Calcitonin gene-related peptide (CGRP) in the female rat urogenital tract

CGRP-immunoreactivity was found throughout the female rat urogenital tract by specific radioimmunoassay, and shown to be present in nerve fibres by immunocytochemistry. The highest concentrations of CGRP-like immunoreactivity were found in the urinary tract, with lower levels in regions of the genitalia. Chromatographic analysis of bladder and vaginal extracts on Sephadex G-50 columns and HPLC revealed at least three CGRP-immunoreactive peaks. The major peak emerged in the same position as synthetic rat CGRP. CGRP nerve fibres were associated mainly with blood vessels, non-vascular smooth muscle, squamous epithelium and uterine and cervical glands, and were particularly abundant in the ureter and bladder. CGRP-immunoreactivity was depleted by neonatal treatment with capsaicin and after surgical section of pelvic and/or hypogastric nerves. Immunocytochemistry demonstrated that depletion occurred predominantly in the mucosal layer of the urogenital tract. These findings indicate a sensory function for most of the CGRP-immunoreactive nerves in the rat urogenital tract.     

Neuropeptide Y-like immunoreactivity in intramural ganglia of the newborn guinea pig urinary bladder

Immunoreactive neuropeptide Y (NPY) was demonstrated in neuronal elements in the urinary bladder wall of the newborn guinea pig. Numerous intramural ganglia were found lying among the smooth muscle bundles and in the submucosa, and NPY-like immunoreactive nerve cell bodies were demonstrated within all of these ganglia. Nerve fibres containing NPY were also richly distributed in the detrusor muscle, submucosa and around blood vessels. In dissociated cell cultures from newborn guinea pig detrusor muscle, a subpopulation (70-85%) of both mononucleate and binucleate intramural neurones was shown to contain NPY-like immunoreactivity. A low percentage (1-6%) of the intramural bladder neurones contained dopamine-beta-hydroxylase. In conclusion, while some NPY-containing nerve fibres in the wall of the bladder are of sympathetic origin, especially those supplying blood vessels, the results of this present study establish that many of these NPY-containing nerve fibres originate from non-adrenergic cell bodies within the intramural bladder ganglia.     

The distribution and colocalization of neuropeptides and catecholamines in nerves supplying the gall bladder of the toad,Bufo marinus

The indirect immunofluorescence technique was used to determine the distribution of peptide-containing axons in the gall bladder of the cane toad, Bufo marinus. In addition, the adrenergic innervation of the gall bladder was examined by use of immunoreactivity to the catecholamine-synthesizing enzyme, tyrosine hydroxylase, and glyoxylic acid-induced fluorescence. On the basis of peptide coexistence, two intrinsic populations of neurones and their projecting fibres could be distinguished substance P neurones and vasoactive intestine peptide neurones. Neither of these two types of neurones contained any other colocalized neuropeptides. Four populations of nerve fibres arising from cell bodies outside the gall bladder were identified: nerves containing colocalized galanin, somatostatin and vasoactive intestinal peptide; nerves containing colocalized calcitonin gene-related peptide and substance P; adrenergic nerves containing neuropeptide Y; and nerves containing only adrenaline.     

Noradrenergic and peptidergic innervation of the mammary gland in the immature pig

The presence and pattern of coexistence of some biologically active substances in nerve fibres supplying the mammary gland in the immature pig were studied using immunohistochemical methods. The substances studied included: protein gene product 9.5 (PGP), tyrosine hydroxylase (TH), somatostatin (SOM), neuropeptide Y (NPY), galanin (GAL), calcitonin gene-related peptide (CGRP) and substance P (SP). The mammary gland was found to be richly supplied by PGP-immunoreactive (PGP-IR) nerve fibres that surrounded blood vessels, bundles of smooth muscle cells and lactiferous ducts. The vast majority of these nerves also displayed immunoreactivity to TH. Immunoreactivity to SOM was observed in a moderate number of nerve fibres which were associated with smooth muscles of the nipple and blood vessels. Immunoreactivity to NPY occurred in many nerve fibres associated with blood vessels and in single nerves supplying smooth muscle cells. Solitary GAL-IR axons supplied mostly blood vessels. Many CGRP-IR nerve fibres were associated with both blood vessels and smooth muscles. SP-IR nerve fibres richly supplied blood vessels only. The colocalization study revealed that SOM, NPY and GAL partly colocalized with TH in nerve fibres supplying the porcine mammary gland.     

