bluuebunny/crossref_metadata_2025 huggingface dataset:
A functional CFTR protein is required for mouse intestinal cAMP‐, cGMP‐ and Ca2+‐dependent HCO3− secretion
Authors: Seidler U. , Blumenstein I. , Kretz A. , Viellard‐Baron D. , Rossmann H. , Colledge W. H. , Evans M. , Ratcliff R. , Gregor M.
Published: September 2004

Most segments of the gastrointestinal tract secrete HCO3−, but the molecular nature of the secretory mechanisms has not been identified. We had previously speculated that the regulator for intestinal electrogenic HCO3− secretion is the cystic fibrosis transmembrane regulator (CFTR) channel. To prove this hypothesis, we have now measured HCO3− secretion by pH‐stat titration, and recorded the electrical parameters of in vitro duodenum, jejunum and ileum of mice deficient in the gene for the CFTR protein (‘CF‐mice’) and their normal littermates.

Basal HCO3− secretory rates were reduced in all small intestinal segments of CF mice. Forskolin, PGE2, 8‐bromo‐cAMP and VIP (cAMP‐dependent agonists), heat‐stable enterotoxin of Escherichia coli (STa), guanylin and 8‐bromo‐cGMP (cGMP‐dependent agonists) and carbachol (Ca2+ dependent) stimulated both the short‐circuit current (ISC) and the HCO3− secretory rate (JHCO3‐) in all intestinal segments in normal mice, whereas none of these agonists had any effect on JHCO3‐ in the intestine of CF mice.

To investigate whether Cl−–HCO3− exchangers, which have been implicated in mediating the response to some of these agonists in the intestine, were similarly active in the small intestine of normal and CF mice, we studied CF gradient‐driven 36Cl− uptake into brush‐border membrane (BBM) vesicles isolated from normal and CF mouse small intestine. Both the time course and the peak value for 4,4’‐diisothiocyanostilbene‐2’,2‐disulphonic acid (DIDS)‐inhibited 36Cl− uptake was similar in normal and CF mice BBM vesicles.

In summary, the results demonstrate that the presence of the CFTR channel is necessary for agonist‐induced stimulation of electrogenic HCO3− secretion in all segments of the small intestine, and all three intracellular signal transduction pathways stimulate HCO3− secretion exclusively via activation of the CFTR channel.
Non‐NMDA receptors modulate respiratory drive in fetal sheep
Authors: Bissonnette John M. , Hohimer A. Roger , Knopp Sharon J.
Published: September 2004

Experiments were carried out in unanaesthetized fetal sheep to evaluate the significance of non‐N‐methyl‐d‐aspartate (non‐NMDA) receptor neurotransmission in the expression of fetal breathing movements. Catheters placed in the trachea and amniotic fluid and electrodes beneath the parietal bones and in the nuchal muscle were used to monitor breath amplitude and frequency and fetal behavioural state.

Experiments were carried out by instillation of neurotransmitter agonists, antagonists or receptor modulators into the cerebrospinal fluid (CSF) of the fourth ventricle by means of a chronic catheter introduced through the foramen magnum.

The non‐NMDA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) decreased respiratory rate in a dose dependent manner by lengthening both inspiratory time (Ti) and expiratory time (Te).

Kainate and (R,S)‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) increased breath amplitude. Instillation of the antagonist 2,3‐dihydro‐6‐nitro‐7‐sulphamoyl‐benzo(f) quinoxaline (NBQX) prior to administering AMPA resulted in apnoea, which was not overcome by the agonist.

Cyclothiazide, which has been shown to prevent desensitization of AMPA receptors, caused an increase in both breath amplitude (152 ± 73%; mean ±s.d.; P= 0.004) and frequency (46 ± 37%; P= 0.049).

These data suggest that glutamate acting at non‐NMDA receptors is an essential component for the expression of fetal breathing movements, and that under resting conditions these non‐NMDA receptors are desensitized following glutamate synaptic release.
Mechanisms for stimulation of rat anterior pituitary cells by arginine and other amino acids
Authors: Villalobos Carlos , Núñez Lucía , García‐Sancho Javier
Published: September 2004

Arginine and other amino acids are secretagogues for growth hormone and prolactin in the intact animal, but the mechanism of action is unclear. We have studied the effects of amino acids on cytosolic free calcium concentration ([Ca2+]i) in single rat anterior pituitary (AP) cells. Arginine elicited a large increase of [Ca2+]i in about 40 % of all the AP cells, suggesting that amino acids may modulate hormone secretion by acting directly on the pituitary.

Cell typing by immunofluorescence of the hormone the cells store showed that the arginine‐sensitive cells are distributed uniformly within all the five AP cell types. The arginine‐sensitive cells overlapped closely with the subpopulation of cells sensitive to thyrotrophin‐releasing hormone.

Other cationic as well as several neutral (dipolar) amino acids had the same effect as arginine. The increase of [Ca2+]i was dependent on extracellular Ca2+ and blocked by dihydropyridine, suggesting that it is due to Ca2+ influx through l‐type voltage‐gated Ca2+ channels. The [Ca2+]i increase was also blocked by removal of extracellular Na+ but not by tetrodotoxin. The substrate specificity for stimulation of AP cells resembled closely that of the amino acid transport system B0,+. We propose that electrogenic amino acid influx through this pathway depolarizes the plasma membrane with the subsequent activation of voltage‐gated Ca2+ channels and Ca2+ entry.

