carbachol 100 nmol/L) autonomous activity is augmented, giving rise to large ( > 10 cmH 2 O) phasic rises in IVP. When the volume was increased, both the amplitude and frequency of the transients increased. When the bladder volume was reduced there was a period of marked inhibition of phasic activity. To explore the mechanisms underlying these changes the possible involvement of local neural reflexes was explored. The neurotoxin tetrodotoxin had no effect on the volumeinduced changes. Sensory nerves are insensitive to tetrodotoxin and thus to assess their possible contribution bladders were exposed to capsaicin (10 m mol/L) to stimulate and eliminate sensory fibres; capsaicin caused complex changes in phasic activity, i.e. an initial increase, a secondary slowing and decrease, followed by a period of recovering amplitude and increased frequency. These changes suggest actions of the sensory nerves on the phasic mechanism indicative of a local axonal reflex. Once the phasic activity had returned to levels before capsaicin, changes in bladder volume still produced increases in activity and inhibition after the volume decrease. Interstitial cells (cells capable of increasing cGMP) are found in the bladder wall; to assess their possible role in the volume-induced changes, bladders were treated with 30 m mol/L ODQ, an inhibitor of guanyl cyclase, for 30-60 min. The volumeinduced rise in frequency was little affected but the inhibition seen on volume reduction was reduced. CONCLUSIONSThese results show that there are components in the bladder wall which respond to distension by affecting phasic activity. This stimulus/response may reflect a volume 'reflex' within the bladder wall, consisting of excitatory and inhibitory components. This local reflex does not appear to involve directly motor or sensory nerves, although the latter can affect phasic activity, and their actions may represent a further reflex mechanism in the bladder wall. The possible involvement of guanyl cyclase in the volume-induced inhibition may indicate a role for interstitial cells. The physiological role of these mechanisms as a component of a motor/ sensor system in the bladder wall is discussed.
OBJECTIVE To describe the distribution of interstitial cells (ICs, defined as cells which show an increase in cGMP in response to nitric oxide, NO) in the isolated mouse bladder, and changes in phasic contractile activity after exposure to a NO donor. MATERIALS AND METHODS The whole bladder was removed from 17 female mice, killed by cervical dislocation. For immunohistochemistry (six mice) the bladder was incubated in carboxygenated Krebs’ solution at 36 °C, containing 1 mm of the phosphodiesterase inhibitor isobutyl‐methyl‐xanthine. Individual pieces of tissue were exposed to 100 µm of the NO donor diethylamine NONOate for 10 min; control tissues remained in Krebs’ solution. Tissues were then fixed in 4% paraformaldehyde and processed for cGMP immunohistochemistry. Bladder pressure was measured in bladders from 11 mice; the bladders were cannulated via the urethra and suspended in a heated chamber containing carboxygenated Tyrode solution at 33–35 °C and intravesical pressure recorded. All drugs were added to the solution bathing the abluminal surface. RESULTS NO induced an increase in cGMP in cells in the outer layers of the bladder wall, forming two distinct types based on their location; cells lying on the surface of the muscle bundles (surface muscle ICs) and cells within the muscle bundles (intramuscular ICs). Cholinergic nerve fibres were identified by the expression of vesicular acetylcholine transporter and neuronal NO synthase (nNOS). Choline acetyltransferase‐ and nNOS‐positive nerves also had high cGMP levels in response to 100 µm diethylamine NONOate. In vitro exposure of an isolated whole unstimulated bladder to 100 µm diethylamine NONOate had no effect on resting bladder pressure. When whole bladders were exposed to muscarinic stimulation (30–100 nm arecaidine) there was an initial large transient rise in pressure followed by complex phasic changes in pressure. Adding 100 µm diethylamine NONOate abolished this phasic activity. Interestingly, the phasic activity was inhibited midway between the peak and trough of a phasic cycle. Such a pattern of inhibition might reflect the complexity of the phasic activity involving both excitatory and inhibitory components. CONCLUSIONS These data show the presence of NO/cGMP‐sensitive ICs in the outer muscle layers of the mouse bladder. Activating these cells alters the pattern of muscarinic‐induced phasic activity. We suggest that the role of the ICs in the outer muscle layers is to generate and modulate phasic activity. If so, then this is the first report of a functional role for ICs in the bladder.
immunofluorescence histochemistry for cGMP, neuronal NO synthase (nNOS), vesicular acetylcholine transferase (VAChT), calcitonin gene-related polypeptide (CGRP) and protein gene product (PGP) 9.5. ICs were identified as non-neuronal cells of appropriate morphology manifesting an increase in cGMP after exposure to the NO donor. RESULTSICs were apparent in the outer muscle, but not the inner muscle or suburothelial region. nNOS-and CGRP-immunoreactive fibres were close to and alongside IC processes. In contrast, nerve fibres containing VAChT were only occasionally found close to ICs and rarely running alongside them. ICs showed no immunoreactivity to c-kit . There was no overt difference in IC cell distribution between young and aged adult specimens. Older mice showed patchy denervation of the detrusor, but ICs were not specifically affected. CONCLUSIONSICs are confined to the outer part of the bladder wall in the mouse and may receive peptidergic and nitrergic innervation, which might serve to modulate their putative functional role. Alterations in the overall IC population do not appear to underlie ageingrelated changes in lower urinary tract function.
1 Peripheral autonomous bladder activity is an incompletely understood property that may be important both in normal bladder function and in functional problems of the lower urinary tract. We describe how a muscarinic agonist, arecaidine, influences intravesical pressure and intramural bladder contractions in the isolated mouse and how response varies in ageing mice. 2 A group of 12 mice aged 3-4 months was compared with an 'ageing' group of 12 mice age 28-34 months. Bladders were microsurgically removed and mounted in whole organ tissue baths. The effects of the muscarinic agonist arecaidine on intravesical pressure and intramural contractions were performed at different bladder volumes. 3 In normal mice, arecaidine elicited tonic and phasic contractions, the latter showing a more substantial increase in amplitude with bladder distension. Localized 'micromotion' contractions were seen in the bladder wall, with regional differences arising after exposure to arecaidine. A background release of acetylcholine was inferred from the pressure increase induced by the cholinesterase inhibitor physostigmine. 4 Both micromotion activity and the phasic component of the arecaidine response were substantially reduced in ageing mice; the tonic component was preserved in the same specimens. 5 We conclude that the enhanced pressure fluctuations seen at high bladder volumes may act as a peripheral determinant of bladder capacity, and that changes in such activity may contribute to altered functional capacity and lower urinary tract symptoms in ageing individuals.
The response to cholinergic stimulation of the isolated bladder has 3 components. The initial tonic peak response increases with bladder distention and it is inhibited by M2 and M3 muscarinic receptor antagonists. The tonic steady state response does not vary with bladder volume and it is inhibited by M2 and M3-receptor antagonists, and by beta3-adrenergic receptor agonists. Phasic fluctuations are minimal at low bladder volume, and with alpha, beta-methylene adenosine triphosphate or an M3-receptor antagonist. Thus, the response to cholinergic stimulation varies with bladder volume. It can be differentially modulated by muscarinic antagonists and also by agents acting through nonmuscarinic receptors.
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