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This study examined the origin of spontaneous activity in neonatal and adult rat bladders and the effect of stretch and muscarinic agonists and antagonists on spontaneous activity. Rats were anesthetized and their bladders were excised, cannulated, and loaded with voltage- and [Ca.sup.2+]-sensitive dyes. Intracellular [Ca.sup.2+] and membrane potential transients were mapped using photodiode arrays in whole bladders, bladder sheets, or cross-section preparations at 37[degrees]C. Intravesical pressure was recorded from whole bladders. In neonatal bladders and sheets, spontaneous [Ca.sup.2+] and electrical signals arose at a site near the dome and spread in a coordinated manner throughout the bladder with different dome-to-neck conduction velocities ([Ca.sup.2+]: 3.7 [+ or -] 0.4 mm/s; membrane potential: 46.2 [+ or -] 3.1 mm/s). In whole bladders, optical signals were associated with spontaneous contractions (10-20 cm[H.sub.2]O). By contrast, in adult bladders spontaneous [Ca.sup.2+] and electrical activity was uncoordinated, originating at multiple sites and was associated with smaller (2-5 cm[H.sub.2]O) contractions. Spontaneous contractions and optical signals were insensitive to tetrodotoxin (2 [micro]M) but were blocked by nifedipine (10 [micro]M). Stretch or low carbachol concentrations (50 nM) applied to neonatal whole bladders enhanced the amplitude (to 20-35 cm[H.sub.2]O) of spontaneous activity, which was blocked by atropine. Bladder cross sections revealed that [Ca.sup.2+] and membrane potential transients produced by stretch or carbachol began near the urothelial-suburothelial interface and then spread to the detrusor. In conclusion, spontaneous activity in neonatal bladders, unlike activity in adult bladders, is highly organized, originating in the urothelium-suburothelium near the dome. Activity is enhanced by stretch or carbachol and this enhancement is blocked by atropine. It is hypothesized that acetylcholine is released from the urothelium during bladder filling to enhance spontaneous activity. optical mapping; pacemaker activity; urothelium; voltage- and [Ca.sup.2+]-sensitive dyes

Normal urinary function is contingent upon a complex hierarchy of CNS regulation. Lower urinary tract afferents synapse in the dorsal horn of the spinal cord and ascend to the midbrain periaqueductal gray (PAG), with a separate nociception path to the thalamus. A spino-thalamo-cortical sensory pathway is present in some primates, including humans. In the brainstem, the pontine micturition center (PMC) is a convergence point of multiple influences, representing a co-ordinating center for voiding. Many PMC neurones have characteristics necessary to categorize the center as a pre-motor micturition nucleus. In the lateral pontine brainstem, a separate region has some characteristics to suggest a 'continence center.' Cerebral control determines that voiding is permitted if necessary, socially acceptable and in a safe setting. The frontal cortex is crucial for decision making in an emotional and social context. The anterior cingulate gyrus and insula co-ordinate processes of autonomic arousal and visceral sensation. The influence of these centers on the PMC is primarily mediated via the PAG, which also integrates bladder sensory information, thereby moderating voiding and storage of urine, and the transition between the two phases. The parabrachial nucleus in the pons is also important in behavioral motivation of waste evacuation. Lower urinary tract afferents can be modulated at multiple levels by corticolimbic centers, determining the interoception of physiological condition and the consequent emotional motor responses. Alterations in cognitive modulation, descending modulation, and hypervigilance are important in functional (symptom-based) clinical disorders. Neurourol. Urodynam. 29: 119-127, 2010. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Functional TRP and ASIC-like channels in cultured urothelial cells from the rat. Am P Physiol Rena! Physiol 296: F892-F90 I, 2009. First published January 21, 2009; doi: 10.11 52/ajprenal.907 18.2008.-Transient receptor potential (TRP) and acid-sensing ion channels (ASIC) are molecular detectors of chemical, mechanical, thermal, and nociceptive stimuli in sensory neurons. They have been identified in the urothehum, a tissue considered part of bladder sensory pathways, where they might play a role in bladder function. This study investigated functional properties of TRP and ASIC channels in cultured urothelial cells from the rat using patch-clamp and fura 2 Ca[sup2+] imaging techniques. The TRPV4 agonist 4a-phorbol-12,13 didecanoate (4ctPDD; 1-5 μM) and the TRPAI/TRPM8 agonist icilin (50-100 μM) elicited transient currents in a high percentage of cells (>70%). 4a-PDD responses were suppressed by the TRPV4 antagonist HC010961 (10 μM). The TRPVI agonist capsaicin (1-100 μM) and the TRPA1ITRPM8 agonist menthol (5-200 μM) elicited transient currents in a moderate percentage of cells (--25%). All of these agonists increased intracellular calcium concentration ([Ca[sup2+]][subi]). Most cells responded to more than one TRP agonist (e.g., capsaicin and 4aPDD), indicating coexpression of different TRP channels. In the presence of the TRPVI antagonist capsazepine (10 μM), changes in pH induced by HCI elicited ionic currents (pH 5.5) and increased [Ca[sup2+]][subi] (pH 6.5) in --50% of cells. Changes in pH using acetic acid (pH 5.5) elicited biphasic-like currents. Responses induced by acid were sensitive to amiloride (10 μM). In summary, urothelial cells express multiple TRP and ASIC channels, whose activation elicits ionic currents and Ca[sup2+] influx. These "neuron-like" properties might be involved in transmitter release, such as ATP, that can act on afferent nerves or smooth muscle to modulate their responses to different stimuli. [ABSTRACT FROM AUTHOR]

