Intercellular communication in a positional field: movement of small ions between insect epidermal cells

The epidermal cells of the developing insect form a two-dimensional isodiffusion network of low resistance to the intercellular movement of at least one small inorganic ion. The spatial gradient in positional information found within the epidermis of each body segment could not be correlated with any quantitative regional or directional differences in intercellular ionic conductance. The lumped resistivity of the intercellular pathway during all stages of development analyzed (450 Ωcm) is only four times greater than the assumed resistivity of cytoplasm alone (100 Ω cm). The specific resistance of junctional membrane is at least three orders of magnitude less than the specific resistance of nonjunctional membrane.Little reduction in ionic conductance could be detected across the cells that form the intersegmental membrane, separating the positional fields of adjacent segments, in the pupa and adult. It is unlikely that a restricted spatial distribution of low-resistance pathways plays a role in the maintenance of the boundaries of neighboring developmental fields.No anteroposterior gradient in bioelectric potential was detected within the segment.     

Intercellular communication in normal and regenerating rat liver: a quantitative analysis

We have compared intercellular communication in the regenerating and normal livers of weanling rats. The electrophysiological studies were conducted at the edge of the liver, and we have found that here as elsewhere in the liver there is a dramatic decrease in the number and size of gap junctions during regeneration. The area of hepatocyte membrane occupied by gap junctions is reduced 100-fold 29-35 h after hepatectomy. By combining observations made with the scanning electron microscope with our freeze fracture data we have estimated the number of communicating interfaces (areas of contact between hepatocytes that include at least one gap junction) formed by hepatocytes in normal and regenerating liver. In normal liver a hepatocyte forms gap junctions with every hepatocyte it contacts (approximately 6). In regenerating liver a hepatocyte forms detectable gap junctions with, on average, only one other hepatocyte. Intercellular spread of fluorescent dye and electric current is reduced in regenerating as compared with normal liver. The incidence of electric coupling is reduced from 100% of hepatocyte pairs tested in control liver to 92% in regenerating liver. Analysis of the spatial dependence of electronic potentials indicates a substantial increase in intercellular resistance in regenerating liver. A quantitative comparison of our morphological and physiological data is complicated by tortuous pattern of current flow and by inhomogeneities in the liver during regeneration. Nevertheless we believe that our results are consistent with the hypothesis that gap junctions are aggregates of channels between cell interiors.     

Intercellular communication between cultured granulosa cells of the marmoset (Callithrix jacchus)

Summary The influence of follicle-stimulating hormone, forskolin, insulin-like growth factor type I, epidermal growth factor, and 12-tetradecanoyl-phorbol-13-acetate on marmoset granulosa cell communication via gap junctions was investigated by morphological means and microinjection of carboxyfluorescein. Gap junctions between neighbouring granulosa cells were present in all groups. The number, but not length, of gap junctions between marmoset granulosa cells increased when the cells had been treated with follicle-stimulating hormone, insulin-like growth factor type I, and follicle-stimulating hormone plus insulin-like growth factor type I. No effect on gap junctions was seen, after exposure of the cells to the other three substances. Carboxyfluorescein and counting of the surrounding labelled cells showed that supplementation with follicle-stimulating hormone, forskolin, insulin-like growth factor type I and epidermal growth factor from the beginning of cultivation led to an increase in stained cells after 48 h. When treatment was started in 48 h cultures the substances reached their maximal activity within 30 min (forskolin and epidermal growth factor) or 3 h (follicle-stimulating hormone and insulin-like growth factor type I). Spreading of the fluorescen dye was inhibited when the medium was supplemented with 12-tetradecanoyl-phorbol-13-acetate. This effect was maximal after 30 min. Additive effects regarding the coupling of the cells were seen by combining of epidermal growth factor with follicle-stimulating hormone, but not with insulin-like growth factor type I or forskolin plus follicle-stimulating hormone.     

Intercellular communication and tissue growth: IX. Junctional membrane structure of hybrids between communication-competent and communication-incompetent cells

Summary The structure of the membrane junctions of the hybrid cell system, examined in the companion paper in respect to competence for communication through cell-to-cell membrane channels, is here examined by freeze-fracture electron microscopy. The junctions of the channel-competent parent cell and of the channel-competent hybrid cells present aggregates of intramembranous particles typical of gap junction; those of the channel-incompetent parent cell and channel-incompetent segregant hybrid cells do not. Competence for junctional communication and for gap junction formation are genetically related. The junctions of the intermediate hybrid cells with incomplete channel-competence (characterized by cell-to-cell transfer of small inorganic ions but not of fluorescein), present special intramembranous fibrillar structures instead of discrete gap-junctional particles. The possibility that these structures may constitute coupling elements with subnormal permeability is discussed in terms of incomplete dominance of the genetic determinants of gap junction.     

