bluuebunny/crossref_metadata_2025 huggingface dataset:
Spontaneous changes in arterial blood pressure and renal interstitial hydrostatic pressure in conscious rats.
Authors: Skarlatos S , Brand P H , Metting P J , Britton S L
Published: December 2014

1. Previous work has demonstrated a positive relationship between experimentally induced changes in arterial pressure (AP) and renal interstitial hydrostatic pressure (RIHP). The purpose of the present study was to test the hypothesis that RIHP is positively correlated with the normal changes in AP that occur spontaneously in conscious rats. 2. Rats were chronically instrumented for the recording of AP (via an aortic catheter) and RIHP. RIHP was measured by implanting a Millar microtransducer, whose tip had been encapsulated in a 35 microns pore polyethylene matrix (5 mm long, 2 mm o.d.), approximately 5 mm below the renal cortical surface. 3. A total of 56 h of simultaneous analog recording of AP and RIHP was obtained from ten rats. Each 1 h segment was digitized and evaluated at frequencies of 1, 0.1, 0.02 and 0.01 Hz. 4. In forty‐nine out of fifty‐six of these 1 h recordings taken at 1 Hz, there were significant positive linear correlations between AP and RIHP (mean r = 0.32) with a mean slope of 0.11 mmHg RIHP/1 mmHg AP. Low‐pass filtering to 0.01 Hz significantly increased the r value to 0.48. 5. These results demonstrate that spontaneous changes in AP and RIHP are positively correlated. The spontaneous coupling of AP and RIHP may be of importance in the regulation of salt and water excretion by the pressure diuresis mechanism.
Somatosensory regulation of renal function in the stroke‐prone spontaneously hypertensive rat.
Authors: Davis G , Johns E J
Published: December 2014

1. Chloralose‐urethane‐anaesthetized Wistar rats and stroke‐prone spontaneously hypertensive rats (SHRSP) were prepared for measuring renal function. 2. Bilateral brachial nerve stimulation for 15 min, at 1.3 Hz (15 V, 0.2 ms), increased blood pressure from 119 +/‐ 2 to 151 +/‐ 6 mmHg (P < 0.01) in Wistar rats and from 181 +/‐ 6 to 203 +/‐ 6 mmHg (P < 0.05) in SHRSP; renal perfusion pressure was mechanically regulated at prestimulus levels but renal blood flow (4.1 +/‐ 0.7 ml min‐1 g‐1 in Wistar rats and 3.4 +/‐ 0.3 ml min‐1 g‐1 in SHRSP) and glomerular filtration rate (1.18 +/‐ 0.09 ml min‐1 g‐1 in Wistar rats and 0.88 +/‐ 0.01 ml min‐1 g‐1 in SHRSP) decreased between 5 and 30% (P < 0.05‐0.001). 3. During brachial nerve stimulation, urine flow (13.8 +/‐ 2.2 microliters min‐1 g‐1) and absolute and fractional sodium excretion (3.70 +/‐ 0.64 mumol min‐1 g‐1 and 2.06 +/‐ 0.23%) decreased by 60‐70% in the Wistar rats (P < 0.01‐0.001) and the responses were unaffected by vagotomy or carotid sinus denervation. In the SHRSP, urine flow rate (9.74 +/‐ 2.52 microliters min‐1 g‐1) and absolute and fractional sodium excretion (1.98 +/‐ 0.59 mumol min‐1 g‐1 and 1.41 +/‐ 0.28%) decreased by 15‐30% (P < 0.05‐0.01), decreases which were significantly (P < 0.05) smaller than those observed in the Wistar rats. Moreover, the responses were some 40‐60% larger in the vagotomized and carotid sinus‐denervated SHRSP. 4. The influence of vagal and carotid sinus afferent nerves on the somatosensory‐induced renal responses in the Wistar rats was small, whereas in the SHRSP they appeared to exert a marked tonic inhibitory effect. It is concluded that there is excessive baroreceptor restraint on neural regulation of renal function in the SHRSP.
Involvement of vasodilator mechanisms in arterial pressure lability after sino‐aortic baroreceptor denervation in rat.
Authors: Zhang Z Q , Barrès C , Julien C
Published: December 2014

1. To examine the regional haemodynamic basis of arterial pressure lability seen after sino‐aortic baroreceptor denervation (SAD), simultaneous beat‐to‐beat recordings of arterial pressure and indices of regional blood flows (Doppler probes around the subdiaphragmatic and lower abdominal aortae and the superior mesenteric artery) were performed in the same conscious rats (n = 7) before, 1 and 14 days after SAD. 2. Acute SAD increased arterial pressure, decreased regional blood flows and vascular conductances, and potentiated the depressor and vasodilator effects of ganglionic blockade with trimethaphan, suggesting sympathetic overactivity. All parameters chronically returned to or near normal. 3. Both acute and chronic SAD increased the variability of arterial pressure and of regional conductances. Arterial pressure lability was characterized by a mixture of depressor and pressor events which were associated with regional vasodilatations and vasoconstrictions, respectively. This haemodynamic pattern was not affected by acute beta‐adrenoceptor blockade with propranolol. 4. In conscious rats, the baroreceptor reflex acts to buffer the spontaneous variability of regional vascular conductances and thereby stabilizes arterial pressure. Sino‐aortic baroreceptor denervation‐induced arterial pressure lability does not depend on the level of sympathetic activation, and is determined by the relative contribution of depressor and pressor events accompanied by extensive vasodilatations and vasoconstrictions, respectively. Vasodilatations are not caused by the stimulation of vascular beta 2‐adrenoceptors.
Post‐spike facilitation of neck EMG by cat tectoreticulospinal neurones during orienting movements.
Authors: Olivier E , Grantyn A , Kitama T , Berthoz A
Published: December 2014

