Upregulation of cyclooxygenase-2 and thromboxane A₂production mediate the action of tumor necrosis factor-α in isolated rat myenteric ganglia

Abstract

Intact myenteric ganglia from 4- to 10-day-old rats were isolated from the small intestine. The preparations were cultured overnight, and drugs were applied within this time frame (20 h). Whole cell patch-clamp technique was used to measure basal membrane potential and carbachol-induced depolarization at neurons within these ganglia. Pretreatment with TNF-alpha (100 ng/ml) hyperpolarized the membrane (from -31.0 +/- 2.7 mV under control conditions to -61.2 +/- 3.2 mV in the presence of the cytokine) and potentiated the depolarization induced by carbachol (from 5.2 +/- 0.7 mV under control conditions to 27.5 +/- 2.0 mV in the presence of the cytokine). These effects were mimicked by carbocyclic thromboxane A2 (10(-6) mol/l), a stable thromboxane A2 agonist. The TNF-alpha action was inhibited by 1-benzylimidazole (2 x 10(-4) mol/l), a thromboxane synthase inhibitor, and BAY U 3405 (5 x 10(-4) mol/l), an inhibitor of thromboxane receptors. Measurements of thromboxane production in the supernatant of the culture revealed an increased concentration of thromboxane B2, the stable metabolite of thromboxane A2, after exposure to TNF-alpha. Immuncytochemical staining for cyclooxygenase-2 (COX-2) and the neuronal marker microtubule-associating protein-2 revealed an upregulation of COX-2 in myenteric neurons after exposure to the cytokine. These results demonstrate the involvement of COX-2 and the subsequent production of thromboxane A2 in the presence of TNF-alpha.

Effect of the stable thromboxane derivative, carbocyclic thromboxane A2, on membrane potential of rat myenteric neurones in culture

“…Because native TXA 2 has only a half‐life of about 30 s in aqueous solutions, 15 all experiments were performed with the stable TXA 2 derivative, cTXA 2 , in which both O‐atoms in the cyclic structure of the molecule are replaced by carbon atoms 16 . In accordance with previous observations, 10 cTXA 2 (10 −6 mol L −1 ) induced a hyperpolarization of the membrane of myenteric neurones from −19.3 ± 2.5 to −29.3 ± 2.3 mV ( n = 9, P < 0.05; Fig. 1A).…”

“…This effect was suppressed by nimesulide, a drug acting as specific COX‐2 inhibitor 2 . A subsequent immunhistochemical investigation revealed an upregulation of COX‐2 in the presence of TNF‐ α 10 . The electrophysiological effects of TNF‐ α seem to be mediated by TXA 2 , as suggested by the sensitivity against a TXA 2 receptor blocker (such as Bay u3405) or a TXA 2 synthase inhibitor (such as 1‐benzylimidazole), and by the ability of carbocyclic TXA 2 (cTXA 2 ), a stable TXA 2 derivative, to mimic the actions of the cytokine 10 .…”

Effects of cyclooxygenase-2 gene inactivation on cardiac autonomic and left ventricular function in experimental diabetes

“…In turn, increased oxidative stress has been shown in hepatic cells lines to increase production of TXB 2 via induction of thromboxane synthase (23). It has been also shown that cytokine-induced COX-2 upregulation is associated with thromboxane release in airway smooth muscle cells, which is blocked by a selective COX-2 inhibitor (34), and that TNF-␣ stimulates production of thromboxane in myenteric neurons by mechanisms involving increased COX-2 and thromboxane synthase activity (48). Thus, although the mechanism(s) of the upregulation of TXB 2 in diabetic mice remain unclear, our data suggest that by modulating myocardial oxidative stress, prostanoid content, and inflammation in response to diabetes, COX-2 gene inactivation may be associated with beneficial effects on cardiovascular function in experimental diabetes.…”

Inflammation and Gut-Brain Axis During Type 2 Diabetes: Focus on the Crosstalk Between Intestinal Immune Cells and Enteric Nervous System

“…This property of TNF-α could explain the negative impact of cytokines on gut motility. In a molecular point of view, the effect of TNF-α is mediated by an upregulation of the Cyclo-Oxygenase (COX-2), which in turn increases the production of an arachidonic acid derived-molecule, Thromboxane A2 (TXA 2 ) ( Rehn et al, 2005 ). In experimental colitis, Chandrasekharan et al (2013) have also demonstrated that TNF-α has an effect on the apoptosis rate of enteric neurons.…”