Intercellular communication and tissue growth: VIII. A genetic analysis of junctional communication and cancerous growth

Summary Normal, proliferating cells are interconnected at their junctions by membrane channels through which molecules can pass from cell to cell (Loewenstein, W.R. 1966.Ann. N.Y. Acad. Sci.137:708). A channel-competent, normally growing cell (human fibroblast) was hybridized with a channel-incompetent cancer cell (mouse L-1d cell), and the segregant hybrid clones were analyzed in a genetic approach to the question of whether the junctional membrane channels are instrumental in transmission of growth-controlling molecular signals. The channel competence of the human parent was characterized by the ability to transfer small inorganic ions (electrical coupling) and fluorescein, and the growth patterns of this cell, by growthin vitro to low saturation densities and nontumorigenicity in immuno-suppressed hosts. The mouse parent cell had the opposite characteristics. The early hybrid generations (which still had a large part of each parent chromosome complement) were of two classes: one class resembled the human parent cell in channel competence,in vitro growth pattern, and low tumorigenicity within 26 days; the other class presented an intermediate expression of channel competence characterized by transfer of small inorganic ions but not of fluorescein. As the hybrid generations lost human chromosomes, there was segregation of several biochemical and morphological traits, but no segregation of channel competence and normal growth traits. Among the segregants were 22 clones which had reverted to the channel-incompetent trait of the mouse parent. In every case, reversion to the channel defect went hand in hand with reversion to the growth defect, just as, in the early-generation hybrids, correction of the channel defect went hand in hand with correction of the growth defect. Thus, the human genetic factor that corrects the channel defect of the mouse parent cell seems closely linked, if not identical, with that correcting the growth defect. This genetic correlation encourages us in the belief that the channel defect may be an etiological factor in this particular cancer form.     

Gap-junctional communication between feeder cells and recipient normal epithelial cells correlates with growth stimulation

LA7 rat mammary tumor cells stimulate the proliferation, in culture, of three normal epithelial cell types, namely mouse mammary, rat mammary, and mouse thymic cells. Gap-junctional communication between LA7 feeders and mouse mammary cells was demonstrated by microinjection of lucifer yellow, which traveled from LA7 to the surrounding mouse mammary cells. The amount of 3H-uridine exchange between feeder and recipient mouse mammary, rat mammary, and mouse thymus cells correlated with the growth rate induced by the feeders. Cells of the Madin Darby canine kidney (MDCK) line, which do not appreciably stimulate mouse mammary cell growth when used as feeder cells, also exchange little 3H-uridine with them. Expression of connexins Cx43, 32, and 26 was studied in all these cell lines and strains by immunocytochemistry. Mouse mammary cells expressed Cx26, and a few mouse thymic cells expressed Cx32. LA7, mouse mammary, mouse thymic, and rat mammary cells all expressed easily detectable amounts of the gap-junction protein Cx43, in contrast to MDCK cells, which expressed only a hint of the protein. These results suggest that gap junctions composed of Cx43 are those by which the normal epithelial cells communicate with the LA feeders. Thus, the ability of feeder cells to stimulate proliferation in recipients correlates with the expression of Cx43 in both members of the feeder/recipient pair and the capacity to form functional gap junctions between these cells.     

Intercellular communication of notochord cells during their differentiation in Cynops orientalis

Intercellular communication of notochord cells during their differentiation was studied by microinjection of a fluorescent dye.Lucifer Yellow,Close correlation existed between the incidences of dye coupling and quantitative evaluation of gap junctions.high incidences of dye coupling and of gap junctions occurred at a stage when notochord cells were active in the change of cell shape and cell arrangement.With the subsidence of cell movements,both dye coupling and gap junctions were reduced to lower levels.It was,therefore,Suggested that intercellular communication via gap junctions played an important role in the coordination of notochord cell movements.Gap Junctions of altered configuration occurred in notochord cells in late taibud stage.The comparison of incidences of dye coupling at this stage with those at other stages strongly suggested that the gap junctions of altered configuration functioned just as those of generalized type.     

Intercellular communication in in vivo- and in vitro-produced bovine embryos.

