bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of high‐energy phosphates on carbachol‐evoked cationic current in single smooth muscle cells from guinea‐pig ileum.
Authors: Bakhramov A
Published: December 2014

1. Single smooth muscle cells from the longitudinal muscle layer of guinea‐pig small intestine were voltage clamped in the whole‐cell recording mode with patch pipettes. The cationic current (Icat) evoked by application of 50 microM carbachol (CCh) was examined when free internal calcium in the cell was ‘clamped’ at 10(‐7) M with 20 mM BAPTA. The effects of varying the composition of the pipette solution were studied. 2. Phosphocreatine (PCr, 6 mM) added to the pipette solution increased Icat by about 7‐fold (to near 620 pA); lower concentrations had similar, generally lesser, effects. Na2ATP (3 or 6 mM) with or without 5 mM MgCl2 was much less effective than phosphocreatine alone. Addition of 3 mM Na2ATP reduced Icat, whether or not phosphocreatine was present. 3. Creatine (6 mM) with or without 2 mM Na2ATP was less effective than phosphocreatine in maintaining Icat. 4. GTP (0.1 mM) did not affect Icat evoked by CCh, whether phosphocreatine was present or not. 5. GTP gamma S (0.2 mM) included in pipette solution mimicked the effect of CCH and evoked Icat independently of whether PCr was present or not in the pipette solution. Including 5 mM ATP in the pipette reduced this current, whereas 5'‐adenylyl imidodiphosphate (AMP‐PNP) and ADP were without effect. 6. The results show that phosphocreatine increases membrane channel responsiveness to receptor activation and that ATP above 2 mM suppresses it.
Action of salicylate on membrane capacitance of outer hair cells from the guinea‐pig cochlea.
Authors: Tunstall M J , Gale J E , Ashmore J F
Published: December 2014

1. The effect of salicylate on membrane capacitance and intracellular pH has been measured in isolated outer hair cells (OHCs) during whole cell recording. Cell membrane capacitance was measured using a lock‐in amplifier technique. 2. Salicylate applied in the bath reduced the fast charge movement, equivalent to a voltage‐dependent membrane capacitance, present in OHCs. Simultaneous measurement of membrane capacitance and voltage‐driven cell length changes showed that salicylate reduced both together. 3. A small effect of salicylate on outward currents at 0 mV was observed. Sodium salicylate (5 mM) reduced the currents by 19% and another weak acid, sodium butyrate (10 mM), reduced outward currents in OHCs by 15%. 4. The ratiometric dye 2,7‐bis(2‐carboxymethyl)‐5,6‐carboxyfluorescein (BCECF) was used to measure pHi changes in OHCs during weak acid exposure. Membrane capacitance and pHi were measured simultaneously in OHCs exposed first to 10 mM sodium butyrate and then to 5 mM sodium salicylate. Although both compounds produced a similar reduction in pHi, butyrate decreased the resting capacitance from a mean resting capacitance of 35 pF (at ‐30 mV) by 5.4 +/‐ 2.1 pF, whereas salicylate decreased it by 15.7 +/‐ 2.3 pF (n = 4). 5. Exposure of OHCs to 10 mM sodium benzoate, an amphiphilic anion, reduced resting membrane capacitance at ‐30 mV by 9.2 +/‐ 3.2 pF (n = 3). Outward currents, measured at 0 mV, were reduced by 0.25 +/‐ 0.05 nA during benzoate application, comparable with the effect of salicylate. 6. Capacitance was measured during slow bath application of salicylate. The resulting dose‐capacitance curve had a Hill coefficient of 3.40 +/‐ 0.85 (n = 4) and a half‐maximal dose of 3.95 +/‐ 0.34 mM. The dose‐capacitance curve was not significantly voltage dependent. 7. Salicylate had no detectable effect on the resting capacitance of Deiters' cells, a non‐sensory cell type of the organ of Corti. 8. It is concluded that many of the described effects of salicylate on hearing may arise from the partitioning of the salicylate molecule into the membrane of the OHC and consequent inhibition of OHC motility.
Response of C fibre nociceptors in the anaesthetized monkey to heat stimuli: correlation with pain threshold in humans.
Authors: Tillman D B , Treede R D , Meyer R A , Campbell J N
Published: December 2014

1. Ramped heat stimuli were used to compare the effects of rate of temperature change on the responses of monkey nociceptors and on heat pain threshold in human subjects. Recordings were made from twenty‐five cutaneous C fibre mechano‐heat nociceptors (CMHs) innervating the hairy skin in the anaesthetized monkey. Heat pain thresholds were determined on the volar forearm of eight human subjects using a converging staircase technique. 2. The heat pain threshold decreased as stimulus ramp rate increased. In contrast, the CMH heat threshold, defined as the surface temperature at which the first action potential occurred, increased as stimulus ramp rate increased. Thus, the properties of the heat stimulus that dictate heat pain threshold are different from the properties of the heat stimulus that govern the initiation of a response in nociceptors. 3. Peak discharge frequency of CMHs during the heat ramp increased with stimulus ramp rate. Heat pain threshold was correlated with achievement of a minimum discharge rate in nociceptors (0.5 Hz), rather than with the threshold for action potential initiation.
Single voltage‐activated Na+ and K+ channels in the somata of rat motoneurones.
Authors: Safronov B V , Vogel W
Published: December 2014

