Improvement of the accuracy by the measurement of the electrical cell membrane parameters.

The electrical parameters of the cell membrane are mostly estimated employing ac methods. The measurement is based on the analysis of the current(s) flowing through an access resistance and the membrane. A current/potential transducer is used at the input of the device. The parameters of this transducer, especially its feedback capacity, degrades the accuracy of the measurement and hence diminishes the suppression of mutual influences of the individual parameters. The paper suggests a possible software correction and is supplemented by remarks for practical application.

Versatile tool for the manipulation of electrophoresis chips.

This communication describes a versatile tool allowing standard operations (i.e. washing, pre-conditioning, separation, inner surface modification of the chip channel) with capillary electrophoresis chips. Through currently designed for a chip of maximal dimensions 30 x 60 mm, other formats of the chip require only a minimum adjustment of the equipment, namely setting of the chip sliding rails and adequate arrangement of the exchangeable heads. The application of the tool is demonstrated by the separation of the standard set of inorganic cations.

A method of resolution improvement by the measurement of cell membrane capacitance.

When measuring cell membrane electrical capacitance in whole cell configuration using alternating currents, the resolution decreases with increasing membrane conductance and pipette resistance. Improved resolution was attained by the dual-frequency method which was modified as to control the voltage amplitude of one of the measuring frequencies. A model circuit was developed for the verification of the method. This circuit allows measurement of calibrated capacitance changes even in the range of 5 to 20 fF. Moreover, the method was applied to capacitance measurements on pancreatic exocrine acinar cells. The results of measurements on the model as well as on pancreatic acinar cells are presented. The principle can also be applied to other hardware and software methods for measuring electrical cell membrane parameters.

Dual-frequency method for synchronous measurement of cell capacitance, membrane conductance and access resistance on single cells.

A dual-frequency method was developed to monitor changes of membrane capacitance, membrane conductance and serial resistance between patch pipette and cytoplasm of the cell in the whole-cell configuration. Measurement of real and imaginary components of cell admittance during excitation with two superimposed sinusoidal voltage of different frequencies provides mathematical solutions for all three variables. The validity of the method was verified with experiments on mast cells and exocrine pancreatic acinar cells. During degranulation of mast cells, induced by GTP gamma S in the pipette solution, a stepwise increase in membrane capacitance could be observed, indicating that the resolution of the method is below 10 fF. Precalibration of the setup allows all calculated data to be expressed as absolute values. The capacitance measurement proved to be rather independent of changes in the access resistance and in the cell membrane resistance over a wide range. The huge changes in membrane conductance of mouse pancreatic acinar cells during hormonal stimulation with acetylcholine produced a relative error of less than 0.3% in the capacitance trace. This allows a clear distinction between changes of membrane conductance and cell capacitance. The method therefore offers great advantages in the study of exocytosis as well as endocytosis in cell types, such as exocrine gland cells, with major changes in membrane conductance during hormonal stimulation.

Simple device for continuous measurement of fluorescent anions and cations in the rat kidney in situ.

A simple device for fluorescence measurements on kidney surface in situ (FKS) is described. The device consists of (1) an illuminating unit, (2) a detector, (3) an evaluation unit. The device was developed for measurement of tissue content of anionic sulfofluorescein and cationic 4(4-dimethylaminostyryl)-N-methylpyridinium (ASP) during its secretion by proximal renal tubular cells.

Early postnatal development of contractile performance and responsiveness to Ca2+, verapamil and ryanodine in the isolated rat heart.

Contractile performance of the isolated perfused rat heart and its inotropic response to Ca2+, verapamil and ryanodine was studied in 1-, 2-, 4-, 7- and 12-day-old animals. Values of the developed force revealed two different phases: a slow decrease from day 1 to day 4 followed by a steep increase up to day 12. A similar biphasic time course was observed in the magnitude of the inotropic effect of Ca2+ (0.6-10.0 mmol.l-1): decrease from day 1 to day 4 followed by an increase up to day 7. The sensitivity to Ca2+ was, however, not changed. An analogous biphasic response was also observed during perfusion with the calcium antagonist verapamil (10(-9) to 3.3 x 10(-7) mol.l-1): the sensitivity to negative inotropic effect rose from day 1 to day 4 and then decreased at day 7 (values of IC50 were 170 +/- 61, 17 +/- 6 and 171 +/- 60 10(-9) mol.l-1 S.E.M. on day 1, 4 and 7, respectively). The contractile response to the inhibitor of calcium release from sarcoplasmic reticulum (SR)--ryanodine (10(-6) mol.l-1)--was surprisingly high already in 1-day-old animals (inhibition of contraction by more than 50%) indicating the presence of functionally active SR in the rat heart just after birth. Our data clearly shows that the early development of contractile function and inotropic responsiveness of the rat heart is not linear and changes dramatically during the first week of life.

