Damage to the bladder neck alters autonomous activity and its sensitivity to cholinergic agonists.

OBJECTIVE: To identify and describe changes to the motor component of the motor/sensory system, which contributes to sensation during the filling phase of the micturition cycle, as a result of surgically induced bladder pathology, i.e. damage to the bladder neck and outlet obstruction. MATERIALS AND METHODS: Adult male guinea pigs (294-454 g) were assigned initially into three groups: (i) normal guinea pigs with no surgical intervention (control, seven); (ii) guinea pigs which, with full surgical anaesthesia, had a silver ring implanted around the bladder neck (obstructed, 13); and (iii) guinea pigs operated to expose the bladder neck but with no implantation of a ring (sham, six). At 2-4 weeks after surgery the bladders were isolated, weighed and the pressure recordings used to identify autonomous activity. RESULTS: The bladder weights in all operated groups, including the sham, were greater than controls. Bladder weights in the obstructed guinea pigs varied considerably, reflecting the degree of pathological change. Consequently, bladders from this group were divided into those with high (OBH) and those with low bladder weight (OBL). The mean (sd) amplitudes of the autonomous contractions were 1.1 (0.1), 10.8 (1.8), 11.4 (2.5) and 17.1 (4.0) cmH(2)O in control, sham, OBL and OBH bladders, respectively, indicating a progressive alteration in function with the pathology. The changes in the sham group suggested that the pathological changes were not the result of obstruction but damage to the bladder neck, the implantation of the silver rings exacerbating the damage. There were episodes of rapid phasic activity (bursts) in 10 of 13 of the ring-implanted bladders, and in two of six in the sham group, but never in controls. Neither the autonomous activity nor the bursts were affected by tetrodotoxin (1 microm) or atropine (3 microm) but they were abolished by noradrenaline (3 microm). In control bladders, adding the muscarinic agonist arecaidine produced a transient acceleration of phasic activity and increased the amplitude of the contractions. There was a similar acceleration of activity in all the operated groups but the concentrations needed to achieve an increase in frequency were significantly lower, the relative sensitivity to arecaidine being OBH >/= OBL > sham > control. CONCLUSION: The mechanism involved in controlling the frequency of the motor component of the motor/sensory system, the 'pacemaker', appears to become progressively 'supersensitive' to cholinergic stimulation with the development of pathology. These observations are discussed in relation to the motor/sensory system and the origins of sensation in the bladder. The argument is proposed that damage to the bladder neck, not obstruction per se, results in altered nonmicturition activity which contributes to increased afferent output. In turn this contributes to the increased sensations of urge associated with bladder dysfunction. The cholinergic regulation of this altered 'pacemaker' might be the target for one of the therapeutic actions of anticholinergic drugs.

The effects of exogenous prostaglandins and the identification of constitutive cyclooxygenase I and II immunoreactivity in the normal guinea pig bladder.