1. The activity of fourteen tectoreticulospinal neurones (TRSNs) was recorded intraaxonally in the caudal pons of alert cats during orienting movements towards visual stimuli. TRSN spikes were used to compute the spike‐triggered average (STA) of rectified EMG of dorsal neck muscles. 2. Eight TRSNs for which 400‐2532 spikes were available were analysed with the STA technique. When the STA was computed from all spikes, significant post‐spike facilitation (PSF) was obtained for six of eighteen cell‐muscle pairs investigated (5 TRSNs). The mean relative amplitude of PSFs was 7.4% (S.D. 3.7). The onset latencies ranged from 1.1 to 5.0 ms and mean duration was 11.4 +/‐ 3.1 ms (mean +/‐ S.D.). 3. Interspike interval distributions were unimodal, with modes between 2.7 and 12.7 ms. Spike trains of TRSNs that produced significant PSFs contained 5‐13% of the interspike intervals < or = 5 ms and 22‐37% of the intervals < or = 10 ms. To evaluate the contribution of short intervals to PSF, STAs were computed separately for spikes preceded by ‘short’ (< or = 5 or < or = 10 ms) and ‘long’ (> 5 or > 10 ms) intervals. 4. When computed from spikes preceded by ‘long’ intervals, PSF amplitudes were small (mean +/‐ S.D., 5.3 +/‐ 2.7%) and onset latencies measured by cusum ranged between 2.4 and 5.4 ms. This is longer than the estimated minimal latency of monosynaptic facilitatory effect on neck EMG (1.9‐2.1 ms). 5. Relative amplitudes of PSF obtained with spikes preceded by ‘short’ intervals were much larger (mean +/‐ S.D., 14.8 +/‐ 7.4%), but cusums indicated negative latencies for four of six PSFs. The unrealistically short onset latencies could be accounted for by the summation of facilitation from the trigger spike with that of the preceding spikes. In four of five TRSNs a large increase of PSF amplitude (from 3.2 to 7.2 times the amplitude obtained from ‘long’ intervals) suggests the presence of frequency‐dependent potentiation of synaptic transmission. 6. This study unequivocally demonstrates that some TRSNs produce significant post‐spike facilitation of neck motoneurones. This facilitation could be mediated by monosynaptic tectomotoneuronal connections although a contribution by disynaptic connections cannot be definitively ruled out. The high instantaneous firing rates of TRSNs produce a potentiation of the otherwise weak facilitatory action of TRSNs that presumably contributes to a rapid recruitment of motoneurones during initiation of head orienting movements.
The opioid peptide dynorphin directly blocks NMDA receptor channels in the rat.
Authors: Chen L , Gu Y , Huang L Y
Published: December 2014

1. The actions of dynorphin on N‐methyl‐D‐aspartate (NMDA) responses were examined in acutely dissociated trigeminal neurons in rat. Whole‐cell and single‐channel currents were recorded using the patch clamp technique. 2. Dynorphins reduced NMDA‐activated currents (INMDA). The IC50 was 0.25 microM for dynorphin (1‐32), 1.65 microM for dynorphin (1‐17) and 1.8 microM for dynorphin (1‐13). 3. The blocking action of dynorphin is voltage independent. 4. The inhibitory action of dynorphin cannot be blocked by high concentration of the non‐selective opioid receptor antagonist naloxone, nor by the specific kappa‐opioid receptor antagonist nor‐Binaltorphimine (nor‐BNI). 5. Single‐channel analyses indicate that dynorphin reduces the fraction of time the channel is open without altering the channel conductance. 6. We propose that dynorphin acts directly on NMDA receptors.
Vasodilatation and smooth muscle membrane potential changes in arterioles from the guinea‐pig small intestine.
Authors: Kotecha N , Neild T O
Published: December 2014

1. Dilatation of arterioles isolated from the guinea‐pig small intestine was evoked by stimulation of a submucous ganglion and the application of acetylcholine, vasoactive intestinal peptide, galanin or dynorphin A. Changes in arteriole diameter and smooth muscle membrane potential were recorded simultaneously. 2. Ganglion stimulation caused vasodilatation and smooth muscle hyperpolarization that varied in both amplitude and time course from one arteriole to another. Vasodilatation could occur without hyperpolarization. 3. Vasodilatation caused by acetylcholine was accompanied by a rapidly developing hyperpolarization that began to decline before the maximum vasodilator effect had developed. 4. Vasoactive intestinal peptide caused dilatation without any change in smooth muscle membrane potential. 5. Galanin and dynorphin caused dilatation and a hyperpolarization of similar time course to the dilatation. 6. In 48% of arterioles tested the dilatation appeared to be mediated solely by acetylcholine. In 31% there was a cholinergic component, but no evidence for the involvement of acetylcholine in the remaining 21%. When the non‐cholinergic dilatation occurred without a hyperpolarization we conclude that it was due to vasoactive intestinal peptide; otherwise it may have been due to either galanin or dynorphin.
Activation of calcium current in voltage‐clamped rat glomerulosa cells by potassium ions.
Authors: Várnai P , Osipenko O N , Vizi E S , Spät A
Published: December 2014

