bluuebunny/crossref_metadata_2025 huggingface dataset:
Excitatory amino acid receptor‐mediated neurotransmission from cutaneous afferents in rat dorsal horn in vitro.
Authors: King A E , Lopez‐Garcia J A
Published: December 2014

1. The cutaneous mechanoreceptive fields (RFs) of forty‐two lumbar dorsal horn neurones have been examined intracellularly using the hemisected spinal cord‐hindlimb preparation of 10 to 14‐day‐old rats. The neurones were classified into three groups on the basis of their excitatory responses to innocuous and noxious mechanical stimulation; the majority (25/42) were activated by noxious and innocuous stimuli and were classed as ‘wide‐dynamic’ type (WDR). ‘Nociceptive‐specific’ neurones (NS) which were excited by noxious stimuli made up the next largest group (12/42) followed by ‘low‐threshold’ neurones (LT, 5/42) which responded only weakly to noxious stimuli. Another fourteen neurones which did not respond to peripheral stimuli were used to test antagonist selectivity against excitatory amino acid agonists. 2. The response to light touch or pinch consisted of an initial EPSP and cell firing followed by subthreshold EPSPs. The mean +/‐ S.E.M. values for the amplitude (mV) and the duration (s) of the EPSP produced by noxious pinch were significantly greater than those to touch; in WDR neurones the respective values were 14.3 +/‐ 0.9 vs. 11.5 +/‐ 0.7 mV (P < 0.01) and 11.9 +/‐ 1.8 vs. 4.8 +/‐ 0.6 s (P < 0.01). 3. The non‐NMDA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 3‐5 microM) antagonized DL‐alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionate (AMPA)‐induced depolarizations. The amplitude and duration of the EPSPs produced in response to low‐ and high‐threshold mechanical stimulation were potently attenuated and cell firing was abolished in WDR, NS, LT neurones. A similar profile of antagonism was produced in five WDR neurones superfused with ACSF containing 1 mM D‐serine. 4. The NMDA‐receptor antagonist D‐aminophosphonovalerate (D‐AP5, 50 microM) attenuated the EPSP amplitude and duration but never abolished cell firing produced by low‐ and high‐intensity cutaneous mechanical stimulation. A preferential effect of D‐AP5 against the EPSP duration resulted in failure of longer latency spikes. 5. The data indicate that non‐NMDA receptors contribute substantially to dorsal horn neurotransmission and somatosensory processing of noxious and innocuous cutaneous stimuli, while the role of NMDA receptors is restricted to longer latency synaptic components.
Na(+)‐K+ pump stimulation elicits recovery of contractility in K(+)‐paralysed rat muscle.
Authors: Clausen T , Andersen S L , Flatman J A
Published: December 2014

1. This study explores the role of active electrogenic Na(+)‐K+ transport in restoring contractility in isolated rat soleus muscles exposed to high extracellular potassium concentration ([K+]o). This was done using agents (catecholamines and insulin) known to stimulate the Na(+)‐K+ pump via different mechanisms. 2. When exposed to Krebs‐Ringer bicarbonate buffer containing 10 mM K+, the isometric twitch and tetanic force of intact muscles decreased by 40‐69%. The major part of this decline could be prevented by the addition of salbutamol (10(‐5) M). In the presence of 10 mM K+, force could be restored almost completely within 5‐10 min by the addition of salbutamol or adrenaline and partly by insulin. 3. In muscles exposed to 12.5 mM K+, force declined by 96%. Salbutamol (10(‐5) M), adrenaline (10(‐6) M) and insulin (100 mU ml‐1) produced 57‐71, 61‐71 and 38‐47% recovery of force within 10‐20 min, respectively. The effects of these supramaximal concentrations of salbutamol and insulin on force recovery were additive. Salbutamol and adrenaline produced significant recovery of contractility at concentrations down to 10(‐8) M (P < 0.005). 4. In soleus, the same agents stimulated 86Rb+ uptake and decreased intracellular Na+. These actions reflect stimulation of active Na(+)‐K+ transport and both showed a highly significant correlation to the recovery of twitch as well as tetanic force (r = 0.80‐0.88; P < 0.001). 5. The force recovery induced by salbutamol, adrenaline and insulin was suppressed by pre‐exposure to ouabain (10(‐5) M for 10 min or 10(‐3) M for 1 min) as well as by tetrodotoxin (10(‐6) M). 6. The observations support the conclusion that the inhibitory effect of high [K+]o on contractility in skeletal muscle can be counterbalanced by stimulation of active electrogenic Na(+)‐K+ transport, the ensuing increase in the clearance of extracellular K+ and in the transmembrane electrochemical gradient for Na+.
Apnoea following normocapnic mechanical ventilation in awake mammals: a demonstration of control system inertia.
Authors: Leevers A M , Simon P M , Xi L , Dempsey J A
Published: December 2014

