A Gaussian-plume based Monte Carlo method for calculating radiation dose in the near field of buildings.

A Monte Carlo (MC) programme was written using the dose point kernel method to calculate doses in the roof zone of a building from nearby releases of radioactive gases. A Gaussian Plume Model (GPM) was parameterised to account for near-field building effects on plume spread and reflection from the roof. Rooftop recirculation zones and building-generated plume spread effects were accounted in a novel Dual Gaussian Plume (DGP) formulation used with the MC model, which allowed for the selection of angle of approach flow, plume release height in relation to the building and position of the release point in relation to the leading edge of the building. Three-dimensional wind tunnel concentration field data were used for the parameterisation. The MC code used the parameterised concentration field to calculate the contributions to effective dose from inhalation, cloud immersion from positron/beta particles, and gamma-ray dose for a wide range of receptor dose positions in the roof zone, including receptor positions at different heights above the roof. Broad trends in predicted radiation dose with angle of approach flow, release position in relation to the building and release height are shown. Alternative approaches for the derivation of the concentration field are discussed.

First Direct Detection Constraints on Planck-Scale Mass Dark Matter with Multiple-Scatter Signatures Using the DEAP-3600 Detector.

Dark matter with Planck-scale mass (≃10^{19} GeV/c^{2}) arises in well-motivated theories and could be produced by several cosmological mechanisms. A search for multiscatter signals from supermassive dark matter was performed with a blind analysis of data collected over a 813 d live time with DEAP-3600, a 3.3 t single-phase liquid argon-based detector at SNOLAB. No candidate signals were observed, leading to the first direct detection constraints on Planck-scale mass dark matter. Leading limits constrain dark matter masses between 8.3×10^{6} and 1.2×10^{19} GeV/c^{2}, and ^{40}Ar-scattering cross sections between 1.0×10^{-23} and 2.4×10^{-18} cm^{2}. These results are interpreted as constraints on composite dark matter models with two different nucleon-to-nuclear cross section scalings.

Do in vitro assays in rat primary neurons predict drug-induced seizure liability in humans?

Drug-induced seizures contribute to the high attrition rate of pharmaceutical compounds in development. The assessment of drug-induced seizure liability generally occurs in later phases of development using low throughput and intensive in vivo assays. In the present study, we evaluated the potential of an in vitro assay for detecting drug-induced seizure risk compared to evaluation in rats in vivo. We investigated the effects of 8 reference drugs with a known seizurogenic risk using micro-electrode array (MEA) recordings from freshly-dissociated rat primary neurons cultured on 48-well dishes for 28 days, compared to their effects on the EEG in anesthetized rats. In addition, we evaluated functional responses and mRNA expression levels of different receptors in vitro to understand the potential mechanisms of drug-induced seizure risk. Combining the functional MEA in vitro data with concomitant gene expression allowed us to identify several potential molecular targets that might explain the drug-induced seizures occurring in both rats and humans. Our data 1) demonstrate the utility of a group of MEA parameters for detecting potential drug-induced seizure risk in vitro; 2) suggest that an in vitro MEA assay with rat primary neurons may have advantages over an in vivo rat model; and 3) identify potential mechanisms for the discordance between rat assays and human seizure risk for certain seizurogenic drugs.

Conversion of simulated radioactive pollutant gas concentrations for a complex building array into radiation dose.

Methods used to convert wind tunnel and ADMS concentration field data for a complex building array into effective radiation dose were developed based on simulations of a site in central London. Pollutant source terms were from positron emitting gases released from a cyclotron and clinical PET radiotracer facility. Five years of meteorological data were analysed to determine the probability distribution of wind direction and speed. A hemispherical plume cloud model (both static and moving) was developed which enabled an expression of gamma-ray dose, taking into account build-up factors in air, in terms of analytic functions in this geometry. The standard building wake model is presented, but this is extended and developed in a new model to cover the concentration field in the vicinity of a roof top structure recirculation zone, which is then related to the concentration in the main building wake zone. For all models presented the effective dose was determined from inhalation, positron cloud immersion and gamma ray plume contributions. Results of applying these models for determination of radiation dose for a particular site are presented elsewhere.

Dispersion of positron emitting radioactive gases in a complex urban building array: a comparison of dose modelling approaches.