1. We examined Ca2+ influx mechanisms using the whole‐cell patch‐clamp technique in primary cultures of rat glomerulosa cells. 2. Depolarization of the plasma membrane, as studied by a stepwise or ramp depolarization technique, activated low‐threshold, transient (T‐type) and high‐threshold, long‐lasting (L‐type) voltage‐dependent calcium channels (VDCCs). 3. Extracellular K+ activated an inward current (Ig1), even in voltage‐clamped cells. This phenomenon was observed within the physiological concentration range, beginning at 4.6 mM K+o (as opposed to the control level of 3.6 mM K+o). Increased cell conductance and increased background noise indicated that Ig1 is evoked by enhanced channel activity. Potassium induced no outward current in the voltage range examined (‐120 to +60 mV). 4. When non‐permeable anions were present only in the pipette and Na+ and Mg2+ were omitted from the bath, K+ still activated the current. Ig1 was blocked by 100 microM cadmium but was insensitive to 2 microM nifedipine or to 300 microM Ni2+. 5. In fluorimetric studies elevation of the cytoplasmic Ca2+ concentration in response to K+ (5.6‐13.6 mM) was reduced only partially when VDCCs were blocked with Ni2+ (200 microM) and nifedipine (2 microM). 6. Elevation of the K+ concentration shifted the threshold potential of the T‐type calcium channel in the negative direction. 7. In summary, K+ as a ligand activates Ca(2+)‐permeable channels in rat glomerulosa cells. This current may contribute to the development of Ca2+ signals in response to stimulation with K+ in the physiological range. The reduction of the activation threshold of the T‐type current by K+ may also be of physiological significance.
Dopamine D1 receptor modulates the voltage‐gated sodium current in rat striatal neurones through a protein kinase A.
Authors: Schiffmann S N , Lledo P M , Vincent J D
Published: December 2014

1. Whole‐cell recordings were made from striatal neurones obtained from neonatal rats and maintained in primary cultures. The effects of dopamine D1 receptor activation were studied on the voltage‐gated sodium current. 2. Bath application of a specific D1 agonist, SKF38393 (1 microM), reduced the neuronal excitability recorded in current‐clamp by increasing the threshold for generation of action potentials. 3. In voltage‐clamp recordings, SKF38393 (1 microM) reversibly reduced the peak amplitude of the sodium current by 37.8 +/‐ 4.95%. This effect was reversed by the D1 antagonist SCH23390 and was blocked by the intracellular loading of GDP‐beta‐S (2 mM) suggesting GTP‐binding protein involvement. 4. The D1 agonist reduced the peak amplitude of the sodium current without significantly affecting (i) the voltage dependence of the current‐voltage relationship, (ii) the voltage dependence of the steady‐state activation and inactivation, (iii) the kinetics of the time‐dependent inactivation, and (iv) the kinetics of recovery from inactivation. 5. The peak amplitude of the sodium current was progressively reduced by intracellular loading of cyclic AMP‐dependent protein kinase (100 U ml‐1). 6. Diffusion of a specific peptide inhibitor of the cyclic AMP‐dependent protein kinase (PKI; 10 microM) into the cytosol of neurones blocked the effect of the D1 agonist on the sodium current amplitude. 7. These results demonstrate that dopamine acting at the D1 receptor reduces the amplitude of the sodium current without modifying its voltage‐ and time‐dependent properties. This effect involves activation of the cyclic AMP‐dependent protein kinase and results in a depression of the striatal neuronal excitability by increasing the threshold for generation of action potentials.
Role of the ferret perigeniculate nucleus in the generation of synchronized oscillations in vitro.
Authors: Bal T , von Krosigk M , McCormick D A
Published: December 2014

1. The cellular mechanisms by which neurons of the ferret perigeniculate nucleus (PGN) participate in the generation of spindle waves and slowed absence seizure‐like oscillations were investigated with intracellular and extracellular recording techniques in geniculate slices maintained in vitro. 2. During spindle wave generation, PGN neurons generated repetitive (2‐9 Hz) high frequency (up to 500 Hz) burst discharges mediated by the activation of a low threshold Ca2+ spike by the arrival of barrages of excitatory postsynaptic potentials (EPSPs). In most PGN cells at resting membrane potentials (‐60 to ‐70 mV) spindle waves were associated with a progressive hyperpolarization that persisted as a prolonged after‐hyperpolarization. 3. The EPSPs occurring in PGN cells were highly synchronized with burst firing in the neighbouring portion of the dorsal lateral geniculate nucleus (LGNd) and were intermixed with short duration inhibitory postsynaptic potentials (IPSPs). After block of GABAergic receptors, the EPSPs occurring during the generation of spindle waves reversed polarity at around 0 mV. In addition, these EPSPs were completely blocked with the bath application of the non‐NMDA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), as was spindle wave generation in both the PGN and LGNd. 4. Slowing the intraspindle frequency to 2‐4 Hz with pharmacological block of GABAA receptors resulted in a marked increase in the intensity of burst firing by PGN cells such that the number of action potentials per burst increased from a maximum of thirteen to a maximum of sixty. Block of GABAA receptors also resulted in a marked increase in the amplitude and duration of the EPSP barrages arriving from the relay laminae during generation of the slowed oscillation. 5. These findings indicate that spindle waves are generated in the ferret LGNd in vitro through an interaction between the GABAergic neurons of the PGN and relay neurons, such that burst firing in relay neurons activates a barrage of EPSPs and a subsequent low threshold Ca2+ spike in PGN cells. This activation of PGN neurons inhibits a substantial number of relay cells, a few of which rebound burst after this IPSP, thus starting the cycle again. Block of GABAA receptors results in a marked enhancement of activity in PGN cells through increased excitation from relay cells and disinhibition from neighbouring PGN cells. This increased activity in PGN neurons results in a markedly enhanced activation of GABAB receptors in relay neurons and the subsequent generation of paroxysmal activity that is similar to that associated with absence seizures.
Inactivation of the cloned potassium channel mouse Kv1.1 by the human Kv3.4 ‘ball’ peptide and its chemical modification.
Authors: Stephens G J , Robertson B
Published: December 2014

