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Effect of intracellular injection of La3+ and Mn2+ on electrical coupling of heart cells.

The effect of intracellular injection of La3+ and Mn2+ on the electrical coupling of canine Purkinje cells investigated. It was found that La3+ causes cell decoupling in about 100 sec, increased the input resistance of the injected cell too. Mn2+ injection reduced drastically the coupling coefficient but did not abolish cell communication. Intracellular injection of La3+ or Mn2+ also caused shortening of the action potential and hyperpolarized the the heart cells. La3+ seems to be as effective as Ca or even stronger than Ca in suppressing intercellular communication in heart fibres.

Action Potentials↗

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↗

Mechanism of pH regulation of connexin 43 expression in MC3T3-E1 cells.

Gap junction (GJ) expression and function allowing cell-cell communication among osteoblasts may be important in new bone formation. An alkaline milieu stimulates mineralization, while extracellular acidification leads to demineralization of bone. It was previously demonstrated that alkaline pH increases, while acid pH decreases GJ intercellular communication by an increase in steady-state GJ connexin 43 (Cx43) mRNA and protein expression. At pH 7.6, transcription of new Cx43 mRNA was significantly higher than that at pH 6.9 but not significantly different at pH 7.2, as assessed by nuclear run-on assay. Transcription of new Cx43 mRNA was higher at pH 7.2 compared to that at pH 6.9. Although Cx43 mRNA half-life tended to be longer at pH 7.6, analysis of variance did not yield a significant difference of the Cx43 mRNA half-life at any of the pHs tested. Likewise, the half-life of Cx43 protein at pHs of 6.9, 7.2, and 7.6 was not significantly different. Plasma membrane and cytosolic Cx43 fractions were proportionately similar at pH 7.2 and 6.9. Thus, the decrease in Cx43 mRNA at low pH compared to high pH is due to a decrease in the transcription rate of Cx43 but not due to an alteration of message stability. The early uncoupling of gap junctions by low pH found previously does not appear to be due to changes in the half-life nor distribution of Cx43 protein between cytosolic and plasma membrane compartments.

Animals↗

Immunolocalization of MP70 in lens fiber 16-17-nm intercellular junctions.

Thin section electron microscopy reveals two different types of membrane interactions between the fiber cells of bovine lens. Monoclonal antibodies against lens membrane protein MP70 (Kistler et al., 1985, J. Cell Biol., 101:28-35) bound exclusively to the 16-17-nm intercellular junctions. MP70 localization was most dramatic in the lens outer cortex and strongly reduced deeper in the lens. In contrast, the 12-nm double membrane structures and single membranes were consistently unlabeled. In freeze-fracture replicas with adherent cortical fiber membranes, MP70 was immunolocalized in the junctional plaques which closely resemble the gap junctions in other tissues. MP70 is thus likely to be associated with intercellular communication in the lens.

Animals↗

An in vitro model of ovarian epithelial carcinogenesis: changes in cell-cell communication and adhesion occurring during neoplastic progression.

To investigate the cellular mechanisms of ovarian epithelial carcinogenesis, a series of progressively transformed rat ovarian surface epithelial (ROSE) cell lines were developed and studied. Transfection of primary ROSE cells and an immortalized ROSE line (ROSE 199) with the pSV3neo plasmid (SV40 T-antigen) yielded transformed lines which retained epithelial morphology. In vivo selection of these pSV3neo cell populations resulted in further phenotypic transformation. Transfection of ROSE 199 with pSV2neo/c-H-rasEJ (rasEJp21) resulted in a malignant line which appeared fibroblast-like and formed invasive sarcomas both in athymic mice and in immunocompetent rats. Gap junctional intercellular communication (GJIC) and cell-cell adhesion were studied in this series of ROSE lines. Both c-H-rasEJ-transformation and in vivo selection resulted in a significant reduction of GJIC between adjoining cells and a transition of in vitro migration as continuous epithelial sheets to the dissociation of individual cells. This apparent shift in cell adhesiveness was associated with reduced expression of the E-cadherin adhesion molecule. Our data suggest that neoplastic progression of the ovarian surface epithelium may be associated with concomitant reductions in GJIC, E-cadherin expression and functional adhesiveness.

Animals↗

Gap junctions and fluid flow response in MC3T3-E1 cells.

