P2X7 receptor antagonist improves gastrointestinal disorders in spontaneously hypertensive rats

Abstract

The purinergic system participates in the control of blood pressure. Hypertension promotes the occurrence of gastrointestinal disorders such as intestinal inflammation and gastric emptying delay. This study aimed i) to investigate the participation of the P2X7 receptor blocker Brilliant Blue G (BBG) on gastric emptying of solids and changes in oxidative stress in the gastric fundus, duodenum, and colon of spontaneously hypertensive rats (SHR) and ii) to study the putative relationship of this effect with the renin-angiotensin system. Rats were divided into five groups: Control, SHR, SHR+BBG, SHR+BBG+ATP, and SHR+BBG+ANG II. In the gastrointestinal tract, we assessed gastric emptying (GE) and oxidative stress markers (NOx, MPO, GSH, SOD). We observed a decrease in the GE rate (P<0.05) in SHR vs control rats (21.8±2.0% vs 42.8±3.5%). The decrease in GE was returned (P<0.05) to control levels by BBG in SHR rats (21.8±2.0% vs 41.6±3.2%). Co-administration of ATP or ANG II together with BBG bypassed the effect of the P2X7 antagonist on GE in SHR (P<0.05) (21.9±5.0% vs 25.6±3.0% vs 41.6±3.2%). The MPO activity increased (P<0.05) in the gastric fundus of SHR compared to control rats (6.12±2.26 vs 0.077±0.02 UMPO/mg tissue); this effect was prevented (P<0.05) by BBG (0.55±0.15 vs 6.12±2.26 UMPO/mg tissue). Data demonstrated that blockage of P2X7 receptors with BBG can improve the GE delay and oxidative stress biomarkers in SHR animals. This preventive effect of BBG on GE delay was abrogated by ANG II and ATP, thus prompting crosstalk between renin-angiotensin and the purinergic signaling systems underlying this phenomenon.

Figure 1. Experimental design. SHR: spontaneously hypertensive rat; BBG: Brilliant Blue G; ANG II: angiotensin II; ATP: adenosine triphosphate.

Figure 2. Gastric emptying of a solid test meal of Wistar vs spontaneously hypertensive rats (SHR), SHR treated with BBG, SHR treated with BBG plus ATP, and SHR treated with BBG plus ANG II. Data are reported as means±SE. *P<0.05 (one-way ANOVA, followed by the Tukey test). BBG: Brilliant Blue G; ANG II: angiotensin II; ATP: adenosine triphosphate.

Figure 3. Glutathione (GSH) concentrations in the gastric fundus (A), duodenum (B), and colon (C) of Wistar rats vs spontaneously hypertensive rats (SHR), SHR treated with BBG, and SHR treated with BBG plus ATP. Myeloperoxidase (MPO) activity in the gastric fundus (D), duodenum (E), and colon (F) of Wistar rats vs SHR, SHR treated with BBG, and SHR treated with BBG plus ATP. Data are reported as means±SE. *P<0.05 (one-way ANOVA, followed by the Tukey test). BBG: Brilliant Blue G; ATP: adenosine triphosphate; NPSH: non-protein sulfhydryl; UMPO: units of MPO.

Figure 4. Reduced glutathione (GSH) concentrations in the gastric fundus (A), duodenum (B), and colon (C) of Wistar rats vs spontaneously hypertensive rats (SHR), SHR treated with BBG, and SHR treated with BBG plus ANG II. Myeloperoxidase (MPO) activity in the gastric fundus (D), duodenum (E), and colon (F) of Wistar rats vs SHR, SHR treated with BBG, and SHR treated with BBG plus ANG II. Data are reported as means±SE. *P<0.05 (one-way ANOVA, followed by the Tukey test). BBG: Brilliant Blue G; ANG II: angiotensin II; NPSH: non-protein sulfhydryl; UMPO: units of MPO.

Figure 5. Nitrite/nitrate (NOx) concentrations in the gastric fundus (A), duodenum (B), and colon (C) of Wistar rats vs spontaneously hypertensive rats (SHR), SHR treated with BBG, and SHR treated with BBG plus ATP. Superoxide dismutase (SOD) activity in the gastric fundus (D), duodenum (E), and colon (F) of Wistar rats vs SHR, SHR treated with BBG, and SHR treated with BBG plus ATP. Data are reported as means±SE. *P<0.05 (one-way ANOVA, followed by the Tukey test). BBG: Brilliant Blue G; ATP: adenosine triphosphate; USOD: units of SOD.

Figure 6. Nitrite/nitrate (NOx) concentrations in the gastric fundus (A), duodenum (B), and colon (C) of Wistar rats vs spontaneously hypertensive rats (SHR), SHR treated with BBG, and SHR treated with BBG plus ANG II. Superoxide dismutase (SOD) activity in the gastric fundus (D), duodenum (E), and colon (F) of Wistar rats vs SHR, SHR treated with BBG, SHR treated with BBG plus ANG II. Data are reported as means±SE. *P<0.05 (one-way ANOVA, followed by the Tukey test). BBG: Brilliant Blue G; ANG II: angiotensin II USOD: units of SOD.

Figure 7. Proposed mechanism of hypertension effects on gastric emptying (GE) and oxidative stress in the gastric fundus. In spontaneously hypertensive rats (SHR), there was a delay in gastric emptying rate, probably due to oxidative stress and inflammation caused by hypertension. Hypertension-induced increases in ANG II acting via the G protein-coupled AT1 receptor subtype favors NADPH oxidase and iNOS activity, which triggers oxidative/nitrosative stress and the release of reactive oxygen and nitrogen species, along with inflammatory lymphocyte recruitment and differentiation. Here, we hypothesized that ANG II may act synergistically with P2X7 receptors located in lymphocytes and other cells by promoting the local release of the danger molecule ATP. Synergism between ANG II and the purinergic system may be either via a direct mechanism or indirectly by increasing inflammatory stress. This inflammatory cascade triggered by ANG II and purinoceptors activation acting together with sympathetic overactivation in hypertensive animals may contribute to the recruitment of immune cells leading to a further increase in neutrophil infiltration and MPO overshooting. No alterations were observed in the activity of SOD and the levels of GSH and NOx in SHR. Blockage of P2X7 receptors with BBG increased the GE rate and reduced both the MPO activity and GSH levels. Interestingly the effect of BBG was “de novo” abrogated by both ATP and ANG II suggesting a crosstalk between ATP-sensitive P2X7 receptors and AT1 receptors activation in this endeavor. ANG II: angiotensin II; AT1: angiotensin II receptor type 1; ATP: adenosine triphosphate; BBG: Brilliant Blue G; GSH: glutathione; iNOS: inducible nitric oxide synthase; NADPH: adenine and nicotinamide dinucleotide phosphate; NE: norepinephrine; MPO: myeloperoxidase; SHR: spontaneously hypertensive rats; SOD: Superoxide dismutase. Red arrows indicate hypertension effects. Green lines indicate antioxidant protection. Light blue arrows indicate BBG actions. Dark blue arrows indicate BBG + ANG II actions. Purple arrows indicate BBG + ATP actions.