1. This study used the whole‐cell patch clamp technique to investigate the action of a 28‐mer ‘inactivation peptide’ based on part of the N‐terminal sequence of the human Kv3.4 K+ channel (hKv3.4 peptide) on the cloned mouse brain K+ channel mKv1.1 expressed in Chinese hamster ovary (CHO) cells, and compared this with the inactivation produced by Shaker B inactivation peptide (ShB peptide). 2. Inclusion of the hKv3.4 peptide in the patch electrode (320 microM) transformed non‐inactivating mKv1.1 into a rapidly inactivating current. The voltage dependence of time constants of decay and steady‐state inactivation induced by hKv3.4 peptide were characteristic of an ‘A‐type’ K+ current. 3. The hKv3.4 peptide had no effect on the voltage dependence of activation of mKv1.1, with a mid‐point of activation of ‐8 mV, and a slope factor of 15 mV. Steady‐state inactivation curves had a mid‐point of inactivation of ‐36 mV and a slope factor of ‐7 mV; the time constant of recovery from inactivation at ‐90 mV was 1.3 s. 4. The chemical modification reagents N‐bromoacetamide (NBA, 100 microM) and chloramine‐T (CL‐T, 500 microM) had no effect on the fast inactivation of mKv1.1 induced by ShB peptide. In contrast, the inactivation caused by hKv3.4 peptide was removed by brief exposure to NBA and CL‐T. 5. Chemical modification resulted in a hyperpolarizing shift of ‐8 mV (CL‐T) and ‐11 mV (NBA) in the voltage dependence of activation of mKv1.1 in the presence of hKv3.4 peptide. 6. Chemical modification was critically dependent on the presence of a cysteine residue at position 6, and not position 24, of hKv3.4 peptide. 7. NBA and CL‐T caused only a slight inhibition of unmodified mKv1.1 current with no significant effect on the voltage dependence of mKv1.1 activation, and also had no effect on channel deactivation at ‐90 mV. 8. Chemical modification experiments were consistent with a selective action on the hKv3.4 peptide itself, specifically at the cysteine residue at position 6.
Classification of ion channels in the luminal and abluminal membranes of guinea‐pig endocardial endothelial cells.
Authors: Manabe K , Ito H , Matsuda H , Noma A , Shibata Y
Published: December 2014

1. The ion channels on both the luminal and abluminal membranes of endocardial endothelial (EE) cells were separately recorded using the patch clamp technique in the guinea‐pig heart. 2. The major population consisted of two types of non‐selective cation channels, which showed open probabilities of 0.21 and 0.33 at the resting potential, and conductances of 36 and 11 pS, respectively. 3. The next major class was Cl‐ channels with an ohmic conductance of 409 pS. The channel was quiescent in the cell‐attached mode but was activated by strong depolarization after excising the patch membrane. 4. The channels activated by intracellular Ca2+ were mainly K+ channels showing a 34 pS slope conductance and, less frequently, Ca(2+)‐dependent K+ channels having a large conductance (210 pS). The inward rectifier K+ channel (32 pS) was also observed. 5. The non‐selective cation channels were recorded on the luminal membrane, but scarcely on the abluminal membrane, suggesting an active transport of K+ and Na+ across the endocardium. 6. The resting membrane conductance of the EE cells may be provided mostly by non‐selective cation channels and 34 pS Ca(2+)‐dependent K+ channels.
Abnormal cardiovascular and catecholamine responses to supine exercise in human subjects with sympathetic dysfunction.
Authors: Smith G D , Watson L P , Pavitt D V , Mathias C J
Published: December 2014

1. The cardiovascular and catecholamine responses to supine leg exercise were measured in fifteen normal subjects (controls) and in three groups with sympathetic dysfunction: fifteen with central failure (Shy‐Drager syndrome; SDS), fifteen with peripheral failure (pure autonomic failure; PAF) and two with isolated dopamine beta‐hydroxylase deficiency (DBH deficiency). 2. With exercise, blood pressure increased in controls, fell markedly in SDS and PAF and was unchanged in DBH deficiency. After exercise, blood pressure rapidly returned to baseline in controls, but remained low in SDS and PAF. With exercise, heart rate increased more in controls than SDS or PAF; the response varied in DBH deficiency. 3. With exercise, cardiac output increased similarly in controls, SDS and PAF, with a larger increase in DBH deficiency. Vascular resistance fell less in controls than SDS, PAF and DBH deficiency. 4. With exercise, plasma noradrenaline increased in controls only; plasma adrenaline remained unchanged in all groups. In DBH deficiency, plasma noradrenaline and adrenaline were undetectable, but plasma dopamine was elevated and rose further with exercise. 5. Supine exercise substantially lowered blood pressure in sympathetic failure due to SDS and PAF. In DBH deficiency blood pressure was unchanged; this lack of fall may have been due to vasoconstriction induced by dopamine and other substances released from otherwise intact sympathetic terminals, or to preserved cardiac vagal function.
Convertible modes of inactivation of potassium channels in Xenopus myocytes differentiating in vitro.
Authors: Ernsberger U , Spitzer N C
Published: December 2014