A radiological assessment was carried out on the release of positron-emitting radioactive gases from a roof-level stack at a central London site. Different modelling approaches were performed to investigate the range of radiation doses to representative persons. Contributions from plume inhalation, gamma shine and immersion to effective dose were taken into account. Dry and wet surface deposition on the roof, and exposure from contamination on the skin of roof-workers, added only a mean 4.7% to effective dose and were neglected. A 1:200 scale model, consisting of the stack and surrounding buildings, was tested in a wind tunnel to simulate pollutant dispersion in the near-field region i.e. rooftop. Concentration field measurements in the wind tunnel were converted into effective dose, including for roof-workers installing glass cladding to the stack building. Changes in the building shape, from addition of the cladding layer, were investigated in terms of the near-field flow pattern and significant differences found between the two cases. Pollutant concentrations were also modelled using Air Dispersion Modelling System (ADMS) and the results used to calculate the effective dose using the same meteorological data set and source release terms. Sector averaged wind tunnel dose estimates were greater than the ADMS figure by approximately a factor of two to three. Different stack release heights were investigated in the wind tunnel and ADMS simulations in order to determine the best height for the replacement flue stack for the building. Other techniques were investigated: building wake models, modified Gaussian plume methods and uniform dilution into a hemispherical volume to show the wide variation in predicted dose possible with different approaches. Large differences found between simpler analytic approaches indicated that more robust radiological assessments, based on more complex modelling approaches, were required to achieve satisfactory estimates of radiation dose to representative groups in adjacent buildings and on the building rooftop.

Occupational radiation exposure during endovascular aortic procedures.

OBJECTIVES: To measure the radiation exposure of the operating team during endovascular aortic procedures, and to determine factors that predict high exposures. MATERIALS AND METHODS: Electronic dosimeters placed over and under protective lead garments, were used to prospectively record radiation exposure during endovascular aortic repairs performed in a designated interventional radiology suite. Univariate and multivariate linear regression analyses of predictors of radiation exposure were performed. RESULTS: A total of 26 infra-renal and 10 thoracic endovascular cases were studied. Median (IQR) patient age and body mass index were 76.0 (70.0-81.8) years and 26.2 (23.9-28.9) kg/m(2) respectively. Over-lead exposure to the operator was higher for thoracic than for infra-renal procedures (421.0 [233.8-597.8] µSv vs. 52.5 [27.8-179.8] µSv, p = .0003), reflecting a significant exposure to unprotected parts of the body. Under-lead exposures for operator and assistant were 5.5 (2.0-14.2) µSv and 1.0 (0.0-2.3) µSv respectively, which for an average caseload would comply with total body effective dose limits. Type of case and percentage of digital subtraction angiography (DSA) time in left anterior oblique angulations predicted dose to the operator (p < .0001). CONCLUSIONS: Thoracic procedures, DSA runs and obliquity of the C-arm are strong predictors of radiation exposure during endovascular aortic repairs. Understanding scatter radiation dynamics and instigating measures to minimise radiation exposure should be mandatory.

Germination of shrub species from Chinese subtropical forests: implications for restoration.

Incorporating native shrubs into restoration projects can improve biodiversity conservation and enhance the sustainability of ecosystem functions. Shrubs grow under different forest canopy structures, having varied microclimatic conditions according to forest type and composition. Currently, there is a lack of information on propagation from seed and planting material availability for the utilization of shrubs in forest restoration. In the present study, we evaluated the effects of temperature and light on germination of ten shrub species (Ardisia japonica, Callicarpa cathayana, Callicarpa giraldii var. subcanescens, Deutzia schneideriana, Fraxinus sieboldiana, Hydrangea chinensis, Maesa japonica, Rhododendron simsii, Spiraea japonica var. fortunei and Weigela japonica var. sinica) occurring in subtropical forests in China. No seeds of any species germinated in the coolest thermal regime (5/10 °C), while optimal temperature requirements varied from 10/20 °C to 25/35 °C. Seeds of small-seeded species had higher germination percentages in the light treatments, while larger seeds were not photoblastic. There was no relationship between germination in the light and the seed shape index. Our results may assist in identification of seed traits and suitable shrub species for restoration in specific forest types, thus aiding native forest recovery of structure and composition. Successful recovery leads to enhanced biodiversity, reestablishment of microhabitats and ecological interactions in the forest understorey.

