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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 °C, gassed with 95% O2 and 5% CO2, and containing 1 mmol/L of the nonspecific phosphodiesterase inhibitor isobutyl‐methyl‐xanthene. Individual pieces of tissue were then exposed to 100 µmol/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 in...

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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.

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.

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The afferent output from the bladder is important for triggering micturition. This study identifies different types of afferent nerve and explores the connections of their collateral fibres on intramural ganglia and potential ganglionic targets. The experiments were performed on tissues from male guinea-pigs (n=16). Fibres positive for choline acetyl transferase (ChAT(+)) were found to originate close to the urothelium, to transit the sub-urothelial interstitial cell layer and to pass into the lamina propria. A different population of fibres, immunopositive for calcitonin gene-related peptide (CGRP), capsaicin receptors or neurofilament protein (NF), were seen to intertwine with the ChAT(+) fibres in the lamina propria. The ChAT(+) fibres did not express NF. Ganglia with ChAT(+) and NF(+) neurones were found in the lamina propria and muscle. ChAT(+) fibres, with pronounced terminal varicosities, were present on the nerve cell bodies. Two types were noted: NF(+) terminals and those with little or no NF (NF(-)) suggesting that their origins were the ChAT(+) afferent collaterals and the adjacent ganglia. Fibres containing CGRP or substance P were seen on the ganglionic cells. alpha1B adrenergic receptors were also found on the neurones indicative of adrenergic synapses. Thus, the ganglia had multiple inputs. Different types of ChAT(+) nerves were seen in the muscle: NF(+) and NF(-). The ChAT(+)/NF(+) nerves may represent a ganglionic output to the muscle. This complex neuronal network may therefore represent the elements generating and modulating bladder sensations. The role of such a scheme in bladder pathology and the therapeutic sites of action of anticholinergic and sympathomimetic drugs are discussed.

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