Multicellular Preparations Research Articles

Action potential and membrane currents of single pacemaker cells of the rabbit heart.

Single, viable pacemaker cells were isolated from sinoatrial (S-A) and atrioventricular (A-V) nodes by treating with collagenase. In normal Tyrode solution containing 1.8 mM Ca2+, these pacemaker cells had a round configuration and contracted rhythmically at a frequency of about 150-260/min. The amplitude, duration, and maximum rate of rise of the spontaneous action potentials recorded using patch clamp electrodes were similar to those obtained from multicellular preparations. Amplitudes of the recorded membrane current were normalized with reference to the surface area of the cell by assuming the cell shape as a plane oblate spheroid. The membrane resistance of the isolated nodal cells was 14.9 +/- 4.0 k omega . cm2 (n = 12) at about -35 mV and the membrane capacitance was 1.30 +/- 0.24 microF/cm2 (n = 18). The inactivation time course of the slow inward current, isi, was fitted with a sum of two exponentials with time constants of 6.7 +/- 0.6 ms and 46.6 +/- 15.3 ms (n = 4) at +10 mV. The amplitude of isi peaked at 0 approximately +10 mV in the current-voltage relationship and was 18.2 +/- 8.4 microA/cm2. The potassium current, iK, was activated in the voltage range positive to -50 mV and was saturated at about +20 mV. The amplitude of the fully-activated iK at -40 mV was 3.3 +/- 1.4 microA/cm2 (n = 10) and showed an inward-going rectification. The activation of the hyperpolarization-activated current was observed at potentials negative to -70 mV in seven of 14 experiments. The current density and membrane capacitance calculated could be overestimated and the membrane resistance underestimated, because of the presence of caveolae on the cell surface. However, these data give the nearest possible estimates of the electrical constants in the nodal cells, which cannot be measured accurately in the conventional multicellular preparations.

COMPARISON OF ISOLATED VENTRICULAR MYOCYTES FROM THREE-WEEK OLD AND ADULT RABBITS

Single, quiescent, electrically stimulable, calcium-tolerant ventricular myocytes isolated by enzymatic digestion from 3-week old (W cells) and 3-month old (M cells) rabbits were examined using a differential interference contrast light microscope. The image of the contracting cell was recorded on video tape and sarcomere length measured. The cell was then fixed and examined with electron microscopy. No evidence of ultrastructural damage was found. Mean length of M cells (N=26) was 147±25 (s.d.) μm and diameter was 21±15 μm. M cells had square or stepped ends. W cells (N=12) were generally smaller: mean length 112±40 pm and diameter 14±9 μm. Several types of W cell were found in the same heart: large cells resembling M cells, and small cells with tapered ends and fewer, larger myofibrils. W cells had widely varying levels of development of transverse tubules and sarcoplasmic reticulum. Rest sarcomere lengths were 1.91±.09 μm for M cells and 1.83±.07 μm for W cells. W cells that had M-cell ultrastructure had sarcomere shortening like that of M cells. Ultrastructurally immature cells had depressed amount and velocity of sarcomere shortening. The amount and velocity of W sarcomere shortening was .17 μm and .63 μm/s and that of M sarcomere shortening was .29 μm and 1.30 μm/s. Thus the ventricle of the three-week old rabbit has cells at various stages of development both structurally and functionally. This inhomogeneity must complicate the assessment of developmental changes of myocardial contractility using measurements obtained from multicellular preparations.

Slow inactivation of a tetrodotoxin-sensitive current in canine cardiac Purkinje fibers

We used the two-microelectrode voltage clamp technique and tetrodotoxin (TTX) to investigate the possible occurrence of slow inactivation of sodium channels in canine cardiac Purkinje fibers under physiologic conditions. The increase in net outward current during prolonged (5-20 s) step depolarizations (range -70 to +5 mV) following the application of TTX is time dependent, being maximal immediately following depolarization, and declining thereafter towards a steady value. To eliminate the possibility that this time-dependent current was due to inadequate voltage control of these multicellular preparations early during square clamp pulses, we also used slowly depolarizing voltage clamp ramps (range 5-100 mV/s) to ensure control of membrane potential. TTX-sensitive current also was observed with these voltage ramps; the time dependence of this current was demonstrated by the reduction of the peak current magnitude as the ramp speed was reduced. Reducing the holding potential within the voltage range of sodium channel inactivation also decreased the TTX-sensitive current observed with identical speed ramps. These results suggest that the TTX-sensitive time-dependent current is a direct measure of slow inactivation of canine cardiac sodium channels. This current may play an important role in modulating the action potential duration.

Electrophysiology of single cardiac cells.

Whatever techniques of isolation one may use, it is certainly true that the isolated single heart cells are very useful in various physiological experiments. For electrophysiology, the application of the single cell serves to test findings previously obtained in the multicellular preparation. Therefore, summary and comparison of the data will become necessary in the near future. Furthermore, the application of the single cell opens new areas for electrophysiology of the heart, because when using only the single cell preparation, one can dialyse intracellular millieu and can also measure single channel analyses.

Characteristics of the second inward current in cells isolated from rat ventricular muscle.

