Purine Participation in the Vasomotor Effect of Hypochlorous Acid in the Porcine Coronary Artery

Abstract

Neutrophils are part of the body’s defence mechanism against microorganisms. They produce their anti-inflammatory action by producing different reactive oxygen species (ROS). Hypochlorous acid (HOCl), generated by neutrophils, plays a major role in killing pathogens and mediating inflammation. It is known that nucleotides and nucleosides are also released during inflammation. Inflammation leads to dysfunctional purinergic signalling, which is linked to cardiovascular diseases such as hypertension, diabetes, atherosclerosis, and thrombosis. However, it is unclear whether purine nucleotides are involved in HOCl's vasomotor response. Understanding vascular control mechanisms is essential for developing new therapeutic approaches to cardiovascular diseases such as hypertension and atherosclerosis. The main purpose of this study was to investigate the direct vasomotor effects of HOCl on the porcine coronary artery (PCA) as well as the mechanisms involved in this response. Further investigation was conducted to examine the possibility that purine receptors were involved in the response produced by HOCl by using different purinergic receptor antagonists. Moreover, the role of pannexin 1 and connexin channels (through which ATP may be released) in PCA's response to HOCl was investigated. It was also investigated whether HOCl released ATP from cultured human coronary artery endothelial cells. Moreover, the pharmacological profile of exogenous ATP and adenosine on PCA was examined for similarities with that of HOCl. Segments of the PCA were placed in an organ bath for isometric tension recording. They were incubated in the absence or presence of different antagonists and inhibitors, pre-contracted with U46619 (thromboxane A2 receptor agonist), then exposed to HOCl (100 and 500 μM). In some segments, the endothelium was removed to investigate the endothelium-dependency of the responses, and in some segments, the perivascular adipose tissue (PVAT) was retained. At 100 μM, HOCl induced an initial transient endothelium-dependent relaxation which returned to baseline; thereafter, a second slow relaxation independent of the endothelium was observed for 60 minutes. A transient endothelium-dependent relaxation response was also induced by 500 μM HOCl, followed by endothelium-dependent contraction; slow endothelium-independent relaxation was observed over 60 minutes. Nitic oxide synthase (NOS) inhibitors also inhibited the rapid relaxation in response to 100 and 500 μM HOCl and the contraction to 500 μM HOCl on the PCA. Based on these findings, it appears that NO might play a role in the rapid relaxation and contraction of HOCl. The HOCl-induced rapid relaxation and slow relaxation over 60 minutes were blocked in tissues precontracted with KCl instead of U46619. This indicates that the relaxation of PCA rings produced by HOCl is mediated by a mechanism that is sensitive to membrane hyperpolarization. Moreover, PVAT exerts an anti-contractile effect on the HOCl response. An adenosine P1 receptor blocker, 8-(p-sulphophenyl) theophylline, inhibited the rapid relaxation of both 100 and 500 μM HOCl and the contraction of 500 μM HOCl, indicating that adenosine receptors may be involved. In the presence of suramin (P2 receptor antagonist), rapid relaxation at 100 μM HOCl was blocked, whereas rapid relaxation at 500 μM HOCl was still evident. However, suramin blocked the contraction to 500 μM HOCl. This may indicate that P2 receptors play a role in the response to HOCl. The study excluded a subset of P2 purinoceptors (P2X1, P2Y1, and P2X4) because specific antagonists for these receptors were found not to affect HOCl responses. The effect of HOCl was not prolonged or altered in any way by ARL67156 (ecto-ATPase inhibitor). However, apyrase (hydrolysis nucleotides) abolished the response to both concentrations of HOCl. Probenecid and carbenoxolone (pannexin 1 and connexin channel blocker) inhibited contraction to 500 μM HOCl, consistent with HOCl-induced purine release via pannexin 1 and/or connexin channels, with purines acting at vasomotor P2 receptors. The concentration of extracellular ATP in samples of bathing fluid from cultured human coronary artery endothelial cells (HCAECs) was measured directly by a luminescence ATP assay kit (Abcam, Cat#ab113849). It found that exposure of HCAECs to 100 μM HOCl resulted in a significant increase in ATP levels within 1 minute. It is consistent with the observation that HOCl-induced rapid endothelium-dependent relaxation of PCA may occur through its actions on P2 and/or P1 receptors (subsequent to the release of ATP from the endothelium and breakdown of ATP to adenosine). Investigation of the responses to exogenous adenosine (30 μM) and ATP (100 μM) showed that the response to adenosine consisted of a largely endothelium-independent relaxation response, whereas the response to ATP additionally included a transient rapid relaxation phase that was partially dependent on the endothelium. Neither L-NAME nor suramin affected the relaxation response to adenosine or ATP. Adenosine and ATP responses were significantly inhibited by 8-SPT and apyrase, which is consistent with the inhibitory effects of 8-SPT and apyrase on HOCl responses in the PCA.Together, these results suggest that P1 and P2 purine receptors may participate in the vasomotor response of the PCA to HOCl. Additionally, the evidence suggests that connexin and pannexin 1 channels are involved in ATP release in response to HOCl. It is evident that purinergic signalling plays an important role in inflammatory responses and vascular contractility as a result of this interaction.