Michel Joffre

Reversible Interruption of Gap Junctional Communication by Testosterone Propionate in Cultured Sertoli Cells and Cardiac Myocytes

Abstract. A direct cell-to-cell exchange of ions and molecules occurs through specialized membrane channels built by the interaction of two half channels, termed connexons, contributed by each of the two adjacent cells. The electrical and diffusional couplings have been investigated by monitoring respectively the cell-to-cell conductance and the fluorescence recovery after photobleaching, in Sertoli and cardiac cells of young rat. In both cell types, a rapid impairment of the intercellular coupling has been observed in the presence of testosterone propionate. This interruption of the cell-to-cell communication through gap junction channels was dose-dependent, observed in the concentration range 1 to 25 μm and was progressively reversed after withdrawing the testosterone ester. Pretreatment with cyproterone acetate, an antiandrogen which blocks the nuclear testosterone receptor by binding, did not prevent the uncoupling action of the androgen ester. This observation, together with the rapid time course of the uncoupling and recoupling, and the rather high effective concentration (micromolar) of the steroid compound, suggests a nongenomic mechanism of action. The uncoupling concentrations were very similar to those of other steroid compounds known to interrupt gap junctional communication. The uncoupling could result from a direct interaction of the steroid with the proteolipidic structure of the membrane, that might alter the conformation of the gap junction channels and their functional state.

Follicle-stimulating hormone increases gap junction communication in Sertoli cells from immature rat testis in primary culture.

The gap junction communication in Sertoli cells from immature rat testes, cultured either in absence or in presence of follicle-stimulating hormone (FSH), was studied by microinjection of a fluorescent dye and by Fluorescence Recovery After Photobleaching (gapFRAP).The cells cultured for 2–4 days in the absence of FSH showed a flattened “epithelial-like” appearance. They were poorly coupled, as judged by the low frequency of cell-to-cell spread of microinjected Lucifer Yellow, and by the value of the rate constant of dye transfer (k) estimated in gapFRAP experiments. However, when two different subpopulations of cells were separately analyzed, namely the cells forming small groups contacting over part of their circumference (“adjoining cells”), and the cells arranged in tight clusters, we found that the value of k in the latter group was much higher, reaching about 75% of that obtained in the presence of FSH.The cells cultured for two days in a medium containing ovine FSH underwent striking morphological changes and presented a rounded, “fibroblast-like” appearance. They were arranged in networks or in clusters. The frequency of cell-to-cell dye diffusion after microinjection and the rate constant of dye transfer were rapidly increased to the same final level by FSH, although they were initially different in these two groups. A concentration dependence of k, in the range 0.05 to 3 ng/ml, was observed in the cells in networks, contrasting with an all-or-none increase in the cells in clusters.Two days after FSH withdrawal, the dye transfer constant returned to prestimulation control values in the cells in clusters, but not in the cells in networks, which maintained a stable degree of coupling comparable to that of the unstimulated cells in clusters. This observation suggests (i) that an initial promoting effect of FSH already exists in the immature rat testis, which is preserved after enzymatic treatment in the cell clusters, but not in the more dispersed cells, and (ii) that the decreased junctional coupling is re-established in the dispersed cells by FSH, through a synthesis or a membrane insertion of connexin.The effects of FSH were mimicked by a brief exposure to 1 m m dibutyryl-cyclicAMP, but not to 10 n m human chorionic gonadotropin (hCG), indicating that the gap junction communication in Sertoli cells is upregulated by FSH through a specific membrane receptor, with cyclicAMP acting as a second messenger.