Evaluation of chronic drug-induced electrophysiological and cytotoxic effects using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).

Introduction: Cardiotoxicity is one of the leading causes of compound attrition during drug development. Most in vitro screening platforms aim at detecting acute cardio-electrophysiological changes and drug-induced chronic functional alterations are often not studied in the early stage of drug development. Therefore, we developed an assay using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) that evaluates both drug-induced acute and delayed electrophysiological and cytotoxic effects of reference compounds with clinically known cardiac outcomes. Methods: hiPSC-CMs were seeded in 48-well multielectrode array (MEA) plates and were treated with four doses of reference compounds (covering and exceeding clinical free plasma peak concentrations -fCmax values) and MEA recordings were conducted for 4 days. Functional-electrophysiological (field-potentials) and viability (impedance) parameters were recorded with a MEA machine. Results: To assess this platform, we tested tyrosine-kinase inhibitors with high-cardiac risk profile (sunitinib, vandetanib and nilotinib) and low-cardiac risk (erlotinib), as well as known classic cardiac toxic drugs (doxorubicin and BMS-986094), ion-channel trafficking inhibitors (pentamidine, probucol and arsenic trioxide) and compounds without known clinical cardiotoxicity (amoxicillin, cetirizine, captopril and aspirin). By evaluating the effects of these compounds on MEA parameters, the assay was mostly able to recapitulate different drug-induced cardiotoxicities, represented by a prolongation of the field potential, changes in beating rate and presence of arrhythmic events in acute (<2 h) or delayed phase ≥24 h, and/or reduction of impedance during the delayed phase (≥24 h). Furthermore, a few reference compounds were tested in hiPSC-CMs using fluorescence- and luminescence-based plate reader assays, confirming the presence or absence of cytotoxic effects, linked to changes of the impedance parameters measured in the MEA assay. Of note, some cardiotoxic effects could not be identified at acute time points (<2 h) but were clearly detected after 24 h, reinforcing the importance of chronic drug evaluation. Discussion: In conclusion, the evaluation of chronic drug-induced cardiotoxicity using a hiPSC-CMs in vitro assay can contribute to the early de-risking of compounds and help optimize the drug development process.

Pulse-shape discrimination against low-energy Ar-39 beta decays in liquid argon with 4.5 tonne-years of DEAP-3600 data.

The DEAP-3600 detector searches for the scintillation signal from dark matter particles scattering on a 3.3 tonne liquid argon target. The largest background comes from 39 Ar beta decays and is suppressed using pulse-shape discrimination (PSD). We use two types of PSD estimator: the prompt-fraction, which considers the fraction of the scintillation signal in a narrow and a wide time window around the event peak, and the log-likelihood-ratio, which compares the observed photon arrival times to a signal and a background model. We furthermore use two algorithms to determine the number of photons detected at a given time: (1) simply dividing the charge of each PMT pulse by the mean single-photoelectron charge, and (2) a likelihood analysis that considers the probability to detect a certain number of photons at a given time, based on a model for the scintillation pulse shape and for afterpulsing in the light detectors. The prompt-fraction performs approximately as well as the log-likelihood-ratio PSD algorithm if the photon detection times are not biased by detector effects. We explain this result using a model for the information carried by scintillation photons as a function of the time when they are detected.

The effect of changes in core body temperature on the QT interval in beagle dogs: a previously ignored phenomenon, with a method for correction.