In the current study, we examined the role of gap junctions in oscillatory fluid flow-induced changes in intracellular Ca(2+) concentration and prostaglandin release in osteoblastic cells. This work was completed in MC3T3-E1 cells with intact gap junctional communication as well as in MC3T3-E1 cells rendered communication deficient through expression of a dominant-negative connexin. Our results demonstrate that MC3T3-E1 cells with intact gap junctions respond to oscillatory fluid flow with significant increases in prostaglandin E(2) (PGE(2)) release, whereas cells with diminished gap junctional communication do not. Furthermore, we found that cytosolic Ca(2+) (Ca) response was unaltered by the disruption in gap junctional communication and was not significantly different among the cell lines. Thus our results suggest that gap junctions contribute to the PGE(2) but not to the Ca response to oscillatory fluid flow. These findings implicate gap junctional intercellular communication (GJIC) in bone cell ensemble responsiveness to oscillatory fluid flow and suggest that gap junctions and GJIC play a pivotal role in mechanotransduction mechanisms in bone.

Animals↗

Energy gradients for VT-signal migration in the CNS: studies on melanocortin receptors, mitochondrial uncoupling proteins and food intake.

The present paper enlightens a new point of view on brain homeostasis and communication, namely how the brain takes advantage of different chemical-physical phenomena such as pressure waves, and temperature and concentration gradients to allow the homeostasis of the brain internal milieu as well as some forms of intercellular communications (volume transmission, VT) at an energy cost much lower than the classical synaptic transmission (the prototype of wiring transmission, WT). The possible melanocortin control of uncoupling protein 2 (UCP2) expression (hence of local brain temperature gradients) has been studied in relation to food intake in male Wistar rats. Osmotic minipumps were subcutaneously (sc) implanted in the midscapular region for intracerebroventricular (icv) infusion. The control rats received an icv infusion of 0.5 microl/h of artificial cerebrospinal fluid (ACSF), while experimental rats received either an icv infusion of 0.16 nmol/h of HS024 or of 0.16 nmol/h of adrenocorticotropin-(1-24) [ACTH-(1-24)]. The ACTH-treated group ate significantly less than the ACSF-treated group during the first three days of infusion, while, subsequently, food intake of the two groups was similar. On the other hand, the HS024-treated group ate significantly more (up to 153% of the control value) than ACSF- and ACTH-treated rats during the entire period. UCP2 mRNA analysis in arcuate nuclei of ACTH, HS024 and ACSF-treated animals showed a significant 75% decrease (p<0.05 vs saline) of the total specific mRNA level in the HS024-treated group vs ACSF-treated animals (control group), while no significant change was observed between ACTH- and ACSF-treated animals. Melanocortin antagonist HS024 via blockade of MCR4 increases food intake and via a reduction of UCP2 expression enhances the food consumption ratio. This result underlines the fact that UCP2 expression and food intake can be differentially regulated. In other words, via a peptidergic control the central nervous system (CNS) can modulate the energy stored from the amount of the food that the animal has eaten and also uncouple the thermal micro-gradients (dependent on UCP2 expression) and hence the VT-signal micro-migrations from the food intake. It should also be noticed that the control of the thermal gradients affects also the neuronal firing rate and hence the transmitter release (likely above all the release of peptides such as neuropeptide Y (NPY), melanin-concentrating hormone (MCH) and beta-endorphin, e.g., in the arcuate nucleus representing signals relevant to energy homeostasis). Thus, WT and VT are both modulated by peptidergic signals that affect thermal gradients.

Adrenocorticotropic Hormone↗

Exocrine pancreas under experimental conditions. IV. Alterations of intercellular junctions between acinar cells following pancreatic duct ligation.

Exocrine pancreatic tissue was investigated during various intervals after pancreatic duct ligation. Concomitant to the increase of the intraluminal pressure, alterations are found in the arrangement of the luminal membrane surface and the zonulae occludentes of the acinar cells. The zonulae occludentes exhibit a disarrangement of their strands. The number of strands diminishes and small interruptions as well as large discontinuities of the strands are observed in most regions of the acinar cells. Furthermore, gap junctions are found very infrequently between acinar cells. The ultrastructural alterations of the zonulae occludentes suggest a gradual adaptation to the increasing unilateral pressure. However, a leakage of the paracellular permeability barrier occurs which contributes to the known shunt between the compartments of the pancreatic juice and the interstitial space following pancreatic duct obstruction. The present investigation is a further example of alterations in the paracellular permeability and intercellular communication of epithelial cells under pathological conditions.