1. Inhibition of inspiratory muscle activity from volume‐related feedback during mechanical ventilation has been shown previously. To determine if this neuromechanical inhibition displays a memory effect, the duration of expiration immediately following cessation of mechanical ventilation was assessed in eight normal subjects. The subjects were passively mechanically ventilated via a nasal mask until the end‐tidal CO2 (PET,CO2) was a minimum of 30 mmHg and inspiratory effort was no longer detected, as evidenced by stabilization of mouth pressure and disappearance of surface diaphragm EMG activity. The ventilator output was held constant at a mean tidal volume (VT) of 1.0 l and breath duration of 4.6 s and PET,CO2 was increased 1‐1.5 mmHg/min (via increased inspired CO2 fraction, FI,CO2) until inspiratory muscle activity returned. The PET,CO2 at which activation first occurred was defined as the CO2 recruitment threshold (PCO2,RT). The mechanical ventilation protocol was repeated and the PET,CO2 increased 1‐1.5 mmHg/min until it was a mean of 1.1 mmHg above spontaneous PET,CO2 and 3.6 mmHg below PCO2,RT. After 4‐6 min of mildly hypercapnic mechanical ventilation, the mechanical ventilation was terminated. 2. Following termination of mechanical ventilation, the duration of the subsequent apnoea was 14.6 +/‐ 2.8 s (mean +/‐ S.E.M.) or 453 +/‐ 123% > spontaneous TE and 178 +/‐ 62% > the TE chosen by the subject during ‘assist control’ ventilation at VT = 1.0 l. 3. To test the hypothesis that the apnoea following cessation of mechanical ventilation was due to a vagally mediated memory effect, the study was repeated in five double‐lung transplant patients with similar PCO2,RT to normal subjects. These pulmonary vagally denervated patients also displayed an apnoea (14.5 +/‐ 4.0 s) upon cessation of mechanical ventilation (at a PET,CO2 2.0 mmHg > eupnoea and 2.4 mmHg < PCO2,RT), that was 367 +/‐ 162% > spontaneous TE. 4. We also found significant apnoea in the awake dog immediately following mildly hypercapnic passive mechanical ventilation, and this was similar before and after bilateral vagal blockade (15.7 +/‐ 1.3 and 19.7 +/‐ 4.7 s, respectively). 5. We conclude that neuromechanical inhibition of inspiratory muscle activity, produced by passive mechanical ventilation at high VT, exhibits a memory effect reflected in TE prolongation, which persists in the face of substantial increases in chemoreceptor stimuli. This effect is not dependent on vagal feedback from lung receptors. 6. We hypothesize that this persistent apnoea represents an inherent ‘inertia’, characteristic of the ventilatory control system.(ABSTRACT TRUNCATED AT 400 WORDS)
Enhancement of ATP‐sensitive potassium current in cat ventricular myocytes by beta‐adrenoreceptor stimulation.
Authors: Schackow T E , Ten Eick R E
Published: December 2014

1. To address the questions of whether beta‐adrenoreceptor stimulation can augment ATP‐sensitive potassium current (IK(ATP)), and what the mechanism of such an effect might be, action potentials and whole‐cell ionic currents were recorded from adult cat cardiac ventricular myocytes using a conventional whole‐cell patch technique. 2. An outwardly directed, ohmic, non‐inactivating, glyburide (10 microM)‐sensitive current reversing near the reversal potential for potassium (EK) developed slowly (10‐25 min) in cells dialysed with an ATP‐free pipette (intracellular) solution. During this time, action potential duration markedly decreased while the resting membrane potential hyperpolarized closer to EK. Extended (> 30 min) periods of internal dialysis with ATP‐free solution eventually resulted in run‐down of the outward current. 3. Externally applied isoprenaline (1 microM) caused a rapidly developing (< or = 60 s), sustained enhancement of a glyburide (10 microM)‐sensitive IK(ATP) in cells internally dialysed with ATP‐free solution. IK(ATP) remained elevated even after the isoprenaline was removed, and subsequent applications of the beta‐agonist failed to increase IK(ATP) further. Half‐maximal isoprenaline stimulation of IK(ATP) occurred at a concentration of approximate of 1.5 nM. 4. Pretreatment with propranolol (1 microM) prevented the enhancement of IK(ATP) by a beta‐agonist. 5. Isoprenaline‐induced IK(ATP) could be blocked by either internal application of GDP‐beta‐S (2‐5 mM) or pretreatment with cholera toxin (1‐10 microgram ml‐1, > 18 h). Pretreatment with pertussis toxin (1‐2 microgram ml‐1, > 18 h) did not attenuate the isoprenaline response, whereas internally applied GTP‐gamma‐S (100 microM) or F‐ (20 mM) caused IK(ATP) to increase rapidly in the absence of the beta‐agonist. 6. Although externally applied forskolin (10 microM) also stimulated IK(ATP), neither 1,9‐dideoxyforskolin (10 microM) nor 8‐(4‐chlorophenylthio)‐cAMP (200 microM) had any effect on the current. Internal application of the adenylate cyclase inhibitor 2'‐deoxyadenosine‐3'‐monophosphate (100 microM) resulted in a reduction in the response to isoprenaline, while internal application of a protein kinase A inhibitor (PKI5‐24, 22.5 microM) did not attenuate the response to the beta‐agonist. 7. IK(ATP) developed slowly during internal dialysis with ATP‐free solution.(ABSTRACT TRUNCATED AT 400 WORDS)
Effect of tonic voluntary activity on the excitability of human motor cortex.
Authors: Mazzocchio R , Rothwell J C , Day B L , Thompson P D
Published: December 2014

1. The threshold for obtaining EMG responses after transcranial magnetic stimulation of the brain is reduced by voluntary contraction of the target muscle. The present experiments tested whether some of this effect is due to increased cortical, as opposed to spinal, excitability during the contraction. 2. Magnetic stimulation was delivered with a figure‐of‐eight coil oriented with the junction region along the interaural line and also (in 4 of 7 subjects) with a circular coil centred at the vertex. The intensity of the conditioning stimulus was subthreshold for evoking a motor response in the relaxed wrist flexor muscles of the forearm. The presence of a small descending corticospinal volley in both the relaxed and active conditions was detected by measuring the facilitation of test H reflexes elicited in the flexor muscles of the forearm. 3. In all subjects, magnetic stimulation with either coil facilitated the H reflex at conditioning‐test intervals of ‐1 to ‐3 ms (median nerve stimulus before magnetic). This was followed by a long‐lasting facilitation. In three of the seven subjects stimulation with the figure‐of‐eight coil elicited an additional, earlier peak of facilitation at a conditioning‐test interval of ‐3 to ‐5 ms. 4. In all subjects, the threshold for obtaining facilitation of the H reflex using a conditioning‐test interval of ‐1 to ‐3 ms was reduced, and the amount of facilitation was larger, if subjects performed a weak tonic voluntary contraction. In contrast, with a conditioning‐test interval of ‐3 to ‐5 ms voluntary contraction had no effect on the threshold. 5. It is suggested that H reflex facilitation at the conditioning‐test interval of ‐1 to ‐3 ms was produced by indirect activation of corticospinal neurones by the magnetic stimulus, whereas at ‐3 to ‐5 ms, the facilitation was produced by direct activation of corticospinal axons. It is concluded that tonic voluntary contraction of a target muscle decreases the threshold for indirect activation of corticospinal neurones but not for direct stimulation of their axons.
Contractile activation and force generation in skinned rabbit muscle fibres: effects of hydrostatic pressure.
Authors: Fortune N S , Geeves M A , Ranatunga K W
Published: December 2014

