ABSTRACT
Patients undergoing chemotherapy with cyclophosphamide experience cystitis due to excretion of a toxic metabolite, acrolein. Cystitis, an inflammation of the bladder, is associated with damage to the integrity of the urothelial barrier. The purinergic P2X7 receptor (P2X7R) is increasingly recognized for its role in inflammation and cell death. P2X7R is expressed abundantly on the bladder urothelium. The aim of this study was to investigate the role of P2X7R in acrolein-induced inflammatory damage in primary cultured porcine bladder urothelial cells. Confluent urothelial cells in culture were treated with acrolein to induce damage; also, with the P2X7R selective antagonist, A804598. Cell viability assay, immunocytochemistry, and trans-epithelial electrical resistance (TEER) studies were carried out to investigate the effect of treatments on urothelial cell function. Acrolein induced a significant reduction in urothelial cell viability, which was protected by the presence of A804598 (10 µM). The urothelial barrier function, indicated by TEER values, was also significantly reduced by acrolein, whereas pre-incubation with P2X7R antagonist significantly protected the urothelial cell barrier from acrolein-induced TEER reduction. The structure of urothelial cell tight junctions was similarly impacted by acrolein treatment, showing the fragmentation of zona occludens-1 (ZO-1) immunoreactivity. Pre-treatment of cells with A804598 countered against the actions of acrolein and maintained ZO-1 expression level and cell structure. The damaging effect of acrolein on urothelial cells integrity could be impaired by inhibition of P2X7R, therefore P2X7R blockade may be a possible therapy in patients with bladder cystitis caused by cyclophosphamide treatment.
ABSTRACT
Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic lower urinary tract condition. Patients with IC/BPS suffer from debilitating pain and urinary urgency. The underlying etiology of IC/BPS is unknown and as such current treatments are mostly symptomatic with no real cure. Many theories have been proposed to describe the etiology of IC/BPS, but this review focuses on the role of inflammation. In IC/BPS patients, the permeability of the urothelium barrier is compromised and inflammatory cells infiltrate the bladder wall. There are increased levels of many inflammatory mediators in patients with IC/BPS and symptoms such as pain and urgency that have been associated with the degree of inflammation. Recent evidence has highlighted the role of purinergic receptors, specifically the P2X7 receptor, in the process of inflammation. The results from studies in animals including cyclophosphamide-induced hemorrhagic cystitis strongly support the role of P2X7 receptors in inflammation. Furthermore, the deletion of the P2X7 receptor or antagonism of this receptor significantly reduces inflammatory mediator release from the bladder and improves symptoms. Research results from IC/BPS patients and animal models of IC/BPS strongly support the crucial role of inflammation in the pathophysiology of this painful disease. Purinergic signaling and purinergic receptors, especially the P2X7 receptor, play an undisputed role in inflammation. Purinergic receptor antagonists show positive results in treating different symptoms of IC/BPS.
ABSTRACT
ATP release from urinary bladder is vital for afferent signaling. The aims of this study were to localize calcium homeostasis modulator 1 (CALHM1) and pannexin-1 expression and to determine their involvement in mediating ATP release in the bladder. To determine gene expression and cellular distribution, PCR and immunohistochemistry were performed, respectively, in the porcine bladder. CALHM1 and pannexin-1-mediated ATP release in response to hypotonic solution (0.45% NaCl)-induced stretch, and extracellular Ca2+ depletion ([Ca2+]0) was measured in isolated urothelial, suburothelial, and detrusor muscle cells. CALHM1 and pannexin-1 mRNA and immunoreactivity were detected in urothelial, suburothelial, and detrusor muscle layers, with the highest expression on urothelium. Hypotonic stretch caused a 2.7-fold rise in ATP release from all three cell populations (P < 0.01), which was significantly attenuated by the pannexin-1 inhibitor, 10Panx1, and by the CALHM1 antibody. Brefeldin A, a vesicular transport inhibitor, and ruthenium red, a nonselective CALHM1 channel blocker, also significantly inhibited stretch-mediated ATP release from urothelial cells. [Ca2+]0 caused a marked, but transient, elevation of extracellular ATP level in all three cell populations. CALHM1 antibody and ruthenium red inhibited [Ca2+]0-induced ATP release from urothelial cells, but their effects on suburothelial and detrusor cells were insignificant. 10Panx1 showed no significant inhibition of [Ca2+]0-induced ATP release in any types of cells. The results presented here provide compelling evidence that pannexin-1 and CALHM1, which are densely expressed in the porcine bladder, function as ATP release channels in response to bladder distension. Modulation of extracellular Ca2+ may also regulate ATP release in the porcine bladder through voltage-gated CALHM1 ion channels.
