Background and Purpose: Asthma is characterized by airway hyperresponsiveness (AHR), allergic inflammation, and airway remodeling. Although recent studies showed that asthma pathophysiology involves P2X purinoceptor 4 (P2X4) activation, a potential link with chronic asthma remains to be explored. We investigated the effect of a novel P2X4 receptor antagonist BR11595 on allergen-induced airway responses in a guinea pig model of chronic asthma. Experimental Approach: Sensitized guinea pigs were exposed to saline or ovalbumin (OVA) once weekly via aerosolization for 12 weeks. BR11595 (10 mg·kg⁻¹) was injected intraperitoneally 5 times per week, for 4 different regimens: all 12 weeks, first 6 weeks, last 6 weeks, or last week only. Airway responsiveness to histamine was assessed 24h before and 6h after OVA exposure in week 1, 6 and 12. Lung tissue Inflammation and remodeling were determined 24h after the last OVA exposure. Key Results: OVA induced AHR at week 1, 6 and 12 compared to saline-challenged animals. The AHR was less pronounced in week 12 compared to week 1. BR11595 significantly reduced OVA-induced AHR in week 6 in guinea pigs treated with BR11595 for 6 weeks. AHR in week 12 was reduced after BR11595 treatment in week 12 only, next to OVA-induced eosinophilia and Goblet cell hyperplasia, indicating an acute role of P2X4 receptors on chronic inflammation. Conclusion and Implications: The P2X4-receptor antagonist BR11595 acutely inhibits AHR, eosinophilia, and Goblet cell hyperplasia after 12 weeks, indicating its potential as a therapeutic target for acute intervention of chronic asthma attacks or exacerbations.

Background and Purpose: We have previously shown that the antidiabetic drug metformin protects hepatocytes against toxicity by various stressors. Chronic or excessive consumption of diclofenac (DF), a pain-relieving drug, leads to drug-induced liver injury via a mechanism involving mitochondrial damage and ultimately apoptotic death of hepatocytes. However, whether metformin protects against DF-induced toxicity is unknown. Recently, we have shown that cAMP elevation is protective against DF-induced apoptotic death in hepatocytes, a protective effect primarily involving the downstream cAMP effector EPAC and preservation of mitochondrial function. This study aimed to investigate whether metformin protects against DF-induced toxicity via cAMP-EPACs. Experimental Approach: Primary rat hepatocytes were exposed to 400 µmol/L DF. CE3F4 or ESI-O5 were used as EPAC-1 or 2 inhibitors, respectively. Apoptosis was measured by caspase-3 activity and necrosis by Sytox green staining. Seahorse X96 assay was used to determine mitochondrial function. Mitochondrial reactive oxygen species (ROS) production was measured using MitoSox, mitochondrial MnSOD expression by immunostaining and mitochondrial morphology (fusion and fission ratio) by 3D refractive index imaging. Key Results: Metformin (1 mmol/L) was protective against DF-induced apoptosis in hepatocytes. This protective effect was EPAC-dependent (mainly EPAC-2). Metformin restored mitochondrial morphology in an EPAC-independent manner. DF-induced mitochondrial dysfunction demonstrated by decreased oxygen consumption rate, increased ROS production, and a reduced MnSOD level were all reversed by metformin in an EPAC-dependent manner. Conclusion and Implications: Metformin protects hepatocytes against DF-induced toxicity via cAMP-dependent EPAC-2. Data available on request from the authors.