1. Voltage‐activated Na+ and K+ channels were investigated in the soma membrane of motoneurones using the patch‐clamp technique applied to thin slices of neonatal rat spinal cord. 2. One type of TTX‐sensitive Na+ channel, with a conductance of 14.0 pS, was found to underlie the macroscopic Na+ conductance in the somata of motoneurones. These channels activated within a potential range between ‐60 and ‐20 mV with a potential of half‐maximal activation (E50) of ‐38.9 mV and steepness factor (k) of 6.1 mV. 3. Kinetics of Na+ channel inactivation could be fitted with a single exponential function at all potentials investigated. The curve of the steady‐state inactivation had the following parameters: a half‐maximal potential (Eh,50) of ‐81.6 mV and k of ‐10.2 mV. 4. Kinetics of recovery of Na+ channels from inactivation at a potential of ‐80 mV were double exponential with fast and slow components of 16.2 (76%) and 153.7 ms (24%), respectively. It is suggested that the recovery of Na+ channels from inactivation plays a major role in defining the limiting firing frequency of action potentials in motoneurones. 5. Whole‐cell K+ currents consisted of transient (A)‐ and delayed‐rectifier (DR)‐components. The A‐component activated between ‐60 and +20 mV with an E50 of ‐33.3 mV and k of 15.7 mV. The curve of steady‐state inactivation was best fitted with an Eh,50 of ‐82.5 mV and k of ‐10.2 mV. The DR‐component of K+ current activated smoothly at more positive potentials. E50 and k for DR‐currents were +1.4 and 16.9 mV, respectively. 6. The most frequent single K+ channel found in the somata of motoneurones was the fast inactivating A‐channel with a conductance of 19.2 pS in external Ringer solution. In symmetrical high‐K+ solutions the conductance was 50.9 and 39.6 pS for inward and outward currents, respectively. The channel activation took place between ‐60 and +20 mV. The curve of steady‐state inactivation of single A‐channels had an Eh,50 of ‐87.1 mV and k of ‐12.8 mV. In high‐Ko+ solution A‐channels demonstrated a rapid deactivation at potentials between ‐110 and ‐60 mV. The time constant of the channel deactivation depended on the membrane potential and changed from 1.5 ms at ‐110 mV to 6.3 ms at ‐60 mV. 7. Delayed‐rectifier K+ channels were found in the soma membrane at a moderate density.(ABSTRACT TRUNCATED AT 250 WORDS)
Different effects on gating of three myotonia‐causing mutations in the inactivation gate of the human muscle sodium channel.
Authors: Mitrovié N , George A L , Lerche H , Wagner S , Fahlke C , Lehmann‐Horn F
Published: December 2014

1. Three mutations at the same site in the inactivation gate of the alpha‐subunit of the human muscle Na+ channel, G1306E, G1306V and G1306A, cause three phenotypes of K(+)‐aggravated myotonia: G1306E as the most severe and G1306A as the most benign form. 2. Recombinant wildtype (WT) and mutant (G1306E, G1306V and G1306A) human Na+ channels were expressed in human embryonic kidney cells (HEK293). G1306E and G1306V channels showed a distinct increase in the time constants of inactivation (tau h1 and tau h2) and in the ratios of steady‐state to peak currents (Iss/Ipeak) (e.g. at 0 mV, G1306E vs. WT; tau h1, 1.29 +/‐ 0.10 vs. 0.52 +/‐ 0.01 ms; Iss/Ipeak, 2.90 +/‐ 0.40 vs. 0.93 +/‐ 0.19%). G1306A channels showed only an increase in tau h1 (0.74 +/‐ 0.07 ms). For G1306E and G1306V channels, the steady‐state inactivation curves, as well as the voltage dependence of the rate of recovery from inactivation, were shifted by +15 mV. For G1306A the h infinity curve was shifted by only +5 mV. 3. G1306E and G1306V channels showed prolonged current rise times and later first openings suggesting slowing of activation. For G1306E channels only, the steady‐state activation curve was shifted by ‐7 mV. For all mutants the deactivation time constants were increased. 4. We conclude that (i) the combination of alterations in inactivation and activation produces the slowing of the current decay, (ii) the slowed inactivation is most responsible for myotonia, and (iii) the shift of the steady‐state activation curve, seen only with G1306E channels, may explain the severity of this phenotype. 5. The results suggest that two of the mutations in the Na+ channel inactivation gate also alter channel activation and deactivation.
Fluid uptake from the renal medulla into the ascending vasa recta in anaesthetized rats.
Authors: MacPhee P J , Michel C C
Published: December 2014

1. We have investigated fluid movements between superficial ascending vasa recta (AVR) and the interstitium of exposed papillae of the renal medullae in 15‐day‐old Sprague‐Dawley rats anaesthetized with Hypnorm and Hypnovel. 2. Using a development of the red cell micro‐occlusion technique, fluid filtration and reabsorption rates per unit area of vessel wall (Jv/A) were determined in 54 single perfused AVR at known microvascular pressures (Pc). The relation between Jv/A and Pc was non‐linear suggesting hydraulic permeabilities (Lp) of 50‐100 x 10(‐7) cm s‐1 cmH2O‐1 when Pc was between 0‐10 cmH2O and 150‐200 x 10(‐7) cm s‐1 cmH2O‐1 when Pc was 10‐15 cmH2O. 3. Rates of fluid reabsorption into the AVR estimated by a densitometric technique in a further fourteen vessels were consistent with Lp values of 50‐100 x 10(‐7) cm s‐1 cmH2O‐1 when Pc was ‐2 to 0 cmH2O. 4. The effective oncotic pressures of perfusates containing bovine serum albumin (BSA) were consistent with minimum values for the reflection coefficients of the walls of the AVR to BSA of between 0.59 and 0.72. 5. The concentration of native serum albumin in the papillary interstitial fluid was 9.1 +/‐ 0.6 mg ml‐1 (mean +/‐ S.E.M., n = 16, from 9 rats), which is approximately 25% of the plasma level. 6. After their microinjection into the medullary interstitium, Patent Blue V and Evans Blue‐albumin cleared within 1 min. There was no evidence of preferential movement of either dye towards the base of the exposed renal medulla. 7. Because Lp of the AVR is high, mean pressures of only approximately 3 cmH2O are necessary to account for the total clearance of fluid from the medullary interstitium into the AVR. From published data and from our own observations, it appears that differences in hydrostatic and oncotic pressure across the walls of the AVR are more than sufficient to provide this driving force. The possibility of the clearance of protein from the interstitium into the AVR is discussed.
Cutaneomuscular reflexes recorded from the lower limb in man during different tasks.
Authors: Gibbs J , Harrison L M , Stephens J A
Published: December 2014