1. Effects of hydrostatic pressure (range 0.1‐10 MPa) on the isometric tension of skinned (rabbit psoas) muscle fibres were examined at 12 degrees C and at different levels of Ca2+ activation (pCa range 4‐7); the effects on both the steady tension and the tension transients induced by rapid pressure release (< 1 ms) are described. 2. The steady tension was depressed by increased pressure (approximately 1% MPa‐1) at a high level of Ca2+ activation (pCa approximately 4) whereas it was potentiated at lower Ca2+ levels (pCa > 6); the effects were reversible. 3. At maximal Ca2+ activation, the tension recovery following pressure release (10 MPa to atmospheric) consisted of a fast (approximately 30 s‐1) and a slow (2‐3 s‐1) phase; the rate and the normalized amplitude (normalized to the steady tension at atmospheric pressure for a particular pCa) of the fast phase were invariant with changes in Ca2+ level. 4. The effects of changing Ca2+ level on the slow phase were complex; its positive amplitude at high Ca2+ levels changed to negative and the rate decreased to approximately 1 s‐1 at low Ca2+ levels (pCa > 6.0). 5. Results are discussed in relation to previous studies on the effect of pressure on intact muscle fibres and the actin‐myosin interaction. This work supports calcium regulation of cross‐bridge recruitment rather than calcium regulation of the rate of a specific step in the cross‐bridge cycle.
The role of sarcoplasmic reticulum in relaxation of mouse muscle; effects of 2,5‐di(tert‐butyl)‐1,4‐benzohydroquinone.
Authors: Westerblad H , Allen D G
Published: December 2014

1. Intracellular calcium concentration ([Ca2+]i) and force were measured from isolated single mouse skeletal muscle fibres at rest and during tetani. The actions of 2,5‐di(tert‐butyl)‐1,4‐benzohydroquinone (TBQ), an inhibitor of the sarcoplasmic reticulum (SR) Ca2+ pump, were examined at a range of concentrations (100‐1000 nM). 2. TBQ increased resting [Ca2+]i, increased tetanic [Ca2+]i and slowed the rate of decline of [Ca2+]i after a tetanus. TBQ produced a small increase in tetanic force and a large slowing of the rate of relaxation after a tetanus. All these effects were reversible. 3. TBQ had no important effects on the Ca2+ sensitivity or the maximum force produced by the myofibrillar proteins. 4. Analysis of the SR Ca2+ pump function confirmed that under control conditions and at very low levels of [Ca2+]i, the relationship between [Ca2+]i and SR pump rate was a 4th power function. TBQ caused a pronounced inhibition of the pump rate and reduced the power function to < 3. 5. Muscle fibres were fatigued by repeated tetani until tetanic [Ca2+]i and force were reduced and the rate of decline of [Ca2+]i after a tetanus was slowed. Under these conditions application of TBQ caused a further slowing of the rate of decline of [Ca2+]i but still increased tetanic [Ca2+]i and force. This result suggests that slowing of the SR pump rate is not the cause of the decline in tetanic [Ca2+]i and force at the late stage of fatigue. 6. A simple model of the interactions of Ca2+, TBQ and pump proteins is described, which predicts the 4th power function of the normal pump, inhibition by TBQ, and the reduced power function in the presence of TBQ. 7. A model of Ca2+ movements and force development in muscle is described, which closely matches the experimental results under control conditions. Inhibition of the SR pump by TBQ using the model of the pump described above simulates qualitatively all the observed effects of TBQ on [Ca2+]i and force. 8. In conclusion, TBQ is a potent, specific and reversible inhibitor of the SR Ca2+ pump in intact mouse skeletal muscle. Inhibition of the pump directly affects intracellular Ca2+ handling and force production.
Variable restriction of albumin diffusion across inflamed cerebral microvessels of the anaesthetized rat.
Authors: Easton A S , Fraser P A
Published: December 2014

1. The possibility of restricted diffusion of macromolecules in single cerebral venular capillaries that have become leaky due to inflammation was investigated by comparing the permeabilities to Lucifer Yellow (457 Da; PLY) and rhodamine‐labelled albumin (69 kDa; PRh‐A). 2. The dyes were trapped between two micro‐occlusion probes and the permeabilities were measured from the rates of decrease in dye fluorescence at low intraluminal hydrostatic pressure. 3. Removal of one probe had little effect on PLY but did reduce PRh‐A, consistent with the influence of convection on diffusion through 22 nm wide transendothelial slits 1 micron deep. 4. Direct comparisons were made over time between PLY and PRh‐A in six vessels while hydrostatic pressure effects were controlled. In all vessels PRh‐A:PLY varied from being similar to the ratio of the free diffusion coefficients to virtually zero even though PLY remained high. The question of the source of this variable restriction to albumin is discussed in terms of the secretion and sloughing of glycosaminoglycans and the possible role of transient formation of transendothelial gaps.
Reflex increase in blood pressure induced by leg compression in man.
Authors: Williamson J W , Mitchell J H , Olesen H L , Raven P B , Secher N H
Published: December 2014

