Effect of estradiol-17 beta and prostaglandins on rat myometrial gap junctions

The effects of estradiol-17 beta and indomethacin on myometrial gap junction development, plasma estradiol levels and uterine PGF2 alpha content were evaluated in immature and/or ovariectomized, mature rats. High doses of estradiol stimulated the development of gap junctions in the myometrium of animals from both groups. Concomitant injections of estradiol and indomethacin to ovariectomized rats potentiated the estradiol stimulation of gap junctions. Plasma estradiol levels were lower in ovariectomized rats treated with both estradiol and indomethacin than in animals treated with estradiol alone. Indomethacin also enhanced the uptake and retention of 3H-estradiol into uterine tissues. Uterine PGF2 alpha content of ovarectomized rats was stimulated with the initial injection of estradiol but thereafter, the PGF2 alpha content declined with repeated injections to values lower than that observed in controls. Prostaglandin F2 alpha content in tissues from rats treated with estradiol plus indomethacin were also higher than that observed in rats treated with indomethacin alone, however, the values obtained in both groups were significantly lower compared to those from control animals. These results are consistent with the hypothesis that steroid hormones and prostaglandins regulate myometrial gap junction formation. Regulation of myometrial gap junctions by prostaglandins is discussed with respect to a down regulation of the steroid-receptor mechanism and effects on cyclo-oxygenase or lipoxygenase products.     

Effect of estradiol-17ß and prostaglandins on rat myometrial gap junctions

The effects of estradiol-17ß and indomethacin on myometrial gap junction development, plasma estradiol levels and uterine PGF_2α content were evaluated in immature and/or ovariectomized, mature rats. High doses of estradiol stimulated the development of gap junctions in the myometrium of animals from both groups. Concomitant injections of estradiol and indomethacin to ovariectomized rats potentiated the estradiol stimulation of gap junctions. Plama estradiol levels were lower in ovariectomized rats treated with both estradiol and indomethacin than in animals treated with estradiol alone. Indomethacin also enhanced the uptake and retention of ³H-estradiol into uterine tissues. Uterine PGF_2α content of ovarectomized rats was stimulated with the initial injection of estradiol but thereafter, the PGF_2α content declined with repeated injections to values lower than that observed in controls. Prostaglandin F_2α content in tissues from rats treated with estradiol plus indomethacin were also higher than that observed in rats treated with indomethacin alone, however, the values obtained in both groups were significantly lower compared to those from control animals. These results are consistent with the hypothesis that steroid hormones and prostaglandins regulate myometrial gap junction formation. Regulation of myometrial gap junctions by prostaglandins is discussed with respect to down regulation of the steroid-receptor mechanism and effects on cyclo-oxygenase or lipoxygenase products.     

Effects of tamoxifen citrate and cycloheximide on estradiol induction of rat myometrial gap junctions

Longitudinal muscle of myometrial tissues from immature rats were examined by quantitative thin section electron microscopy for the presence of gap junctions after treatment with estradiol with and without tamoxifen, and cycloheximide for 1-6 days. Gap junctions were present between myometrial cells on days 4, 5, and 6 after treatment with estradiol (500 micrograms/day). Tamoxifen administered concomitantly with estradiol over the 6-day period completely prevented induction of the junctions. Gap junctions were not detected in the myometrium after treatment with tamoxifen alone. Administration of cycloheximide together with estradiol on day 0 of the 6-day period had no effect on gap junction frequency but resulted in a reduction in gap junction size in the myometrium after continued treatment with the hormone. Treatment with cycloheximide on day 1, however, significantly suppressed the effect of further estradiol treatment on the induction of gap junctions in the myometrium. Junctions were not visible in the tissues from animals treated with cycloheximide alone or in the control groups treated with sesame oil. These results indicate that estradiol influences the presence of gap junctions in the myometrium by regulating the synthesis of gap junction proteins through the steroid receptor mechanism.     