1. Voltage‐dependent inactivating single‐channel potassium currents were recorded in cell‐attached and inside‐out patches from embryonic Xenopus myocytes differentiating in culture. 2. Channels with rapid inactivation (time constants < 25 ms) and with slow inactivation (time constants > 80 ms) recorded after one day in vitro appear to belong to two functionally different classes. Rapidly and slowly inactivating channels show steady‐state inactivation with potentials of half‐inactivation of ‐74 +/‐ 7 and ‐44 +/‐ 9 mV. They exhibit voltage‐dependent activation, with times to half‐maximal activation of 0.79 +/‐ 0.09 and 1.17 +/‐ 0.22 ms when stepped from ‐120 to +40 mV. Rapidly inactivating channels also have a lower open probability than slowly inactivating ones. The channels have similar conductances of 23 +/‐ 6 and 17 +/‐ 4 pS and extrapolated reversal potentials close to the potassium equilibrium potential. 3. In cell‐attached patches, inactivation behaviours of channels with rapid or slow inactivation do not change during recording. After patch excision, rapidly inactivating channels usually switch to a slow inactivation mode. Slowly inactivating channels derived from rapidly inactivating channels after patch excision retain their conductance and extrapolated reversal potential, but are not distinguishable from native slowly inactivating channels with respect to steady‐state inactivation, activation and inactivation times, as well as open probabilities. 4. The change in inactivation behaviour of rapidly inactivating channels after patch excision is reversed by application of reduced dithiothreitol (DTT). In contrast, channels with slow inactivation in the cell‐attached mode do not change in to rapidly inactivating channels after application of DTT in the excised configuration, suggesting that these channels belong to a structurally different class. 5. Frequent observation of superposing channel openings indicates clustering of inactivating potassium channels in the myocyte membrane, since many patches lack channel activity. Clustering does not depend on the presence of differentiating neurones. 6. Channels with rapid inactivation increase 6‐fold in density during the first day in culture in the presence of neurones; channel density decreases in their absence. Channels with slow inactivation increase 2‐fold in density in the presence or absence of differentiating neurones during this period. 7. Channels with rapid or slow inactivation in cell‐attached membrane belong to functionally distinct classes that are developmentally regulated differently. Reversible changes from rapid to slow inactivation mode after patch excision suggest that the channels may be structurally related.
Intracellular calcium and its sodium‐independent regulation in voltage‐clamped snail neurones.
Authors: Kennedy H J , Thomas R C
Published: December 2014

1. We have used both Ca(2+)‐sensitive microelectrodes and fura‐2 to measure the intracellular free calcium ion concentration ([Ca2+]i or its negative log, pCai) of snail neurones voltage clamped to ‐50 or ‐60 mV. Using Ca(2+)‐sensitive microelectrodes, [Ca2+]i was found to be approximately 174 nM and pCai, 6.76 +/‐ 0.09 (mean +/‐ S.E.M.; n = 11); using fura‐2, [Ca2+]i was approximately 40 nM and pCai, 7.44 +/‐ 0.06 (mean +/‐ S.E.M., n = 10). 2. Depolarizations (1‐20 s) caused an increase in [Ca2+]i which was abolished by removal of extracellular Ca2+, indicating that the rise in [Ca2+]i was due to Ca2+ influx through voltage‐activated Ca2+ channels. 3. Caffeine (10‐20 mM) caused an increase in [Ca2+]i in the presence or absence of extracellular Ca2+. The effects of caffeine on [Ca2+]i could be prevented by ryanodine. 4. Thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)‐ATPase, caused a small increase in resting [Ca2+]i and slowed the rate of recovery from Ca2+ loads following 20 s depolarizations. 5. Neither replacement of extracellular sodium with N‐methyl‐D‐glucamine (NMDG), nor loading the cells with intracellular sodium, had any effect on resting [Ca2+]i or the rate of recovery of [Ca2+]i following depolarizations. 6. The mitochondrial uncoupling agent carbonyl cyanide m‐chlorophenylhydrazone (CCmP) caused a small gradual rise in resting [Ca2+]i. Removal of extracellular sodium during exposure to CCmP had no further effect on [Ca2+]i. 7. Intracellular orthovanadate caused an increase in resting [Ca2+]i and prevented the full recovery of [Ca2+]i following small Ca2+ loads, but removal of extracellular sodium did not cause a rise in [Ca2+]i. We conclude that there is no Na(+)‐Ca2+ exchanger present in the cell body of these neurones and that [Ca2+]i is maintained by an ATP‐dependent Ca2+ pump.
Sustained calcium influx activated by basic fibroblast growth factor in Balb‐c 3T3 fibroblasts.
Authors: Munaron L , Distasi C , Carabelli V , Baccino F M , Bonelli G , Lovisolo D
Published: December 2014

1. We have investigated the ionic events elicited in Balb‐c 3T3 fibroblasts by basic fibroblast growth factor (bFGF), a peptide that binds to membrane receptors with tyrosine kinase activity and has a mitogenic action on many cell types. The peptide (0.2‐100 ng ml‐1) caused the appearance of an inward current, as observed in whole‐cell patch‐clamp experiments at a holding potential of ‐50 mV, that could last for tens of minutes and had a peak density of 4.6 +/‐ 2.6 pA pF‐1. The reversal potential was 18.8 +/‐ 16.7 mV. 2. The current was reversibly abolished by removal of bFGF from the external bath. Inhibition of low‐affinity FGF receptors had no effect on the activation of the inward current; it was completely abolished when cells were pre‐incubated with tyrphostin or 5'‐methylthioadenosine (MTA), two inhibitors of the tyrosine kinase activity of the high‐affinity FGF receptors. The inward current was not activated by the emptying of internal calcium stores, as tested with 200 nM thapsigargin. 3. Values of peak current density comparable to control ones were obtained when either all Na+ ions or all Ca2+ ions were removed from the external solution; when both ions were completely removed, no inward current could be observed. The inward current was not affected by 2 microM nifedipine, and was reversibly blocked by the imidazole derivative SK&F 96365‐A. 4. Measurements of free intracellular calcium concentration ([Ca2+]i) with the dye fura‐2 showed that bFGF elicited sustained increases in [Ca2+]i that were completely dependent on external calcium and on the presence of the agonist and could last more than 1 h. 5. Single channel currents (conductance 7.9 pS) in response to bFGF stimulation could be recorded in the cell‐attached configuration with 100 mM CaCl2 in the pipette. When the resting potential was brought near to 0 mV by external perfusion in a high‐K+ solution, Vrev was about 0 mV. 6. We conclude that in Balb‐c 3T3 cells bFGF induces an inward current that is carried at least partially by Ca2+ ions; this current in turn causes a long‐lasting increase in intracellular Ca2+ concentration. The amplitude and time course of these bFGF‐activated ionic events are compatible with their involvement in the control of cell proliferation.
Characterization of single potassium channels in mouse pancreatic acinar cells.
Authors: Schmid A , Schulz I
Published: December 2014