1. We tested the hypotheses that the increase in mean arterial pressure with the application of external leg compression in man is (i) blocked with epidural anaesthesia, and (ii) dependent upon the level of external pressure applied, the quantity of leg muscle mass compressed and the vascular volume of the leg. Fourteen healthy subjects were fitted with an anti‐shock trouser garment to provide three levels (30, 60 and 90 mmHg) of leg compression, while cephalad translocation of fluid was prevented by upper‐thigh cuffs inflated to a supra‐systolic pressure. Cardiovascular responses were recorded during leg compression before and after the administration of epidural anaesthesia in eight subjects, while blood pressure responses from six subjects were compared with their single leg pressor response. 2. Both mean arterial and diastolic pressures were elevated with increasing leg compression, with no changes in heart rate, cardiac output, thoracic impedance, and central venous pressure. The leg compression‐induced blood pressure increases were abolished by epidural anaesthesia. Furthermore, when only one leg was compressed at 90 mmHg, the pressor response was less than that elicited from compression of both legs at the same external pressure. Changes in vascular volume of the leg did not influence the pressor response to leg compression. 3. The results indicate that the mean arterial pressure increases in response to external compression of the legs and that a reflex mechanism, mediated by muscle afferent nerves, is involved. The response is dependent upon both the changes in intramuscular pressure and the quantity of muscle mass compressed.
An in vivo study of the effects of ischaemia on uterine contraction, intracellular pH and metabolites in the rat.
Authors: Harrison N , Larcombe‐McDouall J B , Earley L , Wray S
Published: December 2014

There are no data concerning the functional or metabolic effects of hypoxia in vivo in smooth muscle. We have therefore used 31P‐NMR spectroscopy and intra‐uterine pressure measurements to examine simultaneously, in vivo, the effect of ischaemia on uterine metabolites, intracellular pH (pHi) and force. A 1‐2 cm portion of uterus from day 1 postpartum anaesthetized rats was exteriorized and an NMR surface coil placed on it. A balloon catheter in the uterine lumen recorded intra‐uterine pressure changes from the same area. Reversible occluders were placed around the uterine artery. Occlusion produced a decrease and then abolition of contractions, within 10 min. In four of five animals contraction was abolished within 2 min. Upon reperfusion force was rapidly restored (1 min), in all preparations. The mean level of force was significantly above control (pre‐occlusion) 20‐30 min after reperfusion. The NMR data showed a significant fall in [ATP] (28%) and [phosphocreatine] (34%) during occlusion. Inorganic phosphate doubled in concentration during this period. Metabolites recovered slowly upon reperfusion, taking 20‐30 min to return to pre‐occlusion levels. The mean pHi fell from 7.32 to 7.00 upon occlusion and was rapidly reversed upon reperfusion. The changes in pHi closely correlated with the changes in uterine force. Decreases of pHi of a similar magnitude in vitro have previously been shown to abolish contractions; thus it is suggested that during ischaemia in vivo the depression of contraction is caused by the large fall in pHi.
Influence of phosphate and pH on myofibrillar ATPase activity and force in skinned cardiac trabeculae from rat.
Authors: Ebus J P , Stienen G J , Elzinga G
Published: December 2014

1. The effects of inorganic phosphate (Pi) and pH on maximal calcium‐activated isometric force and MgATPase activity were studied in chemically skinned cardiac trabeculae from rat. ATP hydrolysis was coupled enzymatically to the breakdown of NADH, and its concentration was determined photometrically. Measurements were performed at 2.1 microns sarcomere length and 20 degrees C. ATPase activity and force were also determined when square‐wave‐shaped length changes were applied, with a frequency of 23 Hz and an amplitude of 2.5%. 2. At pH 7.0 without added Pi, the average isometric force (+/‐ S.E.M.) was 51 +/‐ 3 kN m‐2 (n = 23). The average isometric ATPase activity was 0.43 +/‐ 0.02 mM s‐1 (n = 23). During the changes in length ATPase activity increased to 152 +/‐ 3% of the isometric value, while the average force level decreased to 48 +/‐ 2%. 3. Isometric force gradually decreased to 31 +/‐ 2% of the control value when the Pi concentration was increased to 30 mM. Isometric ATPase activity, however, remained constant for Pi concentrations up to 5 mM and decreased to 87 +/‐ 3% at 30 mM Pi. When Pi accumulation inside the preparation due to ATP hydrolysis was taken into account, a linear relationship was found between isometric force and log [Pi]. The decrease in relative force was found to be 44 +/‐ 4% per decade. 4. During the length changes, ATPase activity and average force showed, apart from the increase in ATPase activity and decrease in average force, the same dependence on Pi as the isometric values. Stiffness, estimated from the amplitude of the force responses during the length changes, decreased in proportion to isometric force when the Pi concentration was increased. The changes in the shape of the force responses due to the repetitive changes in length as a function of the Pi concentration were relatively small. These results suggest that the effect of Pi on the transitions which influence ATP turnover is rather insensitive to changes in cross‐bridge strain. 5. Isometric force, normalized to the control value at pH 7.0, increased gradually from 54 +/‐ 1% at pH 6.2 to 143 +/‐ 10% at pH 7.5. ATPase activity remained practically constant for pH values from 6.8 to 7.2 but decreased to 80 +/‐ 1% at pH 6.2 and to 83 +/‐ 5% at pH 7.5. ATPase activity during the length changes was reduced more than the isometric ATPase activity when pH was lowered.(ABSTRACT TRUNCATED AT 400 WORDS)
Inactivation of single Ca2+ channels in rat sensory neurons by extracellular Ca2+.
Authors: Galli A , Ferroni A , Bertollini L , Mazzanti M
Published: December 2014

