Radiation Response of Connexin43-Transfected Cells in Relation to the “Contact Effect”

Some cell lines grown for only two cell doublings as multicell spheroids develop a form of resistance to killing by ionizing radiation that has been called the “contact” effect. While our previous results have implicated a role for higher order chromatin structure in the contact effect, another possible explanation is the presence of intercellular gap junctions that might facilitate communication between cells grown as spheroids and thereby enhance the ability of cells to resist or recover from radiation damage. To examine the role of gap junctions in the contact effect, rat glioma C6 and mouse EMT6 cell lines were transfected with a gene encoding the gap junctional protein connexin43. While C6 glioma cells are deficient in gap junctional communication, cells from spheroids were nonetheless more resistant than monolayers to killing by ionizing radiation, and the contact effect was present to a similar extent in the three transfected clones. For mouse EMT6 cells, radiosensitivity was similar whether cells were grown as monolayers or spheroids. Transfection of EMT6 cells with connexin43 increased gap junctional communication but did not promote development of a contact effect. Tumor volume doubling time in SCID mice increased significantly for one transfected clone; however, doubling timein vitrowas also increased relative to the EMT6 parent. We conclude that extensive gap junctional communication is not a requirement for the increased radiation resistance observed when some cell lines are grown as spheroids.     

Hypophyseal corticosteroids stimulate somatotrope differentiation in the embryonic chicken pituitary gland

Although it is known that glucocorticoids induce differentiation of growth hormone (GH)-producing cells in rodents and birds, the effect of mineralocorticoids on GH mRNA expression and the origin of corticosteroids affecting somatotrope differentiation have not been elucidated. In this study, we therefore carried out experiments to determine the effect of mineralocorticoids on GH mRNA expression in the chicken anterior pituitary gland in vitro and to determine whether corticosteroids are synthesized in the chicken embryonic pituitary gland. In a pituitary culture experiment with E11 embryos, both corticosterone and aldosterone stimulated GH mRNA expression and increased the number of GH cells in both lobes of the pituitary gland in a dose-dependent manner. These effects of the corticosteroids were significantly reversed by pretreatment with mifepristone, a glucocorticoid receptor (GR) antagonist, or spironolactone, a mineralocorticoid receptor (MR) antagonist. Interestingly, an in vitro serum-free culture experiment with an E11 pituitary gland showed that the GH mRNA level spontaneously increased during cultivation for 2 days without any extra stimulation, and this increase in GH mRNA level was completely suppressed by metyrapone, a corticosterone-producing enzyme P450C11 inhibitor. Moreover, progesterone, the corticosterone precursor, also stimulated GH mRNA expression in the cultured chicken pituitary gland, and this effect was blocked by pretreatment with metyrapone. We also detected mRNA expression of enzymes of cytochrome P450 cholesterol side chain cleavage (P450scc) and 3β-hydroxysteroid dehydrogenase1 (3β-HSD1) in the developmental chicken pituitary gland from E14 and E18, respectively. These results suggest that mineralocorticoids as well as glucocorticoids can stimulate GH mRNA expression and that corticosteroids generated in the embryonic pituitary gland by intrinsic steroidogenic enzymes stimulate somatotrope differentiation.     

Studies on arterial mineralocorticoid and glucocorticoid receptors: evidence for the translocation of steroid-cytoplasmic receptor complexes to cell nuclei

The high affinity binders for mineralocorticoids and glucocorticoids, previously reported by us as present in arterial cytosol have been further characterized. The results of this study demonstrate that these binders translocate under appropriate conditions to cell nuclei as complexes with mineralocorticoids and glucocorticoids, respectively. Thus, they exhibit a fundamental property of steroid receptors. This provides evidence for the presence in the arterial wall of a molecular mechanism(s) for the in-situ action of both mineralocorticoids and glucocorticoids.     

Reversible inhibition of intercellular junctional communication by glycyrrhetinic acid

Intercellular gap-junctional communication was measured using metabolic co-operation in co-cultures of argininosuccinate synthetase-deficient and argininosuccinate lyase-deficient human fibroblasts. 18-alpha-glycyrrhetinic acid (AGA) was found to inhibit communication by more than 95% at concentrations as low as 2 microM. Concentrations up to 100 microM were not cytotoxic over a period of 2 hours. Communication inhibition was of rapid onset and was readily reversible. Communication remained continuously yet reversibly blocked in cells cultured in the presence of AGA for 20 days. The related compounds 18-beta-glycyrrhetinic acid and carbenoxolone also caused communication inhibition. The effect is probably not mediated via mineralocorticoid or glucocorticoid receptors since aldosterone and glucocorticoids had no effect on communication. AGA thus has properties of a useful inhibitor in the study of intercellular junctional communication.     

