Per Alm

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

SummaryVIP (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.

Peptidergic (vasoactive intestinal peptide) nerves in the genito-urinary tract.

Abstract The feline genito-urinary tract receives a rich supply of nerve fibers displaying immuno-reactivity like that of vasoactive intestinal peptide. Nerves containing vasoactive intestinal peptide are particularly numerous in the trigonum area of the bladder, around the ureteral openings and in the upper part of the urethra in both sexes, in the epididymis, prostate and vas deferens, and in the uterine cervix. Ganglia located close to or within the wall of the trigonum area and of the upper urethra contain numerous immunoreactive nerve cell bodies, which may be the origin of the fibers containing vasoactive intestinal peptide that innervate these regions.

Distribution, origin and projections of nitric oxide synthase-containing neurons in gut and pancreas

Nitric oxide has been put forward as an important inhibitory neurotransmitter in the gut. Nitric oxide synthase-containing neurons were visualized by immunocytochemistry using antibodies against neuronal nitric oxide synthase or by beta-nicotinamide adenine dinucleotide phosphate diaphorase staining in whole mounts and cryostat sections from the gastrointestinal tract and pancreas of several mammals (mouse, rat, hamster, guinea-pig, cat and man). Nitric oxide synthase-containing neuronal cell bodies were numerous in the myenteric but fewer in the submucous ganglia all along the gut of all species. Varicose nerve terminals formed extensive networks in the circular smooth muscle and the myenteric ganglia. Nitric oxide synthase-containing nerve terminals were frequently found around the Brunner glands in the duodenum; scattered nerve terminals were also found in the gastric and colonic mucosa and around blood vessels in the submucosa all along the gut. In the rat small and large intestine nitric oxide synthase-containing submucous neurons terminated within the mucosa/submucosa and nitric oxide synthase-containing myenteric neurons issued short descending projections, approximately 3 mm, to the smooth muscle and other myenteric ganglia. In the pancreas of all species nitric oxide synthase-containing nerve cell bodies were regularly seen in intrapancreatic ganglia. Positive nerve fibers were mainly found within nerve trunks in interlobular spaces and as delicate fibers within the islets. Double staining for nitric oxide synthase and neuropeptides in intestine and pancreas of rat, guinea-pig and man revealed that only occasionally the nitric oxide synthase-containing nerve cell bodies stored in addition vasoactive intestinal peptide and neuropeptide Y, or enkephalin. However, nitric oxide synthase-containing nerve terminals, particularly those in the circular muscle of the gut, frequently contained vasoactive intestinal peptide/neuropeptide Y (rat and man) or vasoactive intestinal peptide/enkephalin (guinea-pig). In intrapancreatic ganglia few nitric oxide synthase-containing nerve cell bodies were also vasoactive intestinal peptide-immunoreactive. Coexistence of nitric oxide synthase and vasoactive intestinal peptide in nerve terminals could here be detected around blood vessels and interlobular ducts. The distribution of nitric oxide synthase indicates a major role of nitric oxide in the regulation of gut motility; a role in the regulation of blood flow and secretion in both gut and pancreas is also likely.

Nitric oxide synthase in pig lower urinary tract : immunohistochemistry, NADPH diaphorase histochemistry and functional effects

1 The distribution and colocalization of nitric oxide synthase (NOS)‐like immunoreactivity and NADPH diaphorase activity in the pig lower urinary tract were investigated by immunohistochemical and histochemical staining techniques. Functional in vitro studies were performed to correlate the presence of NOS‐immunoreactivity/NADPH diaphorase staining with smooth muscle responses involving the l‐arginine/nitric oxide (NO) pathway. 2 NOS‐immunoreactivity and NADPH diaphorase activity were expressed in nerve trunks and fine nerve fibres in and/or around muscular bundles in the detrusor, trigone and urethra. Thin nerve fibres that dispersed within the muscle bundles were mainly found in the urethral/trigonal area, whereas such fibres were less common in the detrusor. 3 Almost all neuronal structures that were NOS‐immunolabelled were also stained for NADPH diaphorase. In contrast, the urothelium, which was intensively stained by the NADPH diaphorase technique, remained unstained by immunohistochemistry. 4 Electrical field stimulation of pig isolated trigonal and urethral preparations induced relaxations, which were inhibited by tetrodotoxin (1 μm) and NG‐nitro‐l‐arginine (l‐NOARG, 10 μm). 5 l‐Arginine (1 mm), but not d‐arginine, inhibited (25–30%) electrically evoked detrusor contractions. This inhibition was reversed by l‐NOARG (0.1 mm). l‐Arginine did not inhibit detrusor contractions in the presence of scopolamine (1 μm) and had no direct smooth muscle effects per se. 6 Acetylcholine (1 nm − 10 μm) caused concentration‐dependent relaxations of noradrenaline‐induced contractions in pig vesical arteries. Removal of the endothelium practically abolished the acetylcholine‐induced relaxation. Pretreatment with l‐NOARG (0.1 mm and 0.3 mm) caused a rightward shift of the concentration‐response curves to acetylcholine, but the maximal relaxation obtained was significantly reduced (to 65 ± 12%; n = 6; P < 0.05) only at 0.3 mml‐NOARG. 7 In vessel segments contracted with K+ (60 mm), acetylcholine induced concentration‐dependent relaxations. When the vessels were incubated with 0.3 mml‐NOARG and then contracted with K+ (60 mm) all relaxant responses to acetylcholine were abolished. 8 The presence of NO synthesizing enzyme in nerve fibres and the pharmacological evidence for NO‐mediated relaxation of the trigone and urethra suggest that NO or a NO‐related substance may have a role in inhibitory neurotransmission in these regions. In the detrusor, the presence of NO‐synthesizing enzyme in nerves can be demonstrated, but its functional importance is unclear. NO, as well as other endothelium‐derived factors seem to be involved in the endothelium‐dependent acetylcholine‐induced relaxation of pig vesical arteries.