1. Single Ca2+ channels conducting 20 mM Ba2+ from adult rat dorsal root ganglion cells were characterized using the two‐electrode patch‐clamp technique configuration. 2. Channels demonstrating specific characteristics of conductance, voltage dependence and dihydropyridine sensitivity were classified as high‐threshold or L‐type Ca2+ channels. 3. Mean single‐channel current in 20 mM Ba2+ did not show inactivation, but inactivation occurred when using Ca2+ as a permeating ion. 4. Stimulus protocols were delivered alternately in the cell‐attached and whole‐cell electrode, while recording single‐channel activity and total Ca2+ current simultaneously. 5. A mean single‐channel Ba2+ current from a stimulated patch did not show inactivation. However, stimulation of a physiological whole‐cell Ca2+ current induced a marked inactivation of mean single‐channel Ba2+ current. 6. Complete Ca2+ current block by the addition of 200 microM Cd2+ in the external solution removed single‐channel inactivation in patches stimulated through a whole‐cell electrode.
On the mechanism of 4‐aminopyridine action on the cloned mouse brain potassium channel mKv1.1.
Authors: Stephens G J , Garratt J C , Robertson B , Owen D G
Published: December 2014

1. This study used the whole‐cell patch clamp technique to investigate the mechanism of action of the K+ channel blocker 4‐aminopyridine (4‐AP) on the cloned K+ channel mouse Kv1.1 (mKv1.1) expressed in Chinese hamster ovary cells. 2. Cells transfected with mKv1.1 expressed a non‐inactivating, delayed rectifier‐type K+ current. 4‐AP induced a dose‐, voltage‐ and use‐dependent block of mKv1.1. 3. 4‐AP blockade of mKv1.1 was similar whether 4‐AP was administered extracellularly (IC50 = 147 microM) or intracellularly (IC50 = 117 microM). 4. Inclusion of the first twenty amino acids of the N‐terminus sequence of the Shaker B K+ channel (‘inactivation peptide’) in the patch electrode transformed mKv1.1 into a rapidly inactivating current. The time constant of decay for the modified current was dependent on the concentration of inactivation peptide, and under these conditions extracellular 4‐AP had a reduced potency (IC50 values of 471 and 537 microM for 0.5 and 2 mg ml‐1 inactivation peptide, respectively). 5. A permanently charged analogue of 4‐AP, 4‐aminopyridine methiodide (4‐APMI), was found to block mKv1.1 when applied inside the cell, but was without effect when administered externally. 6. Decreasing the intracellular pH (pHi) to 6.4 caused an increase in 4‐AP potency (IC50 = 76 microM), whereas at pHi 9.0, the 4‐AP potency fell (IC50 = 295 microM). Conversely, increasing extracellular pH (pHo) to 9.0 caused an increase in 4‐AP potency (IC50 = 93 microM), whereas at pHo 6.4, 4‐AP potency decreased (IC50 = 398 microM). 7. Taken together, these findings support the hypotheses that the uncharged form of 4‐AP crosses the membrane, and that it is predominantly the cationic form which acts on mKv1.1 channels intracellularly, possibly at or near to the binding site for the inactivation peptide.
Calcium channel subtypes in cat chromaffin cells.
Authors: Albillos A , Artalejo A R , López M G , Gandía L , García A G , Carbone E
Published: December 2014

1. Using the patch‐clamp technique we have investigated the kinetic and pharmacological properties of high‐voltage‐activated (HVA) Ca2+ channels in short‐term‐cultured cat chromaffin cells. 2. In 10 mM Ba2+, HVA currents activated around ‐40 mV, reached maximal amplitude at 0 mV and reversed at about +60 mV. At 0 mV, HVA current activation was fast (mean tau act, 2.45 ms), and followed by either an incomplete inactivation or by a second slow phase of activation (mean tau slow, 36.8 ms) that was lost when Ba2+ was replaced by Ca2+. HVA Ba2+ currents deactivate quickly on repolarization to ‐50 mV (mean tau deact, 0.36 ms). 3. In most cells, HVA currents were sensitive to common dihydropyridine (DHP) derivatives. Nisoldipine blocked the currents maximally at low membrane potentials (mean block 76% at ‐30 mV, 3 microM) and gradually less at higher voltages. Nisoldipine block was clearly time dependent (33 and 56% after 30 and 600 ms, respectively, to 0 mV). 4. Bay K 8644 (3 microM) action was variable and caused (1) a 2‐ to 4‐fold increase of Ba2+ currents at ‐40 to ‐20 mV, (2) a ‐15 mV shift of the current‐voltage relationship and (3) a 10‐ to 20‐fold prolongation of HVA channel deactivation at ‐50 mV. 5. Nisoldipine block and Bay K 8644 potentiation of HVA currents increased markedly in omega‐conotoxin GVIA (omega‐CgTX)‐pretreated cells, suggesting an increased fraction of DHP‐sensitive currents in these cells. Nisoldipine block of residual omega‐CgTX‐resistant currents was almost complete (mean block, 82%) during pulses of 1 s to 0 mV. 6. The degree of inhibition produced by omega‐CgTX (2 microM for 1 min) varied from cell to cell (mean block, 46%) and was partly reversible. Residual omega‐CgTX‐resistant currents exhibited faster activation‐deactivation kinetics than control currents. 7. The slow phase of HVA current activation was abolished if a conditioning prepulse of 40 ms to +70 mV preceded a test pulse to 0 mV. Double‐pulse protocols caused an average current increase (facilitation) of 37% that was voltage dependent and which correlated with the slow phase of Ca2+ channel activation. Facilitation was lost in most omega‐CgTX‐treated cells and was little affected by nisoldipine (3 microM) and Bay K 8644 (1 microM). Facilitation was potentiated in cells dialysed with 100 microM guanosine 5'‐O‐(3‐thiotriphosphate) (GTP‐gamma‐S) and fully prevented by 1 mM guanosine 5'‐O‐(2‐thiodiphosphate) (GDP‐beta‐S).(ABSTRACT TRUNCATED AT 400 WORDS)
Functional role of bicarbonate in propionate transport across guinea‐pig isolated caecum and proximal colon.
Authors: von Engelhardt W , Gros G , Burmester M , Hansen K , Becker G , Rechkemmer G
Published: December 2014