1. Cutaneomuscular reflexes have been recorded in ten adult subjects from extensor digitorum brevis (EDB), tibialis anterior (TA), soleus (Sol), quadriceps femoris (Quad) and erector spinae (ES) following electrical stimulation of the digital nerves of the second toe. 2. Recordings were made while subjects were instructed to activate voluntarily the relevant muscles and also when these muscles were active posturally. 3. Reflex responses could comprise three components: an initial increase in EMG (E1), followed by a decrease (I1), followed by a second increase (E2). E1 and I1 were confined to muscles acting at the ankle and in the foot. E2 was most pronounced in EDB but also found in TA, Sol, Quad (1 subject) and ES. No responses were recorded contralateral to the stimulus. 4. E2 was significantly larger when the reflex was recorded during voluntary contraction of the muscle, rather than when the muscle was active posturally. 5. E1 and I1 components are mediated via spinal pathways. E2 requires the integrity of the dorsal columns, sensorimotor cortex and corticospinal tract (Jenner & Stephens, 1982; Rowlandson & Stephens, 1985b). The present study suggests that one or more of these supraspinal pathways is more active during voluntary contraction of lower limb muscles than when these muscles are active posturally.
Membrane properties of the granule cells of the islands of Calleja of the rat studied in vitro.
Authors: Halliwell J V , Horne A L
Published: December 2014

1. Using patch‐clamp techniques, we have studied granule neurones from the islands of Calleja in vitro: as isolated cells or as groups of varying numbers following enzymic digestion, or within untreated slices of approximately 100 microns thickness. 2. Recordings were made with patch pipettes in conventional or nystatin‐perforated whole‐cell mode. Current‐clamp recordings indicated that these granule cells are excitable and at resting potential produce irregular spontaneous activity. In voltage clamp the transient inward current underlying these action potentials could be evoked. This current had a threshold for activation of about ‐50 mV and was sensitive to TTX. In some cells a TTX‐resistant transient inward current was observed with a threshold for activation of about ‐70 mV. 3. Island of Calleja granule cells also exhibited outward currents. A rapidly activating transient current was observed that was resistant to TEA and sensitive to 4‐AP, and therefore resembled IA. The current was half‐maximally activated at ‐6 mV and steady‐state inactivation was half‐complete at ‐65 mV. 4. More sustained outward currents were also observed. Although some cells appeared to express a Ca(2+)‐activated K+ current, the most common finding was a rapidly activating, slowly inactivating, voltage‐dependent K+ current that was sensitive to TEA and Ba2+. This current resembled M‐current more than delayed rectifier but displayed a number of idiosyncratic kinetic properties. Chief amongst these was the accumulation of an inactivating process when the current was repeatedly evoked from potentials near the cells’ resting value by voltage steps that by themselves produced no observable inactivation during the voltage command; this behaviour was similar to the ‘C‐terminal’ inactivation exhibited by lymphocytes and certain expressed K+ channel clones (Kv1.3). 5. These results indicate that the granule cells of the islands of Calleja are excitable and contain a number of additional regulatory conductances. The implications of these findings in, and the usefulness of this preparation to, the elucidation of the function(s) of the islands of Calleja are discussed.
NMDA receptor‐mediated transmission of carotid body chemoreceptor input to expiratory bulbospinal neurones in dogs.
Authors: Dogas Z , Stuth E A , Hopp F A , McCrimmon D R , Zuperku E J
Published: December 2014

1. This study tested the hypothesis that excitatory amino acid receptors mediate the excitatory response of expiratory bulbospinal neurones to carotid body chemoreceptor inputs. 2. Studies were carried out in thiopental sodium anaesthetized, paralysed, ventilated, vagotomized dogs. 3. Brisk, short‐duration chemoreceptor activation was produced by bilateral bolus injections of CO2‐saturated saline (PCO2 > 700 mmHg) into the autoperfused carotid arteries. A pressurized‐reservoir‐solenoid valve system was used to deliver the CO2 bolus injections just prior to the onset of the neural expiratory phase, as determined from the phrenic neurogram, about once per minute. 4. Multibarrelled micropipettes were used to record neuronal unit activity and deliver neurotransmitter agents. Net responses of expiratory bulbospinal neurones to peripheral chemoreceptor activation were determined by subtracting the mean discharge frequencies (Fn) during three control expiratory cycles from the Fn during administration of a CO2 test bolus. The role of excitatory amino acid receptors in mediating this response was determined by comparing the baseline and bolus expiratory neuronal Fn before, during and after the pressure microejection of the NMDA receptor antagonist 2‐amino‐5‐phosphonovalerate (AP5) or the non‐NMDA receptor antagonist 2,3‐dihydroxy‐6‐nitro‐7‐sulphamoyl‐ benzo(f)quinoxaline (NBQX). Ejection rates of AP5 and NBQX were measured by monitoring the movement of the pipette meniscus. 5. AP5 reduced Fn during both the control and bolus cycles, as well as reducing the change in Fn between control and bolus cycles. NBQX had no effect on either baseline or bolus responses. 6. AP5 did not prevent excitation of expiratory bulbospinal neurones by AMPA. Coadministration of AMPA with AP5 prevented the AP5‐mediated decrease in Fn but not the dose‐dependent reduction in the CO2 bolus response. 7. Taken together, these data strongly suggest that the carotid chemoreceptor‐mediated excitation of expiratory bulbospinal neurones is dependent on NMDA but not non‐NMDA glutamate receptors.
GABA‐induced facilitation of the periodic bursting activity of oxytocin neurones in suckled rats.
Authors: Moos F C
Published: December 2014