In vivo bovine embryos were obtained by nonsurgical flushing of uterine horns of cows submitted to superovulatory treatment, while in vitro embryos were generated from oocytes collected from slaughtered donors. Lucifer Yellow injected into single blastomeres did not diffuse into neighboring cells until the morula stage in in vivo embryos and the blastocyst stage in in vitro embryos. In both cases diffusion was limited to a few cells. In contrast, diffusion was extensive in microsurgically isolated inner cell mass (ICM) but absent in the trophectoderm (TE). At the blastocyst stage, diffusion was always more extensive in in vivo than in in vitro embryos. Ultrastructural analyses confirmed these functional observations, and gap junction-like structures were observed at the blastocyst stage. These structures were diffuse in the ICM of in vivo embryos, scarce in the ICM of in vitro embryos and in the TE of in vivo embryos, and not observed in the TE of in vitro embryos. Blastomeres at all stages of development from the 2-cell stage to the blastocyst stage in in vitro embryos and at the morula and blastocyst stage in in vivo embryos were electrically coupled, and the junctional conductance (Gj) decreased in in vitro embryos from 4.18 +/- 1.70 nS (2-cell stage) to 0.37 +/- 0.12 nS (blastocyst stage). At each developmental stage, in vivo embryos showed a significantly (P < 0. 05) higher Gj than in vitro-produced embryos. Moreover, a significantly (P < 0.01) higher Gj was found in isolated ICM than in the respective blastocyst in both in vivo- and in vitro-produced embryos (3.5 +/- 1.4 vs. 0.7 +/- 0.3 and 2.6 +/- 1.6 vs. 0.37 +/- 0. 12 nS, respectively). The electrical coupling in absence of dye coupling in the early bovine embryo agrees with observations for embryos from other phyla. The late and reduced expression of intercellular communicative devices in in vitro-produced embryos may be one of the factors explaining their developmental low efficiency.     

Intercellular communication within the rat anterior pituitary: relationship between LH-RH neurons and folliculo-stellate cells in the pars tuberalis

The distribution of LH-RH-positive nerve fibers in the median eminence was demonstrated in the 1970s and 1980s. A few LH-RH fibers have been reported to be present in the adjacent pars tuberalis of the pituitary, but their functional significance has not been clarified and still remains enigmatic. Adult male Wistar-Imamichi rats were separated into two groups: one for immunohistochemistry of LH-RH and S-100 protein (for the identification of folliculo-stellate cells) and the other for electron microscopy. For both immunohistochemistry and electron microscopy, the specimens obtained contained the pituitary gland connected with the hypothalamus. Numerous LH-RH-positive fibers were observed as tiny lines with several varicosities both on the primary vascular plexus and in the hypothalamus corresponding to the posterior half of the portal vein area. LH-RH-positive fibers were also noted around S-100-positive cells in the pars tuberalis. Weakly reactive S-100 cells were scattered in the pars tuberalis in the midsagittal plane, while clusters of strong reactive elements occurred 100–300 m from the center. Similar observations were made using fluorescence immunohistochemistry for LH-RH and S-100, and at the electron-microscopic level. At the posterior portion of the portal vein system, bundles of the LH-RH-immunoreactive fibers invaded the pars tuberalis and terminated on agranular cells. Gap junctions were clearly seen among agranular cells corresponding to folliculo-stellate cells. It is postulated that the LH-RH message might be transmitted not only by the established hypophyseal portal vein system but also via the folliculo-stellate cells in the pars tuberalis to aid in the modulation of LH release.     

Ultrastructural study of cholecystokinin-like immunoreactivity in endocrine cells of the insect midgut

Summary Electron-microscopic immunocytochemistry for the demonstration of CCK-like material in basal endocrine cells of the midgut of Aeschna cyanea, Locusta migratoria, Carausius morosus and Periplaneta americana was performed by use of the peroxidase-antiperoxidase procedure and the colloidal gold method. Immunoreactive cells appeared scattered among digestive and regenerative cells of the epithelium. Immunoreactivity was specifically detected over round to oval electron-dense granules whose size appeared rather different from species to species. Thus, the average size of 30% (d30) of the largest granules ranged from 312 nm in Periplaneta americana to 159 nm in Carausius morosus with intermediate values in Aeschna cyanea (d30=195 nm) and Locusta migratoria (d30=225 nm).     

Calcium-containing structures in vertebrate glial cells. Ultrastructural and microprobe analysis

Electron probe microanalysis has revealed that vesicular or cisternal structures containing electron-dense material in frog ependymal glial cells contain deposits of calcium and phosphorus. The so-called "osmiophilic particles" in human astrocytes also contain calcium. It is suggested that these organelles are storage sites of calcium.     

