J. Délèze

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.

RAPID ONSET AND CALCIUM INDEPENDENCE OF THE GAP JUNCTION UNCOUPLING INDUCED BY HEPTANOL IN CULTURED HEART CELLS

The kinetics of the reversible interruption of gap junction communication by the aliphatic alcohol heptanol and the possible mediation of an increase of the cytosolic Ca2+ concentration have been investigated in pairs of myocytes dissociated from neonatal rat ventricles and cultured for 2–3 days. Junctional communication was estimated by measuring either the cell-to-cell electrical conductance with a double wholecell voltage-clamp method, or the rate constant of dye diffusion with the fluorescence recovery after photobleaching (gap FRAP) technique. Electrical coupling was seen to be abruptly interrupted (in less than 0.5 s) by heptanol (1–3 mm). The cytosolic Ca2+ concentration was not affected, even at a saturating heptanol concentration. Heptanol removal allowed a gradual re-opening of gap Junctional channels, as shown by the recovery curve of the cell-to-cell conductance, which is 90 % complete within 90 s. These data are consistent with a direct interaction of heptanol with channel proteins or with their lipid environment.

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.

Synergy between two calcium channel blockers, verapamil and fantofarone (SR33557), in reversing chloroquine resistance in Plasmodium falciparum.

This study describes the synergistic interaction of two calcium channel blockers, verapamil (VR) and SR33557 or fantofarone (SR), in reversing chloroquine resistance in Plasmodium falciparum, the causative agent of human malaria. The two calcium channel blockers exhibited an intrinsic antimalarial activity at 10 and 1 microM for verapamil and fantofarone, respectively. Isobolograms revealed that chloroquine and verapamil, and chloroquine and fantofarone, acted synergistically against chloroquine-resistant strains of P. falciparum. When used at subinhibitory concentrations, verapamil appeared 2 to 3 times more potent than fantofarone in reversing chloroquine resistance. Indeed, verapamil completely reversed the chloroquine resistance in P. falciparum, while fantofarone did so only partially. In the highly chloroquine-resistant strain FcB1, VR and SR acted synergistically to reverse CQ resistance, and the concentrations of VR used in these combinations could be reduced 10- or 100-fold (e.g. 100 nM and 10 nM) those required when this drug was used alone. In the moderately chloroquine-resistant strain K1, a combination of VR and SR for CQ resistance reversal allowed us to reduce the concentration of these chemosensitizers 1000- and 100-fold, respectively. The maximum tolerable plasma level beyond which side-effects occurred when using verapamil is 2.5 microM. Thus, the approach described, which allowed us to lower the doses of chemosensitizers, could well prevent toxic effects in humans and enlighten the advantages of polychemotherapy.

Effect of Antipeptide Antibodies Directed against Three Domains of Connexin43 on the Gap Junctional Permeability of Cultured Heart Cells

Abstract. Cell-to-cell communication can be blocked by intracellular injections of antibodies raised against gap junction proteins, but the mechanism of channel obstruction is unknown. Binding to connexins could lead to a conformational change, interfere with regulatory domains or cause a steric hindrance. To address these questions, the effects on cell-to-cell communication of affinity purified polyclonal antibodies raised against peptides reproducing the intracellular sequences 5–17, 314–322 and 363–382 of rat connexin43 were investigated in cultured rat ventricular cells. The antibodies against sequence 363–382 were characterized by immunoblotting and immunocytochemistry. Characterization of antibodies 5–17 and 314–322 has been previously reported. In a first series of experiments, the effect on gap junctional communication was assessed by injecting a junction-permeant fluorescent dye into cells adjacent to one cell previously microinjected with antibodies. In a second series, junctional permeability was quantitatively determined on records of fluorescence recovery after the photobleaching of 6-carboxyfluorescein-loaded cells. Antibodies 5–17 marked a 43 kDa band on immunoblots, but did not immunolabel gap junctions and had no functional effect. Antibodies 314–322 recognized the 43 kDa protein and labeled the intercalated disks, but failed to interfere with junctional permeability. Antibodies to the nearby sequence 363–382, for which all immunospecific tests had been positive, caused a delayed diffusional uncoupling in 50% of the microinjected cells. It is suggested that the blocking of junctional communication by antibodies results from interference with a regulatory domain of the connexin.

Transforming growth factor β1 (TGFβ1) inhibits gap junctional communication during human trophoblast differentiation

Summary It was recently established that, during trophoblast differentiation, gap junctional communication (GJC) precedes the formation of the syncytiotrophoblast and is required for trophoblast cell fusion. Therefore, the end of cell-to-cell communication through gap junctions appears as a judicious criterion of cell fusion. Although GJC was seen to be stimulated by human Chorionic Gonadotropin (hCG), its regulation remains poorly understood. Transforming Growth Factor-β1 (TGF-β1), a multifunctional cytokine has been shown to modulate trophoblast differentiation. Therefore, the effects of TGF-β1 on intercellular dye diffusion have been investigated in cultured trophoblast of human term placenta, by means of the Fluorescence Recovery After Photobleaching (gap FRAP) technique. In parallel, trophoblast differnetiation, hCG production and human Chorionic Somatomammotropin (hCS, a specific syncytiotrophoblast hormonal product) expression were assessed. The presence of TGF-β1 (5 or 10 ng/ml) in the culture medium for two days partially inhibited syncytium formation. The percentage of coupled cells was significantly decreased (2.8 times) after two days in presence of 10 ng/ml of TGF β1. Simultaneously, hCG release in culture medium was reduced (at this concentration, to 0.65 and 0.79 after respectively two and three days). In these conditions, Western blot analysis of trophoblast cellular proteins revealed that, after two days, hCS expression was reduced by 40% compared to control. Furthermore, the stimulation of trophoblastic GJC by exogenous hCG (500 mIU/ml) was considerably reduced by simultaneous exposure to TGF-β1 (10 mg/ml). The addition of a polyclonal hCG antibody in excess decreased basal GJC. In the presence of hCG antibody, no significant additive inhibition by TGFβ1 was observed. In conclusion, TGF-β1 was found to inhibit intercellular communication and, subsequently, differentiation and concomitant placental hormone secretions.

Chapter 9: A Reexamination of Calcium Effects on Gap Junctions in Heart Myocytes

Publisher Summary The permeability of gap junctions is rapidly (within seconds) and reversibly decreased by an experimental manipulation that decreases the cytosolic pH or increases the cytosolic concentration of free Ca 2+ . Large increases of the cytosolic H + or Ca 2+ concentrations impair the gap junction communication, but in many cell types these changes are slow and often irreversible, and are more akin to pathology than to a physiological regulation. Also, according to both early observations and recent data, the changes in H + or Ca 2+ levels that affect gap junctions are highly variable in different cell systems and may even differ for gap junction channels consisting of the same connexin. In this chapter, the quantitative aspects of the rises in cytosolic Ca 2+ that promote uncoupling in a number of different cell types are critically considered, with particular attention to the myocytes of mammalian heart. The frequently granted hypothesis of a rapid and reversible decrease of the gap junction conductance by increases in the cytosolic Ca 2+ concentration ([Ca 2+ ] i ) in the physiological range cannot be generalized and is not valid for the mammalian heart myocytes. Recent data that show an upregulation of the gap junction conductance in the mammalian heart myocytes by increases in [Ca 2+ ] i quantitatively similar to those occurring during excitationcontraction coupling is also presented in the chapter.

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.