Large dense-core vesicle exocytosis in pancreatic beta-cells monitored by capacitance measurements

This article discusses the currently used methodologies for monitoring exocytosis as changes in cell capacitance. Details are given on composition of solutions, experimental protocols, and how the observed responses can be interpreted physiologically. The concepts are illustrated by examples from our own work on insulin-releasing pancreatic beta-cells. Finally, we consider the feasibility of applying capacitance measurements to endocrine cells in intact pancreatic islets, where the cells are electrically coupled to each other.     

Simplified method for setting the phase angle used in capacitance measurements in studies of exocytosis.

Phase detectors (lock-in amplifiers) have been used in conjunction with patch-clamp apparatus to follow the time course of exo- and endocytotic processes. A critical step is accurate setting of the phase to separate signals due to conductance changes from those due to capacitance changes. It has been the practice to obtain this setting empirically. Analysis of the admittance, and its derivatives, of a model equivalent circuit used commonly in this field shows that the desired phase setting is just twice the phase shift of the current relative to the stimulus voltage, which is easily, accurately and quickly determined. This fact permits rapid and accurate setting of the desire phase angle for those cells to which the equivalent circuit is suitable.     

Fast kinetics of exocytosis revealed by simultaneous measurements of presynaptic capacitance and postsynaptic currents at a central synapse

The rate of release from nerve terminals depends on both the number of release sites and the rate of release at each site. The latter remains largely unknown at central synapses. We addressed this issue by simultaneously measuring the nerve terminal membrane capacitance and the postsynaptic current at single calyceal synapses in rat brainstem. We found that a 10 ms presynaptic step depolarization depleted a releasable pool containing 3300-5200 vesicles. Released vesicles were endocytosed with a time constant of a few seconds to tens of seconds. Release of only one third of this pool saturated both postsynaptic AMPA and NMDA receptors. A release site can release more than three vesicles in 10 ms (>300 vesicles per second). We conclude that both a large number of release sites and a fast release rate at each site enable synapses to release at a high rate.     

Quantifying exocytosis by combination of membrane capacitance measurements and total internal reflection fluorescence microscopy in chromaffin cells

Total internal reflection fluorescence microscopy (TIRF-Microscopy) allows the observation of individual secretory vesicles in real-time during exocytosis. In contrast to electrophysiological methods, such as membrane capacitance recording or carbon fiber amperometry, TIRF-Microscopy also enables the observation of vesicles as they reside close to the plasma membrane prior to fusion. However, TIRF-Microscopy is limited to the visualization of vesicles that are located near the membrane attached to the glass coverslip on which the cell grows. This has raised concerns as to whether exocytosis measured with TIRF-Microscopy is comparable to global secretion of the cell measured with membrane capacitance recording. Here we address this concern by combining TIRF-Microscopy and membrane capacitance recording to quantify exocytosis from adrenal chromaffin cells. We found that secretion measured with TIRF-Microscopy is representative of the overall secretion of the cells, thereby validating for the first time the TIRF method as a measure of secretion. Furthermore, the combination of these two techniques provides a new tool for investigating the molecular mechanism of synaptic transmission with combined electrophysiological and imaging techniques.     

Large dense-core vesicle exocytosis in pancreatic β-cells monitored by capacitance measurements

This article discusses the currently used methodologies for monitoring exocytosis as changes in cell capacitance. Details are given on composition of solutions, experimental protocols, and how the observed responses can be interpreted physiologically. The concepts are illustrated by examples from our own work on insulin-releasing pancreatic β-cells. Finally, we consider the feasibility of applying capacitance measurements to endocrine cells in intact pancreatic islets, where the cells are electrically coupled to each other.     

Molecular identification and reconstitution of depolarization-induced exocytosis monitored by membrane capacitance

Regulated exocytosis of neurotransmitters at synapses is fast and tightly regulated. It is unclear which proteins constitute the "minimal molecular machinery" for this process. Here, we show that a novel technique of capacitance monitoring combined with heterologous protein expression can be used to reconstitute exocytosis that is fast (<0.5 s) and triggered directly by membrane depolarization in Xenopus oocytes. Testing synaptic proteins, voltage-gated Ca2+ channels, and using botulinum and tetanus neurotoxins established that the expression of a Ca2+ channel together with syntaxin 1A, SNAP-25, and synaptotagmin was sufficient and necessary for the reconstitution of depolarization-induced exocytosis. Similar to synaptic exocytosis, the reconstituted release was sensitive to neurotoxins, modulated by divalent cations (Ca2+, Ba2+, and Sr2+) or channel (Lc-, N-type), and depended nonlinearly on divalent cation concentration. Because of its improved speed, native trigger, and great experimental versatility, this reconstitution assay provides a novel, promising tool to study synaptic exocytosis.     

Residential magnetic field measurements in France: comparison of indoor and outdoor measurements.