Transfection with Different Connexin Genes Alters Growth and Differentiation of Human Choriocarcinoma Cells

To examine the role of cell–cell communication via gap junctions in controlling proliferation and differentiation we transfected the malignant trophoblast cell line Jeg-3, which exhibits extremely low cell–cell communication mediated by endogenously expressed connexin40, with connexin26, connexin40, and connexin43, respectively.In vitrogrowth of all cell clones transfected with connexin genes was significantly reduced compared to controls. This effect corresponded to a significant increase in total junctional conductance of all clones. Single-channel conductances for channels formed by the transfected connexins were in the range of the values published previously. Though total junctional conductance varied highly among clones and even within one clone, differentiation of the cells indicated by β-hCG secretion was most prominent in the clones that revealed the largest amount of well-coupled cell pairs. Connexin26 channels enable cells of one clone to reduce drastically growth rate and produce significantly higher secretion of β-hCG. Connexin43 had only moderate effects on the differentiation properties of Jeg-3 cells. These findings suggest that restoration of cell–cell communication plays a role in growth reduction and in differentiation of tumor cells and that different channel proteins might have different effects.     

Role of adrenal hormones in regulating distal nephron structure and ion transport

Mineralocorticoid levels are an important determinant of membrane area and ion transport in the renal initial (ICT) and cortical (CCT) collecting tubules. Adrenalectomy leads to a dramatic and specific decrease in basolateral membrane area of principal (P) cells and depresses sodium reabsorption and potassium secretion. Although aldosterone replacement for 10 days restores basolateral membrane area and ATPase activity to control levels and dramatically elevates ion transport, glucocorticoids have no effect on basolateral membrane area, ion transport, or ATPase. It is suggested that the aldosterone-induced amplification of membrane area occurs as a mechanism whereby cells increase the number of ATPase pumps in the basolateral membrane. An acute (of 2-3 h) increase in aldosterone, but not dexamethasone, also stimulates potassium transport by the ICT. Future studies will have to establish whether the acute stimulation of transport by aldosterone involves a change in basolateral membrane area. It is concluded that mineralocorticoids, but not glucocorticoids, regulate sodium and potassium transport by P cells of the ICT and CCT, in part, by determining the number of ATPase pumps available for transport.     

Extra-adrenal glucocorticoids and mineralocorticoids: evidence for local synthesis, regulation, and function

Glucocorticoids and mineralocorticoids are steroid hormones classically thought to be secreted exclusively by the adrenal glands. However, recent evidence has shown that corticosteroids can also be locally synthesized in various other tissues, including primary lymphoid organs, intestine, skin, brain, and possibly heart. Evidence for local synthesis includes detection of steroidogenic enzymes and high local corticosteroid levels, even after adrenalectomy. Local synthesis creates high corticosteroid concentrations in extra-adrenal organs, sometimes much higher than circulating concentrations. Interestingly, local corticosteroid synthesis can be regulated via locally expressed mediators of the hypothalamic-pituitary-adrenal (HPA) axis or renin-angiotensin system (RAS). In some tissues (e.g., skin), these local control pathways might form miniature analogs of the pathways that regulate adrenal corticosteroid production. Locally synthesized glucocorticoids regulate activation of immune cells, while locally synthesized mineralocorticoids regulate blood volume and pressure. The physiological importance of extra-adrenal glucocorticoids and mineralocorticoids has been shown, because inhibition of local synthesis has major effects even in adrenal-intact subjects. In sum, while adrenal secretion of glucocorticoids and mineralocorticoids into the blood coordinates multiple organ systems, local synthesis of corticosteroids results in high spatial specificity of steroid action. Taken together, studies of these five major organ systems challenge the conventional understanding of corticosteroid biosynthesis and function.     