1. GABAergic innervation of oxytocin neurones is particularly abundant during lactation, but little is known about its functional role. In this study, the role of GABAA receptors in the suckling‐induced bursting activity of oxytocin neurones was investigated in lactating rats. GABAA agonists or antagonists were applied by pressure injection into the immediate neighbourhood of recorded neurones while simultaneous recordings were made from oxytocin neurones in the contralateral supraoptic nucleus. 2. GABA and the GABA agonist isoguvacine decreased the basal electrical activity while application of GABAA antagonists (picrotoxin and gabazine) increased the basal electrical activity. However, in marked and unexpected contrast, application of GABA and isoguvacine facilitated or triggered milk‐ejection reflex bursting activity whereas GABAA antagonists interrupted this reflex activity. 3. Systemic injection of hypertonic saline is known to increase the firing rate of neurones in the supraoptic nucleus and temporarily to interrupt suckling‐induced bursting activity. Application of GABA into one supraoptic nucleus counteracted this inhibitory effect on milk ejection. 4. These observations can be explained if the role of the important GABAergic innervation of oxytocin neurones during lactation is to favour the expression of the stereotyped suckling‐induced bursting activity. It might do this by attenuating inputs unrelated to suckling which are incompatible with bursts.
Facilitation of the arterial baroreflex by the preoptic area in anaesthetized rats.
Authors: Inui K , Nomura J , Murase S , Nosaka S
Published: December 2014

1. Activation of cell bodies in the ventrolateral part of the midbrain periaqueductal grey matter (PAG) facilitates the arterial baroreflex via the nucleus raphe magnus. The facilitatory effects of stimulation within the hypothalamus on the arterial baroreflex and their relation to the PAG and nucleus raphe magnus were studied in urethane‐ and chloralose‐anaesthetized rats. 2. Systematic mapping experiments revealed that the preoptic area (POA) is the principal location in the hypothalamus of neuronal cell bodies that are responsible for the potentiation of the baroreflex. In addition to provoking hypotension and vagal bradycardia, both electrical and chemical stimulation of the POA produced facilitation of baroreflex vagal bradycardia (BVB) that was evoked by electrical stimulation of the aortic depressor nerve. Baroreflex hypotension was slightly augmented during activation of the POA in vagotomized rats. 3. Selective destruction of cell bodies either in the ventrolateral PAG or in the nucleus raphe magnus reduced facilitation of BVB by the POA. Hypotension and bradycardia due to POA stimulation were also markedly attenuated after such selective destruction. 4. In conclusion, the POA, the ventrolateral PAG and the nucleus raphe magnus constitute a functional complex that produces cardiovascular trophotropic effects including hypotension, vagal bradycardia and baroreflex facilitation.
Electrophysiological differences between oxytocin and vasopressin neurones recorded from female rats in vitro.
Authors: Stern J E , Armstrong W E
Published: December 2014

1. Intracellular recordings in vitro from immunochemically identified oxytocin (OT) and vasopressin (VP) neurones in the supraoptic nucleus (SON) of virgin or lactating female rats revealed no differences between neurone types in membrane potential (Vm), input resistance and current‐voltage relationships (I‐V), when taken at resting membrane potentials. 2. When OT (94%), but not VP, neurones (93%) were current clamped at depolarized voltages (above ‐50 mV), small hyperpolarizing pulses revealed a time‐ and voltage‐dependent outward rectification that was present above ‐75 mV and that decreased in amplitude as Vm approached the equilibrium potential for potassium (EK). The rectification was more pronounced when the neurones were held at a more depolarized membrane potential, and was larger the longer the neurone was held depolarized, reaching a maximum at 0.6‐0.9 s. 3. A rebound depolarization followed the offset of hyperpolarizing pulses that were associated with the rectification. The peak amplitude of the rebound showed a time and a voltage dependence. It followed a bell‐shaped curve as the hyperpolarizing commands were made larger, attaining a peak at ‐65 +/‐ 1.5 mV. The rebound amplitude increased with pulse duration, achieving a half‐maximal amplitude at 0.5 +/‐ 0.1 s. 4. The expression of the sustained outward rectification and the rebound in OT neurones was similar in virgin and lactating female rats. 5. These results indicate the presence of significant differences in the intrinsic membrane properties, probably K+ currents, between OT and VP neurones in both lactating and virgin female rats.
Ipsilateral cortical stimulation inhibited the long‐latency response to stretch in the long finger flexors in humans.
Authors: Taylor J L , Fogel W , Day B L , Rothwell J C
Published: December 2014