Amino acids also stimulated prolactin secretion in vitro with a similar substrate specificity to that found for the [Ca2+]i increase. Existing data on the stimulation of secretion of other hormones by amino acids suggest that a similar mechanism could apply to other endocrine glands.
Calibration of an autocorrelation‐based method for determining amplitude histogram reliability and quantal size
Authors: Stratford K. J. , Jack J. J. B. , Larkman A. U.
Published: September 2004

We describe a method, based on autocorrelation and Monte Carlo simulation, for determining the likelihood that peaks in synaptic amplitude frequency histograms could have been a result of finite sampling from parent distributions that were unimodal.

The first step was to calculate an ‘autocorrelation score’ for the histogram to be tested. A unimodal distribution was fitted to the test histogram and subtracted from it. The resulting difference function was smoothed and its autocorrelation function calculated. The amplitude of the first (non‐zero lag) peak in this autocorrelation function was taken as the autocorrelation score for that histogram. The score depends on the sharpness of the histogram peaks, the equality of their spacing and the number of trials.

The second stage was to generate large numbers of random samples, each of the same number of trials as the histogram, from a unimodal generator distribution of similar shape. The autocorrelation score was calculated for each sample and the proportion of samples with scores greater than the histogram gave the likelihood that the histogram peaks could have arisen by sampling artifact.

The method was calibrated using simulated non‐quantal and quantal histograms with different signal‐to‐noise ratios and numbers of trials. For a quantal distribution with four peaks and a signal‐to‐noise ratio of 3, a sample size of about 500 trials was needed for 95% of samples to be distinguished from a non‐quantal distribution.

The ability of the autocorrelation method to distinguish quantal from non‐quantal distributions was compared against two conventional statistical tests, the X2 and the Kolmogorov–Smirnov goodness of fit tests. The autocorrelation method was more specific in extracting quantized responses. The Kolmogorov–Smirnov test in particular could not distinguish quantal distributions with multiple peaks even if the peaks were very sharp.

The improved discrimination of the autocorrelation method proved important when applied to experimental data recorded from hippocampal synapses. Of thirty‐three histograms that were significantly different from smooth distributions by the autocorrelation method (P≤ 0.05), only seventeen were significantly different using the X2 test and only two when using the Kolmogorov–Smirnov test.

The autocorrelation method also gave an estimate of histogram peak spacing or quantal size. Using simulated quantal distributions, we showed that this estimate was likely to be correct within a few per cent for distributions that gave P≤ 0.01 by autocorrelation scoring.
Effect of saponin treatment on the sarcoplasmic reticulum of rat, cane toad and crustacean (Yabby) skeletal muscle
Authors: Launikonis Bradley S. , Stephenson D. George
Published: September 2004

Mechanically skinned fibres from skeletal muscles of the rat, toad and yabby were used to investigate the effect of saponin treatment on sarcoplasmic reticulum (SR) Ca2+ loading properties. The SR was loaded submaximally under control conditions before and after treatment with saponin and SR Ca2+ was released with caffeine.

Treatment with 10 μg ml−1 saponin greatly reduced the SR Ca2+ loading ability of skinned fibres from the extensor digitorum longus muscle of the rat with a rate constant of 0.24 min−1. Saponin concentrations up to 150 μg ml−1 and increased exposure time up to 30 min did not further reduce the SR Ca2+ loading ability of the SR, which indicates that the inhibitory action of 10–150 μg ml−1 saponin is not dose dependent. The effect of saponin was also not dependent on the state of polarization of the transverse‐tubular system.

Treatment with saponin at concentrations up to 100 μg ml−1 for 30 min did not affect the Ca2+ loading ability of SR in skinned skeletal muscle fibres from the twitch portion of the toad iliofibularis muscle but SR Ca2+ loading ability decreased markedly with a time constant of 0.22 min−1 in the presence of 150 μg ml−1 saponin.

The saponin dependent increase in permeability could be reversed in both rat and toad fibres by short treatment with 6 μM Ruthenium Red, a potent SR Ca2+ channel blocker, suggesting that saponin does affect the SR Ca2+ channel properties in mammalian and anuran skeletal muscle.

Treatment of skinned fibres of long sarcomere length (> 6 μM) from the claw muscle of the yabby (a freshwater decapod crustacean) with 10 μg ml−1 saponin for 30 min abolished the ability of the SR to load Ca2+, indicating that saponin affects differently the SR from skeletal muscles of mammals, anurans and crustaceans.

It is concluded that at relatively low concentrations, saponin causes inhibition of the skeletal SR Ca2+ loading ability in a species dependent manner, probably by increasing the Ca2+ loss through SR Ca2+ release channels.
Chemosensory and cholinergic stimulation of fictive respiration in isolated cns of neonatal opossum
Authors: Eugenín Jaime , Nicholls John G.
Published: September 2004

The aim of the present experiments was to characterize the central chemical drive of fictive respiration in the isolated CNS of the newborn opossum, Monodelphis domestica. This opossum preparation, in contrast to those of neonatal rats and mice, produces respiratory rhythm of high frequency in vitro.

Fictive respiration was recorded from C3–C5 ventral roots of the isolated CNS of 4‐ to 14‐day‐old opossums using suction electrodes. At room temperature (21–23 °C) the frequency of respiration was 43 ± 5.3 min−1 (mean ±s.e.m., n= 50) in basal medium Eagle's medium (BMEM) equilibrated with 5% CO2–95% O2, pH 7.37‐7.40. Respiratory discharges remained regular throughout 8 h experiments and continued for more than 20 h in culture.

