We previously showed that adenosine stimulates IL-6 secretion from TtT/GF cells suggesting that adenosine is an important regulator of folliculostellate (FS) cell function. Gap-junction communication is also important for FS cell function so the aim of this study was to investigate the action of adenosine on connexin 43 (Cx 43) expression and intercellular communication.
Adenosine production, as measured by reverse-phase fluorescent HPLC, was easily detectable in MMQ and GH3 cells, respectively 68 and 12 μM/106cells/hr but not in TtT/GF cells. Ecto-5′-nucleotidase (CD73) (the enzyme that cleaves AMP to adenosine) in these cells was demonstrated by HPLC analysis of the conversion of exogenously added fluorescent ethenoAMP to ethenoadenosine. The rate of AMP degradation was the same in all three cell lines with a half-life of around 1 hr; however preincubation with AOPCP or levamisole confirmed that the enzyme mediating this reaction in GH3, but not in MMQ or TtT/GF cells is CD73. The identity of the enzyme mediating AMP degradation in MMQ or TtT/GF cells is unknown.
We investigated the effect of NECA (universal adenosine receptor agonist) on Cx43 expression in TtT/GF cells using Western analysis and showed a time- and dose-related stimulation of both non-phosphorylated and phosphorylated forms. Expression of Cx43 reached a plateau after 4hr exposure to NECA and showed peak stimulation at 1 μM. Similar findings were obtained with adenosine, but not with the non-hydrolysable form of ATP, (ATPγS) which failed to have any effect on Cx43 expression.
Gap-junction transmission in TtT/GF cells was investigated by microinjecting Alexa Fluor 488 into individual cells. 10 μM NECA treatment for 4hrs stimulated the spread of the dye into 10±2.7 (P<0.01) cells compared with 3±1.7 cells in the untreated cultures.
These data show that adenosine, produced by FS cells or neighbouring endocrine cells, stimulates Cx43 expression and increases gap-junction communication.
01 - 05 Apr 2006
European Society of Endocrinology