Cyclic AMP induces rapid increases in gap junction permeability and changes in the cellular distribution of connexin43

The rapid effects of cAMP on gap junction-mediated intercellular communication were examined in several cell types which express different levels of the gap junction protein, connexin43 (Cx43), including immortalized rat hepatocyte and granulosa cells, bovine coronary venular endothelial cells, primary rat myometrial and equine uterine epithelial cells. Functional analysis of changes in junctional communication induced by 8-bromo-cAMP was monitored by a fluorescence recovery after photobleaching assay in subconfluent cultures in the presence or absence of 1.0 mm 1-octanol (an agent which uncouples cells by closing gap junction channels). Communicating cells treated with 1.0 mm 8-bromo-cAMP alone exhibited significant increases in the percent of fluorescence recovery which were detected within 1–3 min depending on cell type, and junctional communication remained significantly elevated for up to 24 hr. Addition of 1.0 mm 8-bromo-cAMP to cultured cells, which were uncoupled with 1.0 mm octanol for 1 min, exhibited partial restoration of gap junctional permeability beginning within 3–5 min. Identical treatments were performed on cultures that were subsequently processed for indirect immunofluorescence to monitor Cx43 distribution. The changes in junctional permeability of cells correlated with changes in the distribution of immunoreactive Cx43. Cells treated for 2 hr with 10 m monensin exhibited a reduced communication rate which was accompanied by increased vesicular cytoplasmic Cx43 staining and reduced punctate surface staining of junctional plaques. Addition of 1.0 mm 8-bromo-cAMP to these cultures had no effect on the rate of communication or the distribution of Cx43 compared to cultures treated with monensin alone. These data suggest that an effect of cyclic AMP on Cx43 gap junctions is to promote increases in gap junctional permeability by increasing trafficking and/or assembly of Cx43 to plasma membrane gap junctional plaques.We acknowledge the technical assistance of Richard Lewis and Meghan Abella. We thank Dr. Hugh Dookwah for contributions to the myometrial cell isolation protocol and Drs. Stephen H. Safe, Timothy D. Phillips, and Evelyn Tiffany-Castiglioni for helpful discussions. This work was funded by NIH (HD-26182, P42-ES04917, ES05871-01A1), the March of Dimes Birth Defects Foundation Basic Research grant #1-0796, and USDA 92-37203-7952.     

4-Hydroxynonenal reduces junctional communication between endothelial cells in culture

The effect of 4-hydroxynonenal (HNE), a lipid peroxidation product, on junctional communication (JC) among cultured vascular endothelial cells was assessed by both study of the transfer of microinjected 6-carboxyfluorescein between neighboring cells and measurement by a "cut-loading and dye transfer" technique. Both methods indicated that at concentrations higher than 10(-9) M and testing times between 6 and 8 h HNE reduces endothelial cell junctional communication. At 10(-8) M, a gradual development of HNE effect appears during 6-8 h of exposure but is followed by a slow recovery completed at 20 h. The reduction in junctional communication is not produced by the inhibition of protein synthesis, as tested by radiolabeled leucine incorporation. The HNE effect might be relevant to pathological processes in which lipid peroxidation is associated with uncontrolled cell proliferation, as in atherogenesis and promotion of carcinogenesis by chronic inflammation.     

Long-Term Treatment with Glucocorticoids Increases Synthesis and Stability of Junctional Acetylcholine Receptors on Innervated Cultured Human Muscle

Abstract: We have studied the effect of long-term treatment with hydrocortisone on the expression of acetylcholine receptors (AChRs) at the neuromuscular junctions of human muscle cultured in monolayer and innervated de novo by fetal rat spinal cord motoneurons. Hydrocortisone increased accumulation of junctional AChRs in a dose-and time-dependent fashion. This increase was due to both decreased degradation and increased synthesis of AChRs. Other glucocorticoids, dexamethasone and prednisolone, exerted similar effects. Our study demonstrates a novel action of glucocorticoids on human junctional AChRs.     

Role of the hypophyseo-adrenal system in regulating the acid-secreting function of the kidneys

The experiments on male albino rats have shown that both stress and hydrocortisone elevate the urinary excretion of hydrogen ions in the form of titrated acids and ammonium ions. Desoxycorticosterone acetate (DOCSA) in intact rats, adrenal- and hypophysectomy lower ammonium ions excretion. Both stress and DOCSA increase the tubular sodium reabsorption, while hydrocortisone, adrenal- and hypophysectomy decrease it. A conclusion is made that both glucocorticoids and mineralocorticoids participate in the renal functional change under stress. It should be underlined that glucocorticoids stimulate the secretion while mineralocorticoids play the leading part in maintenance of sodium homeostasis.     

