Communication-competent Cells Research Articles

Concurrent analysis of intracellular glutathione content and gap junctional intercellular communication

The potential for performing dual analysis of intracellular glutathione levels and assessment of gap junctional intercellular communication with thiol-specific fluorescent probes in anchored cells was evaluated. Gap junction-mediated diffusion of monochlorobimane and 5-chloromethylfluorescein diacetate following intracellular loading and conjugation with glutathione was compared with 5-carboxyfluorescein diacetate (which is routinely used in laser cytometry to monitor intercellular communication) by means of fluorescence recovery after photobleaching using a variety of communication-competent and communication-incompetent cells. The rate of diffusion of fluorescence among communication-competent cells was inversely proportional to the size of the fluorescent probe employed. The thiol-specific probes were also employed to monitor depletion and synthesis of glutathione following treatments to inhibit glutathione synthesis or consume glutathione by adduct formation. Analysis of gap junctional intercellular communication following glutathione depletion revealed a direct correlation between glutathione levels and intercellular communication. These studies support the utility of the thiol-specific probes to monitor the respective role of cellular glutathione and intercellular communication in the mechanisms of cellular injury.

Contact-Dependent Intercellular Communication of Bovine Luteal Cells in Culture*

Mammalian gap junctions permit exchange of nutrients, ions, and regulatory molecules of less than 1.5 kDa among contacting communication-competent cells and may be important for regulation of luteal function and maintenance of luteal homeostasis. The present studies were designed to evaluate gap junction-mediated intercellular communication between bovine luteal cells in culture. Using a dye-coupling technique along with interactive laser cytometry, selected luteal cells were studied for the rate of contact-dependent fluorescence redistribution after photobleaching. The rate of communication, reported as the rate of fluorescence recovery (percentage per min), was determined for steroidogenic cells as follows: 1) small luteal cells contacting only small luteal cells, 2) large luteal cells contacting only small luteal cells, and 3) large luteal cells contacting only large luteal cells. In addition, the effects of known regulators of luteal function [LH, prostaglandin F2 alpha (PGF), and forskolin] on the rate of intercellular communication were determined. Small luteal cells communicated rapidly with each other, exhibiting an initial rate of fluorescence recovery of 4.1 +/- 0.1%/min (n = 187). The rate of small cell-small cell communication was unaffected by LH and PGF. For large luteal cells contacting small luteal cells, however, LH and PGF stimulated (P less than 0.02) the rate of communication compared with no hormone [1.6 +/- 0.2 (n = 18) and 1.5 +/- 0.6 (n = 20) vs. 0.8 +/- 0.3%/min (n = 27), respectively]. LH and PGF in combination, however, did not enhance the rate (0.6 +/- 0.2%/min; n = 19) of large cell-small cell communication. In contrast, forskolin significantly stimulated both small cell-small cell and large cell-small cell communication rates compared with no forskolin [34% increase (n = 48) and 50% increase (n = 23), respectively]. Large luteal cells did not communicate with each other under any condition tested. Transmission electron microscopy revealed the presence of numerous gap junction-like structures in bovine luteal cells in culture. These data suggest that luteal cells are capable of intercellular communication and that the rate of communication may be influenced by hormones. Contact-dependent intercellular communication among luteal cells may, therefore, play a significant role in the regulation of luteal function.

Syncytial organization of cultured rat mesangial cells

To investigate communication competence of cultured rat mesangial cells, Lucifer yellow transfer was studied using microinjection and scrape-loading techniques. Both methods yielded results indicating considerable gap junctional communication between cultured mesangial cells. Gap junctional communication between mesangial cells was upregulated by adenosine 3',5'-cyclic monophosphate (cAMP). Conversely, cell-to-cell communication was attenuated by exposure to the tumor promoter phorbol myristate acetate, the Ca ionophore ionomycin, reduced oxygen intermediates, and cell acidification. Expression of voltage gated calcium channels by mesangial cells was studied microspectrofluorimetrically using fura-2 fluorescence. KCl-induced depolarization, BAY-K 8644, and readdition of calcium to Ca-free depolarizing medium all produced a nifedipine-inhibitable increase in cytosolic calcium concentration. The existence of voltage-gated calcium channels in communication-competent cells suggests the possibility of propagation of depolarizing signals across the syncytium. This was studied by microapplication of KCl to the microenvironment of a single cell and monitoring fura-2 fluorescence in remote cells. This maneuver resulted in propagating calcium waves in communication-competent monolayers; calcium waves could not be evoked in monolayers exposed to an alkanol-type gap junction uncoupler, octanol. It is concluded that cultured rat mesangial cells form a syncytium capable of propagating calcium transients from a single depolarized cell to its coupled neighbors.

Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines.

Connexin43 is a member of the highly homologous connexin family of gap junction proteins. We have studied how connexin monomers are assembled into functional gap junction plaques by examining the biosynthesis of connexin43 in cell types that differ greatly in their ability to form functional gap junctions. Using a combination of metabolic radiolabeling and immunoprecipitation, we have shown that connexin43 is synthesized in gap junctional communication-competent cells as a 42-kD protein that is efficiently converted to a approximately 46-kD species (connexin43-P2) by the posttranslational addition of phosphate. Surprisingly, certain cell lines severely deficient in gap junctional communication and known cell-cell adhesion molecules (S180 and L929 cells) also expressed 42-kD connexin43. Connexin43 in these communication-deficient cell lines was not, however, phosphorylated to the P2 form. Conversion of S180 cells to a communication-competent phenotype by transfection with a cDNA encoding the cell-cell adhesion molecule L-CAM induced phosphorylation of connexin43 to the P2 form; conversely, blocking junctional communication in ordinarily communication-competent cells inhibited connexin43-P2 formation. Immunohistochemical localization studies indicated that only communication-competent cells accumulated connexin43 in visible gap junction plaques. Together, these results establish a strong correlation between the ability of cells to process connexin43 to the P2 form and to produce functional gap junctions. Connexin43 phosphorylation may therefore play a functional role in gap junction assembly and/or activity.

Scrape-loading and dye transfer: A rapid and simple technique to study gap junctional intercellular communication

Gap junction-mediated intercellular communication has been recognized in cells from different tissues of various organisms and has been implicated in a variety of cellular functions and dysfunctions. Here we describe a new, direct and rapid technique with which to study this cellular phenomenon. It employs scrape-loading to introduce a low molecular weight (MW) fluorescent dye, Lucifer yellow CH (MW 457.2) into cells in culture and allows the monitoring of its transfer into contiguous cells. In communication-competent cells the dye transmission occurred within minutes after loading. The involvement of membrane junctions in Lucifer yellow transfer was verified by the concurrent loading of a high MW marker dye conjugate, rhodamine dextran (MW 10000). Once introduced intracellularly the rhodamine dextran is unable to cross the relatively narrow membrane junctions. Chemicals of variable potency known to block junctional communication were tested in Chinese hamster V79 cells and other mammalian cells. The results showed effective blockage of the dye transfer at non-cytotoxic doses. This new technique can be applied to a wide variety of mammalian (including human) cells. In addition, it has the potential to be utilized as a rapid screening assay to detect chemicals that can modulate intercellular communication and to study their mechanism of action.

The regulation of hypoxanthine guanine phosphoribosyl transferase activity through transfer of PRPP by metabolic cooperation

We have developed a method of relating changes in hypoxanthine guanine phosphoribosyl transferase (HGPRTase) activity to the rate of phosphoribosyl pyrophosphate (PRPP) synthesis in isolated cell lines and in co-cultures of different cell lines. Using this approach, we have determined the response of the HGPRTase activity of communication-competent and communication-incompetent cells to changes in PRPP content. The HGPRTase activity of HGPRT + communication-competent NS cells responds to changes of their own PRPP level, as well as to changes of the PRPP level of HGPRT − cells with which they are co-cultured. In contrast, the HGPRTase activity of the HGPRT +, but communication-incompetent L 929 cells responds to changes of their own PRPP content but not to changes of the PRPP content of the cocultured HGPRT − cells. These and other experiments show that PRPP is freely exchangeable between communication-competent cells and that the intracellular activity of HGPRTase in one cell can be regulated by changes in the levels of its substrate in another cell through metabolic cooperation. The results also indicate that HGPRTase normally functions at a small fraction of its total activity, and that this can be greatly increased by raising the intracellular PRPP levels. Furthermore, it is found that when communication-competent cells establish intercellular communication, they share a common pool of PRPP and of purine nucleotides. This approach can be used as the basis of a biochemical method for the quantitation of metabolic cooperation between cells.