Expression and localization of pannexin-1 and CALHM1 in porcine bladder and their involvement in modulating ATP release

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, (10)Panx1, 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. (10)Panx1 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.