Nerves supplying the internal anal sphincter: an immunohistochemical study using donated elderly cadavers.

PURPOSE: Nerves serving the internal anal sphincter (NIAS) have been described as the lower rectal branches of the pelvic autonomic nerve plexus. However, their topographical anatomy and fiber components have remained unclear. METHODS: Using histological sections from ten elderly donated cadavers, we investigated the topographical anatomy and composite fibers of the NIAS using immunohistochemistry for S100 protein, neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP) and tyrosine hydroxylase (TH). RESULTS: At the 2-3 o'clock position in the lower rectum, the NIAS originated from nerves at the posterolateral corner of the prostate in males or in the lower paracolpium in females. The nerves ran inferiorly along the internal aspect of the levator ani muscle, and joined branches of the myenteric plexus at a level slightly above the epithelial junction. The NIAS contained both nNOS-positive parasympathetic nerve fibers and TH-positive sympathetic fibers, but VIP-positive fibers were few in number. CONCLUSIONS: The origin of the NIAS at the posterolateral corner of the prostate as well as in the lower paracolpium might be sacrificed or damaged during radical prostatectomy or tension-free vaginal tape insertion. Low anterior resection of rectal cancer will most likely render damage to the NIAS because of its intersphincteric course. Although the nerve composition of the NIAS is characterized by a higher proportion of sympathetic nerve fibers than the myenteric plexus in the large intestine, their role is unclear. However, evaluation of sphincteric function after surgery would appear to be difficult because of the complex control mechanism independent of nerve supply.

Differential distribution of parvalbumin- and calbindin-D28K-immunoreactive neurons in the rat periaqueductal gray matter and their colocalization with enzymes producing nitric oxide.

The distribution, colocalization with enzymes producing nitric oxide (NO), and the synaptic organization of neurons containing two calcium-binding proteins (CaBPs) - parvalbumin (Parv) and calbindin-D28K (Calb) - were investigated in the rat periaqueductal gray matter (PAG). Parv-immunopositive (ParvIP) neurons were detected in the mesencephalic nucleus and rarely in the PAG. CalbIP neurons were found both in the dorsolateral (PAG-dl) and ventrolateral PAG (PAG-vl); their size ranged from 112.96 µm(2) (PAG-dl) to 125.13 µm(2) (PAG-vl). Ultrastructurally Parv and Calb immunoreactivity was mostly found in dendritic profiles. Axon terminals containing each of the two CaBPs formed symmetric synapses. Moreover both Parv and Calb were used to label a subpopulation of NO-producing neurons. Colocalization was investigated using two protocols: (i) a combination of Calb and Parv immunocytochemistry (Icc) with nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry (Hi) and (ii) neuronal NO synthase-Icc (nNOS) (immunofluorescence). Both techniques demonstrated a complete lack of colocalization of Parv and NADPH-d/nNOS in PAG neurons. Double-labeled (DL) neurons (Calb-NADPH-d; Calb-nNOS) were detected in PAG-dl. NADPH-d-Hi/Calb-Icc indicated that 41-47% of NADPH-d-positive neurons contained Calb, whereas 17-23% of CalbIP cells contained NADPH-d. Two-color immunofluorescence revealed that 53-66% of nNOSIP cells colocalized with Calb and 24-34% of CalbIP neurons contained nNOS. DL neuron size was 104.44 µm(2); neurons labeled only with NADPH-d or Calb measured 89.793 µm(2) and 113.48 µm(2), respectively. Together with previous findings (Barbaresi et al. [2012]) these data suggest that: Therefore the important aspect of the PAG intrinsic organization emerging from this and previous double-labeling studies is the chemical diversity of NO-synthesizing neurons, which is likely related to the different functions in which these neurons are involved.

A QSAR and molecular modeling study on a series of 3, 4-dihydro-1-isoquinolinamines and thienopyridines acting as nitric oxide synthase inhibitors.

A quantitative structure-activity relationship (QSAR) and molecular modeling study were performed on a series of 3,4-dihyro-1-isoquinolinamines and thienopyridines reported by Beaton et al. [Beaton et al. (2001) Bioorg Med Chem Lett 11, 1023-1026, 1027-1030] as potent, highly selective inhibitors of two isoforms of nitric oxide synthase (NOS)--neuronal NOS (nNOS) and endothelial NOS (eNOS), in order to find the physicochemical properties that governed their activity and the mode of interaction with the receptors, so that still more potent compounds in the series could be suggested. A multiple regression analysis revealed that nNOS and eNOS inhibition potency of these compounds could be controlled by their hydrophobic property and molar refractivity, respectively. Thus, nNOS and eNOS inhibition was indicated to involve the hydrophobic interaction and steric effects, respectively, suggesting some structural differences of the two isoforms of NOS. Based on the correlations obtained, some new, more potent compounds belonging to the series were predicted. These compounds were then docked into the receptors to see their interactions and find out the docking scores. The docked structures of two representative compounds, whose interaction energies with nNOS and eNOS, respectively were found to be the lowest, were given as an example to exhibit the possible orientation of the compounds to interact with the receptors.

