Diane F. Matesic

Localization and function of the connexin 43 gap‐junction protein in normal and various oncogene‐expressing rat liver epithelial cells

Clones of rat liver epithelial cells genotypically altered by mutation or by a variety of oncogenes were analyzed by microinjection‐dye transfer, immunofluorescence confocal microscopy, and western blotting to determine at what level and to what degree these transformations disrupted gap‐junctional intercellular communication (GJIC) mediated by connexin 43 (Cx43). Compared with normal rat liver epithelial cells, cells neoplastically transformed by src, neu, ras, and myclras all displayed reduced degrees of GJIC, reduced levels of membrane‐associated Cx43 plaques, and hypophosphorylation of Cx43. Confocal analysis further demonstrated that the Cx43 protein was localized, at least in part, to the nucleus rather than to the plasma membrane in the src‐ and neu‐transformed cells, but not in the ras‐ and myclras‐transformed cells. Nuclei isolated from WB‐neu cells showed substantially higher levels of Cx43 on western blotting than did nuclei from WB‐neo control cells, supporting the idea that the nuclear‐localized immunopositive material detected by confocal microscopy was Cx43 protein. In a GJIC‐deficient mutant rat liver epithelial cell line containing normal numbers of plasma membrane‐localized Cx43 plaques that appeared to be reduced in size, the Cx43 protein was also found to be hypophosphorylated. Cells overexpressing myc, on the other hand, displayed a normal degree of GJIC, increased levels of plasma membrane‐localized Cx43 plaques, and hyperphosphorylation of the Cx43 protein. Cells expressing raf, previously shown to be GJIC competent, showed Cx43 immunostaining patterns similar to those in normal cells, whereas a cell line established from a tumor induced by injection of these raf‐expressing cells into a mouse showed a marked reduction in GJIC and plasma membrane‐associated Cx43 immunostaining. These data suggest that altered localization of the gap‐junction protein Cx43, mediated in part by changes in the phosphorylation of this protein, contributes to the disruption of GJIC in neoplastically transformed rat liver epithelial cells.

Identification of a G-protein in depolarizing rod bipolar cells

Synaptic transmission from photoreceptors to depolarizing bipolar cells is mediated by the APB glutamate receptor. This receptor apparently is coupled to a G-protein which activates cGMP-phosphodiesterase to modulate cGMP levels and thus a cGMP-gated cation channel. We attempted to localize this system immunocytochemically using antibodies to various components of the rod phototransduction cascade, including Gt (transducin), phosphodiesterase, the cGMP-gated channel, and arrestin. All of these antibodies reacted strongly with rods, but none reacted with bipolar cells. Antibodies to a different G-protein, G(o), reacted strongly with rod bipolar cells of three mammalian species (which are depolarizing and APB-sensitive). Also stained were subpopulations of cone bipolar cells but not the major depolarizing type in cat (b1). G(o) antibody also stained certain salamander bipolar cells. Thus, across a wide range of species, G(o) is present in retinal bipolar cells, and at least some of these are depolarizing and APB-sensitive.

Immortalized Hypothalamic Luteinizing Hormone-Releasing Hormone Neurons Express a Connexin 26-like Protein and Display Functional Gap Junction Coupling Assayed by Fluorescence Recovery after Photobleaching

Expression of gap junction proteins was studied in the LHRH neuronal cell line, GT1-7, as a first step in defining the signalling mechanisms responsible for the pulsatile secretion of LHRH. GT1-7 cells were found to express a connexin 26-like protein that comigrated with mouse liver connexin 26 and that reacted with connexin 26-specific antibodies on Western blots. Immunofluorescent staining revealed punctate staining in a fraction of the cells, often present at points of apparent contact with neighboring cell bodies or processes. Fluorescence recovery after photobleaching analysis of 5,6-carboxyfluorescein loaded GT1-7 cells showed dye coupling among 20-30% of cells that made contact with other cells, suggesting the presence of functional gap junctions in this cell line.

