Distribution and colocalization of calcitonin gene-related peptide, tachykinins, and vasoactive intestinal peptide in normal and idiopathic unstable human urinary bladder.

The distribution of nerves containing calcitonin gene-related peptide (CGRP), substance P (SP), neurokinin A (NKA), and vasoactive intestinal peptide (VIP) was examined in the human urinary bladder, using both single- and double-label immunohistochemistry. Nerves containing CGRP and tachykinins were typically present within the subepithelial region, encircling intramural ganglia and around blood vessels. These nerves were sparsely distributed, and only very rarely projected to the smooth muscle bundles of the detrusor. In contrast, VIP-containing nerves formed a dense subepithelial plexus and also projected to the detrusor muscle bundles. The double-label studies revealed that approximately 26% +/- 10% of CGRP-immunoreactive nerves also contained SP and NKA, but that no CGRP fibers coexpressed VIP. Conversely, all SP-immunoreactive fibers also contained CGRP, and many coexpressed NKA. At least three neurochemically distinct populations of nerves can therefore be discerned in the human bladder: VIP/-, CGRP/-, and CGRP/SP/ +/- NKA. The density of CGRP and SP-immunoreactive nerves within the subepithelium of 14 women with urodynamically-proven idiopathic detrusor instability was increased by 82% (p = 0.003) and 94% (p = 0.036), respectively, relative to that of 14 control women with no symptoms of frequency or urgency. This effect was not due to an increase in overall nerve density, because immunoreactivity for the general nerve marker, protein gene product, was not significantly different between the two groups (p = 0.21). The results indicate that at least some patients with detrusor instability demonstrate increases in a specific class of nerve fibers containing CGRP and SP. These peptides are characteristic of sensory neurons in a number of species, suggesting that abnormalities in the afferent arm of the micturition reflex may be associated with detrusor instability.

Distribution of nitric oxide synthase-immunoreactive nerves and identification of the cellular targets of nitric oxide in guinea-pig and human urinary bladder by cGMP immunohistochemistry.

The distribution of nerves with the potential to synthesize nitric oxide was examined within the urinary bladder and proximal urethra of humans and guinea-pigs, using an antibody to nitric oxide synthase. Further experiments identified cells in which cGMP-immunoreactivity was induced following exposure to the nitric oxide donor, sodium nitroprusside. These cells represent the potential physiological targets of neuronally released nitric oxide, since activation of soluble guanylate cyclase, and a consequent rise in intracellular cGMP, mediate many of the effects of this transmitter. Nitric oxide synthase-immunoreactivity was widely distributed in the lower urinary tract. In guinea-pigs, 50-68% of all intrinsic vesical neurons expressed nitric oxide synthase-immunoreactivity, while in humans 72-96% of neurons in the wall of the bladder contained nitric oxide synthase. In both humans and guinea-pigs, varicose nitric oxide synthase-immunoreactive nerve terminals provided a moderate innervation to the detrusor muscle of the bladder body, and a denser innervation to the urethral muscle. Immunoreactive nerves also projected to the subepithelium and around blood vessels, but were rarely observed encircling intramural vesical ganglia. Following stimulation with sodium nitroprusside, smooth muscle cells of the urethra expressed strong cGMP-immunoreactivity, but detrusor muscle cells remained uniformly negative. Although the detrusor muscle fibres did not express cGMP, numerous interstitial cells throughout the bladder body demonstrated an intense induction of cGMP-immunoreactivity by sodium nitroprusside. These cells had long dendritic processes extending parallel to the smooth muscle fibres, and contained vimentin, an intermediate filament expressed by cells of mesenchymal origin. Other cell types in which sodium nitroprusside exposure induced cGMP-immunoreactivity were the uroepithelial cells, vascular smooth muscle cells and pericytes, and a small number of varicose nerve terminals. In the guinea-pig, a minor proportion (less than 10%) of intrinsic neurons in the wall of the bladder also expressed cGMP. No intrinsic neurons were observed in specimens of human bladder processed for cGMP immunohistochemistry. The results provide anatomical evidence that nitric oxide may function as a neurotransmitter in the lower urinary tract. Although nerves with the capacity to produce nitric oxide supply both the detrusor muscle and the urethra, distinct regional differences exist in the effects of nitric oxide on the induction of cGMP. If the nitric oxide-mediated induction of cGMP is a reliable indicator of the physiological responsiveness of a cell to nitric oxide, then smooth muscle cells appear to be the predominant targets of nitric oxide in the urethra, while in the bladder body, interstitial cells may serve this role. These findings support previous studies which have implicated nitric oxide as an inhibitory transmitter involved in the relaxation of the bladder neck. Our experiments further indicate that a number of cell types within the lower urinary tract could potentially mediate the effects of endogenously released nitric oxide.