Superfusion of the brainstem confirmed that solutions of pH 6.3–7.2 increased both the amplitude and frequency of respiration. High pH solutions (7.5–7.7) had the opposite effect and abolished the rhythm at pH7.7. Addition of ACh (50–100 μm) or carbachol (0.01–10 μm) to the brainstem superfusion also increased the amplitude and frequency of respiratory activity, as did physostigmine (50–100 μm) or neostigmine (20–50 μm). Conversely, scopolamine (50–100 μm) reduced the amplitude and frequency of the basal respiratory rhythm by about 30%.

H+ ‐ and cholinergic‐sensitive areas on the surface of the isolated CNS were explored with a small micropipette (outer tip diameter, 100 μm) filled with BMEM (pH 6.5) or 1 μm carbachol. Carabachol applied to H+ ‐ and cholinergic‐sensitive areas in the ventral medulla mimicked the changes of respiratory pattern produced by low pH application. Responses to altered pH and carbachol were abolished by scopolamine (50 μm). Histochemistry demonstrated several medullary groups of neurons stained for acetylcholinesterase. The superficial location of one of these groups coincided with a functional and anatomically well‐defined pH‐ and carbachol‐sensitive area placed medial to the hypoglossal roots.

Exploration of chemosensitive areas revealed that application of drugs or solutions of different pH to a single well‐defined spot could have selective and distinctive effects upon amplitude and frequency of respiratory activity.

These results show that fictive respiration in the isolated CNS of the newborn opossum is tonically driven by chemical‐ and cholinergic‐sensitive areas located on the ventral medulla, the activity of which regulates frequency and amplitude of respiration. They suggest that a cholinergic relay, although not essential for rhythm generation, is involved in the central pH chemosensory mechanism, or that cholinergic and chemical inputs converge upon the same input pathway to the respiratory pattern generator.
Effects of Hypoxia on [Ca2+]i, pHi and Myosin Light Chain Phosphorylation in Guinea‐Pig Taenia Caeci
Authors: Obara Kazuo , Bowman Peggy Sue , Ishida Yukisato , Paul Richard J.
Published: September 2004

Hypoxia (achieved by bubbling with N2 instead of O2) reduces the force of a KCl (40 mm)‐induced contracture to ?10 % of the control value in guinea‐pig taenia caeci. The underlying mechanism of this relaxation in response to hypoxia was investigated by measuring the major cell signalling parameters, intracellular Ca2+ concentration ([Ca2+]i) and myosin regulatory light chain (LC20) phosphorylation (MLC‐Pi), as well as intracellular pH (pHi), a factor often suggested to mediate hypoxic relaxation of muscle.

[Ca2+]i, measured using the ratiometric fluorescent dye fura‐2, increased when 40 mm KCl was added to physiological saline solution (PSS) (peak value assigned 100%), and the steady state after 15 min was 92.8 %. There were no detectable decreases in [Ca2+]i during hypoxia.

MLC‐Pi, measured using isoelectric focusing‐polyacrylamide gel electrophoresis and identified using Western blotting, increased from 9 % of the total LC20 in Ca2+‐free PSS to a peak value of 51 % in 40 mM KCl‐PSS. The steady‐state value in hypoxia of 43 % was not significiantly different from that in control oxygenated conditions at the same point in time.

pHi, measured using the ratiometric fluorescent dye 2′,7′‐bis(carboxyethyl)‐5(6)‐carboxyfluorescein (BCECF), under quiescent conditions (Ca2+‐free PSS) was 7.23 and increased to 7.36 with 40 mM KCl. After imposition of hypoxia pHi remained unchanged despite the known increase in both lactate content and production.

As [Ca2+]i and MLC‐Pi, key factors in activation, were not decreased by hypoxia and changes in pHi were minor, hypoxic relaxation in guinea‐pig taenia caeci appears to be directly related to energy limitation rather than any oxygen‐sensing mechanism.
Spatial Variation in Sympathetic Influences on the Vasculature of the Synovium and Medial Collateral Ligament of the Rabbit Knee Joint
Authors: McDougall Jason J. , Ferrell William R. , Bray Robert C.
Published: September 2004

Laser Doppler perfusion imaging was used to assess the role of the sympathetic nervous system in the control of blood flow to the medial collateral ligament and capsule (synovium and overlying fibrous tissues) of the rabbit knee joint.

Electrical stimulation of the saphenous nerve (width 1 ms; amplitude 20V; 1–30 Hz) produced a frequency‐dependent vasoconstriction of knee joint vasculature. The response was maximal at 30 Hz and gave the greatest fall in perfusion at the femoral insertion of the ligament (by 33.8 ± 7.4%, mean ±s.e.m.; n= 5–6) and the smallest decrease at the tibial insertion of the ligament (by 10.6 ± 2.9%).

Topical application of phentolamine (10−6 mol) had no significant effect on basal knee joint blood flow. However, it abolished the nerve‐mediated constrictor responses in all regions of the medial collateral ligament and synovium at all frequencies.

Topical administration of adrenaline (10−14 to 10−7 mol) caused a dose‐dependent decrease in knee joint blood flow with the highest dose producing > 75% reduction in perfusion at all areas.

There was no evidence of a reactive hyperaemia in the 5 min following a 5 min period of femoral artery occlusion. Artificial manipulation of arterial blood pressure by intravenous infusion or withdrawal of blood caused a proportional change in ligament and synovial blood flow. These observations may indicate a lack of autoregulation in the joint and its exclusion from baroreflex modulation.

