QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 3 February 2004
doi: 10.1242/jcs.00942

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lin, G. C. Articles by Steinberg, T. H. Search for Related Content PubMed PubMed Citation Articles by Lin, G. C. Articles by Steinberg, T. H.

Gap junctional communication modulates agonist-induced calcium oscillations in transfected HeLa cells

¹ Department of Internal Medicine, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110
² Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

View larger version (84K): [in a new window]   Fig. 1. UTP elicits asynchronous calcium oscillations in HeLa cells. (Top) Monolayers of HeLa cells were loaded with the calcium indicator fura-2 and 1 µM UTP was added to the medium while ratio imaging was performed. Time after UTP exposure is indicated on each panel. Scale bar: 40 µm. Oscillating cells (as detected in the bottom panel) are marked with an asterisk. (Bottom) Graph of cytosolic calcium concentration over time for single HeLa cells as shown in the top panel. An initial rise in cytosolic calcium was followed by random, asynchronous calcium oscillations in most cells.

View larger version (38K): [in a new window]   Fig. 2. Protein immunoblot analyses for Cx43, Cx45 and Cx46 in HeLa transfectants. Samples from HeLa and HeLa transfectants were resolved by SDS-PAGE, electrophoretically transferred to PVDF membranes, and then probed with antisera against Cx43, Cx45 or Cx46. Migration of molecular mass standards is indicated in the figures.

View larger version (84K): [in a new window]   Fig. 3. Immunofluorescence localization of connexins in HeLa transfectants. HeLa (a-c), HeLa/Cx43 (d), HeLa/Cx45 (e), or HeLa/Cx46 (f) cells were cultured on glass coverslips, fixed, permeabilized, and then incubated with rabbit antiserum to either Cx43 (a,d), Cx45 (b,e), or Cx46 (c,f) PBS for 1 hour at room temperature. The cells were then washed, labeled with Cy3-conjugated anti-rabbit IgG, and photographed. HeLa cells do not express detectable Cx43 (a) or Cx46 (c), and minimal Cx45 (b). In Cx43 and Cx45 transfected cells, the transfected connexins appear largely in a linear punctate pattern at cell-cell boundaries. In contrast, Cx46 appeared as diffuse linear intercellular staining in HeLa/Cx46 cells. Scale bar: 40 µm.

View larger version (12K): [in a new window]   Fig. 4. Dye transfer in HeLa/Cx43 and HeLa/Cx45 transfectants. Single cells were co-injected with Lucifer Yellow (LY) and DAPI, and dye transfer was assessed as the number of cells receiving dye per injected cell at 4 minutes. Both Cx43 transfected cell lines transferred LY well but did not pass DAPI. Conversely, both Cx45 transfected cell lines transfer DAPI better than LY.

View larger version (14K): [in a new window]   Fig. 5. Mechanically induced calcium waves in HeLa and Cx-transfected HeLa cells are inhibited by desensitization of ATP receptors. Cell monolayers were loaded with fura-2 and single cells were mechanically stimulated before or 5 minutes after desensitization of P2 receptors by addition of 10 µM ATP. Numbers represent the number of cells to which the subsequent calcium wave propagated.

View larger version (25K): [in a new window]   Fig. 6. UTP-mediated calcium responses in connexin-transfected HeLa cells. Cells were loaded with fura-2, exposed to 1 µM UTP, and ratio imaging was performed. Single cell calcium traces were constructed as above. HeLa/Cx45 transfectants exhibited asynchronous calcium oscillations similar to the parent HeLa cells. In contrast HeLa/Cx43 transfectants displayed a calcium rise that was followed by a gradual decline to baseline without much oscillatory behavior. HeLa/Cx46 transfectants displayed dampened oscillatory behavior.

View larger version (67K): [in a new window]   Fig. 7. Anandamide inhibits Cx43-mediated dye transfer. HeLa/Cx43 cells were microinjected with Lucifer Yellow in the presence or absence of anandamide, and dye transfer was assessed. Scale bar: 40 µm.

View larger version (30K): [in a new window]   Fig. 8. Gap junction-mediated effects on calcium oscillations are dependent on the dose of agonist applied. Monolayers of HeLa cells and transfectants were loaded with fura-2 and calcium response to different concentrations of UTP was assessed by video imaging. Single cell traces were categorized as no response, submaximal rise, maximal response without oscillations, or response with oscillations, as described in the text.