1. Single K(+)‐selective channels with a conductance of about 48 pS (pipette, 145 mM KCl; bath, 140 mM NaCl + 4.7 mM KCl) were recorded in the patch‐clamp whole‐cell configuration in isolated mouse pancreatic acinar cells. 2. Neither application of the secretagogues acetylcholine (second messenger, inositol 1,4,5‐trisphosphate) or secretin (second messenger, cAMP), nor addition of the catalytic subunit of protein kinase A to the pipette solution changed the activity of the 48 pS K+ channel. 3. Intracellular acidification with sodium propionate (20 mM) diminished activity of the 48 pS channel, whereas channel open probability was increased by cytosolic alkalization with 20 mM NH4Cl. 4. BaCl2 (5 mM), TEA (10 mM) or apamin (1 microM) added to the bath solution had no obvious effect on the kinetics of the 48 pS channel. Similarly, glibenclamide and diazoxide failed to influence the channel activity. 5. When extracellular NaCl was replaced by KCl, whole‐cell recordings revealed an inwardly rectifying K+ current carried by a 17 pS K+ channel. 6. The inwardly rectifying K+ current was not pH dependent and could largely be blocked by Ba2+ but not by TEA. 7. Since the 48 pS K+ channel is neither Ca2+ nor cAMP regulated, we suggest that this channel could play a role in the maintenance of the negative cell resting potential.
The perforant path projection to hippocampal area CA1 in the rat hippocampal‐entorhinal cortex combined slice.
Authors: Empson R M , Heinemann U
Published: December 2014

1. The perforant path projection from layer III of the entorhinal cortex to CA1 of the hippocampus was studied within a hippocampal‐entorhinal combined slice preparation. We prevented contamination from the other main hippocampal pathways by removal of CA3 and the dentate gyrus. 2. Initially the projection was mapped using field potential recordings that suggested an excitatory sink in stratum lacunosum moleculare with an associated source in stratum pyramidale. 3. However, recording intracellularly from CA1 cells, stimulation of the perforant path produced prominent fast GABAA and slow GABAB IPSPs often preceded by small EPSPs. In a small number of cells we observed EPSPs only. 4. CNQX blocked excitatory and inhibitory responses. This indicated the presence of an intervening excitatory synapse between the inhibitory interneurone and the pyramidal cell. 5. Focal bicuculline applications revealed that the major site of GABAA inhibitory input was to stratum radiatum of CA1. 6. The inhibition activated by the perforant path was very effective at reducing simultaneously activated Schaffer collateral mediated EPSPs and suprathreshold‐stimulated action potentials. 7. Blockade of fast inhibition increased excitability and enhanced slow inhibition. Both increases relied upon the activation of NMDA receptors. 8. Perforant path inputs activated prominent and effective disynaptic inhibition of CA1 cells. This has significance for the output of hippocampal processing during normal behaviour and also under pathological conditions.
Whole‐cell and single‐channel currents activated by GABA and glycine in granule cells of the rat cerebellum.
Authors: Kaneda M , Farrant M , Cull-Candy S G
Published: December 2014

1. Patch‐clamp methods have been used to characterize GABA‐and glycine‐activated channels and spontaneous synaptic currents in granule cells in thin cerebellar slices from 7‐ to 20‐day‐old rats. 2. All granule cells responded to 10 microM GABA, while approximately 60% responded to 100 microM glycine. With repeated against application, whole‐cell responses to GABA, but not those to glycine, declined over a period of minutes unless the pipette solution contained Mg‐ATP. 3. Whole‐cell concentration‐response curves gave EC50 values at 45.2 and 99.6 microM and Hill slopes of 0.94 and 2.6 for GABA and glycine, respectively. At saturating concentrations, currents evoked by GABA were fivefold larger than those evoked by glycine. 4. Whole‐cell current‐voltage (I‐V) relationships of GABA‐ and glycine‐activated currents reversed close to the predicted Cl‐ equilibrium potential. Partial replacement of intracellular Cl‐ with F‐ shifted the GABA reversal potential to a more negative value. ‘Instantaneous’ I‐V relationships produced by ionophoretic application of GABA were linear, while ‘steady‐state’ I‐V relationships produced by ramp changes in potential showed outward rectification. For glycine, ‘steady‐state’ I‐V plots were linear. 5. Responses to GABA were blocked by the GABAA receptor antagonists bicuculline (15 microM), SR‐95531 (10 microM) and picrotoxinin (100 microM) while responses to glycine were selectively blocked by strychnine (200 nM), indicating the presence of two separate receptor types. 6. In outside‐out membrane patches, GABA opened channels with conductances of 16 and 28 pS. The proportion of openings to each of the conductances varied between patches, possibly indicating the activation of two distinct channel types. Glycine‐activated single‐channel currents had conductances of 32, 55 and 104 pS. Single‐channel I‐V relationships were linear. 7. Spontaneous synaptic currents with a rapid rise time and biexponential decay were present in more than half of the cells examined. These currents were eliminated by bicuculline (15 microM) or SR‐95331 (10 microM) and were greatly reduced in frequency by tetrodotoxin (TTX; 300 nM), suggesting that they were mediated by GABA and arose from spontaneous activity in Golgi interneurones. In granule cells where this spontaneous synaptic activity was apparent, glycine and low concentrations of GABA increased the frequency of the synaptic currents.(ABSTRACT TRUNCATED AT 400 WORDS)
The non‐specific ion channel in Torpedo ocellata fused synaptic vesicles.
Authors: Yakir N , Rahamimoff R
Published: December 2014

