bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid ATP‐Dependent Priming of Secretory Granules Precedes Ca2+ ‐Induced Exocytosis in Mouse Pancreatic B‐Cells
Authors: Eliasson Lena , Renström Erik , Ding Wei‐Guang , Proks Peter , Rorsman Patrik 
Published: September 2004

The glucose and ATP dependence of exocytosis were investigated in single mouse pancreatic B‐cells by monitoring changes in cell capacitance evoked by voltage‐clamp depolarizations, infusion of high‐[Ca2+]i buffers or photorelease of caged Ca2+ or ATP.
In intact B‐cells, using the perforated patch whole‐cell technique, glucose (5 mM) increased exocytotic responses evoked by membrane depolarization 5–fold over that observed in the absence of the sugar. Increasing the glucose concentration to 20 mM produced a further doubling of exocytosis. The stimulatory action of glucose was attributable to glucose metabolism and abolished by mannoheptulose, an inhibitor of glucose phosphorylation.
Exocytosis triggered by infusion of high [Ca2+]i and ATP was reduced by 80 % when ATP was replaced by its non‐hydrolysable analogue adenosine 5′‐[β,γ‐methylene]triphosphate (AMP‐PCP) in standard whole‐cell experiments. Exocytosis elicited by GTPγS was similarly affected by replacement of ATP with the stable analogue.
Photoreleasing ATP in the presence of 170 nm [Ca2+]i following the complete wash‐out of endogenous ATP, produced a prompt (latency, < 400 ms) and biphasic stimulation of exocytosis.
Elevation of [Ca2+]i to exocytotic levels by photorelease from Ca2+‐nitrophenyl EGTA preloaded into the cell evoked a biphasic stimulation in the presence of Mg‐ATP. The response consisted of an initial rapid (completed in < 200 ms) phase followed by a slower (lasting ≥ 10 s) sustained component. Replacement of ATP with AMP‐PCP abolished the late component but did not affect the initial phase. The latency between elevation of [Ca2+]i and exocytosis was determined as < 45 ms. Inclusion of N‐ethylmaleimide (NEM; 0.5 mM for 3 min) in the intracellular solution exerted effects similar to those obtained by substituting AMP‐PCP for ATP.
We conclude that the B‐cell contains a small pool (40 granules) of primed granules which are immediately available for release and which are capable of undergoing exocytosis in an ATP‐independent fashion. We propose that this pool of granules is preferentially released during first phase glucose‐stimulated insulin secretion. The short latency between the application of ATP and the onset of exocytosis finally suggests that priming takes place with sufficient speed to participate in the rapid adjustment of the secretory capacity of the B‐cell.
Apparent Ca2+ dissociation constant of Ca2+ chelators incorporated non‐disruptively into intact human red cells
Authors: Tiffert Teresa , Lew Virgilio L. 
Published: September 2004

A recently developed method of measuring cytoplasmic Ca2+ buffering in intact red cells was applied to re‐evaluate the intracellular Ca2+ binding properties of the Ca2+ chelators benz2 and BAPTA. Incorporation of the free chelators was accomplished by incubating the cells with the acetoxymethyl ester forms (benz2 AM or BAPTA AM). The divalent cation ionophore A23187 was used to induce equilibrium distribution of Ca2+ between cells and medium. 45Ca2+ was added stepwise to cell suspensions in the presence and absence of external BAPTA. To induce full Ca2+ equilibration, the plasma membrane Ca2+ pump was inhibited either by depleting the cells of ATP or by adding vanadate to the cell suspension.
The properties of the incorporated chelators were assessed from the difference in cytoplasmic Ca2+ buffering between chelator‐free and chelator‐loaded cells, over a wide range of intracellular ionized calcium concentrations ([Ca2+]i), from nanomolar to millimolar.
Under the experimental conditions applied, incorporation of benz2 and BAPTA into the red cells increased their Ca2+ buffering capacity by 300–600 μmol (340 g Hb)−1. The intracellular apparent Ca2+ dissociation constants (KDi were about 500 nm for benz2 and 800 nm for BAPTA, values much higher than those reported for standard salt solutions (KD) of about 40 and 130 nm, respectively. These results suggest that, contrary to earlier observations, the intracellular red cell environment may cause large shifts in the apparent Ca2+ binding behaviour of incorporated chelators.
The possibility that the observed KD shifts are due to reversible binding of the chelators to haemoglobin is considered, and the implications of the present results for early estimates of physiological [Ca2+]i levels is discussed.
Spatial and temporal frequency selectivity of cells in area 21a of the cat.
Authors: Morley J. W. , Vickery R. M. 
Published: September 2004

The spatial and temporal response properties of single cells in area 21a of the anaesthetized cat were assessed using drifting sinusoidal gratings presented at the optimum orientation for each cell.
Responses to sinusoidal gratings were dominated by an elevation of the mean discharge, with a relatively small modulated component at the temporal frequency of grating drift. The relative modulation ratio for the majority of cells was less than 1, similar to complex cells in the striate cortex.
Of those cells responsive to stimulation with sinusoidal gratings, 94% displayed spatial bandpass characteristics. Values derived from spatial frequency tuning curves were: mean optimum spatial frequency, 0.26 cycles deg−1; mean spatial resolution, 0.86 cycles deg−1; mean spatial bandwidth, 1.8 octaves; and mean normalized bandwidth, 1.3. Two cells (6%) displayed spatial low‐pass characteristics.
Approximately half our sample of cells (44%) displayed temporal low‐pass tuning, while 35% displayed temporal bandpass characteristics. The mean optimum temporal frequency of bandpass cells was 3.3 Hz and the mean temporal bandwidth 1.9 octaves. The remaining cells were classified as temporal broadband (17%) and temporal high‐pass (4%).
We conclude that the dominant functional input to cells with relatively high spatial frequency selectivity and/or temporal low‐pass response properties most probably arises from area 17. The responses of the remaining cells may be explained by input from area 17 or 18.
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.