1. Transcranial magnetic stimulation over the motor cortex of one hemisphere is known to decrease the excitability of the motor cortex of the other hemisphere. We investigated the effect of this interhemispheric or transcallosal inhibition of the motor cortex on the reflex response to stretch in the long flexors of the fingers in human subjects. 2. Stretch of the long finger flexors, through extension of the proximal interphalangeal joints with a torque pulse, resulted in a reflex EMG response with short‐ and long‐latency components. Magnetic stimulation was applied over the motor cortex ipsilateral to the muscles being stretched. When a magnetic shock but not stretch was given, a decrease in background EMG in the ipsilateral finger flexors occurred at a latency of 33 +/‐ 6.2 ms after the stimulus and with a duration of 25 +/‐ 8.5 ms. 3. If the magnetic shock and the stretch were given at appropriate interstimulus intervals, the long‐latency stretch reflex (LLSR) showed inhibition in all subjects. LLSR was reduced to 49.2 +/‐ 19% (S.D.; n = 9) of the area of the control response. 4. The LLSR did not act as a single event in response to the magnetic shock. That is, part of the LLSR could be reduced in amplitude while the remainder was unaffected. The reduction in LLSR had an onset latency of 27 +/‐ 3.8 ms after the magnetic stimulus and a duration of 29‐55 ms. Inhibition was only obvious when this interval after the magnetic stimulus coincided with the LLSR. 5. In most subjects the short‐latency stretch reflex (SLSR) also showed some inhibition (83.4 +/‐ 11.2% of the control). However, this was less than the effect on the LLSR in all subjects. 6. The site of stimulation, over the ipsilateral motor cortex, was specific for inhibition of the LLSR. When the coil was moved anteriorly or to the midline, inhibition was significantly decreased. 7. We suggest that the inhibition of the LLSR of the long flexors of the fingers resulted from a reduction in excitability of the motor cortex produced by an inhibitory transcallosal pathway and conclude that the LLSR in this muscle has a transcortical component.
The effect of histidine and cysteine on zinc influx into rat and human erythrocytes.
Authors: Horn N M , Thomas A L , Tompkins J D
Published: December 2014

1. The effect of histidine and cysteine on the initial rate of zinc influx into rat and human erythrocytes in the presence of bovine serum albumin has been investigated. 2. The L‐enantiomers of both amino acids promoted zinc influx into rat erythrocytes in a dose‐dependent manner. L‐Histidine, but not L‐cysteine, also promoted zinc uptake into human erythrocytes. D‐Histidine did not promote zinc uptake in either rat or human erythrocytes. In rat erythrocytes D‐cysteine was significantly less effective than L‐cysteine. 3. The stimulation of zinc influx into rat erythrocytes by 20 mM L‐histidine was approximately 4.1 times that seen with human erythrocytes. 4. The influx of zinc in the presence of varying concentrations of L‐histidine was linearly related to the calculated concentration of the zinc‐bis‐histidine complex but not to that of the zinc‐mono‐histidine complex or the free ionic zinc concentration. 5. These results are discussed in relation to the nature of the transport mechanisms involved.
The effect of bilateral fetal adrenalectomy on fluid balance in the ovine fetus.
Authors: Benson C A , Wintour E M
Published: December 2014

1. Bilaterally adrenalectomized adult sheep, taken off replacement therapy, exhibit decreased plasma sodium, and increased plasma potassium, urinary sodium excretion and urinary sodium:potassium ratio. The hypothesis tested was that these effects would not be seen in adrenalectomized ovine fetuses, due to the mother being the major regulator of fetal fluid balance. 2. The fetuses of seven Merino ewes were bilaterally adrenalectomized at 120 +/‐ 0.3 days of gestation (term is 150 days) and a bladder cannula inserted. Urine flow and composition were measured six times between 125 and 142 days of gestation, and compared with results in seven control fetuses at the same gestational ages. At between 142 and 145 days of gestation, blood samples were taken from the ewe and fetus, they were killed and the volume and composition of amniotic and allantoic fluids measured. 3. Adrenalectomized fetuses had significantly lower (P < 0.05) plasma aldosterone concentration than that observed in intact fetuses (adrenalectomized, 92 +/‐ 16 pmol l‐1; intact, 224 +/‐ 46 pmol l‐1; P < 0.001). Plasma cortisol level was also lower (adrenalectomized, 8.6 +/‐ 1.4 nmol l‐1; intact, 110.3 +/‐ 24.8 nmol l‐1). Maternal plasma steroids were similar in both groups. 4. There were no significant differences in fetal urine flow rate, osmolality or composition until 139‐142 days of gestation. At this time urine sodium:potassium ratio was significantly greater in adrenalectomized (P < 0.05) than intact fetuses. 5. There were no significant differences in maternal or fetal plasma solutes in the two groups and only minor alterations in the volume and composition of amniotic and allantoic fluid. The conclusion is that the major determinant of fetal fluid and electrolyte balance is placental transfer of water and electrolytes from the mother.
Propagating calcium waves initiated by local caffeine application in rat ventricular myocytes.
Authors: Trafford A W , Lipp P , O'Neill S C , Niggli E , Eisner D A
Published: December 2014

1. Caffeine was applied locally to one region of a resting cell via an extracellular pipette while simultaneously imaging the concentrations of intracellular calcium ([Ca2+]i) and intracellular caffeine ([caffeine]i). 2. Local application of caffeine produced a rise of [caffeine]i which was confined to the region of the cell near the pipette. There was also a local increase of [Ca2+]i which then, in most resting cells, propagated along the cell as a linear Ca2+ wave. The initial magnitude of the rise of [Ca2+]i was greater than that of the electrically stimulated Ca2+ transient. 3. As the wave of increase of [Ca2+]i propagated along the cell it decreased in both amplitude and velocity in cells that had not been treated to elevate the cellular Ca2+ load. 4. In some cells the caffeine response did not propagate significantly. In these cases an increase of the cellular Ca2+ load enabled caffeine‐induced Ca2+ wave propagation along the entire cell length without significant decay in amplitude and velocity. 5. Previous work has shown that an electrically evoked local systolic Ca2+ transient does not propagate. The fact that the caffeine‐evoked response does propagate and the correlation between decay of amplitude and velocity suggest that the transient has to be a certain size before it can propagate. It is suggested that one of the factors which favour propagation of waves under conditions of elevated sarcoplasmic reticulum Ca2+ content is the increased release of Ca2+.
Raised intracellular [Ca2+] abolishes excitation‐contraction coupling in skeletal muscle fibres of rat and toad.
Authors: Lamb G D , Junankar P R , Stephenson D G
Published: December 2014