A study of communication specificity between cells in culture

We have examined the specificity of communication between cells in culture by co-culturing cells derived from mammalian, avian, and arthropod organisms. Both mammalian and avian culture cells have similar gap junctional phenotypes, while the insect (arthropod) cell lines have a significantly different gap junctional structure. Electrophysiological and ultrastructural methods were used to examine ionic coupling and junctional interactions between homologous and heterologous cell types. In homologous cell systems, gap junctions and ionic coupling are present at a high incidence. Also, heterologous vertebrate cells in co-culture can communicate readily. By contrast, practically no coupling (0-8%) is detectable between heterologous insect cell lines (Homopteran or Lepidopteran) and vertebrate cells (mammalian myocardial or 3T3 cells). No gap junctions have been observed between arthropod and vertebrate cell types, even though the heterologous cells may be separated by less than 10 nm. In additional studies, a low incidence of coupling was found between heterologous insect cell lines derived from different arthropod orders. However, extensive coupling was detected between insect cell lines that are derived from the same order (Homoptera). These observations suggest that there is little or no apparent specificity for communication between vertebrate cells in culture that express the same gap junctional phenotype, while there is a definite communication specificity that exists between arthropod cells in culture.     

"Contact inhibition" of alpha-fetoprotein synthesis and junctional communication in adult mouse hepatocyte culture

Synthesis of oncofetal serum protein alpha-fetoprotein (AFP) may be reexpressed in adult differentiated mouse hepatocytes both in regenerating liver and in primary monolayer culture of intact adult liver. We have found that appearance of AFP in these cultures was strongly correlated with the loss of junctional communication between hepatocytes as tested by the dye transfer method. When in hepatocyte culture the gradient of cell density was formed, and the cells in the center of the dense monolayer retained an epithelial morphology and junctional communication and were AFP-negative during 5 days of culture. At the periphery of the monolayer hepatocytes lost junctional communication by the third day of cultivation. They acquired fibroblast-like morphology, formed multilayered sheets, and started to produce AFP. These findings suggest that reexpression of AFP synthesis may be regulated through a process related to "contact inhibition" and junctional communication might play an important role in the phenomenon.     

Influence of the Molecular Structure of Steroids on Their Ability to Interrupt Gap Junctional Communication

17β-estradiol propionate was found to reduce the gap junctional communication in a concentration range similar to that of testosterone propionate, in primary cultures of rat Sertoli cells and cardiac myocytes. Uncoupling was reversible on washing out and occurred without concomitant rise in the intracellular calcium concentration. Esterification was a prerequisite for the activity of extracellularly applied steroid compounds (for example, testosterone was ineffective even at external concentrations up to 100 μm, whereas its intracellular application at 1 μm totally interrupted intercellular communication), but their uncoupling efficiency did not depend on the nature of the ester chain nor on its position on the steroid nucleus. The derivatives of two other androgen hormones (derivatives of the androstane nucleus) were also efficient as junctional uncouplers. Among five steroid molecules belonging to the pregnane family, only one (pregnanediol diacetate) interrupted the junctional communication. Neither cholic acid nor cholesteryl acetate or ouabain showed this effect. Altogether, no correlation with the presence or position of double bonds nor with the trans- or cis-fusion of the A and B rings could be recognized. These results suggest that this reversible, nondeleterious uncoupling effect of steroids is independent of the shape of the molecules and is more probably related to their size and liposolubility, that condition their insertion into the lipid bilayer. Their incorporation into the membrane could disturb the activity of the membrane proteins by a physical mechanism. Received: 10 April 1995/Revised: 27 October 1995     

INTERCELLULAR COMMUNICATION IN RAPIDLY PROLIFERATING AND DIFFERENTIATED C6 GLIOMA CELLS IN CULTURE

Glial cells in the brain are known to provide structural and functional supports to neurons. To sustain such a supportive role, they have developed cell-to-cell communicating gap junctional channels. The authors studied the effect of dbcAMP on gap junctional channels mediated communication in C6 cells, a rat glioma cell line. Quantitative assessment of coupled cells under microscope after microinjection of a fluorescent dye was taken as a measure of junctional permeability. An enhanced coupling between cells was observed following dbcAMP treatment and this elevated coupling was found to be dependent on the duration of exposure of cells to dbcAMP. The studies have focused on a subtle shift in the spatial organization of the functional channels to the processes of dbcAMP induced differentiated cells from the cell cytoplasms and membranes of dbcAMP untreated cells. Immunofluorescence study with affinity purified antibody against gap junction further confirmed the spatial distribution of gap junctional protein(s) in the processes and also showed an increase in the density of the protein at the intercellular spaces in dbcAMP induced differentiated C6 glioma cells.     

