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Early embryos of Patella vulgata have been injected with Lucifer Yellow. No restriction of dye spread was found. We show that later in the development, the larval trochophore stage present evidence of compartments of cell communication. These dye compartments coincide with different presumptive regions.
Organotypic cultures, prepared from young rats, were used to investigate the neuroendocrine properties of anterior pituitary cells. Pituitary cells maintained the features of endocrine cells, up to 7 weeks in vitro. Secretory granules could be seen with electron microscopy, and cells contained immunocytochemically detectable levels of adenohypophyseal hormones. Significant levels of prolactin (PRL), growth hormone and luteinizing hormone were present in the culture media after several weeks in vitro and PRL release could be modulated by dopaminergic agonists or forskolin. The electrophysiological properties of pituitary cells were investigated with both intracellular and patch-clamp recordings after 2 to 7 weeks in vitro. Cellular resting membrane potentials were approximately -50 mV, and spontaneous or depolarization-induced action potentials were found in approximately 50% of cells. Records of voltage-dependent outward membrane currents showed that cells expressed functional voltage-gated channels. Cells remained responsive to hypothalamic neuropeptides, as shown by the outward membrane current triggered by thyrotropin-releasing hormone. Intracellularly injected Lucifer Yellow readily diffused between neighboring cells, suggesting the presence of gap junctions. These data confirm the viability of organotypic cultures of the anterior pituitary gland, and demonstrate that the characteristic properties of this excitable endocrine tissue are conserved. This neuroendocrine preparation is suitable for studying the mechanisms regulating cell-to-cell communication under conditions resembling the in vivo tissue organization.
Epidermal growth factor (EGF) affects follicular steroidogenesis and expression of gonadotropin receptors. The effects of EGF on hCG-induced estradiol and progesterone secretion and ovulation were examined in the in vitro perfused rabbit ovary. We also examined the effects of EGF on hCG-induced progesterone secretion by isolated granulosa cells. In addition, distribution of hCG within the follicle was probed by immunohistochemical means 30 min after its administration to the in vitro perfused ovary. EGF significantly (P less than 0.05) reduced hCG-induced secretion of estradiol (control, 117 +/- 12 pg/min.follicle; 10 ng/ml EGF, 55 +/- 10) and progesterone (control, 18.2 +/- 1.2 ng/min.follicle; 10 ng/ml EGF, 11.9 +/- 0.8) by the perfused ovary. In contrast, EGF did not inhibit hCG-induced progesterone secretion by isolated granulosa cells. Ovulatory efficiency (number of ovulated ova per number of mature follicles x 100) when EGF was given 30 min before hCG was reduced dose-dependently from 58.2% with no EGF to 8.3% with 10 ng/ml EGF (P less than 0.001). Ovulation was not inhibited by EGF when it was given 30 min after hCG. Distribution of hCG in the preovulatory follicle was confined to the basement membrane, thecal cell layer, and a small fraction of the outer granulosa cell layer. These observations suggest that gonadotropin stimulates the follicle through the release of a secondary signal(s) from ligand-bound granulosa cells near the follicle wall to unexposed cells of the inner avascular area. EGF may inhibit the follicular response to hCG by attenuation of this cell to cell communication.
The effects of cAMP-dependent protein kinase A and protein kinase C on cell-cell communication have been examined in primary ovarian granulosa cells microinjected with purified components of these two regulatory cascades. These cells possess connexin43 (alpha 1)-type gap junctions, and are well-coupled electrotonically and as judged by the cell-to-cell transfer of fluorescent dye. Within 2-3 min after injection of the protein kinase A inhibitor (PKI) communication was sharply reduced or ceased, but resumed in about 3 min with the injection of the protein kinase A catalytic subunit. A similar resumption also occurred in PKI-injected cells after exposure to follicle stimulating hormone. Microinjection of the protein kinase C inhibitor protein caused a transient cessation of communication that spontaneously returned within 15-20 min. Treatment of cells with activators of protein kinase C, TPA or OAG for 60 min caused a significant reduction in communication that could be restored within 2-5 min by the subsequent injection of either the protein kinase C inhibitor or the protein kinase A catalytic subunit. With a longer exposure to either protein kinase C activator communication could not be restored and this appeared to be related to the absence of aggregates of connexin43 in membrane as detected immunologically. In cells injected with alkaline phosphatase communication stopped but returned either spontaneously within 20 min or within 2-3 min of injecting the cell with either the protein kinase A catalytic subunit or with protein kinase C. When untreated cells were injected with protein kinase C communication diminished or ceased within 5 min. Collectively these results demonstrate that cell-cell communication is regulated by both protein kinase A and C, but in a complex interrelated manner, quite likely by multiple phosphorylation of proteins within or regulating connexin-43 containing gap junctions.