1. Raising the intracellular [Ca2+] for 10 s at 23 degrees C abolished depolarization‐induced force responses in mechanically skinned muscle fibres of toad and rat (half‐maximal effect at 10 and 23 microM, respectively), without affecting the ability of caffeine or low [Mg2+] to open the ryanodine receptor (RyR)/Ca2+ release channels. Thus, excitation‐contraction coupling was lost, even though the Ca2+ release channels were still functional. Coupling could not be restored in the duration of an experiment (up to 1 h). 2. The Ca(2+)‐dependent uncoupling had a Q10 > 3.5, and was three times slower at pH 5.8 than at pH 7.1. Sr2+ caused similar uncoupling at twenty times higher concentration, but Mg2+, even at 10 mM, was ineffective. Uncoupling was not noticeably affected by removal of ATP or application of protein kinase or phosphatase inhibitors. 3. Confocal laser scanning microscopy showed that the transverse tubular system was sealed in its entirety in mechanically skinned fibres and that its integrity was maintained in uncoupled fibres. Electron microscopy revealed distorted or severed triad junctions and Z‐line aberrations in uncoupled fibres. 4. Only when uncoupling was induced at a relatively slow rate (e.g. over 60 s with 2.5 microM Ca2+) could it be prevented by the protease inhibitor leupeptin (1 mM). Immunostaining of Western blots showed no evidence of proteolysis of the RyR, the alpha 1‐subunit of dihydropyridine receptor (DHPR) or triadin in uncoupled fibres. 5. Fibres which, whilst intact, were stimulated repeatedly by potassium depolarization with simultaneous application of 30 mM caffeine showed reduced responsiveness after skinning to depolarization but not to caffeine. Rapid release of endogenous Ca2+, or raised [Ca2+] under conditions which minimized the loss of endogenous diffusible myoplasmic molecules from the skinned fibre, caused complete uncoupling. Taken together, these results suggest that Ca(2+)‐dependent uncoupling can also occur in intact fibres. 6. This Ca(2+)‐dependent loss of depolarization‐induced Ca2+ release may play an important feedback role in muscle by stopping Ca2+ release in localized areas where it is excessive and may be responsible for long‐lasting muscle fatigue after severe exercise, as well as contributing to muscle weakness in various dystrophies.
Voltage‐dependent potentiation of low‐voltage‐activated Ca2+ channel currents in cultured rat bone marrow cells.
Authors: Publicover S J , Preston M R , El Haj A J
Published: December 2014

1. The whole‐cell patch‐clamp technique was used to study Ca2+ channel currents in stromal cells of 7‐10 day dexamethasone‐treated and control rat bone marrow cultures. In saline containing either 108 mM Ba2+ or a 2.5 mM Ca(2+)‐1 mM Mg2+ mixture, most cells expressed both fast‐inactivating, low‐voltage‐activated (LVA) and slow‐inactivating, high‐voltage‐activated (HVA) currents. 2. Repeated application of 400 ms voltage steps to 60 mV above the holding potential (Vh, ‐90 mV in Ca(2+)‐Mg2+ mixture and ‐60 mV in Ba2+) at a frequency > or = 0.1 Hz resulted in a potentiation of the LVA component of the 2nd and subsequent currents. 3. LVA current potentiation was examined using a two‐pulse (prepulse‐test pulse) method. Prepulses to Vh + 150 mV induced an 80‐100% increase in the amplitude of the LVA component of Ca2+ channel currents in saline containing either Ba2+ or Ca(2+)‐Mg2+. This effect was also seen in non‐dexamethasone‐treated cultures. 4. Potentiation was not modified by omission of ATP and GTP from the pipette saline, and was not inhibited by extracellular application of the broad spectrum kinase inhibitors H‐7 or RK252‐a. 5. Potentiation was dependent on the amplitude and duration of the prepulse. Using the standard protocol, the threshold for potentiation was approximately Vh + 45 mV and saturation occurred at Vh + 150‐180 mV. Further increases in prepulse amplitude did not modify potentiation. With a prepulse to +10 mV (Ba2+ saline) potentiation was half‐maximal with a prepulse duration of 250‐300 ms duration and saturated at 750‐1000 ms. 6. Peak potentiation occurred 1‐2 s after the prepulse. The time for total decay of potentiation varied from 10 to 90 s. 7. Voltage dependency of prepulse‐induced potentiation did not resemble that of inactivation induced by similar prepulses. 8. Current kinetics, I‐V relationship and sensitivity to blockade by Ni2+ and diphenylhydantoin of prepulse‐recruited current resembled those of control LVA current. 9. The amplitude of prepulse‐recruited current was positively correlated with control LVA current amplitude. 10. LVA currents supported regenerative potentials under current clamp. Repeated activation reduced spike latency. 11. It is suggested that current potentiation may be recruited physiologically, possibly in association with activation of stretch‐sensitive channels, causing enhanced activation of HVA Ca2+ currents.
Nitric oxide activates multiple potassium channels in canine colonic smooth muscle.
Authors: Koh S D , Campbell J D , Carl A , Sanders K M
Published: December 2014