Animals↗

Comparative studies of normal and neoplastic ovarian germ cells: 2. Ultrastructure and pathogenesis of dysgerminoma.

A series of dysgerminomas of the ovary was examined by electron microscopy and the findings were compared with germ cells in the normal developing ovary. The ultrastructural characteristics of the neoplastic cells, including prominent reticular nucleoli and sparsely distributed cytoplasmic organelles, indicated a close resemblance to oogonia present in the fetal ovary. Occasional cells had localized collections of glycogen, resembling primitive germ cells, but the irregular ameboid appearance and eccentric nuclear location characteristic of cells in the early stage were not seen. Ultrastructural features of oocyte differentiation, such as synaptinemal complexes and perinuclear arrangement of mitochondria, were not found in any of the tumors. Intercellular bridges, a frequent finding at the oogonial stage in the normal ovary, were absent in all of the tumors. The lack of intercellular communication could be responsible for uncontrolled mitotic division leading to germ cell neoplasia.

Adult↗

An electron microscope study of the development of the exocrine and endocrine pancreas with special reference to intercellular junctions.

Intercellular junctions in pancreatic acinar, duct and endocrine cells were studied by thin section and freeze-fracture methods in developing rats and mice. Undifferentiated cells were joined by the zonula occludens and isolated fragments of tight junctional strands. Small gap junctions were either occasionally associated with tight junctional strands or appeared independent of them. During the morphological differentiation of acinar cells, strands of the zonula occludens developed to form a complicated meshwork while gap junctions rapidly increased in size. Duct cells were joined by the less-developed zonula occludens but gap junctions were rarely seen. In the neonate, intercellular junctions were similar to those in adult acini and intercalated ducts. Endocrine cells were joined by maculae occludentes and small gap junctions. During late prenatal days, the macula occludens increased in size and gap junctions in number. Sometimes tight junctional strands disappeared to leave membrane elevations, some of which were associated with small gap junctions. Maculae occludentes on endocrine cells were gradually fragmentized and diminished during postnatal development. They were completely lost in the rat. These results suggest that intercellular junctions play important roles in pancreatic development. In particular, the transient development of maculae occludentes is associated with endocrine cell development, and intercellular communication mediated by gap junctions may be important for the differentiation of acinar and endocrine cells.

Animals↗

Primary role of Kupffer cell-hepatocyte communication in the expression of oxidative stress in the post-ischaemic liver.

It has been reported that hepatocyte metabolism and function can be modulated by the activated Kupffer cell through the release of different biomolecules like cytokines, eicosanoids, oxygen free radicals and enzymes. In relation to these paracrine factors involved in circuits of intercellular communication, the existence of a hepatic oxygen sensor located in the Kupffer cell has been postulated. According to this postulate the oxygen metabolism of the liver parenchymal cells could be under the control of the Kupffer cells. In order to study the role of the Kupffer cell in the reperfusion syndrome of the liver, a lobular ischaemia-reperfusion model was performed in rats with or without previous treatment with gadolinium chloride to block Kupffer cell function. Spontaneous chemiluminescence of the liver surface, oxygen uptake by tissue slices and tertbutyl hydroperoxide-initiated chemiluminescence determinations were performed to evaluate the oxygen metabolism and the oxy-radical generation by the liver. The lower basal photoemission, in parallel with a lower basal oxygen uptake registered in the hepatic lobes from the animals pretreated with gadolinium chloride clearly indicates that the gadolinium chloride-dependent functional inhibition of Kupffer cell leads to a downregulation of oxygen metabolism by the liver. Moreover, the intensity of oxidative stress exhibited by the postischaemic lobes appears to be closely linked with the Kupffer cell activity. On the basis of the data obtained we propose that a paracrine circuit between activated Kupffer cell and hepatocytes is an early key event in the induction of postischaemic oxidative stress in the liver. Furthermore the interference with the mitochondrial electron flow by some biomolecules released from the activated Kupffer cell, such as tumour necrosis factor, interleukins, eicosanoids, etc., would increase the rate of generation of reactive oxygen species by the inhibited mitochondrial respiratory chain.

Animals↗

Cutting edge: Membrane nanotubes connect immune cells.