In the field of osseous substitution, the possibilities being offered to the surgeons prove sometimes difficult to apply in particular in the case of great losses of osseous substance. For these reasons, it is necessary to develop innovative techniques to satisfy the request increasing for substitutes and to see appearing on the market solutions combining availability, perenniality and biosecurity of the implants. The implantation of stem cells in a biomaterial opens a way of development of therapeutic substitute. Moreover, in order to optimize the rehabitation of the biomaterials by the cells and the host tissues, the second approach consists in modifying the surface of materials by the coating or the grafting of adhesive factors in order to stimulate their colonization. At least, one cannot consider a tissue mechanism of repair without a better knowledge of the respective role of the various cell populations implied in the rebuilding of this tissue and their cell to cell communication processes.
Mammalian ecto ADP-ribosyltransferases (ARTs) constitute a family of structurally related proteins expressed on the cell surface or secreted in the extracellular compartment. Using NAD+ as substrate, they transfer ADP-ribose groups onto target proteins. In contrast to intracellular poly(ADP-ribosyl)transferases (PARPs), these enzymes transfer a single ADPR and are thus mono-ARTs. Five paralogs (ART1-5) have been cloned but only four of them are expressed in human due to a defective ART2 gene, and six in the mouse as the result of ART2 gene duplication. The recent determination of the crystal structure of rat ART2 reveals homologies with bacterial ART toxins and provides a molecular basis for understanding the specificity of ARTs for their targets. A combination of different technological approaches reveals that ecto-ARTs are expressed in different tissues with privileged sites such as heart and skeletal muscles for ART1, T lymphocytes for ART2 or testis for ART5. It also indicates that ART expression is highly regulated. ADP-ribosylation of target proteins on cell surfaces or circulating in body fluids leads to reversible post-translational modifications which can inhibit the targets, as known for bacterial ARTs, or activate them, as in the crosstalk between mouse ART2 and the cytolytic P2X7 receptor on T lymphocytes. ART activity in the extracellular compartment provides sophisticated regulatory mechanisms for cell communication. This designates ecto-ARTs as new candidates for drug targeting.
For many years it has been known that plants perform rapid long-distance signalling using classical action potentials that have impacts on diverse processes in plants. Plants also synthesize numerous neuronal molecules and fulfill some criteria for intelligent behaviour. Analysis of recent breakthrough data from ecophysiology studies has revealed that plant roots can discriminate between 'self' and 'non-self'; in animals, this ability to discriminate is dependent on the activities of neuronal synapses. Here, we propose that plant cells establish modes of information exchange between each other that have properties in common with neuronal synapses. Moreover, plants also assemble adhesive contacts that orchestrate cell-to-cell communication between the host cells when challenged with pathogens, parasites and potential symbionts. We propose that these adhesive contacts resemble the immunological synapses found in animals.
The effects of 8-bromo-cAMP on gap junction expression and intracellular Ca2+ ([Ca2+]i) were examined in an immortalized human myometrial cell line (PHM1-41) generated from myometrial cells obtained from term-pregnant myometrium. PHM1-41 cells express the gap junction protein connexin43 (Cx43). Gap junction-mediated intercellular communication, monitored with a method using fluorescence recovery after photobleaching, revealed low communication rates between cells. Addition of 1.0 mM 8-bromo-cAMP to culture medium resulted in an increase in junctional communication within 5 min, while 30 microM forskolin treatment resulted in increased communication within 10 min. Cells treated with 0.1, 0.5, or 1.0 mM 8-bromo-cAMP for 24 h revealed a dose-dependent increase in communication and an increase in immunostained gap junction plaques at cell-cell contacts. A direct correlation between the rate of dye transfer and the number of plaques was detected. No effect on communication or immunoreactive Cx43 was detected in cells exposed to 10 or 50 nM estradiol-17 beta for 24 h. In experiments analyzing [Ca2+]i, a concentration-dependent increase was induced by oxytocin (1-100 nM) with half-maximal stimulation at approximately 5 nM. Treatment of PHM1-41 cells for 24 h with 0.1, 0.5, or 1.0 mM 8-bromo-cAMP reduced the magnitude of the oxytocin-induced [Ca2+]i response. Thus, 8-bromo-cAMP increased Cx43-containing cell surface gap junctional plaques and up-regulated junctional communication but attenuated the oxytocin-induced [Ca2+]i response. These data indicate that the action of 8-bromo-cAMP in PHM1-41 cells has potentially opposite effects on the contractile and conductive properties of cells, and suggest that up-regulation of myometrial conductivity by agents that increase cAMP could diminish the tocolytic benefits of these agents.