Stimulation of cell proliferation and inhibition of gap junctional intercellular communication by linoleic acid

The effect of linoleic acid (LA) on gap-junction permeability, connexin 43 mRNA level, protein level, and phosphorylation, and the numbers of gap-junctional membrane plaques were studied in the rat liver epithelial cell line WB-F344 to determine whether changes in these parameters correlated with the enhanced cell growth and the inhibition of gap-junction function. When cultured in a medium with low serum (1%), these cells exhibited a slower growth rate than in the high serum medium (7%). Addition of linoleic acid (0.01-3 mg/ml) to the low serum medium increased the growth rate and inhibited gap junctional intercellular communication (GJIC) in a dose-dependent manner. In a comparison of short-term and long-term treatments with LA, GJIC in short-term treated (1 h) WB cells was inhibited at 3 mg/ml LA but readily recovered by washing and removing LA from cells, whereas GJIC in long-term treated (6 days) WB cells did not recover by washing and removing LA from WB cells. Western blot analysis of connexin 43 showed that a short-term incubation with linoleic acid increased the relative amount of unphosphorylated connexin 43 protein, but a long-term incubation with linoleic acid decreased the amount of unphosphorylated connexin 43 protein and increased the relative amount of hyperphosphorylated connexin 43 protein. Connexin 43 and p53 mRNA levels decreased in a time- and dose-dependent manner in linoleic acid-treated cells. These results suggest that growth stimulation and gap junctional intercellular communication inhibition of rat liver epithelial cells by linoleic acid may be mediated in part through modulation of p53 expression and function.

Upregulation of Gap Junctional Intercellular Communication in Immortalized Gonadotropin-Releasing Hormone Neurons by Stimulation of the Cyclic AMP Pathway

Increased gap junctional intercellular communication induced by agents that stimulate the adenylyl cyclase/cAMP pathway was observed in the GnRH-secreting neuronal cell line, GT1-7, and possible underlying mechanisms were examined. A 24-hour treatment of GT1-7 neurons with 100 microM dibutyryl cAMP + 100 microM IBMX or with 2 microM forskolin increased by greater than 2-fold the percentage of cells that were dye coupled, using the noninvasive dye coupling assay, fluorescent recovery after photobleaching (FRAP). Longer treatment times (48 h) and higher concentrations of dibutyryl cAMP (500 microM) did not further increase the percentage of dye-coupled cells, while there was no increase in dye coupling observed between untreated cells and cells treated for 2 h or less. The increase in dye coupling induced by dibutyryl cAMP/IBMX was inhibited by octanol or dieldrin, agents known to block gap junction-mediated intercellular coupling in other cell types. Western blot analysis of total protein or membrane protein-enriched extracts revealed no apparent difference in the cellular levels of connexin 26, a connexin subtype previously shown to be expressed by GT1-7 cells, between untreated cells and cells treated for 24 h with dibutyryl cAMP/IBMX or forskolin. In addition, expression of connexin 32 or 43 protein before or after treatment was not detected. On the other hand, a dramatic increase in both the number of neurites and neurites that immunostained positive for connexin 26 was observed in dibutyryl cAMP/IBMX-treated cells. We hypothesize that the observed increase in dye coupling between GT1-7 neurons following stimulation of the adenylyl cyclase/cAMP pathway results from an augmentation of cell-cell contacts due to an increased number of neurites containing gap junctional plaques, possibly through an effect on cellular differentiation.

Inhibition of gap junctional intercellular communication in heptachlor- and heptachlor epoxide-treated normal human breast epithelial cells

Based on the concern of organochlorides in the environment and in human tissue, this study was designed to determine whether various noncytotoxic levels of heptachlor and heptachlor epoxide could inhibit, reversibly, gap junctional intercellular communication in human breast epithelial cells (HBEC). Cytotoxicity and gap junctional intercellular communication (GJIC) were evaluated by lactate dehydrogenase assay and fluorescence redistribution after photobleaching analysis, respectively. Both heptachlor and heptachlor epoxide were noncytotoxic up to 10 μg/ml. At this concentration, heptachlor and heptachlor epoxide inhibited GJIC of normal human breast epithelial cells after 1 h treatment. Within a 24 h treatment with heptachlor and heptachlor epoxide at 10 μg/ml, recovery of GJIC had not returned. GJIC completely recovered after a 12 h treatment of 1 μg/ml heptachlor epoxide, but it did not recover after a 24 h treatment of 1 μg/ml heptachlor. RT-PCR and Western blots were analyzed to determine whether the heptachlor or heptachlor epoxide might have altered the steady-state levels of gap junction mRNA and/or connexin protein levels or phosphorylation state. No significant difference in the level of connexin 43 (Cx43) message between control and heptachlor-treated cells was observed. Western blot analyses showed hypophosphorylation patterns in cells treated with 10 μg/ml heptachlor and heptachlor epoxide for 1 h with no recovery within 24 h. Immunostaining of Cx43 protein in normal HBEC indicated that heptachlor and heptachlor epoxide caused a loss of Cx43 from the cell membranes at noncytotoxic dose levels. Taken together, these results suggest that heptachlor and heptachlor epoxide can alter GJIC at the post-translational level, and that, under the conditions of exceeding a threshold concentration in the breast tissue containing ‘initiated’ cells for a long time and not being counteracted by anti-tumor-promoting chemicals, they could act as breast tumor promoters.