BACKGROUND AND PURPOSE: Body core temperature (Tc) changes affect the QT interval, but correction for this has not been systematically investigated. It may be important to correct QT intervals for drug-induced changes in Tc. EXPERIMENTAL APPROACH: Anaesthetized beagle dogs were artificially cooled (34.2 degrees C) or warmed (42.1 degrees C). The relationship between corrected QT intervals (QTcV; QT interval corrected according to the Van de Water formula) and Tc was analysed. This relationship was also examined in conscious dogs where Tc was increased by exercise. KEY RESULTS: When QTcV intervals were plotted against changes in Tc, linear correlations were observed in all individual dogs. The slopes did not significantly differ between cooling (-14.85+/-2.08) or heating (-13.12+/-3.46) protocols. We propose a correction formula to compensate for the influence of Tc changes and standardize the QTcV duration to 37.5 degrees C: QTcVcT (QTcV corrected for changes in core temperature)=QTcV-14 (37.5 - Tc). Furthermore, cooled dogs were re-warmed (from 34.2 to 40.0 degrees C) and marked QTcV shortening (-29%) was induced. After Tc correction, using the above formula, this decrease was abolished. In these re-warmed dogs, we observed significant increases in T-wave amplitude and in serum [K(+)] levels. No arrhythmias or increase in pro-arrhythmic biomarkers were observed. In exercising dogs, the above formula completely compensated QTcV for the temperature increase. CONCLUSIONS AND IMPLICATIONS: This study shows the importance of correcting QTcV intervals for changes in Tc, to avoid misleading interpretations of apparent QTcV interval changes. We recommend that all ICH S7A, conscious animal safety studies should routinely measure core body temperature and correct QTcV appropriately, if body temperature and heart rate changes are observed.

Predicting drug-induced changes in QT interval and arrhythmias: QT-shortening drugs point to gaps in the ICHS7B Guidelines.

BACKGROUND AND PURPOSE: The regulatory guidelines (ICHS7B) recommending inhibition of the delayed rectifier K(+) current (I(Kr)), carried by human ether-a-go-go-related gene (hERG) channels in cardiac cells (the hERG test), as a 'first line' test for identifying compounds inducing QT prolongation, have limitations, some of which are outlined here. EXPERIMENTAL APPROACH: hERG current was measured in HEK293 cells, stably transfected with hERG channels; action potential duration (APD) and arrhythmogenic effects were measured in isolated Purkinje fibres and perfused hearts from rabbits. KEY RESULTS: 576 compounds were screened in the hERG test: 58% were identified as hERG inhibitors, 39% had no effect and 3% were classified as stimulators. Of the hERG inhibitors, 92 were tested in the APD assay: 55.4% of these prolonged APD, 28.3% had no effect and 16.3% shortened APD. Of the 70 compounds without effect on hERG channels, 54.3% did not affect APD, 25.7% prolonged, while 20% significantly shortened APD. Dofetilide (hERG inhibitor; IC(50), 29 nM) prolonged QT and elicited early after-depolarizations and/or torsade de pointes (TdP) in isolated hearts. Mallotoxin and NS1643 (hERG current stimulators at 3 microM), levcromakalim and nicorandil (no effect on hERG current), all significantly shortened APD and QT, and elicited ventricular fibrillation (VF) in isolated hearts. CONCLUSION AND IMPLICATIONS: The hERG assay alone did not adequately identify drugs inducing QT prolongation. It is also important to detect drug-induced QT shortening, as this effect is associated with a potential risk for ventricular tachycardia and VF, the latter being invariably fatal, whereas TdP has an approximately 15-25% incidence of death.

Application of optical action potentials in human induced pluripotent stem cells-derived cardiomyocytes to predict drug-induced cardiac arrhythmias.

INTRODUCTION: Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are emerging as new and human-relevant source in vitro model for cardiac safety assessment that allow us to investigate a set of 20 reference drugs for predicting cardiac arrhythmogenic liability using optical action potential (oAP) assay. METHODS: Here, we describe our examination of the oAP measurement using a voltage sensitive dye (Di-4-ANEPPS) to predict adverse compound effects using hiPS-CMs and 20 cardioactive reference compounds. Fluorescence signals were digitized at 10kHz and the records subsequently analyzed off-line. Cells were exposed to 30min incubation to vehicle or compound (n=5/dose, 4 doses/compound) that were blinded to the investigating laboratory. Action potential parameters were measured, including rise time (Trise) of the optical action potential duration (oAPD). RESULTS: Significant effects on oAPD were sensitively detected with 11 QT-prolonging drugs, while oAPD shortening was observed with ICa-antagonists, IKr-activator or ATP-sensitive K+ channel (KATP)-opener. Additionally, the assay detected varied effects induced by 6 different sodium channel blockers. The detection threshold for these drug effects was at or below the published values of free effective therapeutic plasma levels or effective concentrations by other studies. DISCUSSION: The results of this blinded study indicate that OAP is a sensitive method to accurately detect drug-induced effects (i.e., duration/QT-prolongation, shortening, beat rate, and incidence of early after depolarizations) in hiPS-CMs; therefore, this technique will potentially be useful in predicting drug-induced arrhythmogenic liabilities in early de-risking within the drug discovery phase.

