was estimated from the amplitude of fluorescence transients elicited by the rapid application of 20 mM caffeine. 3. Depolarization‐induced [Ca2+]i transients increased in amplitude and duration during superfusion with Na(+)‐free 2 mM Ca2+ solution, independent of changes in peak ICa. Caffeine application confirmed that the SR Ca2+ content increased during this manoeuvre. 4. With increased Ca2+ loading, the fraction of releasable SR Ca2+ involved in depolarization‐induced transients increased, and the gradation in [Ca2+]i transient amplitude produced by beat‐to‐beat variation of voltage clamp pulse duration (10‐100 ms) was progressively lost. This duration dependence of [Ca2+]i transients was maintained during Ca2+ loading when the Ca2+ buffering capacity of the electrode solution was increased with 100 microM BAPTA, 150 microM EGTA, or 60 microM indo‐1. 5. These data suggest that Ca2+ released from the SR during a stimulated [Ca2+]i transient promotes further SR Ca2+ release to a degree which is smoothly graded with SR Ca2+ content. The effects of exogenous Ca2+ buffers suggest that this positive feedback is mediated, at least in part, by [Ca2+]i.
Cyclic AMP prevents activation of a swelling‐induced chloride‐sensitive conductance in chick heart cells.
Authors: Hall S K , Zhang J , Lieberman M
Published: December 2014

1. Changes in myocardial cell volume and whole‐cell currents were measured simultaneously during hyposmotically induced cell swelling. In the conventional patch clamp configuration, hyposmotic challenge caused myocytes to swell continuously and was associated with the development of a sustained, swelling‐induced chloride conductance (ICl). In contrast, perforated patch‐clamped myocytes demonstrated regulatory volume decreases (RVD) during hyposmotic challenge, and ICl was not generated. 2. The swelling‐induced ICl in conventionally patch‐clamped myocytes was inhibited by application of forskolin (15 microM) and was prevented when the pipette filling solution contained cAMP (10 microM) and isobutylmethylxanthine (IBMX, 1 mM). ICl could also be prevented by inhibition of protein phosphatase activity, using okadaic acid (100 nM). Conversely, a swelling‐induced current could be generated in myocytes under perforated patch clamp by inhibition of protein kinase A, using the antagonist Rp‐cAMPS (10 microM). These data demonstrate that cAMP‐dependent protein phosphorylation is both necessary and sufficient to prevent development of ICl during cell swelling. 3. Unlike other chloride currents described previously in heart muscle, generation of the novel swelling‐induced ICl requires dephosphorylation of a cAMP‐dependent protein phosphorylation site; hence it can be prevented by stimulation of cAMP‐dependent protein phosphorylation or by inhibition of protein phosphatase activity.
GABA transport and calcium dynamics in horizontal cells from the skate retina.
Authors: Haugh-Scheidt L , Malchow R P , Ripps H
Published: December 2014

1. Changes in intracellular calcium concentration [Ca2+]i in response to extracellularly applied gamma‐aminobutyric acid (GABA) were studied in isolated horizontal cells from the all‐rod skate retina. 2. Calcium measurements were made using fura‐2 AM, both with and without whole‐cell voltage clamp. Superfusion with GABA, in the absence of voltage clamp, resulted in an increase in [Ca2+]i; the threshold for detection was approximately 50 microM GABA, and a maximal response was elicited by 500 microM GABA. 3. The rise in [Ca2+]i was not mimicked by baclofen nor was it blocked by phaclofen, picrotoxin or bicuculline. However, the GABA‐induced [Ca2+]i increase was completely abolished when extracellular sodium was replaced with N‐methyl‐D‐glucamine. 4. With the horizontal cell voltage clamped at ‐70 mV, GABA evoked a large inward current, but there was no concomitant change in [Ca2+]i. Nifedipine, which blocks L‐type voltage‐gated Ca2+ channels, suppressed the GABA‐induced increase in [Ca2+]i. These findings suggest that the calcium response was initiated by GABA activation of sodium dependent electrogenic transport, and that the resultant depolarization led to the opening of voltage‐gated Ca2+ channels, and a rise in [Ca2+]i. 5. The GABA‐induced influx of calcium appears not to have been the sole source of the calcium increase. The GABA‐induced rise in [Ca2+]i was reduced by dantrolene, indicating that internal Ca2+ stores contributed to the GABA‐mediated Ca2+ response. 6. These observations demonstrate that activation of the GABA transporter induces changes in [Ca2+]i which may have important implications for the functional properties of horizontal cells.
Effect of intracellular magnesium on calcium extrusion by the plasma membrane calcium pump of intact human red cells.
Authors: Raftos J E , Lew V L
Published: December 2014