In mammals, bone marrow is the principal tissue where blood is formed during adult life. Paracrine factors are generally considered to control this process but there is considerable evidence that gap junctions are present in haemopoietic tissues. Gap junctions have been implicated in developmental and patterning roles, and we set out to characterize the cells which are coupled, and to provide evidence for their role(s) in blood cell formation. Direct cell-cell communication, shown by dye-transfer, occurs between haemopoietic cells and certain stromal cells. In culture these stromal cells form a mat in which they retain their dye-coupling properties. Freeze-fracture electron microscopy confirms that this coupling is via gap junctions. When haemopoietic cells are cultured on top of these mats dye spreads upwards from the stromal cells into the haemopoietic cells above. Experiments in which haemopoietic cells were cultured alone, with stromal cell conditioned medium, or in direct contact with stromal cell underlays, were therefore carried out. The results of these experiments provide evidence that gap junctional communication may be playing a vital role in maintaining populations of precursor cells which would otherwise differentiate into end cells, leading to the ultimate demise of the system.
The development of the fetal lung is regulated by fibroblast-type-II cell communications which involve fibroblast pneumonocyte factor (FPF). FPF production is positively regulated by glucocorticoids and negatively regulated by dihydrotestosterone (DHT) and transforming growth-factor beta (TGF-beta). We studied whether DHT or TGF-beta affected other steps in the process of lung maturation, by studying how the developing lung in organ culture would respond to exogenously supplied FPF after DHT or TGF-beta exposure. Fetal rabbit (day 19 of gestation) lung organ cultures were prepared and cultured in the presence of cortisol, DHT or TGF-beta. After seven days, the media were replaced with serum-free medium containing either cortisol or FPF conditioned medium. The incorporation of [14C]glycerol into surfactant lamellar body DSPC was studied over 24 h as the index of surfactant synthesis. Results were compared to simultaneous control cultures. Treatment had no significant effect on tissue protein concentration or on the efficiency of lamellar body recovery. Cortisol stimulated baseline incorporation of glycerol into DSPC. This was inhibited by DHT, such that DHT plus cortisol treatment was no different from untreated controls. FPF stimulated the incorporation of glycerol into DSPC, and did so even after culture treatment with DHT. Cultures treated with TGF-beta exhibited glycerol incorporation similar to untreated controls. After TGF-beta exposure, FPF did not stimulate glycerol incorporation into DSPC. We conclude that DHT interferes with progression of lung development by delaying the appearance of FPF production by the fibroblast. TGF-beta, on the other hand, inhibits other elements of lung maturation besides FPF production. We speculate that TGF-beta interferes with type-II cell development such that the cell cannot respond to FPF.
Several studies have demonstrated that ethanol can increase hepatic O2 uptake (e.g., produce a hypermetabolic state); however, a complete explanation of this important phenomenon remains unclear. Here, the effect of conditioned media from Kupffer cells isolated from rats chronically exposed to ethanol on O2 consumption of normal parenchymal cells was studied to evaluate the possibility that cell-cell communication participates in the mechanism of the hepatic hypermetabolic state. Kupffer cells were isolated from rats fed either a liquid control diet or a diet containing ethanol. Kupffer cells were cultured for 4 h, and conditioned media were incubated with parenchymal cells isolated from untreated rats in a closed chamber with an O2 electrode. O2 consumption of parenchymal cells incubated in fresh media or conditioned media from Kupffer cells from untreated rats was approximately 30 microliters.h-1. 10(6) cells-1; however, values were increased by > 30% by conditioned media from Kupffer cells isolated from rats treated with ethanol. Indomethacin, nisoldipine, and boiling the conditioned media blocked this stimulation, suggesting the involvement of eicosanoids. Indeed, prostaglandin E2 (PGE2) added directly to parenchymal cells increased O2 consumption in a dose-dependent manner by nearly 60%. Furthermore, PGE2 levels in conditioned media from Kupffer cells isolated from ethanol-treated rats were elevated about twofold. The addition of endotoxin to cultured cells caused a similar phenomenon. Taken together, these data support the hypothesis that Kupffer cells are activated by ethanol treatment to release mediators such a PGE2, which stimulate O2 consumption in parenchymal cells, possibly by mechanisms involving bacterial endotoxin.