OBJECTIVES: To establish the functional consequences of exposing the isolated whole bladder preparation to exogenous prostaglandins (PGE(1), PGE(2), PGF(2alpha)) and to determine which cells express cyclooxygenase (COX) types I and II, to generate PG to effect these changes in vivo. MATERIALS AND METHODS: Fifteen female guinea pigs (270-350 g) were used, i.e. seven for structural studies and eight for physiological measurement. For the structural study pieces of the lateral wall were incubated separately in Krebs' solution at 36 degrees C, gassed with 95% O(2) and 5% CO(2) with 1 mm isobutyl-methyl-xanthene. Individual pieces were then exposed to 100 microm of the nitric oxide (NO) donor NONOate for 10 min; control tissues remained in Krebs' solution. Tissues were then fixed in 4% paraformaldehyde. For the physiological experiments bladders were isolated and a cannula inserted into the urethra to monitor intravesical pressure. The bladders were suspended in a chamber containing carboxygenated physiological solution at 33-36 degrees C. All drugs were added to the abluminal bladder surface. RESULTS: In the resting bladder there were small spontaneous transient rises in pressure, i.e. autonomous activity. Exposure to PGE(2) (3-300 nM) resulted in an increase in basal pressure on which were superimposed autonomous activity, which was increased both in amplitude and frequency. The changes in the amplitude and frequency depended on the concentration of PGE(2). After a brief exposure (240 s) to PGE(2) the augmentation of the autonomous activity continued for >60 min despite regular washing. The responses were similar with PGE(1) but the responses to PGF(2alpha) and arachidonic acid were reduced. The augmented activity was reduced by the EP1/EP2 receptor blocking agent AH6809 (10 microm). Using an antibody to the 70 kDa constitutive form (COX I), COX I immunoreactivity (COX I-IR) was located in cells in the basal urothelium, in lamina propria and cells on the surface of the inner muscle bundles. There were few COX I-IR cells associated with the outer muscle bundles. The COX I-IR cells lying within the lamina propria were distinct from the suburothelial cells which respond to NO with an increase in cGMP. The lamina propria COX I-IR cells appeared to form a network surrounding muscle trabeculae within the inner muscle layer. COX II-IR was associated with the nuclei of cells in the urothelium, lamina propria and muscle. CONCLUSIONS: These data show that PGs regulate autonomous activity. Potential sources of endogenous PG were identified. It is unclear how the PGs produced by these cells alter autonomous activity. There might be a direct activation of the muscle by PGs released by the network of superficial muscle interstitial cells, or PG released from the urothelium might influence phasic contractile activity via networks of COX I-IR interstitial cells. The possible roles and importance of this mechanism for bladder physiology and pathology are discussed.

Expression of the cGMP-specific phosphodiesterases 2 and 9 in normal and Alzheimer's disease human brains.

We studied the mRNA expression of cGMP-hydrolysing phosphodiesterases (PDEs) in selected brain areas of normal elderly people and patients with Alzheimer's disease. Using radioactive in-situ hybridization histochemistry we found a widespread distribution of the mRNA for PDE2 and PDE9, whereas no specific hybridization signal was observed for PDE5. We observed PDE2 and PDE9 mRNA in all cortical areas studied (insular cortex, entorhinal cortex and visual cortex), although to a different extent. PDE2 mRNA was high in the claustrum, whereas PDE9 mRNA was moderate. PDE2 and PDE9 mRNAs was present in the putamen. No cGMP-hydrolysing PDE expression was observed in the globus pallidus. PDE2 and PDE9 mRNA was observed in all subareas of the hippocampus; however, there were significant differences in the amount of expression. In the Purkinje and cerebellar granule cells only PDE9 expression was observed. PDE2 and PDE9 mRNA expression was not significantly different in Alzheimer's disease brains.

Interstitial cells and phasic activity in the isolated mouse bladder.

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 degrees C, containing 1 mm of the phosphodiesterase inhibitor isobutyl-methyl-xanthine. Individual pieces of tissue were exposed to 100 microm 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 degrees 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 microm diethylamine NONOate. In vitro exposure of an isolated whole unstimulated bladder to 100 microm 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 microm 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.

Interstitial cells and cholinergic signalling in the outer muscle layers of the guinea-pig bladder.

OBJECTIVES: To explore the relationship between cholinergic mechanisms and interstitial cells (ICs) in the outer muscle layer of the bladder. MATERIALS AND METHODS: In bladder tissue from male guinea-pigs, ICs were identified by their response to nitric oxide (NO) with a rise in cGMP. Sections of the lateral bladder wall were incubated in Krebs' solution containing 1 mm of the nonspecific phosphodiesterase inhibitor isobutyl-methyl-xanthene. Tissues were then exposed to 100 microm of the NO donor NONOate for 10 min, control tissues remained in Krebs' solution. Tissues were then processed for immunohistochemistry for cGMP, choline acetyltransferase (ChAT), neurofilament protein, and the nonspecific neuronal marker protein gene product (PGP) 9.5. RESULTS: cGMP-positive ICs were found mainly in the outer muscle layers of the bladder wall. Three types were identified based on location; on the outer surface of the bladder wall, on the surface of the muscle bundles, and within the muscle bundles. Some of the intramuscular ICs stained for ChAT, but they did not stain with PGP 9.5. Nerve fibres were seen in close contact with the ChAT-positive intramuscular ICs, and these nerves expressed ChAT and neurofilament protein. CONCLUSIONS: A subpopulation of intramuscular ICs can synthesise acetylcholine, and might release acetylcholine onto the underlying muscle. These cells are in close contact with nerves, suggesting that they might be activated by neural inputs. Thus there may be a system in the detrusor involving cholinergic nerves acting on ICs which can activate the smooth muscle via a complex cholinergic input.