Effect of progesterone and oestradiol on expression of connexin43 in cultured human myometrium cells

In human myometrium, the formation of gap junctions at various stages of labour and in correlation with the concentration of progesterone and oestradiol in maternal blood was described previously by electron microscopy and laser confocal microscopy of immunohistochemically stained myometrial sections. The present investigation focused on the effect of continuous exposure of isolated myometrial tissue to progesterone and oestradiol on the number of gap junction plaques in human myometrium cells in vitro. The presence of gap junctions was evaluated by immunocytochemistry with antibodies against gap junction protein, connexin43 (Cx43). Human myometrial cells were isolated from biopsies obtained from term pregnant women who had an elective caesarean operation in the 37th or 40th week of pregnancy. The dispersed myometrial cells that were obtained by limited enzymatic digestion of the myometrial samples were maintained in monolayer culture for 1, 3 and 6 days in the presence of medium that contained foetal bovine serum and the steroids at different concentration. In primary culture, as well as after several passages, the characteristics of these cells were morphologically and biochemically similar to those of smooth muscle cells and myometrial tissue. The obtained results showed that the cells in culture responded synchronously to the increased concentrations of oestradiol/progesterone mixtures. The number of gap junctions increased significantly on days 1, 3 and 6 in culture and showed positive correlation (p < 0.05) with the cell number when the concentration of oestradiol was raised to 1 microgram/mL in the progesterone ratio (1.0 microgram/0.5 microgram/mL). No significant correlation, however, in connexin43 gap junction number versus cell number was observed between the six experimental groups treated with progesterone only.     

Gap junction modulation in rat uterus. II. Effects of antiestrogens on myometrial and serosal cells

The ability of several triphenylethylene antiestrogens to affect the modulation of gap junctions in rat uterine myometrial and serosal cells was examined in animals 60 days following hypophysectomy. Five daily injections of enclomiphene, zuclomiphene, tamoxifen, nafoxidine, CI 628 or CI 680 (500 micrograms per injection) promote uterine luminal epithelial cell hypertrophy characteristic of exogenous estrogen administration. These same compounds, however, fail to induce myometrial cell or increase the number of serosal cell gap junctions, respectively, which is also characteristic of exogenous estrogen treatment. Pretreatment of animals with antiestrogens blocks the ability of estradiol benzoate (E2 B) to induce gap junction formation in myometrial cells when followed by combined injections of E2 B and antiestrogens (both 250 micrograms) administered daily for 5 days. Therefore, with respect to the parameter of myometrial cell gap junction stimulation, all of the antiestrogens examined act as pure estrogen antagonists. These same antiestrogen pretreatments only weakly antagonized the ability of E2 B to modulate serosal cell gap junction membrane. These studies indicate the presence of different mechanisms for the estrogenic modulation of gap junctional membrane in myometrial and serosal cells.     

Sustained inhibition of rat myometrial gap junctions and contractions by lindane

Background  

Gap junctions increase in size and abundance coincident with parturition, forming an intercellular communication network that permits the uterus to develop the forceful, coordinated contractions necessary for delivery of the fetus. Lindane, a pesticide used in the human and veterinary treatment of scabies and lice as well as in agricultural applications, inhibits uterine contractions in vitro, inhibits myometrial gap junctions, and has been associated with prolonged gestation length in rats. The aim of the present study was to investigate whether brief exposures to lindane would elicit sustained inhibition of rat uterine contractile activity and myometrial gap junction intercellular communication.     

Effects of 17 beta-estradiol on myometrial gap junctions and pregnancy in the rat

The effects of estradiol treatment on the development of myometrial gap junctions and premature labour were investigated using timed pregnant rats. In control animals myometrial gap junctions were infrequent between days 17 and 20 of pregnancy, but began to develop on day 21 and were at maximum frequency, size, and membrane area on day 22 during delivery. Gap junctions were completely absent from the myometrium 48 h after delivery. Animals treated with 500 micrograms 17 beta-estradiol/day starting on day 16 of pregnancy developed numerous myometrial gap junctions and delivered their pups prematurely on day 19. Similarly, treatment with 50 micrograms estradiol/day resulted in the development of myometrial gap junctions on day 20 of pregnancy and premature labour. However, treatment with various doses of estradiol up to and including 500 micrograms/day for 3 days beginning 1 day before delivery was not able to maintain the presence of myometrial gap junctions during the postpartum period. These results support the hypothesis that estradiol stimulates the development of myometrial gap junctions and that the presence of gap junctions in the myometrium is a requirement for the occurrence of term, as well as preterm labour. Furthermore, it is evident from this study that the postpartum regression of myometrial gap junctions is not dependent on the decrease in estradiol.     

