Fluorescence recovery after photobleaching.
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Animals↗
Biomedical subjects
J Délèze
Publications and source records attributed to J Délèze.
Conduction block in Purkinje fibers by homogeneous versus localized decrease of the gap junction conductance.
Gap junction channels provide the pathway for the cell-to-cell propagation of cardiac action potential. Impairment of junctional conductance decreases conduction velocity and can cause block, two conditions that favor ventricular arrhythmias and fibrillation by re-entrant excitation. These experiments were designed to examine the effects of homogeneous versus localized decrease of the gap junction conductance on propagation of action potential in Purkinje fibers from sheep hearts. The fibers were mounted in a three-compartment chamber, and cell-to-cell conductance was progressively reduced by applying heptanol either over a central 2-mm segment or over the whole fiber length. The internal resistivities (Ri) at which conduction of the action potential became blocked were determined in both cases. With 3.5 mM heptanol in the central compartment, conduction failed when Ri was increased by only 3-4.6 times the control values. In contrast, when the same concentration of heptanol was added simultaneously to all three compartments, Ri had to rise by a factor of 7.5-9.4 before conduction became decremental and was blocked. In both situations, dV/dt(max) at the time of conduction block was similarly decreased to about 50% of the control values. Other parameters being equal, a moderate decrease of the gap junction conductance and of the fast sodium current, insufficient to block propagation of the action potential when they are homogeneously distributed, become sufficient to interrupt conduction if the action potential merges abruptly into a portion of fiber with normal internal conductivity at the outlet of the area of increased resistance. This greater sensitivity to block is accounted for by the increase in electrical load at the discontinuity in the core conductor between the region of increased internal resistance and the normal part of fiber that follows. Areas of steep transition from high to low input resistances of the core conductor, such as may develop in localized ischemia, therefore appear particularly susceptible to conduction failure.
Action Potentials↗
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.
Animals↗
Reversible interruption of gap junctional communication by testosterone propionate in cultured Sertoli cells and cardiac myocytes.
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 microM 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.
Animals↗
Influence of the molecular structure of steroids on their ability to interrupt gap junctional communication.
17 beta-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 prerequisite for the activity of extracellularly applied steroid compounds (for example, testosterone was ineffective even at external concentrations up to 100 microM, whereas its intracellular application at 1 microM 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.
Calcium↗
Effect of antipeptide antibodies directed against three domains of connexin43 on the gap junctional permeability of cultured heart cells.
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.
Animals↗
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 whole-cell voltage-clamp method, or the rate constant of dye diffusion with the fluorescence recovery after photo-bleaching (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.
4-Aminopyridine↗
The uncoupling effect of diacylglycerol on gap junctional communication of mammalian heart cells is independent of protein kinase C.
Possible regulatory effects on cell-to-cell communication of a synthetic diacylglycerol, an activator of protein kinase C (PKC), were examined in pairs of synchronously beating ventricular myocytes of neonatal rats in primary culture. Junctional communication was estimated by measuring either the rate constant of dye diffusion, with the fluorescence recovery after photobleaching technique, or the cell-to-cell electrical conductance with a double whole-cell voltage clamp. The addition of a freshly prepared emulsion of 1-oleoyl-2-acetyl-sn-glycerol (OAG, 100 micrograms/ml), either in the bath or in the solution filling the patch pipet, was seen to interrupt intercellular communication within approximately 8 to 10 min. This effect is neither mimicked by stimulation of PKC by a phorbol ester, nor prevented by PKC inhibitors, making it unlikely that, in these cells, PKC activation could induce intercellular uncoupling. During OAG exposures, the intracellular calcium concentration was very modestly increased (by a factor 1.5 to 2), which does not suffice to account for uncoupling. OAG might trigger interruption of cell-to-cell communication by a mechanism analogous to that of other lipophilic molecules (such as aliphatic alcohols or long chain unsaturated fatty acids) which interfere with gap junctions.
Animals↗
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 mM dibutyryl-cyclicAMP, but not to 10 nM 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.