1. Nitric oxide (NO), an inhibitory neurotransmitter released from peripheral neurones, hyperpolarizes smooth muscle cells and inhibits contraction. The mechanism of this hyperpolarization is unknown. 2. We have identified three classes of K+ channels activated by NO and NO donors in colonic smooth muscle cells. NO and NO donors increased the open probability of 80 pS channels (KNO1), very small channels (< 4 pS, KNO2), and 270 pS Ca(2+)‐activated K+ channels (BK channels) in cell‐attached patches. 3. Dibutyryl cGMP and 8‐bromo cGMP also increased the open probability of KNO1 and KNO2 in cell‐attached patches. 4. In excised patches of membrane, direct application of NO or the NO donor, S‐nitroso‐N‐acetyl penicillamine (SNAP), increased the open probability of KNO1 and KNO2, but cGMP or dibutyryl cGMP had no effect. SNAP had no effect on the open probability of BK channels in excised patches. 5. The reducing agent dithiothreitol and the alkylating agent N‐ethylmaleimide blocked NO‐induced channel openings. 6. In summary, the hyperpolarization response to NO in smooth muscles may be mediated by multiple K+ channels. At least two of these classes of channels may be activated by dual pathways involving direct activation by NO and cGMP‐mediated mechanisms.
P2 purinoceptor‐mediated depolarization of rat supraoptic neurosecretory cells in vitro.
Authors: Hiruma H , Bourque C W
Published: December 2014

1. Intracellular recordings were obtained from supraoptic magnocellular neurosecretory cells (MNCs) in superfused explants of rat hypothalamus. Application of ATP and UTP, but not adenosine, produced TTX‐insensitive depolarizations accompanied by increases of input conductance. 2. The P2X agonists alpha,beta‐methylene ATP, beta,gamma‐methylene ATP and 2‐methylthio ATP mimicked the effects of ATP in > 77% of the cells tested. Depolarizing responses to ATP were reversibly inhibited by PPADS (pyridoxal‐phosphate‐6‐azophenyl‐2',4'‐ disulphonic acid; IC50 approximately 0.5 microM), a selective P2X antagonist. 3. The reversal potential of responses to ATP (‐37 mV) was not strongly affected by intracellular Cl‐ injection or by removal of Cl‐ from the external solution. The reversal potential of responses to the most potent P2X agonist, alpha,beta‐methylene ATP, was ‐29 mV. These values suggest the involvement of non‐selective cationic channels, a finding which is consistent with the ionotropic cationic channel structure of cloned P2X purinoceptors. 4. The reversal potential of UTP‐mediated responses (‐33 mV) was also consistent with the involvement of non‐selective cationic channels. Since cloned P2U receptors display homology with G‐protein‐coupled receptors, cationic channels modulated by UTP are probably different from those mediating P2X responses.
Characterizations and comparisons of eupnoea and gasping in neonatal rats.
Authors: Wang W , Fung M L , Darnall R A , St John W M
Published: December 2014

1. Our purpose was to characterize the ventilatory patterns of eupnoea and gasping in the neonatal rat. This study was precipitated by reports, using in vitro brainstem spinal cord preparations, that only a single pattern is present in neonatal rats. 2. In anaesthetized or decerebrate rat pups aged less than 13 days, eupnoea was characterized by a sudden onset of inspiratory activity and then a more gradual rise to peak levels. Following vagotomy, frequency fell and peak phrenic activity and tidal volume increased. The rate of rise of inspiratory activity also rose, but peak levels were still achieved during the latter half of inspiration. Vagal efferent activity exhibited bursts during both inspiration and the early expiration. This basic eupnoeic rhythm was not altered after sectioning of the carotid sinus nerves. 3. Upon exposure to hypoxia or anoxia, phrenic activity, tidal volume and frequency initially increased and then declined. In many animals, ventilatory activity then ceased, but later returned with a gasping pattern. 4. Gasping was characterized by a sudden onset of phrenic activity, which reached a peak intensity during the early portion of inspiration. The expiratory burst of vagal activity was eliminated. 5. Reductions of body temperature from 37 to 27 degrees C resulted in prolongations of inspiration and expiration and decreases of phrenic amplitude; phasic phrenic activity completely disappeared in some animals. Upon exposure to anoxia, gasping was observed, even in animals in which phrenic activity had disappeared in hyperoxia. 6. We conclude that, from the day of birth, rats can exhibit eupnoea and gasping patterns which are very similar to those of adult animals. 7. The rhythmic neural activities of the in vitro brainstem‐spinal cord preparation, reported by others, differ markedly from eupnoea but are identical with gasping. We therefore conclude that this preparation is not suitable for investigation of the mechanisms that generate eupnoeic breathing.
Nifedipine‐sensitive intramembrane charge movement in Purkinje cells from mouse cerebellum.
Authors: Melliti K , Bournaud R , Bastide B , Shimahara T
Published: December 2014