1. Unidirectional fluxes of propionate across isolated epithelia from the guinea‐pig caecum and proximal colon were measured under short‐circuit current conditions. In the caecum and proximal colon the serosal‐to‐mucosal propionate flux (JPrsm) was higher than mucosal‐to‐serosal flux (JPrms), resulting in a net secretory flux of propionate. 2. HCO3(‐)‐CO2‐free solution reduced JPrms in the caecum and proximal colon markedly; JPrsm was not (caecum) or little (proximal colon) affected. The subsequent addition of acetazolamide caused a further decrease in JPrms in the proximal colon, but not in the caecum. 3. In HCO3(‐)‐containing solutions acetazolamide or ethoxzolamide inhibited JPrms; JPrsm was not affected. A macromolecular carbonic anhydrase inhibitor, prontosil‐dextran, had no effect on propionate fluxes, indicating that the intracellular carbonic anhydrase is of importance for short‐chain fatty acid transport. 4. Subsequent to carbonic anhydrase inhibition, mucosal addition of amiloride caused a slight further decrease of JPrms in the caecum and proximal colon; JPrsm was not affected. 5. Results support the view that a considerable proportion of short‐chain fatty acids (SCFAs) is absorbed via a SCFA(‐)‐HCO3‐ exchange.
Regulation of the beta‐stimulation of the Na(+)‐K+ pump current in guinea‐pig ventricular myocytes by a cAMP‐dependent PKA pathway.
Authors: Gao J , Cohen I S , Mathias R T , Baldo G J
Published: December 2014

1. The whole‐cell patch‐clamp technique was employed with the free intracellular [Ca2+] fixed at 1.4 microM in order to study the isoprenaline (Iso)‐induced increase in the Na(+)‐K+ pump current (Ip) in acutely isolated guinea‐pig ventricular myocytes. 2. The non‐specific protein kinase inhibitor, H‐7, eliminated the stimulatory effect of Iso, suggesting a phosphorylation step is involved in the beta‐agonist stimulation of Ip. 3. H‐7 or the phosphatase inhibitor calyculin A individually had no effect on basal Ip; however, when Ip was first increased by Iso, H‐7 inhibited and calyculin A further increased Ip. This suggests phosphorylation is not important to the basal regulation of Ip, but does have an effect during beta‐stimulation. 4. The Iso‐induced increase in Ip could be mimicked by adding the membrane‐permanent cAMP analogue chlorophenylthio‐cAMP, blocking cAMP degradation with IBMX or stimulating cAMP production with forskolin. Alternatively the protein kinase A inhibitor PKI blocked the stimulatory effect of Iso. This suggests the Iso‐induced phosphorylation responsible for increasing Ip is mediated by cAMP, which then activates protein kinase A (PKA). 5. We conclude that the beta‐agonist‐induced increase in Ip in the presence of high intracellular [Ca2+] is mediated by a phosphorylation step via the cAMP‐dependent PKA pathway. During beta‐stimulation, this increase in active Na(+)‐K+ transport can serve to offset the effects of increases in passive membrane conductances.
Properties and regulation of basolateral K+ channels in rat duodenal crypts.
Authors: McNicholas C M , Fraser G , Sandle G I
Published: December 2014

1. Patch clamp recording techniques were used to study the properties of K+ channels in the basolateral membrane of rat duodenal crypts, and their regulation by Cl(‐)‐secretory agonists. 2. High conductance (84‐99 pS) K+ channels were activated by 0.1 mM dibutyryl cAMP in 50% of cell‐attached patches, while 0.1 mM carbachol had no effect on channel activity. High conductance K+ channels were voltage independent, Ca2+ insensitive, blocked by 5 mM Ba2+ or 20 mM tetraethylammonium chloride (TEA), and stimulated by 57% when intracellular pH was increased from 7.4 to 7.8. 3. In contrast, low conductance (19‐28 pS) K+ channels were activated by 0.1 mM dibutyryl cAMP in 67% of cell‐attached patches, while 0.1 mM carbachol activated channels in 100% of cell‐attached patches. Low conductance K+ channels were voltage independent, Ca2+ sensitive and pH insensitive. Unlike the high conductance K+ channels, 5 mM Ba2+ had no effect on the activity of low conductance K+ channels, although 20 mM TEA decreased channel activity by 53%. 4. The results show that the basolateral membrane of rat duodenal crypts possesses two discrete populations of K+ channels that may have important roles in sustaining the small intestinal Cl(‐)‐secretory responses triggered by a variety of cAMP‐ and Ca(2+)‐mediated agonists.
Role of cytosolic Ca2+ in inhibition of InsP3‐evoked Ca2+ release in Xenopus oocytes.
Authors: Ilyin V , Parker I
Published: December 2014

1. Calcium liberation induced in Xenopus oocytes by flash photorelease of inositol 1,4,5‐trisphosphate (InsP3) from a caged precursor was monitored by confocal microfluorimetry. The object was to determine whether inhibition of Ca2+ release seen with paired flashes arose as a direct consequence of elevated cytosolic free [Ca2+]. 2. Responses evoked by just‐suprathreshold test flashes were not inhibited by subthreshold conditioning flashes, but were strongly suppressed when conditioning flashes were raised above threshold. 3. Inhibition at first increased progressively as the inter‐flash interval was lengthened to about 2 s and thereafter declined, with a half‐recovery at about 4 s. 4. Intracellular injections of Ca2+ caused relatively slight inhibition of InsP3‐evoked signals, even when cytosolic free [Ca2+] was elevated to levels similar to those at which strong inhibition was seen in paired‐flash experiments. 5. Recovery from inhibition was not appreciably slowed when Ca2+ was injected to raise the free Ca2+ level between paired flashes. 6. We conclude that inhibition of InsP3‐evoked Ca2+ liberation is not directly proportional to cytosolic free Ca2+ level and that recovery from inhibition in paired‐pulse experiments involves factors other than the decline of cytosolic [Ca2+] following a conditioning response.
Blood‐borne interleukin‐1 alpha is transported across the endothelial blood‐spinal cord barrier of mice.
Authors: Banks W A , Kastin A J , Ehrensing C A
Published: December 2014