Adrenergic innervation of the gut musculature in vertebrates

Summary The adrenergic innervation of the gut musculature has been compared in various vertebrates (two teleost fish, an amphibian, a reptile and a mammal) by the fluorescent histochemical localization of certain monoamines. Very few, if any, adrenergic nerves occur within the longitudinal gut muscle of any of these animals, except for the taenia coli of the guinea-pig caecum. In contrast, the circular smooth muscle coat is supplied by varicose adrenergic nerves. These nerve fibres are particularly numerous in the toad large intestine, guinea-pig caecum, and throughout the eel gut, but are generally sparse or absent from the musculature of the stomach and small intestine of the trout, toad, lizard and guinea-pig. The extent of adrenergic innervation of the muscle has been discussed in relation to the physiology of the different muscle coats and to the general structure of the enteric plexuses in the vertebrate gut.     

Immunohistochemical characteristics of suburothelial microvasculature in the mouse bladder

The morphological characteristics of smooth muscle cells (SMCs) and their innervation of the suburothelial microvasculature of the mouse bladder were investigated by immunohistochemistry. Whole mount bladder mucosal preparations were immune-stained for α-smooth muscle actin (α-SMA) and/or neuronal markers and examined using confocal laser scanning microscopy. Suburothelial arterioles consisted of α-SMA-immunopositive circular smooth muscle cells, while the venular wall composed of α-SMA-positive SMCs that displayed several processes which extended from their cell bodies to form an extensive meshwork. In larger venules, a complex meshwork of stellate-shaped SMCs were observed. NG2 chondroitin sulphate proteoglycan-immunoreactive cell bodies of capillary pericytes were not immunoreactive for α-SMA. In the rat bladder suburothelial venules, circular SMCs were the dominant cell type expressing α-SMA-immunoreactivity. Since α-SMA-positive SMCs in suburothelial arterioles and venules in the mouse bladder had quite distinct morphologies, the innervation of both vessels could be examined by double labelling for α-SMA and various neuronal markers. Varicose nerve bundles immunoreactive for tyrosine hydroxylase (sympathetic nerves), choline acetyltransferase (cholinergic nerves) or substance P (primary afferent nerves) were all detected along side suburothelial arterioles. Single varicose nerve fibres positive for these three neuronal markers were also detected around the venules. Thus, whole mount preparations are useful when examining the morphology of α-SMA-positive SMCs of the microvasculature in the suburothelium of mouse bladder as well as their relationship with their innervations. In conclusion, arterioles and venules of the bladder suburothelium are the target of sympathetic, cholinergic and primary afferent nerve fibres.     

Origin and distribution of VIP (Vasoactive Intestinal Polypeptide)-nerves in the genito-urinary tract

Summary VIP (Vasoactive Intestinal Polypeptide)-immunoreactive nerves were found throughout the genito-urinary tract of the cat; they were less numerous in the guinea pig and in the rat. In the cat, VIP nerves were particularly numerous in the neck of the urinary bladder and proximal urethra, in the uterine cervix and in the prostate gland. The nerves were found in smooth muscle, around blood vessels and in the connective tissue immediately beneath the epithelium. Ganglia were found below the trigonum area of the bladder, in the wall of the proximal urethra, and in paracervical tissue. VIP-immunoreactive nerve cell bodies occurred in all these ganglionic formations. These ganglia probably represent the origin of the VIP nerves of the genital tract since their removal in the female cat greatly reduced the VIP nerve supply. Transection of the hypogastric nerves had no overt effect. Transection of the cervix eliminated the VIP nerves above the level of the lesion, except those in the ovaries, supporting the view that the VIP nerves of the uterus and the oviduct are derived from a paracervical source.     

Adrenergic innervation of the lower urinary tract in cattle

Studies were carried out on 8 sexually immature male calves. Sections of the ureters, urinary bladder, and urethra were cut with a freezing microtome and the method of Ky?s?la et al. (1980) was used to visualize the adrenergic nerve fibres. It was found that bovine ureters possessed weak innervation; most of the nerves was located in the muscular membrane, and only in the paravesical part, sparse nerve fibres were found in the submucosa of this one. Apex of the urinary bladder was more weakly supplied with the adrenergic nerves than the corpus, whereas bladder's trigonum and cervix possessed numerous nerve fibres in both muscular and submucosal membranes. The distribution pattern of adrenergic nerves in the urethra was similar to that of urinary bladder's cervix. The presence of adregeneric nerve fibres was found in submucosal layer of both the urinary bladder and the urethra. Part of the nerves was connected with blood vessels of the organs under study.     