Localization of cyclic guanosine 3',5'-monophosphate-hydrolyzing phosphodiesterase type 9 in rat brain by nonradioactive in situ hydridization.

In this chapter, we describe a protocol for the localization of the cyclic guanosine 3',5'-monophosphate-specific phosphodiesterase type 9 (PDE9) mRNA in the adult rat brain that uses digoxigenin-labeled riboprobes in a nonradioactive in situ hybridization (ISH). The three different riboprobes used all showed similar PDE9 mRNA expression patterns, detecting PDE9 in cell bodies throughout the whole brain. By using immunocytochemical double labeling of the ISH sections with the neuronal marker NeuN or the glial cell marker glial fibrillary acidic protein, the cells expressing PDE9 mRNA were further characterized. Double-labeling experiments revealed that PDE9 was predominantly expressed in neurons.

Localization and characterization of cGMP-immunoreactive structures in rat brain slices after NO-dependent and NO-independent stimulation of soluble guanylyl cyclase.

Possible differences in the localization of the cGMP response were investigated in rat brain coronal slices after in vitro incubation and NO-dependent or NO-independent stimulation of soluble guanylyl cyclase (sGC). Dose-dependent stimulation of cGMP synthesis by the NO donors, sodium nitroprusside, S-nitrosoglutathione, 3-morpholinosydnonimine and diethylamino NONOate was studied in the somatoparietal cortex, the hippocampus and the thalamus. cGMP accumulation was evaluated using a radioimmunoassay and by measuring cGMP-immunofluorescence using image analysis. All four NO donors induced similar cGMP staining patterns in the somatoparietal cortex, the hippocampus and the thalamus. NO-mediated cGMP synthesis in the cortical areas colocalized predominantly with the acetylcholine transporter and occasionally with parvalbumin (GABAergic cells) or the neuronal glutamate transporter. Incubation of the slices in the combined presence of a NO donor and the NO-independent activators YC-1 or BAY 41-2272 strongly potentiated cGMP synthesis and induced abundant cGMP-immunoreactivity in cortical GABAergic and glutamatergic cells. These findings indicate that the mechanism of NO release from the NO donors used does not determine the location of the cGMP response. The results suggest that YC-1 and BAY 41-2272 trigger a NO-sensing mechanism in cells in which the sGC is otherwise not sensitive to NO.

cGMP-generating cells in the bladder wall: identification of distinct networks of interstitial cells.

