Electrophysiological study of single Leydig cells freshly isolated from rat testis. II. Effects of ionic replacements, inhibitors and human chorionic gonadotropin on a calcium activated potassium permeability.

Changes in the membrane potential of isolated Leydig cells produced by modified ionic solutions were investigated in vitro either by a total change of the bathing medium (procedure P1) or by a flush of the solution around the impaled cell (procedure P2). In standard Earle's solution, the impalement of 198 cells by a glass microelectrode was accompanied by an hyperpolarization (MP1 = -37.6 +/- 0.7 mV) (means +/- S.E.M.) followed by a gradual depolarization to a steady state potential (MP2 = -25.1 +/- 0.6 mV) (Joffre et al. 1984). Experiments with K modified media (P1) showed that MP2, and to a greater extend MP1, were dependent on the external K. A tenfold increase of K decreased MP2 by 16 mV and MP1 by 25 mV. When the extracellular Cl was reduced (P1) by substituting Cl with a less permeant anion, MP2 was unchanged and MP1 was significantly decreased. A transient depolarization of MP2 occurred when a low Cl medium was flushed (P2). An equimolar Na replacement by choline chloride (P1) did not change MP1 or MP2, during the first 10 min. It suppressed MP1 and decreased MP2 after a 15 min exposure. MP1 reappeared and MP2 increased after the restoration of the normal Na solution (P1 and P2). The modification of external Ca from 0 to 3.6 mM (P1) increased both MP1 and MP2. MP1 was never cancelled in 0 mM Ca. In 18 mM Ca, MP1 was suppressed and MP2 decreased. Restoration of 1.8 mM Ca was rapidly accompanied by the MP1 reappearance.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophysiological study of single Leydig cells freshly isolated from rat testis. I. Technical approach and recordings of the membrane potential in standard solution.

Single Leydig cells were isolated from rat testis by a collagenase digestion procedure and purified through a 21,000 g self generated densities gradient of 35% Percoll. A method including collagen and fibronectin was proposed to attach freshly prepared Leydig cells to the bottom of plastic Petri dishes. Four hours after the isolation of the cells, it was simultaneously possible to determine their membrane potential by a standard electrophysiological technique using intracellular microelectrodes and to judge cellular integrity by direct microscopic observations. In standard Earle's solution, changes of membrane potentials appeared to be biphasic. On 198 impaled cells, 18 +/- 1 S after the impalement was effective, the membrane potential reached a most negative value (MP1) (-37.6 +/- 0.7 mV), followed by a gradual depolarization to a steady state (MP2) (-25.1 +/- 0.6 mV) which remained constant for a few minutes. In standard Earle's solution, the membrane resistance was low or decreasing towards the most negative potential, then it increased towards the steady potential. At this state, the average value of the cell input resistance was 65.9 +/- 6.0 M omega (n = 16). No action potential was observed either in standard Earle's solution or under a depolarizing current state. It was concluded that the electrophysiological characteristics of the Leydig cell are similar to those of fibroblasts and macrophages, three types of cells with the same mesenchymal origin, present in the interstitial tissue of the rat testis. But the resting potential of the Leydig cell is higher and this secreting cell does not elicit hyperpolarizing oscillations at the steady state, under mechanical or electrical stimuli.

Optical studies on contractile structures during activity.

The optical properties of myocardial fibers and skeletal fibers were compared at rest and during activity. Scattering by myocardial trabeculae is more important than by skeletal fibers but birefringence of myocardial trabeculae is less important than that of skeletal fibers. The absorbance curves of both isolated myocardial trabeculae and skeletal fibers in vivo reflect their main components; during contraction the absorbance increases mainly at 285 nm and this increase starts earlier than contraction recorded with a mechanotransducer. An absorbance change in the spectral range of 420-440 nm is also reported which could represent very early biochemical change in the contractile fibers. The technique used, combined with electrophysiological data, allows one to study different problems related to the biophysics and biochemistry of contraction.