Animals↗
Gap junctional communication during human trophoblast differentiation: influence of human chorionic gonadotropin.
During pregnancy, the trophoblast develops from the fusion of cytotrophoblastic cells into a syncytiotrophoblast. As the exchange of molecules through gap junctions is considered to play a role in the control of cell and tissue differentiation, the cell to cell diffusion of a fluorescent dye was investigated in human trophoblastic cells differentiating in culture. The fluorescence recovery after photobleaching technique was used to estimate the transfer of 6-carboxyfluorescein from contiguous cellular elements into photobleached cells. Fluorescence recovery follows a slow exponential time course when the cell to cell exchange process is rate limited by the presence of gap junctional channels between contiguous cells, contrasting with a much faster step-like course in the case of fusion of the plasma membranes. In the presence of 10% fetal calf serum, Percoll-purified cytotrophoblastic cells develop into cellular aggregates, then into a syncytium, within 24-48 h after plating. During this in vitro differentiation, fluorescence recoveries after photobleaching with a time course typical for gap junctions were observed between aggregated cytotrophoblastic cells, between cytotrophoblastic cells and syncytiotrophoblasts, and between contiguous syncytiotrophoblasts. The maximum percentage of gap junctional coupling occurs on the fourth day. This fluorescence recovery is attributed to the diffusion of dye through gap junctions, because it can be interrupted by exposure to a known junctional uncoupler (3 mM heptanol). The effects of hCG on this gap junctional communication during trophoblast differentiation were investigated. In the presence of 500 mIU/ml hCG in the culture medium, the percentage of coupled cells was increased at all stages of culture, and the highest proportion of coupled cells was observed after 2 days of culture vs. 4 days in control medium. Moreover, the diffusion rate constant k (the inverse value of the time constant measured on recovery curves) was also significantly increased in the presence of chorionic hormone. It is concluded that during trophoblast differentiation, the development of a cell to cell communication through gap junctions precedes the formation of a morphological syncytium by cell fusion. This gap junctional communication is promoted by hCG. Furthermore, our study confirms the differentiating role and the autocrine action of hCG in the physiology of the trophoblast.
Cell Communication↗
Cytosolic free calcium in Plasmodium falciparum-infected erythrocytes and the effect of verapamil: a cytofluorimetric study.
The free Ca2+ ion concentration, measured by means of the fluorescent indicators Indo-1 and Fluo-3, has been compared in normal and parasitized erythrocytes from synchronized in vitro cultures of human blood infected with Plasmodium falciparum. The cells were loaded with the calcium probes in the form of their acetoxymethylesters. P. falciparum-infected red blood cells gradually accumulate more free Ca2+ ions than uninfected cells. The increased Ca2+ concentration is preferentially located inside a rather large central area, corresponding to the position and size of the parasite. In contrast, the Ca2+ concentration outside this area is not higher than that in normal red blood cells. This rise in calcium content becomes significant at the end of the ring stage. The concentration measured in 36-hr schizonts reaches two times that measured in uninfected erythrocytes, and it peaks to four times control values in 44-hr schizonts. The Ca2+ channel blocker verapamil (10 to 20 microM), added on the 24th hr of culture, slows down or blocks the parasite's growth at the trophozoite stage. However, the free Ca2+ concentration measured on infected red blood cells at different times after verapamil addition does not differ from that obtained in the absence of verapamil. These results demonstrate that the bulk of the free Ca2+ load of P. falciparum-infected erythrocytes is located inside the parasite or its parasitophorous vacuole. These data also indicate that the increased Ca2+ influx in P. falciparum-infected erythrocytes does not take the route of verapamil-sensitive Ca2+ channels. It also appears that the inhibitory effect of verapamil on the parasite's maturation does not depend on a change in its Ca2+ content.
Animals↗
[Increase by FSH hormone and depression by testosterone of the diffusional coupling between Sertoli cells from immature rat testis in primary culture].