OBJECTIVE: To identify cells which might contribute to the complex physiological responses of the guinea-pig bladder, and specifically to describe the distribution and types of cell in the bladder wall of the guinea pig which respond to nitric oxide (NO) with an increase in intracellular cGMP, i.e. putative interstitial cells (ICs). MATERIALS AND METHODS: The whole bladder was removed from 11 male guinea pigs killed by cervical dislocation. Sections of the bladder wall, from the dome lateral wall and base, were isolated and incubated separately in Krebs' solution at 36 degrees C, gassed with 95% O(2) and 5% CO(2), and containing 1 mmol/L of the nonspecific phosphodiesterase inhibitor isobutyl-methyl-xanthene. Individual pieces of tissue were then exposed to 100 micromol/L of the NO donor NONOate for 10 min; control tissues remained in Krebs' solution. Tissues were then fixed in 4% paraformaldehyde and processed for immunohistochemistry. cGMP and neuronal NO synthase (nNOS) were subsequently visualized using appropriate primary and secondary antibodies. RESULTS: Cells responding to NO with an increase in cGMP were detected in the dome, lateral wall and base, with positive cells in the thin outer surface of the wall (muscle coat), associated with muscle bundles in an outer layer of muscle, and in a region immediately beneath the urothelium. These cells (not urothelium, smooth muscle or vascular) are described as interstitial cells. Superficial urothelial umbrella cells were apparent and were strongly cGMP-positive. A high density of interstitial cells was associated with muscle bundles on the outer aspects of the wall, while few cells were detected on inner bundles. Thus there appeared to be two distinct types of muscle, inner and outer, with no obvious orientation of the fibres in each layer. Both muscle groups contained fibres expressing nNOS. In the outer muscle layer most of these fibres co-localized with cGMP, suggesting that different populations of nerves innervate each layer. There were more nNOS-positive fibres in the base of the bladder than in the dome. Three populations of cGMP-positive interstitial cells were associated with the outer muscle layer; cells in the outer surface (muscle coat interstitial cells, MC-ICs), cells on the surface of the bundles (superficial, SM-ICs) and cells within the muscle bundles (intramuscular, IM-ICs). The IM-ICs form a network in close apposition to the smooth muscle cells while the SM-ICs may connect adjacent muscle bundles and connect to the MC-ICs. Thus, there is a network linking potentially the muscle cells in the outer muscle bundles. cGMP-positive cells were also detected in the suburothelial layer (suburothelial, SU-ICs) which had a different structure to the cells associated with muscle, had a oval cell bodies with bifurcating processes and appeared to form a complex network; they were prevalent in the base and virtually absent in the dome. CONCLUSIONS: There are structures within the bladder wall that can be identified and categorized by the ability of the constituent cells to increase intracellular cGMP in response to NO; these cells have been defined as ICs. Two distinct networks were identified, one associated with the outer muscle layers and another lying immediately beneath the urothelium, predominantly in the base of the bladder. The functions of these cells and networks are unknown; their possible roles in complex motor activity, urothelial signalling and bladder pathophysiology are discussed.

Species differences in the localization of cGMP-producing and NO-responsive elements in the mouse and rat hippocampus using cGMP immunocytochemistry.

The aim of the study was to compare the localization of the nitric oxide (NO)-cGMP pathway in hippocampus of mice and rats using cGMP- and soluble guanylyl cyclase (GC) immunocytochemistry and in situ hybridization of the cGMP-hydrolysing phosphodiesterase types 2, 5 and 9. In vitro incubation of hippocampus slices in the absence of a guanylyl cyclase stimulator or a phosphodiesterase inhibitor resulted in cGMP-positive astrocytes mainly in the CA1 area in mouse slices. In contrast, no cGMP immunoreactivity was observed under these conditions in the rat hippocampus. Treatment with an NO synthase inhibitor or inhibitors of soluble or particulate GC did not abolish cGMP immunoreactivity in astrocytes. Incubation with the NO donors sodium nitroprusside or diethylamino NONOate, or with the NO-independent activators of soluble GC, YC-1 and BAY 41-2272, in combination with phosphodiesterase inhibitors, resulted in an increase in cGMP immunoreactivity in numerous astrocytes throughout the mouse hippocampus. In contrast, under these conditions cGMP immunoreactivity was primarily observed in varicose fibers in rat hippocampus. Comparison of the cellular localization of the beta1 subunit of soluble GC and the mRNAs of PDE2, PDE5 and PDE9 revealed that in both species the beta1 subunit was observed in pyramidal and granule cells, which also expressed the mRNAs of the three phosphodiesterase families. Although the beta1 subunit was observed in astrocytes, none of the phosphodiesterases were detected in these cells. We conclude that, although the expression profiles of the soluble GC beta1 subunit and cGMP-hydrolysing phosphodiesterase mRNAs were identical, the cellular patterns of cGMP immunoreactivity differ between rat and mouse hippocampus.

mRNA expression patterns of the cGMP-hydrolyzing phosphodiesterases types 2, 5, and 9 during development of the rat brain.