These results suggest a potential role for the sympathetic nervous system in the control of knee joint blood flow. Neuromodulation of ligament perfusion appears to predominate at the femoral insertion and this could prove to have functional significance.
Functional electrical properties of the endothelium in lymphatic vessels of the guinea‐pig mesentery
Authors: von der Weid Pierre‐Yves , Van Helden Dirk F.
Published: September 2004

The resting and agonist‐stimulated properties of endothelial cells and electrical communication between the endothelium and smooth muscle were investigated in open segments of guinea‐pig mesenteric lymphatic vessels using intracellular microelectrodes.

Endothelial cells had a mean resting membrane potential (RMP) of – 71.5 ± 0.5mV (n= 100) which was significantly different from the value of −60.8 ± 1.1 mV (n= 75) recorded in smooth muscle.

Acetylcholine (ACh, 5–1 μm) generally evoked an initial hyperpolarization followed by depolarization (mean 3.4 ± 0.5 mV and 15.4 ± 1.0 mV, respectively, n= 75).

Ca2+‐activated K+ channels were likely to underlie the ACh‐induced hyperpolarization as this response exhibited an increase in membrane conductance, was larger in 0.5 mm K+ solution and was blocked by charybdotoxin (50 nm).

The endothelium did not exhibit a response to nitric oxide (NO) as the NO‐donor sodium nitroprusside did not alter the RMP and the electrical responses to ACh were not affected by the NO‐synthase inhibitor Nω‐nitro L‐arginine at a concentration which markedly inhibited smooth muscle hyperpolarization.

Electrical coupling between the endothelium and smooth muscle was not functional as there was extremely limited electrical continuity (1 in 12, endothelial/smooth muscle cell simultaneous recordings) and bradykinin, noradrenaline and isoprenaline caused different electrical responses in the two cell types.

These results provide the first description of RMP and electrical responses to various agonists in the lymphatic endothelium and its lack of functional electrical coupling with the smooth muscle.
The Roles of Adenosine in Regulating the Respiratory and Cardiovascular Systems in Chronically Hypoxic, Adult Rats
Authors: Thomas Teresa , Marshall Janice M.
Published: September 2004

We have investigated the roles of adenosine in regulating the respiratory and cardiovascular systems of rats that were made chronically hypoxic for 3–4 weeks from 6 weeks of age (CH rats) in an hypoxic chamber at 12% O2. They were studied under anaesthesia while breathing 12% O2 and during acute hypoxia (breathing 8% O2 for 5min) before and after addition of the adenosine receptor antagonist 8‐phenyltheophylline (8‐PT, 10 mg kg−1). The results were compared with those obtained from normoxic (N) rats in a previous study.

CH rats breathing 12% O2 had greater minute ventilation (V̇E) than N rats breathing air, but their levels of arterial blood pressure (ABP), heart rate (HR), femoral vascular conductance (FVC) and cerebral vascular conductance (CVC) were fully comparable. 8‐PT increased tidal volume (VT) in CH rats indicating a greater tonic central inhibitory influence of adenosine on VT than in N rats. However, 8‐PT had no effect on cardiovascular variables, indicating no tonic cardioinhibitory or vasodilator influence of adenosine in CH rats.

Acute hypoxia in CH rats increased V̇E such that at the 5th minute of 8% O2 absolute V̇E was comparable to that of N rats breathing 8% O2. Moreover, in CH rats 8‐PT increased VT at the 5th minute of 8% O2 indicating that the central inhibitory influence of adenosine limits the ability to maintain VT in acute hypoxia as it does in N rats.

Eight per cent O2 also produced a fall in ABP in CH rats that was comparable to that induced in N rats by the larger change from air to 8% O2. However, the changes in HR were similar in CH and N rats while the increases in FVC and CVC were smaller in CH rats. This suggests that the ability of the secondary effects of hyperventilation and of the baroreceptor reflex to maintain cardiac output and thereby ABP is reduced in CH rats.

Whereas 8‐PT substantially reduced the hypoxia‐induced increases in FVC and CVC in N rats, it had a small effect in CH rats (P= 0.054 and 0.06, respectively). Further, acute hypoxia in CH rats had no effect on the K+ concentration in the venous efflux of hindlimb K+ (Kv+) before or after 8‐PT treatment. We suggest that in CH rats, the dilator influence of adenosine in acute hypoxia occurs via actions on the blood vessel walls: there was no evidence that adenosine can release dilator concentrations of K+ from skeletal muscle fibres in CH rats as proposed for N rats.
Assessment of the reliability of amplitude histograms from excitatory synapses in rat hippocampal CA1 In Vitro
Authors: Larkman A. U. , Jack J. J. B. , Stratford K. J.
Published: September 2004

Excitatory postsynaptic potentials (EPSPs) were evoked using minimal extracellular stimulation and recorded from pyramidal cells from the CA1 region of slices taken from adult rats and maintained in vitro.

Segments of data were selected that gave EPSP amplitude frequency histograms that showed approximately equally spaced peaks. Selection was performed either on the basis of stationarity of the EPSP mean and standard deviation, or on the trajectory of a graph of the (coefficient of variation)−2 against mean for the EPSP, or, in some cases, by trial and error.

For each histogram, we determined the likelihood that peaks of similar sharpness and equality of spacing could have arisen by sampling artifact from a smooth distribution, using a method based on autocorrelation and Monte Carlo simulation.

Thirty‐three histograms were analysed. For twenty‐six of these, the likelihood of sampling artifact was estimated at 1 in 100 or less, and for eleven histograms the likelihood was less than 1 in 1000. For the histogram with the clearest peaks, the likelihood was less than 1 in 350000. Histograms judged to be reliable by this method could occur when the EPSP mean amplitude was changing.

We conclude that random sampling artifact is very unlikely to be the explanation for the peaks in our data histograms. It seems more likely that they are due to a quantal synaptic transmission mechanism with low quantal variability.