Distribution of PACAP (pituitary adenylate cyclase-activating polypeptide)-like and helospectin-like peptides in the teleost gut

Pituitary adenylate cyclase-activating polypeptide (PACAP) and helospectin are two vasoactive intestinal polypeptide (VIP)-related neuropeptides that have recently been demonstrated in the mammalian gut; the aim of this study was to reveal their occurrence and localisation in the gastrointestinal tract, swimbladder, urinary bladder and the vagal innervation of the gut of teleosts, using immunohistochemical methods on whole-mounts and sections of these tissues from the Atlantic cod, Gadus morhua and the rainbow trout, Oncorhynchus mykiss. Both PACAP-like and helospectin-like peptides were present in the gut wall of the two species. Immunoreactive nerve fibres were found in all layers but were most frequent in the myenteric plexus and along the circular muscle fibres. Immunoreactivity was also demonstrated in nerves innervating the swimbladder wall, the urinary bladder and blood vessels to the gut. Immunoreactive nerve cell bodies were found in the myenteric plexus of the gut and in the muscularis mucosae of the swimbladder. In the vagus nerve, non-immunoreactive nerve cells were surrounded by PACAP-immunoreactive fibres. Double staining revealed the coexistence of PACAP-like and helospectin-like peptides with VIP in all visualized nerve fibres and in some endocrine cells. It is concluded that PACAP-like and helospectin-like peptides coexist with VIP in nerves innervating the gut of two teleost species. The distribution suggests that both PACAP and helospectin, like VIP, are involved in the control of gut motility and secretion.     

Nerves containing substance P,vasoactive intestinal polypeptide,enkephalin or somatostatin in the guinea-pig taenia coli

Summary The guinea-pig taenia coli is rich in peptide-containing nerves. Nerve fibres containing substance P (SP), vasoactive intestinal peptide (VIP), or enkephalin, were numerous in the smooth muscle while somatostatin fibres were very few. Nerve fibres displaying SP or VIP immunoreactivity were numerous in the myenteric plexus. Enkephalin nerve fibres were fairly numerous in the plexus while somatostatin nerve fibres were sparse. Nerve cell bodies containing immunoreactive SP or VIP were regularly seen in the plexus. Delicate varicose elements of the different types of nerve fibres were found to ramify around nerve cell bodies in a manner suggestive of innervation.In the electron microscope the various peptide-storing nerve fibres (i.e., elements containing SP, VIP or enkephalin) were found to contain a varying number of fairly large, electron-opaque vesicles in the varicose swellings. These vesicles represent the storage site of the neuropeptides.The isolated taenia coli responded to electrical nerve stimulation with a contraction. After cholinergic and adrenergic blockade the contractile response was replaced by a relaxation followed by a contraction upon cessation of stimulation. SP contracted the taenia while VIP caused a relaxation. The enkephalins raised the resting tension slightly while somatostatin had no effect. These observations are compatible with a role for SP as an excitatory neurotransmitter and for VIP as an inhibitory one, and with the view that both SP neurones and VIP neurones act as motor neurones. In preparations contracted by SP the electrically induced contractions were reduced in amplitude while the electrically induced relaxations seen after adrenergic and cholinergic blockade were enhanced in amplitude. In preparations relaxed by VIP there was an increased contractile response to electrical stimulation, while in the atropine + guanethidine-treated preparation the electrically induce relaxations were reduced in amplitude. The enkephalins reduced the contractile response to electrical stimulation, while somatostatin induced a very small reduction in the amplitude of such responses. These observations suggest that SP neurones and VIP neurones may play additional roles as interneurones. Somatostatin neurones probably act as interneurones. Enkephalin-containing fibres may serve to modify the release of transmitter from other nerves in the smooth muscle, perhaps through axo-axonal arrangements. Alternatively, the enkephalin nerve fibres in the smooth muscle are afferent elements involved in mediating sensory impulses to the myenteric plexus.     

The distribution of noradrenergic nerves and small,intensely fluorescent (SIF) cells in the cat urinary bladder

Summary Fluorescence and electron microscopy have been used to study the distribution of noradrenergic nerves in the smooth muscle of the cat urinary bladder. Using the former technique, relatively few fluorescent noradrenergic nerves were observed in the body and fundus, while a rich plexus occurred adjacent to muscle cells of the bladder neck. The trigone could not be distinguished neuromorphologically from detrusor muscle in this region. Electron microscopy showed that the majority of noradrenergic terminals in the body and fundus were associated with presumptive cholinergic axons, while in the bladder neck noradrenergic terminals formed typical neuroeffector relationships with individual smooth muscle cells.Numerous ganglia occurred both in the adventitia and among the smooth muscle bundles, particularly in the bladder neck. The majority of the nerve cell bodies were non-fluorescent, although many contained bright orange autofluorescent granules, believed to be lysosomes. A small minority of ganglion cells were associated with fluorescent noradrenergic nerve terminals, thereby providing structural evidence for limited intraganglionic inhibition. In addition, occasional groups of small intensely fluorescent (SIF) cells were observed in some intramural ganglia and these were subsequently identified in the electron microscope. The possibility that these cells may provide a second inhibitory influence on bladder activity was considered.     