The Role of Cellular Coupling in the Spontaneous Generation of Electrical Activity in Uterine Tissue

The spontaneous emergence of contraction-inducing electrical activity in the uterus at the beginning of labor remains poorly understood, partly due to the seemingly contradictory observation that isolated uterine cells are not spontaneously active. It is known, however, that the expression of gap junctions increases dramatically in the approach to parturition, by more than one order of magnitude, which results in a significant increase in inter-cellular electrical coupling. In this paper, we build upon previous studies of the activity of electrically excitable smooth muscle cells (myocytes) and investigate the mechanism through which the coupling of these cells to electrically passive cells results in the generation of spontaneous activity in the uterus. Using a recently developed, realistic model of uterine muscle cell dynamics, we investigate a system consisting of a myocyte coupled to passive cells. We then extend our analysis to a simple two-dimensional lattice model of the tissue, with each myocyte being coupled to its neighbors, as well as to a random number of passive cells. We observe that different dynamical regimes can be observed over a range of gap junction conductances: at low coupling strength, corresponding to values measured long before delivery, the activity is confined to cell clusters, while the activity for high coupling, compatible with values measured shortly before delivery, may spread across the entire tissue. Additionally, we find that the system supports the spontaneous generation of spiral wave activity. Our results are both qualitatively and quantitatively consistent with observations from in vitro experiments. In particular, we demonstrate that the increase in inter-cellular electrical coupling observed experimentally strongly facilitates the appearance of spontaneous action potentials that may eventually lead to parturition.     

Regulation of myometrial beta 2-adrenergic receptors by progesterone and estradiol-17 beta in late pregnant rats

Rat myometrium exhibited a marked decrease in the concentration of beta 2-adrenergic receptors immediately before parturition, i.e., in the last 6 h of pregnancy. This phenomenon continued until the withdrawal of myometrial progesterone (-94% from Day 18 of pregnancy to term) and coincided with the sharp increase (+200%) of the myometrial concentration of estradiol. A linear positive correlation was found (r2 = 0.645) between the concentration of beta 2-adrenergic receptors and the log ratio of myometrial concentration of progesterone/myometrial concentration of estradiol (P/E2), suggesting a modulation of beta 2-adrenergic receptors by steroids. In rats with estrogen-dominated uteri (intact of ovariectomized late pregnant rats injected with estradiol), there was no change either in concentration or affinity of beta 2-adrenergic receptors relative to untreated control pregnant rats. In contrast, rats with progesterone-dominated uteri (intact or ovariectomized late pregnant rats treated with progesterone or ovariectomized rats) have an increased number of beta 2-adrenergic receptors, with a decreased affinity of these receptors compared to untreated control pregnant rats or to estrogen-treated rats. These results suggest that progesterone regulates the number of beta 2-adrenergic receptors in myometrium of late pregnant rats. The mechanisms by which progesterone exerts this regulation remains to be elucidated.     