Residential magnetic field (MF) measurements were performed for the first time in a representative sample of French dwellings. Exposure levels were assessed by two methods: indoor and outdoor measurements. Linear and logistic regression models were used to determine factors associated with the time-weighted average (TWA) home MF. TWA magnetic field magnitudes were approximately log-normally distributed with geometric means under 0.010 microT for both indoor and outdoor measurements. Only 5% of the dwellings presented indoor MF levels greater than 0.120 microT (1.2 mG). Both indoor and outdoor MF variations were explained by three factors: wiring configuration, the dwelling's location (i.e., urban or rural), and housing characteristics (individual houses or apartment building). The reliability of outdoor spot measurements with 30-min bedroom recordings was assessed by an intraclass correlation coefficient. The measurements were accurate in rural areas and small towns. In urban centers, local MF variations spoil the outdoor measurement's reliability. If indoor measurements are taken as the reference method, the use of outdoor instead of indoor measurement leads to an important decrease in statistical power. Further assessment of MF near high power transmission lines is necessary to evaluate the usefulness of outdoor spot recordings near such lines. The urban MF environment also has to be explored to identify extraneous sources.     

Phase tracking: an improved phase detection technique for cell membrane capacitance measurements.

We describe here a technique called phase tracking that greatly improves the accuracy of measurements of the membrane capacitance of single cells. We have modified the original phase detection technique to include a method for creating calibrated changes in the resistance in series with the cell. This provides a method to automate the adjustment of the phase detector to the appropriate phase angle for measuring membrane capacitance. The phase determination depends only on the cell's electrical parameters and does not require matching of the cell impedance with that of the slow capacitance cancellation circuitry of the patch-clamp amplifier. We show here that phase tracking can accurately locate the phase of the capacitance signal and can keep the detector aligned with this signal during measurements of exocytosis in mast cells, irrespective of the large drifts which occur in cell membrane resistance, membrane capacitance, or series resistance. The phase tracking technique is a valuable tool for quantifying exocytosis and endocytosis in single cells.     

Increased cytosolic calcium stimulates exocytosis in bovine lactotrophs. Direct evidence from changes in membrane capacitance

The patch-clamp technique has been used to measure changes in membrane capacitance (Cm) of bovine lactotrophs in order to monitor fluctuations in cell surface area associated with exo- and endocytosis. Cells were prepared by an enrichment procedure and cultured for up to 14 d before use. Under whole-cell recording, cell cytoplasm was dialyzed with various Ca2(+)-containing solutions. The resting Cm of 6.05 +/- 1.68 pF was found to correlate well with squared cell radius, suggesting a specific Cm of 0.8 microF/cm2. Discrete Cm steps of 2-10 fF were recorded, which most likely reflect single fusion and retrieval events of prolactin-containing granules (0.2-0.6 microns in diameter). High Ca2+ resulted in a Cm increase of 20-50% from the resting value, demonstrating a role for [Ca2+]i in stimulus-secretion coupling. Spontaneous Cm changes have also been recorded, which presumably reflect prolactin secretion supported by a tonic influx of Ca2+ through the membrane. This is supported by the following findings: addition of Co2+ diminished or reversed the spontaneous Cm changes and decreased resting [Ca2+]i; and membrane depolarization increased Cm, indicating the role of voltage-activated channels in stimulus-secretion coupling. As bovine lactotrophs have been found to be largely devoid of spontaneous electrical activity, a mechanism involving modulation of a tonic Ca2+ influx is proposed; this is shown to provide adequate control of basal and triggered secretion monitored by Cm.     

A new system for bilayer lipid membrane capacitance measurements: method, apparatus and applications.

A new method and a new apparatus for capacitance measurements on bilayer lipid membranes are described. The membrane is charged and discharged with a constant current during the measurement. The charge-discharge cycle duration, which is proportional to the membrane capacitance, is measured. The measured time period is converted into a binary number by digital systems and then this number is either further converted into a constant capacity-proportional voltage or read out by the computer. The apparatus makes it possible to measure the capacitances of voltage-polarized membranes. Application of the apparatus to capacitance measurements of bilayer lipid membranes during their potential on the capacitance is presented. The capacitances of membranes stimulated by rectangular voltage pulses and of those stimulated by a linearly varying potential were reported.     

System for dynamic measurements of membrane capacitance in intact epithelial monolayers.

Dynamic measurements of exocytosis have been difficult to perform in intact epithelial monolayers. We have designed a system that estimates with +/-1% accuracy (99% confidence) the total membrane capacitance of monolayers represented by a lumped model. This impedance measurement and analysis system operates through a conventional transepithelial electrophysiology clamp, performing all signal measurements as frequently as every 5 s. Total membrane capacitance (the series combination of apical and basolateral membranes) is the inverse of one of three unique coefficients that describe the monolayer impedance. These coefficients are estimated using a weighted, nonlinear, least-squares algorithm. Using the estimated coefficients, solution ranges for individual membrane parameters are calculated, frequently providing results within +/-20% of true values without additional electrophysiological measurements. We determined the measurement system specifications and statistical significance of estimated parameters using 1) analytical testing with circuit simulation software and equation-generated data; 2) a system noise analysis combined with Monte Carlo simulations; and 3) analog model circuits for calibration of the electronic system and to check equation-generated results. Finally, the time course of capacitance changes associated with purinergically stimulated mucin exocytosis are quantified in monolayers of the colonic goblet cell-like cell line HT29-CI.16E.     