Contraceptive gossypol blocks cell-to-cell communication in human and rat cells

Gossypol (a polycyclic lipophilic agent naturally present in cottonseed, known as a potent non-steroid antifertility agent and a non-specific enzyme inhibitor) irreversibly impaired the intercellular communication between homologous pairs of various cultured cells, from man or rat, involved (Sertoli or trophoblastic cells) or not involved (ventricular myocytes) in steroidogenesis, in a dose-dependent manner. In serum-free assays, a rapid junctional uncoupling occurred in non-cytotoxic conditions. At 5 microM (approximately twice the peak plasma concentration measured in human patients during chronic administration), gap junctional communication was interrupted within 4 to 10 min, without concomitant rise in the intracellular Ca2+ concentration. The latter importantly increased when gossypol treatment was prolonged (cytotoxic effect). The short term uncoupling effect of gossypol was prevented by serum proteins, but long-lasting treatments (48 h) with moderate concentrations (3 microM) elicited junctional uncoupling and impeded the in vitro differentiation of human trophoblasts.

INFLUENCE OF THE MOLECULAR STRUCTURE OF STEROIDS ON THEIR ABILITY TO INTERRUPT GAP JUNCTIONAL COMMUNICATION

Abstract. 17β-estradiol propionate was found to reduce the gap junctional communication in a concentration range similar to that of testosterone propionate, in primary cultures of rat Sertoli cells and cardiac myocytes. Uncoupling was reversible on washing out and occurred without concomitant rise in the intracellular calcium concentration.Esterification was a prerequisite for the activity of extracellularly applied steroid compounds (for example, testosterone was ineffective even at external concentrations up to 100 μm, whereas its intracellular application at 1 μm totally interrupted intercellular communication), but their uncoupling efficiency did not depend on the nature of the ester chain nor on its position on the steroid nucleus. The derivatives of two other androgen hormones (derivatives of the androstane nucleus) were also efficient as junctional uncouplers. Among five steroid molecules belonging to the pregnane family, only one (pregnanediol diacetate) interrupted the junctional communication. Neither cholic acid nor cholesteryl acetate or ouabain showed this effect. Altogether, no correlation with the presence or position of double bonds nor with the trans- or cis-fusion of the A and B rings could be recognized.These results suggest that this reversible, nondeleterious uncoupling effect of steroids is independent of the shape of the molecules and is more probably related to their size and liposolubility, that condition their insertion into the lipid bilayer. Their incorporation into the membrane could disturb the activity of the membrane proteins by a physical mechanism.

Characterization and cyclic AMP-dependence of a hyperpolarization-activated chloride conductance in Leydig cells from mature rat testis.

SummaryWe recently described a cyclic AMP-activated current in the membrane of Leydig cells from mature rat testis by using the whole-cell configuration of the patch-clamp technique (Noulin & Joffre, 1992a). In the present study, further experiments were performed in symmetrical CsCl solutions. We show that this current corresponds to a hyperpolarization-activated chloride conductance. Voltage jumps to negative potentials, applied from a holding potential of +60 mV, activated a time-dependent inward current.In control cells, the curve of steady-state current activation typically ranged from +60 mV (0) to −120 mV (1) and had its midpoint at −40 mV. Deactivation at positive potential was characterized by an instantaneous outwardly rectifying current which decayed with time. The kinetics of activation and deactivation were described by a double and a single exponential, respectively.Cyclic AMP, added to the pipette solution, increased both the inward rectification and the amplitude of the steady-state current in the range of 0 to −60 mV. The activation threshold was unchanged, while the V0.5 of the activation curve was shifted by 24 mV to more positive potentials. Consequently, the activation curve was steeper. The two rate constants of activation were increased and were strongly voltage dependent. In parallel, the amplitude of the instantaneous outward current and the rate constant of deactivation were increased.The reversal potential of this current was close to ECl. It did not change with equimolar replacement of cesium by TEA, and shifted with the chloride concentration gradient. This current was inhibited by chloride channel blockers.These results indicate a hyperpolarization-activated chloride conductance in the membrane of Leydig cells which is modulated by cyclic AMP. This nucleotide acts by modifying the kinetics of inward current and both the kinetics and the amplitude of deactivating outward current.