Recent evidence indicates that cGMP plays an important role in neural development and neurotransmission. Since cGMP levels depend critically on the activities of phosphodiesterase (PDE) enzymes, mRNA expression patterns were examined for several key cGMP-hydrolyzing PDEs (type 2 [PDE2], 5 [PDE5], and 9 [PDE9]) in rat brain at defined developmental stages. Riboprobes were used for nonradioactive in situ hybridization on sections derived from embryonic animals at 15 days gestation (E15) and several postnatal stages (P0, P5, P10, P21) until adulthood (3 months). At all stages PDE9 mRNA was present throughout the whole central nervous system, with highest levels observed in cerebellar Purkinje cells, whereas PDE2 and PDE5 mRNA expression was more restricted. Like PDE9, PDE5 mRNA was abundant in cerebellar Purkinje cells, although it was observed only on and after postnatal day 10 in these cells. In other brain regions, PDE5 mRNA expression was minimal, detected in olfactory bulb, cortical layers, and in hippocampus. PDE2 mRNA was distributed more widely, with highest levels in medial habenula, and abundant expression in olfactory bulb, olfactory tubercle, cortex, amygdala, striatum, and hippocampus. Double immunostaining of PDE2, PDE5, or PDE9 mRNAs with the neuronal marker NeuN and the glial cell marker glial fibrillary acidic protein revealed that these mRNAs were predominantly expressed in neuronal cell bodies. Our data indicate that three cGMP-hydrolyzing PDE families have distinct expression patterns, although specific cell types coexpress mRNAs for all three enzymes. Thus, it appears that differential expression of PDE isoforms may provide a mechanism to match cGMP hydrolysis to the functional demands of individual brain regions.

Evaluation of 3-nitrotyrosine as a marker for 3-nitropropionic acid-induced oxidative stress in Lewis and Wistar rats and strain-specific whole brain spheroid cultures.

The present study investigated whether 3-nitrotyrosine is an early marker for neurodegenerative processes involving oxidative stress. We characterized the 3-nitrotyrosine formation after 3-nitropropionic acid (3-NP) exposure in the whole brain spheroid culture model and in a rat model, using Lewis and Wistar rats. Increased 3-nitrotyrosine concentration in spheroid cultures from Lewis rats was observed at lower dose of and shorter exposure time to 3-NP as compared to alterations in glial fibrillary acidic protein concentration, decrease in glutamine synthetase activity or cell loss. Five days of exposure to 3-NP (5 mM) resulted in decreased staining of GABAergic processes, while neuronal nitric oxide synthase staining was preserved. In addition, staining of EAAC1, anti-2',3'-cyclic nucleotide 3'-phosphohydrolase and ED1 was diminished after treatment of spheroid cultures with 3-nitropropionic acid (5 mM), while isolectin B4 staining was increased. Dithiothreitol and vitamin E inhibited the increased formation of 3-nitrotyrosine. Interestingly, N(G)-nitro-L-arginine methyl ester increased the 3-nitrotyrosine formation. No increased 3-nitrotyrosine concentration was shown after exposure to 3-nitropropionic acid during 5 days in spheroid cultures obtained from Wistar rats. In the striatum of 3-NP-exposed Lewis and Wistar rats, no change in 3-nitrotyrosine concentration was observed, whereas only in Wistar rats the glial fibrillary acidic protein concentration was increased in addition to activation of microglial cells. It is concluded that 3-nitrotyrosine was a more sensitive marker for oxidative stress-induced neurodegeneration than glial fibrillary acidic protein and glutamine synthase in spheroid cell cultures of Lewis rats. Finally, the similarities between the 3-NP spheroid model and the vivo model indicate that the spheroid cultures provide a good alternative for chronic exposure of animals to neurotoxins.