Differential LHRH Secretion, Dye Coupling, and Protein Expression in Two Morphologically Distinct Cell Types Identified in GT1–7 Cultures

The immortalized neuronal cell line, GT1–7, has been shown to secrete LHRH in a pulsatile manner and to possess many other characteristics of hypothalamic LHRH neurons in vivo, and thus provides a potential model system for studying biochemical and physiological mechanisms regulating LHRH secretion. In the present study, two morphologically and functionally distinct types of cells have been identified in GT1–7 cultures and each type purified to over 95% homogeneity. One type (N cells) appeared more neuronal with extended neurites and somewhat rounded cell perikarya, while the other type (G cells) had flatter cell perikarya that contained filopodia but no neurites. Growth properties of the two cell types also differed. The doubling time for proliferation of N cells was nearly two‐fold shorter than that for G cells and N cells displayed ‘piling up’ whereas G cells exhibited contact inhibition. Functionally, N cells, but not G cells, were dye‐coupled as measured by a fluorescence photobleaching assay. While both cell types expressed LHRH, N cells released significantly higher levels of LHRH into the culture media and exhibited more intense LHRH immunostaining. The two cell types also showed differences in immunostaining for other proteins. N cells, unlike G cells, immunostained positive for neuron‐specific enolase (NSE), whereas G cells, unlike N cells, stained immunopositive for vimentin. Both cell types expressed SV‐40 T antigen protein, indicating that they were derived from the same transgenic mouse hypothalamic tumour. The physiological significance of these two cell types in GT1–7 cultures remains to be determined, but elucidation of their morphological and biochemical properties is intended to contribute to better understanding and application of this experimentally important neuroendocrine cell line.

The Role of Dendritic Dysfunction in Neurodegeneration

More than a century ago, it had already been recognized that neurons exhibited two morphologically distinctive structures: a long and thin process or “axon” named after the Greek word for axle, and multiple branched protoplasmic processes or “dendrites” named after the Greek word for tree, dendron.’.’ These two anatomically different structures contribute to the classical view: that the dendrites constitute the receiving site of the neuron, while the axon acts to convey neural o u t p ~ t . ~ However, recent physiological findings have led to the new concept that dendrites can not only generate action potentials, but can also be actively involved in information processing and integration within the central nervous system. New evidence from our laboratory and others has suggested that dendritic dysfunction and calcium homeostasis failure may play critical roles in the early phase of pathogenesis. Such defects may contribute significantly to the development of neurological disease. Thus, in order to preserve normal neuronal function, therapeutic agents aimed at preventing dendritic dysfunction should be a primary focus of future research efforts. Excitotoxicity and calcium overload are believed to be major events that lead to neuronal death after injury such as ischemia/hypoxia. However, recent reports have shown that under certain conditions significantly elevated calcium concentrations measured in the somata had almost no harmful effects in cultured cells.’,6 It is well known that the action of excitatory amino acid (EAA) and their receptors, as well as activity-driven calcium changes are targeted at dendrites and dendritic spines of the neurons. Thus, it is conceivable that the confusion may be the consequence of examining the soma/cell body rather than the dendrites. The role of calcium changes with dendritic pathology and cell death remains to be determined. At present, little is known about when, where, and why dendrites degenerate. In the following sections, we will discuss issues that are relevant to calcium homeostasis, dendritic dysfunction, and neuronal survival.