Localisation and measurement of VIP in the genitourinary system of man and animals

VIP is present in the genitourinary system of man and animals. In man the highest concentrations are found in the penis, the uterus and vagina and in the urinary bladder. VIP nerves heavily innervate the erectile tissue of the male external genitalia, the uterine smooth muscle and blood vessels, the seromucous glands of the cervix, and the lamina propria and vaginal epithelium. In the urinary bladder, VIP nerves are located beneath the transitional epithelium, in the lamina propria and in the smooth muscle. Other areas well innervated by VIP nerves include the prostate, seminal vesicles and vasa deferentia. Chemical (phenol- and 6-OHDA) or surgical (hypogastric or pelvic nerve section) extrinsic denervation fail to deplete the genitourinary system of its VIP content, supporting the view that VIP-containing nerves originate from local ganglion cells. Indeed, neuronal cell bodies containing VIP are seen in the paracervical ganglia of the female genitalia, the para- or intramural bladder ganglia and scattered through the base of the cavernosum body, the neck of the bladder and the prostate. The finding of elevated levels of VIP in the local circulation after induced penile erection in man and mammals and the ability of VIP to relax the detrusor muscle of the bladder suggests that the peptide may be involved in penile erection and bladder relaxation, as does the marked VIP depletion in the penis or bladder in patients suffering from diabetic impotence or bladder instability.     

Occurrence of neurotrophin receptors and transmitters in the developing Xenopus gut

The ontogeny of gut innervation in the anuran amphibian Xenopus laevis was studied using immunohistochemistry on sections of whole larvae from NF stages 38-52. Immunoreactivity to acetylated tubulin confirmed the presence of nerve fibres as early as stages 38-39. Actin immunoreactivity was found at stage 41, indicating the presence of smooth muscle cells. Trk-like neurotrophin receptors were occasionally found in nerve fibres as soon as stages 38-39. Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) immunoreactivities coexisted in nerves innervating the gut wall from stages 40-41, and nitric oxide synthase (NOS) from stage 42. Substance P/neurokinin A (SP/NKA) occurred at stage 42. In all these cases, the first fibres were observed in the oesophagus. Calcitonin gene-related peptide (CGRP) was first observed in nerves at stage 48. In general, VIP/PACAP and NOS innervation was denser than the tachykinin innervation. In conclusion, the development of nerve fibres in the Xenopus gut is probably dependent on neurotrophins that may act via Trk-like receptors and occur before the gut wall is fully organised morphologically. Feeding in Xenopus larvae starts at NF stage 45. The study demonstrates that several of the transmitters investigated are expressed in the gut innervation (and in endocrine cells) prior to this stage.     

AChE-positive innervation of the ureters, urinary bladder, and urethra in pigs

Histochemical method of KARNOWSKY and ROOTS (1964) was used to discover the AChE-positive nerves. These nerve fibres were found in all layers of all organs under study. The ureter was weakly innervated, while the urinary bladder and the urethra possessed strong AChE-positive innervation. AChE-positive fibres were most abundant in the bladder trigone. Muscular membrane was the best supplied layer, both in the urinary bladder and in the urethra. Part of AChE-positive nerves was connected with the blood vessels in all organs under discussion.     

Immunoreactivity for high-affinity choline transporter colocalises with VAChT in human enteric nervous system

Cholinergic nerves are identified by labelling molecules in the ACh synthesis, release and destruction pathway. Recently, antibodies against another molecule in this pathway have been developed. Choline reuptake at the synapse occurs via the high-affinity choline transporter (CHT1). CHT1 immunoreactivity is present in cholinergic nerve fibres containing vesicular acetylcholine transporter (VAChT) in the human and rat central nervous system and rat enteric nervous system. We have examined whether CHT1 immunoreactivity is present in nerve fibres in human intestine and whether it is colocalised with markers of cholinergic, tachykinergic or nitrergic circuitry. Human ileum and colon were fixed, sectioned and processed for fluorescence immunohistochemistry with antibodies against CHT1, class III beta-tubulin (TUJ1), synaptophysin, common choline acetyl-transferase (cChAT), VAChT, nitric oxide synthase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP). CHT1 immunoreactivity was present in many nerve fibres in the circular and longitudinal muscle, myenteric and submucosal ganglia, submucosa and mucosa in human colon and ileum and colocalised with immunoreactivity for TUJ1 and synaptophysin confirming its presence in nerve fibres. In nerve fibres in myenteric ganglia and muscle, CHT1 immunoreactivity colocalised with immunoreactivity for VAChT and cChAT. Some colocalisation occurred with SP immunoreactivity, but little with immunoreactivity for VIP or NOS. In the mucosa, CHT1 immunoreactivity colocalised with that for VIP and SP in nerve fibres and was also present in vascular nerve fibres in the submucosa and on epithelial cells on the luminal border of crypts. The colocalisation of CHT1 immunoreactivity with VAChT immunoreactivity in cholinergic enteric nerves in the human bowel thus suggests that CHT1 represents another marker of cholinergic nerves.     

