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 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 Ca depletion ([Ca]) 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 ( < 0.01), which was significantly attenuated by the pannexin-1 inhibitor, 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. [Ca] caused a marked, but transient, elevation of extracellular ATP level in all three cell populations. CALHM1 antibody and ruthenium red inhibited [Ca]-induced ATP release from urothelial cells, but their effects on suburothelial and detrusor cells were insignificant. Panx1 showed no significant inhibition of [Ca]-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 Ca may also regulate ATP release in the porcine bladder through voltage-gated CALHM1 ion channels.

ATP Release Channels

“…Although direct electrophysiological measurements of ATP currents through CALHM1 are lacking, ATP release associated with CALHM1 expression and function has been demonstrated by measurements of ATP release from CALHM1-expressing cells in vitro and in vivo [ 27 , 105 , 145 ]. Heterologous expression of CALHM1 in HeLa cells, COS cells, and Xenopus oocytes led to the cell plasma membranes showing ATP permeability.…”

“…The physiological significance of CALHM1 had remained elusive until the discovery of its ATP-releasing function. CALHM1 has been reported to be expressed in the brain [ 139 , 141 , 146 ], taste buds [ 27 , 143 , 147 ], nasal epithelium [ 105 ], bladder [ 145 ], other tissues [ 139 ]. In the human and mouse brains, its expression has been detected in hippocampal and cortical neurons, where it has been suggested to be involved in the [Ca 2+ ] o regulation of neuronal excitability by modulating membrane conductance [ 141 ], and in memory flexibility by modulating long-term synaptic potentiation via the phosphorylation of NMDA and AMPA receptors mediated by Ca 2+ influx through the channel [ 148 ].…”

“…Other possible roles of CALHM1 as an ATP-release channel have recently been reported in the urinary bladder [ 145 ] and nasal epithelial cells [ 105 ]. In the urinary bladder, the distension of urothelial cells caused by bladder filling leads to the release of ATP, which activates P2X2/3 receptors on the suburothelial sensory nerves and thereby controls pain responses and afferent pathways controlling voiding reflexes [ 165 , 166 , 167 , 168 ].…”

“…However, the mechanisms underlying distension-induced urothelial ATP release are largely unknown. CALHM1 expression was identified in the urothelium as well as the suburothelium and detrusor muscle of the porcine bladder by RT-PCR and immunohistochemistry [ 145 ]. Both hypotonic stress and the depletion of Ca 2+ o induced ATP release from cultured urothelial cells that were significantly inhibited by ruthenium red, a nonspecific CALHM1 blocker, and a specific CALHM1 antibody [ 145 ], suggesting roles for CALHM1 in urinary bladder purinergic signaling along with other ATP-release mechanisms including exocytosis, connexins, and PANX1 that are also present in urothelial cells [ 108 , 170 ].…”

Purinergic P2X7 receptors as therapeutic targets in interstitial cystitis/bladder pain syndrome; key role of ATP signaling in inflammation

“…ATP release from the urothelium is not just occurring through vesicles but also involves the activation of channels and pores in the urothelial cell membrane. One such mechanism involves pannexin channels [116]. Pannexin channels are closed under resting conditions.…”