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Summary The epithelial Na+ channel (ENaC) is a major conductive pathway that transports Na + across the apical membrane of the distal nephron, the respiratory tract, the distal colon and the ducts of exocrine glands. ENaC is regulated by hormonal and humoral factors, among which are extracellular nucleotides that can be available from the epithelial cells themselves.2. Extracellular nucleotides, via the P2Y 2 receptors (P2Y 2 Rs) at the basolateral and apical membrane of epithelia, trigger signalling systems that cause inhibition of the activity of ENaC and activation of Ca 2+ -dependent Cl − secretion.3. Recent data from our laboratory suggest that stimulation of the P2Y 2 Rs at the basolateral membrane inhibits activity of ENaC by a signalling mechanism that involves Gβγ subunits freed from a PTX-sensitive G protein and phospholipase C-β4. A similar signalling mechanism is also partially responsible for inhibition of ENaC during activation of apical P2Y 2 Rs 4. Stimulation of apical P2Y 2 Rs also stimulates an additional signalling mechanism that inhibits ENaC, involving the activated Gα subunit of a PTX-insensitive G protein, and activation of an unidentified PLC. The effect of this PTX-insensitive system requires the activity of the basolateral Na + /K + /2Cl − co-transporter.

Purpose Recent evidence suggests the urothelium functions as a sensory transducer of chemical, mechanical or thermal stimuli that signals to nerve terminals and other cells in the bladder wall. The cellular and molecular basis of neuro-urothelial communication is not easily studied in the intact bladder, which led us to establish a method of co-culturing dorsal root ganglion (DRG) sensory neurons and bladder urothelial cells. Materials and Methods Sensory neurons and urothelial cells obtained from DRG and bladders dissected from adult female Sprague-Dawley rats were isolated by enzyme treatment and mechanical dissociation. These were plated together, or separately, on collagen-coated substrate and cultured in keratinocyte medium for 48–72 h. Retrograde tracer labeling was performed to identify bladder afferents used for functional testing. Results Neurite growth and complexity in neurons co-cultured with urothelial cells was increased relative to neuronal monocultures. The growth-promoting effect of urothelial cells was reduced by the tyrosine kinase inhibitor, K252a, but upstream inhibition of NGF signaling with TrkA-Fc had no effect. Fura-2 calcium imaging of urothelial cells showed responses to ATP (100μM) and activation of TRPV4 (4alpha-PDD, 10μM), but not TRPV1 (capsaicin, 1μM), TRPV3 (farnesyl pyrophosphate, 1μM), or TRPA1 (mustard oil, 100μM). In contrast, co-cultured neurons were activated by all agonists except farnesyl pyrophosphate. Conclusions Co-culturing provides a new methodology for investigating neuro-urothelial interactions in animal models of urological conditions. Our results suggest that neuronal properties are not only maintained in the presence of urothelium but that neurite growth is potentiated by an NGF-independent mechanism.