1. Synaptic vesicles were isolated and fused into large structures with a diameter of more than 20 microns to characterize their ionic channels. The ‘cell’‐attached and inside‐out configurations of the patch clamp technique were used. 2. Two types of ion channels were most frequently observed: a low conductance chloride channel and a high conductance non‐specific channel. 3. The non‐specific channel has a main conducting state and a substate. The main conducting state has a slope conductance of 246 +/‐ 15 pS (+/‐ S.E.M., n = 15), in the presence of different combinations of KCl and potassium glutamate. 4. From the reversal potentials of the current‐voltage (I‐V) relation, it was concluded that this channel conducts both Cl‐ and K+. 5. The non‐specific channel is highly voltage dependent: under steady‐state voltages it has a high open probability near 0 mV and does not inactivate; when the membrane is hyperpolarized (pipette side more positive), the open probability decreases dramatically. 6. Voltage pulses showed that upon hyperpolarization (from holding potentials between ‐20 and + 20 mV), the channels deactivated; when the membrane was stepped back to the holding potential, the channels reactivated rapidly. 7. In a number of experiments, when the pipette side was made more negative than the bath, the open probability also decreased. 8. Frequently, a substate with a conductance of about 44 +/‐ 4% (+/‐ S.E.M., n = 3) of the main state was detected. 9. We speculate that this non‐specific ion channel may have different roles at the various stages of the life cycle of the synaptic vesicle. When the synaptic vesicle is an intracellular structure, it might help its transmitter‐concentrating capacity by dissipating the polarization. After fusion with the surface membrane, it might constitute an additional conductance pathway, taking part in frequency modulation of synaptic transmission.
Tachykininergic slow depolarization of motoneurones evoked by descending fibres in the neonatal rat spinal cord.
Authors: Kurihara T , Yoshioka K , Otsuka M
Published: December 2014

1. In the isolated spinal cord of the neonatal rat, repetitive electrical stimulation of the upper cervical region elicited a prolonged depolarization of lumbar motoneurones (L3‐5) lasting 1‐2 min, which was recorded extracellularly from ventral roots, or intracellularly. 2. This depolarizing response was markedly depressed by the excitatory amino acid receptor antagonists D‐(‐)‐2‐amino‐5‐phosphonovaleric acid (D‐APV, 30 microM) and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 10 microM). The remaining response was further depressed by a 5‐hydroxytryptamine (5‐HT) receptor antagonist, ketanserin (3 microM). 3. In the presence of these antagonists, a small part of the depolarizing response of slow time course remained, and this response was partially blocked by the tachykinin NK1 receptor antagonists GR71251 (0.3‐5 microM) and RP67580 (0.3‐1 microM). In contrast, RP68651 (0.3‐1 microM), the inactive enantiomer of RP67580, had no effect on the depolarizing response. 4. The slow depolarizing response in the presence of D‐APV, CNQX and ketanserin was markedly potentiated by a peptidase inhibitor, thiorphan (1 microM). 5. This descending fibre‐evoked slow depolarization became smaller after prolonged treatment (5‐7 h) with 5,7‐dihydroxytryptamine (10 microM), a neurotoxin for 5‐HT neurones. Under such conditions, the effects of thiorphan and GR71251 on the slow depolarization were virtually absent. 6. Under the action of D‐APV, CNQX and ketanserin, applications of tachykinins, substance P and neurokinin A produced depolarizing responses of lumbar motoneurones, and the responses were depressed by GR71251 and potentiated by thiorphan. 7. These results suggest that tachykinins contained in serotonergic fibres serve as neurotransmitters mediating the descending fibre‐evoked slow excitatory postsynaptic potentials in motoneurones.
In vivo activity of B‐ and C‐neurones in the paravertebral sympathetic ganglia of the bullfrog.
Authors: Ivanoff A Y , Smith P A
Published: December 2014

1. Spontaneous, in vivo synaptic activity was recorded from 146 B‐cells and 60 C‐cells in the IXth and Xth paravertebral sympathetic ganglia of the urethane‐anaesthetized bullfrog. Sympathetic outflow to the blood vessels, which are innervated by C‐cells, is different from that received by targets in the skin, which are innervated by B‐cells. 2. B‐cells were divided into three groups: the first (61 cells) exhibited only action potentials (APs) at 0.01‐0.3 s‐1; the second (59 cells) exhibited APs and EPSPs and the third (26 cells) were silent. In addition to their usual suprathreshold input from the ipsilateral sympathetic chain, 53% of B‐cells received subthreshold input which probably arose from fibres in the contralateral chain. ‘Slow’ B‐cells exhibited less subthreshold activity and a slightly higher AP frequency than ‘fast’ B‐cells. All B‐cells are involved in a sympathetic reflex which is activated by tactile stimulation of the skin of the hindlimb. Activation of this reflex increased AP frequency without promoting long‐lasting depolarization. 3. Sixty‐seven per cent of C‐cells exhibited rhythmic bursting activity with or without small intraburst EPSPs. Bursts tended to correlate with electrocardiographic (ECG) activity. The remainder exhibited an irregular pattern of activity which was not correlated with ECG activity and which included one to three APs and EPSPs interspersed between the bursts. Activity of both types of C‐cell was inhibited following stimulation of the skin. 4. An average of twenty‐three B‐cells and twenty‐one C‐cells discharge simultaneously in vivo. This reflects branching of preganglionic fibres and results in synchrony of discharge in both postganglionic B‐ and C‐fibres.(ABSTRACT TRUNCATED AT 250 WORDS)
Local modulation of adrenergic responses in the hindlimb vasculature of the intact conscious rat.
Authors: DiCarlo S E , Patil R D , Collins H L , Chen C Y
Published: December 2014