Theory of negative capacitance in membrane impedance measurements

Summary Three possible explanations of the negative capacitance seen in theChara corallina membrane impedance are critically examined. These explanations are based on: (1) voltage-dependent channel kinetics; (2) electro-osmosis; and (3) extracellular negative capacitance. It is shown that the first two can produce negative capacitance only with parameters which differ by several orders of magnitude from measured values. The last mechanism can produce a very large magnitude negative capacitance, in the appropriate frequency range. Possible experimental tests are discussed.     

Using branch and basal trunk sap flow measurements to estimate whole-plant water capacitance: a caution

Thermometric sap flow sensors are widely used to measure water flow in roots, stems and branches of plants. Comparison of the timing of flow in branches and stems has been used to estimate water capacitance of large trees. We review studies of sap flow in branches and present our own data to show that there is wide variation in the patterns and timing of sap flow of branches in different parts of the crown, owing to the course of daily solar illuminance. In contiguous forest, east-facing and upper branches are illuminated earlier than west-facing and lower branches and most capacitance studies do not include adequate information about branch sampling regimes relative to the overall pattern of crown illuminance, raising questions about the accuracy of capacitance estimates. Measuring only upper branches and normalising these results to represent the entire crown is dangerous because flows at the stem base likely peak in response to maximum crown illuminance (and transpiration) and this will differ compared to the timing of peak flows in upper branches. We suggest that the magnitude of flow lags between branches and stems needs further study, with careful attention to branch position and method application before a robust understanding of capacitance, particularly in woody tissues of large trees, can be formed. We did not detect flow lags in the world’s tallest and largest tree species Sequoia sempervirens and Sequoiadendron giganteum, despite measurement along large pathlengths (∼57 and 85 m), which raises questions as to why large flow lags are often recorded for much smaller species. One conspicuous possibility is the different methods used among studies. Constant-heating methods such as the thermal dissipation probe (and also heat balance methods) include heat capacitance behaviour due to warming of wood tissues, which delays the response of the sensors to changing sap flow conditions. We argue that methods with intrinsic heat-capacitance present dangers when trying to measure water-capacitance in trees. In this respect heat pulse methods hold an advantage.     

Simultaneous determination of L- and D-methotrexate using a sequential injection analysis/amperometric biosensors system

A sequential injection analysis (SIA) is proposed for the simultaneous determination of L- and D-methotrexate (Mtx) using amperometric biosensors as detectors. A SIA system is proposed due to the highest precision and accuracy and lower consumption of sample and buffer. The amperometric biosensors used as detectors in SIA system were based on L-amino acid oxidase (L-AAOD) or/and L-glutamate oxidase (L-Glox) and horseradish peroxidase (HRP) for the assay of L-Mtx and D-amino acid oxidase (D-AAOD) and HRP for the assay of D-Mtx were selected. The linear concentration ranges are of pmol/l to nmol/l magnitude order, with very low limits of detection. The SIA/biosensors system can be used reliably on-line in synthesis process control, for the simultaneous assay of L- and D-Mtx with a frequency of 34 samples per hour.     

Quantitative measurements of released amines from individual exocytosis events

Chemical analysis of single cells is an area of great interest in the biological sciences. Single-cell systems are being utilized as a model to understand in vivo processes better. One method that is moving to the forefront in cellular analysis is electrochemistry. Owing to their rapid response time and small dimensions, voltammetric microelectrode techniques, such as amperometry and fast-scan voltammetry, have made it possible to monitor minute amounts of biological compounds and transiently occurring chemical events in cellular systems. The application of these methods to the quantitation of individual vesicular release events from single cells is overviewed here. The application of electrochemical monitoring to several types of cultured cells, including bovine adrenal chromaffin cells, rat pheochromocytoma (PC12) cells, beige mouse mast cells, superior cervical ganglion neurons, and human pancreatic β-cells, as well as to the invertebrate systems, the leechHirudo medicinalis, and pond snailPlanorbis corneus has provided a wealth of new information concerning exocytosis. Results obtained from the studies highlight the potential of electrochemical techniques in cellular analysis to contribute to our understanding of molecular and pharmacological effects on exocytosis. This article overviews work done on all the above cell types with an emphasis on PC12 cells.     

Capacitance measurements. An analysis of the phase detector technique used to study exocytosis and endocytosis.

We have studied the admittance of patch-clamped mast cells during exocytosis and found that they are adequately described by a four parameter equivalent circuit. On the basis of these measurements, we show that, contrary to current belief, when using a phase sensitive detector, small capacitance changes due to exocytosis or endocytosis should be studied by measuring current 90 degrees out of phase, relative to the component that corresponds to changes in series resistance. We have extended the theory on phase-detectors to include the errors in the estimation of step changes of membrane capacitance. We show that the measured capacitance of a secretory granule can be up to 80% smaller than its true value, during the course of a typical mast cell degranulation. We also describe a software-based phase-detector that simplifies capacitance measurements.