Brilliant blue G, a P2X7 receptor antagonist, attenuates early phase of renal inflammation, interstitial fibrosis and is associated with renal cell proliferation in ureteral obstruction in rats.

BACKGROUND: Previous study showed that purinergic P2X7 receptors (P2X7R) reach the highest expression in the first week after unilateral ureteral obstruction (UUO) in mice, and are involved in the process of inflammation, apoptosis and fibrosis of renal tissue. We, herein, document the role of purinergic P2X7 receptors activation on the third day of UUO, as assessed by means of BBG as its selective inhibitor. METHODS: We investigated the effects of brilliant blue G (BBG), a P2X7R antagonist, in the third day of kidney tissue response to UUO in rats. For this purpose, male Wistar rats submitted to UUO or sham operated, received BBG or vehicle (V), comprising four groups: UUO-BBG, UUO-V, sham-BBG and sham-V. The kidneys were harvested on day 3 UUO and prepared for histology, immunohistochemistry (P2X7R, PCNA, CD-68, α-sma, TGF-ß1, Heat-shock protein-47, TUNEL assay), quantitative real-time PCR (IL-1ß, procollagens type I, III, and IV) for mRNA quantification. RESULTS: The group UUO-V presented an enhancement in tubular cell P2X7-R expression, increase influx of macrophages and myofibroblasts, HSP-47 and TGF- ß1 expression. Also, upregulation of procollagen types I, III, and IV, and IL-1ß mRNAs were seen. On the other hand, group UUO-BBG showed lower expression of procollagens and IL-1ß mRNAs, as well as less immunoreactivity of HSP-47, TGF-ß, macrophages, myofibroblasts, and tubular apoptosis. This group also presented increased epithelial cell proliferation. CONCLUSION: BBG, a known highly selective inhibitor of P2X7R, attenuated renal inflammation, collagen synthesis, renal cell apoptosis, and enhanced renal cell proliferation in the early phase of rat model of UUO.

P2X3 receptor antagonism reduces the occurrence of apnoeas in newborn rats.

Hyperreflexia of the peripheral chemoreceptors is a potential contributor of apnoeas of prematurity (AoP). Recently, it was shown that elevated P2X3 receptor expression was associated with elevated carotid body afferent sensitivity. Therefore, we tested whether P2X3 receptor antagonism would reduce AoP known to occur in newborn rats. Unrestrained whole-body plethysmography was used to record breathing and from this the frequency of apnoeas at baseline and following administration of either a P2X3 receptor antagonist - AF-454 (5 mg/kg or 10 mg/kg s.c.) or vehicle was derived. In a separate group, we tested the effects of AF-454 (10 mg/kg) on the hypoxic ventilatory response (10 % FiO2). Ten but not 5 mg/kg AF-454 reduced the frequency of AoP and improved breathing regularity significantly compared to vehicle. Neither AF-454 (both 5 and 10 mg/kg) nor vehicle affected baseline respiration. However, P2X3 receptor antagonism (10 mg/kg) powerfully blunted hypoxic ventilatory response to 10 % FiO2. These data suggest that P2X3 receptors contribute to AoP and the hypoxic ventilatory response in newborn rats but play no role in the drive to breathe at rest.

Intra-Articular Blockade of P2X7 Receptor Reduces the Articular Hyperalgesia and Inflammation in the Knee Joint Synovitis Especially in Female Rats.

Synovitis is a key factor in joint disease pathophysiology, which affects a greater proportion of women than men. P2X7 receptor activation contributes to arthritis, but whether it plays a role in articular inflammatory pain in a sex-dependent manner is unknown. We investigated whether the P2X7 receptor blockade in the knee joint of male and female rats reduces the articular hyperalgesia and inflammation induced by a carrageenan knee joint synovitis model. Articular hyperalgesia was quantified using the rat knee joint incapacitation test and the knee joint inflammation, characterized by the concentration of cytokines tumor necrosis factor-α, interleukin-1ß, interleukin-6, and cytokine-induced neutrophil chemoattractant-1, and by neutrophil migration, was quantified using enzyme-linked immunosorbent assay and by myeloperoxidase enzyme activity measurement, respectively. P2X7 receptor blockade by the articular coadministration of selective P2X7 receptor antagonist A740003 with carrageenan significantly reduced articular hyperalgesia, pro-inflammatory cytokine concentrations, and myeloperoxidase activity induced by carrageenan injection into the knee joint of male and estrus female rats. However, a lower dose of P2X7 receptor antagonist was sufficient to significantly induce the antihyperalgesic and anti-inflammatory effects in estrus female but not in male rats. These results suggest that P2X7 receptor activation by endogenous adenosine 5'-triphosphate is essential to articular hyperalgesia and inflammation development in the knee joint of male and female rats. However, female rats are more responsive than male rats to the antihyperalgesic and anti-inflammatory effects induced by P2X7 receptor blockade. PERSPECTIVE: P2X7 receptors could be promising therapeutic targets in the treatment of knee joint disease symptoms, especially in women, who are more affected than men by these conditions.

Physiological roles and potential therapeutic applications of the P2X7 receptor in inflammation and pain.

The P2X7 receptor (P2X7R) is a nonselective cation channel that is activated by extracellular ATP and triggers the secretion of several proinflammatory substances, such as IL-1ß, IL-18, TNF-α, and nitric oxide. Recently, several preclinical studies have demonstrated that this receptor participates in inflammation and pain mechanisms. Taken together, these results indicate that P2X7R is a promising pharmacological target, and compounds that modulate the function of this receptor show potential as new anti-inflammatory medicines. In this review, we discuss aspects of P2X7R pharmacology and the participation of this protein in inflammation and pain and provide an overview of some promising compounds that have been tested as antagonists of P2X7R, with clinical applicability.

Neuronal P2X3 receptor activation is essential to the hyperalgesia induced by prostaglandins and sympathomimetic amines released during inflammation.

We have demonstrated that the activation of P2X3 receptor on peripheral afferent neurons is critical to development of inflammatory hyperalgesia in peripheral tissue, although pharmacological administration of prostaglandin E(2) or sympathomimetic amines is enough to sensitize primary afferent neurons by acting directly in neuronal receptors. Therefore, to clarify this ambiguity this study verifies whether P2X3 receptor activation on primary afferent neurons enables the sensitization induced by prostaglandin E(2) or sympathomimetic amine. Initially, this study confirmed that co-administration of A317491 (60 µg/paw), a selective P2X3 receptor antagonist, or pre-treatment with dexamethasone (1 mg/mL/kg) prevents the mechanical hyperalgesia induced by carrageenan (300 µg/paw) in the rat's hind paw. Sub-threshold doses of PGE(2) (4 ng/paw) or dopamine (0.4 µg/paw), that do not induce hyperalgesia by themselves, when injected just following αßmeATP or carrageenan in rats treated with dexamethasone induced hyperalgesia, which is prevented by A317491 or treatment with periganglionar (DRG-L5) injections of ODN-antisense, against P2X3 receptor. Furthermore, because PKCɛ translocation induces an increase of neuronal susceptibility to inflammatory mediators, this study demonstrates that αßmeATP in peripheral tissue increases the expression of PKCɛ in cell membranes of DRG-L5, and in contrast, the administration of PKCɛ translocation inhibitor (1 µg/paw) in peripheral tissue 45 min before αßmeATP, prevented the hyperalgesia induced by sub-threshold dose of PGE(2) (4 ng/paw). In conclusion, this study suggests that neuronal P2X3 receptor activation and the consequent PKCɛ translocation increase the susceptibility of nociceptor to inflammatory mediators allowing the development of inflammatory hyperalgesia.