培养的内皮细胞和平滑肌细胞之间间隙连接形成及连接通讯的研究

本实验以离子电渗法将荧光黄注入细胞内,观察培养的EC 与SMC 同类及异类细胞间连接通讯现象,证实SMC 与EC 在培养中可形成同类或异类细胞GJ 结构,两种细胞间有接触介导的物质交流。EC-EC 之间的细胞通讯比SMC-SMC 和SMC-EC 之间为强。对SMC 有促增殖作用的LDL(100 μg LDL-蛋白质/ml)及胰岛素(15 mu/ml)对这种连接通讯有抑制作用,促癌剂TPA 几乎完全抑制此种连接通讯。结果提示高浓度LDL 及胰岛素等可能通过抑制SMC 与EC 之间的连接通讯而使SMC 脱离正常控制而大量增殖,促进AS发生、发展。故推论促进细胞连接通讯的因子,可能对AS 有防治作用,值得进一步研究。     

The Sleep-inducing Lipid Oleamide Deconvolutes Gap Junction Communication and Calcium Wave Transmission in Glial Cells

Oleamide is a sleep-inducing lipid originally isolated from the cerebrospinal fluid of sleep-deprived cats. Oleamide was found to potently and selectively inactivate gap junction–mediated communication between rat glial cells. In contrast, oleamide had no effect on mechanically stimulated calcium wave transmission in this same cell type. Other chemical compounds traditionally used as inhibitors of gap junctional communication, like heptanol and 18β-glycyrrhetinic acid, blocked not only gap junctional communication but also intercellular calcium signaling. Given the central role for intercellular small molecule and electrical signaling in central nervous system function, oleamide- induced inactivation of glial cell gap junction channels may serve to regulate communication between brain cells, and in doing so, may influence higher order neuronal events like sleep induction.     

The role of gap junctions in patterning of the chick limb bud

The role of gap junctional communication during patterning of the chick limb has been investigated. Affinity-purified antibodies raised against rat liver gap junctional proteins were used to block communication between limb mesenchyme cells. Co-injection of the antibodies and Lucifer yellow into mesenchyme cultures demonstrated that communication was inhibited almost immediately. When antibodies were loaded into mesenchyme tissue by DMSO permeabilization, [3H]nucleotide transfer was prevented for at least 16 h. Polarizing region tissue from the posterior limb bud margin causes digit duplications when grafted to the anterior margin. Quail polarizing region cells were loaded with gap junction antibody and grafted into chick wing buds. The antibody had no effect on growth or survival of the grafted cells. As very few polarizing region cells are required to initiate duplications, the number of polarizing region cells in the grafts was reduced by diluting 1:9 with anterior mesenchyme tissue. When either polarizing region or anterior mesenchyme tissue in the graft was loaded separately with antibody, there was little effect on respecification of the digit pattern. However, loading both tissues in the graft caused a significant decrease in duplications. This indicates that a major role of gap junctions in limb patterning may be to enable polarizing region cells to communicate directly with adjacent anterior mesenchyme. A role for gap junctional communication between anterior mesenchyme cells cannot be excluded. The results are discussed in relation to the role of retinoic acid as a putative morphogen.     

Cell contacts between follicle cells and the oocyte of Helisoma (Mollusca,Pulmonata)

The cell contacts between follicle cells, and follicle cells and oocytes of egg-laying populations of Helisoma duryi and non-egg-laying populations of H. trivcolvis have been studied. Scanning electron microscopy reveals that four to six follicle cells envelop a single developing oocyte. Thin sections and lanthanum impregnations demonstrate apical zonulae adherentes followed by winding pleated-type septate junctions between follicle cells. Gap junctions and septate junctions have been found between follicle cells and vitellogenic oocytes. Freeze-fracture replicas show relatively wide sinuous rows of septate junctional particles, and nemerous large gap junctional particle aggregates on the P-face between vitellogenic oocytes and follicle cells. Septate and gap junctions between immature or nonvitellogenic oocytes and follicle cells are fewer compared to those in vitellogenic oocytes. Similarly, the junctional complexes are less developed in non-egg-laying H. trivolvis compared to those in egg-laying H. duryi. It is possible that intimate interaction between follicle cells and a developing oocyte is necessary for the maturation of the oocyte. The junctional complexes could be involved in the interaction of the follicle cells and the oocyte, and they must disassemble at the onset of ovulation. Rhombic particle arrays and nonjunctional ridges of particles have been found in the basal part of the oolemma.