A method with enhanced sensitivity for temperature measurement in living tissues.

A system for the evaluation of temperature changes in living tissue at a dimensional level of a single cell is described. A glass micropipette the tip of which is filled with semiconducting glass (Rech et al. 1992), is used as a microsensor. The changes of conductivity of the sensor due to variations of temperature are evaluated by electronic circuitry based on the measurement of an AC current of sinusoidal waveform flowing through the sensor. Temperature changes in the range of 0.01 K can be detected in this way.

Separation of collagens by capillary zone electrophoresis.

Collagen (types I, II, V, IX and XI) constituting polypeptide chains and their polymers and cyanogen bromide-cleaved peptides of collagen type I and type III were investigated by means of capillary zone electrophoresis. Separations were effected in 2.5 mM sodium tetraborate buffer in less than 15 min. A 50 cm x 0.1 mm I.D. fused-silica capillary was used. The separations were run at 18 kV per capillary. The results of the separation were monitored at 220 nm with an on-tube detection system. Using the Offord equation, relative retention times of cyanogen bromide cleavage fragments were plotted against M(2-3)/Z, where M is the molecular mass of a polypeptide and Z its valency. A linear relationship was observed. Collagen alpha-chains and their polymers were also satisfactorily resolved.

Simple apparatus for capillary zone electrophoresis and its application to protein analysis.

The construction of a simple apparatus for capillary zone electrophoresis is described, consisting of an optical system allowing direct absorbance measurement in the capillary in UV light, an evaluating electronic module and a high-potential source. An attempt was made to achieve maximum sensitivity with a simple construction. In the electronics, care was taken to obtain a quiet baseline and to optimize the signal-to-noise ratio. That part of the noise which is of the frequency band of the signal is filtered off. Both suction and electrophoretic sample introduction are possible. According to operator's choice, the apparatus can be run under constant voltage or current and is protected against overloading. The high-potential electrode chamber contains separate buffer and sample compartments and its construction offers an easy interchange between the running and sampling positions. The applicability of the system to the separation of amino acids as phenylthiocarbamyl derivatives, peptides and both artificial and naturally occurring protein mixtures is demonstrated.

Versatile supplement device with remote control for the control of patch clamp experiments.

A versatile device for a patch-clamp amplifier is described. This device contains: (i) an acoustic indicator to monitor the input resistance of the patch pipette, which is used in search-mode to indicate the formation of seals; (ii) two pulse generators; and (iii) a staircase generator to produce various pulse and voltage step programs; (iv) a low-pass filter which is used to filter the output of the patch clamp amplifier; and (v) a remote control which is used to control the entire patch clamp experiment. This remote control is used to switch between search-, current clamp-, and voltage clamp-mode, to activate the respective stimulus potential programs, and to control the tape recorder. This electronic device can be easily connected to patch clamp amplifiers.

A microcomputer-based analyser for evaluation of contractile properties of a cardiac muscle.

A computer controlled equipment is described for computer analysis of isometric contractions of cardiac muscle by different modes of excitation. Both the method and the programme are presented. The following parameters are analysed: half and full contraction time, half relaxation time, maximum force developed during contraction, maximum rate of force development. The same computer is simultaneously used for controlling the stimulation.

A device for microcomputer controlled stimulation of cardiac muscle.

An interface is described which substitutes a stimulator for analysis of contractile properties of a muscle. A solution is presented for use of a computer both for analysis and stimulation control. A programme for such a purpose is described.

Changes of electrodermal properties in the "acupuncture points" on men and rats.

The measurements of skin resistance to electrical current performed in rats and men indicated the occurrence of small skin areas, in which the conductivity for DC and AC was sharply increased. In healthy men, the anatomical localization of these areas of increased skin conductivity (AISC) corresponded to the localization of the so-called "acupuncture points" (AP). In patients, the AISC were also found outside the ordinary AP, mainly in areas of referred pain. The measurements of the size of AISC by multiple electrodes indicated the approximate size of AISC cca 350 microM in the rat, ccs 450 microns in man. The recording of skin conductivity were taken from : a) AISC in man and the rat, b) skin in the close neighbourhood of AISC, c) from the sweating human skin.