The autocorrelation method also gives a measure of the mean peak spacing, and hence the mean quantal size, for each histogram. Quantal sizes ranged from 93 to 285 μV, with a mean ±s.d. of 172 ± 47 μV.

From these quantal sizes and the EPSP mean amplitudes we calculated the mean number of quanta released per trial for each histogram. This ranged from 0.36 to 6.9, with a mean of 3.3 ± 1.67.
Effect of cardiopulmonary C fibre activation on the firing activity of ventral respiratory group neurones in the rat
Authors: Wilson Christopher G. , Bonham Ann C.
Published: September 2004

Cardiopulmonary C fibre receptor stimulation elicits apnoea and rapid shallow breathing, but the effects on the firing activity of central respiratory neurones are not well understood. This study examined the responses of ventral respiratory group neurones: decrementing expiratory (Edec), augmenting expiratory (Eaug), and inspiratory (I) neurones during cardiopulmonary C fibre receptor‐evoked apnoea and rapid shallow breathing.

Extracellular neuronal activity, phrenic nerve activity and arterial pressure were recorded in urethane‐anaesthetized rats. Cardiopulmonary C fibre receptors were stimulated by right atrial injections of phenylbiguanide. Neurones were tested for antidromic activation from the contra‐ and ipsilateral ventral respiratory group (VRG), spinal cord and cervical vagus nerve.

Edecneurones discharged tonically during cardiopulmonary C fibre‐evoked apnoea and rapid shallow breathing, displaying increased burst durations, number of impulses per burst, and mean impulse frequencies. Edecneurones recovered either with the phrenic nerve activity (25 s) or much later (3 min).

By contrast, the firing activity of Eaugand most I neurones was decreased, featuring decreased burst durations and number of impulses per burst and increased interburst intervals. Eaugactivity recovered in ˜3 min and inspiratory activity in ˜1 min.

The results indicate that cardiopulmonary C fibre receptor stimulation causes tonic firing of Edecneurones and decreases in Eaugand I neuronal activity coincident with apnoea or rapid shallow breathing.
Quantal analysis of excitatory synapses in rat hippocampal CA1 In Vitro during low‐frequency depression
Authors: Larkman A. U. , Jack J. J. B. , Stratford K. J.
Published: September 2004

We have performed a detailed quantal analysis of excitatory postsynaptic potentials (EPSPs) evoked by minimal extracellular stimulation in the CA1 region of slices of adult rat hippocampus maintained in vitro.

EPSPs were evoked at 2–5 Hz, and the eight that were analysed all showed at least a 50 % depression of mean peak amplitude during recording.

EPSP amplitude fluctuations were analysed by three methods: the use of amplitude frequency histograms with clear and reliable peaks where available, graphs of the EPSP (coefficient of variation)−2 against EPSP mean, and analysis of EPSP mean and standard deviation assuming simple binomial statistics with the number of release sites (N) kept constant but the quantal size (Q) and the release probability (Pr) allowed to vary over time.

The results of the three analysis procedures were in good agreement. Seven EPSPs showed a substantial reduction in the mean number of quanta released per trial, and in three cases this was the predominant mechanism of the depression. Five EPSPs showed a substantial decrease in Q. Values for N ranged between 3 and 18, with a median of 6; Pr ranged between 0.14 and 0.81 and Q between 66 and 275 μV.

We used the Q estimates from the binomial method to correct the recorded EPSP amplitudes for changes in quantal size over time. For seven out of the eight EPSPs, this rescaling procedure allowed histograms with clear peaks to be obtained from longer runs of data, or improved the sharpness of the peaks in histograms from all the recorded data. The improvement in peak sharpness was assessed using an autocorrelation‐based method. The correction was much less successful if the Q estimates were obtained with a variant of the binomial method in which Pr was held constant and N was allowed to vary.

The only simple explanation for the success of the correction procedure is that changes in quantal size were a major factor in obscuring peaks in histograms based on large numbers of trials, and that the quantal size estimates from the binomial method with N held constant were reasonably accurate.

We conclude that transmission at these synapses was quantal with relatively low quantal variance, but repetitive stimulation often induced substantial changes in the quantal parameters that might prevent the success of conventional quantal analysis approaches.
Paraesthesiae Induced by Prolonged high Frequency Stimulation of Human Cutaneous Afferents
Authors: Kiernan Matthew C. , Hales John P. , Gracies Jean‐Michel , Mogyoros Ilona , Burke David
Published: September 2004

The present study has explored the behaviour of human cutaneous afferents following conduction of prolonged trains of impulses at 200 Hz for 10–20 min, correlating the resultant changes in excitability with the perception of paraesthesiae.

Tetanization for 10 min resulted in activity‐dependent changes in axonal excitability, with an initial period of hyperexcitability, followed by a long‐lasting subexcitability. All subjects experienced paraesthesiae soon after cessation of the tetanic train, and these subsided gradually over 16 min.

Longer tetanic trains of 20 min duration resulted in greater changes in axonal excitability, but with paraesthesiae of a similar time course. The post‐tetanic increase in excitability was abolished when short tetanic trains were delivered > 30 min before long trains, but all subjects still experienced paraesthesiae.

Threshold distributions following tetanic stimulation for both 10 and 20 min established that all axons contributing to the sensory volley underwent a uniform pattern of post‐tetanic threshold changes. There was no evidence of a bimodal distribution with some axons hyper‐polarized and others depolarized, as occurs with motor axons. However, the excitability changes were graded, with axons of lowest threshold undergoing a proportionately greater increase in excitability than axons of higher threshold.