Neuropeptides and neurotransmitter-synthesizing enzymes in intrinsic neurons of the human urinary bladder.

The expression of neuropeptides, and the enzymes nitric oxide synthase and tyrosine hydroxylase were examined in intramural ganglia of human urinary bladder using single label immunocytochemistry. Scattered ganglia composed of between 1-36 neurons (median 4) were observed in all layers of the lateral wall of the bladder. These contained immunoreactivity to vasoactive intestinal peptide, nitric oxide synthase, neuropeptide Y, and galanin. Neurons within the bladder were heterogeneous with regard to their content of these antigens, with the proportion of immunopositive cells ranging from 58-84%. Occasional neurons with immunoreactivity to the catecholamine-synthesizing enzyme, tyrosine hydroxylase, were also observed. No cell somata, however, were immunoreactive for enkephalin, substance P, calcitonin gene-related peptide or somatostatin. Varicose terminals entering the ganglia were seen to form pericellular baskets surrounding some of the principal ganglion cells. The most prominent pericellular varicosities were those containing calcitonin gene-related peptide- or vasoactive intestinal peptide-immunoreactivity, followed by those with immunoreactivity for enkephalin, neuropeptide Y, or galanin. Less common were pericellular varicosities with substance P-immunoreactivity, which may represent collateral processes of unmyelinated primary sensory fibres, and presumptive noradrenergic processes containing tyrosine hydroxylase. Some calcitonin gene-related peptide-immunoreactive varicosities constituted a distinct type, terminating as large pericellular boutons 2-4 microns in diameter. Fibres containing nitric oxide synthase- or somatostatin-immunoreactivity were not associated with the intramural neurons. The results demonstrate that intrinsic neurons within the human urinary bladder express a number of neuroactive chemicals, and could in principle form circuits with the potential to support integrative activity.

Regional differences in the innervation of the human ureterovesical junction by tyrosine hydroxylase-, vasoactive intestinal peptide- and neuropeptide Y-like immunoreactive nerves.

We have used double-label immunohistochemistry to examine the presence and pattern of colocalization of vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), tyrosine hydroxylase (TH) and protein gene product (PGP) in nerve fibers supplying the human ureterovesical junction (UVJ). Several populations of nerve fibers within the UVJ region were identified according to their expression of potential transmitter substances. Presumptive noradrenergic axons containing TH- and NPY-like immunoreactivity (LIR) and non-noradrenergic fibers containing VIP- and NPY-LIR accounted for most of the total (PGP-LIR) innervation and supplied all regions of the UVJ. The distal ureter, Waldeyer's sheath and the trigone were supplied by predominantly noradrenergic TH/NPY-LIR nerve fibers, whereas the majority of fibers supplying the detrusor muscle were non-noradrenergic VIP/NPY-LIR axons. The similarity in innervation of Waldeyer's sheath, ureter and trigone is consistent with the notion that these structures are all derived from a common mesodermal origin. Regional differences in innervation were also noted within the musculature of the distal ureter: TH/NPY-LIR fibers were localized to the outer part of the ureter, while VIP/NPY-LIR fibers supplied the inner part. This finding suggests that the different layers of the ureter may be independently controlled by different populations of nerves. The findings of this study support the view that noradrenergic nerves are important in maintaining the tone of the UVJ, but indicate that other neurotransmitters or neuromodulators may also be involved in the control of this region.

Colocalization of nitric oxide synthase with vasoactive intestinal peptide, neuropeptide Y, and tyrosine hydroxylase in nerves supplying the human ureter.

The distribution and patterns of colocalization of nitric oxide synthase (NOS), vasoactive intestinal peptide (VIP), neuropeptide Y (NPY) and the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) were examined in nerve fibers supplying the human lower ureter using double label immunofluorescence. Many nerve fibers immunoreactive for NOS were observed within the ureter. Positive varicose fibers were seen running longitudinally within the smooth muscle bundles, particularly those of the inner layers of the ureter. Immunoreactive axons were also prominent within the subepithelium, and as plexi surrounding many blood vessels. The colocalization studies indicated that NOS was never present in presumptive sympathetic nerve fibers expressing TH. All fibers containing VIP, however, were also immunoreactive for NOS. In addition, a minor population of NOS fibers did not contain VIP. Neuropeptide Y coexisted with NOS in a significant number of nerve terminals, although fibers expressing only NPY were equally common. Several immunochemically distinct nerve populations can therefore be distinguished in the human ureter: (1) nerves containing NOS either with or without VIP; (2) NOS-immunoreactive fibers with NPY; and (3) those fibers expressing TH or NPY which do not contain NOS. The results indicate that some non-noradrenergic peptide-containing nerves in the human ureter have the capacity to synthesize nitric oxide (NO), and that NO may be involved in the regulation of ureteric motility.