Muscarinic receptors (rnAChRs) have been identified in the urothelium, a tissue that may be involved in bladder sensory mechanisms. This study investigates the expression and function of mAChRs using cultured urothelial cells from the rat. RT-PCR established the expression of all five mAChR subtypes. Muscarinic agonists acetylcholine (ACh; 10 µM), muscarine (Musc; 20 µM), and oxotremonne methiodide (OxoM; 0.001-20 µM) elicited transient repeatable increases in the intracellular calcium concentration ([Ca2+]i) in ~50% of cells. These effects were blocked by the mAChR antagonist atropine methyl nitrate (10 µM). The sources of [Ca2+]1 changes included influx from external milieu in 63% of cells and influx from external milieu plus release from internal stores in 27% of cells. The use of specific agonists and antagonists (10 µM M1 agonist McN-A-343; 10 µM M2, M3 antagonists AF-DX 116, 4-DAMP) revealed that M1, M2, M3 subtypes were involved in [Ca2+]i changes. The PLC inhibitor U-73122 (10 µM) abolished OxoM-elicited Ca2+ responses in the presence of the M2 antagonist AF-DX 116, suggesting that M1, M3, or M5 mediates [Ca2+]i increases via PLC pathway. ACh (0.1 µM), Musc (10 µM), oxotremorine sesquifumarate (20 µM), and McN-A-343 (1 µM) acting on M1, M2, and M3 mAChR subtypes stimulated ATP release from cultured urothelial cells. In summary, cultured urothelial cells express functional M1, M2, and M3 mAChR subtypes whose activation results in ATP release, possibly through mechanisms involving [Ca2+]i changes. [ABSTRACT FROM AUTHOR]

Purinergic mechanisms appear to be involved in motor as well as sensory functions in the urinary bladder. ATP released from efferent nerves excites bladder smooth muscle, whereas ATP released from urothelial cells can activate afferent nerves and urothelial cells. In the present study, we used immunohistochemical techniques to examine the distribution of purinoceptors in the urothelium, smooth muscle, and nerves of the normal cat urinary bladder as well as possible changes in the expression of these receptors in cats with a chronic painful bladder condition termed feline interstitial cystitis (FTC) in which ATP release from the urothelium is increased. In normal cats, a range of P2X (P2X1, P2X2, P2X3, P2X4, P2X5, P2X6, and P2X7) and P2Y (P2Y1, P2Y2, and P2Y4) receptor subtypes was expressed throughout the bladder urothelium. In FIC cats, there is a marked reduction in P2X1 and loss of P2Y1 receptor staining. Both P2X1 and P2Y1 are present in nerves in normal cat bladder, and no obvious differences in staining were detected in FIC. Smooth muscle in the normal bladder did not exhibit P2Y receptor staining but did exhibit P2X (P2X2, P2X1) staining. In the FIC bladder smooth muscle, there was a significant reduction in P2X1 expression. These findings raise the possibility that purinergic mechanisms in the urothelium and bladder smooth muscle are altered in FIC cats. Because the urothelial cells appear to have a sensory function in the bladder, it is possible that the plasticity in urothelial purinergic receptors is linked with the painful bladder symptoms in IC. [ABSTRACT FROM AUTHOR]