Ca-dependent-chloride and potassium currents in rat Leydig cells

Voltage‐clamped membrane currents have been investigated from whole‐cell patch‐clamp recordings performed on single Leydig cells isolated from the adult rat testis. Two outward membrane currents were evoked by depolarizing voltage steps. A potassium current was recorded in cells dialyzed with low (10−9−10−8 M) calcium media. This current was decreased by TEA (10 mM). A chloride current was recorded in cells dialyzed with high (10−7 − 10−6 M) calcium media. This current was decreased by an external exposure to glutamate. Comparison of the currents at low and high internal calcium concentrations suggests that an increase of the intracellular calcium activates a chloride current.

Inhibitory effect of internal sodium and Hepes on the voltage-dependent potassium conductance of rat Leydig cells

We used the whole-cell configuration of the patch-clamp technique to investigate modulation of the voltage-dependent K+ conductance of rat Leydig cells by intracellular pH, Hepes, and Na+. pH (range 6.8-7.4) has no effect on current. Removal of Na+ (from 10 to 0 mM) increases the amplitude of K+ current and abolishes the plateau effect on peak current. Reduction of Hepes (from 25 to 10 mM) increases the amplitude of K+ current and, simultaneously, shifts the threshold for current activation to the left, but the plateau of current is still present. Removal of Na+ cancels the potentiating effect of Hepes on frequency of occurrence of time-dependent inactivation of K+ current. We conclude that the blocking effect of Hepes and Na+ on K+ current may presumably involve two distinct mechanisms, but we can not exclude some cooperativity in their ability to inactivate the voltage-gated potassium conductance of rat Leydig cells.

Glucose- and concentration-dependence of noradrenalin effects on electrical activity in mouse pancreatic β cells

The effects of a wide range of noradrenalin (NA) concentrations (10(-11)-10(-4) M) on the membrane potential and on the glucose-induced electrical activity were investigated with microelectrodes in microdissected mouse islets. In the presence of 11.1 mM glucose, the beta cells exhibited a repetitive activity. NA at more than 10(-7) M induced a rapid hyperpolarization followed by a silent depolarization, then by the appearance of a slowed pace of repetitive activity (dose-dependent effects). NA at less than 10(-7) M did not markedly affect the electrical activity; it only induced a dose-dependent increase in the degree of activity with no change of the potential levels. The glucose-dependence of these effects were then investigated. In the absence of glucose, 10(-8) and 10(-6) M NA did not affect the resting membrane potential. Non-stimulatory glucose concentrations (2.8-7.3 mM) progressively decreased the membrane potential. 10(-8) M NA did not affect it, while 10(-6) M NA induced a dose-dependent and long-lasting hyperpolarization. In the presence of stimulatory glucose concentrations (7.3-30 mM) the degree of activity increased, 10(-8) M NA induced a slight leftward shift and 10(-6) M NA a slight rightward shift of the dose-response curve.

Depressing effect of testosterone and estradiol propionates on the diffusional coupling promoted by FSH on Sertoli cells from immature rat testis in primary culture

The gap junction communication in Sertoli cells from immature rat testes has been studied using the gapFRAP method. The diffusional rate constant k is low in control medium and is greatly increased in the presence of ovine FSH. When the FSH-stimulated Sertoli cells are incubated for 15 minutes with testosterone propionate (25 μM) or estradiol propionate (15 μM), the cell-to-cell coupling is totally abolished. In contrast to its ester, testosterone does not affect cell coupling at the same concentration. A 100 μM concentration of testosterone, during 96 hours, is necessary to uncouple gap junctions of Sertoli cells. This uncoupling effect does not occur in the presence of cyproterone acetate (a antiandrogen), suggesting a mechanism of action different from a genomic effect. It is suggested that this uncoupling effect results from an interaction of the steroid with the lipid bilayer leading to a closure of the gap junctions similar to the action of other lipophilic molecules.