Intercellular communication and cell cooperation in growth control of T-lymphocytes

T-lymphocyte stimulation is strictly dependent on cell cooperation. Cell contact dependent cell cooperation has been described as well as cooperation by soluble mediators, the lymphokines. We here present a unifying hypothesis proposing that both types of cell cooperation are performed by the same mediators.Based on material presented at the Symposium Intercellular Communication Stuttgart, September 16–17, 1982     

Ultrastructural study of cholestasis induced by longterm treatment with estradiol valerate. I. Tight junctional analysis and tracer experiments

Over a period of 20 weeks estradiol valerate (1.5 mg/kg body weight/week) was administered subcutaneously to male Wistar rats from which the livers were examined at four week intervals employing a freeze-fracture technique and colloidal lanthanum tracer studies. In connection with intrahepatic cholestasis, distinct alterations in the tight junctions were observed, consisting of disorganization, rarification and proliferation. Disruption of the tight junctions was not seen and colloidal lanthanum did not penetrate into the bile canalicular lumen. Holding the view that the term "leakiness" of tight junctions should be defined with reference to the tracer employed, we conclude that in the liver one tight junctional strand is sufficient to prevent the escape of larger bile constituents such as bile acids and that a back diffusion of bile acids over the tight junctional barrier does not play a role in the pathogenesis of the estrogen-induced cholestasis. Interruptions of tight junctions, as described by other authors, are interpreted as a secondary mechanical effect. On the other hand, we consider an increased permeability of the tight junctions to water and small solute molecules as probable; possibly this increased permeability is brought about by alterations in the microfilaments. A model for the pathogenesis of the estrogen-induced intrahepatic cholestasis is proposed.     

Freeze-fracture and tracer studies on the intercellular junctions of insect rectal tissues.

Both rectal pads of the cockroach and rectal papillae of the blowfly possess highly infolded lateral borders; these are associated by desmosomes and septate junctions that maintain the physical integrity of the cell layer at the luminal and basal intercellular regions. Adjacent cells are coupled by gap junctions that allow for cell-to-cell communication and which occur at intervals along the undulating lateral clefts. In rectal pads, occluding basal tight junctions are found as well as extensive scalariform junctions. The latter, like the stacked membrane infoldings of rectal papillae, exhibit intercellular columns and numerous intramembranous P face particles; these are undoubtedly involved in ion transport. In the inter-stack clefts of papillae, reticular septate junctions are encountered which, after freeze-fracture, possess a striking network of PF ridges and EF grooves that are discontinuous and not always complementary. These may serve to regulate the speed and extent of distension of the clefts during solute movement to allow for even and effective fluid flow in this transporting epithelium.     

Intercellular communication and some structural aspects of membrane junctions in a simple cell system

Summary AChironomus salivary gland consists of a chain of 30 giant cells (G-cells) and 4 to 6 flat cells (F-cells) spanning a lumen. The surface membranes of these cells are linked by two kinds of organized structures: theseptate junction, taking up nearly the entire surface of cell contact, and thegap junction, occupying a small fraction of this surface. (These junctional structures are examined in the electron microscope after La infiltration.) All cells are joined to their immediate neighbors by septate junctions, the G- to G-cells, the F- to F-cells, and the F- to G-cells; the G-cells, at least, are also joined by gap junctions. All cells are also in communication with each other: small inorganic ions, fluorescein (mol. wt. 330) and Procion Yellow (mol. wt. 550) pass from one cell interior to the next.     

Freeze-fracture study of the epidermal cells of a teleost with particular reference to intercellular junctions and permeability to tracer

The plasmatic membranes, the intercellular junctions and the intercellular spaces of the epidermis of the fish Pimelodus maculatus were studied by freeze-fracture and by lanthanum methods. The observations has confirmed the presence of desmosomes. Gap junctions were not found and the tight junctions can be seen very rarely, arranged to form small discrete maculae. The finger-print pattern due to the microridges of the apical plasma membrane of the superficial cells was studied by direct replicas. The tracer penetrates all the intercellular epidermal spaces but failed to penetrate the dermis, suggesting the presence of a barrier at the dermo-epidermal level.     

Stathmokinetic analysis of human epidermal cells in vitro

Proliferation kinetics of cultured human epidermal cells is characterized in quantitative terms. Three distinct subpopulations of keratinocytes, two of which are cycling, have been discriminated by two parameter DNA/RNA flow cytometry. Based on mathematical modelling, the cell cycle parameters of the cycling subpopulations have been assessed from stathmokinetic data collected at different time points after initiation of cultures (7-15 days). The first subpopulation is composed of low-RNA cells which resemble basal keratinocytes of epidermis and which show some characteristics of stem cells; these cells have a mean generation time of approximately 100 hr. The second subpopulation consists of high-RNA cells, resembling stratum spinosum cells of epidermis, which have an average generation time of approximately 40 hr. The third subpopulation consists of non-cycling cells with G0/G1 DNA content, with cytochemical features similar to those of cells in granular layer of epidermis. The results based on modelling can reproduce with acceptable accuracy the actual growth curve of the cultured cell population. Analysis of kinetics and differentiation of human keratinocytes is of interest in view of the recent application of cultured epidermal cell sheets for transplantation onto burn wounds. The results of this study also reveal the existence of regulatory mechanisms associated with proliferation and differentiation in the cultured epidermal cell population.