1. Previous work has shown that one mechanism by which blood‐borne interleukin‐1 alpha (IL‐1) may be able to affect the central nervous system (CNS) is by direct transport into the brain across the blood‐brain barrier (BBB). The BBB of the brain consists of endothelial (between blood and interstitial fluid) and ependymal (between blood and cerebrospinal fluid) barriers. Which of these barriers IL‐1 can cross has not previously been investigated. At the spinal cord, which could be the site of action for some of the effects of IL‐1 such as analgesia, the BBB consists only of the endothelial barrier. 2. We show here that IL‐1 labelled with 125I (I‐IL) is transported across the BBB of the spinal cord by a saturable system similar to the one previously described for the brain. High performance liquid chromatography (HPLC) showed that most of the material entering the spinal cord represented intact I‐IL. The BBB of the spinal cord was no more leaky to radioactively labelled albumin than the BBB of the brain and was not disrupted by 50 micrograms kg‐1 of IL‐1. 3. Capillary depletion showed that most of the I‐IL entered the parenchymal‐interstitial fluid space of the spinal cord with only a modest amount being sequestered by the endothelial cells of its BBB. 4. I‐IL entered the cervical, thoracic and lumbar regions of the spinal cord equally well. I‐IL entering at the brain and diffusing caudally was estimated only to account for about 1% of the total radioactivity found in the spinal cord after i.v. injection.(ABSTRACT TRUNCATED AT 250 WORDS)
Regulation of intracellular pH in the smooth muscle of guinea‐pig ureter: HCO3‐ dependence.
Authors: Aickin C C
Published: December 2014

1. HCO3(‐)‐dependent mechanisms involved in the regulation of intracellular pH (pHi) were characterized using double‐barrelled pH‐sensitive microelectrodes in smooth muscle cells of the isolated guinea‐pig ureter. 2. Removal of external Cl‐ in the presence of CO2‐HCO3‐ caused a transient alkalosis, consistent with the presence of Cl(‐)‐HCO3‐ exchange, before pHi slowly recovered. Recovery from acidosis in the presence of CO2‐HCO3‐ was not affected, at a time when intracellular Cl‐ would have been maximally depleted, indicating that a counter transport of Cl‐ and HCO3‐ was not involved. The recovery was also not affected by amiloride, indicating that Na(+)‐H+ exchange was not involved. 3. A transient hyperpolarization was associated with the recovery from acidosis in the presence of CO2‐HCO3‐, consistent with rheogenic coupling of Na(+)‐HCO3‐ cotransport. However, depolarization caused by elevation of the extracellular potassium (K+o) concentration, which should favour inward transport by the rheogenic mechanism, caused a fall in pHi and decreased the rate of recovery from acidosis. Furthermore, ouabain abolished the transient hyperpolarization without affecting the recovery of pHi. It is concluded that Na(+)‐HCO3‐ cotransport in the ureter is electroneutral. 4. Recovery from acidosis in the presence of CO2‐HCO3‐ was insensitive to DIDS even after prolonged pre‐equilibriation and extreme acidosis. The results suggest that Na(+)‐HCO3‐ cotransport in the ureter is insensitive to DIDS and that Cl(‐)‐HCO3‐ exchange does not reverse to contribute to the extrusion of acid equivalents. A HCO3‐ conductance may account for the Na(+)‐independent, HCO3(‐)‐dependent recovery from extreme acidosis. 5. Recovery from experimentally induced alkalosis was inhibited by Cl(‐)‐free conditions and by DIDS, indicating that Cl(‐)‐HCO3‐ exchange was involved. 6. It is concluded that pHi in the smooth muscle of guinea‐pig ureter is controlled by three transport mechanisms. By far the most important is an electroneutral Na(+)‐HCO3‐ cotransporter. Na(+)‐H+ exchange appears to play little role in the presence of the physiological buffer. Both of these mechanisms extrude acid equivalents and so protect the cell against its fairly substantial intrinsic intracellular acid loading. Cl(‐)‐HCO3‐ exchange, on the other hand, is stimulated by intracellular alkalosis to transport acid equivalents into the cell and so restore a more normal pHi.
The effects of hyperglycaemic hypoxia on rectification in rat dorsal root axons.
Authors: Grafe P , Bostock H , Schneider U
Published: December 2014

1. Electrotonic responses to 150 ms current pulses were recorded from isolated rat dorsal roots incubated for at least 3 h with either normal (5 mM) or high (25 mM) D‐glucose solutions, and with either normal (25 mM) or low (5 mM) bicarbonate concentrations. 2. On replacement of O2 by N2 for 50 min, all the roots depolarized, but the changes in electrotonus differed systematically. With normal glucose, the depolarization was accompanied by an increase in input conductance. In contrast, for the hyperglycaemic roots the depolarization was slower and accompanied by a fall in input conductance which was exacerbated in low bicarbonate concentrations. 3. The changes induced by hyperglycaemic hypoxia in low bicarbonate could be mimicked by exposure of the roots either to 100% CO2 or to a combination of 3 mM tetraethylammonium chloride and 3 mM 4‐aminopyridine, to block both fast and slow potassium channels. 4. These results indicate that the primary mechanism of hypoxic depolarization of these sensory axons is altered by hyperglycaemia. In normoglycaemia, the changes in electrotonus are consistent with an increase in axonal potassium conductance. The block of potassium channels seen in hyperglycaemic hypoxia is attributed to intra‐axonal acidification by anaerobic glycolysis and may contribute to the pathogenesis of diabetic neuropathy.
Human spinal lateralization assessed from motoneurone synchronization: dependence on handedness and motor unit type.
Authors: Schmied A , Vedel J P , Pagni S
Published: December 2014