We present evidence that nanotubular highways, or membrane nanotubes, facilitate a novel mechanism for intercellular communication in the immune system. Nanotubes were seen to connect multiple cells together and were readily formed between a variety of cell types, including human peripheral blood NK cells, macrophages, and EBV-transformed B cells. Nanotubes could be created upon disassembly of the immunological synapse, as cells move apart. Thus, nanotubular networks could be assembled from transient immunological synapses. Nanotubes were seen to contain GFP-tagged cell surface class I MHC protein expressed in one of the connected cells. Moreover, GPI-conjugated to GFP originating from one cell was transferred onto the surface of another at the connection with a nanotube. Thus, nanotubes can traffic cell surface proteins between immune cells over many tens of microns. Determining whether there are physiological functions for nanotubes is an intriguing new goal for cellular immunology.

Animals↗

Biological markers of early health effects in the assessment of the risk of cancer in people exposed to environmental carcinogens.

Biomarkers of early health effects represent the intermediate stages of the carcinogenesis process, between the initiation and conversion stage and the clinically overt neoplastic disease. The cellular processes indicated by those markers correspond, therefore, to the promotion process. Detection of those cellular processes is extremely important, as the promotion process lasts for many years and bears some signs of being reversible. The promotion process, consisting of a range of consecutive cellular changes involves, among others, activation of proto-oncogens and their transformation into oncogens, and inactivation of the suppressor genes. Therefore, the possibility of observing the trends of those changes by monitoring protein products of the oncogens and suppressor genes in the easily available material (blood, urine) is very useful. Inhibition of intercellular communication seems to play an extremely important role in the complex mechanism of transformation of a normal cell into a neoplastic one. During the carcinogenic process promotion stage, the inhibition is associated with the proteins which form the intercellular junctions and participate in cellular adhesion. That group of proteins includes carcinoembryonal antigen (CEA) and tissue polypeptide antigen (TPA), the tumour antigens which have been known for years. The possible relationship between increased TPA and CEA concentrations is supported not only by the data from the oncology but also by the data from the studies on populations exposed to agents known to increase the risk of neoplastic disease.

Biomarkers, Tumor↗

Patch-clamp study reveals that the importance of connexin43-mediated gap junctional communication for ovarian folliculogenesis is strain specific in the mouse.

Genetic ablation of connexin37 (Cx37) or connexin43 (Cx43), the two gap junction proteins expressed by mouse ovarian granulosa cells, has been shown to result in impaired follicle development. We used patch-clamp techniques to evaluate quantitatively the contribution of these connexins to gap junctional intercellular communication (GJIC) among granulosa cells. The coupling conductance derived from a voltage step-induced capacitive current transient was used as a measure of GJIC in cultured granulosa cells. Using this method, we determined that the conductance of wild-type (84.1 +/- 28.6 nS; n = 6) and Cx37-deficient granulosa cells (83.7 +/- 6.4 nS; n = 11) does not differ significantly (P = 0.35), suggesting a limited contribution, if any, of Cx37 to granulosa cell coupling. In contrast, the conductance between granulosa cells of Cx43-deficient mice (2.6 +/- 0.8 nS; n = 5) was not significantly different from that of single, isolated wild-type granulosa cells (2.5 +/- 0.7 nS, n = 5; P = 0.83), indicating that Cx43-deficient granulosa cells were not electrically coupled. A direct measurement of transjunctional conductance between isolated granulosa cell pairs using a dual patch-clamp technique confirmed this conclusion. Interestingly, a partial rescue of folliculogenesis was observed when the Cx43-null mutation in C57BL/6 mice was crossed into the CD1 strain, and capacitive current measurement demonstrated that this rescue was not due to reestablishment of GJIC. These results demonstrate that folliculogenesis is impaired in the absence of GJIC between granulosa cells, but they also indicate that the severity is dependent on genetic background, a phenomenon that cannot be attributed to the expression of additional connexins.

Animals↗

Stromal-epithelial communications in hyperplastic human endometrium.