Chronic intermittent hypoxia induces NMDA receptor-dependent plasticity and suppresses nitric oxide signaling in the mouse hypothalamic paraventricular nucleus.

Chronic intermittent hypoxia (CIH) is a concomitant of sleep apnea that produces a slowly developing chemosensory-dependent blood pressure elevation ascribed in part to NMDA receptor-dependent plasticity and reduced nitric oxide (NO) signaling in the carotid body. The hypothalamic paraventricular nucleus (PVN) is responsive to hypoxic stress and also contains neurons that express NMDA receptors and neuronal nitric oxide synthase (nNOS). We tested the hypothesis that extended (35 d) CIH results in a decrease in the surface/synaptic availability of the essential NMDA NR1 subunit in nNOS-containing neurons and NMDA-induced NO production in the PVN of mice. As compared with controls, the 35 d CIH-exposed mice showed a significant increase in blood pressure and an increased density of NR1 immunogold particles located in the cytoplasm of nNOS-containing dendrites. Neither of these between-group differences was seen after 14 d, even though there was already a reduction in the NR1 plasmalemmal density at this time point. Patch-clamp recording of PVN neurons in slices showed a significant reduction in NMDA currents after either 14 or 35 d exposure to CIH compared with sham controls. In contrast, NO production, as measured by the NO-sensitive fluorescent dye 4-amino-5-methylamino-2',7'-difluorofluorescein, was suppressed only in the 35 d CIH group. We conclude that CIH produces a reduction in the surface/synaptic targeting of NR1 in nNOS neurons and decreases NMDA receptor-mediated currents in the PVN before the emergence of hypertension, the development of which may be enabled by suppression of NO signaling in this brain region.

Ultrastructural immunogold localization of nitric oxide synthase isoforms in rat and human eosinophils.

The involvement of nitric oxide (NO) as both pro and anti-inflammatory agent in allergic, airway inflammatory, and asthmatic diseases and the active participation of eosinophils in such ailments have been previously suggested. NO modulates eosinophil number, migration and their survival. The microenvironment of NO synthase (NOS) in subcellular organelles determines its rate and efficiency of catalysis, which in turn influences NO generation at distinct intracellular locales. The present study was undertaken to assess the intracellular distribution of NOS isoforms by transmission electron microscopy followed by morphometric analysis in human and rat eosinophils. Rat eosinophils were explored in parallel, and since they are widely used as model systems to mimic human diseases, a comparative study on NOS localization patterns might provide useful information in deciphering NO role in diverse aspects of eosinophil-related inflammatory ailments. The results demonstrated predominance of neuronal NOS (nNOS) in the eosinophilic granules and even distribution of inducible NOS (iNOS) and nNOS in the cytoplasm and nucleus of human eosinophils. In rat eosinophils, however, iNOS was mainly localized in the eosinophilic granules and nucleus, while nNOS was distributed evenly in cytoplasm and nucleus. Distribution of endothelial NOS (eNOS) in eosinophils was scanty. Differences in NOS isoforms and their localization in human and rat cells might have implications in differential mode of catalysis and functional contribution to eosinophil physiology and pathology, warranting detailed investigations. The present study highlights species-specific differences in the relative abundance and distribution pattern of NOS isoforms in rat and human eosinophils, which should be considered cautiously in interpreting the rat data to humans.

The formation of a complex between calmodulin and neuronal nitric oxide synthase is determined by ESI-MS.

Calmodulin (CaM) is an acidic ubiquitous calcium binding protein, involved in many intracellular processes, which often involve the formation of complexes with a variety of protein and peptide targets. One such system, activated by Ca2+ loaded CaM, is regulation of the nitric oxide synthase (NOS) enzymes, which in turn control the production of the signalling molecule and cytotoxin NO. A recent crystallographic study mapped the interaction of CaM with endothelial NOS (eNOS) using a 20 residue peptide comprising the binding site within eNOS. Here the interaction of CaM to the FMN domain of neuronal nitric oxide synthase (nNOS) has been investigated using electrospray ionization mass spectrometry (ESI-MS). The 46 kDa complex formed by CaM-nNOS has been retained in the gas-phase, and is shown to be exclusively selective for CaM.4Ca2+. Further characterization of this important biological system has been afforded by examining a complex of CaM with a 22 residue synthetic peptide, which represents the linker region between the reductase and oxygenase domains of nNOS. This nNOS linker peptide, which is found to be random coil in aqueous solution by both circular dichroism and molecular modelling, also exhibits great discrimination for the form of CaM loaded with 4[Ca2+]. The peptide binding loop is presumed to be configured to an alpha-helix on binding to CaM as was found for the related eNOS binding peptide. Our postulate is supported by gas-phase molecular dynamics calculations performed on the isolated nNOS peptide. Collision induced dissociation was employed to probe the strength of binding of the nNOS binding peptide to CaM.4Ca2+. The methodology taken here is a new approach in understanding the CaM-nNOS binding site, which could be employed in future to inform the specificity of CaM binding to other NOS enzymes.