Patterns of neuronal colocalisation of tyrosine hydroxylase, neuropeptide Y, vasoactive intestinal polypeptide, calcitonin gene-related peptide and substance P in human ureter.

The patterns of colocalisation of neuropeptides, tyrosine hydroxylase (TH), and protein gene product 9.5 (PGP), were studied in nerve fibres supplying the upper and lower human ureter using a double labelling immunofluorescence technique. The majority (85%-95%) of nerve fibres within the ureter contained neuropeptide Y-like immunoreactivity (NPY-LIR), in combination with other peptides. Approximately 52%-63% of the total ureteral innervation was made up of NPY-LIR fibres also expressing TH-LIR, while 21%-42% of fibres contained NPY-LIR in combination with vasoactive intestinal polypeptide (VIP)-LIR. These two immunochemically defined classes did not overlap, since TH- and VIP-LIR were never present within the same nerve fibre. Other minor populations of neurones included those containing calcitonin gene-related peptide (CGRP)-LIR in combination with substance P (SP)-LIR (4%-17%) and those without SP (5%). Rare coexistences were also noted between CGRP- and VIP-LIR (1%-2%), CGRP- and NPY-LIR (< or = 1%), and CGRP- and TH-LIR (< 1%). Regional differences in innervation were found. There were fewer of each class of nerve fibres in the upper ureter compared to the lower ureter. In addition, the proportion of VIP/NPY-LIR fibres of the total innervation was less in the upper ureter, where they were very sparse. Differences in the distribution to various tissue targets were also observed. In the lower ureter, TH/NPY-LIR fibres were localised predominantly to the outer muscle fascicles and adventitia, while VIP/NPY immunoreactive nerves supplied the submucosa and inner smooth muscle fascicles. Both of these populations were also found around blood vessels. A population of presumptive sensory fibres expressing CGRP/SP-LIR were typically present immediately beneath the urinary epithelium and around blood vessels, and only very rarely within muscle fascicles. The finding that TH/NPY- and VIP/NPY-LIR fibres innervate different layers of the ureter raises the possibility that the muscle layers of the ureter may be independently controlled.

Distribution of NADPH-diaphorase-positive nerves supplying the human urinary bladder.

Nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry was used as a marker for neuronal nitric oxide synthase in human bladder tissue. A plexus of NADPHd-containing nerve fibres was observed in bladder biopsies taken from both the lateral wall and trigone regions. Varicose terminals were present in smooth muscle bundles of the detrusor and trigone, and more commonly within the submucosal layer. Reactive fibres were seen running immediately beneath and along the urothelium, and additional nerves formed perivascular plexi around some blood vessels. Fewer positive nerve processes were observed in the trigone region in comparison to the bladder wall. NADPHd-reactive neuronal perikarya were present within intramural ganglia, some of which were in close proximity to NADPHd-stained varicosities. The results indicate that nitric oxide may be involved in the regulation of bladder function in humans.

Presence and regional variation in peptide-containing nerves in the human ureter.

The occurrence, distribution and regional variation of neurones immunoreactive for the neuropeptides, vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), enkephalin (ENK), calcitonin gene-related peptide (CGRP), and substance P (SP) were investigated in human ureters by indirect immunohistochemistry. In addition, immunoreactivities to tyrosine hydroxylase (TH), a marker of noradrenergic neurones and to protein gene product (PGP) 9.5, a general marker of neurones, were also studied. Neurones displaying PGP-, NPY-, VIP- and TH-like immunoreactivity (-LIR) provided a rich innervation to the smooth muscle and blood vessels of the ureter, where they formed dense muscular and perivascular nerve plexuses. In contrast, there was only a moderate to sparse innervation by SP and CGRP-LIR neurones, most of which were distributed to blood vessels and to the sub mucosal layer, and only rarely to smooth muscle bundles. No ENK-LIR was detected in this study. Nerve fibre bundle densities were estimated for each of the localized neurochemicals according to a method described. NPY-LIR nerve fibre bundles were found to account for 80% of the total nerve fibre bundles (i.e. PGP-LIR) in the ureter. On the other hand, TH-LIR and VIP-LIR nerve fibre bundles each accounted for 50% of the total ureteral innervation, whereas SP- and CGRP-LIR nerve fibre bundles each comprised 20% of the total innervation. The abundance and pattern of tissues innervated by these immunoreactive neurones is consistent with the view that some of these neuropeptide substances co-exist with other peptide substances and/or with other known neurotransmitters, such as noradrenaline or acetylcholine. A gradient of innervation was found to exist for all the neurochemicals demonstrated in the ureter, whereby the lower ureter receives a greater density of innervation than the upper ureter. This finding suggests the human ureter is primarily innervated by fibres arising from or via the lower pelvis, i.e. the pelvic plexus. It also supports the view that the lower ureter may perform an important physiological role, such as coordinating the tone of this region during bladder filling and emptying.