The second inward current (Isi) in single cells isolated from ventricular muscle of adult rat hearts was measured in response to step depolarizations under voltage-clamp conditions. The major ion carrying this current was Ca, and Isi was reduced or abolished by Mn, Ni, Cd, nifedipine, nimodipine and D600. Sr and B could substitute for Ca as charge carriers, and reduced the rate of apparent inactivation of Isi. These effects of Sr and Ba, together with the relation between the steady level of apparent inactivation and membrane potential in Ca containing solution, were taken as evidence that inactivation was at least in part dependent on internal Ca. The reduction of external Na to 11% of normal caused a reduction in peak Isi when Ca was present in the external solution, but did not reduce Isi when Ca was replaced by Sr. It therefore seems unlikely that Na is a major charge carrier for Isi under the conditions of our experiments. The time-to-peak and rate of apparent inactivation of Isi were faster than in previous studies that used multicellular preparations. Both the kinetics and peak amplitude of Isi were markedly dependent on temperature (Q10 close to 3). Contraction of the cells, which was monitored optically, was initiated within 3 ms of the peak Isi, reached a maximum level after approximately 40-50 ms, and was about 100 ms in duration.

Piezoelectric translator. A simple and inexpensive device to move microelectrodes and micropipettes small distances rapidly.

A device is described that is capable of rapidly moving microelectrodes and micropipettes over distances up to 15 mu. This piezoelectric transLator uses the diaphragm from virtually any available piezoelectric buzzer in combination with simple physical support and drive electronics. All of the necessary details for the construction of this small device are presented. Each finished unit is about 2 cm long with a diameter of 2 cm and can be readily adapted to existing manipulators. The translator has been found useful in aiding the independent penetration by one or more microelectrodes of single cells or of more complicated multicellular preparations (including those that lie behind a connective tissue layer). This new device offers fine control of microelectrode motion that cannot be obtained by the other methods used to aid microelectrode and micropipette penetration of cell membranes (e.g. capacitance overcompensation--"ringing in"' or "tickling"--or tapping the manipulator base). Finally, the device described in this paper is extremely simple and inexpensive to build.

Voltage clamp with double sucrose gap technique. External series resistance compensation

In this paper we deal with the double sucrose-gap voltage clamp technique. To perform a reliable clamp or to analyze the intracellular potential distribution, any external series resistance in the artificial node must be taken into account for it induces an instability in the external potential as soon as a current develops. A circuit was designed to compensate for this error, it has been found effective on an analog model and on experimental uni- or multicellular preparations. The attenuation in series resistance frequently causes ringing in the step response. This behavior was studied theoretically and also simulated with analog models where a selective bridged-T network was found to represent the electrical characteristics of the preparation when associated with the chamber and control electronics. A residual series resistance was found and is considered to be a part of the preparation. Characteristics necessary to obtain best results are proposed, for a preparation to be studied in experiments utilizing the double sucrose gap technique with external series resistance compensation.

Some technical improvements for the voltage clamp with the double sucrose gap.

A new double sucrose gap chamber using electrodes of high stability and low resistance is described. The size of each sucrose gap and the central node can be modified independently so that this chamber is suitable for research on multicellular or unicellular excitable preparation under voltage clamp conditions. The shape of the central channel is made so as to reduce the series resistance between the preparation and the recording electrode to a minimum. A window made in the floor of the chamber allows to observe the preparation under a microscope.

Axon voltage-clamp simulations. A multicellular preparation

In this paper we extend the simulation of the voltage clamp of a single nerve fiber to a bundle of axons. These simulations included not only the description of the voltage clamp circuit and a single unidimensional cable to represent the preparation in the "node" region of a double sucrose gap used previously but also a series resistance and a shunt pathway. The output of the voltage control amplifier is applied across the membrane plus the series resistance, producing a voltage drop across the series resistance due to the current generated by the membrane in response to a depolarizing voltage step. Since the membrane current has an inward and an outward phase, voltage drops of opposite sign are produced across the series resistance. During the transient current and at all points along an axon, the potential deviation produced by the series resistance is opposite to the deviation produced by the longitudinal gradient. Only at a command potential equal to the sodium equilibrium potential, the membrane potential transiently matches the command potential. For the attempted voltage clamp of an axon, values of series resistance larger than 50 omega-cm2 allowed propagated action potentials in the membrane. In spite of the presence of propagated action potentials at the calbe membrane, the recorded current does not show "notches" and it has a phase of inward current and a phase of outward current. It is concluded that, in a multicellular preparation with series resistance, the recording of a square voltage pulse does not indicate voltage control of the transmembrane potential. The presence of a shunt pathway produces inaccurate values of current density. Neither series or shunt resistance produce "notches" in the current records.

The Radial Exchange of Labelled Water in Maize Roots

Experiments have been performed on the efflux kinetics of labelled water in isolated maize roots. The purpose of this study was to establish the identity of the rate-limiting step in this radial exchange process. Although the efflux curve could be mathematically described by the solution of an appropriate cylindrical diffusion problem, other experimental evidence was incompatible with this model. It was considered that the efflux kinetics of labelled water resulted from the char acteristics of membrane penetration in a heterogeneous multicellular preparation like the root. On this basis we have computed from the efflux curve that the 'typical' membrane permeability of root cells to water was about 4-5 X ioJcm sec-1. Similar efflux studies were performed on 'dead' roots prepared by boiling the normal excised roots for a short time. These experiments indicated that the solution, obtained from a cylindrical diffusion analysis, represented a satisfactory description of the exchange of labelled water in 'dead' roots. A common feature of both efflux curves for normal and 'dead' roots was that each deviates significantly from the theoretical diffusion curves for large values of time. The precise origin of these deviations has not been determined but they do reflect some property of the root tissue.