Correlation between p53, c-erbB-2, and topoisomerase II alpha expression, DNA ploidy, hormonal receptor status and proliferation in 356 node-negative breast carcinomas: prognostic implications

Various new prognostic indicators have been identified for mammary carcinomas, but the issue of their significance remains unsettled. The prognostic impact of p53, c‐erbB‐2, and topoisomerase IIα expression was investigated in relation to standard prognostic factors for carcinomas of the breast and to the tumour cell growth fraction. Paraffin‐embedded specimens of 356 node‐negative infiltrating ductal carcinomas were stained immunohistochemically using a polyclonal antiserum to c‐erb B‐2, and the monoclonal antibodies DO‐1 (p53), Ki‐S4 (topoisomerase IIα), and Ki‐S5 (Ki‐67). The patients were followed for a median duration of 99 months. Both p53 and c‐erb B‐2 were significantly associated with high tumour grade, large tumour size, DNA aneuploidy, lack of steroid hormone receptors, young age, and increased topoisomerase IIα and Ki‐67 expression levels. The correlation of p53 and c‐erb B‐2 was not significant. Topoisomerase IIα and Ki‐67 scores closely paralleled each other, indicating that both reflect the proliferative activity of tumour cells. A univariate analysis of overall (OS), specific (SS), and disease‐free survival (DFS) revealed all the above‐mentioned parameters to be statistically significant except patient age, which was relevant only to overall survival. Multivariate analysis with inclusion of all covariates selected tumour size and proliferation (topoisomerase IIα and Ki‐67) indices as independent predictors of survival in all three models. No additional information was gained by p53 or c‐erb B‐2. It is concluded that the proliferative activity, as assessed by topoisomerase IIα or Ki‐67 immunostaining, is the most useful indicator of breast cancer prognosis, except for tumour size. Copyright

NITRIC OXIDE SYNTHASE-CONTAINING NEURONS IN RAT PARASYMPATHETIC, SYMPATHETIC AND SENSORY GANGLIA : A COMPARATIVE STUDY

SummaryIn rats, the distribution of nerve structures staining for NADPH-diaphorase, and showing immunoreactivities for nitric oxide synthase (NOS), tyrosine hydroxylase and various neuropeptides was studied in sensory ganglia (dorsal root, nodose and trigeminal ganglia), in sympathetic ganglia (superior cervical, stellate, coeliac-superior and inferior mesenteric ganglia), parasympathetic ganglia (sphenopalatine, submandibular, sublingual and otic ganglia), and in the mixed parasympathetic/ sympathetic ganglia (major pelvic ganglia). The coincidence of neuronal cell bodies with strong NOS-immunoreactivity and strong NADPH diaphorase reactivity was almost total. The relative proportions of NOS-immunoreactive nerve cell bodies were largest in parasympathetic ganglia and major pelvic ganglia followed by sensory ganglia. In sympathetic ganglia no NOS-immunoreactive neuronal cell bodies could be detected. In parasympathetic and major pelvic ganglia, there was a very significant neuronal co-localization of immunoreactivities for NOS and vasoactive intestinal polypeptide (VIP). This was almost total in major pelvic ganglia, in which NOS-/VIP-immunoreactive nerve cell bodies were separate from sympathetic (tyrosine hydroxylase-/neuropeptide Y-immunoreactive), suggesting that NOS-/VIP-immuno-reactive neurons might also be parasympathetic.

Nitric oxide synthase in the brain of a teleost

The presence and distribution of the nitric oxide (NO) converting enzyme, NO synthase (NOS), was investigated in the brain of a teleost, the Atlantic salmon. Both NOS immunoreactive and NADPH diaphorase positive, non-neuronal and neuronal cell bodies, fibers and putative nerve terminals were identified throughout the brain. Even so, the staining was not identical in all regions. NO, synthesized by NOS-like enzymes, may play an important role in a diversity of cellular mechanisms in the brain of the salmon, including in neural systems related to olfactory, visual, hypophysiotrophic, viscero-sensoric and motor functions.