Previous studies have suggested that FSH promotes the intercellular coupling of Sertoli cells from immature rat testis in primary culture ([1], [2]). In order to test this hypothesis, we have investigated the diffusional coupling between Sertoli cells in primary culture with the FRAP technique. The coupling is low in unstimulated cells but increases in the presence of FSH. This effect is not reversed by returning to the control medium. Testosterone decreases this coupling, an effect which is reversed by a new exposure to FSH. Taken together these data show that FSH initiates diffusional coupling in Sertoli cells and that testosterone antagonizes this effect.
Animals↗
Segmental electrical uncoupling and conduction blocks after calcium removal and replacement in a mammalian auricle.
The cell-to-cell electrical conduction has been investigated in control conditions, during calcium depletion and after calcium repletion. When rat auricular strips are bathed in a Ca2+-free, EGTA-containing (5 mM) solution, the resting membrane potential slowly decreases to about -35 mV within 20 min. The electrotonic spread of intracellular current pulses remains similar to that observed in control conditions, with length constants of about 215 microns in the fibre direction and 52 microns perpendicular to it. Restoration of calcium ions to the bathing fluid at 37 degrees C induces an irreversible loss of the all-or-none electrical conduction of the action potential, and the auricular fibres become split up into aggregates of electrically coupled cells delimited by border zones where the electrical coupling and the conduction of action potentials are interrupted. Inside each of those islets the resting membrane potential is uniform, but it may vary abruptly (between about -10 and -80 mV) across the border of two islets. Islets with sufficient levels of membrane potential (less than -60 mV) can generate action potentials that do not propagate to adjacent islets. This fragmentation of the cardiac tissue into electrically independent subunits explains the irreversible loss of the propagated electro-mechanical activity (calcium paradox) that is observed after calcium repletion.
Action Potentials↗
Cell-to-cell communication in the heart: structure-function correlations.
The communicating cell junctions that ensure the electrical and diffusional continuity of the intracellular space in the heart fibres can be switched from their normal conducting, or opened state, to an exceptional non-conducting, or closed state. This electrical uncoupling is observed after cell injury in the presence of Ca2+ ions in the extracellular fluid, after metabolic inhibition and in the presence of aliphatic alcohols (C6 to C9). The correlations between electrical uncoupling and gap junction morphology in the heart are briefly reviewed. A decrease of the distance between P-face particles and between the E-face pits has been found in all investigations, but the functional significance of this observation is not understood at present. A quantitatively very similar decrease of the average particle diameter (about -0.7 nm) has been measured in glutaraldehyde-fixed sheep Purkinje fibres and in unfixed, quickly frozen rat auricles that had been electrically uncoupled by three different procedures. About half of this decrease was reversible on short-term electrical recoupling (within 20 min). It is concluded that a measurable decrease of the connexon diameter correlates with electrical uncoupling.
Animals↗
Quantitative gap junction alterations in mammalian heart cells quickly frozen or chemically fixed after electrical uncoupling.
The gap junction morphology was quantified in freeze-fracture replicas prepared from rat auricles that had been either quickly frozen at 6 K or chemically fixed by glutaraldehyde, in a state of normal cell-to-cell conduction or in a state of electrical uncoupling. The general appearance of the gap junctions was similar after both preparative procedures. A quantitative analysis of three gap junctional dimensions provided the following measurements in the quickly frozen conducting auricles (mean +/- SD): P-face particles' diameter 8.27 +/- 0.74 nm (n = 5709), P-face particles' center-to-center distance 10.78 +/- 2.12 nm (n = 4800), and E-face pits' distance 9.99 +/- 2.19 nm (n = 1600). Corresponding values obtained from chemically fixed tissues were decreased by about 3% for the particle's diameter and about 5% for the particles' and pits' distances. Electrical uncoupling by the action of either 1 mM 2-4-dinitrophenol (DNP), or 3.5 mM n-Heptan-1-ol (heptanol), induced a decrease of the particle's diameter, which amounted to -0.69 +/- 0.01 nm (mean +/- SE) in the quickly frozen preparations and -0.71 +/- 0.01 nm in the chemically fixed ones. The particles' distance was decreased by -0.96 +/- 0.04 nm in the quickly frozen samples and by -0.90 +/- 0.03 nm in the chemically fixed ones and the E-face pits' distance was similarly reduced. All differences were statistically significant (P less than 0.001 for all dimensions). Electrical recoupling after the heptanol effect promoted a return of these gap junctional dimensions towards normal values, which was about 50% complete within 20 min. It is concluded that very similar morphological alterations of the gap junctional structure are induced in the mammalian heart by different treatments promoting electrical uncoupling and that these conformational changes appear independently of the preparative procedure. The suggestion that the observed decrease of the particles' diameter is genuinely related to the closing mechanism of the unit cell-to-cell channel set in their centers is thus confirmed.
