Abstract
Adenosine is an endogenous signaling molecule that is highly up-regulated in inflammatory states. Adenosine acts through the A2b receptor, a G protein-coupled receptor that couples positively to Galpha(s) and activates adenylate cyclase. This leads to cAMP-mediated electrogenic chloride secretion in intestinal epithelia. To better understand the regulation of the A2b receptor in intestinal epithelia, we studied the effects of interferon-gamma (IFN-gamma), a potent immunomodulatory cytokine, in the T84 cell line. Pretreatment of cells with 500 units/ml IFN-gamma for 12 h inhibited an adenosine-induced short circuit current (Isc) without affecting the transepithelial resistance. Under these conditions, IFN-gamma did not inhibit the protein expression or membrane recruitment of the A2b receptor, shown to be essential for its function. Interestingly, IFN-gamma inhibited cAMP levels as well as its downstream signaling pathway as shown by the inhibition of adenosine-induced phosphorylation of cAMP response element-binding protein and protein kinase A activity. Similar studies with forskolin, a direct activator of adenylate cyclase, also demonstrated inhibition of cAMP and its downstream response by IFN-gamma. However, IFN-gamma did not affect secretory responses to the calcium-dependent secretagogue carbachol or cAMP analog 8-bromo-cAMP, indicating that normal secretory responses to adequate second messengers in IFN-gamma-treated cells are achievable. Moreover, IFN-gamma inhibited the expression of adenylate cyclase isoforms 5 and 7. In conclusion, we demonstrate that IFN-gamma down-regulates adenosine-mediated signaling possibly through the direct inhibition of adenylate cyclase expression. We propose that IFN-gamma may acutely affect global cAMP-mediated responses in the intestinal epithelia, thereby decreasing secretory responses, which may consequently aggravate inflammatory processes.
Highlights
Intestinal lumen and in tissue during inflammation to as high as 500 – 600 nM [1]
To verify further that adenylate cyclase activity and not the cAMP-dependent transporters involved in adenosineinduced induced short circuit current (Isc) was affected by IFN-␥, we studied the effect of 8-Br-cAMP, a cAMP analog, on the Isc response
We addressed the regulation of the adenosine 2b receptor (A2bR) by IFN-␥, the most critical inflammatory cytokine that is highly up-regulated in acute and chronic colitis in human and is known to play an important role in chloride secretion and barrier function in the intestine [18, 19]
Summary
Intestinal lumen and in tissue during inflammation to as high as 500 – 600 nM [1]. The intestinal adenosine 2b receptor (A2bR), one of the four adenosine receptor subtypes (A1, A2a, A2b, and A3), mediates the biological effects of adenosine [2, 3]. Apical or basolateral stimulation of the A2bR induces an increase in intracellular cAMP and downstream cAMP signaling including phosphorylation and activation of the transcription factor, CREB and the activation of PKA [3, 10] The former is involved in interleukin-6 secretion in the intestine in response to adenosine [11], and PKA is involved in the chloride secretory pathway activated by adenosine [11, 12]. Prior studies using vasoactive intestinal peptide (VIP), cholera toxin (cAMP-mediated Isc), and carbachol (calciummediated Isc) demonstrated significant decrease in transepithelial resistance (TER) and chloride secretion after prolonged treatment with IFN-␥ (Ͼ24 – 48 h) without altering the morphology of cells This inhibition in secretory response has been attributed to decreased synthesis/expression of cystic fibrosis conductance regulator (CFTR) [20], NaϩKϩ-ATPase, and/or NaϩKϩ-2ClϪ cotransporter required for anion secretion [21]. In this study we investigated the effect of acute IFN-␥ exposure on the expression, signaling, and secretory function of the A2bR
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