An immunohistochemical study of the innervation of the large intestine of the toad (Bufo marinus)

The distribution of intrinsic enteric neurons and extrinsic autonomic and sensory neurons in the large intestine of the toad, Bufo marinus, was examined using immunohistochemistry and glyoxylic acid-induced fluoresecence. Three populations of extrinsic nerves were found: unipolar neurons with morphology and location typical of parasympathetic postganglionic neurons containing immunoreactivity to galanin, somatostatin and 5-hydroxytryptamine were present in longitudinally running nerve trunks in the posterior large intestine and projected to the muscle layers and myenteric plexus throughout the large intestine. Sympathetic adrenergic fibres supplied a dense innervation to the circular muscle layer, myenteric plexus and blood vessels. Axons containing colocalized calcitonin gene-related peptide immunoractivity and substance P immunoreactivity distributed to all layers of the large intestine and are thought to be axons of primary afferent neurons. Five populations of enteric neurons were found. These contained immunoreactivity to vasoactive intestinal peptide, which distributed to all layers of the large intestine; galanin/vasoactive intestinal peptide, which projected to the submucosa and mucosa; calcitonin gene-related peptide/vasoactive intestinal peptide, which supplied the circular muscle, submucosa and mucosa; galanin, which projected to the submucosa and mucosa; and enkephalin, which supplied the circular muscle layer.     

Degeneration of adrenergic nerves in the urinary bladder during pregnancy

The adrenergic innervation of the urinary bladder of normal female and pregnant rats has been studied using a fluorescence histochemical method. The bladder is richly innervated by adrenergic nerve fibres as is evidenced by the presence of numerous adrenergic nerves in the adventitia, musculosa and submucosa. However, adrenergic nerve cells could not be observed. During pregnancy, adrenergic nerve fibres showed signs of degeneration, as most of the nerve fibres disappeared and the surviving fibres were much swollen. 10 days after parturition the pattern and density of adrenergic innervation became almost similar to those of the control animals.     

Adrenergic innervation of the ureters, urinary bladder, and urethra in pigs

Studies were conducted on 4 sexually mature and 4 immature pigs. Scraps of the ureters, urinary bladder, and urethra were cut with a freezing microtome. Fluorescence method of Torre and Surgeon (1976) was used to reveal the adrenergic innervation. It was found that the ureters were weakly supplied with the adrenergic nerves; most of the nerves were located in the muscular and submucosal membranes. Apex of the urinary bladder possessed the weakest innervation. More nerves were found in particular layers of the bladder corpus whereas bladder trigonum and cervix possessed numerous nerves. Adrenergic innervation of the urethra was similar to that of the urinary bladder's cervix. Adrenergic nerves were present in the serous and muscular membranes of both the urinary bladder and the urethra. Part of the nerve fibres was connected with blood vessels of the organs under study.     

Pathophysiology of overactive bladder and urge urinary incontinence

Storage symptoms such as urgency, frequency, and nocturia, with or without urge incontinence, are characterized as overactive bladder (OAB). OAB can lead to urge incontinence. Disturbances in nerves, smooth muscle, and urothelium can cause this condition. In some respects the division between peripheral and central causes of OAB is artificial, but it remains a useful paradigm for appreciating the interactions between different tissues. Models have been developed to mimic the OAB associated with bladder instability, lower urinary tract obstruction, neuropathic disorders, diabetes, and interstitial cystitis. These models share the common features of increased connectivity and excitability of both detrusor smooth muscle and nerves. Increased excitability and connectivity of nerves involved in micturition rely on growth factors that orchestrate neural plasticity. Neurotransmitters, prostaglandins, and growth factors, such as nerve growth factor, provide mechanisms for bidirectional communication between muscle or urothelium and nerve, leading to OAB with or without urge incontinence.