In mammals, the bladder stores urine without permitting the passage of urine contents into the bloodstream, a function, in part, of the uroepithelial-associated tight junction complex. The protein constituents that make up this high-resistance barrier in the bladder are currently unknown, although the claudins, a multigene family, are thought to govern paracellular transport in other epithelia. Reverse transcriptase-polymerase chain reaction analysis was used to define that mRNA for claudin-2, -4, -8, -12, and -13 was expressed in mouse bladder tissue. The localization of these claudins, as well as the tight junction-associated proteins zonula occludens-1 (ZO-1) and occludin, within the bladder epithelium was determined by immunofluorescence microscopy. As expected, occludin and ZO-1 were localized to the tight junctions of rat, mouse, and rabbit umbrella cells. Intriguingly, ZO-1 in mouse epithelium, ZO-1 in the dome region of rabbit bladders and occludin in rat and mouse bladders were also expressed in the underlying intermediate and basal cell layers. Claudin-4, -8, and -12 were found in the umbrella cell tight junction; however, additional staining of claudin-4 was observed along the sites of cell-cell contact in the underlying cell layers of rat, mouse, and rabbit tissue. No claudin-2 staining was associated with tight junctions in the uroepithelium. Our results indicate that claudin-4, -8, and -12 are expressed in umbrella cells, where they may impart the high-resistance phenotype associated with this cell type, and that in some instances tight junction proteins are also associated at the sites of cell contact of the underlying cell layers, perhaps playing some role in cell-cell adhesion.

Key points : Neurons can be damaged when tissues are stretched or swollen; while astrocytes can contribute to this process, the mechanosensitive response from neurons is unclear. We show here that isolated retinal ganglion cell neurons respond to mechanical strain with a rapid, sustained release of the neurotransmitter ATP. The conduit for ATP release was through pannexin hemichannels, with probenicid, carbenoxelone and ¹⁰panx inhibiting release. Once released, this ATP acts back on the neurons to autostimulate lethal P2X₇ receptors, as A438079, AZ 10606120 and zinc reduced currents in whole cell patch clamp recordings. Blocking release of ATP through pannexin channels, or activation of P2X₇ receptors, might be neuroprotective for stretched or swollen neurons. Stretch‐dependent release of ATP through neuronal pannexins, combined with the autostimulation of the P2X₇ receptors, provides a new pathway by which neuronal activity and health can be altered by mechanical strain independently of glial activity. Abstract  Mechanical deformation produces complex effects on neuronal systems, some of which can lead to dysfunction and neuronal death. While astrocytes are known to respond to mechanical forces, it is not clear whether neurons can also respond directly. We examined mechanosensitive ATP release and the physiological response to this release in isolated retinal ganglion cells. Purified ganglion cells released ATP upon swelling. Release was blocked by carbenoxolone, probenecid or peptide ¹⁰panx, implicating pannexin channels as conduits. Mechanical stretch of retinal ganglion cells also triggered a pannexin‐dependent ATP release. Whole cell patch clamp recording demonstrated that mild swelling induced the activation of an Ohmic cation current with linear kinetics. The current was inhibited by removal of extracellular ATP with apyrase, by inhibition of the P2X₇ receptor with A438079, zinc, or AZ 10606120, and by pannexin blockers carbenoxolone and probenecid. Probenecid also inhibited the regulatory volume decrease observed after swelling isolated neurons. Together, these observations indicate mechanical strain triggers ATP release directly from retinal ganglion cells and that this released ATP autostimulates P2X₇ receptors. Since extracellular ATP levels in the retina increase with elevated intraocular pressure, and stimulation of P2X₇ receptors on retinal ganglion cells can be lethal, this autocrine response may impact ganglion cells in glaucoma. It remains to be determined whether the autocrine stimulation of purinergic receptors is a general response to a mechanical deformation in neurons, or whether preventing ATP release through pannexin channels and blocking activation of the P2X₇ receptor, is neuroprotective for stretched neurons.