1. Motoneurone synchronization as a means of investigating synaptic connectivity was studied in the extensor carpi radialis muscles of the preferred and non‐preferred arms of healthy right‐ and left‐handed human subjects. The activities of pairs of motor units recorded during voluntary isometric contractions were analysed by cross‐correlation to detect any synchronous motor unit firing in the form of central peaks in the cross‐correlation histograms. 2. The synchronization peaks were compared first in the case of 273 motor unit pairs tested in the preferred and non‐preferred arms of two left‐handed subjects and two right‐handed subjects. The percentage of synchronized motor unit pairs was found to be significantly higher in the preferred arm with synchronization peaks significantly larger and broader than in the non‐preferred arm. The narrow peaks (< 7.5 ms) likely to reflect the activity of common inputs to motoneurones were also found to be significantly larger in the preferred arm of all four subjects. 3. The handedness‐related differences in synchronization were definitely confirmed in a total of 275 pairs of motor units tested in the left extensor carpi radialis muscles of fourteen right‐handed subjects using their non‐preferred arm and six left‐handed subjects using their preferred arm. In order to determine whether the differences in synchronization were dependent on the motor unit type, each motor unit was characterized on the basis of its recruitment threshold and on the basis of the contraction time of its twitch extracted from the overall muscle force using the spike‐triggered averaging method. Two populations of motor units were distinguished, namely the ‘slow’ motor units (recruitment thresholds < 0.4 N, contraction times > 40 ms) and the ‘fast’ motor units (recruitment thresholds > 0.6 N, contraction times < or = 40 ms). 4. In the non‐preferred arm, the synchronization peaks were always fairly narrow, whatever the motor unit's biomechanical properties; whereas in the preferred arm, broad peaks were found to be particularly common among the pairs including one or two fast motor units, which also showed the largest rate of synchronization occurrence. 5. The narrow peaks (< 7.5 ms) were found to be consistently larger in the preferred than the non‐preferred arm whatever the categories of motor unit pairs. In both arms, however, the amplitude of the narrow peaks tended to increase as the recruitment threshold of the motor unit decreased and as their contraction time increased.(ABSTRACT TRUNCATED AT 400 WORDS)
Stable human standing with lower‐limb muscle afferents providing the only sensory input.
Authors: Fitzpatrick R , Rogers D K , McCloskey D I
Published: December 2014

1. This study investigated the sources of sensory information upon which normal subjects’ ability to stand depends. 2. An ‘equivalent body’ was used to simulate the physical properties of each subject's body during standing. The modulation of ankle torque required to support the equivalent body in an upright position was similar to that required to support the subject's own body when standing. However, when balancing the equivalent body, vestibular inputs were excluded from directing the appropriate changes in ankle torque. Thus, stability of stance could be studied with (normal stance) and without (balancing equivalent body) modulation by vestibular inputs. Vision could be excluded by closing the eyes. Sensory input from the feet and ankles could be removed by local anaesthesia from prolonged ischaemia, induced by occluding blood flow with inflated pneumatic cuffs just above the ankles. With vestibular, visual and peripheral sensory inputs negated, standing could rely only upon remaining sensory inputs, notably those from sensory receptors in the leg muscles. 3. Unlike the human body, the equivalent body used to negate vestibular inputs is not segmented. Therefore, the effects on stability of having a segmented body were determined by splinting subjects during standing so that only ankle movement was possible. This was done in the presence and absence of visual stabilization. 4. For each experimental task, either standing or balancing the equivalent body, sway was recorded while posture was unperturbed. Root mean square values of sway amplitude and power spectra were used to compare conditions. 5. Every subject could balance the equivalent body in a stable way when the eyes were closed, and when the feet were anaesthetized.(ABSTRACT TRUNCATED AT 250 WORDS)
Ca2+ inhibition of inositol trisphosphate‐induced Ca2+ release in single smooth muscle cells of guinea‐pig small intestine.
Authors: Zholos A V , Komori S , Ohashi H , Bolton T B
Published: December 2014

1. Single smooth muscle cells from the longitudinal muscle layer of guinea‐pig small intestine were voltage clamped using patch pipettes in the whole‐cell mode. 2. When D‐myo‐inositol 1,4,5‐trisphosphate (InsP3) was released at intervals, by photolysis of ‘caged’ InsP3 within the cell, increases in [Ca2+]i in many cells, as judged from Ca(2+)‐activated K(+)‐current, were all‐or‐none; release of InsP3 before a critical interval had elapsed, which was quite stable for an individual cell, resulted in no response. After Ca(2+)‐induced Ca2+ release had been evoked by depolarization, the InsP3 response was inhibited. Oscillations in [Ca2+]i evoked by muscarinic receptor activation were unaffected by Ruthenium Red; during these oscillations exogenous InsP3 was not effective close to, or shortly after, peak [Ca2+]i but was effective at other times. 3. Reproducible release of Ca2+ and elevation of [Ca2+]i could be produced by brief (up to 0.5 s) pressure applications of 10 mM caffeine at intervals of 10 s or greater but caffeine itself rarely evoked oscillations in [Ca2+]i. Responses to flash release of InsP3 were reduced after caffeine‐induced responses and recovery of caffeine‐induced Ca2+ release was faster than recovery of InsP3‐induced Ca2+ release. 4. The results support the idea that InsP3‐induced Ca(2+)‐store release can be inhibited by a certain level of [Ca2+]i at a time when Ca2+ stores have refilled and can be released by caffeine; they also support the suggestion that during oscillations of [Ca2+]i evoked by muscarinic receptor activation, Ca2+ inhibition of InsP3‐induced Ca2+ release at some critical level of [Ca2+]i allows Ca2+ stores to refill and leads to a fall in [Ca2+]i so contributing to the oscillations which are observed.