Effects of alanine on insulin-secreting cells: patch-clamp and single cell intracellular Ca2+ measurements.

The effects of alanine, glucose and tolbutamide on insulin-secreting cells (RINm5F) have been investigated using patch-clamp and single cell intracellular Ca2+ measurements. When directly challenged with the amino acid L-alanine (2-10 mM) the cells underwent a sharp depolarization, which led to the generation of Ca2+ spike potentials and an increase in [Ca2+]i. The L-alanine-induced depolarization was associated with a net inward membrane current but no measurable change in the resistance of the cell. The latter effect was found to be in contrast to the actions of glucose (5-10 mM) and tolbutamide (100 microM), both of which depolarized cells and raised [Ca2+]i by an increase in the input resistance of the cell membrane, due to the closure of ATP-sensitive potassium channels. In the complete absence of external Na+ (by replacement with 140 mM NMDG+), L-alanine had no effects on either the membrane potential or [Ca2+]i. Similarly, replacing Na+ with NMDG+ in the continued presence of the amino acid resulted in a repolarization of the membrane and an attenuation of the L-alanine-induced rise in [Ca2+]i. The Na+ channel blocker TTX (1-2 microM) had no effects on the alanine-evoked electrical activity. Exchange of the L-form of the amino acid with the D-stereoisomer had similar actions to those of removing external Na+, since D-alanine had no effects on the membrane potential or [Ca2+]i. The actions of L-alanine were also found to be mimicked by the N-methylated amino acid analogue methylamino isobutyric acid (MeAIB) (2-10 mM), suggesting that the A-type electrogenic amino acid cotransport system operates in the RINm5F insulin-secreting cell line.

Both beta-adrenergic receptor stimulation and cardiac tissue type have important roles in elucidating the functional effects of I(Ks) channel blockers in vitro.

INTRODUCTION: Conflicting results associated with the use of I(Ks) blockers on the action potential duration (APD) have raised a question as to whether the variable results arise from the use of different cardiac tissues, beta-adrenergic stimulation, or by the "selectivity" of the chosen I(Ks) blockers. METHODS: We used the highly selective I(Ks) blocker (-)-[3R, 4S] chromanol 293B [(-) chromanol] to mimic drug-induced long QT1 in isolated rabbit Purkinje fibers, papillary muscles, and ventricular trabeculae using the conventional microelectrode technique. RESULTS: I(Ks) block with (-) chromanol at 1 x 10(-5) M did not significantly change the APD at different stimulation rates in all three cardiac tissues. Isoproterenol (Iso:1 x 10(-7) M) shortened APD(90), and (-) chromanol (1 x 10(-5) M) largely prevented this shortening in isolated papillary muscles at 1 Hz [-3% with Iso combined (-) chromanol group versus -16% with iso group; p<0.05] and also at 2 Hz (+7% versus -25% with Iso group; p<0.05), but did not significantly prevent this shortening in isolated Purkinje fibers. In isolated trabeculae, (-) chromanol combined with Iso significantly prolonged the APD(90) by 15% at 1 Hz (versus -10% with Iso group; p<0.05) and by 5% at 2 Hz (versus -11% with Iso group; p<0.05). DISCUSSION: Our study shows that only during beta-adrenoceptor stimulation, pharmacological inhibition of the I(Ks) current plays an important role in the APD recorded from isolated ventricular trabeculae and papillary muscles, but not from Purkinje fibers. These results indicate that the APD prolonging effects of I(Ks)channel blockers during beta-adrenergic receptor stimulation can only be detected from isolated rabbit papillary muscles and ventricular trabeculae, but not Purkinje fibers.

A new method to calculate the beat-to-beat instability of QT duration in drug-induced long QT in anesthetized dogs.