The post‐tetanic excitability changes were greater at the site of stimulation than elsewhere along the peripheral nerve. However, DC polarizing currents applied at this site failed to alter the sensation of paraesthesiae in the post‐tetanic period. Furthermore, local anaesthetic block of the peripheral nerve proximal to the stimulation site failed to suppress the paraesthesiae.

The uniform pattern of post‐tetanic threshold changes for cutaneous afferents differs from the bimodal distribution seen with post‐ischaemic and post‐tetanic motor axons. This difference in behaviour may reflect greater inward rectification and greater expression of a non‐inactivating threshold conductance in cutaneous afferents. It is suggested that the ectopic activity responsible for paraesthesiae in the post‐tetanic period arises from a more central site than the peripheral nerve.
Relationship Between Venous Pressure and tissue Volume During Venous Congestion Plethysmography in Man
Authors: Christ F. , Gamble J. , Baschnegger H. , Gartside I. B.
Published: September 2004

Venous congestion strain‐gauge plethysmography enables the non‐invasive assessment of arterial blood flow, fluid filtration capacity (Kf), venous pressure (Pv)and isovolumetric venous pressure (Pvi)in man. One of the major assumptions of this technique, that cuff pressure (Peuff) applied to the limb equals Pv at the level of the strain gauge, was tested in this study.

In nine healthy male volunteers (mean age, 29.3 ± 1.2 years) the saphenous vein was cannulated with an 18‐gauge catheter proximal to the medial malleolus. The subjects were supine and Pv was continuously measured during the application of small step (8–10 mmHg) increases in congestion Pcuff (up to 70 mm Hg). Pcuff, changes in limb circumference and Pv were recorded by computer for off‐line analysis. Since the determination of Pv is influenced by the changes in plasma oncotic pressure, venous blood samples were obtained at the start of the study, when P& was raised to 30 mmHg and again to 65 mmHg and 4 min after deflation of the cuff.

The relationship between Pv and Pcuff was linear over the range of 10–70 mm Hg (n= 9, 69 measurements, slope 0.9.1, r= 0.97, P. <<0.001). The non‐invasively measured calf Pv based on the intercept of the relationship between the vascular compliance component (Vv) and Pcuff, was 8.0±0.4mmHg, which was not significantly different from the corrected invasively measured Pv value of 8.8±0.3 mmHg P= 0.08).

Venous blood lactate and haemoglobin concentrations, as well as colloid osmotic pressure, total protein and albumin concentrations were unchanged throughout the protocol, whereas significant decreases in Po2 and blood glucose concentration were observed when Pcuff reached 65 mmHg. Assuming a constant oxygen consumption, this may suggest a reduction in tissue perfusion.

This study demonstrates the close correlation between Pcuff and Pvin the saphenous vein. Since the small congestion Pcuff step protocol does not cause significant increase in plasma oncotic pressure, we conclude that Pv as well as Kf can be accurately determined with this venous congestion plethysmography protocol.
Longitudinal distribution of Na+ and Ca2+ channels and β‐adrenoceptors on the sarcolemmal membrane of frog cardiomyocytes
Authors: Jurevičius Jonas , Fischmeister Rodolphe
Published: September 2004

The distribution of L‐type Ca2+ and tetrodotoxin‐sensitive Na+ channels and of β‐adrenergic receptors was examined in frog ventricular myocytes using the whole‐cell patch‐clamp technique and a double capillary for extracellular microperfusion.

Rod‐shaped cells (250–300 μm long) were sealed at both ends to two patch‐clamp pipettes and positioned transversally at different positions between the mouths of two micro‐capillaries separated by a thin wall. A combination of nifedipine (1 μm) and tetrodotoxin (0.3 μm) (blocking solution) was added to one capillary in order to inhibit macroscopic Ca2+ and Na+ currents (ICa and INa, respectively) in the part of the cell exposed to this capillary.

Moving the cell in 10–20 μm steps from the control capillary to the capillary containing the blocking solution induced step decreases in ICa and INa amplitudes. Complete block of both currents occurred when the entire cell was exposed to the blocking solution.

Each step decrease in current was due to the loss of activity of the functional Ca2+ and Na+ channels present in the slice of sarcolemmal membrane newly exposed to the blocking solution. These step current changes allowed longitudinal mapping of current density for Ca2+ and Na+ channels on the sarcolemmal membrane.

Addition of a submaximal concentration of isoprenaline (10 nm) to the control capillary induced a local increase in ICa which enabled examination of the distribution of functional β‐adrenergic receptors as well.

Our results demonstrate that Ca2+ and Na+ channels and β‐adrenergic receptors are equally and essentially uniformly distributed on the sarcolemmal membrane of frog ventricular myocytes.
Evidence Suggesting a Transcortical Pathway from Cutaneous Foot Afferents to Tibialis Anterior Motoneurones in Man
Authors: Nielsen J. , Petersen N. , Fedirchuk B.
Published: September 2004

Stimulation of the superficial peroneal or the sural nerve (3 shocks, 3 ms interval, 1 ms duration, 2.5 × perception threshold) evoked a reflex activation of the tibialis anterior muscle at a latency of approximately 70–95 ms in all of nine healthy human subjects. Stimulation of the medial plantar nerve only rarely produced similar effects. The possibility that a transcortical pathway contributes to these late reflex responses was investigated by combining the cutaneous stimulations and a transcranial magnetic stimulation of the contralateral motor cortex.