1. The intramembrane charge movement was recorded in freshly dissociated Purkinje cells from 14‐ to 18‐day‐old mouse cerebellum using the whole‐cell voltage clamp technique. 2. After pharmacological elimination of all ionic currents, a depolarizing pulse from a holding potential of ‐80 mV revealed a transient capacitive outward current at the onset and a transient inward current at the end of the pulse. The amount of charge transferred at the onset (Qon) was equivalent to that moved at the end of the pulse (Qoff). The decay time course of Qon can be fitted by a single exponential curve with a maximum time constant of 1.89 +/‐ 0.35 ms at 20 mV (n = 11). 3. The charge movement had an S‐shaped dependence on test membrane potential, according to a two‐state Boltzmann function. The maximum amount (Qmax) of Qon that could be moved was 17.46 +/‐ 0.83 nC muF‐1; the membrane potential at which half the charge movement occurred (V) was 13.48 +/‐ 2.20 mV and the slope factor (k) was 16.83 +/‐ 0.84 mV (n = 27). 4. Phenylglyoxal (2 mM), an arginine‐specific modifying reagent, reduced Qmax to 60% of control after 20 min treatment. 5. The charge movement was partially immobilized by nifedipine in a dose‐dependent manner with an IC50 of 70 nM. The fraction of the nifedipine‐sensitive component was 39% of the total charge movement. The potential dependence of the nifedipine‐sensitive charge movement could be expressed by a Boltzmann function with values of 7.00 +/‐ 0.53 nC muF‐1 for Qmax, 31.44 +/‐ 4.23 mV for V and 21.53 +/‐ 3.18 mV for k (n = 8). 6. The P‐type calcium channel specific inhibitor, omega‐Aga IVA (250 nM), had no effect on intramembrane charge movement. 7. The above results show that part of the intramembrane charge movement in Purkinje cells may be related to a conformational change of DHP receptors upon membrane depolarization.
Differential responses of lateral and ventrolateral rat periaqueductal grey neurones to noradrenaline in vitro.
Authors: Vaughan C W , Bandler R , Christie M J
Published: December 2014

1. The action of noradrenaline on the membrane properties of rat periaqueductal grey (PAG) neurones was examined using intracellular recordings in brain slices maintained in vitro. Morphological properties and the anatomical location of neurones were characterized by use of intracellular staining within biocytin. 2. Noradrenaline (0.3‐100 microM) depolarized 66% (81/123) and hyperpolarized 30% (37/123) of neurones. The alpha 1‐ and alpha 2‐adrenoceptor agonists phenylephrine and UK 14304 produced depolarizations and hyperpolarizations in all PAG neurones tested, respectively. Neurones depolarized by noradrenaline were more responsive to phenylephrine, whereas neurones hyperpolarized by noradrenaline were more responsive to UK 14304. 3. The UK 14304‐induced hyperpolarizations reversed polarity at ‐108 +/‐ 2 mV (n = 11). The reversal potential increased when the extracellular potassium concentration was raised (slope = 57.8 mV/log[K+]o mM) in a manner similar to that predicted for potassium conductance. 4. The phenylephrine‐induced depolarizations did not reverse polarity at negative potentials (n = 25), or did so at potentials (‐119 +/‐ 2 mV, n = 13) more negative than the UK 14304‐induced hyperpolarizations. Superfusion with low calcium (0.1 mM), high magnesium (10 mM) and either cobalt (2‐4 mM), or cadmium (100 microM) usually reduced the response to phenylephrine and produced reversals near that predicted for potassium conductance. 5. The majority of the ventrolateral PAG neurones were depolarized by noradrenaline (85%, 62/73). In contrast, almost equal proportions of the lateral PAG neurones were hyperpolarized (54%, 20/37) and depolarized (46%, n = 17/37) by noradrenaline. PAG neurones depolarized or hyperpolarized by noradrenaline could not be differentiated on morphological grounds. 6. These results suggest that the net effect of noradrenaline on lateral and ventrolateral PAG neurones is to bias activity in favour of a ventrolateral PAG‐mediated response pattern, which includes quiescence, hyporeactivity, hypotension and bradycardia.
Long‐term facilitation of phrenic nerve activity in cats: responses and short time scale correlations of medullary neurones.
Authors: Morris K F , Arata A , Shannon R , Lindsey B G
Published: December 2014

1. Stimulation of either peripheral chemoreceptors or nucleus raphe obscurus results in long‐term facilitation of phrenic motoneurone activity. The first objective of this work was to measure the concurrent responses of neurones in the nucleus raphe obscurus, the nucleus tractus solitarii, and the regions of the retrofacial nucleus, nucleus ambiguus and nucleus retroambigualis during induction of long‐term facilitation. A second goal was to assess functional relationships of the chemoresponsive raphe neurones with neurones in the other monitored locations and with phrenic motoneurones. 2. Up to thirty single medullary neurones and phrenic nerve efferent activity were recorded simultaneously in fifteen anaesthetized, paralysed, vagotomized, artificially ventilated adult cats. Carotid chemoreceptors were stimulated by close arterial injection of 200 microliters of CO2‐saturated saline solution. Spike trains were analysed with cycle‐triggered histograms and two statistical tests for respiratory modulation. Peristimulus‐time histograms and cumulative sum histograms were used to assess responses to stimulation. Cross‐correlation was used to test for non‐random temporal relationships between spike trains. Spike‐triggered average histograms provided evidence for functional associations with phrenic motoneurones. 3. One hundred and thirteen of 348 neurones were monitored in the nucleus raphe obscurus. The firing rates of twenty‐nine raphe neurones increased during stimulation; eighteen decreased. In twenty‐one pairs of concurrently monitored raphe neurones, the firing rate of one increased its activity during stimulation then decreased, while the other showed an increase that began as the rate of the former declined. Eighteen chemoresponsive raphe neurones had short time scale features in their phrenic spike‐triggered averages. Short time scale features were found in cross‐correlograms from 184 of 1407 neurone pairs. 4. The data suggest parallel routes by which carotid chemoreceptors influence medullary raphe neurones and support the hypotheses that mid‐line respiratory‐related neuronal assemblies transform information from those receptors and regulate the gain of respiratory motor output.