INTRODUCTION: Instability of QT duration is a marker to predict Torsade de Pointes (TdP) associated with both congenital and drug-induced long QT syndrome. We describe a new method for the quantification of instability of repolarization. METHODS: Female, adult beagle dogs anesthetized with a potent morphinomimetic were treated with either solvent (n=7) or dofetilide (n=7). Poincaré plots with QT(n) versus QT(n+1) were constructed to visualize the beat-to-beat variation in QT intervals from the lead II ECG. Short-term instability (STI), long-term instability (LTI) and total instability (TI) were quantified by calculating the distances of 30 consecutive data-points from the x and y-coordinate to the "centre of gravity" of the data cluster. Dofetilide at 0.0025 to 0.04 mg/kg i.v. (plasma concentrations of 4+/-0.6 to 41+/-2.7 ng/ml), dose-dependently prolonged QT and QTcV (at 0.04 mg/kg i.v.: QT: 280+/-ms versus 236+/-5 ms with solvent; p<0.05 and QTcV: 290+/-9 ms versus 252+/-4 ms with solvent; p<0.05). Concomitantly, the compound induced an increase in the instability parameters in a similar dose-dependent manner (at 0.04 mg/kg i.v.: TI: 6.8+/-0.9 ms versus 1.7+/-0.3 ms; p<0.05, LTI: 3.6+/-0.5 ms versus 1.0+/-0.2 ms; p<0.05 and STI: 4.2+/-0.6 ms versus 1.0+/-0.2 ms; p<0.05). The increases induced by dofetilide were associated with a high incidence of early afterdepolarizations (EADs) in the endocardial monophasic action potential (in 6 out of the 7 compound-treated animals versus 0 out of the 7 solvent animals; p<0.05). CONCLUSION: Quantification of beat-to-beat QT instability by our method clearly detects changes in short-term, long-term and total instability induced by dofetilide, already at pre-arrhythmic doses. Dofetilide administration to anesthetized dogs prolongs ventricular repolarization, concomitantly increases beat-to-beat QT instability and induces early after depolarizations (EADs). As such, the use of these parameters in this in vivo model shows clear potential for risk identification in cardiovascular safety assessment.

Inter-study variability of preclinical in vivo safety studies and translational exposure-QTc relationships--a PKPD meta-analysis.

BACKGROUND AND PURPOSE: Preclinical cardiovascular safety studies (CVS) have been compared between facilities with respect to their sensitivity to detect drug-induced QTc prolongation (ΔQTc). Little is known about the consistency of quantitative ΔQTc predictions that are relevant for translation to humans. EXPERIMENTAL APPROACH: We derived typical ΔQTc predictions at therapeutic exposure (ΔQTcTHER ) with 95% confidence intervals (95%CI) for 3 Kv 11.1 (hERG) channel blockers (moxifloxacin, dofetilide and sotalol) from a total of 14 CVS with variable designs in the conscious dog. Population pharmacokinetic-pharmacodynamic (PKPD) analysis of each study was followed by a meta-analysis (pooling 2-6 studies including 10-32 dogs per compound) to derive meta-predictions of typical ΔQTcTHER . Meta-predictions were used as a reference to evaluate the consistency of study predictions and to relate results to those found in the clinical literature. KEY RESULTS: The 95%CIs of study-predicted ΔQTcTHER comprised in 13 out of 14 cases the meta-prediction. Overall inter-study variability (mean deviation from meta-prediction at upper level of therapeutic exposure) was 30% (range: 1-69%). Meta-ΔQTcTHER predictions for moxifloxacin, dofetilide and sotalol overlapped with reported clinical QTc prolongation when expressed as %-prolongation from baseline. CONCLUSIONS AND IMPLICATIONS: Consistent exposure-ΔQTc predictions were obtained from single preclinical dog studies of highly variable designs by systematic PKPD analysis, which is suitable for translational purposes. The good preclinical-clinical pharmacodynamic correlations obtained suggest that such an analysis should be more routinely applied to increase the informative and predictive value of results obtained from animal experiments.

Acetylcholine and cholecystokinin induce different patterns of oscillating calcium signals in pancreatic acinar cells.