A significant facilitation of short‐latency peaks in the post‐stimulus time histogram of single tibialis anterior motor units evoked by the transcortical magnetic stimulation was observed in eight out of nine subjects following stimulation of the superficial peroneal or sural nerves at the latency of the long‐latency reflex. In contrast such a facilitation was only rarely seen when the medial plantar nerve was stimulated.

With the same timing for the stimuli, the superficial peroneal and sural nerve stimulations also produced a significant increase in the short‐latency, presumed monosynaptic, facilitation of the tibialis anterior H reflex produced by the brain stimulation.

Similar facilitatory effects of the cutaneous stimuli could not be demonstrated when the magnetic stimulation of the cortex was replaced with electrical stimulation, implying that cortical excitability is affected by a conditioning cutaneous stimulation.

It is suggested that the long‐latency reflexes in the tibialis anterior muscle evoked by activation of cutaneous afferents from the human foot are, at least partly, mediated by a transcortical pathway.
Drastic facilitation by α‐latrotoxin of bovine chromaffin cell exocytosis without measurable enhancement of Ca2+ entry or [Ca2+]i
Authors: Michelena Pedro , de la Fuente María‐Teresa , Vega Teresa , Lara Baldomero , López Manuela G. , Gandía Luis , García Antonio G.
Published: September 2004

Latrotoxin (LTX, 1–3 nm) caused a gradual increase of the supontaneous catecholamine release rate in bovine adrenal chromaffin cells superfused with normal Krebs–Hepes solution containing 2.5 mm Ca2+. Ca2+ removal abolished this effect. LTX enhanced also the secretory responses to high K+ (35 or 70 mm) and to acetylcholine (ACh, 30 μm).

The application of Ca2+ pulses to cells previously superfused with a 0 Ca2+ solution (Krebs–Hepes deprived of CaCl2) induced secretory responses that gradually reached 400–800 nA of catecholamines, provided that LTX was present. The responses to ACh or 35 mm K+ pulses (in the presence of Ca2+) were also enhanced by LTX, from around 100–200 nA to over 1000 nA. Though such enhancement remained in the presence of Ca2+ channel blockers, it disappeared upon the lowering of [Na+]0 or in electroporated cells.

Using protocols similar to those of secretion, LTX did not enhance basal 45Ca2+ uptake, whole‐cell Ca2+ currents or basal [Ca2+]j. In fact, LTX attenuated the K+‐ or ACh‐evoked increases in 45Ca2+ uptake and [Ca2+]1.

It is proposed that the secretory response to brief periods of Ca2+ reintroductions is triggered by local subplasmalemmal Ca2+1 tranMents, produced by the Na+‐Ca2+ exchanger of the plasma membrane working in the reverse mode. This situation might be physiologically reproduced during ACh stimulation of chromaffin cells, which is followed by the firing of Na+‐dependent action potentials.
Angiotensin II Inhibition of ATP‐Sensitive K+ Currents in Rat Arterial Smooth Muscle Cells Through Protein Kinase C
Authors: Kubo M. , Quayle J. M. , Standen N. B.
Published: September 2004

The effects of the vasoconstrictor angiotensin II (Ang II) on whole‐cell ATP‐sensitive K+currents (Ik,atp) of smooth muscle cells isolated enzymatically from rat mesenteric arteries were investigated using the patch clamp technique.

Ang II, at a physiological concentration (100 nm), reduced Ik,atp activated by 0.1 mm internal ATP and 10 μm levcromakalim by 36.4 ± 2.3%.

The protein kinase C (PKC) activator 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG, 1 μm) reduced Ik,atp by 44.1 ± 2.7%. GDPβS (1 mm), included in the pipette solution, abolished the inhibition by Ang II, while that by OAG was unaffected.

Pretreatment with the PKC inhibitors staurosporine (100 nm) or calphostin C (500 nm) prevented the Ang II‐induced inhibition of Ik,atp.

Ang II inhibition was unaffected by cell dialysis with PKA inhibitor peptide (5 μm), and the PKA inhibitor Rp‐cAMPS (100 μ) did not reduce Ik,atp.

Our results suggest that Ang II modulates Katp channels through activation of PKC but not through inhibition of PKA.
Kinetic analysis of the phosphorylation‐dependent interactions of synapsin I with rat brain synaptic vesicles
Authors: Stefani Giovanni , Onofri Franco , Valtorta Flavia , Vaccaro Paola , Greengard Paul , Benfenati Fabio
Published: September 2004

Synapsin I, a major synaptic vesicle (SV)‐associated phosphoprotein, is involved in the regulation of neurotransmitter release and synapse formation. By binding to both phospholipid and protein components of SV with high affinity and in a phosphorylation‐dependent fashion, synapsin I is believed to cluster SV and to attach them to the actin‐based cytoskeleton of the nerve terminal.

In the present study we have investigated the kinetic aspects of synapsin I–SV interactions and the mechanisms of their modulation by ionic strength and site‐specific phosphorylation, using fluorescence resonance energy transfer between suitable fluorophores linked to synapsin I and to the membrane bilayer.

The binding of synapsin I to the phospholipid and protein components of SV has fast kinetics: mean time constants ranged between 1 and 4 s for association and 9 and 11s for ionic strength‐induced dissociation at 20°C. The interaction with the phospholipid component consists predominantly of a hydrophobic binding with the core of the membrane which may account for the membrane stabilizing effect of synapsin I.

Phosphorylation of synapsin I by either SV‐associated or purified exogenous Ca2+/calmodulin‐dependent protein kinase II (CaMPKII) inhibited the association rate and the binding to SV at steady state by acting on the ionic strength‐sensitive component of the binding. When dephosphorylated synapsin I was previously bound to SV, exposure of SV to Ca2+/calmodulin in the presence of ATP triggered a prompt dissociation of synapsin I with a time constant similar to that of ionic strength‐induced dissociation.