2,4-Dinitrophenol↗
Effect of several uncouplers of cell-to-cell communication on gap junction morphology in mammalian heart.
Electrical conduction in sheep Purkinje fibers has been blocked by three different procedures: (I) 1 mM 2-4-dinitrophenol, (II) 3.5 mM n-Heptan-1-ol (heptanol), and (III) treatment by a hypotonic (120 mOsmoles) Ca2+-free solution for half an hour, followed by return to normal conditions. The gap junction morphology was analyzed quantitatively in freeze-fracture replicas and compared in electrically conducting and nonconducting fibers. It is found that the three uncouplers of cell-to-cell conduction induce consistent and statistically significant alterations of the gap junction structure. The investigated morphological criteria: (a) P-face junctional particle diameter, control value 8.18 +/- 0.70 nm (mean +/- SD), (b) P-face junctional particles center-to-center spacing, control value 10.23 +/- 1.57 nm, and (c) E-face pits spacing, control value 9.45 +/- 0.98 nm, are, respectively, decreased to 7.46 +/- 0.62 nm, 9.25 +/- 1.34 nm and 8.67 +/- 1.13 nm in Purkinje fibers with complete conduction blocks. All three gap junctional dimensions are seen to decline progressively with time from the onset of an uncoupling treatment towards stable minima reached in half an hour. The observed morphological transitions appear related to the electrical uncoupling for the following reasons: partial electrical uncoupling results in values of the gap junctional dimensions that are intermediate between those measured in electrically coupled and uncoupled preparations, and the three morphological indices are seen to increase again towards control values very soon after electrical conduction has been re-established. It is concluded that the junctional channels closure on electrical uncoupling correlates with a measurable (-0.72 +/- 0.01 nm, difference of the means +/- SE) decrease of the junctional particle diameters.
Animals↗
Permeability of a cell junction during intracellular injection of divalent cations.
Divalent cations are microinjected into Chironomus salivary gland cells while the cell-to-cell passage of fluorescein (330 dalton) and electrical coupling are monitored. Injections of Ca and Mg that substantially depolarize the cells produce block or marked slowing fluorescein passage, accompanied by electrical uncoupling. Injections of Ca, Mg or Sr that cause little depolarization, and presumably smaller elevation of divalent cation concentration in the cytoplasm, produce block or marked slowing of fluorescein passage with little or no detectable electrical uncoupling. This partial uncoupling may reflect total closure of a fraction of the channels in junctional membrane or partial closure of all channels.
Animals↗
The site of healing over after a local injury in the heart.
The fluorochrome, Procion yellow, does not detectably penetrate the intact carciac cells of a living rat auricle, but stains the injured ifbers. When healing over is prevented in a calcium-free solution, the dye that penetrates through a point injury diffuses away from the damaged spot across the intercalated discs over a distance of several cell lengths. However, after an injury of the same size performed in a calcium-containing solution, Procion yellow is taken upt by one cardiac cell only or by a small integral number of cellular units to the exclusion of adjacent cells. It is concluded that the permeability of the discs of an injured cardiac cell decreases during healing over.
Animals↗