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 10 panx inhibiting release.• Once released, this ATP acts back on the neurons to autostimulate lethal P2X 7 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 P2X 7 receptors, might be neuroprotective for stretched or swollen neurons.• Stretch-dependent release of ATP through neuronal pannexins, combined with the autostimulation of the P2X 7 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 10 panx, 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 P2X 7 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 P2X 7 receptors. Since extracellular ATP levels in the retina increase with elevated intraocular pressure, and stimulation of P2X 7 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 J. Xia and J. C. Lim contributed equally to this work. 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 P2X 7 receptor, is neuroprotective for stretched neurons.

As ATP released from astrocytes can modulate many neural signaling systems, the triggers of and pathways for this ATP release are important. Here, the ability of mechanical strain to trigger ATP release through pannexin channels, and the effects of sustained strain on pannexin expression, were examined in rat optic nerve head astrocytes. Astrocytes released ATP when subjected to 5% equibiaxial strain or to hypotonic swelling. While astrocytes expressed mRNA for pannexins 1–3, connexin 43 and VNUT, pharmacological analysis suggested a predominant role for pannexins in mechanosensitive ATP release, with Rho kinases contributing. Astrocytes from panx1−/− mice had reduced baseline and stimulated levels of extracellular ATP, confirming the role for pannexins. Swelling astrocytes triggered a regulatory volume decrease that was inhibited by apyrase or probenecid. The swelling–induced rise in calcium was inhibited by P2X7 receptor antagonists A438079 and AZ10606120, in addition to apyrase and carbenoxelone. Extended stretch of astrocytes in vitro upregulated expression of panx1 and panx2 mRNA. A similar upregulation was observed in vivo in optic nerve head tissue from the Tg-MYOCY437H mouse model of chronic glaucoma; genes for panx1, panx2 and panx3 were increased while immunohistochemistry confirmed increased expression of pannexin 1 protein. In summary, astrocytes released ATP in response to mechanical strain, with pannexin 1 the predominant efflux pathway. Sustained strain upregulated pannexins in vitro and in vivo. Together these findings provide a mechanism by which extracellular ATP remains elevated under chronic mechanical strain, as found in the optic nerve head of patients with glaucoma.

Non-neuronal release of acetylcholine (ACh) has been proposed to play a role in urinary bladder function. These studies investigated the expression and function of the non-neuronal cholinergic system in cultured urothelial cells isolated from the rat urinary bladder. Our findings have revealed that urothelial cells express the high-affinity choline transporter (CHT1) and acetylcholine synthesizing enzymes, choline acetyltransferase (ChAT) and carnitine acetyltransferase (CarAT). In contrast to neurons, urothelial cells do not express the vesicular acetylcholine transporter (VAChT) but do express OCT3, a subtype of polyspecific organic cation transporter (OCT) that is thought to be involved in the release of acetylcholine from nonneuronal cells. Following exposure of cultured urothelial cells to 3 H-choline, radioactivity was detected in the cells and increased release of radioactivity into the eternal media was evoked by mechanical stimulation (exposure of the cells to 50% hypotonic Krebs) or chemical stimulation of purinergic receptors by 100 μM ATP. The present experiments did not establish if the evoked release of radioactivity (termed 3 HACh release in this paper) was due to release of acetylcholine or choline. 3 H-ACh release was not evoked by application of acetylcholine alone, however pretreatment with the non-selective muscarinic receptor antagonist atropine prior to application of acetylcholine facilitated 3 H-ACh release, suggesting that the acetylcholine released from urothelial cells may participate in a negative feedback mechanism by acting on muscarinic receptors to inhibit its own release in the urothelium. Brefeldin, an agent which disrupts vesicular exocytosis, did not block hypotonicevoked 3 H-ACh release. These observations indicate that acetylcholine release from urothelial cells is mediated by different mechanisms than those such as vesicular storage and exocytosis that underlie the release of neurotransmitters from nerves.