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

Abstract

ATP release from urinary bladder is vital for afferent signaling. The aims of this study were to localize CALHM1 and pannexin-1 expression and to determine their involvement in mediating ATP release in the bladder. Gene expression by PCR and immunohistochemistry were performed 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 a 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 CALHM1 antibody. Brefeldin A, a vesicular transport inhibitor, and ruthenium red, a non-selective 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 was 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 that 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.