We have recently described a spontaneous murine model of autoimmune thyroid disease. The disorder was in part characterized by reduced thyroid epithelial cell-cell communication that was associated with abnormalities in three major connexins. To compare whether this finding was a common secondary occurrence in autoimmune thyroid disease, or unique to the spontaneous development in the MRL mice, we induced thyroiditis in Lewis rats. Immunization with thyroid extract and thyroglobulin resulted in extensive lymphocytic infiltration and increased expression of major histocompatibility gene complex (MHC) class II surface antigen in the diseased thyroid. Both experimental and control rat thyroid tissues produced gap junction proteins connexin 43, connexin 32, and connexin 26. The connexins in nondiseased tissue was located in the plasma membrane at points of cell-cell contact and labeled as discrete arrays of punctate fluorescence. The quantity of all three connexins were reduced in the diseased thyroid tissue. More importantly, the connexin proteins were not distributed as gap junctions at contacting cell interfaces. Both nondiseased and diseased thyroid tissue expressed messenger RNA (mRNA) for the three connexins, but the diseased tissue had reduced levels of mRNA for connexin 43 (45%), and to a lesser extent, connexin 26 (25%) and connexin 32 (20%). The reduced connexin mRNA, protein, and lack of assembled gap junctions measured in the diseased tissue were obtained under conditions where the infiltrating cells and their potent cytokine products were continuously present. To determine if this difference persisted when these inflammatory components were absent, primary cultures of thyroid cells from control and experimental rats were established and connexin localization experiments repeated. The diseased thyroid cells, like the diseased tissue, lacked plasma membrane associated connexin protein. The lack of gap junction assembly in the thyrocytes cultured from the diseased tissue was accompanied by a loss of functional coupling. Collectively, the data document that autoimmune diseased thyroid tissue from both the spontaneous mouse and induced rat models have reduced plasma membrane assembled gap junctions and deficient intercellular communication as determined by the inability to transfer lucifer yellow dye to contiguous cells. Nondiseased cultured thyrocyte monolayers and follicles transferred dye to second and third order neighboring cells in 80 and 95% of trials, respectively. In contrast, only 5-10% of the diseased thyrocytes transferred microinjected dye, and in these cases the transit was limited to primary contacting cells. Culturing removed inequities introduced by the infiltrating cells and their products. However, the established cultures of diseased thyroid cells retained their communication deficiency. This suggests that the loss of communication may be a common abnormality in autoimmune disease, and furthermore, this uncoupling could contribute to the loss of coordinated hormonal regulation (hypothyroidism) in the diseased thyroid gland in the absence of thyroid cell destruction.
Emerging evidence indicates that coagulation-related molecular programs are associated with thrombosis, tumor progression, and molecular dysregulation in gastric cancer (GC). However, thrombosis-associated molecular features in GC and their potential links to inherited susceptibility remain insufficiently understood. Integrated analyses of transcriptomic data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets were performed to identify thrombosis-associated genes and establish a machine learning-based prognostic signature. Genome-wide association study (GWAS), expression quantitative trait loci (eQTL), transcriptome-wide association study (TWAS), and Mendelian randomization (MR) analyses were conducted to investigate susceptibility-associated transcriptional programs in GC. Functional assays were used to evaluate candidate genes associated with malignant phenotypes. Single-cell RNA sequencing (scRNA-seq) and cell-cell communication analyses were further performed to characterize cell-type-specific expression patterns and potential intercellular interactions. A total of 22 differentially expressed thrombosis-associated genes were identified, and a prognostic signature comprising 14 genes was established. The signature stratified patients into high- and low-risk groups and showed prognostic performance in both the training and validation cohorts. Integrative GWAS, eQTL, and TWAS analyses identified susceptibility-associated transcriptional programs that were positively correlated with the thrombosis-associated risk score. Silencing ACTN2 and CRYAB significantly reduced GC cell migration and invasion. scRNA-seq analysis revealed relatively high CRYAB expression in neutrophils, and CellChat analysis suggested potential neutrophil-B cell interactions involving COLLAGEN-related signaling. This integrative multi-omics study identified a thrombosis-associated molecular signature linked to prognosis and germline susceptibility-associated transcriptional programs in GC. ACTN2 and CRYAB may represent candidate genes associated with GC cell migration and invasion, while single-cell analysis suggested potential immune-related communication features.