Gap junction modulation in rat uterus. III. Structure-activity relationships of estrogen receptor-binding ligands on myometrial and serosal cells

A number of steroidal and nonsteroidal estrogen receptor-binding ligands were tested for their ability to affect the formation and internalization of gap junctions in hypophysectomized rat uterine myometrial and serosal cells. Potent estrogen, including diethylstilbestrol, estradiol benzoate (EB), estradiol-17 beta, and the weak estrogens, estriol and estrone, stimulate formation of macular and annular gap junctions in myometrium in a dose-dependent fashion when administered in daily injections over 5 days. Induction of annular gap junctions in the uterine serosal epithelium follows a similar dose-dependent pattern of estrogen stimulation but requires lower levels of hormone to initiate the response. In myometrium, differential stimulation of circular and longitudinal myometrial cell layers was observed, with 3 to 5 times more gap junctions detected in the circular than in the longitudinal layer. Progesterone, estriol, or estrone suppress the myometrial gap junction response to EB when administered concurrently with EB. However, the EB-stimulated appearance of myometrial cell gap junctions was blocked when the progesterone-to-estrogen ratio exceeded 100:1. The estrogen receptor-binding androgens, 5 alpha-androstane-3 beta,17 beta-diol (Adiol) and delta 5-androstene-3 beta,17 beta-diol failed to induce myometrial gap junctions at doses up to 5 mg/day for 5 days, whereas Adiol did induce annular gap junctions in the serosal cells at the highest dosage tested. Of the triphenylethylene derivatives and related compounds evaluated, including mixed isomers of tamoxifen and CI 628, the cis (zuclomiphene, ZUC) and trans (enclomiphene) isomers of clomiphene citrate, and a fixed-ring antiestrogen, nafoxidine, only ZUC was able to induce gap junctions in myometrial and serosal cells. These studies indicate that induction of gap junctions in rat uterine myometrial cells is an estrogen-dependent response that requires higher levels of estrogen than other estrogen-dependent target cell responses in the rodent uterus.     

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent^1,2. In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue.Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions^3,4. Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling^3-8. For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation^7,8. However, not only are these studies extremely difficult to perform on neurons with small somata in intact neural retinal tissue, but it can be difficult to adequately control the illumination conditions during the electrophysiological study of single retinal neurons to avoid light-induced changes in gap junction conductance.Here, we present a straightforward method of determining the extent of gap junction tracer coupling between retinal neurons under different illumination conditions and at different times of the day and night. This cut-loading technique is a modification of scrape loading^9-12, which is based on dye loading and diffusion through open gap junction channels. Scrape loading works well in cultured cells, but not in thick slices such as intact retinas. The cut-loading technique has been used to study photoreceptor coupling in intact fish and mammalian retinas^{7, 8,13}, and can be used to study coupling between other retinal neurons, as described here.     

Steroid hormone modulation of prostaglandin secretion in the ruminant endometrium during the estrous cycle

Prostaglandins, produced from membrane phospholipids by the action of phospholipase A2, cyclooxygenase, and specific prostaglandin synthases, are important regulators of ovulation, luteolysis, implantation, and parturition in reproductive tissues. Destruction of the corpus luteum at the end of the estrous cycle in nonpregnant animals is brought about by the pulsatile secretion of prostaglandin F(2alpha) (PGF(2alpha)) from the endometrium. It has been known for many years that progesterone, estradiol, and oxytocin are the hormones responsible for luteolysis. To achieve luteolysis, two independent processes have to be coordinated; the first is an increase in the prostaglandin synthetic capability of the endometrium and the second is an increase in oxytocin receptor number. Although progesterone and estradiol can modulate the expression of the enzymes involved in prostaglandin synthesis, the primary reason for the initiation of luteolysis is the increase in oxytocin receptor on the endometrial epithelial cells. Results of many in vivo studies have shown that progesterone and estradiol are required for luteolysis, but it is still not fully understood exactly how these steroid hormones act. The purpose of this article is to review the recent data related to how progesterone and estradiol could regulate (initiate and then turn off) the uterine pulsatile secretion of PGF(2alpha) observed at luteolysis.     