OBJECTIVE: Our objective was to assess the cell-to-cell contacts in hyperplastic human endometrium and to compare them with contacts in normal endometrium. STUDY DESIGN: Hyperplastic endometrial samples (five with simple hyperplasia and five with complex hyperplasia) without atypia by light microscopy were studied with transmission electron microscopy. The cell-to-cell contacts, basal lamina, and epithelial-to-stromal compartment contacts were studied and compared with these same areas in normal endometrium. RESULTS: Only infrequent gap junctions between epithelial cells were seen. Other epithelial contacts were also greatly reduced when compared with normal epithelium. Cell-to-cell contacts between the epithelial compartment and the stromal compartment were rare and degenerative in configuration. An increase in extracellular matrix and various alterations in the basal lamina appears to contribute to a decrease in cell-to-cell contacts. CONCLUSIONS: Compared with normal endometrium, hyperplastic endometrium demonstrated far fewer cell-to-cell contacts. The basal lamina and extracellular matrix were altered in such a fashion that communication between the stromal and epithelial compartments appeared to be impaired. Stromal and epithelial cells rarely were in contact. Those contacts present were less complex and the majority were degenerative. Hyperplasia without atypia demonstrated a much lower amount of intercellular communication than normal endometrium.

Cell Communication↗

Connexin43 phosphorylation at S368 is acute during S and G2/M and in response to protein kinase C activation.

Phorbol esters such as 12-O-tetradeconylphorbol-13-acetate (TPA) activate protein kinase C, increase Connexin43 (Cx43) phosphorylation, and decrease cell-cell communication via gap junctions in many cell types. Previous work has implicated protein kinase C (PKC) in the direct phosphorylation of Cx43 at S368, which results in a change in single channel behavior that contributes to a decrease in intercellular communication. We have examined Cx43 phosphorylation in several cell lines with an antibody specific for phosphorylated S368. We show that this antibody detects Cx43 only when it is phosphorylated at S368 and, consistent with previous results, TPA treatment causes a dramatic increase in phosphorylation at S368. However, in some cell types, the increased phosphorylation at S368 did not cause a detectable shift in migration as compared with the nonphosphorylated Cx43. Immunofluorescence showed increased S368 immunolabeling in cytoplasmic and plasma membrane structures in response to TPA. Immunoblot analysis of synchronized cells showed increased phosphorylation at S368 during S and G2/M phases of the cell cycle. S-phase cells contained more total Cx43 but assembled fewer functional gap junctional channels than G0-phase cells. Since M-phase cells also communicate poorly and contain few assembled gap junctions, phosphorylation at S368 appears to be negatively correlated with gap junction assembly. Thus, both gap junctional communication and S368 phosphorylation change during S phase and G2/M, implying that phosphorylation at S368 might play a role in key cell-cycle events.

Animals↗

Interaction between primary alveolar macrophages and primary alveolar type II cells under basal conditions and after lipopolysaccharide or quartz exposure.

Intercellular communications between alveolar macrophages (AM) and alveolar epithelial type II (TII) cells have been suggested to be important in cellular responses. The main objective of this study was to improve our understanding of the interactions between AM and TII cells that might occur in the lung. In the present investigation, this interaction was studied under different interaction conditions (transwell or mixed coculture) and different exposure conditions (basal, lipopolysaccharide [LPS] exposure, or silica exposure). Studies also attempted different approaches to identify specific mediator(s) involved in this interaction. The following findings were made: (1) Surfactant released from TII cells appears to exert an inhibitory effect on AM activity. (2) Basal transwell coculture conditions are better than mixed coculture conditions to study AM/TII cell interactions, since the inhibitory effect of the surfactant in the transwell coculture is minimized. (3) AM/TII cell interaction is dependent on cell culture (transwell vs mixed) and exposure conditions. (4) Oxidants, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, prostaglandins, and leukotrienes probably do not independently affect the AM/TII intercellular interaction; instead, they appear to indirectly modulate the complex pathways of AM/TII communication.

Animals↗

The modulation of gap junctional communication by gossypol in various mammalian cell lines in vitro.

The effects of gossypol were examined in several cell lines including hamster V79 lung fibroblasts, WB-344 rat liver oval cells, human osteosarcoma cells and LC540 rat Leydig cells. Gossypol had little cytotoxic effects in these cell lines except at high concentrations. Plasma membrane integrity was maintained in LC540 except at high concentrations of gossypol. Gossypol did not increase mutational frequency as examined by the hypoxanthine guanine phosphoribosyl transferase and Na+,K(+)-ATPase loci in V79 cells. Gossypol inhibited gap junctional intercellular communication in some but not all of the cell lines. This selectivity might be the basis for the sensitivity of certain tissues or organs to gossypol. For example, the Leydig cell, which is the component of a target organ system for toxicity, was sensitive to gossypol. Modulation of gap junctional functions might play a significant role in both pharmacological and toxicological effects of gossypol.

Animals↗