Receptor-activated cytoplasmic calcium (Ca2+) oscillations have been investigated in single pancreatic acinar cells by microfluorimetry (Fura-2 as indicator). At submaximal concentrations of the agonists acetylcholine (ACh) and cholecystokinin octapeptide (CCK-8), both give rise to oscillatory changes in the cytosolic free calcium concentration ([Ca2+]i). The patterns of oscillations are markedly and consistently different for each of these two agonists. The ACh induced oscillations are superimposed upon a median elevation in background [Ca2+]i. The CCK-8 induced oscillations are of longer duration with [Ca2+]i returning to prestimulus levels between the discrete spikes. The ACh induced oscillations are rapidly abolished upon removal of extracellular Ca2+ while the CCK-8 induced oscillations persist for many minutes in the absence of external Ca2+. The CCK-8, but not the ACh, induced oscillations are increased in duration by the protein kinase C (PKC) inhibitor staurosporine and abolished by the PKC activating phorbol ester PMA. It is clear that CCK-8 and ACh do not activate receptor transduction mechanisms in an identical manner to generate oscillating [Ca2+]i signals.

Two different spatiotemporal patterns for Ca2+ oscillations in pancreatic acinar cells: evidence of a role for protein kinase C in Ins(1,4,5)P3-mediated Ca2+ signalling.

The oscillations in cytosolic Ca2+ evoked in pancreatic exocrine acinar cells by submaximal concentrations of the two phosphoinositidase-coupled agonists acetylcholine (ACh) and cholecystokinin octapeptide (CCK-8) have very different temporal patterns. In the present study we use digital video imaging of Fura-2 fluorescence to map the spatial distribution of Ca2+ during the oscillating responses to these two agonists. The spatial patterns induced are very different for each of these agonists. ACh oscillations are sinusoidal and initiated at the secretory pole of these morphologically and functionally polarized cells. As they spread across the cell, pronounced gradients in Ca2+ develop that persist throughout the oscillating response. CCK-8 induces a series of discrete Ca2+ transients of longer duration and lower frequency. These elevations in Ca2+ arise slowly, throughout the cells and without any detectable gradients in Ca2+. We consider that the different spatiotemporal patterns can be explained on the basis of a physiologically relevant interaction between Ins(1,4,5)P3 and protein kinase C in second messenger-mediated Ca2+ signalling.

Ca2+ oscillations in pancreatic acinar cells: spatiotemporal relationships and functional implications.

The pancreatic acinar cells are of particular interest for the study of cytosolic Ca2+ signals, since they are morphologically polarized and generate agonist-specific Ca2+ oscillation patterns. Recent data obtained by combining digital video imaging of Fura-2 fluorescence with patch-clamp whole-cell current recording have provided new information on the spatiotemporal relationships of the cytosolic Ca2+ signals and the Ca(2+)-activated ionic currents. Low agonist concentrations evoke repetitive short-lasting local Ca2+ spikes in the secretory pole region that activate shortlasting current spikes. In the case of acetylcholine stimulation the spikes are confined to this region. When cholecystokinin is used the shortlasting local spikes precede longer Ca2+ transients that spread to the whole of the cell. Infusion of non-metabolizable inositol trisphosphate analogues can mimic these responses. The shortlasting local Ca2+ spikes are particularly sensitive to blockade by the inositol trisphosphate receptor antagonist heparin. These results show that the secretory pole region has a particularly high sensitivity to inositol trisphosphate probably due to clustering of high affinity receptors.

Receptor-activated cytoplasmic Ca2+ oscillations in pancreatic acinar cells: generation and spreading of Ca2+ signals.

Receptor-activated cytoplasmic Ca2+ oscillations have been investigated using both single cell microfluorometry and voltage-clamp recording of Ca(2+)-dependent Cl- current in single internally perfused acinar cells. In these cells there is direct experimental evidence showing that the ACh-evoked [Ca2+]i fluctuations are due to an inositol trisphosphate-induced small steady Ca2+ release which in turn evokes repetitive Ca2+ spikes via a caffeine-sensitive Ca(2+)-induced Ca2+ release process. There is indirect evidence suggesting that receptor-activation in addition to generating the Ca2+ releasing messenger, inositol trisphosphate, also produces another regulator involved in the control of Ca2+ signal spreading. Intracellular inositol trisphosphate or Ca2+ infusion produce short duration repetitive spikes confined to the cytoplasmic area close to the plasma membrane, but these signals can be made to progress throughout the cell by addition of caffeine or by receptor activation.