Staphylococcus aureus-derived peptidoglycan induces Cx43 expression and functional gap junction intercellular communication in microglia

Gap junctions serve as intercellular conduits that allow the exchange of small molecular weight molecules (up to 1 kDa) including ions, metabolic precursors and second messengers. Microglia are capable of recognizing peptidoglycan (PGN) derived from the outer cell wall of Staphylococcus aureus, a prevalent CNS pathogen, and respond with the robust elaboration of numerous pro-inflammatory mediators. Based on recent reports demonstrating the ability of tumor necrosis factor-alpha and interferon-gamma to induce gap junction coupling in macrophages and microglia, it is possible that pro-inflammatory mediators released from PGN-activated microglia are capable of inducing microglial gap junction communication. In this study, we examined the effects of S. aureus-derived PGN on Cx43, the major connexin in microglial gap junction channels, and functional gap junction communication using single-cell microinjections of Lucifer yellow (LY). Exposure of primary mouse microglia to PGN led to a significant increase in Cx43 mRNA and protein expression. LY microinjection studies revealed that PGN-treated microglia were functionally coupled via gap junctions, the specificity of which was confirmed by the reversal of activation-induced dye coupling by the gap junction blocker 18-alpha-glycyrrhetinic acid. In contrast to PGN-activated microglia, unstimulated cells consistently failed to exhibit LY dye coupling. These results indicate that PGN stimulation can induce the formation of a functional microglial syncytium, suggesting that these cells may be capable of influencing neuro-inflammatory responses in the context of CNS bacterial infections through gap junction intercellular communication.     

Myometrial connexin 43 trafficking and gap junction assembly at term and in preterm labor.

Modulation of connexin 43 (cx43) in the myometrium of timed pregnant rats was studied using enzyme-linked immunosorbent assay (ELISA), immunocytochemical localization, and immunoblot. These techniques utilized site-specific antibodies directed against a portion of the carboxyl tail of cx43. We found that cx43 is synthesized several days prior to labor but accumulates within the cytoplasm until parturition, when it is rapidly transported to the plasma membrane and assembled into gap junction plaques at the cell surface. These cx43-positive gap junctions begin to disappear from the plasma membrane within hours of delivery of the last pup and are completely absent within 24 hr following delivery. These structures are apparently internalized and degraded within the cytoplasm. ELISA documents a reduction of total cellular cx43 to baseline levels within 5 days following parturition. While the timing of synthesis, cytoplasmic storage, concentration in apparent Golgi vesicles, and transport to and assembly in the plasma membrane are accelerated in three models of preterm labor, the sequence of these events and the correlation of parturition with the formation of gap junctions are identical to those documented in normal labor. These results support the hypothesis that effective labor requires the synthesis and assembly of cx43 into functional gap junctions at the myometrial cell surface.     

Hormonal modulation of gap junctions in rat ovarian follicles

Summary Homocellular gap junctions between granulosa cells and between theca interna cells, and heterocellular gap junctions between granulosa cells and oocytes persist in rat ovarian follicles for as long as 90 days following hypophysectomy. Gonadotrophic and/or steroid hormones are therefore not required for the maintenance of gap junctions between these cells during early follicular growth. However, replacement therapy with estrogen and human chorionic gonadotrophin results in amplification of gap junctions in granulosa and theca interna cells respectively. Within 24 h following hormonal stimulation, growth of gap junctions is characterized by the appearance of formation plaques as observed in freeze-fracture replicas and by the association of microfilamentous material located subadjacent to gap junction membrane observable in thin-sectioned cells.     

Voltage-clamp studies of gap junctions between uterine muscle cells during term and preterm labor.

Gap junctions between myometrial cells increase dramatically during the final stages of pregnancy. To study the functional consequences, we have applied the double-whole-cell voltage-clamp technique to freshly isolated pairs of cells from rat circular and longitudinal myometrium. Junctional conductance was greater between circular muscle-cell pairs from rats delivering either at term (32 +/- 16 nS, mean +/- SD, n = 128) or preterm (26 +/- 17 nS, n = 33) compared with normal preterm (4.7 +/- 7.6 nS, n = 114) and postpartum (6.5 +/- 10 nS, n = 16); cell pairs from the longitudinal layer showed similar differences. The macroscopic gap junction currents decayed slowly from an instantaneous, constant-conductance level to a steady-state level described by quasisymmetrical Boltzmann functions of transjunctional voltage. In half of circular-layer cell pairs, the voltage dependence of myometrial gap junction conductance is more apparent at smaller transjunctional voltages (< 30 mV) than for other tissues expressing mainly connexin-43. This unusual degree of voltage dependence, although slow, operates over time intervals that are physiologically relevant for uterine muscle. Using weakly coupled pairs, we observed two unitary conductance states: 85 pS (85-90% of events) and 25 pS. These measurements of junctional conductance support the hypothesis that heightened electrical coupling between the smooth muscle cells of the uterine wall emerges late in pregnancy, in preparation for the massive, coordinate contractions of labor.     