1. The effect of varying the concentration of intracellular magnesium on the Ca(2+)‐saturated Ca(2+)‐extrusion rate through the Ca2+ pump (phi max) was investigated in human red blood cells with the aid of the divalent cation ionophore A23187. The aim was to characterize the [Mg2+]i dependence of the Ca2+ pump in the intact cell. 2. The initial experimental protocol consisted of applying a high ionophore concentration to obtain rapid sequential Mg2+ and [45Ca]CaCl2 equilibration, prior to measuring phi max at constant internal [MgT]i by either the Co2+ block method or by ionophore removal. With this protocol, competition between Ca2+ and Mg2+ through the ionophore prevented Ca2+ equilibration at high [Mg2+]o. To provide rapid and comparable Ca2+ loads and maintain intracellular ATP within normal levels it was necessary to separate the Mg2+ and the Ca2+ loading‐extrusion stages by an intermediate ionophore and external Mg2+ removal step, and to use different metabolic substrates during Mg2+ loading (glucose) and Ca2+ loading‐extrusion (inosine) periods. 3. Intracellular Co2+ was found to sustain Ca2+ extrusion by the pump at subphysiological [Mg2+]i. Ionophore removal was therefore used to estimate the [Mg2+]i dependence of the pump at levels below [MgT]i (approximately 2 mmol (340 g Hb)‐1), whereas both ionophore removal and Co2+ block were used for higher [MgT]i levels. 4. [Mg2+]i was computed from measured [MgT]i using known cytoplasmic Mg(2+)‐buffering data. The phi max of the Ca2+ pump increased hyperbolically with [Mg2+]i. The Michaelis parameter (K 1/2) of activation was 0.12 +/‐ 0.04 mmol (1 cell water)‐1 (mean +/‐ S.E.M.). Increasing [MgT]i and [Mg2+]i to 9 mmol (340 g Hb)‐1 and 2.6 mmol (1 cell water)‐1, respectively, failed to cause significant inhibition of the phi max of the Ca2+ pump. 5. The results suggest that within the physiological and pathophysiological range of [Mg2+]i, from 0.3 mmol (1 cell water)‐1 in the oxygenated state to 1.2 mmol (1 cell water)‐1 in the deoxygenated state, the Ca(2+)‐saturated Ca2+ pump remains unaffected by [Mg2+]i at normal ATP levels.
Distribution of Ia effects onto human hand muscle motoneurones as revealed using an H reflex technique.
Authors: Mazzocchio R , Rothwell J C , Rossi A
Published: December 2014

1. The possibility of eliciting H reflexes in relaxed hand muscles using a collision between the orthodromic impulses generated by magnetic cortical stimulation and the antidromic motor volley due to a supramaximal (SM) peripheral nerve stimulus was investigated in seven subjects. 2. Magnetic stimuli, applied through a circular coil (outer diameter, 13 cm) centred at the vertex, evoking EMG responses of 3‐5 mV amplitude in the relaxed abductor digit minimi (ADM) muscle, and SM test stimuli to the ulnar nerve at the wrist producing a direct maximal motor response (Mmax) in the ADM muscle, were given either alone or combined. 3. In all subjects, combined cortical and SM ulnar stimulation produced a response after the Mmax with the latency of an H reflex evoked by the ulnar stimulus. This response occurred only within interstimulus intervals (1‐20 ms) compatible with collision in the motor axons. The response behaved like an H reflex being time‐locked to the SM ulnar stimulus, facilitated by voluntary activation of ADM muscle, depressed by vibration (4 s, 100 Hz) of ADM tendon and by a submotor‐threshold ulnar nerve stimulus applied 50 and 80 ms before the combined stimulation, respectively. 4. In some subjects, it was also possible to distinguish an earlier response preceding the H reflex by 3 ms. Evidence is given that this response is probably of cortical origin. 5. Varying the intensity of magnetic stimulation resulted in a non‐linear relationship between the H reflex size and the size of the cortical response. When the latter was between 5‐25% of Mmax, H reflexes were small (2.5‐7.5% of Mmax); with cortical responses between 25‐50% of Mmax, there was a steep increase in H reflex amplitude (10‐30% of Mmax). We suggest that this behaviour is due to an uneven distribution of Ia effects within the motoneurone pool.
Phenotype‐specific expression of T‐type calcium channels in neurons of the major pelvic ganglion of the adult male rat.
Authors: Zhu Y , Zboran E L , Ikeda S R
Published: December 2014

1. Neurons from the major pelvic ganglia (MPG) of adult male rats were enzymatically dissociated and the neurochemical phenotype and Ca2+ current properties examined. 2. Neurons were divided into two subpopulations based on the presence or absence of low threshold T‐type Ca2+ channels. The subpopulation of neurons expressing T‐type Ca2+ channels was characterized by a mean diameter of 34 microns, a mean membrane capacitance (Cm) of 72 pF, tyrosine hydroxylase immunoreactivity (TH‐IR), a lack of NADPH diaphorase (NADPHd) reactivity and a high degree of alpha 2‐adrenoceptor‐mediated Ca2+ current inhibition (60%). 3. The subpopulation of neurons without overt T‐type Ca2+ channels had a mean diameter of 23 microns, a mean Cm of 30 pF, a lack of TH‐IR and a moderate degree of alpha 2‐adrenoceptor‐mediated Ca2+ current inhibition (27%). About 50% of this subpopulation stained positively for NADPHd. 4. The contribution of high threshold N‐type Ca2+ channels (60‐70%), as determined from omega‐conotoxin GVIA inhibition, and L‐type Ca2+ channels (< 10%), as determined from nifedipine inhibition, to the whole‐cell Ca2+ current was similar for both subpopulations of neurons. 5. These data indicate that the MPG contain at least two subpopulations of postganglionic neurons, i.e. adrenergic and non‐adrenergic, with distinct electrophysiological and neurochemical properties. Furthermore, we propose that the presence or absence of T‐type Ca2+ channels provides an electrophysiological means of identifying adrenergic and non‐adrenergic phenotype, respectively, in neurons of the male rat MPG.