1. Local modulation of adrenergic responses was examined in the hindlimb vasculature of chronically instrumented intact conscious rats. Sprague‐Dawley rats (n = 22) were instrumented with a Doppler flow probe around the right common iliac artery, a polyethylene catheter inserted just distal to the flow probe and a left carotid arterial catheter. 2. The effects of various concentrations of the alpha 1‐adrenergic receptor agonist phenylephrine (0.005‐0.075 microgram kg‐1), the alpha 2‐adrenergic receptor agonist clonidine (0.1‐0.7 microgram kg‐1), and the endogenous adrenergic receptor agonist adrenaline (0.02‐0.08 microgram kg‐1), were investigated under control conditions, and in the presence of the nitric oxide (NO) synthase inhibitor N omega‐nitro‐L‐arginine methyl ester hydrochoride (L‐NAME) (NO‐X, 0.2 mg kg‐1) and the cyclo‐oxygenase inhibitor indomethacin (CO‐X, 10 mg kg‐1). Results were presented as dose‐response curves. 3. Heart rate and arterial pressure were not altered by any of the agents because all were locally injected into the hindlimb vasculature and the selected doses were lower than those which elicited systemic responses. 4. Maximal vasoconstrictor responses to phenylephrine were enhanced in the presence of NO‐X (50 +/‐ 6%) and CO‐X (70 +/‐ 9%). Maximal vasoconstrictor responses to clonidine were also enhanced in the presence of NO‐X (75.3 +/‐ 4.8%) and CO‐X (50.6 +/‐ 5.7%). 5. The responses to adrenaline were biphasic; NO‐X significantly attenuated the vasodilator response (87 +/‐ 6%), and enhanced the vasoconstrictor response (51 +/‐ 7%). CO‐X attenuated the vasoconstrictor response (71 +/‐ 6%). 6. These results demonstrate local modulation of responses to alpha 1‐ and beta‐adrenergic receptor agonists by receptor‐mediated dose‐dependent release of NO and prostaglandins.
Chimeric L‐type Ca2+ channels expressed in Xenopus laevis oocytes reveal role of repeats III and IV in activation gating.
Authors: Wang Z , Grabner M , Berjukow S , Savchenko A , Glossmann H , Hering S
Published: December 2014

1. Chimeric alpha 1 subunits consisting of repeat I and II from the rabbit cardiac (alpha 1C‐a) and repeat III and IV from the carp skeletal muscle Ca2+ channel (alpha 1S) were constructed and expressed in Xenopus laevis oocytes without co‐expressing other channel subunits. Ba2+‐current kinetics of five chimeric channel constructs were studied in Xenopus oocytes using the two‐microelectrode technique. 2. Exchange of repeats III and IV of alpha 1C‐a with sequences of alpha 1S results in a significantly slower and biexponential activation (apparent activation time constants tau 1act = 19.8 +/‐ 1.8 ms and tau 2act = 214 +/‐ 28.7 ms, n = 7) of expressed Ca2+ channel currents; no current inactivation was observable during an 800 ms test pulse to 0 mV. 3. Activation of a chimera consisting of repeats I, II and IV from the alpha 1C‐a subunit and repeat III from alpha 1S was fast and monoexponential (tau 1act = 6.33 +/‐ 1.7 ms, n = 5) and the current inactivated during a 350 ms test pulse to 0 mV (tau inact = 175 +/‐ 22 ms, n = 5). The current kinetics of this construct did not significantly differ from kinetics of a construct consisting of repeats I to IV from alpha 1C‐a (tau 1act = 6.6 +/‐ 2.1 ms; tau inact = 198 +/‐ 14 ms; n = 9).(ABSTRACT TRUNCATED AT 250 WORDS)
Ozone‐induced oxygen radical release from bronchoalveolar lavage cells and airway hyper‐responsiveness in dogs.
Authors: Stevens W H , Conlon P D , O'Byrne P M
Published: December 2014

1. Ozone inhalation causes airway hyper‐responsiveness and airway inflammation in dogs. The purpose of this study was to determine whether these effects are associated with increases in oxygen radical production from bronchoalveolar lavage (BAL) cells. 2. Twelve randomly selected dogs were studied twice, 4 weeks apart. On each study day, acetylcholine (ACh) airway responsiveness was measured before and 1 h after ozone (3 p.p.m., 30 min) or dry air inhalation, followed by BAL. The response to ACh was expressed as the concentration causing an increase in lung resistance of 5 cmH2O l‐1 s‐1 above baseline. Spontaneous and phorbol myristate acetate (PMA) (2.4 mumol l‐1)‐stimulated oxygen radical release from washed BAL cells (4 x 10(6) cells ml‐1) was measured by luminol‐enhanced chemiluminescence in a luminometer at 37 degrees C. 3. Ozone inhalation caused airway hyper‐responsiveness. The concentration of ACh causing an increase in lung resistance of 5 cmH2O l‐1 s‐1 (the ‘provocative’ concentration) fell from 4.68 mg ml‐1 (% S.E.M., 1.43) before, to 0.48 mg ml‐1 (% S.E.M., 1.60) after ozone (P < 0.0001). Spontaneous chemiluminescence area under the curve (AUC) significantly increased after ozone from 4.08 mV (10 min) (% S.E.M., 1.28) after dry air to 8.25 mV (10 min; % S.E.M., 1.29) after ozone (P = 0.007). Ozone inhalation also increased PMA‐stimulated chemiluminescence AUC from 18.97 mV (10 min; % S.E.M., 1.18) after dry air to 144.03 mV (10 min; % S.E.M., 1.45) after ozone (P = 0.0001). The increase in PMA‐stimulated chemiluminescence was significantly correlated with ozone‐induced ACh airway hyper‐responsiveness (r = 0.83, P < 0.001). 4. These results indicate that inhaled ozone increases oxygen radical release from BAL cells and suggest that oxygen radicals are important in causing ozone‐induced airway hyper‐responsiveness.