A pSV3neo-transfected rat ovarian cell line (SV-GC) was developed from a primary granulosa culture (GC) to study gap junctional intercellular communication (GJIC) during Simian virus 40 (SV40) transformation. SV-GC expressed SV40 large T-antigen (T-ag), grew indefinitely in culture without luteinization, was anchorage independent, and formed tumors in nude mice. Ultrastructural analysis identified abundant gap junctional membrane and suggested that SV-GC was arrested at an early stage of differentiation. Functional GJIC, measured by a dye transfer technique (gap FRAP), was comparable to that observed in normal granulosa cells, suggesting that the expression of T-ag alone was insufficient to reduce GJIC. However, there was approximately a 50% loss in the rate of GJIC in the nude mouse SV-GC-tumor derived and G418 selected cell line (T-SV-GC). SV-GC----T-SV-GC also resulted in a transition from migration of cells as an epithelial sheet to the dissociation of individual fibroblastoid cells. Tumor cell detachment was also seen in migrating malignant human (A2780 and 547) and rat (DC3) ovarian cell lines. Co-culture combinations of normal (GC)----transformed (SV-GC)----tumor-derived (T-SV-GC) cells indicated that the rate of heterologous GJIC was characteristic of the least communicating partner. Taken together, these data suggested that SV-GC----T-SV-GC represented progression toward metastasis with concomitant reduction of GJIC and adhesiveness. These sequentially derived cell lines may be a useful in vitro model system for studies focusing on the mechanism involved in the detachment of cells during the progression of ovarian cancer.
In conclusion, many of the activities of IFN are transmissible between cells and may involve more than one transfer mechanism. These mechanisms probably involve secondary messenger molecules which are shared with hormones and act at the transcriptional level. The transfer system seems to be a major amplification process for IFN action. It will be interesting in the future to determine the spectra of IFN activities which are transferable as well as the processes involved.
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The effects of theophylline and di-butyryl cyclic adenosine 3',5'-monophosphate (db-cAMP) on the electrical coupling of heart cells were investigated in rat trabeculae. Theophylline (4 X 10(-4) M) and db-cAMP (5 X 10(-5) M) increased both the space constant and conduction velocity. The time constant of the membrane was not changed by either drug. Measurements of the time constant of the foot of the action potential and conduction velocity were used to calculate the intracellular longitudinal resistance. Both theophylline and db-cAMP were found to enhance cell-to-cell communication in the heart by decreasing the intracellular longitudinal resistance.
Under normal homeostasis, melanocyte growth and behaviour is tightly controlled by the surrounding keratinocytes. Keratinocytes regulate melanocyte behaviour through a complex system of paracrine growth factors and cell-cell adhesion molecules. Pathological changes, leading to development of malignant melanoma, upset this delicate homeostatic balance and can lead to altered expression of cell-cell adhesion and cell-cell communication molecules. In particular, there is a switch from the E-cadherin-mediated keratinocyte-melanocyte partnership to the N-cadherin-mediated melanoma-melanoma and melanoma-fibroblast interaction. Other changes include the alteration in the gap junctions formed between the melanocyte and keratinocyte. Changes in the connexin expression, in particular the loss of connexin 43, may result in a reduction or a loss of gap junctional activity, which is thought to contribute towards tumour progression. In the current review we describe the alterations in cell-cell adhesion and communication associated with melanoma development and progression, and discuss how a greater understanding of these processes may aid the future therapy of this disease.