Involvement of nitric oxide in acute spinal cord injury: an immunocytochemical study using light and electron microscopy in the rat

The possibility that nitric oxide participates in the pathophysiology of spinal cord injury was examined using a constitutive isoform of neuronal nitric oxide synthase immunoreactivity in a rat model. Spinal cord trauma was produced by making an incision into the right dorsal horn of the T10-11 segments. Five h after trauma, a marked upregulation of NOS-immunostained neurons was seen in the perifocal T9 and T12 segments of the cord. The immunolabelling was most pronounced in the dorsal horn of the ipsilateral side. Topical application of an antiserum to nitric oxide synthase (NOS) 2 min after injury prevented the trauma-induced upregulation of NOS-immunoreactivity. In contrast, application of preabsorbed serum or L-NAME, an inhibitor to NOS, was ineffective in reducing the induction of NOS-immunoreactivity. Trauma caused a marked expansion of the cord and resulted in marked cell changes. This expansion and cell reaction was significantly reduced following application of NOS antiserum but it was not seen after application of preabsorbed antiserum or L-NAME. Our results for the first time show that a focal trauma to the spinal cord has the capacity to upregulate neuronal NOS immunoreactivity and that application of NOS antiserum has a neuro protective effect. This indicates that nitric oxide is somehow involved in the pathogenesis of secondary injuries after spinal cord trauma.

CHOLINERGIC NERVES IN HUMAN CORPUS CAVERNOSUM AND SPONGIOSUM CONTAIN NITRIC OXIDE SYNTHASE AND HEME OXYGENASE

PURPOSE To characterize the distribution of cholinergic nerves in the human corpus cavernosum (CC) and spongiosum (CS) using antibodies to the vesicular acetylcholine transporter (VAChT), and to compare this distribution to those of other transmitters/mediators or transmitter/mediator generating enzymes (heme oxygenases: HO-1 and HO-2; neuronal and endothelial NO synthases: nNOS and eNOS; vasoactive intestinal polypeptide: VIP; and tyrosine hydroxylase: TH), and to investigate NO- and carbon monoxide (CO)-mediated effects. MATERIALS AND METHODS Immunocytochemistry, confocal laser scanning microscopy, radioimmunoassay, and functional in vitro studies. RESULTS Along strands of smooth muscle in the CC and CS, rich numbers of VAChT-, nNOS-, VIP-, TH-, and very few HO-1-immunoreactive (-IR) nerve fibers were observed. Immunoreactivities for VAChT and nNOS, VAChT and VIP, and nNOS and VIP, were generally found in the same varicose nerve terminals. TH-IR nerve fibers or terminals did not contain immunoreactivities for VAChT, NOS or VIP. In the endothelium lining penile arteries, immunoreactivities for eNOS, HO-1, and HO-2 were detected. Single endothelial cells, lining the sinusoidal walls of the CC and CS, were found also to contain eNOS and HO-immunoreactivities. Noradrenaline (NA)-contracted preparations of CC and CS were relaxed by NO, CO, carbachol and by electrical stimulation of nerves. Inhibition of NO synthesis abolished electrically- and carbachol-induced relaxation. In NA-activated strips, relaxation induced by exogenously applied NO, but not those by CO, were accompanied by increases in intracellular levels of cyclic GMP. CONCLUSIONS VAChT, NOS and VIP are found in the same nerve terminals within the human CC and CS, suggesting that these terminals comprise a distinct population of parasympathetic, cholinergic nerves. Endothelially derived NO and the HO/CO system may have a complementary role in penile erection.

Protein gene product 9.5 (PGP 9.5)

SummaryThe guinea pig uterus is supplied by different populations of nerves which can be demonstrated by specific immunocytochemical and histochemical techniques. So far, there has been no single marker displaying entire peripheral innervation patterns. Recently, protein gene product (PGP) 9.5, a cytoplasmic protein in neurons and neuroendocrine cells, was found to visualize both different populations and subtypes of nerves. This prompted the present study of using PGP 9.5 for visualization of the whole uterine innervation. This was performed by the indirect immunofluorescence method using antiserum to PGP 9.5 raised in rabbits.PGP-immunoreactivity was present in all neuronal parts of the extrinsic and intrinsic uterine innervation, including different subpopulations of nerves. This was verified by chemical sympathectomy and sensory denervation with 6-hydroxydopamine and capsaicin-treatment respectively, and double immunostaining.By term a disappearance of uterine PGP-nerve-immunoreactivity was observed which was almost complete in fetus-bearing uterine tissue and further strengthens previous assumptions of a general, pregnancy-induced uterine neuronal degeneration.The developmental time-course and morphology of PGP-immunoreactive nerve structures was similar to that for other neuronal markers and support the suggestion of PGP-immunoreactivity as a general marker for the entire uterine innervation, and suggests that the presence of PGP 9.5-immunoreactivity may coincide with functional maturation of uterine innervation.