Ultrastructure, histological distribution, and freeze-fracture immunocytochemistry of gap junctions in rat brain and spinal cord

The historical development of concepts of gap junctions as sites for electrical, ionic, and metabolic coupling is reviewed, from the initial discovery of gap junctions linking heart cells, to the current concepts that gap junctions represent 'electrotonic synapses' between neurons. The ultrastructure and immunocytochemistry of gap junctions in heart, brain, and spinal cord of adult rats is examined using conventional thin sections, negative staining, grid-mapped freeze-fracture replicas, and immunogold-labeled freeze-fracture replicas. We review evidence for neuronal gap junctions at 'mixed' (combined electrical and chemical) synapses throughout adult rat spinal cord. We also show immunogold labeling of connexin43 in astrocyte and ependymocyte gap junctions and of connexin32 in oligodendrocyte gap junctions. Ultrastructural and freeze-fracture immunocytochemical methods have provided for definitive determination of the number, size, histological distribution, and connexin composition of gap junctions between neurons in all regions of the central nervous systems of vertebrate species.     

Electrical Transmission between Mammalian Neurons is Supported by a Small Fraction of Gap Junction Channels

Electrical synapses formed by gap junctions between neurons create networks of electrically coupled neurons in the mammalian brain, where these networks have been found to play important functional roles. In most cases, interneuronal gap junctions occur at remote dendro-dendritic contacts, making difficult accurate characterization of their physiological properties and correlation of these properties with their anatomical and morphological features of the gap junctions. In the mesencephalic trigeminal (MesV) nucleus where neurons are readily accessible for paired electrophysiological recordings in brain stem slices, our recent data indicate that electrical transmission between MesV neurons is mediated by connexin36 (Cx36)-containing gap junctions located at somato-somatic contacts. We here review evidence indicating that electrical transmission between these neurons is supported by a very small fraction of the gap junction channels present at cell-cell contacts. Acquisition of this evidence was enabled by the unprecedented experimental access of electrical synapses between MesV neurons, which allowed estimation of the average number of open channels mediating electrical coupling in relation to the average number of gap junction channels present at these contacts. Our results indicate that only a small proportion of channels (~0.1?%) appear to be conductive. On the basis of similarities with other preparations, we postulate that this phenomenon might constitute a general property of vertebrate electrical synapses, reflecting essential aspects of gap junction function and maintenance.     

Effects of sexual steroid hormones on the functionality of murine peritoneal macrophages.

In the present work, we studied the possible effect of steroid hormones, estradiol, progesterone, and 5 alpha-dihydrotestosterone, on different phenotypic and functional characteristics of peritoneal adherent mononuclear cells. We used female and male mice of Balb/c strain, normal, gonadectomized, and gonadectomized with hormonal replacement. We found that gonadectomy in both sexes produced a significant decrease in the functionality of membrane receptors for the complement and in phagocytic activity of Candida albicans-anti-C albicans system. In addition, the percentages of cells that reduced nitroblue tetrazolium were diminished in castrated animals. Ovariectomized females injected with estradiol presented normal levels of phagocytic and metabolic capacities, but the expression of membrane receptors for complement remained decreased. In contrast, progesterone treatment of ovariectomized animals had the opposite effect. Simultaneous treatment with estradiol plus progesterone gave results similar to those observed with estradiol only. Dihydrotestosterone per se did not affect any of the parameters measured in the conditions used here. These results suggest that female steroids affect macrophage functionality, probably by regulating surface receptors that are involved in phagocytic activity.