In conclusion, the reversible interactions between synapsin I and SV are highly regulated by site‐specific phosphorylation and have kinetics of the same order of magnitude as the kinetics of SV recycling determined in mammalian neurons under comparable temperature conditions. These findings are consistent with the hypothesis that synapsin I associates with, and dissociates from, SV during the exo–endocytotic cycle. The on‐vesicle phosphorylation of synapsin I by the SV‐associated CaMPKII, and the subsequent dissociation of the protein from the vesicle membrane, though not involved in mediating exocytosis of primed vesicles evoked by a single stimulus, may represent a prompt and efficient mechanism for the modulation of neurotransmitter release and presynaptic plasticity.
ATP is released from rabbit urinary bladder epithelial cells by hydrostatic pressure changes–possible sensory mechanism?
Authors: Ferguson D. R. , Kennedy Ian , Burton T. J.
Published: September 2004

The responses of rabbit urinary bladder to hydrostatic pressure changes and to electrical stimulation have been investigated using both the Ussing chamber and a superfusion apparatus. These experiments enabled us to monitor changes in both ionic transport across the tissue and cellular ATP release from it.

The urinary bladder of the rabbit maintains an electrical potential difference across its wall as a result largely of active sodium transport from the urinary (mucosal) to the serosal surface.

Small hydrostatic pressure differences produced by removal of bathing fluid from one side of the tissue caused reproducible changes in both potential difference and short‐circuit current. The magnitude of these changes increases as the volume of fluid removed increases.

Amiloride on the mucosal (urinary), but not the serosal, surface of the membrane reduces the transepithelial potential difference and short‐circuit current with an IC50 of 300 nm. Amiloride reduces the size of, but does not abolish, transepithelial potential changes caused by alterations in hydrostatic pressure.

Field electrical stimulation of strips of bladder tissue produces a reproducible release of ATP. Such release was demonstrated to occur largely from urothelial cells and is apparently non‐vesicular as it increases in the absence of calcium and is not abolished by tetrodotoxin.

It is proposed that ATP is released from the urothelium as a sensory mediator for the degree of distension of the rabbit urinary bladder and other sensory modalities.
Effect of the immunosupressant FK506 on excitation—contraction coupling and outward K+ currents in rat ventricular myocytes
Authors: DuBell William H. , Wright Pamela A. , Lederer W. J. , Rogers Terry B.
Published: September 2004

We examined the effects of the immunosupressant drug FK506 on excitation–contraction coupling in isolated rat ventricular myocytes. [Ca2+]i transients were recorded using the intracellular Ca2+ indicators fluo‐3 and indo‐1 while action potentials (APs) or membrane currents were recorded using patch‐type microelectrodes in the whole cell mode.

FK506 (25 (μm) rapidly and reversibly increased the magnitude of the [Ca2+]i transient in intact cells without changing resting [Ca2+]i or the kinetics of the [Ca2+]i transient, a finding consistent with previous reports that investigated the actions of FK506 on the sarcoplasmic reticulum Ca2+ release channel.

The 36% increase in the [Ca2+]i transient produced by FK506 was accompanied by a 293% increase in AP duration (by 293%). Importantly, the addition of FK506 had no effect on the [Ca2+]i transient when the depolarizing duration was controlled in voltage clamp experiments. The increased AP duration could be explained by a marked inward shift in the net membrane current that was observed in these experiments.

The net inward current change was not directly responsible for a change in Ca2+ influx, since no change in L‐type Ca2+ current (ICa) was observed. Instead, FK506 inhibited both the transient outward K+ current (Ito) and the delayed rectifier K+ current (IK).

We conclude that FK506 increases the [Ca2+]i transient during normal contractions by an indirect action: it prolongs the action potential. This action does not appear to depend on the established action of FK506 on the ryanodine receptor. Instead, the inhibition of outward K+ currents prolongs the AP which secondarily increases Ca2+ influx and/or decreases Ca2+ efflux.
Effects of sympathetic nerve stimulation on membrane potential, [Ca2+]i and force in the arrested sinus venosus of the toad, Bufo marinus
Authors: Cousins Helen M. , Bramich Narelle J.
Published: September 2004

The effects of sympathetic nerve stimulation on membrane potential and on the intracellular concentration of calcium ions, [Ca2+]i, were recorded concurrently from the sinus venosus of the toad, Bufo marinus, in preparations where beating had been abolished by adding an organic calcium antagonist to the physiological saline. In a separate set of experiments the effects of sympathetic nerve stimulation on force production were examined.

Stimulation of the sympathetic nerves caused a membrane depolarization and a simultaneous increase in [Ca2+]i. Both responses were reduced by dihydroergotamine (20 μm).

The membrane depolarization and increase in [Ca2+]i evoked by sympathetic nerve stimulation were abolished by ryanodine (10 μm), or caffeine (3 mm). The effects of caffeine, but not those of ryanodine, were fully reversible.

Although the Ca2+‐ATPase inhibitor thapsigargin (30 μm) itself had little effect on the responses to sympathetic nerve stimulation, in its presence caffeine (3 him) irreversibly abolished the responses.

In the presence of nifedipine (10 μm), sympathetic nerve stimulation caused contractions of the sinus venosus. These responses were abolished by either ryanodine (10 μm) or caffeine (3 mm).

The results suggest that neuronally released transmitter activates a complex biochemical pathway which triggers the release of Ca2+ from internal stores.